Bruce M. McManus

Regulation of the Versican Promoter by the β-Catenin-T-cell Factor Complex in Vascular Smooth Muscle Cells

The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3β, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3β activity by LiCl augmented accumulation of β-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. β-Catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of β-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and β-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of β-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3β pathway via the β-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.

Soluble Recombinant Coxsackievirus and Adenovirus Receptor Abrogates Coxsackievirus B3-Mediated Pancreatitis and Myocarditis in Mice

BACKGROUNDnGroup B coxsackievirus infection can result in organ injury and inflammation. The coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55) have both been identified as receptors for coxsackievirus B3 (CVB3). We have shown elsewhere that early DAF-Fc treatment attenuates CVB3-induced myocarditis and virus replication.nnnMETHODSnCAR was synthesized as a soluble IgG1-Fc fusion protein (CAR-Fc). In vitro, CAR-Fc blocked infection by 2 different strains of CVB3. A/J mice were infected in vivo with CVB3 and were administered CAR-Fc either 3 days before infection, during infection, or 3 days after infection and were compared with mice infected with virus alone and control animals.nnnRESULTSnAll CAR-Fc treatment groups had reduced recoverable infectious virus in the heart. CAR-Fc treatment of mice, either preceding or concurrent with CVB3 infection, resulted in complete inhibition of myocardial lesion area, cell death and inflammation, and viral RNA. Early treatment also completely blocked inflammation and cell death in the pancreas, an organ that is normally very sensitive to infection.nnnCONCLUSIONnTo our knowledge, CAR-Fc is the only protein that has been shown to block coxsackievirus infection of the pancreas. However, regardless of the efficacy of the test protein, target tissue cannot be rescued after day 3 of infection in the A/J mouse model.

Long-term effects of ovariectomy and estrogen replacement treatment on endothelial function in mature rats

OBJECTIVESnEstrogen replacement therapy (ERT) improves blood flow through various mechanisms including an augmented release of nitric oxide (NO). We report on the long-term effects of estrogen loss on vascular function and endothelial regulation.nnnMETHODSnMale, female, ovariectomized and ovariectomized+ERT treated rats were used. Female rats were ovariectomized at 12 weeks of age and received ERT via subcutaneously implanted 90-day release pellets. Vasodilation to acetylcholine (ACh) was studied in tail artery segments; arterial blood was collected for measurements of 17-beta-estradiol and stable metabolites of NO (nitrate/nitrite). Some arterial segments were harvested for TUNEL staining to determine endothelial apoptosis.nnnRESULTSnOvariectomy caused a rapid loss of estradiol that was negated by ERT. Likewise, there was also a loss in plasma NO. Loss of ACh-mediated dilations were age-dependent and were significant in males and untreated ovariectomized rats, with the change being maximal after 12 weeks of ovariectomy. After 12 weeks post-ovariectomy, there were no time dependent changes in ACh sensitivity in either group. Dilations to ACh were maintained in females and age-matched ERT ovariectomized rats over time. TUNEL staining of the endothelium (at 6 months of age) revealed apoptotic changes with the rank order male>ovariectomized>female, or ERT treated ovariectomized female rats.nnnCONCLUSIONSnIn a rat model of surgical menopause, loss of endothelial function is maximal 12 weeks after ovariectomy. Apoptosis of endothelial cells is greatest in arteries from male rats. Our data suggests that early ERT treatment may be an important consideration for reducing endothelium-dependent vascular dysfunction.

Clinical, biochemical, and genetic predictors of coronary artery bypass graft failure

OBJECTIVESnTo identify novel predictors for coronary artery bypass grafting failure, we probed for associations with known clinical and biochemical risk factors for atherosclerosis. We also used microarray analysis to identify novel single nucleotide polymorphisms to better understand the genetics and pathogenesis of graft occlusion.nnnMETHODSnThe present study was a nested case-control substudy of the Radial Artery Patency Study 5-year follow-up data. From 1996 to 2001, 87 patients underwent coronary artery bypass grafting. Of these, 26 patients (29.9%) had an occluded study graft (saphenous vein or radial artery) at 8.0 ± 1.1 years. The clinical parameters, late angiography, blood biomarker levels, and surgical outcomes data were included in a multivariate analysis to determine the independent predictors of graft failure.nnnRESULTSnThe risk factors of graft failure were fibrinogen (odds ratio [OR], 3.94; 95% confidence interval [CI], 1.33-11.63; Pxa0=xa0.01), creatinine (OR, 1.06; 95% CI, 1.02-1.10; Pxa0=xa0.006), and diabetes mellitus (OR, 5.15; 95% CI, 1.08-24.59; Pxa0=xa0.04). High-density lipoprotein (OR, 0.74; 95% CI, 0.53-1.02; Pxa0=xa0.06) was weakly protective; however, low-density lipoprotein and total cholesterol were not predictors. We then identified the association of several human single nucleotide polymorphisms with graft failure, including mutations in glutathione-S-transferase α3. Human coronary arteries and bypass grafts demonstrated increased protein expression of glutathione-S-transferase α3, a known cardioprotective factor, in the atherosclerotic regions and surrounding adventitial tissues.nnnCONCLUSIONSnWe identified diabetes as a potential clinical predictor and plasma fibrinogen, creatinine, and high-density lipoprotein as potential novel biomarkers. These might help risk stratify patients for the development of graft failure. We also demonstrated a novel association between glutathione-S-transferase α3 and graft failure.

