Vitamin C as Scavenger of Reactive Oxygen Species during Healing after Myocardial Infarction.

Currently, coronary artery bypass and reperfusion therapies are considered the gold standard in long-term treatments to restore heart function after acute myocardial infarction. As a drawback of these restoring strategies, reperfusion after an ischemic insult and sudden oxygen exposure lead to the exacerbated synthesis of additional reactive oxidative species and the persistence of increased oxidation levels. Attempts based on antioxidant treatment have failed to achieve an effective therapy for cardiovascular disease patients. The controversial use of vitamin C as an antioxidant in clinical practice is comprehensively systematized and discussed in this review. The dose-dependent adsorption and release kinetics mechanism of vitamin C is complex; however, this review may provide a holistic perspective on its potential as a preventive supplement and/or for combined precise and targeted therapeutics in cardiovascular management therapy.

Engagement of the CXCL12-CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis.

Endothelial progenitor cells (EPCs) are involved in vascular repair and modulate properties of smooth muscle cells (SMCs) relevant for their contribution to neointima formation following injury. Considering the relevant role of the CXCL12-CXCR4 axis in vascular homeostasis and the potential of EPCs and SMCs to release CXCL12 and express CXCR4, we analyzed the engagement of the CXCL12-CXCR4 axis in various modes of EPC-SMC interaction relevant for injury- and lipid-induced atherosclerosis. We now demonstrate that the expression and release of CXCL12 is synergistically increased in a CXCR4-dependent mechanism following EPC-SMC interaction during co-cultivation or in response to recombinant CXCL12, thus establishing an amplifying feedback loop Additionally, mechanical injury of SMCs induces increased release of CXCL12, resulting in enhanced CXCR4-dependent recruitment of EPCs to SMCs. The CXCL12-CXCR4 axis is crucially engaged in the EPC-triggered augmentation of SMC migration and the attenuation of SMC apoptosis but not in the EPC-mediated increase in SMC proliferation. Compared to EPCs alone, the alliance of EPC-SMC is superior in promoting the CXCR4-dependent proliferation and migration of endothelial cells. When direct cell-cell contact is established, EPCs protect the contractile phenotype of SMCs via CXCL12-CXCR4 and reverse cholesterol-induced transdifferentiation toward a synthetic, macrophage-like phenotype. In conclusion we show that the interaction of EPCs and SMCs unleashes a CXCL12-CXCR4-based autoregulatory feedback loop promoting regenerative processes and mediating SMC phenotype control to potentially guard vascular homeostasis.

Endogenous Modulation of Extracellular Matrix Collagen during Scar Formation after Myocardial Infarction.

Myocardial infarction is remains the leading cause of death in developed countries. Recent data show that the composition of the extracellular matrix might differ despite similar heart function and infarction sizes. Because collagen is the main component of the extracellular matrix, we hypothesized that changes in inflammatory cell recruitment influence the synthesis of different collagen subtypes in myofibroblasts, thus changing the composition of the scar. We found that neutrophils sustain the proliferation of fibroblasts, remodeling, differentiation, migration and inflammation, predominantly by IL-1 and PPARγ pathways (n = 3). They also significantly inhibit the mRNA expression of fibrillar collagen, maintaining a reduced stiffness in isolated myofibroblasts (n = 4-5). Reducing the neutrophil infiltration in CCR1-/- resulted in increased mRNA expression of collagen 11, moderate expression of collagen 19 and low expression of collagen 13 and 26 in the scar 4 weeks post infarction compared with other groups (n = 3). Mononuclear cells increased the synthesis of all collagen subtypes and upregulated the NF-kB, angiotensin II and PPARδ pathways (n = 3). They increased the synthesis of collagen subtypes 1, 3, 5, 16 and 23 but reduced the expression of collagens 5 and 16 (n = 3). CCR2-/- scar tissue showed higher levels of collagen 13 (n = 3), in association with a significant reduction in stiffness (n = 4-5). Upregulation of the inflammation-related genes in myofibroblasts mostly modulated the fibrillar collagen subtypes, with less effect on the FACIT, network-forming and globular subtypes (n = 3). The upregulation of proliferation and differentiation genes in myofibroblasts seemed to be associated only with the fibrillar collagen subtype, whereas angiogenesis-related genes are associated with fibrillar, network-forming and multiplexin subtypes. In conclusion, although we intend for our findings to deepen the understanding of the mechanism of healing after myocardial infarction and scar formation, the process of collagen synthesis is highly complex, and further intensive investigation is needed to put together all the missing puzzle pieces in this still incipient knowledge process.

miR155 Deficiency Reduces Myofibroblast Density but Fails to Improve Cardiac Function after Myocardial Infarction in Dyslipidemic Mouse Model.

Myocardial infarction remains the most common cause of heart failure with adverse remodeling. MicroRNA (miR)155 is upregulated following myocardial infarction and represents a relevant regulatory factor for cardiac remodeling by engagement in cardiac inflammation, fibrosis and cardiomyocyte hypertrophy. Here, we investigated the role of miR155 in cardiac remodeling and dysfunction following myocardial infarction in a dyslipidemic mouse model. Myocardial infarction was induced in dyslipidemic apolipoprotein E-deficient (ApoE-/-) mice with and without additional miR155 knockout by ligation of the LAD. Four weeks later, echocardiography was performed to assess left ventricular (LV) dimensions and function, and mice were subsequently sacrificed for histological analysis. Echocardiography revealed no difference in LV ejection fractions, LV mass and LV volumes between ApoE-/- and ApoE-/-/miR155-/- mice. Histology confirmed comparable infarction size and unaltered neoangiogenesis in the myocardial scar. Notably, myofibroblast density was significantly decreased in ApoE-/-/miR155-/- mice compared to the control, but no difference was observed for total collagen deposition. Our findings reveal that genetic depletion of miR155 in a dyslipidemic mouse model of myocardial infarction does not reduce infarction size and consecutive heart failure but does decrease myofibroblast density in the post-ischemic scar.

