OF LIVER FIBROSIS

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of liver fibrosis.

BACKGROUND OF THE INVENTION:

Liver fibrosis is characterized by the loss of parenchyma and deposition of extracellular matrix (ECM) components by activated myofibroblasts, ultimately leading to excessive scar formation and organ failure (1, 2). However, liver fibrosis in response to chronic liver injury is now considered as a dynamic and reversible process even at advanced stages (3). This recovery process requires first, ECM degradation along with resolution of the fibrotic scar, and second, regeneration of the hepatocyte population involving the activation of liver progenitor cells (4, 5). The monocyte -macrophage lineage plays a crucial role in the resolution of fibrosis as well as liver regeneration and bone marrow cell treatment for liver cirrhosis holds considerable therapeutic potential (4, 6, 7). It was recently reported that targeting vascular endothelial growth factor (VEGF) specifically in scar-infiltrating myeloid cells prevented remodeling of the sinusoidal vasculature and abrogated the resolution of murine liver fibrosis. Furthermore, the resolution of liver fibrosis was shown to be associated with increased expression of matrix metalloproteases (MMP)-2 and -14 as well as decreased expression of tissue inhibitor of metalloproteases (TIMP)-l and 2 confined to sinusoidal endothelium, thereby unmasking an unanticipated link between angiogenesis and resolution of fibrosis (8). VEGF expression is at least partially regulated by low oxygen levels through a transcription factor family called Hypoxia-inducible factors (HIF)(9). The von Hippel Lindau protein (VHL) is a negative regulator of HIF and HIF-dependent VEGF expression (9, 10) which results in VEGF overexpression in scar-associated macrophages. However the impact of the VHL protein on liver fibrosis has never been investigated.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of liver fibrosis. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

The inventors have recently shown that targeting Vascular Endothelial Growth Factor (VEGF) specifically in scar-infiltrating myeloid cells prevented remodeling of the sinusoidal vasculature and abrogated the resolution of murine liver fibrosis. Furthermore, the inventors showed that the resolution of liver fibrosis is associated with increased expression of Matrix Metalloproteases (MMP)-2 and -14 as well as decreased expression of Tissue Inhibitor of Metalloproteases (TIMP)-l and 2 confined to sinusoidal endothelium, thereby unmasking an unanticipated link between angiogenesis and resolution of fibrosis. In a gain of function approach, the inventors wanted to test the impact of VEGF overexpression in myeloid cells on fibro lysis. They observe that genetic inactivation of the von Hippel Lindau protein (VHL), a negative regulator of Hypoxia-inducible factors (HIF) in myeloid cells, leads to increased VEGF expression and most importantly, accelerated matrix degradation and fibrosis resolution after CCL4 challenge. This is associated with enhanced expression of MMP-2 and -14 in liver endothelial cells and improved sinusoidal infiltration of the fibrotic scar. In addition, the inventors report overall increased expression of MMP-7, -9 and -13 as well as improved liver regeneration upon ablation of VHL in myeloid cells. Thus, boosting the hypoxic response and HIF signaling in scar infiltrating myeloid cells could represent a promising therapeutic avenue for the treatment of liver fibrosis. Accordingly the present invention relates to a method of treating liver fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of the von Hippel Lindau protein activity or expression.

Liver (hepatic) fibrosis, for example, occurs as a part of the wound- healing response to chronic liver injury. Such damage may be the result of viral activity (e.g., chronic hepatitis types B or C) or other infections (e.g., parasites, bacteria), chemicals (e.g., pharmaceuticals, alcohol, pollutants), immune processes (e.g., autoimmune hepatitis), metabolic disorders (e.g., lipid, glycogen, or metal storage disorders), or cancer growth. Liver fibrosis is characterized by the accumulation of extracellular matrix that can be distinguished qualitatively from that in normal liver. Left unchecked, hepatic fibrosis progresses to cirrhosis (defined by the presence of encapsulated nodules), liver failure, and death.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). As used herein, the term "von Hippel Lindau protein" or "VHL" has its general meaning in the art and refers a protein that in humans is encoded by the VHL gene. The protein is also named HRCA1; RCA1; VHL1; or pVHL.

