High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension
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ABSTRACT: Computational modeling indicated that a pathological level of high shear stress (HSS, 100 dyn/cm2) is generated in distal pulmonary arteries (PA) (100-500 um) in a congenital heart defect with increased PA blood flow causing PA hypertension (PAH), and in idiopathic PAH with occlusive vascular remodeling. The response of human PA endothelial cells (EC) to HSS compared to physiologic laminar shear stress (LSS, 15 dyn/cm2), was therefore assessed. Endothelial-mesenchymal transition (EndMT), a feature of PAH not previously attributed to HSS was observed. HSS did not alter induction of the transcription Krüppel-like factors (KLF) 2/4, but H3K27ac peaks containing motifs for an ETS-family transcription factor (ERG) were reduced, as was the interaction between ERG and KLF2/4 and ERG expression. In PAEC under LSS, reducing ERG by siRNA caused EndMT related to decreased bone morphogenetic protein receptor 2 (BMPR2), cadherin 5 (CDH5) and platelet and endothelial cell adhesion molecule 1 (PECAM1), and increased Snail/Slug (SNAI1/2) and smooth muscle alpha α-2 actin (ACTA2). In PAEC under HSS, transfection of ERG prevented EndMT. We induced HSS in mice by an aorto-caval shunt that causes a progressive increase in PAH over eight weeks and used an adeno-associated viral vector (AAV2-ESGHGYF) to replenish ERG selectively in PAEC. Elevated PA pressure and resistance, EndMT and vascular remodeling assessed by muscularization of peripheral arteries were markedly reduced by ERG delivery in the aorto-caval shunt mice. Thus, agents that restore ERG in the pulmonary vasculature will be of therapeutic benefit in overcoming the adverse effect of HSS on progressive PAH.
Project description:PAH was induced by 60mg/kg MCT and an aorto-caval shunt. At different timepoints of PAH progression (day 14, 21 and 28 after MCT-injection), the left lung with PAH was hemodynamically unloading by unilateral orthotopic transplatation into a syngeneic, healthy recipient. All day 14 and 7/10 day 21 transplanted lungs showed reversal of PAH after LTx. All day 28 and 3/10 day 21 transplanted lungs showed PAH progression after LTx. Lung tissue of Reversible and Irreversible PAH and normal controls, acquired at LTx, was compared using RNA-seq.
Project description:Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common mutant gene in PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxic exposure (reoxygenation), as do mice with EC deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC revealed reduced Foxf1, a transcription factor with relative selectivity for lung EC. Reducing FOXF1 in control PAEC induced DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repaired DNA damage and restored angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repaired DNA damage, induced angiogenesis and reversed pulmonary hypertension.
Project description:The goal of this study was to identify gene signatures in HUVECs exposed to atheroprone low laminar shear stress (LSS) or atheroprotective high laminar shear stress (HSS). The obtained data was used to verify that HSS and LSS application induces gene expression patterns similar to more complex pulsatile and oscillatory flow, respectively.
Project description:Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin a5b1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin a5b1 or depletion of the a5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5β1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5β1 integrin inhibition as a promising therapy for pulmonary hypertension
Project description:Human endogenous retroviral (HERV) proteins are induced by exogenous viruses or other factors that derepress HERV transcription and translation. Previously we showed that HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) proteins are increased in monocytes and macrophages from patients with pulmonary arterial hypertension (PAH). Recombinant HERV-K dUTPase upregulates IL6 in pulmonary arterial endothelial cells (PAECs) and induces pulmonary hypertension in rats. However, it was not known how HERV-K dUTPase released from monocytes engages PAECs to upregulate IL6 or induces other PAH features of PAEC dysfunction. Here we report that HERV-K dUTPase recruits TLR4-myeloid differentiation primary response-88 to increase IL6 and SNAIL, the endothelial-mesenchymal transition (EndMT) transcription factor; HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) upregulates VCAM1. p38 and NF-kB are required to increase expression of all three genes, but in addition, pJNK-pSMAD3 is necessary for SNAIL upregulation, STAT1 for IL6, and pERK1/2-activating transcription factor-2 for VCAM1. Packaging of HERV-K dUTPase in monocyte-derived extracellular vesicles (EVs) induces SNAIL and subsequent EndMT in PAECs, IL6 and VCAM1. Mice infused with EVs from monocytes transfected with HERV-K dUTPase develop pulmonary hypertension. Thus, retroviral proteins delivered in EVs can overtake PAEC signaling and transcriptional machinery to induce dysfunction associated with PAH.
Project description:Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasi-neoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences. To assess whether there is larger scale genomic instability, we performed genome-wide microarray copy number analysis on pulmonary artery endothelial (PAEC) and smooth muscle cells isolated from the lungs of PAH patients. Mosaic chromosomal abnormalities were detected in five of nine PAEC cultures from PAH lungs and zero of four controls. Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and a somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female cases with mosaic loss of the X-chromosome, methylation analysis showed that the active X was deleted. Remarkably, one also showed completely skewed X-inactivation in the non-deleted cells, suggesting the PAEC population was clonal prior to the acquisition of the chromosome abnormality. Our data indicate a high frequency of genetically abnormal sub-clones within the lung vessels of patients with PAH and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH. Cross-sectional study of genomic copy number in cells cultured from the lungs of PAH patients.
Project description:Endothelial-to-mesenchymal transition (EndMT) in which endothelial cells lose their characteristics and acquire mesenchymal property has recently been recognized as a driver of disease progression in wide range of pathologies. However, the regulatory mechanism of EndMT has not been fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induced EndMT. Hence, we analyzed functions of ERG and FLI1 using gene expression microarray and ChIP-seq to elucidate the regulatory mechanism of EndMT.
Project description:Endothelial-to-mesenchymal transition (EndMT) in which endothelial cells lose their characteristics and acquire mesenchymal property has recently been recognized as a driver of disease progression in wide range of pathologies. However, the regulatory mechanism of EndMT has not been fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induced EndMT. Hence, we analyzed functions of ERG and FLI1 using gene expression microarray and ChIP-seq to elucidate the regulatory mechanism of EndMT.
Project description:Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasi-neoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences. To assess whether there is larger scale genomic instability, we performed genome-wide microarray copy number analysis on pulmonary artery endothelial (PAEC) and smooth muscle cells isolated from the lungs of PAH patients. Mosaic chromosomal abnormalities were detected in five of nine PAEC cultures from PAH lungs and zero of four controls. Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and a somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female cases with mosaic loss of the X-chromosome, methylation analysis showed that the active X was deleted. Remarkably, one also showed completely skewed X-inactivation in the non-deleted cells, suggesting the PAEC population was clonal prior to the acquisition of the chromosome abnormality. Our data indicate a high frequency of genetically abnormal sub-clones within the lung vessels of patients with PAH and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH. Cross-sectional study of genomic copy number in cells cultured from the lungs of PAH patients. This study used three Affy SNP chip types: 250kNSP, 250kSTY, and 6.0
Project description:Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasi-neoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences. To assess whether there is larger scale genomic instability, we performed genome-wide microarray copy number analysis on pulmonary artery endothelial (PAEC) and smooth muscle cells isolated from the lungs of PAH patients. Mosaic chromosomal abnormalities were detected in five of nine PAEC cultures from PAH lungs and zero of four controls. Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and a somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female cases with mosaic loss of the X-chromosome, methylation analysis showed that the active X was deleted. Remarkably, one also showed completely skewed X-inactivation in the non-deleted cells, suggesting the PAEC population was clonal prior to the acquisition of the chromosome abnormality. Our data indicate a high frequency of genetically abnormal sub-clones within the lung vessels of patients with PAH and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.