POLYPEPTIDES FOR RESTORING ENDOTHELIAL FUNCTION AND

METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/804,339, filed February 12, 2019, the disclosure of which is expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers R01HL070715 and R01HL105731 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

FIELD

The invention is in the fields of cell and molecular biology, polypeptides, cellular repair, cellular regeneration, and therapeutic methods of use.

BACKGROUND

The endothelium is a fragile layer of cells that line blood vessels. Injury to the endothelium contributes to both acute (malperfusion, edema) and chronic (atherosclerosis, diabetes) pathology. The endothelium represents a model cell type to measure physiologic function in that when the endothelium is healthy, in response to acetylcholine, the cells release nitric oxide (NO) which leads to relaxation of the underlying smooth muscle. When the endothelium is injured (mechanical, chemical, oxidative, inflammatory) either acutely or chronically, endothelial function becomes impaired (endothelial dysfunction), NO production is decreased in response to injury, and there is less relaxation of smooth muscle.

Endothelial function also decreases with age. This may be due to chronic injury or failure of reparative or regenerative responses to injury. Injury leads to activation of stress activated signaling pathways, changes in gene expression, and a unique response in the endoplasmic reticulum (where protein manufacturing and processing occurs). Endoplasmic reticulum can be induced by the antibiotic tunicamycin, and treatment of the endothelium with tunicamycin leads to endothelial dysfunction. Injury also leads to release of ATP which causes endothelial dysfunction. ATP activates purinergic receptors (P2X7R) which subsequently lead to p38 MAPK activation. Activation of p38 MAPK is also associated with endothelial dysfunction. The antibiotic anisomycin activates p38MAPK, and treatment with anisomycin also causes endothelial dysfunction. p38MAPK is activated during inflammation by cytokines such as interleukin- 1 beta (IL-Ib), and treatment with IL-Ib leads to endothelial dysfunction. What is needed are novel polypeptides that can be used for restoring endothelial function and for treating conditions and diseases where improved endothelial function is beneficial.

The compounds, compositions, and methods disclosed herein address these and other needs.

SUMMARY

Disclosed herein are polypeptides and compositions for restoring endothelial function. The inventors have identified novel, non-naturally occurring chimeric polypeptides that restore endothelial function after stretch injury and endoplasmic reticulum stress injury.

In some aspects, disclosed herein is a polypeptide comprising: an amino acid sequence according to the general formula X1-X2; wherein XI comprises a transduction domain; and X2 compri es a polypeptide capable of restoring endothelial function; wherein X2 includes Z3; and wherein Z.3 is selected from a phosphoserine or a phosphoserine analog.

In some embodiments, XI is selected from GRKKRRQRRRPPQ (SEQ ID NO:3); AYARAAARQARA (SEQ ID NO:4); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:5); GWTLN S AGYLLGLINLK AL A AL AKKIL (SEQ ID NO:6); PLSSISRIGDP (SEQ ID NO:7); AAV ALLP A VLL ALL AP (SEQ ID NO:8); AAVLLPVLLAAP (SEQ ID NO:9); VTVL ALGAL AGV GV G (SEQ ID NO: 10); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO: 11); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 12); KLALKLALKALKAALKLA (SEQ ID NO: 13); KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 14); KAFAKLAARLYRKAGC (SEQ ID NO:15); KAFAKLAARLYRAAGC (SEQ ID NO: 16); AAFAKLAARLYRKAGC (SEQ ID NO:17); KAFAALAARLYRKAGC (SEQ ID NO: 18); KAFAKLAAQLYRKAGC (SEQ ID NO: 19), AGGGGY GRKKRRQRRR (SEQ ID NO:20); Y GRKKRRQRRR (SEQ ID NO:21); YARAAARQARA (SEQ ID NO:22); or LTVK (SEQ ID NO:23). In some embodiments, XI comprises YARAAARQARA (SEQ ID NO:22).

In some embodiments, X2 is selected from SPAARRA(pS)AILPG (SEQ ID NO:24); SPARRA(pS)AILPG (SEQ ID NO:25); SPAARRV(pS)AILPG (SEQ ID NO:26);

SPARRV (pS) AILPG (SEQ ID NO:27); SPAARGA(pS)AILPG (SEQ ID NO:28);

SPARGA(pS)AILPG (SEQ ID NO:29); ARRA(pS)AILPG (SEQ ID NO:30); ARRV (pS) AILPG (SEQ ID NO:31); ARGA(pS)AILPG (SEQ ID NO:32); or SPARRA(pS)ALLPG (SEQ ID NO:74). In some embodiments, X2 comprises SPAARRA(pS)AILPG (SEQ ID NO:24).

In some embodiments, Z3 comprises a phospboserine. In some embodiments, Z3 comprises a phospboserine analog.

In some embodiments, the polypeptide comprises the amino acid sequence Y ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO: l).

In some aspects, disclosed herein is a pharmaceutical composition comprising one or more polypeptides of the present invention and a pharmaceutically acceptable carrier.

The polypeptides and compositions disclosed herein comprise non-naturally occurring chimeric polypeptides for use as therapeutic agents for the following: (a) treating or preventing endothelial dysfunction; (b) preventing aging and the consequences of aging (for example, prolonging life (longevity)); (c) treating, preventing and/or reversing atherosclerosis, atherosclerotic lesions, and the consequences of atherosclerosis (myocardial infarction, heart failure, renal failure, stroke, peripheral vascular disease, amputation, death); (d) enhancing techniques for treating atherosclerotic lesions and preventing recurrence (re-stenosis) of atherosclerotic lesions; (e) treating or preventing cardiovascular complications of endothelial dysfunction (angina, myocardial infarction, stroke, death); (f) treating or preventing cardiovascular complications of endothelial dysfunction (angina, myocardial infarction, stroke, death) in patients with metabolic syndrome; (g) treating or preventing arterial stiffness and hypertension and the consequences of arterial stiffness and hypertension (stroke, heart failure); (h) treating or preventing failure of vascular conduits used as bypass grafts; (i) treating or preventing erectile dysfunction; (j) treating or preventing endothelial dysfunction (e.g., acute endothelial dysfunction) associated with injury, burn, acidosis, and/or sepsis and/or (k) to treat or preventing inflammatory diseases (for example, sepsis, rheumatoid arthritis, Crohn’s disease, asthma, COPD), chronic pain, or cancer. In some aspects, disclosed herein is a method for restoring endothelial function, comprising: administering to a subject in need thereof an effective amount of a polypeptide comprising:

an amino acid sequence according to the general formula CΊ-C2; wherein

XI comprises a transduction domain; and

X2 comprises a polypeptide capable of restoring endothelial function;

wherein X2 includes Z3; and

wherein Z3 is selected from a phosphoserine or a phosphoserine analog.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In some aspects, disclosed herein is a method for preventing aging, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising:

an amino acid sequence according to the general formula X1-X2; wherein

XI comprises a transduction domain: and

X2 comprises a polypeptide capable of restoring endothelial function;

wherein X2 includes Z3; and

wherein Z3 is selected from a phosphoserine or a phosphoserine analog.

In some aspects, disclosed herein is a method for restoring endothelial function, comprising: administering to a subject in need thereof an effective amount of a polypeptide comprising: an amino acid sequence according to the general formula XI -X2; wherein XI comprises a transduction domain; and

X2 is SPAARRA(pS)AILPG (SEQ ID NO:24);

wherein pS is phosphoserine.

In another aspect, the present invention provides isolated nucleic acid sequences encoding a polypeptide of the present invention. In further aspects, the present invention provides recombinant expression vectors comprising the nucleic acid sequences of the present invention, and host cells transfected with the recombinant expression vectors of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. FIG. 1 shows that aging leads to decreased endothelial function in rodent blood vessels. Freshly isolated aorta from young (4 months) and old (20 months) rats were suspended in the muscle bath. To determine the effect of injury on endothelial function, phenylephrine (PE)- precontracted tissues were treated with carbachol (CCH; 5xlO ⁷ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. Data are reported as mean responses ± standard deviation. n= 7-8. *p<.05, two-way ANOVA. As seen in Fig. 1, endothelial function is lower in old rats.

FIG. 2 shows that the NiPp polypeptide (SEQ ID NO:l) restores endothelial function in rodent aortic tissue after ATP injury. Freshly isolated rat aorta was treated with 3'-0-(4- Benzoyl)benzoyl adenosine 5'-triphosphate (BzATP, ImM), an analogue of ATP, to induce injury in the absence or presence of NiPp (100 and 500mM) for 1 hour at room temperature. To determine the effect of injury on endothelial function, phenylephrine (PE)-precontracted tissues were treated with carbachol (CCH; 5xlO ⁷ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. Data are reported as mean responses ± standard deviation. n= 5-6. *p<.05, paired t-test. As seen in FIG. 2, ATP injury led to decreased endothelial function that was restored by treatment with NiPp.

FIG. 3 shows that the NiPp polypeptide (SEQ ID NO:l) restores endothelial function in rodent aortic tissue after stretch (mechanical) injury. Freshly isolated rat aorta was subjected to subfailure stretch (to haptic endpoint, approximately 2 times the resting length and treated with NiPp (500mM) for 1 hour at room temperature. To determine the effect of injury on endothelial function, phenylephrine-precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M) and the percent relaxation was determined as a change to the maximal PE- induced contraction. Data are reported as mean responses ± standard deviation. n= 5. *p<.05, two-way ANOVA. As seen in FIG. 3, stretch injury led to decreased endothelial function that was restored by treatment with NiPp.

FIG. 4 shows that the NiPp polypeptide (SEQ ID NO:l) restores endothelial function in rodent aortic tissue after endoplasmic reticulum stress (tunicamycin) injury. Freshly isolated rat aorta was treated with tunicamycin (TM) to induce injury in the absence or presence of NiPp (100 and 500mM) for 2 hours at room temperature. To determine the effect of injury on endothelial function, phenylephrine (PE)-precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. Data are reported as mean responses ± standard deviation. n=6. *p<.05, two-way ANOVA. As seen in FIG. 4, injury led to decreased endothelial function that was restored by treatment with NiPp. FIG. 5 shows that the NiPp polypeptide (SEQ ID NO:l) restores endothelial function in aged human saphenous veins. Human saphenous veins (HSV) were collected from patients undergoing coronary artery bypass grafting procedures and treated with NiPp (500mM) for 1 hour at room temperature. PE-precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M). Percent relaxation was determined as a change to the maximal PE-induced contraction. Data are reported as mean responses ± standard deviation. n= 2. Baseline endothelial function in HSV was low. Treatment with NiPp led to increased endothelial function in human saphenous veins (HSV). As seen in FIG. 5, when normalized to baseline relaxation, NiPp led to 1 83+24% in relaxation responses in HSV.

FIGS. 6A and 6B show that polypeptide restores endothelial function in aged human saphenous veins. FIG. 6A shows human saphenous veins (HSV) were collected from patients undergoing coronary artery bypass grafting procedures (age=66.4 ± 8.8) and treated with NiPp (IOOmM) for 1 hour at room temperature. PE-precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M). Percent relaxation were determined as a change to the maximal PE- induced contraction. Data are reported as mean responses ± standard deviation. n= 10. FIG. 6B shows baseline endothelial function in HSV was low. Treatment with NiPp led to increased endothelial function in HSV. When normalized to baseline relaxation, NiPp led to 255 ± 321.5% in relaxation responses in HSV.

