Endothelial overexpression of TGF-β-induced protein impairs venous thrombus resolution

Endothelial cells play a critical role during venous thrombus remodeling, and unresolved, fibrotic thrombi with irregular vessels obstruct the pulmonary artery in patients with chronic thromboembolic pulmonary hypertension (CTEPH). This study sought to identify endothelial mediators of impaired venous thrombus resolution and to determine their role in the pathogenesis of the vascular obstructions in patients with CTEPH. Endothelial cells outgrown from pulmonary endarterectomy specimens (PEA) were processed for mRNA profiling, and nCounter gene expression and immunohistochemistry analysis of PEA tissue microarrays and immunoassays of plasma were used to validate the expression in CTEPH. Lentiviral overexpression in human pulmonary artery endothelial cells (HPAECs) and exogenous administration of the recombinant protein into C57BL/6J mice after inferior Vena cava ligation were employed to assess their role for venous thrombus resolution. RT2 PCR profiler analysis demonstrated the significant overexpression of factors downstream of transforming growth factor beta (TGFβ), that is TGFβ-Induced Protein (TGFBI or BIGH3) and transgelin (TAGLN), or involved in TGFβ signaling, that is follistatin-like 3 (FSTL3) and stanniocalcin-2 (STC2). Gene expression and immunohistochemistry analysis of tissue microarrays localized potential disease candidates to vessel-rich regions. Lentiviral overexpression of TGFBI in HPAECs increased fibrotic remodeling of human blood clots in vitro, and exogenous administration of recombinant TGFBI in mice delayed venous thrombus resolution. Significantly elevated plasma TGFBI levels were observed in patients with CTEPH and decreased after PEA. Our findings suggest that overexpression of TGFBI in endothelial promotes venous thrombus non-resolution and fibrosis and is causally involved in the pathophysiology of CTEPH

p53 Target Gene SMAR1 Is Dysregulated in Breast Cancer: Its Role in Cancer Cell Migration and Invasion

Tumor suppressor SMAR1 interacts and stabilizes p53 through phosphorylation at its serine-15 residue. We show that SMAR1 transcription is regulated by p53 through its response element present in the SMAR1 promoter. Upon Doxorubicin induced DNA damage, acetylated p53 is recruited on SMAR1 promoter that allows activation of its transcription. Once SMAR1 is induced, cell cycle arrest is observed that is correlated to increased phospho-ser-15-p53 and decreased p53 acetylation. Further we demonstrate that SMAR1 expression is drastically reduced during advancement of human breast cancer. This was correlated with defective p53 expression in breast cancer where acetylated p53 is sequestered into the heterochromatin region and become inaccessible to activate SMAR1 promoter. In a recent report we have shown that SMAR1 represses Cyclin D1 transcription through recruitment of HDAC1 dependent repressor complex at the MAR site of Cyclin D1 promoter. Here we show that downmodulation of SMAR1 in high grade breast carcinoma is correlated with upregulated Cyclin D1 expression. We also established that SMAR1 inhibits tumor cell migration and metastases through inhibition of TGFβ signaling and its downstream target genes including cutl1 and various focal adhesion molecules. Thus, we report that SMAR1 plays a central role in coordinating p53 and TGFβ pathways in human breast cancer

Protein Tyrosine Phosphatase 1B Deficiency in Vascular Smooth Muscle Cells Promotes Perivascular Fibrosis following Arterial Injury

Background Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury. Methods Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ERT2 recombinase under the Myh11 promoter with PTP1B(flox/flox) mice and subjected to FeCl3 carotid artery injury. Results Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA(+) and PDGFR beta(+) myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11-Cre reporter cells in the remodeling adventitia, and SCA1(+) CD45(-) vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor beta (TGF beta) signaling components or enzymes involved in extracellular matrix remodeling and TGF beta liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFR beta and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion. Conclusion Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription

EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia

Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis