Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria

<p>Abstract</p> <p>Background</p> <p>The sun-dried rind of the immature fruit of pomegranate (<it>Punica granatum</it>) is presently used as a herbal formulation (OMARIA, Orissa Malaria Research Indigenous Attempt) in Orissa, India, for the therapy and prophylaxis of malaria. The pathogenesis of cerebral malaria, a complication of the infection by <it>Plasmodium falciparum</it>, is an inflammatory cytokine-driven disease associated to an up-regulation and activity of metalloproteinase-9 and to the increase of TNF production. The <it>in vitro </it>anti-plasmodial activity of <it>Punica granatum (Pg) </it>was recently described. The aim of the present study was to explore whether the anti-malarial effect of OMARIA could also be sustained via other mechanisms among those associated to the host immune response.</p> <p>Methods</p> <p>From the methanolic extract of the fruit rind, a fraction enriched in tannins (<it>Pg</it>-FET) was prepared. MMP-9 secretion and expression were evaluated in THP-1 cells stimulated with haemozoin or TNF. The assays were conducted in the presence of the <it>Pg</it>-FET and its chemical constituents ellagic acid and punicalagin. The effect of urolithins, the ellagitannin metabolites formed by human intestinal microflora, was also investigated.</p> <p>Results</p> <p><it>Pg</it>-FET and its constituents inhibited the secretion of MMP-9 induced by haemozoin or TNF. The effect occurred at transcriptional level since MMP-9 mRNA levels were lower in the presence of the tested compounds. Urolithins as well inhibited MMP-9 secretion and expression. <it>Pg</it>-FET and pure compounds also inhibited MMP-9 promoter activity and NF-kB-driven transcription.</p> <p>Conclusions</p> <p>The beneficial effect of the fruit rind of <it>Punica granatum </it>for the treatment of malarial disease may be attributed to the anti-parasitic activity and the inhibition of the pro-inflammatory mechanisms involved in the onset of cerebral malaria.</p

Functional Alterations of Ion Channels From Cardiac Fibroblasts in Heart Diseases

In an aged population, cardiovascular disease is the leading cause of fatality and morbidity. Age-related fibrotic remodeling of the heart contributes to progressive myocardial dysfunction. Cardiac fibroblasts (CF), responsible for the maintenance of extracellular matrix and fibrosis process, play an important role in cardiac health and disease. CFs influence myocardial function by their chemical, electrical and mechanical interactions with cardiomyocytes through extracellular matrix deposition or secretion of cytokines and growth factors. These, in turn, are modulated by ion channels, macromolecular pores in the plasma membrane that allow selective ionic fluxes of major ions like K+, Ca2+, Na+ or Cl-, which affect membrane potential and cellular signal transduction. The importance of ion channels in modulating various functions of CFs, including proliferation, differentiation, secretion and apoptosis, is being recognized from recent studies of CFs from animal models and tissue from patients with various cardiac pathologies. Understanding the role of ion channels in CFs under physiological conditions and their alterations in age-related cardiac diseases may help facilitate development of novel therapeutic strategies to limit cardiac fibrosis and its adverse effect on myocardial function. This narrative review summarizes the knowledge gained thus far on ion channels in CFs and their relationship with cardiac diseases in human and experimental animal models

Atrial fibrillation is associated with increased susceptibility of mitochondria to permeability transition pore opening

Background/significance: Although atrial fibrillation (AF) is known to result in progressive electrical, contractile and structural remodeling of the atria with gradual cell loss and replacement fibrosis, the molecular basis for the progressive structural alterations is not fully elucidated. Since mitochondria play an essential role in regulation of cell death through the opening of mitochondrial permeability transition pore (mPTP), we hypothesized that the susceptibility of mitochondria from patients with AF to PTP opening is increased contributing to the substrate that promote progression of AF to long-lasting forms. Purpose: The aim of the study was to characterize the sensitivity of mitochondria to Ca2+-induced mPTP opening in human atria from patients with and without AF. Methods: Freshly removed left atrial appendage tissue from patients undergoing cardiac bypass surgery with (AF) and without history of AF (nAF) was used for mitochondrial isolation. The sensitivity of mitochondria towards Ca2+-induced mPTP opening was assessed by exposing the isolated mitochondria to sequential additions of 10 μM of Ca2+ and monitoring simultaneously abrupt mitochondrial Ca2+ release (Fluo-5N fluorescence), mitochondrial depolarization (Safranin O fluorescence), and swelling of mitochondrial matrix (decrease in light scattering). Differences in the expression of proteins participating in mPTP formation, including voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and phosphate carrier protein (PCP) were determined by Western blotting in isolated mitochondria. The protein expression level was expressed in arbitrary units normalized to mitochondrial protein. Results: The sensitivity of mitochondria to mPTP opening was increased in patients with AF compared to nAF (41±12 vs. 66±8 μM Ca2+). Cyclosporine A, an inhibitor of mPTP, increased tolerance of the mitochondria to Ca2+ loading (74±15 vs. 93±9) and reduced mPTP opening in both group of patients. Increased sensitivity towards mPTP opening in patients with AF was associated with a 2 fold downregulation of the expression of PCP (217±66 vs. 437.6±18) and 2 fold increase in expression of VDAC (428±45 vs. 197±19). A trend towards increased level of expression of ANT was observed in those with AF (900±13 vs. 774±29). Conclusion: The increased sensitivity of mitochondria from AF patients to mPTP opening can be partly explained by selective downregulation of PCP protein, putative component of mPTP with upregulation of regulatory VDAC and ANT proteins

TGF-beta1 induced microRNA-21 (miR-21) up-regulation in human cardiac fibroblast precedes their transformation to myofibroblast

Background/significance: The prevalence of atrial fibrillation (AF) increases with aging and aging-associated diseases. Although a common denominator underlying the substrate for aging-related AF is excessive extracellular matrix deposition, the molecular mechanisms regulating human atrial fibrosis are not fully understood. MicroRNAs (miRNA) are small noncoding regulatory RNAs that control gene expression by translational suppression and disruption of target mRNAs. Increased expression level of atrial microRNA-21 (miR-21) has been demonstrated in animal models and patients with AF with positive correlation with atrial collagen content. However, the time course of miR-21 elevation and fibroblast activation that results in collagen deposition in patients at risk for AF has not been characterized. Purpose: The aim of this study was to delineate the temporal association of changes in the cytokine-induced expression of miR-21 to activation of fibroblast isolated from patients with no history or risk factors for AF, with risk factors, but no AF, and those with AF. Methods: Passage 3 cultured human cardiac fibroblasts isolated from patients with no history of or risk factors (hypertension, heart failure, valve disease or age \u3e45 years) for AF (Group 1), with risk factors but no history of AF (Group 2) and those with AF (Group 3) were used for this study. The effect of TGF-beta1 (5ng/ml), a central pathological mediator of fibrosis, on temporal expression of miR-21 and transformation of fibroblast to myofibroblast was determined using Real-time PCR with miR-21 primers and immunohistochemistry with antibodies against Vimentin as a marker for fibroblast and alpha-smooth muscle actin (alpha-SMA) for myofibroblasts. MiR-21 expression and number of alpha- SMA+ cells was compared between TGF-beta1 untreated and treated fibroblasts. Results: TGF-beta1 exposure increased expression of miR-21 and the number of alpha-SMA+ cells in all groups. In fibroblasts from patient with no history of or risk factors for AF the level of expression of miR-21 reached a peak (350% of baseline) within 24 hours of TGF-beta1 treatment, followed by a time-dependent increase in the mRNA expression of alpha-SMA that reached a peak (400% of baseline) in 48 hours. The baseline protein expression level of alpha-SMA in unstimulated fibroblast isolated from patients with persistent AF was 1.5 fold higher than those with no history of AF and 3.0 fold higher than those with no risk factor or history of AF with relatively smaller increase in alpha- SMA expression following TGF-beta1, suggestive of endogenous activation of fibroblasts compared to those without AF. Conclusion: The temporal relationship of miR-21expression following TGF-beta1 stimulation before fibroblast transformation to myofibroblast is suggestive of a regulatory role of miR-21 in fibroblast activation rather than a secondary effect of myofibroblast on miR-21 expression. Patients with persistent AF appear to have endogenously active fibroblast compared to those without risk factors or history of AF

