RESUMO
The kidney vasculature has a complex architecture that is essential for renal function. The molecular mechanisms that direct development of kidney blood vessels are poorly characterized. We identified a regionally restricted, stroma-derived signaling molecule, netrin 1 (Ntn1), as a regulator of renal vascular patterning in mice. Stromal progenitor (SP)-specific ablation of Ntn1 (Ntn1SPKO) resulted in smaller kidneys with fewer glomeruli, as well as profound defects of the renal artery and transient blood flow disruption. Notably, Ntn1 ablation resulted in loss of arterial vascular smooth muscle cell (vSMC) coverage and in ectopic SMC deposition at the kidney surface. This was accompanied by dramatic reduction of arterial tree branching that perdured postnatally. Transcriptomic analysis of Ntn1SPKO kidneys revealed dysregulation of vSMC differentiation, including downregulation of Klf4, which we find expressed in a subset of SPs. Stromal Klf4 deletion similarly resulted in decreased smooth muscle coverage and arterial branching without, however, the disruption of renal artery patterning and perfusion seen in Ntn1SPKO. These data suggest a stromal Ntn1-Klf4 axis that regulates stromal differentiation and reinforces stromal-derived smooth muscle as a key regulator of renal blood vessel formation.
Assuntos
Perfilação da Expressão Gênica , Rim , Camundongos , Animais , Netrina-1/genética , Netrina-1/metabolismo , Rim/fisiologia , Diferenciação Celular/genética , Morfogênese , Miócitos de Músculo LisoRESUMO
In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.
Assuntos
Diafragma , Células Endoteliais , Diafragma/metabolismo , Células Endoteliais/metabolismo , Proteínas de Transporte/metabolismo , Endotélio Vascular/metabolismo , Dissulfetos/metabolismo , Dicroísmo CircularRESUMO
Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor ß. apCAFs directly ligate and induce naive CD4+ T cells into regulatory T cells (Tregs) in an antigen-specific manner. Moreover, treatment with an antibody targeting the mesothelial cell marker mesothelin can effectively inhibit mesothelial cell to apCAF transition and Treg formation induced by apCAFs. Taken together, our study elucidates how mesothelial cells may contribute to immune evasion in pancreatic cancer and provides insight on strategies to enhance cancer immune therapy.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos , Humanos , Neoplasias Pancreáticas/patologia , Linfócitos T Reguladores , Fator de Crescimento Transformador beta/metabolismo , Neoplasias PancreáticasRESUMO
Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1-6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.
Assuntos
Coração/embriologia , Sistema Linfático/citologia , Sistema Linfático/metabolismo , Miocárdio/citologia , Miócitos Cardíacos/citologia , Regeneração , Transdução de Sinais , Animais , Animais Recém-Nascidos , Apoptose , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Integrina beta1/metabolismo , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tamanho do Órgão , Organogênese , Proteína Reelina , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Purpose: Polymorphisms at the caveolin-1/2 locus are associated with glaucoma and IOP risk and deletion of caveolin-1 (Cav1) in mice elevates IOP and reduces outflow facility. However, the specific location/cell type responsible for Cav1-dependent regulation of IOP is unclear. We hypothesized that endothelial Cav1 in the conventional outflow (CO) pathway regulate IOP via endothelial nitric oxide synthase (eNOS) signaling. Methods: We created a mouse with targeted deletion of Cav1 in endothelial cells (Cav1ΔEC) and evaluated IOP, outflow facility, outflow pathway distal vascular morphology, eNOS phosphorylation, and tyrosine nitration of iridocorneal angle tissues by Western blotting. Results: Endothelial deletion of Cav1 resulted in significantly elevated IOP versus wild-type mice but not a concomitant decrease in outflow facility. Endothelial Cav1 deficiency did not alter the trabecular meshwork or Schlemm's canal morphology, suggesting that the effects observed were not due to developmental deformities. Endothelial Cav1 deletion resulted in eNOS hyperactivity, modestly increased protein nitration, and significant enlargement of the drainage vessels distal to Schlemm's canal. L-Nitro-arginine methyl ester treatment reduced outflow in Cav1ΔEC but not wild-type mice and had no effect on the size of drainage vessels. Endothelin-1 treatment decrease the outflow and drainage vessel size in both wild-type and Cav1ΔEC mice. Conclusions: Our results suggest that hyperactive eNOS signaling in the CO pathway of both Cav1ΔEC and global Cav1 knockout mice results in chronic dilation of distal CO vessels and protein nitration, but that Cav1 expression in the trabecular meshwork is sufficient to rescue CO defects reported in global Cav1 knockout mice.
