Gallic acid triphenylphosphonium derivatives TPP+-C10 and TPP+-C12 inhibit mitochondrial function in Candida albicans exerting antifungal and antibiofilm effects.

AIMS: To evaluate the antifungal and antibiofilm activity of gallic acid derivatives TPP+-C10 and TPP+-C12 and their effects on mitochondrial function on two Candida albicans reference strains (ATCC 90029 and ATCC 10231). METHODS AND RESULTS: First, we determined minimal inhibitory concentration (MIC) using a microdilution assay. Both compounds exerted antifungal effects, and their MICs ranged from 3.9 to 13 µM, with no statistically significant differences between them (P > 0.05, t-test). These concentrations served as references for following assays. Subsequently, we measured oxygen consumption with a Clark electrode. Our observations revealed that both drugs inhibited oxygen consumption in both strains with TPP+-C12 exerting a more pronounced inhibitory effect. We then employed flow cytometry with TMRE as a probe to assess mitochondrial membrane potential. For each strain assayed, the compounds induced a decay in transmembrane potential by 75%-90% compared to the control condition (P < 0.05, ANOVA). Then, we measured ATP levels using a commercial kit. TPP+-C12 showed a 50% decrease of ATP content (P < 0.05 ANOVA), while TPP+-C10 exhibited a less pronounced effect. Finally, we assessed the antibiofilm effect using the MTT reduction assay. Both compounds were effective, but TPP+-C12 displayed a greater potency, requiring a lower concentration to inhibit 50% of biofilms viability (P < 0.05, t-test). CONCLUSIONS: Derivatives of gallic acid linked to a TPP+ group exert antifungal and antibiofilm activity through impairment of mitochondrial function in C. albicans.

Interferon-ß decreases LPS-induced neutrophil recruitment to cardiac fibroblasts.

Introduction: Cardiac fibroblasts (CF) are crucial cells in damaged heart tissues, expressing TLR4, IFN-receptor and responding to lipopolysaccharide (LPS) and interferon-ß (IFN-ß) respectively. While CF interact with immune cells; however, their relationship with neutrophils remains understudied. Additionally, theimpact of LPS and IFN-ß on CF-neutrophil interaction is poorly understood. Methods: Isolated CF from adult rats were treated with LPS, with or without IFN-ß. This study examined IL-8 secretion, ICAM-1 and VCAM-1 expression, and neutrophil recruitment, as well as their effects on MMPs activity. Results: LPS triggered increased IL-8 expression and secretion, along with elevated ICAM-1 and VCAM-1 expression, all of which were blocked by TAK-242. Pre-treatment with IFN-ß countered these LPS effects. LPS treated CF showed higher neutrophil recruitment (migration and adhesion) compared to unstimulated CF, an effect prevented by IFN-ß. Ruxolitinib blocked these IFN-ß anti-inflammatory effects, implicating JAK signaling. Analysis of culture medium zymograms from CF alone, and CF-neutrophils interaction, revealed that MMP2 was mainly originated from CF, while MMP9 could come from neutrophils. LPS and IFN-ß boosted MMP2 secretion by CF. MMP9 activity in CF was low, and LPS or IFN-ß had no significant impact. Pre-treating CF with LPS, IFN-ß, or both before co-culture with neutrophils increased MMP2. Neutrophil co-culture increased MMP9 activity, with IFN-ß pre-treatment reducing MMP9 compared to unstimulated CF. Conclusion: In CF, LPS induces the secretion of IL-8 favoring neutrophils recruitment and these effects were blocked by IFN-. The results highlight that CF-neutrophil interaction appears to influence the extracellular matrix through MMPs activity modulation.

SGK1 is necessary to FoxO3a negative regulation, oxidative stress and cardiac fibroblast activation induced by TGF-ß1.