FIGS. 7 A and 7B show that anisomycin-induced phosphorylation of p38 MAPK and decreased carbachol (CCH)-induced relaxation in rat aorta (RA) is prevented by treatment with Niban peptide (NiPp). RA rings were suspended in a muscle bath and incubated with either buffer alone (Ctrl), Aniso (200 mM) for 1 h, or NiPp (500 mM) for 30 min followed by Aniso (200 mM) for 1 h. FIG. 7A shows that percent relaxation induced by 5xlO ⁷ M CCH was calculated. *p<.05 in two-way ANOVA with Tukey post-tests. FIGS. 7B shows that RA rings were snap frozen either untreated or immediately after treatment. Proteins were extracted protein extracted and immunoblotted to examine p38MAPK phosphorylation. Quantification of relative phosphorylation to total p38MAPK level is shown. n=10, *p<.05 in one-way ANOVA with Tukey post-test. NiPp restores endothelial dysfunction and reduced p38 MAPK phosphorylation induced by anisomycin in RA.

FIGS. 8 A and 8B show that NiPp restores endothelial function and reduces p38MAPK phosphorylation in rodent aortic tissue after subfailure stretch injury. Freshly isolated rat aorta (RA) was subjected to subfailure stretch and incubated in PL in the absence (S) or presence of NiPp (500mM) for 1 h at room temperature. FIG. 8A shows that RA were either suspended in the muscle bath, contracted with PE and then treated with escalating doses of carbachol (CCH; 10 ⁸ to 10 ⁵ M). The percent relaxation was determined as a change to the maximal PE-induced contraction n = 5 rats; #p<.05, two-way ANOVA. FIG. 8B shows that RA were snap-frozen after stretch injury and treatment with NiPp (500mM) for lh, protein extracted and immunoblotted to examine P38MAPK phosphorylation. Quantification of relative phosphorylation to total p38MAPK level is shown. N = 10. *p<.05, in one-way ANOVA with Tukey post-test. NiPp restores endothelial dysfunction and reduced p38 MAPK phosphorylation induced by stretch injury in RA.

FIG. 9 shows that NiPp restores endothelial function in rodent aortic tissues after acidosis injury. Freshly isolated rat aorta (RA) was cut into rings and then pretreated in the absence or presence of NiPp (500mM) in PF for 30 min. Tissue rings were then transferred to normal saline (NS) to induce injury in the absence or presence of NiPp (500mM) and continued incubation for 2 h at room temperature. After treatments, RA were suspended in the muscle bath, contracted with PE and then treated with escalating doses of carbachol (CCH; 10 ⁸ to 10 ⁵ M). The percent relaxation was determined as a change to the maximal PE- induced contraction. n= 5-7 rats. *p<.05 in two-way ANOVA with Tukey post-test. NiPp restores NS- induced endothelial dysfunction in RA.

FIG. 10 shows that NiPp restores endothelial function in rodent aortic tissues after cytokines injury. Freshly isolated rat aorta (RA) was cut into rings and suspended in the muscle bath. Tissues were then treated with either IEIb alone (50 ng/ml) or cytomix (IEIb 50 ng/ml, TNFa, 10 ng/ml, IFNg 50 ng/ml) in the absence or presence of NiPp (500 mM) for 2 hrs, contracted with PE, and then treated with escalating doses of carbachol (CCH; 10 ⁸ to 10 ⁵ M). The percent relaxation was determined as a change to the maximal PE-induced contraction. n= 5-7 rats. *p<.05 in two-way ANOVA with Tukey post-test. NiPp restores cytokines-induced endothelial dysfunction in RA.

FIGS. 11A and 11B show that NiPp3 (non-P) or scr3NiPp (scrambled) did not restore BzATP-induced endothelial dysfunction in rat aorta. Freshly isolated rat aorta was treated with 3'-0-(4-Benzoyl)benzoyl adenosine 5'-triphosphate (BzATP, 1 mM), an analogue of ATP, to induce injury in the absence or presence of NiPp, NiPp3, or scr3NiPp (A, 100 and B, 500 mM) for 1 hour at room temperature. To determine the effect of injury on endothelial function, phenylephrine (PE) -precontracted tissues were treated with escalating doses of carbachol (CCH; 10 ¹⁰ to 10 ⁵ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction n = 7. *p<.05, two-way ANOVA with Tukey post-tests. There were significant differences between BzATP-treated vs Ctrl or NiPp-treated RA. NiPp3 (non-P) and scr3NiPp (scrambled) did not restore BzATP-induced endothelial dysfunction in RA indicating the specificity of NiPp activity.

FIG. 12 shows that NiPp restores endothelial function in rodent aortic tissues after cytokine injury. Freshly isolated rat aorta (RA) was cut into rings and suspended in the muscle bath. Tissues were then treated with the cytokine IIAb (50 ng/ml) in the absence or presence of NiPp, NiPp3, or scr3NiPp (100 mM) for 2hrs, contracted with PE, and then treated with escalating doses of carbachol (CCFi; 10 ⁸ to 10 ⁵ M). The percent relaxation was determined as a change to the maximal PE-induced contraction n = 5-7 rats. *p<.05 in two-way ANOVA with Tukey post-tests. There were significant differences between IL 1 b-trcatcd vs Ctrl or NiPp- treated RA. NiPp3 (non-P) and scr3NiPp (scrambled) did not restore IL^-induced endothelial dysfunction in RA indicating the specificity of NiPp activity.

FIG. 13 shows that NiPp3 (non-P) or scr3NiPp (scrambled) did not restore stretch- induced endothelial dysfunction in rat aorta. Freshly isolated rat aorta was subjected to subfailure stretch (to haptic endpoint, approximately 2 times the resting length) and treated with NiPp, NiPp3, or scr3NiPp (500mM) for 1 hour at room temperature. To determine the effect of injury on endothelial function, phenylephrine-precontracted tissues were treated with carbachol (CCFi; 10 ⁸ to 10 ⁵ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. Data are reported as mean responses ± standard deviation. n=8. *p<.05, two-way ANOVA with Tukey post-tests. There were significant differences between Stretch (S) vs. Ctrl or NiPp-treated RA. NiPp3 (non-P) and scr3NiPp (scrambled) did not restore stretch-induced endothelial dysfunction in RA indicating the specificity of NiPp activity.

FIGS. 14A and 14B show kinase profiling of polypeptides. NiPp, scr3NiPp, and NiPp3 were profiled against 490 kinases at lOOuM using the SelectScreen Kinase Profiling Service (www.thermofisher.com). FIG. 14A shows top 2 candidates showing more than 60% inhibition by NiPp. FIG. 14B shows kinase dendrogram showing proportional circle to % inhibition using KinMap (www.kinhub.org). Differential inhibitory activities were demonstrated by the peptides. NiPp inhibits MSK1 and p38MAPK alpha by >60%. These two kinases are central to the p38MAPK kinase signaling cascade that play important roles in stress and inflammatory responses. NiPp-specific inhibitory activity to p38 MAPK were not detected for NiPp3 (non- phosphorylated) or scr3NiPp (scrambled) polypeptides.

FIGS. 15A to 15D show that anisomycin-induced phosphorylation of p38 MAPK and decreased carbachol (CCFi)-induced relaxation in rat aorta (RA) is prevented by treatment with Niban peptide (NiPp). RA rings were suspended in a muscle bath and incubated with either buffer alone (Ctrl), Aniso (200 mM) for 1 h, or NiPp (500 mM) for 30 min followed by Aniso (200 mM) for 1 h. FIG. 15A shows that RA were suspended in the muscle bath, contracted with PE and then relaxed with escalating doses of CCH (10 ⁸ to 10 ⁵ M). The force generated was determined and was adjusted to the weight and length of the tissue. Representative tracings from 1 out of 6 different rats are shown. Orange arrow heads indicate addition of PE (orange) and CCH (blue). FIG. 15B shows that percent relaxation induced by 5 x 10 ⁷ M CCH was calculated. *p<.05 in one-way ANOVA with Tukey post-test. FIG. 15C shows that RA rings were snap frozen either untreated or immediately after treatment. Proteins were extracted and phosphorylation of p38 MAPK was determined by Western blot analysis. Western blots shown are representative of 1 out of 10 rats. FIG. 15D shows quantification of relative phosphorylation to total p38 MAPK level. n=10, *p<.05 in one-way ANOVA with Tukey post-test. Data are expressed as mean ± SD.

FIGS. 16A and 16B show that NiPp restores endothelial function in rodent aortic tissues after acidosis injury and P2X7R activation. FIG. 16A shows that freshly isolated rat aorta (RA) was cut into rings and then pretreated in the absence or presence of NiPp (500 mM) in PL for 30 min. Tissue rings were then transferred to NS and continued incubation in the absence or presence of NiPp (500 mM) for 2 h at room temperature. Control rings (Ctrl) were incubated in PL for 2.5 h. FIG. 16B shows that RA was either left untreated (Ctrl) or treated with BzATP (1 mM) in the absence of presence NiPp (500 mM) in PL for 1 h at room temperature. After treatments, RA were suspended in the muscle bath, contracted with PE and then treated with escalating doses of carbachol (CCH; 10 ⁸ to 10 ⁵ M). The percent relaxation was determined as a change to the maximal PE-induced contraction. Percent relaxation to 5xl0 ⁷ M CCH is shown. n= 5-7 rats. *p<.05 in one-way ANOVA with Tukey post-test. Data are expressed as mean ± SD.

FIGS. 17A and 17B show that NiPp improves endothelial relaxation in human saphenous veins (HSV). FIG. 17A shows that HSV, collected from patients undergoing CABG immediately after surgical harvest, were either incubated in PL in the absence (Ctrl) or presence of NiPp (100 mM) for 2 h at room temperature. HSV were suspended in the muscle bath, contracted with PE and treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M). The percent relaxation was determined as a change to the maximal PE-induced contraction. Percent relaxation to 5x10 ⁶ M CCH is shown. n=10; *p<.05 in paired t-test. FIG. 17B shows patient demographic variables. Data are expressed as mean ± SD.

FIG. 18 shows batch performance of NiPp. NiPp was synthesized at 3 separate times and tested in the muscle bath using rat aorta (RA). RA was either left untreated (Ctrl) or treated with BzATP (1 mM) in the absence of presence of NiPp (500 mM) in PL for 2 h at room temperature. After treatments, RA were suspended in the muscle bath, contracted with PE and then treated with escalating doses of carbachol 5xl0 ⁷ M CCH. The percent relaxation was determined as a change to the maximal PE-induced contraction. The three batches displayed similar bioactivity. n= 6 rats for each batch. *p<.05 in one-way ANOVA with Tukey post-test. Data are expressed as mean ± SD.

FIGS. 19A to 19C show kinase profiling of NiPp. FIG. 19A shows candidates showing more than 40% inhibition by NiPp. FIG. 19B shows kinase dendrogram showing proportional circle to % inhibition using KinMap (www.kinhub.org). FIG. 19C shows the kinase interaction network of NiPp molecular targets in human. Interaction of candidate molecular targets of NiPp identified in the kinase profiling assays were predicted using the STRING system (string-db- org) based on interaction of different type. Five different interactions were revealed among 4 putative molecular targets of NiPp.

FIG. 20 shows comparison of endothelial-dependent relaxation of vascular tissues from different species. Data from previous studies on endothelial function of saphenous veins collected from patients undergoing coronary artery bypass procedures or healthy adult pigs, and thoracic aorta collected from healthy adult rats were plotted for comparison. Basal endothelial-dependent relaxation was determined in the muscle bath by precontracting with sub-maximal doses of phenylephrine and relaxed with 5xl0 ⁷ M CCH.

DETAILED DESCRIPTION

Disclosed herein are polypeptides and compositions for restoring endothelial function. The inventors have identified novel, non-naturally occurring chimeric polypeptides that restore endothelial function after stretch injury and endoplasmic reticulum stress injury.

Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term“comprising” and variations thereof as used herein is used synonymously with the term“including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the term “consisting essentially of’ and“consisting of’ can be used in place of “comprising” and“including” to provide for more specific embodiments and are also disclosed.

The following definitions are provided for the full understanding of terms used in this specification.

Terminology

The single letter designation for amino acids is used predominately herein. As is well known by one of skill in the art, such single letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine.

As used herein, the singular forms“a”,“an” and“the” include plural referents unless the context clearly dictates otherwise. For example, reference to a“polypeptide” means one or more polypeptides.

As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

The term“polypeptide” or“protein” is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds, except where noted. The polypeptides described herein may be chemically synthesized or recombinantly expressed. In some embodiments, the polypeptides of the present invention are chemically synthesized. Synthetic polypeptides, prepared using the known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (N-a-amino protected N-a-t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85: 2149-2154), or the base-labile N-a- amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37 : 3403-3409). Both Fmoc and Boc N-a-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, the polypeptides can be synthesized with other N-a-protecting groups that are familiar to those skilled in this art.

Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35: 161-214, or using automated synthesizers. The polypeptides disclosed herein may comprise D-amino acids (which are resistant to I, -amino acid-specific proteases in vivo), a combination of D- and I, -ami no acids, and various "designer" amino acids (e.g. b-methyl amino acids, C-a- methyl amino acids, and N-a-methyl amino acids, etc.) to convey special properties. Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine.

In addition, the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. For example, a peptide may be generated that incorporates a reduced peptide bond, i.e., Ri— Cth— NH— R2, where Ri and R2 are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a polypeptide would be resistant to protease activity and would possess an extended half-live in vivo.

Conservative substitutions of amino acids in proteins and polypeptides are known in the art. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, He, Feu; Asp, Glu; Asn, Gin; Ser, Thr; Fys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

Substantial changes in protein function or immunological identity are made by selecting substitutions that are less conservative, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.

A“derivative” of a protein or peptide can contain post-translational modifications (such as covalently linked carbohydrate), depending on the necessity of such modifications for the performance of a specific function.

The“fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment possesses a bioactive property (for example, restoring endothelial function).

A“variant” refers to a molecule substantially similar in structure and immunoreactivity. Thus, provided that two molecules possess a common immunoactivity and can substitute for each other, they are considered“variants” as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical. Thus, in one embodiment, a variant refers to a protein whose amino acid sequence is similar to a reference amino acid sequence, but does not have 100% identity with the respective reference sequence. The variant protein has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the variant protein has an amino acid sequence which is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the reference sequence. For example, variant sequences which are at least 95% identical have no more than 5 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence. Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using any available sequence alignment program. An example includes the MEGALIGN project in the DNA STAR program. Sequences are aligned for identity calculations using the method of the software basic local alignment search tool in the BLAST network service (the National Center for Biotechnology Information, Bethesda, Md.) which employs the method of Altschul, S. F., Gish, W„ Miller, W„ Myers, E. W. & Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410. Identities are calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are not ignored when making the identity calculation.

As used herein, the term“capable of restoring endothelial function” refers to agents (for example, polypeptides) that can improve the functioning of endothelial cells, or improve the symptoms associated with defects in endothelial function. In one assay for measuring endothelial function, for example, phenylephrine (PE)-precontracted tissues are treated with carbachol (CCH; with a concentration including, for example, 10 ⁸ to 10 ⁵ M) and the percent relaxation is determined as a change to the maximal PE-induced contraction. In some embodiments, the improvement in restoring endothelial function after injury (for example, stretch injury or endoplasmic reticulum stress injury) can be at least 5% (for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or more) greater than the endothelial function observed in a comparable injury control. In some embodiments, endothelial function can be restored to about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, of the endothelial function of a healthy control (for example, wild- type endothelial function).

The term“about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, 10%, ±5%, or ±1% from the measurable value.

Nucleic Acids and Polypeptides

In some aspects, disclosed herein is a polypeptide comprising;

an amino acid sequence according to the general formula X1-X2; wherein

XI comprises a transduction domain; and

X2 comprises a polypeptide capable of restoring endothelial function;

wherein X2 includes Z3; and

wherein Z3 is selected from a phosphoserine or a phosphoserine analog.

In some embodiments, XI comprises a transduction domain. As used herein, the term "transduction domain" means one or more amino acid sequence or any other molecule that can carry the active domain across cell membranes. These domains can be linked to other polypeptides to direct movement of the linked polypeptide across cell membranes. In some embodiments, the transducing molecules can be covalently linked to the active polypeptide. In some cases, the transducing molecules do not need to be covalently linked to the active polypeptide. In some embodiments, the transduction domain is linked to the rest of the polypeptide via peptide bonding. (See, for example, Cell 55: 1179-1188, 1988; Cell 55: 1189- 1193, 1988; Proc Natl Acad Sci USA 91 : 664-668, 1994; Science 285: 1569-1572, 1999; J Biol Chem. 276: 3254-3261, 2001 ; and Cancer Res 61 : 474-477, 2001). In some embodiments, any of the polypeptides as described herein would include at least one transduction domain. In a further embodiment, XI comprises one or more transduction domains.

In some embodiments, XI is selected from GRKKRRQRRRPPQ (SEQ ID NO:3); AYARAAARQARA (SEQ ID NO:4); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:5); GWTLN S AGYLLGLINLK AL A AL AKKIL (SEQ ID NO:6); PLSSISRIGDP (SEQ ID NO:7); AAV ALLP A VLL ALL AP (SEQ ID NO:8); AAVLLPVLLAAP (SEQ ID NO:9); VTVL ALGAL AGV GV G (SEQ ID NO: 10); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO: 11); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 12); KLALKLALKALKAALKLA (SEQ ID NO: 13); KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 14); KAFAKLAARLYRKAGC (SEQ ID NO:15); KAFAKLAARLYRAAGC (SEQ ID NO: 16); AAFAKLAARLYRKAGC (SEQ ID NO:17); KAFAALAARLYRKAGC (SEQ ID NO: 18); KAFAKLAAQLYRKAGC (SEQ ID NO: 19); AGGGGY GRKKRRQRRR (SEQ ID NO:20); Y GRKKRRQRRR (SEQ ID NO:21); Y ARA A ARQAR A (SEQ ID NO:22); LTVK (SEQ ID NO:23); or a fragment, variant, or derivative thereof.

In some embodiments, XI comprises GRKKRRQRRRPPQ (SEQ ID NO:3). In some embodiments, XI comprises AYARAAARQARA (SEQ ID NO:4). In some embodiments, XI comprises DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:5). In some embodiments, XI comprises GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:6). In some embodiments, XI comprises PLSSISRIGDP (SEQ ID NO:7). In some embodiments, XI comprises AAVALLPAVLLALLAP (SEQ ID NO:8). In some embodiments, XI comprises AAVLLPVLLAAP (SEQ ID NO:9). In some embodiments, XI comprises VTVLALGALAGVGVG (SEQ ID NO: 10). In some embodiments, XI comprises GALFLGWLGAAGSTMGAWSQP (SEQ ID NO: 11). In some embodiments, XI comprises GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 12). In some embodiments, XI comprises KLALKLALKALKAALKLA (SEQ ID NO: 13). In some embodiments, XI comprises KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 14). In some embodiments, XI comprises KAFAKLAARLYRKAGC (SEQ ID NO: 15). In some embodiments, XI comprises KAFAKLAARLYRAAGC (SEQ ID NO:16). In some embodiments, XI comprises

AAFAKLAARLYRKAGC (SEQ ID NO: 17). In some embodiments, XI comprises

KAFAALAARLYRKAGC (SEQ ID NO: 18). In some embodiments, XI comprises KAFAKLAAQLYRKAGC (SEQ ID NO: 19). In some embodiments, XI comprises AGGGGYGRKKRRQRRR (SEQ ID NO:20). In some embodiments, XI comprises YGRKKRRQRRR (SEQ ID NO:21). In some embodiments, XI comprises YARAAARQARA (SEQ ID NO:22). In some embodiments, XI comprises LTVK (SEQ ID NO:23).

In some embodiments, XI comprises a sequence that is at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) identical to an amino acid sequence selected from GRKKRRQRRRPPQ (SEQ ID NO:3); AYARAAARQARA (SEQ ID NO:4);

DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:5);

GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:6); PLSSISRIGDP (SEQ ID NO:7); AAVALLPAVLLALLAP (SEQ ID NO:8); AAVLLPVLLAAP (SEQ ID NO:9); VT VL ALGAL AGV GV G (SEQ ID NO: 10); G ALFLGWLG A AGS TMG A W S QP (SEQ ID NO: 11); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 12);

KLALKLALKALKAALKLA (SEQ ID NO: 13); KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 14); KAFAKLAARLYRKAGC (SEQ ID NO:15); KAFAKLAARLYRAAGC (SEQ ID NO: 16); AAFAKLAARLYRKAGC (SEQ ID NO:17); KAFAALAARLYRKAGC (SEQ ID NO: 18); KAFAKLAAQLYRKAGC (SEQ ID NO: 19), AGGGGYGRKKRRQRRR (SEQ ID NO:20); YGRKKRRQRRR (SEQ ID NO:21); YARAAARQARA (SEQ ID NO:22); and LTVK (SEQ ID NO:23).

Niban (also known as FAM129A) is phosphoprotein involved in apoptosis, cancer, and the endothelial response to stress. Niban phosphorylation decreases in vascular tissues after injury. The inventors have identified certain portions of the Niban protein that are capable of restoring endothelial function. For example, the inventors have identified novel, non-naturahy occurring chimeric polypeptides that restore endothelial function after stretch injury and endoplasmic reticulum stress injury.

In some embodiments, the X2 portion of the chimeric polypeptide comprises a fragment of Niban (or a variant thereof).

In some embodiments, the X2 portion of the chimeric polypeptide comprises a Niban (or FAM129A) homolog. In some embodiments, the X2 sequence can be from a mammal, for example, human, rat, mouse, etc.

