The Effects of a Cd81 Null Mutation on Retinal Pigment Epithelium in Mice

The present study examines the effects of Cd81-null mutation on the development of the retinal pigment epithelium (RPE), specifically cell size and number of cells with multiple nuclei. The outlines of RPE in retinal flat mounts were stained with rhodamine-labeled phalloidin and RPE nuclei with Hoechst stain. The RPE layer was sampled to define the number of cells, the size of the RPE cells and the number of nuclei within the cells. The Cd81-null mutation caused an increase in the number of cells within the RPE layer. The cells were smaller than those in the wild type mice. Furthermore there was an increase in the number of mono-nucleated cells. In the posterior portion of the eye there was a significant increase in the number of multi-nucleated cells. The data indicate that CD81 plays a significant role in the final stages of RPE development, controlling cell number and overall developmental pattern.     

Evidence of decreased adhesion between the neural retina and retinal pigmented epithelium of the Mitf vit (vitiligo) mutant mouse

In order for the retina to function properly, photoreceptor cell outer segments must be in contact with the adjacent retinal pigmented epithelium (RPE). A mouse model homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene manifests disruption of the outer segment/RPE interdigitation and demonstrates progressive loss of the photoreceptor cells. The mouse nevertheless has near normal levels of rhodopsin for many weeks and it is not known whether there is an in vivo loss of adhesion or whether the disruption is visible following tissue processing for histology. To assess this, a mechanical separation experiment was performed in which neural retinas were peeled free from the RPE and examined for the amount of pigment adherent to them. The peeling experiment indicated that control neural retinas retained significant amounts of adherent pigment at all ages examined. Neural retinas of mutant mice at age 2 weeks demonstrated adherent pigment, but older animals retained minimal pigment. Scanning electron microscopy indicated that the RPE cells of control mice were markedly damaged upon peeling and displayed different planes of cleavage, whereas those of mutants showed minimal cellular damage upon peeling, suggestive of decreased adhesion. A recombination experiment revealed that the mutant RPE/eyecup could reappose mutant and control retinas under in vitro conditions, suggesting that RPE fluid transport abilities were intact. The data provide the first direct experimental evidence that the Mitf^vit mutant mouse has a naturally occurring retinal detachment and hence support its value as a model for studies of retina/RPE adhesion.     

The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.     

CD36 deficiency in mice impairs lipoprotein lipase-mediated triglyceride clearance

CD36 is involved in high-affinity peripheral FFA uptake. CD36-deficient (cd36(-)(/)(-)) mice exhibit increased plasma FFA and triglyceride (TG) levels. The aim of the present study was to elucidate the cause of the increased plasma TG levels in cd36(-)(/)(-) mice. cd36(-)(/)(-) mice showed no differences in hepatic VLDL-TG production or intestinal [(3)H]TG uptake compared with wild-type littermates. cd36(-)(/)(-) mice showed a 2-fold enhanced postprandial TG response upon an intragastric fat load (P < 0.05), with a concomitant 2.5-fold increased FFA response (P < 0.05), suggesting that the increased FFA in cd36(-/-) mice may impair LPL-mediated TG hydrolysis. Postheparin LPL levels were not affected. However, the in vitro LPL-mediated TG hydrolysis rate as induced by postheparin plasma of cd36(-)(/)(-) mice in the absence of excess FFA-free BSA was reduced 2-fold compared with wild-type plasma (P < 0.05). This inhibition was relieved upon the addition of excess FFA-free BSA. Likewise, increasing plasma FFA in wild-type mice to the levels observed in cd36(-)(/)(-) mice by infusion prolonged the plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles by 2.5-fold (P < 0.05). We conclude that the increased plasma TG levels observed in cd36(-)(/)(-) mice are caused by decreased LPL-mediated hydrolysis of TG-rich lipoproteins resulting from FFA-induced product inhibition of LPL.     

