Adenosine Diphosphatase Research Articles

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the exvivo thromboelastometric parameters. AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in exvivo thromboelastometric parameters. URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

β‑elemene inhibits oxygen‑induced retinal neovascularization via promoting miR‑27a and reducing VEGF expression.

The present study aimed to investigate the significant role of β-elemene in mouse models of oxygen-induced retinopathy (OIR). C57BL/6J neonatal mice were used to establish OIR models. They were divided into four groups: Normoxia, OIR, OIR control and OIR-treated. Mice in the OIR group were exposed to 75±5% oxygen for 5 days and returned to a normal oxygen environment on postnatal day 12 (P12). The OIR treated group was intravitreally injected with 1 µl β-elemene on P12 and subsequently returned to a normal oxygen environment for 5 days (P12-P17). Retinas were obtained on P17. Retinal neovascularization (RNV) was detected using adenosine diphosphatase staining and analyzed by counting the nuclei of neovascular endothelial cells. Vascular endothelial growth factor (VEGF) expression was determined by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. MicroRNA (miRNA/miR) microarrays were used to screen out differentially expressed miRNAs between the OIR and β-elemene-treated groups. Binding the 3′-untranslated region (UTR) of VEGF and miR-27a was confirmed using luciferase assays. It was found that high oxygen concentrations accelerated RNV and increased the number of preretinal neovascular cells; β-elemene treatment reduced these effects. VEGF mRNA and protein expression was higher in the OIR and OIR control groups, compared with the normoxia and OIR-treated groups. Further, it was shown that miR-22, miR-181a-1, miR-335-5p, miR-669n, miR-190b, miR-27a and miR-93 were upregulated in the OIR-treated group, and downregulated in the OIR group. The prediction websites TargetScan and miRanda revealed that VEGF contained a potential miR-27a binding site in its 3′-untranslated region (UTR). Luciferase assays demonstrated that miR-27a directly bound to the 3′-UTR of VEGF. In vitro experiments demonstrated that miR-27a inhibited VEGF expression. In addition, β-elemene treatment upregulate miR-27a expression in vivo and in vitro. When miR-27a expression was depleted by miR-27a inhibitor, the protective effect of β-elemene on RNV was eliminated. The present study demonstrated that β-elemene reduced RNV in mouse OIR models via miR-27a upregulation, leading to reduced VEGF expression. This finding may contribute to the development of novel therapeutic strategies for human retinopathy.

Inhibition of LY294002 in retinal neovascularization via down-regulation the PI3K/AKT-VEGF pathway in vivo and in vitro.

To investigate the effects of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 on retinal neovascularization (RNV) in the oxygen-induced retinopathy (OIR) mouse model and human umbilical vein endothelial cells (HUVECs). C57BL/6J mice were randomly divided into normoxia-control, OIR-control and LY294002 treatment groups. LY294002 or phosphate-buffered solution was intraperitoneally injected daily into mouse pups from P6 to P9 in LY294002 treatment group or OIR-control group. Morphological and pathological changes in RNV, as well as expression levels of PI3K, serine-threonine kinase (AKT) and vascular endothelial growth factor (VEGF) were observed. HUVECs treating with LY294002 were exposed to hypoxia; the expression of PI3K, AKT and VEGF were examined by Western blot and RT-PCR analyses. Compared with the OIR-control group, LY294002 significantly inhibit RNV. Adenosine diphosphatase (ADPase) staining and hematoxylin and eosin staining indicated that the clock hour scores of neovascularization and the nuclei of pre-retinal neovascular cells in the LY294002 treatment group were clearly less than those in the OIR-control group (1.41±0.52 vs 6.20±1.21; 10.50±1.58 vs 22.25±1.82, both P<0.05). Intravitreal injection of LY294002 (in the LY294002 treatment group) markedly decreased PI3K/AKT-VEGF expression compared with the OIR-control group by immunohistochemistry, Western blotting and RT-PCR (all P<0.05). In HUVECs treated with hypoxia, expression of PI3K, AKT and VEGF were downregulated in the hypoxia-LY294002 group (all P<0.05). The PI3K inhibitor LY294002 can inhibit RNV by downregulating PI3K, AKT, and VEGF expression in vivo and in vitro. LY294002 may provide an effective method for preventing retinopathy of prematurity (ROP).