An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti- sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targerted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor of the present invention inhibits specifically the expression of the protein in myeloid cells. As used herein, the term "myeloid cell" refers to monocytes, neutrophils and macrophages. Yet further, myeloid cell also refers to all stage differentiations of monocytes, macrophages and neutrophils. For instance, the inhibitor can be coupled to an agent that is able to bind to a specific surface receptor of a myeloid cells. For example said receptor can be CD206 and the agent may be selected from anti-CD206 antibodies and macromolecules capable of binding to CD206 such as tilmanocept that consists of dextran 3-[(2-aminoethyl)thio]propyl 17-carboxy- 10,13,16-tris(carboxymethyl)-8-oxo-4-thia-

7,10,13,16-tetraazaheptadec- 1 -yl 3-[[2-[[ 1 -imino-2-(D- mannopyranosylthio)ethyl]amino]ethyl]thio]propyl ether complexes. According to the invention, the inhibitor of the present invention is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. Typically the inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: Transplantation of bone marrow from VHLfl/fl-LysMcre+ mice into C57B16/J mice after CCL4-challenge accelerates fibrosis resolution as compared to mice after reconstitution with wildtype (VHLfl/fl-LysMcre-) bone marrow, (a) VEGF release by macrophages (Macs) from VHLfl/fl-LysMCre- (n=5) and VHL fl/fl- LysMCre+ mice (n=7). (b) Time-schedule for the bone-marrow transplantation of CCL4-treatmed mice, (c) Quantification of Sirius Red-positive area on murine livers sections, (d) Determination of free hydroxyproline in liver tissue samples, (e) Quantification of a-SMA-positive area from the two groups of mice (n=5 for VHL LysMCre- and n=7 for VHL LysMCre+).

Figure 2: Transplantation of bone marrow from VHLfl/fl-LysMcre+ mice into C57B16/J mice after CCL4-challenge accelerates matrix degradation activity (a)

Quantification of DQTM-gelatin-positive areas on liver sections, (b) Quantitative analysis of the VEGFR2 -positive area. Figure 3: Deletion of VHL in myeloid cells during the resolution of liver fibrosis induces the expression of matrix degrading enzymes in liver endothelial cells and whole liver, (a) Quantitative real time-analysis of MMP2, MMP14, and TIMP2-expression, respectively, in liver endothelial cells (LECs). (b) Quantification of MAC-2-expressing macrophages, (c) Quantitative real time-analysis of CXCL9 expression in whole livers, (d) Quantitative real time-analysis of MMP7, MMP9, and MMP13 expression, respectively, in whole livers, (e) Quantitative real time-analysis of MMP7, MMP9, and MMP13 expression, respectively, in isolated liver macrophages (n=5 for VHL LysMCre- and n=7 for VHL LysMCre+). (f) Quantitative real time-analysis of MMP13 expression in peritoneal macrophages (n=5 for VHL LysMCre- and n=6 for VHL LysMCre+). Error bars represent SEM.

Figure 4: Improved liver regeneration in C57B16/J mice after transplantation with bone marrow from VHLfl/fl-LysMcre+ mice after CCL4-challenge compared to mice after transplantation with wildtype (VHLfl/fl-LysMcre-) bone marrow, (a) Quantification of PCNA-positive nuclei per field on liver sections, (b) Quantitative real time-analysis of Hgf expression, respectively, in whole livers, (c) Quantitative analysis of PCK-expressing liver progenitor cells, (d) Quantitative analysis of Dlk-expressing liver progenitor cells, (e) Quantitative real time-analysis of Tweak-expression, respectively, in whole livers.

Error bars represent SEM (n=5 for VHL LysMCre- and n=7 for VHL LysMCre+). Scale bars equal 50 μιη.

Figure 5: Simultaneous targeting of VEGF and VHL in myeloid cells reverses the phenotype of VHL deletion.

(a) Determination of free hydroxyproline in liver tissue samples (n=5). (b) Quantitative analysis of the SMA-positive area on liver sections (n=5). (c) Quantitative analysis of the VEGFR2 -positive area (n=5). (d) Quantitative analysis of PCK-expressing liver progenitor cells (n=5). (e) Quantitative real time-analysis of MMP2, MMP14, and TIMP2-expression, respectively, in liver endothelial cells (LECs) (n=5). (f) Quantitative real time-analysis of MMP13 expression in isolated liver macrophages (n=5). (g) Quantitative real time-analysis of MMP13 expression in peritoneal macrophages (n=5 for VEGF/VHL LysMCre- and n=8 for VEGF/VHL LysMCre+).

Error bars represent SEM. Scale bars equal 100 μιη. Figure 6: Therapy with VHL-deficient macrophages accelerates fibrosis resolution.

(a) Quantitative analysis of the SMA-positive area on liver sections (n=5 for VHL LysMCre- and n=6 for VHL LysMCre+). (b) Quantitative analysis of PCK-expressing liver progenitor cells (n=5 for VHL LysMCre- and n=5 for VHL LysMCre+).

