CD154 Induces Matrix Metalloproteinase-9 Secretion in Human Podocytes.

Matrix remodeling is a key feature of glomerulosclerosis secondary to diabetes or hypertension. Podocytes contribute to glomerular basement membrane (GBM) turnover by producing matrix components and matrix remodelling enzymes, including matrix metalloproteinases (MMPs). The CD40/CD154 signaling pathway modulates matrix remodeling through the synthesis of MMPs and tissue inhibitors of MMPs. Platelets are a primary blood reservoir of CD154. Here we studied, the impact of the CD154/CD40 pathway on MMP-9 expression by cultured human podocytes. The role of CD40/CD154 was evaluated upon exposure of podocytes to recombinant human CD154 (rhCD154) or activated platelet supernatants from healthy human subjects. We first showed by protein and mRNA expression that CD40 was synthesized by podocytes and detectable on kidney tissue sections. CD40 expression was acquired during podocyte differentiation and enhanced upon exposure to rhCD154. In podocytes, rhCD154 induced an increase of MMP-9 production as shown by RT-PCR, Western blot and and gelatin zymography. Activated platelet supernatants induced MMP-9 mRNA synthesis in podocytes, an effect reduced by anti-CD40 antibody. Our results underscore a potential role for platelets through the CD40/CD154 signaling pathway in the control of GBM synthesis and degradation, via its regulatory role on MMP-9 production. CD154 secretion by activated platelets may contribute to GBM alterations in proteinuric nephropathies. J. Cell. Biochem. 117: 2737-2747, 2016. © 2016 Wiley Periodicals, Inc.

Comparative study of membranes induced by PMMA or silicone in rats, and influence of external radiotherapy.

The induced membrane technique has been used for long bone defect reconstruction after traumatism. One of the major drawbacks of this method is the difficult removal of the polymethyl methacrylate spacer after membrane formation. We therefore replaced the stiff PMMA spacer with a semi-flexible medical grade silicone spacer. This study aimed to compare subcutaneously formed membranes, induced by PMMA and silicone, in the irradiated or not irradiated areas within 28 rats that received the spacers. Histological analysis was performed to evaluate the composition of the membrane and to quantify the amount of vessels. Histomorphometric measurements were used to evaluate membranes' thickness, while fibrosis and inflammation were scored. The expression of VEGF and BMP-2 in lysates of the crushed membranes was determined by Western blotting. ALP expression was analyzed in HBMSC cultures in contact with the same lysates. Non-irradiated membranes induced by the two spacer types were non-inflammatory, fibrous and organized in layers. Irradiation did not change the macroscopic properties of membranes that were induced by silicone, while PMMA induced membranes were sensitive to the radiotherapy, resulting in thicker, strongly inflammatory membranes. Irradiated membranes showed an overall reduced osteogenic potential. Medical grade silicone is safe for the use in radiotherapy and might therefore be of great advantage for patients in need of cancer treatment.

The gene expression of human endothelial cells is modulated by subendothelial extracellular matrix proteins: short-term response to laminar shear stress.

Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Tissue engineering strives to produce bio-artificial conduits to provide resistance to thrombosis. The objectives of our study were to culture endothelial cells (EC) on composite assemblies of extracellular matrix proteins, and to evaluate the cellular phenotype under flow. Cell-adhesive assemblies were fabricated on glass slides as combinations of collagen (Co), laminin (LM), and fibronectin (FN), resulting in three samples: Co, Co/LM, and Co/FN. Surface topography, roughness, and wettability were determined. Human saphenous vein EC were harvested from cardiac patients, cultured on the assemblies and submitted to laminar shear stress (SS) of 12 dyn/cm(2) for 40, 80, and 120 min. Cell retention was assessed and qRT-PCR of adhesion genes (VE-cadherin, vinculin, KDR, CD-31 or PECAM-1, ß1-integrins) and metabolic genes (t-PA, NF-κB, eNOS and MMP-1) was performed. Quantitative immunofluorescence of VE cadherin, vinculin, KDR, and vonWillebrand factor was performed after 2 and 6 h of flow. Static samples were excluded from shearing. The cells reached confluence with similar growth curves. The cells on Co/LM and Co/FN were resistant to flow up to 120 min but minor desquamation occurred on Co corresponding with temporary downregulation of VE cadherin and vinculin-mRNA and decreased fluorescence of vinculin. The cells seeded on Co/LM initially more upregulated vinculin-mRNA and also the inflammatory factor NF-κB, and the cells plated on Co/FN changed the expression profile minimally in comparison with the static control. Fluorescence of VE cadherin and vonWillebrand factor was enhanced on Co/FN. The cells cultured on Co/LM and Co/FN increased the vinculin fluorescence and expressed more VE cadherin and KDR-mRNA than the cells on Co. The cells plated on Co/FN upregulated the mRNA of VE cadherin, CD-31, and MMP 1 to a greater extent than the cells on Co/LM and they enhanced the fluorescence of VE cadherin, KDR, and vonWillebrand factor. Some of these changes sustained up to 6 h of flow, as confirmed by immunofluorescence. Combined matrices Co/LM and Co/FN seem to be more suitable for EC seeding and retention under flow. Moreover, Co/FN matrix promoted slightly more favorable cellular phenotype than Co/LM under SS of 2-6 h.

Polyethylene terephthalate membrane grafted with peptidomimetics: endothelial cell compatibility and retention under shear stress.

The present work aimed to treat a polyethylene terephthalate (PET) surface to make the biomaterial more 'attractive' in terms of attachment and shear stress response to endothelial cells with a view to possible applications in vascular grafting. A surface wet-chemistry protocol was applied to graft track-etched PET membranes with RGD peptidomimetics based on the tyrosine template and active at the nano-level vs. isolated human αvß3 receptor, which was monitored by X-ray photoelectron spectroscopy, contact angle measurement and atomic force microscopy for characterization. A primary culture of human saphenous vein endothelial cells was used before and after sterilization of the membranes (heat treatment or γ-ray irradiation) to test the benefit of grafting. The optimal surface concentrations of grafted molecules were around 50 pmol/cm². Compared to GRGDS, the peptidomimetics promoted cell attachment with similar or slightly better performances. Endothelialized grafted supports were further exposed to 2 h of shear stress mimicking arterial conditions. Cells were lost on non-grafted PET whereas cells on grafted polymers sterilized by γ-ray irradiation withstood forces with no significant difference in focal contacts. At the mRNA level, cells on functionalized PET were able to respond to shear stress with NFkB upregulation. Thus, grafting of peptidomimetics as ligands of the αvß3 integrin could be a relevant strategy to improve the adhesion of human endothelial cells and to obtain an efficient endothelialized PET for the surgery of small-diameter vascular prostheses.

uPA and MMP-2 were involved in self-assembled network formation in a two dimensional co-culture model of bone marrow stromal cells and endothelial cells.

Two dimensional (2D) co-cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self-assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-2 (MMP-2) as well tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) were investigated in this 2D co-culture model. We found that the expression of uPA, MMP-2 in the co-cultured cells was significantly higher than those in mono-cultured cells. In opposite, PAI-1, expressed only by HUVECs is not regulated in the co-culture. Inhibition assays confirm that uPA played a critical role in the formation of self-assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP-2 prevented the formation of self-assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP-2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP-2 activity, and may contribute to endothelial cell migration and the formation of this self-assembled network observed in these 2D co-cultured cells.

The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network.