Expression and significance of the Hedgehog signal transduction pathway in oxygen-induced retinal neovascularization in mice.

AimThe aim of the study was to investigate the signal transduction mechanism of Hedgehog–vascular endothelial growth factor in oxygen-induced retinopathy (OIR) and the effects of cyclopamine on OIR.MethodsAn OIR model was established in C57BL/6J mice exposed to hyperoxia. Two hundred mice were randomly divided into a control group, an OIR group, an OIR-control group (treated with isometric phosphate-buffered saline by intravitreal injection), and a cyclopamine group (treated with cyclopamine by intravitreal injection), with 50 mice in each group. The retinal vascular morphology was observed using adenosine diphosphatase and number counting using hematoxylin and eosin-stained image. Quantitative real-time quantitative polymerase chain reaction was used to detect mRNA expression. Protein location and expression were evaluated using immunohistochemistry and Western blot.ResultsThe OIR group and OIR-control group demonstrated large-area pathological neovascularization and nonperfused area when compared with the control group (both P<0.05). The area of nonperfusion and neovascularization in the cyclopamine group was significantly reduced compared with the OIR and OIR-control groups (both P<0.05). Compared with the control group, the OIR and OIR-control groups had more vascular endothelial cells breaking through the inner limiting membrane. The number of new blood vessel endothelial cell nuclei in the cyclopamine group was significantly reduced (both P<0.05) when compared with the OIR and OIR-control groups. The mRNA and protein expressions of Smoothened, Gli1, and vascular endothelial growth factor in the signal pathway of the OIR and OIR-control groups were significantly higher than those of the control group; however, in the cyclopamine group, these factors were reduced when compared with the OIR and OIR-control groups (all P<0.05).ConclusionOur data suggest that abnormal expression of the Hedgehog signaling pathway may be closely associated with the formation of OIR. Inhibiting the Smoothened receptor using cyclopamine could control retinal neovascularization, providing new ideas and measures for the prevention of oxygen-induced retinal neovascularization.

Retinal VEGF levels correlate with ocular circulation measured by a laser speckle-micro system in an oxygen-induced retinopathy rat model.

We used a Laser speckle flowgraphy (LSFG)-micro system to examine the relationship between ocular blood flow and retinal vascular endothelial growth factor (VEGF) at retinopathy onset in oxygen-induced ischemic retinopathy (OIR) model rats. Sixteen 50/10 OIR rats were compared with 17 control rats reared in room air. In postnatal day 14 (P14) and P18 rats, we measured and analyzed the left eye's mean blur rate (MBR) by setting a rubber band on the optic nerve head center, using the LSFG-Micro. At P18, the rats were sacrificed and their left-eye retinas were fixed, flat-mounted and stained with adenosine diphosphatase (ADPase). The right-eye retinas were homogenized; the lysate was centrifuged for an enzyme-linked immunosorbent assay (ELISA). The avascular area was measured as the percentage (%AVA) of the total retinal area. Retinal VEGF was measured by an ELISA. The examination's reproducibility was good. Our multivariate linear mixed model analysis revealed significantly high MBRs in the OIR rats (p=0.0017). In the P18 OIR rats, significant correlations were seen between the MBR and %AVA (r=0.80, p=0.0002) and between the MBR and VEGF (r=0.76, p=0.0006). The LSFG-Micro provided reproducible blood flow measurements in neonatal rats. Because of the vitreous blood vessels, measurement of only the retinal vessels was not possible. However, the MBR was higher in the OIR rats than in the control rats, and the MBR and %AVA were correlated, as were the MBR and retinal VEGF. The MBR may thus serve as an indicator of OIR severity.