Apolipoprotein E4 Is Associated with Right Ventricular Dysfunction in Dilated Cardiomyopathy-An Animal and In-Human Comparative Study.

ApoE abnormality represents a well-known risk factor for cardiovascular diseases. Beyond its role in lipid metabolism, novel studies demonstrate a complex involvement of apoE in membrane homeostasis and signaling as well as in nuclear transcription. Due to the large spread of apoE isoforms in the human population, there is a need to understand the apoE's role in pathological processes. Our study aims to dissect the involvement of apoE in heart failure. We showed that apoE-deficient rats present multiple organ damages (kidney, liver, lung and spleen) besides the known predisposition for obesity and affected lipid metabolism (two-fold increase in tissular damages in liver and one-fold increase in kidney, lung and spleen). Heart tissue also showed significant morphological changes in apoE-/- rats, mostly after a high-fat diet. Interestingly, the right ventricle of apoE-/- rats fed a high-fat diet showed more damage and affected collagen content (~60% less total collagen content and double increase in collagen1/collagen3 ratio) compared with the left ventricle (no significant differences in total collagen content or collagen1/collagen3 ratio). In patients, we were able to find a correlation between the presence of ε4 allele and cardiomyopathy (χ2 = 10.244; p = 0.001), but also with right ventricle dysfunction with decreased TAPSE (15.3 ± 2.63 mm in ε4-allele-presenting patients vs. 19.8 ± 3.58 mm if the ε4 allele is absent, p < 0.0001*) and increased in systolic pulmonary artery pressure (50.44 ± 16.47 mmHg in ε4-allele-presenting patients vs. 40.68 ± 15.94 mmHg if the ε4 allele is absent, p = 0.0019). Our results confirm that the presence of the ε4 allele is a lipid-metabolism-independent risk factor for heart failure. Moreover, we show for the first time that the presence of the ε4 allele is associated with right ventricle dysfunction, implying different regulatory mechanisms of fibroblasts and the extracellular matrix in both ventricles. This is essential to be considered and thoroughly investigated before the design of therapeutical strategies for patients with heart failure.

Phosphatidylserine Supplementation as a Novel Strategy for Reducing Myocardial Infarct Size and Preventing Adverse Left Ventricular Remodeling.

Phosphatidylserines are known to sustain skeletal muscle activity during intense activity or hypoxic conditions, as well as preserve neurocognitive function in older patients. Our previous studies pointed out a potential cardioprotective role of phosphatidylserine in heart ischemia. Therefore, we investigated the effects of phosphatidylserine oral supplementation in a mouse model of acute myocardial infarction (AMI). We found out that phosphatidylserine increases, significantly, the cardiomyocyte survival by 50% in an acute model of myocardial ischemia-reperfusion. Similar, phosphatidylserine reduced significantly the infarcted size by 30% and improved heart function by 25% in a chronic model of AMI. The main responsible mechanism seems to be up-regulation of protein kinase C epsilon (PKC-ε), the main player of cardio-protection during pre-conditioning. Interestingly, if the phosphatidylserine supplementation is started before induction of AMI, but not after, it selectively inhibits neutrophil's activation, such as Interleukin 1 beta (IL-1ß) expression, without affecting the healing and fibrosis. Thus, phosphatidylserine supplementation may represent a simple way to activate a pre-conditioning mechanism and may be a promising novel strategy to reduce infarct size following AMI and to prevent myocardial injury during myocardial infarction or cardiac surgery. Due to the minimal adverse effects, further investigation in large animals or in human are soon possible to establish the exact role of phosphatidylserine in cardiac diseases.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Neutrophils Modulate Fibroblast Function and Promote Healing and Scar Formation after Murine Myocardial Infarction.

AIM: Recruitment of neutrophils to the heart following acute myocardial infarction (MI) initiates inflammation and contributes to adverse post-infarct left ventricular (LV) remodeling. However, therapeutic inhibition of neutrophil recruitment into the infarct zone has not been beneficial in MI patients, suggesting a possible dual role for neutrophils in inflammation and repair following MI. Here, we investigate the effect of neutrophils on cardiac fibroblast function following MI. Methods and Results: We found that co-incubating neutrophils with isolated cardiac fibroblasts enhanced the production of provisional extracellular matrix proteins and reduced collagen synthesis when compared to control or co-incubation with mononuclear cells. Furthermore, we showed that neutrophils are required to induce the transient up-regulation of transforming growth factor (TGF)-ß1 expression in fibroblasts, a key requirement for terminating the pro-inflammatory phase and allowing the reparatory phase to form a mature scar after MI. Conclusion: Neutrophils are essential for both initiation and termination of inflammatory events that control and modulate the healing process after MI. Therefore, one should exercise caution when testing therapeutic strategies to inhibit neutrophil recruitment into the infarct zone in MI patients.

Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial Dysregulation.

OBJECTIVE: The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. APPROACH AND RESULTS: Flow-dependent endothelial dysfunction was induced in apolipoprotein E-deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A-targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [P<0.001] and ≈5× [P<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A-targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side. CONCLUSIONS: Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A-targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.

Cardiomyocyte Hypertrophy in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling.