Background: Immunoglobulins(Igs)play an important role in host defence and were initially thought to be expressed solely by B cells. However, recent data suggest that Igs are also expressed in other lineages. Recently, Ig transcripts were detected in cardiomyocytes, but whether the functional Ig protein is expressed by cardiomyocytes has not been thoroughly elucidated.Methods: Gene Expression Omnibus (GEO) microarray database analysis was used to analyse IgM heavy chain expression in the myocardium of mice. Immunohistochemistry (IHC), ELISA and Western blot were used to identify IgM in cardiomyocytes of both Balb/c mice and mu MT mice (B cell-deficient mice), as well as in cultured cardiomyocytes of neonatal mice and in the myocardial cell line HL-1.Moreover, RT-PCR and cDNA sequencing were used to determine the VDJ rearrangement of the IgM heavy chain.Results: In this study, we first analysed transcription of the IgM heavy chain in heart tissue in mice by mining the GEO database, and we observed that IgM heavy chain transcripts were expressed in heart tissues. Subsequently, IgM was found to be expressed in cardiomyocytes in mice; the IgM was primarily localized on the cell membranes and intercalated discs of murine heart cells and in the cytoplasm and cell membranes of isolated cardiomyocytes and HL-1. Importantly, the functional IgM heavy chain transcripts exhibit a unique VDJ rearrangement pattern. Furthermore, IgM can be secreted and deposited in the extracellular space of the myocardium under ischaemic/hypoxic conditions.Conclusions: Our data indicate for the first time that IgM is expressed by cardiomyocytes in mice and suggest that its physiological function warrants further investigation.

Background: Choroidal neovascularization (CNV), also known as subretinal neovascularization, causes serious damage to the central vision as it happens more commonly in macula. The most important factor involved in angiogenesis is vascular endothelial growth factor (VEGF). By an RNAi technique, VEGF gene knockdown can be used to treat CNV. PEG-conjugated poly (amidoamine) (PEG-PAMAM) dendrimers as a new type of synthetic polymers are very promising to be gene delivery carriers.Methods: To investigate siRNA delivery efficacy of PEG-PAMAM dendrimers, we prepared dendriplexes of PEG-PAMAM dendrimers with a fluorescence-labelled siRNA (PEG-PAMAM/FAM siRNA) or VEGF siRNA (PEG-PAMAM/VEGF siRNA), and studied transfection and downregulation efficacy of the dendriplexes in a cobalt chloride (CoCl2)-induced neovascularization model in retinal vascular endothelial cells (RF/6A).Results: Our results demonstrate that PEG-PAMAM dendrimers had significantly higher transfection efficiency to FAM siRNA than a commercial transfection reagent PEI (1.4-fold, P<0.001) measured by flow cytometry. Compared to the PEI/VEGF siRNA polyplexes, the dendriplexes of the PEG-PAMAM/VEGF siRNA more significantly downregulated VEGF gene expression (P < 0.01) at both mRNA and protein expression level. A tube formation assay also proved that the PEG-PAMAM/VEGF siRNA dendriplexes more significantly inhibited vascular-like formation than PEI/VEGF siRNA did (P < 0.001) in RF/6A.Conclusion: This study demonstrated that G5-PEG was more efficient than PEI in facilitating siRNA delivery, downregulating VEGF expression and inhibiting vascular-like formation on RF/6A.

