RESUMO
BACKGROUND: Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. OBJECTIVE: In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. METHODS AND RESULTS: To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. CONCLUSION: Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19.
Assuntos
COVID-19 , Trombose , Humanos , Receptor PAR-2 , SARS-CoV-2 , Células EndoteliaisRESUMO
Valvular disease is a complex pathological condition that impacts countless individuals around the globe. Due to limited treatments, it is crucial to understand its mechanisms to identify new targets. Valve disease may result in pulmonary venous hypertension, which is linked to compromised functioning of the alveolar and capillary membranes and hindered gas exchange. Nonetheless, the correlation between surfactant proteins (SPs) and valve disease remains unexplored. A total of 44 patients were enrolled in this study, with 36 undergoing aortic valve replacement and 8 needing a second aortic valve substitution due to bioprosthetic valve degeneration. Ten healthy subjects were also included. The results showed that patients who underwent both the first valve replacement and the second surgery had significantly higher levels of immature SP-B (proSP-B) compared to control subjects. The levels of the extra-lung collectin SP-D were higher in patients who needed a second surgery due to bioprosthetic valve degeneration, while SP-A levels remained unchanged. The research also showed that there was no reciprocal relationship between inflammation and SP-D as the levels of inflammatory mediators did not differ between groups. The present study demonstrates that circulating proSP-B serves as a reliable marker of alveolar-capillary membrane damage in patients with valvular heart disease.
Assuntos
Estenose da Valva Aórtica , Valva Aórtica , Calcinose , Proteína B Associada a Surfactante Pulmonar , Humanos , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/cirurgia , Masculino , Feminino , Proteína B Associada a Surfactante Pulmonar/sangue , Proteína B Associada a Surfactante Pulmonar/metabolismo , Idoso , Calcinose/sangue , Valva Aórtica/cirurgia , Valva Aórtica/patologia , Pessoa de Meia-Idade , Biomarcadores/sangue , Estudos de Casos e ControlesRESUMO
BACKGROUND AND AIMS: While the role of PCSK9 in lipid metabolism is well established, its link with endothelial function is less clear. The aim of the present study is to evaluate the relationship between PCSK9 and endothelial dysfunction in the setting of acute myocardial infarction. METHODS AND RESULTS: To this purpose, we analyzed the serum of 74 patients with ST-elevation myocardial infarction (STEMI) at the time of admission and after 5 days. Endothelial dysfunction was evaluated as rate of apoptosis (AR) of human umbilical vein endothelial cells incubated with patients' serum. There was a good correlation between PCSK9 and the apoptosis rate values, both at baseline (r = 0.649) and 5-day (r = 0.648). In the 5 days after STEMI, PCSK9 increased significantly (242-327 ng/ml, p < 0.001), while AR did not (p = 0.491). Overall, 21 (28%) patients showed a reduction of PCSK9, and they had a significantly higher decrease of AR as compared to others (-13.87 vs 5.8%, p = 0.002). At the univariable analysis, the 5-day change of PCSK9 resulted to be the only variable associated with the 5-day change of the apoptosis rate (beta 0.217, 95%CI 0.091-0.344, p = 0.001). CONCLUSION: The variation of endothelial function and PCKS9 in the first days after an acute myocardial infarction are related. Further validation and research are necessary to confirm our findings. CLINICAL TRIAL: NCT02438085.
