A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) - part I: pharmacological characterization.

We have discovered a novel small-molecule (3-phosphinoylpropionic acid) inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa), BX 528, which had an IC (50) of 2 nM in an enzymatic assay and 50 nM in an in-vitro clot lysis assay, with 3,500- to 35,000-fold selectivity against other carboxypeptidases, such as CPN, CPZ and CPD, and 5- and 12-fold selectivity against CPE (CPH) and CPB, respectively. At 10 micro M, BX 528 had no significant activity (<50% inhibition or antagonism) in a panel of 137 enzymes and receptors. It had no effects on blood coagulation and platelet aggregation up to 300 and 10 micro M, respectively. The plasma half-life following intravenous administration was 0.85 hours in rats and 4.5 hours in dogs. No significant metabolism was detected in human, dog or rabbit hepatic microsomes, and no significant inhibition of cytochrome P450 3A4 and 2D6 up to 30 micro M. No cytotoxic or cell proliferative effects were found in three hepatic and renal cell lines up to 300 micro M and no mutagenic activity was seen in the Ames II screen. There were no significant hemodynamic effects in rats and dogs up to 100 and 30 mg/kg with peak plasma drug concentrations of approximately 1,000 and 300 micro M, respectively. In an in-vivo complement activation model in guinea pigs, BX 528 showed minimal inhibition of plasma CPN activity up to 60 mg/kg with peak plasma concentrations up to 250 micro M. Thus, these data demonstrate that BX 528 is a novel, potent, selective and safe TAFIa inhibitor.

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 P2Y(12) 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.

Monocyte chemoattractant protein-1 or macrophage inflammatory protein-1alpha deficiency does not affect angiotensin II-induced intimal hyperplasia in carotid artery ligation model.

BACKGROUND: Angiotensin II (Ang II) promotes atherosclerotic vascular diseases, in which proinflammatory and proliferative effects play a major pathogenic role. Ang II up-regulates chemokines, such as monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha, which are important pro-inflammatory factors mediating infiltration of inflammatory cells into atherosclerotic lesion. The aim of the present study was to determine whether the presence of MCP-1 or MIP-1alpha is essential in Ang II-induced intimal hyperplasia in the carotid artery ligation model. METHODS: Six-month-old male C57BL/6-, MCP-1-, or MIP-1alpha-deficient mice underwent ligation of the common left carotid artery and were randomly assigned to receive either vehicle or Ang II (1.4 mg kg(-1) day(-1)) via a subcutaneously implanted osmotic infusion pump (model 2004, Alzet) for 4 weeks. RESULTS: Ang II not only increased MCP-1 and MIP-1alpha production but also enhanced neo-intimal formation, media thickness, and adventitia development in the ligated carotid arteries in C57BL/6 mice. However, MCP-1 or MIP-1alpha deficiency failed to affect intimal hyperplasia in vascular remodeling. CONCLUSION: These results indicate that MCP-1 or MIP-1alpha may not be essential in mediating the proliferative effects of Ang II, a major pathological changes in intimal hyperplasia in the carotid artery ligation model.

Endothelial NO synthase deficiency promotes smooth muscle progenitor cells in association with upregulation of stromal cell-derived factor-1alpha in a mouse model of carotid artery ligation.

BACKGROUND: Endothelial NO deficiency (endothelial NO synthase [eNOS]-knockout [KO]) enhanced smooth muscle cell (SMC)-rich neointimal lesion formation in a mouse model of carotid artery ligation (CAL). Recent evidence indicated that stromal cell-derived factor-1alpha (SDF-1alpha)-mediated recruitment of circulating SMC progenitor cells substantially contributed to the SMC-rich neointimal hyperplasia induced by vascular injury. The goal of this study was to investigate the effects of eNOS deficiency on the expression of SDF-1alpha and mobilization of circulating SMC progenitor cells in CAL model. METHODS AND RESULTS: Two- to 3-month-old C57BL/6J wild-type (WT) and eNOS-KO mice were evaluated 1, 2, or 4 weeks after CAL. CAL-induced expression of SDF-1alpha, as detected by immunohistochemical staining and further quantified by ELISA in the ligated carotid arteries, was moderate and transient with a peak at 1 week in WT mice. SDF-1alpha expression was significantly higher at 1 week and persisted through 2 weeks in eNOS-KO mice. CAL was associated with increased circulating stem cell antigen-1(+) (Sca-1(+))/c-Kit(-)/Lin- cells (interpreted as SMC progenitor cells), which peaked at 1 week in WT mice. This effect was also significantly greater and longer-lasting in eNOS-KO than WT mice. The number of circulating Sca-1(+)/c-Kit(-)/Lin- cells was positively correlated with the expression of SDF-1alpha but not vascular endothelial growth factor in the ligated carotid arteries. Furthermore, immunostaining showed abundant Sca-1-positive cells in the adventitia of the 1-week ligated carotid arteries from eNOS-KO mice but not in WT mice. We also determined that eNOS deficiency enhanced CAL-induced intimal cell proliferation in the ligated arteries as detected by proliferating cell nuclear antigen staining but did not induce cell apoptosis as detected by staining for active caspase-3. CONCLUSIONS: Our results indicate that eNOS deficiency exacerbates CAL-induced expression of SDF-1alpha and its receptor CXCR4. This is correlated with an increase in Sca-1(+) cells in peripheral blood and adventitia, which may contribute to vascular remodeling and SMC-rich neointimal lesion formation. This suggests that constitutive eNOS inhibits SDF-1alpha expression and provides an important vasculoprotective mechanism for intact endothelium to limit SMC proliferation and recruitment in response to vascular injury.

Enalapril attenuates angiotensin II-induced atherosclerosis and vascular inflammation.

Angiotensin converting enzyme (ACE) inhibitors prevent a wide variety of key events underlying atherogenesis. Whether these actions depend solely on reduction of angiotensin II (Ang II) generation is still to be determined. This study was undertaken to determine whether enalapril, an ACE inhibitor, prevents atherosclerosis and vascular inflammation induced by Ang II in apolipoprotein E-deficient (apoE-KO) mice. Subcutaneous infusion of Ang II (1.44 mg/(kg day)) for 4 weeks increased blood pressure and accelerated atherosclerosis development in the carotid arteries. The expression of the endothelial adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as the chemokines monocyte chemotactic protein-1 (MCP-1) and macrophage-colony stimulating factor (M-CSF) was up-regulated in the aortas of Ang II-treated mice. Enalapril co-treatment (25 mg/(kg day), in drinking water) prevented the development of atherosclerosis without affecting blood pressure or circulating cholesterol. In addition to preventing the Ang II-induced over-expression of adhesion molecules and chemokines in the aorta, enalapril up-regulated the expression of peroxisome proliferator-activated receptors (PPARs)-alpha and -gamma, potential anti-inflammatory transcription factors. In the aortic arch, a lesion-prone site, the co-treatment with enalapril reduced the percentage of arterial wall occupied by macrophages and foam cells, medial sclerosis and elastin reduplication. Together, these data suggest an important role for Ang II-independent mechanisms in the antiatherogenic and anti-inflammatory effects of ACE inhibitors.

17 Beta-estradiol attenuates development of angiotensin II-induced aortic abdominal aneurysm in apolipoprotein E-deficient mice.

