Platelet-derived sTLT-1 is associated with platelet-mediated inflammation in coronary artery disease patients.

The development of coronary artery disease (CAD) depends heavily on platelet activation, and inflammation plays a major role in all stages of atherosclerosis. Platelet-specific soluble triggering receptor expressed on myeloid cells like transcript 1 (sTLT-1) facilitate clot formation and have been linked to chronic inflammation. In this study, we explored the role of platelet-derived sTLT-1 in platelet-mediated inflammation in CAD patients. Plasma levels of sTLT-1 were measured using enzyme-linked immunosorbent assay in CAD patients (n = 163) and healthy controls (n = 99). Correlation analysis was performed to determine the circulatory sTLT-1 levels with platelet activation markers, immune cells, and inflammatory cytokines/chemokines. Increased plasma sTLT-1 levels were observed in CAD patients compared with those in healthy controls (p < 0.0001). A positive correlation was observed between sTLT-1 and platelet activation markers (P-selectin, PAC-1), CD14++ CD16- cells (classical monocytes), Natural killer T (NKT) cells, and platelet-immune cell aggregates with monocytes, neutrophils, dendritic cells, CD11c+ cells, and NKT cells. In contrast, a significant negative correlation was observed with CD8 cells. Furthermore, a significant positive correlation was observed between sTLT-1 and inflammatory markers (TNF-α, IL-1ß, IL-2, IL-6, IL-12p70, IL-18, CXCL-12, and CCL-11). Logistic regression analysis identified sTLT-1 and triglycerides as predictors of CAD. Receiver operating characteristic curve (ROC) analysis showed that sTLT-1 had a higher sensitivity and specificity for predicting CAD. Our findings suggest that platelet activation induces the release of sTLT-1 into the circulation in CAD patients, which aggregates with immune cells and enhances inflammatory responses.

Elevated level of circulatory sTLT1 induces inflammation through SYK/MEK/ERK signalling in coronary artery disease.

The role of inflammation in all phases of atherosclerotic process is well established and soluble TREM-like transcript 1 (sTLT1) is reported to be associated with chronic inflammation. Yet, no information is available about the involvement of sTLT1 in atherosclerotic cardiovascular disease. Present study was undertaken to determine the pathophysiological significance of sTLT1 in atherosclerosis by employing an observational study on human subjects (n=117) followed by experiments in human macrophages and atherosclerotic apolipoprotein E (apoE)-/- mice. Plasma level of sTLT1 was found to be significantly (P<0.05) higher in clinical (2342 ± 184 pg/ml) and subclinical cases (1773 ± 118 pg/ml) than healthy controls (461 ± 57 pg/ml). Moreover, statistical analyses further indicated that sTLT1 was not only associated with common risk factors for Coronary Artery Disease (CAD) in both clinical and subclinical groups but also strongly correlated with disease severity. Ex vivo studies on macrophages showed that sTLT1 interacts with FcÉ£ receptor I (FcÉ£RI) to activate spleen tyrosine kinase (SYK)-mediated downstream MAP kinase signalling cascade to activate nuclear factor-κ B (NF-kB). Activation of NF-kB induces secretion of tumour necrosis factor-α (TNF-α) from macrophage cells that plays pivotal role in governing the persistence of chronic inflammation. Atherosclerotic apoE-/- mice also showed high levels of sTLT1 and TNF-α in nearly occluded aortic stage indicating the contribution of sTLT1 in inflammation. Our results clearly demonstrate that sTLT1 is clinically related to the risk factors of CAD. We also showed that binding of sTLT1 with macrophage membrane receptor, FcÉ£R1 initiates inflammatory signals in macrophages suggesting its critical role in thrombus development and atherosclerosis.

Targeting Peroxisome Proliferator Activated Receptor α (PPAR α) for the Prevention of Mitochondrial Impairment and Hypertrophy in Cardiomyocytes.

BACKGROUND/AIMS: Morphological and biochemical maladaptation of cardiomyocytes are associated with mitochondrial dysfunction and dysregulation in hypertrophic conditions. Peroxisome proliferator activated receptor α (PPARα), a drug target for dyslipidemia, is known to be downregulated in cardiomyocytes in response to hypertrophic stimuli. The current study was undertaken to investigate the role of PPARα signaling in mitochondrial remodeling and thereby dysregulation of cardiomyocytes due to hypertrophy in vitro. METHODS: Rat cardiomyocytes H9c2 (2-1) and neonatal rat ventricular myocytes (NRVMs) were cultured and treated with α1-adrenergic agonist phenylephrine (PE, 100 µM, 24 hours) in the presence or absence of 10 µM fenofibrate or bezafibrate. Cellular hypertrophy was observed by atomic force microscopy and immunofluorescence with F-actin antibody. mRNA levels of hypertrophic marker genes and other genes were examined by quantitative real time PCR. Structural as well as functional remodeling of the mitochondria were evaluated by immunofluorescence (F-actin and COX-I), live cell imaging microscopy (JC-I, mitotracker), mitochondrial complex V activity, MPTP activity and ATP assay. Oxidative stress was measured by using sensitive fluorescent indicator probes. Cellular and mitochondrial calcium were measured by using fluorescent indicator probes Rhod-2 AM and X-rhod-1 AM, respectively. Targetscan prediction analysis was performed to find out miRNAs as putative regulators of VDAC. Luciferase assay was conducted to confirm binding of miR28 with VDAC. RESULTS: Co-treatment of H9c2(2-1) cells with PE and fenofibrate restricted increase in cell size and expression of marker genes such as atrial-natriuretic peptide (ANP), brain-natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC) compared to those with PE alone. Fenofibrate prevented PE-induced down regulation of PPARα-target genes like CPT-I and MCAD. Mitochondrial trans-membrane potential (Δψm) and motility were reduced by PE which were significantly checked by fenofibrate. Increased ROS production and calcium level in PE-treated cells were ameliorated by fenofibrate. Mitochondrial activity and ATP generation were reduced by PE which was rescued by fenofibrate. Fenofibrate also prevented PE-induced down regulation of mitochondrial genes like VDAC-I and COX-IV. Expression of several miRNAs was altered in hypertrophic cardiomyocytes which were restored when co-treated with fenofibrate. miR28 was found to target 3' untranslated region of VDAC-I. CONCLUSION: Overall, the results demonstrate that PPARα signaling is critically involved in mitochondrial dysfunction in hypertrophic cardiomyocytes in which miR28 plays a pivotal role.

Proteomics in India: the clinical aspect.

Proteomics has emerged as a highly promising bioanalytical technique in various aspects of applied biological research. In Indian academia, proteomics research has grown remarkably over the last decade. It is being extensively used for both basic as well as translation research in the areas of infectious and immune disorders, reproductive disorders, cardiovascular diseases, diabetes, eye disorders, human cancers and hematological disorders. Recently, some seminal works on clinical proteomics have been reported from several laboratories across India. This review aims to shed light on the increasing use of proteomics in India in a variety of biological conditions. It also highlights that India has the expertise and infrastructure needed for pursuing proteomics research in the country and to participate in global initiatives. Research in clinical proteomics is gradually picking up pace in India and its future seems very bright.

Cytosolic dynamics of annexin A6 trigger feedback regulation of hypertrophy via atrial natriuretic peptide in cardiomyocytes.

Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca(2+)) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6(ΔC)). Furthermore, stable overexpression of Anxa6(T356D), a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.

Proteomic analysis of human plasma in chronic rheumatic mitral stenosis reveals proteins involved in the complement and coagulation cascade.

BACKGROUND: Rheumatic fever in childhood is the most common cause of Mitral Stenosis in developing countries. The disease is characterized by damaged and deformed mitral valves predisposing them to scarring and narrowing (stenosis) that results in left atrial hypertrophy followed by heart failure. Presently, echocardiography is the main imaging technique used to diagnose Mitral Stenosis. Despite the high prevalence and increased morbidity, no biochemical indicators are available for prediction, diagnosis and management of the disease. Adopting a proteomic approach to study Rheumatic Mitral Stenosis may therefore throw some light in this direction. In our study, we undertook plasma proteomics of human subjects suffering from Rheumatic Mitral Stenosis (n = 6) and Control subjects (n = 6). Six plasma samples, three each from the control and patient groups were pooled and subjected to low abundance protein enrichment. Pooled plasma samples (crude and equalized) were then subjected to in-solution trypsin digestion separately. Digests were analyzed using nano LC-MS(E). Data was acquired with the Protein Lynx Global Server v2.5.2 software and searches made against reviewed Homo sapiens database (UniProtKB) for protein identification. Label-free protein quantification was performed in crude plasma only. RESULTS: A total of 130 proteins spanning 9-192 kDa were identified. Of these 83 proteins were common to both groups and 34 were differentially regulated. Functional annotation of overlapping and differential proteins revealed that more than 50% proteins are involved in inflammation and immune response. This was corroborated by findings from pathway analysis and histopathological studies on excised tissue sections of stenotic mitral valves. Verification of selected protein candidates by immunotechniques in crude plasma corroborated our findings from label-free protein quantification. CONCLUSIONS: We propose that this protein profile of blood plasma, or any of the individual proteins, could serve as a focal point for future mechanistic studies on Mitral Stenosis. In addition, some of the proteins associated with this disorder may be candidate biomarkers for disease diagnosis and prognosis. Our findings might help to enrich existing knowledge on the molecular mechanisms involved in Mitral Stenosis and improve the current diagnostic tools in the long run.

Novel role of peptidoglycan recognition protein 2 in activating NOD2-NFκB inflammatory axis in coronary artery disease.

BACKGROUNDS AND AIMS: The role of inflammation in driving atherosclerosis is well-established. It exerts systemic effects beyond the local site of plaque formation. In the context of coronary artery disease (CAD), the proteins that show altered levels in the plasma, are potentially important for understanding the key regulatory mechanism in the pathogenesis of atherosclerosis. A case-control study revealed that plasma soluble Peptidoglycan Recognition Protein 2 (PGLYRP2) primarily produced by the liver, is increased in subjects with CAD. Furthermore, the concentration of PGLYRP2 in the blood correlates with the severity of coronary artery disease. Thus, it raises interest in understanding the exact role of the protein in aortic inflammation and plaque progression. METHODS: We evaluated the plasma concentration of PGLYRP2 in three distinct groups: patients with CAD (N = 68), asymptomatic individuals (N = 34), and healthy volunteers (N = 20). Furthermore, we investigated the correlation between disease severity and PGLYRP2 levels in CAD patients. To identify potential binding partners of PGLYRP2, we employed computational analysis. We verified the PGLYRP2-NOD2 interaction in macrophage cells and elucidated the inflammatory pathways activated by PGLYRP2 within these cells. To assess the impact of PGLYRP2, we examined its effects in the atherosclerotic mice model (ApoE-/-). RESULTS: In this study, we report for the first time that Nucleotide-binding Oligomerization domain 2 (NOD2) which is expressed on the surface of macrophages, is a receptor of PGLYRP2. The N-terminal domain of PGLYRP2 directly binds to NOD2 and activates the NOD2-RIP2-NFκB cascade that promotes the secretion of proinflammatory cytokines like TNFα, IL1ß, and IL-8. In the atherosclerotic mice model (ApoE-/-) we demonstrate that elevated PGLYRP2 level is parallel with increased proinflammatory cytokines in the plasma when fed a High Cholesterol Diet (HCD). Immunohistochemical analysis reveals that PGLYRP2 is co-localized with NOD2 on the macrophages at the site of the lesion. CONCLUSIONS: Taken together, our data demonstrate that NOD2 acts as a receptor of PGLYRP2 on macrophages, which mediates the activation of the NOD2-RIP2-NFκB pathway and promotes inflammation, thus significantly contributing to the development and progression of atherosclerosis.

Genome characterization, phylogenomic assessment and spatio-temporal dynamics study of highly mutated BA variants from India.

PURPOSE: The emergence of highly mutated and transmissible BA variants has caused an unprecedented surge in COVID-19 infections worldwide. Thorough analysis of its genome structure and phylogenomic evolutionary details will serve as scientific reference for future research. METHOD: Here, we have analyzed the BA variants from India using whole-genome sequencing, spike protein mutation study, spatio-temporal surveillance, phylogenomic assessment and epitope mapping. RESULTS: The predominance of BA.2/BA.2-like was observed in India during COVID-19 third wave. Genome analysis and mutation study highlighted the existence of 2128 amino acid changes within BA as compared to NC_045512.2. Presence of 23 unknown mutation sites (spanning region 61-831) were observed among the Indian BA variants as compared to the global BA strains. Unassigned probable Omicron showed the highest number of mutations (370) followed by BA.1 (104), BA.2.3 (56), and BA.2 (27). Presence of mutations 'Q493R â€‹+ â€‹Q498R â€‹+ â€‹N501Y', and 'K417 â€‹N â€‹+ â€‹E484A â€‹+ â€‹N501Y' remained exclusive to BA.2 as well as unassigned probable Omicron. The time-tree and phylogenomic network assessed the evolutionary relationship of the BA variants. Existence of 424 segregating sites and 113 parsimony informative sites within BA genomes were observed through haplotype network analysis. Epitope mapping depicted the presence of unique antigenic sites within the receptor binding domain of the BA variants that could be exploited for robust vaccine development. CONCLUSION: These findings provide important scientific insights about the nature, diversity, and evolution of Indian BA variants. The study further divulges in the avenues of therapeutic upgradation for better management and eventual eradication of COVID-19.

Corrigendum: Standardised Sonneratia apetala Buch.-Ham. fruit extract inhibits human neutrophil elastase and attenuates elastase-induced lung injury in mice.

[This corrects the article DOI: 10.3389/fphar.2022.1011216.].

Melatonin protects against isoproterenol-induced alterations in cardiac mitochondrial energy-metabolizing enzymes, apoptotic proteins, and assists in complete recovery from myocardial injury in rats.

The present study was undertaken to explore the protective effect of melatonin against isoproterenol bitartrate (ISO)-induced rat myocardial injury and to test whether melatonin has a role in preventing myocardial injury and recovery when the ISO-induced stress is withdrawn. Treatment for rats with ISO altered the activities of some of the key mitochondrial enzymes related to energy metabolism, the levels of some stress proteins, and the proteins related to apoptosis. These changes were found to be ameliorated when the animals were pretreated with melatonin at a dose of 10 mg/kg BW, i.p. In addition to its ability to reduce ISO-induced mitochondrial dysfunction, we also studied the role of melatonin in the recovery of the cardiac tissue after ISO-induced damage. Continuation of melatonin treatment in rats after the withdrawal of ISO treatment was found to reduce the activities of cardiac injury biomarkers including serum glutamate oxaloacetate transaminase (SGOT), lactate dehydrogenase (LDH), and cardio-specific LDH1 to control levels. The levels of tissue lipid peroxidation and reduced glutathione were also brought back to that seen in control animals by continued melatonin treatment. Continuation of melatonin treatment in post-ISO treatment period was also found to improve cardiac tissue morphology and heart function. Thus, the findings indicate melatonin's ability to provide cardio protection at a low pharmacological dose and its role in the recovery process. Melatonin, a molecule with very low or no toxicity may be considered as a therapeutic for the treatment for ischemic heart disease.

Systemic deficiency of vitronectin is associated with aortic inflammation and plaque progression in ApoE-Knockout mice.

Optimal cell spreading and interplay of vascular smooth muscle cells (VSMC), inflammatory cells, and cell adhesion molecules (CAM) are critical for progressive atherosclerosis and cardiovascular complications. The role of vitronectin (VTN), a major cell attachment glycoprotein, in the pathogenesis of atherosclerosis remains elusive. In this study, we attempt to examine the pathological role of VTN in arterial plaque progression and inflammation. We found that, relative expression analysis of VTN from the liver of Apolipoprotein E (ApoE) Knockout mice revealed that atherosclerotic progression induced by feeding mice with high cholesterol diet (HCD) causes a significant downregulation of VTN mRNA as well as protein after 60 days. Promoter assay confirmed that cholesterol modulates the expression of VTN by influencing its promoter. Mimicking VTN reduction with siRNA in HCD fed ApoE Knockout mice, accelerated athero-inflammation with an increase in NF-kB, ICAM-1, and VCAM-1 at the site of the plaque along with upregulation of inflammatory proteins like MCP-1 and IL-1ß in the plasma. Also, matrix metalloprotease (MMP)-9 and MMP-12 expression were increased and collagen content was decreased in the plaque, in VTN deficient condition. This might pose a challenge to plaque integrity. Human subjects with acute coronary syndrome or having risk factors of atherosclerosis have lower levels of VTN compared to healthy controls suggesting a clinical significance of plasma VTN in the pathophysiology of coronary artery disease. We establish that, VTN plays a pivotal role in cholesterol-driven atherosclerosis and aortic inflammation and might be a useful indicator for atherosclerotic plaque burden and stability.

Novel benzoxazinone derivative as potent human neutrophil elastase inhibitor: Potential implications in lung injury.

Neutrophil elastase, a powerful physiological defence tool, may serve as drug target for diverse diseases due to its bystander effect on host cells like chronic obstructive pulmonary disease (COPD). Here, we synthesised seven novel benzoxazinone derivatives and identified that these synthetic compounds are human neutrophil elastase inhibitor that was demonstrated by enzyme substrate kinetic assay. One such compound, PD05, emerged as the most potent inhibitor with lower IC50 as compared to control drug sivelestat. While this inhibition is competitive based on substrate dilution assay, PD05 showed a high binding affinity for human neutrophil elastase (Kd = 1.63 nM) with faster association and dissociation rate compared to notable elastase inhibitors like ONO 6818 and AZD9668, and its interaction with human neutrophil elastase was fully reversible.Preclinical pharmacokinetic studies were performed in vitro where protein binding was found to be 72% with a high recovery rate, aqueous solubility of 194.7 µM, low permeability along with a favourable hERG. Experiments with cell line revealed that the molecule successfully prevented elastase induced rounding and retracted cell morphology and cell cytotoxicity. In mouse model PD05 is able to reduce the alveolar collapse induced by neutrophil elastase. In summary, we demonstrate the in situ, in vitro and in vivo anti-elastase potential of the newly synthesised benzoxazinone derivative PD05 and thus this could be promising candidate for further investigation as a drug for the treatment of COPD.

Standardised Sonneratia apetala Buch.-Ham. fruit extract inhibits human neutrophil elastase and attenuates elastase-induced lung injury in mice.

Chronic obstructive pulmonary disease (COPD) along with asthma is a major and increasing global health problem. Smoking contributes to about 80%-90% of total COPD cases in the world. COPD leads to the narrowing of small airways and destruction of lung tissue leading to emphysema primarily caused by neutrophil elastase. Neutrophil elastase plays an important role in disease progression in COPD patients and has emerged as an important target for drug discovery. Sonneratia apetala Buch.-Ham. is a mangrove plant belonging to family Sonneratiaceae. It is widely found in the Sundarban regions of India. While the fruits of this plant have antibacterial, antifungal, antioxidant and astringent activities, fruit and leaf extracts have been shown to reduce the symptoms of asthma and cough. The aim of this study is to find whether hydro alcoholic fruit extracts of S. apetala inhibit neutrophil elastase and thus prevent the progression of neutrophil elastase-driven lung emphysema. The hydroalcoholic extract, ethanol: water (90:10), of the S. apetala Buch.-Ham. fresh fruits (SAM) were used for neutrophil elastase enzyme kinetic assay and IC50 of the extract was determined. The novel HPLC method has been developed and the extract was standardized with gallic acid and ellagic acid as standards. The extract was further subjected to LC-MS2 profiling to identify key phytochemicals. The standardized SAM extract contains 53 µg/mg of gallic acid and 95 µg/mg of ellagic acid, based on the HPLC calibration curve. SAM also reversed the elastase-induced morphological change of human epithelial cells and prevented the release of ICAM-1 in vitro and an MTT assay was conducted to assess the viability. Further, 10 mg/kg SAM had reduced alveolar collapse induced by neutrophil elastase in the mice model. Thus, in this study, we reported for the first time that S. apetala fruit extract has the potential to inhibit human neutrophil elastase in vitro and in vivo.

Downregulation of PTEN Promotes Autophagy via Concurrent Reduction in Apoptosis in Cardiac Hypertrophy in PPAR α-/- Mice.

