ENOS And ROS Mediated Effect Of Simvastatin On Tumor Vascular Stabilization In Vivo

Part One:Simvastatin increases tumor blood perfusion in LLC and B16 tumor modelsObjective:To investigate effects of simvastatin on structure and function of blood vessels in LLC and B16 tumor models, and the synergistic effect on tumor growth inhibition when simvastatin was used in combination with chemotherapeutics.Methods:Subcutaneously implanted LLC and B16 tumors were randomly selected to receive 0.2 and 10 mg/kg body weight simvastatin for 7 days. FITC-labeled lectin was injected by tail vein, and tumors were harvest, the morphological structure and perfusion of vessels were assessed by co-immunostained for CD31; Contrast-enhanced Doppler imaging was used to evaluate alteration of blood flow functionally. Expression level of HIF-la, detected by immunohistochemistry and western-blot, was used to assess hypoxia, and H&E staining for analysing necrosis in tumor tissues. Tumor-bearing mice were randomly divided into four groups, control group, group that received i.p injection of single chemotherapeutics, group 3 received chemotherapeutics combined with 0.2mg/kg simvastatin and group 4 received chemotherapeutics combined with 10 mg/kg simvastatin. Tumor volume was determined every other day till the end of treatment.Results:Double staining for CD31 and FITC-conjugated lectin revealed that lectin+CD31+vessels were significantly increased in 0.2 and 10 mg/kg simvastatin-treated LLC and B16 tumors (p<0.05), tumor vessels appeared more sharply and discretely outlined. In 0.2 mg/kg simvastatin group, vessel density was increased to some extent, and in 10 mg/kg simvastatin group, the vascularity was markedly reduced. Simvastatin treatment increased vascularity index (VI) and vascularization flow index (VFI) in LLC and B16 tumors at 0.2 and 10 mg/kg/d on day 7 (p<0.05 vs. control for both). When treated with 0.2 and 10 mg/kg/d simvastatin, number of HIF-la-positive cells were reduced in LLC and B16 tumors as compared with untreated controls; western blot data further confirmed that HIF-1αexpression was decreased significantly in both LLC and B16 tumors, and the necrotic area was also markedly reduced (p<0.05). Administration of 0.2 and 10 mg/kg simvastatin followed by chemotherapeutics agents showed remarkably reduced final tumor volume in comparison with control and chemotherapeutics-only groups (p<0.05).Conclusion:Simvastatin could normalize the structure and function of tumor vessels and increase therapeuitic efficacy of chemotherapeutic drugs in tumor models. Part Two:Manifestation and molecular basis of low-and high-dose simvastatin on tumor vessel normalizationObjective:To investigate the patterns of manifestation and molecular basis of low-and high-dose simvastatin on tumor vessel maturation.Methods:The transfer activity of a labelling high-molecular protein (FITC-albumin) across the EC monolayers was measured to determine the efficacy of simvastatin on HUVEC monolayer permeability using cell migration model under hypoxia and normoxia condition. Subcutaneously implanted LLC and B16 tumors were randomly selected to receive 0.2 and 10 mg/kg body weight simvastatin for 7 days. Frozen tissue sections were prepared for co-immunostained with CD31 and a-SMA, and pericyte coverage index was estimated and presented as the percentage of blood vessels stained for CD31 with a-SMA positive pericytes, and eNOS protein level was determined by western blot analysis. Vascular leakage was assessed by i.v. injection of Evans blue dye and allowed to circulate for 20 min before mice were perfused, the tumors were then excised, and the Evans blue dye was extracted from the tumor and quantified by spectrophotometer. Intratumoral ROS production in 0.2/10 mg/kg simvastatin-treated LLC and B16 tumors was measured in situ on tumor slices using a fluorescent ROS probe, H2DCFDA, and the corresponding alteration of VEGF expression was assessed by western blotting. To determined alterations after ROS clearance, tumor bearing mice received an intraperitoneal injection of DPI (a NADPH oxidase inhibitor) for 5 days, vessel density and blood perfusion were measured by double staining for CD31 and lectin, protein level of VEGF and HIF-la in each group was evaluated by western blot analysis.Results:In vitro permeability of HUVEC monolaye demonstrated that simvastatin alleviates hypoxia-induced endothelium permeability in HUVEC in a dose-dependent manner, and the decline was maximal at 5μmol/L simvastatin. Double stained for CD31 and mature pericyte marker a-SMA revealed that tumor vessels that encased compactly with a-SMA+pericytes were remarkably increased in both tumor types (p<0.001) accompanied by a significant up-regulation of eNOS protein level 7 days after 0.2 mg/kg simvastatin treatment in LLC and B16 tumors. In vivo vessel permeability in tumor models showed that Evans blue dye extravasation into low-and high-dose treated tumors tissues were markedly reduced (p<0.05). ROS production in tumor tissues was reduced along with the increase of drug dose, and VEGF expression was remarkably downregulated in 10 mg/kg simvastatin treated tumors versus control and low-concentration groups. We observed a greater proportion of lectin-stained vessels in DPI-treated LLC and B16 tumors accompanied by a remarkably decreased vessel density, and ROS generation was obviously lower than the values in control animals (p<0.05). Moreover, addition of DPI could significantly decease VEGF and HIF-1 a protein level in both tumors.Conclusion:Simvastatin could reduce hypoxia-induced endothelium leakage in a dose-dependent manner in vitro. When given at both low and high concentrations in vivo, simvastatin may induce tumor vascular maturation, however, mediated by probably different underlying mechanisms, as low-concentration simvastatin induced upregulation of eNOS skewed vessels to a pericytes-coated and stable pattern, and high-does simvastatin mainly acted on decreasing ROS-induced HIF-1a and VEGF expression, attenuating VEGF-drived tumor vessel hyperpermeability.