RESUMO
The vasculature of the central nervous system (CNS) forms a selective barrier termed the blood-brain barrier (BBB). Disruption of the BBB may contribute to various CNS diseases. Conversely, the intact BBB restricts efficient penetration of CNS-targeted drugs. Here, we report the BBB-regulatory role of endothelial sphingosine 1-phosphate (S1P) receptor-1, a G protein-coupled receptor known to promote the barrier function in peripheral vessels. Endothelial-specific S1pr1 knockout mice (S1pr1iECKO ) showed BBB breach for small-molecular-mass fluorescence tracers (<3 kDa), but not larger tracers (>10 kDa). Chronic BBB leakiness was associated with cognitive impairment, as assessed by the novel object recognition test, but not signs of brain inflammation. Brain microvessels of S1pr1iECKO mice showed altered subcellular distribution of tight junctional proteins. Pharmacological inhibition of S1P1 function led to transient BBB breach. These data suggest that brain endothelial S1P1 maintain the BBB by regulating the proper localization of tight junction proteins and raise the possibility that endothelial S1P1 inhibition may be a strategy for transient BBB opening and delivery of small molecules into the CNS.
Assuntos
Barreira Hematoencefálica/fisiologia , Endotélio Vascular/fisiologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Transporte Biológico , Encéfalo/irrigação sanguínea , Células Endoteliais/fisiologia , Regulação da Expressão Gênica , Lisofosfolipídeos , Camundongos , Camundongos Knockout , Receptores de Lisoesfingolipídeo/genética , Esfingosina/análogos & derivados , Junções Íntimas/metabolismoRESUMO
BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-ß1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-ß1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa). METHODS: Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels. RESULTS: Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-ß1 levels (r=0.38, p=0.05 for PLAs and r=0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=-0.42, p=0.03 for PLAs and r=-0.37, p=0.06 for single leukocytes). CONCLUSIONS: p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-ß1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-ß1.
Assuntos
Estenose da Valva Aórtica/patologia , Plaquetas/patologia , Leucócitos/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/metabolismo , Plaquetas/metabolismo , Modelos Animais de Doenças , Leucócitos/metabolismo , Camundongos , Fosforilação , Proteína Smad2/análise , Proteína Smad3/análise , Fator de Crescimento Transformador beta1/análiseRESUMO
In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.
Assuntos
Proteína ADAM10 , Antígenos CD , Artrite Experimental , Artrite Reumatoide , Caderinas , Células Endoteliais , Receptores de Esfingosina-1-Fosfato , Animais , Caderinas/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Camundongos , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Antígenos CD/metabolismo , Antígenos CD/genética , Células Endoteliais/metabolismo , Humanos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genética , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Transdução de Sinais , Camundongos Knockout , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Masculino , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolipídeos/metabolismo , Permeabilidade Capilar , FemininoRESUMO
Sphingosine 1-phosphate (S1P), which acts via G protein-coupled S1P receptors (S1PRs), is a bioactive lipid essential for vascular integrity and lymphocyte trafficking. The S1P-S1PR signalling axis is a key component of the inflammatory response in autoimmune rheumatic diseases. Several drugs that target S1PRs have been approved for the treatment of multiple sclerosis and inflammatory bowel disease and are under clinical testing for patients with systemic lupus erythematosus (SLE). Preclinical studies support the hypothesis that targeting the S1P-S1PR axis would be beneficial to patients with SLE, rheumatoid arthritis (RA) and systemic sclerosis (SSc) by reducing pathological inflammation. Whereas most preclinical research and development efforts are focused on reducing lymphocyte trafficking, protective effects of circulating S1P on endothelial S1PRs, which maintain the vascular barrier and enable blood circulation while dampening leukocyte extravasation, have been largely overlooked. In this Review, we take a holistic view of S1P-S1PR signalling in lymphocyte and vascular pathobiology. We focus on the potential of S1PR modulators for the treatment of SLE, RA and SSc and summarize the rationale, pathobiology and evidence from preclinical models and clinical studies. Improved understanding of S1P pathobiology in autoimmune rheumatic diseases and S1PR therapeutic modulation is anticipated to lead to efficacious and safer management of these diseases.
Assuntos
Lúpus Eritematoso Sistêmico , Esclerose Múltipla , Doenças Reumáticas , Humanos , Esclerose Múltipla/tratamento farmacológico , Receptores de Lisoesfingolipídeo/uso terapêutico , Doenças Reumáticas/tratamento farmacológico , Transdução de Sinais , Receptores de Esfingosina-1-FosfatoRESUMO
Transforming growth factor-beta1 (TGF-beta1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations. Platelets contain 40 times as much TGF-beta1 as other cells and secrete it as an inactive (latent) form in complex with latency-associated peptide (LAP), which is disulfide bonded via Cys33 to latent TGF-beta binding protein 1 (LTBP-1). Little is known about how latent TGF-beta1 becomes activated in vivo. Here we show that TGF-beta1 released from platelets or fibroblasts undergoes dramatic activation when subjected to stirring or shear forces, providing a potential mechanism for physiologic control. Thiol-disulfide exchange appears to contribute to the process based on the effects of thiol-reactive reagents and differences in thiol labeling of TGF-beta1 before and after stirring or shear. Activation required the presence of LTBP, as TGF-beta1 contained in complex with only LAP could not be activated by stirring when studied as either a recombinant purified protein complex or in the platelet releasates or sera of mice engineered to contain an LAP C33S mutation. Release and activation of latent TGF-beta1 in vivo was demonstrated in a mouse model 5 minutes after thrombus formation. These data potentially provide a novel mechanism for in vivo activation of TGF-beta1.
Assuntos
Plaquetas/metabolismo , Fator de Crescimento Transformador beta1/sangue , Animais , Fenômenos Biofísicos , Biofísica , Proteínas Sanguíneas/metabolismo , Trombose das Artérias Carótidas/sangue , Linhagem Celular , Meios de Cultivo Condicionados , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Ligação a TGF-beta Latente/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Estresse Mecânico , Compostos de Sulfidrila/sangue , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVE: Immune complex (IC) deposition activates polymorphonuclear neutrophils (PMNs), increases vascular permeability, and leads to organ damage in systemic lupus erythematosus and rheumatoid arthritis. The bioactive lipid sphingosine 1-phosphate (S1P), acting via S1P receptor 1 (S1P1 ), is a key regulator of endothelial cell (EC) barrier function. This study was undertaken to investigate whether augmenting EC integrity via S1P1 signaling attenuates inflammatory injury mediated by ICs. METHODS: In vitro barrier function was assessed in human umbilical vein endothelial cells (HUVECs) by electrical cell-substrate impedance sensing. Phosphorylation of myosin light chain 2 (p-MLC-2) and VE-cadherin staining in HUVECs were assessed by immunofluorescence. A reverse Arthus reaction (RAR) was induced in the skin and lungs of mice with S1P1 deleted from ECs (S1P1 EC-knockout [ECKO] mice) and mice treated with S1P1 agonists and antagonists. RESULTS: S1P1 agonists prevented loss of barrier function in HUVECs treated with IC-activated PMNs. S1P1 ECKO and wild-type (WT) mice treated with S1P1 antagonists had amplified RAR, whereas specific S1P1 agonists attenuated skin and lung RAR in WT mice. ApoM-Fc, a novel S1P chaperone, mitigated EC cell barrier dysfunction induced by activated PMNs in vitro and attenuated lung RAR. Expression levels of p-MLC-2 and disruption of VE-cadherin, each representing manifestations of cell contraction and destabilization of adherens junctions, respectively, that were induced by activated PMNs, were markedly reduced by treatment with S1P1 agonists and ApoM-Fc. CONCLUSION: Our findings indicate that S1P1 signaling in ECs modulates vascular responses to IC deposition. S1P1 agonists and ApoM-Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P1 axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end-organ damage.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Permeabilidade Capilar/genética , Células Endoteliais/metabolismo , Receptores de Lisoesfingolipídeo/genética , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Anilidas/farmacologia , Animais , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Apolipoproteínas M/farmacologia , Reação de Arthus , Caderinas/efeitos dos fármacos , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Miosinas Cardíacas/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Indanos/farmacologia , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Organofosfonatos/farmacologia , Oxidiazóis/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/metabolismo , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato , Tiofenos/farmacologiaRESUMO
Endothelial dysfunction, a hallmark of vascular disease, is restored by plasma high-density lipoprotein (HDL). However, a generalized increase in HDL abundance is not beneficial, suggesting that specific HDL species mediate protective effects. Apolipoprotein M-containing HDL (ApoM+HDL), which carries the bioactive lipid sphingosine 1-phosphate (S1P), promotes endothelial function by activating G protein-coupled S1P receptors. Moreover, HDL-bound S1P is limiting in several inflammatory, metabolic, and vascular diseases. We report the development of a soluble carrier for S1P, ApoM-Fc, which activated S1P receptors in a sustained manner and promoted endothelial function. In contrast, ApoM-Fc did not modulate circulating lymphocyte numbers, suggesting that it specifically activated endothelial S1P receptors. ApoM-Fc administration reduced blood pressure in hypertensive mice, attenuated myocardial damage after ischemia/reperfusion injury, and reduced brain infarct volume in the middle cerebral artery occlusion model of stroke. Our proof-of-concept study suggests that selective and sustained targeting of endothelial S1P receptors by ApoM-Fc could be a viable therapeutic strategy in vascular diseases.