Endothelial HIFα/PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension
Fatima Z. Saddouk, Andrew Kuzemczak, Junichi Saito, Daniel M. Greif
Fatima Z. Saddouk, Andrew Kuzemczak, Junichi Saito, Daniel M. Greif
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Research Article Pulmonology Vascular biology

Endothelial HIFα/PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension

Abstract

Mechanisms underlying maintenance of pathological vascular hypermuscularization are poorly delineated. Herein, we investigated retention of smooth muscle cells (SMCs) coating normally unmuscularized distal pulmonary arterioles in pulmonary hypertension (PH) mediated by chronic hypoxia with or without Sugen 5416, and reversal of this pathology. With hypoxia in mice or culture, lung endothelial cells (ECs) upregulated hypoxia-inducible factor 1α (HIF1-α) and HIF2-α, which induce platelet-derived growth factor B (PDGF-B), and these factors were reduced to normoxic levels with re-normoxia. Re-normoxia reversed hypoxia-induced pulmonary vascular remodeling, but with EC HIFα overexpression during re-normoxia, pathological changes persisted. Conversely, after establishment of distal muscularization and PH, EC-specific deletion of Hif1a, Hif2a, or Pdgfb induced reversal. In human idiopathic pulmonary artery hypertension, HIF1-α, HIF2-α, PDGF-B, and autophagy-mediating gene products, including Beclin1, were upregulated in pulmonary artery SMCs and/or lung lysates. Furthermore, in mice, hypoxia-induced EC-derived PDGF-B upregulated Beclin1 in distal arteriole SMCs, and after distal muscularization was established, re-normoxia, EC Pdgfb deletion, or treatment with STI571 (which inhibits PDGF receptors) downregulated SMC Beclin1 and other autophagy products. Finally, SMC-specific Becn1 deletion induced apoptosis, reversing distal muscularization and PH mediated by hypoxia with or without Sugen 5416. Thus, chronic hypoxia induction of the HIFα/PDGF-B axis in ECs is required for non–cell-autonomous Beclin1-mediated survival of pathological distal arteriole SMCs.

Authors

Fatima Z. Saddouk, Andrew Kuzemczak, Junichi Saito, Daniel M. Greif

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Figure 2

HIFα is upregulated with hypoxia and downregulated with re-normoxia.

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HIFα is upregulated with hypoxia and downregulated with re-normoxia. (A)...
(A) Wild-type mice were exposed to either normoxia or hypoxia for 21 days with or without subsequent re-normoxia for 7 or 14 days. Lung lysates were analyzed by qRT-PCR to assess Hif1a, Hif2a, and Bnip3 levels. n = 3–4 mice (2 males, 1–2 females) per experimental group. (B and C) Wild-type mice were exposed to normoxia or hypoxia for 21 days and then were or were not subjected to normoxia for 14 days. Whole-lung lysates were analyzed by Western blot for HIF1-α, HIF2-α, and GAPDH protein (B), with densitometry relative to GAPDH and normalized to normoxia (C). n = 4 mice (2 males, 2 females) per experimental group. (D) Acta2-CreERT2 ROSA26RZs/+ mice were induced with tamoxifen (1 mg/day for 5 days), rested for 5 days, exposed to hypoxia for 21 days, and then re-normoxia for 10 days. Zs+ cells were isolated by FACS, and Hif1a expression levels were measured by qRT-PCR. n = 3 mice (1 male, 2 females) per experimental group. (E–G) Wild-type mice were exposed to normoxia or hypoxia for 21 days followed by normoxia for 10 days. (E) Lung CD31+CD45– ECs were isolated by FACS, and transcript levels of Hif1a and Hif2a were measured by qRT-PCR. n = 3 (1 male, 2 females) per experimental group. (F and G) Lung ECs were isolated with anti-CD31–coated beads. Western blot analysis of EC lysates for HIF1-α, HIF2-α, and GAPDH protein are shown (F) with densitometry relative to GAPDH and normalized to normoxia (G). n = 5 mice (2 males, 3 females) per experimental group. Multifactor ANOVA with Tukey’s multiple-comparison test was used (A, C–E, and G). N, normoxia; H, hypoxia; H+N, hypoxia followed by normoxia.