Distal vessel stiffening is an early and pivotal mechanobiological regulator of vascular remodeling and pulmonary hypertension

Abstract

Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2-derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I₂ analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.

Figure 1. Increased PA stiffness in rat pulmonary hypertension models and human PAH.

(A and C) Sprague-Dawley rats were treated with monocrotaline (MCT) or PBS (n = 6 per group) and lungs harvested after 4 weeks. (B and D) Sprague-Dawley rats were treated with SU5416 (S) or vehicle (V), exposed to hypoxia (H) or normoxia (N) for 3 weeks, and then returned to normoxia for an additional 5 weeks (n = 4 per group). Pulmonary arterioles (PA) < 100 μm (A and B) and lung parenchyma (C and D) were mechanically characterized via AFM microindentation. Horizontal lines represent the mean shear modulus of each group, and each symbol corresponds to 1 PA (A and B) or parenchymal region (C and D). Statistical significance was determined by a mixed model with treatment as a fixed effect and individual rats as a random effect. (E and F) AFM microindentation was used to mechanically characterize PAs (E) and lung parenchyma (F) in human lung samples from PAH (n = 17; IPAH n = 8, FPAH n = 3, APAH n = 6) and control subjects (n = 7). Horizontal lines represent the mean shear modulus of each group, and each symbol corresponds to 1 PA (E) or parenchymal region (F). Statistical significance was determined by a mixed model with disease state as a fixed effect and subgroups and individual patients as nested fixed effects.

Figure 2. Increased matrix stiffness leads to remodeling responses in PASMC and PAEC.

Human PASMC and PAEC were plated on polyacrylamide substrates with stiffness of 0.1–25.6 kPa. After 48 hours, (A) cell density was determined and fold change expressed as the ratio of cell number at 48 hours vs. 4 hours (n = 7–10; statistical significance determined by the Kruskal-Wallis 1-way ANOVA). (B) Apoptosis was quantified and percent apoptosis normalized to maximum apoptosis at 0.1 kPa (n = 2 independent experiments performed in triplicate; statistical significance determined by the Mann-Whitney U test). (C) qPCR was performed for COL1A1 and FN1 in PASMC and normalized to GAPDH expression (n = 3–6 per stiffness; statistical significance determined by the Mann-Whitney U test). (D) Traction forces were quantified in PASMC by measuring fluorescent bead position displacement and calculating root-mean-square traction (n = 25 cells per stiffness; P < 0.0001 by the Kruskal-Wallis 1-way ANOVA, ***P < 0.001 by Dunn’s post test). (E) BMPR2 and control PASMC were plated on gels with stiffness of 0.1–25.6 kPa (n = 2 independent experiments performed in triplicate; statistical significance determined by 2-way ANOVA, P < 0.0116 stiffness, P < 0.0044 genotype, P = 0.5911 interaction. (F) PASMC were plated on discrete stiffness gels and exposed to normoxia or hypoxia. Cell density was determined at 48 hours and normalized to 4 hours (n = 3 independent experiments performed in triplicate; statistical significance determined by 2-way ANOVA, P < 0.0001 stiffness, P < 0.0001 treatment, P = 0.0358 interaction). Data represent the mean and SEM.

Figure 3. Distal PA stiffening occurs early in MCT-induced pulmonary hypertension.

Male Sprague-Dawley rats were treated with monocrotaline (MCT) or PBS (n = 6–8 per time point) and harvested at serial time points following MCT. (A) RVSP and (B) Fulton’s index were measured weekly for 4 weeks following MCT. Quantification of wall thickness of (C) PAs < 100 μm and (D) PAs > 100 μm. Data represent 25th–75th percentiles (box), median (line), and 5th and 95th percentiles (whiskers). Statistical significance was determined by one-way ANOVA followed by Dunn’s post test for multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001). AFM microindentation was used to mechanically characterize (E) PAs < 100 μm and (F) PAs > 100 μm. Horizontal lines represent the mean shear modulus of each group, and each symbol corresponds to 1 PA. Statistical significance was determined by a mixed model with treatment as a fixed effect and individual rats as a random effect followed by Dunnett’s test for multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4. Distal PA stiffening occurs early in the sugen hypoxia PH model.

Sprague-Dawley rats were treated with SU5416 (S) or vehicle (V), exposed to hypoxia (H) or normoxia (N) (n = 4–8 per time point) for 1, 2, or 3 weeks. Animals exposed to 3 weeks of hypoxia were returned to normoxia for an additional 2, 5, or 9 weeks. PAs < 100 μm (A) and PAs > 100 μm (B) were mechanically characterized via AFM microindentation. Horizontal lines represent the mean shear modulus of each group, and each symbol corresponds to 1 individual PA. Statistical significance was determined by a mixed model with treatment as a fixed effect and individual rats as a random effect followed by Dunnett’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001). RVSP (C), Fulton’s index (D), and PA wall thickness (E and F) were assessed at serial time points. (G–J) Echocardiography was performed (n = 4–7 per group) and measurements of RVFW thickness, PAAT, TAPSE, and RVID made at 5 and 8 weeks. Data represent the mean ± SEM. Statistical significance was determined by the Mann-Whitney U test for pairwise comparisons or by 1-way ANOVA followed by Dunn’s post test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5. Increased matrix stiffness leads to decreased COX-2 expression and prostanoid production.

Human PASMC (A) and PAEC (B) were cultured on polyacrylamide substrates with stiffness of 0.1–25.6 kPa. After 48 hours, RNA was isolated and reverse transcribed to cDNA, and qPCR was performed for PTGS2 (COX-2). Results were normalized to GAPDH expression (n = 2–5; statistical significance determined by 1-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 by Dunnett’s post tests). (C) PASMC were transiently transfected with a COX-2 promoter luciferase construct (–976/+56) via electroporation, and transfected cells were plated on polyacrylamide substrates with stiffness of 0.1–25.6 kPa. After 24 hours, cells were harvested and luciferase and β-galactosidase assays performed (n = 3; statistical significance determined by 1-way ANOVA). (D and E) After 48 hours, levels of 6-keto PGF_1α and PGE₂ were measured in media from (D) PASMC and (E) PAEC by ELISA (n = 3; statistical significance determined by 1-way ANOVA). (F and G) Lipid mediators were extracted from lungs of PBS- and MCT-treated rats at 1, 2, and 3 weeks (n = 4–5 per group) and prostanoids assessed using liquid chromatography–tandem mass spectrometry (LC-MS-MS). Prostanoid levels are expressed as pg/100 mg of lung tissue. Statistical significance was determined by 1-way ANOVA followed by Dunn’s post test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001). Data represent the mean ± SEM.

Figure 6. Iloprost attenuates stiffness-dependent remodeling responses and traction forces in PASMC.

PASMC were plated on discrete stiffness gels and treated with iloprost (3 or 10 μmol/l) or vehicle for 48 hours. (A) Cell density was determined after 48 hours and normalized to 4 hours (n = 3–5; statistical significance determined by 2-way ANOVA, P < 0.0001 stiffness, P < 0.0001 treatment, P = 0.1020 interaction). (B) Collagen concentration was measured in the media of vehicle- and iloprost-treated PASMC after 48 hours and normalized to cell number (n = 2–4; statistical significance determined by 2-way ANOVA, P = 0.0203 stiffness, P = 0.0161 treatment, P = 0.2960 interaction). (C–J) Representative images of immunofluorescent staining for procollagen I (C–F) and EDA fibronectin (G–J) in vehicle- and iloprost-treated PASMC. Scale bar: 100 μm. Quantitation of immunostaining for procollagen I (K) (P = 0.0082 stiffness, P = 0.0084 treatment, P = 0.0184 interaction by 2-way ANOVA) and EDA fibronectin (L) (P < 0.0001 stiffness, P = 0.0043 treatment, P = 0.0047 interaction by 2-way ANOVA). (M–R) PASMC were plated on polyacrylamide gels with discrete shear moduli of 0.4, 1.6, and 6.4 kPa and, after 24 hours, treated with iloprost (10 μmol/l) or vehicle for 30 minutes. Representative traction fields following treatment with vehicle (row 1, M–O) or iloprost (row 2, P–R). Each column represents discrete substrate stiffness. Color scale indicates magnitude of traction in kPa. Scale bars: 50 μm. (S) Quantification of traction forces (n = 17–20 cells per group) exerted by vehicle- and iloprost-treated PASMC on discrete stiffness gels (P < 0.0001 stiffness, P < 0.0001 treatment, P < 0.0001 interaction by 2-way ANOVA). Data represent the mean ± SEM.

Figure 7. Treprostinil prevents PA stiffening in the MCT model.

Sprague-Dawley rats were treated with MCT or vehicle and, after 2 weeks, had s.c. minipumps implanted to deliver i.v. treprostinil (90 ng/kg/min) or saline. PAs < 100 μm (A) and PAs > 100 μm (B) were mechanically characterized via AFM microindentation. Horizontal lines represent the mean shear modulus of each group (n = 4–6 per group), and each symbol corresponds to 1 individual PA. Statistical significance was determined by a mixed model with treatment as a fixed effect and individual rats as a random effect, followed by Dunnett’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001). Lungs were harvested (n = 4–6 per group) and qPCR performed for (C) COL1A1, (D) COL3A1, (E) FN1, and (F) LOX and normalized to 18S ribosomal RNA expression. Data represent 25th–75th percentiles (box), median (line), and 5th and 95th percentiles (whiskers). Statistical significance was determined by 1-way ANOVA followed by Dunn’s post test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 8. Inhibition of PA stiffening with treprostinil attenuates MCT-induced pulmonary hypertension.

Sprague-Dawley rats were treated with monocrotaline (MCT) or vehicle and, after 2 weeks, were treated with i.v. treprostinil or saline for 2 weeks. (A) RVSP and (B) Fulton’s index were measured. Quantification of wall thickness of (C) PAs < 100 μm and (D) PAs > 100 μm. Representative H&E images of PAs < 100 μm (E–H) and PAs > 100 μm (I–L) from MCT- and MCT+ treprostinil–treated rats. Scale bars: 100 μm. Data represent the mean ± SEM. Statistical significance was determined by 1-way ANOVA followed by Dunn’s post test (*P < 0.05; **P < 0.01; ***P < 0.001).