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Human Cardiac Progenitor Cells Enhance Exosome Release and Promote Angiogenesis Under Physoxia.

ABSTRACT: Studies on cardiac progenitor cells (CPCs) and their derived exosomes therapeutic potential have demonstrated only modest improvements in cardiac function. Therefore, there is an unmet need to improve the therapeutic efficacy of CPCs and their exosomes to attain clinically relevant improvement in cardiac function. The hypothesis of this project is to assess the therapeutic potential of exosomes derived from human CPCs (hCPCs) cultured under normoxia (21% O2), physoxia (5% O2) and hypoxia (1% O2) conditions. hCPCs were characterized by immunostaining of CPC-specific markers (NKX-2.5, GATA-4, and c-kit). Cell proliferation and cell death assay was not altered under physoxia. A gene expression qPCR array (84 genes) was performed to assess the modulation of hypoxic genes under three different oxygen conditions as mentioned above. Our results demonstrated that very few hypoxia-related genes were modulated under physoxia (5 genes upregulated, 4 genes down regulated). However, several genes were modulated under hypoxia (23 genes upregulated, 9 genes downregulated). Furthermore, nanoparticle tracking analysis of the exosomes isolated from hCPCs under physoxia had a 1.6-fold increase in exosome yield when compared to normoxia and hypoxia conditions. Furthermore, tube formation assay for angiogenesis indicated that exosomes derived from hCPCs cultured under physoxia significantly increased tube formation as compared to no-exosome control, 21% O2, and 1% O2 groups. Overall, our study demonstrated the therapeutic potential of physoxic oxygen microenvironment cultured hCPCs and their derived exosomes for myocardial repair.

SUBMITTER: Dougherty JA

PROVIDER: S-EPMC7068154 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Human Cardiac Progenitor Cells Enhance Exosome Release and Promote Angiogenesis Under Physoxia.

Dougherty Julie A JA Patel Nil N Kumar Naresh N Rao Shubha Gururaja SG Angelos Mark G MG Singh Harpreet H Cai Chuanxi C Khan Mahmood M

Frontiers in cell and developmental biology 20200306

Studies on cardiac progenitor cells (CPCs) and their derived exosomes therapeutic potential have demonstrated only modest improvements in cardiac function. Therefore, there is an unmet need to improve the therapeutic efficacy of CPCs and their exosomes to attain clinically relevant improvement in cardiac function. The hypothesis of this project is to assess the therapeutic potential of exosomes derived from human CPCs (hCPCs) cultured under normoxia (21% O<sub>2</sub>), physoxia (5% O<sub>2</sub ...[more]

PMID: 32211408

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Project description:ObjectivesThe goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the treatment of myocardial infarction.BackgroundRegenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation, and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration.MethodshCPCs isolated from the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence.ResultshCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery.ConclusionsGenetic engineering of hCPCs with Pim-1 enhanced repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of hCPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in pre-clinical settings.

Project description:Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell-cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.

Project description:BACKGROUND:Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. METHODS:A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. RESULTS:In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. CONCLUSION:Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular structures in vitro and in vivo also in the absence of proteases activity, performing a new type of neovascularization: the "amoeboid angiogenesis". uPAR is indispensable for ECs and ECFCs to perform an efficient amoeboid angiogenesis. Therefore, uPAR silencing or the block of its integrin-interaction, together with standard treatment against VEGF, could be a possible solution for angiogenesis inhibition.

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Human Cardiac Progenitor Cells Enhance Exosome Release and Promote Angiogenesis Under Physoxia.

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Human Cardiac Progenitor Cells Enhance Exosome Release and Promote Angiogenesis Under Physoxia.

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