Assuntos
Infarto do Miocárdio , Intervenção Coronária Percutânea , Infarto do Miocárdio com Supradesnível do Segmento ST , Células Endoteliais da Veia Umbilical Humana , Humanos , Pró-Proteína Convertase 9RESUMO
Critical limb ischemia (CLI) is a severe manifestation of peripheral artery disease characterized by ischemic pain, which is frequently associated with diabetes and non-healing lesions to inferior limbs. The clinical management of diabetic patients with CLI typically includes percutaneous transluminal angioplasty (PTA) to restore limb circulation and surgical treatment of diabetic foot ulcers (DFU). However, even after successful treatment, CLI patients are prone to post-procedure complications, which may lead to unplanned revascularization or foot surgery. Unfortunately, the factors predicting adverse events in treated CLI patients are only partially known. This study aimed to identify potential biomarkers that predict the disease course in diabetic patients with CLI. For this purpose, we measured the circulating levels of a panel of 23 molecules related to inflammation, endothelial dysfunction, platelet activation, and thrombophilia in 92 patients with CLI and DFU requiring PTA and foot surgery. We investigated whether these putative biomarkers were associated with the following clinical endpoints: (1) healing of the treated DFUs; (2) need for new revascularization of the limb; (3) appearance of new lesions or relapses after successful healing. We found that sICAM-1 and endothelin-1 are inversely associated with DFU healing and that PAI-1 and endothelin-1 are associated with the need for new revascularization. Moreover, we found that the levels of thrombomodulin and sCD40L are associated with new lesions or recurrence, and we show that the levels of these biomarkers could be used in a decision tree to assign patients to clusters with different risks of developing new lesions or recurrences.
Assuntos
Diabetes Mellitus , Pé Diabético , Amputação Cirúrgica , Biomarcadores , Isquemia Crônica Crítica de Membro , Pé Diabético/terapia , Endotelina-1 , Humanos , Inflamação , Isquemia/terapia , Inibidor 1 de Ativador de Plasminogênio , Estudos Retrospectivos , Trombomodulina , Resultado do TratamentoRESUMO
BACKGROUND: Biomarkers can be used to detect the presence of endothelial and/or alveolar epithelial injuries in case of ARDS. Angiopoietin-2 (Ang-2), soluble intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1), P-selectin and E-selectin are biomarkers of endothelial injury, whereas the receptor for advanced glycation end-products (RAGE) reflects alveolar epithelial injury. The aims of this study were to evaluate whether the plasma concentration of the above-mentioned biomarkers was different 1) in survivors and non-survivors of COVID-19-related ARDS and 2) in COVID-19-related and classical ARDS. METHODS: This prospective study was performed in two COVID-19-dedicated Intensive Care Units (ICU) and one non-COVID-19 ICU at Ferrara University Hospital. A cohort of 31 mechanically ventilated patients with COVID-19 ARDS and a cohort of 11 patients with classical ARDS were enrolled. Ang-2, ICAM-1, VCAM-1, P-selectin, E-selectin and RAGE were determined with a bead-based multiplex immunoassay at three time points: inclusion in the study (T1), after 7 ± 2 days (T2) and 14 ± 2 days (T3). The primary outcome was to evaluate the plasma trend of the biomarker levels in survivors and non-survivors. The secondary outcome was to evaluate the differences in respiratory mechanics variables and gas exchanges between survivors and non-survivors. Furthermore, we compared the plasma levels of the biomarkers at T1 in patients with COVID-19-related ARDS and classical ARDS. RESULTS: In COVID-19-related ARDS, the plasma levels of Ang-2 and ICAM-1 at T1 were statistically higher in non-survivors than survivors, (p = 0.04 and p = 0.03, respectively), whereas those of P-selectin, E-selectin and RAGE did not differ. Ang-2 and ICAM-1 at T1 were predictors of mortality (AUROC 0.650 and 0.717, respectively). At T1, RAGE and P-selectin levels were higher in classical ARDS than in COVID-19-related ARDS. Ang-2, ICAM-1 and E-selectin were lower in classical ARDS than in COVID-19-related ARDS (all p < 0.001). CONCLUSIONS: COVID-19 ARDS is characterized by an early pulmonary endothelial injury, as detected by Ang-2 and ICAM-1. COVID-19 ARDS and classical ARDS exhibited a different expression of biomarkers, suggesting different pathological pathways. Trial registration NCT04343053 , Date of registration: April 13, 2020.