CCR7/p-ERK1/2/VEGF signaling promotes retinal neovascularization in a mouse model of oxygen-induced retinopathy.

To investigate the role of CCR7/p-ERK1/2/VEGF signaling in the mouse model of oxygen-induced retinopathy (OIR). Neonatal C57BL/6J mice were evenly randomized into four groups: normoxia, OIR, OIR control (treated with scramble siRNA), and OIR treated (treated with CCR7 siRNA). Normoxia group was not specially handled. Postnatal day 7 (P7) mice in the OIR group were exposed to 75%±5% oxygen for 5d (P7-P12) and then maintained under normoxic conditions for 5d (P12-P17). Mice in the OIR control and OIR treated groups were given injections of scramble or CCR7 siRNA plasmid on P12 before returning to normoxic conditions for 5d (P12-P17). Retina samples were collected from all mice on P17, stained with adenosine diphosphatase (ADPase), and retinal neovascularization (RNV) was assessed. Retinas were also stained with hematoxylin and eosin (H&E) for RNV quantitation. The distribution and expression of CCR7, p-ERK1/2 and vascular endothelial growth factor (VEGF) were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). High oxygen promoted retinal neovascularization (P<0.05) and increased the number of endothelial nuclei in new vessels extending from the retina to the vitreous body; CCR7 promoted this process (P<0.05). CCR7 and VEGF mRNA were expressed at higher levels in the OIR and OIR control groups than in the normoxia and OIR treated groups. CCR7, p-ERK1/2, and VEGF protein were expressed in the retinas of mice in the OIR and OIR control groups. Intravitreal injection of CCR7 siRNA significantly reduced CCR7, p-ERK1/2, and VEGF expression in the OIR mouse model (all P<0.05). CCR7 significantly enhanced the neovascularization and non-perfusion areas in the OIR group (P<0.05). CCR7 siRNA significantly reduced levels of p-ERK1/2 and VEGF as compared to OIR controls (P<0.05). These results suggest that CCR7/p-ERK 1/2/VEGF signaling plays an important role in OIR. CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.

P2Y6 receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 &amp; CXCL8 mRNA expression in respiratory epithelium

One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-β, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (β-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y6R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca2+ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y6 purinergic receptors.

Expression of Ecto-nucleoside Triphosphate Diphosphohydrolases-2 and -3 in the Enteric Nervous System Affects Inflammation in Experimental Colitis and Crohn's Disease.

Recent studies have suggested that the enteric nervous system can modulate gut immunity. Ecto-nucleoside triphosphate diphosphohydrolases [E-NTPDases] regulate purinergic signalling by sequential phosphohydrolysis of pro-inflammatory extracellular adenosine 5'-triphosphate [ATP]. Herein, we test the hypothesis that E-NTPDases modulate gut inflammation via neuro-immune crosstalk. We determined expression patterns of NTPDase2 and NTPDase3 in murine and human colon. Experimental colitis was induced by dextran sodium sulphate [DSS] in genetically engineered mice deficient in NTPDase2 or NTPDase3. We compared plasma adenosine diphosphatase [ADPase] activity from Crohn's patients and healthy controls, and linked the enzyme activity to Crohn's disease activity. NTPDase2 and -3 were chiefly expressed in cells of the enteric nervous system in both murine and human colon. When compared with wild type, DSS-induced colitis was exacerbated in Entpd2, and to a lesser extent, Entpd3 null mice as measured by disease activity score and histology, and marked anaemia was seen in both. Colonic macrophages isolated from Entpd2 null mice displayed a pro-inflammatory phenotype compared with wild type. In human plasma, Crohn's patients had decreases in ADPase activity when compared with healthy controls. The drop in ADPase activity was likely associated with changes in NTPDase2 and -3, as suggested by inhibitor studies, and were correlated with Crohn's disease activity. NTPDase2 and -3 are ecto-enzymes expressed in the enteric nervous system. Both enzymes confer protection against gut inflammation in experimental colitis and exhibit alterations in Crohn's disease. These observations suggest that purinergic signalling modulated by E-NTPDases governs neuro-immune interactions that are relevant in Crohn's disease.

