RESUMO
For decades, bovine jugular vein conduits (BJV) and classic cryopreserved homografts have been the two most widely used options for pulmonary valve replacement (PVR) in congenital heart disease. More recently, decellularized pulmonary homografts (DPH) have provided an alternative avenue for PVR. Matched comparison of patients who received DPH for PVR with patients who received bovine jugular vein conduits (BJV) considering patient age group, type of heart defect, and previous procedures. 319 DPH patients were matched to 319 BJV patients; the mean age of BJV patients was 15.3 (SD 9.5) years versus 19.1 (12.4) years in DPH patients (p = 0.001). The mean conduit diameter was 24.5 (3.5) mm for DPH and 20.3 (2.5) mm for BJV (p < 0.001). There was no difference in survival rates between the two groups after 10 years (97.0 vs. 98.1%, p = 0.45). The rate of freedom from endocarditis was significantly lower for BJV patients (87.1 vs. 96.5%, p = 0.006). Freedom from explantation was significantly lower for BJV at 10 years (81.7 vs. 95.5%, p = 0.001) as well as freedom from any significant degeneration at 10 years (39.6 vs. 65.4%, p < 0.001). 140 Patients, matched for age, heart defect type, prior procedures, and conduit sizes of 20-22 mm (± 2 mm), were compared separately; mean age BJV 8.7 (4.9) and DPH 9.5 (7.3) years (p = n.s.). DPH showed 20% higher freedom from explantation and degeneration in this subgroup (p = 0.232). Decellularized pulmonary homografts exhibit superior 10-year results to bovine jugular vein conduits in PVR.
Assuntos
Cardiopatias Congênitas , Valva Pulmonar , Humanos , Bovinos , Animais , Lactente , Adolescente , Criança , Valva Pulmonar/transplante , Veias Jugulares/transplante , Resultado do Tratamento , Cardiopatias Congênitas/cirurgia , Aloenxertos , Estudos RetrospectivosRESUMO
von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.
Assuntos
Células Endoteliais/citologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Fator de von Willebrand/metabolismo , Animais , Anticorpos Monoclonais/química , Regulação da Expressão Gênica , Hemostasia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Knockout , Adesividade Plaquetária , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/química , TromboseRESUMO
OBJECTIVES: Mechanical composite valve grafts (MCVG) are the first-line therapy for aortic root replacement in young adults. Decellularized aortic homografts (DAH) present a promising novel alternative due to their lower thrombogenicity. We aimed to compare both treatment options regarding survival and valve-related adverse events. METHODS: This study was designed as a single-center retrospective cohort study including patients who underwent root replacement with MCVG or DAH between 2000 and 2022. Urgent or emergent procedures were excluded. RESULTS: The study cohort included 289 patients (MCVG n = 216, DAH n = 73) with a mean age of 48.5 ± 12 years (MCVG 49 ± 12 years vs DAH 47 ± 11 years; p = 0.23) and a median EuroScore II of 1.7% [1.2, 2.6]. The 30-day mortality was 1% (n = 3). Cumulative survival at 3 years was 99% for DAH and 94% for MCVG, respectively (p = 0.15). Mean follow-up was 98.9 ± 72.7 months. Bleeding events (n = 14, 6.5%) and thromboembolism (n = 14, 6.5%) were only observed in the MCVG group (p = 0.19 and 0.09 respectively). Four cases (5%) of moderate structural valve deterioration occurred, all in the DAH group (p = <0.001). The cumulative incidence of a composite end-point of valve-related adverse events was significantly higher in the MCVG group (p = 0.0295). CONCLUSIONS: Aortic root replacement with mechanical composite valve grafts and decellularized aortic homografts showed low mortality in an elective setting. Patients in the homograft cohort demonstrated significantly higher freedom from valve-related adverse events. DAH present a promising treatment option for young patients requiring root replacement, however, data on long-term durability are needed.
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The 5-hydroxytryptamine type 4 receptor (5-HT(4)R) regulates many physiological processes, including learning and memory, cognition, and gastrointestinal motility. Little is known about its role in angiogenesis. Using mouse hindlimb ischemia model of angiogenesis, we observed a significant reduction of limb blood flow recovery 14 days after ischemia and a decrease in density of CD31-positive vessels in adductor muscles in 5-HT(4)R(-/-) mice compared to wild type littermates. Our in vitro data indicated that 5-HT(4)R endogenously expressed in endothelial cells (ECs) may promote angiogenesis. Inhibition of the receptor with 5-HT(4)R antagonist RS 39604 reduced EC capillary tube formation in the reconstituted basement membrane. Using Boyden chamber migration assay and wound healing "scratch" assay, we demonstrated that RS 39604 treatment significantly suppressed EC migration. Transendothelial resistance measurement and immunofluorescence analysis showed that a 5-HT(4)R agonist RS 67333 led to an increase in endothelial permeability, actin stress fiber and interendothelial gap formation. Importantly, we provided the evidence that 5-HT(4)R-regulated EC migration may be mediated by Gα13 and RhoA. Our results suggest a prominent role of 5-HT(4)R in promoting angiogenesis and identify 5-HT(4)R as a potential therapeutic target for modulating angiogenesis under pathological conditions.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Receptores 5-HT4 de Serotonina/metabolismo , Compostos de Anilina/farmacologia , Animais , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Músculos/irrigação sanguínea , Músculos/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Piperidinas/farmacologia , Propano/análogos & derivados , Propano/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Agonistas do Receptor 5-HT4 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT4 de Serotonina/farmacologia , Cicatrização/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: decellularized aortic homografts (DAH) represent a promising alternative for aortic valve replacement in young adults due to their low immunogenicity and thrombogenicity. Herein, we report our midterm, single-center experience in adult patients with non-frozen DAH from corlife. METHODS: safety, durability, and hemodynamic performance were evaluated according to current guidelines in all consecutive patients who had received a DAH at our center since 03/2016. RESULTS: seventy-three (mean age 47 ± 11 years, 68.4% (n = 50) male) patients were enrolled. The mean diameter of the implanted DAH was 24 ± 2 mm. Mean follow-up was 36 ± 27 months, with a maximum follow-up of 85 months and cumulative follow-up of 215 years. No cases of stenosis were observed, in four (5.5%) cases moderate aortic regurgitation occurred, but no reintervention was required. No cases of early mortality, non-structural dysfunction, reoperation, valve endocarditis, or thrombosis were observed. Freedom from bleeding and thromboembolic events was 100%; freedom from re-intervention was 100%; survival was 98.6% (n = 72). CONCLUSIONS: early and mid-term results showed low mortality and 100% freedom from reoperation, thromboembolic events, and bleeding at our center. However, in order for this novel approach to be established as a valid alternative to aortic valve replacement in young patients, long-term data are required.
RESUMO
The Ross-Personalized External Aortic Root Support procedure is a surgical aortic valve replacement technique in which the autologous pulmonary valve is transposed in the aortic position to replace the malfunctioning aortic valve and a homograft is implanted in the pulmonary position. To prevent autograft dilatation, a Personalized External Aortic Root Support prosthesis is included in the proximal autograft anastomosis and wrapped around the ascending aorta. The aorta is transected transversely, the aortic valve is resected, and the coronary arteries are mobilized and cut out of the sinuses, leaving a rim. The pulmonary autograft is harvested by transecting the pulmonary artery and part of the right ventricular outflow tract. The autograft is approximated to the aortic root and inverted inside the ventricle. The proximal anastomosis is performed including the prosthesis between the aortic root and the autograft. The coronary buttons are threaded through appropriately positioned and sized holes in the prosthesis and reimplanted into the autograft. The ascending aorta is appropriately adapted and anastomosed with the distal autograft. When the patient is off cardiopulmonary bypass, the prosthesis can be closed longitudinally and is anchored to the distal aortic adventitia.
Assuntos
Insuficiência da Valva Aórtica , Estenose da Valva Aórtica , Implante de Prótese de Valva Cardíaca , Valva Pulmonar , Humanos , Autoenxertos/cirurgia , Aorta Torácica/cirurgia , Transplante Autólogo , Valva Aórtica/cirurgia , Aorta/cirurgia , Estenose da Valva Aórtica/cirurgia , Insuficiência da Valva Aórtica/cirurgia , Valva Pulmonar/transplante , Implante de Prótese de Valva Cardíaca/métodos , ReoperaçãoRESUMO
PPP protein phosphatases are an important enzyme family involved in a variety of aspects of cellular signalling and metabolism. PPPs are ubiquitous in eukaryotes, and are also present in many bacteria. Canonical eukaryotic PPP phosphotases are represented by five major subfamilies (PP1, PP2A, calcineurin, PP5 and PPEF/PP7). We previously reported that three "bacterial-like" PPP groups span the prokaryote-eukaryote boundary, including "Shewanella-like" phosphatases (Shelphs), which are in the focus of this study. Here we predict possible biological functions and functional partners of Shelphs by examining composition of bacterial operons and expression data for eukaryotes available in public databases. In Arabidopsis thaliana, the predicted possible roles include light-dependent regulation of chloroplast functions, signalling between the nucleus and the chloroplast, and defence responses. In Plasmodium falciparum, Shelphs are predicted to be associated with host cell invasion. One isoform has been located in the apical complex, essential for the interaction with the host cell. This makes P. falciparum Shelphs obvious potential candidates for therapeutic targets. Shelphs are also present in bacteria that constitute a considerable proportion of symbiotic microflora in humans. The predicted involvement of bacterial Shelphs in sensing and import of nutrients and extrusion of toxins may be relevant to the links between physiology of humans and our symbionts. Thus, despite the absence of Shelphs in animals, including humans, they may have a direct relationship to human health. Some predicted biological processes and potential functional partners of Shelphs are common between different bacterial and/or eukaryotic lineages, suggesting evolutionary conservation of some Shelph regulatory modules.
Assuntos
Proteínas de Bactérias/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Shewanella/enzimologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Regulação Enzimológica da Expressão Gênica , Humanos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologiaRESUMO
Background and Aim: The use of antibiotics is prohibited in poultry feeding in many countries worldwide, which has resulted in the emergence of antibiotic-resistant bacteria. Therefore, probiotic supplements are a good alternative in these circumstances. Probiotics, including Bifidobacterium adolescentis and Lactobacillus acidophilus, can reduce the development of resistance and stimulate the growth of broiler ducklings. This study aimed to evaluate properties of the probiotic supplement containing these bacteria. Materials and Methods: We studied the response of broiler ducklings to the addition of a dietary probiotic supplement containing lactobacilli and bifidobacteria until they reached 7 weeks of age. All birds participating in the experiment received probiotic supplements on days 1-7, 15-21, 29-35. The state of the ducklings was assessed on day 21. At the age of 21 and 42 days, 4-5 ml of blood was drawn from the wing vein of 10 randomly selected birds (5 in each group). Blood samples were analyzed for total protein, concentration of glucose, hemoglobin, calcium and inorganic phosphorus, as well as the number of erythrocytes and leukocytes. Enzymatic calorimetric method, molybdate method, and haemocytometry according to Nutt method and haemoglobin cyanide method were used to analyze blood samples. Results: The live weight of the experimental ducklings increased by 5.0%, showing a positive effect of probiotic supplementation, whereas their feed consumption per kilogram of weight gain decreased. Their hemoglobin content and red blood cell count increased. Although the number of lactobacilli and bifidobacteria increased, the number of Escherichia coli cells decreased 2.15-fold (p<0.05). The addition of the probiotic supplement contributed to improving the digestibility of protein and fat by 1.6%, fiber by 3.4%, and nitrogen-free extractive substances by 4.7%. The broiler ducklings had high meat quality indicators, including dressing percentage, which increased by 5.4%, whereas the fat content decreased by 1.3%. Conclusion: The introduction of the probiotic supplement in the diet of broiler ducklings improved their growth indicators and increased the number of lactobacilli and bifidobacteria while decreasing the number of E. coli cells in the intestine. It not only improved the meat quality but also increased the profit from $0.392 per bird in the control group to $0.472 per bird in the experimental group. Therefore, this probiotic supplement is a good alternative for raising ducklings in large enterprises and farms. The study limitations may be that the results are only applicable to broiler ducklings. The use of lactobacilli may yield different results in other bird breeds or broiler ducklings in different age groups.