EXAMPLE:

Experimental Procedures Animals

The Animal Care and Use Committee of the Bezirksregierung Dusseldorf, Germany, approved all procedures performed on mice. Myeloid cell-specific knock out of VHL was achieved by breeding male Mice (C57/B6), with both alleles of VHL flanked by loxP sites (VHL+ ^f /+ ^f ) with female mice (C57/B6) homozygous for the floxed VHL allele expressing Cre recombinase driven by the lysozyme M promoter (LysMCre+/VHL+ ^f /+ ^f )(12). As bone marrow donor mice we used mice carrying two floxed VHL alleles and positive for cre-expression (LysMCre+/VHL+ ^f /+ ^f ) and female littermates negative for cre expression (LysMCre - /VHL+ ^f /+ ^f ) served as wildtype controls. All animals received care according to the "Guide for the care and use of laboratory animals"

Induction of hepatic fibrosis and tissue preparation

For the induction of hepatic fibrosis female mice were treated with CCU intraperitoneally (240 μΐ CCU suspended in olive oil per kg body weight 3 times a week) for 12 weeks. Control mice received i.p. injections of 100 μΐ olive oil. Mice were sacrificed at indicated time points and livers harvested for further analysis. For histology, livers were fixed in 4 % (w/v) PFA overnight and embedded in paraffin or alternatively frozen in O.C.T. tissue TEK. For R A and protein isolation livers were separated and snap-frozen in liquid nitrogen.

Immunofluorescence and immunohistochemistry

5-μιη sections were deparaffmized with xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed by boiling the sections in low-pH citrate buffer for 15 minutes. Sections were treated with 3 % (v/v) H2O2 for 10 min at RT, blocked with 5 % normal goat serum (Sigma) for 1 hour at RT and incubated with the primary antibody overnight at 4 °C. Antigens of interest were visualized using the Vectastain ABC kit (Vector Laboratories) or by species-specific fluorochrome-conjugated secondary antibodies. For stainings with mouse- derived antibodies, Mouse on mouse (M.O.M.) Basic Kit (Vector Laboratories) was used following the kit instructions. Cell nuclei were stained with DAPI (Invitrogen) and coverslips were mounted with Mounting Medium (Dako). The following antibodies were used in this study: mouse MAC-2 at 1 : 100 dilution (Cedarlane), mouse a -SMA at 1 :500 dilution (Chemikon), rabbit VEGFR-2 at 1 : 100 dilution (Cell signaling), rabbit VEGF (Calbiochem) at 1 :200, mouse pro-MMP2 (Chemicon) at 1 : 100, mouse PCNA (Sigma) at 1 :200, PCK (Abeam) at 1 :200 and Dlk (Abeam) at 1 : 150. Sinus Red staining

Liver tissues were stained for collagen using the Picrosirius Red Stain kit (Polysciences Inc.) following the manufacturer's instructions. For quantitative analysis of, a minimum of 10 non-overlapping fields of each section were photographed (Nikon Eclipse El 000 microscope and the Nikon DS-Ril camera system) at 200x. The percentage of Sirius red staining was measured with Image J (National Institute of Health).

In situ zymography

A mixture of DQ™-gelatin and reaction buffer (Invitrogen) was applied on top of OCT- embedded frozen sections and incubated at 37 °C for 24 h in a dark humid chamber. The gelatinolytic activity was observed as green fluorescence by fluorescence microscopy (excitation: 342 nm; emission: 441 nm).

Quantification of histology markers

For quantitative analysis of immunohistochemical markers, sections were photographed into JPEG images (Nikon Eclipse E1000 microscope and the Nikon DS-Ril camera system). The number of pixels marked above a threshold by each marker was measured using the ImageJ program (National Institute of Health) and calculated as the percentage of the total area covered by DAPI. For assessment of MAC-2 and PCNA , positive cells were counted in each field. RNA extraction and qPCR-analysis

Total RNA was isolated by the phenol/chloroform extraction method. cDNA was synthesized from 1 μg of DNA-free total RNA in a 25 μΐ reaction volume using M-MLV Reverse Transkriptase (Promega) and oligo-dT-primers (Life Technologies). Gene-specific transcription levels were determined in a 20 μΐ reaction volume in duplicate using SYBR Green Mastermix (Promega) and an IQ5 real-time PCR machine (Bio-Rad). Quantification was done in a two-step real-time PCR with a denaturation step at 95 °C for 10 min and followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. Standard cDNA samples with 10-fold serial dilutions were used for PCR efficiency calculations. Data were normalizedby the level of 16S mRNA expression.