Assuntos
Biomarcadores/análise , Lesão Pulmonar/diagnóstico , Respiração Artificial/efeitos adversos , Idoso , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/sangue , Área Sob a Curva , COVID-19/sangue , COVID-19/prevenção & controle , Estudos de Coortes , Selectina E/análise , Selectina E/sangue , Feminino , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/sangue , Lesão Pulmonar/sangue , Lesão Pulmonar/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/análise , Proteínas Quinases Ativadas por Mitógeno/sangue , Selectina-P/análise , Selectina-P/sangue , Estudos Prospectivos , Curva ROC , Respiração Artificial/normas , Respiração Artificial/estatística & dados numéricos , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/fisiopatologia , Versicanas/análise , Versicanas/sangue , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/sangueRESUMO
The aim of this study (NCT04343053) is to investigate the relationship between platelet activation, myocardial injury, and mortality in patients affected by Coronavirus disease 2019 (COVID-19). Fifty-four patients with respiratory failure due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were enrolled as cases. Eleven patients with the same clinical presentation, but negative for SARS-CoV-2 infection, were included as controls. Blood samples were collected at three different time points (inclusion [T1], after 7 ± 2 days [T2] and 14 ± 2 days [T3]). Platelet aggregation by light transmittance aggregometry and the circulating levels of soluble CD40 ligand (sCD40L) and P-selectin were measured. Platelet biomarkers did not differ between cases and controls, except for sCD40L which was higher in COVID-19 patients (p = .003). In COVID-19 patients, P-selectin and sCD40L levels decreased from T1 to T3 and were higher in cases requiring admission to intensive care unit (p = .004 and p = .008, respectively). Patients with myocardial injury (37%), as well as those who died (30%), had higher values of all biomarkers of platelet activation (p < .05 for all). Myocardial injury was an independent predictor of mortality. In COVID-19 patients admitted to hospital for respiratory failure, heightened platelet activation is associated with severity of illness, myocardial injury, and mortality.ClinicalTrials.gov number: NCT04343053.
Assuntos
Plaquetas/metabolismo , COVID-19 , Traumatismos Cardíacos , Miocárdio , Insuficiência Respiratória , SARS-CoV-2/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Ligante de CD40/sangue , COVID-19/sangue , COVID-19/mortalidade , COVID-19/patologia , Feminino , Traumatismos Cardíacos/sangue , Traumatismos Cardíacos/mortalidade , Traumatismos Cardíacos/patologia , Traumatismos Cardíacos/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miocárdio/patologia , Selectina-P/sangue , Agregação Plaquetária , Insuficiência Respiratória/sangue , Insuficiência Respiratória/mortalidade , Insuficiência Respiratória/patologia , Insuficiência Respiratória/virologiaRESUMO
From January 2020, coronavirus disease (COVID-19) originated in China has spread around the world. The disease is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The presence of myocarditis, cardiac arrest, and acute heart failure in COVID-19 patients suggests the existence of a relationship between SARS-CoV-2 infection and cardiac disease. The Notch signalling is a major regulator of cardiovascular function and it is also implicated in several biological processes mediating viral infections. In this report we discuss the possibility to target Notch signalling to prevent SARS-CoV-2 infection and interfere with the progression of COVID-19- associated heart and lungs disease.
Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/fisiopatologia , Cardiopatias/tratamento farmacológico , Cardiopatias/etiologia , Pneumopatias/tratamento farmacológico , Pneumopatias/etiologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/fisiopatologia , Receptores Notch/antagonistas & inibidores , Proteína ADAM17/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2 , Betacoronavirus/efeitos dos fármacos , COVID-19 , China , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Progressão da Doença , Furina/metabolismo , Parada Cardíaca/etiologia , Parada Cardíaca/patologia , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Humanos , Interleucina-6/imunologia , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Miocardite/etiologia , Miocardite/patologia , Pandemias , Peptidil Dipeptidase A/deficiência , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Receptores Notch/metabolismo , SARS-CoV-2 , Transdução de Sinais/efeitos dos fármacosRESUMO
Background Myeloperoxidase (MPO) is an enzyme with a recognized prognostic role in coronary artery disease (CAD), which is also emerging as a promising biomarker for cardiac risk stratification. However, the lack of a consensus method for its quantification has hindered its implementation in clinical practice. The aim of our work was to optimize an absolute sensitive assay for active MPO without external standards, to validate the method in the clinical context of CAD patients, and to estimate the enzyme specific activity. Methods In order to determine the MPO concentration using fluorescence readings, this ELISA assay exploits the activity of the enzyme recognized by specific antibodies. The assay was validated in a small cohort of patients that included: healthy subjects (n=60); patients with acute myocardial infarction (AMI, n=25); patients with stable CAD (SCAD, n=25) and a concomitant chronic obstructive pulmonary disease (COPD). Then, total MPO concentration and specific activity (activity/total MPO) were determined. Results The assay showed an intra- and inter-assay coefficient of variation of 5.8% and 10.4%, respectively, with a limit of detection (LoD) of 0.074 µU. Both AMI and SCAD patients had higher active and total MPO than controls (p<0.0001 and p<0.01, respectively). The specific activity of MPO was higher in SCAD patients compared to both controls and AMI (p<0.0001). Conclusions The study presents a robust and sensitive method for assaying MPO activity in biological fluids with low variability. Moreover, the determination of the specific activity could provide novel insight into the role of MPO in cardiovascular diseases (CVDs).
