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.

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.

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

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.

Cardiac FGF23: new insights into the role and function of FGF23 after acute myocardial infarction.

OBJECTIVE: We aimed to elucidate the local role of FGF23 after myocardial infarction in a mouse model induced by left anterior descending artery (LAD) ligation. APPROACH AND RESULTS: (C57BL/6 N) mice underwent MI via LAD ligation and were sacrificed at different time-points post MI. The expression and influence of FGF23 on fibroblast and macrophages was also analyzed using isolated murine cells. We identified enhanced cardiac FGF23 mRNA expression in a time-dependent manner with an early increase, already on the first day after MI. FGF23 protein expression was abundantly detected in the infarcted area during the inflammatory phase. While described to be primarily produced in bone or macrophages, we identified cardiac fibroblasts as the only source of local FGF23 production after MI. Inflammatory mediators, such as IL-1ß, IL-6 and TNF-α, were able to induce FGF23 expression in these cardiac fibroblasts. Interestingly, we were not able to detect FGF23 at later time points after MI in mature scar tissue or remote myocardium, most likely due to TGF-ß1, which we have shown to inhibit the expression of FGF23. We identified FGFR1c to be the most abundant receptor for FGF23 in infarcted myocardium and cardiac macrophages and fibroblasts. FGF23 increased migration of cardiac fibroblast, as well as expression of Collagen 1, Periostin, Fibronectin and MMP8. FGF23 also increased expression of TGF-ß1 in M2 polarized macrophages. CONCLUSION: In conclusion, cardiac fibroblasts in the infarcted myocardium produce and express FGF23 as well as its respective receptors in a time-dependent manner, thus potentially influencing resident cell migration. The transitory local expression of FGF23 after MI points towards a complex role of FGF23 in myocardial ischemia and warrants further exploration, considering its role in ventricular remodeling.

Hemocompatibility of plasma electrolytic oxidation (PEO) coated Mg-RE and Mg-Zn-Ca alloys for vascular scaffold applications.

Percutaneous transluminal coronary angioplasty and subsequent vascular scaffold implantation remains the prevalent invasive treatment of coronary heart disease. In-stent restenosis remained a problem with bare metal stents, until drug-eluting stents were introduced. The inhibition of the healing process by the antimitotic drug coating and the permanent metallic remnant can promote sub-acute and delayed stent thrombosis. Thus, the development of biodegradable stents emerged as a subject of research. Magnesium-based bioabsorbable devices can provide sufficient radial force in the acute phase of vessel-treatment and degrade thoroughly in aqueous environment, making them potential new candidates for vascular scaffold applications. Magnesium alloys tend to degrade very quickly due to their high electrochemical corrosion potential. Plasma Electrolytic Oxidation modification of magnesium alloys improves interface and degradadation properties and may therefore enhance the performance and suitability for vascular scaffold applications of these materials. Assuring the hemocompatibility and foremost assessing the thrombogenicity of new biomaterials prior to their use is essential in order to avoid adverse effects. The goal was to assess thrombocyte adhesion on coated Mg-RE and Mg-Zn-Ca alloys. Static experiments with human blood were carried out on the plasma-electrolytically treated or corresponding untreated Mg alloy in order to assess quantity and quality of thrombocyte adhesion via standardized SEM imaging. In a second step, a parallel plate flow chamber was designed in order to examine thrombocyte adhesion under dynamic flow conditions. During flow chamber experiments the test-materials were exposed to human thrombocyte concentrate and the number of adherent thrombocytes was assessed. The flow chamber was additionally perfused with human blood and thrombocyte adhesion was semiquantitatively and qualitatively assessed via SEM imaging and subsequent scoring. In conclusion, a new parallel plate flow chamber design simulating blood-circulation was successfully established, enabling the further assessment of platelet adhesion on bioabsorbable materials under dynamic flow conditions. Static and dynamic experiments showed, that plasma-electrolytically treated specimens showed low thrombocyte adhesion on both alloys, proposing their potential use in vascular scaffolds. The uncoated magnesium alloys showed rapid degradation along with gas formation due to the chemically active surface and therefore give concern regarding their safety and suitability for vascular applications.

Blocking CCL5-CXCL4 heteromerization preserves heart function after myocardial infarction by attenuating leukocyte recruitment and NETosis.

Myocardial infarction (MI) is a major cause of death in Western countries and finding new strategies for its prevention and treatment is thus of high priority. In a previous study, we have demonstrated a pathophysiologic relevance for the heterophilic interaction of CCL5 and CXCL4 in the progression of atherosclerosis. A specifically designed compound (MKEY) to block this CCL5-CXCR4 interaction is investigated as a potential therapeutic in a model of myocardial ischemia/reperfusion (I/R) damage. 8 week-old male C57BL/6 mice were intravenously treated with MKEY or scrambled control (sMKEY) from 1 day before, until up to 7 days after I/R. By using echocardiography and intraventricular pressure measurements, MKEY treatment resulted in a significant decrease in infarction size and preserved heart function as compared to sMKEY-treated animals. Moreover, MKEY treatment significantly reduced the inflammatory reaction following I/R, as revealed by specific staining for neutrophils and monocyte/macrophages. Interestingly, MKEY treatment led to a significant reduction of citrullinated histone 3 in the infarcted tissue, showing that MKEY can prevent neutrophil extracellular trap formation in vivo. Disrupting chemokine heterodimers during myocardial I/R might have clinical benefits, preserving the therapeutic benefit of blocking specific chemokines, and in addition, reducing the inflammatory side effects maintaining normal immune defence.

Minimal invasive surgical procedure of inducing myocardial infarction in mice.

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

Identification of the Vasoconstriction-Inhibiting Factor (VIF), a Potent Endogenous Cofactor of Angiotensin II Acting on the Angiotensin II Type 2 Receptor.

BACKGROUND: The renin-angiotensin system and especially the angiotensin peptides play a central role in blood pressure regulation. Here, we hypothesize that an as-yet unknown peptide is involved in the action of angiotensin II modulating the vasoregulatory effects as a cofactor. METHODS AND RESULTS: The peptide with vasodilatory properties was isolated from adrenal glands chromatographically. The effects of this peptide were evaluated in vitro and in vivo, and the receptor affinity was analyzed. The plasma concentration in humans was quantified in patients with chronic kidney disease, patients with heart failure, and healthy control subjects. The amino acid sequence of the peptide from bovine adrenal glands was HSSYEDELSEVL EKPNDQAE PKEVTEEVSSKDAAE, which is a degradation product of chromogranin A. The sequence of the peptide isolated from human plasma was HSGFEDELSEVLENQSSQAELKEAVEEPSSKDVME. Both peptides diminished significantly the vasoconstrictive effect of angiotensin II in vitro. Therefore, we named the peptide vasoconstriction-inhibiting factor (VIF). The vasoregulatory effects of VIF are mediated by the angiotensin II type 2 receptor. VIF impairs angiotensin II-induced phosphorylation of the p38 mitogen-activated protein kinase pathway but not of extracellular-regulated kinase 1/2. The vasodilatory effects were confirmed in vivo. The plasma concentration was significantly increased in renal patients and patients with heart failure. CONCLUSIONS: VIF is a vasoregulatory peptide that modulates the vasoconstrictive effects of angiotensin II by acting on the angiotensin II type 2 receptor. It is likely that the increase in VIF may serve as a counterregulatory effect to defend against hypertension. The identification of this target may help us to understand the pathophysiology of renal and heart failure and may form a basis for the development of new strategies for the prevention and treatment of cardiovascular disease.

Blockade of CCR3 retains the neutrophils, preserving their survival during healing after myocardial infarction.

