Eleni Bagli

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 IC50 of about 5 μmol/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 G1 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.

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.

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.

Central corneal thickness in subjects with glaucoma and in normal individuals (with or without pseudoexfoliation syndrome).

Objective: The evaluation of central corneal thickness (CCT) in subjects with pesudoexfoliation glaucoma (PEXG), primary open-angle glaucoma (POAG), and in normotensive individuals with or without pseudoexfoliation syndrome (PXS). Study design/patients and methods: CCT was evaluated with ultrasound pachymetry in a total of 179 individuals: 32 had bilateral PEXG, 55 had bilateral POAG, 35 had PXS, and 57 were healthy individuals without PXS. Results: CCT in PEXG eyes (526.00 ± 34.30 μm) was significantly thinner compared to POAG eyes (549.36 ± 39.3 μm) (P = 0.027) and normal control eyes with (550.64 ± 39.0 μm) or without PXS (547.36 ± 33.1 μm), (P = 0.039 and 0.048 respectively). No statistically significant difference was found comparing CCT values of POAG eyes to control group eyes. Conclusion: The evaluation of CCT is necessary in all patients with glaucoma and especially in those with PEXG due to the thinner cornea and the risk of underestimation of intraocular pressure.

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

BackgroundIncreased 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.ResultsIn 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).Conclusions6-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.

Comparison of one-site versus two-site phacotrabeculectomy without the use of antimetabolites intraoperatively in patients with pseudoexfoliation glaucoma and primary open-angle glaucoma

Purpose: The comparison of one-site versus two-site phacotrabeculectomy in patients with pseudoexfoliation glaucoma (PEXG) and primary open-angle glaucoma (POAG), with respect to intraocular pressure (IOP), antiglaucomatous medication (AM) requirements and visual acuity (VA). Methods: Forty-seven patients (eyes) with cataract and POAG and 46 sex- and age-matched patients with cataract and PEXG were randomized to one- or two-site phacotrabeculectomy and reviewed with a follow-up at three years. Results: Mean preoperative IOP was 22.04 ± 2.27 mmHg and 22.92 ± 2.35 mmHg in POAG and PEXG groups (p > 0.05) using a mean of 2.76 ± 0.74, 2.74 ± 0.69 AM, respectively (p > 0.05). After three years of follow-up, the mean IOP was 15.04 ± 1.57 mmHg in the one-site, 15.04 ± 1.99 mmHg in the two-site group with POAG, 15 ± 1.8 mmHg in the one-site, 15.32 ± 1.31 mmHg in the two-site group with PEXG, using a mean of 0.68 ± 0.69, 0.84 ± 0.75, 0.96 ± 0.67, and 0.8 ± 0.62 AM, respectively. Mean IOP and AM postoperatively were significantly less compared to preoperatively for each group (p < 0.05). No difference was observed in mean IOP and AM between the four groups at three years postoperatively (p > 0.05). VA improved similarly in four groups at the three-year follow-up (p < 0.05). Intraoperative, postoperative complications, and need for 5-fluorouracil injections were similar between the surgical groups. Conclusion: One-site and two-site phacotrabeculectomy without the use of antimetabolites intraoperatively were similarly safe and effective in IOP control over a three-year follow-up period in patients with POAG and PEXG.

Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells.

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis.

Neuroprotective Agents in Glaucoma

1.1 Glaucoma as a neurodegenerative disorder Glaucoma is a multifactorial disease in which multiple genetic, systemic and environmental factors interact to precipitate the disease. Increased intraocular pressure (IOP) is believed to be one of the major factors responsible for glaucomatous cell death. (Guo et al., 2005). The axons of the optic nerve make a twisting exit through the fenestrated collageneous barrier (the lamina cribrosa) before entering the brain. It has been proposed that increased IOP causes mechanical stress to the lamina cribrosa, which in turn exerts pressure on the axons that pass through it. This pressure may block the passage of essential material through the axons; which would lead to a slow degeneration of the axons of the retinal ganglion cells (RGCs) (Howell et al., 2007). With increased IOP, gradual withdrawal of trophic factor from RGCs has been observed in a mouse model. Also apoptosis was observed to be a significantly delayed in RGCs in such conditions (Johnson et al., 2000). It was therefore proposed that with increased IOP and mechanical stress on the lamina cribrosa, axons of RGCs are lost much earlier than the cell body of the RGCs. Thus RGCs become ineffective in their visual function much earlier than actual phenotypic changes are observed in the retina/optic disc, as they are incapable of sending visual signals to the visual center of the brain (Whitmore et al., 2005). The primary goal of glaucoma treatment is to preserve vision. Elevated IOP as an important risk factor for glaucoma has continued to be a clinical focus for several reasons, including limited knowledge of the factors causing optic nerve damage, ease of measurement, the number of available IOP-reducing therapies, and the relationship of elevated IOP to disease progression. However, the relationship between IOP reduction and glaucoma damage is less clear, and such ambiguities suggest that factors other than IOP may be responsible for some of the long-term damage from glaucoma. Ocular blood flow in various tissues (e.g. retina, iris, optic nerve and choroid) was found to be reduced in glaucoma patients (Flammer et al., 2002). The blood flow reduction was more pronounced in Normal Tension Glaucoma (NTG) patients (Mozaffarieh et al., 2008). Interestingly, reduction of blood flow was also observed in the nailbed capillaries of fingers in glaucoma patients; suggesting that global vascular dysregulation is involved in Primary Open Angle Glaucoma (POAG) especially in NTG cases. Glaucoma is therefore no longer diagnosed by elevated IOP levels, and is now recognized as a neuropathy defined by characteristic optic disc and visual field change (Walland et al., 2006). IOP level is no longer relied on as a diagnostic criterion because 20% to 30% of glaucoma patients have IOP in the normal range (typically 10-21 mm Hg) (Sommer et al., 1991). Furthermore, there is annual progression even in patients treated with IOP-lowering

Pseudoexfoliation syndrome without glaucoma: White matter abnormalities detected by conventional MRI and diffusion tensor imaging

PURPOSE To assess macro- and microstructural brain changes in patients with pseudoexfoliation syndrome (PXS). MATERIALS AND METHODS Comprehensive ophthalmic examination and brain MRI were conducted on 20 patients with PXS without glaucoma (aged 62.75 ± 0.4 years) and 20 controls (aged 62 ± 0.6 years). White matter (WM) integrity was evaluated on FLAIR and single-shot multisection SE-EPI diffusion tensor imaging (DTI) sequences. The presence and the number of white matter hyperintensities (WMHIs) on FLAIR images was compared between all patients and control subjects. Microstructural WM changes on DTI was evaluated using Tract-based spatial statistics (TBSS). DTI metrics of the optic tracts were assessed by the region-of-interest (ROI) method. RESULTS A significantly higher number of WMHIs was found in the patients with PXS than in the control subjects (P ≤ 0.002). On DTI the patients showed bilateral increase in the mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD) values in the anterior thalamic radiation, the inferior fronto-occipital fasciculus, the superior longitudinal fasciculus, the inferior longitudinal fasciculus and the forceps minor. TBSS revealed no significant difference in fractional anisotropy (FA) values, but ROIs analysis of the optic tracts revealed decreased FA values in the patients. CONCLUSION MRI in patients with PXS detects abnormalities in the brain and the optic tracts at a subclinical stage. Early detection of microstructural changes could be useful to guide appropriate treatment to impede the disease process.

Medium-term Electrophysiologic Effects of a Cellularized Scaffold Implanted in Rats After Myocardial Infarction

Background Cardiac repair strategies are being evaluated for myocardial infarctions, but the safety issues regarding their arrhythmogenic potential remain unresolved. By utilizing the in-vivo rat model, we have examined the medium-term electrophysiologic effects of a biomaterial scaffold that has been cellularized with spheroids of human adipose tissue, derived from mesenchymal stem cells and umbilical vein endothelial cells. Methods Mesenchymal stem cells, which exhibit adequate differentiation capacity, were co-cultured with umbilical vein endothelial cells and were seeded on an alginate based scaffold. After in-vitro characterization, the cellularized scaffold was implanted in (n=15) adult Wistar rats 15 min post ligation of the left coronary artery, with an equal number of animals serving as controls. Two weeks thereafter, monophasic action potentials were recorded and activation-mapping was performed with a multi-electrode array. An arrhythmia score for inducible ventricular tachyarrhythmias was calculated after programmed electrical stimulation. Results The arrhythmia score was comparable between the treated animals and controls. No differences were detected in the local conduction at the infarct border and in the voltage rise in monophasic action potential recordings. Treatment did not affect the duration of local repolarization, but tended to enhance its dispersion. Conclusions The fabricated bi-culture cellularized scaffold displayed favorable properties after in-vitro characterization. Medium-term electrophysiologic assessment after implantation in the infarcted rat myocardium revealed low arrhythmogenic potential, but the long-term effects on repolarization dispersion will require further investigation.