Search

Background and Aims: COVID-19 is associated with liver injury and elevated interleukin-6 (IL-6). We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19., Methods: Coagulopathy, endotheliopathy, and alanine aminotransferase (ALT) were retrospectively analyzed in a subset (n = 68), followed by a larger cohort (n = 3,780) of patients with COVID-19. Liver histology from 43 patients with COVID-19 was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism., Results: Factor VIII, fibrinogen, D-dimer, von Willebrand factor (vWF) activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in patients with COVID-19 and liver injury (elevated ALT). IL-6 positively correlated with vWF antigen (p = 0.02), factor VIII activity (p = 0.02), and D-dimer (p <0.0001). On liver histology, patients with COVID-19 and elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet (p <0.0001) and vWF (p <0.01) staining, and IL-6 levels positively correlated with vWF staining (p <0.01). IL-6 trans-signaling leads to increased expression of procoagulant (factor VIII, vWF) and proinflammatory factors, increased cell surface vWF (p <0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble glycoprotein 130 (IL-6 trans-signaling inhibitor), the JAK inhibitor ruxolitinib, and STAT1/3 small-interfering RNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling., Conclusion: IL-6 trans-signaling drives the coagulopathy and hepatic endotheliopathy associated with COVID-19 and could be a possible mechanism behind liver injury in these patients., Lay Summary: Patients with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in patients with COVID-19. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies., Methods and Results: We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Krűppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models., Conclusions: Our results demonstrate that strategies to augment MEF2 activity hold potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH., (© 2014 American Heart Association, Inc.)

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.

Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how β-arrestins, intracellular proteins that regulate agonistmediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. We performed both in vitro studies with primary endothelial cells subjected to β-arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of β-arrestin 1 and β-arrestin 2. We found that β-arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of β-arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. β-arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, β-arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR-II), whose disruption similarly led to an impaired endothelial shear response. β-arrestins also regulated Smad transcription factor phosphorylation by BMPR-II. Mice with endothelial specific deletion of β-arrestin 1 and β-arrestin 2 were found to have impaired retinal angiogenesis. In conclusion, we have identified a novel role for endothelial β-arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR-II and contribute to vascular development. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling associated with obliteration of pulmonary arterioles and formation of plexiform lesions composed of hyperproliferative endothelial and vascular smooth-muscle cells. Here we describe a microRNA (miRNA)-dependent association between apelin (APLN) and fibroblast growth factor 2 (FGF2) signaling in pulmonary artery endothelial cells (PAECs). APLN deficiency in these cells led to increased expression of FGF2 and its receptor FGFR1 as a consequence of decreased expression of miR-424 and miR-503, which directly target FGF2 and FGFR1. miR-424 and miR-503 were downregulated in PAH, exerted antiproliferative effects in PAECs and inhibited the capacity of PAEC-conditioned medium to induce the proliferation of pulmonary artery smooth-muscle cells. Reconstitution of miR-424 and miR-503 in vivo ameliorated pulmonary hypertension in experimental models. These studies identify an APLN-dependent miRNA-FGF signaling axis needed for the maintenance of pulmonary vascular homeostasis. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "The ERG-APLNR Axis Controls Pulmonary Venule Endothelial Proliferation in Pulmonary Veno-Occlusive Disease" by C. Lathen and colleagues.

Background--Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. Methods and Results--We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Kruppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models. Conclusions--Ourresults demonstrate thatstrategies to augmentMEF2activityholdpotentialtherapeuticvalueinPAH.Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH. [ABSTRACT FROM AUTHOR]

