RESUMO
Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1ß production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Hipóxia/complicações , Inflamação/complicações , Infecções por Mycoplasma/complicações , Mycoplasma/patogenicidade , Succinatos/metabolismo , Animais , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/metabolismo , Metabolismo Energético , Glioblastoma/etiologia , Glioblastoma/metabolismo , Metaboloma , Camundongos , Células Tumorais CultivadasRESUMO
Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Estresse Mecânico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Artérias/citologia , Capilares/citologia , Sobrevivência Celular , Ativação Enzimática , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/biossíntese , Veias/citologiaRESUMO
Vascular endothelial growth factor (VEGF) is critical for angiogenesis, but also has pleiotropic effects on several nonvascular cells. Our aim was to investigate the role of VEGF in brown adipose tissue (BAT). We show that VEGF expression increases 2.5-fold during differentiation of cultured murine brown adipocytes and that VEGF receptor-2 is phosphorylated, indicating VEGF signaling. VEGF increased proliferation in brown preadipocytes in vitro by 70%, and blockade of VEGF signaling using anti-VEGFR2 antibody DC101 increased brown adipocyte apoptosis, as determined by cell number and activation of caspase 3. Systemic VEGF neutralization in mice, accomplished by adenoviral expression of soluble Flt1, resulted in 7-fold increase in brown adipocyte apoptosis, mitochondrial degeneration, and increased mitophagy compared to control mice expressing a null adenovirus. Absence of the heparan sulfate-binding VEGF isoforms, VEGF164 and VEGF188, resulted in abnormal BAT development in mice at E15.5, with fewer brown adipocytes and lower mitochondrial protein compared to wild-type littermates. These results suggest a role for VEGF in brown adipocytes and preadipocytes to promote survival, proliferation, and normal mitochondria and development.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/embriologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Vascular endothelial cells (ECs) are continuously exposed to blood flow that contributes to the maintenance of vessel structure and function; however, the effect of hemodynamic forces on transforming growth factor-ß (TGF-ß) signaling in the endothelium is poorly described. We examined the potential role of TGF-ß signaling in mediating the protective effects of shear stress on ECs. APPROACH AND RESULTS: Human umbilical vein ECs (HUVECs) exposed to shear stress were compared with cells grown under static conditions. Signaling through the TGF-ß receptor ALK5 was inhibited with SB525334. Cells were examined for morphological changes and harvested for analysis by real-time polymerase chain reaction, Western blot analysis, apoptosis, proliferation, and immunocytochemistry. Shear stress resulted in ALK5-dependent alignment of HUVECs as well as attenuation of apoptosis and proliferation compared with static controls. Shear stress led to an ALK5-dependent increase in TGF-ß3 and Krüppel-like factor 2, phosphorylation of endothelial NO synthase, and NO release. Addition of the NO donor S-nitroso-N-acetylpenicillamine rescued the cells from apoptosis attributable to ALK5 inhibition under shear stress. Knockdown of TGF-ß3, but not TGF-ß1, disrupted the HUVEC monolayer and prevented the induction of Krüppel-like factor 2 by shear. CONCLUSIONS: Shear stress of HUVECs induces TGF-ß3 signaling and subsequent activation of Krüppel-like factor 2 and NO, and represents a novel role for TGF-ß3 in the maintenance of HUVEC homeostasis in a hemodynamic environment.
Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hemodinâmica/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta3/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Quinoxalinas/farmacologia , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse MecânicoRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. In advanced AMD, new vessels from choriocapillaris (CC) invade through the Bruch's membrane (BrM) into the retina, forming choroidal neovascularization (CNV). BrM, an elastic lamina that is located between the retinal pigment epithelium (RPE) and CC, is thought to act as a physical and functional barrier against CNV. The BrM of patients with early AMD are characterized by decreased levels of antiangiogenic factors, including endostatin, thrombospondin-1 (TSP-1), and pigment epithelium-derived factor (PEDF), as well as by degeneration of the elastic layer. Motivated by a previous report that heat increases elastin expression in human skin, we examined the effect of heat on human ARPE-19 cell production of BrM components. Heat treatment stimulated the production of BrM components, including TSP-1, PEDF, and tropoelastin in vitro and increased the antiangiogenic activity of RPE measured in a mouse corneal pocket assay. The effect of heat on experimental CNV was investigated by pretreating the retina with heat via infrared diode laser prior to the induction of CNV. Heat treatment blocked the development of experimental CNV in vivo. These findings suggest that heat treatment may restore BrM integrity and barrier function against new vessel growth.
Assuntos
Neovascularização de Coroide/prevenção & controle , Temperatura Alta/uso terapêutico , Epitélio Pigmentado da Retina/irrigação sanguínea , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Lâmina Basilar da Corioide/irrigação sanguínea , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/patologia , Linhagem Celular , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Endostatinas/genética , Endostatinas/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Humanos , Lasers Semicondutores/uso terapêutico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/terapia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Serpinas/genética , Serpinas/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo , Tropoelastina/metabolismo , Degeneração Macular Exsudativa/genética , Degeneração Macular Exsudativa/metabolismo , Degeneração Macular Exsudativa/patologia , Degeneração Macular Exsudativa/prevenção & controleRESUMO
Age-related macular degeneration (AMD) is one of the most common causes of visual impairment in the elderly, with a complex and still poorly understood etiology. Whole-genome association studies have discovered 34 genomic regions associated with AMD. However, the genes and cognate proteins that mediate the risk, are largely unknown. In the current study, we integrate levels of 4782 human serum proteins with all genetic risk loci for AMD in a large population-based study of the elderly, revealing many proteins and pathways linked to the disease. Serum proteins are also found to reflect AMD severity independent of genetics and predict progression from early to advanced AMD after five years in this population. A two-sample Mendelian randomization study identifies several proteins that are causally related to the disease and are directionally consistent with the observational estimates. In this work, we present a robust and unique framework for elucidating the pathobiology of AMD.
Assuntos
Degeneração Macular , Proteogenômica , Idoso , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Análise da Randomização Mendeliana , Fatores de RiscoRESUMO
The small GTPase RhoA and its downstream effectors, ROCK1 and ROCK2, regulate a number of cellular processes, including cell motility, proliferation, survival, and permeability. Pharmacological inhibitors of the Rho pathway reportedly block angiogenesis; however, the molecular details of this inhibition are largely unknown. We demonstrate that vascular endothelial growth factor-A (VEGF) rapidly induces RhoA activation in endothelial cells (ECs). Moreover, the pharmacological inhibition of ROCK1/2 using 10 microM Y-27632 (the IC(50) for this compound in ECs) strongly disrupts vasculogenesis in pluripotent embryonic stem cell cultures, VEGF-mediated regenerative angiogenesis in ex vivo retinal explants, and VEGF-mediated in vitro EC tube formation. Furthermore, using small interfering RNA knockdown and mouse heterozygote knockouts of ROCK1 and ROCK2, we provide data indicating that VEGF-driven angiogenesis is largely mediated through ROCK2. These data demonstrate that Rho/ROCK signaling is an important mediator in a number of angiogenic processes, including EC migration, survival, and cell permeability, and suggest that Rho/ROCK inhibition may prove useful for the treatment of angiogenesis-related disorders.
Assuntos
Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Apoptose , Western Blotting , Bovinos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Microscopia de Fluorescência , Piridinas/farmacologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinases Associadas a rho/genéticaRESUMO
Aberrant transforming growth factor-beta (TGF-beta) signaling plays a clear role in a number of pathologies, in particular, fibrotic diseases and cancer. Accumulating evidence also suggests that TGF-beta is required for vascular homeostasis, shedding light on the role of circulating TGF-beta and the expression of TGF-beta receptors in the adult vasculature, in the absence of any pathologic processes. In human pathologies such as hereditary hemorrhagic telangiectasia (HHT) and preeclampsia, TGF-beta signaling is abnormal. Studies from these disorders have helped elucidate the complex and multiple roles of this ubiquitously-expressed growth factor. The aim of this review is to collate some of the recent evidence that demonstrates a non-redundant role for TGF-beta signaling in maintaining vessel structure and function.
Assuntos
Homeostase/fisiologia , Microvasos/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , HumanosRESUMO
OBJECTIVE: Motivated by the central roles that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-beta signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. METHODS AND RESULTS: VEGF or TGF-beta inhibition, accomplished using adenovirus expression of soluble Flt1 (Ad-sFlt1) or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-beta resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-beta was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin alphaIIbbetaIII, or by leukocyte expression of L-selectin. Soluble vascular cell adhesion molecule (VCAM)-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. CONCLUSIONS: VEGF or TGF-beta neutralization leads to impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-beta in maintaining the endothelium in a nonactivated state and have implications for therapeutic approaches that neutralize VEGF or TGF-beta.
Assuntos
Endotélio Vascular/citologia , Leucócitos/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Vasodilatação/fisiologia , Animais , Adesão Celular , Selectina E/biossíntese , Endotélio Vascular/metabolismo , Contagem de Leucócitos , Leucócitos/citologia , Veias Mesentéricas/citologia , Veias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Selectina-P/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: By the development of a novel retinal microvascular endothelial and pericyte cell coculture system, this study determined the effects of pulsatile flow on the activation of the endothelial cell markers nitric oxide (NO), prostacyclin (PGI2), and endothelin (ET)-1. METHODS: Monocultured bovine retinal endothelial cells (BRECs) and cocultured BRECs with bovine retinal pericytes (BRPs) were exposed to low flow (flow rate, 0.3 mL/min; pulse pressure, 6 mmHg; shear stress, 0.5 dyne/cm2) or high flow (flow rate, 25 mL/min; pulse pressure, 56 mmHg; shear stress, 23 dynes/cm2) for 24 hours, by using a novel perfused transcapillary culture system. The cells were characterized by immunohistochemistry and electron and confocal microscopy. Endothelial nitric oxide synthase (eNOS) and phosphorylated-eNOSSer1179 (pp-eNOS) were determined by Western blot analysis. Nitrate, PGI2, and ET-1 levels were quantified in the medium perfusate by using fluorometric and enzyme-linked immunosorbent assays, respectively. Activation of cyclooxygenase (COX)-2 in BRECs was determined by measuring COX-2 promoter activity with a luciferase reporter assay. RESULTS: The presence of BRPs and BRECs was confirmed by Western blot, immunocytochemistry, and scanning electron microscopy. Phosphorylated eNOS (pp-eNOS) protein levels in BRECs were significantly increased from low to high flow in both mono- and cocultures, concomitant with a significant increase in nitrate levels in the conditioned medium after exposure to pulsatile flow. In parallel cultures, PGI2 levels were also significantly enhanced concomitant with an increase in the transactivation of a COX-2 promoter BREC after exposure to pulsatile flow. ET-1 levels were also increased in both mono- and cocultured cells. conclusions. In this study a novel, functioning, in vitro model of retinal microvascular endothelial and pericyte cells that respond to changes in pulsatile flow was established.
