BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation.
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ABSTRACT:
SUBMITTER: Diao H
PROVIDER: S-EPMC9332509 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rationale: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure. Methods: We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. In vitro TGFβ1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the Rxfp1 promoter region in MCECs. Results: Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFβ-pSMAD2/3 signaling in endothelial cells. Conclusions: This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.
| S-EPMC7086357 | biostudies-literature
Project description:The molecular mechanisms responsible for the development and progression of atherosclerotic lesions have not been fully established. Here, we investigated the role played by endothelial-to-mesenchymal transition (EndMT) and its key regulator FGF receptor 1 (FGFR1) in atherosclerosis. In cultured human endothelial cells, both inflammatory cytokines and oscillatory shear stress reduced endothelial FGFR1 expression and activated TGF-? signaling. We further explored the link between disrupted FGF endothelial signaling and progression of atherosclerosis by introducing endothelial-specific deletion of FGF receptor substrate 2 ? (Frs2a) in atherosclerotic (Apoe(-/-)) mice. When placed on a high-fat diet, these double-knockout mice developed atherosclerosis at a much earlier time point compared with that their Apoe(-/-) counterparts, eventually demonstrating an 84% increase in total plaque burden. Moreover, these animals exhibited extensive development of EndMT, deposition of fibronectin, and increased neointima formation. Additionally, we conducted a molecular and morphometric examination of left main coronary arteries from 43 patients with various levels of coronary disease to assess the clinical relevance of these findings. The extent of coronary atherosclerosis in this patient set strongly correlated with loss of endothelial FGFR1 expression, activation of endothelial TGF-? signaling, and the extent of EndMT. These data demonstrate a link between loss of protective endothelial FGFR signaling, development of EndMT, and progression of atherosclerosis.
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Project description:Endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to cardiac fibrosis and heart failure (HF). Recent studies have demonstrated that EndMT is regulated by autophagy, and we previously showed suppression of excessive autophagy and alleviation of cardiac fibrosis in HF mice with inactivated receptor for advanced glycation end products (RAGE). Thus, we investigated whether reduced cardiac fibrosis due to RAGE knockout occurred by inhibiting EndMT mediated by excessive autophagy. We found a decrease in endothelial cells (CD31+/VE-Cadherin+) and an increase in cells co-expressing CD31 and α-smooth muscle actin (α-SMA, myofibroblast marker) at 8 weeks in heart tissue of mice subjected to transverse aortic constriction (TAC), which implied EndMT. Knockout RAGE decreased EndMT accompanied by decreased expression of autophagy-related proteins (LC3BII/I and Beclin 1), and alleviated cardiac fibrosis and improved cardiac function in TAC mice. Moreover, 3-methyladenine (3-MA) and chloroquine (CQ), inhibitors of autophagy, attenuated EndMT, and cardiac fibrosis in TAC mice. Importantly, EndMT induced by AGEs could be blocked by autophagy inhibitor in vivo and in vitro. These results suggested that AGEs/RAGE-autophagy-EndMT axis involved in the development of cardiac fibrosis and knockout RAGE ameliorated cardiac fibrosis through decreasing EndMT regulated by autophagy, which could be a promising therapeutic strategy for HF.
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Project description:Growing evidence suggests close associations between periodontitis and atherosclerosis. To further understand the pathological relationships of these associations, we developed periodontitis with ligature placement around maxillary molars or ligature placement in conjunction with Porphyromonas gingivalis lipopolysaccharide injection at the ligature sites (ligature/P.g. LPS) in Apolipoprotein E knock out mice and studied the atherogenesis process in these animals. The mice were fed with high fat diet for 11 weeks and sacrificed for analyzing periodontitis, systemic inflammation, and atherosclerosis. Controls did not develop periodontitis or systemic inflammation and had minimal lipid deposition in the aortas, but mice receiving ligature or ligature/P.g. LPS showed severe periodontitis, systemic inflammation, and aortic plaque formation. The aortic plaque contained abundant macrophages and cells expressing both endothelial and mesenchymal cell markers. The severity of periodontitis was slightly higher in mice receiving ligature/P.g. LPS than ligature alone, and the magnitude of systemic inflammation and aortic plaque formation were also notably greater in the mice with ligature/P.g. LPS. These observations indicate that the development of atherosclerosis is due to systemic inflammation caused by severe periodontitis. In vitro, P.g. LPS enhanced the secretion of pro-inflammatory cytokines from macrophages and increased the adhesion of monocytes to endothelial cells by upregulating the expression of adhesion molecules from endothelial cells. Moreover, secretory proteins, such as TNF-?, from macrophages induced endothelial-mesenchymal transitions of the endothelial cells. Taken together, systemic inflammation induced by severe periodontitis might exacerbate atherosclerosis via, in part, causing aberrant functions of vascular endothelial cells and the activation of macrophages in mice.
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Project description:Rationale: Nicotine exposure via cigarette smoking is strongly associated with atherosclerosis. However, the underlying mechanisms remain poorly understood. The current study aimed to identify whether endothelial to mesenchymal transition (EndMT) contributes to nicotine-induced atherosclerosis. Methods: ApoE-/- mice were administered nicotine in their drinking water for 12 weeks. The effects of nicotine on EndMT were determined by immunostaining on aortic root and RNA analysis in aortic intima. In vitro nicotine-treated cell model was established on human aortic endothelial cells (HAECs). The effects of nicotine on the expression of EndMT-related markers, ERK1/2 and Snail were quantified by real-time PCR, western blot and immunofluorescent staining. Results: Nicotine treatment resulted in larger atherosclerotic plaques in ApoE-/- mice. The vascular endothelial cells from nicotine-treated mice showed mesenchymal phenotype, indicating EndMT. Moreover, nicotine-induced EndMT process was accompanied by cytoskeleton reorganization and impaired barrier function. The ?7 nicotine acetylcholine receptor (?7nAChR) was highly expressed in HAECs and its antagonist could effectively relieve nicotine-induced EndMT and atherosclerotic lesions in mice. Further experiments revealed that ERK1/2 signaling was activated by nicotine, which led to the upregulation of Snail. Blocking ERK1/2 with inhibitor or silencing Snail by small interfering RNA efficiently preserved endothelial phenotype upon nicotine stimulation. Conclusion: Our study provides evidence that EndMT contributes to the pro-atherosclerotic property of nicotine. Nicotine induces EndMT through ?7nAChR-ERK1/2-Snail signaling in endothelial cells. EndMT may be a therapeutic target for smoking-related endothelial dysfunction and cardiovascular disease.
| S-EPMC7196288 | biostudies-literature
Project description:Despite many decades of research, complications of atherosclerosis resulting from the rupture or erosion of unstable plaques remain the leading cause of death worldwide. Advances in cellular lineage tracing techniques have allowed researchers to begin investigating the role of individual cell types in the key processes regulating plaque stability, including maintenance of the fibrous cap, a protective collagen-rich structure that underlies the endothelium. This structure was previously thought to be entirely derived from smooth muscle cells (SMC), which migrated from the vessel wall. However, recent lineage tracing studies have identified endothelial cells (EC) as an essential component of this protective barrier through an endothelial-to-mesenchymal transition (EndoMT), a process that has previously been implicated in pulmonary, cardiac, and kidney fibrosis. Although the presence of EndoMT in atherosclerotic plaques has been shown by several laboratories using EC-lineage tracing mouse models, whether EndoMT is detrimental (i.e., worsening disease progression) or beneficial (i.e., an athero-protective response that prevents plaque instability) remains uncertain as there are data to support both possibilities, which will be further discussed in this review.
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Project description:Chronic allograft dysfunction (CAD) is the major cause of late graft loss in long-term renal transplantation. In our previous study, we found that epithelial-mesenchymal transition (EMT) is a significant event in the progression of renal allograft tubulointerstitial fibrosis, and impaired autophagic flux plays a critical role in renal allograft fibrosis. Everolimus (EVR) has been reported to be widely used to prevent the progression of organ fibrosis and graft rejection. However, the pharmacological mechanism of EVR in kidney transplantation remains to be determined. We used CAD rat model and the human kidney 2 (HK2) cell line treated with tumor necrosis factor-α (TNF-α) and EVR to examine the role of EVR on TNF-α-induced EMT and transplanted renal interstitial fibrosis. Here, we found that EVR could attenuate the progression of EMT and renal allograft interstitial fibrosis, and also activate autophagy in vivo. To explore the mechanism behind it, we detected the relationship among EVR, autophagy level, and TNF-α-induced EMT in HK2 cells. Our results showed that autophagy was upregulated upon mTOR pathway inhibition by EVR, which could significantly reduce expression of TNF-α-induced EMT. However, the inhibition of EVR on TNF-α-induced EMT was partly reversed following the addition of autophagy inhibitor chloroquine. In addition, we found that TNF-α activated EMT through protein kinase B (Akt) as well as nuclear factor kappa B (NF-κB) pathway according to the RNA sequencing, and EVR's effect on the EMT was only associated with IκB-α stabilization instead of the Akt pathway. Together, our findings suggest that EVR may retard impaired autophagic flux and block NF-κB pathway activation, and thereby prevent progression of TNF-α-induced EMT and renal allograft interstitial fibrosis.
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Project description:Statins may decrease chronic kidney diseases (CKDs) risk, but their underlying molecular mechanisms are not completely understood. Recent studies indicate Endothelial-to-mesenchymal transition (EndMT) plays an important role contributing to renal interstitial fibrosis. In the present study, we first investigated whether lovastatin could ameliorate renal fibrosis via suppression of EndMT and its possible mechanism. In vitro experiments, lovastatin significantly ameliorated microalbuminuria and pathologic changes in diabetic rats. Double labeling immunofluorescence showed lovastatin could inhibit EndMT in glomeruli. Furthermore, lovastatin could inhibit oxidative stress and down-regulate TGF-β1-Smad signaling. Consistent alterations were observed in vivo that lovastatin substantially suppressed EndMT and TGF-β1 signaling induced by high glucose in glomerular endothelial cells (GEnCs). These data indicated that lovastatin could ameliorate EndMT in glomeruli in diabetic nephropathy, the mechanism of which might be at least partly through suppression of oxidative stress and TGF-β1/Smad signaling pathway.
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Project description:BACKGROUND:Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels. METHODS:Over the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial-mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-β signaling by immunoblotting for Smad2/3 and Smad7. RESULTS:A decrease in SA-β-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-β pathway, contributed to EndMT induction. CONCLUSIONS:We revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy. Video abstract.
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Project description:Macroautophagy/autophagy plays a vital role in the homeostasis of diverse cell types. Vascular endothelial cells contribute to vascular health and play a unique role in vascular biology. Here, we demonstrated that autophagy defects in endothelial cells induced IL6 (interleukin 6)-dependent endothelial-to-mesenchymal transition (EndMT) and organ fibrosis with metabolic defects in mice. Inhibition of autophagy, either by a specific inhibitor or small interfering RNA (siRNA) for ATG5 (autophagy related 5), in human microvascular endothelial cells (HMVECs) induced EndMT. The IL6 level was significantly higher in ATG5 siRNA-transfected HMVECs culture medium compared with the control HMVECs culture medium, and neutralization of IL6 by a specific antibody completely inhibited EndMT in ATG5 siRNA-transfected HMVECs. Similar to the in vitro data, endothelial-specific atg5 knockout mice (Atg5 Endo; Cdh5-Cre Atg5 flox/flox mice) displayed both EndMT-associated kidney and heart fibrosis when compared to littermate controls. The plasma level of IL6 was higher in Atg5 Endo compared to that of control mice, and fibrosis was accelerated in Atg5 Endo treated with a HFD; neutralization of IL6 by a specific antibody inhibited EndMT and fibrosis in HFD-fed Atg5 Endo associated with the amelioration of metabolic defects. These results revealed the essential role of autophagy in endothelial cell integrity and revealed that the disruption of endothelial autophagy could lead to significant pathological IL6-dependent EndMT and organ fibrosis. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; EndMT: endothelial-to-mesenchymal transition; HES: hematoxylin and eosin stain; HFD: high-fat diet; HMVECs: human microvascular endothelial cells; IFNG: interferon gamma; IL6: interleukin 6; MTS: Masson's trichrome staining; NFD: normal-fat diet; siRNA: small interfering RNA; SMAD3: SMAD family member 3; TGFB: transforming growth factor β; TNF: tumor necrosis factor; VEGFA: vascular endothelial growth factor A.
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