Bone marrow isolation and adoptive cell transfer

Hind limbs of donor mice were removed and cleaned. Both tops of the femur were cut off and each bone flushed with 5 ml RPMI 1640 containing 2 % FBS, 10 units/ml heparin, penicillin and streptomycin. The solution was filtered through a sterile 40 μιη cell strainer (BD Bioscience), washed twice and the cells directly used for injection. 1 week before and 2 weeks after irradiation (10 Gray), recipient mice were given acidified water (pH 2.6) supplemented with 10 mg/ml Neomycin and 25 mg/ml Polymyxin B (Sigma). The animals were irradiated 48 hours after the last CCL4-injection and adoptive transfer of bone marrow cells was performed 24 hours after irradiation. For adoptive transfer, 5x106 cells of the isolated bone marrow were injected into the tail-vain of the recipient mice. The mice were subsequently left for bone marrow reconstitution and recovery for 4 weeks, a time point that had been determined as suitable in pilot experiments. Isolation of liver endothelial cells and macrophages

For isolation of liver endothelial cells, freshly harvested murine livers were homogenized by mechanical disaggregation and digested in cell lysis buffer (DMEM + 2 mg/ml collagenase type III) for 1 hour at 37 °C. Single cell suspensions were generated by passing the cells through a 40-μιη cell strainer, followed by resuspension in MACS-buffer according to the manufacturer's instructions (Miltenyi Biotec) and incubation of 4xl0 ⁷ cells with mouse CD31- or F4/80-antibodies for 45 min at 4 °C. The cell suspensions were washed with MACS-buffer and incubated with secondary IgG microbeads for 15 min at 4 °C before positive selection using an automated MACS separator (Miltenyi Biotec). The purity of the cell isolates was tested by immunocytochemistry for VEGFR2 and MAC2 after fixing an aliquot of isolated cells on a slide with 4% PFA in order to identify endothelial cells and macrophages, respectively.The purity for endothelial cells was 86.78 % ± 1.507 (n=14) and 89.51 ± 1.281 N=16 for macrophages. For the isolation of peritoneal macrophages mice were injected with thiogly collate intraperitoneally and cells were harvested by peritoneal lavage(12). Mouse VEGF-A ELISA

Thioglycollate-elicited peritoneal macrophages were cultured for 12 hours. Mouse VEGF-A ELISA on supernatants was performed with Quantikine ELISA, Mouse VEGF Immunoassay, R&D Systems. The results are represented as pg/mL VEGF per mg of whole protein

Hydroxyproline-assay

Hepatic hydroxyproline content was quantified colorimetrically using snap frozen liver samples. Tissue (-100 mg) was homogenized in distilled water and protein was precipitated using trichloroacitic acid. Samples were washed with ethanol, dried and hydrolyzed in 6 M HCl at 110°C for 18 hours. The hydrolysate was filtered and neutralized with 10 M NaOH. Samples were then incubated with freshly prepared chloramine T solution. Ehrlich's solution was added and samples incubated for 20 minutes at 65°C. The optical density of each sample and serial dilutions of trans-4-hydroxy-L-proline standard (Sigma, Saint Louis, MO) was measured at 550 nm. Hepatic hydroxyproline content is expressed as μ g hydroxyproline per gram liver

Statistical analysis

Statistical analysis was done using Prism 6.0 software (GraphPad Software). Statistical significance was determined by unpaired students-t test. Data are expressed as mean +/- SEM. Statistical significance is indicated as * p<0.05, ** p<0.01, *** pO.001, **** p<0.0001.n=5 for VHL LysMCre- BM and n=7 for VHL LysMCre+ BM.

Results Genetic targeting of VHL in myeloid cells increases VEGF expression and accelerates the resolution of fibrosis

VEGF is emerging as a driver of fibrolysis and we have recently identified myeloid cells as an essential source of VEGF upon fibrosis resolution (8, 11). Here, we tested the therapeutic potential of genetically inactivating the von Hippel Lindau protein (VHL), a negative regulator of Hypoxia-inducible factors (HIF) and HIF-dependent VEGF expression (9, 10) specifically in myeloid cells (VHL ^fl/fl -LysMCre+ mice) using a foxP-flanked Vhl allele crossed to the lysozyme M promoter-driven Cre recombinase (12) which results in VEGF overexpression in macrophages (Fig. 1 A). To investigate the impact of this gain of function approach specifically on the resolution of fibrosis, we subjected CCL ₄ -treated C57B16/J mice to whole body irradiation (10 Gy) and subsequent transplantation of bone marrow (BM) from VHL - LysMCre+ mice or wildtype (VHL^-LysMCre-) mice (as depicted in Figure 1 B).