Allergen-induced airway hyperreactivity is diminished in CD81-deficient mice

We demonstrated previously that CD81(-/-) mice have an impaired Th2 response. To determine whether this impairment affected allergen-induced airway hyperreactivity (AHR), CD81(-/-) BALB/c mice and CD81(+/+) littermates were sensitized i.p. and challenged intranasally with OVA. Although wild type developed severe AHR, CD81(-/-) mice showed normal airway reactivity and reduced airway inflammation. Nevertheless, OVA-specific T cell proliferation was similar in both groups of mice. Analysis of cytokines secreted by the responding CD81(-/-) T cells, particularly those derived from peribronchial draining lymph nodes, revealed a dramatic reduction in IL-4, IL-5, and IL-13 synthesis. The decrease in cytokine production was not due to an intrinsic T cell deficiency because naive CD81(-/-) T cells responded to polyclonal Th1 and Th2 stimulation with normal proliferation and cytokine production. Moreover, there was an increase in T cells and a decrease in B cells in peribronchial lymph nodes and in spleens of immunized CD81(-/-) mice compared with wild-type animals. Interestingly, OVA-specific Ig levels, including IgE, were similar in CD81(-/-) and CD81(+/+) mice. Thus, CD81 plays a role in the development of AHR not by influencing Ag-specific IgE production but by regulating local cytokine production.     

A study of photoreceptor-retinal pigment epithelium complex: age-related changes in monkeys

Retinas of 4-, 10-, and 20-year-old monkeys were studied by light microscopy, electron microscopy, and scanning electron microscopy. Sections from the midperipheral region of every retina were selected for comparison. Although no significant differences were found between 4- and 10-year-old retinas, four major changes were found in 20-year-old monkey retinas: (i) increased number of displaced photoreceptor cells (DPC), (ii) increased number of macrophages of different morphology in subretinal space, (iii) increase in pigment granules in retinal pigment epithelium (RPE) cells, and (iv) altered morphology of Muller cells. DPC included both rods and cones. Their location and morphology depended on the stage of their displacement. These cells were usually oval or rounded in shape and were found either among the outer segments of other photoreceptor cells, having stalks extending into the outer nuclear layer, or were located in the subretinal space and had no stalk. A narrow space around the DPC stalks, indicating a change in the intercellular connection between photoreceptor cells and Muller cells, was observed. Furthermore, the Muller cells related to DPC had shortened and markedly reduced microvilli. Two types of macrophages were found in the subretinal space of aged monkey retinas. One type was similar in morphology to RPE cells. Some of these cells were noticed detaching from RPE. Other types of macrophages were nonpigmented. The modifications in RPE were closely related to the changes in the associated neuroretina. The RPE cells in aged retina were devoid of microvilli or had a few thin microvilli. The pleomorphic pigment granules were dispersed throughout the cytoplasm. These cells varied in their size, shape, and surface features. These changes could significantly alter the retinal metabolic equilibrium and may be indicative of age related degenerative processes.     

Altered visual function in monocarboxylate transporter 3 (Slc16a8) knockout mice

To meet the high-energy demands of photoreceptor cells, the outer retina metabolizes glucose through glycolytic and oxidative pathways, resulting in large-scale production of lactate and CO(2). Mct3, a proton-coupled monocarboxylate transporter, is critically positioned to facilitate transport of lactate and H(+) out of the retina and could therefore play a role in pH and ion homeostasis of the outer retina. Mct3 is preferentially expressed in the basolateral membrane of the retinal pigment epithelium and forms a heteromeric complex with the accessory protein CD147. To examine the physiological role of Mct3 in the retina, we generated mice with a targeted deletion in Mct3 (slc16A8). The overall retinal histology of 4- to 36-wk-old Mct3(-/-) mice appeared normal. In the absence of Mct3, expression of CD147 was lost from the basolateral but not apical RPE. The saturated a-wave amplitude (a(max)) of the scotopic electroretinogram (ERG) was reduced by approximately twofold in Mct3(-/-) mice relative to wild-type mice. A fourfold increase in lactate in the retina suggested a decrease in outer-retinal pH. In single-cell recordings from superfused retinal slices, saturating amplitudes of single rod photocurrents (J(max)) were comparable indicating that Mct3(-/-) mouse photoreceptor cells were inherently healthy. Based on these data, we hypothesize that disruption of Mct3 leads to a potentially reversible decrease in subretinal space pH, thereby reducing the magnitude of the light suppressible photoreceptor current.     