A putative adenosine kinase family protein possesses adenosine diphosphatase activity

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as "adenosine-specific kinase" family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the "adenosine-specific kinase" family and suggests a need to reexamine its functional annotation.

Formononetin, an active compound of Astragalus membranaceus (Fisch) Bunge, inhibits hypoxia-induced retinal neovascularization via the HIF-1α/VEGF signaling pathway.

BackgroundIt has been reported that formononetin (FMN), one of the main ingredients from famous traditional Chinese medicine “Huang-qi” (Astragalus membranaceus [Fisch] Bunge) for Qi-tonifying, exhibits the effects of immunomodulation and tumor growth inhibition via antiangiogenesis. Furthermore, A. membranaceus may alleviate the retinal neovascularization (NV) of diabetic retinopathy. However, the information of FMN on retinal NV is limited so far. In the present study, we investigated the effects of FMN on the hypoxia-induced retinal NV and the possible related mechanisms.Materials and methodsThe VEGF secretion model of acute retinal pigment epithelial-19 (ARPE-19) cells under chemical hypoxia was established by the exposure of cells to 150 μM CoCl2 and then cells were treated with 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1, a potent HIF-1α inhibitor, 1.0 μg/mL) or different concentrations of FMN (0.2 μg/mL, 1.0 μg/mL, and 5.0 μg/mL). The supernatants of cells were collected 48 hours later to measure the VEGF concentrations, following the manufacturer’s instruction. The mRNA expressions of VEGF, HIF-1α, PHD-2, and β-actin were analyzed by quantitative reverse transcription polymerase chain reaction, and the protein expressions of HIF-1α and PHD-2 were determined by Western blot analysis. Furthermore, the rats with retinopathy were treated by intraperitoneal administration of conbercept injection (1.0 mg/kg) or FMN (5.0 mg/kg and 10.0 mg/kg) in an 80% oxygen atmosphere. The retinal avascular areas were assessed through visualization of the retinal vasculature by adenosine diphosphatase staining and hematoxylin and eosin staining.ResultsFMN can indeed inhibit the VEGF secretion of ARPE-19 cells under hypoxia, downregulate the mRNA expression of VEGFA and PHD-2, and decrease the protein expression of VEGF, HIF-1α, and PHD-2 in vitro. Furthermore, FMN can prevent hypoxia-induced retinal NV in vivo.ConclusionFMN can ameliorate retinal NV via the HIF-1α/VEGF signaling pathway, and it may become a potential drug for the prevention and treatment of diabetic retinopathy.

Bevacizumab treatment reduces retinal neovascularization in a mouse model of retinopathy of prematurity.

To evaluate the effect of different bevacizumab concentrations on retinal neovascularization in a retinopathy of prematurity (ROP) mouse model. A total of 60 of C57BL/6 J mice were exposed to 75%±2% oxygen from postnatal d7 to postnatal d12. Fifteen nonexposed mice served as negative controls (group A). On d12, 30 mice (group C) were injected with 2.5 µg intravitreal bevacizumab (IVB), 30 mice (group D) were injected with 1.25 µg IVB in one eye. The contralateral eyes were injected with balanced salt solution (BSS) (control group=group B). The adenosine diphosphatase (ADPase) histochemical technique was used for retinal flat mount to assess the oxygen-induced changes of retinal vessels. Neovascularization was quantified by counting the endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina. Histological changes were examined by light microscopy. The mRNA levels of vascular endothelial growth factor (VEGF) were quantified by Real-time PCR. Western-blotting analysis was performed to examine the expression of P-VEGFR. Comparing with the control group B, regular distributions and reduced tortuosity of vessels were observed in our retinal flat mounts in groups C and D. The endothelial cell count per histological section was lower in groups C (P<0.0001) and D (P<0.0001) compared with the control group B. Histological evaluation showed no retinal toxicity in any group. In all oxygen treated groups VEGF mRNA expression was significantly increased as compared to age-matched controls. No significant change in VEGF mRNA expression could be achieved in either of the treatments or the oxygen controls. The results of the Western blot were consistent with that of the Real-time PCR analysis. An intravitreal injection of Bevacizumab is able to reduce angioproliferative retinopathy in a mouse model for oxygen-induced retinopathy.

