血小板反应素1在STZ诱导大鼠糖尿病性视网膜病变的表达研究

探讨TSP1表达在糖尿病视网膜病变中的作用和机制,为治疗和预防糖尿病视网膜病变提供新的实验和理论依据。用链脲佐菌素(STZ)腹腔注射建立糖尿病模型8周后,采用免疫组织化学、RT-PCR及实时荧光定量PCR法,分析TSP1在早期链尿佐菌素诱导的糖尿病SD大鼠视网膜中的表达。结果显示在早期糖尿病大鼠的视网膜表面血管、神经节细胞层、内外核层中均有明显的TSP1表达,糖尿病视网膜组TSP1 mRNA表达要高于对照组,其中实时荧光定量PCR CT值的结果显示糖尿病组TSP1 mRNA表达量较对照组要高约3．48倍,二组间差别有显著性意义(P<0．01),提示TSP1在视网膜组织中的表达与糖尿病视网膜病变的发生密不可分,TSP1表达的增加可能在糖尿病视网膜病变的发生和发展中起重要作用。     

Alterations in the Retinal Dopaminergic Neuronal System in Rats with Streptozotocin-Induced Diabetes

Neurochemical alterations, which may be associated with the development of diabetic retinal dysfunction, were investigated using streptozotocin (STZ)-induced hyperglycemia in rats. Young male Wistar rats, weighing 100-150 g, were made diabetic with daily intraperitoneal injections of STZ (30 mg/kg) for 5 days. This treatment caused a continuous hyperglycemia (400-600 mg/dl) and suppressed gain in body weight. Nine weeks after the STZ treatment, a significant increment in retinal valine and a decline in phenylalanine were noted, while the concentrations of other neuroactive amino acids, such as gamma-aminobutyric acid and aspartic acid, in the retina remained unchanged. On the other hand, the concentration of retinal dopamine (DA) was found to decrease significantly from the third week of hyperglycemia, when [3H]spiperone binding showed a tendency to increase in the retinal particulate fraction. However, the activities of tyrosine hydroxylase and aromatic L-amino acid decarboxylase (AADC) and the uptake of [3H]tyrosine showed no alteration in the retina of diabetic rats. The accumulation rate of 3,4-dihydroxyphenylalanine (DOPA) in vivo in the retina of diabetic rats, measured following the administration of the AADC inhibitor m-hydroxybenzyl-hydrazine (100 mg/kg i.p.), was also unchanged. Although [3H]DA uptake by retinal tissue was similar in control and diabetic animals, the spontaneous efflux of [3H]DA from the retina was found to be significantly accelerated in STZ-treated animals. In addition, the release of preloaded [3H]DA, elicited by repeated photic stimulation, was significantly attenuated in retina from diabetic rats. These results suggest that an accelerated efflux of DA, possibly leading to the depletion of DA from the retinal DA system, may account for early retinal dysfunctions known to occur in diabetic subjects.     

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).     

Environmental Enrichment Protects the Retina from Early Diabetic Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Available treatments are not completely effective. We analyzed the effect of environmental enrichment on retinal damage induced by experimental diabetes in adult Wistar rats. Diabetes was induced by an intraperitoneal injection of streptozotocin. Three days after vehicle or streptozotocin injection, animals were housed in enriched environment or remained in a standard environment. Retinal function (electroretinogram, and oscillatory potentials), retinal morphology, blood-retinal barrier integrity, synaptophysin, astrocyte and Müller cell glial fibrillary acidic protein, vascular endothelial growth factor, tumor necrosis factor-α, and brain-derived neurotrophic factor levels, as well as lipid peroxidation were assessed in retina from diabetic animals housed in standard or enriched environment. Environmental enrichment preserved scotopic electroretinogram a-wave, b-wave and oscillatory potential amplitude, avoided albumin-Evan''s blue leakage, prevented the decrease in retinal synaptophysin and astrocyte glial fibrillary acidic protein levels, the increase in Müller cell glial fibrillary acidic protein, vascular endothelial growth factor and tumor necrosis factor-α levels, as well as oxidative stress induced by diabetes. In addition, enriched environment prevented the decrease in retinal brain-derived neurotrophic factor levels induced by experimental diabetes. When environmental enrichment started 7 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that enriched environment could attenuate the early diabetic damage in the retina from adult rats.     

