published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Physiology / Master / Master of Medical Sciences

Background: Regulation of vascular tone is complex. Various complementary signaling pathways causing contraction and relaxation of vascular smooth muscle take place to ensure proper blood flow within the vasculature. Mitogen activated protein kinase (MAPK) signaling cascade is observed to be one of the many signaling pathways that regulate vascular tone. Aim: This study examines the role of the following MAPK: mitogen-activated extracellular-regulated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p38 MAPK in the regulation of relaxation in the endothelium and smooth muscle. Method: Isometric tension of isolated porcine coronary artery rings were measured with organ chamber setup. The effects of MEK inhibitor, PD98059 (30 μM), ERK inhibitor, U0126 (10 μM) and p38 MAPK inhibitor, SB203580 (10 μM), on relaxations induced by bradykinin (a vasodilating peptide), SKA-31 [an activator of small and intermediate conductance calcium-activated potassium channels (SKCa and IKCa,, respectively)], Deta NONOate (a nitric oxide donor) and forskolin (an adenylate cyclase activator) were examined in arteries with and without endothelium, contracted with an thromboxane A2 analog, U46619 (300 nM – 1 μM). In some experiments, rings were also incubated with the following pharmacological inhibitors, indomethacin (cyclooxygenase inhibitor, 10 μM), L-NAME (nitric oxide synthase inhibitor, 300 μM), TRAM34 (IKCa blocker, 1 μM), and UCL1684 (SKCa blocker, 1 μM), alone or in combination. Results: 1. Bradykinin-induced relaxation was potentiated by MEK and ERK inhibition but not by p38 MAPK inhibition. 2. SKA-31-induced relaxation was potentiated by MEK and p38 MAPK inhibition but not by ERK inhibition. 3. Deta NONOate-induced relaxation was potentiated by MEK, p38 MAPK inhibition, but not by ERK inhibition except in the presence of indomethacin, TRAM-34 plus UCL1684. 4. Forskolin-induced relaxation was potentiated by MEK and p38 MAPK inhibition, but not by ERK inhibition. Discussion: MAPK plays a role in regulating the vascular tone in both the endothelium and smooth muscle of porcine coronary arteries. MEK appears to have an inhibitory action on relaxation that is downstream of the generation of endothelium-derived nitric oxide, activation of IKCa and SKCa and activation of adenylate cyclase. ERK are unlikely to be the downstream target of MEK for inhibiting relaxation, in view of the lack of effects of its inhibitor on endothelium-derived hyperpolarizing factor (EDHF)-mediated and endothelium-independent relaxations. The involvement of ERK in relaxation pathways in the endothelium appears to be complicated, since U0126 caused opposing effects (inhibition and potentiation) on bradykinin-induced relaxation in the presence of indomethacin without and with L-NAME or TRAM-34 plus UCL1684. As inhibition of p38 MAPK results in potentiation of relaxations to all relaxing agents tested except bradykinin, this MAPK may have opposing action in the endothelium and smooth muscle; endothelial p38 MAPK may facilitate relaxation while smooth muscle p38 MAPK attenuates it. In conclusion, this study provided additional information on the influences of MEK, ERK and p38 MAPK on relaxation; this knowledge may contribute to the understanding of the mechanisms underlying the development of vascular disorders. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

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Neointimal proliferation is a key process underlying many cardiovascular diseases such as atherosclerosis and angioplasty-induced restenosis. Vascular smooth muscle cells (SMC) are significant contributors to the development and stability of the neointimal lesion. This is due, in part, to their capacity to be phenotypically modulated, facilitating SMC proliferation in response to mechanical injury, their subsequent migration, and deposition of extracellular matrix. The aim of this thesis was to characterize the function of two genes identified in our laboratory to be upregulated shortly after mechanical injury of vascular SMC and their exposure to fibroblast growth factor (FGF)-2, an injury-induced cytokine. The first is activation transcription factor (ATF) 4, which is upregulated by FGF-2 and mechanical injury in vascular SMC in vitro, and by balloon-injury in the artery wall. The induction of ATF4 by FGF-2 was shown to be mediated through the PI3K pathway, and preceded by phoshorylation of eIF2alpha, a known upstream effector of ATF4 activation. Knock-down of ATF4 expression inhibited balloon-injury induced neointimal hyperplasia, suggesting that ATF4 is a key player in the SMC response to injury. Furthermore, microarray analysis identified several genes whose transcription in response to FGF-2 may be regulated by ATF4. In particular, this work demonstrates that ATF4 is necessary for VEGF-A upregulation in SMC in response to FGF-2 and mechanical injury in vitro and in the artery wall following balloon-injury. The second is a translation factor, YrdC203. Using confocal fluorescence microscopy, YrdC203 was found to localize partially to the ER, and with RPL12, a component of the 60S ribosomal subunit. Immunoprecipitation studies demonstrate that YrdC203 also interacts with an initiation factor, eIF5B. Mutation of an initiation factor’s signature on the exterior of YrdC203 perturbed its interaction with RPL12 and eIF5B, and inhibited the increase in protein synthesis observed with overexpression of YrdC203. This implicates YrdC203 as a translation factor responsible for ensuring protein synthesis in vascular SMC in response to injury. The present work provides evidence for new molecular mechanisms, transcriptional and translational, regulating the response of vascular SMC to injury. This would provide leads for future therapeutic targets.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 14, 2009) Includes bibliographical references.