In some embodiments, X2 is selected from SPAARRA(pS)AILPG (SEQ ID NO:24); SPARRA(pS)AILPG (SEQ ID NO:25); SPAARRV(pS)AILPG (SEQ ID NO:26);

SPARRV (pS) AILPG (SEQ ID NO:27); SPAARGA(pS)AILPG (SEQ ID NO:28);

SPARGA(pS)AILPG (SEQ ID NO:29); ARRA(pS)AILPG (SEQ ID NO:30); ARRV (pS) AILPG (SEQ ID N0:31); ARGA(pS)AILPG (SEQ ID NO:32); or SPARRA(pS)ALLPG (SEQ ID NO:74); or a fragment, variant, or derivative thereof.

In some embodiments, X2 comprises SPAARRA(pS)AILPG (SEQ ID NO:24). In some embodiments, X2 comprises SPARRA(pS)AILPG (SEQ ID NO:25). In some embodiments, X2 comprises SPAARRV(pS)AILPG (SEQ ID NO:26). In some embodiments, X2 comprises SPARRV(pS) AILPG (SEQ ID NO:27). In some embodiments, X2 comprises

SPAARGA(pS)AILPG (SEQ ID NO:28). In some embodiments, X2 comprises SPARGA(pS)AILPG (SEQ ID NO:29). In some embodiments, X2 comprises

ARRA(pS)AILPG (SEQ ID NO:30). In some embodiments, X2 comprises ARRV(pS)AILPG (SEQ ID NO:31). In some embodiments, X2 comprises ARGA(pS)AILPG (SEQ ID NO:32). In some embodiments, X2 comprises SPARRA(pS)ALLPG (SEQ ID NO:74).

In some embodiments, X2 comprises a sequence that is at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) identical to an amino acid sequence selected from SPAARRA(pS) AILPG (SEQ ID NO:24); SPARRA(pS)AILPG (SEQ ID NO:25); SPAARRV(pS)AILPG (SEQ ID NO:26); SPARRV (pS) AILPG (SEQ ID NO:27); SPAARGA(pS)AILPG (SEQ ID NO:28); SPARGA(pS)AILPG (SEQ ID NO:29); ARRA(pS)AILPG (SEQ ID NO:30); ARRV (pS) AILPG (SEQ ID NO:31); ARGA(pS)AILPG (SEQ ID NO:32); or SPARRA(pS)ALLPG (SEQ ID NO:74).

In some embodiments, the polypeptide is selected from

GRKKRRQRRRPPQSPAARRA(pS)AILPG (SEQ ID NO:33);

AY ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO:34);

DAATATRGRSAASRPTERPRAPARSASRPRRPVESPAARRA(pS)AILPG (SEQ ID NO:35); GWTLNSAGYLLGLINLKALAALAKKILSPAARRA(pS)AILPG (SEQ ID NO:36); PLSSISRIGDPSPAARRA(pS)AILPG (SEQ ID NO:37);

AAVALLPAVLLALLAPSPAARRA(pS)AILPG (SEQ ID NO:38);

AAVLLPVLLAAPSPAARRA(pS)AILPG (SEQ ID NO:39);

VT VL ALGAL AGV GV GSP A ARR A(pS) AILPG (SEQ ID NO:40);

GALFLGWLGAAGSTMGAWSQPSPAARRA(pS)AILPG (SEQ ID NO:41);

GWTLNSAGYLLGLINLKALAALAKKILSPAARRA(pS)AILPG (SEQ ID NO:42);

KLALKLALKALKAALKLASPAARRA(pS)AILPG (SEQ ID NO:43);

KETWWETWWTEWSQPKKKRKVSPAARRA(pS)AILPG (SEQ ID NO:44);

KAFAKLAARLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:45);

KAFAKLAARLYRAAGCSPAARRA(pS)AILPG (SEQ ID NO:46); AAFAKLAARLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:47);

KAFAALAARLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:48);

KAFAKLAAQLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:49),

AGGGGYGRKKRRQRRRSPAARRA(pS)AILPG (SEQ ID NO: 50);

Y GRKKRRQRRRSPAARRA(pS) AILPG (SEQ ID NO:51);

Y ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO:l); or LTVKSPAARRA(pS)AILPG (SEQ ID NO:52).

In some embodiments, the polypeptide comprises the amino acid sequence

Y ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO:l).

In some embodiments, any of the XI transduction domains (for example, SEQ ID NOs:3 to 23) can be used in combination with any of the X2 polypeptides (for example, SEQ ID NOs:24 to 32, or 74).

In some aspects, the XI and X2 polypeptide sequences can be reversed. Thus, in some aspects, disclosed herein is a polypeptide comprising:

an amino acid sequence according to the general formula X2-X1 ; wherein

XI comprises a transduction domain; and

X2 comprises a polypeptide capable of restoring endothelial function;

wherein X2 includes Z3; and

wherein Z3 is selected from a phosphoserine or a phosphoserine analog.

In some embodiments, the polypeptide comprises a sequence that is at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) identical to an amino acid sequence selected from

GRKKRRQRRRPPQSPAARRA(pS)AILPG (SEQ ID NO:33);

AY ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO:34);

DAATATRGRSAASRPTERPRAPARSASRPRRPVESPAARRA(pS)AILPG (SEQ ID NO:35); GWTLNSAGYLLGLINLKALAALAKKILSPAARRA(pS)AILPG (SEQ ID NO:36); PLSSISRIGDPSPAARRA(pS)AILPG (SEQ ID NO:37);

AAVALLPAVLLALLAPSPAARRA(pS)AILPG (SEQ ID NO:38);

AAVLLPVLLAAPSPAARRA(pS)AILPG (SEQ ID NO:39);

VT VL ALGAL AGV GV GSP A ARR A(pS) AILPG (SEQ ID NO:40);

GALFLGWLGAAGSTMGAWSQPSPAARRA(pS)AILPG (SEQ ID NO:41);

GWTLNSAGYLLGLINLKALAALAKKILSPAARRA(pS)AILPG (SEQ ID NO:42);

KLALKLALKALKAALKLASPAARRA(pS)AILPG (SEQ ID NO:43);

KETWWETWWTEWSQPKKKRKVSPAARRA(pS)AILPG (SEQ ID NO:44); KAFAKLAARLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:45);

KAFAKLAARLYRAAGCSPAARRA(pS)AILPG (SEQ ID NO:46);

AAFAKLAARLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:47);

KAFAALAARLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:48);

KAFAKLAAQLYRKAGCSPAARRA(pS)AILPG (SEQ ID NO:49),

AGGGGYGRKKRRQRRRSPAARRA(pS)AILPG (SEQ ID NO: 50);

Y GRKKRRQRRRSPAARRA(pS) AILPG (SEQ ID NO:51);

Y ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO:l); or LTVKSPAARRA(pS)AILPG (SEQ ID NO:52).

In some embodiments, the polypeptide is selected from

GRKKRRQRRRPPQARRA(pS)AILPG (SEQ ID NO:53);

AY ARAAARQARAARRA(pS) AILPG (SEQ ID NO:54);

DAATATRGRSAASRPTERPRAPARSASRPRRPVEARRA(pS)AILPG (SEQ ID NO:55); GWTLNSAGYLLGLINLKALAALAKKILARRA(pS)AILPG (SEQ ID NO:56);

PLSSISRIGDPARRA(pS)AILPG (SEQ ID NO:57);

AAV ALLP A VLL ALL AP ARR A(pS) AILPG (SEQ ID NO:58);

AAVLLPVLLAAPARRA(pS)AILPG (SEQ ID NO:59);

VT VL ALGAL AGV GV GARRA(pS) AILPG (SEQ ID NO:60);

GALFLGWLGAAGSTMGAWSQPARRA(pS)AILPG (SEQ ID NO:61);

GWTLNSAGYLLGLINLKALAALAKKILARRA(pS)AILPG (SEQ ID NO:62);

KLALKLALKALKAALKLAARRA(pS) AILPG (SEQ ID NO: 63);

KETWWETWWTEWSQPKKKRKVARRA(pS) AILPG (SEQ ID NO: 64);

KAFAKLAARLYRKAGCARRA(pS)AILPG (SEQ ID NO:65);

KAFAKLAARLYRAAGCARRA(pS)AILPG (SEQ ID NO:66);

AAFAKLAARLYRKAGCARRA(pS)AILPG (SEQ ID NO:67);

KAFAALAARLYRKAGCARRA(pS)AILPG (SEQ ID NO:68);

KAFAKLAAQLYRKAGCARRA(pS)AILPG (SEQ ID NO:69),

AGGGGY GRKKRRQRRRARRA(pS) AILPG (SEQ ID NO:70);

Y GRKKRRQRRRARRA(pS) AILPG (SEQ ID NO:71);

Y ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO:l);

Y ARA A ARQ ARA ARR A(pS) AILPG (SEQ ID NO:76); or LTVKARRA(pS)AILPG (SEQ ID NO:72).

In some embodiments, the polypeptide comprises a sequence that is at least 60% (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) identical to an amino acid sequence selected from GRKKRRQRRRPPQARRA(pS)AILPG (SEQ ID NO:53);

AY ARAAARQARAARRA(pS) AILPG (SEQ ID NO:54);

DAATATRGRSAASRPTERPRAPARSASRPRRPVEARRA(pS)AILPG (SEQ ID NO:55); GWTLNSAGYLLGLINLKALAALAKKILARRA(pS)AILPG (SEQ ID NO:56);

PLSSISRIGDPARRA(pS)AILPG (SEQ ID NO:57);

AAV ALLP A VLL ALL AP ARR A(pS) AILPG (SEQ ID NO:58);

AAVLLPVLLAAPARRA(pS)AILPG (SEQ ID NO:59);

VT VL ALGAL AGV GV GARRA(pS) AILPG (SEQ ID NO:60);

GALFLGWLGAAGSTMGAWSQPARRA(pS)AILPG (SEQ ID NO:61);

GWTLNSAGYLLGLINLKALAALAKKILARRA(pS)AILPG (SEQ ID NO:62);

KLALKLALKALKAALKLAARRA(pS) AILPG (SEQ ID NO: 63);

KETWWETWWTEWSQPKKKRKVARRA(pS) AILPG (SEQ ID NO: 64);

KAFAKLAARLYRKAGCARRA(pS)AILPG (SEQ ID NO:65);

KAFAKLAARLYRAAGCARRA(pS)AILPG (SEQ ID NO:66);

AAFAKLAARLYRKAGCARRA(pS)AILPG (SEQ ID NO:67);

KAFAALAARLYRKAGCARRA(pS)AILPG (SEQ ID NO:68);

KAFAKLAAQLYRKAGCARRA(pS)AILPG (SEQ ID NO:69),

AGGGGY GRKKRRQRRRARRA(pS) AILPG (SEQ ID NO:70);

Y GRKKRRQRRRARRA(pS) AILPG (SEQ ID NO:71);

Y ARAAARQARASPAARRA(pS) AILPG (SEQ ID NO: l);

Y ARA A ARQ ARA ARR A(pS) AILPG (SEQ ID NO:76); or LTVKARRA(pS)AILPG (SEQ ID NO:72).

In some aspects, disclosed herein is a polypeptide comprising:

an amino acid sequence according to the general formula XI-X2; wherein

XI comprises a transduction domain; and

X2 is SPAARRA(pS)AILPG (SEQ ID NO:24); and

wherein pS is phosphoserine.