Platelet Function and Hyperglycemia in Acute Coronary Syndrome

The literature provides strong evidence showing that platelet function is altered in patients with diabetes mellitus (DM) and that the platelet hyperactivation associated with hyperglycemia is produced by a number of thromboxane A2-independent pathways, such as those responsible for the release of adenosine diphosphatase, thrombin, epinephrine, and von Willebrand factor, among others. Being that they are independent of thromboxane A2, none of these platelet activation pathways are affected by the potential beneficial effect of the classical antiplatelet therapy with acetylsalicylic acid (ASA) or more modern compounds like clopidogrel. For this reason, the search for novel therapeutic approaches designed to control platelet reactivity, especially in diabetic individuals and not only in atherosclerosis not associated with DM, has been an object of interest in recent years, particularly if we take into consideration that diabetics have a certain resistance to the antiplatelet effect of ASA. Diabetic thrombocytopathy is due to the activation of multiple pathways that play a role in platelet function, the most relevant of which are the modification of platelet membrane components, the alteration of calcium and magnesium homeostasis (increasing and decreasing mobilization, respectively), increases in arachidonic acid metabolism and in thromboxane A2 synthesis, a decrease in prostacyclin and nitric oxide synthesis, and increased expression of certain adhesion molecules such as glycoprotein IIb/IIIa (GPIIb/IIIa) and P-selectin. The platelets of patients with DM, both type 1 and type 2, exhibit increased reactivity in early stages of the disease, and these phenomena are relevant because they contribute to the excessive rates of cardiovascular morbidity and mortality associated with this disease. Thus, the knowledge of these plateletrelated phenomena from the pathophysiological point of view, and of their eventual normalization, has been of great interest; in fact, the clinical practice guidelines on DM have incorporated, to a greater or lesser extent, recommendations concerning antiplatelet therapy. The platelets of diabetic patients express greater amounts of adhesion molecules such as activated GPIIb/IIIa, lysosomal GP53, thrombospondin, and P-selectin. In addition to supporting greater platelet aggregability, this may play a role in

Hydrogen Peroxide Accumulation in the Choroid During Intermittent Hypoxia Increases Risk of Severe Oxygen-Induced Retinopathy in Neonatal Rats

Extremely low gestational age neonates (ELGANs) requiring oxygen therapy often experience frequent episodes of intermittent hypoxia (IH) and are at high risk for severe retinopathy of prematurity (ROP). Using an established model for oxygen-induced retinopathy (OIR), we examined the hypothesis that there is a critical number of daily brief IH episodes which will result in irreversible retinal oxidative damage. Newborn rats were exposed to increasing daily clustered IH episodes (12% O₂ with 50% O₂) from postnatal day (P) 0 to P7 or P0 to P14, or placed in room air (RA) until P21 following 7- or 14-day IH. RA littermates at P7, P14, and P21 served as controls. A group exposed to constant 50% O₂ (CH) served as a second control. Blood gases, eye opening at P14, retinal, and choroidal oxidative stress and lipid peroxidation (8-isoPGF(2α)), oxidants (H₂O₂) and antioxidants (catalase and SOD), retinal pathology (adenosine diphosphatase (ADPase)-stained retinal flatmounts), and mitochondria-related genes were assessed. pO₂ levels were higher with increasing IH episodes and remained elevated during the reoxygenation period. High SO₂ levels were associated with most severe OIR. Levels of all measured biomarkers peaked with six IH episodes and decreased with 8 to 12 episodes. H₂O₂ accumulated in the choroid during the reoxygenation period with irreversible retinal damage. Our data suggest that six is the maximum number of IH episodes that the retina can sustain. Accumulation of H₂O₂ in the choroid may result in high levels being delivered to the entire retina, ultimately resulting in irreversible retinal oxidative damage.

