WO2007049096A1

WO2007049096A1 - A method of expansion of endothelial progenitor cells

Info

Publication number: WO2007049096A1
Application number: PCT/IB2005/003620
Authority: WO
Inventors: David Smadja; Pascale Gaussem; Martine Aiach; Georges Uzan
Original assignee: Inserm (Institut National De La Sante Et De La Recherche Medicale)
Priority date: 2005-10-25
Filing date: 2005-10-25
Publication date: 2007-05-03

Abstract

The invention relates to an in vitro method for expanding endothelial progenitor cells, which method comprises culturing immature mononuclear cells from a biological sample, in a culture medium that comprises endothelial growth factors and an activator of PAR-1 receptor, whereby endothelial progenitor cells are produced and grow. It also relates to the cell culture that can be obtained by such method.

Description

A method of expansion of endothelial progenitor cells

The invention relates to a method for expanding endothelial progenitor cells, that is of particular interest for autologous cell therapy.

Angiogenesis is essential for embryonic development and has been implicated in wide range of pathological situations such as vascular diseases and tumor growth. In adults, angiogenesis was thought to result exclusively from the proliferation, migration and sprouting of preexisting endothelial cells, but the recent discovery of endothelial progenitor cells (EPCs) has challenged this view (Asahara et al., 1997). These cells originate from bone marrow, are found in peripheral blood, and contribute to postnatal angiogenesis (Asahara et al., 1997 ; Asahara et al., 1999 ; Shi et al., 1998 ; Rafii et al., 2003). A common precursor of hematopoietic and endothelial cells (the hemangioblast), initially restricted to embryonic development, was recently described in adults (Pelosi et al., 2002). When CD34+ cells isolated from human umbilical cord blood are expanded in a medium containing endothelial growth factors, they differentiate into endothelial cells.

Bone marrow EPCs circulate in human peripheral blood (Asahara et al., 1997 ; Asahara et al., 1999 ; Shi et al., 1998) and can reach an angiogenic target and participate in the vascularization process in animal models of ischemia (Kalka et al., 2000 ; Kawamoto et al., 2001 ; Edelberg et al., 2002). Bone marrow mononuclear cells have been used in most clinical trials conducted to date, but circulating EPCs may be an interesting alternative source of a cell therapy product. In particular, such progenitor cells hold great promise as autologous cell therapy products for patients with vascular diseases associated with cardiac and leg ischemia, for example (Tateishi-Yuyama et al., 2002 ; Britten et al., 2003 ; Assmus et al., 2002). However, EPCs are present in a small numbers in peripheral blood, and would have to be expanded before use.

The inventors have now demonstrated the presence of PAR-1 receptor on the surface of these cells. PAR-1 is a protease-activated G protein coupled receptor specifically cleaved by thrombin at its extracellular N-terminus. The inventors have further shown that activation of PAR-1 induced the survival and proliferation of these cells in a concentration-dependent manner, far more potently than with human umbilical vein endothelial cells. PAR-1 activation enhanced EPC chemotaxis along a SDF-1 gradient via an upregulation of CXCR-4 and chemokinesis along a VEGF gradient . PAR-1 activation on EPCs also led to actin cytoskeleton reorganization and spontaneous migration of EPCs, as well as to differentiation into capillary-like structures.

On that basis, the invention thus provides an in vitro (or ex vivo) method for expanding endothelial progenitor cells, which method comprises culturing immature mononuclear CD34⁺ cells from a biological sample, in a culture medium that comprises at least one endothelial and/or angiogenic growth factor and an activator of PAR-1 receptor, whereby endothelial progenitor cells are produced and grow.

PAR-1 activators :

PAR-1 has been cloned and characterized as receptor for thrombin, the major serine effector protease involved in coagulation, vascular injury and inflammation. PAR-1 is known to be coupled to G_q and Gj proteins. PAR-1 activation results in the stimulation of phospholipase (PLC) activity, leading to the formation of inositol triphosphate (IP₃) and diacylglycerol (DAG) followed by calcium mobilization and activation of proteine kinase C (PKC). PAR-1 is also involved in the activation of tyrosine kinase (Src family), PI3 (PI3K), protein kinase B (Akt) and mitogen-activated protein kinase (MAPK), e.g. ERK.

In the context of the present invention, the "PAR-1 activator" refers to any molecule or substance that induces or enhances the activation pathway governed by PAR-1. Such activation may be checked by monitoring the formation of IP₃ or diacylglycerol for instance, or the phosphorylation of ERK MAP kinase.

In a particular embodiment of the invention, the PAR-1 activator is a peptide designed after the amino acid sequence of the proteolytically-exposed tethered ligands that can activate PAR-1 in the absence of proteases. More particularly, the activator may be a peptide that consists of the amino acid sequence SFLLRX (SEQ ID NO:1 ), with X being OH or an aminoacid sequence having between 1 to 10 aminoacids, wherein the C- terminus of the SFLLRX peptide can be substituted with a carboxylic acid protecting group.

Preferably, it is a peptide that consists of the aminoacid sequence SFLLRN.

Suitable protecting groups are described in Green and Wuts (1991 ). The carboxyl group at the C-terminus can be protected, for example, by an amide (i.e. the hydroxyl group at the C-terminus is replaced with NH₂, NHR' and NR'R") or ester (Ae., the hydroxyl group at the C-terminus is replaced with -OR'). R' and R" are independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group. In addition, taken together with the nitrogen atom, R' and R" can form a C₄ to Cs heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examples of suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include --NH₂, -NHCH₃, -N(CH₃)₂, -NH(ethyl), ~N(ethyl)₂, --N(methyl) (ethyl), -NH(benzyl), -N(C₁-C₄ alkyl)(benzyl), -NH(phenyl), -N(Ci-C₄ alkyl)(phenyl), -OCH₃, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(iso-propyl), -O-(sec-butyl), -O-(t-butyl), -O-benzyl and -O-phenyl.

In another embodiment, the PAR-1 activator is thrombin. It may be recombinant or isolated thrombin, or it may be thrombin adsorbed to a fibrin network. Accordingly, mononuclear cells of a patient may be cultured in vitro on a fibrin network, preferably an autologous fibrin network obtained by coagulation of a blood sample from the same patient.

