A second study used allogeneic swine APCs (sAPCs). CMR imaging captured at 5?d (7) or 45?d post\MI (8). Format: Windows Media Video file (WMV). JAH3-7-e006727-s001.pdf (459K) GUID:?28FAA70C-EDD2-4F8C-8391-B94E037124CC Abstract Background Transplantation of adventitial pericytes (APCs) promotes cardiac repair in murine models of myocardial infarction. The aim of present study was to confirm the benefit of APC therapy in a large animal model. Methods and Results We performed a blind, randomized, AB05831 placebo\controlled APC therapy trial in a swine model of reperfused myocardial infarction. A first study used human APCs (hAPCs) from patients undergoing coronary artery bypass graft surgery. A second study used allogeneic swine APCs (sAPCs). Main AB05831 end points were (1) ejection portion as assessed by cardiac magnetic resonance imaging and (2) myocardial vascularization and fibrosis as determined by immunohistochemistry. Transplantation of hAPCs reduced fibrosis but failed to improve the other efficacy end points. Incompatibility of the xenogeneic model was suggested by the occurrence of a cytotoxic response following in?vitro challenge of hAPCs with swine spleen lymphocytes and the failure to retrieve hAPCs in transplanted hearts. We next considered sAPCs as an alternative. Circulation cytometry, immunocytochemistry, and functional/cytotoxic assays show that sAPCs are a surrogate of hAPCs. Transplantation of allogeneic sAPCs benefited capillary density and fibrosis but did not improve cardiac AB05831 magnetic resonance imaging indices of contractility. Transplanted cells were detected in the border zone. Conclusions Immunologic barriers limit the applicability of a xenogeneic swine model to assess hAPC efficacy. On the other hand, we newly show that transplantation of allogeneic sAPCs is usually feasible, safe, and immunologically acceptable. The approach induces proangiogenic and antifibrotic benefits, though these effects were not enough to result in functional improvements. probes used in the molecular biology studies are shown in Table?S3. Differentiation and clonogenic assays Adipogenic and osteogenic differentiation studies were conducted as previously explained.9 In addition, single\cell cloning was performed on 2 sAPC lines at P3, using a motorized device connected to the flow cytometric sorter (Cyclone, Beckman Coulter). Sorted cells were placed into each well of a 96\well culture plate (Greiner Bio\one, UK) and cultured up to 4?weeks in endothelial cell growth medium\2 for quantification of colonies generated from a single cell. Analysis of vascular endothelial growth factor A production The levels of vascular endothelial growth factor A (VEGF\A) protein were decided in CM by an anti\human ELISA kit (R&D System, cat n#: DY293B). To this aim, sAPCs (N=3) were cultured in a T25 flask and exposed to normoxia or Mouse monoclonal to TLR2 hypoxia for 48?hours in 2.5\mL serum\free, endothelial basal medium 2. In addition, a Western blot analysis was performed to detect the same protein in concentrated CM and unconditioned media (endothelial cell growth medium\2). Network formation The capacity of forming networks on Matrigel was assessed using sAPCs or swine pulmonary artery endothelial cells (sPAECs) alone or both in coculture (N=3 biological replicates run in triplicate). In addition, the network formation capacity of sPAECs was assessed following activation with sAPC CM or unconditioned media (endothelial cell growth medium\2). Immunogenic Activity of APCs Studies were carried out to compare the capacity of xenogeneic hAPCs and allogeneic sAPCs (N=3 biological replicates) to induce immune responses upon challenge with swine spleen T lymphocytes. In Vivo Transplantation of APCs Study design Experiments were performed in a total of 42 female Large\White swine. A feasibility/efficacy study was conducted in 32 swine according to the protocol summarized in Physique?1A. In brief, reperfused MI was induced at day 0 (vide infra). A cardiac magnetic resonance (CMR) scan was performed 5?days after MI induction, immediately before randomization to intramyocardial injection of vehicle, hAPCs, or sAPCs. A follow\up CMR scan was performed at 45?days. Immediately after the last CMR scan, animals were euthanized and myocardial tissue samples from your infarct, peri\infarct, and remote areas were collected for histology, immunohistochemistry, and molecular biology studies. Open in a separate window Physique 1 Study design. A, In the efficacy study, swine were subjected to closed\chest 50\minute balloon occlusion of the mid\LAD artery to induce acute MI. At day 5 post\MI, they underwent a comprehensive basal CMR study. Animals that did not show a transmural infarction (at least 50% of the wall thickness infarcted) were excluded. Immediately after day 5 CMR, animals were randomized to receive intramyocardial vehicle or APC injection via minithoracotomy. CMR was repeated at day 45 post\MI and hearts were harvested for histology and other tests AB05831 explained in the Materials and Methods section. B, A similar protocol was used to assess cell engraftment with hearts being collected 5?days after vehicle.
A second study used allogeneic swine APCs (sAPCs)