Assuntos
Doença da Artéria Coronariana/sangue , Peroxidase/sangue , Biomarcadores/sangue , Estudos de Coortes , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/enzimologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Prognóstico , Reprodutibilidade dos TestesRESUMO
Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004; p < 0.0001). Moreover, we isolated human aortic valve interstitial cells (AVICs) from AVs and found that COX-2 expression is decreased in AVICs from calcific valves compared to AVICs from non-calcific AVs. Furthermore, we observed that COX-2 inhibition with celecoxib induces AVICs trans-differentiation towards a myofibroblast phenotype, and increases the levels of TGF-ß-induced apoptosis, both processes able to promote the formation of calcific nodules. We conclude that reduced COX-2 expression is a characteristic of human AVICs prone to calcification and that COX-2 inhibition may promote aortic valve calcification. Our findings support the notion that celecoxib may facilitate CAVD progression.
Assuntos
Estenose da Valva Aórtica/tratamento farmacológico , Valva Aórtica/patologia , Calcinose/tratamento farmacológico , Ciclo-Oxigenase 2/genética , Inflamação/tratamento farmacológico , Fator de Crescimento Transformador beta/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Valva Aórtica/efeitos dos fármacos , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Apoptose/efeitos dos fármacos , Calcinose/genética , Calcinose/patologia , Celecoxib/administração & dosagem , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , RNA Mensageiro/genéticaRESUMO
Ticagrelor is a powerful P2Y12 inhibitor with pleiotropic effects in the cardiovascular system. Consistently, we have reported that in patients with stable coronary artery disease (CAD) and concomitant chronic obstructive pulmonary disease (COPD) who underwent percutaneous coronary intervention (PCI), 1-month treatment with ticagrelor was superior in improving biological markers of endothelial function, compared with clopidogrel. The objective of this study was to investigate the mechanisms underlying these beneficial effects of ticagrelor by conducting molecular analyses of RNA isolated from peripheral blood cells of these patients. We determined mRNAs levels of markers of inflammation and oxidative stress, such as RORγt (T helper 17 cells marker), FoxP3 (regulatory T cells marker), NLRP3, ICAM1, SIRT1, Notch ligands JAG1 and DLL4, and HES1, a Notch target gene. We found that 1-month treatment with ticagrelor, but not clopidogrel, led to increased levels of SIRT1 and HES1 mRNAs. In patients treated with ticagrelor or clopidogrel, we observed a negative correlation among changes in both SIRT1 and HES1 mRNA and serum levels of Epidermal Growth Factor (EGF), a marker of endothelial dysfunction found to be reduced by ticagrelor treatment in our previous study. In conclusion, we report that in stable CAD/COPD patients ticagrelor positively regulates HES1 and SIRT1, two genes playing a protective role in the context of inflammation and oxidative stress. Our observations confirm and expand previous studies showing that the beneficial effects of ticagrelor in stable CAD/COPD patients may be, at least in part, mediated by its capacity to reduce systemic inflammation and oxidative stress.
Assuntos
Células Sanguíneas/efeitos dos fármacos , Doença da Artéria Coronariana/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Sirtuína 1/genética , Ticagrelor/farmacologia , Fatores de Transcrição HES-1/genética , Células Sanguíneas/metabolismo , Células Cultivadas , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Fatores de Transcrição HES-1/metabolismoRESUMO
Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/- mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.
Assuntos
Aorta/metabolismo , Aterosclerose/genética , Endotélio Vascular/metabolismo , Proteína KRIT1/genética , Mutação com Perda de Função , Animais , Aorta/patologia , Apoptose , Aterosclerose/metabolismo , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Proteína KRIT1/deficiência , Proteína KRIT1/metabolismo , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Receptor Notch1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17ß-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor ß (ERß), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERß. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERß to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.
Assuntos
Apoptose , Endotélio Vascular/metabolismo , Estradiol/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor Notch1/agonistas , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND/AIMS: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. METHODS: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo - Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. RESULTS: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. CONCLUSION: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.
Assuntos
Colágeno/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Hipóxia Celular , Células Cultivadas , Colágeno/análise , Colágeno/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Células-Tronco Mesenquimais/citologia , Regiões Promotoras GenéticasRESUMO
It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Insuficiência Cardíaca/sangue , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Receptores Notch/metabolismo , Soro/metabolismo , Transdução de Sinais , Idoso , Colágeno/farmacologia , Citocinas/sangue , Feminino , Géis/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H2O2) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H2O2 transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H2O2 was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H2O2 intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H2O2-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation.
Assuntos
Aquaporinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Leucemia/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aquaporinas/biossíntese , Aquaporinas/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Leucemia/patologia , NADPH Oxidases/genética , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Calcification of the aortic valve (CAVDS) is a major cause of aortic stenosis (AS) leading to loss of valve function which requires the substitution by surgical aortic valve replacement (SAVR) or transcatheter aortic valve intervention (TAVI). These procedures are associated with high post-intervention mortality, then the corresponding risk assessment is relevant from a clinical standpoint. This study compares the traditional Cox Proportional Hazard (CPH) against Machine Learning (ML) based methods, such as Deep Learning Survival (DeepSurv) and Random Survival Forest (RSF), to identify variables able to estimate the risk of death one year after the intervention, in patients undergoing either to SAVR or TAVI. We found that with all three approaches the combination of six variables, named albumin, age, BMI, glucose, hypertension, and clonal hemopoiesis of indeterminate potential (CHIP), allows for predicting mortality with a c-index of approximately 80 % . Importantly, we found that the ML models have a better prediction capability, making them as effective for statistical analysis in medicine as most state-of-the-art approaches, with the additional advantage that they may expose non-linear relationships. This study aims to improve the early identification of patients at higher risk of death, who could then benefit from a more appropriate therapeutic intervention.
Assuntos
Estenose da Valva Aórtica , Valva Aórtica , Calcinose , Aprendizado Profundo , Humanos , Valva Aórtica/cirurgia , Valva Aórtica/patologia , Calcinose/cirurgia , Calcinose/mortalidade , Feminino , Masculino , Idoso , Estenose da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/mortalidade , Substituição da Valva Aórtica Transcateter/mortalidade , Idoso de 80 Anos ou mais , Análise de Sobrevida , Fatores de Risco , Modelos de Riscos Proporcionais , Medição de Risco/métodos , Implante de Prótese de Valva Cardíaca/mortalidade , Implante de Prótese de Valva Cardíaca/métodos , Pessoa de Meia-IdadeRESUMO
Although the epidermal growth factor receptor 2 (ErbB2) and Notch1 signaling pathways have both significant roles in regulating cardiac biology, their interplay in the heart remains poorly investigated. Here, we present evidence of a crosstalk between ErbB2 and Notch1 in cardiac cells, with effects on autophagy and proliferation. Overexpression of ErbB2 in H9c2 cardiomyoblasts induced Notch1 activation in a post-transcriptional, p38-dependent manner, while ErbB2 inhibition with the specific inhibitor, lapatinib, reduced Notch1 activation. Moreover, incubation of H9c2 cells with lapatinib resulted in stalled autophagic flux and decreased proliferation, consistent with the established cardiotoxicity of this and other ErbB2-targeting drugs. Confirming the findings in H9c2 cells, exposure of primary neonatal mouse cardiomyocytes to exogenous neuregulin-1, which engages ErbB2, stimulated proliferation, and this effect was abrogated by concomitant inhibition of the enzyme responsible for Notch1 activation. Furthermore, the hearts of transgenic mice specifically overexpressing ErbB2 in cardiomyocytes had increased levels of active Notch1 and of Notch-related genes. These data expand the knowledge of ErbB2 and Notch1 functions in the heart and may allow better understanding the mechanisms of the cardiotoxicity of ErbB2-targeting cancer treatments.
