RESUMO
The value of anti-CTLA-4 antibodies in cancer therapy is well established. However, the broad application of currently available anti-CTLA-4 therapeutic antibodies is hampered by their narrow therapeutic index. It is therefore challenging and attractive to develop the next generation of anti-CTLA-4 therapeutics with improved safety and efficacy. To this end, we generated fully human heavy chain-only antibodies (HCAbs) against CTLA-4. The hIgG1 Fc domain of the top candidate, HCAb 4003-1, was further engineered to enhance its regulatory T (Treg) cell depletion effect and to decrease its half-life, resulting in HCAb 4003-2. We tested these HCAbs in in vitro and in vivo experiments in comparison with ipilimumab and other anti-CTLA4 antibodies. The results show that human HCAb 4003-2 binds human CTLA-4 with high affinity and potently blocks the binding of B7-1 (CD80) and B7-2 (CD86) to CTLA-4. The results also show efficient tumor penetration. HCAb 4003-2 exhibits enhanced antibody-dependent cellular cytotoxicity function, lower serum exposure, and more potent anti-tumor activity than ipilimumab in murine tumor models, which is partly driven by a substantial depletion of intratumoral Tregs. Importantly, the enhanced efficacy combined with the shorter serum half-life and less systemic drug exposure in vivo potentially provides an improved therapeutic window in cynomolgus monkeys and preliminary clinical applications. With its augmented efficacy via Treg depletion and improved safety profile, HCAb 4003-2 is a promising candidate for the development of next generation anti-CTLA-4 therapy.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Imunoterapia , Neoplasias , Linfócitos T Reguladores , Animais , Citotoxicidade Celular Dependente de Anticorpos , Antígeno CTLA-4/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/farmacologia , Ipilimumab/farmacologia , Camundongos , Neoplasias/patologia , Neoplasias/terapiaRESUMO
The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/farmacologia , Microscopia Crioeletrônica , Humanos , Glicoproteínas de Membrana , Camundongos , Testes de Neutralização , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope ViralRESUMO
Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Fenótipo Secretor Associado à Senescência , Via de Sinalização Wnt , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Proteína Wnt3A/metabolismoRESUMO
Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell - pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells.
Assuntos
Células Endoteliais/patologia , Imageamento Tridimensional , Microscopia Intravital , Pericitos/patologia , Animais , Comunicação Celular , Linhagem Celular Tumoral , Camundongos , Análise Espaço-TemporalRESUMO
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) exerts beneficial effects in a variety of cardiovascular disease states. Studies on the benefit of eNOS activity in pressure-overload cardiac hypertrophy and dysfunction produced by aortic stenosis are equivocal, which may be due to different expression levels of eNOS or different severities of pressure-overload. Consequently, we investigated the effects of eNOS-expression level on cardiac hypertrophy and dysfunction produced by mild or severe pressure-overload. To unravel the impact of eNOS on pressure-overload cardiac dysfunction we subjected eNOS deficient, wildtype and eNOS overexpressing transgenic (eNOS-Tg) mice to 8weeks of mild or severe transverse aortic constriction (TAC) and studied cardiac geometry and function at the whole organ and tissue level. In both mild and severe TAC, lack of eNOS ameliorated, whereas eNOS overexpression aggravated, TAC-induced cardiac remodeling and dysfunction. Moreover, the detrimental effects of eNOS in severe TAC were associated with aggravation of TAC-induced NOS-dependent oxidative stress and by further elevation of eNOS monomer levels, consistent with enhanced eNOS uncoupling. In the presence of TAC, scavenging of reactive oxygen species with N-acetylcysteine reduced eNOS S-glutathionylation, eNOS monomer and NOS-dependent superoxide levels in eNOS-Tg mice to wildtype levels. Accordingly, N-acetylcysteine improved cardiac function in eNOS-Tg but not in wildtype mice with TAC. In conclusion, independent of the severity of TAC, eNOS aggravates cardiac remodeling and dysfunction, which appears due to TAC-induced eNOS uncoupling and superoxide production.
Assuntos
Cardiomegalia/enzimologia , Cardiomegalia/genética , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico/metabolismo , Remodelação Ventricular , Acetilcisteína/farmacologia , Animais , Aorta/cirurgia , Cardiomegalia/etiologia , Cardiomegalia/patologia , Constrição Patológica/complicações , Constrição Patológica/cirurgia , Ativação Enzimática , Feminino , Sequestradores de Radicais Livres/farmacologia , Deleção de Genes , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Índice de Gravidade de Doença , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismoRESUMO
Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1(-/-)). Parallel to the strong reduction in PLTP activity in plasma of ABCA1(-/-) mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1(-/-) mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1(-/-) mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1(-/-), apoE(-/-) and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1(-/-), apoE(-/-) and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.
