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Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia inducible factor-1 alpha at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

Venous neointimal hyperplasia (VNH) is responsible for hemodialysis vascular access malfunction. Here we tested whether VNH formation occurs, in part, due to vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP)-9 gene expression causing adventitial fibroblast transdifferentiation to myofibroblasts (α-SMA-positive cells). These cells have increased proliferative and migratory capacity leading to VNH formation. Simvastatin was used to decrease VEGF-A and MMP-9 gene expression in our murine arteriovenous fistula model created by connecting the right carotid artery to the ipsilateral jugular vein. Compared to fistulae of vehicle-treated mice, the fistulae of simvastatin-treated mice had the expected decrease in VEGF-A and MMP-9 but also showed a significant reduction in MMP-2 expression with a significant decrease in VNH and a significant increase in the mean lumen vessel area. There was an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and decreases in α-SMA density, cell proliferation, and HIF-1α and hypoxyprobe staining. This latter result prompted us to determine the effect of simvastatin on fibroblasts subjected to hypoxia in vitro. Simvastatin-treated fibroblasts had a significant decrease in myofibroblast production along with decreased cellular proliferation, migration, and MMP-9 activity but increased caspase 3 activity suggesting increased apoptosis. Thus, simvastatin results in a significant reduction in VNH, with increase in mean lumen vessel area by decreasing VEGF-A/MMP-9 pathway activity.

It is a challenge to develop a universal single drug that can treat breast cancer at single or multiple-stage complications, yet remains nontoxic to normal cells. The challenge is even greater when breast cancer-specific estrogen-based drugs are being developed which cannot act against multi-staged breast cancer complications owing to cells’ differential ER expression status and their possession of drug-resistant and metastatic phenotypes. We report here the development of a first cationic lipid-conjugated estrogenic derivative (ESC8) that kills breast cancer cells independent of their estrogen receptor (ER) expression status. This ESC8 molecule apparently is nontoxic to normal breast epithelial cells, as well as to other non-cancer cells. ESC8 induces apoptosis through an intrinsic pathway in ER-negative MDA-MB-231 cells. In addition, ESC8-treatment induces autophagy in these cells by interfering with the mTOR activity. This is the first example of an estrogen structure-based molecule that co-induces apoptosis and autophagy in breast cancer cells. Further in vivo study confirms the role of this molecule in tumor regression. Together, our results open new perspective of breast cancer chemotherapy through a single agent, which could provide the therapeutic benefit across all stages of breast cancer.

VEGF induces vascular permeability (VP) in ischemic diseases IntroductionVascular permeability (VP) plays an integral role in the pathology of cardiovascular disease, stroke, and cancer. VEGF was originally discovered as "vascular permeability factor" described as being a tumor-secreted factor that promotes microvascular permeability potently. 1 It was later discovered separately as VEGF, an endothelial mitogen 2 essential for the development of blood vessels. [3][4][5] Ischemia resulting from cardiac infarction or stroke promotes VEGF expression, which leads to hyperpermeability, edema, and tissue damage. 6,7 Angiogenesis is a key restorative mechanism in response to ischemia, 8,9 creating the therapeutical challenge of regulating the negative and beneficial actions of VEGF temporally to reduce edema and improve ischemic tissue. In cancer, VEGFmediated VP promotes tumor-cell extravasation through damaged endothelial cell junctions, often leading to widespread metastasis. 10 In addition, VEGF expression induces tumor angiogenesis through extravasation of plasma proteins into the surrounding tissue to develop a provisional matrix capable of supporting vascular sprouting and tumor growth. 1 Despite the prevalent role of VEGF-mediated VP in the pathophysiology of cardiac and cerebral diseases and cancer, the genetic regulation of VEGF-induced VP remains unclear because of the lack of an adequate, highthroughput in vivo model with which to assess quantitatively the cumulative contributions of receptors and transduction molecules to VEGF-mediated VP.New methods are necessary to elucidate genetic regulators of VP. Present in vivo VP models, such as the Miles assay, 11,12 the dual-isotopemodified Miles assay, 13 the in vivo peripheral permeability assay, 14 and intravital microscopy, 15-17 have various disadvantages, including the requirement for expensive and time-intensive murine knockout models for genetic studies. The zebrafish (Danio rerio) is a vertebrate with an optically clear embryo that allows high-resolution live imaging and is amenable to high-throughput genetic manipulation, but has yet to be used as an in vivo model to study VEGF-induced VP. In the present study, we undertook the development of a heat shock-inducible zebrafish VEGF model through which VP can be visualized and quantitated in real-time using microangiography of fluorophoreconjugated dextrans. Protein translation-blocking morpholinos (MO) microinjected into VEGF-inducible zebrafish embryos represent a relatively inexpensive and high-throughput means of identifying regulators of VEGF-mediated VP. We demonstrate the utility of this newly developed approach by identifying phospholipase C␤3 (PLC␤3) as a regulator of VEGF-mediated VP.VEGF mediates its activities through 2 receptor tyrosine kinases, VEGFR2 and VEGFR1, [18][19][20] with neuropilin acting as a coreceptor. [21][22][23] Downstream signaling events induced by VEGF include the serine phosphorylation of PLC␤3 24 and tyrosine phosphorylation of PI3K and PLC␥. 25 PLC isoforms mediate the h...

