METHODS, SYSTEMS, AND COMPOSITIONS RELATING TO TREATMENT OF NEUROLOGICAL CONDITIONS, DISEASE, OR INJURIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application serial no.

61/750,166, filed January 8, 2013, which is hereby incorporated by reference in full.

SUPPORT

[0002] Some of the work described herein was supported by National Institute on Aging grant ROl AG031811, National Institute of Neurological Disorders and Stroke grants POl NS23393 (MC) and 1R41NS064708, and/or American Heart

Association grant 09GRNT2300151 and 12SDG9300009. The government may have certain rights in some of the embodiments.

TECHNICAL FIELD

[0003] Without limitation, some embodiments comprise methods, systems, and

compositions relating to treatment of neurological conditions, disease, or injuries, and the use of same in the research, diagnosis, and treatment of injury or disease.

BACKGROUND

[0004] Stroke is a major cause of cerebral white matter and vascular damage, which induces long-term disability as a result of limited axonal regeneration (axon- regrowth or sprouting) and vascular remodeling (e.g., neovascularization and vascular stabilization) in the inhibitory environment of the adult mammalian central nervous system. Axonal damage and degeneration are prominent components of acute neurological disorders such as stroke and other neurological conditions, diseases, or injuries. Successful axonal outgrowth in the adult central nervous systems ("CNS") is central to the process of nerve regeneration and brain repair.

[0005] Some patient populations have an increased risk of stroke. As one nonlimiting example, diabetes mellitus ("DM") is a major health problem, and DM patients have a 3-4 fold higher risk of experiencing ischemic stroke. DM adversely influences the post-stroke level of disability, increasing the extent of the cerebral injured area and promoting worse outcome compared to the general population. Diabetes also induces neuroaxonal dystrophy, synaptic dysplasia and defective axonal regeneration. Restriction of axonal regeneration and neuro-plasticity contributes to the worse functional recovery after stroke.

[0006] Current treatments are limited in their abilities to address underlying causes of neurological conditions, disease, or injuries, as nonlimiting examples, after stroke, brain and spinal cord injury, neural injury, multiple sclerosis and

neurodegenerative disease, and/or to be suitable for use across patient populations in need of treatment. Thus, an unmet need remains for pharmacological therapeutic approaches designed to remodel axons, promote white matter remodeling, and/or to promote brain plasticity (neurorestoration) to reduce and/or improve neurological deficits after stroke or other neurological conditions, disease or injury in patient populations in need of such treatment.

SUMMARY

[0007] The following examples of some embodiments are provided without limiting the invention to only those embodiments described herein and without waiving or disclaiming any embodiments or subject matter:

[0008] Some embodiments provide methods, systems, and compositions for promoting, increasing, and/or improving white matter remodeling, neurite outgrowth, and/or neurological function in a patient in need thereof , including in mammals, and specifically in human beings. Some embodiments comprise the administration to the subject in need thereof of a composition comprising a pharmaceutically effective amount of one or more of a group comprising, or consisting of,

Angiopoietin-1, a promoter of Angiopoietin 1 expression, D-4F, human umbilical cord blood cells ("HUCBCs"), Niacin Niaspan, and GW3965 to a patient in need of treatment. Some embodiments provide a medicament comprising a

pharmaceutically effective amount of one or more of Angiopoietin-1, a promoter of Angiopoietin 1 expression, D-4F, HUCBCs, Niacin/Niaspan, and GW3965, and/or use of such a medicament in treating a patient with respect to the patient's neurological condition, disease or injury, including but not limited to, in conjunction with stroke. BRIEF DESCRIPTION OF DRAWINGS

[0009] Some embodiments will now be described, by way of example only and without disclaimer of other embodiments, with reference to the accompanying drawings, in which:

[0010] FIG. 1 is comprised of images and a data representation showing that

Angiopoietin 1 ("Angl") increases neurite outgrowth in cultured primary cortical neurons ("PCN").

[0011] FIG. 2 is data representations showing that Angl treatment decreases

oligodendrocyte ("OL") death and increases OL differentiation

[0012] FIG. 3 is images showing that D-4F treatment increases Angl expression in

cultured PCN and oligodendrocytes.

[0013] FIG. 4 is data representations and images showing that D-4F treatment of stroke promotes white matter ("WM") remodeling and dose-dependently improves functional outcome in WT mice.

[0014] FIG. 5 is images showing that D-4F treatment of stroke significantly increases

Angl expression in the ischemic brain.

[0015] FIG. 6 is data representations and images and graphs showing D-4F has a

neurorestorative effect and promotes WM remodeling in WT mice.

[0016] FIG. 7 is images and data representations showing that HUCBC treatment of stroke in Type two diabetic (T2DM) rats improves functional outcome, and increases Angl and decreases "receptor of advanced glycation end- products""("RAGE") expression in the ischemic brain.

[0017] FIG. 8 is data representations showing that TlDM-MCAo rats have worse

neurological outcome after stroke compared to WT-MCAo rats (p<0.05).

[0018] FIG. 9 is images and data representation showing that TlDM-MCAo rats exhibit decreased axonal density in the ischemic brain compared to WT-MCAo rats.

[0019] FIG. 10 is images and data representations showing that Niaspan treatment of stroke in T1DM rats promotes axonal remodeling and synaptic plasticity.

[0020] FIG. 11 is images and data representations showing that Niacin increases Angl expression in cultured hypoxic PCN; Niacin and Angl increase neurite outgrowth under HG conditioned media; and inhibition of the Angl, but not Tie2-FC, decreased neurite outgrowth in cultured hypoxic PCN under HG conditions.

[0021] FIG. 12 is data representations showing that GW3965 treatment increases HDL-C levels and improves functional outcome in mice 14 days after MCAo.

[0022] FIG. 13 is images and data representations showing that GW3965 treatment increases Synaptophysin expression and axonal and myelin growth and decreases axon damage in the IBZ 14 days after MCAo.

[0023] FIG. 14 is images and data representations showing that GW3965 treatment increases angiogenesis, arteriogenesis, and vascular stabilization in the IBZ 14 days after MCAo.

[0024] FIG. 15 is images and data representations showing that GW3965 treatment increases Angl and Tie2 expression in the IBZ 14 days after MCAo.

[0025] FIG. 16 is images and data representations showing t GW3965 increases neurite outgrowth, capillary-like tube formation, and artery explant cell migration.

DETAILED DESCRIPTION

[0026] Without limitation to only those embodiments expressly disclosed herein and without disclaiming or waiving any embodiments or subject matter, some embodiments comprise the administration to a mammalian subject in need thereof of a composition comprising a pharmaceutically effective amount of agent which provides or promotes Angiopoetin-1 activity and provides, increases, and/or improves white matter remodeling, neurite outgrowth, and/or neurological function in the subject. Such agents may comprise, without limitation,

Angiopoietin-1, a promoter of Angiopoietin-1 expression, D-4F, HUCBCs, Niaspan/Niacin, and GW3965.

[0027] We have discovered unexpectedly that in some embodiments, without limitation, increasing Angiopoietin-1 signaling activity promotes white matter remodeling and neurorestorative effects after stroke in subjects in need of treatment. In accordance with some embodiments, Angiopoietinl treatment promotes neurite outgrowth in cultured primary cortical neurons and increases oligodendrocyte differentiation in cultured premature oligodendrocytes. Treatment of stroke with agents or cells that increase Angiopoietin-1 expression, as nonlimiting examples, bone marrow stromal cells (BMSCs), human umbilical cord blood cells

(HUCBCs), D-4F (a reconstituted fragment of HDL, 18-amino acid peptide that mimics the tertiary structure of ApoA-I)(SEQ ID NO: 1 : Ac-D-W-F-K-A-F-Y-D- -V-A-E-K-F-K-E-A-F-NH2 (4F)),or Niaspan (prolonged released Niacin), and/or agents that increase HDL, including without limitation, GW3965 (synthetic liver x receptor agonist, elevates high density lipoprotein cholesterol), significantly promotes vascular and white matter remodeling and thereby improves functional outcome after stroke.

[0028] As a nonlimiting example, using PCN culture, we have discovered unexpectedly that treatment of PCN with Angiopoietin-1 significantly increased neurite outgrowth compared to non-treated controls. Premature oligodendrocytes treated with Angiopoietin-1 significantly decreased cell death and increased cell differentiation and myelin basic protein ("MBP") and CNPase gene and protein expression, markers of mature oligodendrocytes, compared to non-treatment controls. Treatment of stroke with restorative agents, as nonlimiting examples, HUCBCs, D-4F or Niaspan (prolonged released Niacin), all significantly increase Angiopoietinl expression and Angiopoietinl/Tie2 activity in the ischemic brain, promote vascular and white matter remodeling, and subsequently improve functional outcome after stroke.

[0029] Our data indicate that increasing Angiopoietin-1 upregulates neurite outgrowth and oligodendrocyte differentiation which promote white matter remodeling and thereby induce neurorestorative effects after stroke.

[0030] In accordance with some embodiments, without limitation, we have discovered unexpectedly that increasing Angiopoietin-1 and/or angiopoietin activity promotes neurite outgrowth and oligodendrocytes differentiation and white matter remodeling as well as enhances neurorestorative effects.

[0031] Angiopoietin-1, an endothelial growth factor, mediates vascular remodeling, promotes pericyte recruitment, remodeling, maturation, and stabilization of blood vessels, and prevents plasma leakage in the ischemic brain. Although

Angiopoietin-1 has been shown to promote vascular integrity and angiogenesis anti-inflammatory effects, before our discovery, to our knowledge, a direct effect of Angiopoietin-1 on white matter structure and remodeling had not been demonstrated. We have now shown, for the first time, that the Angiopoietin-1 signaling pathway not only regulates vascular remodeling but also promotes neurite outgrowth and oligodendrocyte differentiation. This is a highly novel approach to brain plasticity, and our data indicate that Angiopoietin-1 has a profound therapeutic neurorestorative effect on cerebral tissue. Our data show that Angiopoietin-1 peptide treatment of primary cortical neurons in normal glucose and high glucose conditions after oxygen glucose deprivation ("OGD") significantly increased neurite outgrowth compared to non-treatment control. In addition, Angiopoietin-1 treatment significantly increased differentiation of premature oligodendrocytes into mature oligodendrocytes and decreased cell death under OGD conditions. In addition, we also found that treatment of stroke with restorative therapies, such as, BMSCs, HUCBCs, D-4F, Niaspan (prolonged released Niacin), and GW3965, all significantly increase Angiopoietin-1 expression and Angl/Tie2 activity in the ischemic brain, increase white matter remodeling as well as improve functional outcome after stroke when treatment initiated at 24h after MCAo. In some embodiments, Angiopoietin-1 or agents which increase Angiopoietin-1, or which increase Angiopoietin-1 related signaling activity, will likely promote white matter remodeling which improves neurological function after stroke, brain and spinal cord injury, neural injury, multiple sclerosis and neurodegenerative disease, including without limitation, Alzheimer's disease, vascular dementia, and peripheral neuropathies.

In some embodiments, without limitation, white matter remodeling is increased, thereby reducing neurological deficits after stroke, neural trauma, multiple sclerosis, Alzheimer's disease, vascular dementia, and peripheral neuropathies, and neurodegenerative disease. Therapies are needed to remodel the brain which will enhance WM remodeling and recovery of neurological function after an injury, or in other neurological conditions or diseases. Our discovery that

Angiopoietin-1 promotes neurite outgrowth and oligodendrocyte differentiation indicate that Angiopoietin-1 or agents which increase Angiopoietin-1 expression and its activity promote white matter remodeling after stroke with or without diabetes, brain injury and neurodegenerative disease and thereby improve neurological function after treatment of these neurological diseases and injury.

[0033] Neurodegenerative disease, stroke and neural injury attack millions of Americans annually, and are the most common form of pathology and the leader in loss of quality of life among all diseases. Thus, in accordance with some embodiments, treatment of neurological disease and injury with agents that increase

Angiopoietin-1 and its activity will provide an effective therapy for these pervasive neurological insults.

[0034] In some embodiments, without limitation, Angiopoietin-1, Angiopoietin-1

mimetics, and agents which promote increase of Angiopoietin-1 are or will be restorative therapies which improve neurological function after stroke in the diabetes and non-diabetes population, neural injury and neurodegenerative disease. We have found that Angiopoietin-1 increases neurite outgrowth and oligodendrocyte differentiation and decreases cell death and promotes white matter remodeling. Angiopoietin-1 or related agents which increase

Angiopoietin-1 signaling activity may be administered to patients before or after the onset of injury or disease to reduce the neurological deficits associated with disease and possibly aging.

[0035] Some embodiments use Angiopoietin-1, or any agent that increases Angiopoietin- 1 signaling activity, to improve neurological function. To our knowledge, no one has reported that these agents have the property of increasing axonal and white matter remodeling. In some embodiments, treatment of neurological disease with such agents is likely to improve neurological function in subjects in need of treatment. In accordance with some embodiments, Angiopoietin-1 or any agents which increase Angiopoietin-1 signaling activity promote neurological recovery after stroke, brain injury, multiple sclerosis, Alzheimer's disease, vascular dementia, peripheral neuropathies, and neurodegenerative disease. EXAMPLES

[0036] The following examples of some embodiments are provided without limiting the invention to only those embodiments described herein and without waiving or disclaiming any embodiments or subject matter.

EXAMPLE 1:

[0037] Our data indicate that increasing Angiopoietin-1 upregulates neurite outgrowth and oligodendrocyte differentiation which promote white matter remodeling and thereby induce neurorestorative effects after stroke.

[0038] In accordance with some embodiments, without limitation, we have discovered unexpectedly that increasing Angiopoietin-1 and/or angiopoietin activity promotes neurite outgrowth and oligodendrocyte differentiation and white matter remodeling as well as enhances neurorestorative effects.

[0039] Angiopoietin- 1 , an endothelial growth factor, mediates vascular remodeling, promotes pericyte recruitment, remodeling, maturation, and stabilization of blood vessels, and prevents plasma leakage in the ischemic brain. However, although Angiopoietin- 1 has been shown to promote vascular integrity and angiogenesis anti-inflammatory effects, to our knowledge, before our discovery, a direct effect of Angiopoietin-1 on white matter structure and remodeling had not been demonstrated. Thus, we have discovered unexpectedly that the Angiopoietin-1 signaling pathway not only regulates vascular remodeling but also promotes neurite outgrowth and oligodendrocyte differentiation. This is a highly novel approach to brain plasticity and our data indicate that Angiopoietin-1 may have a profound therapeutic neurorestorative effect on cerebral tissue. Our data show that Angiopoietin-1 peptide treatment of primary cortical neurons in normal glucose and high glucose conditions after oxygen glucose deprivation significantly increased neurite outgrowth compared to non-treatment control. In addition, Angiopoietin-1 treatment significantly increased differentiation of premature oligodendrocytes into mature oligodendrocytes and decreased cell death under OGD conditions.

[0040] As part of our work, we discovered that Angiopoietin-1 ("Angl") increases neurite outgrowth in cultured primary cortical neurons ("PCN"). FIG. 1 comprises images and a data representation showing these discoveries. FIG. 1 shows Tujl immunostaining in control and Angl - treated PCN and quantitative data. PCN were subjected to 2 hours ("h") of oxygen-glucose deprivation ("OGD") and cultured in high glucose ("HG", 37.5 mmol/1 glucose) conditions and were then treated with or without Angl (200ng/ml) for 24h. TUJ1 immunostaining was performed. The total length of neurite outgrowth of TUJ1 positive cells was measured. FIG. 1 shows that Angl treatment significantly increases neurite outgrowth compared to non-treatment HG-controls.

[0041] We also discovered that Angl treatment decreases OL death and increases OL differentiation. FIG. 2 is a data representation showing these discoveries, with FIG. 2A showing LDH assay, and FIG. 2B showing MBP real time PCR. To evaluate whether Angl regulates WM remodeling, an immortalized mouse premature oligodendrocyte cell line (N20.1) was used. Cultured premature OLs were subjected to 2h of OGD and then were treated with: 1) control; 2) Angl (200ng/ml) for 3 days in normal glucose (lOmmol/1 glucose); and 3) HG

(37.5mmol/l glucose) conditions. FIG. 2A shows that HG significantly increases OL death measured by lactate dehydrogenase (LDH) compared to normal glucose condition, while Angl significantly decreases cell death in both normal and HG conditions. To evaluate whether Angl regulates OL differentiation, premature-OL were subjected 2h of OGD and were then cultured in differentiation media and treated with: 1) control; or 2) Angl (200ng/ml) for 10 days in normal glucose condition. Myelin basic protein ("MBP") is a mature OL marker. Using real time PCR, FIG. 2B shows that Angl significantly increased MBP gene expression compared to non-treatment controls. These data indicate that Angl decreases OL cell death and increases OL differentiation.

[0042] We also discovered that D-4F treatment increases Angl expression in cultured PCN and oligodendrocytes. To evaluate whether D-4F treatment regulates Angl expression, PCN and an immortalized mouse premature oligodendrocyte cell line (N20.1) were treated with D-4F (lOOng/ml) for 3 days. Angl expression was measured by Western blot. The data shown in FIG. 3 indicate that D-4F treatment increases Angl expression in cultured PCN and OL. [0043] We also discovered that D-4F treatment of stroke promotes WM remodeling and dose-dependently improves functional outcome in WT mice. To evaluate the effect of D-4F treatment on stroke, adult male C57BL/6J (2-3m) mice were subjected to right temporal (1 hour) MCAo using the filament model. Mice were gavaged with: saline as control and different doses of D-4F (2mg/kg, 4mg kg, 8mg kg, 16mg/kg and 32mg/kg) starting 2h after MCAo and daily for 7 days. Modified neurological severity score ("mNSS") and Foot-fault tests were performed at 1, 3 and 7 days after MCAo by an investigator who was blinded to the experimental groups. FIGS. 4A-B shows that D-4F treatment of stroke dose-dependently improves functional outcome after stroke compared to WT-MCAo control, and the doses of 16mg/kg and

32mg/kg induce significant functional outcome after stroke. To evaluate whether D- 4F regulates WM change, the density of luxol fast blue ("LFB", a myelin marker) in the ischemic border area ("IBZ") of the striatal bundles was measured. FIGS. 4C-E shows that D-4F (16mg/kg) treatment significantly increases LFB density in the ipsilateral striatal bundles compared to no-treatment control. The data indicate that D-4F dose-dependently improves functional outcome and promotes WM remodeling in the ischemic brain.

[0044] We also discovered that D-4F treatment of stroke significantly increases Ang-1 expression in the ischemic brain. To evaluate whether D-4F treatment regulates Angl expression in the ischemic brain, adult male C57BL/6J (2-3m) mice were subjected to MCAo. Mice were gavaged with: saline as control and different doses of D-4F (16mg/kg) starting 2h after MCAo and daily for 7 days. Ang-1 was measured by Western blot assay. As shown in FIG. 5, our data indicated that D- 4F treatment of stroke significantly increases Angl expression in the ischemic brain.

[0045] We also discovered that D-4F has a neurorestorative effect and promotes WM remodeling in WT mice. To evaluate whether D-4F treatment promotes neurorestoration after stroke, male C57BL/6J mice (2-3m) were subjected to extraluminal distal MCAo (dMCAo) and were treated with or without D-4F (16mg/kg, gavaged) starting 24h after dMCAo daily for 28 days. The Food pellet reaching test was performed by an investigator blinded to the experimental groups. FIG. 6A shows that D-4F treatment does not decrease lesion volume (D4F-group:10.1±2.5%;Control:l 1.5±2.3%) but significantly improves long term functional outcome at 21 and 28 days after dMCAo compared to non-treatment control. To evaluate whether D-4F regulates WM remodeling, the density of Bielschowsky silver ("BS," a marker of axons) in the ischemic border area ("IBZ") of the corpus callosum ("CC") were measured. FIGS. 6 B-D show that the positive area of BS in the CC was significantly increased in D-4F treated group compared to non-treatment control (p<0.05). Thus, our data indicated that D-4F has a neurorestorative effect and promotes WM remodeling in WT mice.

[0046] We also discovered that HUCBC treatment of stroke in Type two diabetic

("T2DM") rats improves functional outcome, increases Angl expression, and decreases "receptor of advanced glycation end-products" (RAGE) expression in the ischemic brain. To evaluate whether HUCBC treatment improves functional outcome after stroke in T2DM rats, adult male Wistar rats were fed a high-fat diet D 12492 for 3 weeks and were then injected with a single low dose of

streptozotocin ("STZ") (40 mg/kg, ip) to induce T2DM. T2DM rats were subjected to temporal (2 hour) MCAo and were randomized to intravenous injection via tail vein with: 1) PBS as control; 2) HUCBCs (5xl0 ⁶ ) at 3 days after MCAo. FIG. 7A shows that HUCBC treatment of stroke significantly improves functional outcome in T2DM rats compared to T2DM-MCAo controls (p<0.05). To examine whether HUCBC regulates Angl and RAGE expression, immunostaining and Western blot were performed. FIGS. 7B-C shows that HUCBC treatment significantly increases Angl and decreases RAGE expression in the ischemic border zone ("IBZ") compared to T2DM-MCAo control at 28 days after MCAo (p<0.05), respectively. Western blot (FIG. 7D) shows that HUCBC treatment increases Angl and decreases RAGE level in the IBZ compared to DM-MCAo control.

EXAMPLE 2:

[0047] We also investigated axonal plasticity in the bilateral motor cortices and the long term therapeutic effect of Niaspan on axonal remodeling after stroke in type-1 diabetic ("T1DM") rats. Among other results, we discovered unexpectedly that Niaspan increases axonal remodeling after stroke in type 1 diabetes rats. [0048] Axonal damage and degeneration are prominent components of some acute neurological disorders, including but not limited to, stroke. Successful axonal outgrowth in the adult central nervous system ("CNS") is central to the process of nerve regeneration and brain repair. Diabetes mellitus ("DM") is a major health problem, and DM patients have a 3-4 fold higher risk of experiencing ischemic stroke. DM adversely influences the post-stroke level of disability, increasing the extent of the cerebral injured area and promoting worse outcome compared to the general population. Diabetes also induces neuroaxonal dystrophy, synaptic dysplasia and defective axonal regeneration. Restriction of axonal regeneration and neuro-plasticity contributes to the worse functional recovery after stroke.

[0049] Angl, a family of endothelial growth factors, promotes migration, sprouting, and survival of endothelial cells and mediates vascular remodeling. Angl plays a role in the recruitment of vascular smooth muscle cells ("VSMCs") and pericytes during vascular maturation and the remodeling processes. However,

overexpression of Angl in the brain not only increases vascularization but also alters neuronal dendrite configuration. Angl promotes neuronal differentiation in neural progenitor cells and neurite outgrowth in cultured dorsal root ganglion cells and in PC 12 cells. In addition, neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD-95 correlates with Ang-1 levels in culture.

[0050] Niacin (nicotinic acid) is an effective medication in clinical use for increasing high density lipoprotein ("HDL") cholesterol and is safely used in patients with diabetes. Niacin improves endothelial function, reduces inflammation, and has been used to improve endothelium-dependent vasodilatation in coronary heart disease patients.

[0051] In our work, we evaluated the effect of Niaspan treatment on axonal remodeling and functional outcome after stroke in type one diabetes rats ("T1DM") and the role of Angl as underlying the axonal remodeling induced by Niaspan. Our general approach was as follows: T1DM was induced in young adult male Wistar rats via injection of streptozotocin. T1DM rats were subjected to 2 h transient middle cerebral artery occlusion ("MCAo") and were treated with 40 mg/kg Niaspan or saline starting 24 h after MCAo and daily for 28 days. Anterograde tracing using biotinylated dextran amine (BDA) injected into the contralateral motor cortex was performed to assess axonal sprouting in the ipsilateral motor cortex area. Functional outcome, SMI-31 (a pan-axonal microfilament marker), Bielschowsky silver and synaptophysin expression were measured. In vitro studies using primary cortical neuron (PCN) cultures and in vivo BDA injection into the brain to anterogradely label axons and terminals were employed.

[0052] In summary, we discovered unexpectedly that Niaspan treatment of stroke in TIDM-MCAo rats significantly improved functional outcome after stroke and increased SMI-31, Bielschowsky silver and synaptophysin expression in the ischemic brain compared to saline treated TIDM-MCAo rats (p<0.05). Using BDA to anterograde label axons and terminals, Niaspan treatment significantly increased axonal density in ipsilateral motor cortex in TIDM-MCAo rats (p<0.05, n=7/group). Niacin treatment of PCN significantly increased Angl expression under high glucose condition. Niacin and Angl significantly increased neurite outgrowth, and anti-Angl antibody marginally attenuated Niacin induced neurite outgrowth (p=0.06, n=6/group) in cultured PCN under high glucose condition. Thus, Niaspan treatment increased ischemic brain Angl expression and promoted axonal remodeling in the ischemic brain as well as improved functional outcome after stroke, wherein Angl may partially contribute to Niaspan-induced axonal remodeling after stroke in TIDM-rats.

[0053] Materials and methods

[0054] All experiments were strictly conducted in accordance with Henry Ford Hospital Institutional Animal Care and Use Committee guidelines. Rats were housed individually in an enriched environment and in a temperature-controlled (70°- 72 °F), humidity-controlled (30%-50%) vivarium and maintained with free access to food and water. After surgery, rats were monitored continuously until they fully recovered from anesthesia and twice during the first 24 h after stroke.

[0055] Diabetes induction:

[0056] Adult male Wistar rats (225-250 g) purchased from Charles River (Wilmington, MA) were used. T1DM was induced by a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg, Sigma Chemical Co., St. Louis, MO) to rats. The fasting blood glucose level was tested by using a glucose analyzer (Accu-Chek Compact System; Roche Diagnostics, Indianapolis, IN) and rats with fasting blood glucose >300 mg/dl were identified as diabetic. Animals were subjected to MCAo 2 weeks after diabetes induction.

[0057] MCAo model and experiment groups:

[0058] Wild type ("WT") non-diabetic Wistar rats and STZ-induced TIDM rats were anesthetized and transient (2 h) MCAo was induced by using a previously described method of intraluminal vascular occlusion. Briefly, rats were initially anesthetized with 3.5% isoflurane and maintained with 1.0 to 2.0% isoflurane in 70% N ₂ 0 and 30% 0 ₂ by a face mask. Rectal temperature was maintained at 37 °C throughout the surgical procedure by means of a feedback-regulated water heating system. A 4-0 nylon suture with its tip rounded by heating near a flame was inserted into the external carotid artery ("ECA") through a small puncture. The length of nylon suture, determined according to the animal's weight, was gently advanced from the ECA into the lumen of the internal carotid artery ("ICA") until the suture blocked the origin of the middle cerebral artery

("MCA"). After 2 h of MCAo, animals were reanesthetized with isoflurane, and restoration of blood flow was performed by withdrawal of the filament. Rats were randomly separated to three groups 24 h after MCAo and were gavaged with: 1) saline WT-MCAo control; 2) saline for TIDM-MCAo-control; 3) T1DM- MCAo-+Niaspan treatment: 40 mg/kg Niaspan (Kos Pharmaceuticals, Inc.

Cranbury, NJ; dissolved in saline) started 24 h after MCAo and administered daily until sacrifice. A battery of functional test was performed. One set of rats was sacrificed at 14 days after MCAo for immunostaining (n=8/group). Another set of animals (n=7/group) was employed for biotinylated dextran amine ("BDA") injection in the contralateral hemisphere at 14 days after MCAo and rats were sacrificed at 28 days to measure axonal density in the ipsilateral hemisphere.

[0059] Functional tests: [0060] A battery of behavioral tests (modified neurological severity score, mNSS) and foot-fault tests were performed prior to and at 1, 7, 14, 21 and 28 days after MCAo by an investigator who was blinded to the experimental groups.

[0061] mNSS:

[0062] Neurological function was graded on a scale of 0 to 18 (normal score 0; maximal deficit score 18. mNSS is a composite of motor, sensory, reflex and balance tests)

[0063] Foot-fault test:

[0064] For locomotor assessment, rats were tested for placement dysfunction of

forelimbs with the modified foot-fault test. The percentage of foot-faults of the left paw to the total number steps was determined.

[0065] Blood glucose measurement:

[0066] Blood glucose was measured at 24 h after MCAo and at 28 days prior to sacrifice by using test strips for glucose (Polymer Technology System, Inc. Indianapolis, IN 46268) according to the manufacturer's instructions. The data are presented as milligrams per deciliter.

