TNFR1 Contributes to Activation-Induced Cell Death of Pathological CD4+ T Lymphocytes During Ischemic Heart Failure.

CD4+ T cells turn pathological during heart failure (HF). We show that the expression of tumor necrosis factor (TNF)-α and tumor necrosis factor receptor (TNFR1) increases in HF-activated CD4+ T cells. However, the role of the TNF-α/TNFR1 axis in T-cell activation/proliferation is unknown. We show that TNFR1 neutralization during T-cell activation (ex vivo) or the loss of TNFR1 in adoptively transferred HF-activated CD4+ T cells (in vivo) augments their prosurvival and proliferative signaling. Importantly, TNFR1 neutralization does not affect CD69 expression or the pathological activity of HF-activated TNFR1-/- CD4+ T cells. These results show that during HF TNFR1 plays an important role in quelling prosurvival and proliferative signals in CD4+ T cells without altering their pathological activity.

Nanocurcumin accords protection against acute hypobaric hypoxia induced lung injury in rats.

Decline in oxygen availability experienced under hypobaric hypoxia (HH) mediates imbalance in lung fluid clearance and is a causative agent of acute lung injury. Here, we investigate the pathological events behind acute HH mediated lung injury and assess the therapeutic efficacy of nanocurcumin in its amelioration. We assess the protective efficacy of nanotized curcumin (nanocurcumin) in ameliorating HH induced lung injury and compare to curcumin. Rats exposed to acute HH (6, 12, 24, 48 and 72 h) were subjected to histopathology, blood-gas analysis and clinical biochemistry, cytokine response and redox damage. HH induced lung injury was analysed using markers of lung injury due to pulmonary vasoconstriction (ET-1/2/3 and endothelin receptors A and B) and trans-vascular fluid balance mediator (Na+/K+ ATPase). The protective efficacy of nanocurcumin was analysed by examination of Akt/Erk signalling cascade by western blot. HH induced lung injury was associated with discrete changes in blood analytes, differential circulatory cytokine response and severe pulmonary redox damages. Up-regulation of ET-1/2/3 and its receptors along with down-regulation of Na+/K+ ATPase confirmed defective pulmonary fluid clearance which promoted edema formation. Nanocurcumin treatment prevented lung edema formation and restored expression levels of ET-1/2/3 and its receptors while restoring the blood analytes, circulatory cytokines and pulmonary redox status better than curcumin. Modulation in Akt/Erk signalling pathway in rat lungs under HH confirmed the protective efficacy of nanocurcumin.

Nanocurcumin Prevents Hypoxia Induced Stress in Primary Human Ventricular Cardiomyocytes by Maintaining Mitochondrial Homeostasis.

Hypoxia induced oxidative stress incurs pathophysiological changes in hypertrophied cardiomyocytes by promoting translocation of p53 to mitochondria. Here, we investigate the cardio-protective efficacy of nanocurcumin in protecting primary human ventricular cardiomyocytes (HVCM) from hypoxia induced damages. Hypoxia induced hypertrophy was confirmed by FITC-phenylalanine uptake assay, atrial natriuretic factor (ANF) levels and cell size measurements. Hypoxia induced translocation of p53 was investigated by using mitochondrial membrane permeability transition pore blocker cyclosporin A (blocks entry of p53 to mitochondria) and confirmed by western blot and immunofluorescence. Mitochondrial damage in hypertrophied HVCM cells was evaluated by analysing bio-energetic, anti-oxidant and metabolic function and substrate switching form lipids to glucose. Nanocurcumin prevented translocation of p53 to mitochondria by stabilizing mitochondrial membrane potential and de-stressed hypertrophied HVCM cells by significant restoration in lactate, acetyl-coenzyme A, pyruvate and glucose content along with lactate dehydrogenase (LDH) and 5' adenosine monophosphate-activated protein kinase (AMPKα) activity. Significant restoration in glucose and modulation of GLUT-1 and GLUT-4 levels confirmed that nanocurcumin mediated prevention of substrate switching. Nanocurcumin prevented of mitochondrial stress as confirmed by c-fos/c-jun/p53 signalling. The data indicates decrease in p-300 histone acetyl transferase (HAT) mediated histone acetylation and GATA-4 activation as pharmacological targets of nanocurcumin in preventing hypoxia induced hypertrophy. The study provides an insight into propitious therapeutic effects of nanocurcumin in cardio-protection and usability in clinical applications.

Nanocurcumin protects cardiomyoblasts H9c2 from hypoxia-induced hypertrophy and apoptosis by improving oxidative balance.