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca2+ signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca2+ entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by ELISA of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP-BDNF vector. Exposure to the pro-inflammatory cytokine TNFα (20 ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10µM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca2+ (EGTA; 1mM) or intracellular Ca2+ (BAPTA; 5µM) significantly reduced secreted BDNF, as did knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca2+ influx in regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given BDNF effects on Ca2+ and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.

Purpose: Various studies have shown the importance of the GAIP interacting protein, COOHterminus (GIPC, also known as Synectin) as a central adaptor molecule in different signaling pathways and as an important mediator of receptor stability. GIPC/Synectin is associated with different growth-promoting receptors such as insulin-like growth factor receptor I (IGF-IR) and integrins. These interactions were mediated through its PDZ domain. GIPC/Synectin has been shown to be overexpressed in pancreatic and breast cancer. The goal of this study was to show the importance of GIPC/Synectin in pancreatic cancer growth and to evaluate a possible therapeutic strategy by using a GIPC-PDZ domain inhibitor. Furthermore, the effect of targeting GIPC on the IGF-I receptor as one of its associated receptors was tested. Experimental Design: The in vivo effects of GIPC/Synectin knockdown were studied after lentiviral transduction of luciferase-expressing pancreatic cancer cells with short hairpin RNA against GIPC/Synectin. Additionally, a GIPC-PDZ^targeting peptide was designed. This peptide was tested for its influence on pancreatic cancer growth in vitro and in vivo. Results: Knockdown of GIPC/Synectin led to a significant inhibition of pancreatic adenocarcinoma growth in an orthotopic mouse model. Additionally, a cell-permeable GIPC-PDZ inhibitor was able to block tumor growth significantly without showing toxicity in a mouse model. Targeting GIPC was accompanied by a significant reduction in IGF-IR expression in pancreatic cancer cells. Conclusions: Our findings show that targeting GIPC/Synectin and its PDZ domain inhibits pancreatic carcinoma growth and is a potential strategy for therapeutic intervention of pancreatic cancer.

Vascular endothelial growth factor (VEGF)-induced receptor phosphorylation is the crucial step for initiating downstream signaling pathways that lead to angiogenesis or related pathophysiological outcomes. Our previous studies have shown that the neurotransmitter dopamine could inhibit VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), endothelial cell proliferation, migration, microvascular permeability, and thus, angiogenesis. In this study, we address the mechanism by which VEGFR-2 phosphorylation is regulated by dopamine. Here, we demonstrate that D2 dopamine receptor (D2DR) colocalizes with VEGFR-2 at the cell surface. Dopamine pretreatment increases the translocation and colocalization of Src-homology-2-domain-containing protein tyrosine phosphatase (SHP-2) with D2DR at the cell surface. Dopamine administration leads to increased VEGF-induced phosphorylation of SHP-2 and this increased phosphorylation parallels the increased phosphatase activity of SHP-2. Active SHP-2 then dephosphorylates VEGFR-2 at Y951, Y996 and Y1059, but not Y1175. We also observe that SHP-2 knockdown impairs the dopamine-regulated inhibition of VEGF-induced phosphorylation of VEGFR-2 and, subsequently, Src phosphorylation and migration. Our data establish a novel role for SHP-2 phosphatase in the dopamine-mediated regulation of VEGFR-2 phosphorylation.