Complement system dysregulation and inflammation in the retinal pigment epithelium of a mouse model for Stargardt macular degeneration

Accumulation of vitamin A-derived lipofuscin fluorophores in the retinal pigment epithelium (RPE) is a pathologic feature of recessive Stargardt macular dystrophy, a blinding disease caused by dysfunction or loss of the ABCA4 transporter in rods and cones. Age-related macular degeneration, a prevalent blinding disease of the elderly, is strongly associated with mutations in the genes for complement regulatory proteins (CRP), causing chronic inflammation of the RPE. Here we explore the possible relationship between lipofuscin accumulation and complement activation in vivo. Using the abca4(-/-) mouse model for recessive Stargardt, we investigated the role of lipofuscin fluorophores (A2E-lipofuscin) on oxidative stress and complement activation. We observed higher expression of oxidative-stress genes and elevated products of lipid peroxidation in eyes from abca4(-/-) versus wild-type mice. We also observed higher levels of complement-activation products in abca4(-/-) RPE cells. Unexpectedly, expression of multiple CRPs, which protect cells from attack by the complement system, were lower in abca4(-/-) versus wild-type RPE. To test whether acute exposure of healthy RPE cells to A2E-lipofuscin affects oxidative stress and expression of CRPs, we fed cultured fetal-derived human RPE cells with rod outer segments from wild-type or abca4(-/-) retinas. In contrast to RPE cells in abca4(-/-) mice, human RPE cells exposed to abca4(-/-) rod outer segments adaptively increased expression of both oxidative-stress and CRP genes. These results suggest that A2E accumulation causes oxidative stress, complement activation, and down-regulation of protective CRP in the Stargardt mouse model. Thus, Stargardt disease and age-related macular degeneration may both be caused by chronic inflammation of the RPE.     

Control of IFN-alphaA by CD73: implications for mucosal inflammation

Inflammatory diseases influence tissue metabolism, altering regulation of extracellular adenine nucleotides, with a resultant protective influence of adenosine. Ecto-5'-nucleotidase (CD73) is a central surface enzyme generating extracellular adenosine. Thus, we hypothesized that CD73 is protective in mucosal inflammation as modeled by trinitrobenzene sulfonate (TNBS) colitis. Initial studies revealed a >3-fold induction of CD73 mRNA levels after TNBS colitis. Additionally, the severity of colitis was increased, as determined by weight loss and colonic shortening, in cd73(-/-) mice relative to cd73(+/+) controls. Likewise, enteral administration of the selective CD73 inhibitor alpha,beta-methylene ADP to cd73(+/+) mice resulted in a similar increase in severity of TNBS colitis. Gene array profiling of cytokine mRNA expression, verified by real-time PCR, revealed a >90% down-regulation of IFN-alphaA in cd73(-/-) mice and alpha,beta-methylene ADP-treated cd73(+/+) mice, compared with cd73(+/+) mice. Exogenous administration of recombinant IFN-alphaA partially protected TNBS-treated cd73(-/-) mice. Cytokine profiling revealed similar increases in both IFN-gamma and TNF-alpha mRNA in colitic animals, independent of genotype. However, IL-10 mRNA increased in wild-type mice on day 3 after TNBS administration, whereas cd73(-/-) mice mounted no IL-10 response. This IL-10 response was restored in the cd73(-/-) mice by exogenous IFN-alphaA. Further cytokine profiling revealed that this IL-10 induction is preceded by a transient IFN-alphaA induction on day 2 after TNBS exposure. Together, these studies indicate a critical regulatory role for CD73-modulated IFNalphaA in the acute inflammatory phase of TNBS colitis, thereby implicating IFN-alphaA as a protective element of adenosine signaling during mucosal inflammation.     

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.     

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.     

Age-dependent accumulation of lipofuscin in perivascular and subretinal microglia in experimental mice