Assuntos
Humor Aquoso/metabolismo , Caveolina 1/genética , DNA/genética , Células Endoteliais/metabolismo , Glaucoma/genética , Pressão Intraocular/fisiologia , Polimorfismo Genético , Animais , Western Blotting , Caveolina 1/metabolismo , Modelos Animais de Doenças , Células Endoteliais/patologia , Glaucoma/metabolismo , Glaucoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Proper organ development depends on coordinated communication between multiple cell types. Retinoic acid (RA) is an autocrine and paracrine signaling molecule essential for the development of most organs, including the lung. Despite extensive work detailing effects of RA deficiency in early lung morphogenesis, little is known about how RA regulates late gestational lung maturation. Here, we investigate the role of the RA catabolizing protein Cyp26b1 in the lung. Cyp26b1 is highly enriched in lung endothelial cells (ECs) throughout development. We find that loss of Cyp26b1 leads to reduction of alveolar type 1 cells, failure of alveolar inflation and early postnatal lethality in mouse. Furthermore, we observe expansion of distal epithelial progenitors, but no appreciable changes in proximal airways, ECs or stromal populations. Exogenous administration of RA during late gestation partially mimics these defects; however, transcriptional analyses comparing Cyp26b1-/- with RA-treated lungs reveal overlapping, but distinct, responses. These data suggest that defects observed in Cyp26b1-/- lungs are caused by both RA-dependent and RA-independent mechanisms. This work reports crucial cellular crosstalk during lung development involving Cyp26b1-expressing endothelium and identifies a novel RA modulator in lung development.
Assuntos
Epitélio/embriologia , Pulmão/embriologia , Alvéolos Pulmonares/embriologia , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/fisiologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Células Endoteliais/citologia , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/efeitos dos fármacos , Gravidez , Prenhez , Transdução de Sinais , Células-Tronco/citologia , Tretinoína/farmacologiaRESUMO
Sphingosine-1-phosphate (S1P) produced by sphingosine kinases (SPHK1 and SPHK2) is a signaling molecule involved in cell proliferation and formation of cellular junctions. In this study, we characterized the retinas of Sphk1 knockout (KO) mice by electron microscopy and immunocytochemistry. We also tested cultured Müller glia for their response to S1P. We found that S1P plays an important role in retinal and retinal pigment epithelial (RPE) structural integrity in aging mice. Ultrastructural analysis of Sphk1 KO mouse retinas aged to 15 months or raised with moderate light stress revealed a degenerated outer limiting membrane (OLM). This membrane is formed by adherens junctions between neighboring Müller glia and photoreceptor cells. We also show that Sphk1 KO mice have reduced retinal function in mice raised with moderate light stress. In vitro assays revealed that exogenous S1P modulated cytoskeletal rearrangement and increased N-cadherin production in human Müller glia cells. Aged mice also had morphological degeneration of the RPE, as well as increased lipid storage vacuoles and undigested phagosomes reminiscent of RPE in age-related macular degeneration. These findings show that SPHK1 and S1P play a vital role in the structural maintenance of the mammalian retina and retinal pigmented epithelium by supporting the formation of adherens junctions.
Assuntos
Junções Aderentes/metabolismo , Envelhecimento/metabolismo , Membrana Celular/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Retina/metabolismo , Junções Aderentes/ultraestrutura , Animais , Caderinas/metabolismo , Endotélio/metabolismo , Células Ependimogliais/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Camundongos Knockout , Fenótipo , Retina/ultraestrutura , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The brain, spinal cord, and retina are supplied by capillaries that do not permit free diffusion of molecules between serum and parenchyma, a property that defines the blood-brain and blood-retina barriers. Exceptions to this pattern are found in circumventricular organs (CVOs), small midline brain structures that are supplied by high permeability capillaries. In the eye and brain, high permeability capillaries are also present in the choriocapillaris, which supplies the retinal pigment epithelium and photoreceptors, and the ciliary body and choroid plexus, the sources of aqueous humor and cerebrospinal fluid, respectively. We show here that (1) endothelial cells in these high permeability vascular systems have very low beta-catenin signaling compared to barrier-competent endothelial cells, and (2) elevating beta-catenin signaling leads to a partial conversion of permeable endothelial cells to a barrier-type state. In one CVO, the area postrema, high permeability is maintained, in part, by local production of Wnt inhibitory factor-1.
Assuntos
Permeabilidade Capilar , Corioide/fisiologia , Órgãos Circunventriculares/fisiologia , Regulação da Expressão Gênica , Transdução de Sinais , beta Catenina/metabolismo , Animais , Barreira Hematoencefálica , Barreira Hematorretiniana , Células Endoteliais/fisiologia , CamundongosRESUMO
Cerebral microcirculation is critical for the preservation of brain health, and vascular impairment is associated with age-related neurodegenerative diseases. Because the retina is a component of the central nervous system, cellular changes that occur in the aging retina are likely relevant to the aging brain, and the retina provides the advantage that the entire vascular bed is visible, en face. In this study, we tested the hypothesis that normal, healthy aging alters the contractile vascular smooth muscle cell (VSMC) coverage of retinal arterioles. We found that aging results in a significant reduction of contractile VSMCs in focal patches along arterioles. Focal loss of contractile VSMCs occurs at a younger age in mice deficient in the senescence-associated protein, caveolin-1. Age-related contractile VSMC loss is not exacerbated by genetic depletion of insulin-like growth factor-1. The patchy loss of contractile VSMCs provides a cellular explanation for previous clinical studies showing focal microirregularities in retinal arteriolar responsiveness in healthy aged human subjects and is likely to contribute to age-related retinal vascular complications.
Assuntos
Envelhecimento , Caveolina 1/fisiologia , Contração Muscular , Músculo Liso Vascular/fisiologia , Artéria Retiniana/fisiologia , Animais , Apoptose , Arteríolas/fisiologia , Caveolina 1/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Although major progress in our understanding of the genes and mechanisms that regulate lymphatic vasculature development has been made, we still do not know how lumen formation and maintenance occurs. Here, we identify the Ras-interacting protein Rasip1 as a key player in this process. We show that lymphatic endothelial cell-specific Rasip1-deficient mouse embryos exhibit enlarged and blood-filled lymphatics at embryonic day 14.5. These vessels have patent lumens with disorganized junctions. Later on, as those vessels become fragmented and lumens collapse, cell junctions become irregular. In addition, Rasip1 deletion at later stages impairs lymphatic valve formation. We determined that Rasip1 is essential for lymphatic lumen maintenance during embryonic development by regulating junction integrity, as Rasip1 loss results in reduced levels of junction molecules and defective cytoskeleton organization in vitro and in vivo We determined that Rasip1 regulates Cdc42 activity, as deletion of Cdc42 results in similar phenotypes to those seen following the loss of Rasip1 Furthermore, ectopic Cdc42 expression rescues the phenotypes in Rasip1-deficient lymphatic endothelial cells, supporting the suggestion that Rasip1 regulates Cdc42 activity to regulate cell junctions and cytoskeleton organization, which are both activities required for lymphatic lumen maintenance.
