Tsz Kin Ng

HTRA1 variants in exudative age-related macular degeneration and interactions with smoking and CFH.

PURPOSE Mapping the genes for age-related macular degeneration (AMD) had not been successful until recent genome-wide association studies revealed Tyr402His in CFH and rs11200638 in HTRA1 as AMD-related genetic variants. This study was conducted to identify other critical factors in HTRA1 that are associated with exudative AMD. METHODS The promoter, splice regions, and coding exons of HTRA1 were sequenced in 163 patients with exudative AMD and 183 sex- and age-matched control subjects. Also documented were the CFH genotype and smoking status. RESULTS Four significant SNPs were found in the promoter and the first exon of HTRA1: rs11200638 (-625G>A), rs2672598 (-487T>C), rs1049331 (102C>T, Ala34Ala), and rs2293870 (108G>T, Gly36Gly) with respective P = 1.7 x 10(-14), 3.0 x 10(-10), 3.7 x 10(-12), and 3.7 x 10(-12). Among them, rs11200638 is the most significant associated SNP with a high odds ratio (OR) of 7.6 (95% CI: 3.94-14.51). One risk haplotype block across the promoter and exon 1, ACCTT, significantly predisposes to AMD (P = 6.68 x 10(-14)). In both models, significant independent additive effects were identified with smoking and rs800292 (184G>A, Val62Ile) of CFH. Smoking and rs11200638 (HTRA1) combined caused a 15.7-fold increased risk, whereas combined rs800292 and rs11200638 caused a 23.3-fold increased risk. An extremely high population attributable risk (PAR) of 78% was also found. CONCLUSIONS A high impact of the additive effect of CFH and HTRA1 in the development of exudative AMD was shown. The HTRA1-smoking additive effect found in this study further suggests the importance of this environmental risk factor in AMD.

Progress of mesenchymal stem cell therapy for neural and retinal diseases

Complex circuitry and limited regenerative power make central nervous system (CNS) disorders the most challenging and difficult for functional repair. With elusive disease mechanisms, traditional surgical and medical interventions merely slow down the progression of the neurodegenerative diseases. However, the number of neurons still diminishes in many patients. Recently, stem cell therapy has been proposed as a viable option. Mesenchymal stem cells (MSCs), a widely-studied human adult stem cell population, have been discovered for more than 20 years. MSCs have been found all over the body and can be conveniently obtained from different accessible tissues: bone marrow, blood, and adipose and dental tissue. MSCs have high proliferative and differentiation abilities, providing an inexhaustible source of neurons and glia for cell replacement therapy. Moreover, MSCs also show neuroprotective effects without any genetic modification or reprogramming. In addition, the extraordinary immunomodulatory properties of MSCs enable autologous and heterologous transplantation. These qualities heighten the clinical applicability of MSCs when dealing with the pathologies of CNS disorders. Here, we summarize the latest progress of MSC experimental research as well as human clinical trials for neural and retinal diseases. This review article will focus on multiple sclerosis, spinal cord injury, autism, glaucoma, retinitis pigmentosa and age-related macular degeneration.

MicroRNA-145 Regulates Human Corneal Epithelial Differentiation

Background Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium. Methodology/Principal Findings Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs. Conclusion/Significance We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

Nicotine Alters MicroRNA Expression and Hinders Human Adult Stem Cell Regenerative Potential

Adult stem cells are critical for the healing process in regenerative medicine. However, cigarette smoking inhibits stem cell recruitment to tissues and delays the wound-healing process. This study investigated the effect of nicotine, a major constituent in the cigarette smoke, on the regenerative potentials of human mesenchymal stem cells (MSC) and periodontal ligament-derived stem cells (PDLSC). The cell proliferation of 1.0 μM nicotine-treated MSC and PDLSC was significantly reduced when compared to the untreated control. Moreover, nicotine also retarded the locomotion of these adult stem cells. Furthermore, their osteogenic differentiation capabilities were reduced in the presence of nicotine as evidenced by gene expression (RUNX2, ALPL, BGLAP, COL1A1, and COL1A2), calcium deposition, and alkaline phosphatase activity analyses. In addition, the microRNA (miRNA) profile of nicotine-treated PDLSC was altered; suggesting miRNAs might play an important role in the nicotine effects on stem cells. This study provided the possible mechanistic explanations on stem cell-associated healing delay in cigarette smoking.

Differentiation of exudative age-related macular degeneration and polypoidal choroidal vasculopathy in the ARMS2/HTRA1 locus.

PURPOSE differentiate the associations of exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) with the ARMS2/HTRA1 locus. METHODS The entire ARMS2 sequence was sequenced and HTRA1 rs11200638 genotyped in 568 unrelated Chinese individuals: 156 exudative AMD patients, 164 PCV patients, and 248 controls. A meta-analysis was performed to examine the effects of rs10490924 and rs11200638 at the ARMS2/HTRA1 locus in PCV. RESULTS In total, 31 polymorphisms in ARMS2 were identified. Significant associations with both exudative AMD and PCV were observed in 11 of them and HTRA1 rs11200638, with different genotypic distributions between exudative AMD and PCV (P < 0.001). After adjusting for rs11200638, ARMS2 rs10490924 remained significantly associated with exudative AMD (P = 0.011), but not with PCV (P = 0.077). Meta-analysis showed consistent allelic associations of rs10490924 and rs11200638 with PCV in different study populations. CONCLUSIONS There is a strong and consistent association of the ARMS2/HTRA1 locus with both exudative AMD and PCV, suggesting the two disorders share, at least partially, similar molecular mechanisms. Different effect sizes indicate the existence of additional genetic and environmental factors affecting them to different extents.

Alterations in Serum Fatty Acid Concentrations and Desaturase Activities in Bietti Crystalline Dystrophy Unaffected by CYP4V2 Genotypes

PURPOSE To evaluate the serum fatty acid changes in Chinese patients with Bietti crystalline dystrophy (BCD) in association with CYP4V2 mutation. METHODS Sixteen Chinese patients with BCD confirmed with CYP4V2 mutation were recruited. Peripheral venous blood was obtained after fasting and serum fatty acid concentrations were measured and compared with those in 13 control subjects. Delta-9-desaturase and Delta-5-desaturase activities were estimated based on serum fatty acid compositions. Serum insulin and glucagon concentrations and their correlations with fatty acid and desaturase activities were also evaluated. Fatty acid concentrations were compared among patients with BCD with different genotypes or phenotypes. RESULTS Patients with BCD were found to have a significantly higher concentration of octadecanoic acid (18:0) than that in control subjects (18.28% versus 13.52%, P = 0.007), as well as a lower concentration of octadecadienoic acid (18:1n-9) than that in control subjects (10.97% vs. 14.88%, P = 0.007). The total monounsaturated fatty acid concentration was significantly lower in BCD than in the control (11.82% vs. 15.85%, P = 0.012). The activity of Delta-9-desaturase was also significantly lower in BCD (0.71 vs. 1.14, P = 0.004). Serum glucagon was significantly associated with increased total unsaturated fatty acid and decreased polyunsaturated fatty acid in control subjects but not in patients with BCD. No significant difference in the fatty acid concentration and desaturase activities was found in patients with different genotypes or phenotypes. CONCLUSIONS Abnormal serum fatty acid composition with reduced Delta-9-desaturase activity was detected in patients with BCD, and the metabolic derangement was unaffected by CYP4V2 mutations. The findings suggest that systemic abnormality in fatty acid metabolism occurs in patients with BCD independent of CYP4V2 genotype.

Cigarette smoking hinders human periodontal ligament-derived stem cell proliferation, migration and differentiation potentials

Cigarette smoking contributes to the development of destructive periodontal diseases and delays its healing process. Our previous study demonstrated that nicotine, a major constituent in the cigarette smoke, inhibits the regenerative potentials of human periodontal ligament-derived stem cells (PDLSC) through microRNA (miRNA) regulation. In this study, we hypothesized that the delayed healing in cigarette smokers is caused by the afflicted regenerative potential of smoker PDLSC. We cultured PDLSC from teeth extracted from smokers and non-smokers. In smoker PDLSC, we found significantly reduced proliferation rate and retarded migration capabilities. Moreover, alkaline phosphatase activity, calcium deposition and acidic polysaccharide staining were reduced after BMP2-induced differentiation. In contrast, more lipid deposition was observed in adipogenic-induced smoker PDLSC. Furthermore, two nicotine-related miRNAs, hsa-miR-1305 (22.08 folds, p = 0.040) and hsa-miR-18b (15.56 folds, p = 0.018), were significantly upregulated in smoker PDLSC, suggesting these miRNAs might play an important role in the deteriorative effects on stem cells by cigarette smoke. Results of this study provide further evidences that cigarette smoking affects the regenerative potentials of human adult stem cells.

Association of C2 and CFB Polymorphisms with Anterior Uveitis

PURPOSE Association of rs800292 (I62V) in the complement factor H (CFH) gene with anterior uveitis (AU) was identified in our previous study. We proceeded to investigate whether polymorphisms of two tightly linked genes in the complement pathway, complement component 2 (C2) and complement factor B (CFB), are associated with AU. METHODS Five single-nucleotide polymorphisms (SNPs), rs1048709, rs537160, rs4151657, rs2072633 in CFB, and rs3020644 in C2, were examined using genotyping assays in 98 Chinese AU patients and 291 unrelated controls. Adjustments and stratifications were given for sex, clinical manifestations, and HLA-B27 status. RESULTS There were significant increases in the frequency of A allele and AA homozygosity for CFB-rs1048709 in AU patients compared with that of controls (P value after Bonferroni correction [P(corr)] = 2.67 × 10⁻⁴, P(corr) = 0.001, respectively). No association was found between AU and the other four SNPs after adjustment for multiple testing. Logistic regression analysis showed none of the 5 SNPs had significant interaction with sex. Stratified analyses showed that only rs1048709 was significantly associated with AU in HLA-B27-positive patients but not in HLA-B27-negative patients. No association was found in the 5 tested SNPs with clinical manifestations. A haplotype block across CFB (AATA) was significantly predisposed to AU with increased risk of 1.97 (P(corr) = 0.0005). Additive effect of CFB-rs1048709 and CFH-rs800292 was identified with an odds ratio of 7.48. CONCLUSIONS Our results revealed an association between AU and CFB-rs1048709. The influence on AU might differ depending on HLA-B27 status. The joint effect in CFB and CFH strengthens the concept that the complement system plays an important role in the pathogenesis of AU.

Assessing sodium iodate-induced outer retinal changes in rats using confocal scanning laser ophthalmoscopy and optical coherence tomography.

PURPOSE Sodium iodate induces RPE atrophy and photoreceptor degeneration, as seen in the pathogenesis of many retinal diseases. We investigated a new approach of analyzing retinal images using confocal scanning laser ophthalmoscopy (cSLO) that allows longitudinal assessment of sodium iodate-induced lesions in the retina of living rats. METHODS A single dose of sodium iodate (25-75 mg/kg) was given intravenously to adult Sprague-Dawley rats. Control animals were given normal saline or sodium iodide. The retina was examined by cSLO and optical coherence tomography (OCT) in living rats, which were then killed for histologic assessments. RESULTS Confocal scanning laser ophthalmoscopy revealed the appearance of dark patchy blots in planar images of the retina 7 days after intravenous injection of sodium iodate (25-75 mg/kg). This finding coincided with the observations of degenerative changes in the outer retinal layers in OCT images and in histology of the retina. Further analyses showed a concomitant localization of degenerative profiles in histologic preparations of this retina, suggesting that the blots corresponded to the deteriorating photopigments and outer nuclear layer (ONL). In histologic sections, these degenerative profiles appeared as irregular folds or rosettes in the ONL. Quantitative analyses showed that the changes in blot number were dose dependent, which again coincided with results showing a dose-dependent lesion in the photopigment layer and ONL in histologic sections of the retina. CONCLUSIONS Sodium iodate-induced degenerative changes can be assessed quantitatively and reliably by in vivo retinal imaging using cSLO in adult rats, allowing efficient evaluation of lesions in a large area of retina in longitudinal studies.

Interactive Expressions of HtrA1 and VEGF in Human Vitreous Humors and Fetal RPE Cells

PURPOSE High-temperature requirement factor A1 (HtrA1) is associated with exudative age-related macular degeneration, an angiogenic retinal disease related to vascular endothelial growth factor (VEGF). This study investigates the interactive relationship between the expressions of HtrA1 and VEGF. METHODS; The vitreous humor levels of HtrA1, VEGF, and pigment epithelium-derived factor were determined in 55 unrelated Han Chinese patients who underwent ocular surgeries. Expressions of HTRA1 and VEGFA were studied interactively and under stress conditions in primary human fetal retinal pigment epithelial (RPE) cells to evaluate their regulations. RESULTS Vitreous levels of HtrA1 were significantly associated with that of VEGF in vitreous samples from all patients (Pearsons correlation coefficient test, r = 0.650, P = 7.91 × 10(-8)) and from patients with retinal detachment (r = 0.835, P = 2.14 × 10(-7)). On stress induction, HTRA1 and VEGFA were upregulated in human fetal RPE cells treated by tunicamycin and dithiothreitol, but reduced after treatment by MG132. However, HtrA1 and VEGF did not regulate each other in their expressions. CONCLUSIONS This study revealed an association between HtrA1 and VEGF in human vitreous humors and RPE cells. They are both related to stress and inflammatory conditions.