Angiogenesis is very important for vascularized tissue engineering. In this study, we found that a two-dimensional co-culture of human bone marrow stromal cell (HBMSC) and human umbical vein endothelial cell (HUVEC) is able to stimulate the migration of co-cultured HUVEC and induce self-assembled network formation. During this process, expression of vascular endothelial growth factor (VEGF165) was upregulated in co-cultured HBMSC. Meanwhile, VEGF165-receptor2 (KDR) and urokinase-type plasminogen activator (uPA) were upregulated in co-cultured HUVEC. Functional studies show that neutralization of VEGF165 blocked the migration and the rearrangement of the cells and downregulated the expression of uPA and its receptor. Blocking of vascular endothelial-cadherin (VE-cad) did not affect the migration of co-cultured HUVEC but suppressed the self-assembled network formation. In conclusion, co-cultures upregulated the expression of VEGF165 in co-cultured HBMSC; VEGF165 then activated uPA in co-cultured HUVEC, which might be responsible for initiating the migration and the self-assembled network formation with the participation of VE-cad. All of these results indicated that only the direct contact of HBMSC and HUVEC and their respective dialogue are sufficient to stimulate secretion of soluble factors and to activate molecules that are critical for self-assembled network formation which show a great application potential for vascularization in tissue engineering.

Role of neural-cadherin in early osteoblastic differentiation of human bone marrow stromal cells cocultured with human umbilical vein endothelial cells.

In our previous studies, roles of gap junction and vascular endothelial growth factor in the cross-talking of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) have been extensively studied. The present study focused on the investigation of the roles of neural (N)-cadherin in early differentiation of HBMSCs in direct-contact cocultures with HUVECs for 24 and 48 h. Quantitative real-time polymerase chain reaction, immunofluorescence, Western blot, as well as functional studies were applied to perform the studies at both protein and gene levels. Results showed that cocultured cells expressed much higher N-cadherin than monocultured cells after 24 and 48 h of culture. We observed that N-cadherin concentrated in the membrane of cocultured HBMSCs (co-HBMSCs) while distributed within the cytoplasm of monocultured HBMSCs, which indicated that the cell-cell adhesion was improved between cocultured cells. In addition, more beta-catenin was found to translocate into the cocultured cells nuclei and more T cell factor-1 (TCF-1) were detected in cocultured cells than in the monocultured cells. Moreover, mRNA levels of early osteoblastic markers including alkaline phosphatase (ALP) and type I collagen (Col-I) of co-HBMSCs were significantly upregulated, whereas neutralization of N-cadherin led to a downregulation of ALP and Col-I in both of the HBMSCs and co-HBMSCs compared with untreated cells. Taking our findings together it can be concluded that cocultures of HBMSCs with HUVECs increased N-cadherin expression and improved cell-cell adhesion. Whether this applies only to osteoprogenitor cells or to all the cell types in the culture will need to be determined by further studies. Subsequently, signaling transduction might be induced with the participation of beta-catenin and TCF-1. With the N-cadherin-mediated cell-cell adhesion and signaling transductions, the early osteoblastic differentiation of co-HBMSCs was significantly upregulated.

The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect.

Bone regeneration seems to be dependant on cell communication between osteogenic and endothelial cells arising from surrounding blood vessels. This study aims to determine whether endothelial cells can regulate the osteogenic potential of osteoprogenitor cells in vitro and in vivo, in a long bone defect, when co-immobilized in alginate microspheres. Alginate is a natural polymer widely used as a biomaterial for cell encapsulation. Human osteoprogenitors (HOP) from bone marrow mesenchymal stem cells were immobilized alone or together with human umbilical vein endothelial cells (HUVEC) inside irradiated, oxidized and RGD-grafted alginate microspheres. Immobilized cells were cultured in dynamic conditions and cell metabolic activity increased during three weeks. The gene expression of alkaline phosphatase and osteocalcin, both specific markers of the osteoblastic phenotype, and mineralization deposits were upregulated in co-immobilized HOPs and HUVECs, comparing to the immobilization of monocultures. VEGF secretion was also increased when HOPs were co-immobilized with HUVECs. Microspheres containing co-cultures were further implanted in a bone defect and bone formation was analysed by muCT and histology at 3 and 6 weeks post-implantation. Mineralization was observed inside and around the implanted microspheres containing the immobilized cells. However, when HOPs were co-immobilized with HUVECs, mineralization significantly increased. These findings demonstrate that co-immobilization of osteogenic and endothelial cells within RGD-grafted alginate microspheres provides a promising strategy for bone tissue engineering.

