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A2886 - Eps-15 Homology Domains: Role In The Pathogenesis Of Pulmonary Arterial Hypertension
Author Block: S. A. Predescu1, S. Qin1, C. Bardita2, D. N. Predescu1; 1Rush University Medical Center, Chicago, IL, United States, 2Pulmonary Critical Care, University Of Alabama, Birmingham, AL, United States.
Rationale: Eps-15 homology (EH) domains confine caveolae to the plasma membrane and upregulate compensatory endocytic pathways in cells with impaired vesicular trafficking. The observations suggest that the EH domains of the general endocytic protein intersectin-1 (ITSN), shown to possess endothelial cells (EC) proliferative potential and to be expressed in the lungs and ECs of pulmonary arterial hypertension (PAH) patients may upregulate alternative transport pathways to compensate for caveolae dysfunction induced by ITSN deficiency and as a result, to contribute to the pathogenesis of PAH.
Methods: The studies used the novel murine model of plexiform PAH - ITSN heterozygous mice (K0ITSN+/-) transduced with the Myc-tagged EHITSN, pulmonary artery EC of PAH patients (ECPAH; ITSN-deficient and expressing the EHITSN) and ECs stably-transfected with Myc-EHITSN. The monocrotaline-induced rat and mouse models of PAH shown to express the EHITSN as well as untreated animals and ECs were used for comparison. Perfusion of the murine and rat lung microvasculature with 6-nm gold- and dinitrophenylated-albumin and a biotin assay for caveolae internalization followed by electron microscopy surveys and biochemical quantification of transendothelial tracer transport and biotin internalization were employed to address the effects of ITSN deficiency and EHITSN expression on lung endothelial barrier and caveolae endocytosis. Biochemical and morphological approaches were used to evaluate the expression and interaction of EH-binding protein-1, a protein that links caveolae to the actin cytoskeleton, with ITSN. Phalloidin staining was used to assess the status of cellular actin.
Results: ITSN-1s deficiency and expression of Myc-EHITSN cooperate in upregulating alternative transport pathways to compensate for deficient vesicular trafficking caused by decreased ITSN expression. Quantification of biotin internalization in Myc-EHITSN-transduced ECs show values comparable to untreated cells while ECPAH show a 34% increase. In vivo studies demonstrate that the Myc-EHITSN-transduced K0ITSN+/- mice and monocrotaline-treated animals showed 2.9-fold increase in DNP-BSA transport compared to untreated animals. Electron microscopy revealed increased occurrence of non-conventional endocytic/transcytotic structures and interstitial edema suggesting leakiness of the interendothelial junctions. Moreover, ITSN deficiency and EHITSN expression interfere with subcellular distribution and function of EH-binding protein-1, an ITSN-interacting protein, leading to actin reorganization and loss of junctional integrity.
Conclusion: Our studies implicate ITSN-1s deficiency and EHITSN expression in the vascular pathology and progression of PAH by upregulation of alternative endocytic pathways to support ECs’ proliferation and overgrowth and by disruption of lung endothelial barrier and vascular leak, which under inflammatory conditions associated with PAH lead to endothelial instability and vascular remodeling.