Peroxisome Proliferator-Activated Receptor-β/δ Regulates Angiogenic Cell Behaviors and Oxygen-Induced Retinopathy

To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR). HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining. GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV. PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.

Expression and function of Delta-like ligand 4 in a rat model of retinopathy of prematurity.

The Delta-like ligand 4/Notch signaling pathway was shown to participate in the process of retinal development and angiogenesis. However, the function of the Delta-like ligand 4/Notch signaling pathway in retinopathy of prematurity requires further study. Retinopathy of prematurity was induced in 5-day-old Sprague-Dawley rats exposed to hyperoxia for 7 days, and then returned to room air. Reverse transcription-PCR and western blot revealed that Delta-like ligand 4 levels decreased at postnatal day 12 and increased at postnatal day 17 in retinopathy of prematurity rats. Flat-mounted adenosine diphosphatase stained retina and hematoxylin-eosin stained retinal tissue slices showed that the clock hour scores and the nuclei counts in retinopathy of prematurity rats were significantly different compared to normal control rats. After retinopathy of prematurity rats were intravitreally injected with Delta-like ligand 4 monoclonal antibody to inhibit the Delta-like ligand 4/Notch signaling pathway, there was a significant increase in the severity of retinal neovascularization (clock hours) in the intravitreally injected eyes. The nuclei count was highly correlated with the clock hour score. These results suggest that Delta-like ligand 4/Notch signaling plays an essential role in the process of physiological and pathological angiogenesis in the retina.

Atherosclerosis and atherosensitivity in two southwest Algerian desert rodents, Psammomys obesus and Gerbillus gerbillus, and in Rattus norvegicus.

Cardiovascular disease, including atherosclerosis, is the leading cause of death in patients with diabetes worldwide; thus, it is a major medical concern. The endothelium contributes to the control of many vascular functions, and clinical observations show that it is a primary target for diabetic syndrome. To get better insight into the mechanisms underlying atherosclerosis, we studied the interspecific differences in the arterial metabolisms of two, Psammomys obesus and Gerbillus gerbillus, as well as Rattus norvegicus (Wistar rat), well known for its atheroresistance. Twenty-two enzymatic activities and six macromolecular substances were histochemically compared in the two desert species and in Wistar aortas (abdominal and thoracic) and arteries (femoral and caudal) embedded in a common block. In the healthy adult rodents, enzyme activities were very intense. They demonstrated that aortic myocytes are capable of various synthesis and catabolism processes. However, considering the frequency of atherosclerosis and its phenotypes, significant differences appeared between the species studied. Our comparative study shows that aortic atherosensitive animals have several common metabolic characteristics, which are found in Psammomys rich in metachromatic glycosaminoglycans (involved in the inhibition of lipolysis and in calcification of the organic matrix), reduced activity in enzymes related to the Krebs cycle (weakening energetic power), and low lipolytic enzyme, adenosine triphosphatase, and adenosine diphosphatase activities. However, the most fundamental pathophysiological difference is the low lipolytic power of the aorta of Psammomys when compared to Wistar rats. This characteristic determines its atherosensitivity and makes this animal model more applicable to the experimental development of atherosclerosis.

