Endothelial to mesenchymal transition via transforming growth factor-beta1/Smad activation is associated with portal venous stenosis in idiopathic portal hypertension

Abstract

Idiopathic portal hypertension (IPH) represents noncirrhotic portal hypertension of unknown etiology, mainly due to stenosis of peripheral portal veins. This study was performed to clarify the mechanism of portal venous stenosis in IPH from the viewpoint of the contribution of the endothelial to mesenchymal transition of the portal vein endothelium via transforming growth factor-beta1 (TGF-beta1)/Smad activation. In vitro experiments using human dermal microvascular endothelial cells demonstrated that TGF-beta1 induced myofibroblastic features in human dermal microvascular endothelial cells, including spindle cell morphology, reduction of CD34 expression, and induction of S100A4, alpha-smooth muscle actin, and COL1A1 expression, as well as the increased nuclear expression of phospho-Smad2. Bone morphogenic protein-7 preserved the endothelial phenotype of human dermal microvascular endothelial cells. Immunohistochemical analysis showed that endothelial cells of the peripheral portal veins in IPH were characterized by the decreased expression of CD34 and the enhanced nuclear expression of phospho-Smad2; these results also confirmed the expression of S100A4 and COL1A1 in the portal vein endothelium. Serum TGF-beta1 levels in patients with IPH were significantly higher than those of healthy volunteers and patients with chronic viral hepatitis/liver cirrhosis, while an elevation of serum bone morphogenic protein-7 levels was not observed. These results suggest that the endothelial to mesenchymal transition of the portal venous endothelium via TGF-beta1/Smad activation is associated with portal venous stenosis in IPH, and bone morphogenic protein-7 may therefore be a suitable therapeutic candidate for IPH.

Figure 1

Effects of TGF-β1 and BMP7 on cellular phenotype of HMVECs at the mRNA level. HMVECs were treated with TGF-β1 (10 ng/ml) alone or in combination with BMP7 (100 ng/ml) for 5 days, and phenotypic changes were examined using RT-PCR as described in the Materials and Methods. HMVECs expressed receptors for TGF-β (TβR-I, TβR-II) and BMP7 (BMP receptor type IA, BMP type II receptor) (A). RT-PCR (B) and real-time PCR (C) showed that treatment of HMVECs with TGF-β1 reduced the expression of CD34 mRNA, and induced the mRNA expression of S100A4, α-SMA, and COL1A1. All of these changes of HMVECs following TGF-β1 treatment were inhibited by the addition of BMP7 in the culture medium (B, C). The data represent three independent experiments (A, B), and the mean ± SD of six per group (C). *P < 0.01; **P < 0.05.

Figure 2

Effects of TGF-β1 and BMP7 on cellular phenotype of HMVECs at the protein level. HMVECs were treated with TGF-β1 (10 ng/ml) alone or in combination with BMP7 (100 ng/ml) for 5 days, and phenotypic changes were examined by Western blotting using protein extracts from HMVEC. Consistent with the RT-PCR results, TGF-β1 reduced the expression of CD34 protein, and induced the protein expression of S100A4, α-SMA and COL1A1, and the addition of BMP7 inhibited these phenotypic changes (A). Semiquantitative analysis of the Western blotting confirmed this tendency (B). BMP7 inhibited TGF-β1-induced COL1A1 protein expression in a dose-dependent manner (C). The data represent five independent experiments (A), and the mean ± SD of five per group (B, C). *P < 0.01; **P < 0.05.

Figure 3

Morphological and phenotypic alterations of HMVECs by TGF-β1 and BMP7. HMVECs grew in a form of epithelioid, sheet-like appearance under the phase-contrast microscopy, and a 5-day treatment with TGF-β1 (10 ng/ml) changed the cellular morphology of HMVECs from epithelioid into spindle-shaped appearance. Immunostaining showed that the spindle-shaped HMVECs following TGF-β1 treatment showed reduced expression of CD34, and increased expression of S100A4, α-SMA, COL1A1, and pSmad2. Addition of BMP7 (100 ng/ml) in the culture medium inhibited the phenotypic changes of HMVECs by TGF-β1. The protein expression was visualized by a Vector Red reaction under immunofluorescence confocal microscopy, and nuclei were stained with 4′6-diamidino-2-phenylindole (blue). Original magnification ×1000.