In some aspects, disclosed herein is a polypeptide comprising:

an amino acid sequence according to the general formula X1-X2; wherein

XI comprises a transduction domain; and

X2 is ARRA(pS)AILPG (SEQ ID NO:30); and

wherein pS is phosphoserine. In some embodiments, the one or more polypeptides disclosed herein are phosphorylated.

In some embodiments, Z3 comprises a phosphoserine or a phosphoserine analog. In some embodiments, Z3 comprises a phosphoserine. In some embodiments, Z3 comprises a phosphoserine analog.

According to various embodiments of the polypeptides of the invention, a“pS” residue may be a phosphoserine or a phosphoserine analog (or phosphoserine mimic). Examples of phosphoserine analogs/mimics include, but are not limited to, sulfoserine, amino acid mimics containing a methylene substitution for the phosphate oxygen, 4- phosphono(difluoromethyl)phenylanaline, and L-2-amino-4-(phosphono)-4,4-difuorobutanoic acid. Other phosphoserine mimics can be made by those of skill in the art; for example, see Otaka et al., Tetrahedron Letters 36: 927-930 (1995). In some embodiments, a phosphoserine analog contains a non-hyrirolysahle linkage to the phosphate group, e.g., a CF _? . group. See. e.g., U.S. Fat. Mo. 6,309,863.

In embodiments where the S residue is phosphorylated, the peptide can be synthesized using a phosphorylated amino acid (or phospho-mimic) during polypeptide synthesis, or the S residue can be phosphorylated after its addition to the polypeptide chain.

In another aspect, the present disclosure provides isolated nucleic acid sequences encoding a polypeptide of the present invention. In further aspects, the present disclosure provides recombinant expression vectors comprising the nucleic acid sequences of the present invention, and host cells transfected with the recombinant expression vectors of the present invention.

In some embodiments, the polypeptides are isolated. In some embodiments, the polypeptides are synthetic. In some embodiments, the polypeptides are recombinant. In some embodiments, the nucleic acid sequences are isolated. In some embodiments, the nucleic acid sequences are synthetic. In some embodiments, the nucleic acid sequences are recombinant.

The transduction of peptide motifs that modulate endothelial function provides for novel peptide-based therapeutics. One of the advantages of this approach is the evolutionary specificity of downstream protein targets. Receptor based modulation of signaling cascades leads to amplifying enzymatic activities. Thus, exploiting specific post-translational modifications of proteomic targets can be more stoichiometric and thus suitable for finer regulation of cellular processes. This approach also has advantages over gene therapy in that there are no delays in protein production or difficulties with regulating the amount of protein expression. Finally, this approach may be feasible for the treatment of specific modalities that are refractory to activation of upstream receptors or signaling cascades. For example, endothelial dysfunction associated with injury occurs coincident with downregulation of the expression of phosphorylated Niban and the polypeptides disclosed herein can recapitulate the endogenous phosphorylated Niban and restore endothelial function.

Compositions

In another aspect, the present disclosure provides pharmaceutical compositions, comprising one or more of the polypeptides disclosed herein, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are especially useful for carrying out the methods of the invention described below.

For administration, the polypeptides are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, dextran sulfate, heparin- containing gels, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as polyethylene glycol, palmitic acid and octadecanedioic acid. Such linkage can be covalent or non-covalent as is understood by those of skill in the art.

The polypeptides or pharmaceutical compositions thereof may be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. Embodiments for administration vary with respect to the condition being treated.

The polypeptides may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The polypeptides of the invention may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the polypeptides, and are not harmful for the proposed application.

Methods

The polypeptides and compositions disclosed herein comprise non-naturally occurring chimeric polypeptides for use as therapeutic agents for the following: (a) treating or preventing endothelial dysfunction; (b) preventing aging and the consequences of aging (for example, prolonging life (longevity)); (c) treating, preventing and/or reversing atherosclerosis, atherosclerotic lesions, and the consequences of atherosclerosis (myocardial infarction, heart failure, renal failure, stroke, peripheral vascular disease, amputation, death); (d) enhancing techniques for treating atherosclerotic lesions and preventing recurrence (re-stenosis) of atherosclerotic lesions; (e) treating or preventing cardiovascular complications of endothelial dysfunction (angina, myocardial infarction, stroke, death); (f) treating or preventing cardiovascular complications of endothelial dysfunction (angina, myocardial infarction, stroke, death) in patients with metabolic syndrome; (g) treating or preventing arterial stiffness and hypertension and the consequences of arterial stiffness and hypertension (stroke, heart failure); (h) treating or preventing failure of vascular conduits used as bypass grafts; (i) treating or preventing erectile dysfunction; (j) treating or preventing endothelial dysfunction (e.g., acute endothelial dysfunction) associated with injury, burn, acidosis, and/or sepsis and/or (k) to treat or preventing inflammatory diseases (for example, sepsis, rheumatoid arthritis, Crohn’s disease, asthma, COPD, chronic pain, cancer).

In some aspects, disclosed herein is a method for restoring endothelial function, comprising: administering to a subject in need thereof an effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for restoring endothelial function, comprising: administering to a subject in need thereof an effective amount of a polypeptide comprising:

an amino acid sequence according to the general formula XI-X2; wherein

XI comprises a transduction domain; and

X2 comprises a polypeptide capable of restoring endothelial function;

wherein X2 includes Z3; and

wherein Z3 is selected from a phosphoserine or a phosphoserine analog. In some aspects, disclosed herein is a method for preventing aging, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for preventing aging, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide comprising:

an amino acid sequence according to the general formula X1-X2; wherein

XI comprises a transduction domain: and

X2 comprises a polypeptide capable of restoring endothelial function;

wherein X2 includes Z3; and

wherein Z3 is selected from a phosphoserine or a phosphoserine analog.

Aging leads to endothelial function and endothelial function contributes to many of the diseases of aging such as coronary artery disease, stroke, hypertension, and diabetes. Thus, preventing or reversing endothelial dysfunction can decrease the morbidity and mortality of these diseases of aging and can increase lifespan.

In some aspects, disclosed herein is a method for restoring endothelial function, comprising: administering to a subject in need thereof an effective amount of a polypeptide comprising: an amino acid sequence according to the general formula X1 -X2; wherein XI comprises a transduction domain; and

X2 is SPAARRA(pS)AILPG (SEQ ID NO:24);

wherein pS is phosphoserine.

In some aspects, disclosed herein is a method for treating, preventing and/or reversing atherosclerosis, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating atherosclerotic lesions and preventing recurrence (re-stenosis) of atherosclerotic lesions, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating or preventing cardiovascular complications of endothelial dysfunction, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating or preventing arterial stiffness and/or hypertension, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein. In some aspects, disclosed herein is a method for treating or preventing failure of vascular conduits used as bypass grafts, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating or preventing endothelial dysfunction (e.g., acute endothelial dysfunction) associated with injury, acidosis, burn, or sepsis, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating or preventing inflammatory diseases (for example, sepsis, rheumatoid arthritis, Crohn’s disease, asthma, COPD), comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating or preventing chronic pain, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some aspects, disclosed herein is a method for treating or preventing cancer, comprising: administering to a subject in need thereof a therapeutically effective amount of a polypeptide disclosed herein.

In some embodiments, the inflammatory diseases, chronic pain, and/or cancer are associated with elevated activation levels and/or increased amount of a p38MAPK kinase. The p38MAPK can be, for example, r38a, r38b, r38g, or r38d. In some embodiments, the p38MAPK is p38MAPKa.

In another aspect, the disclosure provides methods for the use of a composition comprising a polypeptide comprising an amino acid sequence according to general formula

XI -X2; wherein

XI comprises a transduction domain; and X2 comprises a polypeptide capable of restoring endothelial function; wherein X2 includes Z3; and wherein Z3 is selected from a phosphoserine or a phosphoserine analog, for the preparation of a medicament for carrying out one or more of the following therapeutic uses: (a) treating or preventing endothelial dysfunction; (b) preventing aging and the consequences of aging (for example, prolonging life (longevity)); (c) treating, preventing and/or reversing atherosclerosis, atherosclerotic lesions, and the consequences of atherosclerosis (myocardial infarction, heart failure, renal failure, stroke, peripheral vascular disease, amputation, death); (d) enhancing techniques for treating atherosclerotic lesions and preventing recurrence (re-stenosis) of atherosclerotic lesions; (e) treating or preventing cardiovascular complications of endothelial dysfunction (angina, myocardial infarction, stroke, death); (f) treating or preventing cardiovascular complications of endothelial dysfunction (angina, myocardial infarction, stroke, death) in patients with metabolic syndrome; (g) treating or preventing arterial stiffness and hypertension and the consequences of arterial stiffness and hypertension (stroke, heart failure); (h) treating or preventing failure of vascular conduits used as bypass grafts; (i) treating or preventing erectile dysfunction; (j) treating or preventing endothelial dysfunction (e.g., acute endothelial dysfunction) associated with injury, burn, acidosis, and/or sepsis and/or (k) to treat or preventing inflammatory diseases (sepsis, rheumatoid arthritis, Crohn’s disease, asthma, COPD), chronic pain, or cancer.

As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s). The terms disorder and disease are used interchangeably herein.

As used herein, the term "inhibit" or "inhibiting" means to limit the disorder in individuals at risk of developing the disorder.

As used herein, the term“preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.

As used herein, "administering" includes in vivo administration, as well as administration directly to tissue ex vivo, such as vein grafts.

In some embodiments of the methods disclosed herein, such as reducing atherosclerotic lesions, the administering may be direct, by contacting a blood vessel in a subject being treated with one or more polypeptides of the invention. For example, a liquid preparation of one or more polypeptides according to the invention can be forced through a porous catheter, or otherwise injected through a catheter to the injured site, or a gel or viscous liquid containing the one or more polypeptides according to the invention can be spread on the injured site. In some embodiments of direct delivery, one or more polypeptides according to the invention can be delivered into cells at the site of injury or intervention. In some embodiments, delivery into cells is accomplished by using the one or more polypeptides according to the invention that include at least one transduction domain to facilitate entry into the cells. In various other embodiments of the methods disclosed herein, particularly those that involve reducing atherosclerotic lesions, the method is performed on a subject who has undergone, is undergoing, or will undergo a procedure selected from the group consisting of angioplasty, vascular stent placement, endarterectomy, atherectomy, bypass surgery (such as coronary artery bypass surgery; peripheral vascular bypass surgeries), vascular grafting, organ transplant, prosthetic device implanting, micro vascular reconstructions, plastic surgical flap construction, and catheter emplacement.

In a further embodiment, the methods disclosed herein are used for treating or preventing endothelial dysfunction. Endothelial dysfunction is associated with aging, diabetes, atherosclerosis, injury, burn, acidosis, and sepsis. Thus, the invention may be used for treating or preventing the complications of diabetes, atherosclerosis, aging, injury, burn, acidosis, and sepsis such as arterial lesions that lead to coronary artery disease, myocardial infarction, heart failure, renal failure stroke, limb loss, vascular hyperpermeability leading to malperfusion/edema, erectile dysfunction, and death.

