Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Myocardial-Treg Crosstalk: How to Tame a Wolf.

The immune system plays a vital role in maintaining tissue integrity and organismal homeostasis. The sudden stress caused by myocardial infarction (MI) poses a significant challenge for the immune system: it must quickly substitute dead myocardial with fibrotic tissue while controlling overt inflammatory responses. In this review, we will discuss the central role of myocardial regulatory T-cells (Tregs) in orchestrating tissue repair processes and controlling local inflammation in the context of MI. We herein compile recent advances enabled by the use of transgenic mouse models with defined cardiac antigen specificity, explore whole-heart imaging techniques, outline clinical studies and summarize deep-phenotyping conducted by independent labs using single-cell transcriptomics and T-cell repertoire analysis. Furthermore, we point to multiple mechanisms and cell types targeted by Tregs in the infarcted heart, ranging from pro-fibrotic responses in mesenchymal cells to local immune modulation in myeloid and lymphoid lineages. We also discuss how both cardiac-specific and polyclonal Tregs participate in MI repair. In addition, we consider intriguing novel evidence on how the myocardial milieu takes control of potentially auto-aggressive local immune reactions by shaping myosin-specific T-cell development towards a regulatory phenotype. Finally, we examine the potential use of Treg manipulating drugs in the clinic after MI.

The Fractalkine Receptor CX3CR1 Links Lymphocyte Kinetics in CMV-Seropositive Patients and Acute Myocardial Infarction With Adverse Left Ventricular Remodeling.

Aims: Latent cytomegalovirus (CMV) infection is associated with adverse cardiovascular outcomes. Virus-specific CX3CR1+ effector memory T-cells may be instrumental in this process due to their pro-inflammatory properties. We investigated the role of CX3CR1 (fractalkine receptor) in CMV-related lymphocyte kinetics and cardiac remodeling in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (pPCI). Methods and Results: We retrospectively analysed lymphocyte count, troponin, and survival in 4874 STEMI/pPCI patients, evaluated lymphocyte kinetics during reperfusion in a prospective cohort, and obtained sequential cardiac MRI (cMRI) to assess remodeling. Pre-reperfusion lymphopenia independently predicted mortality at 7.5 years. Prior to reperfusion, CCR7+ T-lymphocytes appeared to be depleted. After reperfusion, T-lymphocytes expressing CX3CR1 were depleted predominantly in CMV-seropositive patients. During ischaemia/reperfusion, a drop in CX3CR1+ T-lymphocytes was significantly linked with microvascular obstruction in CMV+ patients, suggesting increased fractalkine-receptor interaction. At 12 weeks, CMV+ patients displayed adverse LV remodeling. Conclusion: We show that lymphopenia occurs before and after reperfusion in STEMI by different mechanisms and predicts long-term outcome. In CMV+ patients, increased fractalkine induction and sequestration of CX3CR1+ T-cells may contribute to adverse remodeling, suggesting a pro-inflammatory pathomechanism which presents a novel therapeutic target.

Terminally Differentiated CD4+ T Cells Promote Myocardial Inflammaging.

The cardiovascular and immune systems undergo profound and intertwined alterations with aging. Recent studies have reported that an accumulation of memory and terminally differentiated T cells in elderly subjects can fuel myocardial aging and boost the progression of heart diseases. Nevertheless, it remains unclear whether the immunological senescence profile is sufficient to cause age-related cardiac deterioration or merely acts as an amplifier of previous tissue-intrinsic damage. Herein, we sought to decompose the causality in this cardio-immune crosstalk by studying young mice harboring a senescent-like expanded CD4+ T cell compartment. Thus, immunodeficient NSG-DR1 mice expressing HLA-DRB1*01:01 were transplanted with human CD4+ T cells purified from matching donors that rapidly engrafted and expanded in the recipients without causing xenograft reactions. In the donor subjects, the CD4+ T cell compartment was primarily composed of naïve cells defined as CCR7+CD45RO-. However, when transplanted into young lymphocyte-deficient mice, CD4+ T cells underwent homeostatic expansion, upregulated expression of PD-1 receptor and strongly shifted towards effector/memory (CCR7- CD45RO+) and terminally-differentiated phenotypes (CCR7-CD45RO-), as typically seen in elderly. Differentiated CD4+ T cells also infiltrated the myocardium of recipient mice at comparable levels to what is observed during physiological aging. In addition, young mice harboring an expanded CD4+ T cell compartment showed increased numbers of infiltrating monocytes, macrophages and dendritic cells in the heart. Bulk mRNA sequencing analyses further confirmed that expanding T-cells promote myocardial inflammaging, marked by a distinct age-related transcriptomic signature. Altogether, these data indicate that exaggerated CD4+ T-cell expansion and differentiation, a hallmark of the aging immune system, is sufficient to promote myocardial alterations compatible with inflammaging in juvenile healthy mice.

Angiotensin II receptor type 1 blockade improves hyporesponsiveness to vasopressors in septic shock.

Sepsis activates the renin-angiotensin system and the production of angiotensin II, which has a key role in the regulation of blood pressure through AT1 receptors. However, excessive activation of AT1 receptor is associated with deleterious effects. We investigated the consequences of a differential blockade of AT1 receptor caused by two doses of losartan (0.25 mg/kg or 15 mg/kg, s.c), a selective AT1 receptor antagonist on sepsis outcome. These doses reduced the effect of angiotensin II in normal rats by 30% and >90% 8 h after administration, respectively, but only the higher dose maintained its inhibitory effect (~70%) 24 h after injection. Sepsis was induced by cecal ligation and puncture (CLP). Losartan was injected 2 h after CLP and parameters were evaluated 6 and 24 h after CLP. Septic rats developed hypotension and hyporesponsiveness to vasoconstrictors, an intense inflammatory process and increase in plasma markers of organ dysfunction. The lower dose of losartan improved the vasoconstrictive response to phenylephrine and angiotensin II, reduced lung myeloperoxidase and prevented leukopenia 24 h after CLP, but it did not reduce NOS-2 expression, plasma IL-6 levels or organ injury parameters of septic rats. On the other hand, the higher dose of losartan worsened the response to vasoconstrictors, potentiated the hypotension and increased further levels of creatine, urea and lactate in septic rats. Therefore, an early and partial blockade of AT1 receptor with a low dose of losartan may counteract sepsis-induced refractoriness to vasoconstrictors thus providing an opportunity to improve the outcome of this condition.

Pleiotropic cardiac functions controlled by ischemia-induced lncRNA H19.

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation.

The apoptosis clearance signal phosphatidylserine inhibits leukocyte migration and promotes inflammation resolution in vivo.

Previous studies have shown that phagocytosis of apoptotic cells can tune the macrophage phenotype and trigger the resolution of inflammation. This mechanism is largely dependent on the recognition of phosphatidylserine (PS) residues on the outer membrane of dying cells. Therefore, we sought to assess the effects of PS-containing liposomes (mimics of apoptotic cells) on the leukocyte migration profile during the inflammatory process in vivo. Inflammation was induced by carrageenan injection into air pouches created on the dorsal region of mice, as this model enables convenient access to the exudates for further investigation. Mice were treated with PBS, PS-containing or phosphatidylcholine (PC)-containing liposomes (10, 30 or 100 mg/kg intraperitoneally [i.p.]). Starting 8 h after carrageenan injection, the level of leukocyte infiltration was monitored over three days. The PS-containing, but not PC-containing, liposomes reduced the polymorphonuclear (PMN) and mononuclear (MN) leukocyte influx into the inflamed pouches in a dose-dependent fashion. Most notably, these effects could also be adoptively transferred; that is, they were also found in mice injected with a liposome-free peritoneal lavage obtained from the mice that had received the intraperitoneal PS-liposome treatment. The effect of treatment with the PS-induced soluble mediators (PS-ISMs) was found to be dependent on the presence of peritoneal macrophages and was susceptible to heat, trypsin degradation, and cycloheximide treatment. The PS-containing liposomes promoted the reduction of PMN leukocyte influx by triggering the release of anti-inflammatory autacoids with a proteinaceous nature that were produced de novo after PS exposure.

Myocardial infarction triggers cardioprotective antigen-specific T helper cell responses.