Interestingly, reconstitution with BM from VHL ^fl/fl -LysMCre+ mice results in reduced liver collagen content compared to mice reconstituted with wildtype (VHL ^fl/fl -LysMCre -) bone marrow (Figure 1 C, D) as assessed by quantitative analysis of the sirius red-positive area on liver sections (Figure 1 C, D) as well as by determination of the total liver hydroxy-proline content (Figure 1 D). Consistent with the notion that ECM degradation itself can contribute to myofibroblast contraction upon fibrosis regression (15, 16), we observe reduced numbers of a- SMA-expressing myofibroblasts after reconstitution with VHLfl/fl-LysMCre+ BM (Fig. 1 E). Taken together, this indicates that boosting the hypoxic response in myeloid cells by deleting VHL accelerates the resolution of fibrosis.

Deletion of VHL in myeloid cells upon resolution enhances ECM degradation activity

The resolution of fibrosis requires the breakdown of the ECM network. Hence, we performed an in situ zymography by incubating liver sections with fluorescein-labeled gelatin (DQ-gelatin™). This fluorogenic substrate yields a bright fluorescent signal upon proteolytic digestion and allows the in situ detection of ECM degradation (data not shown). Quantitative analysis of the fluorescent signal revealed increased zymographic activity in mice with BM from VHL ^fl/fl -LysMCre+ mice compared to mice after reconstitution with wildtype (VHL^- LysMCre-) bone marrow (Figure 2A). Accelerated resolution of the fibrotic scar in VHLfl/fl- LysMCre+ BM-reconstituted mice was indeed associated with a more homogenous pattern of sinusoids and a reduction of vascular density (Fig. 2B).This further suggests that mice reconstituted with BM from VHL ^fl/fl -LysMCre+ mice are more efficient in breaking down ECM and resolving liver fibrosis.

Boosting the hypoxic response in myeloid cells promotes sinusoidal remodeling and the expression of matrix degrading enzymes

We have previously shown that the fibrotic scar is mostly devoid of sinusoids, suggesting sinusoidal rarification in this area (8). Upon regression of the fibrotic scar, though, the fibrotic areas become revascularized in a VEGF-dependent manner, resulting in a more homogenous distribution of sinusoidal vessels. This was linked to a proresolution phenotype of the liver endothelium, involving increased expression of MMP-2 and -14 as well as reduced expression of TIMP-1 and -2 in response to myeloid cell-derived VEGF (8). In order to determine whether targeting of VHL in myeloid cells translates into vascular changes, we performed simultaneous detection of sinusoidal vessels and the fibrotic scar by means of double immunofluorescence for VEGFR2 and SMA on liver sections from both genotypes. Sinusoidal infiltration of the fibrotic scar was more advanced in livers from VHL ^fl/fl -LysMCre+ BM- reconstituted mice (data not shown). Strikingly, this was associated with enhanced expression of MMP-2 and -14 in sorted liver endothelial cells (Fig. 3 A), thus further substantiating the role of VEGF as a driver of fibrolysis. Noteworthy, the expression of the chemokine CXCL9 that regulates macrophage infiltration (11) and the number of infiltrating macrophages were not affected (Fig. 3 B and C). Noteworthy, we also observed increased expression of MMP-7, -9 and -13 in whole livers after reconstitution with VHL ^fl/fl -LysMCre + bone marrow (Fig.3 D). Isolated F4-80-positive macrophages from fibrotic livers showed upregulation of MMP-13 expression upon VHL deletion (Fig. 3 E), whereas MMP-7 and -9 expression in isolated liver macrophages remained similar across genotypes (Fig. 3 E), pointing towards another, non- macrophage source for these MMPs in our particular setting. Consistently, peritoneal macrophages isolated from VHLfl/fl-LysMCre+ mice also show increased levels of MMP-13 transcripts (Fig. 3 F). Taken together, this suggests that targeting the hypoxic response in myeloid cells may contribute to the resolution of fibrosis in a much broader sense and not only through VEGF-dependent effects on the liver vasculature.

In addition to macrophages, dendritic cells, Natural Killer (NK) cells and neutrophils have been shown to participate in the regression of liver fibrosis (17-19). Flow cytometry analysis (data not shown) of fibrotic livers at endpoint revealed that the number of MHCII+/CD11C+ dendritic cells (data not shown), NKp46+/NKl .l+ NK cells (data not shown) and CDl lb+/Ly6G+ neutrophils (data not shown) were similar across genotypes. However, reconstitution with VHLfl/fl-LysMCre+ BM resulted in decreased numbers of F4/80- expressing macrophages (data not shown), possibly as a consequence of overall decreased fibrosis at endpoint.