[0067] Biotinylated dextran amine ("BDA") injection and measurements of axon density:

[0068] To investigate whether TlDM-MCAo affects axonal length and axonal sprouting, BDA was injected into the contralateral cortex at 14 days after MCAo

(n-7/group) for anterograde labeling of axons and terminals in the ipsilateral hemisphere. These rats were sacrificed 28 days after MCAo. Briefly, the animals were placed in a stereotaxic device, and a unilateral craniotomy was performed on the skull overlying the right cerebral cortex. BDA solution (100 nL; a 10% solution dissolved in 0.1 mol/1 phosphate-buffered saline (PBS) (pH 7.4), molecular weight 10,000 mw; Molecular Probes, Eugene, OR) was injected through a finely drawn glass capillary into the right stereotaxic coordinates (+0.5 mm or -0.5 mm rostral to the bregma, 2.5 mm lateral to the midline, 1.5 mm depth from the surface of the cortex) over a 3-min time period. The micropipette remained in place for 4-min after the injection was administered.

[0069] Tissue preparation:

[0070] Two weeks after BDA injection, animals were anesthetized with ketamine and transcardially perfused with saline, followed by 4% paraformaldehyde. The brain tissues were processed to acquire adjacent 100-mm-thick coronal sections using a vibratome. Ten sections from the center of the lesion (bregma -2 mm to +2 mm) were used to detect BDA labeling. Briefly, free-floating sections were incubated with 0.5% H ₂ 0 ₂ in 50 mmol/1 Tris-buffered saline (pH 7.4) for 20 min, and washed with Tris-buffered saline containing 0.25% Triton X-100 three times for 30 min each at room temperature. Then the sections were incubated with avidin- biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) in Tris- buffered saline/Triton X-100 at 41 °C for 3 days, and BDA labeling was visualized with 3, 30-diaminobenzidine-nickel (Vector Laboratories) for light microscopy examination. Image was used for subsequent quantitative

measurements of BDA-positive axonal densities in the ipsilateral cortex (six fields per section) from 10 sections from each individual animal divided by the total mean density in all animals and shown as percentage of proportional areas.

[0071] Histological and immunohistochemical assessment:

[0072] The brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde before being embedded in paraffin. For immunostaining, a standard paraffin block was obtained from the center of the lesion (bregma -1 mm to +1 mm). A series of 6 μπι thick sections were cut from the block. Every 10th coronal section for a total of 5 sections was used for immunohistochemical staining. Antibody against synaptophysin (monoclonal antibody; dilution 1 :1000, Chemicon, Temecula, CA) and SMI-31 (a pan-axonal neurofilament marker), neurofilaments, phosphorylated monoclonal antibody, 1 : 1000, Covance, CA) was employed. Bielschowsky silver immunostaining was used to demonstrate axons. Briefly, for Bielschowsky staining, slides were placed in 20% silver nitrate in the dark, then ammonium hydroxide was added, and were then treated with NaOH and sodium thiosulfate. Control experiments consisted of staining brain coronal tissue sections as outlined above, but non-immune serum was substituted for the primary antibody. The immunostaining analysis was performed by an investigator blinded to the experimental groups.

[0073] Bielschowsky silver, SMI-31 and synaptophysin expression quantification: [0074] For quantitative measurements of Bielschowsky silver, SMI-31 and synaptophysin, five slides from each brain, with each slide containing 4 fields from striatum for Bielschowsky silver, and 8 fields from the ischemic border (1BZ, cortex and striatum) for SMI-31, synaptophysin were digitized under a 20 x objective (Olympus BX40) using a 3 -CCD color video camera (Sony DXC- 970MD) interfaced with an MCID image analysis system (Imaging Research, St. Catharines, Canada). Positive areas of immunoreactive cells were measured in the IBZ. Data were analyzed in a blinded manner.

[0075] Double immunohistochemical staining:

[0076] To identify cell type of Angl -reactive cells, double immunofluorescence staining (Angl /SMI-31) was performed. SMI-31 and Angl (rabbit polyclonal lgG, 1 :2000, Abeam, Cambridge, MA, USA) were used. FITC (Calbiochem, Darmstadt, Germany) and cyanine-5.18 (CY5, Jackson Immunoresearch, West Grove, PA) were used for double-label immunostaining. Each coronal section was first incubated with the primary and anti-SMI-31 antibodies with Cy5, and was then followed by Angl antibody with FITC. Control experiments consisted of staining brain coronal tissue sections as outlined above, but omitted the primary antibodies.

[0077] PCN culture and quantification of dendrite outgrowth in PCN:

[0078] PCNs were obtained from pregnant 17 day Wistar rat embryos and cultured with Neuralbasal-A medium (GIBCO) containing 2% B27 medium-supplement (GIBCO). One hour of oxygen-glucose deprivation ("OGD") was induced.

[0079] To evaluate whether Niacin regulates Angl expression, the OGD-PCN cultures were treated without or with Niacin (1 mM) under high glucose ("HG") condition (HG, 37.5 mmol/1 glucose) for 3 days. Angl immunostaining was performed (n=4/group). The percentage of Angl positive cell number in total cells was presented.

[0080] Our previous studies found that high glucose ("HG") significantly decreases PCN neurite outgrowth. To evaluate whether Niacin regulates neurite outgrowth under HG condition, the OGD-PCN cultures were then divided into (n-6/group): 1) HG control (HG, 37.5 mmol/1 glucose); 2) HG+Niacin (1 mM); 3) HG+Angl (100 ng/ml, Chemicon, Temecula, CA); 4) HG+Niacin+anti-Angl antibody (1 μg/ml, Abeam, Cambridge, MA); 5) HG+Niacin+Tie2-FC (recombinant mouse Tie2/FC, 2 μ^ηιΐ, Chimera, R&D System, Cambridge, MA) for 3 days. The PCN cultures were performed for TUJ1 immuno fluorescent staining using a monoclonal anti- TUJ1 antibody (1 : 1000, Covance, Princeton, NJ) with Cy3 for dendrite outgrowth measurement. To trace the dendrite outgrowth of fluorescently labeled neurons, the fluorescent photomicrographs were captured at 20x magnification with a digital camera. The total dendrites of 20 TUJ1 positive neurons were measured using MCID analysis system. The average length of neuronal dendrite outgrowth was presented.

[0081] Statistical analysis:

[0082] All measurements and analyses were performed by normality of distribution, and the homogeneity of variances was tested including the functional outcome, biochemistry, immunostaining and cell culture. One-way ANOVA was used for the evaluation of functional tests, immunostaining and neurite outgrowth analysis, respectively. Pearson's partial correlation after bivariate correlation analysis was used to analyze the correlation of neurologic functional outcome with axonal density in the ischemic brain. All data are presented as mean±standard error of the mean ("SE").

[0083] We discovered that Niaspan treatment in TlDM-MCAo rats does not alter blood glucose levels but increases HDL levels after stroke in TIDM rats. There were no significant differences in blood glucose levels between the two groups prior to treatment (TlDM-MCAo control: 367.2±43.6 mg/dl; TlDM-MCAo+Niaspan: 379.2±53.1 mg/dl) and after treatment at 28 days after MCAo (TlDM-MCAo control: 423.7±65.9 mg/dl; Tl DM-MCAo+Niaspan: 458.6±59.1 mg/dl). Niaspan treatment significantly increased HDL level (39.3±2.2 mg/dl) compared to TlDM-MCAo control (31.6±1.3 mg/dl, p<0.05).

[0084] We also discovered unexpectedly that Niaspan treatment of stroke in TIDM rats significantly improves functional outcome after stroke (FIG. 8). To evaluate whether Niaspan treatment regulates long term functional outcome after stroke, a battery of behavioral tests was performed. FIGS. 8A-B show that TlDM-MCAo rats exhibit significantly worse functional outcome until one month after MCAo compared to WT-MCAo rats (p<0.05). Niaspan treatment of stroke in TlDM- MCAo rats significantly attenuated neurological deficit on mNSS (FIG. 8A) and foot-fault (FIG. 8B) tests compared to non-treatment TlDM-MCAo rats (p<0.05, n=7/group). As shown in the data of FIG. 8, TlDM-MCAo rats have worse neurological outcome after stroke compared to WT-MCAo rats (p<0.05). Niaspan treatment of stroke in T1DM rats significantly improves functional outcome after stroke. FIG. 8A: mNSS test; FIG. 8B: Foot-fault tests. *p<0.05 compared to TlDM-MCAo. n=7/group.

We also discovered that cerebral axonal density is decreased in TlDM-MCAo rats, and that Niaspan treatment of TlDM-MCAo rats increases cerebral axonal density (FIG. 9). To investigate whether TlDM-MCAo affects axonal density, BDA was injected into the contralateral cortex at 14 days after MCAo and rats were sacrificed at 28 days after MCAo to anterogradely label axons and terminals. FIG. 9 shows that the density of BDA positive axons was significantly decreased in TlDM-MCAo rats compared to WT-MCAo rats (p<0.05, n=7/group).

However, Niaspan treatment significantly increased the axonal density in the ischemic brain compared to TlDM-MCAo control rats (p<0.05, n=7/group). Correlation coefficient analysis shows significant negative correlations between mNSS score (28 days after MCAO) and axonal density (D, r=-0.72, PO.05), and foot-fault (28 days after MCAO) and axonal density (D, r=-0.74, P<0.05) in stroke animals. These data indicate that TlDM-MCAo rats exhibit decreased ability of axonal outgrowth after stroke compared to WT-MCAo rats and that Niaspan treatment significantly increases axonal density in TlDM-MCAo rats. In addition, the significant correlation between axonal density and functional response suggests that axonal density contributes to neurological recovery. As shown in FIG. 9, TlDM-MCAo rats exhibit decreased axonal density in the ischemic brain compared to WT-MCAo rats. Niaspan treatment of stroke in TlDM-MCAo rats significantly increased axonal density in the ischemic brain. FIG. 9A: Low magnification of BDA injection in the contralateral cortex (brown color, right side) and labeled axon in the ipsilateral cortex (brown color, left side). FIG. 9B: Quantitative data of axonal density (n=7/group). FIG. 9C: Axons are labeled with BDA in the ipsilateral hemisphere in WT-MCAo, TIDM-MCAo and TlDM-MCAo+Niaspan treatment animals. FIG. 9D: Correlation analysis of neurological outcome and axonal density.

[0086] We also discovered that Niaspan treatment promotes axonal remodeling and synaptic plasticity in T1DM rats after MCAo (FIGS. lOA-C). Synaptophysin is a marker for presynaptic plasticity and synaptogenesis. SMI-31 is a pan-axonal neurofilament marker; Bielschowsky silver is a marker for axons. FIG. lOA-C show that T1DM rats exhibit significantly decreased SMI-31 expression in the striatum of the ischemic border zone (IBZ) compared to WT-MCAo control rats (p<0.05). TIDM-MCAo did not significantly decrease synaptophysin and Bielschowsky silver density compared to WT-MCAo rats (p>0.05). Niaspan treatment significantly increased SMI-31 , Bielschowsky silver and synaptophysin expression in the ischemic hemisphere in TlDM-MCAo+Niaspan rats compared to non-treatment TIDM-MCAo control rats (p<0.05). As shown in the data of FIG. 10, Niaspan treatment of stroke in T1DM rats promotes axonal remodeling and synaptic plasticity. FIG. 10A: Bielschowsky silver (a marker for axons) immuno staining and quantitative data. FIG. 10B: SMI-31 (a pan-axonal neurofilament marker) immunostaining and quantitative data; FIG. IOC:

Synaptophysin (a marker for presynaptic plasticity) immunostaining and quantitative data.

[0087] In addition, we discovered that Niaspan treatment increases Angl expression in TIDM-MCAo rats (FIGS. 10D-E). Axons and blood vessels share molecular signals for purposes of navigation, regeneration and terminal arborization. Angl regulates vascular maturation. In our previous study, we found that T2DM- MCAo mice exhibit significantly decreased Angl protein expression in the ischemic brain and Niaspan treatment increased Angl expression in T1DM stroke rats. In the present study, we found that Angl colocalized with SMI-31 (FIG. 10D) in the ischemic brain, indicating that Angl is expressed in neurons. FIG. 10D: Angl/SMI31 double immunostaining. [0088] Our data also showed unexpectedly that Niacin and Angl increase neurite outgrowth in cultured PCN (FIG. 11). To evaluate whether Angl participates in Niaspan-induced axonal outgrowth, in vitro primary cortical neuron (PCN) culture was employed. Under conditions of high glucose, Niacin significantly increased PCN Angl expression compared to non- treatment HG-control (FIGS. 11 A-C, p<0.05). Both Niacin and Angl treatment increased neurite outgrowth in cultured PCN under condition of HG (FIGS. 1 1E-F) compared to non- treatment control (FIG. 1 ID, p<0.05). Anti-Angl marginally decreased Niacin-induced neurite outgrowth (FIG. 11G, p=0.06). However, Tie2-FC did not decrease Niacin-induced neurite outgrowth (FIG. 1 1H, p>0.05). These data indicate that Angl partially contributes to Niacin-induced neurite outgrowth, while it is not mediated by the Tie2 pathway. Thus, as shown in FIG. 1 1, Niacin increases Angl expression in cultured hypoxic PCN, and Niacin and Angl increase neurite outgrowth under HG conditioned media. Inhibition of the Angl, but not Tie2-FC, decreased neurite outgrowth in cultured hypoxic PCN under HG conditions. FIG. 1 1 A-B: Angl immunostaining in cultured hypoxic PCN treated without (A) or with Niacin (B). FIG. 1 1C: Angl expression quantitative data (n=4/group). FIG. 1 1D-H: Neurite outgrowth measured by TUJ1 immunostaining in cultured hypoxic PCN under HG condition control (D); HG+Angl (E); HG+Niacin (F); HG+Niacin+anti-Angl (G); HG+Niacin+Tie-2-FC (H) and neurite outgrowth quantitative data (I). N=6/group.

[0089] In our work, we discovered unexpectedly that TIDM-MCAo rats exhibit

significantly decreased axonal density after stroke compared to WT-MCAo rats. Niaspan treatment of stroke in T1DM rats increased Angl expression, promoted axonal remodeling in the IBZ, and improved functional outcome after stroke. High glucose (HG) decreased neurite outgrowth in PCN cultures. Niaspan treatment increased Angl expression in the ischemic brain and in the cultured PCN. Niacin and Angl significantly increased neurite outgrowth in PCN culture under HG condition. Inhibition of Angl , but not Tie2-FC, partially attenuated Niacin-induced PCN dendrite outgrowth. Therefore, Niacin-induced increase of Angl may partially contribute to axonal remodeling in T1DM rats after stroke. [0090] As indicated herein, axonal density is decreased in TIDM rats after stroke compared to WT-MCAo rats, while Niaspan treatment of stroke increases axonal density in TIDM-MCAo rats. Generally, functional recovery following acute CNS injury in humans, such as stroke, is exceptionally limited, leaving the affected individual with life-long neurological deficits. Axonal remodeling is related with functional outcome after stroke. Enhancement of plasticity by induction of axonal density has been shown to compensate for formerly lost function in spinal cord injury, as well as in stroke models. In our work, anterograde tracing with BDA injected into the right motor cortex was used to assess axonal sprouting in the contralateral motor cortex and ipsilateral rostral forelimb area. We found that TIDM-MCAo rats show decreased axonal density in the ipsilateral in TIDM-MCAo rats compared to WT-MCAo rats. Niaspan treatment significantly attenuated the decreased axonal density in TIDM-MCAo rats. Cortical neurons surviving in the peri-infarct motor cortex undergo axonal sprouting to restore connections between different cerebral areas after stroke, and spontaneous functional recovery after stroke maybe attributed to axonal remodeling in the corticospinal system. Therefore, the increased axonal remodeling of some embodiments may contribute to the Niaspan-induced functional outcome after stroke in TIDM-MCAo rats. Moreover, functional benefit is present from 7 days after Niaspan treatment and persists to at least 28 days after treatment. The long-term beneficial effect after stroke is related to axonal remodeling. Without limitation to any particular mechanism of action, the early beneficial effect may be related to vascular protection/remodeling, such as decrease of BBB leakage and brain hemorrhage. Previous studies have found that axonal remodeling starts from 2 to 3 weeks after stroke and could be detected at 28 days after MCAo . Our data also indicate that functional outcome after stroke is significantly correlated with axonal density in the ischemic brain at 28 days after MCAo. Thus, axonal remodeling may be needed for the maintenance of improved neurological benefit.

[0091] We also found that Angl partially contributes to Niaspan-induced axonal

outgrowth in TIDM-MCAo rats. Many factors may regulate axonal remodeling after stroke, such as brain-derived neurotrophic factor ("BDNF") and nerve growth factor ("NFG") and Angl, among others. In our previous studies, we found that Niaspan significantly increases both BDNF/TrkB and Angl expression in WT-stroke rats. However, we found that Niaspan significantly increases Angl expression, but does not increase BDNF expression in the ischemic brain in diabetes stroke rats (data not shown). The response to stroke and treatment differs between diabetic and wild-type stroke animals. In addition, Angl is of interest because of its complementary and important role in angiogenesis and vascular maturation. Angl not only promotes angiogenesis and vascular maturation, but is also a neurotrophic/neuritotrophic factor. In our current work, we found that TIDM-MCAo rats exhibit decreased Angl expression as well as decreased axonal density compared to WT-MCAo rats. Inhibition of Angl using an anti- Angl antibody in cultured PCN partially decreases Niacin-induced neurite outgrowth. In addition, HG treatment in cultured PCN also decreases neurite outgrowth. In our current work, we found that Angl treatment in cultured PCN in vitro attenuates the HG induced decrease of neurite outgrowth. Therefore decreased Angl expression in TIDM-MCAo rats may contribute to the increased axon damage. Although the mechanism of the observed Angl -induced rescue of axonal outgrowth on HG has not been specifically elucidated, our work indicates that it is not mediated via Tie2 pathway because function-blocking experiments using Tie2-FC did not affect neurite outgrowth on Niacin treated group.

In our current work, we found that Niaspan treatment in DM stroke rats increases Angl expression. Angl is an angiogenic factor and also promotes vascular stabilization and maturation. We found that Angl not only regulates vascular change but also directly promotes neurite outgrowth in cultured primary cortical neurons. Therefore, without limitation to any particular mechanism, Angl may couple the vascular and axon remodeling. In addition, vascular remodeling includes angiogenesis, vascular stabilization and maturation. Increase of vascular stabilization and maturation will decrease BBB leakage and brain hemorrhagic transformation. In our previous studies, we found that diabetes significantly decreased Angl, but increased Ang2 and MMP9 expression, which may contribute to the increased vascular damage, BBB leakage and brain hemorrhagic transformation. Increase of BBB leakage and brain hemorrhage may induce neurotoxicity into the ischemic brain and thereby promotes axonal and white matter damage. Therefore, without limitation to any particular mechanism, the increase of Angl may decrease BBB leakage and brain hemorrhage and thereby indirectly decrease axonal damage in the ischemic brain. Thus, Angl may have both direct and indirect effects on the regulation of axonal remodeling.

[0093] In summary, we have discovered unexpectedly that treatment of stroke in TIDM rats with Niaspan at 24 h after stroke significantly increased synaptic plasticity and axonal remodeling as well as long-term functional outcome, with Angl partially contributing to Niaspan-induced synaptic plasticity and axonal remodeling after stroke in TIDM rats.

[0094] EXAMPLE 3:

[0095] GW3965, a synthetic liver X receptor agonist, elevates high-density lipoprotein

("HDL") cholesterol and has antiatherosclerosis and anti-inflammation properties. In our work, we evaluated whether GW3965 treatment of stroke increases vascular remodeling, promotes synaptic protein expression and axonal growth in the ischemic brain, and improves functional outcome in mice.

[0096] Stroke is a major cause of cerebral white matter and vascular damage, which

induces long-term disability as a result of the limited axonal regeneration (axon- regrowth or sprouting) and vascular remodeling (neovascularization and vascular stabilization) in the inhibitory environment of the adult mammalian central nervous system. Successful axonal outgrowth in the adult is central to the process of nerve regeneration and brain repair. Vascular remodeling plays an important role in neurological functional recovery after stroke.

[0097] Liver X receptor ("LXR") activates reverse cholesterol transport and raises high- density lipoprotein cholesterol ("HDL-C"). Increasing HDL-C improves functional outcome after stroke. Treatment of stroke in rats with Niacin, an effective medication in current clinical use for increasing HDL-C, significantly increases blood HDL-C and improves functional outcome. Treatment of stroke in mice with T0901317, an agonist of LXRa, increases serum HDL-C as well as improves functional outcome. However, high doses of Niacin produce adverse side effects of skin flushing, stomach upset, and liver damage, and T0901317 concurrently increases total blood cholesterol and triglycerides and may induce severe liver damage. In contrast, GW3965, a synthetic LXRJ3 selective agonist, raises HDL-C but without inducing hepatic steatosis and hypertriglyceridemia in rodents. Treatment stroke with GW3965 from early-onset (10 minutes to 2 hours) induces neuroprotection by antineuroinflammation and stabilizes the blood-brain barrier (BBB) integrity in the ischemic brain. However, many neuroprotective treatments have failed in clinical trials because stroke patients are very rarely treated within minutes of stroke onset. In our work, we evaluated the effect of GW3965, as a subacute treatment (24 hours after stroke), on HDL-C and functional outcome and mechanisms underlying the restorative response of brain to this drug on axonal outgrowth and vascular remodeling in a mouse stroke model.

[0098] Generally, mice were subjected to transient middle cerebral artery occlusion and treated without or with different doses of GW3965 (5, 10, or 20 mg/kg) starting 24 hours after middle cerebral artery occlusion daily for 14 days. Neurological functional tests, blood high-density lipoprotein cholesterol measurement, and immunostaining were performed. Mouse brain endothelial cells, primary cultured artery explants, and primary cortical neurons cultures were also used in vitro.

[0099] In summary, we discovered unexpectedly that GW3965 treatment of stroke

significantly increased blood high-density lipoprotein cholesterol level, synaptic protein expression, axonal density, angiogenesis and arteriogenesis, and

Angiopoietinl, Tie2, and occludin expression in the ischemic brain, and improved functional outcome compared with middle cerebral artery occlusion control animals (n=10; P<0.05). In vitro, GW3965 and high-density lipoprotein cholesterol also significantly increased capillary-like tube formation and artery explant cell migration as well as neurite outgrowth. Inhibition of Angiopoietin-1 attenuated GW3965-induced tube- formation, artery cell migration, and neurite outgrowth (n=6 per group; P<0.05). [0100] Thus, we have discovered unexpectedly that GW3965 promotes synaptic protein expression and axonal growth and increases vascular remodeling, which may contribute to improvement of functional outcome after stroke. Increasing

Angiopoietin-l/Tie2 signaling activity may play an important role in GW3965- induced brain plasticity and neurological recovery from stroke.

[0101] Materials and Methods

[0102] All experiments were conducted in accordance with the standards and procedures of the American Council on Animal Care and Institutional Animal Care and Use Committee of Henry Ford Health System.

[0103] Animal Model and Experimental Group:

[0104] Adult male C57BL/6J mice aged 2 to 3 months (Charles River) were subjected to 2.5 hours of right middle cerebral artery occlusion ("MCAo") by a filament method. Mice were gavaged starting 24 hours after MCAo with the following: (1) saline for vehicle control; (2) different doses of GW3965 (Sigma, 5, 10, or 20 mg/kg) daily for 14 days. All mice received bromodeoxyuridine (BrdU, 50 mg/kg, Sigma) intraperitoneal injections to label proliferating cells starting 24 hours after MCAo and daily for 14 days. The blood level of HDL-C, total cholesterol ("T- CH"), and triglyceride, lesion volume calculation, immunostaining, Western blot, and real-time PCR ("RT-PCR") were performed 14 days after MCAo. An additional 2 mice were euthanized 24 hours after MCAo to harvest artery explants for the cell migration assay.

[0105] Functional Tests:

[0106] Modified neurological severity score ("mNSS") and left foot-fault tests were

performed before MCAo and at 1, 7, and 14 days after MCAo, as known to the skilled artisan.

[0107] HDL-C, T-CH, and Triglyceride Measurement:

[0108] Blood levels of HDL-C, T-CH, and triglyceride were measured at 14 days after MCAo using CardioChek Ρ·Α analyzer and HDL-C, T-CH, and triglyceride test strips (Polymer 285 Technology System) according to the manufacturer's instructions. Data are presented as mg/dl values. : [0109] Histological and Immunohistochemical Assessment and Lesion Volume

Measurement:

[0110] The brains were fixed by transcardial perfusion with saline followed by 4%

paraformaldehyde before being embedded in paraffin. The cerebral tissues were cut into 7 equally spaced (1 mm) coronal blocks. A series of adjacent 6^m-thick sections were cut from each block and stained with hematoxylin and eosin ("H&E") for the lesion volumes calculation, as known to the skilled artisan. Every 10th coronal section cut from the center of the lesion (bregma -1 mm to +1 mm) for a total 5 sections was used for immunohistochemical staining.

Immunostaining for Synaptophysin (1 : 1000, Chemicon), Amyloid precursor protein ("APP", 1 :50, Cell Signaling Technology), Angiopoietinl (Angl , 1 :2,000, Abeam), von Willebrand Factor ("vWF", 1 :400; Dako), alpha smooth muscle actin ("aSMA", 1 :800, Dako), and histochemical-staining for Bielschowsky silver and Luxol Fast Blue, single immuno fluorescent-staining for SMI31 (1 : 1000, Covance), Tie2 (1 :80, Santa Cruz Biotechnology), and occludin (1 :200, Zymed) conjugated with Cy3 (1 :200, Jackson Immunoresearch Laboratories), and double immunofluorescent-staining for BrdU ( 1 : 100, Boehringer Mannheim) with vWF or aSMA were used. Control experiments consisted of staining brain coronal tissue sections as outlined above, but nonimmune serum was substituted for the primary antibody.

[0111] Photo Acquisition and Immunostaining Quantitation:

[0112] Images were acquired from 5 slides each brain, with each slide containing 8 fields view within the cortex and striatum from the ischemic boundary zone (see e.g., FIG13, "IBZ,") and analyzed with a Micro Computer Imaging Device ("MCID") imaging analysis system (Imaging Research), as known to the skilled artisan.

[0113] The following were calculated in the IBZ: (1) the percentage of Synaptophysin- or Angl -positive area in the cortex; (2) the percentage of APP-, Bielschowsky silver-, SMI31-, or Luxol Fast Blue-immunoreactive area in the bundles of the striatum; (3) the percentage of Tie2- or occludin-positive area in vessels; (4) the vascular density by the total number of vWF-vessels per mm ² ; the average vascular perimeter (μπι) from a total of 20 enlarged thin walled vessels; (5) the arterial density by the total numbers of aSMA-arteries with regard to small and large vessels (mean diameter >10 μηι) per mm ^" ; (6) the average arterial diameter from 10 largest arteries; (7) for cell proliferation, the percentage of BrdU- positive endothelial cells (EC) and smooth muscle cells (SMC) in the vessels and arteries.

[0114] Primary Cortical Neuron and Neurite Outgrowth Measurements:

[0115] Primary cortical neurons ("PCNs") were subjected to 1 hour of oxygen and

glucose deprivation followed by 24 hours of reperfusion. The hypoxic PCNs were then treated with (n=6 wells per group) the following: (1) nontreatment for control; (2) Angl 100 ng/ml (mouse Angl peptide, Millipore); (3) HDL 80 μg ml (Calbiochem); (4) GW3965 1 μΜ; (5) GW3965 1 μΜ + Anti-Angl (1 μ^πιΐ, Rabbit anti-Angl affinity purified polyclonal antibody, Millipore) for 24 hours. Then, the PCN cultures were performed TUJ 1 -staining (a phenotypic marker of neural cells, 1 : 1000, Covance) with Cy3 for neurite outgrowth measurement. Photomicrographs at x20 were captured, and neurite length was measured and averaged.

[0116] Mouse Brain EC Culture and Capillary- Like Tube Formation Assay:

[0117] Mouse brain ECs (MBECs; 2 10 ⁴ cells, ATCC, CRL-2299) were incubated in DMEM medium and were randomly divided into (n=6 wells per group) the following: (1) Nontreatment for control; (2) Angl 100 ng ml; (3) HDL 80 μg/ml; (4) GW3965 1 μηιοΙ/L; (5) GW3965 1 μΜ + Anti-Angl 1 μ^πιΐ treatment for 5 hours. Capillary-like tube formation was quantitated.

[0118] Primary Artery Explant Culture and Artery Cell Migration Measurement:

[0119] The ipsilateral common carotid arteries were surgically removed from mice 24 hours after MCAo. The common carotid arteries were cut into 1 mm ³ and randomly divided into 5 groups as follows: (1) Nontreatment for control; (2) Angl 100 ng/ml; (3) HDL 80 (4) GW3965 1 μΜ; (5) GW3965 1 μΜ + Anti-Angl 1 μg/ml. The artery explants were placed in the center of Matrigel and the arterial cultures were allowed to grow for 5 days before being photographed, and the 10 longest distances of neurite outgrowth were measured under a microscope at x4 magnification and averaged (n=6 wells per group).