T cell autoreactivity is a hallmark of autoimmune diseases but can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of myocardial infarction (MI), the leading cause of death worldwide. After screening more than 150 class-II-restricted epitopes, we found that myosin heavy chain alpha (MYHCA) was a dominant cardiac antigen triggering post-MI CD4+ T cell activation in mice. Transferred MYHCA614-629-specific CD4+ T (TCR-M) cells selectively accumulated in the myocardium and mediastinal lymph nodes (med-LN) of infarcted mice, acquired a Treg phenotype with a distinct pro-healing gene expression profile, and mediated cardioprotection. Myocardial Treg cells were also detected in autopsies from patients who suffered a MI. Noninvasive PET/CT imaging using a CXCR4 radioligand revealed enlarged med-LNs with increased cellularity in MI-patients. Notably, the med-LN alterations observed in MI patients correlated with the infarct size and cardiac function. Taken together, the results obtained in our study provide evidence showing that MI-context induces pro-healing T cell autoimmunity in mice and confirms the existence of an analogous heart/med-LN/T cell axis in MI patients.

Lymphocytes at the Heart of Wound Healing.

The adult mammalian heart displays negligible regenerative capacity. Therefore, myocardial infarction (MI) often results in an irreversible loss of contractile tissue, leading to a collagenous scar formation, progressive remodelling and heart failure (HF). Over the past few years, emerging evidences indicate that a myocardial ischemic injury mobilizes not only sterile unspecific inflammation but also lymphocyte-mediated immune responses to cardiac auto-antigens. In the current chapter, we depict the infarcted heart as a "wounded" tissue and focus on the dynamic events leading to myocardial repair, with special emphasis on the role played by lymphocytes in this process.

Myocardial aging as a T-cell-mediated phenomenon.

In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4+ Foxp3- (forkhead box P3) IFN-γ+ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.

The autoimmune nature of post-infarct myocardial healing: oral tolerance to cardiac antigens as a novel strategy to improve cardiac healing.

Following a myocardial infarction, lymphocytes have been suggested to react with the damaged heart tissue, which can impair proper tissue healing. In the present work, we investigate whether ingestion of a myocardial homogenate and the consequent development of immunological tolerance can modify the course of post-infarction myocardial repair. Infarction-like myocardial lesions were induced in Wistar rats by injecting high doses of isoproterenol. The healing process was evaluated morphologically and functionally for 60 days. Cardiac function was evaluated using isolated and perfused heart (Langendorff) preparations. At day 14 after isoproterenol treatment, lymphocytes from the mediastinal lymph nodes proliferated when exposed in vitro to myocardial homogenate. Moreover, TNF-α, IFN-γ and CCL-5, but not FOXP3+ expression, was increased in draining lymph nodes in isoproterenol-injured animals, indicating that the observed lymphocyte population that proliferated in response to cardiac components presented a pro-inflammatory and pro-fibrotic profile. In contrast, lymphocytes from draining lymph nodes of rats given a heart homogenate by gavage 7 days before isoproterenol did not proliferate. Furthermore, the group rendered tolerant expressed cardiac FOXP3+ earlier than did the control group, and showed a milder inflammatory infiltrate, lower MMP-9 expression, less collagen deposition, and improved cardiac performance when compared to animals that received only isoproterenol administration. The present findings suggest that the establishment of oral tolerance to heart components prior to myocardial infarction may drive the cardiac healing process to proceed with less inflammation and fibrosis, thus preserving contractile organ function.

Inflammation as an animal development phenomenon.

Inflammation is a term that has been used throughout history in different contexts; it may represent a simple collection of clinical symptoms for which drugs are developed, a disease mechanism, or even a defense mechanism against microbes validating Pasteur's studies on bacteriology and Darwin's proposed struggle for survival. Thus, an explanation of this term must also consider the scientific questions addressed. In this study, I propose that several of the inflammatory events typically described in immunological, pathological, and pharmacological contexts can also be perceived as mechanisms of animal development. Thus, by recognizing that the generation of an animal form, its conservation, and its regeneration after tissue damage are phenomena of the same nature, inflammation can be addressed through the approach of developmental biology, thereby acquiring a much neglected physiological counterpart.