IL-33/ST2 receptor-dependent signaling in the development of pulmonary hypertension in Sugen/hypoxia mice

Abstract

Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH is characterized by pulmonary artery remodeling, elevated right ventricular pressure (RVP) and, ultimately, cardiac failure. Pulmonary endothelial cells can sense danger or damage caused by mechanical injury or pathogens through alarmin cytokines. These cytokines can signal proliferation to restore barrier integrity or aberrant hyperproliferation and remodeling. We hypothesized that IL-33 signals pulmonary artery endothelial cells to proliferate under hypertensive conditions during the remodeling response and rise in RVP. To test this hypothesis, pulmonary hypertension (PH) was induced in C57Bl/6J, IL-33 receptor gene deleted (ST2^-/- ) and MYD88 gene deleted (MYD88^-/- ) mice by exposure to 10% O₂ and SU5416 injections (SUHX). RVP, arterial wall thickness, endothelial cell proliferation and IL-33 levels and signaling were evaluated. In response to SUHX. RVP increased in C57Bl/6J mice in response to SUHX (49% male and 70% female; p < 0.0001) and this SUHX response was attenuated in ST2^-/- mice (29% male p = 0.003; 30% female p = 0.001) and absent in MYD88^-/- mice. Wall thickness was increased in SUHX C57Bl/6J mice (p = 0.005), but not in ST2^-/- or MYD88^-/- mice. Proliferating cells were detected in C57Bl/6J mice by flow cytometry (CD31⁺ /BrDU⁺ ; p = 0.02) and immunofluorescence methods (Ki-67+). IL-33 was increased by SUHX (p = 0.03) but a genotype effect was not observed (p = 0.76). We observed that in hPAECs, IL-33 expression is regulated by both IL-33 and DLL4. These data suggest IL-33/ST2 signaling is essential for the endothelial cell proliferative response in PH.

Keywords: endothelial cells; interleukin-33 receptor; pulmonary hypertension.

FIGURE 1

Pulmonary hypertension parameters in SUHX mice. Developed right ventricular pressures in (a) C57Bl/6J, (b) ST2^−/−, and (c) MYD88^−/− mice in response to SUHX and DMSO/RA. Right ventricle contractility [(RV) + dP/dt] in response to SUHX and DMSO/RA treatments in (d) C57Bl/6J, (e) ST2^−/−, and (f) MYD88^−/− mice. Right ventricle relaxation [(RV) − dP/dt] in response to SUHX and DMSO/RA in (g) C57Bl/6J, (h) ST2^−/−, and (i) MYD88^−/− mice. Values are presented as mean ± SD (n = 15 male, n = 8 female DMSO/RA; n = 14 male, n = 6 female SUHX). *Significant difference between SUHX and DMSO/RA within a sex (p < 0.05)

FIGURE 2

Pulmonary vascular remodeling‐wall thickness in the small arteries (<50 μm in diameter) is shown in lung sections stained with hematoxylin and eosin (a) C57Bl/6J, (b) ST2^−/− and (c) MYD88^−/− mice under DMSO/RA and SUHX treatments. Quantitation of wall thickness in (d) C57Bl/6J, (e) ST2^−/− and (f) MYD88^−/− under DMSO/RA and SUHX conditions. Values are presented as mean ± SD (n = 9–14 DMSO; n = 14–17 SUHX). **Statistical significance between DMSO/RA and SUHX treatments within each genotype, p < 0.05

FIGURE 3

Vascular remodeling in small arteries and arterioles. Endothelial cell proliferation was evaluated by immunohistochemical and flow cytometry analysis of proliferating CD31⁺ cells. The percentage of proliferating endothelial cells (% CD31⁺/BrDU⁺) in (a) C57Bl/6J and (b) ST2^−/− mice under DMSO/RA and SUHX conditions. Localization of proliferating (Ki‐67) cells detected in SMA positive small arteries of (c) C57Bl/6J, (d) ST2^−/−, and (e) MYD88^−/− mice under DMSO/RA and SUHX conditions. Scale bars represent 50 μm. Values are presented as the means ± SD (n = 5–15 DMSO/RA, n = 12–17 SUHX). **Statistical significance between DMSO/RA and SUHX treatments, p < 0.01

FIGURE 4

IL‐33 protein levels in whole lungs of mice exposed to SUHX (a) Western Blot representation of IL‐33, ST2, and MYD88 protein in lungs of C57BL/6J, ST2^−/−, and MYD88^−/− mice exposed to either SUHX or DMSO/RA conditions (all n = 8). Equal loading of protein was confirmed using α‐Tubulin. (b) Quantitative analysis of IL‐33 in whole lungs. Values are presented as the means ± SD

FIGURE 5

Endothelial IL‐33 autocrine expression in the presence of Notch ligand (DLL4). IL‐33 message (a) and protein levels (b) in human pulmonary artery endothelial cells after a 48 h response to exogenous IL‐33, culture on a gelatin matrix or gelatin/DLL4 matrix. Experiments were performed in triplicates with each triplicate repeated three times. Values are presented as the means ± SD. *Statistical significance between gelatin and IL‐33 and/or DLL4 treatments, p < 0.05