ALKBH5 inhibitors as a potential treatment strategy in heart failure-inferences from gene expression profiling.

Heart Failure (HF) is a complex clinical syndrome in which the heart is unable to provide enough blood flow to meet metabolic needs and lacks efficient venous return. HF is a major risk factor for morbidity and mortality with cardiovascular diseases globally. Despite enormous research, the molecular markers relevant to disease prognosis and management remain not well understood. Here, we analyzed the whole transcriptomes of 18 failing hearts and 15 non-failing hearts (predominantly of Caucasian origin), by applying the standard in silico tools. The analyses revealed novel gene-markers including ALKBH5 of mRNA demethylation and KMT2E of histone modification processes, significantly over-expressed in the HF compared with the non-failing hearts (FDR < 0.05). To validate the over-expression of ALKBH5, we determined the global m6A level in hypoxic H9c2 cells using a dot blot assay. The global m6A level was found markedly lower in the hypoxic H9c2 cells than in the control cells. Additionally, the expression of ALKBH5 in the H9c2 cells was quantified by the qPCR and found to be 1.18 times higher at 12 h (p < 0.05), and 1.67 times higher at 24 h of hypoxia (p < 0.01) compared with the control cells, indicating a likely role of ALKBH5 in the failing cardiac cells. Furthermore, we identified several compounds through the virtual screening of 11,272 drug-like molecules of the ZINC15 database to inhibit the ALKBH5 in a molecular docking process. Collectively, the study revealed novel markers potentially involved in the pathophysiology of HF and suggested plausible therapeutic molecules for the management of the disease.

Epigenetic Regulation of Macrophage Polarization in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of hospitalization and death worldwide, especially in developing countries. The increased prevalence rate and mortality due to CVDs, despite the development of several approaches for prevention and treatment, are alarming trends in global health. Chronic inflammation and macrophage infiltration are key regulators of the initiation and progression of CVDs. Recent data suggest that epigenetic modifications, such as DNA methylation, posttranslational histone modifications, and RNA modifications, regulate cell development, DNA damage repair, apoptosis, immunity, calcium signaling, and aging in cardiomyocytes; and are involved in macrophage polarization and contribute significantly to cardiac disease development. Cardiac macrophages not only trigger damaging inflammatory responses during atherosclerotic plaque formation, myocardial injury, and heart failure but are also involved in tissue repair, remodeling, and regeneration. In this review, we summarize the key epigenetic modifications that influence macrophage polarization and contribute to the pathophysiology of CVDs, and highlight their potential for the development of advanced epigenetic therapies.

Frequency of complete remission after standard 3+7 induction therapy in patients with acute myeloid leukemia.

Objectives: To find the frequency of complete remission rate after standard 3+7 induction therapy in patients with Acute Myeloid Leukemia (AML) among different clinicopathological groups. Methods: Non-probability purposive sampling technique was used to collect data from July 2016 to Jan 2017, conducted at Department of Oncology, Jinnah Hospital Lahore. Sample size of 50 cases was calculated with 95% confidence level, 14% margin of error and taking expected percentage of complete remission (CR) 57% in AML patients after 3+7 induction therapy. Results: Out of 46 patients, majority had AML-M2. AML- Not Otherwise Specified (NOS) disease. Most common pathological presentation was Total Leucocyte Count (TLC) of <50,000/mm3 (60%) as compared to patients with hyperleukocytosis i.e., TLC >50,000/mm3 (40%). Twenty-two percent had low Lactate Dehydrogenase (LDH) level involvement and 78% had high LDH level involvement. As compared to target of 57 %, complete response was observed in 54% patients, (p<0.02) with better results in younger age group, male patients with low LDH and TLC level. Conclusion: It was concluded that 3+7 induction chemotherapy has 54% CR rates in patients with AML. Whereas, in AML-M5, AML-M6, AML-NOS patients especially, with high LDH and TLC and patients with advanced age, CR rate is low and needs more aggressive treatment.

Potential therapeutic strategies for myocardial infarction: the role of Toll-like receptors.

Myocardial infarction (MI) is a life-threatening condition among patients with cardiovascular diseases. MI increases the risk of stroke and heart failure and is a leading cause of morbidity and mortality worldwide. Several genetic and epigenetic factors contribute to the development of MI, suggesting that further understanding of the pathomechanism of MI might help in the early management and treatment of this disease. Toll-like receptors (TLRs) are well-known members of the pattern recognition receptor (PRR) family and contribute to both adaptive and innate immunity. Collectively, studies suggest that TLRs have a cardioprotective effect. However, prolonged TLR activation in the response to signals generated by damage-associated molecular patterns (DAMPs) results in the release of inflammatory cytokines and contributes to the development and exacerbation of myocardial inflammation, MI, ischemia-reperfusion injury, myocarditis, and heart failure. The objective of this review is to discuss and summarize the association of TLRs with MI, highlighting their therapeutic potential for the development of advanced TLR-targeted therapies for MI.

GSK-3ß-mediated activation of NLRP3 inflammasome leads to pyroptosis and apoptosis of rat cardiomyocytes and fibroblasts.

