Une des propriétés majeures de la thrombine est le caractère pléiotropique de ses effets physiologiques et pathologiques à la fois dans le compartiment sanguin et tissulaire de la paroi. La voie de signalisation RhoA est activée par la fixation de la thrombine aux récepteurs PARs et cette voie est un régulateur principal de la mécanotransduction et de la plasticité cellulaire. Le facteur d’échange de RhoA, Arhgef1, est impliqué dans le développement de l’hypertension dépendante de l’angiotensine II et dans l’athérothrombose. Notre hypothèse est que le contrôle de la signalisation intracellulaire de RhoA par Arhgef1 est un élément régulateur de la coagulation plasmatique et pourrait participer aux modifications phénotypiques des plaquettes et des cellules vasculaires et ainsi contribuer à l’augmentation de la génération de thrombine tissulaire. Les objectifs ont été de caractériser la génération de thrombine et la fonction plaquettaire depuis leur activation jusqu’à leurs implications dans un modèle de thrombose tissulaire et d’étudier le rôle prothrombotique des cellules musculaires lisses vasculaires (CMLVs) chez des souris Arhgef1 -/-. Résultats : Les souris Arhgef1-/- ont une numération plaquettaire normale mais présentent une diminution significative de l’activation plaquettaire, de la génération de thrombine en sang total et en présence de plaquettes (mais pas en plasma pauvre en plaquettes) et de l’adhérence plaquettaire par rapport aux souris contrôles. Ces modifications se traduisent, in vivo, par un plus grand nombre d’arrêts transitoires de l’écoulement sanguin dans le modèle de saignement à la queue et un allongement du temps de survenue du thrombus occlusif carotidien en réponse au FeCl3 chez les souris Arhgef1 -/- comparées aux contrôles. Les CMLVs des souris Arhgef1 -/- génèrent moins de thrombine à leur surface et ont une prolifération diminuée par rapport aux CMLVs des souris contrôles. En conclusion, les résultats démontrent le rôle d’Arhgef1 dans les fonctions plaquettaires et dans la régulation du phénotype des CMLVs. Le mécanisme principal fait intervenir la Rho GTPase dans l’adhésion plaquettaire et la génération de thrombine à la surface CMLVs qui contrôlent la formation du thrombus. Ces résultats suggèrent que ce facteur d’échange est capable d’amplifier la thrombose artérielle et pourrait être impliqué via les récepteurs à la thrombine dans le couplage thrombine tissulaire-rigidité cellulaire via les plaquettes et les CMLVs dans les pathologies vasculaires. / One of the major properties of thrombin is the pleiotropic character of its physiological and pathological effects in both the blood and the tissue compartment of the vessel wall. The RhoA signaling pathway is activated by the binding of thrombin to the PARs receptors and this pathway is a major regulator of mechanotransduction and cellular plasticity. The RhoA exchange factor, Arhgef1, is involved in the development of angiotensin II-dependent hypertension and in atherothrombosis. Our hypothesis is that the control of intracellular RhoA signaling by Arhgef1 is a regulatory element of plasma coagulation and could participate in phenotypic modifications of platelets and vascular cells and thus contribute to the increase of tissue thrombin generation. The objectives were to characterize thrombin generation and platelet function from their activation to their implications in a model of tissue thrombosis, and to study the prothrombotic role of vascular smooth muscle cells (VSMCs) in Arhgef1 -/- mice. Results: Arhgef1 -/- mice had a normal platelet count but showed a significant decrease in platelet activation, thrombin generation in whole blood and in the presence of platelets (but not in platelet poor plasma) and platelet adhesion compared to control mice. These modifications result, in vivo, by a greater number of transitory stopping of the blood flow in the tail bleeding model and an increase in the time of occurrence of the carotid occlusive thrombus in response to FeCl3 in Arhgef1 -/- mice compared to controls. The VSMCs of Arhgef1 -/- mice generate less thrombin at their surface and have decreased proliferation compared to VSMCs of the control mice. In conclusion, the results demonstrate the role of Arhgef1 in platelet function and in the regulating of the phenotype of VSMCs. The main mechanism involves Rho GTPase in platelet adhesion and in thrombin generation at the VSMC surface that control thrombus formation. These results suggest that this exchange factor is able to amplify aterial thrombosis and could be involved via thrombin receptors in tissue thrombin-cell stiffness coupling via platelets and VSMCs in vascular pathologies.

Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

Abstract not available

Na+/Ca2+ exchanger (NCX) is a membrane protein which can mediate either Ca2+ entry (reverse mode) or exit (forward mode) in cells. As one of the major Ca2+ transport systems, NCX is postulated to play a critical role in the vascular smooth muscle cell. The aims of the present study are to firstly demonstrate the functional existence of NCX in vascular smooth muscle (including aorta and arteriole); to clarify the modulation of NCX; to explore the selectivity of NCX inhibitor KB-R7943; and lastly to investigate the role of NCX in the myogenic response. KB-R7943 has been widely used as a NCX inhibitor. The study investigated its pharmacological actions in rat aorta on a variety of Ca2+ dependent systems. Rat aortic rings were used. The constriction to low extracellular [Na+] is a functional response mediated by NCX operating in reverse mode. The data demonstrate that 10 µM KB-R7943 inhibited L-type Ca2+ channel, the capacitative Ca2+ entry and  adrenergic receptor pathway. Nevertheless, KB-R7943 can be used as a selective inhibitor of NCX at the lower concentration of 1 µM in rat aortic rings. The study investigated whether the endothelium could modulate NCX in rat aortic rings. Lowering extracellular [Na+] to 1.18 mM induced constriction in endothelium denuded rat aortic rings, but only a small constriction in endothelium intact rat aortic rings. In endothelium intact rat aortic rings, the guanylate cyclise inhibitor ODQ (1 µM) and the nitric oxide synthase inhibitor L-NAME (50 µM) greatly amplified the vasoconstriction to lowering extracellular [Na+], but had no effect when the endothelium was removed. The adenylate cyclise inhibitor SQ 22536 (100 µM) and the cyclooxygenase inhibitor indomethacin (10 M) showed no significant effect on the low-Na+ induced vasoconstriction in either endothelium denuded or intact aortic rings. The results suggest that endothelium modulated the NCX operation via the nitric oxide/guanylate cyclase, not the adenylate cyclase system; further prostanoids including prostacyclin was not involved. The interaction between nitric oxide and NCX was furt her explored using the nitric oxide donor sodium nitroprusside. Endothelium denuded rat aortic rings were preconstricted to the same extent with either low Na+ (1.18 mM), or the thromboxane A2 agonist U46619 (0.1 µM) or high K+ (80 mM). The vasorelaxation of SNP (30 nM) in low Na+ constriction was significantly larger compared to other agents. This indicates that NO has a special antagonism of low Na+ constriction and a hypothesis is proposed involving Na+/K+ ATPase. The investigation of NCX is mainly conducted in large vessels; much less evidence is available for small resistance vessels. The study investigated the role of NCX on myogenic response in pressurized cremaster muscle arterioles. Reducing extracellular [Na+] resulted in graded vasoconstriction which was inhibited by NCX inhibitor SEA0400 (1 µM). Myogenic vasoconstriction and the concomitant rise in internal [Ca2+] were induced by a transmural pressure increase from 70 to 120 mmHg which was prevented by NCX inhibitor: SEA0400 (1 µM). In conclusion, the present study suggests that NCX contributes to the myogenic response in cremaster arteriole.

<i>In vivo</i> and <i>in vitro</i> studies on numerous types of blood vessels obtained from a variety of vascular beds and species have demonstrated that oxygen free radicals (OFRs) can evoke both vasodilation and vasoconstriction. Specific OFRs have been shown to elicit different and often times opposite effects on vascular smooth muscle. Therefore, this thesis attempts to define the vascular actions and mechanism of oxygen free radicals (OFRs) [superoxide anion (O₂^-), hydrogen peroxide (HO₂) and hydroxyl radical (OH)] on isolated rabbit aorta. This thesis will examine the role of OH in Ach- and nitroglycerin (NTG)-induced relaxation of isolated rabbit aorta. Superoxide anions generated by xanthine (X) plus xanthine oxidase (XO) produced concentration-dependent contractions of isolated rabbit aorta. The contractile response to O₂^- was completely abolished in preparations denuded of endothellum or pretreated with superoxide dismutase (SOD), a scavenger of O₂^-. The contractile response was reduced by indomethacin (I), a cyclooxygenase inhibitor. These results suggest that O₂^- mediated by vasoconstrictor arachidonic acid metabolites. Hydrogen peroxide generated by glucose and glucose oxidase produced contraction (low concentrations) and relaxation followed by contraction (high concentrations) in isolated rabbit aorta. The contractile response was abolished in the presence of catalase, a scavenger of H₂O₂ however the relaxant effect was exaggerated. Indomethacin markedly reduced the H₂O₂-induced contraction. Relaxation was completely prevented by de-endothelialization or pretreatment with N^G-monomethyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthetase. These results suggest that H₂O₂ in large concentrations produces a biphasic response, relaxation followed by contraction. Relaxation is endothelium dependent and is mediated by endothelium-derived relaxing factor (EDRF), nitric oxide (NO). The contractile response is endothelium independent and is mediated by vasoconstrictor arachidonic acid metabolites of smooth muscle. Hydroxyl radicals generated by dihydroxyfumarate (DHF), ferric chloride (FeCl₃) and adenosine diphosphate (ADP) (DHF/FeCl₃-ADP) produced concentration dependent relaxations of NE-precontracted rabbit aorta. Mannitol (Ml) completely inhibited OH-induced relaxation. Relaxation was markedly reduced in aortic rings mechanically denuded of endothelium. The relaxant effect was reduced by an inhibitor of NO synthesis (LNMMA), by an inhibitor of guanylate cyclase (methylene blue), by an inhibitor of cyclooxygenase (indomethacin) and by an inhibitor of an ATP-sensitive K⁺ channel blocker (glyburide). These results indicate that OH produces relaxation that is endothelium-dependent and partially mediated by an endothelium-derived relaxing factor (NO), vasodilatory arachidonic acid metabolites and an ATP-sensitive K⁺ channel. We hypothesized that Ach-induced vascular relaxation is mediated by OH derived from the interaction of NO and O₂^-. To test this hypothesis we investigated the effect of Ach and NTG on NE-precontracted isolated rabbit aortic preparations in the absence or presence of scavengers of O₂^- (SOD), and OH (dimethylthiourea (DMTU) or mannitol or Garlicin). The OFR scavengers (SOD, dimethylthiourea, mannitol, garlicin and histidine) alone or the combination of SOD and DMTU markedly reduced Ach- or NTG-induced relaxation. However, the combination of histidine, (a ¹O₂ scavenger) SOD and DMTU completely abolished Ach-induced relaxation.