Figure 4

Reduction of CD34 expression and induction of pSmad2 expression in portal vein endothelium of IPH livers. Immunostaining of TGF-β receptors (TβR-I, TβR-II), CD34 and pSmad2 was performed for liver sections of normal liver (NL), chronic viral hepatitis/liver cirrhosis (CVH/LC) and idiopathic portal hypertension (IPH). TβR-I and TβR-II were diffusely expressed in the portal vein endothelium of IPH (A), as well as NL and CVH/LC. Endothelial cells of peripheral portal vein of IPH showed reduced expression of CD34, and increased expression of pSmad2 (B). The CD34-reduction index and the pSmad2-labeling index of portal vein endothelium were determined as described in the Materials and Methods section. Portal vein endothelium of IPH showed a significant reduction of CD34 expression (C) and a significant induction of pSmad2 (D) when compared with those of NL and CVH/LC. The data of white circles represent those from the cases of IPH complicated with systemic sclerosis (C, D). The CD34-reduction index and the pSmad2-labeling index showed a fine liner correlation (E). Arrowheads indicate nuclei of pSmad2-positive portal vein endothelium (B). HA, hepatic artery. PV, portal vein. White bars = 50 μm; black bars = 20 μm. *P < 0.01.

Figure 5

Colocalization of CD34 and mesenchymal markers in portal vein endothelium of IPH livers. Double immunofluorescence staining of CD34 and S100A4 protein, and CD34 and COL1A1 was performed for liver sections of normal liver (NL), chronic viral hepatitis/liver cirrhosis (CVH/LC), and idiopathic portal hypertension (IPH). Figures shown were merged images in which the expression of CD34 was colored in red, and the expression of S100A4 and COL1A1 was colored in green. Nuclei were stained with 4′6-diamidino-2-phenylindole. Coexpression of CD34 and S100A4 was observed in portal vein endothelium of IPH (arrow), but the expression was limited in a small number of portal veins of IPH. Portal vein endothelium of NL and CVH/LC typically lacked double-positive signals of CD34 and S100A4. Portal vein endothelium of IPH occasionally showed colocalization of CD34 and COL1A1 (arrowheads). PV, portal vein. White bars = 30 μm.

Figure 6

Elevation of circulating TGF-β1 levels in IPH. The serum TGF-β1 and BMP7 levels of 66 samples obtained from 57 patients with idiopathic portal hypertension (IPH), 16 healthy volunteers, and 19 patients with chronic viral hepatitis/liver cirrhosis (CVH/LC) were determined using an enzyme-linked immunosorbent assay. Serum obtained from IPH patients contained a significantly high level of TGF-β1 when compared with that of healthy controls and CVH/LC (A), while the serum BMP7 level in patients with CVH/LC showed a significant increase compared with those of the other two groups (B). When the serum TGF-β1 level was plotted against the serum BMP7 level for all cases examined, a significant inverse correlation was observed between them (C). *P < 0.01; **P < 0.05.

Figure 7

Proposed mechanism of the portal venous stenosis of IPH. TGF-β1 induces endothelial to mesenchymal transition (EndMT) of endothelial cells of peripheral portal vein of idiopathic portal hypertension (IPH). Endothelial cells acquire myofibroblastic features via Smad activation, and produce extracellular matrix molecules including collagen. Collagen depositions in peripheral portal tracts compress the portal veins, resulting in portal venous stenosis and presinusoidal portal hypertension. BMP7 may act as an inhibitor of EndMT by antagonizing the effects of TGF-β1.