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OBJECTIVES: Early results from the prospective ESPOIR Trial have indicated excellent results for pulmonary valve replacement using decellularized pulmonary homografts (DPH). METHODS: A 5-year analysis of ESPOIR Trial patients was performed to provide an insight into the midterm DPH performance. ESPOIR Trial and Registry patients were matched with cryopreserved homografts (CH) patients considering patient age, type of heart defect and previous procedures to present the overall experience with DPH. RESULTS: A total of 121 patients (59 female) were prospectively enrolled (8/2014-12/2016), median age 16.5 years (interquartile range 11.2-29.8), and median DPH diameter 24 mm. One death (73 year-old) occurred during a median follow-up of 5.9 years (5.4-6.4), in addition to 2 perioperative deaths resulting in an overall mortality rate of 2.5%. One case of endocarditis in 637 patient-years was noticed, resulting in an incidence of 0.15% per patient-year. At 5 years, the mean peak gradient was 19.9 mmHg (9.9), mean regurgitation 0.9 (0.6, grade 0-3) and freedom from explantation/any reintervention 97.5% (1.5). The combined DPH cohort, n = 319, comprising both Trial and Registry data, showed significantly better freedom from explantation for DPH 95.5% (standard deviation 1.7) than CH 83.0% (2.8) (P < 0.001) and less structural valve degeneration at 10 years when matched to 319 CH patients [DPH 65.5% (standard deviation 4.4) and CH 47.3% (3.7), P = 0.11]. CONCLUSIONS: The 5-year data of the prospective ESPOIR Trial show excellent performance for DPH and low rates of adverse events. ESPOIR Registry data up to 15 years, including a matched comparison with CH, demonstrated statistically significant better freedom from explantation.
Assuntos
Implante de Prótese de Valva Cardíaca , Próteses Valvulares Cardíacas , Valva Pulmonar , Humanos , Feminino , Adolescente , Idoso , Valva Pulmonar/transplante , Estudos Prospectivos , Resultado do Tratamento , Sistema de Registros , Aloenxertos/cirurgia , Próteses Valvulares Cardíacas/efeitos adversos , Implante de Prótese de Valva Cardíaca/métodos , Valva Aórtica/cirurgia , SeguimentosRESUMO
Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. The function of VASP in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP(-/-) mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP(-/-) mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may be correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function.
Assuntos
Permeabilidade Capilar , Moléculas de Adesão Celular/deficiência , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Pulmão/irrigação sanguínea , Proteínas dos Microfilamentos/deficiência , Fosfoproteínas/deficiência , Receptor PAR-1/metabolismo , Trombina/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Impedância Elétrica , Humanos , Junções Intercelulares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Neuropeptídeos/metabolismo , Fosfoproteínas/genética , Interferência de RNA , Fatores de Tempo , Transfecção , Regulação para Cima , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We review the evidence suggesting the involvement of Cadherin 13 (CDH13, T-cadherin, H-cadherin) in various cancers. CDH13 is an atypical member of the cadherin family, devoid of a transmembrane domain and anchored to the exterior surface of the plasma membrane via a glycosylphosphatidylinositol anchor. CDH13 is thought to affect cellular behavior largely through its signaling properties. It is often down-regulated in cancerous cells. CDH13 down-regulation has been associated with poorer prognosis in various carcinomas, such as lung, ovarian, cervical and prostate cancer. CDH13 re-expression in most cancer cell lines inhibits cell proliferation and invasiveness, increases susceptibility to apoptosis, and reduces tumor growth in in vivo models. These properties suggest that CDH13 may represent a possible target for therapy in some cancers. At the same time, CDH13 is up-regulated in blood vessels growing through tumors and promotes tumor neovascularization. In contrast to most cancer cell lines, CDH13 overexpression in endothelial cells promotes their proliferation and migration, and has a pro-survival effect. We also discuss molecular mechanisms that may regulate CDH13 expression and underlie its roles in cancer.
