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 9

Nrp1 deletion in smooth muscle cells results in a reduction of TGF-β1–pSMAD1/5/8 signaling.

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Nrp1 deletion in smooth muscle cells results in a reduction of TGF-β1–p...
(A) Aortic smooth muscle cells were isolated from Nrp1fl/fl (WT) or Nrp1fl/fl/SM22αCre+ (Nrp1SM22KO) mice. After overnight serum starvation, cells were stimulated with 10 ng/mL TGF-β1 for 30 minutes, and pSMAD1/5/8 levels were assessed by Western blot. Representative Western blot shows TGF-β1 stimulation (TGF-β1) increased pSMAD1/5/8 compared with unstimulated control (UNS) cells from WT mice but not in cells from Nrp1SM22KO mice with no change in pSMADs 2 and 3. (B and C) Densitometric analysis of NRP-1 (B) and pSMAD1/5/8 (C) were performed using NIH ImageJ software. Data are shown as mean ± SEM of n = 3. (D–G) Immunostaining analysis of AVF inflow arteries (GA) and contralateral control arteries (CA) at 14 days after AVF creation. All images were captured using 10× magnification. Scale bar: 50 μm. Arrows indicate the brown stained nuclei of pSMAD1/5/8 (D), pSMAD2 (F), and pSMAD3 (G). The intensity of brown stain pSMAD1/5/8 (E), pSMAD2 (H), and pSMAD3 (I) was measured as described and presented as mean ± SEM of n = 5–8 animals. Two-way ANOVA was performed. *P < 0.05.