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

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.

Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes.

Chloride intracellular channel (CLICs) proteins show 60-70% sequence identity to each other, and exclusively localize to the intracellular organelle membranes and cytosol. In support of our recent publication, "Molecular identity of cardiac mitochondrial chloride intracellular channel proteins" (Ponnalagu et al., 2016) [1], it was important to characterize the specificity of different CLIC paralogs/ortholog (CLIC1, CLIC4, CLIC5 and DmCLIC) antibodies used to decipher their localization in cardiac cells. In addition, localization of CLICs in the other organelles such as endoplasmic reticulum (ER) of cardiomyocytes was established. This article also provides data on the different primers used to show the relative abundance of CLIC paralogs in cardiac tissue and the specificity of the various CLIC antibodies used. We demonstrate that the predominant CLICs in the heart, namely CLIC1, CLIC4 and CLIC5, show differential distribution in endoplasmic reticulum. CLIC1 and CLIC4 both show co-localization to the endoplasmic reticulum whereas CLIC5 does not.

Invadolysin acts genetically via the SAGA complex to modulate chromosome structure.

Identification of components essential to chromosome structure and behaviour remains a vibrant area of study. We have previously shown that invadolysin is essential in Drosophila, with roles in cell division and cell migration. Mitotic chromosomes are hypercondensed in length, but display an aberrant fuzzy appearance. We additionally demonstrated that in human cells, invadolysin is localized on the surface of lipid droplets, organelles that store not only triglycerides and sterols but also free histones H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aberrantly structured chromosomes of invadolysin mutants? We have identified a genetic interaction between invadolysin and nonstop, the de-ubiquitinating protease component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-remodelling complex. invadolysin and nonstop mutants exhibit phenotypic similarities in terms of chromosome structure in both diploid and polyploid cells. Furthermore, IX-14(1)/not(1) transheterozygous animals accumulate mono-ubiquitinated histone H2B (ubH2B) and histone H3 tri-methylated at lysine 4 (H3K4me3). Whole mount immunostaining of IX-14(1)/not(1) transheterozygous salivary glands revealed that ubH2B accumulates surprisingly in the cytoplasm, rather than the nucleus. Over-expression of the Bre1 ubiquitin ligase phenocopies the effects of mutating either the invadolysin or nonstop genes. Intriguingly, nonstop and mutants of other SAGA subunits (gcn5, ada2b and sgf11) all suppress an invadolysin-induced rough eye phenotype. We conclude that the abnormal chromosome phenotype of invadolysin mutants is likely the result of disrupting the histone modification cycle, as accumulation of ubH2B and H3K4me3 is observed. We further suggest that the mislocalization of ubH2B to the cytoplasm has additional consequences on downstream components essential for chromosome behaviour. We therefore propose that invadolysin plays a crucial role in chromosome organization via its interaction with the SAGA complex.