TGF-ß1 decreases CHOP expression and prevents cardiac fibroblast apoptosis induced by endoplasmic reticulum stress.

Transforming growth factor-beta 1 (TGF-ß1) is a cytokine with marked pro-fibrotic action on cardiac fibroblasts (CF). TGF-ß1 induces CF-to-cardiac myofibroblast (CMF) differentiation, defined by an increase in α-smooth muscle cells (α-SMA), collagen secretion and it has a cytoprotective effect against stimuli that induce apoptosis. In the Endoplasmic Reticulum (ER) lumen, misfolded protein accumulation triggers ER stress and induces apoptosis, and this process plays a critical role in cell death mediated by Ischemia/Reperfusion (I/R) injury and by ER stress inducers, such as Tunicamycin (Tn). Here, we studied the regulation of CHOP, a proapoptotic ER-stress-related transcription factor in CF under simulated I/R (sI/R) or exposed to Tn. Even though TGF-ß1 has been shown to participate in ER stress, its regulatory effect on CF apoptosis and ER stress-induced by sI/R or TN has not been evaluated yet. CF from neonatal rats were exposed to sI/R, and cell death was evaluated by cell count and apoptosis by flow cytometry. ER stress was assessed by western blot against CHOP. Our results evidenced that sI/R (8/24) h or Tn triggers CF apoptosis and an increase in CHOP protein levels. TGF-ß1 pre-treatment partially prevented apoptosis induced by sI/R or Tn. Furthermore, TGF-ß1 pre-treatment completely prevented CHOP increase by sI/R or Tn. Additionally, we found a decrease in α-SMA expression induced by sI/R and in collagen secretion induced by Tn, which were not prevented by TGF-ß1 treatment. In conclusion, TGF-ß1 partially protects CF apoptosis induced by sI/R or Tn, through a mechanism that would involve ER stress.

In cardiac fibroblasts, interferon-beta attenuates differentiation, collagen synthesis, and TGF-ß1-induced collagen gel contraction.

Cardiac fibroblasts (CF) play a key role in the homeostasis of the extracellular matrix in cardiac tissue and are newly recognized as inflammatory supporter cells. Besides, CF-to-Cardiac myofibroblast differentiation is commanded by TGF-b, through SMAD signaling pathways, and these last cells are strongly implicated in cardiac fibrosis. In the heart IFN-ß is produced by CF; however, the role of IFN-ß, STAT proteins, and STAT-homo or heterodimers in the regulation of CF function with or without a fibrotic environment is unknown. CF were isolated from hearts of adult rats, and by western blot analysis we studied STAT1, STAT2, and STAT3 phosphorylation and through specific siRNA against these proteins we analyzed their role in CF functions such as differentiation (α-SMA expression); and pro-collagen type-I synthesis and secretion expression levels; collagen gels contraction and CF migration. In cultured adult rats CF, IFN-ß increases phosphorylation of STAT1, STAT2, and STAT3. Both STAT1 and STAT2 were involved in decreasing α-SMA and CF migration induced by TGF-ß1. Also, IFN-ß through STAT1 regulated pro-collagen type-I protein expression levels, and collagen gels contraction induced by TGF-ß1. STAT3 was not involved in any effects of IFN-ß studied. In conclusion, IFN-ß through STAT1 and STAT2 shows antifibrotic effects on CF TGF-ß1-treated, whereas STAT3 did not participate in such effect.

Cardiac fibroblasts as sentinel cells in cardiac tissue: Receptors, signaling pathways and cellular functions.

Cardiac fibroblasts (CF) not only modulate extracellular matrix (ECM) proteins homeostasis, but also respond to chemical and mechanical signals. CF express a variety of receptors through which they modulate the proliferation/cell death, autophagy, adhesion, migration, turnover of ECM, expression of cytokines, chemokines, growth factors and differentiation into cardiac myofibroblasts (CMF). Differentiation of CF to CMF involves changes in the expression levels of various receptors, as well as, changes in cell phenotype and their associated functions. CF and CMF express the ß2-adrenergic receptor, and its stimulation activates PKA and EPAC proteins, which differentially modulate the CF and CMF functions mentioned above. CF and CMF also express different levels of Angiotensin II receptors, in particular, AT1R activation increases collagen synthesis and cell proliferation, but its overexpression activates apoptosis. CF and CMF express different levels of B1 and B2 kinin receptors, whose stimulation by their respective agonists activates common signaling transduction pathways that decrease the synthesis and secretion of collagen through nitric oxide and prostacyclin I2 secretion. Besides these classical functions, CF can also participate in the inflammatory response of cardiac repair, through the expression of receptors commonly associated to immune cells such as Toll like receptor 4, NLRP3 and interferon receptor. The activation by their respective agonists modulates the cellular functions already described and the release of cytokines and chemokines. Thus, CF and CMF act as sentinel cells responding to a plethora of stimulus, modifying their own behavior, and that of neighboring cells.

H2 O2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and Ca(2+) signals in human periodontal fibroblasts.

BACKGROUND: The mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown. OBJECTIVE: To determine the effect of H2 O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFκB) and Ca(2+) -mediated signals in human periodontal ligament fibroblasts. MATERIAL AND METHODS: Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5-10 µm) of H2 O2 or control media. NFκB involvement was evaluated through immunofluorescence of p65 subunit, using the NFκB blocking peptide SN50 and catalase. Ca(2+) signals were analyzed by loading the cells with Fluo4-AM and recording the fluorescence changes in a confocal microscope before and after the addition of H2 O2 . 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl was used to chelate intracellular Ca(2+) . The activity and levels of MMP-2 and MMP-9 were analyzed by gelatin zymogram and densitometric scanning, and enzyme-linked immunosorbent assay, respectively. Statistical analysis was performed with stata V11.1 software using the ANOVA test. RESULTS: H2 O2 at concentrations of 2.5-5 µm induced Ca(2+) signaling and NFκB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2 O2 at 2.5-5 µm significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls. CONCLUSION: Sublethal H2 O2 induces Ca(2+) -dependent NFκB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.

4-Phenylbutyric acid prevent cytotoxicity induced by thapsigargin in rat cardiac fibroblast.

Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail. Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot. In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA. Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.

Simvastatin alters fibroblastic cell responses involved in tissue repair.

BACKGROUND AND OBJECTIVE: Statins have been used to control hypercholesterolemia. However, these drugs also exert pleiotropic effects that include the modulation of inflammation and cell signaling. The present study has analyzed the effects of simvastatin on several cell responses involved in tissue repair, including cell adhesion, cell migration and invasion, actin cytoskeleton remodeling and cell viability. MATERIAL AND METHODS: Primary cultures of gingival fibroblasts were stimulated with simvastatin. Cell adhesion was evaluated using a colorimetric assay. Cell spreading was evaluated microscopically. Cell migration and invasion were assessed using a scratch wound-healing assay and a bicameral cell culture system, respectively. Changes in actin cytoskeleton and focal adhesion assembly were evaluated through immunofluorescence for actin, vinculin and active ß1 integrin. Rac activation was evaluated by means of a pull-down assay. Cell viability was assessed using a colorimetric assay that determines mitochondrial functionality. Data analysis was performed using the Mann-Whitney U-test. RESULTS: Simvastatin diminished cell adhesion and spreading over a fibronectin matrix. It also altered the closure of scratch wounds induced on cell monolayers and cell invasion through a Transwell system. Simvastatin-treated cells displayed an altered lamellipodia with poorly developed focal adhesion contacts and reduced levels of ß1 integrin activation. During cell spreading, simvastatin diminished Rac activation. CONCLUSION: The present study shows that simvastatin may alter cell migration by disrupting the cell signaling networks that regulate the actin cytoskeleton dynamics. This mechanism may affect the response of gingival mesenchymal cells during wound healing.

Reversal of angiotensin II-stimulated collagen gel contraction in cardiac fibroblasts by aminopeptidase inhibition.

The purpose of this investigation was to determine whether aminopeptidase inhibition could affect the angiotensin II-stimulated collagen gel contraction in basal (control) and TGF-beta1-treated cardiac fibroblasts (or myofibroblasts). The tested aminopeptidase inhibitors were the broad range aminopeptidase inhibitor bestatin, the specific inhibitor of alanine aminopeptidase leuhistin, and the specific inhibitor of arginine aminopeptidase arphamenine A. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and incubated with(out) 400 pmol/L TGF-beta1 in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). These fibroblasts were then further incubated in a floating collagen gel lattice with the tested products (angiotensin II, bestatin, leuhistin, or arphamenine A) for 3 days in DMEM without FBS. The contraction of the collagen gel lattice by cardiac fibroblasts was determined by measuring the gel volume with tritiated water. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Angiotensin II (100 nmol/L) reduced the gel volume in control and TGF-beta1-treated cardiac fibroblasts. The angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated fibroblasts was almost completely reversed by leuhistin and arphamenine A (100 micromol/L). Bestatin (100 micromol/L) only partially inhibited the angiotensin II-stimulated collagen gel contraction in control fibroblasts, although it did not affect the angiotensin II-induced contraction in TGF-beta1-treated fibroblasts. In control and TGF-beta1-treated cardiac fibroblasts, 100 micromol/L leuhistin or arphamenine A only partially inhibited alanine aminopeptidase activity, whereas bestatin (100 micromol/L) completely inhibited the alanine aminopeptidase activity. Arginine aminopeptidase activity was only partially inhibited by leuhistin and arphamenine A at 100 micromol/L in control and TGF-beta1-treated fibroblasts. Bestatin, however, completely blocked the arginine aminopeptidase activity in control fibroblasts and only partially in TGF-beta1-treated fibroblasts at 100 micromol/L. Our data suggest that both alanine and arginine aminopeptidases are involved in the reversal of the angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated cardiac fibroblasts or myofibroblasts.

A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes.

In all cell types, the maintenance of normal cell volume is an essential homeostatic function. Relatively little is known about the induction of apoptosis by hyperosmotic stress and its molecular mechanism in terminally differentiated cardiac myocytes. We compared the apoptotic response of cultured neonatal rat cardiomyoctes to hyperosmotic stress by sorbitol (SOR) with those induced by doxorubicin (Doxo) or angiotensin II (Ang II). We also examined the apoptotic-signaling pathway stimulated by the hyperosmotic stress. Apoptosis was assessed by the observation of: (1) cell viability, (2) DNA fragmentation detected by the TUNEL method and by agarose gel electrophoresis, and (3) poly(ADP-ribose)polymerase (PARP) degradation, and Bcl-XS and Bcl-XL levels by Western blot analysis. Exposure of cardiomyocytes to 0.3 M SOR for 24 h resulted in decreased cell viability and increased generation of oligosomal DNA fragments (2.5-fold of controls). At this time, 83 +/- 5% of SOR-treated myocytes were TUNEL-positive (vs 23.7 +/- 6.8% in controls; P<0.01). PARP levels also decreased by approximately 42% when cardiac myocytes were exposed to SOR. Hyperosmotic stress induced a more rapid and stronger apoptotic response in cardiomyocytes than Doxo or Ang II. In addition, SOR increased 3.2-fold Bcl-XS proapoptotic protein without changes in Bcl-XL antiapoptotic protein levels and in the p53-transactivating activity. Taken together, these results strongly suggest that hyperosmotic stress triggers cardiac myocyte apoptosis in a p53-independent manner, being earlier and stronger than apoptosis induced by Doxo and Ang II.

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes.

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.

IGF-1 regulates apoptosis of cardiac myocyte induced by osmotic-stress.

Insulin-like growth factor-1 (IGF-1) is a natural protectant of cardiac myocytes that has been shown to improve cardiac function. The role of IGF-1 in attenuating apoptosis induced by osmotic stress (sorbitol, SOR) or by other known apoptotic stimuli (doxorubicin, angiotensin II, and serum withdrawal) was determined in cultured cardiac myocytes. After 6 h of exposure to SOR, apoptosis was initiated, concomitant with a decrease in cell survival and increases in poly-[ADP-ribose] polymerase (PARP) degradation and DNA fragmentation. These effects were maximal after 24 h. IGF-1 partially attenuated apoptosis induced by sorbitol but not that induced by angiotensin II, doxorubicin, or serum withdrawal. In cells preincubated with IGF-1 before the addition of SOR, we detected an increase in the number of viable cells, a decrease in the generation of DNA fragments on agarose gel electrophoresis and in the percentage of positive TUNEL cells, and a reduction on PARP levels. These results suggest that IGF-1 prevents apoptosis induced by osmotic stress in cardiac myocytes but not apoptosis induced by doxorubicin and angiotensin II.

Scavenging of the one-electron reduction product from nisoldipine with relevant thiols: electrochemical and EPR spectroscopic evidences.

PURPOSE: To determine the formation of the one-electron reduction product from nisoldipine and its reactivity with relevant thiols in mixed medium. METHODS: Cyclic voltammetry (CV) and electron paramagnetic resonance (EPR) techniques were used to determine the one-electron reduction product corresponding to the nitro radical anion. CV was employed to assess both the rate constants corresponding to the decay of the radicals and its interaction with relevant thiols. RESULTS: The nisoldipine radical anion follows second order kinetics, with an association rate constant of 283+/-16 l mol(-1) sec(-1). Nitro radical anion from nisoldipine significantly reacted with thiol compounds. This reactivity was significantly higher than the natural decay of the radical in mixed medium. EPR spectra recorded in situ using DMF/ 0.1 N NaOH (pH 13) confirmed the formation of the nitro radical anion, giving a well-resolved spectra in 35 lines using 0.1 G modulation. CONCLUSIONS: Electrochemical and EPR data indicated that all the tested thiols scavenged the nitro radical anion from nisoldipine. The following tentative order of reactivity towards the thiols can be proposed: cysteamine approximately glutathione > N-acetylcysteine > captopril > penicillamine.

Antioxidant effects of 1,4-dihydropyridine and nitroso aryl derivatives on the Fe+3/ascorbate-stimulated lipid peroxidation in rat brain slices.

1. Lipid peroxidation in rat brain slices was induced by Fe+3/ascorbate. 2. Brain lipid peroxidation, as measured by malondialdehyde formation, was inhibited by all the tested nitro aryl 1,4-dihydropyridine derivatives over a wide range of concentrations. The time-course antioxidant effects of the most representative agents were assessed. On the basis of both time-course and IC50 experiments the tentative order of antioxidant activity on rat brain slices could be: nicardipine>nisoldipine> (R,S/S,R)-furnidipine > (R,R/S,S)-furnidipine>nitrendipine>nimodipine> nifedipine. 3. 1,4-Dihydropyridine derivatives that lack of a nitro group in the molecule (isradipine, amlodipine) also inhibited lipid peroxidation in rat brain slices but at higher concentrations than that of nitro-substituted derivatives. 4. All the tested nitroso aryl derivatives [2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicar. boxylic acid dimethyl ester (NTP), nitrosotoluene, nitrosobenzene] were more potent inhibitors of lipid peroxidation than were the parent nitro compounds. In conclusion, on the basis of the IC50 values determined, the rank order of antioxidant potency for these derivatives can be established as: ortho-nitrosotoluene>NTP>nitrosobenzene.

Isradipine and lacidipine: effects in vivo and in vitro on Trypanosoma cruzi epimastigotes.

1. Isradipine and lacidipine, two new drugs that are members of the nitro-aryl-1,4-dihydropyridine family, produced inhibition of both growth cultures and oxygen consumption on epimastigotes of Trypanosoma cruzi Tulahuen strain, at micromolar concentrations. 2. Isradipine was found to be the most potent derivative in both, in growth cultures (I50 = 20.8 microM) and in vivo oxygen uptake (I50 = 31.1 microM). 3. Diltiazem and verapamil, two well-known calcium channel antagonists, lacked inhibitory activity, even at a 100 microM concentration. 4. The present findings indicate that the trypanocide effects exerted by isradipine and lacidipine are not related with a disruption of the calcium homeostasis of the parasite.