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Purpose: Previously, we identified increased retinal degeneration and cytokine response in a mouse model of dry age-related macular degeneration (AMD) in the presence of systemic inflammation from rheumatoid arthritis (RA). Histone deacetylases (HDACs) regulate cytokine production by reducing acetylation and are found to be dysregulated in inflammatory diseases, including RA and AMD. Therefore, this current study investigates the effect of HDAC inhibition on AMD progression in the presence of systemic inflammation. Methods: Collagen induced arthritis (CIA) was induced in C57BL6J mice, followed by sodium iodate (NaIO3)-induced retinal degeneration. Mice were treated with a selective HDAC class I inhibitor, MS-275, and retinal structure [optical coherence tomography (OCT)], function (electroretinography), and molecular changes quantitative real-time polymerase chain reaction (RT-qPCR, Western Blot) were assessed. Results: NaIO3 retinal damage was diminished in CIA mice treated with MS-275 (P ≤ 0.05). While no significant difference was observed in retinal pigment epithelium (RPE) function, a trend in increased c-wave amplitude was detected in CIA + NaIO3 mice treated with MS-275. Finally, we identified decreased Hdac1, Hdac3, and Cxcl9 expression in CIA + NaIO3 mouse RPE/choroid when treated with MS-275 (P ≤ 0.05). Conclusions: Our data demonstrate that HDAC inhibition can reduce the additive effect of NaIO3-induced retinal degeneration in the presence of systemic inflammation by CIA as measured by OCT analysis. In addition, HDAC inhibition in CIA + NaIO3 treated mice resulted in reduced cytokine production. These findings are highly innovative and provide additional support to the therapeutic potential of HDAC inhibitors for dry AMD treatment.
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Modelos Animales de Enfermedad , Histona Desacetilasa 1 , Inhibidores de Histona Desacetilasas , Inflamación , Yodatos , Ratones Endogámicos C57BL , Piridinas , Tomografía de Coherencia Óptica , Animales , Ratones , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/uso terapéutico , Inflamación/tratamiento farmacológico , Yodatos/administración & dosificación , Yodatos/toxicidad , Piridinas/farmacología , Piridinas/administración & dosificación , Piridinas/uso terapéutico , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/metabolismo , Benzamidas/farmacología , Benzamidas/administración & dosificación , Benzamidas/uso terapéutico , Histona Desacetilasas/metabolismo , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/patología , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/patología , Masculino , Electrorretinografía , Compuestos de Piridinio/farmacología , Compuestos de Piridinio/administración & dosificación , Atrofia Geográfica/tratamiento farmacológicoRESUMEN
Purpose: Age-related macular degeneration (AMD) shares similar risk factors and inflammatory responses with rheumatoid arthritis (RA). Previously, we identified increased risk for dry AMD among patients with RA compared to control subjects, using retrospective data analysis. In this current study, we investigate the role of systemic inflammation triggered in a murine model of arthritis on choroidal neovascularization and retinal pigment epithelium (RPE) degeneration mouse models. Methods: Collagen-induced arthritis (CIA) was induced in C57BL/6J mice prior to laser-induced choroidal neovascularization (CNV; wet AMD model) or sodium iodate-induced retinal degeneration (NaIO3; dry AMD model). CNV lesion size and retinal thickness were quantified by optical coherence photography (OCT), visual function was analyzed using optokinetic response and electroretinography, RPE morphology was examined by immunohistochemistry, and inflammatory gene expression was analyzed by quantitative PCR. Results: CIA mice demonstrated decreased spatial acuity and contrast sensitivity, whereas no difference was observed in the RPE-generated c-wave. CNV lesion size was decreased in CIA mice. NaIO3 decreased c-wave amplitude, as well as retinal thickness, which was augmented by CIA. NaIO3 treatment resulted in loss of normal RPE hexagonal shape, which was further aggravated by CIA. Increased Cxcl9 expression was observed in the presence of CIA and CIA combined with AMD. Disease severity differences were observed between sexes. Conclusions: Our data suggest systemic inflammation by CIA results in increased pathology in a dry AMD model, whereas it reduces lesions in a wet AMD model. These findings highlight the need for additional investigation into the role of secondary inflammation and sex-based differences on AMD.
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Inflamación/complicaciones , Degeneración Macular/etiología , Animales , Artritis Experimental/complicaciones , Neovascularización Coroidal/etiología , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Femenino , Inflamación/etiología , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia ÓpticaRESUMEN
Prevalence of age-related macular degeneration (AMD), the leading cause of blindness in the United States, increases greatly with age. While age is the greatest risk factor of AMD, other risks such as smoking, family history, complement pathway activation, and race exist. Various systemic inflammatory diseases share many of these risk factors with AMD, as well as a similar inflammatory profile. Due to these similarities, patient database studies analyzing the association between patients with various systemic diseases and AMD have been performed. In this review we will summarize recent finding on this subject and discuss the implications on AMD diagnosis. By gaining greater insight into the association between chronic systemic inflammation and AMD, implications for novel therapeutic approaches to treat AMD pathology may be identified.
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Inflamación/complicaciones , Degeneración Macular/complicaciones , Comorbilidad , Humanos , Prevalencia , Factores de RiesgoRESUMEN
Purpose: AMD is the leading cause of blindness in the United States. The role of secondary inflammatory disease on AMD progression is largely unknown. Here we investigate the association between AMD and rheumatoid arthritis (RA), using MarketScan data for patients aged ≥65 years on Medicare. Methods: Baseline data were extracted for subjects with at least two International Classification, Ninth Revision (ICD-9) diagnosis codes of RA and control subjects (no RA) and were matched at baseline by propensity score. Matched cohort data were extracted post-baseline time and examined up to 4.5 years of follow-up for ICD-9 diagnosis code AMD records. Multivariable regression models compared risk of an AMD diagnosis post-baseline for RA subjects and matched controls. Days until first AMD diagnosis between RA patients and controls was examined using survival analysis. Results: Risk of new AMD diagnosis was elevated in RA patients (odds ratio [OR] 2.08; 95% confidence interval [CI] 1.98-2.18), with an increase in nonexudative AMD patients (P < 0.0001). Risk was elevated in female (n = 27,548) (OR 1.11; 95% CI 1.05-1.17) compared with male (n = 9704; P < 0.001) patients. The time to first AMD diagnosis was shorter for RA subjects than controls (P < 0.0001). Conclusions: Our analysis provides support of association between RA diagnosis and increased nonexudative AMD diagnosis.
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Artritis Reumatoide/epidemiología , Atrofia Geográfica/epidemiología , Medicare/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Antirreumáticos/uso terapéutico , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/tratamiento farmacológico , Bases de Datos Factuales , Exudados y Transudados , Femenino , Estudios de Seguimiento , Atrofia Geográfica/diagnóstico , Humanos , Clasificación Internacional de Enfermedades , Masculino , Estudios Retrospectivos , Encuestas y Cuestionarios , Análisis de Supervivencia , Estados Unidos/epidemiologíaRESUMEN
Age-related macular degeneration (AMD) is the leading cause of blindness in the US. Polymorphisms in complement components are associated with increased AMD risk, and it has been hypothesized that an overactive complement system is partially responsible for AMD pathology. Choroidal neovascularization (CNV) has two phases, injury/angiogenesis and repair/fibrosis. Complement activation has been shown to be involved in the angiogenesis phase of murine CNV, but has not been investigated during repair. Anaphylatoxin (C3a and C5a) signaling in particular has been shown to be involved in both tissue injury and repair in other models. CNV was triggered by laser-induced photocoagulation in C57BL/6 J mice, and lesion sizes measured by optical coherence tomography. Alternative pathway (AP) activation or C3a-receptor (C3aR) and C5a-receptor (C5aR) engagement was inhibited during the repair phase only of CNV with the AP-inhibitor CR2-fH, a C3aR antagonist (N2-[(2,2-diphenylethoxy)acetyl]-l-arginine, TFA), or a C5a blocking antibody (CLS026), respectively. Repair after CNV was also investigated in C3aR/C5aR double knockout mice. CR2-fH treatment normalized anaphylatoxin levels in the eye and accelerated regression of CNV lesions. In contrast, blockade of anaphylatoxin-receptor signaling pharmacologically or genetically did not significantly alter the course of lesion repair. These results suggest that continued complement activation prevents fibrotic scar resolution, and emphasizes the importance of reducing anaphylatoxins to homeostatic levels. This duality of complement, playing a role in injury and repair, will need to be considered when selecting a complement inhibitory strategy for AMD.
