Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca2+) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca2+-ATPase (SERCA) which is involved in the regulation of the intracellular Ca2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes

Generation of bi-allelic MYBPC3 truncating mutant and isogenic control from an iPSC line of a patient with hypertrophic cardiomyopathy

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms

PMEA polymer-coated PVC tubing maintains anti-thrombogenic properties during in vitro whole blood circulation

Poly(2-methoxyethylacrylate) (PMEA) is a new coating material that appears to reduce protein and platelet adsorption. However, the exact performance of PMEA coated circuit remains to be revealed in well-controlled experiments. Therefore, we compared its hemocompatibility with covalent-bound heparin-, and non-coated circuits during 6 hours of in vitro circulation, using donor blood from six volunteers. In our model, simple tubing circuits containing one-way ball valve were placed on the rotary table, which moved alternatively to generate pulsatile recirculation of heparinized human blood inside the tubing. Using this model, we expected fine assessment of the material surface, because we could reduce blood damage by avoiding air and a blood pump. Moreover, the small capacity of circuit allowed us to compare three kinds of circuits using a single unit of donor blood, eliminating effects by possible variations between blood donors. The anti-thrombin capacity of the PMEA-coated circuits was maintained even after six hours blood circulation, whereas surface thrombin generation increased markedly after use in non-coated circuits (P <0.05). Deposition of fibrin onto PMEA circuits was reduced more than 30% compared with heparin and non-coated circuits (P <0.05). However, the increase of plasma Factor XIIa was similar in all circuits. Increase of CD11b expression on circulating leukocytes and of plasma C3a was ameliorated in the heparin- and PMEA-coated circuits (P <0.05). PMEA-coated circuits appear to maintain their anti-thrombogenicity during use, otherwise PMEA-coated and heparin-coated circuits showed a similar character in hemocompatibility. This long-standing anti-thrombogenicity might be attributable to less adsorption of activated blood components onto the surface

Evaluation of MYBPC3 trans-splicing and gene replacement as therapeutic options in human iPSC-derived cardiomyocytes

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ~50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 50 or 30 pre-trans-splicing molecules represented ~1% of total MYBPC3 transcripts in healthy hiPSCCMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ~2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation

Evaluation of MYBPC3 trans-splicing and gene replacement as therapeutic options in human iPSC-derived cardiomyocytes

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation