Valdeci da Cunha

Fasudil, a Rho-Kinase Inhibitor, Attenuates Angiotensin II–Induced Abdominal Aortic Aneurysm in Apolipoprotein E–Deficient Mice by Inhibiting Apoptosis and Proteolysis

Background—Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein–coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II–induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. Methods and Results—Six-month-old apolipoprotein E–deficient mice were infused with Ang II (1.44 mg · kg−1 · d−1) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg · kg−1 · d−1 in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. Conclusions—These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II–induced AAA through inhibition of both apoptosis and proteolysis pathways.

Anti-atherosclerotic effect of simvastatin depends on the presence of apolipoprotein E.

Low density lipoprotein receptor deficient (LDLR-KO) and apolipoprotein E deficient (apo E-KO) mice both develop hyperlipidemia and atherosclerosis by different mechanisms. The aim of the present study was to compare the effects of simvastatin on cholesterol levels, endothelial dysfunction, and aortic lesions in these two models of experimental atherosclerosis. Male LDLR-KO mice fed a high cholesterol (HC; 1%) diet developed atherosclerosis at 8 months of age with hypercholesterolemia. The addition of simvastatin (300 mg/kg daily) to the HC diet for 2 more months lowered total cholesterol levels by approximately 57% and reduced aortic plaque area by approximately 15% compared with the LDLR-KO mice continued on HC diet alone, P<0.05. Simvastatin treatment also improved acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in isolated aortic rings, which was associated with an increase in NOS-3 expression by approximately 88% in the aorta measured by real time polymerase chain reaction (PCR), P<0.05. In contrast, in age-matched male apo E-KO mice fed a normal diet, the same treatment of simvastatin elevated serum total cholesterol by approximately 35%, increased aortic plaque area by approximately 15%, and had no effect on endothelial function. These results suggest that the therapeutic effects of simvastatin may depend on the presence of a functional apolipoprotein E.

A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) – Part II: Enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis

We have discovered a novel small-molecule TAFIa inhibitor, BX 528, which is potent, highly selective against other carboxypeptidases and safe. The present study was to determine if BX 528 can enhance exogenous and endogenous thrombolysis in four different animal models. In the first three models, a thrombus was induced by FeCl (2) (dogs) or laser (rats) injury of the femoral artery, or formed ex vivo and implanted in the jugular vein in rabbits. A low dose of exogenous t-PA was given to induce a low-level thrombolysis on an established thrombus. Co-treatment with BX 528 further enhanced the thrombolytic effects induced by the exogenous t-PA and, thus, reduced thrombosis in all three animal models. In a second rat model, fibrin deposition in the lungs was induced by batroxobin, which was spontaneously resolved in 30 minutes due to the activation of endogenous fibrinolysis. Pre-treatment with lipopolysaccharide (LPS) attenuated this spontaneous fibrinolysis. Co-treatment with 10 mg/kg BX 528 prevented the LPS-induced attenuation of endogenous fibrinolysis. Thus, these studies demonstrated that inhibition of TAFIa by BX 528, our newly discovered small-molecule TAFIa inhibitor, enhanced both the exogenous (induced by a low dose of t-PA) and endogenous (LPS-induced resistance) thrombolysis without increasing the bleeding risk in four different animal models of thrombosis in different species (rat, dog and rabbit) employing different thrombogenic stimuli (FeCl (2) , laser, ex vivo and batroxobin) to induce thrombus formation in different tissues (artery, vein and lung microcirculation).

A novel P2Y12 adenosine diphosphate receptor antagonist that inhibits platelet aggregation and thrombus formation in rat and dog models

Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y(12) receptor antagonist, BX 667. The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y(12) receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats, we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis. BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl(2), both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model. Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination. These data demonstrate that the reversible P2Y(12) receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.

Increased nitric oxide accounts for decreased basal vascular tone and responsiveness in the resistance vessels of high-cholesterol-fed rabbits.

The objective of this study was to determine the effects of hypercholesterolemia on basal vascular tone and vascular responses to pharmacologic agents in hindquarter resistance vessels. Blood pressure and hindquarter blood flow were measured in conscious rabbits fed a high cholesterol diet (1%) for 17 weeks (HC) compared to age-matched rabbits fed a normal diet (control). Basal hindquarter blood flow and vascular conductance were significantly higher in HC than in control rabbits. Administration of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 mg/kg) decreased basal hindquarter blood flow and vascular conductance in a greater magnitude in HC than in control rabbits, thus, abolished the differences in both the flow and conductance between 2 groups, indicating that increased NO was responsible for reduced basal vascular tone in the HC rabbits. L- (30 mg/kg), a selective inducible NOS (iNOS) inhibitor had no effects on either flow or conductance. This result does not support the involvement of iNOS. In separate experiments, animals were anesthetized and instrumented with an extracorporeal circuit to measure perfusion pressure under constant blood flow to the hindquarter vascular bed. In the HC group, vascular responses to acetylcholine, S-nitroso-N-acetyl-penicillamine and phenylephrine were all attenuated when compared to the responses in the control rabbits. These results indicate that local overproduction of NO due to hypercholesteremia could desensitize smooth muscle reactivity, thus causing general vascular hyporesponsiveness to vasoactive agents.