Novel strategies to delineate matrix metalloproteinase (MMP)-substrate relationships and identify targets to block MMP activity.

Adverse extracellular matrix (ECM) remodeling contributes to fibrotic disorders in the kidney, lung, and heart. Matrix metalloproteinases (MMPs) are key enzymes regulating ECM turnover, and MMP inhibition attenuates remodeling. Recent technological developments allow MMP-substrate relationships to be identified and explored as novel therapeutic targets. This review summarizes current and novel strategies to block MMP activity.

Matrix metalloproteinase (MMP)-7 activates MMP-8 but not MMP-13.

Matrix Metalloproteinases (MMPs) are a class of zinc-dependent enzymes that degrade extracellular matrix components, particularly collagen. MMPs have been implicated in a diverse list of pathological processes, including cancer and cardiovascular disease. Recent efforts to bring MMP inhibitors to clinical trials, however, have proved disappointing. These failures are attributed, in part, to the non-selective nature of current inhibitors. The possibility also exists, however, that inhibition of a particular MMP type will lead to feedback accumulation of parallel MMP members. MMP-7, also known as matrilysin, has a broad list of substrates, including denatured collagen and other MMPs involved in the collagenolytic pathway, namely MMP-1, MMP-2, and MMP-9. Whether the additional collagenases, MMP-8 and MMP-13, are also activated by MMP-7 has not been explored. We show here that recombinant active MMP-7 was able to process MMP-8 to its active form in vitro, but did not activate MMP-13. In the left ventricles of mice lacking the MMP-7 gene, MMP-8 levels increased while MMP-13 levels decreased in vivo. The switch in MMP profile was not accompanied by a change in left ventricular dimensions or wall thickness. Together, these data suggest that MMP-8 is an in vivo substrate of MMP-7, and that the accumulation of pro-MMP-8 in the absence of MMP-7 downregulates pro-MMP-13 levels in order to maintain baseline collagenolytic function. The interplay between MMP-8 and MMP-13 suggest that these MMPs may play reciprocal roles. The design of selective MMP inhibitors, therefore, must take into consideration changes in parallel MMP types as a potential compensatory mechanism.

Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice.

Background- To investigate the role of endothelial nitric oxide synthase (NOS3) in left ventricular (LV) remodeling after myocardial infarction (MI), the impact of left anterior descending coronary artery ligation on LV size and function was compared in 2- to 4-month-old wild-type (WT) and NOS3-deficient mice (NOS3(-/-)). Methods and Results- Two days after MI, both strains of mice had a similar LV size, fractional shortening, and ejection fraction by echocardiography. Twenty-eight days after MI, both strains had dilated LVs with decreased fractional shortening and lower ejection fractions. Although the infarcted fraction of the LV was similar in both strains, LV end-diastolic internal diameter, end-diastolic volume, and mass were greater, but fractional shortening, ejection fraction, and the maximum rate of developed LV pressure (dP/dt(max)) were lower in NOS3(-/-) than in WT mice. Impairment of diastolic function, as measured by the time constant of isovolumic relaxation (tau) and the maximum rate of LV pressure decay (dP/dt(min)), was more marked in NOS3(-/-) than in WT mice. Mortality after MI was greater in NOS3(-/-) than in WT mice. Long-term administration of hydralazine normalized blood pressure in NOS3(-/-) mice, but it did not prevent the LV dilatation, impaired systolic and diastolic function, and increased LV mass that followed MI. In WT mice, capillary density and myocyte width in the nonischemic portion of the LV did not differ before and 28 days after MI, whereas in NOS3(-/-) mice, capillary density decreased and myocyte width increased after MI, whether or not hydralazine was administered. Conclusions- These results suggest that the presence of NOS3 limits LV dysfunction and remodeling in a murine model of MI by an afterload-independent mechanism, in part by decreasing myocyte hypertrophy in the remote myocardium.

IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury.

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.

Interleukin 6 induction in the canine myocardium after cardiopulmonary bypass.

OBJECTIVE: Interleukin 6 is a proinflammatory cytokine with a plasma concentration that has been noted to increase in response to cardiopulmonary bypass. The source of interleukin 6 after cardiopulmonary bypass is unknown. This study examined the myocardium as a potential source of interleukin 6 in this context. METHODS: Dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After rewarming, they were reperfused with the chest open for either 3 (n = 4) or 6 (n = 4) hours, at the end of which myocardial samples were obtained. Four additional animals undergoing open thoracotomy without bypass served as time-matched controls. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization were used to examine the myocardium for the induction of interleukin 6 and intercellular adhesion molecule-1. RESULTS: Northern blot analysis and reverse transcriptase-polymerase chain reaction demonstrated a marked increase in myocardial interleukin 6 messenger RNA in 3 of 4 dogs at 3 hours after bypass and 3 of 4 dogs at 6 hours after bypass, which was not present in sham-bypass control animals. Northern blots at 3 hours after cardiopulmonary bypass also demonstrated myocardial intercellular adhesion molecule-1 induction. In situ hybridization studies confirmed that cardiac myocytes were a principal source of interleukin 6 messenger RNA early after cardiopulmonary bypass. Northern blots of messenger RNA extracted from isolated neutrophils and mononuclear leukocytes obtained from blood samples before bypass, at the end of bypass, and 3 hours after bypass failed to demonstrate interleukin 6 induction. CONCLUSION: Despite protection with cold cardioplegic arrest, the myocardium was a significant source of interleukin 6 synthesis after cardiopulmonary bypass. Local production of interleukin 6 may play a pivotal role in postoperative myocardial function.

beta-blockade prevents sustained metalloproteinase activation and diastolic stiffening induced by angiotensin II combined with evolving cardiac dysfunction.

Angiotensin II (Ang II)-mediated sympathostimulation may worsen the progression of cardiac failure, although the nature and mechanisms of such interactions are largely unknown. We previously demonstrated that Ang II combined with evolving cardiodepression (48-hour tachycardia pacing, 48hP) induces marked chamber stiffening and increases metalloproteinases (MMPs). Here, we test the hypothesis that both abnormalities stem from sympathostimulatory effects of Ang II. Forty-eight dogs were instrumented to serially assess conscious ventricular mechanics, MMP abundance and activity, and myocardial histopathology. 48hP combined with 5 days of Ang II (15+/-5 ng. kg(-1). min(-1) IV) more than doubled chamber stiffness (end-diastolic pressure >25 mm Hg, P<0.001), whereas stiffness was unchanged by Ang II or 48hP alone. In vitro and in situ zymography revealed increased MMP abundance and activity (principally 92-kDa gelatinase) from Ang II+48hP. Both stiffening and MMP changes were prevented by cotreatment with high-dose atenolol (which nearly fully inhibited isoproterenol-induced inotropy) but not partial beta-blockade. Myocellular damage with fibroblast/neutrophil infiltration from Ang II+48hP was also inhibited by high- but not low-dose atenolol, whereas collagen content was not elevated with either dose. These data support a role of sympathostimulation by Ang II in modulating myocardial MMP abundance and activity and diastolic stiffening in evolving heart failure and suggest a novel mechanism by which beta-blockade may limit chamber remodeling and diastolic dysfunction.

Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion.

BACKGROUND: Myocardial infarction is associated with an intense inflammatory reaction leading to healing and scar formation. Because mast cells are a significant source of fibrogenic factors, we investigated mast cell accumulation and regulation of stem cell factor (SCF), a potent growth and tactic factor for mast cells, in the healing myocardium. METHODS AND RESULTS: Using a canine model of myocardial ischemia and reperfusion, we demonstrated a striking increase of mast cell numbers during the healing phase of a myocardial infarction. Mast cell numbers started increasing after 72 hours of reperfusion, showing maximum accumulation in areas of collagen deposition (12.0+/-2.6-fold increase; P<0.01) and proliferating cell nuclear antigen (PCNA) expression. The majority of proliferating cells were identified as alpha-smooth muscle actin-positive myofibroblasts or factor VIII-positive endothelial cells. Mast cells did not appear to proliferate. Using a nuclease protection assay, we demonstrated induction of SCF mRNA within 72 hours of reperfusion. Immunohistochemical studies demonstrated that a subset of macrophages was the source of SCF immunoreactivity in the infarcted myocardium. SCF protein was not found in endothelial cells and myofibroblasts. Intravascular tryptase-positive, FITC-avidin-positive, CD11b-negative mast cell precursors were noted in the area of healing and in the cardiac lymph after 48 to 72 hours of reperfusion. CONCLUSIONS: Mast cells increase in number in areas of collagen deposition and PCNA expression after myocardial ischemia. The data provide evidence of mast cell precursor infiltration into the areas of cellular injury. SCF is induced in a subset of macrophages infiltrating the healing myocardium. We suggest an important role for SCF in promoting chemotaxis and growth of mast cell precursors in the healing heart.

Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion.

BACKGROUND: Neutrophil-induced cardiomyocyte injury requires the expression of myocyte intercellular adhesion molecule (ICAM)-1 and ICAM-1-CD11b/CD18 adhesion. We have previously demonstrated interleukin (IL)-6 activity in postischemic cardiac lymph; IL-6 is the primary stimulus for myocyte ICAM- 1 induction. Furthermore, we found that induction of IL-6 mRNA occurred very early on reperfusion of the infarcted myocardium. We hypothesized that the release of a preformed upstream cytokine induced IL-6 in leukocytes infiltrating on reperfusion. METHODS AND RESULTS: Constitutive expression of TNF-alpha and not IL-1beta was demonstrated in the normal canine myocardium and was localized predominantly in cardiac mast cells. Mast cell degranulation in the ischemic myocardium was documented by demonstration of a rapid release of histamine and TNF-alpha in the cardiac lymph after myocardial ischemia. Histochemical studies with FITC-labeled avidin demonstrated degranulating mast cells only in ischemic samples of canine myocardium. Immunohistochemistry suggested that degranulating mast cells were the primary source of TNF-alpha in the ischemic myocardium. In situ hybridization studies of reperfused myocardium localized IL-6 mRNA in infiltrating mononuclear cells and in mononuclear cells appearing in the postischemic cardiac lymph within the first 15 minutes of reperfusion. Furthermore, isolated canine mononuclear cells incubated with postischemic cardiac lymph demonstrated significant induction of IL-6 mRNA, which was partially blocked with a neutralizing antibody to TNF-alpha. CONCLUSIONS: Cardiac mast cells degranulate after myocardial ischemia, releasing preformed mediators, such as histamine and TNF-alpha. We suggest that mast cell-derived TNF-alpha may be a crucial factor in upregulating IL-6 in infiltrating leukocytes and initiating the cytokine cascade responsible for myocyte ICAM-1 induction and subsequent neutrophil-induced injury.

Intercellular adhesion molecule-1 regulation in the canine lung after cardiopulmonary bypass.

OBJECTIVE(S): Neutrophil sequestration in the lung after cardiopulmonary bypass has been shown to be dependent on the adhesion molecule CD18. Thus we sought to determine whether endothelial expression of intercellular adhesion molecule-1 (a ligand for CD18) in pulmonary capillaries mediates neutrophil adhesion in this setting. METHODS: Seven adult mongrel dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After warming, dogs were reperfused for up to 9 hours and lung biopsy specimens were obtained. Lung tissue was examined by Northern and Western blot analysis and by immunohistologic methods. Three sham-operated dogs served as time-matched controls. RESULTS: Northern blots demonstrated increased expression of intercellular adhesion molecule-1 messenger ribonucleic acid within 5 minutes of cessation of bypass (or approximately 30 minutes after aortic crossclamp release), which persisted at 9 hours of recovery and was not present in controls. Western blots showed intercellular adhesion molecule-1 protein expression before bypass but a measurable increase in intercellular adhesion molecule-1 protein in four of seven dogs in the bypass group by the ninth hour of recovery. Pulmonary neutrophil accumulation 9 hours after cardiopulmonary bypass was greater in those dogs with an increased intercellular adhesion molecule-1 protein expression. Immunoelectron microscopy demonstrated the pulmonary capillary endothelium capable of increased intercellular adhesion molecule-1 protein expression at the 9-hour time point. CONCLUSIONS: Cardiopulmonary bypass resulted in intercellular adhesion molecule-1 induction in the canine lung during recovery. An increased expression of intercellular adhesion molecule-1 protein in the lung was associated with an increased accumulation of neutrophils in affected animals. Thus intercellular adhesion molecule-1 expression may serve as a mechanism that predisposes the lungs to inflammatory cell-mediated injury postoperatively.