Assuntos
Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Embrião de Mamíferos/embriologia , Células Endoteliais/metabolismo , Vasos Linfáticos/embriologia , Junções Íntimas/metabolismo , Animais , Proteínas de Transporte/genética , Citoesqueleto/genética , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Vasos Linfáticos/citologia , Camundongos , Camundongos Transgênicos , Junções Íntimas/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Endothelial cell (EC) activation and vascular injury are hallmark features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Caveolin-1 (Cav-1) is highly expressed in pulmonary microvascular ECs and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine if the lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) promotes priming of ECs via Cav-1 depletion and if this contributes to the onset of pulmonary vascular remodeling. To test the hypothesis that depletion of Cav-1 primes ECs to respond to profibrotic signals, C57BL6 wild-type (WT) mice (Tie2.Cre-;Cav1fl/fl ) were exposed to nebulized LPS (10 mg; 1 h daily for 4 days) and compared with EC-specific Cav1-/- (Tie2.Cre+;Cav1fl/fl ). After 96 h of LPS exposure, total lung Cav-1 and bone morphogenetic protein receptor type II (BMPRII) expression were reduced in WT mice. Moreover, plasma albumin leakage, infiltration of immune cells, and levels of IL-6/IL-6R and transforming growth factor-ß (TGF-ß) were elevated in both LPS-treated WT and EC-Cav1-/- mice. Finally, EC-Cav1-/- mice exhibited a modest increase in microvascular thickness basally and even more so on exposure to LPS (96 h). EC-Cav1-/- mice and LPS-treated WT mice exhibited reduced BMPRII expression and endothelial nitric oxide synthase uncoupling, which along with increased TGF-ß promoted TGFßRI-dependent SMAD-2/3 phosphorylation. Finally, human lung sections from patients with ARDS displayed reduced EC Cav-1 expression, elevated TGF-ß levels, and severe pulmonary vascular remodeling. Thus EC Cav-1 depletion, oxidative stress-mediated reduction in BMPRII expression, and enhanced TGF-ß-driven SMAD-2/3 signaling promote pulmonary vascular remodeling in inflamed lungs.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Caveolina 1/metabolismo , Células Endoteliais/patologia , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Remodelação Vascular , Actinas/metabolismo , Lesão Pulmonar Aguda/complicações , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Adulto , Idoso , Animais , Líquido da Lavagem Broncoalveolar , Forma Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/farmacologia , Lipopolissacarídeos , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Biológicos , Óxido Nítrico Sintase Tipo III/metabolismo , Proteólise/efeitos dos fármacos , Artéria Pulmonar/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Síndrome do Desconforto Respiratório/complicações , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/patologia , Fatores de Tempo , Remodelação Vascular/efeitos dos fármacosRESUMO
Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., "lipid rafts") have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signaling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function.
Assuntos
Barreira Hematorretiniana , Cavéolas/metabolismo , Caveolinas/metabolismo , Glaucoma/metabolismo , Pressão Intraocular/fisiologia , Mecanotransdução Celular/fisiologia , Fenômenos Fisiológicos Oculares , Animais , Glaucoma/fisiopatologia , HumanosRESUMO
Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1-/-) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm's canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1-/- outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1-/- eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology.
Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Glaucoma/metabolismo , Pressão Intraocular , Malha Trabecular/metabolismo , Animais , Cavéolas/patologia , Caveolina 1/genética , Glaucoma/genética , Glaucoma/patologia , Glaucoma/fisiopatologia , Humanos , Camundongos , Camundongos Knockout , Malha Trabecular/patologia , Malha Trabecular/fisiopatologiaRESUMO
AIMS/HYPOTHESIS: We aimed to determine whether plasma lipoproteins, after leakage into the retina and modification by glycation and oxidation, contribute to the development of diabetic retinopathy in a mouse model of type 1 diabetes. METHODS: To simulate permeation of plasma lipoproteins into retinal tissues, streptozotocin-induced mouse models of diabetes and non-diabetic mice were challenged with intravitreal injection of human 'highly-oxidised glycated' low-density lipoprotein (HOG-LDL), native- (N-) LDL, or the vehicle PBS. Retinal histology, electroretinography (ERG) and biochemical markers were assessed over the subsequent 14 days. RESULTS: Intravitreal administration of N-LDL and PBS had no effect on retinal structure or function in either diabetic or non-diabetic animals. In non-diabetic mice, HOG-LDL elicited a transient inflammatory response without altering retinal function, but in diabetic mice it caused severe, progressive retinal injury, with abnormal morphology, ERG changes, vascular leakage, vascular endothelial growth factor overexpression, gliosis, endoplasmic reticulum stress, and propensity to apoptosis. CONCLUSIONS/INTERPRETATION: Diabetes confers susceptibility to retinal injury imposed by intravitreal injection of modified LDL. The data add to the existing evidence that extravasated, modified plasma lipoproteins contribute to the propagation of diabetic retinopathy. Intravitreal delivery of HOG-LDL simulates a stress known to be present, in addition to hyperglycaemia, in human diabetic retinopathy once blood-retinal barriers are compromised.
