Expression and activation of the ketone body receptor HCAR2/GPR109A promotes preservation of retinal endothelial cell barrier function.

Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.

Protective effects of agonists of growth hormone-releasing hormone (GHRH) in early experimental diabetic retinopathy.

The potential therapeutic effects of agonistic analogs of growth hormone-releasing hormone (GHRH) and their mechanism of action were investigated in diabetic retinopathy (DR). Streptozotocin-induced diabetic rats (STZ-rats) were treated with 15 µg/kg GHRH agonist, MR-409, or GHRH antagonist, MIA-602. At the end of treatment, morphological and biochemical analyses assessed the effects of these compounds on retinal neurovascular injury induced by hyperglycemia. The expression levels of GHRH and its receptor (GHRH-R) measured by qPCR and Western blotting were significantly down-regulated in retinas of STZ-rats and in human diabetic retinas (postmortem) compared with their respective controls. Treatment of STZ-rats with the GHRH agonist, MR-409, prevented retinal morphological alteration induced by hyperglycemia, particularly preserving survival of retinal ganglion cells. The reverse, using the GHRH antagonist, MIA-602, resulted in worsening of retinal morphology and a significant alteration of the outer retinal layer. Explaining these results, we have found that MR-409 exerted antioxidant and anti-inflammatory effects in retinas of the treated rats, as shown by up-regulation of NRF-2-dependent gene expression and down-regulation of proinflammatory cytokines and adhesion molecules. MR-409 also significantly down-regulated the expression of vascular endothelial growth factor while increasing that of pigment epithelium-derived factor in diabetic retinas. These effects correlated with decreased vascular permeability. In summary, our findings suggest a neurovascular protective effect of GHRH analogs during the early stage of diabetic retinopathy through their antioxidant and anti-inflammatory properties.

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Regulation of the cholesterol efflux transporters ABCA1 and ABCG1 in retina in hemochromatosis and by the endogenous siderophore 2,5-dihydroxybenzoic acid.

Hypercholesterolemia and polymorphisms in the cholesterol exporter ABCA1 are linked to age-related macular degeneration (AMD). Excessive iron in retina also has a link to AMD pathogenesis. Whether these findings mean a biological/molecular connection between iron and cholesterol is not known. Here we examined the relationship between retinal iron and cholesterol using a mouse model (Hfe(-/-)) of hemochromatosis, a genetic disorder of iron overload. We compared the expression of the cholesterol efflux transporters ABCA1 and ABCG1 and cholesterol content in wild type and Hfe(-/-) mouse retinas. We also investigated the expression of Bdh2, the rate-limiting enzyme in the synthesis of the endogenous siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA) in wild type and Hfe(-/-) mouse retinas, and the influence of this siderophore on ABCA1/ABCG1 expression in retinal pigment epithelium. We found that ABCA1 and ABCG1 were expressed in all retinal cell types, and that their expression was decreased in Hfe(-/-) retina. This was accompanied with an increase in retinal cholesterol content. Bdh2 was also expressed in all retinal cell types, and its expression was decreased in hemochromatosis. In ARPE-19 cells, 2,5-DHBA increased ABCA1/ABCG1 expression and decreased cholesterol content. This was not due to depletion of free iron because 2,5-DHBA (a siderophore) and deferiprone (an iron chelator) had opposite effects on transferrin receptor expression and ferritin levels. We conclude that iron is a regulator of cholesterol homeostasis in retina and that removal of cholesterol from retinal cells is impaired in hemochromatosis. Since excessive cholesterol is pro-inflammatory, hemochromatosis might promote retinal inflammation via cholesterol in AMD.

Retinal expression of the serine protease matriptase-2 (Tmprss6) and its role in retinal iron homeostasis.

PURPOSE: Matriptase-2 (also known as TMPRSS6) is a critical regulator of the iron-regulatory hormone hepcidin in the liver; matriptase-2 cleaves membrane-bound hemojuvelin and consequently alters bone morphogenetic protein (BMP) signaling. Hemojuvelin and hepcidin are expressed in the retina and play a critical role in retinal iron homeostasis. However, no information on the expression and function of matriptase-2 in the retina is available. The purpose of the present study was to examine the retinal expression of matriptase-2 and its role in retinal iron homeostasis. METHODS: RT-PCR, quantitative PCR (qPCR), and immunofluorescence were used to analyze the expression of matriptase-2 and other iron-regulatory proteins in the mouse retina. Polarized localization of matriptase-2 in the RPE was evaluated using markers for the apical and basolateral membranes. Morphometric analysis of retinas from wild-type and matriptase-2 knockout (Tmprss6(msk/msk) ) mice was also performed. Retinal iron status in Tmprss6(msk/msk) mice was evaluated by comparing the expression levels of ferritin and transferrin receptor 1 between wild-type and knockout mice. BMP signaling was monitored by the phosphorylation status of Smads1/5/8 and expression levels of Id1 while interleukin-6 signaling was monitored by the phosphorylation status of STAT3. RESULTS: Matriptase-2 is expressed in the mouse retina with expression detectable in all retinal cell types. Expression of matriptase-2 is restricted to the apical membrane in the RPE where hemojuvelin, the substrate for matriptase-2, is also present. There is no marked difference in retinal morphology between wild-type mice and Tmprss6(msk/msk) mice, except minor differences in specific retinal layers. The knockout mouse retina is iron-deficient, demonstrable by downregulation of the iron-storage protein ferritin and upregulation of transferrin receptor 1 involved in iron uptake. Hepcidin is upregulated in Tmprss6(msk/msk) mouse retinas, particularly in the neural retina. BMP signaling is downregulated while interleukin-6 signaling is upregulated in Tmprss6(msk/msk) mouse retinas, suggesting that the upregulaton of hepcidin in knockout mouse retinas occurs through interleukin-6 signaling and not through BMP signaling. CONCLUSIONS: The iron-regulatory serine protease matriptase-2 is expressed in the retina, and absence of this enzyme leads to iron deficiency and increased expression of hemojuvelin and hepcidin in the retina. The upregulation of hepcidin expression in Tmprss6(msk/msk) mouse retinas does not occur via BMP signaling but likely via the proinflammatory cytokine interleukin-6. We conclude that matriptase-2 is a critical participant in retinal iron homeostasis.

L-2-oxothiazolidine-4-carboxylic acid attenuates oxidative stress and inflammation in retinal pigment epithelium.

PURPOSE: Oxidant- and inflammation-induced damage to retinal pigment epithelial (RPE) cells is central to the pathogenesis of age-related macular degeneration (AMD). Thus, developing novel strategies to protect these cells is important. We reported previously on the robust antioxidant and therefore cell-protective effects of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) in cultured human RPE cells. New reports citing a novel anti-inflammatory role for OTC in addition to the known glutathione-stimulating and antioxidant properties emerged recently; however, this role has not been evaluated in RPE cells or in intact retina. Given the crucial causative roles of oxidative stress and inflammation in AMD pathogenesis, knowing whether OTC might exhibit a similar benefit in this cell and tissue type has high clinical relevance; thus, we evaluated OTC in the present study. METHODS: ARPE-19 and primary RPE cells isolated from wild-type, Gpr109a(-/-) , or Slc5a8(-/-) mouse eyes were exposed to TNF-α in the presence or absence of OTC, followed by analysis of IL-6 and Ccl2 expression with real-time quantitative polymerase chain reaction or enzyme-linked immunosorbent assay. Cellular and molecular markers of inflammation and oxidative stress (i.e., IL-1ß, TGF-ß, ABCG1, ABCA1, reduced glutathione, and dihydroethidium) were evaluated in Ccl2(-/-)/Cx3cr1(-/-) double knockout mice on rd8 background (DKO rd8) treated with OTC (10 mg/ml) in drinking water for a period of 5 months. RESULTS: OTC treatment significantly inhibited the expression and secretion of IL-6 and Ccl2 in TNF-α-stimulated ARPE-19 cells. Studies conducted using DKO rd8 animals treated with OTC in drinking water confirmed these findings. Cellular and molecular markers of inflammation were significantly suppressed in the retinas of the OTC-treated DKO rd8 animals. Subsequent in vitro and in vivo studies of the possible mechanism(s) to explain these actions revealed that although OTC is an agonist of the anti-inflammatory G-protein coupled receptor GPR109A and a transportable substrate of the sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), these properties may play a role but do not explain entirely the anti-inflammatory effects this compound elicits in cultured RPE cells and the intact mouse retina. CONCLUSIONS: This study represents, to our knowledge, the first report of the suppressive effects of OTC on inflammation in cultured RPE cells and on inflammation and oxidative stress in the retina in vivo.

Iron-mediated retinal degeneration in haemojuvelin-knockout mice.

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv-/- mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-/- mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv-/- mice and examined their growth patterns. Hjv-/- RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-/- RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the 'transporter proper' subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv-/- RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.

Regulation of proton-coupled folate transporter in retinal Müller cells by the antipsoriatic drug monomethylfumarate.

Fumaric acid esters are used to treat psoriasis, an inflammatory skin disease characterized by keratinocyte proliferation. Inflammation and proliferation are hallmarks of retinal disease; hence, fumaric acid esters may have therapeutic value in retinal pathology. In diseased retinas, Müller glial cells (MCs) undergo reactive gliosis, a hyperproliferative state. MCs take up folate, a vitamin necessary for cell proliferation, via the proton-coupled folate transporter (PCFT). Here we examined the effect of monomethylfumarate (MMF), the active metabolite of fumaric acid esters, on expression and function of PCFT in MCs. Primary MCs, isolated from neonatal mouse retinas, were treated with MMF, and PCFT function was monitored by measuring uptake of radiolabeled methyltetrahydrofolate (MTF) at pH 5.5. Dose-response and time-course analyses were performed to identify optimal conditions for maximal effect. The influence of MMF treatment on kinetic parameters of PCFT was studied, and PCFT expression was analyzed at the mRNA and protein level. MTF uptake in MCs decreased by ˜50% following 18 h treatment with 1 mM MMF. This effect was specific to fumaric acid esters. MMF treatment decreased the maximal velocity of the transporter without altering substrate affinity. The decrease in PCFT function following MMF treatment was accompanied by attenuated PCFT expression. This is the first report that an antipsoriatic compound can regulate folate transport in MCs and may have potential for the treatment of reactive gliosis in retinal disease.

Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells.

Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Fas(lpr)) or FasL-deficient (Fas(gld)) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation.

Autonomous regulation of retinal insulin biosynthesis in diabetes.

Diabetic retinopathy (DR) is a neurodegenerative disease that results as a complication of dysregulated glucose metabolism, or diabetes. The signaling of insulin is lost or dampened in diabetes, but this hormone has also been shown to be an important neurotrophic factor which supports neurons of the brain. The role of local insulin synthesis and secretion in the retina, however, is unclear. We have investigated whether changes in local insulin synthesis occur in the diabetic retina and in response to stressors known to initiate retinal neurodegenerative processes. The expression of insulin and its cleavage product, c-peptide, were examined in retinas of a Type I diabetes animal model and human postmortem donors with DR. We detected mRNAs for insulin I (Ins1), insulin II (Ins2) and human insulin (Ins) by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Using an ex-vivo system, isolated neuroretinas and retinal pigmented epithelium (RPE) layers were exposed to glycemic, oxidative and inflammatory environments to measure insulin gene transcripts produced de novo in the retina under disease-relevant conditions. The expression of insulin in the retina was altered with the progression of diabetes in STZ mice and donors with DR. Transcription factors for insulin, were simultaneously expressed in a pattern matching insulin genes. Furthermore, de novo insulin mRNA in isolated retinas was induced by acute stress. RPE explants displayed the most pronounced changes in Ins1 and Ins2. This data reveals that the retina, like the brain, is an organ capable of producing local insulin and this synthesis is altered in diabetes.

Blockade of dendritic cell development by bacterial fermentation products butyrate and propionate through a transporter (Slc5a8)-dependent inhibition of histone deacetylases.

Mammalian colon harbors trillions of bacteria, yet there is no undue inflammatory response by the host against these bacteria under normal conditions. The bacterial fermentation products acetate, propionate, and butyrate are believed, at least in part, to be responsible for these immunosuppressive effects. Dendritic cells play an essential role in presentation of antigens to T lymphocytes and initiation of adaptive immune responses. Here we report that butyrate and propionate block the generation of dendritic cells from bone marrow stem cells, without affecting the generation of granulocytes. This effect is dependent on the Na(+)-coupled monocarboxylate transporter Slc5a8, which transports butyrate and propionate into cells, and on the ability of these two bacterial metabolites to inhibit histone deacetylases. Acetate, which is also a substrate for Slc5a8 but not an inhibitor of histone deacetylases, does not affect dendritic cell development, indicating the essential role of histone deacetylase inhibition in the process. The blockade of dendritic cell development by butyrate and propionate is associated with decreased expression of the transcription factors PU.1 and RelB. Butyrate also elicits its biologic effects through its ability to activate the G-protein-coupled receptor Gpr109a, but this mechanism is not involved in butyrate-induced blockade of dendritic cell development. The participation of Slc5a8 and the non-involvement of Gpr109a in butyrate effects have been substantiated using bone marrow cells obtained from Slc5a8(-/-) and Gpr109a(-/-) mice. These findings uncover an important mechanism underlying the anti-inflammatory functions of the bacterial fermentation products butyrate and propionate.

Transport of hepcidin, an iron-regulatory peptide hormone, into retinal pigment epithelial cells via oligopeptide transporters and its relevance to iron homeostasis.

Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC50 values in the range of 0.4-1.7 µM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.

Retinal transfer of nicotinate by H+ -monocarboxylate transporter at the inner blood-retinal barrier.

Nicotinic acid is a constituent of the coenzymes NAD and NADP. It also serves as an agonist for the G-protein-coupled receptor GPR109A. Nicotinic acid is widely used at high doses as a lipid-lowering drug, which is associated with an ocular side effect known as niacin maculopathy. Here we investigated the mechanism by which nicotinate is transferred into retina across the inner blood-retinal barrier (BRB). In vivo the blood-to-retina transport of [(3)H]-nicotinate was studied using the carotid artery injection technique. The characteristics of nicotinate transport at the inner BRB were examined in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), an in vitro model of inner BRB. The expression of transporters in TR-iBRB2 cells was determined by reverse transcription-polymerase chain reaction. In vivo [(3)H]-nicotinate uptake by the retina was 5.4-fold greater than that of [(14)C]-sucrose, a BRB impermeable vascular space marker. Excess amounts of unlabeled nicotinate and salicylate significantly decreased the in vivo retinal uptake of [(3)H]-nicotinate. [(3)H]-Nicotinate was taken up by TR-iBRB2 cells via an H(+)-dependent saturable process with a Michaelis constant of ~7 mM. Na(+) had minimal effect on the uptake. The H(+)-dependent uptake was significantly inhibited by endogenous monocarboxylates such as lactate and pyruvate, and monocarboxylic drugs such as valproate, salicylate, and ibuprofen. These characteristics are consistent with those of H(+)-coupled monocarboxylate transporters (MCTs). MCT1, MCT2, and MCT4 mRNAs were expressed in TR-iBRB2 cells. The Na(+)-dependent monocarboxylate transporters SMCT1 and SMCT2 were not expressed in these cells. In conclusion, transfer of nicotinate from blood to retina across the inner BRB occurs primarily via H(+)-coupled monocarboxylate transporters.

Oxidative Stress and Inflammation in Retinal Degeneration.

Inflammation and oxidative stress play prominent roles in the pathogenesis of many degenerative diseases of the retina, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinal vein occlusion, and retinitis pigmentosa [...].

Pharmacological and Metabolic Significance of Bile Acids in Retinal Diseases.

Bile acids (BAs) are amphipathic sterols primarily synthesized from cholesterol in the liver and released in the intestinal lumen upon food intake. BAs play important roles in micellination of dietary lipids, stimulating bile flow, promoting biliary phospholipid secretion, and regulating cholesterol synthesis and elimination. Emerging evidence, however, suggests that, aside from their conventional biological function, BAs are also important signaling molecules and therapeutic tools. In the last decade, the therapeutic applications of BAs in the treatment of ocular diseases have gained great interest. Despite the identification of BA synthesis, metabolism, and recycling in ocular tissues, much remains unknown with regards to their biological significance in the eye. Additionally, as gut microbiota directly affects the quality of circulating BAs, their analysis could derive important information on changes occurring in this microenvironment. This review aims at providing an overview of BA metabolism and biological function with a focus on their potential therapeutic and diagnostic use for retinal diseases.

Exacerbation of AMD Phenotype in Lasered CNV Murine Model by Dysbiotic Oral Pathogens.

Emerging evidence underscores an association between age-related macular degeneration (AMD) and periodontal disease (PD), yet the biological basis of this linkage and the specific role of oral dysbiosis caused by PD in AMD pathophysiology remains unclear. Furthermore, a simple reproducible model that emulates characteristics of both AMD and PD has been lacking. Hence, we established a novel AMD+PD murine model to decipher the potential role of oral infection (ligature-enhanced) with the keystone periodontal pathogen Porphyromonas gingivalis, in the progression of neovasculogenesis in a laser-induced choroidal-neovascularization (Li-CNV) mouse retina. By a combination of fundus photography, optical coherence tomography, and fluorescein angiography, we documented inflammatory drusen-like lesions, reduced retinal thickness, and increased vascular leakage in AMD+PD mice retinae. H&E further confirmed a significant reduction of retinal thickness and subretinal drusen-like deposits. Immunofluorescence microscopy revealed significant induction of choroidal/retinal vasculogenesis in AMD+PD mice. qPCR identified increased expression of oxidative-stress, angiogenesis, pro-inflammatory mediators, whereas antioxidants and anti-inflammatory genes in AMD+PD mice retinae were notably decreased. Through qPCR, we detected Pg and its fimbrial 16s-RrNA gene expression in the AMD+PD mice retinae. To sum-up, this is the first in vivo study signifying a role of periodontal infection in augmentation of AMD phenotype, with the aid of a pioneering AMD+PD murine model established in our laboratory.

RAD51AP1 Loss Attenuates Colorectal Cancer Stem Cell Renewal and Sensitizes to Chemotherapy.

DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)-induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin /+ ). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment. IMPLICATIONS: This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.

Expression and function of iron-regulatory proteins in retina.

Iron is essential for cell survival and function; yet excess iron is toxic to cells. Therefore, the cellular and whole-body levels of iron are regulated exquisitely. At least a dozen proteins participate in the regulation of iron homeostasis. Hemochromatosis, a genetic disorder of iron overload, is caused by mutations in at least five genes, namely HFE, hemojuvelin, Transferrin receptor 2, ferroportin, and hepcidin. Retina is separated from systemic circulation by inner and outer blood-retinal barriers; therefore it is widely believed that this tissue is immune to changes in systemic circulation. Even though hemochromatosis is associated with iron overload and dysfunction of a variety of systemic organs, little is known on the effects of this disease on the retina. Recent studies have shown that all five genes that are associated with hemochromatosis are expressed in the retina in a cell type-specific manner. The retinal pigment epithelium, which forms the outer blood-retinal barrier, expresses all of these five genes. It is therefore clearly evident that iron homeostasis in the retina is maintained locally by active participation of various iron-regulatory proteins. Excess iron is detrimental to the retina as evidenced from human studies and from mouse models of iron overload. Retinal iron homeostasis is disrupted in various clinical conditions such as hemochromatosis, aceruloplasminemia, age-related macular degeneration, and bacterial and viral infections.

Expression of the iron-regulatory protein haemojuvelin in retina and its regulation during cytomegalovirus infection.

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE [HLA-like protein involved in iron (Fe) homoeostasis], transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Expression of the first four genes coding for these proteins in retina has been established. Here we report on the expression of HJV. Since infection of retina with CMV (cytomegalovirus) causes blindness, we also investigated the expression of HJV and other iron-regulatory proteins in retina during CMV infection. HJV (HJV gene) mRNA was expressed in RPE (retinal pigment epithelium)/eyecup and neural retina in mouse. In situ hybridization and immunohistochemistry confirmed the presence of HJV mRNA and protein in RPE, outer and inner nuclear layers, and ganglion cell layer. Immunocytochemistry with cell lines and primary cell cultures showed HJV expression in RPE and Müller cells. In RPE, the expression was restricted to apical membrane. Infection of primary cultures of mouse RPE with CMV increased HJV mRNA and protein levels. Under similar conditions, HFE (HFE gene) mRNA levels were not altered, but HFE protein was decreased. Hepcidin expression was, however, not altered. These findings were demonstrable in vivo with CMV-infected mouse retina. The CMV-induced up-regulation of HJV in RPE was independent of changes in HFE because the phenomenon was also seen in HFE-null RPE cells. CMV-infected primary RPE cells showed evidence of iron accumulation and oxidative stress, as indicated by increased levels of ferritin and hydroxynonenal. The observed changes in HJV expression and iron status during CMV infection in retina may have significance in the pathophysiology of CMV retinitis.

Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: role of cystine/glutamate exchanger.

Haemochromatosis is an iron-overload disorder with age-dependent oxidative stress and dysfunction in a variety of tissues. Mutations in HFE (histocompatability leucocyte antigen class I-like protein involved in iron homoeostasis) are responsible for most cases of haemochromatosis. We demonstrated recently that HFE is expressed exclusively in the basal membrane of RPE (retinal pigment epithelium). In the present study, we used Hfe-/- mice to examine ferritin levels (an indirect readout for iron levels) and morphological changes in retina. We found increased ferritin accumulation in retina in 18-month-old, but not in 2-month-old, mice with considerable morphological damage compared with age-matched controls. The retinal phenotype included hypertrophy and hyperplasia of RPE. RPE cells isolated from Hfe-/- mice exhibited a hyperproliferative phenotype. We also compared the gene expression profile between wild-type and Hfe-/- RPE cells by microarray analysis. These studies showed that many cell cycle-related genes were differentially regulated in Hfe-/- RPE cells. One of the genes up-regulated in Hfe-/- RPE cells was Slc7a11 (where Slc is solute carrier) which codes for the 'transporter proper' xCT in the heterodimeric cystine/glutamate exchanger (xCT/4F2hc). This transporter plays a critical role in cellular glutathione status and cell-cycle progression. We confirmed the microarrray data by monitoring xCT mRNA levels by RT (reverse transcription)-PCR and also by measuring transport function. We also found increased levels of glutathione and the transcription factor/cell-cycle promoter AP1 (activator protein 1) in Hfe-/- RPE cells. Wild-type mouse RPE cells and human RPE cell lines, when loaded with iron by exposure to ferric ammonium citrate, showed increased expression and activity of xCT, reproducing the biochemical phenotype observed with Hfe-/- RPE cells.