Assuntos
Endotelina-1/metabolismo , Endotélio Vascular/metabolismo , Epoprostenol/metabolismo , Óxido Nítrico Sintase/metabolismo , Pericitos/metabolismo , Fluxo Pulsátil/fisiologia , Vasos Retinianos/fisiologia , Animais , Western Blotting , Bovinos , Células Cultivadas , Técnicas de Cocultura , Ciclo-Oxigenase 2 , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Técnica Indireta de Fluorescência para Anticorpo , Isoenzimas/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Óxido Nítrico Sintase Tipo III , Pericitos/citologia , Fosforilação , Prostaglandina-Endoperóxido Sintases/metabolismo , Vasos Retinianos/citologiaRESUMO
PURPOSE: The role of VEGF-A in the normal ciliary body is largely unexplored. The ciliary body is similar in many respects to the choroid plexus of the brain, and we demonstrated previously the importance of VEGF-A in maintenance of choroid plexus vasculature and ependymal cells. Therefore, the role of VEGF-A in ciliary body homeostasis was explored. METHODS: Swiss-Webster mice (VEGF-LacZ) were used to determine VEGF-A expression during ciliary body development and in the adult. VEGFR2 expression was determined in adult wild type C56BL/6J mice. Systemic VEGF-A neutralization in vivo was achieved with adenovirus-mediated overexpression of soluble VEGFR1 (sFlt1). Following VEGF-A neutralization, the ciliary epithelium was analyzed by light microscopy and transmission electron microscopy (TEM). The effect of VEGF-A blockade on ciliary body function also was assessed by measuring intraocular pressure. RESULTS: VEGF-A expression was detected at embryonic day 18.5 (E18.5), the onset of ciliary process formation. In the adult ciliary body, VEGF-A was expressed by the pigmented epithelium, whereas VEGFR2 was localized primarily to the capillary endothelium and nonpigmented epithelium. Systemic VEGF-A neutralization led to a thinning of the nonpigmented epithelium, vacuolization of the pigmented epithelium, loss of capillary fenestrations, and thrombosis. These changes were associated with impaired ciliary body function, as evidenced by decreased intraocular pressure in sFlt1-overexpressing animals (15.31 ± 2.06 mm Hg) relative to controls (18.69 ± 1.49 mm Hg). CONCLUSIONS: VEGF-A has an important role in ciliary body homeostasis. Potential for undesired off-target effects should be considered with the chronic use of anti-VEGF-A therapies.
Assuntos
Corpo Ciliar/metabolismo , Prenhez , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Corpo Ciliar/embriologia , Corpo Ciliar/crescimento & desenvolvimento , Ensaio de Imunoadsorção Enzimática , Epitélio/metabolismo , Epitélio/ultraestrutura , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão e Varredura , GravidezRESUMO
AIMS: Aberrant retinal blood flow is a hallmark of various retinopathies and may be a causative factor in the pathology associated with these conditions. We examined the effects of pulsatile flow on bovine retinal endothelial cell (BREC) and bovine retinal pericyte (BRP) apoptosis and proliferation. METHODS AND RESULTS: Co-cultured BRECs and BRPs were exposed to low (0.3 mL/min) or high (25 mL/min) pulsatile flow for 72 h using a perfused transcapillary culture system. Pulsatile flow increased BREC nitric oxide synthase (eNOS) and cyclooxygenase-2 (COX-2) expression and activity concomitant with a significant decrease in pre-pro-endothelin-1 (ET-1) mRNA and peptide. BREC apoptosis was significantly attenuated following exposure to high flow. The inhibition of NOS, COX, and ET receptors significantly reduced the pro-survival effects of flow on BREC. In contrast, BRP apoptosis was significantly enhanced following exposure to high flow. The inhibition of COX and ET receptors significantly attenuated the high flow-induced increase in BRP apoptosis when compared with untreated controls. Treatment of static BREC with NO donor (S-nitroso-N-acetylpenicillamine, SNAP), ET-1, or iloprost inhibited serum deprivation-induced apoptosis, whereas treatment of BRP with ET-1 and iloprost, but not SNAP, was ineffective. High pulsatile flow decreased BRP proliferation, in the absence of any changes in BREC proliferation. CONCLUSION: Increased pulsatile flow promotes BREC survival and enhances BRP apoptosis through the activation of endothelial-derived vasoactive substances. Altered pulsatile flow does not alter BREC proliferation in co-culture with BRP, whereas BRP proliferation was significantly decreased at high flow rates. These interactions have important implications for vessel growth and regression during retinal vascular pathogenesis.
Assuntos
Apoptose/fisiologia , Células Endoteliais/citologia , Microcirculação/fisiologia , Pericitos/citologia , Fluxo Pulsátil/fisiologia , Vasos Retinianos/citologia , Animais , Bovinos , Divisão Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/fisiologia , Endotelina-1/metabolismo , Epoprostenol/metabolismo , Óxido Nítrico/metabolismo , Pericitos/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Retina/citologia , Retina/fisiologia , Vasos Retinianos/fisiologia , Estresse MecânicoRESUMO
PURPOSE: Aberrant retinal blood flow is a hallmark of retinopathies and may be a causative factor in their pathophysiology. In this study, the effects of pulsatile flow on hedgehog and Notch control of retinal endothelial cell and pericyte apoptosis were examined. METHODS: The levels of hedgehog and Notch signaling components in bovine retinal endothelial cells (BRECs) and pericytes (BRPs) were examined in vitro under static conditions and after exposure to pulsatile flow, with a perfused transcapillary co-culture system. Notch and hedgehog signaling was examined by immunocytochemistry, immunoblot, and real-time PCR. RESULTS: Notch and hedgehog proteins were present in BRECs and BRPs in vitro and in human retinal vasculature in vivo. Inhibition of hedgehog with cyclopamine and Notch with DAPT decreased hedgehog target gene levels and Notch intracellular receptor expression, respectively, concomitant with an increase in BREC and BRP apoptosis. Sonic hedgehog (Shh) mediated upregulation of Notch1 receptor levels was attenuated after cyclopamine treatment in both cell types. Exposure of co-cultured BRECs and BRPs to pulsatile flow increased apoptosis in the BRPs while concurrently decreasing apoptosis in the BRECs. These changes were concomitant with increased expression of Notch and hedgehog signaling components in the BRECs and reduced expression in the BRPs. The flow-induced decrease in apoptosis in the BRECs was associated with increased Notch receptor expression and was reversed after inhibition of hedgehog signaling with cyclopamine and inhibition of Notch signaling after ectopic expression of the CBF-1/RBP-Jκ-binding protein, RPMS-1. CONCLUSIONS: Pulsatile flow promotes BREC survival and enhances BRP apoptosis through modulation of Notch and hedgehog pathways. These interactions have important implications for the pathogenesis of retinopathies.
Assuntos
Apoptose/fisiologia , DNA/genética , Endotélio Vascular/citologia , Proteínas Hedgehog/genética , Pericitos/citologia , Receptores Notch/genética , Retina/citologia , Animais , Western Blotting , Bovinos , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Proteínas Hedgehog/biossíntese , Humanos , Imuno-Histoquímica , Pericitos/metabolismo , Reação em Cadeia da Polimerase , Receptores Notch/biossíntese , Retina/metabolismo , Transdução de SinaisRESUMO
Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-beta signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-beta signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-beta signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-beta signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-beta signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-beta signaling. These results implicate constitutive TGF-beta signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-beta signaling in vasoproliferative and vascular degenerative retinal diseases.
Assuntos
Endotélio Vascular/fisiologia , Homeostase , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Animais , DNA Complementar , Endotélio Vascular/ultraestrutura , Marcação In Situ das Extremidades Cortadas , Camundongos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Although the terms ischemia and hypoxia are often used interchangeably, they represent distinct processes that result in different modulatory effects at the cellular level. Hypoxia is a reduction in oxygen delivery below tissue demand, whereas ischemia is a lack of perfusion, characterized not only by hypoxia but also by insufficient nutrient supply. Hypoxia can be either acute or chronic, and both are centrally regulated by hypoxia-inducible factor, a transcription factor that governs the expression of key response genes such as vascular endothelial growth factor and erythropoietin. Whereas severe chronic hypoxia can cause cell death, less-severe hypoxia can protect against subsequent damage, a phenomenon known as hypoxic conditioning. Several important processes are characterized by hypoxia, including ischemia-reperfusion, tumor growth and progression, inflammation, myocardial ischemia, and a number of ocular pathologies.