Assuntos
Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/sangue , Retinopatia Diabética/etiologia , Animais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Eletrorretinografia , Humanos , Lipoproteínas LDL/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Caveolin-1 associates with the endo/lysosomal machinery of cells in culture, suggesting that it functions at these organelles independently of its contribution to cell surface caveolae. Here we explored mice lacking caveolin-1 specifically in the retinal pigment epithelium (RPE). The RPE supports neighboring photoreceptors via diurnal phagocytosis of spent photoreceptor outer segment fragments. Like mice lacking caveolin-1 globally, (RPE)CAV1(-/-) mice developed a normal RPE and neural retina but showed reduced rod photoreceptor light responses, indicating that lack of caveolin-1 affects photoreceptor function in a non-cell-autonomous manner. (RPE)CAV1(-/-) RPE in situ showed normal particle engulfment but delayed phagosome clearance and reversed diurnal profiles of levels and activities of lysosomal enzymes. Therefore, eliminating caveolin-1 specifically impairs phagolysosomal degradation by the RPE in vivo. Endogenous caveolin-1 was recruited to maturing phagolysosomes in RPE cells in culture. Consistent with these in vivo data, a moderate increase (to â¼ 2.5-fold) or decrease (by half) of caveolin-1 protein levels in RPE cells in culture was sufficient to accelerate or impair phagolysosomal digestion, respectively. A mutant form of caveolin-1 that fails to reach the cell surface augmented degradation like wild-type caveolin-1. Acidic lysosomal pH and increased protease activity are essential for digestion. We show that halving caveolin-1 protein levels significantly alkalinized lysosomal pH and decreased lysosomal enzyme activities. Taken together, our results reveal a novel role for intracellular caveolin-1 in modulating phagolysosomal function. Moreover, they show, for the first time, that organellar caveolin-1 significantly affects tissue functionality in vivo.
Assuntos
Caveolina 1/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Catepsina D/metabolismo , Linhagem Celular , Ritmo Circadiano , Lisossomos/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Fagossomos/metabolismo , Transporte Proteico , Proteólise , Ratos , Receptores da Transferrina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sus scrofa , Visão OcularRESUMO
Caveolin-1 (Cav-1), the scaffolding protein of caveolae, is expressed in several retinal cell types and is associated with ocular pathologies. Cav-1 modulates neuroinflammatory/neuroprotective responses to central nervous system injury. We have shown that loss of Cav-1 results in a blunted cytokine response in retinas challenged with inflammatory stimuli. As neuroinflammatory and neuroprotective signaling overlap in their cytokine production and downstream signaling pathways, we hypothesized that loss of Cav-1 may also suppress neuroprotective signaling in the retina. To test this, we subjected mice in which Cav-1 was deleted specifically in the retina to a neurodegenerative insult induced by sodium iodate (NaIO3) and measured STAT3 activation, a measure of neuroprotective signaling. Our results show that Cav-1 ablation blunts STAT3 activation induced by NaIO3. STAT3 activation in response to intravitreal administration of the IL-6 family cytokine, leukemia inhibitory factor (LIF), was not affected by Cav-1 deletion indicating a competent gp130 receptor response. Thus, Cav-1 modulates neuroprotective signaling by regulating the endogenous production of neuroprotective factors.
