Cells in the body respond to mechanical loads in ways that are crucial to normal and disease physiology. Understanding these processes is difficult due to the complex mechanical environment in vivo. In this research, we have developed several cell-stretching devices capable of subjecting cell cultures to non-uniform stretch distributions in order to investigate pathological responses of vascular smooth muscle cells to physiologic stretches. 10T1/2 cells were cyclically stretched with these devices for 24 hours upon silicone membranes, PDMS tubes, and within 3D PEGDA hydrogels. After stretching, altered cell behaviors were measured, including orientation, proliferation (quantified by BrdU incorporation), and gene expression (quantified by real-time, RT-PCR). Cells demonstrated marked changes in orientation, proliferation, and mRNA expression, which all varied with cellular location in the non-uniform environment. More specifically, increased orientation, increased proliferation, and more dramatically altered mRNA expression were found in regions of high, uniaxial stretch, relative to regions of low, near-equibiaxial stretch. These findings demonstrate the capabilities of graded stretch distributions to produce graded cell responses, indicating potentially localized smooth muscle cell behavior in a diseased artery. The novel devices employed herein will hopefully improve our understanding of these complicated cellular pathways, ultimately allowing for improved treatment or prevention of vascular disease.

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

Previous studies have shown that L-tryptophan treatment has been known to reduce blood pressure (BP) in hypertensive rats. L-tryptophan is converted to serotonin (5-HT), a potent vasoconstrictor agonist. The direct vascular effects of L-tryptophan, an essential amino acid, and the mechanism that contributes to the fall in BP have not been fully explored. The present study aims to examine the direct vascular responses to both D- and L- tryptophan using perfused mesenteric vascular bed, an ex-vivo preparation that represents the resistance function of circulation. Perfusion was maintained at a constant flow rate (5 mL/min) with Krebs buffer (pH 7.4, 37˚C) after isolation from 12 to 14 week old male Sprague-Dawley rats. The basal perfusion pressure (PP) (mean ± SEM) was 27 ± 3 mmHg. Inclusion of D- and L-isomers in the perfusion medium led to concentration-dependent increase in PP. While the maximal response (Emax) was similar, D-tryptophan (EC50: 0.25 ± 0.12* µmol; Emax: 128 ± 8 mmHg) was more potent (lower EC50 value; *p < 0.01) than L-tryptophan (EC50: 0.79 ± 0.30 µmol; Emax: 141 ± 7 mmHg). Inclusion of increasing concentrations (2, 5 and 10 nM) of the 5-HT2A selective antagonist, ketanserin, led to parallel right-ward shifts in the concentration-response curves to D- and L-tryptophan with restoration of their Emax. In contrast, the α1 selective agonist, methoxamine (30 µM), constricted preparations, both D- (IC50: 0.94 ± 0.30* µmol; Imax: 96 ± 2%) and L-tryptophan (IC50: 2.8 ± 1.0 µmol Imax: 88± 1%) evoked concentration-dependent vasodilatation, an effect that was resistant to blockade by either ketanserin or other 5-HT antagonists. Again, D-tryptophan was more potent than L-tryptophan in the presence of 5-HT antagonist (*p < 0.05). Neither the removal of endothelium nor incubation with selective inhibitors of dilatory mediators released from the endothelium, failed to alter the vasodilator responses to D- and L-tryptophan. In potassium chloride depolarized preparations, L-tryptophan evoked an additive vasoconstrictor response. The vasodilator responses to L-tryptophan persisted in the presence of glibenclamide, a KATP channel inhibitor, or tetraethyl ammonium, a BKCa channel inhibitor, or BaCl2, a Kir channel inhibitor, or ouabain, a Na+-K+-ATPase pump inhibitor. These data confirm that the essential amino acid, L-tryptophan, as well as its D-isomer, evoke a biphasic vasoconstrictor and vasodilator responses in the resistance type mesenteric vascular bed. While the vasoconstrictor responses are mediated by activation of vascular 5-HT receptors, the endothelium-independent vasodilator responses are not linked to activation of vascular 5-HT receptors, vascular potassium channels, Na+-K+-ATPase pump or via inhibition of voltage-operated Ca2+-channels. Plasma concentration of L-tryptophan is about 90 - 120 µM. The endothelium/5-HT independent direct vasodilator responses characterized here for the first time could account for the antihypertensive/ BP lowering effect of L-tryptophan reported earlier by other laboratories.

The research presented here deals with synthetic materials for application in tissue engineering, primarily poly(ethylene glycol) (PEG) and poly(dimethyl siloxane)star (PDMS)star. Tissue engineering seeks to repair or replace damaged tissue through implantation of cell encapsulated in an artificial scaffold. Cell differentiation and extracellular matrix (ECM) deposition can be influenced through a wide variety of in vitro culture techniques including biochemical stimuli, cell-cell interactions, mechanical conditioning and scaffold physical properties. In order to systematically optimize in vitro conditions for tissue engineering experiments, the individual effects of these different components must be studied. PEG hydrogels are a suitable scaffold for this because of their biocompatibility and biological "blank slate" nature. This dissertation presents data investigating: the effects of glycosaminoglycans (GAGs) as biochemical stimuli on pig vocal fold fibroblasts (PVFfs); the effects of mechanical conditioning and cell-cell interactions on smooth muscle cells (SMCs); and the effects of scaffold physical properties on SMCs. Results show that GAGs influence PVFf behavior and are an important component in scaffold design. Hyaluronic acid (HA) formulations showed similar production in collagen I and III as well as reduced levels of smooth muscle a-actin (SMa-actin), while chondroitin sulfate (CSC) and heparin sulfate showed enriched collagen III environments with enhanced expression of SMa-actin. A physiological flow system was developed to give comprehensive control over in vitro mechanical conditioning on TEVGs. Experiments performed on SMCs involved creating multi-layered TEVGs to mimic natural vascular tissue. Constructs subjected to mechanical conditioning with an endothelial cell (EC) layer showed enhanced expression of SMC differentiation markers calponin h1 and myocardin and enhanced deposition of elastin. Consistent with other studies, EC presence diminished overall collagen production and collagen I, specifically. Novel PDMSstar-PEG hydrogels were studied to investigate the effects of inorganic content on mesenchymal stem cell differentiation for use in TEVGs. Results agree with previous observations showing that a ratio of 5:95 PDMSstar: PEG by weight enhances SMC differentiation markers; however, statistically significant conclusions could not be made. By studying and optimizing in vitro culture conditions including scaffold properties, mechanical conditioning and multi-layered cell-cell interactions, TEVGs can be designed to maximize SMC differentiation and ECM production.

Arteries possess the ability to grow and remodel in response to sustained alterations in biomechanical loading, likely via mechanisms that are similarly involved in diverse arterial pathologies and responses to treatment. In particular, myriad experminental observations suggest that cell and matrix turnover within vasoaltered states enable arteries to adapt to sustained changes in mechanical stimuli. The goal herein is to show explicitly how altered smooth muscle contractility and matrix growth and remodeling work together to adapt the geometry, structure, stiffness, and function of a representative basilar artery. This work seeks to illustrate the importance of complementary vasoactivity and matrix remodeling for basilar arteries in response to sustained alterations in mechanical stimuli. Toward this end, an extended constrained mixture model of the arterial wall is employed whereby the mass fractions, material properties, and natural configurations of individual constituents can evolve separately and thereby dictate overall growth and remodeling. This approach accounts for fundamentally important behaviors. Simulations provide important intuition and insight regarding constitutive functional forms and model parameters.

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 199-207). Also available on the Internet.

Cyclic adenosine monophosphate (cAMP) is an important intracellular secondary messenger. The major target of cAMP was traditionally considered as protein kinase (PK) A. This belief has been challenged by the discovery of exchange protein activated by cAMP 1 (Epac1), a cAMP-dependent guanine-nucleotide-exchange factor (GEF). Epac1 is ubiquitously expressed in all tissues and plays important roles particularly in the cardiovascular system. As cAMP activates both PKA and Epac1, the development of 8-pCPT-2'-O-Me-cAMP (8-pCPT), which has 107-fold higher affinity to bind and activate Epac1 than PKA, aids the researches on Epac1-mediated responses. In the present study, the protein expressions of Epac1 in the porcine coronary arteries and rat aortas were confirmed by Western blot analysis. In organ chambers, 8-pCPT induced acute relaxations in isolated porcine coronary arteries contracted to thromboxane receptor (TP-receptor) antagonists, and the relaxation was endothelium-independent. The 8-pCPT-induced Epac1 activation selectively altered the vasoactive responses to the TP-receptor agonists. The Epac1-mediated relaxation was found not related to PKA, PKG and the opening of ATP-sensitive potassium channels. Although Epac1 was first cloned as a Rap-linked GEF, in the porcine coronary artery, small GTPase Rac1 is the downstream target of Epac1 instead of Rap1 for relaxation. Activation of TP-receptors stimulates Rho-kinase to cause contraction, and the 8-pCPT-induced relaxation was Rho-kinase dependent, probably through pathway that is distinct from Rac1. Activation of Epac1 also inhibited the contraction to PKC, which is also downstream of TP-receptor but independent to Rho-kinase activity. On the contrary, in the aorta from male Sprague-Dawley rats aged 10-12 weeks, 8-pCPT induced relaxation in rings contracted to phenylephrine (PE) and the relaxation was endothelium-dependent. The relaxation depended mainly on endothelial nitric oxide synthase (eNOS) and partly on cyclooxygenase (COX). Western blot analysis found that 8-pCPT did not enhance eNOS phosphorylation, which is one of the mechanisms for eNOS activation. Activation of Epac1 also did not alter the phosphorylation of Akt and ERK1/2 which play important roles in cAMP-dependent eNOS. More experiments are needed to examine whether or not Epac1 alters nitric oxide (NO) and prostanoids synthesis, which are the major endothelium-derived mediators responsible for vascular tone regulation. In summary, the selective Epac activator 8-pCPT induced significant relaxations by distinct mechanisms in porcine coronary arteries and rat aortas. It is most likely that the relaxing effects of Epac1 activator are tissue and/or species specific. Owing to the effects of 8-pCPT on vascular relaxation, Epac1 might be an alternative therapeutic target for the treatment of vasospasm and hypertension. Further studies are necessary to explore the detailed mechanisms of Epac1 and its in vivo effects and in diseased models. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy