Transcriptome analysis of cardiac endothelial cells after myocardial infarction reveals temporal changes and long-term deficits.

Endothelial cells (ECs) have essential roles in cardiac tissue repair after myocardial infarction (MI). To establish stage-specific and long-term effects of the ischemic injury on cardiac ECs, we analyzed their transcriptome at landmark time points after MI in mice. We found that early EC response at Day 2 post-MI centered on metabolic changes, acquisition of proinflammatory phenotypes, initiation of the S phase of cell cycle, and activation of stress-response pathways, followed by progression to mitosis (M/G2 phase) and acquisition of proangiogenic and mesenchymal properties during scar formation at Day 7. In contrast, genes involved in vascular physiology and maintenance of vascular tone were suppressed. Importantly, ECs did not return to pre-injury phenotypes after repair has been completed but maintained inflammatory, fibrotic and thrombotic characteristics and lost circadian rhythmicity. We discovered that the highest induced transcript is the mammalian-specific Sh2d5 gene that promoted migration and invasion of ECs through Rac1 GTPase. Our results revealed a synchronized, temporal activation of disease phenotypes, metabolic pathways, and proliferation in quiescent ECs after MI, indicating that precisely-timed interventions are necessary to optimize cardiac tissue repair and improve outcomes. Furthermore, long-term effects of acute ischemic injury on ECs may contribute to vascular dysfunction and development of heart failure.

The BDNF rs6265 Polymorphism is a Modifier of Cardiomyocyte Contractility and Dilated Cardiomyopathy.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality.

Aims: Macrophage phagocytosis of dead cells is a prerequisite for inflammation resolution. Because CXCL4 induces macrophage phagocytosis in vitro, we examined the impact of exogenous CXCL4 infusion on cardiac wound healing and macrophage phagocytosis following myocardial infarction (MI). Methods and results: CXCL4 expression significantly increased in the infarct region beginning at Day 3 post-MI, and macrophages were the predominant source. Adult male C57BL/6J mice were subjected to coronary artery occlusion, and MI mice were randomly infused with recombinant mouse CXCL4 or saline beginning at 24 h post-MI by mini-pump infusion. Compared with saline controls, CXCL4 infusion dramatically reduced 7 day post-MI survival [10% (3/30) for CXCL4 vs. 47% (7/15) for saline, P < 0.05] as a result of acute congestive heart failure. By echocardiography, CXCL4 significantly increased left ventricular (LV) volumes and dimensions at Day 5 post-MI (all P < 0.05), despite similar infarct areas compared with saline controls. While macrophage numbers were similar at Day 5 post-MI, CXCL4 infusion increased Ccr4 and Itgb4 and decreased Adamts8 gene levels in the infarct region, all of which linked to CXCL4-mediated cardiac dilation. Isolated Day 5 post-MI macrophages exhibited comparable levels of M1 and M4 markers between saline and CXCL4 groups. Interestingly, by both ex vivo and in vitro phagocytosis assays, CXCL4 reduced macrophage phagocytic capacity, which was connected to decreased levels of the phagocytosis receptor CD36. In vitro, a CD36 neutralizing antibody (CD36Ab) significantly inhibited macrophage phagocytic capacity. The combination of CXCL4 and CD36Ab did not have an additive effect, indicating that CXCL4 regulated phagocytosis through CD36 signalling. CXCL4 infusion significantly elevated infarct matrix metalloproteinase (MMP)-9 levels at Day 5 post-MI, and MMP-9 can cleave CD36 as a down-regulation mechanism. Conclusion: CXCL4 infusion impaired macrophage phagocytic capacity by reducing CD36 levels through MMP-9 dependent and independent signalling, leading to higher mortality and LV dilation.

The Mouse Heart Attack Research Tool 1.0 database.

The generation of big data has enabled systems-level dissections into the mechanisms of cardiovascular pathology. Integration of genetic, proteomic, and pathophysiological variables across platforms and laboratories fosters discoveries through multidisciplinary investigations and minimizes unnecessary redundancy in research efforts. The Mouse Heart Attack Research Tool (mHART) consolidates a large data set of over 10 yr of experiments from a single laboratory for cardiovascular investigators to generate novel hypotheses and identify new predictive markers of progressive left ventricular remodeling after myocardial infarction (MI) in mice. We designed the mHART REDCap database using our own data to integrate cardiovascular community participation. We generated physiological, biochemical, cellular, and proteomic outputs from plasma and left ventricles obtained from post-MI and no-MI (naïve) control groups. We included both male and female mice ranging in age from 3 to 36 mo old. After variable collection, data underwent quality assessment for data curation (e.g., eliminate technical errors, check for completeness, remove duplicates, and define terms). Currently, mHART 1.0 contains >888,000 data points and includes results from >2,100 unique mice. Database performance was tested, and an example is provided to illustrate database utility. This report explains how the first version of the mHART database was established and provides researchers with a standard framework to aid in the integration of their data into our database or in the development of a similar database. NEW & NOTEWORTHY The Mouse Heart Attack Research Tool combines >888,000 cardiovascular data points from >2,100 mice. We provide this large data set as a REDCap database to generate novel hypotheses and identify new predictive markers of adverse left ventricular remodeling following myocardial infarction in mice and provide examples of use. The Mouse Heart Attack Research Tool is the first database of this size that integrates data sets across platforms that include genomic, proteomic, histological, and physiological data.

Periodontal-induced chronic inflammation triggers macrophage secretion of Ccl12 to inhibit fibroblast-mediated cardiac wound healing.

Chronic inflammatory diseases, such as periodontal disease, associate with adverse wound healing in response to myocardial infarction (MI). The goal of this study was to elucidate the molecular basis for impaired cardiac wound healing in the setting of periodontal-induced chronic inflammation. Causal network analysis of 168 inflammatory and extracellular matrix genes revealed that chronic inflammation induced by a subseptic dose of Porphyromonas gingivalis lipopolysaccharide (LPS) exacerbated infarct expression of the proinflammatory cytokine Ccl12. Ccl12 prevented initiation of the reparative response by prolonging inflammation and inhibiting fibroblast conversion to myofibroblasts, resulting in diminished scar formation. Macrophage secretion of Ccl12 directly impaired fibronectin and collagen deposition and indirectly stimulated collagen degradation through upregulation of matrix metalloproteinase-2. In post-MI patients, circulating LPS levels strongly associated with the Ccl12 homologue monocyte chemotactic protein 1 (MCP-1). Patients with LPS levels ≥ 1 endotoxin units (EU)/ml (subseptic endotoxemia) at the time of hospitalization had increased end diastolic and systolic dimensions compared with post-MI patients with < 1 EU/ml, indicating that low yet pathological concentrations of circulating LPS adversely impact post-MI left ventricle (LV) remodeling by increasing MCP-1. Our study provides the first evidence to our knowledge that chronic inflammation inhibits reparative fibroblast activation and generates an unfavorable cardiac-healing environment through Ccl12-dependent mechanisms.

Transgenic overexpression of macrophage matrix metalloproteinase-9 exacerbates age-related cardiac hypertrophy, vessel rarefaction, inflammation, and fibrosis.

Advancing age is an independent risk factor for cardiovascular disease. Matrix metalloproteinase-9 (MMP-9) is secreted by macrophages and robustly increases in the left ventricle (LV) with age. The present study investigated the effect of MMP-9 overexpression in macrophages on cardiac aging. We compared 16- to 21-mo-old C57BL/6J wild-type (WT) and transgenic (TG) male and female mice (n = 15-20/group). MMP-9 overexpression amplified the hypertrophic response to aging, as evidenced by increased LV wall thickness and myocyte cross-sectional areas (P < 0.05 for both). MMP-9 overexpression reduced LV expression of the angiogenesis-related factors ICAM-1, integrins α3 and ß3, platelet/endothelial cell adhesion molecule-1, thrombospondin-1, tenascin-c, and versican (all P < 0.05). Concomitantly, the number of vessels in the TG was lower than WT LV (P < 0.05). This led to a mismatch in the muscle-to-vessel ratio and resulted in increased cardiac inflammation. Out of 84 inflammatory genes analyzed, 16 genes increased in the TG compared with WT (all P < 0.05). Of the elevated genes, 14 were proinflammatory genes. The increase in cardiac inflammation resulted in greater accumulation of interstitial collagen in TG (P < 0.05). Fractional shortening was similar between groups, indicating that global cardiac function was still preserved at this age. In conclusion, overexpression of MMP-9 in macrophages resulted in exacerbated cardiac hypertrophy in the setting of vessel rarefaction, which resulted in enhanced inflammation and fibrosis to augment the cardiac-aging phenotype. Our results provide evidence that macrophage-derived MMP-9 may be a therapeutic target in elderly subjects.NEW & NOTEWORTHY The present study was the first to use mice with transgenic overexpression of matrix metalloproteinase-9 (MMP-9) in macrophages to examine the effects of macrophage-derived MMP-9 on cardiac aging. We found that an elevation in macrophage-derived MMP-9 induced a greater age-dependent cardiac hypertrophy and vessel rarefaction phenotype, which enhanced cardiac inflammation and fibrosis.

