Neuropilin-1 deficiency in vascular smooth muscle cells is associated with hereditary hemorrhagic telangiectasia arteriovenous malformations
Sreenivasulu Kilari, Ying Wang, Avishek Singh, Rondell P. Graham, Vivek Iyer, Scott M. Thompson, Michael S. Torbenson, Debabrata Mukhopadhyay, Sanjay Misra
Sreenivasulu Kilari, Ying Wang, Avishek Singh, Rondell P. Graham, Vivek Iyer, Scott M. Thompson, Michael S. Torbenson, Debabrata Mukhopadhyay, Sanjay Misra
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Research Article Vascular biology

Neuropilin-1 deficiency in vascular smooth muscle cells is associated with hereditary hemorrhagic telangiectasia arteriovenous malformations

Abstract

Patients with hereditary hemorrhagic telangiectasia (HHT) have arteriovenous malformations (AVMs) with genetic mutations involving the activin-A receptor like type 1 (ACVRL1 or ALK1) and endoglin (ENG). Recent studies have shown that Neuropilin-1 (NRP-1) inhibits ALK1. We investigated the expression of NRP-1 in livers of patients with HHT and found that there was a significant reduction in NRP-1 in perivascular smooth muscle cells (SMCs). We used Nrp1SM22KO mice (Nrp1 was ablated in SMCs) and found hemorrhage, increased immune cell infiltration with a decrease in SMCs, and pericyte lining in lungs and liver in adult mice. Histologic examination revealed lung arteriovenous fistulas (AVFs) with enlarged liver vessels. Evaluation of the retina vessels at P5 from Nrp1SM22KO mice demonstrated dilated capillaries with a reduction of pericytes. In inflow artery of surgical AVFs from the Nrp1SM22KO versus WT mice, there was a significant decrease in Tgfb1, Eng, and Alk1 expression and phosphorylated SMAD1/5/8 (pSMAD1/5/8), with an increase in apoptosis. TGF-β1–stimulated aortic SMCs from Nrp1SM22KO versus WT mice have decreased pSMAD1/5/8 and increased apoptosis. Coimmunoprecipitation experiments revealed that NRP-1 interacts with ALK1 and ENG in SMCs. In summary, NRP-1 deletion in SMCs leads to reduced ALK1, ENG, and pSMAD1/5/8 signaling and reduced cell death associated with AVM formation.

Authors

Sreenivasulu Kilari, Ying Wang, Avishek Singh, Rondell P. Graham, Vivek Iyer, Scott M. Thompson, Michael S. Torbenson, Debabrata Mukhopadhyay, Sanjay Misra

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Figure 2

Direct connection of artery to vein in the lung and dilated vessels in lung and liver of Nrp1SM22KO mice.

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Direct connection of artery to vein in the lung and dilated vessels in l...
(A) Lung tissue sections were stained for H&E, which shows heterogenic thickness of the vessel (open and closed arrows) in Nrp1SM22KO animals. (B) Verhoeff–Van Gieson–stained sections from Nrp1fl/fl (WT) and Nrp1fl/fl/SM22αCre+ (Nrp1SM22KO) mice were used to distinguish the artery (closed arrow) and vein (open arrow). Ar, artery; V, vein; AW, airway in lungs. Scale bar: 50 μm. (C–F) vascular cross-sectional area was determined in CD31-stained lung (C and D) liver tissue sections (E and F). All images were captured at 10× magnification using a Zeiss Axio imager M2 equipped with an Axiocam 503 camera and a motorized stage. Scale bar: 500 μm. The area enclosed with the dotted line box was digitally enlarged to show as inset panel. The solid black arrows indicate CD31+ cells lining the blood vessels. Vessels were identified manually and shaded in green, as shown in right panels. The average vessel area in lung (C) and liver (E) was measured using Fiji-ImageJ software, and the average cross-sectional area in lung (D) and in liver (F) were shown as bar graphs. Data are shown as mean ± SEM of n = 5. There is a significant increase in average cross-sectional area of vessels in lung (D) and liver (E) from Nrp1SM22KO mice compared with WT animals. Nonparametric Mann-Whitney U test was performed. *P < 0.05.