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Neovascularización Coroidal/inmunología , Vía Alternativa del Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Receptor de Anafilatoxina C5a/inmunología , Receptores de Complemento/inmunología , Regeneración/inmunología , Animales , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Vía Alternativa del Complemento/genética , Proteínas del Sistema Complemento/genética , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Receptor de Anafilatoxina C5a/genética , Receptores de Complemento/genética , Regeneración/genéticaRESUMEN
Complement activation plays a significant role in age-related macular degeneration (AMD) pathogenesis, and polymorphisms interfering with factor H (fH) function, a complement alternative pathway (AP) inhibitor, are associated with increased AMD risk. We have previously validated an AP inhibitor, a fusion protein consisting of a complement receptor 2 fragment linked to the inhibitory domain of fH (CR2-fH) as an efficacious treatment for choroidal neovascularization (CNV) when delivered intravenously. Here we tested an alternative approach of AAV-mediated delivery (AAV5-VMD2-CR2-fH or AAV5-VMD2-mCherry) using subretinal delivery in C57BL/6J mice. Secretion of CR2-fH was confirmed in polarized retinal pigment epithelium (RPE) cells. A safe concentration of AAV5-VMD2-CR2-fH was identified using electroretinography, optical coherence tomography (OCT), RPE morphology, and antibody profiling. One month after gene delivery, CNV was induced using argon laser photocoagulation. OCT assessment demonstrated reduced CNV with AAV5-VMD2-CR2-fH administration. Bioavailability studies revealed that gene-therapy delivered similar levels of CR2-fH to the RPE/choroid as treatment by intravenous injections, and C3a ELISA verified reduced CNV-associated ocular C3a production. These results contribute to existing data illustrating the importance of the AP of complement in CNV development and its potential role in AMD treatment. Demonstration of AAV-vector efficacy opens new avenues for the development of treatment strategies.
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PURPOSE: The membrane attack complex (MAC) in choriocapillaris (CC) and retinal pigment epithelium (RPE) increase with age and disease (age-related macular degeneration). MAC assembly can be inhibited by CD59, a membrane-bound regulator. Here we further investigated the role of CD59 in murine choroidal neovascularization (CNV), a model involving both CC and RPE, and tested whether CR2-CD59, a soluble targeted form of CD59, provides protection. METHODS: Laser-induced CNV was generated in wild type and CD59a-deficient mice (CD59-/-). CNV size was measured by optical coherence tomography, and CR2-CD59 was injected intraperitoneally. Endogenous CD59 localization and MAC deposition were identified by immunohistochemistry and quantified by confocal microscopy. Cell-type-specific responses to MAC were examined in retinal pigment epithelial cells (ARPE-19) and microvascular endothelial cells (HMEC-1). RESULTS: CD59 levels were severely reduced and protein was mislocalized in the RPE surrounding the lesion. CNV lesion size and subretinal fluid accumulation were exacerbated in CD59-/- when compared with those in WT mice, and an increase in MAC deposition was noted. In contrast, CR2-CD59 significantly reduced both structural features of CNV severity. In vitro, MAC inhibition in ARPE-19 cells prevented barrier function loss and accelerated wound healing and cell adhesion, whereas in HMEC-1 cells, CR2-CD59 decelerated wound healing and cell adhesion. CONCLUSION: These data further support the importance of CD59 in controlling ocular injury responses and indicate that pharmacological inhibition of the MAC with CR2-CD59 may be a viable therapeutic approach for reducing complement-mediated ocular pathology.
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Antígenos CD59/metabolismo , Neovascularización Coroidal/tratamiento farmacológico , Inactivadores del Complemento/farmacología , Células Endoteliales/patología , Rayos Láser , Receptores de Complemento 3d/metabolismo , Animales , Antígenos CD59/administración & dosificación , Antígenos CD59/deficiencia , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Inactivadores del Complemento/administración & dosificación , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Complemento 3d/administración & dosificación , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
PURPOSE: Morphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP. METHODS: Mice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR. RESULTS: The CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM. CONCLUSIONS: Taken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD.
