Interferon-γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)-1 and transforming growth factor (TGF) β2

Abstract

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN-α or IFN-γ downregulated Fli1 and VE-cadherin. In contrast, IFN-α and IFN-γ exerted opposite effects on the expression of α-SMA, CTGF, ET-1, and TGFβ2, with IFN-α downregulating and IFN-γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN-γ-mediated changes in Fli1, VE-cadherin, CTGF, and ET-1 levels, whereas upregulation of α-SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN-γ, including downregulation of α-SMA and TGFβ2, suggesting that activation of the ET-1 pathway plays a main role in the IFN-γ responses in HDMECs. IFN-γ induced expression of selected genes related to endothelial-to-mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN-γ on EndoMT was mediated via TGFβ2 and ET-1 signaling pathways. This study demonstrates distinct effects of IFN-α and IFN-γ on the biology of vascular endothelial cells. IFN-γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT.

Fig. 1

IFN-α and IFN-γ up-regulate the specific target genes in a dose- and time-dependent manner in HDMVECs. HDMVECs were stimulated with either IFN-α (A, B) or IFN-γ(C, D) at different dosage (50, 100, 500, 1, 000, and 2,000 U/ml) for 24 h (A, C) or at the concentration of100 U/ml for different timepoints (24, 48,72, 96, and 120 h) (B, D). mRNA expression was analyzed by real-time PCR. Values represent mean ± SEM from three independent cell lines. ^*P ≤0.05

Fig. 2

IFN-γ decreases Fli1 and VE-cadherin mRNA expression in HDMVECs. HDMVECs were stimulated with either IFN-α (A, B) or IFN-γ (C, D) at different dosage (50,100, 500, 1,000, and 2,000U/ml) for 24 h (A, C) or at the concentration of 100 U/ml for different timepoints (24, 48, 72,96, and 120 h) (B, D). Fli1 and VE-cadherin mRNA expression was analyzed by real-time PCR. Values are represented as mean± SEM (n=3). *P ≤ 0.05

Fig. 3

Distinct effects of IFN-α and IFN-γ on the profibrotic gene expression in HDMVECs. HDMVECs were stimulated with 100 U/ml of IFN-α (A)or 1,000 U/ml of IFN-γ(B), or left untreated (UT)for 24 h. The mRNA expression was determined by real-time PCR. Values are represented as mean ± SEM (n =5).^*P ≤0.05. Cell lysates were analyzed for the indicated proteins by Western blotting with GAPDH used as a loading control (C). The Western blot images are representative of the three independent experiments. ET-1 was measured by ELISA (D).

Fig. 4

TGFβ and ET-1 mediate the profibrotic effects of IFN-γ in HDMVECs. A, B: Human dermal microvascular endothelial cells were pretreated for 1 h with either the α-TGFβ1/2/3 neutralizing antibody (α-T) or the inhibitor of TGFβ type I receptor (ALK5) (SB431542) at the concentration of 2.5 μg/ml and 20 μM, respectively, followed by the stimulation with IFN-γ(1,000 U/ml) or TGFβ1 (1 ng/ml) for 24 h. Fli1, VE-cadherin, SMA, CTGF, ET-1, and TGFβ2 gene expression was analyzed by real-time PCR. C: HDMVECs were pretreated for 3 h with dual endothelin receptor antagonist (Bosentan) at the concentration of 10 μM, followed by the stimulation with IFN-γ(1,000 U/ml) for 24 h, or left untreated (UT). Fli1, VE-cadherin, SMA, CTGF, ET-1, and TGFβ2 gene expression was analyzed by real-time PCR. Values represent mean ± SEM (n =6 or n =3). ^*P ≤0.05. D: Western blot images are representative of the three independent experiments. E: Average protein was calculated using ImageJ densitometry software. Values represent mean ± SEM (n =3).^#P ≤ 0.05 when compared to UT.^*P≤0.05 when compared SB +TGFβ1 to TGFβ1. ^**P ≤0.05 when compared SB +IFN-γ and Bos +IFN-γ to IFN-γ.

Fig. 5

IFN-γ-activated HDMVECs induce matrix production in fibroblasts HDMVECs were stimulated with IFN-α (100 U/ml), IFN-γ(1,000 U/ml), or TGFβ1 (1 ng/ml) for 12 h, followed by media change and incubation in the fresh starvation media (0.1% BSA) for 36 h. The conditioned media from three independent HDMVECs (CM 1–3) were collected and added to the confluent human dermal fibroblasts for 24 h. A: The mRNA expression of the indicated profibrotic genes was assessed in stimulated fibroblasts by real-time PCR. Values represent mean ± SEM ^*P ≤0.05 (n =3). B: Collagen secretion in fibroblasts stimulated with CM 1–3 was examined by Western blotting. Bottom part (ctrl) shows collagen secretion in fibroblasts stimulated directly with IFN-α (100 U/ml), IFN-γ (1,000 U/ml), or TGFβ1 (1 ng/ml) for 24 h.

Fig. 6

IFN-γ regulates selected genes involved in the process of EndoMT via upregulation of ET-1 and TGFβ2 signaling pathway (A) cDNA obtained from the IFN-γ-stimulated HDMVECs (1,000 U/ml) for 24 h was subjected to the EMT Profiler PCR Array System (SABiosciences). Gene validation was performed by real-time PCR. Values represent mean ± SEM from three experiments. ^*P ≤0.05. B: Immunofluorescence staining of VE-cadherin (green) and Phalloidin-F-actin (red) in HDMVECs treated for 48 h with 1,000 U/ml of IFN-γ, 1 ng/ml of TGFβ1 or left untreated (UT). Representative photographs are shown from three experiments. C: HDMVECs were pretreated for 1 h with either ALK5 kinase inhibitor (SB431542) or dual endothelin receptor antagonist (Bosentan) at the concentration of 2.5 μg/ml, 20 μM, and 10 μM, respectively, followed by the stimulation with IFN-γ(1,000 U/ml) or TGFβ1 (1 ng/ml) for 24 h. Selected EMT-specific genes were analyzed by real-time PCR. Values represent mean ± SEM (n =3–6). ^#P ≤0.05 when compared to UT. ^*P ≤0.05 when compared SB +TGFβ1 to TGFβ1. ^**P ≤0.05 when compared SB +IFN-γ and Bos +IFN-γ to IFN-γ. D, E: Immunofluorescence staining of VE-cadherin (green) and Phalloidin-F-actin (red) in HDMVECs pretreated for 1 h with SB431542 or bosentan at the concentration of 20 μM and 10 μM, respectively, followed by the stimulation with IFN-γ(1,000 U/ml) or TGFβ2 (1 ng/ml) for 48 h or left untreated (UT).