We previously demonstrated that GSK-3ß mediates NLRP3 inflammasome activation and IL-1ß production in cardiac fibroblasts (CFs) after myocardial infarction (MI). In this study, we show how GSK-3ß-mediated activation of the NLRP3 inflammasome/caspase-1/IL-1ß pathway leads to apoptosis and pyroptosis of cardiomyocytes (CMs) and CFs. Administration of lipopolysaccharide (LPS)/ATP to primary newborn rat cardiac fibroblasts (RCFs) led to increase in proteins of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, IL-1ß, and IL-18. Additionally, the expression of caspase-3 and N-terminal fragments of gasdermin D (N-GSDMD) and the Bax/Bcl-2 ratio increased. Administration of the GSK-3ß inhibitor SB216763 reduced the levels of apoptosis- and pyroptosis-related proteins regulated by NLRP3 inflammasome activation in RCFs. Next, we transferred the culture supernatant of LPS/ATP-treated RCFs to in vitro primary newborn rat cardiomyocytes (RCMs). The results showed that SB216763 attenuate the upregulation of the ratios of Bax/Bcl-2 and the expression of caspase-3 and N-GSDMD in RCMs. Direct stimulation of RCMs and H9c2 cells with recombinant rat IL-1ß increased the p-GSK-3ß/GSK-3ß and Bax/Bcl-2 ratios and the expression of caspase-3 and N-GSDMD, while both SB216763 and TLR1 (an IL-1ß receptor inhibitor) markedly reduced these effects, as assessed using propidium iodide positive staining and the lactate dehydrogenase release assay. The caspase-11 inhibitor wedelolactone decreased the expression level of N-GSDMD but did not alter the p-GSK-3ß/GSK-3ß ratio. Lastly, we established a Sprague-Dawley rat MI model to confirm that SB216763 diminished the increase in caspase-3 and N-GSDMD expression and the Bax/Bcl-2 ratio in the ischemic area. These data demonstrate that GSK-3ß regulates apoptosis and pyroptosis of RCMs and RCFs due to NLRP3 inflammasome activation in RCFs.

The caspase-1 inhibitor VX765 upregulates connexin 43 expression and improves cell-cell communication after myocardial infarction via suppressing the IL-1ß/p38 MAPK pathway.

Connexin 43 (Cx43) is the most important protein in the gap junction channel between cardiomyocytes. Abnormalities of Cx43 change the conduction velocity and direction of cardiomyocytes, leading to reentry and conduction block of the myocardium, thereby causing arrhythmia. It has been shown that IL-1ß reduces the expression of Cx43 in astrocytes and cardiomyocytes in vitro. However, whether caspase-1 and IL-1ß affect connexin 43 after myocardial infarction (MI) is uncertain. In this study we investigated the effects of VX765, a caspase-1 inhibitor, on the expression of Cx43 and cell-to-cell communication after MI. Rats were treated with VX765 (16 mg/kg, i.v.) 1 h before the left anterior descending artery (LAD) ligation, and then once daily for 7 days. The ischemic heart was collected for histochemical analysis and Western blot analysis. We showed that VX765 treatment significantly decreased the infarct area, and alleviated cardiac dysfunction and remodeling by suppressing the NLRP3 inflammasome/caspase-1/IL-1ß expression in the heart after MI. In addition, VX765 treatment markedly raised Cx43 levels in the heart after MI. In vitro experiments were conducted in rat cardiac myocytes (RCMs) stimulated with the supernatant from LPS/ATP-treated rat cardiac fibroblasts (RCFs). Pretreatment of the RCFs with VX765 (25 µM) reversed the downregulation of Cx43 expression in RCMs and significantly improved intercellular communication detected using a scrape-loading/dye transfer assay. We revealed that VX765 suppressed the activation of p38 MAPK signaling in the heart tissue after MI as well as in RCMs stimulated with the supernatant from LPS/ATP-treated RCFs. Taken together, these data show that the caspase-1 inhibitor VX765 upregulates Cx43 expression and improves cell-to-cell communication in rat heart after MI via suppressing the IL-1ß/p38 MAPK pathway.

Targeting Glycogen Synthase Kinase 3 Beta Regulates CD47 Expression After Myocardial Infarction in Rats via the NF-κB Signaling Pathway.

The aim of this study was to investigate the effects of the GSK-3ß/NF-κB pathway on integrin-associated protein (CD47) expression after myocardial infarction (MI) in rats. An MI Sprague Dawley rat model was established by ligating the left anterior descending coronary artery. The rats were divided into three groups: Sham, MI, and SB + MI (SB216763) groups. Immunohistochemistry was used to observe the changes in cardiac morphology. A significant reduction in the sizes of fibrotic scars was observed in the SB + MI group compared to that in the MI group. SB216763 decreased the mRNA and protein expression of CD47 and NF-κB during MI. Primary rat cardiomyocytes (RCMs) and the H9c2 cell line were used to establish in vitro hypoxia models. Quantitative real-time PCR and western blotting analyses were conducted to detect mRNA and protein expression levels of CD47 and NF-κB and apoptosis-related proteins, respectively. Apoptosis of hypoxic cells was assessed using flow cytometry. SB216763 reduced the protein expression of CD47 and NF-κB in RCMs and H9c2 cells under hypoxic conditions for 12 h, and alleviated hypoxia-induced apoptosis. SN50 (an NF-κB inhibitor) also decreased CD47 protein expression in RCMs and H9c2 cells under hypoxic conditions for 12 h and protected cells from apoptosis. GSK-3ß upregulates CD47 expression in cardiac tissues after MI by activating NF-κB, which in turn leads to myocardial cell damage and apoptosis.

Potential regulatory role of epigenetic RNA methylation in cardiovascular diseases.

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide, especially in developing countries. To date, several approaches have been proposed for the prevention and treatment of CVDs. However, the increased risk of developing cardiovascular events that result in hospitalization has become a growing public health concern. The pathogenesis of CVDs has been analyzed from various perspectives. Recent data suggest that regulatory RNAs play a multidimensional role in the development of CVDs. Studies have identified several mRNA modifications that have contributed to the functional characterization of various cardiac diseases. RNA methylation, such as N6-methyladenosine, N1-methyladenosine, 5-methylcytosine, N7-methylguanosine, N4-acetylcytidine, and 2'-O-methylation are novel epigenetic modifications that affect the regulation of cell growth, immunity, DNA damage, calcium signaling, apoptosis, and aging in cardiomyocytes. In this review, we summarize the role of RNA methylation in the pathophysiology of CVDs and the potential of using epigenetics to treat such disorders.

Glycogen synthase kinase-3ß inhibition alleviates activation of the NLRP3 inflammasome in myocardial infarction.

Inflammasome-promoted sterile inflammation following cardiac damage is critically implicated in heart dysfunction after myocardial infarction (MI). Glycogen synthase kinase-3 (GSK-3ß) is a prominent mediator of the inflammatory response, and high GSK-3 activity is associated with various heart diseases. We investigated the regulatory mechanisms of GSK-3ß in activation of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in a rat model with successful induction of MI on days 2-28. An in vitro investigation was performed using newborn rat/human cardiomyocytes and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. GSK-3ß inhibition markedly improved myocardial dysfunction and prevented remodeling, with parallel reduction in the parameters of NLRP3 inflammasome activation after MI. GSK-3ß inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes. GSK-3ß's interaction with activating signal cointegrator (ASC) as well as GSK-3ß inhibition reduced ASC phosphorylation and oligomerization at the tissues and cellular levels. Taken together, these data show that GSK-3ß directly mediates NLRP3 inflammasome activation, causing cardiac dysfunction in MI.

MicroRNAs: Emerging biomarkers for atrial fibrillation.

Atrial fibrillation (AF) causes severe cardiac dysrhythmia among patients with cardiovascular diseases. AF increases the risk of stroke and heart failure and is a growing public health concern. AF is also associated with various disease conditions such as hypertension, coronary artery disease, aging, and diabetes mellitus. The mechanism underlying AF is not completely understood due to its complexity. However, experimental and clinical data have revealed that the prevalence of this disease is associated with atrial arrhythmogenic remodeling. Currently, there are no biomarkers that are available for the early diagnosis of AF. Several studies have proposed microRNAs (miRNAs) as useful biomarkers for the diagnosis of AF due to their stability and easy availability both in atrial tissue and circulating blood. miRNAs play an important role in the development of the heart. The dysregulation of miRNA expression is associated with cardiac remodeling. Genetic factors strongly contribute to the pathogenesis of AF. Recently, single nucleotide polymorphisms (SNPs) in various genes and miRNAs have been reported to be associated with AF. The aim of this review was to discuss the correlation between SNPs in miRNAs and AF, including those miRNAs that are commonly reported as potential biomarkers for AF.

Antimicrobial activity of Prunella Vulgaris extracts against multi-drug resistant Escherichia Coli from patients of urinary tract infection.

BACKGROUND AND OBJECTIVE: Escherichia Coli is the most common etiological agent of UTI and accounts for more then 100, 0000 hospitalization annually. The objective of this study was to investigate the antimicrobial activity of aqueous and ethanolic extracts of Prunella vulgaris against E. coli from urinary tract infection patients. METHODS: Urine samples of forty four suspected patients from Tertiary Care Hospital Faisalabad were used in this study. Ethanolic and aqueous extracts of Prunella vulgris (PV), a medicinal plant was evaluated for its ability to inhibit the growth of 38 resistant isolates of Escherichia coli strains and compared to Ciprofloxacin, Ofloxacin, Cefixime and Tobramycin by well diffusion method. Minimum inhibitory concentration was measured by using broth micro dilution method. RESULTS: PV showed antibacterial activity against Escherichia coli strains, however Tobramycin at 10 microgram (10µg) inhibited the resistant E. coli to a greater extent as compared to other antibiotics and was resistant to twice less number of strains, about 82% of E. coli isolates have MDR pattern. CONCLUSION: Ciprofloxacin has more efficacy than PV and no synergistic effect with extracts of PV. Cefixime is least efficacious against resistant E. coli, however it has synergistic effect with extracts of PV.