Cardiac fibroblasts (CFs) activation is a common response to most pathological conditions affecting the heart, characterized by increased cellular secretory capacity and increased expression of fibrotic markers, such as collagen I and smooth muscle actin type alpha (α-SMA). Fibrotic activation of CFs induces the increase in tissue protein content, with the consequent tissue stiffness, diastolic dysfunction, and heart failure. Therefore, the search for new mechanisms of CFs activation is important to find novel treatments for cardiac diseases characterized by fibrosis. In this regard, TGF-ß1, a cytokine with proinflammatory and fibrotic properties, is crucial in the CFs activation and the development of fibrotic diseases, whereas its molecular targets are not completely known. Serum and glucocorticoid-regulated kinase (SGK1) is a protein involved in various pathophysiological phenomena, especially cardiac and renal diseases that curse with fibrosis. Additionally, SGK1 phosphorylates and regulates the activity and expression of several targets, highlighting FoxO3a for its role in the regulation of oxidative stress and CFs activation induced by TGF-ß1. However, the regulation of SGK1 by TGF-ß1 and its role in CFs activation have not been studied. In this work, we evaluate the role of SGK1 in CFs isolated from neonatal Sprague-Dawley rats. The participation of SGK1 in the fibrotic activation of CFs induced by TGF-ß1 was analyzed, using an inhibitor or siRNA of SGK1. In addition, the role of SGK1 on the regulation of FoxO3a and oxidative stress induced by TGF-ß1 was analyzed. Our results indicate that TGF-ß1 increased both the activity and expression of SGK1 in CFs, requiring the activation of MAPKs, ERK1/2, p38 and JNK, while inhibition and silencing of SGK1 prevented TGF-ß1-induced fibrotic activation of CFs. In addition, SGK1 inhibition prevented FoxO3a inactivation and expression reduction, catalase and SOD2 expression decrease, and the increase of oxidative stress induced by TGF-ß1. Taken together, our results position SGK1 as an important regulator of CFs activation driven by TGF-ß1, at least in part, through the regulation of FoxO3a and oxidative stress.

Lipoxin A4 prevents high glucose-induced inflammatory response in cardiac fibroblast through FOXO1 inhibition.

Cardiac cells respond to various pathophysiological stimuli, synthesizing inflammatory molecules that allow tissue repair and proper functioning of the heart; however, perpetuation of the inflammatory response can lead to cardiac fibrosis and heart dysfunction. High concentration of glucose (HG) induces an inflammatory and fibrotic response in the heart. Cardiac fibroblasts (CFs) are resident cells of the heart that respond to deleterious stimuli, increasing the synthesis and secretion of both fibrotic and proinflammatory molecules. The molecular mechanisms that regulate inflammation in CFs are unknown, thus, it is important to find new targets that allow improving treatments for HG-induced cardiac dysfunction. NFκB is the master regulator of inflammation, while FoxO1 is a new participant in the inflammatory response, including inflammation induced by HG; however, its role in the inflammatory response of CFs is unknown. The inflammation resolution is essential for an effective tissue repair and recovery of the organ function. Lipoxin A4 (LXA4) is an anti-inflammatory agent with cytoprotective effects, while its cardioprotective effects have not been fully studied. Thus, in this study, we analyze the role of p65/NFκB, and FoxO1 in CFs inflammation induced by HG, evaluating the anti-inflammatory properties of LXA4. Our results demonstrated that HG induces the inflammatory response in CFs, using an in vitro and ex vivo model, while FoxO1 inhibition and silencing prevented HG effects. Additionally, LXA4 inhibited the activation of FoxO1 and p65/NFκB, and inflammation of CFs induced by HG. Therefore, our results suggest that FoxO1 and LXA4 could be novel drug targets for the treatment of HG-induced inflammatory and fibrotic disorders in the heart.

Angiotensin II Triggers NLRP3 Inflammasome Activation by a Ca2+ Signaling-Dependent Pathway in Rat Cardiac Fibroblast Ang-II by a Ca2+-Dependent Mechanism Triggers NLRP3 Inflammasome in CF.

Angiotensin II (Ang-II) is a widely studied hypertensive, profibrotic, and pro-inflammatory peptide. In the heart, cardiac fibroblasts (CF) express type 1 angiotensin II receptors (AT1R), Toll-like receptor-4 (TLR4), and the NLRP3 inflammasome complex, which play important roles in pro-inflammatory processes. When activated, the NLRP3 inflammasome triggers proteolytic cleavage of pro-IL-1, resulting in its activation. However, in CF the mechanism by which Ang-II assembles and activates the NLRP3 inflammasome remains not fully known. To elucidate this important point, we stimulated TLR4 receptors in CF and evaluated the signaling pathways by which Ang-II triggers the assembly and activity. In cultured rat CF, pro-IL-1ß levels, NLRP3, ASC, and caspase-1 expression levels were determined by Western blot. NLRP3 inflammasome complex assembly was analyzed by immunocytochemistry, whereas by ELISA, we analyzed NLRP3 inflammasome activity and [Formula: see text] release. In CF, Ang-II triggered NLRP3 inflammasome assembly and caspase-1 activity; and in LPS-pretreated CF, Ang-II also triggered [Formula: see text] secretion. These effects were blocked by losartan (AT1R antagonist), U73221 (PLC inhibitor), 2-APB (IP3R antagonist), and BAPTA-AM (Ca2+ chelator) indicating that the AT1R/PLC/IP3R/Ca2+ pathway is involved. Finally, bafilomycin A1 prevented Ang-II-induced [Formula: see text] secretion, indicating that a non-classical protein secretion mechanism is involved. These findings suggest that in CF, Ang-II by a Ca2+-dependent mechanism triggers NLRP3 inflammasome assembly and activation leading to [Formula: see text] secretion through a non-conventional protein secretion mechanism.

Statins change the cytokine profile in Trypanosoma cruzi-infected U937 macrophages and murine cardiac tissue through Rho-associated kinases inhibition.

Introduction: Chronic Chagasic cardiomyopathy (CCC), caused by the protozoan Trypanosoma cruzi, is the most severe manifestation of Chagas disease.CCC is characterized by cardiac inflammation and fibrosis caused by a persistent inflammatory response. Following infection, macrophages secrete inflammatory mediators such as IL-1ß, IL-6, and TNF-α to control parasitemia. Although this response contains parasite infection, it causes damage to the heart tissue. Thus, the use of immunomodulators is a rational alternative to CCC. Rho-associated kinase (ROCK) 1 and 2 are RhoA-activated serine/threonine kinases that regulate the actomyosin cytoskeleton. Both ROCKs have been implicated in the polarization of macrophages towards an M1 (pro-inflammatory) phenotype. Statins are FDA-approved lipid-lowering drugs that reduce RhoA signaling by inhibiting geranylgeranyl pyrophosphate (GGPP) synthesis. This work aims to identify the effect of statins on U937 macrophage polarization and cardiac tissue inflammation and its relationship with ROCK activity during T. cruzi infection. Methods: PMA-induced, wild-type, GFP-, CA-ROCK1- and CA-ROCK2-expressing U937 macrophages were incubated with atorvastatin, or the inhibitors Y-27632, JSH-23, TAK-242, or C3 exoenzyme incubated with or without T. cruzi trypomastigotes for 30 min to evaluate the activity of ROCK and the M1 and M2 cytokine expression and secretion profiling. Also, ROCK activity was determined in T. cruzi-infected, BALB/c mice hearts. Results: In this study, we demonstrate for the first time in macrophages that incubation with T. cruzi leads to ROCK activation via the TLR4 pathway, which triggers NF-κB activation. Inhibition of ROCKs by Y-27632 prevents NF-κB activation and the expression and secretion of M1 markers, as does treatment with atorvastatin. Furthermore, we show that the effect of atorvastatin on the NF-kB pathway and cytokine secretion is mediated by ROCK. Finally, statin treatment decreased ROCK activation and expression, and the pro-inflammatory cytokine production, promoting anti-inflammatory cytokine expression in chronic chagasic mice hearts. Conclusion: These results suggest that the statin modulation of the inflammatory response due to ROCK inhibition is a potential pharmacological strategy to prevent cardiac inflammation in CCC.

Communication Between Cardiomyocytes and Fibroblasts During Cardiac Ischemia/Reperfusion and Remodeling: Roles of TGF-ß, CTGF, the Renin Angiotensin Axis, and Non-coding RNA Molecules.

Communication between cells is a foundational concept for understanding the physiology and pathology of biological systems. Paracrine/autocrine signaling, direct cell-to-cell interplay, and extracellular matrix interactions are three types of cell communication that regulate responses to different stimuli. In the heart, cardiomyocytes, fibroblasts, and endothelial cells interact to form the cardiac tissue. Under pathological conditions, such as myocardial infarction, humoral factors released by these cells may induce tissue damage or protection, depending on the type and concentration of molecules secreted. Cardiac remodeling is also mediated by the factors secreted by cardiomyocytes and fibroblasts that are involved in the extensive reciprocal interactions between these cells. Identifying the molecules and cellular signal pathways implicated in these processes will be crucial for creating effective tissue-preserving treatments during or after reperfusion. Numerous therapies to protect cardiac tissue from reperfusion-induced injury have been explored, and ample pre-clinical research has attempted to identify drugs or techniques to mitigate cardiac damage. However, despite great success in animal models, it has not been possible to completely translate these cardioprotective effects to human applications. This review provides a current summary of the principal molecules, pathways, and mechanisms underlying cardiomyocyte and cardiac fibroblast crosstalk during ischemia/reperfusion injury. We also discuss pre-clinical molecules proposed as treatments for myocardial infarction and provide a clinical perspective on these potential therapeutic agents.

Resolvin-D1 attenuation of angiotensin II-induced cardiac inflammation in mice is associated with prevention of cardiac remodeling and hypertension.

AIMS: Despite the broad pharmacological arsenal to treat hypertension, chronic patients may develop irreversible cardiac remodeling and fibrosis. Angiotensin II, the main peptide responsible for the Renin-Angiotensin-Aldosterone-System, has been closely linked to cardiac remodeling, hypertrophy, fibrosis, and hypertension, and some of these effects are induced by inflammatory mediators. Resolvin-D1 (RvD1) elicits potent anti-inflammatory and pro-resolving effects in various pathological models. In this study, we aimed to examine whether RvD1 ameliorates cardiac remodeling and hypertension triggered by angiotensin II. METHODS AND RESULTS: Alzet® osmotic mini-pumps filled with angiotensin II (1.5 mg/kg/day) were implanted in male C57BL/6 J mice for 7 or 14 days. RvD1 (3 µg/kg/day, i.p) was administered one day after the surgery and during the complete infusion period. Blood pressure and myocardial functional parameters were assessed by echocardiography. At the end of the experimental procedure, blood and heart tissue were harvested, and plasma and histological parameters were studied. After 7 and 14 days, RvD1 reduced the increase of neutrophil and macrophage infiltration triggered by angiotensin II, and also reduced ICAM-1 and VCAM-1 expression levels. RvD1 also reduced cytokine plasma levels (IL-1ß, TNF-α, IL-6, KC, MCP-1), cardiac hypertrophy, interstitial and perivascular fibrosis, and hypertension. CONCLUSIONS: This study unveils novel cardioprotective effects of RvD1 in angiotensin II-induced hypertension and cardiac remodeling by attenuating inflammation and provides insights into a potential clinical application.

FoxO1 is required for high glucose-dependent cardiac fibroblasts into myofibroblast phenoconversion.

In the normal heart, cardiac fibroblasts (CFs) maintain extracellular matrix (ECM) homeostasis, whereas in pathological conditions, such as diabetes mellitus (DM), CFs converse into cardiac myofibroblasts (CMFs) and this CFs phenoconversion increase the synthesis and secretion of ECM proteins, promoting cardiac fibrosis and heart dysfunction. High glucose (HG) conditions increase TGF-ß1 expression and FoxO1 activity, whereas FoxO1 is crucial to CFs phenoconversion induced by TGF-ß1. In addition, FoxO1 increases CTGF expression, whereas CTGF plays an active role in the fibrotic process induced by hyperglycemia. However, the role of FoxO1 and CTGF in CFs phenoconversion induced by HG is not clear. In this study, we investigated the effects of FoxO1 pharmacological inhibition on CFs phenoconversion in both in vitro and ex vivo models of DM. Our results demonstrate that HG induces CFs phenoconversion and FoxO1 activation. Moreover, AS1842856, a pharmacological inhibitor of FoxO1 activity, prevents CFs phenoconversion and CTGF expression increase induced by HG, whereas these results were corroborated by FoxO1 silencing. Additionally, K252a, a pharmacological blocker of CTGF receptor, prevents HG-induced CFs phenoconversion, which was corroborated with CTGF expression knockdown. Furthermore, through CFs isolation from heart of diabetic rats, we showed that hyperglycemia induces FoxO1 activation, the increase of CTGF expression and CFs phenoconversion, whereas the FoxO1 activity inhibition reverses the effects induced by hyperglycemia on CFs. Altogether, our results demonstrate that FoxO1 and CTGF are necessary for CFs phenoconversion induced by HG and suggest that both proteins are likely to become a potential targeted drug for fibrotic response induced by hyperglycemic conditions.