Induction of monocyte chemoattractant protein-1 in the small veins of the ischemic and reperfused canine myocardium.

BACKGROUND: Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area. Since augmented monocyte influx has been implicated as a potential mechanism for improved healing after reperfusion, we wished to study the induction of monocyte chemoattractant protein-1 (MCP-1) during reperfusion. METHODS AND RESULTS: The cDNA for MCP-1 was cloned from a canine jugular vein endothelial cell (CJVEC) library and exhibited 78% identity with the deduced amino acid sequence of human MCP-1. Samples of myocardium were taken from control and ischemic segments after 1 hour of ischemia and various times of reperfusion; total RNA was isolated from myocardial samples and probed with a cDNA probe for canine MCP-1. Induction of MCP-1 mRNA occurred only in previously ischemic segments within the first hour of reperfusion, peaked at 3 hours, and persisted throughout the first 2 days of reperfusion. In the absence of reperfusion, no significant MCP-1 induction was seen. Both ischemic (but not preischemic) cardiac lymph and human recombinant TNF-alpha induced MCP-1 in CJVECs. MCP-1 was identified by immunostaining on infiltrating cells and venular (but not arterial) endothelium by 3 hours. In contrast, in situ hybridization showed MCP-1 mRNA to be confined to the endothelium of small veins (venules) 10 to 70 microns in diameter. CONCLUSIONS: MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.

Phagocytes in ischemia injury.

We are now developing the means to evaluate components of this inflammatory response that may facilitate healing. A key event in the change in the inflammatory response is the development of a cytokine cascade that promotes phenotypic changes in the infiltrating leukocytes, which endow them with the ability to promote fibroblast proliferation and collagen deposition, the hallmarks of healing.

Clinical versus administrative data bases for CABG surgery. Does it matter?

This study compared the ability of a clinical and administrative data base in New York State to predict in-hospital mortality and to assess hospital performance for coronary artery bypass graft surgery. The results indicated that the clinical data base, the Cardiac Surgery Reporting System, is substantially better at predicting case-specific mortality than the administrative data base, the Statewide Planning and Research Cooperative System. Also, correlations between hospital mortality rates that are risk-adjusted using the two systems were only moderately high (0.75 to 0.80). The addition of new risk factors from the Statewide Planning and Research Cooperative System improved the predictive power of both systems but did not diminish the difference in effectiveness of the two systems. The three unique clinical risk factors in the Cardiac Surgery Reporting System (ejection fraction, reoperation, and more than 90% narrowing of the left main trunk) seemed to account for much of the difference in effectiveness of the two systems.

A longitudinal analysis of the relationship between in-hospital mortality in New York State and the volume of abdominal aortic aneurysm surgeries performed.

This study uses New York State hospital discharge data to examine the relationship between in-hospital mortality for a patient receiving an abdominal aortic aneurysm resection and the volume of aneurysm operations performed in the previous year at the hospital where the operation took place and by the surgeon performing the operation. Previous research on this topic is extended in several respects: (1) A three-year data base is used to examine the manner in which hospital and surgeon volume jointly affect mortality rate and to examine ruptured and unruptured aneurysms separately; (2) a six-year data base is used to study the "practice makes perfect" hypothesis and the "selective referral" hypothesis; and (3) the degree of specialization of high-volume surgeons is contrasted with that of other surgeons. The results demonstrate a significant inverse relationship between hospital volume and mortality rate for unruptured aneurysms. Further, very few surgeons substantially increased their aneurysm surgery volumes in the six-year study period. Weak selective referral effects were found for both surgeons and hospitals, and higher-volume aneurysm surgeons tended to have much higher specialization rates.