Andriy Yabluchanskiy

Matrix metalloproteinase-9: Many shades of function in cardiovascular disease.

Matrix metalloproteinase (MMP)-9, one of the most widely investigated MMPs, regulates pathological remodeling processes that involve inflammation and fibrosis in cardiovascular disease. MMP-9 directly degrades extracellular matrix (ECM) proteins and activates cytokines and chemokines to regulate tissue remodeling. MMP-9 deletion or inhibition has proven overall beneficial in multiple animal models of cardiovascular disease. As such, MMP-9 expression and activity is a common end point measured. MMP-9 cell-specific overexpression, however, has also proven beneficial and highlights the fact that little information is available on the underlying mechanisms of MMP-9 function. In this review, we summarize our current understanding of MMP-9 physiology, including structure, regulation, activation, and downstream effects of increased MMP-9. We discuss MMP-9 roles during inflammation and fibrosis in cardiovascular disease. By concentrating on the substrates of MMP-9 and their roles in cardiovascular disease, we explore the overall function and discuss future directions on the translational potential of MMP-9 based therapies.

Neutrophil roles in left ventricular remodeling following myocardial infarction

Polymorphonuclear granulocytes (PMNs; neutrophils) serve as key effector cells in the innate immune system and provide the first line of defense against invading microorganisms. In addition to producing inflammatory cytokines and chemokines and undergoing a respiratory burst that stimulates the release of reactive oxygen species, PMNs also degranulate to release components that kill pathogens. Recently, neutrophil extracellular traps have been shown to be an alternative way to trap microorganisms and contain infection. PMN-derived granule components are also involved in multiple non-infectious inflammatory processes, including the response to myocardial infarction (MI). In this review, we will discuss the biological characteristics, recruitment, activation, and removal of PMNs, as well as the roles of PMN-derived granule proteins in inflammation and innate immunity, focusing on the MI setting when applicable. We also discuss future perspectives that will direct research in PMN biology.

A Novel Collagen Matricryptin Reduces Left Ventricular Dilation Post-Myocardial Infarction by Promoting Scar Formation and Angiogenesis

BACKGROUND Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). OBJECTIVES This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). METHODS Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. RESULTS In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mouse LV, at levels that inversely correlated to MMP-9 levels. We synthesized a peptide beginning at the cleavage site (p1158/59, amino acids 1159 to 1173) to investigate its biological functions. In vitro, p1158/59 stimulated fibroblast wound healing and robustly promoted angiogenesis. In vivo, early post-MI treatment with p1158/59 reduced LV dilation at day 7 post-MI by preserving LV structure (p < 0.05 vs. control). The p1158/59 stimulated both in vitro and in vivo wound healing by enhancing basement membrane proteins, granulation tissue components, and angiogenic factors. CONCLUSIONS Collagen Iα1 matricryptin p1158/59 facilitates LV remodeling post-MI by regulating scar formation through targeted ECM generation and stimulation of angiogenesis.

Temporal Neutrophil Polarization Following Myocardial Infarction

AIMS Although macrophage phenotypes have been well studied in the myocardial infarction (MI) setting, this study investigated temporal neutrophil polarization and activation mechanisms. METHODS AND RESULTS Neutrophils isolated from the infarcted left ventricle (LV) of mice showed high expression of proinflammatory markers at Day 1 and anti-inflammatory markers at Days 5 and 7 post-MI, indicating distinct neutrophil phenotypes along the post-MI time continuum. Flow cytometry analysis revealed that although proinflammatory N1 neutrophils were always predominant (>80% of total neutrophils at each time point), the percentage of N2 neutrophils increased post-MI from 2.4 ± 0.6% at Day 1 to 18.1 ± 3.0% at Day 7. In vitro, peripheral blood neutrophils were polarized to proinflammatory N1 by lipopolysaccharide and interferon-γ or anti-inflammatory N2 by interleukin-4, indicating high plasticity potential. The in vivo post-MI relevant LV damage-associated molecular patterns (DAMPs) polarized neutrophils to a proinflammatory N1 phenotype by activating toll-like receptor-4. Transforming growth factor-β1 inhibited proinflammatory production in neutrophils. N1 neutrophils positively correlated with infarct wall thinning at Day 7 post-MI, possibly due to high production of matrix metalloproteinases-12 and -25. CONCLUSION This study is the first to identify the existence of N1 and N2 neutrophils in the infarct region and reveals that N1 polarization could be mediated by DAMPs.

Cardiac aging is initiated by matrix metalloproteinase-9-mediated endothelial dysfunction

Aging is linked to increased matrix metalloproteinase-9 (MMP-9) expression and extracellular matrix turnover, as well as a decline in function of the left ventricle (LV). Previously, we demonstrated that C57BL/6J wild-type (WT) mice > 18 mo of age show impaired diastolic function, which was attenuated by MMP-9 deletion. To evaluate mechanisms that initiate the development of cardiac dysfunction, we compared the LVs of 6-9- and 15-18-mo-old WT and MMP-9 null (Null) mice. All groups showed similar LV function by echocardiography, indicating that dysfunction had not yet developed in the older group. Myocyte nuclei numbers and cross-sectional areas increased in both WT and Null 15-18-mo mice compared with young controls, indicating myocyte hypertrophy. Myocyte hypertrophy leads to an increased oxygen demand, and both WT and Null 15-18-mo mice showed an increase in angiogenic signaling. Plasma proteomic profiling and LV analysis revealed a threefold increase in von Willebrand factor and fivefold increase in vascular endothelial growth factor in WT 15-18-mo mice, which were further elevated in Null mice. In contrast to the upregulation of angiogenic stimulating factors, actual LV vessel numbers increased only in the 15-18-mo Null LV. The 15-18-mo WT showed amplified expression of inflammatory genes related to angiogenesis, including C-C chemokine receptor (CCR)7, CCR10, interleukin (IL)-1f8, IL-13, and IL-20 (all, P < 0.05), and these increases were blunted by MMP-9 deletion (all, P < 0.05). To measure vascular permeability as an index of endothelial function, we injected mice with FITC-labeled dextran. The 15-18-mo WT LV showed increased vascular permeability compared with young WT controls and 15-18-mo Null mice. Combined, our findings revealed that MMP-9 deletion improves angiogenesis, attenuates inflammation, and prevents vascular leakiness in the setting of cardiac aging.

Deriving a cardiac ageing signature to reveal MMP-9-dependent inflammatory signalling in senescence

AIMS Cardiac ageing involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by MMP-9. Here, we report a cardiac ageing signature that encompasses macrophage pro-inflammatory signalling in the left ventricle (LV) and distinguishes biological from chronological ageing. METHODS AND RESULTS Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac ageing signature. The decline in early-to-late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, whereas LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages and a decrease in cardiac M2 macrophages in WT mice; of which, both were prevented by MMP-9 deletion. In vitro, MMP-9 directly activated young macrophage polarization to an M1/M2 mid-transition state. CONCLUSION Our results define the cardiac ageing inflammatory signature and assign MMP-9 roles in mediating the inflammaging profile by indirectly and directly modifying macrophage polarization. Our results explain early mechanisms that stimulate ageing-induced cardiac fibrosis and diastolic dysfunction.

Myocardial Infarction Superimposed on Aging: MMP-9 Deletion Promotes M2 Macrophage Polarization

In this study, we examined the combined effect of aging and myocardial infarction on left ventricular remodeling, focusing on matrix metalloproteinase (MMP)-9-dependent mechanisms. We enrolled 55 C57BL/6J wild type (WT) and 85 MMP-9 Null (Null) mice of both sexes at 11-36 months of age and evaluated their response at Day 7 post-myocardial infarction. Plasma MMP-9 levels positively linked to age in WT mice (r = .46, p = .001). MMP-9 deletion improved survival (76% for WT vs 88% for Null, p = .021). Post-myocardial infarction, there was a progressive increase in left ventricular dilation with age in WT but not in Null mice. By inflammatory gene array analysis, WT mice showed linear age-dependent increases in three different proinflammatory genes (C3, CCl4, and CX3CL1; all p < .05), whereas Null mice showed increases in three proinflammatory genes (CCL5, CCL9, and CXCL4; all p < .05) and seven anti-inflammatory genes (CCL1, CCL6, CCR1, IL11, IL1r2, IL8rb, and Mif; all p < .05). Compared with WT, macrophages isolated from Null left ventricle infarct demonstrated enhanced expression of anti-inflammatory M2 markers CD163, MRC1, TGF-β1, and YM1 (all p < .05), without affecting proinflammatory M1 markers. In conclusion, MMP-9 deletion stimulated anti-inflammatory polarization of macrophages to attenuate left ventricle dysfunction in the aging post-myocardial infarction.

