Boldine prevents the inflammatory response of cardiac fibroblasts induced by SGK1-NFκB signaling pathway activation.

Cardiac fibroblasts (CF) are mesenchymal-type cells responsible for maintaining the homeostasis of the heart's extracellular matrix (ECM). Their dysfunction leads to excessive secretion of ECM proteins, tissue stiffening, impaired nutrient and oxygen exchange, and electrical abnormalities in the heart. Additionally, CF act as sentinel cells in the cardiac tissue microenvironment, responding to various stimuli that may affect heart function. Deleterious stimuli induce an inflammatory response in CF, increasing the secretion of cytokines such as IL-1ß and TNF-α and the expression of cell adhesion molecules like ICAM1 and VCAM1, initially promoting damage resolution by recruiting immune cells. However, constant harmful stimuli lead to a chronic inflammatory process and heart dysfunction. Therefore, it is necessary to study the mechanisms that govern CF inflammation. NFκB is a key regulator of the cardiac inflammatory process, making the search for mechanisms of NFκB regulation and CF inflammatory response crucial for developing new treatment options for cardiovascular diseases. SGK1, a serine-threonine protein kinase, is one of the regulators of NFκB and is involved in the fibrotic effects of angiotensin II and aldosterone, as well as in CF differentiation. However, its role in the CF inflammatory response is unknown. On the other hand, many bioactive natural products have demonstrated anti-inflammatory effects, but their role in CF inflammation is unknown. One such molecule is boldine, an alkaloid obtained from Boldo (Peumus boldus), a Chilean endemic tree with proven cytoprotective effects. However, its involvement in the regulation of SGK1 and CF inflammation is unknown. In this study, we evaluated the role of SGK1 and boldine in the inflammatory response in CF isolated from neonatal Sprague-Dawley rats. The involvement of SGK1 was analyzed using GSK650394, a specific SGK1 inhibitor. Our results demonstrate that SGK1 is crucial for LPS- and IFN-γ-induced inflammatory responses in CF (cytokine expression, cell adhesion molecule expression, and leukocyte adhesion). Furthermore, a conditioned medium (intracellular content of CF subject to freeze/thaw cycles) was used to simulate a sterile inflammation condition. The conditioned medium induced a potent inflammatory response in CF, which was completely prevented by the SGK1 inhibitor. Finally, our results indicate that boldine inhibits both SGK1 activation and the CF inflammatory response induced by LPS, IFN-γ, and CF-conditioned medium. Taken together, our results position SGK1 as an important regulator of the CF inflammatory response and boldine as a promising anti-inflammatory drug in the context of cardiovascular diseases.

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.

Senescent cardiac fibroblasts: A key role in cardiac fibrosis.

Cardiac fibroblasts are a cell population that controls the homeostasis of the extracellular matrix and orchestrates a damage response to maintain cardiac architecture and performance. Due to these functions, fibroblasts play a central role in cardiac fibrosis development, and there are large differences in matrix protein secretion profiles between fibroblasts from aged versus young animals. Senescence is a multifactorial and complex process that has been associated with inflammatory and fibrotic responses. After damage, transient cellular senescence is usually beneficial, as these cells promote tissue repair. However, the persistent presence of senescent cells within a tissue is linked with fibrosis development and organ dysfunction, leading to aging-related diseases such as cardiovascular pathologies. In the heart, early cardiac fibroblast senescence after myocardial infarction seems to be protective to avoid excessive fibrosis; however, in non-infarcted models of cardiac fibrosis, cardiac fibroblast senescence has been shown to be deleterious. Today, two new classes of drugs, termed senolytics and senostatics, which eliminate senescent cells or modify senescence-associated secretory phenotype, respectively, arise as novel therapeutical strategies to treat aging-related pathologies. However, further studies will be needed to evaluate the extent of the utility of senotherapeutic drugs in cardiac diseases, in which pathological context and temporality of the intervention must be considered.

Multicellular regulation of miR-196a-5p and miR-425-5 from adipose stem cell-derived exosomes and cardiac repair.

Cardiac transplantation of adipose-derived stem cells (ASC) modulates the post-myocardial infarction (post-MI) repair response. Biomolecules secreted or shuttled within extracellular vesicles, such as exosomes, may participate in the concerted response. We investigated the exosome's microRNAs due to their capacity to fine-tune gene expression, potentially affecting the multicellular repair response. We profiled and quantified rat ASC-exosome miRNAs and used bioinformatics to select uncharacterized miRNAs down-regulated in post-MI related to cardiac repair. We selected and validated miR-196a-5p and miR-425-5p as candidates for the concerted response in neonatal cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages using a high-content screening platform. Both miRNAs prevented cardiomyocyte ischemia-induced mitochondrial dysfunction and reactive oxygen species production, increased angiogenesis, and polarized macrophages toward the anti-inflammatory M2 immunophenotype. Moreover, miR-196a-5p reduced and reversed myofibroblast activation and decreased collagen expression. Our data provide evidence that the exosome-derived miR-196a-5p and miR-425-5p influence biological processes critical to the concerted multicellular repair response post-MI.

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.

Toll-Like Receptor 4 Activation Prevents Rat Cardiac Fibroblast Death Induced by Simulated Ischemia/Reperfusion.

Death of cardiac fibroblasts (CFs) by ischemia/reperfusion (I/R) has major implications for cardiac wound healing. In in vivo models of myocardial infarction, toll-like receptor 4 (TLR4) activation has been reported as a cardioprotector; however, it remains unknown whether TLR4 activation can prevent CF death triggered by simulated I/R (sI/R). In this study, we analyzed TLR4 activation in neonate CFs exposed to an in vitro model of sI/R and explored the participation of the pro-survival kinases Akt and ERK1/2. Simulated ischemia was performed in a free oxygen chamber in an ischemic medium, whereas reperfusion was carried out in normal culture conditions. Cell viability was analyzed by trypan blue exclusion and the MTT assay. Necrotic and apoptotic cell populations were evaluated by flow cytometry. Protein levels of phosphorylated forms of Akt and ERK1/2 were analyzed by Western blot. We showed that sI/R triggers CF death by necrosis and apoptosis. In CFs exposed only to simulated ischemia or only to sI/R, blockade of the TLR4 with TAK-242 further reduced cell viability and the activation of Akt and ERK1/2. Preconditioning with lipopolysaccharide (LPS) or treatment with LPS in ischemia or reperfusion was not protective. However, LPS incubation during both ischemia and reperfusion periods prevented CF viability loss induced by sI/R. Furthermore, LPS treatment reduced the sub-G1 population, but not necrosis of CFs exposed to sI/R. On the other hand, the protective effects exhibited by LPS were abolished when TLR4 was blocked and Akt and ERK1/2 were inhibited. In conclusion, our results suggest that TLR4 activation protects CFs from apoptosis induced by sI/R through the activation of Akt and ERK1/2 signaling pathways.

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.

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.

hsa-miR-4443 inhibits myocardial fibroblast proliferation by targeting THBS1 to regulate TGF-ß1/α-SMA/collagen signaling in atrial fibrillation

Fibrosis caused by the increase in extracellular matrix in cardiac fibroblasts plays an important role in the occurrence and development of atrial fibrillation (AF). The aim of this study was to investigate the role of hsa-miR-4443 in AF, human cardiac fibroblast (HCFB) proliferation, and extracellular matrix remodeling. TaqMan Stem-loop miRNA assay was used to measure hsa-miR-4443 expression in patients with persistent AF (n=123) and healthy controls (n=100). Patients with AF were confirmed to have atrial fibrosis by late gadolinium enhancement. At the cellular level, after hsa-miR-4443 mimic and inhibitor were transfected with HCFBs, proliferation, apoptosis, migration, and invasion were analyzed. Lastly, hsa-miR-4443-targeted gene and transforming growth factor (TGF)-β1/α-SMA/collagen pathway were evaluated by dual-luciferase reporter assay and western blot, respectively. In patients with AF, hsa-miR-4443 decreased significantly and collagen metabolism level increased significantly. Logistic regression analysis showed that low hsa-miR-4443 level was a risk factor of AF (P<0.001). The receiver operating characteristic curve revealed that hsa-miR-4443 was useful for predicting AF (area under the curve: 0.828, sensitivity: 0.71, specificity: 0.78, P<0.001). In HCFBs, hsa-miR-4443 targeted thrombospondin-1 (THBS1) and downregulated TGF-β1/α-SMA/collagen pathway. The inhibition of hsa-miR-4443 expression promoted HCFB proliferation, migration, invasion, myofibroblast differentiation, and collagen production. The significant reduction of hsa-miR-4443 can be used as a biomarker for AF. hsa-miR-4443 protected AF by targeting THBS1 and regulated TGF-β1/α-SMA/collagen pathway to inhibit HCFB proliferation and collagen synthesis.

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.

Synthesis and Biological Evaluation of Novel Thiazolyl-Coumarin Derivatives as Potent Histone Deacetylase Inhibitors with Antifibrotic Activity.

New histone deacetylases (HDAC) inhibitors with low toxicity to non-cancerous cells, are a prevalent issue at present because these enzymes are actively involved in fibrotic diseases. We designed and synthesized a novel series of thiazolyl-coumarins, substituted at position 6 (R = H, Br, OCH3), linked to classic zinc binding groups, such as hydroxamic and carboxylic acid moieties and alternative zinc binding groups such as disulfide and catechol. Their in vitro inhibitory activities against HDACs were evaluated. Disulfide and hydroxamic acid derivatives were the most potent ones. Assays with neonatal rat cardiac fibroblasts demonstrated low cytotoxic effects for all compounds. Regarding the parameters associated to cardiac fibrosis development, the compounds showed antiproliferative effects, and triggered a strong decrease on the expression levels of both α-SMA and procollagen I. In conclusion, the new thiazolyl-coumarin derivatives inhibit HDAC activity and decrease profibrotic effects on cardiac fibroblasts.

Trypanosoma cruzi activates mouse cardiac fibroblasts in vitro leading to fibroblast-myofibroblast transition and increase in expression of extracellular matrix proteins.

BACKGROUND: Cardiac fibrosis is a consequence of chronic chagasic cardiomyopathy (CCC). In other cardiovascular diseases, the protagonist role of fibroblasts in cardiac fibrosis is well established. However, the role of cardiac fibroblasts (CFs) in fibrosis during the CCC is not clear. Here, our aim was to investigate the effect of Trypanosoma cruzi, the etiological agent of Chagas disease on CFs activation. METHODS: Cardiac fibroblasts were purified from primary cultures of mouse embryo cardiac cells. After two passages, cells were infected with T. cruzi (Y strain) and analyzed at different times for determination of infectivity, activation and production of extracellular matrix components (fibronectin, laminin and collagen IV) by immunofluorescence and western blot. RESULTS: At second passage, cultures were enriched in CFs (95% of fibroblasts and 5% of cardiomyocytes), as revealed by presence of alpha-smooth muscle actin (α-SMA) and discoidin domain receptor 2 (DDR2) and absence of sarcomeric tropomyosin (ST) protein expression. Trypanosoma cruzi infection induced fibroblast-myofibroblast transition, with increased expression of α-SMA after 6 and 24 h post-infection (hpi). Fibronectin was increased at 6, 24 and 48 hpi, laminin was increased at 6 and 24 hpi and collagen IV was increased at 6 hpi. CONCLUSIONS: Our results showed that T. cruzi activates CFs, inducing activation and exacerbates ECM production. Furthermore, our data raise the possibility of the involvement of CFs in heart fibrosis during Chagas disease.

SDF-1α (Stromal Cell-Derived Factor 1α) Induces Cardiac Fibroblasts, Renal Microvascular Smooth Muscle Cells, and Glomerular Mesangial Cells to Proliferate, Cause Hypertrophy, and Produce Collagen.

BACKGROUND: Activated cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs) proliferate, cause hypertrophy, and produce collagen; in this way, activated CFs contribute to cardiac fibrosis, and activated PGVSMCs and GMCs promote renal fibrosis. In heart and kidney diseases, SDF-1α (stromal cell-derived factor 1α; endogenous CXCR4 [C-X-C motif chemokine receptor 4] receptor agonist) levels are often elevated; therefore, it is important to know whether and how the SDF-1α/CXCR4 axis activates CFs, PGVSMCs, or GMCs. METHODS AND RESULTS: Here we investigated whether SDF-1α activates CFs, PGVSMCs, and GMCs to proliferate, hypertrophy, or produce collagen. DPP4 (dipeptidyl peptidase 4) inactivates SDF-1α and previous experiments show that growth-promoting peptides have greater effects in cells from genetically-hypertensive animals. Therefore, we performed experiments in the absence and presence of sitagliptin (DPP4 inhibitor) and in cells from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats. Our studies show (1) that spontaneously hypertensive and Wistar-Kyoto rat CFs, PGVSMCs, and GMCs express CXCR4 receptors and DPP4 activity; (2) that chronic treatment with physiologically relevant concentrations of SDF-1α causes concentration-dependent increases in the proliferation (cell number) and hypertrophy (3H-leucine incorporation) of and collagen production (3H-proline incorporation) by CFs, PGVSMCs, and GMCs; (3) that sitagliptin augments these effects of SDF-1α; (4) that interactions between SDF-1α and sitagliptin are greater in spontaneously hypertensive rat cells; (5) that CXCR4 antagonism (AMD3100) blocks all effects of SDF-1α; and (6) that SDF-1α/CXCR4 signal transduction likely involves the RACK1 (receptor for activated C kinase 1)/Gßγ/PLC (phospholipase C)/PKC (protein kinase C) signaling complex. CONCLUSIONS: The SDF-1α/CXCR4 axis drives proliferation and hypertrophy of and collagen production by CFs, PGVSMCs, and GMCs, particularly in cells from genetically hypertensive animals and when DPP4 is inhibited.

Expression and function of toll-like receptor 4 and inflammasomes in cardiac fibroblasts and myofibroblasts: IL-1ß synthesis, secretion, and degradation.

UNLABELLED: Cardiac inflammation can be produced by pathogen-associated molecular patterns (PAMPs), from parasitic, bacterial or viral origin; or by danger-associated molecular patterns (DAMPs), released from dead cells after cardiac tissue damage, for example by cardiac infarction. Both, PAMPS and DAMPS activate TLR4 on resident immune cells and heart tissue cells, triggering an inflammatory process necessary to begin the wound healing process. Cardiac fibroblasts (CF) are the most abundant cells in the heart and are critical to wound healing, along with cardiac myofibroblasts (CMF), which are differentiated from CF through a TGF-ß1-mediated process. While TLR4 and the inflammasome complex are known to play important roles in CF function, the effects of TGF-ß1 on TLR4 and inflammasome expression and activity remain unknown. To elucidate this important point, we evaluated the effect of TGF-ß1 on TLR4, and the inflammasome protein expression and activity through activation by LPS, mimicking a myocarditis condition by bacterial origin. We found that TGF-ß1 increased TLR4 expression in CF and that the process was mediated by the TGFßRI and p38 signaling pathways. In both CF and CMF, LPS triggered ERK1/2, PI3K-Akt, and p65-NF-κB phosphorylation. All of these effects were blocked by TAK-242, a TLR4 signaling pathway inhibitor. LPS increased pro-IL-1ß levels, which were dependent on the ERK1/2, PI3K-Akt, and NF-κB signaling pathways, and levels were higher in CF than CMF. NLRP3 and ASC levels were similar in CF and CMF, while pro-caspase-1 levels and caspase-1 activity were higher in CMF. LPS+ATP treatment induced inflammasome complex assembly and activation, triggering the release of IL-1ß in both CMF and CF. Finally, the unsecreted pro-IL-1ß in the CF was degraded by autophagy. CONCLUSION: TGF-ß1 increases TLR4 expression in CF. Despite different pro-IL-1ß and caspase-1 activity levels in CF versus CMF, the two cell types secreted similar levels of IL-1ß after LPS+ATP treatment. These findings suggest that both cell types are active participants in inflammation.