Development of an LC-MS/MS assay for quantification of intact INSL3 in rat plasma.

Background: Relatively large disulfide-linked polypeptides can serve as signaling molecules for a diverse array of biological processes and may be studied in animal models to investigate their function in vivo. The aim of this work was to develop an LC-MS/MS assay to measure a model peptide, INSL3, in rat plasma. Results: A dual enrichment strategy incorporating both protein precipitation and solid phase extraction was utilized to isolate INSL3 from rat plasma, followed by targeted LC-MS/MS detection. The method was able to measure full-length INSL3 (6.1 kDa) down to 0.2 ng/ml with acceptable accuracy and precision. Conclusion: The final assay was applied to support an exploratory pharmacokinetic study to evaluate steady-state concentrations of dosed INSL3 in rat plasma.

A Structured Approach to Optimizing Animal Model Selection for Human Translation: The Animal Model Quality Assessment.

Animal studies in pharmaceutical drug discovery are common in preclinical research for compound evaluation before progression into human clinical trials. However, high rates of drug development attrition have prompted concerns regarding animal models and their predictive translatability to the clinic. To improve the characterization and evaluation of animal models for their translational relevance, the authors developed a tool to transparently reflect key features of a model that may be considered in both the application of the model but also the likelihood of successful translation of the outcomes to human patients. In this publication, we describe the rationale for the development of the Animal Model Quality Assessment tool, the questions used for the animal model assessment, and a high-level scoring system for the purpose of defining predictive translatability. Finally, we provide an example of a completed Animal Model Quality Assessment for the adoptive T-cell transfer model of colitis as a mouse model to mimic inflammatory bowel disease in humans.

Activation of the Amino Acid Response Pathway Blunts the Effects of Cardiac Stress.

BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

Soluble epoxide hydrolase inhibition does not prevent cardiac remodeling and dysfunction after aortic constriction in rats and mice.

Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress.

Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.

CCR2 receptor antagonists: optimization of biaryl sulfonamides to increase activity in whole blood.

A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.

Chronic inhibition of hypoxia-inducible factor prolyl 4-hydroxylase improves ventricular performance, remodeling, and vascularity after myocardial infarction in the rat.

BACKGROUND: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI). METHODS AND RESULTS: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group). CONCLUSIONS: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.

Pharmacological inhibition of C-C chemokine receptor 2 decreases macrophage infiltration in the aortic root of the human C-C chemokine receptor 2/apolipoprotein E-/- mouse: magnetic resonance imaging assessment.

UNLABELLED: Purpose- This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. METHODS AND RESULTS: Apolipoprotein E(-/-) mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05). CONCLUSIONS: An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

Assessment of macrophage infiltration in a murine model of abdominal aortic aneurysm.

PURPOSE: To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model. MATERIALS AND METHODS: Osmotic pumps were implanted subcutaneously in apoE(-/-) mice for continuous infusion of Angiotensin II (Ang-II). Weekly bright-blood gradient echo scans were performed on the suprarenal abdominal aorta to evaluate aneurysm development. Once an AAA was detected, animals were administered 1000 mumol/kg of the USPIO contrast agent ferumoxtran-10 (Combidex) followed by in vivo scanning 24 h post-USPIO administration. After in vivo imaging, aortas were harvested for ex vivo imaging, histology, iron quantification, and gene expression analysis. RESULTS: Reduced signal intensity was evident in the post-USPIO transverse images of the abdominal aorta. The areas of reduced signal were primarily along the aneurysm shoulder and outer perianeurysm areas and corresponded to regions of macrophage infiltration and colocalized USPIO determination by means of histological staining. The absolute iron content measured significantly correlated to the area of signal reduction in the ex vivo images (r = 0.9; P < 0.01). In the AAA tissue, the macrophage-driven cytokine gene expression was up-regulated along with a matrix metalloproteinase known to mediate extracellular matrix breakdown in this disease model. CONCLUSION: These results demonstrate the feasibility of using an USPIO contrast agent as a surrogate for detecting the acute inflammatory process involved in the development of abdominal aneurysms.

