Association of MMP2-1306C/T Polymorphism with Ischemic Retinal Vein Occlusion.

PURPOSE: To investigate the possible association of the matrix metalloproteinase 2 (MMP2)-1306C/T polymorphism with the risk of ischemic retinal vein occlusion (iRVO). METHODS: A total of 69 patients with RVO were enrolled in this study (43 with non-iRVO and 26 with iRVO). All subjects were screened for hypertension, diabetes mellitus, hyperlipidemia, history of stroke, anticoagulant medication, smoking status and glaucoma. The genotyping of MMP2-1306C/T polymorphism was performed using PCR-RFLP-based methods. RESULTS: MMP2-1306C/T T allele carriers (CT+TT) were statistically significant associated with a higher risk of iRVO compared to CC genotype in the overall RVO group (odds ratio = 3.91, p = 0.015, 95% confidence interval:1.30-11.79). Analysis, following stratification by age revealed that T allele carriers had a statistically significant increased risk of iRVO compared to C allele carriers only in RVO patients <75 years old. CONCLUSION: Our results demonstrated that MMP2-1306C/T polymorphism is a likely predisposing factor for iRVO in patients <75 years old. This is the first study attempting association of a gene polymorphism with the prevalence of iRVO.

Intravitreal ranibizumab versus vitrectomy for recurrent vitreous haemorrhage after pars plana vitrectomy for proliferative diabetic retinopathy: a prospective study.

PURPOSE: To compare prospectively intravitreal ranibizumab treatment and pars plana vitrectomy (PPV) in patients with recurrent vitreous haemorrhage (VH) due to proliferative diabetic retinopathy (PDR), who were previously treated with PPV. METHODS: Participants in this prospective study were 37 patients (37 eyes) with PDR, previously treated with PPV. All patients presented recurrent VH and were treated with either ranibizumab (n = 18) or PPV (n = 19). All participants were examined at week 2 post-treatment and every month thereafter for 1 year. Main outcomes were the need of PPV, the rate of recurrence of VH and the change in visual acuity by the end of the 12-month follow-up. RESULTS: At month 12, there was statistically significant improvement in visual acuity in both groups compared to baseline, but the two groups did not differ regarding the change in visual acuity. In ranibizumab group, two patients presented recurrent VH during the follow-up and one patient needed PPV to clear the VH by month 12. In PPV group, two patients had mild recurrent VH, which cleared itself. No statistically significant difference was noticed regarding the rate of recurrent VH and the need of PPV between the two groups. CONCLUSION: Intravitreal ranibizumab seems to be a safe and effective treatment alternative in patients with recurrent VH secondary to PDR, who had been previously treated with PPV.

Genetic polymorphisms associated with the prevalence of retinal vein occlusion in a Greek population.

PURPOSE: To investigate possible associations of single-nucleotide polymorphisms (SNPs) from five genes with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). METHODS: A total of 69 patients with retinal vein occlusion-RVO (24 with BRVO and 45 with CRVO), and 82 controls, were enrolled in this study. All subjects were screened for hypertension, diabetes mellitus, hyperlipidemia, glaucoma, anticoagulant medication, smoking status and history of stroke. The genotyping of AGTR1-A1166C, adiponectin + 276 G/T, MMP2-1306C/T, Gpla/lla-C807T/G873A and VKORC1-G1639A polymorphisms was performed using restriction fragment length polymorphism or allele-specific polymerase chain reaction. RESULTS: The percentage of the AGTR1-A1166C C allele carriers and Gpla/lla-C807T/G873A T/A carriers was significantly higher in the CRVO patients than in the controls (P = 0.00001 and P = 0.0004, respectively). At the multiple logistic regression analysis, the AGTR1-A1166C C allele carrier status and the Gpla/lla-C807T/G873A T/A allele carrier status were found to be associated with an increased risk of CRVO. Moreover, adiponectin + 276 G/T T allele carriers had a significantly increased risk of RVO in subjects ≥ 75 years old. There was no significant difference between the BRVO patients and controls concerning the genotype or the allele frequency distributions of these SNPs. The genotype distributions or allelic frequencies of the other evaluated polymorphisms did not significantly differ between the patients with RVO and the control subjects. CONCLUSIONS: AGTR1 A1166C and Gpla/lla C807T/G873A polymorphisms are likely to be risk factors for CRVO. Adiponectin + 276 G/T SNP is likely to predispose to RVO in older subjects.