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 PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Thesis advisor: William P. Fay. Includes bibliographical references

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Τα αγγειακά λεία μυικά κύτταρα (ΑΛΜΚ) αποτελούν το κυρίαρχο στοιχείο του μέσου χιτώνα των αιμοφόρων αγγείων, ενώ συμμετέχουν ενεργά και στο σχηματισμό και την ωρίμανση του καρδιαγγειακού συστήματος. Η δομή τους εξυπηρετεί την εκτέλεση της σημαντικότερης λειτουργίας τους, που είναι η συστολή. Αξιοσημείωτο χαρακτηριστικό των αγγειακών λείων μυικών κυττάρων αποτελεί η φαινοτυπική τους πλαστικότητα, δηλαδή η ικανότητα στροφής από το συσταλτικό σε έναν περισσότερο συνθετικό φαινότυπο, που λαμβάνει χώρα υπό προϋποθέσεις. Οι αλληλεπιδράσεις με τα υπόλοιπα κυτταρικά στοιχεία του τοιχώματος των αρτηριών και των έμμορφων συστατικών του αίματος, αλλά και η φαινοτυπική πλαστικότητα καθιστούν καθοριστικό το ρόλο των αγγειακών λείων μυικών κυττάρων στην παθογένεια της αθηροσκλήρωσης. / Vascular smooth muscle cells (VSMCs) comprise the main element of the tunica media of blood vessels, while they actively participate in the formation and maturation of the cardiovascular system. Their structure serves their basic function, which is contraction. An interesting feature of vascular smooth muscle cells is their phenotypic plasticity, the ability to shift from a contractile to a more synthetic phenotype, under certain conditions. The interaction with other cellular elements within the vascular wall or in the bloodstream, as well as their phenotypic plasticity, give vascular smooth muscle cells a decisive role in the pathogenesis of atherosclerosis.

Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

Arterial remodeling is a process by which arteries respond to sustained changes in their mechanical environment. This process occurs in a way such that an artery's local mechanical environment (circumferential, shear, and axial stress) is maintained at a homeostatic level. However, most studies utilize a methodology that controls the global parameters (pressure, flow rate, and axial stretch). This approach often confounds the results since the actual drivers of remodeling are not independently isolated. This research involved developing a methodology and system capable of independently controlling each of the local parameters and examining the effect of axial stress on remodeling. An organ culture system capable of monitoring and controlling the three global parameters and calculating the cross sectional geometry was developed. This combination of hardware was incorporated into LabVIEW which afforded the user the ability to define desired values for the local mechanical parameters. Porcine common carotid arteries were cultured for seven days in this system under physiologically normal circumferential and shear stresses and a constant axial stress of either 150 kPa or 300 kPa. Material response, general arterial morphology, tissue viability, and collagen synthesis were examined in order to gauge the effectiveness of the organ culture system and assess any arterial remodeling. The results of this study demonstrate the ability of the organ culture system in achieving and maintaining target values of stress throughout the culture period. Cell viability, general arterial morphology, and collagen synthesis rates were maintained for all arteries. The elevated axial stress appeared to cause a softening of the artery in both the axial and circumferential direction. It was hypothesized that this softening was the result of a changing collagen structure. Additional softening seen in arteries was attributed to the effects of the culture system.