Hypoxia-inducible factor-1α in pulmonary artery smooth muscle cells lowers vascular tone by decreasing myosin light chain phosphorylation

Abstract

Rationale: Hypoxia-inducible factor-1α (HIF-1α), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1α in modulating pulmonary vascular tone.

Objective: To investigate the role of smooth muscle cell (SMC)-specific HIF-1α in regulating pulmonary vascular tone.

Methods and results: Mice with an SMC-specific deletion of HIF-1α (SM22α-HIF-1α(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1α modulates pulmonary vascular tone and the response to hypoxia. SM22α-HIF-1α(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22α-HIF-1α(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1α decreased myosin light chain phosphorylation in HIF-1α-null SMCs.

Conclusions: In both normoxia and hypoxia, PASMC HIF-1α maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1α expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

Figure 1. Smooth muscle specific deletion of HIF-1a in SM22a-HIF-1a^−/− mice and HIF-1a^−/− SMC

Pulmonary artery and aorta tissues from SM22a-HIF-1a^−/− (B and D, respectively) and WT (SM22a-HIF-1a^+/+) mice (A and C, respectively) were stained with X-Gal. Scale bar = 100mm. (E) Real-time quantitative RT-PCR and (F) Western immunoblot to detect HIF-1α mRNA and protein in AoSMC and PASMC isolated from SM22α-HIF-1α^−/− and WT mice (left and right panels, respectively). Bars represent means ± SEM (n=3).

Figure 2. Smooth muscle specific loss of HIF-1α increases pulmonary vascular tone

(A) RVSP in SM22α-HIF-1α^−/− and WT mice in normoxia and following chronic hypoxia (10% O₂, 3 wks) (n=14-18 per group). (B) Percent of muscularized peripheral PA to total PA (n=5-9 per group). (C) RVSP in SM22α-HIF-1α^−/− and WT mice under baseline, in response to hypoxia for 15 minutes, and after recovery (n=12-16 per group). Bars represent means ± SEM. * p < 0.05 and ** p < 0.01, SM22α-HIF-1α^−/− vs. WT; §§ p < 0.01 and §§§ p < 0.001, hypoxia vs. normoxia for each genotype.

Figure 3. Loss of HIF-1α in PASMC increases myosin light chain phosphorylation

(A) pMLC was measured in PASMC isolated from SM22α-HIF-1α^−/− and WT mice by Western immunoblot under normoxia and after exposure to acute hypoxia. Bars represent means ± SEM (n=3). * p < 0.05 and ** p < 0.01, HIF-1α^−/− vs. WT; § p < 0.05 and §§ p < 0.01, hypoxia vs. normoxia. (B) Expression of pMLC in hPASMC transfected with siNC (non-targeted control siRNA) or siHIF-1a. Nuclear extracts demonstrate expression of active HIF-1a in siNC-transfected cells. TATA bp serves as a nuclear loading control. * p < 0.05 and ** p < 0.01, siHIF-1a vs. siNC; §§§ p < 0.001, hypoxia vs. normoxia. (C) Expression of pMLC in mPASMC null for HIF-1a expression, HIF-1a^−/−, transfected with empty vector, pcDNA3, or constitutively active HIF-1a, HIF-1a (CA). * p< 0.05 and *** p < 0.001, HIF-1a (CA) vs. pcDNA3.

Figure 4. Loss of HIF-1α in PASMC increases myosin light chain phosphorylation in vivo

Lung tissues from SM22α-HIF-1α^−/− and WT mice exposed to normoxia or chronic hypoxia were examined for pMLC by Western immunoblot. Bars represent means ± SEM (n=3). ** p < 0.01, SM22α-HIF-1α^−/− vs. WT; §§ p < 0.01 and §§§ p < 0.001, hypoxia vs. normoxia.