Deletion of VHL in myeloid cells accelerates liver regeneration

Recovery from chronic liver injury also requires regeneration of the liver parenchyma involving the proliferation of hepatocytes as well as the activation of liver progenitor cells(4, 7). VEGF has been implicated in hepatocyte proliferation and liver regeneration(13, 14). However, analyzing the number of PCNA-positive proliferating hepatocytes did not reveal differences between genotypes (Figure 4 A). Expression of the hepatocyte mitogen Hepatocyte Growth Factor (HGF) also remained unchanged (Figure 4 B). Expression of the cytokine TWEAK (tumor necrosis factor-like weak inducer of apoptosis), along with expansion of resident liver progenitor cells has recently been shown to contribute to liver regeneration after CCL4-challenge (7). Therefore, we wanted to investigate the effect of VHL deletion in myeloid cells on liver progenitor cell compartment. Interestingly, reconstitution with VHL^-LysMCre + bone marrow results in increased numbers of pancytokeratin- and Dlk-expressing liver progenitor cells, indicating improved liver regeneration along with accelerated fibrolysis (Fig. 4 C, D). However, this was not associated with differences in TWEAK expression between genotypes (Figure 4 E).

Given the current efforts of macrophage-based therapy for liver fibrosis, we propose that enhancing the hypoxic response in scar infiltrating macrophages could represent a therapeutic avenue for the treatment of liver fibrosis and improvement of liver regeneration.

The effects of VHL deletion in myeloid cells depend largely on VEGF expression

Ablation of VHL results in constitutive activation of HIF and expression of HIF target genes other than VEGF. Therefore additional, VEGF -independent but HIF-dependent effects may contribute to enhanced fibrolysis in our study. In order to dissect out VEGF-dependent from VEGF-independent effects upon VHL inactivation in myeloid cells, we generated mice with a simultaneous deletion of VHL and VEGF in myeloid cells (VHLfl/fl/VEGFfl/fl- LysMCre+) and transplanted their BM along with the appropriate controls (VHLf /fl/VEGFfl/fl-LysMCre-) into CC14-treated C57B16/J mice after whole body irradiation (10 Gy), followed by a 4 week recovery phase. As shown in Fig. 5, reconstitution with BM from VHLfl/fl/VEGFfl/fl-LysMCre+ mice results in liver collagen contents (Fig. 5 A) and numbers of a-SMA-expressing myofibroblasts (Fig. 5 B) that are as high as in mice reconstituted with wildtype (VHLfl/ fl/VEGFfl/ fl-LysMCre-) BM. In addition, the density of VEGFR2 (+) liver sinusoids (Fig. 5 C) as well as the number of endogenous liver progenitor cells (Fig. 5 D) are similar across genotypes. Therefore, simultaneous deletion of VEGF and VHL in myeloid cells reverses the phenotype observed after VHLfl/fl-LysMCre+ BM transplantation (Fig. 1, 2, 3 and 4), indicating that the improved outcome upon VHL deletion in myeloid cells depends largely on increased VEGF expression. Consistently, the double knockout of VHL and VEGF in myeloid cells partially prevents the induction of a proresolution phenotype in the liver endothelium as illustrated by similar expression expression of MMP-14 and TIMP-2 in sorted liver endothelial cells (Fig. 5 E). Although, the expression of MMP-2 in the liver endothelium was still significantly increased in mice reconstituted with VHLfl/fl/VEGFfl/fl-LysMCre+ (Fig. 5 E), it was lower than in VHLfi/fl-LysMCre+ BM reconstitution setting. Noteworthy, MMP-13 expression in isolated F4/80-positive macrophages from fibrotic livers of VHLfl/fl/VEGFfl/fl-LysMCre+ BM-reconstituted mice (Fig. 5 F) as well as in VHL/VEGF-deficient peritoneal macrophages (Fig. 5 G) was lower than in the VHLfl/fl-LysMCre+ setting, yet still significantly higher than under wildtype (VHLfl/fl/VEGFfl/fl-LysMCre-) conditions (Fig. 5 F and G, respectively). This indicates that upon VHL deletion in myeloid cells, the proresolution phenotype in the liver endothelium largely depends on VEGF, whereas increased MMP-13 expression in scar associated macrophages seems to be rather VEGF-independent but HIF-dependent. Therapy with VHL-deficient macrophages enhances fibroly sis

Macrophage infusion is an emerging immunotherapeutic tool for the treatment of liver fibrosis. In murine models, the transfer of F4/80-positive bone marrow-derived but otherwise unmanipulated macrophages has been shown to foster scar resolution as well as liver regeneration (6). Given the superior outcome on fibro lysis and concomitant regeneration of fibrotic livers after reconstitution with VHLfl/fl-LysMCre+ BM along with increased expression of VEGF and MMP-13 in VHL-deficient peritoneal macrophages, we wanted to evaluate the therapeutic potential of boosting the hypoxic response in a setting of macrophage therapy. To this end, we isolated peritoneal macrophages from VHLfl/fl-LysMCre- and VHLfl/fl-LysMCre+ mice injected CC14-treated C57B16/J mice which yielded a CD l ib- and F4/80-positive macrophage population regardless of the genotype (data not shown). After fluorescent labelling with carboxyfluorescein succinimidyl ester (CFSE) (data not shown), 1x106 macrophages of each genotype were injected intravenously into CC14-treated C57B16/J mice and livers were analyzed at day 2 and 5 post-infusion (7). The transferred CFSE-labelled macrophages incorporate into fibrotic liver and are present at day 2 post-infusion to similar extent across genotypes (data not shown).

It has been shown that the success of macrophage therapy depends on additional recruitment of host immune cells (6) as well as the presence of CDl lBhigh F4/80int Ly6Clo "restorative" macrophages with crucial proresolution properties. Flow cytometry analysis of fibrotic livers at day 2 and 5 post-infusion revealed that the number of MHCII+/CD11C+ dendritic cells (data not shown), NKp46+/NKl .l+ NK cells (data not shown) and CDl lb+/Ly6G+ neutrophils (data not shown) and F4/80-expressing macrophages (data not shown) were similar across genotypes. Moreover, we confirm the CD1 lBhigh F4/80int Ly6Clo restorative macrophage as the predominant cell type during early fibro lysis (data not shown). Yet, the number of CD1 lBhigh F4/80int Ly6Clo restorative macrophages was similar across genotypes (data not shown). However, we observe reduced numbers of a-SMA-expressing myofibroblasts and subtle but significantly increased expansion of endogenous liver progenitor cells at day 5 after therapy with VHL-deficient macrophages (Fig. 6 A and B, respectively), indicating improved scar resolution and liver regeneration in this setting.

Taken together, this indicates that boosting the hypoxic response in macrophages exogenous ly may be effective in macrophage therapy for liver fibrosis.

Discussion Mouse studies have shown that transfer of untreated cells of the monocyte-macrophage lineage is able to reduce fibrosis as well as to foster liver regeneration (7) and clinical testing of this approach is on the way (4). We have previously shown that sinusoidal angiogenesis driven by myeloid cell-derived VEGF along with upregulation of MMP-2 and MMP-14 in sinusoidal endothelial cells is required the resolution of liver fibrosis (8). Here we show that the resolution of fibrosis can be accelerated by deleting VHL, a negative regulator of HIFs and VEGF, specifically in myeloid cells. Consistently, this was associated with increased VEGF expression in macrophages, increased expression of MMP-2 and MMP-14 in sinusoidal endothelial cells upon recovery and finally enhanced ECM degradation.

However, ablation of VHL results in constitutive activation of HIF and expression of HIF target genes other than VEGF. Therefore additional, VEGF-independent but HIF- dependent effects may contribute to enhanced fibrolysis in our study. Indeed, macrophages and neutrophils are the major sources of MMP-13 and MMP-9 (7), respectively, in the context of liver fibrosis and both MMPs are HIF targets (15). Consistent with this, we observe overall increased expression of MMP-9 and MMP-13 in livers from mice reconstituted VHLfl/fl- LysMCre + bone marrow. Therefore, enhanced expression of MMP-9 and MMP-13 upon deletion of VHL specifically in myeloid cells - including macrophages and neutrophils - could contribute the accelerated resolution of fibrosis in our study.

Moreover, the two major iso forms HIF-1 and HIF-2 have been shown to play an important role in M1/M2 macrophage polarization (16). Hence, despite the fact that M1/M2 characterization does not accurately reflect the macrophage phenotypes observed in the context of liver fibrosis (7, 17), it will be important to dissect out the presumably non-overlapping roles of HIF-1 and HIF-2 in myeloid cells during fibrosis resolution.

A recent mouse study showed that infusion and transient engraftment of BM-derived macrophages improves fibrosis resolution by recruiting additional endogenous macrophages and neutrophils as well as by paracrine induction of a fibrolytic phenotype in those host-derived cells (7). In addition, deletion of VHL in myeloid cells can enhance immune cell recruitment (12). In our study, however, we do observe enhanced expression of MMP-9 and MMP-13 in the absence of increased macrophage recruitment after reconstitution with VHLfl/fl-LysMCre + bone marrow. This further suggests that VHL deletion in the myeloid cell compartment induces a profibrotic myeloid cell phenotype rather than enhancing recruitment of additional immune cells.