Assuntos
Lipoproteínas HDL/sangue , Proteínas de Transferência de Fosfolipídeos/sangue , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Receptores X do Fígado , Macrófagos/metabolismo , Camundongos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (F(max)). However, only exercise training restored myofilament Ca(2+)-sensitivity and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca(2+) homeostasis, as myofilament F(max) actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and l-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament F(max). In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling."
Assuntos
Terapia por Exercício , Infarto do Miocárdio/terapia , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Esforço Físico , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular , Acetilcisteína/farmacologia , Citoesqueleto de Actina/metabolismo , Animais , Antioxidantes/farmacologia , Arginina/metabolismo , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Miocárdica , Infarto do Miocárdio/complicações , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/fisiopatologia , Óxido Nítrico Sintase Tipo III/genética , Estresse Oxidativo , Fosforilação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Regulação para Cima , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologia , Pressão Ventricular , Remodelação Ventricular/efeitos dos fármacosRESUMO
Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to vascular maintenance by participating in angiogenesis, re-endothelialization, and remodeling. Myeloid progenitor cells in the BM are functionally and quantitatively an important precursor pool for cells that contribute to these processes. However, these precursor pools in the BM also give rise to important effector cells of the innate immune system, such as macrophages and dendritic cells. We hypothesized that the disturbed repair responses that are being observed in diabetes mellitus are also related to an effect on functional and differentiation characteristics at the level of this bone marrow precursor pool. Indeed, we observed that bone marrow differentiation cultures for EPC, macrophages (Mph), or dendritic cells (DC) from hyperglycemic BM yielded 40% fewer EPC and 50% more Mph compared with control BM. These changes were directly related to the hemoglobin A(1C) levels of the donor mice. BM-derived DC numbers were not affected by hyperglycemia. The composition of the BM was not altered; in particular, the numbers of CD31+/Ly6C+ cells, which serve as common progenitors for EPC, Mph, and DC, were unaffected. In addition, BM-derived EPC from hyperglycemic mice were less angiogenic and more proinflammatory in regards to endocytosis, T-cell activation, and interleukin 12 production. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibition by statin supplementation of the culture medium counteracted these hyperglycemia-induced changes. Our study results show that hyperglycemia alters the differentiation fate of BM precursor cells, reducing the potential to generate vascular regenerative cells and favoring the development of proinflammatory cells.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Hiperglicemia/imunologia , Hiperglicemia/fisiopatologia , Fenótipo , Células-Tronco/citologia , Células-Tronco/imunologia , Animais , Células Cultivadas , Células Endoteliais/citologia , Citometria de Fluxo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperglicemia/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/efeitos dos fármacos , Estreptozocina/farmacologia , Cordão Umbilical/citologiaRESUMO
Angiotensin II (ATII)-mediated hypertension increases the risk for acute coronary events, which may be caused by augmented collagen degradation. Interstitial fibers of collagen type I in the plaque can be degraded by MMP8 and MMP13 specifically. Indeed high MMP8 levels have been correlated with ruptured plaques in patients. To study the contribution of ATII in plaque rupture, we evaluated its effect on MMP8 and MMP13 activity on the vulnerable lesions using an extravascular device that induces regions of pro-atherogenic shear stress in the carotid arteries of ApoE KO mice. This triggers the growth of lesions with a "vulnerable" macrophage-rich phenotype (referred to as upstream lesions) and lesions with a "stable" fibrotic phenotype (referred to as downstream lesions). ATII administration increased mean blood pressure, and increased the incidence of intra-plaque hemorrhages (IPH) from 30% to 73% of the animals in the upstream segments. The area of IPH was also increased by 5-fold. No IPHs were observed in the downstream lesions of the control group or the ATII group. In addition, ATII treatment doubled the size of upstream and downstream lesions. Upstream lesions in the ATII group were decreased in collagen content by 3-fold, contained 2-fold higher MMP8 and MMP13 levels, with a 2- and 3-fold increase in collagen type I degradation by MMP8 and MMP13 respectively compared to the upstream lesions in the control group. Gene expression analysis showed general increase in procollagens and TIMPs expression in response to ATII. However, ATII also decreased procollagen 5alpha3 expression in downstream lesions and decreased TIMP4 expression in upstream lesions. These data show that ATII promotes a "stable" fibrotic phenotype by inducing severe intra-plaque hemorrhages, characterized by increased degradation of interstitial collagen I via an MMP-mediated (MMP8 and MMP13) mechanism.
Assuntos
Angiotensina II/metabolismo , Doenças das Artérias Carótidas/enzimologia , Artéria Carótida Primitiva/enzimologia , Colágeno Tipo I/metabolismo , Hemorragia/etiologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Pró-Colágeno/metabolismo , Angiotensina II/administração & dosagem , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Pressão Sanguínea , Doenças das Artérias Carótidas/complicações , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/fisiopatologia , Artéria Carótida Primitiva/patologia , Artéria Carótida Primitiva/fisiopatologia , Colágeno Tipo I/genética , Modelos Animais de Doenças , Ativação Enzimática , Fibrose , Perfilação da Expressão Gênica , Hemorragia/enzimologia , Hemorragia/patologia , Bombas de Infusão Implantáveis , Infusões Subcutâneas , Macrófagos/enzimologia , Macrófagos/patologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 8 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Pró-Colágeno/genética , Índice de Gravidade de Doença , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
BACKGROUND: Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerotic lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP. METHODS AND FINDINGS: Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR-/-) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt-->LDLR-/- mice (2.3-fold) and even further in the huPLTPtg/tg-->LDLR-/- mice (4.5-fold) compared with the control PLTPwt/wt-->LDLR-/- mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages. CONCLUSIONS: We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size.
Assuntos
Aterosclerose/metabolismo , Células da Medula Óssea/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Transplante de Medula Óssea , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Transferência de Fosfolipídeos/biossíntese , Proteínas de Transferência de Fosfolipídeos/sangue , Receptores de LDL/genética , Receptores de LDL/fisiologiaRESUMO
OBJECTIVE: A transgenic mouse model was generated that allows conditional expression of human PLTP, based on the tetracycline-responsive gene system, to study the effects of an acute increase in plasma PLTP activity as may occur in inflammation. METHODS AND RESULTS: The effects of an acute elevation of plasma PLTP activity on the metabolism of apolipoprotein B-containing lipoproteins and on diet-induced pre-existing atherosclerosis were determined in mice displaying a humanized lipoprotein profile (low-density lipoprotein receptor knockout background). Induced expression of PLTP strongly increases plasma VLDL levels in LDL receptor knockout mice, whereas VLDL secretion is not affected. The elevation in plasma triglyceride levels is explained by a PLTP-dependent inhibition of VLDL catabolism, which is caused, at least partly, by a decreased lipoprotein lipase activity. Together with the decreased plasma HDL levels, the acutely increased PLTP expression results in a highly atherogenic lipoprotein profile. Induction of PLTP expression leads to a further increase in size of pre-existing atherosclerotic lesions, even on a chow diet. In addition, the lesions contain more macrophages and less collagen relative to controls, suggesting a less stable lesion phenotype. CONCLUSIONS: In conclusion, acute elevation of PLTP activity destabilizes atherosclerotic lesions and aggravates pre-existing atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptores de LDL/metabolismo , Animais , Apolipoproteínas B/sangue , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/sangue , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Lipase/sangue , Lipase Lipoproteica/sangue , Lipoproteínas VLDL/sangue , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Transferência de Fosfolipídeos/sangue , Proteínas de Transferência de Fosfolipídeos/genética , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Triglicerídeos/sangue , Regulação para CimaRESUMO
Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.
Assuntos
Endotélio Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Resistência Vascular , Vasodilatação , Sistema Vasomotor/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Aorta/enzimologia , Aorta/metabolismo , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Óxidos N-Cíclicos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Nitroprussiato/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel , Marcadores de Spin , Regulação para Cima , Resistência Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Sistema Vasomotor/efeitos dos fármacos , Sistema Vasomotor/enzimologiaRESUMO
We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.
Assuntos
Aterosclerose/etiologia , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/etiologia , Quimiocinas/fisiologia , Resistência ao Cisalhamento , Animais , Apolipoproteínas E/genética , Aterosclerose/patologia , Receptor 1 de Quimiocina CX3C , Artérias Carótidas/química , Doenças das Artérias Carótidas/patologia , Quimiocinas/genética , Expressão Gênica , Camundongos , Camundongos Mutantes , Receptores de Citocinas/análise , Receptores de HIV/análise , Estresse MecânicoRESUMO
BACKGROUND: Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies. METHODS AND RESULTS: We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38+/-0.68 versus 0.22+/-0.04); contained fewer smooth muscle cells (1.9+/-1.6% versus 26.3+/-9.7%), less collagen (15.3+/-1.0% versus 22.2+/-1.0%), and more lipids (15.8+/-0.9% versus 10.2+/-0.5%); and showed more outward vascular remodeling (214+/-19% versus 117+/-9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II-induced intraplaque hemorrhages occurred in the lowered shear stress regions only. CONCLUSIONS: Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.