PAR-1 activation can also be induced via an indirect mechanism. For instance, activated protein C is known to activate PAR-1 and thereby to promote angiogenesis (Riewald M et al, 2002 ; Uchiba M et al, 2004). The starting cells :

The starting cells are mononuclear cells that can be of any source. They are preferably of human origin. The mononuclear cells may be obtained from a patient. They may be prepared from any biological sample, such as blood, bone marrow or cord blood. Only a small supply of cells is necessary. For instance, one may perform the method starting with 50 ml of cord blood. Blood samples may be normal or pathological Peripheral Blood.

These starting cells are immature cells, which means that they have not, or at least not fully, differentiated. Especially they do not show an endothelial phenotype, and therefore do not include endothelial cells like HUVEC (human umbilical vein endothelial cells).

These immature cells are notably characterized by expression of CD34. The starting cells in the method of the invention are thus CD34+ mononuclear cells. This does not mean that the population of starting cells is always enriched in CD34+ cells by a selection using antibodies directed against CD34. This being said, enrichment in CD34+ cells may be advantageous. In that case the immature mononuclear CD34+ cells are a population of mononuclear cells enriched in CD34+ cells.

For that purpose, the mononuclear cells can be enriched using commercially available antibodies that bind to CD34, using methods known to those of skill in the art. For example, the antibodies may be conjugated to magnetic beads and immunological procedures utilized to recover the desired cell type, can be achieved by a number of different methods. The most widely used method for separating CD34+ cells from mononuclear cells is a positive immunological selection based on binding of these cells to anti-CD34- antibodies immobilized on a solid support (Cellpro, Baxter; Minimacs, Miltenyi Biotec, stem sep CD34, Stem Cells). Other selection methods include negative selection where all cells not expressing CD34 are isolated away from the CD34+ cells based on their expression of lineage specific cell surface antigens. Endothelial progenitor cells may also be differentiated from stems cells (MAPC, multipotent adult progenitor cells). Other methods for expanding stems cells can be performed, using other stem cells growth factors, such as Hox B4. Enrichment in CD133⁺ or VEGFR-2* cells may also be advantageous. In a specific embodiment, the starting cells may also be immature mononuclear cells without any cell selection, from peripheral blood mobilized by the use of any authorized hematopoietic cytokine in humans, or a subpopulation of CD34⁺ cells (e.g. expressing KDR, CD146 antigens).

The culture medium :

A conventional basal medium for endothelial cell culture may be used. Since endothelial cells are adhesive cells, the culture is performed on a solid support, such a plastic culture dishes, or in teflon bags. Preferably, the matrix used to coat the support can be a polymer having cell adhesive activity, i.e. gelatin, collagen, fibrinectin, proteoglycans, or a fibrin network.

For example the culture medium may be E-BM, in particular EBM-2, BME, D-MEM, MEM alpha, Eagle MEM, M199, F-10, F-12 and RPMI1641 , MCDB107, MCDB131, MCDB152 and MCDB153, X VIVO 10 and 15, or EGM. All these culture media are well known to the skilled person, and are commercially available.

Preferably, serum albumin, e.g. human or bovine serum albumin, and/or fetal calf serum is added to the medium. Serum or plasma can be added at a concentration of 5 to 50 %. However it is preferably a serum-free medium. Growth factors are added to the culture medium. These include non specific and endothelial and/or angiogenic growth factors such as those contained in autologous or foetal calf serum.

As endothelial and/or angiogenic growth factors, one may use in particular the different isoforms of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) such as acidic and basic FGFs, VEGF-related molecules, such as PIGF, EPO, SDF-1 , HoxB4, inhibitors of HMG-CoA reductase (e.g. statins), and so on.

Other growth factors include the different isoforms of transforming growth factor (TGF), and of tumor necrosis factor (TNF) and related molecules, angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), granulocyte colony stimulating factor (G- CSF), hepatocyte growth factor scatter factor (HGF/SF), pleiotrophin, proliferin, follistatin, placental growth factor (PIGF), midkine, platelet-derived growth factor-BB (PDGF-BB), fractalkine. Peptides or antibodies that specifically induce angiogenesis may also be added to the culture medium.

Expansion of endothelial progenitor cells : The method of the invention is aimed at obtaining (i.e. producing) endothelial progenitor cells (EPCs) in a great number.

In the context of the invention, the terms "expansion" or "expanding" refer to both the obtention and the proliferation of the endothelial progenitor cells. The term "endothelial progenitor cells" or "EPCs" refer to cells that derive from mononuclear cells and express specific markers that identify them as endothelial cells. However, although these cells have undergone some differentiation steps, they still have the properties of immature cells. They are also called EPDC (Endothelial Progenitor Derived Cells) in the literature (Bompais et al., 2004) or "early" and "late" endothelial progenitor cells, OEC (outgrowth endothelial cells), BOEC (blood outgrowth endothelial cells).

The time period in which the number of cells are increased is, at least in part, a function of the cell type and of the specific culture method used, including culture medium and the matrix. Routine procedures known to those of ordinary skill in the art can be used to determine the number of cells in culture as a function of increasing incubation time of the cultured cells. Typically, expansion (increase in cell number) is measured by counting the cell numbers by, for example, measuring incorporation of a specific dye or incorporation of

H³ thymidine or BrdU in the nucleus. Thus, the length of cell culture incubation period varies and depends on the degree of desired expansion.

In general, expansion is evaluated by the increase in total number of cells from the start of incubation.

Approximately, the mononuclear cells are cultured during about 20 to about 40 days, preferably about 30 days. In a particular embodiment, the method of the invention may comprise one step of culturing the mononuclear cells in the presence of endothelial growth factors, followed by the step of activating PAR-1 receptor. Alternatively, the method of the invention may comprise one step only, wherein the mononuclear cells are cultured in the presence of endothelial growth factors and in the presence of an activator of the PAR-1 receptor.