PURPOSE:Recent research has provided novel but contrary insight into the function of Slit2-Robo signaling in angiogenesis. Although the role of Robo in choroidal neovascularization (CNV) has been studied, the effect of its ligand, Slit2, on CNV development is unclear. This study investigated the role of endogenous Slit2 in CNV and the possible mechanisms.METHODS:Laser-induced CNV in Slit2 transgenic and wild-type mice was used to study the effects of endogenous Slit2 on angiogenesis in vivo. Fluorescein angiography was performed to evaluate the leakage area of each lesion. Plasmid-based gene transfer technology was used to increase Slit2 expression and to study its effects on human umbilical vein endothelial cells (HUVECs) in vitro. Cell proliferation, migration, and tube formation were assessed. Quantitative real-time PCR and Western blot were used to measure expression in the extracellular signal-related kinase 1/2 (ERK1/2), protein kinase B (AKT), and p38 mitogen-activated protein kinase (p38 MAPK) molecular pathways.RESULTS:Laser treatment led to more CNV and vascular leakage in Slit2 transgenic mice compared with wild-type mice. Upregulation of Slit2, Robo1, VEGF receptor 2 (VEGFR2), and phosphorylated ERK1/2 (p-ERK1/2) were detected in retina and choroidal tissue of laser-treated transgenic mice. After transfection of HUVECs with a Slit2 overexpression plasmid, cell proliferation, migration, and tube formation capacities were promoted. Slit2, Robo1, VEGFR2, and p-ERK1/2 were elevated in transfected HUVECs.CONCLUSION:Slit2 overexpression promoted angiogenic effects in both a laser-induced CNV mouse model and HUVECs and promoted the biological activity of endothelial cells. Slit2 may promote angiogenesis by upregulating Robo1 and activating the VEGFR2-ERK1/2 pathway.

Objective To assess associations between the aqueous humour concentration of interleukin IL-1β, IL-6, IL-8, IL-10 and IL-12p, tumor necrosis factor α (TNF-α) and vascular endothelial growth factor (VEGF) and axial length in eyes with cataract. Methods The hospital-based investigation included patients who underwent cataract surgery between March 2014 and April 2014. Using aqueous humour collected at the start of cataract surgery, the interleukins IL-1β, IL-6, IL-8, IL-10 and IL-12p, TNF-α and VEGF were examined using a cytometric bead array. Axial length was determined by partial coherence laser interferometry (IOL Master). Results The study included 33 patients with cataract (33 eyes) with a mean age of 69.2±10.8 years (range:50–87 years) and a mean axial length of 24.7±1.9 mm (range:22.6–31.5 mm). Lower aqueous concentration of VEGF was significantly associated with longer axial length (VEGF concentration (pg/mL) = -5.12 x Axial Length (mm) + 163; correlation coefficient r = -0.41; P<0.001) and more myopic refractive error (VEGF concentration (pg/mL) = 1.27xspherical equivalent (diopters)+44.8; r = 0.383; P = 0.002). The aqueous concentrations of all other substances were not significantly (all P>0.10) associated with axial length or refractive error. Conclusions Higher intravitreal concentrations of VEGF were measured in eyes with a longer axial length, while the intraocular concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12p and TNF-α were not correlated with axial length. The lower concentration of VEGF in axially elongated eyes may be one of the reasons for the lower prevalence of age-related macular degeneration and diabetic retinopathy in myopic eyes.

Citation: Li S, Huang L, Sun Y, et al. Slit2 promotes angiogenic activity via the Robo1-VEGFR2-ERK1/2 pathway in both in vivo and in vitro studies. Invest Ophthalmol Vis Sci. 2015;56:5210–5217. DOI:10.1167/ iovs-14-16184 PURPOSE. Recent research has provided novel but contrary insight into the function of Slit2Robo signaling in angiogenesis. Although the role of Robo in choroidal neovascularization (CNV) has been studied, the effect of its ligand, Slit2, on CNV development is unclear. This study investigated the role of endogenous Slit2 in CNV and the possible mechanisms.

PURPOSEAge-related macular degeneration (AMD) is the main cause of visual impairment and legal blindness in older individuals. COL8A1 rs13095226 variants have recently been implicated associated with neovascular age-related macular degeneration (nAMD) and Polypoidal Choroidal Vasculopathy (PCV) in American studies. The aim of this study was to investigate the association between the COL8A1 rs13095226 Polymorphism and neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in Chinese people.METHODS900 Chinese subjects-300 cases with nAMD, 300 cases with PCV and 300 controls, were enrolled in a cross-sectional observational study. The diagnoses of nAMD and PCV were confirmed by Fundus photography, Fluorescence Fundus Angiography (FFA) and Indocyanine Green Angiography (ICGA). Genomic DNA was extracted from venous blood leukocytes and genotypes of rs13095226 were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Differences in allele distribution between cases and controls were tested by chi-square tests, with age and gender adjusted by logistic regression analysis.RESULTThe COL8A1 rs13095226 polymorphism was not statistically significantly different from the nAMD or PCV to the normal controls (P>0.05) in Chinese Population. The association remained insignificant after adjustment for age and gender differences (P>0.05).CONCLUSIONSThis case-control study indicated that the COL8A1 rs13095226 polymorphism is not associated with nAMD or PCV, which suggesting this gene maybe not a susceptibility gene locus for nAMD or PCV in Chinese subjects.

