Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

Abstract

Aims: Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH.

Methods and results: We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH.

Conclusion: Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Figure 1

Pulmonary TSP1 and CD47 are upregulated in human subjects with PAH and in experimental PAH. (A) Lung tissue from a historical cohort of non-PAH, IPAH or SCD was probed by western blot for TSP1 and β-actin. (western blot is the only representative). Densitometry is the result of 10 non-PAH patients, five SCD, 5 IPAH patients and is presented as mean ratio of TSP1 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with non-PAH patients. (B) Western blot analysis of lung samples from a prospective cohort of non-PAH and PAH patients probed for CD47 and β-actin. Densitometry is the result of three non-PAH and five PAH patients. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with non-PAH patients. (C) TSP1 and CD47 mRNA expression levels in lung samples from the prospective cohort. Representative data from three independent experiments are presented. mRNA levels are expressed as fold change over non-PAH and normalized with 18S ribosomal subunit levels as endogenous non-PAH. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with control. Hash (#) indicates statistically significant difference (P < 0.05) compared with non-PAH. (D) Representative western blot of lung tissue from normoxic and chronically hypoxic (21 days normobaric 10% O₂) C57BL/6 wild-type and TSP1 null mice probed against TSP1 and β-actin. Densitometry is presented as mean ratio of TSP1 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxic mice and is based on an analysis of eight mice per group. (E) Representative western blot probed against TSP1 and α-tubulin from lung samples from normoxic and acute hypoxia (normobaric 7.5% O₂) challenged wild-type mice. Representative data from three independent experiments are presented. Densitometry is presented as mean ratio of TSP1 to α-tubulin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared to normoxia and is based on four mice at each time point. (F) TSP1 mRNA expression levels in lung samples from wild-type and TSP1 null mice exposed to normoxia or hypoxia (normobaric 7.5% O₂) for the indicated time points. Representative data from three independent experiments are presented. mRNA levels are expressed as fold change over normoxic conditions and normalized with HPRT levels as endogenous control. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (G) Representative western blot of wild-type and TSP1 null pulmonary microvascular endothelial cells exposed to normoxia or hypoxia (normobaric 1% O₂) for 12 h probed against CD47 and α-tubulin. Representative data from three independent experiments are presented. Densitometry is presented as mean ratio of CD47 to α-tubulin (± SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (H) Representative western blot from wild-type mice exposed to acute hypoxia for the indicated time points probed against CD47 and β-actin. Densitometry from analysis of western results from four mice in each treatment group is presented as mean ratio of CD47 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia.

Figure 2

Activated CD47 promotes hypoxia-stimulated PAH. Wild-type and TSP1 null mice were exposed to 21 days chronic hypoxia (normobaric 10% O₂) or room air. At the end of the exposure period, (A) right ventricular systolic pressure (RVSP), and (B) right ventricular hypertrophy (Fulton Index, RV/LV + S) were determined. Data represent mean ± SD (n = 8 per group). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with hypoxic wild-type. (C) Representative images of pulmonary tissue sections from normoxic and hypoxic male C57BL/6 wild-type and TSP1 null mice demonstrating pulmonary arterioles stained for α-smooth muscle actin (red arrows). Scale bar represents 50 µm. Sections of lungs were analysed for partial and full muscularization of arterioles by an observer blinded to strain and treatment. Quantification was based on 20 random 40x fields per section and three sections per mouse. n = 8 mice per group, analysis is the result of 10 vessels per section. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxic wild-type. Hash (#) indicates statistically significant difference (P < 0.05) compared with hypoxic wild-type. (D) Quantification of arteriole wall thickness of lung sections by a blinded observer based on 20 random 40x fields per section. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxic wild-type. Hash (#) indicates statistically significant difference (P < 0.05) compared with hypoxic wild-type.

Figure 3

Activated CD47 regulates hypoxic eNOS by altering Cav-1CD47 co-association. (A) Western blot of lung tissue from wild-type and TSP1 null mice exposed to chronic hypoxia or room air probed against total eNOS protein, eNOS phosphorylation at serine-1176 (murine residue), Cav-1, and β-actin. Quantification of densitometric analysis of the ratio of (B) total eNOS to β-actin, (C) p-eNOS to total eNOS, (D) Cav-1 to β-actin. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with wild-type normoxic. Hash (#) indicates statistically significant difference (P < 0.05) compared with wild-type normoxic. All ratios represent mean ± SD and are the result of the total cohort (n = 8 animals per group). (E) Co-immunoprecipitation in human pulmonary microvascular endothelial cells of Cav-1 and CD47. Immunoprecipitation was with monoclonal antibodies to Cav-1, CD47, or an isotype-matched control IgG antibody. Results presented are representative of four separate experiments. (F) Human pulmonary microvascular endothelial cells were serum starved and then treated with TSP1 (2.2 nmol/L) ± hypoxia (1% O₂ for 4 h), lysate prepared and immunoprecipitation for CD47 performed with protein blotted for Cav-1. Results are presented as the quantification of densitometric analysis from three separate experiments of the ratio of Cav-1 to CD47 following immunoprecipitation. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (G) Western blot of pulmonary Cav-1 from lung lysate from chronically hypoxic (21 days normobaric 10% O₂) wild-type (n = 3) and CD47 null (n = 4) mice. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with wild-type.

Figure 4

Activated CD47 promotes oxidative stress in hypoxic PAH. (A) Representative native gel electrophoresis of lung lysates probed against eNOS to determine eNOS monomer to dimer ratio in lung lysates from wild-type and TSP1 null mice after chronic hypoxia or room air. (B) Densitometry is presented as monomer to dimer ratio of eNOS expression and are presented as the mean ± SD (n = 4). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxic control. (C) Representative photomicrographs of lung tissue from wild-type and TSP1 null mice exposed to chronic hypoxia or normoxia stained using an antibody against 3-nitrotyrosine. Immunohistochemistry was performed on two sections from four mice in each group. Scale bar represents 100 µm.

