[PubMed] [Google Scholar] 13. the ERK/MAPK signalling pathway can reduce the appearance of VSMCs osteogenic phenotype. Our findings may gamma-secretase modulator 2 present a novel therapeutic approach to prevent of vascular calcification after venous thrombosis. test for unpaired means. Fisher’s exact test was used to analyse the differential expression of osteogenic markers in human specimens. A value 0.05 was considered statistically significant. 3.?RESULTS 3.1. Venous calcification in human thrombosis vein walls During the treatment, we in the beginning observed that obvious calcification spots could be detected in the venous wall from CTV of PTS patients (Physique?1A), suggesting that this calcified remodelling of the venous wall may Rabbit Polyclonal to GIMAP2 be involved in the occurrence and development of PTS. We collected human thrombotic and non\thrombotic superficial vein wall specimens after venous thrombosis 0.5 to 2?months, during which patients exhibited a high incidence of vessel wall remodelling. In order to clarify whether venous thrombosis gamma-secretase modulator 2 could promote the appearance of venous wall calcification, we measured calcium deposition in the venous wall by using Alizarin Red S staining. Compared with the non\thrombotic vein wall, calcium deposition was significantly increased in the thrombotic vein gamma-secretase modulator 2 wall (Physique?1B). In addition, unlike the arterial calcification that mainly manifests as intima or medial calcification, the site of calcium deposition within the venous wall after thrombosis was mainly gathered at the media and adventitia, and the spotty calcification in the media presented spindle shape, similar to the morphology of VSMCs (black arrows). We assumed that calcium deposition was caused by VSMCs, which subsequently gave rise to the decreased compliance of the vein wall and further exacerbated damage to the wall vein after thrombosis. Open in a separate window Physique 1 Venous calcification in human thrombosis vein walls. A: Representative CTV images of venous calcification of PTS patients. (A, B) were coronal images. (C, D) were axial images. Red arrows marked arterials and blue arrows signed the veins. As visualization of the deep vein adjacent to the arterial of image b was obscure, no indicators were marked the deep vein. The yellow arrows indicated venous calcification. The green lines marked the section site of axial plane in coronal images. A: anterior, P: posterior, L: left, R: right. B: aCc and dCg showed Alizarin Red S staining of non\thrombotic (as internal comparison) and thrombotic venous wall in different magnification, respectively. h represented Alizarin Red S staining of strong vein wall tissue, as a negative control. I: intimal, M: medial, A:?adventitial 3.2. VSMCs are involved in venous calcification in patients with venous thrombosis To explore the molecular mechanism related to calcification of the post\thrombotic venous wall, we applied microarray\based gene expression analysis to compare the differentially expressed genes in 4 pairs of human thrombotic and non\thrombotic superficial veins specimens and specifically analysed the expression of genes that are associated with the osteogenesis process. The heatmap showed that a a part of genes regulating the osteogenesis process was changed (Physique?2A). To verify the heatmap results, we investigated changes in osteogenesis\related genes of the vein wall in the mRNA level and found osteogenic transcription factors (and and and (and and and in the SB group, as well as and (Physique?4A). Consistent with the mRNA results, the immunofluorescence showed the expression of Runx2 went up in the SB group (Physique?4B). The present results strongly suggested that VSMCs dedifferentiate and transform into osteoblast\like cells. So far, we have verified that by activating the TNF signalling pathway and inhibiting the TGF signalling pathway, VSMCs could be effectively induced by dedifferentiation into an osteogenic phenotype. Furthermore, Alizarin Red S staining showed that the calcium deposit was affected in the SB group (Physique?4C). Taken together, these results exhibited that stimulating VSMCs with TNF can reduce contractile phenotypic markers expression, subsequently inhibiting the TGF signalling pathway to promote VSMCs to gain osteogenic phenotype markers and to accelerate the rate of calcium deposition. It was suggested that this combined effect of TGF and TNF signalling pathways might be an important regulatory factor for the transformation of VSMCs to osteoblast\like cells in the early stage of PTS. Open in a separate window Physique 4 Expressions and distributions of osteogenic markers by TNF activation and TGF downregulation in rat VSMCs. (A) qPCR was performed for VSMCs osteogenic markers (and and and and and and and em Ocn /em ) showed an opposite pattern (Physique?5D). We concluded that after the activation.