Mechanical stress affects methylation pattern of GNAS isoforms and osteogenic differentiation of hAT-MSCs.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth.

BACKGROUND: Increased consumption of plant-based diets has been linked to the presence of certain phytochemicals, including polyphenols such as flavonoids. Several of these compounds exert their protective effect via inhibition of tumor angiogenesis. Identification of additional phytochemicals with potential antiangiogenic activity is important not only for understanding the mechanism of the preventive effect, but also for developing novel therapeutic interventions. RESULTS: In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have screened a set of hitherto untested phytoestrogen metabolites concerning their anti-angiogenic effect, using endothelial cell proliferation as an end point. Here, we show that a novel phytoestrogen, 6-methoxyequol (6-ME), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVE) cells, whereas VEGF-induced migration and survival of HUVE cells remained unaffected. In addition, 6-ME inhibited FGF-2-induced proliferation of bovine brain capillary endothelial (BBCE) cells. In line with its role in cell proliferation, 6-ME inhibited VEGF-induced phosphorylation of ERK1/2 MAPK, the key cascade responsible for VEGF-induced proliferation of endothelial cells. In this context, 6-ME inhibited in a dose dependent manner the phosphorylation of MEK1/2, the only known upstream activator of ERK1/2. 6-ME did not alter VEGF-induced phosphorylation of p38 MAPK or AKT, compatible with the lack of effect on VEGF-induced migration and survival of endothelial cells. Peri-tumor injection of 6-ME in A-431 xenograft tumors resulted in reduced tumor growth with suppressed neovasularization compared to vehicle controls (P < 0.01). CONCLUSIONS: 6-ME inhibits VEGF- and FGF2-induced proliferation of ECs by targeting the phosphorylation of MEK1/2 and it downstream substrate ERK1/2, both key components of the mitogenic MAPK pathway. Injection of 6-ME in mouse A-431 xenograft tumors results to tumors with decreased neovascularization and reduced tumor volume suggesting that 6-ME may be developed to a novel anti-angiogenic agent in cancer treatment.

Renin-angiotensin-aldosterone system genes and nonarteritic anterior ischemic optic neuropathy.

PURPOSE: Recent literature suggests a genetic component for non-arteritic anterior ischemic optic neuropathy (NAION). We examined the association of the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene, of the M235T polymorphism of the angiotensinogen gene, and of the A1166C polymorphism of the angiotensin II type 1 receptor gene with NAION. METHODS: Forty-seven patients with NAION and 76 controls, age- and gender-matched, were recruited and genotyped for renin-angiotensin-aldosterone system (RAAS) genes. Genotypes were determined by polymerase chain reaction and restriction enzyme analysis. NAION and control groups were compared in regard to the prevalence of renin-angiotensin-aldosterone system polymorphisms, and further stratified by age and gender. RESULTS: NAION occurrence was not associated with the M235T polymorphism of the angiotensinogen gene and the A1166C polymorphism of the angiotensin II, type 1 receptor gene. Regarding the angiotensin-converting enzyme insertion/deletion polymorphism, our findings suggest that the II genotype could be a risk factor for NAION in younger male patients when compared to all cases and controls (p=0.033, odds ratio=5.71, confidence interval=1.152¨C28.35 and p=0.03, odds ratio=5.33, confidence interval=1.17¨C24.31 respectively). Furthermore I allele was present in all male patients younger than 55 years, making this allele a likely predisposing factor for NAION in young males. CONCLUSIONS: Since NAION may occur when compromised watershed microcirculation is combined with insufficient autoregulation of systematic circulation, polymorphisms of genes involved in systematic circulation, such as the RAAS genes, may be associated with NAION occurrence. Large-scale, multicentered, controlled prospective studies are needed to further explore the effects of RAAS polymorphisms or other genetic factors on NAION susceptibility.