Assuntos
Hipóxia/patologia , Traumatismo por Reperfusão/patologia , Reperfusão , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Eritropoese/fisiologia , Regulação da Expressão Gênica , Humanos , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Neovascularização Fisiológica/fisiologia , Recuperação de Função Fisiológica/fisiologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismoRESUMO
Angiogenesis is largely controlled by hypoxia-driven transcriptional up-regulation and secretion of vascular endothelial growth factor (VEGF) and its binding to the endothelial cell tyrosine receptor kinases, VEGFR1 and VEGFR2. Recent expression analysis suggests that VEGF is expressed in a cell-specific manner in normoxic adult tissue; however, the transcriptional regulation and role of VEGF in these tissues remains fundamentally unknown. In this report we demonstrate that VEGF is coordinately up-regulated during terminal skeletal muscle differentiation. We reveal that this regulation is mediated in part by MyoD homo- and hetero-dimeric transcriptional mechanisms. Serial deletions of the VEGF promoter elucidated a region containing three tandem CANNTG consensus MyoD sites serving as essential sites of direct interaction for MyoD-mediated up-regulation of VEGF transcription. VEGF-null embryonic stem (ES) cells exhibited reduced myogenic differentiation compared with wild-type ES cells, suggesting that VEGF may serve a role in skeletal muscle differentiation. We demonstrate that VEGFR1 and VEGFR2 are expressed at low levels in myogenic precursor cells and are robustly activated upon VEGF stimulation and that their expression is coordinately regulated during skeletal muscle differentiation. VEGF stimulation of differentiating C2C12 cells promoted myotube hypertrophy and increased myogenic differentiation, whereas addition of sFlt1, a VEGF inhibitor, resulted in myotube hypotrophy and inhibited myogenic differentiation. We further provide evidence indicating VEGF-mediated myogenic marker expression, mitogenic activity, migration, and prosurvival functions may contribute to increased myogenesis. These data suggest a novel mechanism whereby VEGF is coordinately regulated as part of the myogenic differentiation program and serves an autocrine function regulating skeletal myogenesis.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/embriologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Proteína MyoD/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Although the role of vascular endothelial growth factor (VEGF) in developmental and pathological angiogenesis is well established, its function in the adult is less clear. Similarly, although transforming growth factor (TGF) beta is involved in angiogenesis, presumably by mediating capillary (endothelial cell [EC]) stability, its involvement in quiescent vasculature is virtually uninvestigated. Given the neurological findings in patients treated with VEGF-neutralizing therapy (bevacizumab) and in patients with severe preeclampsia, which is mediated by soluble VEGF receptor 1/soluble Fms-like tyrosine kinase receptor 1 and soluble endoglin, a TGF-beta signaling inhibitor, we investigated the roles of VEGF and TGF-beta in choroid plexus (CP) integrity and function in adult mice. Receptors for VEGF and TGF-beta were detected in adult CP, as well as on ependymal cells. Inhibition of VEGF led to decreased CP vascular perfusion, which was associated with fibrin deposition. Simultaneous blockade of VEGF and TGF-beta resulted in the loss of fenestrae on CP vasculature and thickening of the otherwise attenuated capillary endothelium, as well as the disappearance of ependymal cell microvilli and the development of periventricular edema. These results provide compelling evidence that both VEGF and TGF-beta are involved in the regulation of EC stability, ependymal cell function, and periventricular permeability.
Assuntos
Plexo Corióideo/metabolismo , Epêndima/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenoviridae/genética , Animais , Permeabilidade Capilar , Plexo Corióideo/ultraestrutura , Células Endoteliais/metabolismo , Epêndima/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical. METHODS AND FINDINGS: Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective. CONCLUSIONS: These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.
Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Visão Ocular/genética , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Apoptose/genética , Comunicação Autócrina/genética , Comunicação Autócrina/fisiologia , Sobrevivência Celular/genética , Óperon Lac , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retina/fisiologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neurônios Retinianos/metabolismo , Neurônios Retinianos/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
During the last decade, the presumed etiology of glaucoma has moved from a pure pressure concept to a combined mechanical and vascular theory. Evidence of a localized vascular insufficiency leading to perfusion deficits of ocular structures, including the optic nerve head, the retina, the choroid, and the retrobulbar vessels, is now clear. This article evaluates the role of vasospasm as the primary cause of such a vascular failure. The role of both ocular and systemic vasospasms and their clinical correlations are discussed. At a cellular level, the function of the modulating role of the vascular endothelium is reviewed. Evidence of abnormalities of the vascular endothelium and its vasoactive peptides as a conduit for vasospasm is mounting. Herein lies exciting prospects for potential pharmacologic targets in future glaucoma management.