Extending and encoding existing biological terminologies and datasets for use in the reasoned semantic web

BackgroundClinical phenotypes and disease-risk stratification are most often determined through the direct observations of clinicians in conjunction with published standards and guidelines, where the clinical expert is the final arbiter of the patient’s classification. While this human approach is highly desirable in the context of personalized and optimal patient care, it is problematic in a healthcare research setting because the basis for the patients classification is not transparent, and likely not reproducible from one clinical expert to another. This sits in opposition to the rigor required to execute, for example, Genome-wide association analyses and other high-throughput studies where a large number of variables are being compared to a complex disease phenotype. Most clinical classification systems and are not structured for automated classification, and similarly, clinical data is generally not represented in a form that lends itself to automated integration and interpretation. Here we apply Semantic Web technologies to the problem of automated, transparent interpretation of clinical data for use in high-throughput research environments, and explore migration-paths for existing data and legacy semantic standards.ResultsUsing a dataset from a cardiovascular cohort collected two decades ago, we present a migration path - both for the terminologies/classification systems and the data - that enables rich automated clinical classification using well-established standards. This is achieved by establishing a simple and flexible core data model, which is combined with a layered ontological framework utilizing both logical reasoning and analytical algorithms to iteratively lift clinical data through increasingly complex layers of interpretation and classification. We compare our automated analysis to that of the clinical expert, and discrepancies are used to refine the ontological models, finally arriving at ontologies that mirror the expert opinion of the individual clinical researcher. Other discrepancies, however, could not be as easily modeled, and we evaluate what information we are lacking that would allow these discrepancies to be resolved in an automated manner.ConclusionsWe demonstrate that the combination of semantically-explicit data, logically rigorous models of clinical guidelines, and publicly-accessible Semantic Web Services, can be used to execute automated, rigorous and reproducible clinical classifications with an accuracy approaching that of an expert. Discrepancies between the manual and automatic approaches reveal, as expected, that clinicians do not always rigorously follow established guidelines for classification; however, we demonstrate that personalized ontologies may represent a re-usable and transparent approach to modeling individual clinical expertise, leading to more reproducible science.

Cyclosporine treatment preserves coronary resistance artery function in rat cardiac allografts.

BACKGROUNDnA marked decline in vascular myogenic response occurs during the course of rat cardiac allograft rejection. Two important contributory features are an inducible nitrous oxide synthase (iNOS)-catalyzed, NO-mediated vasodilation and a loss of smooth muscle function. In this study, we examine the effect of cyclosporine immunosuppressive therapy on the alleviation of arterial dysfunction of coronary resistance arteries in allografts using pressure myography.nnnMETHODSnRats receiving heterotopic abdominal cardiac transplantation were treated with cyclosporine (5 mg/kg), Cremophore or distilled water. Coronary septal arteries (internal diameter 200 microm) were dissected from isograft (Lewis to Lewis) and allograft (Fisher to Lewis) rat hearts at Day 21 post-transplantation and mounted on a pressure myograph. Pressure-induced vasoconstriction was measured before and after iNOS inhibition with aminoguanidine (AG; 100 micromol/liter). Both endothelium-based (ACh-induced) and endothelium-independent (sodium nitroprusside-induced) vasorelaxation were also recorded in each group.nnnRESULTSnPressure-induced myogenic contraction was reduced in allograft coronary arteries at Day 21 post-transplantation compared with matched isografts (p < 0.05). AG potentiated myogenic tone in allograft arteries, but had no effect on untreated Day 21 isograft vessels, indicating the presence of iNOS-based relaxation only in allograft vessels. Depolarization-induced vasoconstriction was lower in allograft compared with isograft arteries (p < 0.05). Cyclosporine therapy also improved depolarization-induced constriction in allograft vessels compared with untreated groups (p < 0.05). Furthermore, cyclosporine therapy preserved endothelium-based and endothelium-independent vasorelaxation in allograft arteries at Day 21 post-transplantation.nnnCONCLUSIONSnCyclosporine immunosuppressive therapy has a significant effect on the alleviation of early endothelial and smooth muscle dysfunction in coronary allograft arteries.

Cellular and Immunological Regulation of Viral Myocarditis

Since the discovery of coxsackievirus type B3 (CVB3) more than 50 years ago there has been considerable progress in the understanding of viral heart disease. Coxsackievirus was first discovered as a filterable agent associated with a paralytic syndrome, so named for its identification in Coxsackie, New York (coxsackievirus type A) (Dalldorf and Sickles, 1948). Coxsackievirus type B (CVB) was isolated the following year from patients with aseptic meningitis and by the mid-1950s an association with acute myocarditis in humans was apparent (Melnick et al., 1949). Many other viruses have since been shown to cause myocarditis and its long term sequelae, arrhythmias, dilated cardiomyopathy (DCM) and heart failure. By example, adenovirus, herpes viruses, and influenza can cause myocarditis in humans and models of heart failure. The viral agent most often reported as being the cause of viral myocarditis is CVB3. For example, CVB3 RNA can be detected in the heart muscle of 10 35 % of DCM patients (Feldman and McNamara, 2000; Hosenpud et al., 2001), depending on the study cohort.

Response to Letter Regarding Article, “Ablation of Matrix Metalloproteinase-9 Increases Severity of Viral Myocarditis in Mice”

It is now clear that release of metalloproteinase–9 (MMP-9)2 inhibition by tissue inhibitor of MMP-1 (TIMP-1)1 is beneficial during the acute phase of CVB3 myocarditis, but chronic MMP activation is detrimental3 after infection has been cleared. We investigated the course of coxsackievirus B3 (CVB3)–induced viral myocarditis within a murine MMP-9 knockout model.2 On accumulating initial data, it was clear that a detailed study was required to support our observation that MMP-9 was protective during acute CVB3 infection and myocarditis. We noted that CVB3 infection within the pancreas and liver was also higher in the MMP-9 …