BACKGROUND: Chemokines are critical mediators in controlling and monitoring the healing and ventricular remodeling after myocardial infarction (MI). They proved to be valuable targets for therapeutic measures to reduce the scar formation and to preserve heart function in patients suffering MI. In the present study, the role of CCR3 in myocardial ischemia/reperfusion was established. METHODS AND RESULTS: One week after infarct induction in a mouse coronary ligation model, the functional and morphological parameters of the heart were analyzed. Isolated-heart Langendorff perfusion showed no significantly differences in heart function, infarction size and post infarction angiogenesis after CCR3 blockade. Apoptotic, proliferation signals as well as collagen synthesis were not affected in CCR3 antagonist treated mice. Notably, CCR3 inhibition was accompanied by massive neutrophil infiltration, while leaving the presence of other immune cell subsets in heart unaffected. CONCLUSION: Since neutrophils represents one of the most widely explored therapeutic targets in the treatment of cardiac disease, this study may open a new perspective for a better understanding of the physiology and homeostasis of neutrophils and points out new directions for intervention in acute MI.

Nano-level morphology of scar tissue after myocardial infarction.

Atomic force microscopy (AFM) is a pioneer imaging technique commonly employed by biological researchers in detection of the properties of biological membranes over the last decade. The AFM findings distinguish its applicability from the conventional methods, such as: confocal, multi-photons, electron microscopy, etc. as well as from the mechanical methods (compression and indentation test, extensiometry, etc.). With its high resolution (below 10 nm), AFM has emerged as a powerful tool in obtaining the nanostructural details and biomechanical properties of heart tissue. The composition of extracellular matrix is essential for heart compliance and its mechanical function. Here, we illustrate the surface morphology, its structural assembling and the mechanical properties of a myocardial infarction scar section aquired via AFM, in dry conditions. The cross section through the mature myocardial scar of mice after myocardial infarction shows that the embedded fibrils into the tissue matrix of a mature scar overlap at some sites, and form network-like structures. The nano-fibrils surface shows defined structural periodicity. A cross-section along the axial fibrilar direction gives an average D-periodic banding pattern of approximately 50,3 nm (± 6,2 nm std.). As future perspective, yet uncovered morphological and mechanical investigations, correlated with functional studies, open a new window for understanding pathological mechanisms.

Aging- and activation-induced platelet microparticles suppress apoptosis in monocytic cells and differentially signal to proinflammatory mediator release.

BACKGROUND: Platelet microparticles (PM) are the most abundant cell-derived microparticles in the blood, and accumulate in thrombo-inflammatory diseases. Platelets produce PM upon aging via an apoptosis-like process and by activation with strong agonists. We previously showed that long-term treatment of monocytic cells with apoptosis-induced PM (PMap) promotes their differentiation into resident macrophages. Here we investigated shorter term effects of various types of PM on monocyte signalling and function. METHODS AND RESULTS: Flow cytometry and scanning electron microscopy revealed that PM formed upon platelet aging (PMap) or ultra-sonication (PMsonic) expressed activated αIIbß3 integrins and tended to assemble into aggregates. In contrast, PM formed upon platelet activation with thrombin (PMthr) or Ca(2+) ionophore (PMiono) had mostly non-activated αIIbß3 and little aggregate formation, but had increased CD63 expression. PM from activated and sonicated platelets expressed phosphatidylserine at their surface, while only the latter were enriched in the receptors CD40L and CX3CR1. All PM types expressed P-selectin, interacted with monocytic cells via this receptor, and were internalised into these cells. The various PM types promoted actin cytoskeletal rearrangements and hydrogen peroxide production by monocytic cells. Markedly, both aging- and activation-induced PM types stimulated the phosphoinositide 3-kinase/Akt pathway, suppressing apoptosis induced by several agonists, in a P-selectin-dependent manner. On the other hand, the PM types differentially influenced monocyte signalling in eliciting Ca(2+) fluxes (particularly PMap) and in releasing secondary mediators (complement factor C5a with PMap, and pro-inflammatory tumour necrosis factor-α with PMthr). CONCLUSIONS: In spite of their common anti-apoptotic potential via Akt activation, aging- and activation-induced PM cause different Ca(2+) signalling events and mediator release in monocytic cells. By implication, aging and activated platelets may modulate monocyte function in different way by the shedding of different PM types.