Cardiac hypertrophy is characterized by an increase in the size of the cardiomyocytes which is initially triggered as an adaptive response but ultimately becomes maladaptive with chronic exposure to different hypertrophic stimuli. Prolonged cardiac hypertrophy is often associated with mitochondrial dysfunctions and cardiomyocyte cell death. Peroxisome proliferator activated receptor alpha (PPAR α), which is critical for mitochondrial biogenesis and fatty acid oxidation, is down regulated in hypertrophied cardiomyocytes. Yet, the role of PPAR α in cardiomyocyte death is largely unknown. To assess the role of PPAR α in chronic hypertrophy, isoproterenol, a ß-adrenergic receptor agonist was administered in PPAR α knock out (PPAR α-/-) mice for 2 weeks and hypertrophy associated changes in cardiac tissues were observed. Echocardiographic analysis ensured the development of cardiac hypertrophy and compromised hemodynamics in PPAR α-/- mice. Proteomic analysis using high resolution mass spectrometer identified about 1,200 proteins enriched in heart tissue. Proteins were classified according to biological pathway and molecular functions. We observed an unexpected down regulation of apoptotic markers, Annexin V and p53 in hypertrophied heart tissue. Further validation revealed a significant down regulation of apoptosis regulator, PTEN, along with other apoptosis markers like p53, Caspase 9 and c-PARP. The autophagy markers Atg3, Atg5, Atg7, p62, Beclin1 and LC3 A/B were up regulated in PPAR α-/- mice indicating an increase in autophagy. Similar observations were made in a high cholesterol diet fed PPAR α-/-mice. The results were further validated in vitro using NRVMs and H9C2 cell line by blocking PPAR α that resulted in enhanced autophagosome formation upon hypertrophic stimulation. The results demonstrate that in the absence of PPAR α apoptotic pathway is inhibited while autophagy is enhanced. The data suggest that PPAR α signaling might act as a molecular switch between apoptosis and autophagy thereby playing a critical role in adaptive process in cardiac hypertrophy.

Interaction of annexin A6 with alpha actinin in cardiomyocytes.

BACKGROUND: Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state. RESULTS: To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells. CONCLUSIONS: In overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

Exploring the possible metabolism mediated interaction of Glycyrrhiza glabra extract with CYP3A4 and CYP2D6.

The rhizome of Glycyrrhiza glabra L. (licorice) is used very widely in Indian and Chinese traditional medicine, and it is a popular flavor ingredient of drinks, sweets and candies. Its medicinal uses include treating bronchitis, dry cough, respiratory infections, liver disorders and diabetes. Glycyrrhizin is normally considered to be its biologically active marker, so a rapid RP-HPLC method was developed for the quantitative estimation of glycyrrhizin in the extract. The effect of the standardized extract and its marker on drug metabolizing enzymes was evaluated through CYP3A4 and CYP2D6 inhibition assays to evaluate the safety through its drug interaction potential. The inhibition of CYP3A4 and CYP2D6 isozymes was analysed by the fluorescent product formation method. In the CYP450-CO assay, the interaction potential of the standardized extract and pooled microsomes (percentage inhibition 23.23 ± 1.84%), was found to be less than the standard inhibitor. In the fluorimetric assay, G. glabra extracts showed higher IC(50) values than their positive inhibitors, ketoconazole and quinidine for CYP3A4 and CYP2D6, respectively. Furthermore, the interaction potential of the plant extract was greater than the pure compound. The results demonstrate that G. glabra and its principle bioactive compound, glycyrrhizin, when co-administered with conventional medicines showed only a weak interaction potential with drug metabolizing enzymes.

Melatonin protects against isoproterenol-induced myocardial injury in the rat: antioxidative mechanisms.

The present study was undertaken to explore the protective effect of melatonin against isoproterenol bitartrate (ISO)-induced myocardial injury in rat. Treatment of rats with ISO increased the level of lipid peroxidation products and decreased the reduced glutathione levels in cardiac tissue indicating that this synthetic catecholamine induces oxidative damage following oxidative stress. Pretreatment of ISO-injected rats with melatonin at a dose of 10 mg/kg body weight, i.p. prevented these changes. Additionally, melatonin also restored the activities and the levels of antioxidant enzymes which were found to be altered by ISO treatment. Treatment of rats with ISO resulted into an increased generation of hydroxyl radicals with melatonin pretreatment significantly reducing their production. Finally, treatment of rats with ISO caused a lowering of systolic pressure with reduced cardiac output and diastolic dysfunction whereas melatonin pretreatment significantly restored many of these parameters to normal. The findings document melatonin's ability to provide cardio protection at a low pharmacological dose. Melatonin has virtually no toxicity which raises the possibility of this indole being a therapeutic treatment for ischemic heart disease.

Anti-allergic activity of standardized extract of Albizia lebbeck with reference to catechin as a phytomarker.

BACKGROUND AND AIM: The major groups of phytonutrients found in plants include polyphenols, flavonoids, terpenes, amines, etc., all of which are observed to have potential anti-allergic activity. In this study, we evaluated the anti-allergic activity of the standardized extract of Albizia lebbeck with respect to the catechin, a polyphenolic phytomarker. MATERIAL AND METHODS: The percentage of catechin in the ethanolic extract was found to be 14.72 mg/g. We administered Albizia lebbeck (50-300 mg/kg) and 50 mg/kg of catechin to mice to evaluate the mast cell stabilization and estimation of histamine elevation in the plasma. RESULTS AND CONCLUSION: Results support the conclusion that Albizia lebbeck at different concentrations has got potent mast cell stabilizing property and the IC(50) value of Albizia lebbeck was found to be 85 microg/ml. This inhibitory potential of catechin from Albizia lebbeck is perhaps due to modulation of two important effector's functions, histamine release and cytokine expression of antigen -IgE activated mast cells.

PARIS-DJ-1 Interaction Regulates Mitochondrial Functions in Cardiomyocytes, Which Is Critically Important in Cardiac Hypertrophy.

Mitochondrial dysfunction is one of the major pathological attributes of cardiac hypertrophy and is associated with reduced expression of PGC1α in cardiomyocytes. However, the transcriptional regulation of PGC1α remains elusive. Here, we show that parkin interacting substrate (PARIS), a KRAB zinc finger protein, prevented PGC1α transcription despite the induction of cardiomyocytes with hypertrophic stimuli. Moreover, PARIS expression and its nuclear localization are enhanced in hypertrophy both in vitro and in vivo Knocking down PARIS resulted in mitochondrial biogenesis and improved respiration and other biochemical features that were compromised during hypertrophy. Furthermore, a PARIS-dependent proteome showed exclusive binding of a deSUMOylating protein called DJ-1 to PARIS in control cells, while this interaction is completely abrogated in hypertrophied cells. We further demonstrate that proteasomal degradation of DJ-1 under oxidative stress led to augmented PARIS SUMOylation and consequent repression of PGC1α promoter activity. SUMOylation-resistant mutants of PARIS failed to repress PGC1α, suggesting a critical role for PARIS SUMOylation in hypertrophy. The present study, therefore, proposes a novel regulatory pathway where DJ-1 acts as an oxidative stress sensor and contributes to the feedback loop governing PARIS-mediated mitochondrial function.

Novel Mechanism of Cholesterol Transport by ABCA5 in Macrophages and Its Role in Dyslipidemia.

Cholesterol homeostasis results from a delicate interplay between influx and efflux of free cholesterol primarily mediated by ABCA1. Here we report downregulation of ABCA1 in hyper-cholesterol conditions in macrophages, which might be responsible for compromised reverse cholesterol transport and hyperlipidemia. Surprisingly, this is countered by the upregulation of a lesser known family member ABCA5 to maintain cholesterol efflux. The relative contribution of ABCA1 and ABCA5 toward cholesterol efflux was evaluated and revealed ABCA5 as the primary efflux mediator under high cholesterol load. These observations were correlated to cholesterol load in circulation in vivo, and we observed an inverse expression profile in mice models of atherosclerosis (ApoE-/-) and hyperlipidemia (PPARα-/-) in response to high cholesterol diet. Observations were further validated in human plasma samples. Simulation studies revealed a unique conformation of ABCA5 proposing a favored route for cholesterol loading onto high-density lipoproteins for reverse cholesterol transport. Thus, our study implicates a functional complementation between these two transporters, formulating an efficient strategy to maintain efflux in cholesterol excess conditions in macrophages.