Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival.

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

Macrophage-based therapeutic approaches for cardiovascular diseases.

Despite the advances in treatment options, cardiovascular disease (CVDs) remains the leading cause of death over the world. Chronic inflammatory response and irreversible fibrosis are the main underlying pathophysiological causes of progression of CVDs. In recent decades, cardiac macrophages have been recognized as main regulatory players in the development of these complex pathophysiological conditions. Numerous approaches aimed at macrophages have been devised, leading to novel prospects for therapeutic interventions. Our review covers the advancements in macrophage-centric treatment plans for various pathologic conditions and examines the potential consequences and obstacles of employing macrophage-targeted techniques in cardiac diseases.

Radiotherapy-induced Immune Response Enhanced by Selective HDAC6 Inhibition.

Radiotherapy is a curative cancer treatment modality that imparts damage to cellular DNA, induces immunogenic cell death, and activates antitumor immunity. Despite the radiotherapy-induced direct antitumor effect seen within the treated volume, accumulating evidence indicates activation of innate antitumor immunity. Acute proinflammatory responses mediated by anticancer M1 macrophages are observed in the immediate aftermath following radiotherapy. However, after a few days, these M1 macrophages are converted to anti-inflammatory and pro-cancer M2 phenotype, leading to cancer resistance and underlying potential tumor relapse. Histone deacetylase 6 (HDAC6) plays a crucial role in regulating macrophage polarization and innate immune responses. Here, we report targeting HDAC6 function with a novel selective inhibitor (SP-2-225) as a potential therapeutic candidate for combination therapy with radiotherapy. This resulted in decreased tumor growth and enhanced M1/M2 ratio of infiltrating macrophages within tumors. These observations support the use of selective HDAC6 inhibitors to improve antitumor immune responses and prevent tumor relapse after radiotherapy.

Deciphering Pro-angiogenic Transcription Factor Profiles in Hypoxic Human Endothelial Cells by Combined Bioinformatics and in vitro Modeling.

Background: Constant supply of oxygen is crucial for multicellular tissue homeostasis and energy metabolism in cardiac tissue. As a first response to acute hypoxia, endothelial cells (ECs) promote recruitment and adherence of immune cells to the dysbalanced EC barrier by releasing inflammatory mediators and growth factors, whereas chronic hypoxia leads to the activation of a transcription factor (TF) battery, that potently induces expression of growth factors and cytokines including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). We report a hypoxia-minded, targeted bioinformatics approach aiming to identify and validate TFs that regulate angiogenic signaling. Results: A comprehensive RNA-Seq dataset derived from human ECs subjected to normoxic or hypoxic conditions was selected to identify significantly regulated genes based on (i) fold change (normoxia vs. hypoxia) and (ii) relative abundancy. Transcriptional regulation of this gene set was confirmed via qPCR in validation experiments where HUVECs were subjected to hypoxic conditions for 24 h. Screening the promoter and upstream regulatory elements of these genes identified two TFs, KLF5 and SP1, both with a potential binding site within these regions of selected target genes. In vitro, siRNA experiments confirmed SP1- and KLF5-mediated regulation of identified hypoxia-sensitive endothelial genes. Next to angiogenic signaling, we also validated the impact of TFs on inflammatory signaling, both key events in hypoxic sensing. Both TFs impacted on inflammatory signaling since endogenous repression led to increased NF-κB signaling. Additionally, SP1 silencing eventuated decreased angiogenic properties in terms of proliferation and tube formation. Conclusion: By detailed in silico analysis of promoter region and upstream regulatory elements for a list of hypoxia-sensitive genes, our bioinformatics approach identified putative binding sites for TFs of SP or KLF family in vitro. This strategy helped to identify TFs functionally involved in human angiogenic signaling and therefore serves as a base for identifying novel RNA-based drug entities in a therapeutic setting of vascularization.

Faster skin wound healing predicts survival after myocardial infarction.

Both skin wound healing and the cardiac response to myocardial infarction (MI) progress through similar pathways involving inflammation, resolution, tissue repair, and scar formation. Due to the similarities, we hypothesized that the healing response to skin wounding would predict future response to MI. Mice were given a 3-mm skin wound using a disposable biopsy punch and the skin wound was imaged daily until closure. The same set of animals was given MI by permanent coronary artery ligation 28 days later and followed for 7 days. Cardiac physiology was measured by echocardiography at baseline and MI days 3 and 7. Animals that survived until day 7 were grouped as survivors, and animals that died from MI were grouped as nonsurvivors. Survivors had faster skin wound healing than nonsurvivors. Faster skin wound healing predicted MI survival better than commonly used cardiac functional variables (e.g., infarct size, fractional shortening, and end diastolic dimension). N-glycoproteome profiling of MI day 3 plasma revealed α2-macroglobulin and ELL-associated factor 1 as strong predictors of future MI death and progression to heart failure. A second cohort of MI mice validated these findings. To investigate the clinical relevance of α2-macroglobulin, we mapped the plasma glycoproteome in patients with MI 48 h after admission and in healthy controls. In patients, α2-macroglobulin was increased 48 h after MI. Apolipoprotein D, another plasma glycoprotein, detrimentally regulated both skin and cardiac wound healing in male but not female mice by promoting inflammation. Our results reveal that the skin is a mirror to the heart and common pathways link wound healing across organs.NEW & NOTEWORTHY Faster skin wound healers had more efficient cardiac healing after myocardial infarction (MI). Two plasma proteins at D3 MI, EAF1 and A2M, predicted MI death in 66% of cases. ApoD regulated both skin and cardiac wound healing in male mice by promoting inflammation. The skin was a mirror to the heart and common pathways linked wound healing across organs.

Novel Paired Normal Prostate and Prostate Cancer Model Cell Systems Derived from African American Patients.

Prostate cancer is the most frequently diagnosed solid malignancy in men. African American (AA) men are at greater risk for developing prostate cancer, and experience higher mortality rates, as compared with Caucasian American men. However, mechanistic studies to understand this health disparity have been limited by the lack of relevant in vitro and in vivo models. There is an urgent need for preclinical cellular models to investigate molecular mechanisms underlying prostate cancer in AA men. We collected clinical specimens from radical prostatectomies of AA patients and established 10 paired tumor-derived and normal epithelial cell cultures from the same donors, which were further cultivated to extend the growth under "conditional reprogramming." Clinical and cellular annotations characterized these model cells as intermediate risk and predominantly diploid. Immunocytochemical analyses demonstrated variable expression levels of luminal (CK8) and basal (CK5, p63) markers in both normal and tumor cells. However, expression levels of TOPK, c-MYC, and N-MYC were markedly increased only in tumor cells. To determine cell utility for drug testing, we examined viability of cells following exposure to the antiandrogen (bicalutamide) and two PARP inhibitors (olaparib and niraparib) and observed decreased viability of tumor-derived cells as compared with viability of normal prostate-derived cells. Significance: Cells derived from prostatectomies of AA patients conferred a bimodal cellular phenotype, recapitulating clinical prostate cellular complexity in this model cell system. Comparisons of viability responses of tumor derived to normal epithelial cells offer the potential for screening therapeutic drugs. Therefore, these paired prostate epithelial cell cultures provide an in vitro model system suitable for studies of molecular mechanisms in health disparities.

18Oxygen Substituted Nucleosides Combined with Proton Beam Therapy: Therapeutic Transmutation In Vitro.

PURPOSE: Proton therapy precisely delivers radiation to cancers to cause damaging strand breaks to cellular DNA, kill malignant cells, and stop tumor growth. Therapeutic protons also generate short-lived activated nuclei of carbon, oxygen, and nitrogen atoms in patients as a result of atomic transmutations that are imaged by positron emission tomography (PET). We hypothesized that the transition of 18O to 18F in an 18O-substituted nucleoside irradiated with therapeutic protons may result in the potential for combined diagnosis and treatment for cancer with proton therapy. MATERIALS AND METHODS: Reported here is a feasibility study with a therapeutic proton beam used to irradiate H2 18O to a dose of 10 Gy produced by an 85 MeV pristine Bragg peak. PET imaging initiated >45 minutes later showed an 18F decay signal with T1/2 of ∼111 minutes. RESULTS: The 18O to 18F transmutation effect on cell survival was tested by exposing SQ20B squamous carcinoma cells to physiologic 18O-thymidine concentrations of 5 µM for 48 hours followed by 1- to 9-Gy graded doses of proton radiation given 24 hours later. Survival analyses show radiation sensitization with a dose modification factor (DMF) of 1.2. CONCLUSIONS: These data support the idea of therapeutic transmutation in vitro as a biochemical consequence of proton activation of 18O to 18F in substituted thymidine enabling proton radiation enhancement in a cancer cell. 18O-substituted molecules that incorporate into cancer targets may hold promise for improving the therapeutic window of protons and can be evaluated further for postproton therapy PET imaging.

Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology.

BACKGROUND: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. METHODS AND RESULTS: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. CONCLUSION: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.

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.

MiRNA-181a is a novel regulator of aldosterone-mineralocorticoid receptor-mediated cardiac remodelling.

AIM: The aldosterone-mineralocorticoid receptor (Aldo-MR) pathway is activated during cardiac stress, such as hypertension, myocardial infarction (MI), and heart failure. The importance of Aldo and MR in the pathogenesis of cardiac diseases is well established; however, the regulatory mechanisms behind Aldo/MR-induced cardiac remodelling remain uncertain. We here investigated potential miRNA-mediated regulation of the Aldo-MR pathway to improve mechanistic understanding. METHODS AND RESULTS: High-throughput screening of 2,555 miRNAs using an MR responsive stable cardiomyocyte cell line (MMTV-GFP-HL-1) identified miR-181a as a potential regulator of Aldo-MR pathway. MiR-181a was found to downregulate the expression of Ngal (lipocalin-2), a well-established downstream effector molecule of Aldo-MR. In addition, Aldo-induced cellular hypertrophy decreased significantly upon miR-181a overexpression. Genetic miR-181 knockout in murine MI model led to deteriorated cardiac function, cardiac remodelling, and activation of Aldo-MR pathway while AAV9-mediated miR-181a overexpression improved cardiac function and deactivated Aldo-MR pathway proving a cardio-protective role of miR-181a. Global RNA sequencing of cells under Aldo treatment with/without miR-181a overexpression identified potential miR-181a targets functionally contributing to Aldo-MR pathway. Adamts1, a direct target of miR-181a, was found to be downregulated with miR-181a overexpression and upregulated with inhibition. Similar to miR-181a overexpression, siRNA-mediated inhibition of Adamts1 inhibited Aldo-MR pathway. CONCLUSION: We here show that miR-181a is a novel regulator of the Aldo-MR pathway regulating the levels of Ngal via direct targeting of Adamts1. This new insight establishes miR-181a as a factor of immense value participating in downstream networks of Aldo-MR pathway. Our in vivo studies further confirmed miR-181a as cardio-protective under MI stress. Thus, miR-181a's involvement in Aldo-MR-mediated cardiac remodelling confers it with tremendous potential to be developed further as a new therapeutic target.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Systematic Review with Network Meta-Analysis: Comparative Efficacy and Safety of Combination Therapy with Angiotensin II Receptor Blockers and Amlodipine in Asian Hypertensive Patients.

BACKGROUND: Hypertension (HTN) is the leading risk factor for cardiovascular mortality globally. The WHO estimates a 60% increase in Asian HTN patients between 2000 and 2025. Numerous studies have compared safety and efficacy between antihypertensive classes, but in-class comparisons of angiotensin II receptor blockers (ARBs) in combination therapy (CT) (fixed-dose combination or dual combination) with a calcium channel blocker (CCB) are lacking in Asia. OBJECTIVE: To compare the efficacy and safety of the various ARB-amlodipine CTs and amlodipine (AML) monotherapy for treatment of HTN in Asian population. METHODS: A systematic literature review sourced Asian randomized controlled trials (RCTs) from PubMed and Cochrane Libraries to inform a network meta-analysis (NMA). We considered the ARB-AML CT. The primary efficacy and safety endpoints were short-term (8-12 weeks) treatment response and treatment-emergent adverse events (TEAEs), respectively. AML monotherapy was used as a comparator to allow for indirect treatment effect estimation in the absence of direct RCTs evidence comparing the different ARB-AML CTs. RESULTS: The analysis included 1198 Asian HTN patients from seven studies involving six ARB-AML CTs: azilsartan (AZL), candesartan (CAN), fimasartan (FIM), losartan (LOS), olmesartan (OLM), and telmisartan (TEL). Compared to AML monotherapy, CT of AZL-AML had five times greater odds of prompting a treatment response (OR 5.2, 95% CI: 2.5, 11.2), while CAN-AML had 3.9 (95% CI: 2.5, 6.4), FIM-AML had 3.4 (95% CI: 1.4, 8.5), TEL-AML had 3.3 (95% CI: 1.6, 7.1), OLM-AML had 2.7 (95% CI: 1.6, 5.0), and LOS-AML had 2.0 (95% CI: 0.6, 7.3). All ARB-AML CTs had safety profiles comparable to AML monotherapy except TEL-AML, which had significantly lower odds of TEAEs (0.26 (95% CI: 0.087, 0.70)). CONCLUSION: This study suggests that all ARB-AML CTs compared favorably to AML monotherapy regarding short-term treatment response in uncomplicated HTN patients of Asian origin. AZL-AML prompted the most favorable treatment response. Safety profiles among the ARB-AML CTs were largely comparable. Due to the limited study size and small number of trials (direct evidence), our findings should best be interpreted as an exploratory effort importance to inform future research direction.

African-American Prostate Normal and Cancer Cells for Health Disparities Research.

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.

In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens.

Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5' target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.

The Effects of a Mobile Wellness Intervention with Fitbit Use and Goal Setting for Workers.

Background and Introduction: There is strong evidence that worksite wellness programs can significantly improve the health profile of participating workers. To date, little is known about research on the effects of mobile wellness interventions in worksite settings. Furthermore, no studies have been conducted to evaluate mobile wellness interventions with activity trackers and tailoring strategies for physically inactive workers in manufacturing companies. This study aimed to examine the effects of a mobile wellness intervention with Fitbit and goal setting using brief counseling and text messaging among workers. Materials and Methods: A total of 79 (n = 79) workers from large manufacturing companies were allocated into an experimental group (n = 41) and a control group (n = 38). All participants were asked to wear an activity tracker (Fitbit Charger HR) during all waking hours for 5 weekdays. Participants in the experimental group received Fitbit, daily motivational text messaging, and biweekly counseling with a specifically designed workbook for 12 weeks, whereas Fitbit was only provided to the control group. Results: At the 12-week measurement, there were significant differences between the experimental group and control group on wellness (p < 0.001), physical activity behavior (p < 0.001), daily walking steps (p < 0.001), and physical activity self-efficacy (p < 0.001). Discussion and Conclusions: Although Fitbit facilitates an individual's activities by providing information about daily steps, the tracker itself, without additional goal-setting techniques, may be insufficient to encourage behavior change. These findings indicate that the mobile wellness intervention with Fitbit and goal setting using brief counseling and tailored text messaging is more effective for physically inactive workers.

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.

Close yet so far away: a look into the management strategies of genetic imprinting disorders.

Genetic imprinting is the process of epigenetic labelling or silencing of particular genes, based on the maternal or paternal origin of the gene, in a heritable pattern. The incidence of imprinting disorders has become a growing concern due to the potential association between these congenital syndromes and assisted reproductive technologies (ARTs). This review presents a general summary of the imprinting process as well as the current knowledge surrounding the genetic and epigenetic underpinnings of the most prevalent imprinting disorders: Beckwith-Wiedemann syndrome (BWS), Silver-Russell syndrome (SRS), Prader-Willi syndrome (PWS), and Angelman syndrome (AS). As research continues to elucidate the molecular pathways that characterize genetic imprinting, efforts have been made to establish guidelines that incorporate phenotypic manifestations as well as genetic testing to ensure safe and effective management of symptoms. While these efforts are likely to benefit future clinical management, their efficacy cannot yet be generalized to all patients diagnosed with these syndromes, as many of the genetic abnormalities and the associated phenotypic manifestations have yet to be characterized. Furthermore, future advances in the knowledge of epigenetic processes and genetic loci involved in the development of these syndromes may allow for the development of curative therapies.

Inflammatory cells and their non-coding RNAs as targets for treating myocardial infarction.

Myocardial infarction triggers infiltration of several types of immune cells that coordinate both innate and adaptive immune responses. These play a dual role in post-infarction cardiac remodeling by initiating and resolving inflammatory processes, which needs to occur in a timely and well-orchestrated way to ensure a reestablishment of normalized cardiac functions. Thus, therapeutic modulation of immune responses might have benefits for infarct patients. While such strategies have shown great potential in treating cancer, applications in the post-infarction context have been disappointing. One challenge has been the complexity and plasticity of immune cells and their functions in cardiac regulation and healing. The types appear in patterns that are temporally and spatially distinct, while influencing each other and the surrounding tissue. A comprehensive understanding of the immune cell repertoire and their regulatory functions following infarction is sorely needed. Processes of cardiac remodeling trigger additional genetic changes that may also play critical roles in the aftermath of cardiovascular disease. Some of these changes involve non-coding RNAs that play crucial roles in the regulation of immune cells and may, therefore, be of therapeutic interest. This review summarizes what is currently known about the functions of immune cells and non-coding RNAs during post-infarction wound healing. We address some of the challenges that remain and describe novel therapeutic approaches under development that are based on regulating immune responses through non-coding RNAs in the aftermath of the disease.

Proteomic analysis of the cardiac extracellular matrix: clinical research applications.

INTRODUCTION: The cardiac extracellular matrix (ECM) provides anatomical, biochemical, and physiological support to the left ventricle. ECM proteins are difficult to detect using unbiased proteomic approaches due to solubility issues and a relatively low abundance compared to cytoplasmic and mitochondrial proteins present in highly prevalent cardiomyocytes. Areas covered: Proteomic capabilities have dramatically improved over the past 20 years, due to enhanced sample preparation protocols and increased capabilities in mass spectrometry (MS), database searching, and bioinformatics analysis. This review summarizes technological advancements made in proteomic applications that make ECM proteomics highly feasible. Expert commentary: Proteomic analysis of the ECM provides an important contribution to our understanding of the molecular and cellular processes associated with cardiovascular disease. Using results generated from proteomics approaches in basic science applications and integrating proteomics templates into clinical research protocols will aid in efforts to personalize medicine.

Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing after myocardial infarction.

Matrix metalloproteinase (MMP)-9 increases in the myocardium with advanced age and after myocardial infarction (MI). Because young transgenic (TG) mice overexpressing human MMP-9 only in macrophages show better outcomes post-MI, whereas aged TG mice show a worse aging phenotype, we wanted to evaluate the effect of aging superimposed on MI to see if the detrimental effect of aging counteracted the benefits of macrophage MMP-9 overexpression. We used 17- to 28-mo-old male and female C57BL/6J wild-type (WT) and TG mice ( n = 10-21 mice/group) to evaluate the effects of aging superimposed on MI. Despite similar infarct areas and mortality rates at day 7 post-MI, aging TG mice showed improved diastolic properties and remodeling index compared with WT mice (both P < 0.05). Macrophage numbers were higher in TG than WT mice at days 0 and 7 post-MI, and the post-MI increase was due to elevated cluster of differentiation 18 protein levels (all P < 0.05). RNA sequencing analysis of cardiac macrophages isolated from day 7 post-MI infarcts identified 1,276 statistically different (all P < 0.05) genes (994 increased and 282 decreased in TG mice). Reduced expression of vascular endothelial growth factor A, platelet-derived growth factor subunit A, and transforming growth factor-ß3, along with elevated expression of tissue inhibitor of MMP-4, in macrophages revealed mechanisms of indirect downstream effects on fibroblasts and neovascularization. While collagen accumulation was enhanced in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both), the post-MI collagen cross-linking ratio was higher in WT mice ( P < 0.05), consistent with increased diastolic volumes. Vessel numbers [by Griffonia ( Bandeiraea) simplicifolia lectin I staining] were decreased in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both). In conclusion, macrophage-derived MMP-9 improved post-MI cardiac wound healing through direct and indirect mechanisms to improve diastolic physiology and remodeling. NEW & NOTEWORTHY Aging mice with macrophage overexpression of matrix metalloproteinase-9 have increased macrophage numbers 7 days after myocardial infarction, resulting in improved diastolic physiology and left ventricular remodeling through effects on cardiac wound healing.