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Vía Alternativa del Complemento/genética , Regulación de la Expresión Génica , Degeneración Macular/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Recuperación de la Función , Retina/fisiopatología , Humo/efectos adversos , Animales , Vía Alternativa del Complemento/efectos de los fármacos , ADN/genética , Modelos Animales de Enfermedad , Electrorretinografía , Degeneración Macular/inducido químicamente , Degeneración Macular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Retina/ultraestructura , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Agudeza Visual/fisiologíaRESUMEN
Neovascular age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). An overactive complement system is associated with AMD pathogenesis, and serum pro-inflammatory cytokines, including IL-17, are elevated in AMD patients. IL-17 is produced by complement C5a-receptor-expressing T-cells. In murine CNV, infiltrating γδT- rather than Th17-cells produce the IL-17 measurable in lesioned eyes. Here we asked whether C5a generated locally in response to CNV recruits IL-17-producing T-cells to the eye. CNV lesions were generated using laser photocoagulation and quantified by imaging; T-lymphocytes were characterized by QRT-PCR. CNV resulted in an increase in splenic IL-17-producing γδT- and Th17-cells; yet in the CNV eye, only elevated levels of γδT-cells were observed. Systemic administration of anti-C5- or anti-C5a-blocking antibodies blunted the CNV-induced production of splenic Th17- and γδT-cells, reduced CNV size and eliminated ocular γδT-cell infiltration. In ARPE-19 cell monolayers, IL-17 triggered a pro-inflammatory state; and splenocyte proliferation was elevated in response to ocular proteins. Thus, we demonstrated that CNV lesions trigger a systemic immune response, augmenting local ocular inflammation via the infiltration of IL-17-producing γδT-cells, which are presumably recruited to the eye in a C5a-dependent manner. Understanding the complexity of complement-mediated pathological mechanisms will aid in the development of an AMD treatment.
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Linfocitos T CD8-positivos/inmunología , Coroides/inmunología , Neovascularización Coroidal/inmunología , Complemento C5a/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Células Th17/inmunología , Inmunidad Adaptativa , Animales , Anticuerpos Neutralizantes/farmacología , Linfocitos T CD8-positivos/patología , Línea Celular , Movimiento Celular/efectos de los fármacos , Coroides/patología , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/etiología , Neovascularización Coroidal/genética , Complemento C5a/antagonistas & inhibidores , Expresión Génica , Humanos , Inmunidad Innata , Inyecciones Intravenosas , Interleucina-17/genética , Interleucina-17/inmunología , Fotocoagulación/efectos adversos , Ratones , Ratones Endogámicos C57BL , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/inmunología , Bazo/inmunología , Bazo/patología , Células Th17/patologíaRESUMEN
PURPOSE: Complement factor B (CFB) is a required component of the alternative pathway (AP) of complement, and CFB polymorphisms are associated with age-related macular degeneration (AMD) risk. Complement factor B is made in the liver, but expression has also been detected in retina and retinal pigment epithelium (RPE)-choroid. We investigated whether production of CFB by the RPE can promote AP activation in mouse choroidal neovascularization (CNV). METHODS: Transgenic mice expressing CFB under the RPE65 promoter were generated and crossed onto factor B-deficient (CFB-KO) mice. Biological activity was determined in vitro using RPE monolayers and in vivo using laser-induced CNV. Contribution of systemic CFB was investigated using CFB-KO reconstituted with CFB-sufficient serum. RESULTS: Transgenic mice (CFB-tg) expressed CFB in RPE-choroid; no CFB was detected in serum. Cultured CFB-tg RPE monolayers secreted CFB apically and basally upon exposure to oxidative stress that was biologically active. Choroidal neovascularization sizes were comparable between wild-type and CFB-tg mice, but significantly increased when compared to lesions in CFB-KO mice. Injections of CFB-sufficient serum into CFB-KO mice resulted in partial reconstitution of systemic AP activity and significantly increased CNV size. CONCLUSIONS: Mouse RPE cells express and secrete CFB sufficient to promote RPE damage and CNV. This further supports that local complement production may regulate disease processes; however, the reconstitution experiments suggest that additional components may be sequestered from the bloodstream. Understanding the process of ocular complement production and regulation will further our understanding of the AMD disease process and the requirements of a complement-based therapeutic.