[0120] RT-PCR: [0121] The ipsilateral brain tissue and MBECs were harvested, total RNA was isolated, and quantitative PCR was performed. The following primers for RT-PCR were designed using Primer Express software (ABI). GAPDH: Fwd, AGA ACA TCA TCC CTG CAT CC (SEQ ID NO: 2); Rev: CAC ATT GGG GGT AGG AAC AC (SEQ ID NO: 3). Angl : Fwd, TAT TTT GTG ATT CTG GTG ATT (SEQ ID NO: 4) Rev, GTT TCG CTT TAT TTT TGT AATG (SEQ ID NO: 5). Tie2: Fwd, CGG CCA GGT ACA TAG GAG GAA (SEQ ID NO: 6); Rev, TCA CAT CTC CGA ACA ATC AGC (SEQ ID NO: 7).

[0122] Western Blot:

[0123] Equal amounts of cell lysate were subjected to Western blot. The following

primary antibodies were used: anti-Angl (1 :2,000, Abeam), anti-Synaptophysin (1 : 1000, Chemicon), anti-p-actin (1 :2000; Santa Cruz).

[0124] Statistical Analysis:

[0125] Independent 2-sample t test was used to assess the lesion volume,

immunostaining, Western blot, and RT-PCR measurement. Pearson partial correlations after bivariate correlation were used to analyze the correlation of the blood HDL level with the neurological functional outcome. One-way ANOVA and Tukey test after post hoc test were performed for functional outcome, HDL- C, T-CH, and triglyceride, neurite outgrowth, tube-formation, and artery explant cell migration analysis. All data are presented as mean±SE. All measurements and functional evaluations were performed in a blinded manner.

[0126] Our results showed unexpectedly that GW3965 treatment of stroke increases blood HDL-C level and improves neurological outcome. No significant benefit was detected in the 5 mg/kg GW3965 treatment group compared with the MCAo control group. However, 10 mg/kg and 20 mg/kg of GW3965 treatment significantly improved mNSS and left foot-fault 14 days after MCAo. Moreover, 10 mg/kg of GW3965 treatment significantly decreased mNSS score 7 days after MCAo compared with MCAo-control or 5 mg/kg of GW3965 treatment group (Figure 1 A, P<0.05, n=10/group). Therefore, in our work, we selected 10 mg/kg as the optimal treatment dose for lesion volume measurement, immunostaining, Western blot, and RT-PCR assay. As shown in FIG. 12, GW3965 treatment increases HDL-C levels and improves functional outcome in mice 14 days after MCAo. FIG.12A: mNSS and left foot-fault test. FIG. 12B: HDL-C, triglyceride, and T-CH in blood. FIG. 12C: Correlation analysis between HDL-C and mNSS or foot-fault. n=10 per group. HDL-C indicates high-density lipoprotein cholesterol; MCAo, middle cerebral artery occlusion; mNSS, modified neurological severity score; and T-CH, total cholesterol.

[0127] FIG. 12B shows that 10 mg/kg and 20 mg/kg GW3965-treatment of stroke

significantly increased blood HDL-C level (average increased 18.9 mg/dl) but did not significantly increase triglyceride level. However, we found that 10 mg/kg and 20 mg/kg GW3965 significantly increased T-CH level (average 127.1±5.2) compared with MCAo-control animals (107.8±3.6; P=0.028). To investigate the cause of GW3965 treatment-induced increase in T-CH, we subtracted HDL-C from T-CH and found that there is no significant difference in T-CH level after subtraction of HDL-C (after subtraction of HDL-C level, MCAo-control:

54.8±2.84; GW3965-treatment: 58.0±5.31 ; P=0.69). Therefore, the data indicate that the increased T-CH is attributed to the increase of HDL-C. Correlation analysis (FIG. 12C) showed that the level of blood HDL is significantly negatively correlated with mNSS score (r=-0.899; P<0.01 ) and the percentage of left foot-fault (r=-0.764; P<0.05). These data indicate that GW3965 treatment of stroke increases HDL-C and thereby improves functional outcome.

[0128] No significant differences of lesion volumes in 10 mg/kg GW3965-treatment

(16.1 1%±1.22%) were detected compared with MCAo-control (17.96%±1.86%; P=0.419, n=10 per group).

[0129] We also discovered unexpectedly that GW3965 treatment of stroke decreases axon damage and increases synaptic protein expression and axon density. FIGS. 13B-D shows that GW3965 treatment significantly increased Synaptophysin (a marker for presynaptic plasticity and synaptogenesis) positive area in the IBZ, the density of Bielschowsky sliver (a marker for axons), SMI31 (a marker of nondamaged phosphorylated neurofilament), and LBF (a myelin marker) but decreased APP (a marker of axonal damage) positive area in the striatal bundles compared with MCAo-control (n=10; P<0.05). Western blot assay also showed GW3965 treatment significantly increased the protein level of Synaptophysin in the 1BZ (n=4; P<0.05). These data indicate that GW3965 treatment decreases axon damage, increases axon, neurofilament, and myelin densities in the striatal bundles, and promotes synaptic protein expression in the ischemic brain after stroke. As shown in FIG. 13, GW3965 treatment increases Synaptophysin expression and axonal and myelin growth and decreases axon damage in the IBZ 14 days after MCAo. GW3965 treatment increases Synaptophysin expression and axonal and myelin growth and decreases axon damage in the IBZ 14 days after MCAo. GW3965 treatment increases Synaptophysin expression and axonal and myelin growth and decreases axon damage in the IBZ 14 days after MCAo. FIG. 13 A: Schematic map showing the IBZ and quantified regions. FIG. 13B:

Synaptophysin-immunostaining, Western blot, and quantitative data. FIG. 13C: Bielschowsky silver and SMI31 immunostaining and quantitative data. FIG. 13D: LFB and APP immunostaining and quantitative data. Scare bar, 100 im. n=10 per group in immunostaining, n=4 per group in Western blot. APP = amyloid precursor protein; IBZ = ischemic boundary zone; LFB = Luxol Fast Blue; and MCAo = middle cerebral artery occlusion.

We also discovered unexpectedly that GW3965 treatment increases angiogenesis, arteriogenesis, and vascular stabilization in the ischemic brain. FIG. 14 shows that compared with MCAo-control, GW3965 treatment significantly increased the following: (1) the vascular density and perimeter of vWF-vessels; (2) the arterial density and diameter of aSMA-arteries; (3) the percentage of BrdU-ECs in vessels and BrdU-SMCs in arteries; and (4) the expression of occludin (a tight junction protein of critical component of BBB) in the IBZ (n=10; P<0.05). These data indicate that GW3965 treatment increases neovascularization (angiogenesis and arteriogenesis) and vascular stabilization in the ischemic brain. (Figure 14: GW 3965 treatment increases angiogenesis, arteriogenesis, and vascular stabilization in the IBZ 14 days after MCAo. FIG, 14 A, vWF-immuno staining and quantitative data. FIG. 14B, ccSMA-immunostaining and quantitative data. FIG. 14C, BrdU double-immunofluorescent staining with vWF and SMA and quantitative data. FIG. 14D, Occludin-immunofluorescent staining and quantitative data. Scare bar in A and B, 100 im; n=10 per group. BrdU indicates bromodeoxyuridine; IBZ, ischemic boundary zone; MCAo, middle cerebral artery occlusion; SMA, smooth muscle actin; and vWF, von Willebrand factor.

[0131] We also found that GW3965 treatment significantly increased Angl and Tie2 expression in the ischemic brain as measured by immunostaining in the IBZ compared with MCAo control (FIG. 15A and FIG. 15B; n=10; P<0.05). In addition, GW3965 treatment significantly increased Angl protein expression analyzed by Western blot and Angl/Tie2 mRNA level measured by RT-PCR in the IBZ (Figure 15C and 15D; n=4; P<0.05). (FIG 15: GW 3965 treatment increases Angl and Tie2 expression in the IBZ 14 days after MCAo. FIG. 15A, Angl-immunohistostaining and quantitative data. FIG. 15B, Tie2- immunofluorescent staining and quantitative data. FIG. 15C, Western blot showing Angl protein expression and quantitative data. FIG. 15D, Angl and Tie2 gene expression measured by RT-PCR. Scare bar, 100 im (A), 50 im (B). n=10 per group in A and B; n=4 per group in C and D. Ang indicates angiopoietin; IBZ, ischemic boundary zone; and MCAo, middle cerebral artery occlusion.)

[0132] We also discovered unexpectedly that GW3965 increases neurite outgrowth, capillary-like tube formation, and artery explant cell migration in vitro. FIG. 16 shows that compared with nontreatment control, Angl and HDL and GW3965 treatment significantly increased the following: (1) the neurite outgrowth in the hypoxic PCNs; (2) the capillary-like tube formation in the cultured MBECs; and (3) the artery explant cell migration in the primary cultured arteries. However, anti-Angl significantly attenuated GW3965-induced neurite outgrowth, capillary tube formation, and artery explant cell migration (n=6; P<0.05). Consistent with the in vivo data, HDL and GW3965 significantly increased Angl mRNA expression, and GW3965 significantly increased Tie2 mRNA expression in the cultured MBECs (n=6; P<0.05). (FIG. 16: GW3965 increases neurite outgrowth, capillary-like tube formation, and artery explant cell migration. HDL and

GW3965 increase Angl, GW3965 also increases Tie2 gene expression in cultured MBECs. FIG. 16A, TUJl-immunostaning in PCNs of 1 hour OGD and neurite outgrowth quantitative data. FIG. 16B, Capillary-like tube formation in MBECs and quantitative data. FIG. 16C, Angl and Tie2 gene expression in MBECs. FIG. 16D, Artery explant cell migration in CCAs and quantitative data, n = 6 per group. Ang indicates Angiopoietin; CCA, common carotid arteries; HDL, high- density lipoprotein; MBEC, mouse brain endothelial cell; OGD, oxygen and glucose deprivation; and PCN, primary cortical neurons.)

