Adipose mesenchymal stem cell-derived soluble factors, produced under hypoxic condition, efficiently support in vivo angiogenesis

Abstract

Tissue regeneration or healing both require efficient vascularization within a tissue-damaged area. Based on this concept, a remarkable number of strategies, aimed at developing new tools to support re-vascularization of damaged tissue have emerged. Among the strategies proposed, the use of pro-angiogenic soluble factors, as a cell-free tool, appears as a promising approach, able to overcome the issues concerning the direct use of cells for regenerative medicine therapy. Here, we compared the effectiveness of adipose mesenchymal stem cells (ASCs), use as cell suspension, ASC protein extract or ASC-conditioned-medium (i.e., soluble factors), combined with collagenic scaffold, in supporting in vivo angiogenesis. We also tested the capability of hypoxia in increasing the efficiency of ASC to promote angiogenesis, via soluble factors, both in vivo and in vitro. In vivo studies were performed using the Integra® Flowable Wound Matrix, and the Ultimatrix in sponge assay. Flow cytometry was used to characterize the scaffold- and sponge-infiltrating cells. Real-time PCR was used to evaluate the expression of pro-angiogenic factors by stimulating Human Umbilical-Vein Endothelial Cells with ASC-conditioned media, obtained in hypoxic and normoxic conditions. We found that, in vivo, ACS-conditioned media can support angiogenesis similar to ASCs and ASC protein extract. Also, we observed that hypoxia increases the pro-angiogenic activities of ASC-conditioned media, compared to normoxia, by generating a secretome enriched in pro-angiogenic soluble factors, with bFGF, Adiponectine, ENA78, GRO, GRO-a, and ICAM1-3, as most regulated factors. Finally, ASC-conditioned media, produced in hypoxic condition, induce the expression of pro-angiogenic molecules in HUVECs. Our results provide evidence that ASC-conditioned-medium can be proposed as a cell-free preparation able to support angiogenesis, thus providing a relevant tool to overcome the issues and restrictions associated with the use of cells.

Human Dental Pulp Mesenchymal Stem Cell-Derived Soluble Factors Combined with a Nanostructured Scaffold Support the Generation of a Vascular Network In Vivo

“…Both histological and SEM analysis show that FWM and DPSCs and FWM and DPSC-CM are infiltrated by erythrocytes, platelets, and leukocytes. These results are similar to those from our previous results obtained on ASCs and their CM [4,16,34].…”

“…We found DPSC-secretome, here named conditioned medium (CM), was efficient in inducing the generation of mature blood vessels and in supporting the infiltration of proangiogenic M2 macrophages, as well as the DPSC cellular preparation, thus, demonstrating that the system supports angiogenesis similar to what is already observed with adipose stem cells (ASCs) [16,34].…”

“…For all these reasons, researchers focused their attention on the development of devices consisting of bio-scaffolds, in combination cellular products, such as MSCs or, even better, their cellular derivatives such as the conditioned medium [1][2][3][4][5]8,10,[15][16][17]34,35].…”

“…It was established that the paracrine pathway serves as the primary mechanism to facilitate vessel sprouting, and the use of MSCs, as direct cell source, is not mandatory and may potentially introduce additional unpredictable occurrences. This scenario provides the rationale to employ cell-free derivatives, such as cell-conditioned media (here referred to also as secretome) composed of growth factors, soluble factors, and cytokines secreted by cells with pro-angiogenic features [15][16][17][18]. These factors play a pivotal role in controlling the proliferation, adhesion, and differentiation of stem and progenitor cells, as well as contributing to the establishment of fully functional vascular networks, essential during tissue regeneration [15][16][17][18].…”

“…As mentioned, an efficient vascularization is necessary to support tissue regeneration of the injured area; for this reason, several strategies were developed, involving the use of MSC-secretome, extracellular matrix component, and biochemical cues [4,[7][8][9][10][15][16][17][18]33,34]. MSCs represent the ideal source to obtain the cell-secretome, since they can be easily isolated and maintained in starvation up to 72 h [17,35].…”