IL-33 Initiates Vascular Remodelling in Hypoxic Pulmonary Hypertension by up-Regulating HIF-1α and VEGF Expression in Vascular Endothelial Cells

Abstract

IL-33 may play a role in the vascular remodelling of hypoxic pulmonary hypertension (PH) but the precise mechanisms are still unclear. We hypothesized that hypoxia promotes expression of IL-33 and its receptor ST2 on vascular endothelial cells, which in turn leads to dysfunction of vascular endothelial cells and smooth muscle cells contributing to PH. Immunohistochemistry showed that immunoreactivity for IL-33 and ST2 was significantly increased in lung tissue of murine model of hypoxia-induced PH (HPH) and of subjects with bronchiectasis-PH. trans-Thoracic echocardiography showed that haemodynamic changes and right ventricular hypertrophy associated with HPH were significantly abrogated in St2^-/- compared with WT mice. Administration of IL-33 further exacerbated these changes in the hypoxia-exposed WT mice. In vitro, hypoxia significantly increased IL-33/ST2 expression by human pulmonary arterial endothelial cells (HPAECs), while exogenous IL-33 enhanced proliferation, adhesiveness and spontaneous angiogenesis of HPAECs. Knockdown of endogenous Il33 or St2 using siRNA transfection significantly suppressed these effects in both normoxic and hypoxic culture-conditions. Deletion of the St2 gene attenuated hypoxia-induced, elevated lung expression of HIF-1α/VEGFA/VEGFR-2/ICAM-1, while administration of exogenous VEGFA partially reversed the attenuation of the haemodynamic indices of PH. Correspondingly, knockdown of the St2 or Hif1α genes almost completely abrogated IL-33-induced expression of HIF-1α/VEGFA/VEGFR-2 by HPAECs in vitro. Further, IL-33-induced angiogenesis by HPAECs was extensively abrogated by knockdown of the Hif1α/Vegfa or Vegfr2 genes. These data suggest that hypoxia induces elevated expression of IL-33/ST2 by HPAECs which, at least partly by increasing downstream expression of HIF-1α and VEGF initiates vascular remodelling resulting in HPH.

Keywords: Hypoxia inducible factor 1; Hypoxic pulmonary hypertension; IL-33; Vascular endothelial growth factor.

Fig. 1

Increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) and elevated expression of IL-33 and ST2 immunoreactivity in lung sections from wild type mice exposed to normoxia(N4W)or to hypoxia (H4W) for 4 weeks. (A) RVSP and RVHI in murine models of hypoxia-induced murine PH (H4W) compared with control mice (N4W) (n = 6 each group). Data are presented as the mean ± SEM. ***p < 0.001. (B) Immunoreactivity for IL-33 and ST2 (brown) in murine lung tissue sections. (C) Western blot analysis of IL-33 and ST2 proteins (fold changes compared with β-actin) in lung tissue of murine models (n = 4–6 in each group). *p < 0.05.

Fig. 2

Elevated expression of IL-33 and ST2 in patients with PH. (A and B) Immunoreactivity for IL-33 and ST2 (brown) in lung tissues from patients with bronchiectasis with PH and normal controls (n = 3 for each group). The arrows show pulmonary artery endothelia cells which are immunoreactive for IL-33 and ST2. Data are presented as the mean ± SEM. **p < 0.01; *p < 0.05.

Fig. 3

Deletion of the St2 gene reversed hypoxia-induced PH while IL-33 aggravated hypoxic pulmonary vascular remodelling. (A) Comparison of pulmonary vascular remodelling in wild type and St2^−/− mice under conditions of normoxia and hypoxia: (top left panel) H&E staining of pulmonary arterioles; (bottom left panel) Immunofluorescent staining of α-SMA showing pulmonary arterioles; (right) Percentage medial thicknesses (%MT) of pulmonary arterioles categorised by external diameter into four groups (0–25 μm, 26–50 μm, 51–75 μm and 76–100 μm). ***p < 0.001 (vs N4W-WT: WT mice exposed to normoxia for 4 weeks); ^###p < 0.001 (vs H4W-WT: WT mice exposed to hypoxia for 4 weeks) (mean ± SEM of all arterioles in the entire left lung sections, n = 6 mice in each category). (B) Sections of cardiomyocytes from wild type and St2^−/− mice exposed to normoxia and hypoxia. Cardiomyocyte hypertrophy was evident in the right ventricle (left panel), but not the left ventricle (right panel). ***p < 0.001 (vs WT mice under normoxia), ^###p < 0.001 (vs WT mice under hypoxia) (data show mean ± SEM of the diameter of 200 cardiomyocytes per section, n = 6 mice in each category). (C) RVSP, RVHI and RV/body weight ratio of wild type and St2^−/− mice exposed to normoxia compared to hypoxia. *** p < 0.001 (vs wild type mice under normoxia), ^###p < 0.001 (vs wild type mice under hypoxia) (mean ± SEM, n = 6–10 each group). (D) Administration of IL-33 aggravated hypoxic pulmonary vascular remodelling. (left panel) RVSP and RVHI of wild type mice exposed to hypoxia with or without IL-33. (middle panel) H&E staining of pulmonary arterioles from mice under hypoxia with or without exogenous IL-33. (right panel) %MT of pulmonary arterioles of mice exposed to hypoxia with or without exogenous IL-33 grouped by external diameter (mean ± SEM of all arterioles in the entire left lung sections, n = 6 mice in each group). ***p < 0.001, **p < 0.01.

Fig. 4

Effects of exogenous IL-33 or of depletion of ST2 on RV wall thickness and the peak velocity of blood flow. IL-33 aggravates hypoxic pulmonary vascular remodelling. Magnetic resonance imaging (MRI) and M-mode trans-thoracic echocardiography showing typical cross sectional images of the heart (A), the thickness of the RV wall (B) and the PA peak flow velocity (C) in mice exposed to normobaric hypoxia concomitantly with exogenous IL-33 or normal saline for 4 weeks. Typical images showing changes of thickness of the RV wall (D, as indicated by arrow) and PA peak velocity (E) in WT and St2^−/− mice exposed to normoxia or hypoxia. The results were measured by M-mode trans-thoracic echocardiography.

Fig. 5

Effects of exposure to hypoxia on expression of IL-33 by human pulmonary arterial endothelial cells (HPAECs) and effects of exogenous IL-33 on HPAECs. (A) Hypoxia-induced expression of IL-33 mRNA (left) and protein (right) by HPAECs (n = 4). *p < 0.05, **p < 0.01. (B) Effects of IL-33 on the cell cycle positions and DNA synthetic activities of HPAECs determined by analysing total DNA and incorporated BrdU using flow cytometry. The cells were treated with or without IL-33 for 24 h under normoxia. Gate P3 represents cells in G0/G1 phase, P4 is S phase, P5 is G2 + M phase, and P6 is apoptotic cells. *p < 0.05, n = 3. (C) (left panel) Adhesion of HPAECs to culture plate wells in the presence of various concentrations of IL-33 under conditions of normoxia or hypoxia. Adherent cells were counted and expressed as the numbers of cells per high power field (5 wells for each group, and 6 HPFs in each well were counted). ***p < 0.001, ^###p < 0.001. (right panel) Adhesion of HPAECs following IL-33 gene knock down by siRNA under conditions of normoxia or hypoxia (n = 5). **p < 0.01, ***p < 0.001. (D) (left panel) Spontaneous formation of capillaries (angiogenesis) by HPAECs cultured with IL-33 at various concentrations under conditions of normoxia or hypoxia. (right panel) Angiogenesis by HPAECs following IL-33 knock down by siRNA under conditions of normoxia or hypoxia. The results are expressed as the mean total tube lengths (percentages of medium control values) and numbers of tubes per field, respectively (n = 6). **p < 0.01, ***p < 0.001, ^###p < 0.001.

Fig. 6

IL-33 regulates the function of HPAECs via ST2. (A) Effects of hypoxia on expression of St2 mRNA (left) and protein (right) by HPAECs (n = 4). *p < 0.05, **p < 0.01. (B) Effects of knock down of ST2 expression with siRNA on hypoxia- and IL-33-induced adhesion of HPAECs (mean ± SEM, n = 5). The results are expressed as numbers of cells per high power field. ***p < 0.001, ^###p < 0.001. (C) Effects of knock down of ST2 expression with siRNA on hypoxia- and IL-33-induced angiogenesis by HPAECs (n = 6). The results are expressed as the mean total tube lengths (percentages of medium control values) and numbers of tubes per field, respectively. ***p < 0.001, ^###p < 0.001.

Fig. 7

Effects of co-culture with HPAECs on proliferation and migration of HPASMCs in the presence/absence of IL-33. (A) IL-33 alone did not affect proliferation of HPASMCs, while positive control PDGF promoted the proliferation of HPASMCs (n = 6 each group). ***p < 0.001, ^###p < 0.001. (B) The effect of IL-33 on proliferation of HPASMCs (labelled SMC) when co-cultured with HPAECs (labelled EC) (n = 6 each group). ***p < 0.001, ^###p < 0.001. (C) The effect of IL-33 on migration of HPASMCs (labelled SMC) when co-cultured with HPAECs (labelled EC) (n = 6 each group). ***p < 0.001.

Fig. 8

Attenuated hypoxia-induced pulmonary vascular remodelling in St2^−/− mice reflects reduced expression of HIF-1α/VEGFA/VEGFR-2. (A) Western blot analysis of HIF-1α, VEGFA, VEGFR-2 and ICAM-1 in lung tissues of WT and St2^−/− mice following exposure to normoxia and hypoxia in vivo (n = 3–7). *p < 0.05, **p < 0.01. (B) RVSP, RVHI and RV/body weight ratios of WT mice exposed to normoxia and hypoxia, and St2^−/− mice exposed to hypoxia with or without exogenous VEGFA (n = 6–8 each group). *p < 0.05, **p < 0.01, ***p < 0.001, ^##p < 0.01, ^###p < 0.001. (C) Pulmonary vascular remodelling in WT mice exposed to normoxia and hypoxia, and St2^−/− mice exposed to hypoxia with or without exogenous VEGFA (n = 4 each group). (left) H&E staining of pulmonary arterioles. (right) Percentage medial thickness (%MT) of pulmonary arterioles grouped according to external diameter (μm). **p < 0.01, ***p < 0.001, ^###p < 0.001.

Fig. 9

IL-33/ST2 enhances angiogenetic activity of HPAECs through the HIF-1α/VEGFA/VEGFR-2 axis. (A) Western blot analysis of HIF-1α, VEGFA and VEGFR-2 protein expression in HPAECs incubated with exogenous IL-33 (10 ng/mL) or medium control following transfection with siRNA encoding ST2 or control (n = 4–7). *p < 0.05, **p < 0.01, ^#p < 0.05, ^##p < 0.01. (B) Western blot analysis of VEGFA and VEGFR-2 protein expression in HPAECs treated with exogenous IL-33 following transfection with siRNA encoding HIF-1α or control (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001. (C) Angiogenetic ability of HPAECs treated with exogenous IL-33 following transfection with siRNAs encoding HIF-1α, VEGFA or VEGFR-2 (n = 6). The results are expressed as the mean total tube lengths (percentages of control values) and numbers of tubes per field. ***p < 0.001, ^###p < 0.001.