INHIBITORY ROLE FOR VITRONBCTIN IN ANGIOTBNSIN II-INDUCED VASCULAR FIBROSIS Rodney J. Dilley, Natalie Kvalheim, Stefan Koschnick and David J. Loskutoff. The Baker Medical Research Institute, Prahran Australia and The Scripps Research Institute, La Jolla USA

The vasoconstrictor angiotensifi II is a potent stimulator of cardiac fibrosis, producing interstitial and perivascular collagen deposition. The role of the fibrinolytic system in cardiac fibrosis was examined using angiotensin treated mice deficient in plasminogen activator inhibitor-l (PAI-1) and its cofactor vitronectin (Vn) and compared to wild type mice. Mice were infused with angiotensin II (20 ng/min) from osmotic minipumps for 12 days. Blood pressures were measured by tail-cuff method and cardiovascular tissues sampled for histology after perfusion fixation. Blood pressures were elevated in all angiotensin treated mice when compared to vehicle treated controls. There was also significant ventricular hypertrophy in all angiotensin treated groups. Cardiac fibrosis occurred in wildtype mice, with increased interstitial collagen (microscars) in both left and right ventricles and increased perivascular collagen. PAI- deficient mice exhibited some perivascular expansion and microscar formation, particularly in the right ventricle. In Vn deficient mice there was a substantial increase in vascular damage, with aggressive extension of perivascular fibrosis into the interstitium and neointima formation. These changes suggest that Vn plays an inhibitory role in vascular damage or repair in this model. The mechanism of action is unknown however as Vn is a cofactor for PAI-l’s antifibrinolytic action it may be necessary for anchoring PAI- in the tissues, inhibiting migration of cells into the perivascular region or regulating collagen degradation.

REGULATION OF ND AND FREE RADICAL GENERAlION IN THE STlMULATED RAT POLYMORPHONUCLEAR LEUCOCYTES

M. Dixit, P. Shama and M. K. Bharthwal Pharmacology Division, Central Drug Research Institute, Lucknow - 226001, INDIA

Previous study from this lab have shown NO mediated modulation of free radical generation from polymorphonuclear leukocytes (PMNs) following hypoxic- reoxygenation as well as in the normoxic cells. The present study is an attempt to investigate further the regulation of NO and free radical generation by calcium and oxygen. PMNs were isolated from the rat blood using Ficoll-Hypaque. Nitric oxide synthase (NOS) activity was measured by estimating nitrite content or by the conversion of radioactive L-Arginine to L-Citrultiie. while free radical generation was measured by flowcytometry. Following stimulation PMNs mediied free radical generation was decreased in time dependent manner while NO synthesis was decree& iWaUy but gradually increased time dependentty. Modulation of calcium levels although a%cted the free radical generation from PMNs, however, it had no effect on the NO synthesis. Catmodulin antagonist inhibited both free radical-and NO generation. in byperoxic medium decreased in NO synthesis was not observed. The result obtained thus indicate that augmentation of free radical generation from rat PMNs following stimulation is dependent on the availability of oxygen in the medium and PMNs NO generation is not regulated by the intracellular calcium.

HEAT SHOCK PROTEINS AND PROTECTION AGAINST ISCHEMIC INJURY Dillmann W.H., University of California, San Diego, San Diego, Catifornia, U.S.A.

MyocardA ischemia leads to increased expression of members of the heat shock protein @ISP) family. The protective effects of inducible HSWO, the mitcchondrial chaperones HSP60 and HSPlO and members of the small heat shock proteins family especially aB4ystallin has been explored by us using transgenic mice or adenoviral vectors to express bansgenes in cardiac myocytfs We could demonstrate in transgenic mice overexpressing inducible HSWO that infarct site was much smaller and recovery from brief episodes of ischemia resulting in stunning was much improved. Similarly, we constructed mice in which CrB-crystallin was expresed in cardiac myocytes. Transgene overexpmssion of uEt- crystdin confers simultaneous protection against cardiac myocyte apoptosis and necrosis during myocAial ischemia and reperfusion. ln more recent studies adenoviral vectors were constructed which overexpressed the mitochondrisl chaperone HSP60 or the mitochondrial chaperone HSPlO or a combination of HSP60 end HSPlO in cardiic myocytes. Cardiac myocytes were submitted to simulated ischemia Overexpression of the combination of HSP60 and HSPIO and HSP60 or HSPlO individually protected cardiac myocytes against apoptosis led to decreased mitcchontial cytochrome~ release and increased complex III and complex IV function in mitochondria after simulated ischemia/reoxygenation. These findings therefore indicate that members of different heat shock protein families including HSP70 aB-cry.stallin and HSP60 and 10 exert prottive effects against ischemia reoxygenation in cardiac myocytes by mechanisms which appear to be specific for diierent types of heat shock proteins.

CARDIAC PHENOTYPES IN RELAXIN KNOCKOUT MICE: IMPORTANCE OF THE GENDER ‘Xiao-Jun Du, ‘Ling Zhao, ‘Xiao-Ming Gao, IGeoffrey W. Tregear, ‘Baker Medical Research lnstitutc, and ‘Howard Florey Institute, Melbourne, Australia

Specific relaxin (RLX) binding sites exist in the heart mediating functional respaces to exogenous RLX. However, cardiac actions of endogenous RLX remain unclear. We performed echocardiography aud catheterization in mak aad female mice (15-20 m, S-IO/group) with RLXknockout (4) vs. wildtype (+/+) and heterozygous (+/-) littamats. When anesthetized, n&er heart rate, blood pressure, left ventricular (LV) dimensions, ti-actional shortening, LV dP/dt were different among tbc 3 groups of either gender, nor was responses to isoprotormol. These findings indicate unaltered cardiac function in RLX - /- mice. However, compared to +/+ and +/- males, 4 males had 15% increase in LV mass (by echo and weight), 30% increase in atria1 weight and IO-30% increase iu weights of lungs and liver, and a higher LVEDP (all P<O.O5). Hydroxyproline concentration was higher in the LV of 4 vs. +/+ (+17%, P=O.O65) and +/- males (+22%, P<O.O2), indicating higher collagen content. None of t&e parameters was differeat among female groups. Thus, RLX kuockout in male mice results in moderate cardiac hypertrophy, impeded venous return and modest qan congestion, most like+ due to increased LV collagen content and chamber stiIfi~ess. These cardiac phenotypa by RLX knockout are absatt in the female gender.

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