Hypoxia-induced cardiomyocyte hypertrophy is evident; however, the distinct molecular mechanism underlying the oxidative stress-mediated damages to cardiomyocytes remains unknown. Curcumin (diferuloylmethane) is known for anti-hypertrophic effects, but low bioavailability makes it unsuitable to exploit its pharmacological properties. We assessed the efficacy of nanotized curcumin, i.e. nanocurcumin, in ameliorating hypoxia-induced hypertrophy and apoptosis in H9c2 cardiomyoblasts and compared it to curcumin. H9c2 cardiomyoblasts were challenged with 0.5 % oxygen, for 24 h to assess hypoxia-induced oxidative damage, hypertrophy and consequent apoptosis. The molecular mechanism underlying the protective efficacy of nanocurcumin was evaluated in regulating Raf-1/Erk-1/2 apoptosis by caspase-3/-7 pathway and oxidative stress. Nanocurcumin ameliorated hypoxia-induced hypertrophy and apoptosis in H9c2 cells significantly (p ≤ 0.01), by downregulating atrial natriuretic factor expression, caspase-3/-7 activation, oxidative stress and stabilizing hypoxia-inducible factor-1α (HIF-1α) better than curcumin. Nanocurcumin provides insight into its use as a potential candidate in curing hypoxia-induced cardiac pathologies by restoring oxidative balance.

Novel vascular endothelial growth factor blocker improves cellular viability and reduces hypobaric hypoxia-induced vascular leakage and oedema in rat brain.

Vascular endothelial growth factor (VEGF) is an important cerebral angiogenic and permeability factor under hypoxia. There is a need to find effective molecules that may ameliorate hypoxia-induced cerebral oedema. In silico identification of novel candidate molecules that block VEGF-A site were identified and validated with a Ramachandran plot. The active site residues of VEGF-A were detected by Pocketfinder, CASTp, and DogSiteScorer. Based on in silico data, three VEGF-A blocker (VAB) candidate molecules (VAB1, VAB2, and VAB3) were checked for improvement in cellular viability and regulation of VEGF levels in N2a cells under hypoxia (0.5% O2 ). Additionally, the best candidate molecule's efficacy was assessed in male Sprague-Dawley rats for its ameliorative effect on cerebral oedema and vascular leakage under hypobaric hypoxia 7260 m. All experimental results were compared with the commercially available VEGF blocker sunitinib. Vascular endothelial growth factor-A blocker 1 was found most effective in increasing cellular viability and maintaining normal VEGF levels under hypoxia (0.5% oxygen) in N2a cells. Vascular endothelial growth factor-A blocker 1 effectively restored VEGF levels, decreased cerebral oedema, and reduced vascular leakage under hypobaric hypoxia when compared to sunitinib-treated rats. Vascular endothelial growth factor-A blocker 1 may be a promising candidate molecule for ameliorating hypobaric hypoxia-induced vasogenic oedema by regulating VEGF levels.

Amlodipine protects rat ventricular cardiomyoblast H9c2 From hypoxia-induced apoptosis and restores oxidative balance by Akt-1-dependent manner.

BACKGROUND: Hypoxia-induced rise in intracellular calcium concentration is a causative agent of apoptosis and oxidative damage in cardiomyocytes. We examined the efficacy of calcium channel blocker amlodipine in preventing hypoxia-induced apoptosis in H9c2 cells and restoring oxidative balance. METHODS: H9c2 cells were exposed to hypoxia (0.5% oxygen) to evaluate the efficacy of amlodipine in restoring cellular calcium levels. Cellular markers of apoptosis (Bax/Bcl2 and caspase-3, -7, and -9) and pro-survival markers (Akt/p-Akt levels) were evaluated under hypoxia. Redox damage was evaluated by assessing markers of oxidative damage, that is, glutathione reduced, glutathione oxidized, lipid peroxidation, reactive oxygen species, and manganese superoxide dismutase activity. Cellular adenosine triphosphate (ATP) pool and AMPKα levels were measured to evaluate regulation of metabolism under hypoxia. RESULTS: Amlodipine treatment at 25 nM prevented apoptosis and restored cellular calcium levels and oxidative damage in cardiomyocytes. Stabilization of caspase-3, -7, and -9 along with restoration of Akt/p-Akt levels depicted pro-survival efficacy of amlodipine. Also, restoration of cellular ATP and AMPKα levels indicates that amlodipine prevents cardiomyocytes from hypoxia-induced metabolic stress. CONCLUSIONS: Amlodipine thus protects H9c2 cells from hypoxia-induced apoptosis by regulating Akt/p-Akt-mediated caspase-3, -7, and -9 activation and restoring cellular ATP and redox status.

In-silico screening and in-vitro validation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) inhibitors.

VEGFR-2 tyrosine kinase receptor draws attention of the scientific fraternity in drug discovery for its important role in cancer, cardiopulmonary, cardiovascular diseases etc. Hence there is a need for novel VEGFR-2 inhibitors screening and testing for their biological activities. The 3D-structure was collected from PDB and stability was checked by using WHATIF and PROCHECK programs and subjected for virtual screening on Zinc database. We used virtual screening method to screen new VEGFR-2 blocker molecules based on their binding energies and then docked with active site on the receptor with the help of AUTODOCK software. Based on the results obtained top three molecules (VRB1-3) were selected and tested in Cardiomyocytes H9c2 cells for cell viability under hypoxic condition. The invitro studies showed VRB2 as the best molecule among the selected three molecules as well as with a standard commercial drug Sunitinib.