OBJECTIVE: Angiotensin II (Ang II) promotes vascular inflammation, accelerates atherosclerosis, and induces abdominal aortic aneurysm (AAA). These changes were associated with activation of nuclear factor (NF)-kappaB-mediated induction of proinflammatory genes. The incidence of AAA in this model was higher in male than in female mice, and the vascular effects of estrogen may be associated with anti-inflammatory actions. The present study was undertaken to test the hypothesis that estrogen can attenuate Ang II-induced AAA in apolipoprotein E-deficient mice via its anti-inflammatory mechanism. METHODS AND RESULTS: Infusion of Ang II (1.44 mg/kg per d for 1 month) induced AAA in 90% of the animals (n=20) with an expansion of the suprarenal aorta (diameter 1.9+/-0.14 mm versus <1 mm in normal mice). In mice treated with 17beta-estradiol (E2, 0.25-mg subcutaneous pellets), Ang II induced AAA only in 42% of the animals (n=19) with a significant reduction of average diameters of the suprarenal aorta (1.5+/-0.14 mm). E2 also decreased the expressions of intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, E-selectin, monocyte chemotactic protein-1, and macrophage-colony stimulating factor in the aorta. CONCLUSIONS: These data suggest that attenuation of AAA by E2 is associated with inhibition of proinflammatory gene expression.

Noninvasive measurement of abdominal aortic aneurysms in intact mice by a high-frequency ultrasound imaging system.

Mouse models of abdominal aortic aneurysm (AAA) have been commonly used in many laboratories for studying molecular mechanisms of AAA formation and development, as well as for testing novel therapeutic agents in the treatment of AAA. However, because of the small size of the animal, the quantification and characterization of AAA development and progress is difficult, time-consuming and requires the sacrifice of the experimental animals. We report here a noninvasive method to detect and measure AAA in mice using a high-frequency ultrasound (US) imaging system specifically designed for microimaging of the mice (Vevo 660; VisualSonics, Toronto, ONT, Canada). A total of 21 male apolipoprotein-E-deficient mice were chronically infused with angiotensin II (1.44 mg/kg daily) for 28 days to induce AAA formation. A 2-D echo image of the abdominal aorta was acquired at longitudinal and transverse planes, followed immediately by post mortem dissection of the abdominal aorta for direct measurements. The US images clearly showed a bulge-like expansion localized specifically in the suprarenal region of the abdominal aorta, with a shape strikingly similar to that of the aorta dissected post mortem. In addition, the US images can also provide measurements of the luminal diameter and wall thickness of the abdominal aorta. The average dimensions of the abdominal aorta were not significantly different between the US and post mortem measurements, nor between the transverse and longitudinal US images. The different types of the measurements are also highly correlated with each other, with a linear correlation (r) between 0.7 and 0.9. Thus, we have established and validated a novel application to noninvasively measure AAA development and progress in a mouse model using a high-frequency US imaging system that has the advantages of low cost, rapid imaging speed, reproducibility and high resolution, and makes repeated monitoring of the progress of AAA development over a time-course possible.

Angiotensin II is associated with activation of NF-kappaB-mediated genes and downregulation of PPARs.

Angiotensin II (ANG II) promotes vascular inflammation through nuclear factor-kappaB (NF-kappaB)-mediated induction of pro-inflammatory genes. The role of peroxisome proliferator-activated receptors (PPARs) in modulating vascular inflammation and atherosclerosis in vivo is unclear. The aim of the present study was to examine the effects of ANG II on PPARs and NF-kappaB-dependent pro-inflammatory genes in the vascular wall in an in vivo model of atherosclerosis and aneurysm formation. Six-month-old male apolipoprotein E-deficient (apoE-KO) mice were treated with ANG II (1.44 mg/kg per day for 30 days). ANG II enhanced vascular inflammation, accelerated atherosclerosis, and induced formation of abdominal aortic aneurysms. These effects of ANG II in the aorta were associated with downregulation of both PPAR-alpha and PPAR-gamma mRNA and protein and an increase in transcription of monocyte chemotactic protein-1 (MCP-1), macrophage-colony stimulating factor (M-CSF), endothelial-selectin (E-selectin), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) throughout the entire aorta. ANG II also activated NF-kappaB with increases in both p52 and p65 NF-kappaB subunits. In summary, these in vivo results indicate that ANG II, through activation of NF-kappaB-mediated pro-inflammatory genes, promotes vascular inflammation, leading to acceleration of atherosclerosis and induction of aneurysm in apoE-KO mice. Downregulation of PPAR-alpha and -gamma by ANG II may diminish the anti-inflammatory potential of PPARs, thus contributing to enhanced vascular inflammation.

