RESUMO
The prevalence of calcific aortic valve disease (CAVD) remains substantial while there is currently no medical therapy available. Forkhead box O1 (FOXO1) is known to be involved in the pathogenesis of cardiovascular diseases, including vascular calcification and atherosclerosis; however, its specific role in calcific aortic valve disease remains to be elucidated. In this study, we identified FOXO1 significantly down-regulated in the aortic valve interstitial cells (VICs) of calcified aortic valves by investigating clinical specimens and GEO database analysis. FOXO1 silencing or inhibition promoted VICs osteogenic differentiation in vitro and aortic valve calcification in Apoe-/- mice, respectively. We identified that FOXO1 facilitated the ubiquitination and degradation of RUNX2, which process was mainly mediated by SMAD-specific E3 ubiquitin ligase 2 (SMURF2). Our discoveries unveil a heretofore unacknowledged mechanism involving the FOXO1/SMURF2/RUNX2 axis in CAVD, thereby proposing the potential therapeutic utility of FOXO1 or SMURF2 as viable strategies to impede the progression of CAVD.
Assuntos
Estenose da Valva Aórtica , Valva Aórtica , Calcinose , Subunidade alfa 1 de Fator de Ligação ao Core , Proteína Forkhead Box O1 , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Calcinose/metabolismo , Calcinose/patologia , Calcinose/genética , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/genética , Masculino , Osteogênese/genética , Modelos Animais de Doenças , Diferenciação CelularRESUMO
Rationale: While reactive oxygen species (ROS) has been recognized as one of the main causes of cardiac injury following myocardial infarction, the clinical application of antioxidants has shown limited effects on protecting hearts against ischemia-reperfusion (I/R) injury. Thus, the precise role of ROS following cardiac injury remains to be fully elucidated. Objective: We investigated the role of mitsugumin 53 (MG53) in regulating necroptosis following I/R injury to the hearts and the involvement of ROS in MG53-mediated cardioprotection. Methods and Results: Antioxidants were used to test the role of ROS in MG53-mediated cardioprotection in the mouse model of I/R injury and induced human pluripotent stem cells (hiPSCs)-derived cardiomyocytes subjected to hypoxia or re-oxygenation (H/R) injury. Western blotting and co-immunoprecipitation were used to identify potential cell death pathways that MG53 was involved in. CRISPR/Cas 9-mediated genome editing and mutagenesis assays were performed to further identify specific interaction amino acids between MG53 and its ubiquitin E3 ligase substrate. We found that MG53 could protect myocardial injury via inhibiting the necroptosis pathway. Upon injury, the generation of ROS in the infarct zone of the hearts promoted interaction between MG53 and receptor-interacting protein kinase 1 (RIPK1). As an E3 ubiquitin ligase, MG53 added multiple ubiquitin chains to RIPK1 at the sites of K316, K604, and K627 for proteasome-mediated RIPK1 degradation and inhibited necroptosis. The application of N-acetyl cysteine (NAC) disrupted the interaction between MG53 and RIPK1 and abolished MG53-mediated cardioprotective effects. Conclusions: Taken together, this study provided a molecular mechanism of a potential beneficial role of ROS following acute myocardial infarction. Thus, fine-tuning ROS levels might be critical for cardioprotection.