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Coroides/patología , Neovascularización Coroidal/genética , Factor B del Complemento/genética , Vía Alternativa del Complemento/genética , Regulación de la Expresión Génica , ARN Mensajero/genética , Animales , Western Blotting , Células Cultivadas , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Factor B del Complemento/biosíntesis , Modelos Animales de Enfermedad , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Rayos Láser/efectos adversos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/fisiopatología , Tomografía de Coherencia ÓpticaRESUMEN
Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly. To study potential underlying mechanisms of AMD, animal models are utilized, focusing mostly on mice. Recently, genomic and phenotypic differences between the so-called control substrains, C57BL/6J and C57BL/6N, have been described in models of ocular and non-ocular diseases. In particular, the rd8 mutation of the Crb1 gene present in the C57BL/6N has been shown to impact certain ocular phenotypes and appears to augment phenotypes generally associated with inflammation. Here, we investigated angiogenic factor and cytokine expression using pathway arrays as well as the susceptibility to laser-induced choroidal neovascularization (CNV), a model of wet AMD, in the two substrains. Age-matched 3-month-old C57BL/6J and C57BL/6N animals differed in gene expression levels for angiogenic factors and cytokines, with 6N animals expressing higher levels of inflammatory markers than 6Js. Yet laser-induced CNV was comparable in size between the two substrains. This lack of difference in CNV size was correlated with a gene expression profile that was comparable between the two substrains, due to the fact that the degree of change in gene expression of inflammatory markers after CNV was blunted in 6N mice. In summary, significant gene expression differences exist between C57BL/6J and C57BL/6N animals, reinforcing the notion that appropriate litter-mate controls or genetic background controls need to be used. Contrary to our expectation, CNV was not augmented in 6N animals, suggesting that low chronic inflammation in the RPE might provide a level of pre-conditioning and protection against stress.
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Quimiocinas/genética , Neovascularización Coroidal/genética , Regulación de la Expresión Génica , ARN/genética , Animales , Quimiocinas/biosíntesis , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Rayos Láser/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
Complement activation is associated with age-related macular degeneration (AMD), with the retinal pigment epithelium (RPE) being one of the main target tissues. In AMD, disease severity is correlated with the formation of the membrane attack complex (MAC), the terminal step in the complement cascade, as well as diminished RPE expression of CD59, a membrane-bound regulatory protein of MAC formation. This has prompted the search for therapeutic strategies based on MAC inhibition, and soluble forms of CD59 (sCD59) have been investigated in mouse laser-induced choroidal neovascularization, a model for "wet" AMD. Unlike membrane-bound CD59, sCD59 provides relatively poor cell protection from complement, and different strategies to increase sCD59 activity at the cell membrane level have been investigated. These include increasing the circulatory half-life of sCD59 by the addition of an Fc moiety; increasing the half-life of sCD59 in target tissues by modifying CD59 with a (non-specific) membrane-targeting domain; and by locally overexpressing sCD59 via adenoviral vectors. Finally, a different strategy currently under investigation employs complement receptor (CR)2-mediated targeting of CD59 exclusively to membranes under complement attack. CR2 recognizes long-lasting membrane-bound breakdown activation fragments of complement C3. CR2-CD59 may have greater therapeutic potential than other complement inhibitory approaches, since it can be administered either systemically or locally, it will bind specifically to membranes containing activated complement activation fragments, and dosing can be regulated. Hence, this strategy might offer opportunities for site-specific inhibition of complement in diseases with restricted sites of inflammation such as AMD.
