The six-transmembrane protein Stamp2 ameliorates pulmonary vascular remodeling and pulmonary hypertension in mice

Abstract

Six-transmembrane protein of prostate (Stamp2) protects from diabetes and atherosclerosis in mice via anti-inflammatory mechanisms. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PAH), we investigated the role of Stamp2. Stamp2 expression was substantially reduced in the lung of humans with idiopathic PAH, as well as in experimental PAH. In Stamp2-deficient mice, hypoxia modestly aggravated pulmonary vascular remodeling and right ventricular pressure compared to WT. As endothelial cell (EC) and pulmonary arterial smooth muscle cell (PASMC) phenotypes drive remodeling in PAH, we explored the role of Stamp2. Knock-down of Stamp2 in human EC neither affected apoptosis, viability, nor release of IL-6. Moreover, Stamp2 deficiency in primary PASMC did not alter mitogenic or migratory properties. As Stamp2 deficiency augmented expression of inflammatory cytokines and numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages may drive vascular remodeling. Thus, PASMC responses were assessed following treatment with conditioned media of primary Stamp2^-/- or WT macrophages. Stamp2^-/- supernatants induced PASMC proliferation and migration stronger compared to WT. A cytokine array revealed CXCL12, MCP-1 and IL-6 as most relevant candidates. Experiments with neutralizing antibodies confirmed the role of these cytokines in driving Stamp2's responses. In conclusion, Stamp2 deficiency aggravates pulmonary vascular remodeling via cross-talk between macrophages and PASMC. Despite a substantial pro-inflammatory response, the hemodynamic effect of Stamp2 deficiency is modest suggesting that additional mechanisms apart from inflammation are necessary to induce severe PAH.

Keywords: Inflammation; Macrophages; Pulmonary hypertension; Stamp2; Vascular remodeling.

Fig. 1

Stamp2 expression is reduced in human and experimental PAH. a Immunoblot and densitometric analyses, demonstrating Stamp2 expression in lung tissue from hypoxia-challenged mice compared to normoxic control mice (n = 3,3). b Stamp2 mRNA expression in lung tissue of the above mentioned mice (n = 3,4,4). c Stamp2 protein expression and densitometric analysis (n = 4,4) and d mRNA expression in lung tissue of Sugen5416/hypoxia (SuHx)-treated rats compared to healthy control rats (n = 5,5). e Stamp2 expression in lung tissue from IPAH patients as compared to healthy donors (n = 10,8). f Immunohistochemical stainings demonstrating reduced Stamp2 expression in pulmonary vessels from IPAH patients and from experimental PAH versus controls (400 × magnification). All data represent means ± SD.*p < 0.05, ****p < 0.0001 as assessed by two-tailed students t-test

Fig. 2

Lack of Stamp2 augments hypoxia-induced PAH in mice. Pulmonary and systemic hemodynamics and pulmonary vascular remodeling of WT versus Stamp2-deficient mice exposed to hypoxia (10% O₂ for 21 days). a Immunohistochemical stainings of small pulmonary arteries from Stamp2^−/− and WT mice. Shown are representative images of lung sections immunostained for von Willebrand factor (vWF) (brown) and α-smooth muscle actin (purple) (400 × magnification). b Quantitative morphometric analysis of the muscularization of small (< 80 µm) pulmonary arteries. Shown is the percentage of fully (M), partially (P) and non-muscularized (N) vessels (analyses of at least 50/animal, n = 4,5,5,5). c Right ventricular systolic pressure (RVSP, [mmHg]), measured by Millar microtip catheters (1F) inserted into the right ventricle via the jugular vein (n = 7,9,9,20). d Right ventricular (RV) hypertrophy shown as RV/LV + S ratio (n = 7,9,9,20). e Systemic arterial pressure (SAP, [mmHg]) measured using a Millar microtip catheter (1F), inserted into the left carotid artery (n = 6,7,9,19). f Heart rate (HR, [bpm]) (n = 7,9,9,20). All data represent means ± SD. *p < 0.05, **p < 0.01, ***p < 0,001 as assessed by ANOVA or two-tailed students t-test

Fig. 3

Hypoxia promotes downregulation of Stamp2 expression in various cell types. Expression of Stamp2 a protein and b mRNA in human MVEC in response to 0, 24, 48, 72 h of hypoxia (1% O₂) (n = 4). Expression of Stamp2 protein (c) and mRNA (d) in hypoxia-exposed murine PASMC at the indicated time points (n = 3). Stamp2 expression protein (e) and mRNA (f) in murine macrophages (n = 3). All data represent means ± SD. *p < 0.05, **p < 0.01, as assessed by two-tailed students t-test

Fig. 4

Stamp2 deficiency/downregulation does not affect PASMC and MVEC responses. a BrdU incorporation in primary Stamp2-deficient and WT PASMC in response to FCS [10%] and IL-6 [15 ng/ml] (n = 6)) and b PASMC-migration in response to FCS [10%] (n = 5). c Apoptosis (n = 9) and d cellular viability (n = 6) of human MVEC transfected with either siRNA targeting Stamp2 or non-silencing siRNA for 48 h. All data represent means ± SD

Fig. 5

Stamp2 deficiency leads to increased pulmonary inflammation. a Representative immunohistochemical stainings demonstrating increased presence of CD68⁺ macrophages in the wall of small pulmonary vessels of normoxia- and hypoxia-exposed Stamp2-deficient mice versus WT controls (400 × magnification). b CD68⁺ vessels in percentage to the total number of vessels per defined area (60 × magnification) (n = 5,5,5,5). mRNA expression of c MCP-1, d TNF-α, e IL-1ß and f IL-6 in lung tissue from Stamp2-deficient and WT control mice after exposure to 0 days, 3 days or 3 weeks of hypoxia (n = 4,4,4,4). All data represent means ± SD of at least 4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 as assessed by two-tailed students t-test

Fig. 6

Macrophage supernatants of Stamp2-deficient mice promote PASMC proliferation and migration. a Proliferation (n = 4) and b migration of WT-PASMC in response to supernatants of primary, thioglycollate-elicited Stamp2-deficient or WT macrophages (MΦ) assessed by BrdU-incorporation and scratch assay (n = 3), respectively. Data are represented as means ± SD. *p < 0.05, **p < 0.01, as assessed by two-tailed students t-test. c Densitometric quantification of a cytokine array from Stamp2-deficient or WT MΦ supernatants (n = 2) d mRNA expression of CXCL12 in lung tissue from Stamp2-deficient and WT mice after exposure to 0 days, 3 days or 3 weeks of hypoxia (n = 3). All data represent means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 as assessed by two-tailed students t-test

Fig. 7

CXCL12, MCP-1 and IL-6 mediate cellular cross-talk between macrophages and PASMC. Migration (scratch assay) of WT-PASMC in response to a IL-6 (100 ng/ml) alone or in combination with an IL-6-neutralizing antibody (NAB) (n = 4), b CXCL12 (100 ng/ml) alone or with a CXCL12-NAB (n = 4) c MCP-1 (100 ng/ml) alone or with a MCP-1-NAB (n = 4,) d IL-6 (100 ng/ml) + CXCL12 (100 ng/ml) + MCP-1 (100 ng/ml) alone and in combination with their corresponding NABs (n = 4,4,3,3). e BrdU incorporation of PASMC in response to MCP-1 (100 ng/ml) and CXCL12 (100 ng/ml) alone and together with their corresponding NABs or with combined MCP-1 (100 ng/ml) and CXCL12 (100 ng/ml) either alone or with both NABs (n = 5). f BrdU incorporation (n = 5) of PASMC in response to macrophage supernatants of thioglycollate-elicited Stamp2-deficient primary macrophages in the absence and presence of MCP-1- or CXCL12-neutralizing antibodies (NAB). g Migration (scratch assay) of WT PASMC in response to supernatants of thioglycollate-elicited Stamp2-deficient primary macrophages (MΦ) in the absence or presence of NAB against CXCL12 + MCP-1 + IL-6 (n = 3). Data are represented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 as assessed by two-tailed students t-test