WO2011058182A1

WO2011058182A1 - Osteopontin-conditioned medium for the treatment of vascular diseases

Info

Publication number: WO2011058182A1
Application number: PCT/EP2010/067515
Authority: WO
Inventors: Timothy O'brien; Angela Duffy; Erin Vaughan
Original assignee: National University of Ireland Galway NUI; National University of Ireland
Current assignee: National University of Ireland Galway NUI; National University of Ireland
Priority date: 2009-11-13
Filing date: 2010-11-15
Publication date: 2011-05-19
Anticipated expiration: 2012-05-13

Abstract

A pharmaceutical composition comprising osteopontin-conditioned culture medium from endothelial progenitor cells together with a pharmaceutically acceptable carrier or excipient and a method of producing an osteopontin-conditioned culture medium from endothelial progenitor cells for use as a pharmaceutical comprising culturing endothelial progenitor cells in the presence of osteopontin, or culturing endothelial progenitor cells which have been modified to express or overexpress osteopontin. Method of treatment of vascular diseases or diabetes -associated vascular complications.

Description

Title

Osteopontin-conditioned medium for the treatment of vascular diseases

Field of the Invention

The present invention relates to the use of osteopontin-conditioned medium for the treatment of vascular diseases or complications. In particular, the invention provides compositions and methods of treatment based on osteopontin-conditioned medium and to methods of producing the osteopontin-conditioned medium.

Background to the Invention

The discovery of endothelial progenitor cells (EPCs) by Asahara et al in 1997 has provided an insight into the pathogenesis of many vascular disease states such as ischaemia, restenosis and pulmonary hypertension^1"6. Urbich et al have recently defined EPCs as highly proliferative non-endothelial cells which are capable of transdifferentiating into endothelial cells⁷. EPCs can be isolated from various sources, including bone marrow, peripheral blood and umbilical cord blood^8"10. There are two phenotypes of EPCs (early versus late EPCs) which both have distinct proliferative and angiogenic potential⁸'¹¹. The ability to adhere to matrix molecules such as fibronectin, incorporate acLDL and bind lectin remains the commonly used definition for EPCs, but, increasingly, further flow cytometry analysis and immuno staining with various markers such as haematopoeitic markers and endothelial markers are utilised to define EPCs^12"15.

Patients with type l ¹⁶ and type 2^17,18 diabetes mellitus have a lower number of EPCs as compared to healthy volunteers. Patients with type 2 diabetes complicated with peripheral vascular disease have even lower number of EPCs compared to those without complications¹⁸. EPC number in these patients inversely correlates with glycaemic control^16" ¹⁸. EPCs isolated from patients with type 2 diabetes had decreased adhesion to activated endothelial cells, and to matrix molecules such as collagen and fibronectin¹⁷. EPCs derived from patients with both types of diabetes have impaired ability to form tubules in vitro¹⁶'¹¹. Furthermore, bone marrow mononuclear cells derived from streptozotocin induced diabetic mouse differentiate less efficiently into EPCs in vitro and are less likely to form tubules than those derived from non-diabetic mice¹⁹. The conditioned media from EPCs isolated from patient with type 1 diabetes has a reduced angiogenic capacity and may contain inhibitors of tubule formation in vitro¹⁶. The phenotype of EPCs derived from patients with type 1 diabetes also remains unchanged even after culture in normoglycaemic conditions¹⁶.

Osteopontin (OPN) is an arginine-glycine-aspartic acid (RGD)-containing glycoprotein. It is involved in cell migration, cell survival, regulation of immune cell function, inhibition of calcification and control of tumor cell phenotype^23"25. Osteopontin enhances tumour growth²⁶, and its progression²⁷. In the setting of primary non-small cell lung cancer, over expression of OPN increases the aggressiveness of the tumour²⁸. Inhibition of OPN expression by either an inducible short-hairpin R A vector²⁹, R A interference³⁰ or antisense oligonucleotides³¹ attenuates the aggressiveness of various tumours. Therefore, the lower osteopontin level in diabetic EPCs may explain why diabetic subjects are more prone to vascular complications.

Patients with Type 1 Diabetes Mellitus (TIDM) often demonstrate an ineffective angiogenic response to ischemia. This may be due to a decrease in the number and function of their endothelial progenitor cells (EPC). EPC are promising cells for autologous cell transplantation in the treatment of TIDM hence it is imperative to understand the underlying cause of their dysfunction. Therefore, we identified proteins that are dysregulated in EPC from patients with TIDM. Osteopontin (OPN), a secreted protein involved in cell migration, cell survival, and tumour growth was found to be down-regulated. Thus, we believe that OPN down regulation in TIDM EPC contributes to a decreased angiogenic response.

Object of the Invention

It is an object of the invention to provide therapies for the prevention and treatment of vascular diseases or diabetes associated vascular complications, in particular to stimulate angiogenesis and for the treatment of diabetes. Such therapies may be cell-based. A further object is to provide pharmaceutical compositions for the prevention and treatment of vascular diseases or diabetes associated vascular complications, in particular to stimulate angiogenesis and for the treatment of diabetes

Summary of the Invention

According to the present invention there is provided pharmaceutical composition comprising osteopontin-conditioned culture medium together with a pharmaceutically acceptable carrier or excipient.

The osteopontin-conditioned culture medium may be derived from endothelial progenitor cells which have been cultured in the presence of OPN. Alternatively the osteopontin- conditioned culture medium may be derived from endothelial progenitor cells which have been modified to express or overexpress OPN.

OPN in this context includes but is not limited to native OPN. recombinant OPN, OPN containing a purification tag, or OPN fused to a carrier molecule.

Suitably the culture medium is cell-free. In some embodiments the composition may further comprise endothelial progenitor cells, osteopontin, endothelial progenitor cells which have been modified to express or

overexpress OPN, or combinations thereof.

Preferably the culture medium is derived from EPCs which have been exposed to OPN for at least 12 hours. Culturing may take place, more preferably, for at least 24 hours.

Such a composition finds use in the treatment of vascular diseases or diabetes associated vascular complications arising from such diseases. As used herein the term 'vascular diseases or diabetes associated vascular complications' includes conditions such as myocardial infarction, peripheral vascular disease, ischemia, cerebrovascular disease which may be due to the presence of risk factors for these vascular diseases such as diabetes mellitus, dyslipidaemia and hypertension.

The invention also provides a method of producing an OPN-conditioned medium for use as a pharmaceutical comprising culturing EPC cells in the presence of osteopontin for at least 12 hours. Culturing may take place for, more preferably, at least 24 hours.

Preferably the EPCs are cultured in the presence of OPN and then washed and allowed to grow for a further at least 6 hours. In some cases culturing may take place, more preferably, for at least 12 hours. In other cases, culturing may take place, most preferably, at least 48 hours before the medium is harvested. The cells may be washed with a buffer such as phosphate buffered saline or similar buffer known to the skilled person.

In an alternative embodiment the OPN-conditioned medium is derived from EPCs which have been modified to express or overexpress OPN and which have been cultured for at least 12 hours.

In a further aspect the invention provides a method of assaying OPN-conditioned medium for the presence of angiogenesis-inducing compounds comprising assaying the subject compound. The assay may be a matrigel tubule formation assay.

In a still further aspect the invention provides a method of treatment of vascular diseases or diabetes associated vascular complications comprising administering to a subject in need of such treatment a pharmaceutically effective amount of :

OPN-conditioned medium;

OPN-conditioned medium together with endothelial progenitor cells;

OPN-conditioned medium together with endothelial progenitor cells which have been modified to express or overexpress OPN;

OPN-conditioned medium together with OPN;

OPN-conditioned medium together with OPN and endothelial progenitor cells; or OPN-conditioned medium together with with OPN and endothelial progenitor cells which have been modified to express or overexpress OPN.

Suitably the method of treatment may further comprise administration of one or more of FGFa (fibroblast growth factor), 11-6 (interleukin 6), TGF-a (transforming growth factor alpha) and IL-8 (interleukin-8). Suitably, any combination of any of these factors may be advantageously used.

The method of treatment may also further comprise administration of mesenchymal stem cells.

As defined herein OPN-conditioned medium means culture medium derived from cultured EPCs which have been grown in the presence of OPN or culture medium which has been derived from cultured EPCs which have been modified to express or overexpress EPC.

As used herein the term CAC (circulating angiogenic cells) is equivalent to EPC.

Brief Description of the Drawings

Figure 1. Patients with Type I Diabetes have fewer EPC than healthy patients.

Peripheral blood was isolated from healthy controls (Healthy) and patients with Type I Diabetes Mellitus (TIDM). EPCs were isolated and plated on fibronectin coated plates and cultured in normal glucose concentrations. Media was changed at day 4 removing nonadherent cells and EPC were counted at day 7. *p < .05.