Fundus autofluorescence (AF) imaging by confocal scanning laser ophthalmoscopy has been widely used by ophthalmologists in the diagnosis/monitoring of various retinal disorders. It is believed that fundus AF is derived from lipofuscin in retinal pigment epithelial (RPE) cells; however, direct clinicopathological correlation has not been possible in humans. We examined fundus AF by confocal scanning laser ophthalmoscopy and confocal microscopy in normal C57BL/6 mice of different ages. Increasingly strong AF signals were observed with age in the neuroretina and subretinal/RPE layer by confocal scanning laser ophthalmoscopy. Unlike fundus AF detected in normal human subjects, mouse fundus AF appeared as discrete foci distributed throughout the retina. Most of the AF signals in the neuroretina were distributed around retinal vessels. Confocal microscopy of retinal and choroid/RPE flat mounts demonstrated that most of the AF signals were derived from Iba-1+ perivascular and subretinal microglia. An age-dependent accumulation of Iba-1+ microglia at the subretinal space was observed. Lipofuscin granules were detected in large numbers in subretinal microglia by electron microscopy. The number of AF+ microglia and the amount of AF granules/cell increased with age. AF granules/lipofuscin were also observed in RPE cells in mice older than 12 months, but the number of AF+ RPE cells was very low (1.48 mm(-2) and 5.02 mm(-2) for 12 and 24 months, respectively) compared to the number of AF+ microglial cells (20.63 mm(-2) and 76.36 mm(-2) for 6 and 24 months, respectively). The fluorescence emission fingerprints of AF granules in subretinal microglia were the same as those in RPE cells. Our observation suggests that perivascular and subretinal microglia are the main cells producing lipofuscin in normal aged mouse retina and are responsible for in vivo fundus AF. Microglia may play an important role in retinal aging and age-related retinal diseases.     

Completion of meiosis is not always required for acrosome formation in HSP70-2 null mice.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase. However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.     

Differential developmental deficits in retinal function in the absence of either protein tyrosine sulfotransferase-1 or -2

To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1(-/-) and Tpst2(-/-) mice, retinal function was compromised during early development. However, Tpst1(-/-) retinas became electrophysiologically normal by postnatal day 90 while Tpst2(-/-) mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina.     

绿色荧光蛋白基因修饰的人视网膜色素上皮细胞异种移植的长期观察

探讨利用视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞移植介导目的基因转移至视网膜的可行性。人的RPE细胞在体外经携带绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因的逆转录病毒感染及G4 1 8筛选后 ,手术显微镜直视下经睫状体平坦部注射到兔眼视网膜下间隙。术后通过活体荧光眼底照相 ,荧光显微镜、共聚焦显微镜及透射电镜等观察眼球铺片及切片 ,发现经 gfp基因修饰的人RPE细胞在兔眼视网膜下间隙可存活一年以上。移植的RPE gfp细胞不仅仅局限于移植部位 ,大多扩散到超过 2～ 3个象限的眼底 ,镶嵌于宿主RPE细胞之间或呈单层排列在宿主色素上皮与神经视网膜之间 ,并持续高水平表达GFP。移植后早期玻璃体内每周注射免疫抑制剂普乐可复 (FK5 0 6 )可明显改善移植细胞的存活状态     

Light-Induced Dopamine Release from Teleost Retinas Acts as a Light-Adaptive Signal to the Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules migrate in and out of the cells' long apical projections in response to changes in light condition. When the RPE is in its normal association with the retina, light onset induces pigment granules to disperse into the apical projections; dark onset induces pigment granules to aggregate into the cell bodies. However, when the RPE is separated from the retina, pigment granule movement in the isolated RPE is insensitive to light onset. It thus seems likely that a signal from the retina communicates light onset to the RPE to initiate pigment dispersion. We have examined the nature of this retina-to-RPE signal in green sunfish, Lepomis cyanellus. In isolated retinas with adherent RPE, light-induced pigment dispersion in the RPE is blocked by treatments known to block Ca2+-dependent transmitter release in the retina. In addition, the medium obtained from incubating previously dark-adapted retinas in the light induces light-adaptive pigment dispersion when added to isolated RPE. In contrast, the medium obtained from incubating dark-adapted retinas in constant darkness does not affect pigment distribution when added to isolated RPE. These results are consistent with the idea that RPE pigment dispersion is triggered by a substance that diffuses from the retina at light onset. The capacity of the conditioned medium from light-incubated retinas to induce pigment dispersion in isolated RPE is inhibited by a D2 dopamine antagonist, but not by D1 or alpha-adrenergic antagonists. Light-induced pigment dispersion in whole RPE-retinas is also blocked by a D2 dopamine antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of retinal vascular development and neovascularization during oxygen-induced ischemic retinopathy in Bcl-2-/- mice

Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, we investigated the physiological role of bcl-2 in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibited a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2-/- mice. We observed, in bcl-2-/- mice, delayed development of retinal vasculature and remodeling, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2-/- mice exhibited a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization was significantly reduced in bcl-2-/- mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR.     

Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5'-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39(-/-) mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39(-/-) or cd73(-/-) mice with soluble apyrase or 5'-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5'-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.     

Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.