QS153. The Critical Importance of Nitric Oxide in Regulating Human Bone Marrow Stem Cell VEGF Production

328 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS manumycin A had no effect, inhibition of rho with C3 limited both PI3-K and akt activation. PDK1 responses were unchanged by inhibition of GTPases. Inhibition of oxygen free radical generation with N-acetylcysteine and of EGFR with AG1478 inhibited PDK1 activation in response to S-1-P. Conclusion: S-1-P mediated migration is akt dependent. S-1-P mediated akt phosphorylation is controlled by G␤␥ dependent, PI3-K activation, which requires the GTPase rho and G␤␥ dependent PDK1 activation which requires oxygen free radical generation and EGFR activation. QS152. C-REACTIVE PROTEIN INDUCES ENDOTHELIAL DYSFUNCTION THROUGH DOWNREGULATION OF ENOS EXPRESSION AND INCREASE OF OXIDATIVE STRESS IN PORCINE PULMONARY ARTERIES. Suman Annambhotla, Xinwen Wang, Ajay Dhahwal, Peter Lin, Qizhi Yao, Changyi J. Chen; Baylor College of Medicine, Houston, TX Introduction: C-reactive protein (CRP) is a potent acute phase reactant released in the setting of an inflammatory response. It is also associated with the pathogenesis of vascular disease and potentiates the progression of pulmonary pathology in acute lung injury, sepsis, pulmonary artery hypertension and acute respiratory distress syndrome (ARDS). However, it is unclear whether CRP directly induces endothelial dysfunction in the pulmonary system. The objective of this study was to determine the effect and molecular mechanisms of CRP on endothelial functions in porcine pulmonary arteries (PPA). Methods: PPA 5-mm rings were incubated with increasing concentrations of human CRP (1, 10, and 25 ␮g/mL). A myograph tension system was utilized to evaluate vasomotor function of the PPA rings. Each PPA group was evaluated for vessel contraction induced by a thromboxane analog (U46619) and vasorelaxation by bradykinin or sodium nitroprusside (SNP). The endothelial nitric oxide synthase (eNOS) expression was measured using real time PCR. Superoxide anion (O ⫺ 2) production was analyzed by the lucegenin-enhanced chemiluminescence method. Results: In response to 10 ⫺6 M bradykinin, endothelium-dependent vasorelaxation of the PPA rings decreased by 25%, and 53% at CRP concentrations of 10 and 25 ␮g/mL, respectively, compared with untreated controls (P⬍0.05). CRP did not alter maximum vessel contraction in response to U46619. Endothelium-independent relaxation in response to SNP did not show significant changes between treated and control groups. Expression of eNOS mRNA was reduced by 14% and 58% at CRP concentrations of 10 and 25 ␮g/mL, respectively, compared with controls in PPA. O ⫺ 2 production was increased by 70% and 146% in similarly treated PPA rings at equivalent CRP concentrations of 10 and 25 ␮g (P⬍0.05). Conclusions: CRP at clinically relevant concentrations impairs endothelium-dependent vasorelaxation in PPA. This endothelial dysfunction is associated with decreased eNOS expression as well as increased free radical production in CRP-treated PPA. These findings suggest that CRP may propagate pulmonary artery endothelial dysfunction during inflammation, thereby contributing to the pulmonary pathology. QS153. THE CRITICAL IMPORTANCE OF NITRIC OXIDE IN REGULATING HUMAN BONE MARROW STEM CELL VEGF PRODUCTION. Yue Wang, Meijng Wang, Paul Crisostomo, Troy Markel, John Brown, Keith D. Lillemoe, Daniel R. Meldrum; Indiana University, Indianapolis, IN Introduction: Nitric oxide (NO) is an important intracellular signaling molecule that has been involved in wide variety of biological process, such as smooth muscle relaxation, blood pressure regulation and platelet aggregation. Myocardial ischemia results in the production of substantial amounts of NO through the reduction of nitrite, which represents a circulating and tissue storage form of NO. It has been reported that bone marrow stem cells (BMSCs) protect ischemia heart, in part, by producing growth factors, such as vascular

endothelial growth factor (VEGF). However, it is unknown whether NO alters the production of VEGF from BMSCs. We hypothesized that NO would enhance BMSC growth factor production. Methods: Human BMSCs were plated and cultured at 1,000,000 cells per well. Those cells were treated with NO donor (FK409) for 24 hours at concentrations of 0 nM (vehicle control), 1 nM, 10 nM, 20 nM and 100 nM. After 24 hours incubation, the supernatants were collected and measured for production of VEGF via enzyme linked immunosorbent assay (ELISA). All values represent the mean ⫹/⫺ standard error of the mean (SEM). Statistical differences between the control groups and those obtained under various treatment conditions were determined by using a Student’s t-test. Values of P ⬍ 0.05 were judged to be statistically significant. Results: FK409 significantly suppressed the production of VEGF from human BMSCs. The amount of VEGF was 453.4 ⫹/⫺ 39.5 pg/ml, 523.6 ⫹/⫺ 32.0 pg/ml, 481.1 ⫹/⫺ 35.6 pg/ml and 406.2 ⫹/⫺ 25.3 pg/ml in the supernatants of cells treated by FK409 at 1 nM, 10 nM, 20 nM and 100 nM. All values were significantly lower compared to controls (657.0 ⫹/⫺ 12.2). NO donor also abolished the augmentation of VEGF production induced by tumor necrosis factor alpha (TNF) or lipo-polysaccharide (LPS). The amount of VEGF in the supernatant was 175.5 ⫹/⫺ 7.7 pg/ml when cells were treated by NO donors and TNF (50 ng/ml), which was significantly lower than groups treated by TNF alone (590.9 ⫹/⫺ 2.7 pg/ml) or controls (436.8 ⫹/⫺ 31.3 pg/ml). Similarly, when cells were treated with NO donor and LPS, the VEGF production was decreased to control values (471.0 ⫹/⫺ 18.0 pg/ml), which was significantly lower compared to LPS treated group (566.3 ⫹/⫺ 4.4 pg/ml). Conclusion: This abstract constitutes the first report regarding the effect of NO on BMSC growth factor production. Myocardial ischemia leads to the substantial increase in myocardial tissue NO through the reduction of nitrite. The appreciation that NO profoundly inhibits growth factor production represents another important piece of the stem cell puzzle. This understanding may be important clinically in situations where it may desirable to either enhance of reduce BMSC growth factor production. QS154. EFFECT OF ESTROGEN AND PROGESTERONE ON MATRIX METALLOPROTEINASE GENE REGULATION IN HUMAN AORTIC VASCULAR SMOOTH MUSCLE CELLS. Oscar H. Grandas, Deidra H. Mountain, Stacy S. Kirkpatrick, David C. Cassada, Scott L. Stevens, Michael B. Freeman, Mitchell H. Goldman; University of Tennessee Medical center at Knoxville, Knoxville, TN Objectives: Postmenopausal women receiving hormone replacement therapy (HRT) have more adverse outcomes after vascular reconstructions. Estrogen-binding receptors have been identified on vascular smooth muscle cells (VSMCs), indicating that vascular function may be under direct hormonal control. Vascular remodeling modified by matrix metalloproteinases (MMPs) and chemotactic cytokines, due to their ability to degrade components of the extracellular matrix and direct chemotactic cell migration, may be regulated by estrogen and progesterone. Here we studied the effect of estrogen and progesterone on MMP gene expression in human VSMCs. Methods and Results: VSMCs were incubated with Est (5ng/mL), Prog (50ng/mL), Est⫹Prog combination (Est/Prog), interleukin⫺1␤ (100 U/ml; IL⫺1␤) and/or doxycycline (40␮g/ml; Doxy). Gene array analysis indicated Est⫹IL⫺1␤ modulates the expression of numerous genes, notably increasing the expression of CCL20 and MMP-3. RT-PCR analyses revealed CCL20 and MMP-3 mRNA levels were significantly increased by Est/Prog⫹IL⫺1␤ treatment (62⫾9% and 60⫾8%, respectively; P⬍0.05 vs. control, n⫽3). RT-PCR analyses revealed MT1-MMP mRNA levels were significantly increased by Est/Prog⫹IL⫺1␤ treatment (105⫾17%; P⬍0.05 vs. control). The addition of doxycycline had no significant effect in mRNA expression. IL⫺1 ␤ treated groups had the largest effect on MMP-3.