PURPOSE To evaluate the relationship between aqueous inflammation cytokines and cytomegalovirus (CMV) particles in patients with cytomegalovirus retinitis (CMVR), and evaluate the changes in aqueous inflammation cytokines during multiple intravitreal injections of antiviral drugs for CMVR. METHODS There were 10 patients (12 eyes; 16 courses of treatment per eye) who underwent continued intravitreal ganciclovir or foscarnet for treatment of CMVR. Before each intravitreal injection, 50-100 μL of aqueous humor was removed and sent to the laboratory to examine the concentration of the CMV DNA load by using polymerase chain reaction and to examine the concentration of interleukin (IL)-1β, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and IL-12p70 using a cytometric bead array. RESULTS A Kendall correlation test showed that the concentration of the CMV DNA load in the aqueous humor was significantly associated with the aqueous level of IL-6 (P<0.001, r=0.327) and IL-8 (P<0.001, r=0.381), but not significantly associated with IL-1β, IL-10, IL-12p70, and TNF-α. The boxplots showed that the concentration of the aqueous CMV DNA load, IL-8 and IL-10 continuously declined after multiple intravitreal injections of antiviral drugs, and the decline trend of IL-8 was most remarkable. IL-1β, IL-10, TNF-α, and IL-12p70 were negative in some of the aqueous levels of CMVR patients throughout the course of treatment (25.0%-62.5%). CONCLUSIONS Our study showed that IL-8 was significantly associated with the aqueous level of CMV copies and continuously declined during a course of treatment that involved multiple intravitreal injections of antiviral drugs. IL-8 may be considered a good quantitative laboratory indicator of the recovery of CMVR.

Currently there is no material clinically available as a long-term vitreous substitute. In this study, an insitu gelation system based on alpha-poly(ethylene glycol) methacrylate (alpha-PEG-MA) and a redox-initiated radical polymerization/crosslinking reaction was evaluated for this purpose. Ammonium persulfate CAPS) and N,N,N',N'-tetramethyl ethylene diamine (TMEDA) were used as initiators. The gelation time, rheological properties, reaction kinetics and swelling profiles were studied in detail and the system with 10 wt% of alpha-PEG-MA and 8 mM APS/TMEDA was chosen as the optimal material for in vivo studies. Using the rabbit as the animal model, we showed that the system did form a space-filling and transparent gel in the vitreous cavity, and the inflammation response could be controlled to an acceptable level. (C) 2014 Elsevier Ltd. All rights reserved.

Purpose: rs1999930 and rs4711751 have recently been identified as novel variants associated with advanced age-related macular degeneration (AMD) in populations of European ancestry. We aimed to investigate whether these two single nucleotide polymorphisms (SNPs) were associated with neovascular AMD (nAMD) or with polypoidal choroidal vasculopathy (PCV), a variant of AMD in Asians, using a Chinese case-control study. Methods: A total of 900 subjects, including 300 controls, 300 cases with nAMD and 300 cases with PCV, were included in the present study. Genomic DNA was extracted from venous blood leukocytes. The allelic variants of rs1999930 and rs4711751 were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differences in allele distribution between cases and controls were tested by a χ2 test, with additional adjustments for age and gender using logistic regression. The statistical power was also calculated. Values of p < 0.05 were considered statistically significant. Results: No statistically significant association was observed between the two polymorphisms of nAMD or PCV phenotype (p > 0.05 for all comparisons). The difference remained insignificant after correction for age and gender (p > 0.05 for all comparisons). The statistical powers to detect the association between these two SNPs and nAMD or PCV range from 0.05 to 0.36, assuming conventional levels of statistical significance. Conclusions: In the present study, we could not replicate the reported association of these two SNPs and either nAMD or PCV in a Chinese population, suggesting that they are unlikely to be a major AMD and PCV susceptibility gene locus in the Chinese population. Considering the low power value, a large sample size is required to draw more reliable conclusions.