Assuntos
Caderinas/fisiologia , Genes Supressores de Tumor , Neoplasias/patologia , Movimento Celular , Proliferação de Células , HumanosRESUMO
Coronavirus disease 2019 (COVID-19) radically modified the organization of healthcare systems with shutdown of routine activities and outpatient clinics. Herein, we report our institutional experience with a Telemonitoring and Care Program (TC-Program) to monitor and support left ventricular assist device (LVAD) patients during COVID-19 outbreak. This single-arm cohort study analyzed 156 patients who entered the TC-Program at our institution between April and August 2020. The TC-Program was based on routine phone calls to patients and a 24/7 emergency line. In November 2020, patients were asked for feedback on the TC-Program and checked for survival, transplant, or explant. The primary endpoint was the rate of TC-Program-driven interventions. Patients (males: 82.8%) were 61 years old (interquartile range [IQR]: 53.0-67.5) and on LVAD support for 1,266 days (IQR: 475-2,211). Patients were included in the TC-Program for a median time of 99 days (min:15, max:120) and received a median number of six phone calls (min:1, max:14). Twenty-three patients (14.7%) were referred for clinical evaluation after phone contact. Two patients (1.27%) were diagnosed with COVID-19: one of them died after intensive care, and one remained paucisymptomatic and recovered. Three patients asked to exit the program considering it not useful while the others gave high rates in terms of usefulness (median: 9, IQR: 8-10), information (median: 9, IQR: 8-10), good medical care (median: 9, IQR: 8-10), and psychologic support (median: 8, IQR: 7-10). A TC-Program based on the four ICSA principles (Inform, Care, Support, and Adapt) is feasible in LVAD patients and can be rapidly implemented during the COVID-19 pandemic.
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COVID-19/prevenção & controle , Insuficiência Cardíaca/terapia , Coração Auxiliar/estatística & dados numéricos , Controle de Infecções/organização & administração , Telemedicina , COVID-19/epidemiologia , Estudos de Coortes , Surtos de Doenças , Feminino , Insuficiência Cardíaca/epidemiologia , Coração Auxiliar/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/prevenção & controle , SARS-CoV-2RESUMO
T-cadherin is an atypical member of the cadherin family, which lacks the transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. Unlike canonical cadherins, it is believed to function primarily as a signaling molecule. T-cadherin is highly expressed in endothelium. Using transendothelial electrical resistance measurements and siRNA-mediated depletion of T-cadherin in human umbilical vein endothelial cells, we examined its involvement in regulation of endothelial barrier. We found that in resting confluent monolayers adjusted either to 1% or 10% serum, T-cadherin depletion modestly, but consistently reduced transendothelial resistance. This was accompanied by increased phosphorylation of Akt and LIM kinase, reduced phosphorylation of p38 MAP kinase, but no difference in tubulin acetylation and in phosphorylation of an actin filament severing protein cofilin and myosin light chain kinase. Serum stimulation elicited a biphasic increase in resistance with peaks at 0.5 and 4-5 h, which was suppressed by a PI3 kinase/Akt inhibitor wortmannin and a p38 inhibitor SB 239063. T-cadherin depletion increased transendothelial resistance between the two peaks and reduced the amplitude of the second peak. T-cadherin depletion abrogated serum-induced Akt phosphorylation at Thr308 and reduced phosphorylation at Ser473, reduced phosphorylation of cofilin, and accelerated tubulin deacetylation. Adiponectin slightly improved transendothelial resistance irrespectively of T-cadherin depletion. T-cadherin depletion also resulted in a reduced sensitivity and delayed responses to thrombin. These data implicate T-cadherin in regulation of endothelial barrier function, and suggest a complex signaling network that links T-cadherin and regulation of barrier function.
Assuntos
Caderinas/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Acetilação , Fatores de Despolimerização de Actina/metabolismo , Adiponectina/metabolismo , Caderinas/genética , Permeabilidade Capilar/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Células Cultivadas , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Humanos , Quinases Lim/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais , Trombina/metabolismo , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PPEF/PP7 represents one of the five subfamilies of the PPP protein Ser/Thr phosphatases. Studies published in recent years point to a role of plant PP7 at a crossroad of different pathways of light and stress signalling. In animals, PPEFs are highly expressed in sensory neurons, and Drosophila PPEF phosphatase, rdgC, is essential for dephosphorylation of rhodopsin. Expression profiling suggests that mammalian PPEF may play a role in stress-protective responses, cell survival, growth, proliferation, and oncogenesis. Despite structural similarities of the catalytic domains and the fact that some of these phosphatases are involved in light perception both in animals and in plants, the plant and non-plant representatives of this group have distinct domain architecture and appear not to be orthologues.