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.

Lipopolysaccharide attenuates thrombolysis in batroxobin-induced lung vasculature fibrin deposition but not in ferrous chloride-induced carotid artery thrombus in rats: role of endogenous PAI-1.

In this study, we investigated if elevation of endogenous plasminogen activator inhibitor type 1 (PAI-1) by lipopolysaccharide (LPS) can retard thrombolysis in both a rat model of lung vasculature fibrin deposition and a platelet-rich thrombus model induced by endothelial injury. By 3 h following an intravenous bolus injection of 0.5 mg/kg LPS, the plasma PAI-1 level had increased to approximately 8 ng/ml. 125I-labeled fibrinogen was injected intravenously followed by an injection of batroxobin. Batroxobin converts fibrinogen into insoluble fibrin, which was then deposited in the lungs within 5 min, followed by spontaneous fibrinolysis that completely cleared fibrin deposition in the lungs by 30 min. In rats pre-treated with LPS, spontaneous fibrinolysis was significantly retarded. In the endothelial injury model, topical application of FeCl2 on the carotid artery induced an occlusive platelet-rich thrombus, which was not sensitive to endogenous thrombolysis. Exogenous tissue-type plasminogen activator (tPA) was required to recanalize the occlusive thrombus in a dose-dependent manner. Pre-treatment with LPS did not alter the dose-response curve of exogenous tPA-induced thrombolysis. These data indicate that batroxobin-induced lung vasculature fibrin deposition in rats, unlike the FeCl2 model, is sensitive to the impact of endogenous PAI-1 on fibrinolysis.

Reduction of cardiac functional reserve and elevation of aortic stiffness in hyperlipidemic Yucatan minipigs with systemic and coronary atherosclerosis.

To test the hypothesis that cardiac functional reserve is reduced in animals with severe atherosclerosis, Yucatan minipigs were fed a high-cholesterol diet (Chol) for 8 months. Half of them was made diabetic, an additional risk factor for atherosclerosis, with streptozotocin (STZ). Another group of age-matched minipigs were fed a normal diet as controls. At the end of the treatment period, animals were instrumented for the measurement of cardiovascular hemodynamic parameters under isoflurane anesthesia. Cardiac functional reserve was measured by the magnitude of the inotropic response to isoproterenol stress. Hyperlipidemic minipigs developed severe atherosclerotic plaques in the aorta, coronary and iliac artery, accompanied by an increase in the aortic stiffness indexed by increases in pulse wave velocity and augmentation index. These vascular changes were more severe in STZ-induced insulin-dependent diabetes mellitus. The isoproterenol-induced increase in left ventricular contractility (dP/dt) and relaxation (-dP/dt) and, consequently, cardiac output were also significantly reduced in both the Chol groups with or without STZ, compared to control group. Thus, cardiac functional reserve measured by isoproterenol-stimulated responses was reduced in atherosclerotic minipigs, which was further diminished in diabetes.

Interferon-beta attenuates angiotensin II-accelerated atherosclerosis and vascular remodeling in apolipoprotein E deficient mice.

Atherosclerotic vascular disease is an inflammatory disease. Interferon-beta (IFN-beta) is an important immune modulator. However, the role of IFN-beta in atherosclerotic vascular disease is still not clear. The present study is designed to determine the effects of IFN-beta on atherosclerosis, abdominal aortic aneurysm (AAA) formation and proliferative vascular remodeling in apolipoprotein E (apoE) deficient mice. Six-month-old male apoE deficient mice fed a normal chow underwent ligation of the common left carotid artery, and were randomly assigned to receive either vehicle or angiotensin II (Ang II, 1.4 mg/kg daily) via a subcutaneously implanted osmotic infusion pump. The animals were further assigned to groups that were subjected to subcutaneous injection of vehicle or murine IFN-beta (10 MIU/kg, daily). Ang II increased atherosclerotic area in the non-ligated carotid artery and aortic arch, induced AAA, and exacerbated ligation-induced adventitial proliferation and neointimal hyperplasia characterized by smooth muscle cell (SMC) proliferation and macrophage infiltration in the ligated carotid artery. Co-treatment with IFN-beta, had no effects by itself, significantly attenuated Ang II-accelerated increase in the areas of neointima, adventitia, SMC and macrophage in the ligated carotid artery and suppressed Ang II-exacerbated atherosclerosis, but did not affect Ang II-induced AAA formation. These data indicate that IFN-beta can play a prominent anti-atherosclerosis, anti-inflammation, and anti-proliferation role of vasculoprotection.

Urokinase-type plasminogen activator deficiency (uPA-KO) prevented carotid artery ligation-induced vascular remodeling in mice.

It has been demonstrated that urokinase-type plasminogen activator (uPA) plays an important role in vascular remodeling. This study was designed to determine whether uPA deficiency (KO) affects carotid artery ligation-induced vessel remodeling and the interaction with angiotensin II (Ang II). Ligation of the left common carotid artery in 6-month-old wild-type (C57 black/6J) mice for 4 weeks induced a concentric remodeling with vessel wall thickening, characterized by cell proliferation in neointima, media, and adventitia, and with lumen narrowing without a significant enlargement of overall vessel dimension. Intima lesions were characterized by alpha-actin positive smooth muscle cell (SMC) proliferation in a matrix background. No detectable presence of MAC-3 positive macrophages existed in the vascular wall. The ligation-induced vascular neointimal formation and adventitial proliferation, but not lumen narrowing and media expansion, were completely prevented in age-matched uPA-KO mice. Chronic infusion of Ang II (1.44 mg/kg per day) via a subcutaneously implanted osmotic minipump did not significantly affect the gross morphology of the nonligated carotid artery from both wild-type and uPA-KO mice, but it enhanced ligation-induced vascular remodeling. However, in the presence of Ang II, uPA deficiency had no effects on ligation-induced mophermetric change, but it partially and significantly reduced cell proliferation. These data indicate that uPA may play a critical role in ligation-induced vessel remodeling. Ang II may activate other mechanisms independent of uPA to exacerbate ligation-induced vascular remodeling.

Gait analysis in a murine model of collagen-induced arthritis.

Murine collagen-induced arthritis (CIA) has become a valuable animal model for elucidating pathogenic mechanisms and evaluating therapeutic effects for rheumatoid arthritis. Recent advances in digital imaging and computer technology have enabled gait analysis to develop into a powerful tool for objectively detecting functional deficits in human and animal models. The present study explored the use of non-invasive video-capture gait analysis in the evaluation of a murine CIA model. CIA was induced in 45 female DBA/1LacJ mice (8 to 10 weeks old) by immunization with lyophilized bovine articular type II collagen. Gait parameters were determined by ventral plane videography and were correlated to traditional arthritis clinical scores. Our results showed that increases in clinical scores that measure the severity of CIA corresponded to changes in multiple gait parameters that reflect both morphologic (increases in paw area) and functional (increase in stride frequency, decrease in stride length, hind-limb paw placement angle, as well as stride, stance, and braking times) deficits. Our work indicated that the non-invasive video-capture device may be used as a simple and objective data acquisition system for quantifying gait disturbances in CIA mice for the investigation of mechanisms and the evaluation of therapeutic agents.

Antiviral and myocyte protective effects of murine interferon-beta and -{alpha}2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice.