Culture of the EPCs is stopped when they have sufficiently proliferated and have acquired an "angiogenic phenotype". Such "angiogenic phenotype" includes properties that favor, induce or enhance angiogenesis. These properties can be tested in vitro. They include EPC migration in response to the activation of PAR-1 , in presence of an endothelial growth factor such as VEGF, promotion of actin polymerization, or formation of capillary-like structures in a standard in vitro Matrigel model.

In a specific embodiment, it is provided an in vitro method for expanding endothelial progenitor cells, which method comprises culturing mononuclear cells enriched in CD34+ cells, in a culture medium that comprises endothelial growth factors and both the peptide SFLLRN and thrombin adsorbed on a fibrin network, during about 20 to about 40 days, whereby endothelial progenitor cells are produced and grow.

Cell culture :

A further subject of the invention is a cell culture that comprises cells obtainable by the methods as described above.

Such cells display specific features, are not stem cells anymore but are committed to the endothelial lineage.

The endothelial progenitor cells are particularly useful in cell therapy, e.g. for enhancing angiogenesis in a subject in need thereof. As used herein, the term "angiogenesis" refers to the process by which new blood vessels are formed into a tissue or organ with accompanying increased blood circulation. The process of angiogenesis involves migration and proliferation of endothelial cells, which line the lumen of blood vessels.

Angiogenesis is observed in humans and animals only in restricted situations, for example, in wound healing, fetal and embryonal development and formation of the endometrium and placenta. Unregulated angiogenesis occurs in a number of pathological conditions, such as tumor metastasis. By "enhancing angiogenesis" it means stimulating, accelerating or potentiating the process of blood vessel formation of large and small vessels, as well as capillaries. The terms "enhancing angiogenesis" and "increased vascularization" are used herein interchangeably. The term "subject" is taken to mean any animal subject, preferably, a human subject. "A subject in need of enhanced angiogenesis" refers to a subject suffering a condition where vascularization is inadequate and angiogenesis is clinically required, for example, in the treatment of damaged tissues or organs, or in keeping the transplanted tissues or organs alive. The present invention particularly contemplates subjects with peripheral vascular disease, i.e. critical leg ischaemia, or diabetics with peripheral vascular pathologies, and patients with infertility due to inadequate vascularization of the uterine endometrium.

The present invention also concerns patients with cardiac disease, for example, a patient who has undergone transplantation of a heart or heart tissue or bypass surgery. For patients who are likely to suffer cardiac attacks, administration of a preparation of cell culture obtainable by the method of the invention can prevent heart attacks by increasing the blood circulation through new blood vessels to the anginal cardiac tissue before the tissue becomes infarcted. For patients who have already suffered myocardiac infarction, enhancing angiogenesis can speed recovery. Thus, the cell culture can be supplied to patients before, during or after infarction.

The present invention also contemplates subjects suffering tissue damage due to surgery, burns, fracture, laceration, or infection. The tissues to which the present methods of enhancing angiogenesis are applicable include skin, gastrointestinal tract, urinal tract, as well as avascular tissues resistant to vascularization such as the meniscus of the knee or the wrist, or the end of the clavicle, or the temporomandibular joint.

Another type of subject contemplated by the present invention include those who have suffered stroke or an ischemic attack. Angiogenesis is desirable to enhance blood flow to the nervous system, such as the cerebral cortex and spinal cord. The cell culture obtainable by the method of the invention is also useful in promoting vascularization of relatively avascular tumors for enhanced delivery of anti-tumor substances.

The figures and below examples illustrate the invention without limiting its scope.

LEGENDS TO THE FIGURES

Figure 1: Analysis of PAR-1 expression by flow cytometry

A. Results are shown as fluorescence histograms (PAR-1 expression; IgG control).

B. Surface density of PAR-1 on EPC during a 5-week expansion period, by comparison with HUVEC. A mean of 15 colonies were tested at each time point. Boxes represent the median values with 25th and 75th percentiles, and the bar chart shows 90th and 10th percentiles.

Figure 2: PAR-1 activation promotes EPC survival and proliferation

A. The effect of SFLLRN on EPC survival was evaluated by measuring 3H-thymidine incorporation in serum-free conditions. D= day of first colony replating. The increase in 3H-thymidine incorporation by SFLLRN-treated cells was calculated relative to untreated control cells (arbitrarily = 1 ).

B. In serum-free conditions EPC survival was enhanced by SFLLRN (75 or 100 μmol/L), more strongly than HUVEC.

C. The MEK inhibitor PD98059 inhibited 3H-thymidine incorporation by EPC. Quiescent EPC were grown in serum-free medium without (control) or with 100 μmol/L SFLLRN in the presence of 10 μmol/L MEK inhibitor PD98059.

D. The effect of SFLLRN on EPC and HUVEC proliferation was evaluated by cell counting in EBM-2-containing serum, 96 hours after SFLLRN stimulation (mean ± SD). EPC proliferation was significantly stronger than HUVEC proliferation. E. Effect of SFLLRN on EPC and HUVEC proliferation as evaluated by release of pNPP (405 nm) in EBM-2-containing serum (mean ± SD). EPC proliferation was also significantly stronger than HUVEC proliferation. * : p< 0.05, **: p<0.001 , ***: p<0.0001 Figure 3: PAR-1 activation induces changes in F-actin and in EPC chemotaxis.

A. Confocal images of changes in F-actin organization in EPC treated with no agonist (left panel) or with SFLLRN 75 μmol/L (right panel).

B. Flow cytometric analysis of the time course of F-actin content. EPC were treated with SFLLRN 75 μmol/L for the times indicated (X axis). Data indicate the -fold increase in F-actin content.

C. Spontaneous migration was measured in a modified Boyden chamber assay. PAR-1 activation on EPC and HUVEC resulted in a concentration-dependent increase in cell migration towards EBM-2 medium not supplemented with growth factors. Data represent the -fold increase in the number of migrating cells by comparison to the control (untreated cells, arbitrarily = 1). Bars represent the mean ± SD of three independent experiments. *: p=0.0203 and *: p=0.009

Figure 4: SFLLRN enhances EPC migration and CXCR-4 expression

A. VEGF induced EPC chemotaxis in a Boyden chamber migration assay. PAR-1 activation on EPC resulted in a concentration-dependent increase in cell migration towards VEGF (10 ng/mL).

Data represent the -fold increase in the number of migrating cells by comparison to untreated control cells (arbitrarily = 1). * : p< 0.05 ; **: p<0.001 ; ***p<0.0001

B. SDF-1 -induced EPC chemotaxis in a Boyden chamber migration assay. PAR-1 activation on EPC resulted in a concentration-dependent increase in cell migration towards SDF-1 (100 ng/mL).

Data represent the -fold increase in the number of migrating cells by comparison to untreated control cells (arbitrarily = 1 ), after substraction of spontaneous migration attributable to the effect of SFLLRN. * : p< 0.05 ; ^**: p<0.001 ; ***p<0.0001

C. Analysis of CXCR-4 expression by flow cytometry on unstimulated EPC and after 24 hours of stimulation with SFLLRN 75 μmol/L. The gray line histogram represents the control (lgG2a). D. Inhibition of the chemotactic response of SFLLRN-activated EPC and HUVEC toward SDF-1 (100 ng/ml) by mab 12G5 (respectively p=0.0011 and 0.001) and PD98059 pretreatment (respectively p=0.0008 and 0.0017). The mean and SEM of three experiments are shown.

Figure 5: CXCR-4 or SDF-1 blockade inhibits SFLLRN-induced tubule formation. EPC were stimulated with SFLLRN for 4 hours before being used in the tubule formation assay on Matrigel for 18 hours. EPC were plated on Matrigel in the presence or absence of a monoclonal antihuman antibody against CXCR-4 (clone 12G5, 10 μg/mL) or against SDF-1 (clone 79014, 100 μg/mL) or pretreatment by PD98059 before SFLLRN activation. A: Photos (original magnification, X20) are representative of three independent experiments. B: Quantitative analysis of network length. ***: p<0.0001

EXAMPLES :

Example 1 : Expansion of EPCs

In this study the inventors examined the expression and function of PAR-1 by human late EPC expanded from cord blood CD34+ cells. METHODS

EPC culture

Mononuclear cells were isolated from human cord blood by density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, Saint-Quentin Fallavier, France). Plastic-non adherent cells were enriched in CD34+ cells by magnetic activated cell sorting on MiniMacs columns (Miltenyi Biotec, Paris, France) following the manufacturer's instructions. Cells were plated on 0.2% gelatin-coated 24-well plastic culture dishes at a density of 5x105/ml and maintained in endothelial basal medium (EBM-2; BioWhittaker, Cambrex, France) supplemented with EGM SingleQuots and 5% FBS. The medium was changed 4 days after plating, and non adherent cells were removed by thoroughly washing with culture medium. Thereafter, media were changed every 4 days and the cultures were monitored daily for the emergence of small compact colonies. When an EPC colony became microscopically visible, the cells were trypsinized and replated in a 6-well plate. When these expanded EPC became confluent, they were trypsinized, counted and replated in T75 flasks. Human endothelial cells (HUVEC) were isolated from human umbilical veins as described by Jaffe et al, 1973, and were maintained in endothelial basal medium (EBM-2) supplemented with EGM SingleQuots and 5% FBS at 37°C in humidified 5% CO2/air.

All culture reagents were from Gibco (Paisley, Scotland). To assess cell surface antigen expression, the inventors used fluorescence-activated cell sorter (FACS) analysis as previously described by Bompais et al, 2004. PAR-1 expression on the EPC surface was quantified with a calibrator (Qifikit, Dako, Trappes, France) containing a mixture of five calibration beads coated with increasing densities of mouse IgG. Details of immunocytochemistry and confocal immunofluorescence staining are given below.

To test the effect of PAR-1 activation on EPC, all the following experiments were performed after a culture period of 16h in unsupplemented EBM-2 medium. Cells were then activated with SFLLRN peptide from Stago Recherche (Gennevilliers, France). The MEK inhibitor PD98059 (Calbiochem) was added 15 minutes before the agonists. All assays were performed in triplicate.

Flow cytometry

Cultured cells were detached with collagenase (Boehringer Mannheim, Meylan, France), washed in HBSS containing 10% FBS, resuspended in 50 μl of PBS-1 % BSA, and incubated for 30 min at 4°C with primary mouse monoclonal antibodies (mAb) against VEGF receptor (VEGFR-2, Sigma- Aldrich), Tie-2 receptor (BD Pharmingen, Grenoble, France), CD31 (PECAM-1 , Immunotech, Marseille, France), CD34-PCy5 (lotest, Beckman Coulter), PAR-1 and PAR-1 -PE (clone WEDE 15, Immunotech, Marseille, France) and SDF-1 receptor (CXCR-4 clone 12G5, R&D systems) at saturating concentrations. Isotype-matched mouse IgGI or lgG2a used as a negative control were purchased from the same manufacturer as the immune antibodies. For quantitative flow cytometry, the staining reagent was a polyclonal FITC- conjugated f(ab')2 fragment of a goat antimouse antibody (Dako). Ten thousand events were acquired on a FACScan flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software (Becton Dickinson). PAR-1 expression on the EPC surface was quantified with a calibrator (Qifikit, Dako, Trappes, France) containing a mixture of five calibration beads coated with increasing densities of mouse IgG (~3000 to 600 000 molecules). Surface molecule numbers were derived from the calibration curve, after subtracting the negative isotype control value. Immunofluorescence staining

Cells were seeded on glass coverslips coated with collagen in 12-well plates. They were fixed with 4% paraformaldehyde and incubated with 50 mM NH4CI. For internalization experiments, cells were incubated at 37°C for 30 minutes prior to fixation with 10 μg/ml DiI-Ac-LDL (Molecular Probes). After fixation, cells were permeabilized with 0.1% Triton-X-100 in PBS and non specific binding sites were saturated with PBS-10% FBS for 30 minutes. Cells were then incubated with the primary antibodies in PBS-1 % FBS. The monoclonal antibody against CD31 was from Dako. Cells were then incubated with goat secondary antibodies coupled to either AlexaFluor 488 or AlexaFluor 555 (Molecular Probes). Actin was visualized by using phalloidin coupled to Bodipy 558/568 (Molecular Probes). Nuclei were stained with ToPro-3 (Molecular Probes). Coverslips were mounted with Mowiol, and observed with a Leica TCS SP2 confocal microscope equipped with a 488-nm argon laser, a 543-nm HeNe laser and a 633-nm HeNe laser (Leica Microsystems). Images were acquired with a x63/1.32 PL APO objective. lmmunocytochemistry