Does ghrelin explain accelerated gastric emptying in the early stages of diabetes mellitus?

During the early stages of diabetes, gastric emptying is often accelerated, rather than delayed. The mechanism of accelerated gastric emptying in diabetes has not been fully studied. A recent study showed that plasma ghrelin levels were elevated in diabetes. As postprandial antropyloric coordination plays an important role in mediating solid gastric emptying, we hypothesize that the elevated plasma ghrelin levels increase postprandial antropyloric coordination to accelerate emptying in the early stages of diabetes. To test this hypothesis, rats were made diabetic by streptozotocin (STZ; 50 mg/kg) injection, and, 2 wk later, pre- and postprandial plasma ghrelin levels, antropyloric coordination, and solid gastric emptying were determined. In control rats, plasma ghrelin levels were immediately reduced after feeding. In contrast, plasma ghrelin levels remained within the fasted levels in STZ rats after feeding. In STZ rats, gastric emptying was significantly accelerated (77.4 +/- 3.2%, n = 6), compared with that of control rats (58.8 +/- 2.5%, n = 6, P < 0.05). Treatments with anti-ghrelin antibodies attenuated accelerated gastric emptying in STZ rats (50.1 +/- 3.5%, n = 6, P < 0.05), while having little effect in vehicle control rats. The incidence of postprandial antropyloric coordination was significantly increased in STZ rats, compared with that of control rats (P < 0.05). Treatments with anti-ghrelin antibodies suppressed this enhanced antropyloric coordination in STZ rats. Our study suggests that elevated endogenous ghrelin enhances antropyloric coordination, which accelerates gastric emptying in the early stages of diabetes.     

Hepatic calcium-binding protein regucalcin concentration is decreased by streptozotocin-diabetic state and ethanol ingestion in rats

The alteration in calcium-binding protein regucalcin in the liver and serum of rats with streptozotocin (STZ)-diabetic state or ethanol ingestion was investigated. STZ (6.0 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 3 weeks later they were sacrificed by bleeding. Liver regucalcin mRNA levels were not clearly altered by the diabetic state, as evidenced by Northern blotting using regucalcin cDNA (0.9 kb of open reading frame). Based on enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG, hepatic regucalcin concentration was decreased about 50% of control levels by STZ treatment. However, serum regucalcin concentration was not significantly altered by STZ treatment. Meanwhile, when rats ingested ethanol (10 and 30%) in the drinking water for 2 weeks, liver regucalcin mRNA levels were clearly increased, although hepatic regucalcin concentration was significantly decreased. Serum regucalcin concentration was not appreciably altered. Serum transaminases (GOT and GPT) activities were significantly increased at 1 or 3 weeks after STZ administration in rats, while their activities were not altered by ethanol ingestion. The present study demonstrates that hepatic regucalcin concentration is decreased independent of mRNA expression in the STZ-diabetes and during ethanol ingestion in rats.     

Early changes in pituitary adenylate cyclase-activating peptide, vasoactive intestinal peptide and related receptors expression in retina of streptozotocin-induced diabetic rats

The retinal expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) and their receptors was investigated in early streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection (60mg/kg i.p.). PACAP, VIP and their receptors in nondiabetic control and diabetic retinas were assayed by quantitative real-time PCR and Western blot 1 and 3 weeks after STZ injection. Effects of intravitreal treatment with PACAP38 on the expression of the two apoptotic-related genes Bcl-2 and p53 were also evaluated. PACAP and VIP, as well as VPAC1 and VPAC2 receptors, but not PAC1 mRNA levels, were transiently induced in retinas 1week following STZ. These findings were confirmed by immunoblot analyses. Three weeks after the induction of diabetes, significant decreases in the expression of peptides and their receptors were observed, Bcl-2 expression decreased and p53 expression increased. Intravitreal injection of PACAP38 restored STZ-induced changes in retinal Bcl-2 and p53 expression to nondiabetic levels. The initial upregulation of PACAP, VIP and related receptors and the subsequent downregulation in retina of diabetic rats along with the protective effects of PACAP38 treatment, suggest a role for both peptides in the pathogenesis of diabetic retinopathy.     

Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice.

Experimental prevention of basal lamina (BL) thickening of retinal capillaries ameliorates early vascular changes caused by diabetes. Connective tissue growth factor (CTGF) is upregulated early in diabetes in the human retina and is a potent inducer of expression of BL components. We hypothesize that CTGF is causally involved in diabetes-induced BL thickening of retinal capillaries. To test this hypothesis, we compared the effects of streptozotocin (STZ)-induced diabetes on retinal capillary BL thickness between wild-type mice (CTGF+/+) and mice lacking one functional CTGF allele (CTGF+/-). Differences in BL thickness were calculated by quantitative analysis of electron microscopic images of transversally sectioned capillaries in and around the inner nuclear layer of the retina. We show that BL thickening was significant in diabetic CTGF+/+ mice compared with control CTGF+/+ mice, whereas diabetes did not significantly induce BL thickening in CTGF+/- mice. We conclude that CTGF expression is necessary for diabetes-induced BL thickening and suggest that reduction of CTGF levels may be protective against the development of diabetic retinopathy.     

吡格列酮治疗STZ诱导糖尿病大鼠视网膜中TSP-1的变化

观察吡格列酮在治疗STZ诱导糖尿病大鼠视网膜中血小板反应素Ⅰ(thmmbospondin-1,TSP-1)的变化,探讨噻唑烷二酮类药物在糖尿病视网膜病变中的影响和可能作用机制。用链脲佐菌素(STZ)腹腔注射建立大鼠糖尿病模型,随机分正常对照组(C组),糖尿病安慰剂组(D组)和糖尿病吡格列酮治疗组(DP组),8周后取视网膜组织行免疫组织化学、RT-PCR半定量检测TSP- 1mRNA水平的变化,结果显示在糖尿病早期视网膜神经节细胞层、内核层中均有明显的TSP1表达,对比于糖尿病对照组,糖尿病治疗组视网膜单位面积表达TSP1的细胞数量要较二组对照组高;糖尿病治疗组、糖尿病对照组和正常对照组间TSP-1的荧光灰度值分别为528.2±47.78、493.4±39.36和417.7±28.74:内参照GAPDH的荧光灰度值分别为796.4±21.18、811.2±36.80和832.4±43.18,各组间TSP-1 mRNA表达有显著性差异,提示吡格列酮对糖尿病早期视网膜中TSP-1的表达变化有影响,吡格列酮可能在延缓糖尿病视网膜病变的发生发展中起一定的作用。     

Reduced Levels of Brain Derived Neurotrophic Factor (BDNF) in the Serum of Diabetic Retinopathy Patients and in the Retina of Diabetic Rats

Diabetic retinopathy (DR) is widely recognized as a neurovascular disease. Retina, being a neuronal tissue of the eye, produces neurotrophic factors for its maintenance. However, diabetes dysregulates their levels and thereby may damage the retina. Among neurotrophins, brain derived neurotrophic factor (BDNF) is the most abundant in the retina. In this study, we investigated the level of BDNF in the serum of patients with DR and also in the serum and retina of streptozotocin-induced diabetic rats. The level of BDNF was significantly decreased in the serum of proliferative diabetic retinopathy patients as compared to that of non-diabetic healthy controls (25.5 ± 8.5–10.0 ± 8.1 ng/ml, p < 0.001) as well as compared to that of diabetic patients with no retinopathy (21.8 ± 4.7–10.0 ± 8.1 ng/ml, p < 0.001), as measured by ELISA techniques. The levels of BDNF in the serum and retina of diabetic rats were also significantly reduced compared to that of non-diabetic controls (p < 0.05). In addition, the expression level of tropomyosin-related kinase B (TrkB) was significantly decreased in diabetic rat retina compared to that of non-diabetic controls as determined by Western blotting technique. Caspase-3 activity was increased in diabetic rat retina after 3 weeks of diabetes and remained elevated until 10 weeks, which negatively correlated with the level of BDNF (r = ?0.544, p = 0.013). Our results indicate that reduced levels of BDNF in diabetes may cause apoptosis and neurodegeneration early in diabetic retina, which may lead to neuro-vascular damage later in DR.     

