Donald J. Voelker

Matrix metalloproteinase activity expression in infarcted, noninfarcted and dilated cardiomyopathic human hearts.

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.

Differential gene expression of extracellular matrix components in dilated cardiomyopathy

Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13‐21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP‐1). Northern blot analysis using MMP‐1 cDNA probe indicated that MMP‐1 was induced three‐ to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP‐1 and MMP‐1 levels were 37 ± 8 ng/mg and 9 ± 2 ng/mg in normal tissue specimens (P < 0.01) and 2 ± 1 ng/mg and 45 ± 11 ng/mg in DCM tissue (P < 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP‐1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP‐1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic‐fibroblast cell ECM‐proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy.

Extracellular matrix regulation of metalloproteinase and antiproteinase in human heart fibroblast cells.

Following myocardial infarction, extracellular matrix (ECM) is disrupted, which leads to the generation of collagen‐ and elastin‐derived peptides (CDPs and EDPs, respectively). To investigate whether ECM‐derived peptides (i.e., CDPs and EDPs) induce extracellular proteinases in human heart fibroblast (HHF) cells, we isolated CDP and EDP using gelfiltration and antibody affinity column chromatography. The CDP and EDP were characterized by their intrinsic fluorescence due to cross‐link structure (pyridinoline and desmosine, respectively) and by immunoblot analysis using anti‐desmosine antibody. Neutrophil elastase and cathepsin G were identified using selective chromogenic substrates and by their specific inhibition with α1‐proteinase inhibitor and α1‐antichymotrypsin, respectively. Elastase and cathepsin G were elevated in the infarcted tissue. Selective inhibition of matrix metalloproteinase (MMP) by a higher concentration of tetracycline or doxycycline in zymographic gels elicited an inhibition constant (IC50) of 278 ± 10 μM and indicated that majority of MMP in the infarcted tissue is from fibroblast cells. The HHF proliferation was measured using an acid‐phosphatase assay. The EDP and CDP induce HHF cell proliferation. After EDP treatment phenotypic (formation of pseudopodia) changes were observed in HHF cells. To measure whether phenotypic changes by EDP or CDP are associated with MMP and tissue inhibitor of metalloproteinase (TIMP) expression in HHF cells, we measured MMP and TIMP expression by zymographic and Northern blot (mRNA) analyses. The expression of MMP and TIMP were upregulated at both the protein and gene transcription levels. These results suggested that during ischemic cardiomyopathy, initially neutrophil proteinase activates latent myocardial MMP which can degrade ECM, which continuously degrades if not controlled by TIMP, leading to ventricular dilatation and dysfunction.

Aspirin and clopidogrel hyporesponsiveness and nonresponsiveness in patients with coronary artery stenting.

Patients undergoing coronary artery stenting receive an antiplatelet regimen to reduce the risk of antithrombotic complications. Current guidelines recommend the use of acetyl salicylic acid (aspirin) and clopidogrel as evidenced by large clinical trials. There has been a concern about variable responses of patients to aspirin and clopidogrel which may predispose them to subacute stent thrombosis or late stent thrombosis. Up to 25% of patients with acute myocardial infarction (AMI) undergoing percutaneous coronary intervention (PCI) were found to have hyporesponsiveness or resistance to clopidogrel which may predispose them to recurrent events. Dual antiplatelet regimen is a standard therapy in these patients and there is always a concern about variable responses to aspirin and clopidogrel predisposing them to acute coronary syndrome (ACS). Prevalence of this hyporesponsiveness or resistance may be due to noncompliance, genetic mutations, co-morbid situations and concomitant use of other drugs. This issue is of considerable importance in the era of coronary drug eluting stents when a long-term dual antiplatelet regimen is needed. This paper is a review for clinicians taking care of such patients with hyporesponsiveness or nonresponsiveness to dual antiplatelet regimen.

Cardiac risk stratification: Role of the coronary calcium score

Coronary artery calcium (CAC) is an integral part of atherosclerotic coronary heart disease (CHD). CHD is the leading cause of death in industrialized nations and there is a constant effort to develop preventative strategies. The emphasis is on risk stratification and primary risk prevention in asymptomatic patients to decrease cardiovascular mortality and morbidity. The Framingham Risk Score predicts CHD events only moderately well where family history is not included as a risk factor. There has been an exploration for new tests for better risk stratification and risk factor modification. While the Framingham Risk Score, European Systematic Coronary Risk Evaluation Project, and European Prospective Cardiovascular Munster study remain excellent tools for risk factor modification, the CAC score may have additional benefit in risk assessment. There have been several studies supporting the role of CAC score for prediction of myocardial infarction and cardiovascular mortality. It has been shown to have great scope in risk stratification of asymptomatic patients in the emergency room. Additionally, it may help in assessment of progression or regression of coronary artery disease. Furthermore, the CAC score may help differentiate ischemic from nonischemic cardiomyopathy.

Evolving role of platelet function testing in coronary artery interventions

The substantial reduction in ischemic events provided by the dual antiplatelet regimen with aspirin and clopidogrel is well documented in patients with acute coronary syndrome and patients undergoing percutaneous coronary intervention. Recently the variable response to the antiplatelet agents has received considerable attention after several “boxed warnings” on clopidogrel. This led to intense controversy on pharmacokinetic, pharmacodynamic, and pharmacogenomic issues of antiplatelet drugs, especially clopidogrel. Research use of platelet function testing has been successfully validated in identifying new antiplatelet drugs like prasugrel and ticagrelor. These platelet function assays are no longer regarded just as a laboratory phenomenon but rather as tools that have been shown to predict mortality in several clinical trials. It is believed that suboptimal response to an antiplatelet regimen (pharmacodynamic effect) may be associated with cardiovascular, cerebrovascular, and peripheral arterial events. There has been intense controversy about this variable response of antiplatelet drugs and the role of platelet function testing to guide antiplatelet therapy. While the importance of routine platelet function testing may be uncertain, it may be useful in high-risk patients such as those with diabetes mellitus, diffuse three vessels coronary artery disease, left main stenosis, diffuse atherosclerotic disease, and those with chronic renal failure undergoing percutaneous coronary intervention. It could also be useful in patients with suspected pharmacodynamic interaction with other drugs to assure the adequacy of platelet inhibition. While we wait for definitive trials, a predictive prognostic algorithm is necessary to individualize antiplatelet therapy with P2Y12 inhibitors based on platelet function assays and genetic testing.

Role of extracellular matrix metalloproteinases in cardiac remodelling

The latent collagenolytic system is an intrinsic part of normal myocardium. Controlled activation of this system becomes necessary in ventricular chamber remodeling following inflammation and injury, such as dilated cardiomyopathy and myocardial infarction. Evidence exists to indicate activation of collagenolytic enzymes in patients with congestive heart failure and dilated, dysfunctional ventricles due to cardiomyopathy and ischemic heart disease.

Platelet function testing to predict hyporesponsiveness to clopidogrel in patients with chest pain seen in the emergency department

Background A dual antiplatelet regimen has been shown to reduce the risk of major adverse cardiovascular events after percutaneous coronary intervention. However, there is little information available on inhibition of platelet aggregation in patients with a prior coronary stent presenting with chest pain. This study evaluated the prevalence of hyporesponsiveness to clopidogrel and factors associated with this in patients presenting to our emergency department with chest pain who had previously undergone coronary stent placement and were prescribed dual antiplatelet therapy. Methods Responsiveness to clopidogrel was evaluated in a cohort of 533 consecutive stented patients presenting to the emergency department with chest pain. P2Y12 reaction units (PRU) and percent P2Y12 inhibition with clopidogrel were measured in all patients. Of 533 patients, 221 (41.6%) had PRU ≥ 230. A multivariate logistic regression model was used to determine the relationship between hyporesponsiveness to clopidogrel (defined as PRU ≥ 230) and several potential risk factors, ie, gender, age, race, type 1 or type 2 diabetes, hypertension, smoking, chronic renal failure, and obesity. Results There was a greater risk of hyporesponsiveness in African Americans than in non-African American patients (adjusted odds ratio [OR] = 2.165), in patients with type 2 diabetes than in those without (adjusted OR = 2.109), and in women than in men (adjusted OR = 1.813), as well as a greater risk of hyporesponsiveness with increasing age (adjusted OR = 1.167 per decade). Conclusion There was a high prevalence of hyporesponsiveness to clopidogrel in patients presenting with chest pain and a prior coronary stent. Non-insulin-dependent diabetes mellitus and African American race were the strongest predictors of hyporesponsiveness to clopidogrel, followed by gender and age.

Coronary computed tomographic angiography (CCTA) in community hospitals: "current and emerging role".

Coronary computed tomographic angiography (CCTA) is a rapidly evolving test for diagnosis of coronary artery disease. Although invasive coronary angiography is the gold standard for coronary artery disease (CAD), CCTA is an excellent noninvasive tool for evaluation of chest pain. There is ample evidence to support the cost-effective use of CCTA in the early triage process of patients presenting with chest pain in the emergency room. CCTA plays a critical role in the diagnosis of chest pain etiology as one of potentially fatal conditions, aortic dissection, pulmonary embolism, and myocardial infarction. This ‘triple rule out’ protocol is becoming an increasingly practicable and popular diagnostic tool in ERs across the country. In addition to a quick triage of chest pain patients, it may improve quality of care, decrease cost, and prevent medico-legal risk for missing potentially lethal conditions presenting as chest pain. CCTA is also helpful in the detection of subclinical and vulnerable coronary plaques. The major limitations for wide spread acceptance of this test include radiation exposure, motion artifacts, and its suboptimal imaging with increased body mass index.

Proteinases and Restenosis: Matrix Metalloproteinase and their Inhibitor and Activator

Remodeling of the extracellular matrix (ECM) is the central part of normal tissue development and is involved in tumor growth and metastasis, angiogenesis, wound healing/repair, inflammation, embryonic development, post-partum involution of the uterus, bone and growth plate remodeling, ovulation, and the progression of certain diseases.1,2 The process of connective tissue restructuring represents a balance between matrix production and its degradation. The breakdown of this highly organized extracellular environment is controlled, for the most part, by a gene family of matrix metalloproteinases (MMPs) which collectively can degrade virtually all components of connective tissues.3,4