Cells fixed in methanol for 10 min at -20⁰C were washed once in distilled water, twice in TBS (Tris 50 mM, NaCI 138 mM, KCI 2.7 mM, pH 8.0) and once in TBS, 1% BSA, 0.1% sodium azide. They were then incubated with an anti-human von Willebrand factor (vWF) mAb (Dako, diluted 1/30) or an IgGI isotype control (diluted 1/50) for 30 min at room temperature. The secondary biotinylated antibody (goat anti-mouse IgG) was applied for 15 min, and streptavidin-alkaline phosphatase and substrate-chromogen solution were then added as recommended by the manufacturer (Dako LSAB System, Alkaline Phosphatase). The counterstain was hematoxylin-eosin. Levamisole 2 mM was used to inhibit endogenous alkaline phosphatase.

Cell survival assay EPC were activated in serum-free EBM-2 medium containing SFLLRN

(50, 75 or 100 μmol/L). DNA synthesis was determined by measuring incorporation of 5'-[3H]-thymidine ([3H]-Tdr, Amersham, Les Ulis, France) with a Betamatic counter (1900 CA Packard) during 4 hours. Results are expressed as the increase in thymidine incorporation over control (EBM-2 without SFLLRN). The values of the SFLLRN-treated samples were subsequently normalized such that the untreated control value was 1. Cell proliferation assay

The effects of various concentrations of SFLLRN peptide or SDF-1 on EPC proliferation were examined by cell counting with a phase-contrast microscope or by measuring cell phosphatase activity based on the release of paranitrophenol (pNPP, Sigma) at 405 nm (Flustar optima, BMG labtech, Champigny Sur Marne, France) after 4 days of incubation. EPC and HUVEC were activated in 10% FBS EBM-2 medium containing SFLLRN (25, 50, 75, 100 or 150 μmol/L). Real-time quantitative RT-PCR

Real-time quantitative RT-PCR was performed using the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems). Briefly, total RNA is reverse-transcribed before realtime PCR amplification. Quantitative values are obtained from the threshold cycle (Ct) number at which the increase in the signal associated with exponential growth of PCR products begins to be detected using PE Biosystems analysis software, according to the manufacturer's manuals. The precise amount of total RNA added to each reaction mix (based on optical density) and its quality (i.e. lack of extensive degradation) are both difficult to assess. The inventors therefore also quantified transcripts of the TBP gene which encodes the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) as the endogenous RNA control, and each sample was ormalized on the basis of its TBP content. Results, expressed as N-fold differences in target gene expression relative to the TBP gene, termed Ntarget, were determined with the formula: Ntarget = 2? Ctsample, where the ?Ct value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the TBP gene. The Ntarget values of the samples were subsequently normalized such that the untreated control sample Ntarget values were 1. Primers for TBP and the 10 target genes were chosen with the assistance of the Oligo 5.0 computer program (National Biosciences, Plymouth, MN). To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 65°C for 1 min. ELISA measurement of secreted SDF-1 and VEGF Cells were incubated for 24 hours in EBM-2, 5% FBS at 37⁰C with 75 μmol/L SFLLRN. SDF-1 and VEGF concentrations were measured with Quantikine ELISA kits (R&D systems). In vitro capillary-like growth assay

Cells were activated for 4 hours in EBM-2 medium containing 75 μmol/L SFLLRN. Cells were then seeded on Matrigel (3x104 cells/well) and cultured for 18 h at 37⁰C with 5% CO2. Capillary-like structures were examined by phase-contrast microscopy and endothelial cell networks formed by EPC were quantified by computer-assisted analysis (VIDEOMET 5.4.0). Cell migration assay

EPC migration was measured by using modified Boyden chambers

(Costar, Avon, France) with 8-μm pore-size filters. EPC were seeded at a density of 5x104 per well in 200 μL of migration medium (EBM-2/1% SVF), and were allowed to migrate for 5 hours at 37⁰C. Recombinant human VEGF or

SDF-1 (R&D systems) was diluted in EBM-2 medium supplemented with 1%

FBS and placed in the lower chamber of the modified Boyden chamber, in a volume of 600 μl. When checkerboard analysis was used to evaluate chemotaxis and chemokinesis, 0, 1 , 10, 100 ng/ml VEGF was added to the upper and/or lower chamber.

Filamentous actin (F-actin) measurement

After 16 hours of growth-factor deprivation, EPC were stimulated with 75 μmol/L SFLLRN in EBM-2, 5% FBS at 37°C for various times. F-actin was visualized by immunofluorescence (see above) or flow cytometry. The cells were permeabilized with 0.1% saponin for 10 min, washed twice in HBSS containing 10% FBS, stained with 1 unit of Alexa-Phalloidin (Interchim, Montlucon, France) for 30 min, then washed and analyzed by flow cytometry. Statistical analysis

Data are shown as means ± SD. Significant differences were identified by ANOVA followed by Fisher's protected least-significant difference test, lntergroup comparisons of PAR-1 density on the EPC surface were based on the Mann and Whitney non parametric test. AH statistical tests were performed using the Stat View software package (SAS, Cary, NC, USA). Differences with p values <0.05 were considered significant.

RESULTS

Late endothelial progenitor cells express PAR-1 When cultured in the presence of specific endothelial growth factors

(EGM-2 medium), human cord blood CD34+ cells (purity 86.0 ± 5.7%) yielded small colonies that appeared within 13.5 days (SD: 3.3 days, median: 14 days; 25 cultures). At confluence, EPC exhibited the cobblestone morphology and monolayer growth pattern typical of the endothelial lineage. The endothelial phenotype of expanded EPC (so-called late EPC) (Hur et al, 2004) was further characterized by positive staining for acetylated low-density lipoprotein uptake, expression of endothelial markers such as Tie-2, von Willebrand factor, CD31 , and VEGFR-2, and uptake of DiI-Ac-LDL. EPC retained a high proliferative potential and expressed CD133 with low mRNA levels during 40 days of expansion. Flow cytometry also showed the expression of the thrombin receptor PAR-1 on 96.0±1.4% of expanded EPC, 85.1 ±6.7% of which were also positive for CD34. Interestingly, 90.2±1.1% of freshly purified CD34+ cells also expressed PAR-1 , pointing to early expression of this receptor (Figure 1A)₌ EPC were expanded for 5 weeks after the first passage, corresponding to a total of 45 to 60 days of culture. Mean PAR-1 density measured by means of quantitative flow cytometry was 13 700 sites per cell, a value similar to that found on HUVEC (19 720 sites, p>0.1). PAR-1 density varied strongly among EPC colonies, but the median expression level remained constant throughout the 5-week expansion period (FigureiB).

PAR-1 activation promotes EPC survival and proliferation

Specific PAR-1 activation of EPC was induced by the peptide SFLLRN, which mimics thrombin activation without cleaving the receptor. In order to explore the effect of PAR-1 activation on EPC viability, the cells were deprived of serum and growth factors (EBM-2 medium) for 16 h before adding

SFLLRN. In these conditions, SFLLRN induced a concentration-dependent increase in late EPC proliferation, as quantified by 3H-thymidine incorporation, with a maximal effect between 20 and 30 days of culture (Figure 2A). Thus, all subsequent experiments were done within the first 30 days of culture. At optimal concentrations (75 and 100 μmol/L), SFLLRN induced markedly stronger 3H-thymidine incorporation by EPC than by HUVEC (p=0.027 and 0.0009, respectively; Figure 2B). To investigate the involvement of ERK phosphorylation in EPC signaling and in the effect of SFLLRN on EPC proliferation, the inventors used the MAPK kinase (MEK) inhibitor PD98059 to inhibit threonine and tyrosine phosphorylation on ERK1 and ERK2. Pretreatment of EPC with PD98059 (10 μmol/L) inhibited SFLLRN-induced EPC proliferation by 85% (Figure 2C). These results suggest that EPC proliferation can be triggered by PAR-1 activation in the absence of other specific growth factors, and that this effect is associated with ERK phosphorylation, as in HUVEC (Olivot et al, 2001 ).

As shown in Figure 2D, SFLLRN also increased the proliferation of late EPC cultured in serum containing medium, in a concentration-dependent manner, with a maximal effect at 75 and 100 μmol/L. EPC proliferation was significantly stronger than HUVEC proliferation (p=0.0045, 0.006 and 0.0003 for SFLLRN concentrations of 75, 100 and 150 μmol/L, respectively, Figure 2D). These results were confirmed by measuring pNPP release at optimal SFLLRN concentrations (75 and 100 μmol/L) (Figure 2E). Effect of PAR-1 activation on pro-angiogenic cytokine gene expression

In order to examine the transcriptional effect of PAR-1 activation, the inventors used real-time quantitative RT-PCR to measure the mRNA levels of several angiogenic factors, including VEGF isoforms and SDF-1 , and their receptors. EPC contained low basal levels of SDF-1 , CXCR-4, VEGF-A, -B, -C, - D, VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1 mRNA. SFLLRN induced a slight increase in VEGF-A isoform mRNA after 4 hours. No significant increase in the mRNA expression of the VEGF receptors or the co-receptor NRP-1 was observed (Table 1 ).

Table 1 : Effect of SFLLRN 75 umol/L on the mRNA levels of VEGF and SDF-1 and their receptors.

EPC were stimulated with SFLLRN for 4 or 8 hours after 16 hours of serum and growth-factor privation. mRNAs were measured by real-time quantitative RT-PCR and normalized to TBP mRNA (mean ± SD, n=3). *p<0.05 and **p<0.001 , compared with unstimulated cells.

Table 1

4 hours 8 hours

SDF-I 10.1 ± 0.7** 7,0 ± 2.3**

CXCR-4 2.8 ± 0.3** 3,6 ± 0.9* *

VEGF-A 2.8 ± 0.2* 1.6 ± 0.2

VEGF-B 1.2 ±0.0 1.3 ± 0.7

VEGF-C LO ± 0.6 0.6 ± 0.3

VEGF-D Li ±0.2 0.9 ± 03

VEGFR-1 1.3 ± 0.2 2.5 ± 0.3

VECFR-2 1.2 ±0.4 1.8 ± 0.8

VEGFR-3 LO ± 0.1 1.3 ± 0.2

NRP-1 1.3 ±0.2 1.8 ± 0.3

In contrast, SFLLRN markedly increased the mRNA expression of both SDF-1 and its receptor CXCR-4. The increase in SDF-1 mRNA was 12-fold after 4 hours of stimulation, and 7-fold after 8 hours. In parallel, the CXCR4 mRNA level increased significantly upon PAR-1 stimulation, to reach a maximum of 4-fold at 8 hours. These effects were totally abrogated by the MEK inhibitor PD98059, suggesting that enhancement of SDF-1/CXCR-4 is dependent on the ERK pathway.

The inventors then examined the effect of PAR-1 activation on SDF-1 and VEGF secretion by EPC. EPC supernatants were collected after incubation with SFLLRN for 24 hours, and cytokine levels were measured with ELISA kits. SDF-1 release increased two-fold at 75 μmol/L SFLLRN (1200 ± 210 pg/106 vs 618 ±257 pg/10⁶ in untreated controls, p =0.1 ; n=3). Only trace amounts of VEGF were detected even after 72 hours of activation by SFLLRN. PAR-1 activation induces actin cytoskeleton reorganization and spontaneous migration of late EPC

Non activated EPC displayed a faint ring of polymerized actin at their periphery when stained with phalloidin (Figure 3A). Addition of 75 μmol/L SFLLRN to EPC for 5 and 30 min induced a strong increase in fluorescence intensity, striking reorganization of the actin cytoskeleton, and an increase in stress fiber formation (Figure 3A). The significant increase in F-actin cell content upon SFLLRN stimulation was confirmed and quantified by flow cytometry with alexa-phalloidin (Figure 3B). To examine spontaneous migration linked to PAR-1 activation, EPC were treated for 4 hours with increasing concentrations of SFLLRN before being placed in the upper compartment of a Boyden chamber, the lower compartment of which contained EBM-2 medium. SFLLRN (50, 75, and 100 μmol/L) promoted EPC migration through the membrane in a concentration-dependent manner, with a two-fold increase at the maximal concentration tested (100 μmol/L, p=0.023, Figure 3C).

Together, these findings imply that PAR-1 activation supports motility of late EPC.

SFLLRN increases SDF-1 EPC migration through CXCR-4 expression EPC and HUVEC were treated with increasing SFLLRN concentrations then allowed to migrate towards VEGF or SDF-1. With VEGF, a significant effect was observed with both EPC and HUVEC (Figure 4A) exposed to concentrations of 75 μmol/l or 100 μmol/l (p=0.018 and p=0.008 respectively) but not to the lowest concentration (50 μmo!/l). In contrast, all SFLLRN concentrations induced strong migration toward SDF-1 (p<0.001), the effect being far more potent on EPC than on HUVEC (Figure 4B).

To explain the effect of PAR-1 activation on migration towards chemoattractants, the inventors explored the expression of their respective receptors on the EPC surface. In keeping with the mRNA results, flow cytometry showed a 3-fold increase in CXCR-4 protein expression on EPC upon SFLLRN 75 μmol/L treatment (Figure 4C, p<0.01 ). Interestingly, cell- surface VEGFR-2 expression was not significantly affected by SFLLRN treatment, in keeping with the lack of a significant increase in VEGFR-2 mRNA expression. Thus, the motility of EPC toward SDF-1 may result from increased expression of functional CXCR-4. To confirm the role of CXCR-4, the inventors used 12G5, an mAb recognizing an epitope located in the second extracellular loop of CXCR4 (Murohara et al, 2002), which indeed inhibited EPC and HUVEC migration towards SDF-1 by more than 80%. Interestingly, the MEK inhibitor PD98059 also inhibited about 80% of SDF-1 -directed migration of SFLLRN-activated EPC. Taken together, these data strongly suggest that SDF- 1 -induced migration of SFLLRNactivated

EPC is mediated by an increase in CXCR4 expression, the molecular pathway of which involves MEK.

The anti-CXCR4 antibody, however, did not inhibit the chemotactic response of endothelial cells towards VEGF, implying that the effect of this growth factor on SFLLRN-activated EPC is not dependent on SDF-1. In the absence of VEGF receptor induction, it is important to discriminate between chemotaxis (directional motility) and chemokinesis (random motility) to explain the effect of VEGF. The inventors therefore tested various concentrations of VEGF in the upper and/or lower wells of the Boyden chamber. With equal concentrations of VEGF below and above the membrane, a significant enhancement of SFLLRN-activated EPC migration was observed, indicating a chemokinetic effect of VEGF. This chemokinesis represented about 40% of the migratory effect (Table 2). Table 2 : Checkerboard migration analysis of SFLLRN-activated EPC (75 uM) towards VEGF

Various concentrations of VEGF were added to the upper and/or lower chamber, and SFLLRN activated EPC were allowed to migrate for 6 hours. Results are the numbers of migrated cells. ^*

Statistically significant compared with corresponding values without VEGF in the lower chamber.

* : p< 0.05 ; **: p<0.001 ; ***: p<0.0001

CXCR4/SDF-1 pathway blockade inhibits EPC tube formation induced by PAR-1 activation The inventors used a Matrigel model to examine the capacity of

SFLLRN-activated EPC to differentiate into capillary-like structures. When EPC were cultured for 16 h without serum, they formed few capillary-like structures (Figure 5A, left panel), whereas HUVEC were no longer able to form pseudo- tubes. Treatment with SFLLRN (75 μmol/L) promoted EPC organization into branched structures and pseudo-tubes with enclosed areas (network length: 857 ± 160 μm in untreated controls versus 3111 ± 95 μm in SFLLRN-treated cells, p<0.0001 ) (Figure 5). Given the role of PAR-1 activation in CXCR- 4/SDF-1 induction, the inventors explored the involvement of this system in tubule morphogenesis by using blocking anti-CXCR-4 and anti-SDF-1 antibodies and the MEK inhibitor PD98059. The increase in tube formation in Matrigel induced by SFLLRN was blocked by these antibodies, as well as by PD98059, but not by the irrelevant isotypic control antibody (Figure 5B) or by a VEGFR-2 inhibitor (data not shown). To rule out the possibility of SDF-1- mediated proliferation in Matrigel, the inventors checked that SDF-1 concentrations ranging from 10 to 100 ng/ml did not increase expanded EPC numbers (1.024, 1.021 and 0.879-fold increases, respectively, at 10 ng/ml, 50 ng/ml and 100 ng/ml; untreated control values were 1). The results of these experiments imply that PAR-1 activation enhances EPC organization into pseudovascular structures in vitro through an autocrine mechanism involving the SDF-1/CXCR-4 pathway.

Effect of thrombin on EPC proliferation and chemotaxis

Fibrin matrix preparation A fibrin network was generated in microplates by adding 0.025 M

CaCI₂ for 1h at 37°C to platelet-depleted plasma obtained after 15 min of centrifugation at 2300 g. We used 24-well and 6-well microplates containing 600 μl and 1000 μl of plasma , for migration and proliferation assays, respectively. Thrombin activity on the matrix surface was quantified by measuring the kinetics of the thrombin selective chromogenic substrate S2238 (Chromogenix) hydrolysis at 405 nm using a calibration curve constructed with serial dilutions of purified human thrombin prepared by Bernard Le Bonniec

The physiological PAR-1 activator is thrombin, an enzyme formed during activation of coagulation pathways. Most thrombin formed during the clotting process is adsorbed to the fibrin network. To determine whether fibrin- bound thrombin activates PAR-1 on EPCs, the inventors prepared a fibrin matrix by recalcification of human platelet-depleted plasma at 37°C in microplate wells. Thrombin activity on the matrix surface was confirmed by hydrolysis of S2238, a thrombin-specific chromogenic substrate; the mean thrombin concentration was 4.2 nM per well. EPCs strongly adhered to and proliferated on the fibrin matrix. The fibrin matrix was then treated with the specific thrombin inhibitor hirudin, and thrombin inhibition was confirmed by the lack of S2238 hydrolysis.

The inventors examined whether EPCs were able to migrate towards a fibrin matrix in a modified Boyden chamber. Fibrin attracted EPCs through the membrane, and this effect was inhibited by pretreatment of the fibrin network with hirudin (mean inhibition 66%, three experiments; p<0.001 ).

Finally, this model of fibrin matrix, more than explaining thrombus resolution by thrombin, may represent a new autologous matrix for EPC expansion.

CONCLUSION:

SFLLRN had a strong, concentration-dependent effect on late EPC survival and proliferation during the first 40 days of culture. Interestingly, EPC proliferated far more strongly than HUVEC in response to SFLLRN, independently of PAR-1 density (similar in the two cell types).

To better characterize the effect of PAR-1 activation on EPC, the inventors quantified the mRNA levels of the main pro-angiogenic cytokines and their receptors by using real-time quantitative RTPCR.

Interestingly, PAR-1 activation induced a marked increase in CXCR-4 and SDF-1 mRNA, associated with CXCR-4 overexpression on the EPC membrane and with SDF-1 release into the culture medium.

Using a standard Matrigel model developed to mimic vascular tube formation, the inventors found that PAR-1 activation induced human EPC to adopt an "angiogenic" phenotype. This effect involved the SDF-1 /CXCR-4 pathway, as it was completely abrogated by anti-CXCR-4 and anti-SDF-1 antibodies as well as the MEK inhibitor. Altogether, the present data suggest that SDF-1 and CXCR-4 overexpression results from transcriptional upregulation upon PAR-1 activation and directly influences vascular tube formation. Vascular tube formation results from a finely tuned balance between proliferation, migration and differentiation. As migration is essential for EPC homing to ischemic tissues, the inventors explored the influence of PAR-1 activation on EPC migration in Boyden chamber assays. SFLLRN promoted spontaneous EPC migration in a concentration-dependent manner, an effect involving actin cytoskeleton reorganization. The inventors also found that SFLLRN induced EPC migration along a VEGF gradient in a concentration- dependent manner, and even more potently along an SDF-1 gradient. SDF-1 and VEGF are both markedly upregulated in hypoxic tissues, and this may contribute significantly to EPC chemoattraction. CXCR-4 upregulation is a possible mechanism underlying the migratory response of SFLLRN-treated EPC towards SDF-1. The inventors found that SFLLRN enhanced the expression of CXCR-4 and its unique ligand SDF-1 , suggesting that the pro- angiogenic effect of PAR-1 activation may be mediated by an autocrine mechanism involving SDF-1/CXCR-4.

PAR-1 activation on HUVEC has been shown to upregulate both VEGF synthesis (Dupuy et al, 2003) and the expression of the main VEGF receptor VEGFR-2 (Tsopanoglou et al, 1999). However, the inventors observed no activation of the VEGF/VEGFR-2 pathway and no inhibition of vascular tube formation in vitro in the presence of a VEGFR-2 inhibitor. Indeed, the mRNA and protein expression of VEGF isoforms and receptors did not rise significantly after SFLLRN treatment. Moreover, VEGF-induced migration was far less potent than SDF-1 -induced migration. Thus, PAR-1 enhancement of SDF-1/CXCR-4- mediated angiogenesis, occurring independently of VEGF, may be another specific feature of late EPC.

This study provides the first experimental proof of PAR-1 expression on EPC. Activation of PAR-1 with peptide SFLLRN confers proangiogenic properties on EPC, an effect mediated by SDF-1 /CXCR-4 pathway enhancement. The present data support the use of the SFLLRN peptide to expand EPC ex vivo.

Moreover, properties of thrombin adsorbed on fibrin clot on EPC biology indicate that autologous fibrin clot can constitute a new matrix for in vitro expansion of EPC. REFERENCES

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Claims

1. An in vitro method for expanding endothelial progenitor cells, which method comprises culturing immature mononuclear CD34+ cells from a biological sample, in a culture medium that comprises at least one endothelial and/or angiogenic growth factor and an activator of PAR-1 receptor, whereby endothelial progenitor cells are produced and grow.

2. The method of claim 1 , wherein the activator is a peptide that consists of the amino acid sequence SFLLRX (SEQ ID NO:1), with X being OH or an aminoacid sequence having between 1 to 10 aminoacids, wherein the C- terminus of the SFLLRX peptide can be substituted with a carboxylic acid protecting group.

3. The method of claim 2, wherein the activator is a peptide that consists of the aminoacid sequence SFLLRN.

4. The method of claim 1 , wherein the activator is thrombin.

5. The method of claim 4, wherein the activator is thrombin adsorbed to a fibrin network.

6. The method according to any of claims 1 to 5, wherein the immature mononuclear CD34+ cells are a population of mononuclear cells enriched in CD34+ cells.

7. The method according to any of claims 1 to 6, wherein the mononuclear cells are cultured during about 20 to about 40 days.

8. The method according to any of claims 1 to 7, which comprises one step of culturing the mononuclear CD34+ cells in the presence of endothelial growth factors, followed by the step of activating PAR-1 receptor, by culturing the cells in the presence of the activator of PAR-1 receptor.

9. The method according to any of claims 1 to 7, which comprises culturing mononuclear cells enriched in CD34⁺ cells, in a culture medium that comprises endothelial growth factors and both the peptide SFLLRN and thrombin adsorbed to a fibrin network, during about 20 to about 40 days, whereby endothelial progenitor cells are produced and grow.

10. A cell culture that comprises cells obtainable by the method of any of claims 1 to 9.