RESUMO
Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinase known to play essential roles in the nervous tissue. Myocardial upregulation of UCHL1 was observed in human dilated cardiomyopathy and several animal models of heart disease, but the (patho)physiological significance of UCHL1 in cardiomyocytes remains undefined. Hence, we conducted this study to fill this critical gap. We produced cardiomyocyte-restricted Uchl1 knockout (CKO) by coupling the Uchl1-floxed allele with transgenic Myh6-Cre in C57B/6J inbred mice. Mice transgenic for Myh6-Cre were used as controls (CTL). Myocardial Uchl1 proteins were markedly reduced in CKO mice but they did not display discernible abnormal phenotype. Ten-week old CTL or CKO mice were subjected to left anterior descending artery ligation (myocardial infarction, MI) or sham surgery (Sham) and characterized at 7- and 28-day after surgery. Compared with Sham mice, significant increases in myocardial UCHL1 proteins were detected in CTL MI but not in CKO MI mice. MI-induced left ventricular (LV) chamber dilation, reduction of ejection fraction (EF) and fractional shortening (FS), and LV anterior wall thinning detected by echocardiography were comparable between the CTL MI and CKO MI groups 7-day post-MI. However, by 28-day post-MI, MI-induced LV chamber dilatation, EF and FS reduction, increases of myocardial ubiquitin conjugates, and increases in the heart weight to body weight ratio and the ventricular weight to body weight ratio were significantly more pronounced in CKO MI than CTL MI mice. As further revealed by LV pressure-volume relationship analyses, CKO MI mice but not CTL MI mice displayed significant decreases in stroke volume, cardiac output, and the maximum rates of LV pressure rising or declining and of LV volume declining, as well as significant increases in LV end-diastolic pressure and Tau, compared with their respective Sham controls. LC3-II flux assays reveal that autophagic flux is decreased in CKO mouse myocardium as well as in cultured Uchl1-deficient cardiomyocytes. In conclusion, UCHL1 of cardiomyocytes is dispensable for development but promotes macroautophagy in cardiomyocytes. Upregulation of UCHL1 in post-MI hearts occurs primarily in the cardiomyocytes and protects against post-MI cardiac remodeling and malfunction likely through supporting autophagic flux and proteostasis during a stress condition.
RESUMO
Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling.
Assuntos
Traumatismos Cardíacos , Células-Tronco Pluripotentes Induzidas , Infarto do Miocárdio , Animais , Escherichia coli , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Transdução de Sinais , Ubiquitina Tiolesterase/genéticaRESUMO
OBJECTIVE: Cavin3 is a putative tumor suppressor protein. However, its molecular action on tumor regulation is largely unknown. The aim of the current study is to explore the implication of cavin3 alteration, its clinical significance, and any potential molecular mechanisms in the regulation of breast cancer (BC). METHODS: TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) data bases, and 17 freshly paired BC and adjacent normal tissues were analyzed for mRNA levels of Cavin3. Furthermore, cavin3 protein expression from 407 primary BC samples were assessed by immunohistochemistry (IHC) and measured by H-score. The clinical significance of cavin3 expression was explored by Kaplan-Meier analysis and the Cox regression method. In vitro biological assays were performed to elucidate the function and underlying mechanisms of cavin 3 in BC cell lines. RESULTS: Cavin3 mRNA was dramatically down-regulated in BC compared with the negative control. The median H-score of cavin3 protein by IHC was 50 (range 0-270). There were 232 (57%) and 175 (43%) cases scored as low (H-score≤50) and high (H-score >50) levels of cavin3, respectively. Low cavin3 was correlated with a higher T and N stage, and worse distant metastasis-free survival (DMFS) and overall survival (OS). Multivariate survival analysis revealed low cavin3 was an independent fact for worse DMFS. In BC cells, an overexpression of cavin3 could inhibit cell migration and invasion, and significantly decreased the level of p-Akt. Knockout of cavin3, meanwhile, promoted cell invasion ability and increased the level of p-AKT. CONCLUSION: Cavin3 expression is significantly lower in BC and is correlated with distant metastasis and worse survival. Cavin3 functions as a metastasis suppressor via inhibiting the AKT pathway, suggesting cavin3 as a potential prognostic biomarker and a target for BC treatment.
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Corneal wounds usually heal quickly; but diabetic patients have more fragile corneas and experience delayed and painful healing. In the present study, we compared the healing capacity of corneal epithelial cells (CECs) between normal and diabetic conditions and the potential mechanisms. Primary murine CEC derived from wild-type and diabetic (db/db) mice, as well as primary human CEC were prepared. Human CEC were exposed to high glucose (30 mM) to mimic diabetic conditions. Cell migration and proliferation were assessed using Scratch test and MTT assays, respectively. Reactive oxygen species (ROS) production in the cells was measured using dichlorofluorescein reagent. Western blot was used to evaluate the expression levels of Akt. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) expression were used to determine tight junction integrity. We found that the diabetic CEC displayed significantly slower cell proliferation and migration compared with the normal CEC from both mice and humans. Furthermore, ROS production was markedly increased in CEC grown under diabetic conditions. Treatment with an antioxidant N-acetyl cysteine (NAC, 100 µM) significantly decreased ROS production and increased wound healing in diabetic CEC. Barrier function was significantly reduced in both diabetic mouse and human CEC, while NAC treatment mitigated these effects. We further showed that Akt signaling was impaired in diabetic CEC, which was partially improved by NAC treatment. These results show that diabetic conditions lead to delayed wound-healing capacity of CEC and impaired tight junction formation in both mice and human. Increased ROS production and inhibited Akt signaling may contribute to this outcome, implicating these as potential targets for treating corneal wounds in diabetic patients.