Assuntos
Caveolina 1/genética , Neuroproteção/genética , Retina/metabolismo , Transdução de Sinais/genética , Animais , Western Blotting , Caveolina 1/deficiência , Feminino , Imuno-Histoquímica , Injeções Intraperitoneais , Iodatos/administração & dosagem , Iodatos/farmacologia , Fator Inibidor de Leucemia/administração & dosagem , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Knockout , Neuroproteção/efeitos dos fármacos , Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: Caveolin-1 (Cav-1), the signature protein of caveolae, modulates inflammatory responses, and innate immunity. However, Cav-1's role in retinal inflammation has not been rigorously tested. In this study, we examined the effect of Cav-1 ablation on the sensitivity of the retina to inflammation. METHODS: Cav-1 knockout (KO) mice were challenged by intravitreal injection of lipopolysaccharide (LPS) and inflammatory cell recruitment was assessed by flow cytometry and immunohistochemistry. Leukostasis was assessed in retinal flatmounts after perfusion with FITC-labeled Concanavalin A (FITC-ConA). Chemoattractants were measured by multiplex immunoassays. Blood-retinal barrier (BRB) breakdown was assessed quantitatively by a FITC-dextran permeability assay. The ratio of extravascular to total immune cells was determined by CD45 immunohistochemistry of retinal flatmounts. RESULTS: Inflammatory challenge resulted in significant blunting of proinflammatory cytokine (monocyte chemoattractant protein-1 [MCP-1/CCL2], CXCL1/KC, IL-6, and IL-1ß) responses as well as reduced inflammatory BRB breakdown in Cav-1 KO retinas. Paradoxically, Cav-1 deficiency resulted in significantly increased recruitment of immune cells compared with controls as well as increased leukostasis. A similar ratio of extravascular/total leukocytes were found in Cav-1 KO and wild-type (WT) retinas suggesting that Cav-1 deficient leukocytes were as competent to extravasate as those from WT mice. We found increased levels of circulating immune cells in naïve (not challenged with LPS) Cav-1 KO mice compared with controls. CONCLUSIONS: Caveolin-1 paradoxically modulates inflammatory signaling and leukocyte infiltration through distinct mechanisms. We hypothesize that Cav-1 expression may enhance inflammatory signaling while at the same time supporting the physical properties of the BRB.
Assuntos
Barreira Hematorretiniana/fisiologia , Caveolina 1/genética , Fatores Quimiotáticos/metabolismo , Regulação da Expressão Gênica , Imunidade Inata/genética , RNA/genética , Uveíte/genética , Animais , Western Blotting , Caveolina 1/biossíntese , Modelos Animais de Doenças , Citometria de Fluxo , Imuno-Histoquímica , Leucócitos/imunologia , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Uveíte/imunologia , Uveíte/metabolismoRESUMO
Pericyte degeneration is an early event in diabetic retinopathy and plays an important role in progression of diabetic retinopathy. Clinical studies have shown that fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has robust therapeutic effects on diabetic retinopathy in type 2 diabetic patients. We evaluated the protective effect of PPARα against pericyte loss in diabetic retinopathy. In streptozotocin-induced diabetic mice, fenofibrate treatment significantly ameliorated retinal acellular capillary formation and pericyte loss. In contrast, PPARα(-/-) mice with diabetes developed more severe retinal acellular capillary formation and pericyte dropout, compared with diabetic wild-type mice. Furthermore, PPARα knockout abolished the protective effect of fenofibrate against diabetes-induced retinal pericyte loss. In cultured primary human retinal capillary pericytes, activation and expression of PPARα both significantly reduced oxidative stress-induced apoptosis, decreased reactive oxygen species production, and down-regulated NAD(P)H oxidase 4 expression through blockade of NF-κB activation. Furthermore, activation and expression of PPARα both attenuated the oxidant-induced suppression of mitochondrial O2 consumption in human retinal capillary pericytes. Primary retinal pericytes from PPARα(-/-) mice displayed more apoptosis, compared with those from wild-type mice under the same oxidative stress. These findings identified a protective effect of PPARα on retinal pericytes, a novel function of endogenous PPARα in the retina.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Fenofibrato/farmacologia , PPAR alfa/agonistas , Pericitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/metabolismo , Pericitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismoRESUMO
Caveolin-1 (Cav-1), the signature protein of caveolae is expressed in several cell types in the adult retina and is linked to ocular pathologies including uveitis, diabetic retinopathy, and primary open angle glaucoma. Genetic ablation of Cav-1 causes retinal functional deficits due to disruptions in environmental homeostasis. To better understand Cav-1 function in the retina, we examined its expression/localization during postnatal retinal development. From P0-P5, Cav-1 was detected only in the developing superficial retinal vessels, in hyaloid and choroidal vasculature, and in the retinal pigment epithelium (RPE). At P7, staining began to be observed centrally in radial cells in the neuroretina, and this staining increased dramatically by P9/10 in identifiable Müller glia. Prominent vascular staining continued throughout development. These results support the idea that Cav-1 is an indicator of Müller glial differentiation and suggests that it plays an important role in Müller cell function.
Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Células Ependimogliais/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Animais , Diferenciação Celular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/citologia , Vasos Retinianos/metabolismoRESUMO
Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.