The polypeptides may be administered systemically or via sustained release systemic administration to treat or prevent endothelial dysfunction or the consequences of endothelial dysfunction and atherosclerosis such as angina, myocardial infarction, stroke, death, limb loss, renal failure, sepsis, or erectile dysfunction. Atherosclerosis is a response to injury over time and is a leading cause of death. Hence, the polypeptides may be administered systemically or via sustained release systemic administration to treat or prevent aging and the consequences of aging resulting in the prolongation of a healthier life (longevity, health span). Prolonged impaired relaxation of blood vessels due to endothelial dysfunction leads to arterial stiffness. Thus, the polypeptides may be used to treat arterial stiffness and the consequences of arterial stiffness such as hypertension, stroke and heart failure.

To treat arterial lesions in coronary, renal, and peripheral artery, bypass grafts composed of prosthetic (dacron, PTFE, etc.) or autogenous (such as saphenous vein) materials may be used. The polypeptides may be used to improve both short and long-term graft success by systemic or local (graft) administration around the time period of graft implantation.

By the term“effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is“effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact“effective amount.” However, an appropriate“effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an“effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

An“effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

As used herein, a“therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a“prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.

The term“therapeutically effective amount” can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

As used herein, the term“pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term“pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

An effective amount of the polypeptides that can be employed ranges generally between about 0.01 pg/kg body weight and about 10 mg/kg body weight ( or between about 0.05 m g/kg and about 5 mg/kg body weight). Flowever, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.

As used herein, the term“subject” or“host” can refer to living organisms such as mammals, including, but not limited to humans, livestock, dogs, cats, and other mammals. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

Devices

In another aspect, the disclosure provides improved biomedical devices, wherein the biomedical devices comprise one or more of the polypeptides disclosed herein disposed on or in the biomedical device.

As used herein, a "biomedical device" refers to a device to be implanted into a subject, for example, a human being, in order to bring about a desired result. Example biomedical devices according to this aspect of the invention include, but are not limited to, patches, microneedles, stents, grafts, shunts, stent grafts, fistulas, angioplasty devices, balloon catheters, implantable drug delivery devices, wound dressings such as films (e.g., polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross- linked polymers containing about at least 60% water), foams (hydrophilic or hydrophobic), calcium alginates (nonwoven composites of fibers from calcium alginate), cellophane, and biological polymers.

As used herein, the term "graft" refers to both natural and prosthetic grafts and implants. In some embodiments, the graft is a vascular graft.

As used herein, the term "stent" includes the stent itself, as well as any sleeve or other component that may be used to facilitate stent placement.

As used herein, "disposed on or in" means that the one or more polypeptides can be either directly or indirectly in contact with an outer surface, an inner surface, or embedded within the biomedical device.

"Direct" contact refers to disposition of the polypeptides directly on or in the device, including but not limited to soaking a biomedical device in a solution containing the one or more polypeptides, spin coating or spraying a solution containing the one or more polypeptides onto the device, implanting any device that would deliver the polypeptide, and administering the polypeptide through a catheter directly on to the surface or into any organ. "Indirect" contact means that the one or more polypeptides do not directly contact the biomedical device. For example, the one or more polypeptides may be disposed in a matrix, such as a gel matrix or a viscous fluid, which is disposed on the biomedical device. Such matrices can be prepared to, for example, modify the binding and release properties of the one or more polypeptides as required.

The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

EXAMPLES

The following examples are set forth below to illustrate the polypeptides, compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Example 1. Introduction

The inner lining of the vascular wall consists of a monolayer of endothelial cells. Mechanical forces (blood flow disturbances, mechanical stretch), chemical stressors (glycemic, oxidative, osmotic, acidosis), inflammation, and aging are associated with dysfunction of this fragile endothelial monolayer. A common physiologic sequela of endothelial injury is impaired endothelial- dependent relaxation, often referred to as“endothelial dysfunction".

While multiple mechanisms have been implicated in promulgating endothelial injury, one of the common underlying themes is disruption of the endothelial membrane. Measurement of endothelial membrane injury has been performed by measuring extracellular release of biomolecules that have high intracellular concentrations. Adenosine triphosphate (ATP), in which there is a large gradient between intracellular (1-10 mM) and extracellular concentrations (1-10 mM), was one of the first markers of membrane injury. More recently, lactate dehydrogenase (LDH) release has been used as a marker of membrane injury. Loss of endothelial membrane integrity also leads to changes in transcellular resistance which can be measured with impedance (transepithelial/endothelial electrical resistance, TEER). Exposure of veins to acidic saline solutions such as Normal Saline (NS) that is widely used as a resuscitation fluid, or mechanical stretch during surgical harvest leads to endothelial injury and release of ATP, LDH and decreased TEER. Prolonged exposure to high concentrations of ATP activates the purinergic receptor, P2X7R. P2X7R activation is one of the most potent activators of the inflammasome. P2X7R modulates responses to injury via activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. p38 MAPK is also activated by environmental stress and inflammatory cytokines. p38 MAPK modulates a myriad of physiological processes through transcriptional regulation and/or activation of downstream kinases. Thus, increased extracellular ATP after injury is not only a marker of injury, but may also play a role in propagating the vascular“response to injury.”

A clinically relevant model of human vascular injury is the process of surgical harvest and preparation of human saphenous vein (HSV) prior to implantation as an autologous transplanted vascular graft. HSV is injured by mechanical stretch during harvest and pressure distention, storage in acidic NS solution, and orientation marking with surgical skin markers. To understand the response to surgical injury, segments of HSV removed atraumatically were compared to cognate segments after to harvest and preparation injury. Injured HSV segments demonstrated impaired endothelial dependent relaxation which was associated with a decrease in Niban phosphorylation. The Niban gene, also known as FAM129A, was first identified as a gene upregulated in cancer. Niban is involved in the regulation of cancer progression, cell proliferation, apoptosis and endoplasmic reticulum (ER) stress responses. Ji et al reported that Akt-dependent phosphorylation of Niban is involved in ultra-violet (UV)-induced cell apoptosis. In Niban knockout mice, the ER stress response pathway was affected as phosphorylation of eukaryotic translational initiation factor (elF) 2a, p70 ribosomal S6 subunit kinase (S6K) 1, and eukaryotic initiation factor 4E- binding protein (4E-BP) were altered, implicating a role of Niban in modulating translation in cell death signaling. In a rat aorta model of subfailure stretch injury, decreased Niban phosphorylation was associated with an increase in p38 MAPK phosphorylation, supporting the interplay between p38 MAPK after acute vascular injury. Taken together, these data indicate that Niban plays a protective role in response to cellular injury.

In the examples herein, the relationship between p38 MAPK and Niban phosphorylation and the mechanistic interplay of these molecules that contributes to endothelial dysfunction was investigated. Cell-permeant phospho-peptide mimetics of Niban (NiPp) were designed, synthesized, and characterized to function as a therapeutic for treating endothelial dysfunction. Example 2. Materials and Methods.

Materials. All chemicals and reagents were purchased from Sigma unless otherwise described. The peptide (NiPp) used in this study was synthesized by f-moc chemistry and purified using high-performance chromatography by EZBiolab (NJ).

Tissue procurement. Aorta (RA) was procured from 250-300 g, Sprague Dawley rats. Animal procedures followed study protocols approved by the Vanderbilt Institutional Animal Care and Use Committee and adhered to National Institute of Health guidelines for care and use of laboratory animals. Immediately after euthanasia by CO2, the thoracic and abdominal RA was isolated via an incision along the mid-abdomen, placed in heparinized PlasmaLyte (PL; 10 unit heparin/mL PlasmaLyte, Baxter, Deerfield IL) and transported to the laboratory for immediate testing.

Human saphenous veins (HSV) was obtained under approval from the Institutional Review Board of Vanderbilt University Medical Center from consented patients undergoing coronary artery bypass grafting procedures. HSV segments were collected immediately following surgical harvest and transported to the laboratory in PL for immediate experimentation.

Measurement of endothelial- dependent relaxation. Rings of HSV or RA (1-2 mm) were suspended in a muscle bath containing a bicarbonate buffer (120 mM sodium chloride, 4.7 mM potassium chloride, 1.0 mM magnesium sulfate, 1.0 mM monosodium phosphate, 10 mM glucose, 1.5 mM calcium chloride, and 25 mM sodium bicarbonate, pH 7.4) equilibrated with 95% 0 ₂ /5% CO2 at 37 °C for 1 h at a resting tension of 1 g, manually stretched to three times the resting tension, and maintained at resting tension for an additional 1 h.

This produced the maximal force tension relationship as previously described. After equilibration, the rings were primed with 110 mM potassium chloride (with equimolar replacement of sodium chloride in bicarbonate buffer) to determine functional viability. Viable rings were then tested for contractile response to a dose of phenylephrine (PE) to yield submaximal contraction (approximately 60-70% of maximum KC1; 5xl0 ^-6 M for HSV and 1- 5xl0 ^-7 M for RA) and relaxed with carbachol (CCH, 5xl0 ⁷ M), an acetylcholine analogue, to determine endothelial-dependent relaxation responses. Lorce measurements were obtained using the Radnoti force transducer (model 159901A, Radnoti LLC, Monrovia, CA) interfaced with a PowerLab data acquisition system and LabChart software (AD Instruments Inc., Colorado Springs, CO). Contractile responses were defined by stress, calculated using force generated by tissues as follows: stress (xlO ⁵ N/m ² ) = force (g) x 0.0987/area, where area = wet weight (mg)/ at maximal length (mm)]/1.055. Relaxation was calculated as percent change in stress compared to the maximal PE- induced contraction (set as 100%). Each data point was averaged from at least two rings from the same specimen. To determine concurrent signaling events, tissues were frozen in liquid nitrogen at relevant timepoints.

p38 MAPK activation with Anisomycin. To activate p38 MAPK, thoracic RA were cut into rings and suspended in the muscle bath. Rings were left untreated, treated with anisomycin (200 mM) for lh, or pretreated with NiPp (500 pM) for 30 min followed by anisomycin (200pM) for 1 h. After treatments, endothelial-dependent relaxation was determined or tissues were snap-frozen for Western blot analysis.

Vascular injury and treatment ofRA. Abdominal RA was subjected to subfailure stretch to the haptic endpoint (-200% the resting length) for 10s and repeated twice as previously described and a segment was reserved as non-stretched control. The tissues were then cut into 1-2 mm rings and incubated for 1 h at room temperature in PL with or without NiPp (500 pM). To induce acidotic injury, thoracic RA were cut into 1 -2mm rings and pretreated in the absence or presence of NiPp (500 pM) in PL for 30 min. Tissue rings were then transferred to NS and continued incubation in the absence or presence of NiPp (500 pM) for 2 h. Control rings were incubated in PL for 2.5 h.

To induce P2X7R associated endothelial dysfunction, thoracic RA rings were incubated with the P2X7R agonist 2'(3')-0-(4-Benzoylbenzoyl)adenosine 5'- triphosphate (BzATP; 1 mM) in the absence or presence of NiPp (500 pM) in PL for lh at room temperature. After treatments, RA were either suspended in the muscle bath to determine endothelial-dependent relaxation or snap-frozen for Western blot analysis.