Figure 5

Blocking CD47 activation upregulates Cav-1 to inhibit eNOS-derived superoxide production in hypoxic pulmonary arterial endothelial cells. (A) Representative western blot of hPAEC exposed to normoxia, hypoxia (1% O₂ for 12 h), or hypoxia plus a CD47-blocking antibody (αCD47, clone B6H12, 1 µg/mL). Quantification of densitometric analysis of the ratio of (B) TSP1 to β-actin and (C) Cav-1 to β-actin. Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic control. (D) Superoxide production in hPAEC exposed to normoxia, hypoxia (1% O₂ for 12 h), hypoxia plus L-NAME (100 mmol/L), or hypoxia plus a CD47-blocking antibody (clone B6H12, 1 µg/mL). Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic control. (E) Western blot analysis for Cav-1 expression in hPAEC transfected with control or Cav-1 siRNA. (F) Superoxide production in hPAEC transfected with control or Cav-1 siRNA then exposed to 24 h hypoxia (1% O₂) with or without a CD47-blocking antibody. Data represent three independent experiments. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with hypoxia control alone. Hash (#) indicates statistically significant difference (P < 0.05) compared with hypoxia alone. (G) Wild-type and TSP1 null pulmonary microvascular endothelial cells harvested from 10-week-old male animals were grown to 80% confluence, treated with normoxia or hypoxia (1% O₂) ± TSP1 (2.2 or 22 nmol/L) as indicated for 24 h, cell lysate prepared and western blot analysis for Cav-1 expression performed. A representative blot and accompanying densitometry from three separate experiments are presented. Quantification of densitometric analysis of the mean ratio of Cav-1 to α-tubulin. Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic untreated. Hash (#) indicates statistically significant difference (P < 0.05) from normoxic wild-type + 22 nmol/L TSP1 and hypoxic wild-type. (H) Western blot analysis for phospho-Cav-1^Tyr14 expression in normoxic hPAEC treated with the indicated amounts of TSP1 (2.2 nmol/L) ± a CD47 antibody (B6H12, 1 µg/mL) for 30 m. Quantification of densitometric analysis of mean ratio p-Cav-1 to total Cav-1 of three separate experiments. Asterisk (*) indicates statistically significant difference (P < 0.05) from untreated and antibody alone. Hash (#) indicates statistically significant difference (P < 0.05) from TSP1 treated.

Figure 6

Blockade of CD47 activation upregulates Cav-1 and prevents experimental PAH. Age-matched male Sprague Dawley rats were treated with monocrotaline (mct, 50 mg/kg) ± a CD47-blocking antibody (clone OX101, 1 µg/gram body weight on day 1 and day 14 via i.p. injection). Animals were euthanized after 4 weeks. Lung lysates were prepared and proteins separated by SDS–PAGE electrophoresis. Blots were probed with a monoclonal antibody to (A) TSP1, (B) CD47, or (C) p-eNOS. Expression was quantified by densitometry and is presented as the mean ± SD. Densitometry results are from four animals in each group. (A) Asterisk (*) indicates statistically significant difference (P < 0.05) mct compared with control; Hash (#) indicates statistically significant difference (P < 0.05) mct compared with mct + CD47 antibody (Ab). (B) Asterisk (*) indicates statistically significant difference (P < 0.05) mct compared with control. (C) Asterisk (*) indicates statistically significant difference (P < 0.05) control compared with mct + CD47 antibody (Ab). (D) Age-matched male Sprague Dawley rats were treated with monocrotaline (mct) ± CD47 monoclonal antibody (Ab) (clone OX101, 1 µg/gram body weight) and right ventricular systolic pressure and RV/LV + S ratio (Fulton Index) measured at 4 weeks. Quantification of pulmonary arteriole wall thickness was performed by a blinded reviewer from tissue section images using an image analysis programme (Metamorph) with percent wall thickness defined as the wall thickness normalized to the vessel diameter and was specifically calculated by the following formula: wall thickness (%) = area external−area internal/area external × 100, where area external and area internal are the areas bounded by external elastic lamina and the lumen, respectively. Normalization was performed to account for increases in vessels size and wall thickness as a function of vessel diameter. Results are the mean ± SD of four animals in each group. Asterisk (*) indicates statistically significant difference (P < 0.05) mct compared with control. Hash (#) indicates statistically significant difference (P < 0.05) mct compared with mct + CD47 antibody (Ab). (E) Representative western blot of rat lung lysate from treated animals probed for Cav-1 and β-actin. Expression was quantified by densitometry presented as the mean ± SD from four animals in each group. Asterisk (*) indicates statistically significant difference (P < 0.05) control compared with mct. Hash (#) indicates statistically significant difference (P < 0.05) mct compared with mct + CD47 antibody (Ab).

Figure 7

Activated CD47 promotes PAH through suppressing constitutive Cav-1 inhibition of eNOS. In health and under normoxic conditions, CD47 constitutively associates with Cav-1 on the cell membrane of pulmonary arterial endothelial cells. In hypoxia and PAH, the CD47 ligand TSP1 is upregulated. On binding with TSP1, the cell receptor CD47 is activated leading to disruption of the constitutive interaction between CD47 and Cav-1. This in turn leads to decreased Cav-1 and increased eNOS activity. Monomeric hyperactive eNOS then produces superoxide rather than NO resulting in tissue oxidation and nitration.