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Fosfoproteínas Fosfatases/fisiologia , Animais , Sítios de Ligação , Motivos EF Hand , Humanos , Fosfoproteínas Fosfatases/química , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Estrutura Terciária de ProteínaRESUMO
This study aims to compare the immunogenicity of fresh decellularized with cryopreserved native heart valve allografts to identify potential immunological risks in subsequent organ transplantations. We measured the induction of allogeneic HLA class I and II specific antibodies and characterized donor-specific antibodies by Luminex-based single beads assay in both groups. Serum samples were collected before valve replacement, at 3 and 24 months postoperatively. Donor-specific HLA antibodies were assessed positive if the mean fluorescent intensity (MFI) was >1000. Between November 2016 and April 2017 patients with fresh decellularized homografts (n = 4) and cryopreserved native homografts (n = 4) were analyzed. Patients receiving cryopreserved native allografts reacted with broad HLA-specific antibody response. Antibodies were directed against mismatched HLA antigens of the donors but also against HLA specificities not present on the homograft with many antibodies having mean fluorescence intensity values >10 000. While HLA class I specific antibodies showed a significant increase (P = .002) in their MFI values on day 90, HLA class II specific antibodies did not show a significant increase (P = .069). In the fresh decellularized homografts group, no significant antibody induction was observed. Consequently, the native group presented significantly higher MFIs for HLA antibodies on day 90 compared with the patients receiving decellularized allografts (P = .021). No detectable HLA antibody response was observed after implantation of decellularized in comparison with cryopreserved native allografts. Lower immunogenicity as compared with native homografts might increase the chance of receiving a transplant if will be required later in the life of the patients.
Assuntos
Antígenos HLA , Doadores de Tecidos , Alelos , Aloenxertos , Antígenos HLA/genética , Humanos , Transplante HomólogoRESUMO
T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in tumor-associated neovascularization and is deregulated in many cancers. Unlike canonical cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the plasma membrane have recently been identified. We examined T-cadherin location in human endothelial cells using confocal microscopy and subcellular fractionation. We found that a considerable proportion of T-cadherin is located in the nucleus and in the centrosomes. T-cadherin colocalized with a centrosomal marker gamma-tubulin uniformly throughout the cell cycle at least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently concentrated in the midbody and was apparently degraded. Its overexpression resulted in an increase in the number of multinuclear cells, whereas its downregulation by small interfering RNA led to an increase in the number of cells with multiple centrosomes. These findings indicate that deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or centrosomal replication.
Assuntos
Caderinas/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Citocinese/fisiologia , Células Endoteliais/metabolismo , Western Blotting , Imunofluorescência , Humanos , Imunoprecipitação , Microscopia Confocal , Transporte Proteico/fisiologia , RNA Interferente Pequeno , Tubulina (Proteína)/metabolismoRESUMO
G alpha12 constitutes, along with G alpha13, one of the four families of alpha subunits of heterotrimeric G proteins. We found that the N terminus of G alpha12, but not those of other G alpha subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous G alpha12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N-terminal sequence of G alpha12 fused to GFP efficiently targeted the fusion protein to mitochondria. G alpha12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by G alpha12/13, inhibited mitochondrial motility, while depletion of endogenous G alpha12 increased mitochondrial motility. G alpha12Q229L variants uncoupled from RhoGEFs (but not fully functional activated G alpha12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined G alpha12Q229L variants reduced phosphorylation of Bcl-2 at Ser-70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl-2. These G alpha12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein alpha subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.
Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Potencial da Membrana Mitocondrial , Movimento , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
Protein phosphorylation is an important mechanism implicated in physiology of any organism, including parasitic protozoa. Enzymes that control protein phosphorylation (kinases and phosphatases) are considered promising targets for drug development. This review attempts to provide the first account of the current understanding of the structure, regulation and biological functions of protein Ser/Thr phosphatases in unicellular parasites. We have examined the complements of phosphatases ("phosphatomes") of the PPP and PPM families in several species of Apicomplexa (including malaria parasite Plasmodium), as well as Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis and a microsporidium Encephalitozoon cuniculi. Apicomplexans have homologues (in most cases represented by single isoforms) of all human PPP subfamilies. Some apicomplexan PPP phosphatases have no orthologues in their vertebrate hosts, including a previously unrecognised group of pseudo-phosphatases with putative Ca(2+)-binding domains, which we designate as EFPP. We also describe the presence of previously undetected Zn finger motifs in PPEF phosphatases from kinetoplastids, and a likely case of convergent evolution of tetratricopeptide repeat domain-containing phosphatases in G. lamblia. Among the parasites examined, E. cuniculi has the smallest Ser/Thr phosphatome (5 PPP and no PPM), while T. vaginalis shows the largest expansion of the PPP family (169 predicted phosphatases). Most protozoan PPM phosphatases cluster separately from human sequences. The structural peculiarities or absence of human orthologues of a number of protozoan protein Ser/Thr phosphatases makes them potentially suitable targets for chemotherapy and thus warrants their functional assessment.
Assuntos
Eucariotos/enzimologia , Fosfoproteínas Fosfatases , Animais , Eucariotos/classificação , Eucariotos/genética , Evolução Molecular , Humanos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Filogenia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The aim of this review is to provide a synthesis of the published experimental data on protein tyrosine phosphatases from parasitic protozoa, in silico analysis based on the availability of completed genomes and to place available data for individual phosphatases from different unicellular parasites into the comparative and evolutionary context. We analysed the complement of protein tyrosine phosphatases (PTP) in several species of unicellular parasites that belong to Apicomplexa (Plasmodium; Cryptosporidium, Babesia, Theileria, and Toxoplasma), kinetoplastids (Leishmania and Trypanosoma spp.), as well as Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis and a microsporidium Encephalitozoon cuniculi. The analysis shows distinct distribution of the known families of tyrosine phosphatases in different species. Protozoan tyrosine phosphatases show considerable levels of divergence compared with their mammalian homologues, both in terms of sequence similarity between the catalytic domains and the structure of their flanking domains. This potentially makes them suitable targets for development of specific inhibitors with minimal effects on physiology of mammalian hosts.
Assuntos
Eucariotos/enzimologia , Proteínas Tirosina Fosfatases , Animais , Apicomplexa/enzimologia , Kinetoplastida/enzimologia , Microsporídios não Classificados/imunologia , Fosforilação , Proteínas Tirosina Fosfatases/classificação , Proteínas Tirosina Fosfatases/metabolismo , RNA de Protozoário/metabolismoRESUMO
Apoptosis signal-regulating kinase (ASK1) is a mitogen-activated protein kinase (MAPK) that transduces apoptotic signals from a variety of stresses. We have shown previously that alpha subunits of heterotrimeric G12 and G13 proteins stimulate ASK1 kinase activity and ASK1-dependent apoptosis. Here, we report a novel mechanism of G-protein-dependent regulation of ASK1. We demonstrated that G alpha13 forms a complex with ASK1 in an activation-independent manner. Both N- and C-terminal regulatory domains of ASK1 were essential for the efficient interaction, while its kinase domain was not required. Formation of the G alpha13-ASK1 complex was enhanced by JNK-interacting leucine zipper protein, JLP. Constitutively activated G alpha13Q226L increased ASK1 expression. Short-term activation of a serotonin 5-HT4 receptor that is coupled to G alpha13 also increased ASK1 expression. Importantly, prolonged activation of 5-HT4 receptor in COS-7 cells or prolonged treatment of human umbilical vein endothelial cells with thrombin concomitantly down-regulated both G alpha13 and ASK1. Data showed that G alpha13Q226L reduced the rate of ASK1 degradation, decreased ASK1 ubiquitination, and reduced association of ASK1 with an E3 ubiquitin ligase CHIP, previously shown to mediate ASK1 degradation. Our findings indicate that ASK1 expression levels can be regulated by G alpha13, at least in part via control of ASK1 ubiquitination and degradation.