Assuntos
Doença da Artéria Coronariana , Programas de Rastreamento/métodos , Pico do Fluxo Expiratório , Ativação Plaquetária , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Ticagrelor/administração & dosagem , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/terapia , Erros de Diagnóstico/prevenção & controle , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Efeitos Adversos de Longa Duração/diagnóstico , Efeitos Adversos de Longa Duração/etiologia , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Intervenção Coronária Percutânea/métodos , Inibidores da Agregação Plaquetária/administração & dosagem , Testes de Função Plaquetária , Prognóstico , Doença Pulmonar Obstrutiva Crônica/complicações , Medição de Risco/métodos , Inquéritos e QuestionáriosRESUMO
There is a growing interest in arterial and heart valve calcifications, as these contribute to cardiovascular outcome, and are leading predictors of cardiovascular and kidney diseases. Cardiovascular calcifications are often considered as one disease, but, in effect, they represent multifaced disorders, occurring in different milieus and biological phenotypes, following different pathways. Herein, we explore each different molecular process, its relative link with the specific clinical condition, and the current therapeutic approaches to counteract calcifications. Thus, first, we explore the peculiarities between vascular and valvular calcium deposition, as this occurs in different tissues, responds differently to shear stress, has specific etiology and time courses to calcification. Then, we differentiate the mechanisms and pathways leading to hyperphosphatemic calcification, typical of the media layer of the vessel and mainly related to chronic kidney diseases, to those of inflammation, typical of the intima vascular calcification, which predominantly occur in atherosclerotic vascular diseases. Finally, we examine calcifications secondary to rheumatic valve disease or other bacterial lesions and those occurring in autoimmune diseases. The underlying clinical conditions of each of the biological calcification phenotypes and the specific opportunities of therapeutic intervention are also considered and discussed.
RESUMO
Chronic rhinosinusitis with nasal polyps is a widespread pathology characterized by persistent inflammation of nasal and paranasal mucosa. Although it represents one of the most frequent diseases of the nasal cavities, its etiology is still not completely elucidated. There is evidence suggesting that the Notch signaling, a highly conserved intercellular pathway known to regulate many cellular processes, including inflammation, is implicated in nasal polyps formation. The purpose of this study was to investigate the expression of genes of the Notch pathway in nasal polyps from patients with chronic rhinosinusitis. Nasal polyps and adjacent mucosa tissue were obtained from 10 patients. RNA was analyzed by quantitative reverse transcriptase-polymerase chain reaction for the expression level of (1) Notch pathway components such as receptors (NOTCH1-4), ligands (DLL4, JAGGED-1), and target genes (HEY1, 2, and HES1) and (2) genes providing information on the pathogenesis of polyposis (C-MYC and SCGB1A1) and on eosinophils content (CCL26, IL5, and SAA2). We report a Notch-driven gene expression pattern in nasal polyps which correlates with the expression of genes highly expressed in eosinophils, whose presence is an important parameter to define the pathophysiologic diversity characterizing nasal polyps. Taken together, our results suggest a role for Notch signaling in the pathophysiology of polyposis. Further studies are needed to elucidate the role of Notch in nasal polyps formation and to establish whether it could represent a novel therapeutic target for this pathology.