Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia.
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ABSTRACT:
SUBMITTER: Beegle JR
PROVIDER: S-EPMC5003097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutant MetRS in MSCs allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia
2021-09-07 | PXD026954 | Pride
Project description:AimsNovel cell-based therapeutic angiogenic treatments for patients with critical limb ischemia may afford limb salvage. Mesenchymal stem cells (MSCs) do not overexpress E-selectin; however, we have previously demonstrated the cell-adhesion molecule's vital role in angiogenesis and wound healing. Thus, we created a viral vector to overexpress E-selectin on MSCs to increase their therapeutic profile.Methods and resultsFemoral artery ligation induced hind limb ischemia in mice and intramuscular injections were administered of vehicle or syngeneic donor MSCs, transduced ex vivo with an adeno-associated viral vector to express either GFP+ (MSCGFP) or E-selectin-GFP+ (MSCE-selectin-GFP). Laser Doppler Imaging demonstrated significantly restored reperfusion in MSCE-selectin-GFP-treated mice vs. controls. After 3 weeks, the ischemic limbs in mice treated with MSCE-selectin-GFP had increased footpad blood vessel density, hematoxylin and eosin stain (H&E) ischemic calf muscle sections revealed mitigated muscular atrophy with restored muscle fiber size, and mice were able to run further before exhaustion. PCR array-based gene profiling analysis identified nine upregulated pro-angiogenic/pro-repair genes and downregulated Tumor necrosis factor (TNF) gene in MSCE-selectin-GFP-treated limb tissues, indicating that the therapeutic effect is likely achieved via upregulation of pro-angiogenic cytokines and downregulation of inflammation.ConclusionThis innovative cell therapy confers increased limb reperfusion, neovascularization, improved functional recovery, decreased muscle atrophy, and thus offers a potential therapeutic method for future clinical studies.
| S-EPMC8841646 | biostudies-literature
Project description:The therapeutic potential of mesenchymal stem cell (MSC) transplantation for the treatment of ischemic conditions such as coronary artery disease, peripheral arterial disease, and stroke has been explored in animal models and early-phase clinical trials. A substantial database documents the safety profile of MSC administration to humans in a large number of disease states. The mechanism of the therapeutic effect of MSC transplantation in ischemic disease has been postulated to be due to paracrine, immunomodulatory, and differentiation effects. This review provides an overview of the potential role of MSC-based therapy for critical limb ischemia (CLI), the comparison of MSC cellular therapy with angiogenesis gene therapy in CLI, and the proposed mechanism of action of MSC therapy. Preclinical efficacy data in animal models of hindlimb ischemia, current early-phase human trial data, and considerations for future MSC-based therapy in CLI will also be discussed.
| S-EPMC3580466 | biostudies-literature
Project description:Critical limb ischemia (CLI), the terminal stage of peripheral arterial disease (PAD), is characterized by an extremely high risk of amputation and vascular issues, resulting in severe morbidity and mortality. In patients with severe limb ischemia with no alternative therapy options, such as endovascular angioplasty or bypass surgery, therapeutic angiogenesis utilizing cell-based therapies is vital for increasing blood flow to ischemic regions. Mesenchymal stem cells (MSCs) are currently considered one of the most encouraging cells as a regenerative alternative for the surgical treatment of CLI, including restoring tissue function and repairing ischemic tissue via immunomodulation and angiogenesis. The regenerative treatments for limb ischemia based on MSC therapy are still considered experimental. Despite recent advances in preclinical and clinical research studies, it is not recommended for regular clinical use. In this study, we review the immunomodulatory features of MSC besides the current understanding of different sources of MSC in the angiogenic treatment of CLI subjects and their potential applications as therapeutic agents. Specifically, this paper concentrates on the most current clinical application issues, and several recommendations are provided to improve the efficacy of cell therapy for CLI patients.
| S-EPMC9449296 | biostudies-literature
Project description:Numerous signaling molecules are altered following nerve injury, serving as a blueprint for drug delivery approaches that promote nerve repair. However, challenges with achieving the appropriate temporal duration of recombinant protein delivery have limited the therapeutic success of this approach. Genetic engineering of mesenchymal stem cells (MSCs) to enhance the secretion of proangiogenic molecules such as vascular endothelial growth factor (VEGF) may provide an alternative. We hypothesized that the administration of VEGF-expressing human MSCs would stimulate neurite outgrowth and proliferation of cell-types involved in neural repair. When cultured with dorsal root ganglion (DRG) explants in vitro, control and VEGF-expressing MSCs (VEGF-MSCs) increased neurite extension and proliferation of Schwann cells (SCs) and endothelial cells, while VEGF-MSCs stimulated significantly greater proliferation of endothelial cells. When embedded within a 3D fibrin matrix, VEGF-MSCs maintained overexpression and expressed detectable levels over 21 days. After transplantation into a murine sciatic nerve injury model, VEGF-MSCs maintained high VEGF levels for 2 weeks. This study provides new insight into the role of VEGF on peripheral nerve injury and the viability of transplanted genetically engineered MSCs. The study aims to provide a framework for future studies with the ultimate goal of developing an improved therapy for nerve repair.
