Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.570
Filtrar
1.
FASEB J ; 35(11): e21942, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34670018

RESUMO

Atherosclerosis is a chronic inflammatory disease. Pathophysiological similarities between chronic infections and atherosclerosis triggered interests between these conditions. The seroepidemiological study showed that Helicobacter pylori strains that express cytotoxin-associated gene A (CagA), an oncoprotein and a major virulence factor, was positively correlated with atherosclerosis and related clinical events. Nevertheless, the underlying mechanism is poorly understood. In this study, the seroprevalence of infection by H. pylori and by strains express CagA assessed by enzyme-linked immunosorbent assay (ELISA) showed that the prevalence of CagA strains rather than H. pylori in patients was positively correlated with atherogenesis. Correspondingly, we found that CagA augmented the growth of plaque of ApoE-/- mice in the early stage of atherosclerosis and promoted the expression of adhesion molecules and inflammatory cytokines in mouse aortic endothelial cells (MAECs). Mechanistically, both si-NLRP3 and si-IL-1ß mitigated the promoting effect of CagA on the inflammatory activation of HAECs. In vivo, the inhibition of NLRP3 by MCC950 significantly attenuated the promoting effect of CagA on plaque growth of ApoE-/- mice. We also propose NLRP3 as a potential therapeutic target for CagA-positive H. pylori infection-related atherosclerosis and emphasize the importance of inflammation in atherosclerosis pathology.


Assuntos
Antígenos de Bactérias/metabolismo , Aorta/patologia , Aterosclerose/sangue , Proteínas de Bactérias/metabolismo , Caspase 1/metabolismo , Células Endoteliais/metabolismo , Infecções por Helicobacter/sangue , Helicobacter pylori/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Placa Aterosclerótica/sangue , Idoso , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Aorta/metabolismo , Aterosclerose/microbiologia , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Feminino , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Placa Aterosclerótica/microbiologia , Estudos Soroepidemiológicos , Células THP-1
2.
Arterioscler Thromb Vasc Biol ; 41(11): 2671-2680, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34615376

RESUMO

The aorta is highly heterogeneous, containing many different types of cells that perform sophisticated functions to maintain aortic homeostasis. Recently, single-cell RNA sequencing studies have provided substantial new insight into the heterogeneity of vascular cell types, the comprehensive molecular features of each cell type, and the phenotypic interrelationship between these cell populations. This new information has significantly improved our understanding of aortic biology and aneurysms at the molecular and cellular level. Here, we summarize these findings, with a focus on what single-cell RNA sequencing analysis has revealed about cellular heterogeneity, cellular transitions, communications among cell populations, and critical transcription factors in the vascular wall. We also review the information learned from single-cell RNA sequencing that has contributed to our understanding of the pathogenesis of vascular disease, such as the identification of cell types in which aneurysm-related genes and genetic variants function. Finally, we discuss the challenges and future directions of single-cell RNA sequencing applications in studies of aortic biology and diseases.


Assuntos
Aorta/metabolismo , Aneurisma Aórtico/genética , Perfilação da Expressão Gênica , Análise de Célula Única , Transcriptoma , Animais , Aorta/patologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Dilatação Patológica , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , RNA-Seq
3.
Am J Physiol Heart Circ Physiol ; 321(4): H756-H769, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34506228

RESUMO

Inflammation caused by infiltrating macrophages and T cells promotes plaque growth in atherosclerosis. Cadherin-11 (CDH11) is a cell-cell adhesion protein implicated in several fibrotic and inflammatory diseases. Much of the research on CDH11 concerns its role in fibroblasts, although its expression in immune cells has been noted as well. The objective of this study was to assess the effect of CDH11 on the atherosclerotic immune response. In vivo studies of atherosclerosis indicated an increase in Cdh11 in plaque tissue. However, global loss of Cdh11 resulted in increased atherosclerosis and inflammation. It also altered the immune response in circulating leukocytes, decreasing myeloid cell populations and increasing T-cell populations, suggesting possible impaired myeloid migration. Bone marrow transplants from Cdh11-deficient mice resulted in similar immune cell profiles. In vitro examination of Cdh11-/- macrophages revealed reduced migration, despite upregulation of a number of genes related to locomotion. Flow cytometry revealed an increase in CD3+ and CD4+ helper T-cell populations in the blood of both the global Cdh11 loss and the bone marrow transplant animals, possibly resulting from increased expression by Cdh11-/- macrophages of major histocompatibility complex class II molecule genes, which bind to CD4+ T cells for coordinated activation. CDH11 fundamentally alters the immune response in atherosclerosis, resulting in part from impaired macrophage migration and altered macrophage-induced T-cell activation.NEW & NOTEWORTHY Cadherin-11 is well known to contribute to inflammatory and fibrotic disease. Here, we examined its role in atherosclerosis progression, which is predominantly an inflammatory process. We found that while cadherin-11 is associated with plaque progression, global loss of cadherin-11 exacerbated the disease phenotype. Moreover, loss of cadherin-11 in bone marrow-derived immune cells resulted in impaired macrophage migration and an unexplained increase in circulating helper T cells, presumably due to altered macrophage function without cadherin-11.


Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Caderinas/deficiência , Quimiotaxia , Macrófagos/metabolismo , Placa Aterosclerótica , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Aorta/imunologia , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Transplante de Medula Óssea , Caderinas/genética , Modelos Animais de Doenças , Feminino , Ativação Linfocitária , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/imunologia
4.
Am J Physiol Heart Circ Physiol ; 321(5): H825-H838, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34533401

RESUMO

Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.


Assuntos
Amidoidrolases/biossíntese , Aorta/enzimologia , Pressão Sanguínea , Ventrículos do Coração/enzimologia , Hipertensão/enzimologia , Hipertrofia Ventricular Esquerda/enzimologia , Remodelação Vascular , Função Ventricular Esquerda , Remodelação Ventricular , Amidoidrolases/genética , Angiotensina II , Animais , Aorta/patologia , Aorta/fisiopatologia , Modelos Animais de Doenças , Indução Enzimática , Fibrose , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hipertensão/induzido quimicamente , Hipertensão/patologia , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Tempo , Vasodilatação
5.
Arterioscler Thromb Vasc Biol ; 41(11): 2730-2739, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34587757

RESUMO

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.


Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Suplementos Nutricionais , Ácido N-Acetilneuramínico/administração & dosagem , Ácidos Neuramínicos/administração & dosagem , Placa Aterosclerótica , Ração Animal , Animais , Anticorpos/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/imunologia , Ácidos Neuramínicos/metabolismo , Pan troglodytes , Receptores de LDL/genética , Receptores de LDL/metabolismo , Sialadenite/metabolismo , Sialadenite/patologia , Células THP-1
6.
PLoS One ; 16(9): e0256278, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34516570

RESUMO

INTRODUCTION: Aneurysms of the ascending aorta (AA) correspond to a dilatation of the ascending aorta that progressively evolves over several years. The main complication of aneurysms of the ascending aorta is type A aortic dissection, which is associated with very high rates of morbidity and mortality. Prophylactic ascending aorta replacement guidelines are currently based on maximal AA diameter. However, this criterion is imperfect. Stretching tests on the aorta carried out ex-vivo make it possible to determine the elastic properties of healthy and aneurysmal aortic fragments (tension test, resistance before rupture). For several years now, cardiac magnetic resonance imaging (MRI) has provided another means of evaluating the elastic properties of the aorta. This imaging technique has the advantage of being non-invasive and of establishing aortic compliance (local measurement of stiffness) without using contrast material by measuring the variation of the aortic surface area during the cardiac cycle, and pulse wave velocity (overall stiffness of the aorta). MATERIALS AND METHODS: Prospective single-center study including 100 patients with ascending aortic aneurysm requiring surgery. We will perform preoperative cine-MRI and biomechanical laboratory stretching tests on aortic samples collected during the cardiac procedure. Images will be acquired with a 3T MRI with only four other acquisitions in addition to the conventional protocol. These additional sequences are a Fast Low Angle Shot (FLASH)-type sequence performed during a short breath-hold in the transverse plane at the level of the bifurcation of the pulmonary artery, and phase-contrast sequences that encodes velocity at the same localization, and also in planes perpendicular to the aorta at the levels of the sino-tubular junction and the diaphragm for the descending aorta. For ex-vivo tests, the experiments will be carried out by a biaxial tensile test machine (ElectroForce®). Each specimen will be stretched with 10 times of 10% preconditioning and at a rate of 10 mm/min until rupture. During the experiment, the tissue is treated under a 37°C saline bath. The maximum elastic modulus from each sample will be calculated. RESULTS: The aim of this study is to obtain local patient-specific elastic modulus distribution of the ascending aorta from biaxial tensile tests and to assess elastic properties of the aorta using MRI, then to evaluate the correlation between biaxial tests and MRI measurements. DISCUSSION: Our research hypothesis is that there is a correlation between the evaluation of the elastic properties of the aorta from cardiac MRI and from stretching tests performed ex-vivo on aorta samples collected during ascending aorta replacement.


Assuntos
Aorta/patologia , Aneurisma Aórtico/patologia , Elasticidade , Imagem Cinética por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Fenômenos Biomecânicos , Suspensão da Respiração , Humanos , Estudos Prospectivos , Análise de Onda de Pulso
7.
Cells ; 10(9)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34572082

RESUMO

Aortic diseases comprise aneurysms, dissections, and several other pathologies. In general, aging is associated with a slow but progressive dilation of the aorta, along with increased stiffness and pulse pressure. The progression of aortic disease is characterized by subclinical development or acute presentation. Recent evidence suggests that inflammation participates causally in different clinical manifestations of aortic diseases. As of yet, diagnostic imaging and surveillance is mainly based on ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI). Little medical therapy is available so far to prevent or treat the majority of aortic diseases. Endovascular therapy by the introduction of covered stentgrafts provides the main treatment option, although open surgery and implantation of synthetic grafts remain necessary in many situations. Because of the risks associated with surgery, there is a need for identification of pharmaceutical targets interfering with the pathophysiology of aortic remodeling. The participation of innate immunity and inflammasome activation in different cell types is common in aortic diseases. This review will thus focus on inflammasome activities in vascular cells of different chronic and acute aortic diseases and discuss their role in development and progression. We will also identify research gaps and suggest promising therapeutic targets, which may be used for future medical interventions.


Assuntos
Aorta , Doenças da Aorta , Inflamassomos/metabolismo , Aorta/citologia , Aorta/patologia , Aorta/fisiologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/fisiopatologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/fisiopatologia , Doenças da Aorta/metabolismo , Doenças da Aorta/fisiopatologia , Proteínas de Ligação a DNA/metabolismo , Sistemas de Liberação de Medicamentos , Células Endoteliais/metabolismo , Humanos , Imuno-Histoquímica , Inflamassomos/fisiologia , Inflamação/metabolismo , Inflamação/fisiopatologia , Interleucina-1beta/metabolismo , Linfócitos/metabolismo , Macrófagos/metabolismo , Miócitos de Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
8.
Sci Rep ; 11(1): 15790, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349174

RESUMO

The mechanisms leading to cardiac remodeling in Marfan syndrome (MFS) are a matter of debate since it could be either due to structural dysfunction of the myocardial extracellular matrix or to increased afterload caused by the dilated aorta. We aim to characterize the presence of abnormal myocardial function in MFS and to investigate its potential association with increased afterload. Aorta, left ventricle (LV) and the postsystolic thickening (PST) were analyzed in echocardiography in Fbn1C1039G/+ mice and in patients with MFS in comparison with wild type (WT) mice and healthy humans. PST was more frequent in MFS than in WT mice (p < 0.05). MFS mice with PST showed larger aorta than those without PST. Patients with MFS showed larger aorta, poorer LV function and a higher prevalence of PST (56%) than did the healthy controls (23%); p = 0.003. Blood pressure was similar. The higher prevalence of PST in an experimental murine model and in MFS patients, regardless of systemic arterial pressure, suggests an increased afterload on the LV myocardium. This finding supports the use of PST as an indicator of myocardial damage and encourage searching for novel early preventive therapy.


Assuntos
Síndrome de Marfan/patologia , Miocárdio/patologia , Animais , Aorta/patologia , Modelos Animais de Doenças , Ventrículos do Coração/patologia , Humanos , Síndrome de Marfan/fisiopatologia , Camundongos Endogâmicos C57BL , Função Ventricular Esquerda
9.
Arch Biochem Biophys ; 710: 109000, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34343486

RESUMO

Impaired endothelium-mediated vasodilation and/or increased sensitivity to vasoconstrictors lead to vascular smooth muscle cell (VSMC) dysfunction in individuals with diabetes. Diabetic nephropathy is associated with a considerably higher risk of cardiovascular disease and death than their nondiabetic counterparts. We studied the activity of Cullin 3 RING ubiquitin ligase (CRL3) and its substrates in mice using an intraperitoneal injection of streptozotocin (STZ) and db/db mice. The levels of CRL3 adaptors, including Kelch-like 2/3 (KLHL2/3) and Rho-related BTB domain-containing protein 1, were significantly decreased in the aortic tissues and heart of the STZ group, whereas the levels of Cullin 3 (CUL3) and its neddylated derivatives were substantially increased. Decreased KLHL3 expression and significantly increased expression of NEDD8 conjugates were observed in the kidneys of db/db mice. The neddylation inhibitor MLN4924 decreased the degradation of KLHL2/KLHL3 under high-glucose conditions with/without insulin, and transfection with KLHL2 promoted the degradation of its substrates with-no-lysine (WNK) kinases. Increased abundance of WNK3, RhoA/ROCK activity and phosphodiesterase 5 enhanced the sensibility to vasoconstrictors and impaired vasodilation. Moreover, WNK3 localized in VSMCs undergoing cell division, and high-glucose medium increased WNK3 signaling in VSMCs undergoing mitosis, which might explain the increased thickness of aortic tissues in subjects with diabetes. Increases in WNK4 abundance resulted in increased sodium reabsorption in the distal renal tubules. Thus, KLHL2/RhoBTB1/KLHL3 inactivation in the aortic tissues and kidney is a result of excessive activation of neddylation in hyperglycemia and hyperinsulinemia, which affects vascular tone and sodium reabsorption.


Assuntos
Proteínas Culina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Vasoconstrição/fisiologia , Animais , Aorta/metabolismo , Aorta/patologia , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
10.
Eur Rev Med Pharmacol Sci ; 25(15): 5063-5069, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34355379

RESUMO

OBJECTIVE: Vaccine-induced immune thrombocytopenia (VITT) is a new syndrome occurring primarily in healthy young adults, with a female predominance, after receiving the first dose of ChAdOx1 nCoV-19 vaccine. We describe VITT syndrome characterized by severe thrombosis and thrombocytopenia found in our patient, with fatal outcome. CASE REPORT: A 58-year-old man, after 13 days from the first administration of ChAdOx1 nCoV-19 vaccine (AstraZeneca), presented with abdominal pain, diarrhea and vomitus. Laboratory tests revealed a severe thrombocytopenia, low fibrinogen serum levels and marked increase of D-dimer serum levels. The patient quickly developed a multiple organ failure, till death, three days after the hospital admission. RESULTS: At histology, in the lungs, interalveolar septa appeared thickened with microthrombi in the capillaries and veins. Interalveolar septa appeared thickened and showed vascular proliferation. Thrombi were detected in the capillaries of glomerular tufts. In the hearth, thrombi were observed in veins and capillaries. In the liver, voluminous fibrin thrombi were diffusely observed in the branches of the portal vein. Microthrombi were also found in the vasa vasorum of the wall of abdominal aorta. In the brain, microthrombi were observed in the capillaries of the choroid plexuses. Diffuse hemorrhagic necrosis was observed in the intestinal wall with marked congestion of the venous vessels. CONCLUSIONS: In our patient, the majority of data necessary for a VITT final diagnosis were present: thrombocytopenia and thrombosis in pulmonary, portal, hepatic, renal and mesenteric veins, associated with a marked increase of D-dimer serum levels. The finding of cerebral thrombosis in choroid plexuses, is a new finding in VITT. These features are suggestive for a very aggressive form of VITT.


Assuntos
Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Púrpura Trombocitopênica Idiopática/etiologia , Trombose/etiologia , Aorta/patologia , COVID-19/sangue , Vacinas contra COVID-19/administração & dosagem , Plexo Corióideo/patologia , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Íleo/patologia , Rim/patologia , Fígado/patologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Púrpura Trombocitopênica Idiopática/sangue , Trombose/sangue
11.
Cells ; 10(7)2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34359889