Effects of Subconjunctival Injection of Anti-Vascular Endothelial Growth Factor Antibody on Oxygen-Induced Ischemic Retinopathy in a Neonatal Rat Model

The present study investigated the effects of subconjunctival injections of an anti-rat vascular endothelial growth factor (anti-VEGF) antibody on oxygen-induced retinopathy (OIR) in a neonatal rat model. OIR was induced by daily cycles of 80% oxygen (20.5h), room air (0.5h), and a progressive return to 80% oxygen (3h) for 12 days [until postnatal day (P) 12]. On P12, rats received subconjunctival injections in their right eye of 0.1 or 1.0μg anti-VEGF antibody (or 1.0μg goat IgG as a control). No injections were made into the left eye. On P18, rats were killed and their retinas were removed and flat-mounted before being stained with adenosine diphosphatase. Retinal neovascularization (NV) was scored and the extent of avascular areas, as a percentage of total retinal area (%AVA), was determined using image analysis. Although there was a tendency for lower mean NV scores in eyes injected with 0.1 and 1.0μg anti-VEGF compared with control (4.3±1.1, 2.3±1.0, and 6.7±1.3, respectively; n=10–13), the difference failed to reach statistical significance. Similarly, although there was a tendency for mean %AVA to be lower in the injected eyes for both the 0.1 and 1.0μg anti-VEGF groups compared with control (15±3%, 13±3%, and 25±4%, respectively; n=10–13), the differences were not significant. Similar tendencies were observed in the contralateral eyes. Although further studies using larger numbers of rats are needed to obtain statistically significant results, the results of the present study suggest that the subconjunctival injection of anti-VEGF antibody may prove to be a useful route of administration in conjunction with intravitreal injections, which are the generally used method at present. However, careful attention should be paid to the possibility of systemic side effects.

Efficacy of Troxerutin on Streptozotocin-induced Rat Model in the Early Stage of Diabetic Retinopathy

The vascular changes associated with early diabetic retinopathy, which include the formation of microaneurysms and acellular capillaries, vessel dilation, vascular endothelial growth factor expression, were investigated experimentally in streptozotocin-induced diabetic rats treated with antioxidants: troxerutin (trihydroxy-ethylrutoside, CAS 7085-55-4), Vaccinium myrtillus, and calcium dobesilate (hydroquinone calcium sulfonate, CAS 20123-80-2). The development and progression of retinopathy was followed using fundus photography. After 3 months, the rats were sacrificed and half of the eyes were prepared for neovascularization analysis, and the other half were used for VEGF (vascular endothelial growth factor) analysis. The results from fundus photography and ADPase (adenosine diphosphatase) staining were quantified by the percentage area of the retinal vasculature using a commercial image analyzer. The VEGF protein in the retinal homogenates was assessed using an ELISA (enzyme linked immunosorbent assay) kit and VEGF-mRNA by RT-PCR (reverse transcription polymerase chain reaction). In the ADPase stain, the retinal vascular percent area increased significantly in the diabetic control. Neovascularization and aneurysms were observed in the diabetic control and were attenuated by 50 mg/kg troxerutin, but the retinal vascular percentage area was not significantly different from the diabetic control. The VEGF protein concentration was higher in diabetic rats than in the nondiabetic rats (21.5 +/- 2.1 vs 27.7 +/- 5.8 pg/mg, p < 0.05), and this increase was attenuated by 10 mg/kg troxerutin (24.5 +/- 3.8 pg/mg, p < 0.05) and prevented by 50 mg/kg troxerutin (19.5 +/- 2.2 pg/mg, p < 0.05). However, there were no significant differences between the groups. The VEGF-mRNA density showed a increasing tendency by 20% in the diabetic rats compared with the non-diabetic rats (1.0 +/- 0.1 vs 1.2 +/- 0.1 VEGF/beta-actin), and this increase was corrected by 10 mg/kg troxerutin (1.0 +/- 0.1 VEGF/beta-actin), 50 mg/kg troxerutin (0.9 +/- 0.1 VEGF/beta-actin) and Vaccinium myrtillus (1.1 +/- 0.1 VEGF/beta-actin). Oxidative stress might be involved in the upregulation of retinal VEGF during early diabetes, and it is likely that troxerutin has comparatively effective antioxidant properties. Therefore, troxerutin might be a useful treatment for attenuating diabetic retinopathy.

