Wang, Ying

Abstract Vascular smooth muscle cells (VSMCs) in the normal arterial media continually express contractile phenotypic markers which are reduced dramatically in response to injury. Tripartite motif‐containing proteins are a family of scaffold proteins shown to regulate gene silencing, cell growth, and differentiation. We here investigated the biological role of tripartite motif‐containing 28 (TRIM28) and tripartite motif‐containing 27 (TRIM27) in VSMCs. We observed that siRNA‐mediated knockdown of TRIM28 and TRIM27 inhibited platelet‐derived growth factor (PDGF)‐induced migration in human VSMCs. Both TRIM28 and TRIM27 can regulate serum response element activity and were required for maintaining the contractile gene expression in human VSMCs. At the same time, TRIM28 and TRIM27 knockdown reduced the expression of PDGF receptor‐β (PDGFRβ) and the phosphorylation of its downstream signaling components. Immunoprecipitation showed that TRIM28 formed complexes with TRIM27 through its N‐terminal RING‐B boxes‐Coiled‐Coil domain. Furthermore, TRIM28 and TRIM27 were shown to be upregulated and mediate the VSMC contractile marker gene and PDGFRβ expression in differentiating human bone marrow mesenchymal stem cells. In conclusion, we identified that TRIM28 and TRIM27 cooperatively maintain the endogenous expression of PDGFRβ and contractile phenotype of human VSMCs.

ABSTRACT Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aβ peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aβ1‐42 oligomers can readily enter a senescence phenotype. To determine the effect of Aβ oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aβ1‐42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aβ1‐42 monomers, and 5 µM Aβ1‐42 fibrils, respectively. Brain ECs treated with Aβ1‐42 oligomers showed increased senescence‐associated β‐galactosidase staining and increased senescence‐associated p21/ p53 expression. Treatment with either Aβ1‐42 monomer or Aβ1‐42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aβ1‐42 oligomer‐treated ECs, and these cells showed significantly increased VEGFR‐1 expression and decreased VEGFR‐2 levels. Overexpression of VEGFR‐1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR‐1 signaling events in this paradigm. More importantly, small interfering RNA‐mediated knockdown of VEGFR‐1 expression in brain ECs was able to prevent up‐regulation of p21 protein expression and significantly reduced induction of senescence following Aβ1‐42 oligomer treatment. Our studies show that exposure to Aβ1‐42 oligomers may impair vascular functions by altering VEGFR‐1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR‐1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.—Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor‐1 modulates amyloid β 1–42 oligomer‐induced senescence in brain endothelial cells. FASEB J. 33, 4626–4637 (2019). www.fasebj.org

ABSTRACT Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin‐1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA–mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen‐inducible endothelial cell–specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR‐219‐5p in HUVECs. Although the endothelial cell–specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low‐dose angiotensin II infusion–induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low‐dose angiotensin II–induced increases in blood pressure.—Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin‐1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II–induced hypertension. FASEB J. 33, 494–500 (2019). www.fasebj.org

Mitochondria are dynamic organelles that are able to change their morphology and cellular distribution by either fission or fusion. However, the molecular mechanisms controlling mitochondrial dynamics in vascular endothelial cells (ECs) remain largely unknown. In this study, we observed that knockdown of microtubule‐associated tumor suppressor 1 (MTUS1) in ECs inhibited tube formation and migration, accompanied with decreased promigratory signalings. We showed that MTUS1 was localized in the outer membrane of mitochondria in ECs. Knockdown of MTUS1 disturbed the elongated mitochondrial network and induced the formation of perinuclear clusters of mitochondria. Importantly, mitochondrial motility and fusion were suppressed, whereas generation of reactive oxygen species was increased in MTUS1 knockdown ECs. Mechanistically, we showed that the N‐terminal coiled‐coil domain of MTUS1 interacted with the mitochondrial membrane proteins, mitofusin‐1 and mitofusin‐2, to maintain mitochondrial morphology in ECs. This study illustrated a novel role of MTUS1 in mitochondrial morphology and EC angiogenic responses.—Wang Y., Huang Y., Liu, Y., Li, J., Hao, Y., Yin, P., Liu, Z., Chen J., Wang Y., Wang N., Zhang P., Microtubule associated tumor suppressor 1 interacts with mitofusins to regulate mitochondrial morphology in endothelial cells. FASEB J . 32, 4504–4518 (2018). www.fasebj.org

ABSTRACT Angiogenesis and inflammation are regarded as important factors in the pathogenesis of chronic inflammation, cancer, and wound healing. Recent studies have supported prior evidence that common signaling pathways are involved in angiogenesis and inflammatory responses; however, key factors controlling both processes remain unclear. Although tripartite motif‐containing (TRIM)‐28 is known to have an immunosuppressive role in immune cells, its expression level and role in endothelial cells (ECs) are still unclear. In this study, we investigated the role of TRIM28 in inflammatory responses and angiogenic activity of ECs for the first time. We showed that TRIM28 is the most abundant TRIM family member and is localized in nuclei of ECs. Small interfering RNA‐mediated knockdown of TRIM28 strikingly suppressed expression of TNF receptor (TNFR)‐1 and −2, decreased TNFα‐induced phosphorylation of IKKα/β and IκBα and degradation of IκBα and nuclear translocation of p65, and suppressed basal level and TNF‐α‐induced expression of chemokines and adhesion molecules, including VCAM‐1, IL‐6, ICAM‐1, E‐selectin, and monocyte chemoattractant protein (MCP)‐1. Unexpectedly, IL‐8 was potentiated by TRIM28 knockdown in ECs in an NF‐κB‐inducing kinase–dependent manner. Meanwhile, knockdown of TRIM28 inhibited expression of VEGF receptor 2 and suppressed VEGF‐induced proliferation and tube formation by ECs. Finally, knockdown of TRIM28 suppressed recruitment of ECs in vivo in a murine synthetic basement membrane model. In summary, we found that TRIM28 acts as a central factor in controlling endothelial inflammatory responses and angiogenic activities by retaining expression of TNFR‐1 and −2 and VEGF receptor 2 in ECs.—Wang, Y., Li, J., Huang Y., Dai, X., Liu, Y., Liu, Z., Wang, Y., Wang, N., Zhang, P. Tripartite motif–containing 28 bridges endothelial inflammation and angiogenic activity by retaining expression of TNFR1 and −2 and VEGFR2 in endothelial cells. FASEB J. 31, 2026–2036 (2017). www.fasebj.org

ABSTRACT Inflammatory response of blood–brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1–CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1–IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.

Abstract Background: The Tumor Growth Factor-β (TGF-β) pathway is a potent tumor suppressor pathway. Its inactivation has been implicated in hepatocarcinogenesis. However, in patients with advanced stage Hepatocellular Carcinoma (HCC), TGF-β1 serum concentrations are elevated and correlated to tumor size, recurrence and shortened survival. The mechanisms mediating TGF-β1 ligand overexpression in HCC are unknown. Material and Methods: Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Array. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analysis. shRNA was used for gene silencing. Transcription factor DNA binding was evaluated by chromatin immunoprecipitation. For promoter analysis, transcription factor binding site containing DNA fragments were cloned into the enhancer or promoter position of luciferase expression vectors and subsequent transfected. Results: In up to 68% of advanced stage HCC tumors Stem Cell Factor (SCF) is overexpressed, and SMAD2 and STAT3 are constitutively activated. SCF expression in human HCC tumors is intratumorally and intracellularly correlated to expression of pSer465/467SMAD2 and pTyr705STAT3. In HCC cell lines, SCF stimulation induces TGF-β1 transcription, expression and secretion, all of which are abrogated by genetic silencing of STAT3. TGF-β1 stimulation of human HCC cells induces TGF-β1 transcription, and genetic silencing of SCF prevents TGF-β1 autoregulation. We identified two STAT3 recognition sites within the TGF-β1 gene. In human HCC cells, TGF-β1 stimulation results in STAT3 binding to one of the identified recognition sites. TGF-β1 activates luciferase expression vectors under control of the corresponding STAT3 binding site but not in its absence. TGF-β1-induced STAT3 binding and expression vector activation is abrogated in HCC cells with genetically silenced SCF. TGF-β1 induces SCF expression but SMAD2 knockdown inhibits TGF-β1-induced SCF transcription and expression. We identified 7 putative SMAD2-binding elements (SBE) within the SCF promoter. TGF-β1 stimulation results in SMAD2 binding to the SCF promoter and activation of luciferase expression vectors regulated by the SCF promoter; conversely, genetic silencing of SMAD2 abrogates TGF-β1-induced SCF promoter activation. Importantly, knockdown of SCF restored the antiproliferative functions of TGF-β, and prevented TGF-β-induced tumor cell migration and invasion. Conclusion: In advanced stage HCC, SCF drives TGF-β1 autoregulation through STAT3-mediated regulation of the TGF-β1 promoter. Thereby, SCF and TGF-β1 form a positive feedback loop that functionally switches the TGF-β pathway from a tumor suppressor to a tumor promoter in advanced stage HCC. Citation Format: Pingyu Zhang, Andres Rojas, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. A positive feedback loop between TGF-β and SCF mediates TGF-β1 ligand overexpression in advanced hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2015-3911

Vasculogenesis and angiogenesis are controlled by vascular permeability factor/vascular endothelial growth factor A (VEGF). Dysregulation of these physiological processes contributes to the pathologies of heart disease, cancer and stroke. Rho GTPase proteins play an integral role in VEGF-mediated formation and maintenance of blood vessels. The regulatory functions of RhoA and RhoB in vasculogenesis and angiogenesis are well defined, whereas the purpose of RhoC remains poorly understood. Here, we describe how RhoC promotes vascular homeostasis by modulating endothelial cell migration, proliferation, and permeability. RhoC stimulates proliferation of human umbilical vein endothelial cells (HUVEC) by stabilizing nuclear β-catenin, which promotes transcription of cyclin D1 and subsequently drives cell cycle progression. RhoC negatively regulates endothelial cell migration through MAPK and downstream MLC-2 signaling and decreases vascular permeability through downregulation of the phospholipase Cγ (PLCγ)/Ca2+/eNOS cascade in HUVEC. Using a VEGF-inducible zebrafish (Danio rerio) model, we observed significantly less vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Taken together, our findings suggest RhoC is a key regulator of vascular homeostasis in endothelial cells.