Overexpression of CD44 in Neural Precursor Cells Improves Trans- Endothelial Migration and Facilitates Their Invasion of Perivascular Tissues In Vivo

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional in vivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Internalization of nanopolymeric tracers does not alter characteristics of placental cells

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases.     

Endothelial cell injury by high glucose and heparanase is prevented by insulin, heparin and basic fibroblast growth factor

Background

Uncontrolled hyperglycemia is the main risk factor in the development of diabetic vascular complications. The endothelial cells are the first cells targeted by hyperglycemia. The mechanism of endothelial injury by high glucose is still poorly understood. Heparanase production, induced by hyperglycemia, and subsequent degradation of heparan sulfate may contribute to endothelial injury. Little is known about endothelial injury by heparanase and possible means of preventing this injury.

Objectives

To determine if high glucose as well as heparanase cause endothelial cell injury and if insulin, heparin and bFGF protect cells from this injury.

Methods

Cultured porcine aortic endothelial cells were treated with high glucose (30 mM) and/or insulin (1 U/ml) and/or heparin (0.5 μg/ml) and /or basic fibroblast growth factor (bFGF) (1 ng/ml) for seven days. Cells were also treated with heparinase I (0.3 U/ml, the in vitro surrogate heparanase), plus insulin, heparin and bFGF for two days in serum free medium. Endothelial cell injury was evaluated by determining the number of live cells per culture and lactate dehydrogenase (LDH) release into medium expressed as percentage of control.

Results

A significant decrease in live cell number and increase in LDH release was found in endothelial cells treated with high glucose or heparinase I. Insulin and/or heparin and/or bFGF prevented these changes and thus protected cells from injury by high glucose or heparinase I. The protective ability of heparin and bFGF alone or in combination was more evident in cells damaged with heparinase I than high glucose.

Conclusion

Endothelial cells injured by high glucose or heparinase I are protected by a combination of insulin, heparin and bFGF, although protection by heparin and/or bFGF was variable.     

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice

Background aimsWe recently showed that co-transplantation of mesenchymal stromal cells (MSCs) improves islet function and revascularization in vivo. Pre-transplant islet culture is associated with the loss of islet cells. MSCs may enhance islet cell survival or function by direct cell contact mechanisms and soluble mediators. We investigated the capacity of MSCs to improve islet cell survival or β-cell function in vitro using direct and indirect contact islet-MSC configurations. We also investigated whether pre-culturing islets with MSCs improves islet transplantation outcome.MethodsThe effect of pre-culturing islets with MSCs on islet function in vitro was investigated by measuring glucose-stimulated insulin secretion. The endothelial cell density of fresh islets and islets cultured with or without MSCs was determined by immunohistochemistry. The efficacy of transplanted islets was tested in vivo using a syngeneic streptozotocin-diabetic minimal islet mass model. Graft function was investigated by monitoring blood glucose concentrations.ResultsIndirect islet-MSC co-culture configurations did not improve islet function in vitro. Pre-culturing islets using a direct contact MSC monolayer configuration improved glucose-stimulated insulin secretion in vitro, which correlated with superior islet graft function in vivo. MSC pre-culture had no effect on islet endothelial cell number in vitro or in vivo.ConclusionsPre-culturing islets with MSCs using a direct contact configuration maintains functional β-cell mass in vitro and the capacity of cultured islets to reverse hyperglycemia in diabetic mice.     

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Endothelial Differentiation of Mesenchymal Stromal Cells

Human mesenchymal stromal cells (hMSCs) are increasingly used in regenerative medicine for restoring worn-out or damaged tissue. Newly engineered tissues need to be properly vascularized and current candidates for in vitro tissue pre-vascularization are endothelial cells and endothelial progenitor cells. However, their use in therapy is hampered by their limited expansion capacity and lack of autologous sources. Our approach to engineering large grafts is to use hMSCs both as a source of cells for regeneration of targeted tissue and at the same time as the source of endothelial cells. Here we investigate how different stimuli influence endothelial differentiation of hMSCs. Although growth supplements together with shear force were not sufficient to differentiate hMSCs with respect to expression of endothelial markers such as CD31 and KDR, these conditions did prime the cells to differentiate into cells with an endothelial gene expression profile and morphology when seeded on Matrigel. In addition, we show that endothelial-like hMSCs are able to create a capillary network in 3D culture both in vitro and in vivo conditions. The expansion phase in the presence of growth supplements was crucial for the stability of the capillaries formed in vitro. To conclude, we established a robust protocol for endothelial differentiation of hMSCs, including an immortalized MSC line (iMSCs) which allows for reproducible in vitro analysis in further studies.     

Increased Migration of Human Mesenchymal Stromal Cells by Autocrine Motility Factor (AMF) Resulted in Enhanced Recruitment towards Hepatocellular Carcinoma

Background and Aims

Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC.

Methods

Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated.

Results

AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro.

Conclusion

AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.     

Mesenchymal stem cells as a gene therapy carrier for treatment of fibrosarcoma

Background aimsCell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment.MethodsIn this study, we injected intravenously (i.v.) 1 × 10⁶ MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals.ResultsWe observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group.ConclusionsiNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors.     

Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts

Background

Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin''s lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL.

Methodology/Principal Findings

The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin''s lymphomas with an indolent (EBV⁻ Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV⁺ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures.

Conclusions/Significance

Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.     

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells

Background aimsHuman mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populationsMethodsGiven these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD modelResultsOur study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD.ConclusionsOur results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.     

Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.     

Endothelial cells interact with the core protein of basement membrane perlecan through beta 1 and beta 3 integrins: an adhesion modulated by glycosaminoglycan

Aortic endothelial cells adhere to the core protein of murine perlecan, a heparan sulfate proteoglycan present in endothelial basement membrane. We found that cell adhesion was partially inhibited by beta 1 integrin-specific mAb and almost completely blocked by a mixture of beta 1 and alpha v beta 3 antibodies. Furthermore, adhesion was partially inhibited by a synthetic peptide containing the perlecan domain III sequence LPASFRGDKVTSY (c-RGD) as well as by GRGDSP, but not by GRGESP. Both antibodies contributed to the inhibition of cell adhesion to immobilized c-RGD whereas only beta 1-specific antibody blocked residual cell adhesion to proteoglycan core in the presence of maximally inhibiting concentrations of soluble RGD peptide. A fraction of endothelial surface-labeled detergent lysate bound to a core affinity column and 147-, 116-, and 85-kD proteins were eluted with NaCl and EDTA. Polyclonal anti-beta 1 and anti-beta 3 integrin antibodies immunoprecipitated 116/147 and 85/147 kD surface-labeled complexes, respectively. Cell adhesion to perlecan was low compared to perlecan core, and cell adhesion to core, but not to immobilized c-RGD, was selectively inhibited by soluble heparin and heparan sulfates. This inhibition by heparin was also observed with laminin and fibronectin and, in the case of perlecan, was found to be independent of heparin binding to substrate. These data support the hypothesis that endothelial cells interact with the core protein of perlecan through beta 1 and beta 3 integrins, that this binding is partially RGD- independent, and that this interaction is selectively sensitive to a cell-mediated effect of heparin/heparan sulfates which may act as regulatory ligands.     

Endothelial microparticles reduce ICAM‐1 expression in a microRNA‐222‐dependent mechanism

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced.     

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [³H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.     

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.     

Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity

Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF₁₆₅ in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF₁₆₅, VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.     

Injection of mesenchymal stromal cells into a mechanically stimulated in vitro model of cardiac fibrosis has paracrine effects on resident fibroblasts

Background aimsMyocardial infarction results in the formation of scar tissue populated by myofibroblasts, a phenotype characterized by increased contractility and matrix deposition. Mesenchymal stromal cells (MSC) delivered to the myocardium can attenuate scar growth and restore cardiac function, though the mechanism is unclear.MethodsThis study describes a simple yet robust three-dimensional (3D) in vitro co-culture model to examine the paracrine effects of implanted MSC on resident myofibroblasts in a controlled biochemical and mechanical environment. The fibrosis model consisted of fibroblasts embedded in a 3D collagen gel cultured under defined oxygen tensions and exposed to either cyclic strain or interstitial fluid flow. MSC were injected into this model, and the effect on fibroblast phenotype was evaluated 48 h after cell injection.ResultsAnalysis of gene and protein expression of the fibroblasts indicated that injection of MSC attenuated the myofibroblast transition in response to reduced oxygen and mechanical stress. Assessment of vascular endothelial growth factor and insulin-like growth factor-1 levels demonstrated that their release by fibroblasts was markedly upregulated in hypoxic conditions but attenuated by strain or fluid flow. In fibroblast-MSC co-cultures, vascular endothelial growth factor levels were increased by hypoxia but not affected by mechanical stimuli, whereas insulin-like growth factor-1 levels were generally low and not affected by experimental conditions.ConclusionsThis study demonstrates how a 3D in vitro model of the cardiac scar can be used to examine paracrine effects of MSC on the phenotype of resident fibroblasts and therefore illuminates the role of injected progenitor cells on the progression of cardiac fibrosis.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.