Figure 2. EPC from patients with TIDM exhibit decreased binding to activated endothelial cells. To examine functional differences between EPC isolated from healthy volunteers (□) and patients with diabetes (■) adherence assays to matrix molecules and endothelial cells were performed. (A) lxlO⁵ EPC were added to fibronectin or collagen coated plates and incubated for one hour. Cells were then washed and stained with .1% crystal violet. Optical density of the media was then measured. (B) HUVEC were plated to confluency on a 4 well chamber slide and they were pre-treated with control media or lng/ml TNF-a for 12 hours. lxlO⁵ Dil labelled EPC were incubated with the HUVEC for three hours and nonattached cells were removed and adherent cells were counted by a blinded observer. ***p< .001 between healthy and TIDM (activated HUVECs)

Figure 3. EPC from diabetic patients exhibit impaired tubule formation.

Four- well chamber slides were coated with matrigel and 4xl0⁴ HUVEC were co-plated with 2xl0⁴ EPC isolated from either healthy volunteers or volunteers with TIDM. 12 hours later the number of tubules (structures exhibiting 4 times greater length than width) was determined by a blinded observer. ** p ^.01 Figure 4. Blood flow and micovascular density is impaired in ischemic hindlimbs of knockout mice. Laser Doppler blood flow analysis was performed on wild type and OPN knockout mice and representative images are shown (A). The color scale illustrates areas of minimal blood flow (black) to maximal (red). The ratio of blood flow in the ischemic limb to the non-ischemic limb in wild type mice (*) and OPN knockout mice (■) is quantified. Microvascular density is quantified is both ischemic (□) and non-ischemic limbs (■). **p < .01

Figure 5. Incubation with osteopontin increases the angiogenic potential of EPC in vitro. To determine if incubation with OPN would increase the angiogenic potential of EPC the cells were incubated with or without OPN (5μg/ml) for 24 hours. 2xl0⁴ EPC were co- plated with 4xl0⁴ HUVEC or HUVEC were plated alone as a control (HUVEC) on matrigel and tubule formation was assessed by a blinded counter 12 hours later. *p ^.05 ; *** p <. 001 when compared to HUVEC alone.

Figure 6. Identification of Murine EPC. Direct fluorescent staining was used to identify EPC obtained from murine bone marrow. Hoechst (blue) was used to identify the nucleus and lectin binding (green) and Ac-LDL (red) uptake was visualized at 20x.

Figure 7. Incubation with OPN increases adhesion to activated endothelial cells in OPN knockout EPCs. Ac-LDL BODIPY labelled EPCs (wild type, OPN knockout, and OPN knockout cells pre- incubated with OPN for 24 hours) were plated on a confluent layer of TNF-a activated endothelial cells. Cells were then washed and adherent cells were counted by a blinded counter. (A) Images were taken at 4x. (B) Quantification of counted EPC. ** p< .001 *** p <_ .001 when compared too wild type.

Figure 8 : shows tubule formation in HUVEC, WT, KO and KO plus OPN mice.

Figure 9 : Decreased EPC Number in Patients With Type I Diabetes

Figure 10 : EPCs from Diabetic Patients Do Not Adhere as Well to Activated Endothelial Cells

Figure 11 : EPCs from Diabetic Patients Show Decreased Angiogenic Potential

Figure 12 : Osteopontin

Figure 13 : Quantitave PCR Shows a Decrease in OPN RNA in Diabetic EPCs

Figure 14 : Osteopontin Knockout EPCs are Dysfunctional

Figure 15 : Conditioned Media From EPCs Enhances Angiogenesis In Vitro

Figure 16 : OPN Does Not Increase Angiogeneis Directly

Figure 17 : OPN Depleted CM Does Not Decrease Angiogenic Potential Figure 18 : Incubation With OPN Increases Adhesion

Figure 19 : Exposure to OPN Increases Angiogenic Potential of KO EPCs

Figure 20: Assay Used to Determine if Incubation with OPN Results in the Secretion of

Angiogenic Factors from OPN KO Cells.

Figure 21: Conditioned Media From KO Cells Incubated with OPN Enhances Angiogenic Potential

