Alteration of pulmonary artery integrin levels in chronic hypoxia and monocrotaline-induced pulmonary hypertension

Abstract

Background: Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats.

Methods and results: Immunoblot showed increased α(1)-, α(8)- and α(v)-integrins, and decreased α(5)-integrin levels in PAs of both models. β(1)- and β(3)-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α(1)- and α(5)-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α(1)-ligand collagen IV. Moreover, α(1)-integrin binding hexapeptide GRGDTP elicited an enhanced Ca(2+) response, whereas the response to α(5)-integrin binding peptide GRGDNP was reduced in CH-PASMCs.

Conclusion: Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression.

Fig. 1

Representative immunoblots of integrin proteins in endothelium-denuded PA (a) and aorta (c) of rats exposed to chronic hypoxia and normoxia. Each lane represents protein isolated from an individual animal. b, d Densitometric data obtained from immunoblots (n = 12–15 animals). Twelve animals per treatment group were used to compile the data in b for α₁ and α₈ integrins, while 15 were used for all others. Data are normalized to the average of the normoxic controls. Asterisks show significant changes in integrin protein expression upon exposure to chronic hypoxia (* p < 0.05, ** p < 0.01 vs. control).

Fig. 2

Representative immunoblots of integrin proteins in endothelium-denuded PA (a) and aorta (c) of rats treated with MCT (60 mg/kg) for 24 days versus control. b, d Densitometric data obtained from immunoblots (n = 11–12 animals). Data are normalized to the average of the controls. Asterisks show significant changes in integrin protein expression upon treatment with MCT (* p < 0.05, ** p < 0.01 vs. control).

Fig. 3

Immunostaining of integrin and smooth muscle α-actin in small PAs in lung sections of control rats. Confocal images show fluorescent signals from lung sections double-stained with a α₁ (a), α₅ (b), α₈ (c), β₁ (d) and β₃ (e) integrin-specific antibody, and an α-actin-specific antibody. Transmission images show the lung structures, while the yellow color indicates the regions in which there were overlaying of signals of integrin protein and α-actin in PAs. The images were taken with a Plan Neofluar ×20 objective (numerical aperture = 0.5), pinhole size = 1 airy, and zoom was between 2.7 and 3.6. Scale bars = 20 μm (a, b); 10 μm (c–e).

Fig. 4

Confocal images of immuno-fluorescent signals (red) of α₁ (a) and α₅ integrin (b) in PASMCs isolated from normoxic and chronic hypoxic rats. Transmitted light images (right panels) were included for reference. The images were taken with a Plan Neofluar ×40 oil objective (numeric aperture = 1.3), pinhole size = 1 airy, and zoom = 4. Laser power, sensitivity and gain were set at the same level for confocal imaging of the normoxic and hypoxic cells.

Fig. 5

a Representative immunoblots of phosphorylated FAK and total FAK in endothelium-denuded PAs from normoxic and hypoxic animals. Each lane represents protein isolated from an individual animal. b A bar graph summarizes the averaged signal ratio of phosphorylated FAK over total FAK in PAs (n = 6 animals in each group). c The signal ratio of phosphorylated FAK over total FAK in PASMCs isolated from normoxic and hypoxic rats that were transiently cultured in culture dish coated with collagen IV or fibronectin (n = 4 experiments with cells from 4 different animals in each group). Asterisks show significant changes in increase in FAK phosphorylation upon exposure to chronic hypoxia (* p < 0.05 vs. normoxia).

Fig. 6

[Ca²⁺]_i transients elicited by integrin-binding peptides in PASMCs from normoxic and chronic hypoxic rats. a Time course of GRGDTP-induced [Ca²⁺]_i response in normoxic (black squares) or chronic hypoxic (gray circles) PASMCs. n = 29 dishes of PASMCs, from 3 rats on 3 separate days. b Bar graph summarizing the peak and plateau phases of Δ[Ca²⁺]_i at 3.5 and 10 min after application of GRGDTP. c Time course of GRGDNP-elicited [Ca²⁺]_i response in normoxic (black squares) or chronic hypoxic (gray circles) PASMCs. n = 30 dishes of PASMCs, from 3 rats on 3 separate days. d Bar graph summarizing the peak and plateau phases of Δ[Ca²⁺]_i at 3.5 and 13 min postapplication of GRGDNP.