TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

Abstract

Aims: Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1^-/- mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1-CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy.

Methods and results: We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1-CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47^-/- mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47^-/- pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47^+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1-CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters.

Conclusions: In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Keywords: CD47; Clinical pulmonary hypertension; ET-1; Thrombospondin-1; cMyc.

Figure 1

TSP1 and CD47 are upregulated in human PH and contribute to PH-related arterial vasculopathy and dysfunction. (A) Western analysis of lysates from parenchyma and (C) 5th-order PAs from normal (non-PH) and PH human lungs was performed. Representative blots are demonstrated. Densitometry analysis of (A) parenchyma and (D) vessels is presented as mean ± SD (n = 4–8 normal and 5–15 PH samples). *P < 0.05, **P < 0.01, ***P < 0.001. (B) mRNA expression of TSP1, CD47, cMyc, ET-1, and ETA in parenchyma and (E) 5th-order PAs from normal (non-PH) and PH human lungs. Representative data from n = 5 normal and n = 8 PH lungs is presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Immunofluorescent staining of 5th-order PA vessels from normal and PH lungs. TSP1 and CD47 are stained red and green, respectively. Original magnification ×10; scale bar 200 μm. Blinded quantitative analysis of intensity of staining was performed and presented as mean ± SD, n = 5–7 patients per group; for each tissue section the hpf was divided into three to six approximately equal quadrants and fluorescence calculated via the integrated density function found in ImageJ. ****P < 0.0001. mRNA expression of collagen matrix genes in n = 5–8 normal and n = 6–12 PH lungs. Distal 5th-order PAs were dissected from normal (non-PH) human and end-stage PH lungs and mounted on a dual pin myography. (G) Analysis of vasodilation or vasoconstriction of non-PH vessels to the indicated doses of acetylcholine (Ach), sodium nitroprusside (SNP), and phenylephrine (PE) is presented. Results are the mean ± SD of n = 4 vessels per treatment group. (H) Myography results of distal 5th-order PA from normal human lungs to a log dose of SNP ± TSP1 (2.2 nM) ± CD47 antibody (clone B6H12, 1 µg/ml) or ET-1 ± TSP1 (2.2 nM). Results are the mean ± SD of n = 3–5 vessels per treatment group, *P < 0.05. (I) Myography results of distal 5th-order PAs from end-stage PH lungs to a log dose of Ach and SNP ± CD47 antibody (clone B6H12, 1 µg/ml). As absolute changes in pressure varied in each instance, representative graphs from n = 2–5 separate experiments in each group are presented. *P < 0.05, ***P < 0.001.

Figure 2

Absence of CD47 protects from hypoxia-mediated PH. Male age-matched C57BL/6 WT (CD47^+/+) and CD47^−/− mice were exposed to normoxia (Nx) or hypoxia (Hx, FiO₂ 10%, 3 weeks), and underwent (A, B) analysis of the indicated cardiopulmonary parameters and determination of the RV free wall weight and Fulton index. (C) Determination of the change in total body weight and tibia length of WT and CD47^−/− mice under normoxia and hypoxia. Data shown are mean ± SD, n = 4–6 animals per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) Representative lung sections prepared from each treatment group were stained for α-smooth muscle actin (brown) and blinded quantitative analysis of vascular wall thickness was performed on vessels between 50 and 100 μm in cross-sectional diameter. Wall thickness was measured at four equally separate points around the circumference of each vessel. Scale bars 50 μm; original magnification ×10 (D) and ×20 (E). Quantification is presented as mean ± SD, n = 4–5 tissue sections per treatment group, ****P < 0.0001.

Figure 3

CD47 is required for hypoxia-mediated induction of pulmonary ET-1/ETA. (A) Age-matched male WT (CD47^+/+), TSP1^−/− and CD47^−/− mice were challenged with normoxia (Nx) or short-term hypoxia (Hx) (10% FiO₂, 24 h). Lung tissue lysates from WT and TSP1^−/− mice (n = 3–5 per group) were prepared and protein separated via SDS–PAGE electrophoresis. Densitometry is presented as the mean ratio of target protein to α-tubulin ± SD; **P < 0.01, ***P < 0.001, ****P < 0.0001. Whole lung mRNA expression of TSP1 and CD47 from Nx or acutely Hx WT, TSP1^−/−, and CD47^−/− mice. Data from n = 3–5 mice per group are presented as mean ± SD, **P < 0.01. (B) Age-matched male WT and CD47^−/− mice were challenged with Nx or chronic Hx (10% FiO₂, 3 weeks). Lung tissue homogenates were prepared and protein separated by SDS–PAGE. Representative western immunoblots from treatment groups (n = 5–12 per group) for indicated proteins are presented. Densitometry is presented as the mean ratio of target protein to β-actin ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ET-1 levels in lung homogenates from WT and CD47^−/− mice under Nx or chronic Hx were measured by ELISA. Data are presented as mean ± SD from n = 4–8 samples per group, ***P < 0.001, ****P < 0.0001. (D) mRNA expression of TSP1, CD47, cMyc, ET-1, ETA, and ETB in whole lung homogenates. Data from n = 6–8 mice per group are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Platelet poor plasma TSP1 levels from WT and CD47^−/− mice under Nx or chronic Hx. Data are presented as the mean ± SD from n = 5–9 mice per group, *P < 0.05, **P < 0.01. (F) Myography results of proximal PA vessels from age-matched male WT mice treated with TSP1 (2.2 nM) and the indicated concentrations of acetylcholine (Ach), sodium nitroprusside (SNP), or endothelin-1 (ET-1). Results are the mean ± SD of n = 3–4 vessels per treatment, ^#P = 0.05, *P < 0.05, ***P < 0.001.

Figure 4

TSP1, via CD47, suppresses cMyc to increase ET-1/ETA signaling in pulmonary vascular cells. (A) Representative immunofluorescence image of cultured rat-1a Myc/ER fibroblast cells expressing the c-Myc/ER fusion protein display nuclear c-Myc (green), DAPI (blue), phalloidin (red); scale bar 50 μm, original magnification ×63. Rat-1a fibroblasts were treated with 4-hydroxytamoxifen at the designated concentrations for 12 h. mRNA expression of CD47, ET-1, and ETA was determined. Data from n = 4 experiments with each run in triplicate are presented as mean ± SD, ****P < 0.0001. (B) Murine pulmonary endothelial cells were isolated from WT and CD47^−/− mice. mRNA expression for CD47, cMyc, and ET-1 was determined. Data from n = 6 independent experiments are presented as mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Human pulmonary arterial endothelial cells (hPAEC) (passage 3–6) were cultured to 80% confluence then treated with normoxia (Nx), hypoxia (Hx, FiO₂ 1%), or hypoxia + CD47 blocking antibody (1 μg/ml) for 24 h. mRNA expression of CD47, ET-1, and ETA was determined. ET-1 level in cell culture supernatants was measured by ELISA. Representative data from four experiments are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) hPAEC were transfected with control (CTRL) or cMyc siRNA for 72 h and transcript levels of CD47, cMyc, ET-1, and ETA measured. ET-1 level in cell culture supernatants was measured by ELISA. Data are represented as mean ± SD from n = 5 experiments; **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Human pulmonary vascular smooth muscle cells (hPVSMC) were subjected to Nx or Hx (FiO₂ 1%, 24 h) and protein expression for TSP1 and cMyc determined. Representative blots are shown and data from n = 4 independent experiments are presented as mean ± SD, *P < 0.05. (F) hPVSMC were subjected to Nx or Hx (FiO₂ 1%, 24, 48, and 72 h) and mRNA transcript levels determined for TSP1, CD47, and ETA. Data from n = 4–5 experiments are presented as mean ± SD, *P < 0.05 Nx versus Hx at indicated time point. (G) Pulmonary VSMC from WT, TSP1^−/−, and CD47^−/− mice were isolated and subjected to Nx or Hx (FiO₂ 1%, 24 h). Cell lysate was prepared and protein and mRNA analysis performed. Representative western immunoblots for the indicated proteins are shown. Densitometry from n = 4 independent experiments for cMyc and n = 4–5 independent experiments for TSP1 is presented as the mean ratio of target protein to tubulin ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001. mRNA data are presented as the mean ratio of target protein to the housekeeping gene (± SD), *P < 0.05 Nx versus Hx. (H) hPVMSC were incubated with ET-1 (1 μM) with or without a CD47 blocking antibody (clone B6H12, 1 μg/ml) for 24 h. Cell size was analyzed by FACS. Data from n = 3–5 independent experiments are presented as mean ± SD, *P < 0.05, **P < 0.01.