Purpose Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES). Methods M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection. Results The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models. Conclusion The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.

PURPOSE:To investigate whether 11 variants in complement factor H gene contributed differently in patients with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) of Chinese descent.METHODS:We performed a case-control study in a group of Chinese patients with nAMD (n = 344) or PCV (n = 368) and contrasted the results against an independent control group comprising 511 mild cataract patients without any evidence of age-related maculopathy. Association analysis of allele and genotype frequencies was performed for 11 haplotype-tagging single-nucleotide polymorphisms (SNPs) at the CFH locus (rs1061170, rs1329428, rs1410996, rs2284664, rs375396, rs529825, rs551397, rs7540032, rs800292, rs2274700, and rs1065489). Multinomial logistic regression analyses were performed to estimate and compare the effect of these 11 CFH polymorphisms on AMD and PCV, using the wild-type genotype as reference. Differences in the observed genotypic distributions between cases and controls were tested by using χ(2) tests, with age and sex adjusted for using logistic regression.RESULTS:CFH rs1065489 was not significantly associated with the nAMD phenotype in Chinese collections either on univariate or multivariate analysis (P > 0.05 for all comparisons). The other 10 SNPs of CFH were significantly associated with the nAMD phenotype. As for PCV, all 11 SNP markers were significantly associated with risk of PCV before or after correction for age and sex differences. Eight of the 11 SNP markers showed significant evidence of heterogeneity between AMD and PCV (P < 0.05 for all comparisons).CONCLUSIONS:Our data suggest that the genetic architecture at the CFH locus is complex with some markers showing significant skewing of the genotypes toward nAMD or PCV in Asians. This further supports the clinical observation that nAMD and PCV could have distinct pathogenesis mechanisms, which will require larger studies to accurately dissect.

Zwitterionic polymers have shown ultra-low biofouling properties at surfaces and excellent biocompatibility as implant. In this study, an in situ-forming zwitterionic hydrogel was designed and evaluated, both in vitro and in vivo, as a long-term vitreous substitute. The zwitterionic polymer poly(MPDSA-co-AC) was designed as a copolymer of the sulfobetaine methacrylamide and acryloyl cystamine monomers, providing the zwitterionic components and the thiol functional groups, respectively. The in situ gelation was via the thiol-ene Michael addition reaction with α-PEG-MA as the crosslinker. The gelation time, rheological properties, swelling profiles, and the transparency of zwitterionic hydrogels were studied in detail. Two systems with different crosslinker concentrations were tested in a rabbit model, and the one with the thiol-ene ratio of 2 : 1 showed excellent biocompatibility in vivo, formed space-filling hydrogels and remained transparent in the vitreous cavity for the 2 month implantation period. Therefore, in situ-forming zwitterionic hydrogels represent a promising material system as a vitreous substitute and possibly for other soft tissue replacements.

Purpose: rs1999930 and rs4711751 have recently been identified as novel variants associated with advanced age-related macular degeneration (AMD) in populations of European ancestry. We aimed to investigate whether these two single nucleotide polymorphisms (SNPs) were associated with neovascular AMD (nAMD) or with polypoidal choroidal vasculopathy (PCV), a variant of AMD in Asians, using a Chinese case-control study. Methods: A total of 900 subjects, including 300 controls, 300 cases with nAMD and 300 cases with PCV, were included in the present study. Genomic DNA was extracted from venous blood leukocytes. The allelic variants of rs1999930 and rs4711751 were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differences in allele distribution between cases and controls were tested by a χ2 test, with additional adjustments for age and gender using logistic regression. The statistical power was also calculated. Values of p < 0.05 were considered statistically significant. Results: No statistically significant association was observed between the two polymorphisms of nAMD or PCV phenotype (p > 0.05 for all comparisons). The difference remained insignificant after correction for age and gender (p > 0.05 for all comparisons). The statistical powers to detect the association between these two SNPs and nAMD or PCV range from 0.05 to 0.36, assuming conventional levels of statistical significance. Conclusions: In the present study, we could not replicate the reported association of these two SNPs and either nAMD or PCV in a Chinese population, suggesting that they are unlikely to be a major AMD and PCV susceptibility gene locus in the Chinese population. Considering the low power value, a large sample size is required to draw more reliable conclusions.