There is emerging evidence that the aberrant pulmonary vascular remodeling that occurs in pulmonary arterial hypertension (PAH) is at least in part driven by the transformation of endothelial cells to mesenchymal cells (EndoMT), but the mechanism driving this pathobiology remains to be fully elucidated. Bone morphogenetic protein (BMP) signaling has been implicated to be involved in EndoMT in different vascular contexts, but the exact mechanism in the pulmonary vasculature remains to be defined. We describe the role of BMP receptor type 1A (BMPR1A or ALK3) as a key factor for maintenance of endothelial fate and suppression of EndoMT in the pulmonary vasculature. We found that inducible endothelial specific deletion of Bmpr1a in mice (Bmpr1aiECKO) resulted in spontaneous EndoMT, with significant increase in smooth muscle actin (SMA) positive cells, associated with extensive pulmonary vascular remodeling and fibrosis. Bmpr1aiECKO mice developed spontaneous pulmonary hypertension (PH) that was mediated by augmented TGF-β signaling driven by increased TGFBR2 expression, resulting in aberrant SMAD2/3 activation. Increased TGFBR2 expression in BMPR1A deficient state was secondary to increased activity of the transcription factor TCF3, whose negative inhibition by ID2 is abrogated in BMPR1A deficient state. EndoMT and PH secondary to loss of endothelial BMPR1A was effectively rescued by concurrent knockdown of TGFBR2. Overall, these studies define a mechanism of EndoMT driven by loss of endothelial BMPR1A, and demonstrate the efficacy of inhibiting EndoMT as a potential novel therapeutic strategy in PAH and other EndoMT-related vascular disorders. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary arterial hypertension (PAH) is a progressive and ultimately life-limiting disease that is associated with dysregulated endothelial function. The epigenetic factor p300 is a transcriptional coactivator and histone acetyltransferase known to acetylate transcription factors in endothelial cells that are key to vascular health. Here we set out to characterize the role of p300 in the pulmonary vasculature. Methods: Mice with conditional, endothelial specific deletion of p300 (p300iEC-/-) were generated and exposed to 2 weeks of hypoxia. Right heart catheterizations were performed and lung sections were collected for immunostaining. RNA-seq of CD31+ cells isolated from p300iEC-/- mouse lung was performed on an Ilumina HiSeq 2500 system and the data analyzed using Cufflinks. Pathway analysis was performed using Qiagen's Ingenuity Pathway Analysis. Results and conclusions: p300iEC-/- mice exposed to hypoxia developed significantly worse PH (n=15, RVSP 41.2 +/- 2.3 mmHg) compared to control mice (n=15, 32.8 mmHg +/- 1.1 mmHg, p=0.0027) (A) as well as increased right ventricular (RV) mass ratio (n=6, 0.35 +/- 0.03, compared to n=10, 0.28 +/- 0.01, p=0.0164). Immunostaining of lung sections demonstrated significant increase in muscularization of the distal pulmonary vessels (B) associated with decreased staining of eNOS, which has been implicated to be protective against PH. RNA-seq of endothelial cells harvested from p300iEC-/- mice was performed, and 75 significantly differentially expressed genes identified. Ingenuity pathway analysis of these genes found multiple canonical pathways in inflammatory function, such as natural killer cell signaling, phagocytosis, and leukocyte extravasation. Overall, our results suggest p300 is important in maintaining endothelial health in the pulmonary vasculature, and loss of p300 leads to pathogenesis through endothelial and immunologic dysregulation. [ABSTRACT FROM AUTHOR]

The considerable atheroprotection afforded by statins (HMG-CoA reductase inhibitors) is thought to be above and beyond that expected from their cholesterol lowering capabilities, which is known as statin pleiotropy. Endothelial microRNAs are emerging as key mediators of cellular functions that can dictate vascular homeostasis. We hypothesized that statins induce differential expression of microRNAs in endothelial cells, which may define novel signaling pathways by which statins exert their pleiotropic effects. We carried out a microRNA expression array using HUVECs subjected to treatment with simvastatin. Of endothelial microRNAs that are differentially regulated in response to statin, we found that miR-335 was the most significantly downregulated, in a KLF2 dependent manner. MiR-335 overexpression was found to increase expression of inflammatory markers in the endothelial cells and augment monocyte adhesion. These effects were mediated by miR-335 induced NF-κB activation, as demonstrated by significantly increased NF-κB reporter activity in cells overexpressing miR-335. To identify potential mechanism by which miR-335 regulates NF-κB activation, we conducted a gene expression profile analyses and cross-referenced the down-regulated genes in our expression profiling analyses with those genes that are predicted to be targeted by miR-335-5p. We identified CHFR (checkpoint with forkhead and ring finger domain) as a direct 3' UTR target of miR-335, which in turn negatively regulates NF-κB activity. Similar to the effect of miR-335 overexpression, CHFR silencing led to significant increase expression of inflammatory markers in the endothelial cells, which was mediated by CHFR silencing induced NF-κB activation. Finally, in vivo analysis using miR335 deficient mice found them to be metabolically protected, and on the Apoe deficient background found that these mice were protected against high fat diet induced atherosclerosis (Figure 1). Overall, we identify miR-335 as a critical microRNA downregulated by statins that potentiates a number of pro-inflammatory signaling sequelae and may define an important node in statin pleiotropy. [ABSTRACT FROM AUTHOR]