Resolvin D1 and E1 promote resolution of inflammation in rat cardiac fibroblast in vitro.

Cardiac fibroblasts (CFs) have a key role in the inflammatory response after cardiac injury and are necessary for wound healing. Resolvins are potent agonists that control the duration and magnitude of inflammation. They decrease mediators of pro-inflammatory expression, reduce neutrophil migration to inflammation sites, promote the removal of microbes and apoptotic cells, and reduce exudate. However, whether resolvins can prevent pro-inflammatory-dependent effects in CFs is unknown. Thus, the present work was addressed to study whether resolvin D1 and E1 (RvD1 and RvE1) can prevent pro-inflammatory effects on CFs after lipopolysaccharide (LPS) challenge. For this, CFs were stimulated with LPS, in the presence or absence of RvD1 or RvE1, to analyze its effects on intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), monocyte adhesion and the cytokine levels of tumor necrosis factor alpha (TNF-α), interleukin-6(IL-6), interleukin-1beta (IL-1ß), monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10). Our results showed that CFs are expressing ALX/FPR2 and ChemR23, RvD1 and RvE1 receptors, respectively. RvD1 and RvE1 prevent the increase of ICAM-1 and VCAM-1 protein levels and the adhesion of spleen mononuclear cells to CFs induced by LPS. Finally, RvD1, but not RvE1, prevents the LPS-induced increase of IL-6, MCP-1, TNF-α, and IL-10. In conclusion, our findings provide evidence that in CFs, RvD1 and RvE1 might actively participate in the prevention of inflammatory response triggered by LPS.

Role of FoxO3a as a negative regulator of the cardiac myofibroblast conversion induced by TGF-ß1.

Cardiac fibroblasts (CFs) are necessary to maintain extracellular matrix (ECM) homeostasis in the heart. Normally, CFs are quiescent and secrete small amounts of ECM components, whereas, in pathological conditions, they differentiate into more active cells called cardiac myofibroblasts (CMF). CMF conversion is characteristic of cardiac fibrotic diseases, such as heart failure and diabetic cardiomyopathy. TGF-ß1 is a key protein involved in CMF conversion. SMADs are nuclear factor proteins activated by TGF-ß1 that need other proteins, such as forkhead box type O (FoxO) family members, to promote CMF conversion. FoxO1, a member of this family protein, is necessary for TGF-ß1-induced CMF conversion, whereas the role of FoxO3a, another FoxO family member, is unknown. FoxO3a plays an important role in many fibrotic processes in the kidney and lung. However, the participation of FoxO3a in the conversion of CFs into CMF is not clear. In this paper, we demonstrate that TGF-ß1 decreases the activation and expression of FoxO3a in CFs. FoxO3a regulation by TGF-ß1 requires activated SMAD3, ERK1/2 and Akt. Furthermore, we show that FoxO1 is crucial in the FoxO3a regulation induced by TGF-ß1, as shown by overexpressed FoxO1 enhancing and silenced FoxO1 suppressing the effects of TGF-ß1 on FoxO3a. Finally, the regulation of TGF-ß1-induced CMF conversion was enhanced by FoxO3a silencing and suppressed by inhibited FoxO3a degradation. Considering these collective findings, we suggest that FoxO3a acts as a negative regulator of the CMF conversion that is induced by TGF-ß1.

The Association of Ascorbic Acid, Deferoxamine and N-Acetylcysteine Improves Cardiac Fibroblast Viability and Cellular Function Associated with Tissue Repair Damaged by Simulated Ischemia/Reperfusion.

Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2',7'-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphomaextra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-ß1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.

TGF-ß1 induced up-regulation of B1 kinin receptor promotes antifibrotic activity in rat cardiac myofibroblasts.

Cardiac myofibroblast (CMF) are non-muscle cardiac cells that play a crucial role in wound healing and in pathological remodeling. These cells are mainly derived of cardiac fibroblast (CF) differentiation mediated by TGF-ß1. Evidence suggests that bradykinin (BK) regulates cardiac fibroblast function in the heart. Both B1 and B2 kinin receptors (B1R and B2R, respectively) mediate the biological effects of kinins. We recently showed that both receptors are expressed in CMF and its stimulation decreases collagen secretion. Whether TGF-ß1 regulates B1R and B2R expression, and how these receptors control antifibrotic activity in CMF remains poorly understood. In this work, we sought to study, the regulation of B1R expression in cultured CMF mediated by TGF-ß1, and the molecular mechanisms involved in B1R activation on CMF intracellular collagen type-I levels. Cardiac fibroblast-primary culture was obtained from neonatal rats. Hearts were digested and CFs were attached to dishes and separated from cardiomyoctes. CMF were obtained from CF differentiation with TGF-ß1 5 ng/mL. CF and CMF were treated with B1R and B2R agonists and with TGF-ß1 at different times and concentrations, in the presence or absence of chemical inhibitors, to evaluate signaling pathways involved in B1R expression, collagen type-I and prostacyclin levels. B1R and collagen type-I levels were evaluated by western blot. Prostacyclin levels were quantified by an ELISA kit. TGF-ß1 increased B1R expression via TGFß type I receptor kinase (ALK5) activation and its subsequent signaling pathways involving Smad2, p38, JNK and ERK1/2 activation. Moreover, in CMF, the activation of B1R and B2R by their respective agonists, reduced collagen synthesis. This effect was mediated by the canonical signaling pathway; phospholipase C (PLC), protein kinase C (PKC), phospholipase A2 (PLA2), COX-2 activation and PGI2 secretion and its autocrine effect. TGF-ß1 through ALK5, Smad2, p38, JNK and ERK1/2 increases B1R expression; whereas in CMF, B1R and B2R activation share common signaling pathways for reducing collagen synthesis.

IFN-ß Plays Both Pro- and Anti-inflammatory Roles in the Rat Cardiac Fibroblast Through Differential STAT Protein Activation.

Cardiac fibroblasts (CFs) contribute to theinflammatory response to tissue damage, secreting both pro- and anti-inflammatory cytokines and chemokines. Interferon beta (IFN-ß) induces the phosphorylation of signal transducer and activator of transcription (STAT) proteins through the activation of its own receptor, modulating the secretion of cytokines and chemokines which regulate inflammation. However, the role of IFN-ß and STAT proteins in modulating the inflammatory response of CF remains unknown. CF were isolated from adult male rats and subsequently stimulated with IFN-ß to evaluate the participation of STAT proteins in secreting chemokines, cytokines, cell adhesion proteins expression and in their capacity to recruit neutrophils. In addition, in CF in which the TRL4 receptor was pre-activated, the effect of INF-ß on the aforementioned responses was also evaluated. Cardiac fibroblasts stimulation with IFN-ß showed an increase in STAT1, STAT2, and STAT3 phosphorylation. IFN-ß stimulation through STAT1 activation increased proinflammatory chemokines MCP-1 and IP-10 secretion, whereas IFN-ß induced activation of STAT3 increased cytokine secretion of anti-inflammatory IL-10. Moreover, in TLR4-activated CF, IFN-ß through STAT2 and/or STAT3, produced an anti-inflammatory effect, reducing pro-IL-1ß, TNF-α, IL-6, MCP-1, and IP-10 secretion; and decreasing neutrophil recruitment by decreasing ICAM-1 and VCAM-1 expression. Altogether, our results indicate that IFN-ß exerts both pro-inflammatory and anti-inflammatory effects in non-stimulated CF, through differential activation of STAT proteins. When CF were previously treated with an inflammatory agent such as TLR-4 activation, IFN-ß effects were predominantly anti-inflammatory.

Elastin degradation products in acute lung injury induced by gastric contents aspiration.

BACKGROUND: Gastric contents aspiration is a high-risk condition for acute lung injury (ALI). Consequences range from subclinical pneumonitis to respiratory failure, depending on the volume of aspirate. A large increment in inflammatory cells, an important source of elastase, potentially capable of damaging lung tissue, has been described in experimental models of aspiration. We hypothesized that in early stages of aspiration-induced ALI, there is proteolytic degradation of elastin, preceding collagen deposition. Our aim was to evaluate whether after a single orotracheal instillation of gastric fluid, there is evidence of elastin degradation. METHODS: Anesthesized Sprague-Dawley rats received a single orotracheal instillation of gastric fluid and were euthanized 4, 12 and 24 h and at day 4 after instillation (n = 6/group). We used immunodetection of soluble elastin in lung tissue and BALF and correlated BALF levels of elastin degradation products with markers of ALI. We investigated possible factors involved in elastin degradation and evaluated whether a similar pattern of elastin degradation can be found in BALF samples of patients with interstitial lung diseases known to have aspirated. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. RESULTS: We found evidence of early proteolytic degradation of lung elastin. Elastin degradation products are detected both in lung tissue and BALF in the first 24 h and are significantly reduced at day 4. They correlate significantly with ALI markers, particularly PMN cell count, are independent of acidity and have a similar molecular weight as those obtained using pancreatic elastase. Evaluation of BALF from patients revealed the presence of elastin degradation products not present in controls that are similar to those found in BALF of rats treated with gastric fluid. CONCLUSIONS: A single instillation of gastric fluid into the lungs induces early proteolytic degradation of elastin, in relation to the magnitude of alveolar-capillary barrier derangement. PMN-derived proteases released during ALI are mostly responsible for this damage. BALF from patients showed elastin degradation products similar to those found in rats treated with gastric fluid. Long-lasting effects on lung elastic properties could be expected under conditions of repeated instillations of gastric fluid in experimental animals or repeated aspiration events in humans.

Changes in the pattern of fibrosis in the rat lung with repetitive orotracheal instillations of gastric contents: evidence of persistent collagen accumulation.

Recurrent aspiration of gastric contents has been associated with several interstitial lung diseases. Despite this association, the pathogenic role of aspiration in these diseases has been poorly studied and little is known about extracellular matrix (ECM) changes in animal models of repetitive events of aspiration. Our aim was to study the repair phase of lung injury induced by each of several instillations of gastric fluid in Sprague-Dawley rats to evaluate changes in ECM and their reversibility. Anesthetized animals received weekly orotracheal instillations of gastric fluid for 1, 2, 3, and 4 wk and were euthanized at day 7 after last instillation. For reversibility studies, another group received 7 weekly instillations and was euthanized at day 7 or 60 after last instillation. Biochemical and histological measurements were used to evaluate ECM changes. Lung hydroxyproline content increased progressively and hematoxylin and eosin, Masson's trichrome, and alpha-SMA stains showed that after a single instillation, intra-alveolar fibrosis predominated, whereas with repetitive instillations this fibrosis pattern became less prominent and interstitial fibrosis progressively became evident. Both type I and III collagen increased in intra-alveolar and interstitial fibrosis. Imbalance between matrix metalloproteinase-2 (MMP-2) activity and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression was observed, favoring either collagen degradation or accumulation depending on the number of instillations. Caspase-3 activation was also dose dependent. ECM changes were partially reversible at long-term evaluation, since Masson bodies, granulomas, and foreign body giant cells disappeared, whereas interstitial collagen accumulated. In conclusion, repetitive lung instillations of gastric fluid induce progressive fibrotic changes in rat lung ECM that persist at long-term evaluation.

Expression and function of TLR4- induced B1R bradykinin receptor on cardiac fibroblasts.

Cardiac fibroblasts (CF) are key cells for maintaining extracellular matrix (ECM) protein homeostasis in the heart, and for cardiac repair through CF-to-cardiac myofibroblast (CMF) differentiation. Additionally, CF play an important role in the inflammatory process after cardiac injury, and they express Toll like receptor 4 (TLR4), B1 and B2 bradykinin receptors (B1R and B2R) which are important in the inflammatory response. B1R and B2R are induced by proinflammatory cytokines and their activation by bradykinin (BK: B2R agonist) or des-arg-kallidin (DAKD: B1R agonist), induces NO and PGI2 production which is key for reducing collagen I levels. However, whether TLR4 activation regulates bradykinin receptor expression remains unknown. CF were isolated from human, neonatal rat and adult mouse heart. B1R mRNA expression was evaluated by qRT-PCR, whereas B1R, collagen, COX-2 and iNOS protein levels were evaluated by Western Blot. NO and PGI2 were evaluated by commercial kits. We report here that in CF, TLR4 activation increased B1R mRNA and protein levels, as well as COX-2 and iNOS levels. B1R mRNA levels were also induced by interleukin-1α via its cognate receptor IL-1R1. In LPS-pretreated CF the DAKD treatment induced higher responses with respect to those observed in non LPS-pretreated CF, increasing PGI2 secretion and NO production; and reducing collagen I protein levels in CF. In conclusion, no significant response to DAKD was observed (due to very low expression of B1R in CF) - but pre-activation of TLR4 in CF, conditions that significantly enhanced B1R expression, led to an additional response of DAKD.

Acute lung injury by gastric fluid instillation: activation of myofibroblast apoptosis during injury resolution.

BACKGROUND: Gastric contents aspiration in humans has variable consequences depending on the volume of aspirate, ranging from subclinical pneumonitis to respiratory failure with up to 70% mortality. Several experimental approaches have been used to study this condition. In a model of single orotracheal instillation of gastric fluid we have shown that severe acute lung injury evolves from a pattern of diffuse alveolar damage to one of organizing pneumonia (OP), that later resolves leaving normal lung architecture. Little is known about mechanisms of injury resolution after a single aspiration that could be dysregulated with repetitive aspirations. We hypothesized that, in a similar way to cutaneous wound healing, apoptosis may participate in lung injury resolution by reducing the number of myofibroblasts and by affecting the balance between proteases and antiproteases. Our aim was to study activation of apoptosis as well as MMP-2/TIMP-2 balance in the sub-acute phase (4-14 days) of gastric fluid-induced lung injury. METHODS: Anesthesized Sprague-Dawley rats received a single orotracheal instillation of gastric fluid and were euthanized 4, 7 and 14 days later (n = 6/group). In lung tissue we studied caspase-3 activation and its location by double immunofluorescence for cleaved caspase-3 or TUNEL and alpha-SMA. MMP-2/TIMP-2 balance was studied by zymography and Western blot. BALF levels of TGF-ß1 were measured by ELISA. RESULTS: An OP pattern with Masson bodies and granulomas was seen at days 4 and 7 that was no longer present at day 14. Cleaved caspase-3 increased at day 7 and was detected by immunofluorescence in Masson body-alpha-SMA-positive and -negative cells. TUNEL-positive cells at days 4 and 7 were located mainly in Masson bodies. Distribution of cleaved caspase-3 and TUNEL-positive cells at day 14 was similar to that in controls. At the peak of apoptosis (day 7), an imbalance between MMP-2 activity and TIMP-2 expression was produced by reduction in TIMP-2 expression. CONCLUSIONS: Apoptosis is activated in Masson body-alpha-SMA-positive and -negative cells during the sub-acute phase of gastric fluid-induced lung injury. This mechanism likely contributes to OP resolution, by reducing myofibroblast number and new collagen production. In addition, pre-formed collagen degradation is favored by an associated MMP-2/TIMP-2 imbalance.

Heparan sulfate potentiates leukocyte adhesion on cardiac fibroblast by enhancing Vcam-1 and Icam-1 expression.

Cardiac fibroblasts (CF) act as sentinel cells responding to chemokines, cytokines and growth factors released in cardiac tissue in cardiac injury events, such as myocardial infarction (MI). Cardiac injury involves the release of various damage-associated molecular patterns (DAMPs) including heparan sulfate (HS), a constituent of the extracellular matrix (ECM), through the TLR4 receptor activation triggering a strong inflammatory response, inducing leukocytes recruitment. This latter cells are responsible of clearing cell debris and releasing cytokines that promote CF differentiation to myofibroblast (CMF), thus initiating scar formation. CF were isolated from adult male rats and subsequently stimulated with HS or LPS, in the presence or absence of chemical inhibitors, to evaluate signaling pathways involved in ICAM-1 and VCAM-1 expression. siRNA against ICAM-1 and VCAM-1 were used to evaluate participation of these adhesion molecules on leukocytes recruitment. HS through TLR4, PI3K/AKT and NF-ΚB increased ICAM-1 and VCAM-1 expression, which favored the adhesion of spleen mononuclear cells (SMC) and bone marrow granulocytes (PMN) to CF. These effects were prevented by siRNA against ICAM-1 and VCAM-1. Co-culture of CF with SMC increased α-SMA expression, skewing CF towards a pro-fibrotic phenotype, while CF pretreatment with HS partially reverted this effect. CONCLUSION: These data show the dual role of HS during the initial stages of wound healing. Initially, HS enhance the pro-inflammatory role of CF increasing cytokines secretion; and later, by increasing protein adhesion molecules allows the adhesion of SMC on CF, which trigger CF-to-CMF differentiation.