Differential effects of p38 mitogen-activated protein kinase and cyclooxygenase 2 inhibitors in a model of cardiovascular disease.

The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1beta. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.

p38 MAPK inhibition reduces aortic ultrasmall superparamagnetic iron oxide uptake in a mouse model of atherosclerosis: MRI assessment.

OBJECTIVE: Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation. The purpose of this study was to noninvasively evaluate USPIO uptake in aorta of apoE-/- mice and to determine the effects of Angiotensin II (Ang II) infusion and chronic antiinflammatory treatment with a p38 MAPK inhibitor on this uptake. METHODS AND RESULTS: ApoE-/- mice were administered saline or Ang II (1.44 mg/kg/d) for 21 days. In vivo MRI assessment of USPIO uptake in the aortic arch was observed in all animals. However, although the Ang II group had significantly higher absolute iron content (increased 103%, P<0.001) in the aortic arch compared with the saline group, the p38 MAPK inhibitor (SB-239063, 150 mg/kg/d) treatment group did not (increased 6%, NS). The in vivo MRI signal intensity was significantly correlated to the absolute iron content in the aortic arch. Histological evaluation of the aortic root lesion area showed colocalization of USPIO with macrophages and a reduction in USPIO but not macrophage content with SB-239063 treatment. CONCLUSIONS: The present study demonstrates that noninvasive assessment of USPIO uptake, as a marker for inflammation in murine atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the uptake of USPIO in aorta of Ang II-infused apoE-/- mice.

In vivo serial assessment of aortic aneurysm formation in apolipoprotein E-deficient mice via MRI.

BACKGROUND: Hyperlipidimic mice administered angiotensin II have been used for the study of abdominal aortic aneurysms (AAAs). The purpose of this study was to examine the use of MRI for studying AAA development and for examining the effects of pharmacological intervention on AAA development in the apolipoprotein E-deficient mouse. METHODS AND RESULTS: Suprarenal aortic aneurysms were generated in apolipoprotein E-deficient mice administered angiotensin II (1000 ng/kg per min) for up to 28 days. In vivo MRI was performed serially (once weekly) to assess AAA development and rupture. Comparison of AAA size as measured by in vivo and ex vivo MRI resulted in excellent agreement (r=0.96, P<0.0001). In addition, MRI correlated with histology-derived AAA area assessment (in vivo versus histology: r=0.84, P<0.0001; ex vivo versus histology: r=0.89, P<0.0001). In a separate study, angiotensin II-administered apolipoprotein E-deficient mice were treated with doxycycline (broad-based matrix metalloproteinase inhibitor; 30 mg/kg per day for 28 days). MRI was able to noninvasively assess a reduced rate of AAA development (46% versus 71%, P<0.05), a decreased AAA area (2.56 versus 4.02 mm(2), P<0.01), and decreased incidence of rupture (43% versus 100%) in treated versus control animals. Inhibition of aorta matrix metalloproteinase 2/9 activity was observed in the treated animals. CONCLUSIONS: These results demonstrate the use of MRI to noninvasively and temporally assess AAA development on pharmacological intervention in this preclinical cardiovascular disease model.

PPARdelta activation normalizes cardiac substrate metabolism and reduces right ventricular hypertrophy in congestive heart failure.

Previously, it was shown that selective deletion of peroxisome proliferator activated receptor delta (PPARdelta) in the heart resulted in a cardiac lipotoxicity, hypertrophy, and heart failure. The aim of the present study was to determine the effects of chronic and selective pharmacological activation of PPARdelta in a model of congestive heart failure. PPARdelta-specific agonist treatment (GW610742X at 30 and 100 mg/kg/day for 6-9 weeks) was initiated immediately postmyocardial infarction (MI) in Sprague-Dawley rats. Magnetic resonance imaging/spectroscopy was used to assess cardiac function and energetics. A 1-(13)C glucose clamp was performed to assess relative cardiac carbohydrate versus fat oxidation. Additionally, cardiac hemodynamics and reverse-transcription polymerase chain reaction gene expression analysis was performed. MI rats had significantly reduced left ventricle (LV) ejection fractions and whole heart phosphocreatine/adenosine triphosphate ratio compared with Sham animals (reduction of 43% and 14%, respectively). However, GW610742X treatment had no effect on either parameter. In contrast, the decrease in relative fat oxidation rate observed in both LV and right ventricle (RV) following MI (decrease of 58% and 54%, respectively) was normalized in a dose-dependent manner following treatment with GW610742X. These metabolic changes were associated with an increase in lipid transport/metabolism target gene expression (eg, CD36, CPT1, UCP3). Although there was no difference between groups in LV weight or infarct size measured upon necropsy, there was a dramatic reduction in RV hypertrophy and lung congestion (decrease of 22-48%, P<0.01) with treatment which was associated with a >7-fold decrease (P<0.05) in aterial natriuretic peptide gene expression in RV. Diuretic effects were not observed with GW610742X. In conclusion, chronic treatment with a selective PPARdelta agonist normalizes cardiac substrate metabolism and reduces RV hypertrophy and pulmonary congestion consistent with improvement in congestive heart failure.

Selective PPARdelta agonist treatment increases skeletal muscle lipid metabolism without altering mitochondrial energy coupling: an in vivo magnetic resonance spectroscopy study.

Peroxisome proliferator-activated receptor-delta (PPARdelta) activation results in upregulation of genes associated with skeletal muscle fatty acid oxidation and mitochondrial uncoupling. However, direct, noninvasive assessment of lipid metabolism and mitochondrial energy coupling in skeletal muscle following PPARdelta stimulation has not been examined. Therefore, in this study we examined the response of a selective PPARdelta agonist (GW610742X at 5 or 100 mg.kg(-1).day(-1) for 8 days) on skeletal-muscle lipid metabolism and mitochondrial coupling efficiency in rats by using in vivo magnetic resonance spectroscopy (MRS). There was a decrease in the intramyocellular lipid-to-total creatine ratio as assessed by in vivo (1)H-MRS in soleus and tibialis anterior muscles by day 7 (reduced by 49 and 46%, respectively; P < 0.01) at the high dose. Following the (1)H-MRS experiment (day 8), [1-(13)C]glucose was administered to conscious rats to assess metabolism in the soleus muscle. The relative fat-vs.-carbohydrate oxidation rate increased in a dose-dependent manner (increased by 52 and 93% in the 5 and 100 mg.kg(-1).day(-1) groups, respectively; P < 0.05). In separate experiments where mitochondrial coupling was assessed in vivo (day 7), (31)P-MRS was used to measure hindlimb ATP synthesis and (13)C-MRS was used to measure the hindlimb tricarboxylic acid cycle flux (V(tca)). There was no alteration, at either dose, in mitochondrial coupling efficiency measured as the ratio of unidirectional ATP synthesis flux to V(tca). Soleus muscle GLUT4 expression was decreased by twofold, whereas pyruvate dehydrogenase kinase 4, carnitine palmitoyl transferase 1a, and uncoupling protein 2 and 3 expression was increased by two- to threefold at the high dose (P < 0.05). In summary, these are the first noninvasive measurements illustrating a selective PPARdelta-mediated decrease in muscle lipid content that was consistent with a shift in metabolic substrate utilization from carbohydrate to lipid. However, the mitochondrial-energy coupling efficiency was not altered in the presence of increased uncoupling protein expression.

Rosiglitazone protects against ischemia/reperfusion-induced leukocyte adhesion in the zucker diabetic fatty rat.

Increased susceptibility to atherosclerosis increases the risk of mortality in type 2 diabetic patients. Leukocyte adhesion to the endothelium is a critical step in atherogenesis. In addition to its insulin-sensitizing effects, rosiglitazone (RSG) possesses anti-inflammatory properties. However, the effects of RSG on the initial phase of leukocyte recruitment (rolling, adhesion) have not been studied in vivo. This study tested the hypothesis that RSG treatment of Zucker diabetic fatty (ZDF) rats inhibits ischemia/reperfusion-induced leukocyte adhesion to the endothelium. Male ZDF rats (16 weeks) were treated with RSG (3 mg/kg/day, p.o.) 7 days before experimentation. Leukocyte-endothelial interactions in cremaster venules were recorded using intravital microscopy prior to 30 min of ischemia and during a 90-min reperfusion period. Although blood pressure, plasma glucose, and insulin were not different between treatment groups, RSG treatment was associated with reduced leukocyte rolling and inhibition of leukocyte adhesion throughout the reperfusion period (P < 0.01). Cremaster mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) was reduced by 35% in RSG-treated animals (P < 0.01), whereas P- and E-selectin and intercellular adhesion molecule-1 (ICAM-1) were unchanged. Immunostaining for P-selectin, E-selectin, and VCAM-1 was reduced by 21, 61, and 50%, respectively (for all, P < 0.05), in RSG-treated animals. Inhibition of ischemia/reperfusion-induced leukocyte adhesion might contribute to the utility of RSG as a therapy for vascular disease.

Synthetic LXR agonists increase LDL in CETP species.

Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7alpha-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism.

Differential uptake of ferumoxtran-10 and ferumoxytol, ultrasmall superparamagnetic iron oxide contrast agents in rabbit: critical determinants of atherosclerotic plaque labeling.

PURPOSE: To compare atherosclerotic plaque uptake of a first (ferumoxtran-10) and second generation (ferumoxytol) ultrasmall superparamagnetic iron oxide (USPIO) contrast agent with different pharmacokinetic/pharmacodynamic properties. MATERIALS AND METHODS: New Zealand White rabbits maintained on a high cholesterol/fat diet were subjected to balloon injury to the abdominal aorta. Ferumoxtran-10 or ferumoxytol (500 micromol/kg) was administered at 2, 4, and 8 weeks following injury. In vivo magnetic resonance imaging (MRI) was performed immediately prior to, immediately after, and 6 days post-contrast administration. Ex vivo MRI, histologic, and inductively coupled plasma-mass spectrometry (ICP-MS) iron analyses were performed on the excised vessels. RESULTS: The blood pool clearance of ferumoxytol (t(1/2) < or = 6 hours) was more rapid than that of ferumoxtran-10 (t(1/2) < or = 48 hours). Decreased in vivo MRI signal intensity in the abdominal aorta was observed at 2, 4, and 8 weeks following injury with ferumoxtran-10, but not with ferumoxytol. Consistent with these observations, ex vivo MRI signal intensity was decreased in the ferumoxtran-10 vessels, and to a lesser degree in the ferumoxytol vs. control vessels (- contrast agent). In contrast, in vitro macrophage phagocytosis of USPIO was four to six fold greater with ferumoxytol than with ferumoxtran-10. Additionally, the absolute iron content correlated with ex vivo MRI signal intensity in all vessels (r = -0.86, P < 0.0001). CONCLUSIONS: These data suggest that the exposure period of atherosclerotic plaque to USPIO rather than the kinetics of the USPIO uptake by plaque alone is a critical criterion for experimental design of in vivo studies.

Hypertensive target organ damage is attenuated by a p38 MAPK inhibitor: role of systemic blood pressure and endothelial protection.

OBJECTIVE: Evidence suggests important relationships among chronic inflammatory processes, endothelial dysfunction, hypertension and target organ damage. The present study examined the effects of chronic treatment with an anti-inflammatory p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-239063AN) in the N(omega)-nitro-l-arginine methyl ester-treated spontaneously hypertensive rat (SHR+l-NAME) model of severe hypertension and accelerated target organ damage. METHODS: SHRs were divided into control (n=16), l-NAME (n=26) and l-NAME+SB-239063AN (n=24) groups. l-NAME was delivered by the drinking water ad lib (50 mg/L) and SB-239063AN was administered by the diet (1200 ppm) for 4 weeks. Arterial blood pressure (telemetry) and target organ damage (kidney, heart, and vasculature) were examined. RESULTS: The introduction of l-NAME to the drinking water elicited a severe/sustained increase in blood pressure and significant morbidity and mortality. Chronic treatment with SB-239063AN had no effect on the initial blood pressure response (7 days) to l-NAME but attenuated subsequent increases in diastolic blood pressure and significantly reduced morbidity/mortality (42% vs. 5%, p<0.002). Renal dysfunction characterized by increased total protein and albumin excretion was apparent within 2 weeks in the SHR+l-NAME groups. Treatment with SB-239063AN delayed the onset of proteinuria and albuminuria. SB-239063AN treatment also significantly reduced l-NAME-induced interstitial fibrosis in the kidney and restrictive concentric hypertrophy in the left ventricle (end-diastolic volume 0.24+/-0.05 vs. 0.41+/-0.05 ml; p<0.05). Endothelial dysfunction was also not altered by SB-239063AN treatment (Rmax 49+/-6% vs. 45+/-9%). CONCLUSIONS: The results demonstrate that morbidity/mortality and accelerated target organ damage induced by inhibition of nitric oxide synthase in SHR was attenuated by treatment with a selective p38 MAPK inhibitor, SB-239063AN. The organ protection observed in the heart and kidney was not associated with preservation of endothelial function.

Cathepsin S expression is up-regulated following balloon angioplasty in the hypercholesterolemic rabbit.

OBJECTIVE: Neointimal development following balloon angioplasty involves many factors including smooth muscle cell (SMC) migration and proliferation and extracellular matrix (ECM) remodeling. Further, in hypercholesterolemic (HC) conditions, there is an influx of macrophage foam cells (FCs) into the restenotic lesion, which also involves degradation of the basement membrane and surrounding ECM. The ECM remodeling that occurs during restenosis has been shown to be mediated by various proteases. Here we have investigated the role of cathepsin S (CatS), a cysteine protease, in this process. METHODS AND RESULTS: We have demonstrated by Taqman quantitative PCR, Western blot, and immunohistochemistry that CatS is up-regulated in restenotic lesions of HC rabbits following balloon injury of the iliofemoral artery. CatS mRNA expression was elevated 28-fold in balloon-injured vessels relative to uninjured contralateral vessels in HC rabbits 8 weeks post-angioplasty (p<0.05). CatS protein expression was detected within 1 day post-injury, persisted throughout the entire time course evaluated (60 days post-injury), and was co-localized with SMCs, macrophages, and FCs. In contrast, cystatin C (CysC), the endogenous inhibitor of cathepsins, was only minimally up-regulated following injury. CysC mRNA expression was elevated 3.5-fold in balloon-injured vessels relative to uninjured contralateral vessels in HC rabbits 8 weeks post-angioplasty (p<0.005), and up-regulation of protein expression was not detected until days 28 and 60 post-injury. Additional biochemical studies using recombinant rabbit CatS revealed that rabbit CatS digests laminin, fibronectin, and type I collagen. Further, CatS expression was evaluated in SMCs that were induced to migrate through a matrix-coated Boyden chamber upon platelet-derived growth factor (PDGF) stimulation. The addition of a selective CatS inhibitor reduced SMC migration dose-dependently with an 80% reduction in migration at 30 nM (p<0.005). Additionally, we have shown that CatS protein expression by human macrophages was increased upon stimulation with oxidized low density lipoprotein (ox-LDL), implying augmentation of CatS production during foam cell formation. CONCLUSION: Taken together, our results indicate an enhanced expression of CatS during neointima formation and it is associated with invading SMCs, macrophages, and FCs, highlighting the importance of CatS in the pathogenesis of restenosis.