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.

Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress-related proteins, such as the GTPase RhoA and NADPH oxidase protein p47phox in young (5-6 mo) and aged (26-28 mo) FVB wild-type (WT) and cardiac-specific metallothionein transgenic mice. Metallothionein mice showed a longer life span (by approximately 4 mo) than FVB mice evaluated by the Kaplan-Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR(90), reduced tolerance to high stimulus frequency, and slowed intracellular Ca2+ decay, all of which were nullified by metallothionein. Aging increased superoxide generation, active RhoA abundance, cytochrome c release, and p47phox expression and suppressed aconitase activity without affecting protein nitrotyrosine formation in the hearts. These aging-induced changes in oxidative stress and related protein biomarkers were attenuated by metallothionein. Aged metallothionein mouse myocytes were more resistant to the superoxide donor pyrogallol-induced superoxide generation and apoptosis. In addition, aging-associated prolongation in TR90 was blunted by the Rho kinase inhibitor Y-27632. Collectively, our data demonstrated that metallothionein may alleviate aging-induced cardiac contractile defects and oxidative stress, which may contribute to prolonged life span in metallothionein transgenic mice.

Purpose-A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. We tested the hypothesis that there is increased gene expression of hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor-A (VEGF-A) and its receptors (VEGFR-1, -2), matrix metalloproteinase-2 (MMP-2), -9 (MMP-9), tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2), and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis.Materials and methods-Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal insufficiency and characterized in eight mice. Twenty eight days later, an AVF (n=11) was created from the right carotid artery to ipsilateral jugular vein and the mice were sacrificed at day 7 (n=4) and day 14 (n=4). The outflow and control veins were removed for gene expression. Three mice were sacrificed at day 28 for histologic analysis.

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.

Our recent study defined the chemokine induced human monocyte signaling under normoglycemic condition. To explore the hyperglycemia induced monocyte signaling, we performed adhesion, migration and transmigration assays on human monocytes obtained from THP-1 cell line in the presence of normal (5mM) and high (10 and 20mM) glucose concentrations without chemokines. We observed augmented (p<0.01) monocyte adhesion to HUVEC monolayer at 10mM than 5mM glucose with no further increase at 20mM glucose concentration (p<0.03 vs. 10mM; p<0.01 vs. 5mM). But incremental increase in monocyte migration (p<0.01), transmigration (p<0.01) and stress fibre response (p<0.01) were observed at 10 and 20mM glucose concentrations in comparison to 5mM glucose concentrations. We found gradational increase (p<0.01) in phosphorylation of Akt S473 and glycogen synthase kinase (GSK3β S9) in hyperglycemia (10 and 20mM) when compared to 5mM glucose. Furthermore hyperglycemia (both 10 mM and 20 mM) treated monocyte showed upregulated phosphorylation of p101 and p110γ subunits of PI-3 kinase in comparison to 5mM glucose. Hyperglycemia induced monocyte migration was restored to basal levels in the presence of PI-3 kinase inhibitor, LY. These observations imply that modest hyperglycemia per se, as is commonly observed in diabetic individuals, is a potent stimulator of monocyte activity even without chemokines.