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.

共著者あり。共著者名:Munakata Mitsuru, Sato Atsuko, Amishima Masaru, Homma Yukihiko, Kawakami Yoshikazu. / Hokkaido University (北海道大学) / 博士 / 医学

<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.

Bidens pilosa L. is a traditional Chinese herbal medicine of which was considered as a potential COX2 inhibitor and anti-inflammatory agent. The objective of this study is to discriminate the protective effect of aqueous extract of Bidens pilosa L. leaves (BPLAE) against TAA-induced live fibrosis using an animal model. The herb extracts were administrated via intraperitoneal injection once per week (1.25, 2.5 g/kg), and thioacetamide (200 mg/kg) was injected three times per week and the mice were sacrificed at week 4 and week 8, respectively. Immunohistochemistry staining, Hematoxylin-eosin (HE) staining, Sirius red staining were carried out to evaluate the pathological alterations of mouse livers; in addition, Western blotting was performed to measure the differential expression of £\-smooth muscle actin (£\-SMA) between different treatment groups (vehicle, week 4 and week 8). Hepatic hydroxyproline was also detected in order to compare difference in collagen formation of each group. The results showed that Bidens pilosa L. effectively reduced amount of hepatic hydroxyproline and £\-SMA protein in mice with fibrotic liver induced by TAA. Moreover, in histiopathological exam, the BPLAE treated mice demonstrated a lower collagen and £\-SMA expression, which indicated that BPLAE might reduce degree and severity of liver fibrosis in mice. In conclusion, these results suggested that BPLAE potentially against fibrogenesis in TAA- induced mice liver fibrosis. Additionally, we found that BPLAE might involve in the signaling pathway of MAPK (ERK1/ERK2), which reduced the phosporylation level of p44 but not p42. Further studies using cell base assay to confirm the inhibiting role of BPLAE against cell proliferation or migration is warrant.

Despite efforts by clinicians and scientists world-wide, coronary artery disease remains to be the leading cause of morbidity and mortality in industrialized nations. Development of a tissue engineered coronary by-pass graft with low thrombogenicity and immune responses, suitable mechanical properties, and a capacity to remodel to their environment could have a significant impact on the treatment of coronary artery disease. While many methods for the tissue engineering of blood vessels have been developed, one promising approach is the self-assembly method. Using autologous cells that produce an endogenous extracellular matrix (ECM), the potential for therapeutic success is high due to biocompatibility. However, despite these advantages, improvements can be made which will give the grafts an even higher rate of patency. This dissertation presents a study of the characterization of the biaxial mechanical properties of self-assembly tissue engineered blood vessels (SA-TEBV), as well as developing a framework for fabrication strategies of SA-TEBV. Native arteries are exposed to multiaxial mechanical loads, including (a pulsatile) blood pressure that causes the vessel to cyclically distend circumferentially, blood flow that induces a shearing load along the luminal surface, and an axial extending load; the latter is relieved upon excision, causing the vessel to retract. These mechanical loads introduce intramural wall stresses and flow induced wall shear stresses that play a key role in mechano-biological signaling and tissue homeostasis. Until now, the mechanical properties of SA-TEBV have only been characterized in the circumferential direction (i.e. burst pressure and circumferential elastic modulus). The objective of this work is to characterize the biaxial mechanical properties of SA-TEBV to quantify their mechanical behavior and local intramural stresses under physiological loading. The work will show that while the global mechanical response of the SA-TEBV is similar to that of native arteries (and potentially sufficient), the local intramural stresses (using the current fabrication techniques) differ greatly from native coronary arteries. Therefore, a novel approach to fabricate the self-assembly derived tissue sheets is developed and tested which utilizes biaxial mechanical stimulation to alter the microstructure, thereby controlling their mechanical response.

The engineering of human vascular grafts is an intense area of study since there is crucial need for alternatives to native vein or artery for vascular surgery. This current study sought to prove that a tissue engineered blood vessel (TEBV) 1mm in diameter could be developed from human smooth muscle cells and that endothelial progenitor cells (EPCs) could be cultured and used to endothelialize these grafts. This project had four specific aims: the isolation and characterization of EPCs, the seeding of a novel scaffold with EPCs and exposure to physiologic shear stress in vitro, the development of TEBV from human smooth muscle cells that are strong enough to implant in vivo, and the in vivo implantation of TEBV into the rat aortic model with a comparison of EPC seeded TEBVs pretreated with shear stress and unseeded TEBVs. The results yielded isolation of four EPC lines and a flow system design capable of seeding EPCs onto a novel scaffold with preliminary studies indicating that it is capable of exposing the EPCs to physiologic shear stress, although further studies require more optimization. The development of mechanically strong TEBV was highly successful, yielding TEBVs comparable to native vessels in collagen density and burst pressure, but with much lower compliance. Current implantation studies indicated that unseeded TEBV grafts implanted into the rat aorta without anticoagulation is highly thrombogenic. However, anticoagulation using Plavix may be capable of maintaining graft patency. These TEBVs did not rupture or form aneurysm in vivo and the future completion of the in vivo studies are likely to demonstrate the high potential of these grafts.

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

Studies on the perfused rabbit ear artery preparation showed that withdrawal of Mg 2+ from extracellular fluid potentiated the responses to histamine and ATP but not to catecholamines. Similar results were obtained in [2xCa2+] Krebs solution. Increases in [Mg 2+] decreased responses to the three agonists to a similar extent. In subsequent experiments attempts were made to alter the availability of calcium for contraction induced by these agonists either by changing the [Ca 2+] of the Krebs solution or by using Ca 2+ influx inhibitors, ouabain and ryanodine. The effects of these agonists were compared to those observed when Mg2+ was altered. In general, the results obtained in perfused rabbit ear artery supported the hypothesis that changes in extracellular [Mg2+] affect the availability of calcium for contraction but were not consistent with the suggestion that Mg2+ alters Ca2+ influx. In a second type of preparation tension responses of superfused rings of ear artery were studied. Responses to changes in extracellular [Ca2+] and[ Mg2+] were found to differ slightly from those obtained in the perfused artery. A simultaneously perfused and superfused arterial preparation showed that responses to changes in [ Mg2+] and[Ca2+] were different if the agonist was administered to the adventitial surface of the vessel rather than via the intimal surface. The effects of alterations in extracellular [Mg 2+] were studied in mesenteric arteries from weight matched normotensive and spontaneously hypertensive rats (SHR). No differences in response to NA or ATP when extracellular [Mg 2+ ] was either increased or reduced were observed in the SHR compared to the normotensive animal. However, a difference in calcium dependence was demonstrated between the two types of vessels to NA. In contrast to mesenteric arteries, experiments on aortae from normotensive rats and SHR showed no differences in the calcium dependence of NA responses between normotensive and SHR vessels, whereas, [4xMg2+ ] Krebs solution reduced the responses of normotensive aorta to NA more than SHR. These results in the rat were not consistent with the hypothesis that alteration in [Mg 2+] can be explained in terms of altered calcium availability. Attempts to increase intracellular cyclic AMP with theophylline showed that the response to ED50 NA in both mesenteric arteries and aortae from normotensive were reduced more than SHR. It is concluded that the effect of changes in extracellular [Mg2+] on the reactivity of vascular muscle varies depending on the type of vessel and species of animal from which the vessel is taken. In addition when all the experimental results are considered, it is not possible to explain all the actions of altered [ Mg2+ ] simply in terms of changed calcium availability.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences