Epsin15 Homology Domains: Role in the Pathogenesis of Pulmonary Arterial Hypertension.

Dan Predescu,Shanshan Qin,Rabia Bhalli,Monal Patel,Cristina Bardita,Sanda Predescu

doi:10.3389/fphys.2018.01393

Abstract

Intersectin-1s (ITSN) deficiency and expression of a biologically active ITSN fragment, result of granzyme B cleavage under inflammatory conditions associated with pulmonary arterial hypertension (PAH), are characteristics of lung tissue of human and animal models of PAH. Recently, we have shown that this ITSN fragment comprising two Epsin15 homology domains (EHITSN) triggers endothelial cell (EC) proliferation and the plexiform arteriopathy in PAH. Limited evidence also indicates that the EH domains of endocytic proteins such as ITSN, upregulate compensatory endocytic pathways in cells with impaired vesicular trafficking. Thus, we sought to investigate whether the EHITSN may be involved in this compensatory mechanism for improving the EC endocytic dysfunction induced by ITSN deficiency and possibly contribute to PAH pathogenesis. We used stably-transfected human pulmonary artery ECs expressing the Myc-EHITSN (ECEH-ITSN) and ITSN knockout heterozygous mice (K0ITSN+/-) transduced with the Myc-EHITSN, in conjunction with functional assays: the biotin assay for caveolae internalization and 8 nm gold (Au)- and dinitrophenylated (DNP)-albumin perfusion of murine lung microvasculature. Pulmonary artery ECs of PAH patients (ECPAH), ITSN knockdown ECs (ECKD-ITSN), the monocrotaline (MCT)-induced mouse and rat models of PAH, as well as untreated animals, served as controls. ELISA via streptavidin-HRP or anti-DNP antibody (Ab), applied on ECs and lung lysates indicated greater than 30% increase in biotin internalization in ECEH-ITSN compared to ECCtrl. Despite their endocytic deficiency, ECPAH internalized biotin similar to ECCtrl which is twofold higher compared to ECKD-ITSN. Moreover, the lung microvascular bed of Myc-EHITSN-transduced mice and MCT-treated animals showed greater than twofold increase in DNP-BSA transendothelial transport, all compared to untreated controls. Electron microscopy (EM) revealed the increased occurrence of non-conventional endocytic/transcytotic structures (i.e., caveolae clusters, tubulo-vesicular and enlarged endocytic structures, membranous rings), usually underrepresented. Most of these structures were labeled by Au-BSA, consistent with their involvement in the transendothelial transport. Furthermore, ITSN deficiency and EHITSN expression alter the subcellular localization of the EH-binding protein 1 (EHBP1) and cortical actin organization, altogether supporting the increase occurrence/trafficking of the alternative endocytic structures. Thus, the EHITSN by shifting the physiological vesicular (caveolae) transport toward the alternative endocytic pathways is a significant contributor to the dysfunctional molecular phenotype of ECPAH.

Highlights

Patho-physiologically, pulmonary arterial hypertension (PAH) is defined by mean pulmonary arterial pressure exceeding the upper limits of typical values, i.e., ≥ 25 mmHg at rest, as result of extensive pulmonary vascular dysfunction (Stenmark et al, 2009; Sehgal and Lee, 2011)
Two significant differences were noted in the pattern of biotin/neutrAvidin Alexa Fluor 594 labeling among the experimental conditions used: (i) the fine punctate labeling, indicative of biotin association with endothelial vesicular carriers, was less pronounced in ECKD−ITSN and ECPAH due to the reduced caveolae number, and ii) the frequency of the large fluorescent puncta showed an increase in both ECEH−ITSN and ECPAH, compared to ECCtrl
Our present studies extend these observations as we found that prolonged EHITSN expression cooperates with ITSN deficiency in ECPAH and experimental animal models of PAH in decreasing caveolae number, impairing their transport function while boosting the upregulation of alternative transport pathways

Summary

Introduction

Patho-physiologically, pulmonary arterial hypertension (PAH) is defined by mean pulmonary arterial pressure exceeding the upper limits of typical values, i.e., ≥ 25 mmHg at rest, as result of extensive pulmonary vascular dysfunction (Stenmark et al, 2009; Sehgal and Lee, 2011). PAH affects all segments of the pulmonary vascular bed (Chazova et al, 1995). Concentric and eccentric intimal thickening, medial hypertrophy, lumen obliteration and recanalization affect the pulmonary arteries. Modest venous hypertrophy due to increase in the intimal and adventitial thickness and capillary bed rarefaction are among the structural changes of the pulmonary vasculature in PAH, as well (Chazova et al, 1995). The plexiform lesions, hallmark of established PAH, are found at branching points in the small pulmonary arterioles; they are glomeruloid-like vascular structures, lumen-obliterating and composed predominantly of actively dividing and apoptosisresistant endothelial cell (ECs) (Tuder et al, 1994; Sakao et al, 2005)

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