Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium

Abstract

Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis.

A Novel p38 Mitogen-activated Protein Kinase/Elk-1 Transcription Factor-dependent Molecular Mechanism Underlying Abnormal Endothelial Cell Proliferation in Plexogenic Pulmonary Arterial Hypertension

“…All animals were sacrificed 8 days after the last MCT administration. Lungs (free of blood) were excised and used to prepare lung lysates as described previously (31). Repeated MCT administration and prolonged treatment compensate for strain-and species-specific differences between hepatic cytochrome P450 enzyme required for MCT processing to active MCT pyrrole and allow consistent, reproducible induction of PAH in mice (30,32,33).…”

Intersectin‐1s: An Important Regulator of Cellular and Molecular Pathways in Lung Injury

“…2A, 2a1, 2B [arrows and outlined area], 2D). 105 Interestingly, the functionality of this alternative transport increased at 24 days of KD ITSN , leading to partial resealing of interendothelial junctions and reduced interstitial edema. At this time point, mice still displayed increased microvascular permeability, a sequela of ALI, but neither the gold albumin (18-20-nm diameter) nor the DNP-BSA (dinitrophenol-bovine serum albumin; 6-10-nm diameter) tracers were able to penetrate the inter- endothelial junctions, meaning that resealing mechanisms were returning the endothelial barrier to its perm-selective status.…”

“…At this time point, mice still displayed increased microvascular permeability, a sequela of ALI, but neither the gold albumin (18-20-nm diameter) nor the DNP-BSA (dinitrophenol-bovine serum albumin; 6-10-nm diameter) tracers were able to penetrate the inter- endothelial junctions, meaning that resealing mechanisms were returning the endothelial barrier to its perm-selective status. 105 Patchy areas of mild septal edema were still present, implying that the resealing did not return the structure of the junctional complexes to their initial status, because of continuous ITSN-1s deficiency; additional studies are needed to fully define this intermediary state of interendothelial junction opening (>3 nm but <6 nm). KD ITSN in mouse lungs had a dual outcome: (1) acute (3 days) KD ITSN -induced impairment in caveolae transport function, significant disruption of junctional integrity, and acute pulmonary edema and (2) chronic (24 days): KD ITSN upregulated alternative transport pathways to compensate for deficient caveolae transport function and partially restored the endothelial barrier integrity, maintaining, however, a mild edema.…”

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

“…12,14 Mice were sacrificed at 2, 6, 12, and 18 days; three mice [control wild-type (wt), vehicle and nonspecific (scrambled) siRNA treated, as well as ITSN siRNA treated] were used per experimental condition and per experiment, with experiments repeated at least three times. Equal doses of the scrambled siRNA and ITSN siRNA were used.…”