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Antígenos CD59/metabolismo , Neovascularización Coroidal/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Degeneración Macular/metabolismo , Animales , Antígenos CD59/genética , Antígenos CD59/inmunología , Neovascularización Coroidal/inmunología , Neovascularización Coroidal/terapia , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Modelos Animales de Enfermedad , Terapia Genética/métodos , Degeneración Macular/inmunología , Degeneración Macular/terapia , RatonesRESUMEN
Organic anion transporter 6 (Oat6; Slc22a20), a member of the OAT family, was demonstrated previously to mediate the transport of organic anions (Am J Physiol Renal Physiol 291:F314-F321, 2006). In the present study, we sought to further delineate the function of murine Oat6 (mOat6) by analyzing the effect of select organic anions on mOat6-mediated transport by using a Chinese hamster ovary (CHO) cell line stably expressing mOat6 (CHO-mOat6). When examined, kinetic analysis demonstrated that the mechanism of inhibition of mOat6 and mOat3 was competitive. Homovanillic acid, 5-hydroxyindole acetic acid, 2,4-dihydroxyphenylacetate, hippurate, and dehydroepiandrosterone sulfate (DHEAS) each significantly reduced mOat6 activity with inhibitory constant (K(i)) values of 3.0 +/- 0.5, 48.9 +/- 10.3, 61.4 +/- 7.1, 59.9 +/- 4.9, and 38.8 +/- 3.1 microM, respectively. Comparison to K(i) values determined for mOat3 (67.8 +/- 7.2, 134.5 +/- 27.0, 346.7 +/- 97.9, 79.3 +/- 4.0, and 3.8 +/- 1.1 microM, respectively) revealed that there are significant differences in compound affinity between each transporter. Fluoroquinolone antimicrobials and reduced folates were without effect on mOat6-mediated uptake. Investigation of testicular cell type-specific expression of mOat6 by laser capture microdissection and quantitative polymerase chain reaction revealed significant mRNA expression in Sertoli cells, but not in Leydig cells or spermatids. Overall, these data should aid further refinements to the interpretation and modeling of the in vivo disposition of OAT substrates. Specifically, expression in Sertoli cells suggests Oat6 may be an important determinant of blood-testis barrier function, with Oat6-mediated transport of estrone sulfate and DHEAS possibly representing a critical step in the maintenance of testicular steroidogenesis.
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Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Células de Sertoli/metabolismo , Animales , Barrera Hematotesticular/fisiología , Células CHO , Cricetinae , Cricetulus , Sulfato de Deshidroepiandrosterona/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Cinética , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Microdisección , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/efectos de los fármacos , Espermátides/metabolismo , Especificidad por SustratoRESUMEN
Recently, a novel Slc22 gene family member expressed in murine olfactory mucosa was identified and based on sequence homology proposed to be an organic anion transporter [Oat6 (Slc22a20); J. C. Monte, M. A. Nagle, S. A. Eraly, and S. K. Nigam. Biochem Biophys Res Commun 323: 429-436, 2004]. However, no functional data for Oat6 was reported. In the present study, we demonstrate that murine Oat6 mediates the inhibitable transport of estrone sulfate using both Xenopus oocyte expression assay and Chinese hamster ovary (CHO) cells stably transfected with mOat6 (CHO-mOat6). Uptake was virtually eliminated by probenecid and the anionic herbicide 2,4-dichlorophenoxyacetate. The organic anions ochratoxin A, salicylate, penicillin G, p-aminohippurate, and urate inhibited mOat6-mediated accumulation to varying degrees. Transport of estrone sulfate by mOat6 was demonstrated to be saturable, and K(m) estimates of 109.8 +/- 22.6 microM in oocytes and 44.8 +/- 7.3 microM in CHO-mOat6 cells were obtained. Inhibitory constants for 2,4-dichlorophenoxyacetate (15.7 +/- 2.0 microM), salicylate (49.0 +/- 4.4 microM), probenecid (8.3 +/- 2.5 microM), and penicillin G (1,450 +/- 480 microM) were also determined. Accumulation of estrone sulfate mediated by mOat6 was significantly trans-stimulated by glutarate, indicating that mOat6 functions as an organic anion/dicarboxylate exchanger. These data demonstrate for the first time that the novel murine gene Oat6 (Slc22a20) encodes a functional organic anion transporter and mOat6 is indeed the newest member of the OAT gene family.