Figure 22: OPN Induces Expression of Angiogenic Proteins

Detailed Description of the Drawings

To elucidate the role of OPN in EPC, EPC were isolated from OPN knockout (KO) mice and wild type mice (WT) for a matrigel tubule assay to assess in vitro angiogenic potential. KO EPC induced significantly less tubule formation than WT EPC (p<.05, n=3). Knockout EPC that were pre-incubated with recombinant OPN induced tubule formation at levels similar to WT and significantly higher than KO cells that were not incubated with OPN (p<.05, n=3). Further, conditioned media (CM) from WT cells induced tubule formation to the same levels as the EPCs themselves suggesting that secreted proteins are responsible for the angiogenic effect. Interestingly, when KO EPC were pre-incubated with OPN, washed, and fed with new media the CM media induced tubule formation to WT levels (n=3), even though there was no OPN directly in the media. Hence, we further hypothesized that OPN is acting on EPC to induce the secretion of angiogenic cytokines. To test this, a chemi-array was performed on EPC from WT, KO and KO exposed to OPN. WT EPCs expressed FGFa at a much higher level than KO cells. Expression of FGF was restored to WT levels when KO cells were pre-incubated with OPN. Further, WT cells expressed 11-6 and TGF-a whereas KO cells did not express these proteins at detectable levels. Interestingly, when KO cells are exposed to OPN both IL-6 and TGF-a are expressed at WT levels. Taken together, this data suggests that OPN expression increases the angiogenic potential of EPCs via an autocrine mechanism whereby OPN is secreted by the EPC and subsequently induces the expression of a variety of angiogenic proteins.

MATERIALS AND METHODS

Conditioned Media Collection: Media was changed on Day 7 EPCs and collected after 48 hours (day 9) for use in the assays. For EPCs pre-exposed to OPN, on day 6 EPCs were incubated with OPN for 24 hours. At that time they were washed 3 times and fresh media was placed on the cells and collected after 48 hours (day 9). Tubule Formation Assay-Conditioned Media

HUVECs (endothelial cells) in lOOul of Endothelial Basal Medium-2 and lOOul of conditioned media were added to matrigel coated wells and placed in the incubator for 24 hours. At 24 hours tubule formation was measured.

Secreted Angiogenic Protein Array

Secreted angiogenic proteins were measured using an angiogenic protein array purchased from Panomics. Chemilumensce was determined using Image J and all proteins were normalized to the positive controls on each membrane. Conditioned media was obtained from KO EPCs, KO EPCs pre-incubated with OPN for 24 hours and wild type EPCs.

Subject recruitment

Patients with poorly controlled type I diabetes (as defined by HbAi/_c > 10%) who were on insulin for more than one year and not on any other medications were recruited from the Diabetes Day Centre, University College Hospital Galway, Ireland. Patients with micro- or macrovascular complications were excluded from the study. Microvascular complications were defined as the presence of microalbumia, diabetic retinopathy and neuropathy. Macrovascular complications were defined as the presence of any previous history acute coronary syndrome, peripheral vascular disease and cerebrovascular disease. Four patients with type I diabetes (mean HbAi/_c =12.5+4.3%; mean duration of diabetes= 7+ 2.3 years) and four gender and age matched (22.3+4.3 vs 22.8+2.1 years old) healthy volunteers. All subjects were recruited with ethical approval from University College Hospital Galway Clinical Research and Ethical Committee.

Isolation of EPC

EPC were cultured according to previously described techniques with some modifications (1). Briefly, peripheral blood mononuclear cells (PBMNC) were isolated by Ficollpaque (GE Healthcare) density centrifugation. After purification with three washing steps, lOxlO⁶ or 2x10⁶ were plated on fibronectin coated 6-well plates or 4-well glass slides respectively. Cells were cultured in endothelial cell basal medium-2 (Clonetics) supplemented with EGM- 2 single aliquots (Clonetics) consisting of 5% FBS, vascular endothelial growth factor, fibroblast growth factor-2, epidermal growth factor, insulin like growth factor, insulin like growth factor- 1 and ascorbic acid. EPC were confirmed by dual staining with Dil- acetylated low-density lipoprotein (Invitrogen) and FITC-lectin (Sigma).

EPC Adhesion to Matrix Molecules

Adhesion to matrix molecules was previously described (la). Briefly, collagen or fibronectin was coated onto 24 well plates for two hours at 37°C. Wells were then blocked with 1% BSA in PBS for two hours. 1 xlO⁵ EPC were added to each well for one hour.

Adherent cells were stained with 0.1% crystal violet and rinsed with 10% acetic acid.

Attached cells were quantified by analyzing the optical density of the media at 600nm with a microtiter plate reader.

Adhesion to Mature Endothelial Cells

Adhesion to mature endothelial cells was determined according to previously described techniques (la). Briefly, a monolayer of human umbilical vein endothelial cells (HUVEC) was prepared 48 hours before the assay by plating 2 xlO⁵ cells (passage 5 to 10) in each well of a four well glass slide. Twelve hours before the assay the HUVEC were treated with

TNF-alpha (BD Biosciences) lng/ml or media. After the 12 hours, lxlO⁵ EPCs labelled with dil were added to the HUVEC and incubated for 3 hours. Nonattached cells were then gently removed with PBS and adherent cells were fixed with 4% PFA and counted by a blinded observer.