| S-EPMC4830493 | biostudies-literature
Project description:Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to poor cell quality (ie, inability to generate a sufficient number of angiogenic MSCs) or to the inadequate retention and viability of MSCs after delivery, or both. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine the effect of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared with MSCs from healthy donors.MSCs were obtained from the tibia of four CLI patients (ISC) and four ISC patients with diabetes mellitus (ISC+DM) undergoing major amputation. Healthy MSCs were aspirated from the iliac crest of four young and healthy donors. MSCs were isolated and expanded in culture with PL or FBS. MSCs from passage 3 to 6 were used for phenotypic marker expression and for adipogenic and osteogenic differentiation and were tested for their in vitro angiogenic activity on human microdermal endothelial cells. In parallel MSCs were cultured to passage 11 for population-doubling calculations.MSCs from ISC and ISC+DM patients and from healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared with FBS-stimulated MSCs in all groups. Biologically active amounts of angiogens were identified in the secretome of all MSCs without consistent trends among groups. PL expansion did not adversely affect the angiogenic activity of MSCs compared with FBS. The ISC and ISC+DM MSCs demonstrated angiogenic effects on endothelial cells similar to those of healthy and ISC MSCs.PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity compared with their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of ISC and ISC+DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.
| S-EPMC4996777 | biostudies-literature
Project description:ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and resultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.
| S-EPMC8278824 | biostudies-literature
Project description:The aim of this study was to investigate the in vitro osteogenic capacity of bone morphogenetic protein 7 (BMP-7) overexpressing adipose-derived (Ad-) mesenchymal stem cells (MSCs) sheets (BMP-7-CS). In addition, BMP-7-CS were transplanted into critical-sized bone defects and osteogenesis was assessed. BMP-7 gene expressing lentivirus particles were transduced into Ad-MSCs. BMP-7, at the mRNA and protein level, was up-regulated in BMP-7-MSCs compared to expression in Ad-MSCs. Osteogenic and vascular-related gene expressions were up-regulated in BMP-7-CS compared to Ad-MSCs and Ad-MSC sheets. In a segmental bone-defect model, newly formed bone and neovascularization were enhanced with BMP-7-CS, or with a combination of BMP-7-CS and demineralized bone matrix (DBM), compared to those in control groups. These results demonstrate that lentiviral-mediated gene transfer of BMP-7 into Ad-MSCs allows for stable BMP-7 production. BMP-7-CS displayed higher osteogenic capacity than Ad-MSCs and Ad-MSC sheets. In addition, BMP-7-CS combined with demineralized bone matrix (DBM) stimulated new bone and blood vessel formation in a canine critical-sized bone defect. The BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. Thus, BMP-7-CS and DBM have therapeutic potential for the treatment of critical-sized bone defects and could be used to further enhance clinical outcomes during bone-defect treatment.
| S-EPMC6073206 | biostudies-literature
Project description:Critical limb ischemia (CLI), an end-stage manifestation of peripheral artery disease (PAD), still lacks effective therapeutic strategies. Recently, dental pulp-derived mesenchymal stem cells (DP-MSCs) have been attracting more and more attentions in therapeutic applications due to their high proliferation ability, powerful osteogenic differentiation potential, and effective anti-inflammatory effects. In this study, we compared the therapeutic effects of MSCs derived from different sources in a femoral artery-ligated preclinical ischemic model. We found that treatments with MSCs, including bone marrow- (BM-), adipose- (AD-), dental pulp- (DP-), and umbilical cord- (UC-) derived MSCs, improved limb functions, reduced inflammatory responses, increased angiogenesis, and promoted regeneration of muscle fiber. Among them, DP-MSCs and BM-MSCs produced much more impressive effects in restoring limb functions and promoting angiogenesis. The flow velocity restored to nearly 20% of the normal level at 3 weeks after treatments with DP-MSCs and BM-MSCs, and obvious capillary proliferation and collateral development could be observed. Although neovascularization was induced in the ischemic limb after ligation, MSCs, especially DP-MSCs, significantly enhanced the angiogenesis. In vitro experiments showed that serum deprivation improved the expression of angiogenic factors, growth factors, and chemokines in DP-MSCs and UC-MSCs, but not in BM-MSCs and AD-MSCs. However, DP-MSCs produced stronger therapeutic responses than UC-MSCs, which might be due to the higher expression of hepatocyte growth factor (HGF) and hypoxia-inducible factor-1 α (HIF-1α). We speculated that DP-MSCs might stimulate angiogenesis and promote tissue repair via expressing and secreting angiogenic factors, growth factors, and chemokines, especially HGF and HIF-1α. In conclusion, DP-MSCs might be a promising approach for treating CLI.
| S-EPMC8427677 | biostudies-literature
Project description:BackgroundCell therapy has been proposed for patients with critical limb ischemia (CLI). Autologous bone marrow derived cells (BMCs) have been mostly used, mesenchymal stem cells (MSCs) being an alternative. The aim of this study was to characterize two types of MSCs and evaluate their efficacy.MethodsMSCs were obtained from CLI-patients BMCs. Stimulated- (S-) MSCs were cultured in endothelial growth medium. Cells were characterized by the expression of cell surface markers, the relative expression of 6 genes, the secretion of 10 cytokines and the ability to form vessel-like structures. The cell proangiogenic properties was analysed in vivo, in a hindlimb ischemia model. Perfusion of lower limbs and functional tests were assessed for 28 days after cell infusion. Muscle histological analysis (neoangiogenesis, arteriogenesis and muscle repair) was performed.ResultsS-MSCs can be obtained from CLI-patients BMCs. They do not express endothelial specific markers but can be distinguished from MSCs by their secretome. S-MSCs have the ability to form tube-like structures and, in vivo, to induce blood flow recovery. No amputation was observed in S-MSCs treated mice. Functional tests showed improvement in treated groups with a superiority of MSCs and S-MSCs. In muscles, CD31+ and αSMA+ labelling were the highest in S-MSCs treated mice. S-MSCs induced the highest muscle repair.ConclusionsS-MSCs exert angiogenic potential probably mediated by a paracrine mechanism. Their administration is associated with flow recovery, limb salvage and muscle repair. The secretome from S-MSCs or secretome-derived products may have a strong potential in vessel regeneration and muscle repair. Trial registration NCT00533104.
| S-EPMC6688282 | biostudies-literature