PURPOSE:Age-related macular degeneration (AMD) and Alzheimer disease (AD) are age-related neurodegenerative diseases that share similar environmental risk factors, cellular pathologies, and genetic backgrounds. Recently, the rs2075650 single nucleotide polymorphism in the translocase of outer mitochondrial membrane 40 homolog (TOMM40) gene was identified as a risk factor for AMD and Alzheimer disease. We aimed to examine the associations between the TOMM40 rs2075650 polymorphism and neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a Chinese population.METHODS:The study consisted of 900 subjects, including 300 controls, 300 cases with nAMD, and 300 cases with PCV. Genomic DNA was extracted from venous blood leukocytes. The allelic variant of rs2075650 was determined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Differences in the observed genotypic distributions between the case and control groups were tested using chi-square tests, with age and gender adjusted using logistic regression analysis.RESULTS:The TOMM40 rs2075650 polymorphism was not statistically significantly associated with the nAMD or PCV phenotype (p>0.05). The difference remained insignificant after correction for age and gender differences based on the logistic regression models (p>0.05).CONCLUSIONS:Our data provide no evidence to support an association of rs2075650 in TOMM40 with nAMD or PCV, suggesting that this gene is unlikely to be a major AMD and PCV susceptibility gene locus in the Chinese population.

Z E R S R S O O β D 1 O O β D 2 O O β D 3 4 11 Lagerstroemia balansae

Radix Astragali and Radix Hedysari, two famous traditional Chinese medicines (TCM), were considered to possess the same efficacy in TCM traditionally. However, modern pharmacological and phytochemical investigations showed that they were different in efficacy and chemical constituents to some degree. In this study, the principal constituents of the two drugs, isoflavonoids and saponins, were comparatively analysed using HPLC-UV and HPLC-ELSD methods. The results showed that there were some common compounds, especially flavonoids, in both drugs, while the differences in the types and contents of their chemical constituents, especially in saponins, were also obvious. The similarities and differences of the chemicals may be responsible, at least partially, for the similarities and differences in their efficacies or bioactivities.

OBJECTIVE To study the polyphenols of Potentilla discolor Bunge.METHODS The crude drug of Potentilla discolor was extracted with 95% ethanol and purified by repeated chromatographic methods.The structures of the isolated compounds were elucidated by physicochemical and spectral analysis.RESULTS Eleven compounds were yielded and identified as: kaempferol(1),apigenin(2),luteolin(3),quercetin(4),quercetrin(5),tribuloside(6),kaempferol-3-O-β-D-glucuronide(7),ellagic acid(8),ellagic acid-3-O-methyl ether(9),ellagic acid-3-O-methyl ether-4-O-α-L-rhamnopyranoside(10) and gallic acid(11).CONCLUSION Compound 2,3 and 9 were obtained from Potentilla discolor Bunge for the first time,and compound 9 was yielded from the genus of Potentilla for the first time.

Three new triterpenoid saponins, named julibroside J(16) (2), julibroside J(17) (3), and julibroside J(21) (4), each of which possesses an oleanane triterpenoid aglycone of acacic acid, two monoterpenoid acids, and nine sugar moieties, together with one known saponin, julibroside II (1), were isolated from the stem bark of Albizia julibrissin by chromatographic methods. Their structures were established by chemical and spectroscopic means. Saponins 1, 2, and 4 showed inhibitory activities against Bel 7402 human cancer cell line in vitro.

A phytochemical investigation of the roots of Symplocos caudata Wall (Symplocaceae) resulted in the isolation and characterization of two optical isomers of a neolignan glycoside (1) and a new cerebroside (2). Their structures were elucidated as (7R,8S)-erythro-7,9,9'-trihydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan-4-O-β-D-glucopyranoside, (7S,8R)-erythro-7,9,9'-trihydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan-4-O-β-Dglucopyranoside (1), and 1-O-β-D-glucopyranosyl-(2S,3S,4R,8Z,12E)-2-N-[(2'R)-2'-hydroxyheptacosanoyl]-8,12-docosadiene-1,3,4-triol (2), respectively, on the basis of spectroscopic data (1D and 2D NMR, MS and CD).