Matrigel Tubule Assay

The assay was performed according to previously described techniques (la). Briefly, Matrigel (Sigma) was thawed on ice and placed in 4-well glass slides and incubated at room temperature for 30 minutes to allow for solidification. EPC were incubated at 37° for 24 hours with or without 5μg/ml OPN (Sigma) and labelled with Dil. 2x10⁴ labelled EPC were co-plated with 4xl0⁴ HUVEC and tubule formation was determined at 12 hours. Tubule formation was defined as a structure exhibiting a length 4 times its width. The number of tubules formed was assessed by a blinded counter.

RNA Extraction

Total RNA was isolated from day 4 EPC using RNA Mini Kit (Qiagen) as described by the manufacturer. The concentration of isolated total RNA was analyzed using NanoDrop counter. Quantlt DNA, High Sensitivity kit (Invitrogen) was used to detect the presence of any genomic DNA in the total RNA samples.

Primer Sequences

Primers for OPN (CAGAGEPCAGCATCGTCGG; GGCAAAAGCAAATEPCTGCAA) and the house keeping gene, Cyclophilin A

(TGCTGGACCCAAEPCAAATG;CATGCCTTCTTTEPCTTTGCC) (Sigma Genosys) were designed using PrimerExpress software.

Real Time PCR

The ABI Prisim 7000 Sequence Detection System (Applied Biosystems) with One Step QuantiTect SYBR Green PCR kit (Qiagen) was used for all PCR experiments. The reactions were performed according to the manufacturer's instructions with minor modifications. A sample volume of 25ml was incubated for 30 minutes at 50°C; 95°C for 15 minutes; followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C. The dissociation curves were generated using a temperature range between 60°C and 95°C. All reactions were further analyzed by electrophoresis on 2% agarose gels stained with SyBrGreen dye to confirm the PCR results. Each sample was analyzed in triplicate.

Murine Hind Limb Ischemia Model

C57BL/6 (WT) and OPNVOPN^" (OPN-KO) mice were purchased from Charles River Lab and Jackson Laboratory respectively. OPN-KO and WT mice aged between 8-10 weeks of age were used. The mice were housed at the animal facility in the Regenerative Medicine Institute (REMEDI), NCBES, NUIG. All procedures were approved by the Minister of Health and Children under the Cruelty to Animal Acts, 1876. Unilateral hind limb ischemia was created in WT and OPN-KO mice as previously described (2a). In brief, an incision was performed in the skin overlying the middle portion of the left hind limb. After ligation of the proximal end of the femoral artery, the distal portion of the saphenous artery the artery as well as all side branches were dissected free and excised. The skin was closed using an absorbable suture. Animals were anesthetized with ketamine and xylazine and maintained with isoflurane if required. For experiments involving the injection of EPCs, KO Mice were injected with lxlO⁶ EPCs in 200ul of EMB-2 with no additional supplements or with just EBM-2 alone. Injections were performed at the time ischemia was induced. For each animal there were four injections into the muscle in the area immediately surrounding where the femroal artery was ligated.

Laser Doppler Blood Flow Assessment (LDBF)

The blood flow in both hind limbs and feet were measured using a laser Doppler blood flow analyzer (PeriScan PIMII, Perimed Inc) immediately before and after surgery as well as on post operative days 7,14 and 28. Microvasulature blood flow was displayed in a color coded image. Analysis was performed by comparing blood flow in the ischemic limb (left) to the non-ischemic limb (right) thereby accounting for variations due to ambient light and temperature.

Isolation and Identification of Murine Endothelial Progenitor Cells

Murine EPC were isolated from the bone marrow of WT and OPN-KO mice. The hind limbs were separated from the body and the epiphyses were removed from both ends. The marrow was flushed from the bone in 5mls of EGM-2 and the media with bone marrow was collected and centrifuged at 1700 rpm for 25 minutes. The supernatant was removed and cells were resuspended in one ml of red cell lysis buffer (Sigma). Cells were collected at 1700 rpm for five minutes and washed twice in PBS. Cells were resuspended in 4mls EBM- 2 and plated on a fibronectin coated plate. EPC were identified by direct fluorescent staining to detect Fluorescein Ulex europaeus agglutinin (Company) and Dil-acLDL (Invitrogen) as described (3 a).

Results

EPC number and function is impaired in patients with type 1 diabetes.

In order to better understand EPC dysfunction we sought to establish a disease model with impaired EPC function. It has been suggested that EPC play a role in wound repair and it is well established that patients with diabetes have an impaired angiogenic response to ischemic injury. Thus, we sought to confirm that EPC from patients with type 1 diabetes melitus (T1DM) were dysfunctional. EPC were isolated from healthy volunteers and from age gender matched patients with type 1 diabetes. The cells were grown in normal glucose media for seven days and the number of EPC was then assessed. Patients with type 1 diabetes were found to have significantly fewer EPC than healthy controls (Figure 1).

In order to determine if EPC dysfunction in T1DM is linked to simply a lower number of EPC or to actual impairment of the cells a series of functional assays was performed. To determine if diabetic EPC have defects in their ability to adhere to matrix molecules and to sites of injury a set of in vitro assay were performed. Adhesion to matrix molecules, fibronectin and collagen, was not significantly different between control EPC and EPC from T1DM (Figure 2a). Further, adhesion to quiescent endothelial cells was not significantly different between the two groups. Interestingly, when an injury is mimicked, by activation of the endothelial cells with TNF-a, adherence was significantly impaired in EPC from T1DM (Figure 2b).

In addition to adhering to the site of the injury EPC are believed to play a role, either directly or indirectly, in angiogenesis. To assess the angiogenic potential of EPC from controls and patients with T1DM a tubule formation assay was used. EPC derived from patients with T1DM exhibited a diminished ability to form tubules (Figure 3).

Osteopontin expression is downregulated in EPC from patients with T1DM

Taken together, the impaired ability to adhere to activated endothelial cells and the attenuated ability to form tubules suggests that T1DM EPC can serve as a model of EPC dysfunction. Therefore, several candidate genes were identified that we hypothesized could contribute to the observed defects in EPC from diabetic patients. Quantitative real-time PCR was used to determine the expression levels in EPC isolated from both controls and T1DM. Osteopontin was discovered to be downregulated in T1DM EPC.

OPN Increases Tubule Formation in EPC

To determine if osteopontin can play a role in angiogenesis, healthy human EPC were incubated with OPN for 24 hours and then used in a tubule formation assay. EPC that were incubated with osteopontin significantly increased tubule formation (Figure 4).

Angiogenesis is Impaired in Osteopontin Knockout Mice

To assess the role osteopontin plays in angiogenesis in vivo a murine model of hind limb ischemia was used. Ischemia was created in the left limb of wild type mice (control) and osteopontin knockout mice and laser doppler blood flow (LDBF) analysis was used to assess blood flow in the limbs immediately following surgery and then 7,14 and 28 days post procedure (Figure 4a). The LDBF ratio was also calculated (Figure 4b) and indicates that restoration of perfusion in the knockout mice was significantly impaired at days 7, 14 and 28 when compared to wild type. In addition, microvascular density was also calculated (Figure 4c). Microvascular density increased in response to ischemia in the wildtype mice while conversely it decreased in the knockout mice further supporting the LDBF data.

Interestingly, the observation that OPN incubation with EPC results in increased tubule formation taken together with the decreased angiogenesis observed in the murine model of hind limb ischemia supports a critically important role for osteopontin in the response to ischemia. However, this data does not directly link osteopontin expression in EPC specifically to the angiogenic response. Therefore, EPC were isolated from wild type and OPN knockout mice to explore the potential role ostepontin expression in EPC plays in wound healing.

Osteopontin Knockout Mice Exhibit Decreased Adherence to Activated Endothelial Cells

EPC were isolated from the bone marrow of both wild type and OPN knockout mice and dual stained to confirm uptake of both actylated LDL and UEA-1 lectin (Figure 6). Day seven EPC obtained from the marrow were then used to determine if OPN expression plays a role in the ability of the cells to adhere to sites of injury.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

References

la. Tepper, O.M., Galiano, R.D., Capla, J.M., Kalka, C, Gagne, P.J., Jacobowitz, G.R., Levine, J.P., and Gurtner, G.C. 2002. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation 106:2781-2786.

2a. Rivard, A., Silver, M., Chen, D., Kearney, M., Magner, M., Annex, B., Peters, K., and Isner, J.M. 1999. Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF. Am J Pathol 154:355-363.

3a. Hu, Z., Zhang, F., Yang, Z., Yang, N., Zhang, D., Zhang, J., and Cao, K. 2008. Combination of simvastatin administration and EPC transplantation enhances angiogenesis and protects against apoptosis for hindlimb ischemia. J Biomed Sci 15:509-517.