The synaptophysin/synaptobrevin interaction critically depends on the cholesterol content

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.     

Increased synaptophysin expression through whisker stimulation in rat

1. Synaptophysin is responsible for the cycling of the synaptic vesicles containing the neurotransmitter, and it can be phosphorylated.2. This study examined whether repeated whisker stimulation alters the expression of synaptophysin mRNA in the rat barrel cortex, and found induced expression of synaptophysin mRNA in the contralateral barrel cortex compared to that in the ipsilateral hemisphere.3. This result suggests that synaptophysin is involved in the modulation of the synaptic plasticity.     

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the genes in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 expression was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.     

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the genes in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 expression was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.     

Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina subject to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mmHg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 microl of 100 microM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI.     

Tactile stimulation-induced rapid elevation of the synaptophysin mRNA expression level in rat somatosensory cortex

Synaptophysin is an integral membrane protein abundant in the synaptic vesicle and is found in nerve terminals throughout the brain. It was recently suggested that synaptophysin is also involved in the modulation of activity-dependent synapse formation. In this study, we examined at the individual level whether tactile stimulation selectively influenced the synaptophysin mRNA expression level in the somatosensory cortex of rats. Anesthetized rats were caressed on the back by an experimenter's palms for 20 min and the mRNA expression levels in the somatosensory and the visual cortices 5 min afterwards were determined using quantitative PCR methodology. The synaptophysin mRNA expression level was selectively higher in the experimental group than in the control group in the somatosensory cortex but not in the visual cortex. This suggests that the mRNA expression level of synaptophysin induced by neuronal activity is related to the regulation of synapse formation or remodeling or both.     

The retinal pigment epithelium of Cr-deficient rats

The purpose of this study is to investigate the effect of Cr deficiency on the rat retina. Three-week-old Wistar Kyoto rats were divided into 2 groups. Cr-deficient rats were fed AIN-93G diet without Cr and deionized distilled water. Control rats were fed AIN-93G diet and deionized distilled water. The Cr and sugar concentrations in the whole blood and cholesterol concentration in the serum were measured. We observed the retina with an electron microscope, and counted phagocytized lamellar structures in the retinal pigment epithelium (RPE) before and after the start of light exposure on negative electron microscopic films. The whole blood Cr level of Cr-deficient rats was less than 0.2 microg/l. The blood sugar level of Cr-deficient rats was significantly higher than that of normal rats (p < 0.05). There were significantly more phagocytized lamellar structures in the RPE of Cr-deficient rats 1, 2, 7, 11 and 12 h after the start of light exposure than in that of normal rats (p < 0.05). However, no morphological abnormalities were found in the photoreceptor cells of Cr-deficient rats. Phagocytosis in the photoreceptor outer segment discs in the RPE was accelerated, but the pattern of the retinal circadian rhythm with maximum phagocytosis 2 h after exposure to light was unchanged. The Cr-deficient state may cause the membrane to degenerate, and phagocytosis of the photoreceptor outer segment discs in the RPE may be accelerated. This study provided an evidence of the nutritional importance of Cr in rat retina.     

Deficiency of neuropilin 2 suppresses VEGF-induced retinal neovascularization

Vascular endothelial growth factor (VEGF) plays a central role in the development of ocular neovascularization (NV) and is an excellent target for therapeutic intervention. VEGF acts through several receptors, including VEGF receptor 1, VEGF receptor 2, neuropilin-1 (Npn1), and Npn2, but the exact role of these receptors in the development of retinal NV is unknown. In this study, we investigated the expression of npn2 mRNA during new blood vessel growth in the retina and used npn2 knockout mice to assess the impact of deficiency of Npn2 on retinal NV. The level of npn2 mRNA in the retina increased during retinal vascular development, after exposure to hyperoxia, and after the onset of retinal ischemia. Immunohistochemistry showed colocalization of Npn2 with a vascular marker in retinal NV. Compared with littermate controls, mice deficient in Npn2 had significantly less ischemia-induced retinal NV and very little subretinal NV due to expression of a Vegf transgene. These data suggest that Npn2 facilitates VEGF-induced retinal NV and may constitute a useful target for therapeutic intervention in ocular diseases complicated by NV.     

Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.