Building a better infarct: Modulation of collagen cross-linking to increase infarct stiffness and reduce left ventricular dilation post-myocardial infarction

Matrix metalloproteinase-9 (MMP-9) deletion attenuates collagen accumulation and dilation of the left ventricle (LV) post-myocardial infarction (MI); however the biomechanical mechanisms underlying the improved outcome are poorly understood. The aim of this study was to determine the mechanisms whereby MMP-9 deletion alters collagen network composition and assembly in the LV post-MI to modulate the mechanical properties of myocardial scar tissue. Adult C57BL/6J wild-type (WT; n=88) and MMP-9 null (MMP-9(-/-); n=92) mice of both sexes underwent permanent coronary artery ligation and were compared to day 0 controls (n=42). At day 7 post-MI, WT LVs displayed a 3-fold increase in end-diastolic volume, while MMP-9(-/-) showed only a 2-fold increase (p<0.05). Biaxial mechanical testing revealed that MMP-9(-/-) infarcts were stiffer than WT infarcts, as indicated by a 1.3-fold reduction in predicted in vivo circumferential stretch (p<0.05). Paradoxically, MMP-9(-/-) infarcts had a 1.8-fold reduction in collagen deposition (p<0.05). This apparent contradiction was explained by a 3.1-fold increase in lysyl oxidase (p<0.05) in MMP-9(-/-) infarcts, indicating that MMP-9 deletion increased collagen cross-linking activity. Furthermore, MMP-9 deletion led to a 3.0-fold increase in bone morphogenetic protein-1, the metalloproteinase that cleaves pro-collagen and pro-lysyl oxidase (p<0.05) and reduced fibronectin fragmentation by 49% (p<0.05) to enhance lysyl oxidase activity. We conclude that MMP-9 deletion increases infarct stiffness and prevents LV dilation by reducing collagen degradation and facilitating collagen assembly and cross-linking through preservation of the fibronectin network and activation of lysyl oxidase.

IGF-1 has sexually dimorphic, pleiotropic, and time-dependent effects on healthspan, pathology, and lifespan

Reduced circulating levels of IGF-1 have been proposed as a conserved anti-aging mechanism that contributes to increased lifespan in diverse experimental models. However, IGF-1 has also been shown to be essential for normal development and the maintenance of tissue function late into the lifespan. These disparate findings suggest that IGF-1 may be a pleiotropic modulator of health and aging, as reductions in IGF-1 may be beneficial for one aspect of aging, but detrimental for another. We postulated that the effects of IGF-1 on tissue health and function in advanced age are dependent on the tissue, the sex of the animal, and the age at which IGF-1 is manipulated. In this study, we examined how alterations in IGF-1 levels at multiple stages of development and aging influence overall lifespan, healthspan, and pathology. Specifically, we investigated the effects of perinatal, post-pubertal, and late-adult onset IGF-1 deficiency using genetic and viral approaches in both male and female igff/f C57Bl/6 mice. Our results support the concept that IGF-1 levels early during lifespan establish the conditions necessary for subsequent healthspan and pathological changes that contribute to aging. Nevertheless, these changes are specific for each sex and tissue. Importantly, late-life IGF-1 deficiency (a time point relevant for human studies) reduces cancer risk but does not increase lifespan. Overall, our results indicate that the levels of IGF-1 during development influence late-life pathology, suggesting that IGF-1 is a developmental driver of healthspan, pathology, and lifespan.

Matrix metalloproteinases: Drug targets for myocardial infarction

Myocardial infarction (MI) remains a major cause of morbidity and mortality worldwide. Rapid advances in the treatment of acute MI have significantly improved short-term outcomes in patients, due in large part to successes in preventing myocardial cell death and limiting infarct area during the time of ischemia and subsequent reperfusion. Matrix metalloproteases (MMPs) play key roles in post-MI cardiac remodeling and in the development of adverse outcomes. This review highlights the importance of MMPs in the injury and remodeling response of the left ventricle and also discusses their potential as therapeutic targets Additional pre-clinical and clinical research is needed to further investigate and understand the cardioprotective effects of MMPs inhibitors.