TGF-beta1 activates mitochondrial biogenesis and energetic remodeling of NIH/3T3 fibroblasts

Background/significance: Differentiation of fibroblasts (FB) into myofibroblasts (myoFB) is the key event in the wound healing process of damaged tissue. Following tissue injury and initial migration and intensive proliferation, FB differentiate into myoFB, cells managing remodeling of extracellular matrix and depositing collagen. Phenotypic switch from proto-myoFB into myoFB is accompanied by remodeling cell metabolism and conversion of non-excitable precursors into the contracting cells capable of synthesis and deposition of structural proteins which are orchestrated by metalloproteinase(s) into extracellular matrix. In addition to remodeling of extracellular matrix myoFB also became excitable cells, capable of mechanical contraction. Purpose: We hypothesized that TGF-beta1 mediated differentiation of naive NIH/3T3 FB into myoFB will be associated with energetic remodeling and increased presence of mitochondria as more efficient source of intracellular ATP. Methods: NIH/3T3 cell line obtained from ATCC. Current study used confocal microscopy, Seahorse Extracellular Flux Respirometry, multiwell plate reader and standard biochemical and biophysical protocols. Vimentin, alpha-SMA and mitochondrial proteins were determined using Western blotting mt-DNA was quantified with Mitochondrial DNA kit; matrix metalloproteinase(s) were determined using Zymogel. Results: TGF-beta1 exposure induced differentiation of naïve NIH 3T3 cells into differentiated myofibroblasts as verified by increased expression of characteristic alpha-SMA from 0.87±0.2 to 1.44±0.27 RU (normalized to housekeeping GAPDH). At the same time, TGF-beta1 exposure increased the number of mitochondria per cell, doubled baseline respiration of differentiated cells, increased average mitochondrial DNA/cell as well as expression of mitochondria specific proteins such as ANT. TGF-beta1 exposure increased baseline respiration of NIH/3T3 cells from 1.13±0.1 nmol O2/min/106 cells in naïve culture to 2.25±0.03 nmol O2/min/106 cells in TGF-beta1 treated cells. Mitochondrial DNA more than doubled from 307±9 μg DNA/106 cells in control to 530±12 μg DNA/106 cells in TGF-beta1 exposed cells and was paralleled by increased expression of mitochondrial specific proteins: VDAC 58 % and ANT by 57 %. Conclusion: TGF-beta1 dependent differentiation of NIH/3T3 fibroblasts results in energetic remodeling and activation of mitochondrial biogenesis due to increased energy demand, which is compensated and fulfilled by increased mitochondrial biogenesis as the source of more efficient source of ATP

Colonic inflammation alters Src kinase-dependent gating properties of single Ca2+ channels via tyrosine nitration

Nitration of L-type calcium channels during colonic inflammation impairs phosphorylation by the tyrosine kinase, Src kinase. This results in decreased calcium currents. The purpose of this study was to determine the mechanism of the downregulation of Ca2+ currents in colonic inflammation. In whole cell voltage clamp of mouse single smooth muscle cells, long-duration depolarization produced noninactivating calcium currents that were significantly reduced by the Src kinase inhibitor, protein phosphatase 2 (PP2). Unitary Ba2+ currents were recorded upon repolarization from positive potentials in cell-attached patches of smooth muscle and hCav1.2b-transfected cells to assess the properties of the single channels attributed to the noninactivating open state. Repolarization to −40 mV from 0 mV resulted in single-channel events with conductance of ∼23 pS. The ensemble average of the tail currents from 1,000 sweeps was 337 ± 27 fA in control and 218 ± 49 fA (P < 0.05) in inflamed cells. Neither open-probability nor open-time constants were significantly different between control and inflamed cells. However, the transition to the open state measured as channel availability was significantly reduced from 19 ± 3% to 6.4 ± 1%. Similarly, peak ensemble average current and channel availability were significantly reduced by PP2 and treatment with peroxynitrite in control cells. Mutation of COOH-terminal tyrosine residues in hCav1.2b Chinese hamster ovarian cells also decreased peak ensemble average tail currents and availability. The present findings suggest that the transition of Ca2+ channels to the noninactivating open state is Src kinase dependent. Tyrosine nitration prevents Src-mediated transitions, leading to decreased calcium currents