The present study tested the hypothesis that murine (m)IFN-beta or mIFN-alpha(2) can eliminate cardiac viral load and protect cardiomyocytes from injury in animals infected with coxsackievirus B3 (CVB3). CVB3-inoculated male Balb/c mice exhibited signs of illness, including lethargy, progressive weight loss, and death (10% on day 3 and 100% on day 8). Cardiac viral load was high [4,277 +/- 1,009 plaque-forming units and 25 +/- 5 copies CVB3/hypoxanthine guanine phosphoribosyl transferase 1 mRNA] on day 4. The cardiac tissue exhibited severe inflammatory infiltration and myocyte damage with an average myocarditis integrated pathology score of 2.1 +/- 0.2 on day 7. Most of the mice infected with CVB3 also developed epicarditis, and 55% had intraventricular thrombi present. Treatment with mIFN-beta [2.5 to 10 million international units (MIU)/kg] dose-dependently improved the general health status in CVB3-inoculated mice, as evidenced by reduction in weight loss, prevention of death, elimination of cardiac viral load, protection of myocytes from injury, decrease in inflammatory cell infiltration, and attenuation of intraventricular thrombus formation. Treatment with 10 MIU/kg mIFN-alpha(2) resulted in a similar level of efficacy as that induced by 5 MIU/kg mIFN-beta, with the exception that mIFN-alpha(2) did not reduce cardiac CVB3 mRNA. However, mIFN-alpha(2) , but not any dose group of mIFN-beta, significantly attenuated CVB3-induced epicarditis. These data demonstrate antiviral effects for both mIFN-beta and mIFN-alpha(2), which lead to protection of the mice from CVB3-induced myocarditis. However, the potential mechanisms leading to a differential host response for the two isoforms of mIFN remain to be elucidated.

Reduced cardiac functional reserve in apolipoprotein E knockout mice.

Several years ago, the authors reported that aortic flow velocity under resting conditions was significantly higher in apolipoprotein E knockout (apoE-KO) mice than in age-matched C57Black/6J wildtype (WT) controls. The goal of this study was to examine whether the cardiac functional reserve is impacted in response to a pharmacological stress agent in apoE-KO mice. Cardiac function was measured noninvasively by the Doppler ultrasound method at baseline and at 1 min, 5 min, 10 min, and 20 min after intraperitoneal injection of dobutamine at the doses of 1 microg/g, 3 microg/g, or 10 microg/g in 16-month-old male apoE-KO (n = 9) and WT (n = 10) mice under light anesthesia with 1.5% isoflurane via inhalation. The baseline peak and mean aortic flow velocities were 39% to 48% higher, and left ventricular contractility measured by peak acceleration rate of aortic flow velocity was 24% higher in apoE-KO compared with WT mice (P < 0.01). Dobutamine stress dose-dependently increased cardiac function, which, however, was significantly smaller with a right shift of the dose-response curve in apoE-KO mice compared with WT controls. The hypotensive response to dobutamine was not significantly different between the 2 groups. Thus, despite an elevated resting aortic flow velocity and left ventricular contractility, cardiac functional reserve in response to dobutamine stress was significantly reduced in apoE-KO mice, which could be the consequence of coronary atherosclerosis and endothelial dysfunction that limits blood supply to the heart.

Interaction between mild hypercholesterolemia, HDL-cholesterol levels, and angiotensin II in intimal hyperplasia in mice.

Two month old C57BL/6 mice were placed on three different diets: 1) normal diet (NC; 0.025% cholesterol), 2) hypercholesterolemic Western-type diet (HC-W; 0.2% cholesterol), and 3) hypercholesterolemic Paigen-type diet (HC-P; 1.25% cholesterol plus 0.5% cholic acid). At 6 months of age, the animals underwent ligation of the left carotid artery and were randomly assigned to vehicle (PBS, subcutaneous) or angiotensin II (Ang II; 1.4 mg/kg/day, subcutaneous) treatment for 4 weeks. Low density lipoprotein-cholesterol levels were similarly increased in both HC diets (NC, 4 +/- 3 mg/dl; HC-W, 123 +/- 17 mg/dl; HC-P, 160 +/- 14 mg/dl). However, the levels of high density lipoprotein-cholesterol (HDL-C) were reduced only in animals fed the HC-P diet (NC, 82 +/- 6 mg/dl; HC-W, 79 +/- 7 mg/dl; HC-P, 58 +/- 7 mg/dl). In Ang II-treated mice, carotid artery ligation induced intimal smooth muscle cell proliferation to a similar extent in NC- and HP-W-fed animals. However, a significantly larger intimal area developed in ligated vessels from Ang II-treated mice fed the HC-P diet (3.6-fold higher than in Ang II-treated NC mice). Together, these results show the accelerating effect of mild hypercholesterolemia, reduced HDL-C levels, and Ang II on intimal hyperplasia after carotid artery ligation in mice.