1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Science. 1997;275:964-7.

2. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T. Proc Natl Acad Sci USA. 2000;97:3422-7.

3. Kubota Y, Kishi K, Satoh H, Tanaka T, Nakajima H, Nakajima T. Cell Transplant. 2003;12:647-57.

4. Griese DP, Ehsan A, Melo LG, Kong D, Zhang L, Mann MJ, Pratt RE, Mulligan RC, Dzau VJ. Circulation. 2003;108:2710-5.

5. He T, Smith LA, Harrington S, Nath KA, Caplice NM, Katusic ZS. Stroke.

2004;35:2378-84.

6. Takahashi M, Nakamura T, Toba T, Kajiwara N, Kato H, Shimizu Y. Tssue Eng. 2004;10:771-9.

7. Urbich C, Dimmeler S. Trends Cardiovasc Med. 2004; 14:318-22.

8. Gulati R, Jevremovic D, Peterson TE, Chatterjee S, Shah V, Vile RG, Simari RD. Ore Res. 2003;93: 1023-5.

9. Eggermann J, Kliche S, Jarmy G, Hoffmann K, Mayr-Beyrle U, Debatin KM, Waltenberger J, Beltinger C. Cardiovasc Res. 2003;58:478-86.

10. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Clin Invest. 2000; 105:71-7.

11. Hur J, Yoon CH, Kim HS, Choi JH, Kang HJ, Hwang KK, Oh BH, Lee MM, Park YB. Arterioscler Thromb Vase Biol. 2004;24:288-93. 12. Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ, Gilley D, Yoder MC. Blood. 2004;104:2752-60.

13. Walenta K, Friedrich EB, Sehnert F, Werner N, Nickenig G. Biochem Biophys Res Commun. 2005;333:476-82.

14. Bellik L, Ledda F, Parenti A. FEB S Lett. 2005;579:2731-6.

15. Liew A, Barry F, O'Brien T. Bioessays. 2006;28:261-70.

16. Loomans CJ, de Koning EJ, Staal FJ, Rookmaaker MB, Verseyden C, de Boer HC, Verhaar MC, Braam B, Rabelink TJ, van Zonneveld AJ. Diabetes. 2004;53: 195-9.

17. Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC. Circulation. 2002;106:2781-6.

18. Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, Avogaro A. J Am Coll Cardiol. 2005;45: 1449-57.

19. Willing M, Sowers M, Aron D, Clark MK, Burns T, Bunten C, Crutchfield M, DAgostino D, Jannausch M. J Bone Miner Res. 1998;13:695-705.

20. Asnaghi V, Lattanzio R, Mazzolari G, Pastore MR, Ramoni A, Maestroni A, Ruggieri D, Luzi L, Brancato R, Zerbini G. Diabetologia. 2006.

21. Fadini GP, Sartore S, Baesso I, Lenzi M, Agostini C, Tiengo A, Avogaro A.

Diabetes Care. 2006;29:714-6.

22. Loomans CJ, De Koning EJ, Staal FJ, Rabelink TJ, Zonneveld AJ. Antioxid Redox Signal. 2005;7: 1468-75.

23. Denhardt DT, Lopez CA, RoUo EE, Hwang SM, An XR, Walther SE. Ann N Y Acad Sci. 1995;760: 127-42.

24. Haubner R, Weber WA, Beer AJ, Vabuliene E, Reim D, Sarbia M, Becker KF, Goebel M, Hein R, Wester HJ, Kessler H, Schwaiger M. PLoSMed. 2005;2:e70.

25. Uede T, Katagiri Y, Iizuka J, Murakami M. Microbiol Immunol. 1997;41 :641-8. 26. Hirama M, Takahashi F, Takahashi K, Akutagawa S, Shimizu K, Soma S,

Shimanuki Y, Nishio K, Fukuchi Y. Cancer Lett. 2003;198: 107-17.

27. Zhou Y, Dai DL, Martinka M, Su M, Zhang Y, Campos EI, Dorocicz I, Tang L, Huntsman D, Nelson C, Ho V, Li G. J Invest Dermatol. 2005;124: 1044-52.

28. Hu Z, Lin D, Yuan J, Xiao T, Zhang H, Sun W, Han N, Ma Y, Di X, Gao M, Ma J, Zhang J, Cheng S, Gao Y. Clin Cancer Res. 2005; 11 :4646-52.