Western blot Analysis. Lrozen tissues were pulverized, and proteins were extracted in modified RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 1 mM EDTA, 1 mM EDTA) supplement with protease and phosphatase inhibitors. Protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane followed by immunoblotting with the antibodies against phospho-p38 MAPK-Thrl80/Tyrl82, p38 MAPK, (Cell Signaling Technology, CA), and GAPDH (Millipore, MA). The blots were then incubated with IRDye labeled secondary antibodies (LI-COR Biosciences, NE). The protein-antibody complexes were visualized and quantified using the Odyssey Infrared Imaging System. Phosphorylation was calculated as a ratio of the phosphorylated protein to total protein and was then normalized to the untreated tissues (Ctrl) with the control value set as 1.0.

Kinase profiling. NiPp was dissolved in DMSO, screened at a single concentration of lOOpM using in vitro kinase assays (SelectScreen Kinase Profiling Service; ThermoLisher Scientific, Madison WI) including the Z’LYTE and Adapta kinase activity assays and the LanthaScreen Eu Kinase Binding Assay). Candidate interactions were predicted using STRING Version 11 (www.string-db-org) with the human database at confidence=0.7.

Peptide Synthesis and Purification. Peptides were synthesized using standard f-moc chemistry and purified using high performance liquid chromatography (HPLC) by EZ Biolabs (Carmel, IN).

Muscle bath studies. Tissue rings were suspended in bicarbonate buffer in the muscle bath at 37°C, equilibrated with 95% 02 / 5% C02 at 37°C for 1 hour. Rings were manually stretched to 3 grams of tension, followed by a resting tension of 1 gram for an additional 1 hour to produce a maximal force-tension relationship. Next, rings were contracted with 110 mM potassium chloride to determine functional viability. The tissues were then precontracted with phenylephrine and relaxed with carbachol, an acetylcholine analogue. Force measurements were obtained using the Radnoti force transducer (model 159901A, Radnoti LLC, Monrovia, CA) interfaced with a PowerLab data acquisition system and LabChart software (AD Instruments Inc., Colorado Springs, CO). Relaxation was calculated as percent change in stress compared to the maximal PE-induced contraction (set as 100%).

Statistical Analysis. Data were reported as mean responses ± standard deviation. Outliers, normality, and statistical significance (p value) were determined using GraphPad Prism version 5.0. Differences among groups were determined by paired t test for experiments with dependent (matched) pairs. One-way or two-way ANOVA with post hoc tests were used to determine differences among multiple, dependent samples from the same animal. A p-value < 0.05 was considered statistically significant.

Example 3. Aging leads to decreased endothelial function in rodent blood vessels.

This example illustrates that aging leads to decreased endothelial function in rodent blood vessels. Freshly isolated aorta from young (4 months) and old (20 months) rats were suspended in the muscle bath. To determine the effect of injury on endothelial function, phenylephrine (PE)-precontracted tissues were treated with carbachol (CCH; 5xl0 ⁷ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. The result of this experiments is illustrated in FIG. 1. This study shows that old rats have decreased endothelial function. Example 4. Restoring endothelial function in rodent aortic tissue after ATP injury.

This experiment demonstrates that the NiPp polypeptide [Y ARAA ARQARASPAARRA(pS) AILPG (SEQ ID NO: l); where X 1 = YARAAARQARA (SEQ ID NO:22) and X2= SPAARRA(pS)AILPG (SEQ ID NO:24)] restores endothelial function in rodent aortic tissue after ATP injury. Freshly isolated rat aorta was treated with 3'- 0-(4-Benzoyl)benzoyl adenosine 5'-triphosphate (BzATP, ImM), an analogue of ATP, to induce injury in the absence or presence of NiPp (100 and 500mM) for 1 hour at room temperature. To determine the effect of injury on endothelial function, phenylephrine (PE)- precontracted tissues were treated with carbachol (CCH; 5xlO ⁷ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. The result of the experiment is illustrated in FIG. 2. The experiment demonstrates that ATP injury leads to decreased endothelial function that is restored by treatment with NiPp. As control sequences, the portion of the NiPp polypeptide corresponding to the X2 Niban polypeptide was scrambled or containing non-phosphorylated Serine and used as controls (scr3NiPp=SEQID No. 79, [Y ARA AARQARAAPA(pS) ARIALPGSR (SEQ ID NO:2); where X 1 = Y ARA A ARQ ARA (SEQ ID NO:22) and X2= APA(pS)ARIALPGSR (SEQ ID NO:73) and NiPp3 = SEQ ID NO:77]. The result of the experiment is illustrated in FIGS. 11 A and B.

Example 5. Restoring endothelial function in rodent aortic tissue after stretch (mechanical) injury.

This study shows that the NiPp polypeptide (SEQ ID NO: l) restores endothelial function in rodent aortic tissue after stretch (mechanical) injury. Freshly isolated rat aorta was subjected to subfailure stretch (to haptic endpoint, approximately 2 times the resting length) and treated with NiPp (500mM) for 1 hour at room temperature. To determine the effect of injury on endothelial function, phenylephrine -precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. The result of the experiments is illustrated in FIG. 3. The result of this study demonstrates that stretch injury leads to decreased endothelial function that is restored by treatment with NiPp. Control peptides (SEQ ID NOs:77 and 79) did not restore BzATP-induced endothelial dysfunction in RA indicating the specificity of NiPp activity. The result of the experiment is illustrated in FIG. 13. Example 6. Restoring endothelial function in rodent aortic tissue after endoplasmic reticulum stress (tunicamycin) injury.

This experiment shows that the NiPp polypeptide (SEQ ID NO: l) restores endothelial function in rodent aortic tissue after endoplasmic reticulum stress (tunicamycin) injury. Freshly isolated rat aorta was treated with tunicamycin (TM) to induce injury in the absence or presence of NiPp (100 and 500mM) for 2 hours at room temperature. To determine the effect of injury on endothelial function, phenylephrine (PE) -precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M) and the percent relaxation was determined as a change to the maximal PE-induced contraction. The result of the experiments is illustrated in FIG. 4. The experiment shows that injury leads to decreased endothelial function that is restored by treatment with NiPp.

Example 7. Restoring endothelial function in aged human saphenous veins (HSV).

This study illustrates that the NiPp polypeptide (SEQ ID NO:l) restores endothelial function in aged human saphenous veins. Human saphenous veins (HSV) were collected from patients undergoing coronary artery bypass grafting procedures and treated with NiPp (500mM) for 1 hour at room temperature. PE-precontracted tissues were treated with carbachol (CCH; 10 ⁸ to 10 ⁵ M). The result of the experiments is illustrated in FIG. 5. The result of this study shows that baseline endothelial function in human saphenous veins (HSV) is low. The result of this study also demonstrates that treatment with NiPp leads to increased endothelial function in HSV. When normalized to baseline relaxation, NiPp leads to 183+24% in relaxation responses in HSV.

Example 8. Design of phosphomimetic of Niban, NiPp.

Phosphorylation of Niban at serine 602 is downregulated during UV-induced cell death and injured vascular tissues. A peptide (Niban phosphopeptide,“NiPp”) was designed to contain the enhanced protein transduction domain TAT, conjugated to a phosphopeptide analog of the region surrounding serine 602 of Niban (YARAAARQARASPAARRA(pS)AILPG (SEQ ID NO: l); bold=Niban sequence). Multiple batches were synthesized and displayed similar bioactivity (FIG. 18). Example 9. Anisomycin treatment activates p38 MAPK and impairs endothelial function of intact rat aortic tissues.

Intact strips of rat aorta (RA) were treated with anisomycin, an antibiotic produced by Streptomyces griseolus known to induce p38 MAPK activation in endothelial cells. Anisomycin treatment led to increases in the phosphorylation of p38 MAPK and impaired endothelial dependent relaxation (FIGS. 15A to 15D).

Example 10. NiPp reduced p38 MAPK phosphorylation and restored endothelial function in anisomycin-treated rat aorta

Using the model of impaired endothelial-dependent relaxation in anisomycin treated vessels, RA was treated with buffer alone, anisomycin or NiPp for 30 minutes followed by anisomycin. Pre-treatment with NiPp improved endothelial-dependent relaxation and decreased anisomycin-induced increases in p38 MAPK phosphorylation (FIGS. 15A to 15D).

Example 11. NiPp restored endothelial function and reduced p38 MAPK phosphorylation after subfailure stretch injury in rat aorta.

To determine if NiPp restores endothelial function after other types of injury, RA was subjected to stretch injury and treated with NiPp. NiPp restored endothelial function after stretch injury (FIG. 8A) and reduced p38 MAPK phosphorylation (FIGS. 8B and 8C).

Example 12. NiPp restored endothelial function after acidosis injury and P2X7R activation in rat aorta.

An additional, clinically relevant type of injury is exposure to acidic NS solution commonly used clinically for intravenous resuscitation and for storage of vascular tissues prior to use as autologous vascular reconstruction conduits. Pre-treatment with NiPp improved endothelial function in RA incubated in NS (FIG. 16 A).

Both stretch and NS induced injury lead to release of ATP and activation of P2X7R. RA were treated with the ATP analogue, BzATP, a potent and specific P2X7R agonist in the presence of NiPp. NiPp co-treatment prevented endothelial dysfunction induced by BzATP (FIG. 16B) Example 13. NiPp improved endothelial function after cytokine injury in rat aorta.

Freshly isolated rat aorta (RA) was cut into rings and suspended in the muscle bath. Tissues were then treated with either IIAb alone (50 ng/ml) or cytomix (IL 1 b 50 ng/ml, TNFa, 10 ng/ml, IFNg 50 ng/ml) in the absence or presence of NiPp (500 mM) for 2 hrs, contracted with PE, and then treated with escalating doses of carbachol (CCH; 10-8 to 10-5 M). The NiPp3 treatment group exhibited significant improvement of endothelial relaxation in comparison to IIAb or cytomix treatment group, indicating that NiPp improved endothelial function after cytokine injury (FIG. 10).

Freshly isolated rat aorta (RA) was cut into rings and suspended in the muscle bath. Tissues were then treated with the cytokine IIAb (50 ng/ml) in the absence or presence of NiPp, NiPp3, or scr3NiPp (100 mM) for 2hrs, contracted with PE, and then treated with escalating doses of carbachol (CCH; 10 ⁸ to 10 ⁵ M). NiPp3 (SEQ ID NO:77) is a control polypeptide containing non-phosphorylated sequence of X2 of NiPp. scr3NiPp3 (SEQ ID NO:79) is a control polypeptide containing scrambled sequence of X2 of NiPp. The NiPp3 treatment group exhibited significant improvement of endothelial relaxation, whereas scr3NiPp3 and NiPp3 failed to restore endothelial function (FIG. 12).

Example 14. NiPp improved endothelial function in human saphenous vein harvested for coronary artery bypass surgery.

To determine the effect of NiPp on human tissues with endogenous impaired endothelial function, segments of HSV were harvested at the time of coronary artery bypass surgery. The tissues were either untreated or incubated in the presence of NiPp for 2 hours and endothelial responses were determined. Treatment with NiPp improved endothelial-dependent relaxation (FIGS. 17A and 17B).

Example 15. NiPp is a kinase inhibitor.

As injury leads to increased p38 MAPK phosphorylation and decreased Niban phosphorylation, one of the mechanisms by which NiPp restores function after endothelial injury can be kinase inhibition. A kinase profiling was performed using three different profiling platforms that measure activity and kinase/substrate binding. Kinases of which activity or substrate binding were inhibited by NiPp (IOOmM) at >40% are listed and shown in a kinase dendrogram (FIGS. 14A,14B, 19A and 19B).

The top two candidate kinase targets of NiPp were mitogen-and stress- activated kinase 1 (MSK1 ; also known as RPS6KA5) and p38 MARKa (MAPK14), which were inhibited by 66% and 61%, respectively. MSK1 is an AGC kinase of the RSK family that is phosphorylated by ERK and p38 MAPK in response to cellular stress. Other kinases inhibited by the NiPp include CKD18 of the CMGC kinase family), FGFR1 of the TK family, and the PIK3R3 kinase (FIGS. 19A and 19B).

Example 16. Discussion.

p38 MAPK activation after vascular injury is associated with endothelial dysfunction and decreased Niban phosphorylation, indicating a link between the two proteins. This study demonstrated that pharmacological activation of p38 MAPK with anisomycin was modulated by NiPp, a phosphopeptide mimetic of Niban. NiPp pre-treatment reduced p38 MAPK phosphorylation and prevented endothelial dysfunction, implicating that NiPp can ameliorate injury responses that involve p38 MAPK signaling. p38 MAPK is activated in response to multiple cellular stressors including infection, UV exposure, and ischemic injury of the brain, kidney, liver, and heart. p38 MAPK also responds to inflammatory cytokines and is a key mediator of inflammatory responses. In the vascular wall, p38 MAPK is activated following balloon or bypass grafting related injuries and promotes neointima formation. Niban is characterized as an ER stress-related, anti-apoptotic protein. A number of studies demonstrate that ER stress plays a role in endothelial dysfunction and leads to p38 MAPK activation. Acidosis, P2X7R activation, and mechanical stretch are also known to induce ER stress signaling in various cell types. The present investigation further shows that impaired endothelial responses caused by with these injuries in RA, all of which lead to increases in p38 MAPK phosphorylation, were restored with NiPp treatment. Impaired endothelial function is associated with aging, atherosclerosis, diabetes and renal failure. Saphenous vein harvested for coronary reconstructions were obtained from an aged patient population (66.4 ± 8.8 yrs old) with systemic atherosclerosis and multiple co-morbidities (FIGS. 17A and 17B). Human saphenous veins (HSV) obtained from this population displays impaired endothelial dependent relaxation (10-15% at 5 x 10 ⁷ M CCH) compared to normal healthy tissues from young animals such as pigs or rats (-50-70%; FIG. 20). Thus, HSV represents a model of endogenous endothelial dysfunction in human tissue.

Treatment of HSV with NiPp resulted in improvement of endothelial-dependent relaxation (FIGS. 17A and 17B). Given that injury to HSV is associated with decreases in Niban phosphorylation, this finding indicates that Niban can be involved in cellular signaling events that regulate the response to vascular injury and that NiPp can be used to improve endothelial function in diseased human tissues. Since NiPp treatment was associated with decreased phosphorylation of p38 MAPK in RA, a mechanism of NiPp function can kinase inhibition. Therefore, a kinase profiling assay was performed to determine the effect of NiPp on the activity of a panel of kinases in vitro. The top two kinases inhibited by NiPp were MSK1 and p38 MAPKa. Inhibition to the p38 MAPKa isoform appeared to be specific as inhibition by NiPp to the other isoforms (b, d, and g) in the kinaseprofiling screen were only at 5, 3, and 3% respectively. Despite high sequence homology, the isoforms have difference in tissue expression, upstream kinase activators and downstream substrates. The p38 MAPKa isoform, initially identified as a protein that underwent phosphorylation in response to endotoxin treatment and hyperosmolarity shock, plays a role in endothelial dysfunction in that inhibition of this isoform leads to improved endothelial function in animal models of cardiovascular diseases including cardiac hypertrophy, balloon injury, salt/fat induced hypertension and in hypercholesterolemic patients. The a-isoform is also a key regulator of pro-inflammatory cytokine production and itself can be activated by ILl-b. MSK1, a downstream kinase of the p38 MAPK activation (FIG. 19), has complex cell-dependent roles in inflammatory responses. In endothelial cells, MSK1 promotes CREB activation in response to TNFa.

Niban gene expression is altered in several cancers and acute pancreatitis. Niban gene expression is also upregulated by ILl-b and has been implicated in steroid-responsive inflammatory responses in asthma. Further analyses of the STRING database revealed that the candidate targets of NiPp are involved in in 39 KEGG pathways that are implicated in cancer development, stress responses, and cytoskeletal regulation. These data show Niban as a stress response protein that participates in cellular injury responses.

While p38 MAPK activation plays a role in the response to injury, subsequent downregulation after injury is necessary to restore cellular homeostasis, uncontrolled p38 MAPK responses can contribute to aberrant downstream p38 MAPK-dependent signaling. Activation of p38 MAPK occurs via phosphorylation of the Thr-Gly-Tyr motif by upstream MKKKs and MKK3/MKK6. Typically, protein phosphatases interact with kinases to downregulate activation. More recently, microRNAs were also reported to regulate p38 MAPK. A number of protein phosphatases have been identified to carry out this function on p38 MAPK. The finding that NiPp reduces p38 MAPK phosphorylation and restores function after vascular injury indicates that Niban can be an endogenous down-regulator of p38 MAPK that restores cellular homeostasis after injury. This is consistent with some of the known functions of Niban as a stress responsive molecule. Very few endogenous kinase inhibitors have been identified and characterized to date. Protein kinase inhibitor (PKI), which inhibits Protein Kinase A, is an anti-inflammatory and anti-proliferative protein regulator in endothelial and vascular smooth muscle cells, respectively. Another endogenous kinase inhibitor, secretoneurin, inhibits Calcium/Calmodulin-Dependent Protein Kinase II and attenuates calcium- dependent arrhythmias as well as playing a critical role in neural vasculature. Thus, while few endogenous kinase inhibitors have been identified, they do exist and phosphorylated Niban may, at least in part, modulate cellular responses via p38 MAPK inhibition.

p38 MAPK has been a prime target for the development of small molecule therapeutics; however, given its central role in many organs, toxicity has been a major limitation due to the crosstalk between different intracellular pathways that p38 MAPK regulates. The investigation and determination that NiPp possesses an isoform-specific inhibitory property to p38 MAPK offers a therapeutic that mimics endogenous signaling, in a tissue specific manner. Moreover, NiPp modulation of the p38 MAPK signaling pathway can attenuate injury responses that occur during traumatic injuries such as surgery, sepsis, or inflammatory diseases. In addition, the unique approach of utilizing a cell permeant peptide analogue of phosphorylated Niban to elucidate the function of this molecule and its role in p38 MAPK signaling cascade is relevant to intact vascular and other tissues where genetic engineering approaches are less optimal due to low cellular turnover.

Example 17. NiPp is a p38MAPKa inhibitor.

The p38 mitogen- activated protein kinase (MAPK) family consist of four isoforms: r38a, r38b, r38g, and r38d. In response to extracellular stimuli such as cytokines and stress, p38MAPK is activated by phosphorylation at threonine- 180 and tyrosine- 182 and phosphorylates downstream mediators such as other kinases and transcription factors. p38MAPK kinase signaling plays an important role in inflammation and other physiological processes and deregulation of these signaling pathways contribute to progression of cardiovascular diseases, inflammatory diseases, chronic pain, and cancer. Specific targeting of p38MAPK can be therapeutic strategies.

The data in FIGS. 7B, 8B, 14A, 14B, 15C, 15D, 19A and 19D show that NiPp is a p38MAPKa inhibitor. Such function can be applied for therapeutic use to treat or prevent inflammatory diseases (sepsis, rheumatoid arthritis, Crohn’s disease, asthma, COPD), chronic pain, and cancers. Table 1: Composition of polypeptides. Composition of polypeptides consisting of a sequence according to the general formula: XI -X2. Sequences of polypeptides indicated by single-letter amino acid code. (pS) denotes phosphoserine. NiPp2 is a polypeptide containing shortened sequence of X2 of NiPp (SEQ ID NO:76). NiPp3 is a control polypeptide containing non- phosphorylated sequence of X2 of NiPp (SEQ ID NO:77). scr3NiPp3 is a control polypeptide containing scrambled sequence of X2 of NiPp (SEQ ID NO:79).

SEQUENCES

Human Niban (FAM129A) Protein Sequence (SEQ ID NO:75)

MGGSASSQLDEGKCAYIRGKTEAAIKNFSPYYSRQYSVAFCNHVRTEVEQQR

DLTSQFLKTKPPLAPGTILYEAELSQFSEDIKKWKERYVVVKNDYAVESYENKEAYQ

RGAAPKCRILPAGGKVLTSEDEYNLLSDRHFPDPLASSEKENTQPFVVLPKEFPVYL

WQPFFRHGYFCFHEAADQKRFSALLSDCVRHLNHDYMKQMTFEAQAFLEAVQFFR

QEKGHYGSWEMITGDEIQILSNLVMEELLPTLQTDLLPKMKGKKNDRKRTWLGLLE

EAYTLVQHQVSEGLSALKEECRALTKGLEGTIRSDMDQIVNSKNYLIGKIKAMVAQP

AEKSCLESVQPFLASILEELMGPVSSGFSEVRVLFEKEVNEVSQNFQTTKDSVQLKE H

LDRLMNLPLHSVKMEPCYTKVNLLHERLQDLKSRFRFPHIDLVVQRTQNYMQELME

NAVFTFEQLLSPHLQGEASKTAVAIEKVKLRVLKQYDYDSSTIRKKIFQEALVQITL P

TVQKALASTCKPELQKYEQFIFADHTNMIHVENVYEEILHQILLDETLKVIKEAAIL K

KHNLFEDNMALPSESVSSLTDLKPPTGSNQASPARRASAILPGVLGSETLSNEVFQE S

EEEKQPEVPSSLAKGESLSLPGPSPPPDGTEQVIISRVDDPVVNPVATEDTAGLPGT CS

SELEFGGTLEDEEPAQEEPEPITASGSLKALRKLLTASVEVPVDSAPVMEEDTNGES H

VPQENEEEEEKEPSQAAAIHPDNCEESEVSEREAQPPCPEAHGEELGGFPEVGSPAS PP

ASGGLTEEPLGPMEGELPGEACTLTAHEGRGGKCTEEGDASQQEGCTLGSDPICLSE S

QVSEEQEEMGGQSSAAQATASVNAEEIKVARIHECQWVVEDAPNPDVLLSHKDDVK

EGEGGQESFPELPSEE

Sequences for Niban (also known as FAM129A) homologs can include the following sequences as identified by their accession numbers, for example: Human NP_443198.1; Rat NP_071578.2; Mouse NP_071301.2; Pig, NP_001230148.1 ; or Dog, XP_537163.2.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.