In addition to accelerated fibrosis resolution we observe improved liver regeneration in mice reconstituted VHLfl/fl-LysMCre + bone marrow. VEGF has been implicated in early liver regeneration by stimulating hepatocyte proliferation, either directly or in an angiocrine manner (13, 14) e.g., by stimulating endothelial HGF release. Therefore, increased myeloid cell VEGF expression upon VHL deletion could foster hepatocyte proliferation. Analyzing the number of PCNA-positive proliferating hepatocytes as well as hepatic HGF expression did not reveal differences between genotypes. Taken together, this argues against a crucial role of myeloid cell-derived VEGF in hepatocyte proliferation.

However, recently Thomas J.A. et al. (7) successfully used macrophage therapy to improve liver regeneration. In this study, parenchymal regeneration was entirely driven by liver progenitor cell expansion and this was associated with increased expression of VEGF and the cytokine TWEAK (7). Similarly, we do observe significant expansions of liver progenitor cells in mice reconstituted VHLfl/fl-LysMCre + bone marrow, although, in the absence of increased TWEAK expression. In this context it is important to mention that (human) hepatic progenitors express VEGFR1, opening up a possible direct role for VEGF in liver progenitor expansion. Finally, scar degradation by itself can facilitate progenitor cell expansion (18). Thus, it is likely that accelerated scar resolution as observed in mice reconstituted VHLfl/fl-LysMCre + bone marrow indirectly promotes the process of liver regeneration.

Taken together, our results suggest that broad activation of the hypoxic response in myeloid cells along with augmented VEGF expression upon recovery is favorable with regard to scar resolution and liver regeneration. This has potential implication for the use of HIF- inducing agents like prolyl hydroxylase inhibitors in the setting of autologous cell therapy, e.g. ex vivo treatment of cells of the monocyte/macrophage lineage with such compounds prior to transfer. The data reported here could inform the design of clinical studies involving prolyl hydroxylase inhibitors to improve the efficacy of autologous cell therapy in chronic liver disease. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Desmouliere A, Darby IA, Gabbiani G. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest 2003;83: 1689-1707.

2. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell

C, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest 1998;102:538-549.

3. Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA. Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 2014; 14:181-

194.

4. Forbes SJ, Newsome PN. New horizons for stem cell therapy in liver disease. J Hepatol 2012;56:496-499.

5. Friedman SL, Sheppard D, Duffield JS, Violette S. Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med 2013 ;5 : 167sr 161.

6. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, et al. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 2005; 115:56-65.

7. Thomas JA, Pope C, Wojtacha D, Robson AJ, Gordon- Walker TT, Hartland S, Ramachandran P, et al. Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function. Hepatology 2011;53:2003-2015.

8. Kantari-Mimoun C, Castells M, Klose R, Meinecke AK, Lemberger UJ, Rautou PE, Pinot-Roussel H, et al. Resolution of liver fibrosis requires myeloid cell-driven sinusoidal angiogenesis. Hepatology 2014.

9. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell

2012;148:399-408.

10. Kaelin WG, Jr. The von Hippel-Lindau tumour suppressor protein: 02 sensing and cancer. Nat Rev Cancer 2008;8:865-873. 11. Yang L, Kwon J, Popov Y, Gajdos GB, Ordog T, Brekken RA, Mukhopadhyay D, et al. Vascular Endothelial Growth Factor Promotes Fibrosis Resolution and Repair in Mice. Gastroenterology 2014.

12. Cramer T, Johnson RS. A novel role for the hypoxia inducible transcription factor HIF-lalpha: critical regulation of inflammatory cell function. Cell Cycle 2003;2: 192-

193.

13. Ding BS, Nolan DJ, Butler JM, James D, Babazadeh AO, Rosenwaks Z, Mittal V, et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 2010;468:310-315.

14. LeCouter J, Moritz DR, Li B, Phillips GL, Liang XH, Gerber HP, Hillan KJ, et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 2003;299:890-893.

15. Yang S, Kim J, Ryu JH, Oh H, Chun CH, Kim BJ, Min BH, et al. Hypoxia- inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction. Nat Med 2010;16:687-693.

16. Takeda N, O'Dea EL, Doedens A, Kim JW, Weidemann A, Stockmann C, Asagiri M, et al. Differential activation and antagonistic function of HIF- {alpha} isoforms in macrophages are essential for NO homeostasis. Genes Dev 2010;24:491-501.

17. Ramachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, Hartland SN, et al. Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis. Proc Natl Acad Sci U S A 2012;109:E3186-3195.

18. Kallis YN, Robson AJ, Fallowfield JA, Thomas HC, Alison MR, Wright NA, Goldin RD, et al. Remodelling of extracellular matrix is a requirement for the hepatic progenitor cell response. Gut 2011;60:525-533.