Expression and significance of hypoxia-inducible factor-1α in retinal neovascularization induced by metabolic acidosis in newborn rats

Objective To investigate the expression and significance of hypoxia-inducible factor-1a （HIF -1a） in the retinal neovascularization by metabolic acidosis in newborn rats. Methods One hundred and twenty newborn SD rats were randomly divided into acidosis （experiment） and normoxia （control） groups. A total of 60 newborn rats in experiment group underwent tubal feeding of NH4Cl at the dose of 535 mg/（kg · d） from the second day after birth, twice a day for 6 days and followed by a period of recovery. The rats in the two groups were sacrificed at the 3rd, 5th, 8th, 10th, 13th and 20th day after birth, respectively. The morphologic changes of retinal vessels were estimated by observing the vascular pattern in adenosine diphosphatase stained retina flat mounts. The newborn vessels were quantified by HE staining. Immunohistochemieal method was used to detect HIF-1a expression. Results In experiment group, numerous neovascufarization and un-perfused area at the periphery of vessels occurred on the 10th day. The result of HE staining showed that in experiment group of 10 day old, the number of neovascular nuclei extending into the vireo was 28. 78± 7.53, and that of the control group was 1.22 ±1.48 （t= 11. 169,P〈0. 01）. The results of immunohistochemistry revealed that the expression of HIF-1a protein were stronger in the experiment group than in the control group on the 8th, 10th and 13th day, and there were significant differences between the two groups （108. 87±15.21, 183. 68 ± 26.58 and 129.42 ± 9.85 vs 74.98±4.50, 76.38± 3.38 and 74.78±1.86, t=4.625, 9.023 and 9. 672,P〈0.05）. Conclusions HIF-1a might play an important role in retinal neovascularization. Key words: Retinal neovascularization; Hypoxia-inducihle factor 1, alpha subunit; Acidosis; Animals, newborn; Rats

The effects of Buyang Huanwu Decoction on hemorheological disorders and energy metabolism in rats with coronary heart disease

Buyang Huanwu Decoction (BYHWD), a traditional Chinese medicine (TCM) formula, has been recognized as a clinical treatment for coronary heart disease (CHD) with qi deficiency and blood stasis syndrome. The effects of BYHWD on hemorheological disorders and energy metabolism in CHD with qi deficiency and blood stasis syndrome are still unclear. To investigate whether the ameliorative effects of BYHWD on CHD rats with qi deficiency and blood stasis syndrome are associated with the regulation of hemorheological disorders and energy metabolism. The rats were lavaged with 25.68, 12.84 and 6.42 g/kg BYHWD (g weight of mixed crude drugs/kg body weight), respectively, once a day for 21 days. The body weight, exhaustive swimming time and tongue characters were observed and recorded. The whole blood viscosity and plasma viscosity were determined by hematology analyzer. The level of fibrinogen (Fbg) in plasma was determined by using Fbg assay kit. The platelet aggregation induced by adenosine diphosphatase was measured by semi-automatic whole blood platelet analyzer. The level of blood glucose (BG) was determined by LifeScan. The activity of Na(+)-K(+)-ATPase in heart tissues was detected by spectrophotometer. BYHWD improved the exterior signs of qi deficiency and blood stasis syndrome in rats with CHD, including the body weight, exhaustive swimming time and tongue quality. The whole blood viscosity in rats treated with 25.68 g/kg BYHWD decreased at the shear rate of 10s(-1) (P<0.05) and the plasma viscosity decreased in rats treated with 25.68 and 12.84 g/kg BYHWD (P<0.05). The plasma Fbg level and the platelet aggregation decreased in rats treated with 25.68 g/kg BYHWD (P<0.01). The results also revealed that the BG level decreased and the Na(+)-K(+)-ATPase activity in heart tissues increased in rats treated with 25.68 and 12.84 g/kg BYHWD (P<0.01). The results suggest that the ameliorative effects of BYHWD on CHD rats with qi deficiency and blood stasis syndrome are mediated by the improvement of hemorheological disorders and energy metabolism.