VEGF autoregulates its proliferative and migratory ERK1/2 and p38 cascades by enhancing the expression of DUSP1 and DUSP5 phosphatases in endothelial cells.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual-specificity phosphatase (DUSP) genes DUSP1 and DUSP5 in endothelial cells. Using fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence cross-correlation spectroscopy (FCCS), we found that DUSP1/mitogen-activated protein kinases phosphatase-1 (MKP-1) was localized in both the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (effective dissociation constant, K(D)(eff), values of 294 and 197 nM, respectively), whereas DUSP5 was localized in the nucleus of endothelial cells in complex with ERK1/2 (K(D)(eff) 345 nM). VEGF administration affected differentially the K(D)(eff) values of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation.

Activin A suppresses neuroblastoma xenograft tumor growth via antimitotic and antiangiogenic mechanisms.

The tumor suppressor function of activin A, together with our findings that activin A is an inhibitor of angiogenesis, which is down-regulated by the N-MYC oncogene, prompted us to investigate in more detail its role in the malignant transformation process of neuroblastomas. Indeed, neuroblastoma cells with restored activin A expression exhibited a diminished proliferation rate and formed smaller xenograft tumors with reduced vascularity, whereas lung metastasis rate remained unchanged. In agreement with the decreased vascularity of the xenograft tumors, activin A inhibited several crucial angiogenic responses of cultured endothelial cells, such as proteolytic activity, migration, and proliferation. Endothelial cell proliferation, activin A, or its constitutively active activin receptor-like kinase 4 receptor (ALK4T206D), increased the expression of CDKN1A (p21), CDKN2B (p15), and CDKN1B (p27) CDK inhibitors and down-regulated the expression of vascular endothelial growth factor receptor-2, the receptor of a key angiogenic factor in cancer. The constitutively active forms of SMAD2 and SMAD3 were both capable of inhibiting endothelial cell proliferation, whereas the dominant-negative forms of SMAD3 and SMAD4 released the inhibitory effect of activin A on endothelial cell proliferation by only 20%. Thus, the effects of activin A on endothelial cell proliferation seem to be conveyed via the ALK4/SMAD2-SMAD3 pathways, however, non-SMAD cascades may also contribute. These results provide novel information regarding the role of activin A in the malignant transformation process of neuroblastomas and the molecular mechanisms involved in regulating angiogenesis thereof.

Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3'-kinase activity.

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC(50) of about 5 mumol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3'-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G(1) progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound.

Luteolin reduces lipopolysaccharide-induced lethal toxicity and expression of proinflammatory molecules in mice.

Luteolin is a flavonoid that has been shown to reduce proinflammatory molecule expression in vitro. In the present study, we have tested the ability of luteolin to inhibit lipopolysaccharide (LPS)- induced lethal toxicity and proinflammatory molecule expression in vivo. Mice receiving LPS (Salmonella enteriditis LPS, 32 mg/kg, intraperitoneally) exhibited high mortality with only 4.1% of the animals surviving seven days after the LPS challenge. On the contrary, mice that had received luteolin (0.2 mg/kg, intraperitoneally) before LPS showed an increased survival rate with 48% remaining alive on Day 7. To investigate the mechanism by which luteolin affords protection against LPS toxicity we measured intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF-alpha) production in response to LPS in the presence or absence of luteolin pretreatment. Treatment of animals with LPS increased serum TNF-alpha levels in a time-dependent manner. The increase in peak serum TNF-alpha levels was sensitive to luteolin pretreatment. Luteolin pretreatment also reduced LPS-stimulated ICAM-1 expression in the liver and abolished leukocyte infiltration in the liver and lung. We conclude that luteolin protects against LPS-induced lethal toxicity, possibly by inhibiting proinflammatory molecule (TNF-alpha, ICAM-1) expression in vivo and reducing leukocyte infiltration in tissues.