RESUMO

Blocking tumor vascularization has not yet come to fruition to the extent it was hoped for, as angiogenesis inhibitors have shown only partial success in the clinic. We hypothesized that under-appreciated vascular wall-resident stem and progenitor cells (VW-SPCs) might be involved in tumor vascularization and influence effectiveness of anti-angiogenic therapy. Indeed, in patient samples, we observed that vascular adventitia-resident CD34+ VW-SPCs are recruited to tumors in situ from co-opted vessels. To elucidate this in detail, we established an ex vivo model using concomitant embedding of multi-cellular tumor spheroids (MCTS) and mouse aortic rings (ARs) into collagen gels, similar to the so-called aortic ring assay (ARA). Moreover, ARA was modified by removing the ARs' adventitia that harbors VW-SPCs. Thus, this model enabled distinguishing the contribution of VW-SPCs from that of mature endothelial cells (ECs) to new vessel formation. Our results show that the formation of capillary-like sprouts is considerably delayed, and their number and network formation were significantly reduced by removing the adventitia. Substituting iPSC-derived neural spheroids for MCTS resulted in distinct sprouting patterns that were also strongly influenced by the presence or absence of VW-SPCs, also underlying the involvement of these cells in non-pathological vascularization. Our data suggest that more comprehensive approaches are needed in order to block all of the mechanisms contributing to tumor vascularization.


Assuntos
Túnica Adventícia/patologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Células-Tronco/patologia , Animais , Antígenos CD34/metabolismo , Aorta/patologia , Capilares/patologia , Humanos , Camundongos , Modelos Biológicos , Neovascularização Patológica , Neovascularização Fisiológica , Ratos , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo
12.
Cells ; 10(8)2021 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-34440796

RESUMO

The purpose of this study is to investigate the role of platelet bone morphogenetic proteins (BMP)-4 during vascular inflammation and remodeling in a mouse model of carotid wire injury. Transgenic mice with a platelet-specific deletion of BMP-4 (BMP4Plt-/-) were generated. Intravital microscopy was performed to evaluate leukocyte adhesion to the vessel wall. Expression of adhesion molecules and chemokines were analyzed. Platelet-leukocyte aggregates (PLAs) were evaluated using flow cytometry. For carotid wire injury, BMP4Plt-/- mice were further crossed with LDLr-/- mice (BMP4Plt-/-/LDLr-/-) and fed with a high cholesterol diet for 2-weeks. Carotid wire injury was performed, and re-endothelialization and neointimal formation were evaluated. In comparison to the control mice, stimulation with TNFα resulted in fewer rolling and adherent leukocytes to the vessel wall in the BMP4Plt-/- mice. mRNA and protein expression of P-selectin and adhesion molecules were reduced in the aorta of the BMP4Plt-/- mice. In platelets from the BMP4Plt-/- mice, the expression of P-selectin was reduced, and fewer PLA formations were measured than in the control mice. Loss of platelet BMP-4 further prevented neointima formation after carotid wire injury. Endothelial regeneration after injury was decelerated in the BMP4Plt-/- mice, and confirmed in-vitro, where the deletion of platelet BMP-4 inhibited endothelial cell proliferation and migration. We demonstrate for the first time that platelet BMP-4 is involved during vascular inflammation and remodeling. This is partially mediated by the inhibition of platelet activation, reduced expression of adhesion molecules and inflammatory responses. Our findings identify platelet BMP-4 as a mediator of vascular inflammation in early atherosclerosis and restenosis.


Assuntos
Aorta/patologia , Plaquetas/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Lesões das Artérias Carótidas/metabolismo , Inflamação/metabolismo , Neointima/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Lesões das Artérias Carótidas/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Expressão Gênica , Inflamação/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
13.
Radiology ; 301(2): 311-319, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34374587

RESUMO

Background Recent imaging techniques show that patients with aortic intramural hematoma (IMH) have various sizes of focal intimal disruptions (FIDs) that may affect clinical outcomes. Purpose To evaluate the relationship between size of FIDs detected at multidetector CT and disease progression in patients with type B IMH. Materials and Methods This retrospective study analyzed consecutive patients with type B IMH who underwent multidetector CT at admission within 24 hours after symptom onset from January 2011 to March 2017. FID was defined as a focal contrast material-filled pouch projecting outside of the aorta lumen with a communicating orifice greater than 3 mm. Large FID was defined as an FID of at least 10 mm in length and width and at least 5 mm in depth. Aorta-related events were defined as a composite of aortic rupture, surgical or endovascular aortic repair, progressive aortic enlargement, and development of aortic dissection. Results Seventy-six patients (mean age, 72 years ± 11 [standard deviation]; 54 men) were evaluated. Fifty patients (66%; 50 of 76) had 91 FIDs. Twenty-eight patients had large FIDs and 22 patients had small FIDs. Aorta-related events occurred in 15 of 28 patients with large FIDs, two of 22 patients with small FIDs, and four of 26 patients with no FID. Patients with large FIDs showed lower aorta-related event-free survival rates than did patients with small FIDs or no FID (small FIDS, 63% ± 10 vs 90% ± 7 at 3 years, respectively [P = .001]; and no FIDS, 63% ± 10 vs 84% ± 7 at 3 years, respectively [P = .005]). Multivariable analysis showed that a maximum aortic diameter of at least 40 mm (hazard ratio, 4.8; 95% CI: 1.8, 12.6; P = .001) and large FID (hazard ratio, 3.2; 95% CI: 1.1, 8.9; P = .03) were the independent predictors of aorta-related events. Conclusion A large portion of patients with B-intramural hematoma (IMH) had focal intimal disruption (FID) detected at CT at admission. Large FIDs were associated with disease progression of IMH. © RSNA, 2021 See also the editorial by Raptis and Braverman in this issue. Online supplemental material is available for this article.


Assuntos
Aneurisma Dissecante/diagnóstico por imagem , Doenças da Aorta/diagnóstico por imagem , Hematoma/diagnóstico por imagem , Tomografia Computadorizada Multidetectores/métodos , Túnica Íntima/diagnóstico por imagem , Idoso , Aorta/diagnóstico por imagem , Aorta/patologia , Doenças da Aorta/patologia , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Masculino , Estudos Retrospectivos , Sensibilidade e Especificidade , Túnica Íntima/patologia
14.
Toxicology ; 458: 152841, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34216699

RESUMO

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1ß, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/efeitos da radiação , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Lisofosfatidilcolinas/metabolismo , Fosfolipases A2/metabolismo , Fosfolipases A2/efeitos da radiação , Animais , Aorta/patologia , Aorta/efeitos da radiação , Citocinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/efeitos da radiação , Radiação Ionizante , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo
15.
Mayo Clin Proc ; 96(7): 1874-1887, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34218860

RESUMO

OBJECTIVE: To elucidate sex differences in valve morphology, disease phenotype, progression, and outcomes among children and young adults with bicuspid aortic valve (BAV). PATIENTS AND METHODS: This is a retrospective cohort study examining all children and young adults (aged ≤22 years) with isolated BAV diagnosed, by excluding patients with concomitant congenital heart defects or genetic syndromes, from January 1, 1990, through December 1, 2016, at Mayo Clinic in Rochester, Minnesota. RESULTS: Of 1010 patients with BAV, 558 had isolated BAV. Distributions of morphology were right-left in 65.8% (n=367), right-noncoronary in 34% (n=190), and left-noncoronary cusp fusion in 0.2% (n=1) of patients; with no sex differences. Male to female ratio was 3:1. At the first echocardiographic evaluation in the study, there were no sex differences in terms of frequency of aortic valve stenosis or regurgitation. However, males had significantly higher grades of aortic valve regurgitation at 17 years of age onward (P<.0001). Males had significantly larger mid-ascending aorta (P=.01) and sinus of Valsalva dimensions (z score; P=.0001) as compared with females, with a novel finding of peak aortic dimensions around 8 years of age. Males also had more than 2-fold higher risk for sinus of Valsalva dilation (z score >2) as compared with females (odds ratio, 2.3; 95% CI, 1.2 to 4.2; P=.01). There were no significant sex differences in the primary cardiac outcomes of interventions on aortic valve and/or aorta, aortic dissection, or death. CONCLUSION: In children and young adults with BAV, males have a higher grade of aortic regurgitation in late adolescence, significantly larger aortic dimensions, different patterns of aortic growth, and more frequent sinus of Valsalva dilation as compared with females. Overall, the rate of primary cardiac events is lower in young patients, with no significant sex differences.


Assuntos
Aorta , Valva Aórtica , Doença da Válvula Aórtica Bicúspide , Ecocardiografia/métodos , Fatores Sexuais , Seio Aórtico , Adolescente , Fatores Etários , Aorta/diagnóstico por imagem , Aorta/crescimento & desenvolvimento , Aorta/patologia , Valva Aórtica/anormalidades , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/fisiopatologia , Insuficiência da Valva Aórtica/diagnóstico , Insuficiência da Valva Aórtica/etiologia , Doença da Válvula Aórtica Bicúspide/diagnóstico , Doença da Válvula Aórtica Bicúspide/fisiopatologia , Variação Biológica da População , Criança , Progressão da Doença , Feminino , Humanos , Masculino , Tamanho do Órgão , Seio Aórtico/diagnóstico por imagem , Seio Aórtico/patologia
16.
Am J Hum Genet ; 108(9): 1578-1589, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34265237

RESUMO

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.


Assuntos
Aneurisma da Aorta Torácica/genética , Diabetes Mellitus Tipo 2/genética , Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Locos de Características Quantitativas , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Aorta/metabolismo , Aorta/patologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Estudos de Casos e Controles , Caspase 3/genética , Caspase 3/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Células Endoteliais/patologia , Regulação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Íntrons , Michigan , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Mutação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
17.
Biomolecules ; 11(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202347

RESUMO

Atherosclerosis is a multifactorial disease, for which the etiology is so complex that we are currently unable to prevent it and effectively lower the statistics on mortality from cardiovascular diseases. Parallel to modern analyses in molecular biology and biochemistry, we want to carry out analyses at the level of micro- and macroelements in order to discover the interdependencies between elements during atherogenesis. In this work, we used the Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) to determine the content of calcium, magnesium, iron, copper, chromium, zinc, manganese, cadmium, lead, and zinc in the aorta sections of people who died a sudden death. We also estimated the content of metalloenzymes MMP-9, NOS-3, and SOD-2 using the immunohistochemical method. It was observed that with the age of the patient, the calcium content of the artery increased, while the content of copper and iron decreased. Very high correlations (correlation coefficient above 0.8) were observed for pairs of parameters in women: Mn-Ca, Fe-Cu, and Ca-Cd, and in men: Mn-Zn. The degree of atherosclerosis negatively correlated with magnesium and with cadmium. Chromium inhibited absorption of essential trace elements such as Cu and Fe due to its content being above the quantification threshold only if Cu and Fe were lower. Moreover, we discussed how to design research for the future in order to learn more about the pathomechanism of atherosclerosis and the effect of taking dietary supplements on the prevalence of cardiovascular diseases.


Assuntos
Aorta/química , Aorta/metabolismo , Aterosclerose/metabolismo , Espectrofotometria Atômica/métodos , Oligoelementos/análise , Oligoelementos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Aorta/patologia , Aterosclerose/patologia , Cádmio/análise , Cádmio/metabolismo , Cálcio/análise , Cálcio/metabolismo , Cobre/análise , Cobre/metabolismo , Feminino , Humanos , Ferro/análise , Ferro/metabolismo , Magnésio/análise , Magnésio/metabolismo , Masculino , Manganês/análise , Manganês/metabolismo , Pessoa de Meia-Idade
18.
J Am Heart Assoc ; 10(15): e021707, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34325521

RESUMO

Background Abdominal aortic aneurysm (AAA) is a life-threatening vascular disorder characterized by chronic inflammation of the aortic wall, which lacks effective pharmacotherapeutic remedies and has an extremely high mortality. Nuclear receptor NR4A1 (Nur77) functions in various chronic inflammatory diseases. However, the influence of Nur77 on AAA has remained unclear. Herein, we sought to determine the effects of Nur77 on the development of AAA. Methods and Results We observed that Nur77 expression decreased significantly in human and mice AAA lesions. Deletion of Nur77 accelerated the development of AAA in mice, as evidenced by increased AAA incidence, abdominal aortic diameters, elastin fragmentation, and collagen content. Consistent with genetic manipulation, pharmacological activation of Nur77 by celastrol showed beneficial effects against AAA. Microscopic and molecular analyses indicated that the detrimental effects of Nur77 deficiency were associated with aggravated macrophage infiltration in AAA lesions and increased pro-inflammatory cytokines secretion and matrix metalloproteinase (MMP-9) expression. Bioinformatics analyses further revealed that LOX-1 was upregulated by Nur77 deficiency and consequently increased the expression of cytokines and MMP-9. Moreover, rescue experiments verified that LOX-1 notably aggravated inflammatory response, an effect that was blunted by Nur77. Conclusions This study firstly demonstrated a crucial role of Nur77 in the formation of AAA by targeting LOX-1, which implicated Nur77 might be a potential therapeutic target for AAA.