Assuntos
Movimento Celular/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Células Epiteliais/metabolismo , Transdução de Sinais/fisiologia , Junções Íntimas/metabolismo , Cicatrização/fisiologia , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Córnea/citologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The cornea plays an important role in transmitting light and providing protection to the eye, but is susceptible to injury and infection. Standard treatments for corneal wounds include topical lubricants, antibiotics, bandage contact lens, and surgery. However, these measures are often ineffective. Here we show that MG53, a protein with an essential role in cell membrane repair, contributes to the corneal injury-repair process. Native MG53 is present in the corneal epithelia, tear film, and aqueous humor, suggesting its potential function in corneal homeostasis. Knockout of MG53 in mice causes impaired healing and regenerative capacity following injury. Exogenous recombinant human MG53 (rhMG53) protein protects the corneal epithelia against mechanical injury and enhances healing by promoting migration of corneal fibroblasts. Using in vivo alkaline-induced injury to the rat cornea, we show that rhMG53 promotes re-epithelialization and reduces post-injury fibrosis and vascularization. Finally, we show that rhMG53 modulates TGF-ß-mediated fibrotic remodeling associated with corneal injury. Overall, our data support the bi-functional role of MG53 in facilitating corneal healing and maintaining corneal transparency by reducing fibrosis and vascularization associated with corneal injuries.
Assuntos
Córnea/metabolismo , Lesões da Córnea/genética , Proteínas de Membrana/genética , Cicatrização/genética , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Córnea/efeitos dos fármacos , Córnea/patologia , Lesões da Córnea/metabolismo , Lesões da Córnea/fisiopatologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Humanos , Proteínas de Membrana/metabolismo , Camundongos Knockout , Ratos , Proteínas Recombinantes/farmacologia , Regeneração/efeitos dos fármacos , Regeneração/genética , Roedores/genética , Roedores/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
BACKGROUND: Myocardial viability assessment is typically performed in patients with coronary artery disease (CAD) and severe left ventricular (LV) dysfunction to identify those who might benefit from revascularization and assist in decision making process. However, the prognostic value of myocardial viability testing remains a debating issue. METHODS: Positron Emission Tomography using 18F-fluorodeoxyglucose (18FDG-PET) was performed in 81 patients with ischemic LV dysfunction [ejection fraction (EF) ≤35%] for myocardial viability assessment prior to coronary artery bypass surgery. Fifty-three of them received finally coronary artery bypass grafting and were divided into two groups according to the extent of myocardial scar: one group with scar burden ≥10% (n=30) and the other with scar burden <10% (n=23). The remaining patients were contraindicated for CABG and received optimal medical treatment (OMT, n=28). All patients were followed up and the primary endpoint was all-cause mortality and the secondary endpoint was a composite of all-cause mortality and major adverse cardiovascular and cerebrovascular events (MACCE). RESULTS: 18FDG-PET revealed a different profile of myocardial viability among three groups with respect to the extent of myocardial scar, the hibernating myocardium (both P<0.01), some echocardiographic parameters such as left ventricular diastolic dimension (LVDD) and EF were also significantly different (both P<0.05). Nevertheless, the baseline prevalence of comorbidities and functional classifications were comparable. The per-procedural parameters were not significantly different between two CABG groups. In a median follow-up time of 32 months, Kaplan Meier analysis uncovered no significant difference in terms of overall survival (P=0.74) and MACCE-free survival (P=0.66) among three groups. CONCLUSIONS: Myocardial viability assessment using 18FDG-PET is of limited prognostic value in patients with CAD and severe LV dysfunction. In patients with substantial myocardial scar burden despite the existence of considerable hibernating myocardium, functional recovery following surgical revascularization is not necessarily translated to survival benefits.