Angiotensin II induces histomorphologic features of unstable plaque in a murine model of accelerated atherosclerosis.

BACKGROUND: We explored the role of angiotensin II in determining the histomorphometric features of plaque stability in apolipoprotein E-deficient mice submitted to ligation of the carotid artery. METHODS: Six-month-old apolipoprotein E-deficient mice underwent ligation of the common left carotid artery and were immediately assigned to receive either angiotensin II (1.4 mg . kg(-1) . d(-1) subcutaneously) or vehicle (phosphate-buffered saline; control) via a subcutaneous osmotic minipump for 4 weeks. RESULTS: Ligated arteries from control animals developed intimal lesions composed of macrophage foam cell plaques, which accumulated adjacent to the internal elastic lamina and were surrounded by a fibromuscular layer. Angiotensin II-treated mice had a greater intimal area (threefold), which was accompanied by a fivefold increase in the foam cell area. Lesions from angiotensin II-treated mice also displayed complex morphology characterized by intralesional neovasculature and hemorrhage. The content of active matrix metalloproteinase 2, mainly colocalized with macrophage foam cells, and the production of the inflammatory mediators monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 were also increased by angiotensin II treatment. Although angiotensin II induced vessel expansion and lumen loss to a similar extent, only vessel enlargement correlated with intimal area. CONCLUSIONS: Taken together, this study's results support a role of angiotensin II in plaque vulnerability by promoting intraplaque neovascularization/hemorrhage, inflammation, and expansive remodeling.

Inhibition of rho-kinase by hydroxyfasudil prevents vasopressin-induced myocardial ischemia in Donryu rats by attenuating coronary vasoconstriction.

BACKGROUND: Inhibition of rho-kinase has been shown to attenuate vasopressin (AVP)-induced myocardial ischemia measured as S-wave depression in Donryu rats. This has been attributed to a direct inhibitory effect on AVP-induced coronary vasoconstriction. However, since AVP also increased mean arterial blood pressure (MAP) which was attenuated by the rho-kinase inhibitors used, the prevention of myocardial ischemia could have been due to effects on afterload. RESULTS: The purpose of this study was to determine if rho-kinase inhibition prevents S-wave depression independent of the effects on blood pressure. In anesthetized Donryu rats (200-340 g), infusion of AVP (0.1 IU/kg) resulted in a sustained increase in MAP (DeltaMAP=46+/-7 mm Hg) and a transient S-wave depression (-90+/-20 microV). Infusion of phenylephrine titrated to achieve a comparable pressor response (DeltaMAP=52+/-2 mm Hg) resulted in a significantly smaller S-wave depression (-30+/-20 microV). Pretreatment with the rho-kinase inhibitor, hydroxyfasudil (3 mg/kg), decreased MAP by -28+/-2 mm Hg and significantly attenuated AVP-induced S-wave depression (-10+/-10 microV) compared to AVP. When rats were pretreated with phenylephrine titrated to maintain MAP, hydroxyfasudil still significantly attenuated AVP-induced S-wave depression (-14+/-12 microV). Hydralazine (1 mg/kg), which lowered MAP by -36+/-5 mm Hg, had no significant effect on AVP-induced S-wave depression (-105+/-32 microV). CONCLUSION: These data indicate that inhibition of rho-kinase with hydroxyfasudil attenuates AVP-induced myocardial ischemia independent of changes in MAP and are consistent with an inhibitory effect on coronary vasoconstriction.