Assuntos
Aorta , Inflamação/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores Depuradores Classe E/metabolismo , Animais , Aorta/imunologia , Aorta/patologia , Aneurisma da Aorta Abdominal/metabolismo , Citocinas/metabolismo , Descoberta de Drogas , Elastina/metabolismo , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Tamanho do Órgão , Transdução de Sinais , Remodelação Vascular/imunologia
19.
PLoS One ; 16(7): e0255238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34310653

RESUMO

INTRODUCTION: Aortic dissection (AD) is a life-threatening emergency, and lumican (LUM) is a potential Biomarker for AD diagnosis. We investigated LUM expression patterns in patients with AD and explored the molecular functions of Lum in AD mice model. METHODS: LUM expression patterns were analyzed using aortic tissues of AD patients, and serum soluble LUM (s-LUM) levels were compared between patients with acute AD (AAD) and chronic AD (CAD). Lum-knockout (Lum-/-) mice were challenged with ß-aminopropionitrile (BAPN) and angiotensin II (Ang II) to induce AD. The survival rate, AD incidence, and aortic aneurysm (AA) in these mice were compared with those in BAPN-Ang II-challenged wildtype (WT) mice. Tgf-ß/Smad2, Mmps, Lum, and Nox expression patterns were examined. RESULTS: LUM expression was detected in the intima and media of the ascending aorta in patients with AAD. Serum s-LUM levels were significantly higher in patients with AAD than CAD. Furthermore, AD-associated mortality and thoracic aortic rupture incidence were significantly higher in the Lum-/- AD mice than in the WT AD mice. However, no significant pathologic changes in AA were observed in the Lum-/- AD mice compared with the WT AD mice. The BAPN-Ang II-challenged WT and Lum-/- AD mice had higher Tgf-ß, p-Smad2, Mmp2, Mmp9, and Nox4 levels than those of non-AD mice. We also found that Lum expression was significantly higher in the BAPN-Ang II-challenged WT in comparison to the unchallenged WT mice. CONCLUSION: LUM expression was altered in patients with AD display increased s-LUM in blood, and Lum-/- mice exhibited augmented AD pathogenesis. These findings support the notion that LUM is a biomarker signifying the pathogenesis of injured aorta seen in AAD. The presence of LUM is essential for maintenance of connective tissue integrity. Future studies should elucidate the mechanisms underlying LUM association in aortic changes.


Assuntos
Aneurisma Dissecante/patologia , Lumicana/sangue , Doença Aguda , Aminopropionitrilo/farmacologia , Aneurisma Dissecante/metabolismo , Aneurisma Dissecante/mortalidade , Angiotensina II/farmacologia , Animais , Aorta/metabolismo , Aorta/patologia , Ruptura Aórtica/epidemiologia , Ruptura Aórtica/patologia , Biomarcadores/sangue , Doença Crônica , Modelos Animais de Doenças , Humanos , Incidência , Estimativa de Kaplan-Meier , Lumicana/deficiência , Lumicana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/efeitos dos fármacos
20.
J R Soc Interface ; 18(180): 20210336, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34314650

RESUMO

Hypertension induces significant aortic remodelling, often adaptive but sometimes not. To identify immuno-mechanical mechanisms responsible for differential remodelling, we studied thoracic aortas from 129S6/SvEvTac and C57BL/6 J mice before and after continuous 14-day angiotensin II infusion, which elevated blood pressure similarly in both strains. Histological and biomechanical assessments of excised vessels were similar at baseline, suggesting a common homeostatic set-point for mean wall stress. Histology further revealed near mechano-adaptive remodelling of the hypertensive 129S6/SvEvTac aortas, but a grossly maladaptive remodelling of C57BL/6 J aortas. Bulk RNA sequencing suggested that increased smooth muscle contractile processes promoted mechano-adaptation of 129S6/SvEvTac aortas while immune processes prevented adaptation of C57BL/6 J aortas. Functional studies confirmed an increased vasoconstrictive capacity of the former while immunohistochemistry demonstrated marked increases in inflammatory cells in the latter. We then used multiple computational biomechanical models to test the hypothesis that excessive adventitial wall stress correlates with inflammatory cell infiltration. These models consistently predicted that increased vasoconstriction against an increased pressure coupled with modest deposition of new matrix thickens the wall appropriately, restoring wall stress towards homeostatic consistent with adaptive remodelling. By contrast, insufficient vasoconstriction permits high wall stresses and exuberant inflammation-driven matrix deposition, especially in the adventitia, reflecting compromised homeostasis and gross maladaptation.


Assuntos
Túnica Adventícia , Hipertensão , Túnica Adventícia/patologia , Animais , Aorta/patologia , Aorta Torácica/patologia , Modelos Animais de Doenças , Fibrose , Hipertensão/patologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...