Figure 5

RV TSP1–CD47 signaling is increased in experimental PH. WT and CD47^−/− mice were challenged with Nx or chronic Hx (FiO₂ 10%, 3 weeks). (A) Homogenates of right ventricle (RV) were prepared and protein separated by SDS–PAGE. Representative western immunoblots from treatment groups (n = 3–6 per group) for indicated proteins are presented. Densitometry is presented as the mean ratio of target protein to β-actin ± SD, *P < 0.05, **P < 0.01. (B) mRNA expression of TSP1, CD47, cMyc, ET-1, and ETA in RV. Data from n = 3–6 mice per group are presented as mean ± SD, ***P < 0.001, ****P < 0.0001. (C) Immunofluorescent staining of RV from WT and CD47^−/− mice subjected to Nx or chronic Hx. Type I collagen and myocytes are stained green and red, respectively. Scale bars 50 μm, original magnification ×40. Blinded quantitative analysis of fibrosis (total length × intensity of matrix/total surface area of myocytes) was performed and presented as mean ± SD, n = 5–7 tissue sections from three different mice per treatment group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis.

Figure 6

Disrupting TSP1–CD47 signaling induces pulmonary cMyc and mitigates some cardiopulmonary effects in established PH. (A) Dual-pin myography results of proximal PA vessels from male Sprague-Dawley rats treated with the indicated concentrations of KCl (100 nM), Ach and SNP ± TSP1 (2.2 nM), SNP ± a CD47 specific peptide (7N3, 10 μM). Results are the mean ± SD of n = 4 vessels per treatment group, *P < 0.05, ***P < 0.001. (B) Sprague-Dawley rats (CD47^+/+) were treated with monocrotaline (MCT, 50 mg/kg). Two weeks post-MCT animals were treated with a single dose of CD47 antagonist antibody (clone OX101, 0.4 µg/g body weight), n = 3–5 per group. Representative western immunoblots (B) for the indicated proteins are presented. Lung tissue lysate was prepared and (C) protein and (D) mRNA analysis of TSP1, CD47, cMyc, ET-1, and ETA performed. Densitometry is presented as the mean ratio of target protein to β-actin ± SD, *P < 0.05, **P < 0.01. (E) Analysis of the RV and LV-free wall weight and Fulton index from the indicated treatment groups. Data shown are mean ± SD, n = 4–5 animals per group, *P < 0.05, **P < 0.01, ****P < 0.0001. (F) Analysis of the indicated cardiac parameters via open chest Millar catheter. Data shown are mean ± SD, n = 4–5 animals per group, **P < 0.01.

Figure 7

TSP1–CD47 signaling limits pulmonary cMyc to upregulate ET-1/ETA and promote PH. Hypoxia-mediated induction of TSP1 in the pulmonary vasculature activates endothelial CD47 to suppress cMyc allowing upregulation of ET-1/ETA. ET-1 then targets the pulmonary arterial vascular smooth muscle cell (VSMC) compartment to promote vascular remodeling and cell hypertrophy, decreased vasodilation and increased vasoconstriction. Conversely, treatment with a CD47 antibody, that blocks TSP1 activation of CD47, limits ET-1-mediated VSMC hypertrophy and improves PA sensitivity to NO pathway activators.