Early matrix metalloproteinase-9 inhibition post-myocardial infarction worsens cardiac dysfunction by delaying inflammation resolution.

Matrix metalloproteinase-9 (MMP-9) is robustly elevated in the first week post-myocardial infarction (MI). Targeted deletion of the MMP-9 gene attenuates cardiac remodeling post-MI by reducing macrophage infiltration and collagen accumulation through increased apoptosis and reduced inflammation. In this study, we used a translational experimental design to determine whether selective MMP-9 inhibition early post-MI would be an effective therapeutic strategy in mice. We enrolled male C57BL/6J mice (3-6months old, n=116) for this study. Mice were subjected to coronary artery ligation. Saline or MMP-9 inhibitor (MMP-9i; 0.03µg/day) treatment was initiated at 3h post-MI and the mice were sacrificed at day (D) 1 or 7 post-MI. MMP-9i reduced MMP-9 activity by 31±1% at D1 post-MI (p<0.05 vs saline) and did not affect survival or infarct area. Surprisingly, MMP-9i treatment increased infarct wall thinning and worsened cardiac function at D7 post-MI. While MMP-9i enhanced neutrophil infiltration at D1 and macrophage infiltration at D7 post-MI, CD36 levels were lower in MMP-9i compared to saline, signifying reduced phagocytic potential per macrophage. Escalation and prolongation of the inflammatory response at D7 post-MI in the MMP-9i group was evident by increased expression of 18 pro-inflammatory cytokines (all p<0.05). MMP-9i reduced cleaved caspase 3 levels at D7 post-MI, consistent with reduced apoptosis and defective inflammation resolution. Because MMP-9i effects on inflammatory cells were significantly different from previously observed MMP-9 null mechanisms, we evaluated pre-MI (baseline) systemic differences between C57BL/6J and MMP-9 null plasma. By mass spectrometry, 34 plasma proteins were significantly different between groups, revealing a previously unappreciated altered baseline environment pre-MI when MMP-9 was deleted. In conclusion, early MMP-9 inhibition delayed inflammation resolution and exacerbated cardiac dysfunction, highlighting the importance of using translational approaches in mice.

Defining the sham environment for post-myocardial infarction studies in mice.

The purpose of this study was to evaluate the effect of sham surgery in a minimally invasive surgical model of permanent coronary artery occlusion used to generate myocardial infarction (MI) in mice. Adult male C57BL/6J mice (3-6 mo old) were divided into five groups: day (D) 0 (no surgical operation), D1 Sham, D1 MI, D7 Sham, and D7 MI. A refined MI surgery technique was used to approach the coronary artery without the ribs being cut. Both sham and MI mice had the left ventricle (LV) exposed through a small incision. To test the effects of surgery alone, the suture was passed around the coronary artery but not ligated. The MI mice were subjected to permanent coronary artery ligation. The mice were killed at D1 or D7 postsurgical procedure. Compared with D0 no surgery controls, the D1 and D7 sham groups exhibited no surgical mortality and similar necropsy and echocardiographic variables. Surgery alone did not induce an inflammatory cell response, as evidenced by the lack of leukocyte infiltration in the sham groups. Analysis of 165 inflammatory cytokines and extracellular matrix factors in sham revealed that a minor gene response was initiated but not translated to protein levels. Collagen deposition did not occur in the absence of MI. In contrast, the D1 and D7 MI groups showed the expected robust inflammatory and scar formation responses. When a minimally invasive procedure to generate MI in mice was used, the D0 (no surgical operation) control was an adequate replacement for the use of sham surgery groups.

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-ß1 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.

CD36 Is a Matrix Metalloproteinase-9 Substrate That Stimulates Neutrophil Apoptosis and Removal During Cardiac Remodeling.

BACKGROUND: After myocardial infarction, the left ventricle undergoes a wound healing response that includes the robust infiltration of neutrophils and macrophages to facilitate removal of dead myocytes as well as turnover of the extracellular matrix. Matrix metalloproteinase (MMP)-9 is a key enzyme that regulates post-myocardial infarction left ventricular remodeling. METHODS AND RESULTS: Infarct regions from wild-type and MMP-9 null mice (n=8 per group) analyzed by glycoproteomics showed that of 541 N-glycosylated proteins quantified, 45 proteins were at least 2-fold upregulated or downregulated with MMP-9 deletion (all P<0.05). Cartilage intermediate layer protein and platelet glycoprotein 4 (CD36) were identified as having the highest fold increase in MMP-9 null mice. By immunoblotting, CD36 but not cartilage intermediate layer protein decreased steadily during the time course post-myocardial infarction, which identified CD36 as a candidate MMP-9 substrate. MMP-9 was confirmed in vitro and in vivo to proteolytically degrade CD36. In vitro stimulation of day 7 post-myocardial infarction macrophages with MMP-9 or a CD36-blocking peptide reduced phagocytic capacity. Dual immunofluorescence revealed concomitant accumulation of apoptotic neutrophils in the MMP-9 null group compared with wild-type group. In vitro stimulation of isolated neutrophils with MMP-9 decreased neutrophil apoptosis, indicated by reduced caspase-9 expression. CONCLUSIONS: Our data reveal a new cell-signaling role for MMP-9 through CD36 degradation to regulate macrophage phagocytosis and neutrophil apoptosis.

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.

Plasma Glycoproteomics Reveals Sepsis Outcomes Linked to Distinct Proteins in Common Pathways.

OBJECTIVE: Sepsis remains a predominant cause of mortality in the ICU, yet strategies to increase survival have proved largely unsuccessful. This study aimed to identify proteins linked to sepsis outcomes using a glycoproteomic approach to target extracellular proteins that trigger downstream pathways and direct patient outcomes. DESIGN: Plasma was obtained from the Lactate Assessment in the Treatment of Early Sepsis cohort. N-linked plasma glycopeptides were quantified by solid-phase extraction coupled with mass spectrometry. Glycopeptides were assigned to proteins using RefSeq (National Center of Biotechnology Information, Bethesda, MD) and visualized in a heat map. Protein differences were validated by immunoblotting, and proteins were mapped for biological processes using Database for Annotation, Visualization and Integrated Discovery (National Institute of Allergy and Infectious Diseases, National Institutes of Health; Bethesda, MD) and for functional pathways using Kyoto Encyclopedia of Genes and Genomes (Kanehisa Laboratories, Kyoto, Japan) databases. SETTING: Hospitalized care. PATIENTS: Patients admitted to the emergency department were enrolled in the study when the diagnosis of sepsis was made, within 6 hours of presentation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 501 glycopeptides corresponding to 234 proteins were identified. Of these, 66 glycopeptides were unique to the survivor group and corresponded to 54 proteins, 60 were unique to the nonsurvivor group and corresponded to 43 proteins, and 375 were common responses between groups and corresponded to 137 proteins. Immunoblotting showed that nonsurvivors had increased total kininogen; decreased total cathepsin-L1, vascular cell adhesion molecule, periostin, and neutrophil gelatinase-associated lipocalin; and a two-fold decrease in glycosylated clusterin (all p < 0.05). Kyoto Encyclopedia of Genes and Genomes analysis identified six enriched pathways. Interestingly, survivors relied on the extrinsic pathway of the complement and coagulation cascade, whereas nonsurvivors relied on the intrinsic pathway. CONCLUSION: This study identifies proteins linked to patient outcomes and provides insight into unexplored mechanisms that can be investigated for the identification of novel therapeutic targets.

Cardiac extracellular proteome profiling and membrane topology analysis using glycoproteomics.

PURPOSE: Extracellular proteins are easily accessible, which presents a subproteome of molecular targets that have high diagnostic and therapeutic potential. Efforts have been made to catalog the cardiac extracellular matridome and analyze the topology of identified proteins for the design of therapeutic targets. Although many bioinformatics tools have been developed to predict protein topology, topology has been experimentally validated for only a very small portion of membrane proteins. The aim of this study was to use a glycoproteomics and MS approach to identify glycoproteins in the extracellular matridome of the infarcted left ventricle (LV) and provide experimental evidence for topological determination. EXPERIMENTAL DESIGN: Glycoproteomics analysis was performed on eight biological replicates of LV samples from wild-type mice at 7 days following myocardial infarction using SPE of glycopeptides, followed by mass spectrometric identification of N-linked glycosylation sites for topology assessment. RESULTS: We identified hundreds of glycoproteins, and the identified N-glycosylation sites provide novel information on the correct topology for membrane proteins present in the infarct setting. CONCLUSIONS AND CLINICAL RELEVANCE: Our data provide the foundation for future studies of the LV infarct extracellular matridome, which may facilitate the discovery of drug targets and biomarkers.