[0133] HDL-C is related to stroke recovery. Low levels of HDL-C predict high mortality and rapidly progressive stroke; higher levels of HDL-C are associated with better cognitive recovery after stroke. LXRs belong to the nuclear receptor superfamily that can regulate important lipid metabolic pathways. GW3965 increased expression of the reverse cholesterol transporter ABCAl and increased the plasma concentrations of HDL-C. In our current work, we found that GW3965 treatment significantly increases blood HDL-C level and improves functional outcome after stroke, and the increased HDL-C is significantly correlated with functional outcome. Therefore, increasing HDL-C by GW3965 treatment may contribute to functional outcome.

[0134] Without limitation to a particular mechanism, stroke-induced white matter injury may help explain the failure of neuroprotective drugs in clinical trials for stroke because these drugs were rarely characterized for their ability to protect white matter. Cellular cholesterol modulates axon and dendrite outgrowth and neuronal polarization under culture conditions. LXRs are essential for maintenance of motor neurons in the spinal cord and dopaminergic neurons in the substantia nigra. LXRP regulates the formation of superficial cortical layers and migration of later-born neurons. LXR knockout mice exhibit excessive lipid deposits, proliferation of astrocytes, loss of neurons and their dendrites, and disorganized myelin sheaths. LXR activators induce neuronal differentiation in rat

pheochromocytoma cells and stimulate neurite outgrowth. In our work, we found that GW3965 treatment of stroke significantly decreased APP expression in the ischemic brain. APP is a transmembrane glycoprotein that is widely expressed in mammalian tissues and is transported through axons. Axonal damage evokes a disturbance of fast axonal transport, can occur even in the early stage of white matter lesions, and cannot transport APP. Therefore, the decrease of APP expression by GW3965 treatment of stroke may reflect the decreased axonal damage in the ischemic brain. Axonal plasticity parallels functional recovery after cortical injury, including stroke. In our current work, we demonstrate for the first time that GW3965 treatment starting at 24 hours after MCAo significantly increases Synaptophysin expression and axon, myelin, and neurofilament density in the ischemic brain; in addition, GW3965 increases neurite outgrowth in the PCNs. GW3965-induced axonal plasticity may contribute to functional improvement after stroke.

Recovery of neurological function after stroke is mediated by many coupled events, including neurogenesis, synaptogenesis, and vascular remodeling. The BBB contributes to the maintenance of brain cholesterol metabolism and protects this uniquely balanced system from exchange with plasma lipoprotein cholesterol. We found that GW3965 treatment increases occludin expression in vessels in the ischemic brain, which is consistent with previous findings that GW3965 maintains HDL-C homeostasis at the BBB and stabilizes the BBB. Angiogenesis involves the capillary sprouting, branching, splitting, and differential growth of vessels in the primary plexus to form the mature vascular system. Brain capillary ECs, representing a physiological barrier to the central nervous system, express apolipoprotein A-I, the major HDL-C, and promote cellular cholesterol mobilization. HDL-C decreases platelet aggregation and inhibits EC apoptosis. HDL-C also enhances EC migration and angiogenesis. Intravenous injection of reconstituted HDL stimulates differentiation of endothelial progenitor cells and enhances ischemia-induced angiogenesis. Arteriogenesis during

neovascularization, supporting cells such as pericytes and SMCs are recruited to the vessels to provide structural support and stability for the vascular walls. LXR knockout mice exhibit enlarged brain blood vessels with weak staining of aSMA and excessive lipid accumulation around the abnormal vessels, which lose their contractile ability and are susceptible to rupture. In our work, we found that GW3965 treatment of stroke induces angiogenesis and arteriogenesis identified by increasing EC/SMC proliferation and vascular density/perimeter/diameter in vessels in the ischemic brain. GW3965 also increases MBEC capillary-like tube formation and artery cell migration in vitro. GW3965 treatment-induced angiogenesis/arteriogenesis may contribute to the functional outcome after stroke.

[0136] Angl, an angiopoietic factor, and its receptor Tie2 play an important role in

neovascularization. Angl also promotes synaptic plasticity and axon remodeling. Angl stimulates neuronal differentiation and supports neurite outgrowth and synaptogenesis in neuronal progenitor cells, sensory neurons, and PC 12 cells, and Niacin increases Angl gene and protein expression after stroke. In our current work, GW3965 treatment increases Angl/Tie2 protein expression in the ischemic brain and Angl/Tie2 mRNA expression in cultured MBECs. Angl also promotes GW3965-induced capillary-like tube formation, artery explant cell migration, and neurite outgrowth in vitro, which in concert indicate that the Angl/Tie2 pathway mediates GW3965-induced brain plasticity after stroke.

[0137] In summary, we have found unexpectedly that GW3965 has neurorestorative benefits in stroke treatment. GW3965 treatment starting 1 day after stroke did not decrease lesion volume but did increase synaptic protein expression, axonal growth, and vascular remodeling in the ischemic brain as well as improves functional outcome. Increasing HDL and upregulation of Angl/Tie2 activity appears to contribute to the GW3965-induced brain plasticity after stroke.

[0138] Thus, without limitation and without waiver or disclaimer of embodiments or subject matter, some embodiments provide methods, systems, and compositions which provide, increase, or promote Angl or Angl signaling activity and provide, , increase, or improve white model remodeling, neurite outgrowth, and/or

neurological function in treated subjects. Some embodiments comprise administration of a composition comprising a pharmaceutically effective amount of one or more of Angl, a promoter of Angl expression, D-4F, HUCBCs, Niacin/Niaspan, and GW3965 (the above referred to convenience only as "Angl- related composition(s)" or "Angl -related composition administration") to prevent, control, or alleviate neurological conditions, disease, or injuries in subjects needing such treatment. In accordance with some embodiments, without limitation, one may inhibit such illness or injury through Angl -related composition administration for a finite interval of time, thereby limiting the development or course of such condition, disease or injury.

[0139] In accordance with some embodiments, there is a high likelihood that the duration of therapy comprising Angl -related composition administration would be relatively brief and with a high probability of success. Prophylactic Angl -related composition administration of some embodiments may greatly reduce the incidence of damage associated with some forms of illness or injury.

[0140] Any appropriate routes of Angl -related composition administration known to those of ordinary skill in the art may comprise some embodiments.

[0141] Angl -related compositions of some embodiments would be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. In accordance with some embodiments, experience with dose levels in animals is known and dose levels acceptable for safe human use are determinable or scalable in accordance with such information and/or good medical practice. The "pharmaceutically effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement, including but not limited to, decrease in damage or injury, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.

[0142] In accordance with some embodiments, Angl-related compositions can be

administered in various ways. They can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles. The Angl-related compositions can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneal, and intranasal administration as well as intrathecal and infusion techniques, or by local administration or direct inoculation to the site of disease or pathological condition. Implants of the Angl-related compositions may also be useful. The patient being treated is a warm-blooded animal and, in particular, mammals including humans. The pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active components of some embodiments. In some embodiments, Angl- related compositions may be altered by use of antibodies to cell surface proteins or ligands of known receptors to specifically target tissues of interest.

[0143] Since the use of Angl -related composition administration in accordance with some embodiments specifically targets the evolution, expression, or course of associated conditions or pathologies, it is expected that the timing and duration of treatment in humans may approximate those established for animal models in some cases. Similarly, the doses established for achieving desired effects using such compounds in animal models, or for other clinical applications, might be expected to be applicable in this context as well. It is noted that humans are treated generally longer than the experimental animals exemplified herein which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses may be single doses or multiple doses over periods of time. The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.

[0144] When administering the Angl -related compositions of some embodiments

parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0145] When necessary, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for such Angl -related composition compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to some embodiments, however, any vehicle, diluent, or additive used would have to be compatible with the Angl -related compositions.

[0146] Sterile injectable solutions can be prepared by incorporating Angl -related

compositions utilized in practicing the some embodiments in the required amount of the appropriate solvent with various of the other ingredients, as desired.

[0147] A pharmacological formulation of some embodiments may be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the inhibitor(s) utilized in some embodiments may be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0148] In some embodiments, without limitation, the Angl -related compositions may be administered initially by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used. The quantity to be administered and timing of administration may vary for the patient being treated.

[0149] Additionally, in some embodiments, without limitation, Angl -related

compositions may be administered in situ to bring internal levels to a suitable level. The patient's levels are then maintained as appropriate in accordance with good medical practice by appropriate forms of administration, dependent upon the patient's condition .The quantity to be administered and timing of administration may vary for the patient being treated.

While some embodiments have been particularly shown and described with reference to the foregoing preferred and alternative embodiments, it should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the invention without departing from the spirit and scope of the invention as defined in the following claims. It is intended that the following claims define the scope of the invention and that the method and apparatus within the scope of these claims and their equivalents be covered thereby. This description of some embodiments should be understood to include all novel and non-obvious combinations of elements described herein, and claims may be presented in this or a later application to any novel and non- obvious combination of these elements. The foregoing embodiments are illustrative, and no single feature or element is essential to all possible

combinations that may be claimed in this or a later application. Where the claims recite "a" or "a first" element of the equivalent thereof, such claims should be understood to include incorporation of one or more such elements, neither requiring nor excluding two or more such elements.

SEQUENCES

SEQ ID NO: 1

D-4F, generally described as:

Ac-D-W-F- -A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (4F) SEQ ID NO: 2:

AGA ACA TCA TCC CTG CAT CC SEQ ID NO: 3:

CAC ATT GGG GGT AGG AAC AC SEQ ID NO: 4:

TAT TTT GTG ATT CTG GTG ATT SEQ ID NO: 5:

GTT TCG CTT TAT TTT TGT AATG SEQ ID NO: 6:

CGG CCA GGT ACA TAG GAG GAA SEQ ID NO: 7

TCA CAT CTC CGA ACA ATC AGC