29. Ito T, Hashimoto Y, Tanaka E, Kan T, Tsunoda S, Sato F, Higashiyama M, Okumura T, Shimada Y. Clin Cancer Res. 2006;12: 1308-16. 30. Wai PY, Mi Z, Guo H, Sarraf-Yazdi S, Gao C, Wei J, Marroquin CE, Clary B, Kuo PC. Carcinogenesis. 2005;26:741-51.

31. Muramatsu T, Shima K, Ohta K, Kizaki H, Ro Y, Kohno Y, Abiko Y, Shimono M. Cancer Lett. 2005;217:87-95.

32. Rivard A, Silver M, Chen D, Kearney M, Magner M, Annex B, Peters K, Isner JM. Am J Pathol. 1999; 154: 355-63

33. Ballard VL, Sharma A, Duignan I, Holm JM, Chin A, Choi R, Hajjar KA, Wong SC, Edelberg m.Faseb J. 2006; 20:717-9 ().

Claims

1. A pharmaceutical composition comprising osteopontin-conditioned culture medium from endothelial progenitor cells together with a pharmaceutically acceptable carrier or excipient.

2. A pharmaceutical composition as claimed in claim 1 wherein the osteopontin- conditioned culture medium is derived from endothelial progenitor cells which have been cultured in the presence of osteopontin.

3. A pharmaceutical composition as claimed in claim 1 or 2 wherein the culture medium is derived from endothelial progenitor cells which have been exposed to

osteopontin for at least 12 hours, more preferably at least 24 hours.

4. A pharmaceutical composition as claimed in claim 1 wherein the osteopontin- conditioned culture medium is derived from endothelial progenitor cells which have been modified to express or overexpress osteopontin.

5. A pharmaceutical composition as claimed in any preceding claim wherein the culture medium is cell-free.

6. A pharmaceutical composition as claimed in any preceding claim further comprising endothelial progenitor cells, osteopontin, endothelial progenitor cells which have been modified to express or overexpress osteopontin, mesenchymal stem cells or combinations thereof.

7. A method of producing an osteopontin-conditioned culture medium from endothelial progenitor cells for use as a pharmaceutical comprising culturing endothelial progenitor cells in the presence of osteopontin, or culturing endothelial progenitor cells which have been modified to express or overexpress osteopontin.

8. A method as claimed in claim 7 wherein the osteopontin-conditioned culture medium is derived from endothelial progenitor cells which have been exposed to osteopontin for at least 6 hours, more preferably at least 12 hours, more preferably at least 24 hours.

9. A method as claimed in claim 7 or 8 wherein the endothelial progenitor cells are cultured in the presence of osteopontin and then washed and allowed to grow for a further at least 12 hours, more preferably at least 24 hours, before the medium is harvested.

10. A method as claimed in claim 9 wherein the endothelial progenitor cells are washed with a buffer such as phosphate buffered saline or similar buffer.

11. A method as claimed in claim 7 wherein the osteopontin-conditioned medium is derived from endothelial progenitor cells which have been modified to express or overexpress osteopontin and which have been cultured for at least 12 hours.

12. A method of assaying osteopontin-conditioned medium for the presence of angiogenesis-inducing compounds comprising assaying a subject compound isolated from the medium.

13. A method as claimed in claim 12 wherein the subject compound is assayed in a matrigel tubule formation assay.

14. A method of treatment of vascular diseases or diabetes-associated vascular complications comprising administering to a subject in need of such treatment a pharmaceutically effective amount of :

osteopontin-conditioned medium;

osteopontin-conditioned medium together with endothelial progenitor cells;

osteopontin-conditioned medium together with endothelial progenitor cells which have been modified to express or overexpress osteopontin;

osteopontin-conditioned medium together with osteopontin;

osteopontin-conditioned medium together with osteopontin and endothelial progenitor cells; or,

osteopontin-conditioned medium together with with osteopontin and endothelial progenitor cells which have been modified to express or overexpress osteopontin.

15. A method of treatment as claimed in claim 14 further comprising administration of one or more of FGFa (fibroblast growth factor), 11-6 (interleukin 6), TGF-a (transforming growth factor alpha) and IL-8 (interleukin-8).

16. A method of treatment as claimed in claim 14 or 15 further comprising administration of mesenchymal stem cells.

17. A pharmaceutical composition substantially as described herein with reference to the accompanying drawings.

18. A method of producing an osteopontin-conditioned culture medium substantially as described herein with reference to the accompanying drawings.

19. A method of assaying osteopontin-conditioned medium substantially as described herein with reference to the accompanying drawings.

20. A method of treatment of vascular diseases or diabetes associated vascular complications substantially as described herein with reference to the accompanying drawings.