Responsiveness of human bone marrow stromal cells to shear stress.

We examined the hypothesis that human mesenchymal stem cells detect physiological mechanical signals. Human bone marrow stromal cells (HBMSCs) were exposed to fluid shear stress of 12 dynes/cm(2) and analysed for their ability to express osteoblast-specific markers and associated signalling pathways. HBMSCs showed a significant increase in alkaline phosphatase (ALP) gene expression and a marked decrease in type I collagen, while no effect on Cbfa1/Runx2 was detected. This regulation is related to p38 and ERK1/2 activation, although the use of specific inhibitors to these two MAP kinases suggests that ALP mRNA induction is especially dependent on p38 activity, while type I collagen downregulation is ERK1/2-dependent. Interestingly, the expression of connexin43, which is involved in cell-to-cell communication of osteoblastic cells through gap junction formation, and its distribution through the cells, were modified by fluid flow (FF). HBMSCs are sensitive to shear stress and it appears essential to take their responsiveness into consideration before associating these regenerative cells with a bioactive biomaterial in a new bone tissue-engineering strategy.

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells.

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross-talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co-culture between osteoblastic and endothelial cells. Through a well defined direct contact co-culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular-like structures. VEGF(165) gene expression was detected in the HOPs, was up-regulated in the co-cultured HOPs and both Flt-1 and KDR gene expression increased in co-cultured HUVECs. However, the cell rearrangement observed in co-culture was promoted by a combination of soluble chemoattractive factors and not by VEGF(165) alone. Despite having no observable effect on endothelial cell tubular-like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co-culture-stimulated osteoblastic phenotype. This co-culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering.

Capability of human umbilical cord blood progenitor-derived endothelial cells to form an efficient lining on a polyester vascular graft in vitro.

One of the goals of vascular tissue engineering is to create functional conduits for small-diameter bypass grafting. The present biocompatibility study was undertaken to check the ability of cord blood progenitor-derived endothelial cells (PDECs) to take the place of endothelial cells in vascular tissue engineering. After isolation, culture and characterization of endothelial progenitor cells, the following parameters were explored, with a commercial knitted polyester prosthesis (Polymaille C, Laboratoires Pérouse, France) impregnated with collagen: cell adhesion and proliferation, colonization, cell retention on exposure to flow, and the ability of PDECs to be regulated by arterial shear stress via mRNA levels. PDECs were able to adhere to commercial collagen-coated vascular grafts in serum-free conditions, and were maintained but did not proliferate when seeded at 2.0 x 10(5) cm(-2). Cellularized conduits were analyzed by histology and histochemical staining, demonstrating collagen impregnation and the endothelial characteristics of the colonizing cells. Thirty-six hours after cell seeding the grafts were maintained for 6 h of either static conditions (controls) or application of pulsatile laminar shear stress, which restored the integrity of the monolayer. Finally, quantitative real-time RT-PCR analysis performed at 4 and 8 h from cells lining grafts showed that MMP1 mRNA only was increased at 4h whereas vWF, VE-cadherin and KDR were not significantly modified at 4 and 8 h. Our results show that human cord blood PDECs are capable of forming an efficient lining and to withstand shear stress.

The effect of cyclo-DfKRG peptide immobilization on titanium on the adhesion and differentiation of human osteoprogenitor cells.

This study takes place in the field of development of a bioactive surface of titanium alloys. In this paper, titanium was functionalized with cyclo-DfKRG peptide by coating or grafting using different anchors (thiol or phosphonate) as spacers between the surface and the peptide. Cell adhesion, and differentiation of human osteoprogenitor (HOP) cells arising from human bone marrow were investigated. Our results seem to demonstrate that cyclo-DfKRG peptide coating with a phosphonate anchor and grafting procedure contributes to higher cell adhesion and a strong ALP and Cbfa1 mRNA expression, after 10 days of cell seeding. At the contrary, this peptide coated with a thiol anchor stimulates differentiation of HOP within 3 days of culture.