Brolucizumab en degeneración macular neo vascular asociada a la edad / Brolucizumab in neovascular macular degeneration age-associated

CONTEXTO: La degeneración macular asociada a la edad (DMAE) es una de las principales causas de ceguera en el mundo, siendo su prevalencia en adultos mayores de 50 años del 5,6%, seguida de retinopatia diabética (2,5%). En los países de Latinoamérica se calcula una prevalencia en la población general del 10% y se estima que aproximadamente 39 millones de latinoamericanos padecerán alguna forma de DMAE para el 2040. Se trata de una enfermedad de la retina progresiva, crónica y multifactorial que lleva al deterioro de la visión y ceguera afectando principalmente personas mayores de 60 años. Afecta la mácula (porción central de la retina) resultando en la pérdida de la visión central lo que impediría conducir, leer y realizar actividades habituales de la vida diaria. Progresa desde estadíos iniciales a formas tardías y se puede subdividir en dos formas: neo vascular (húmeda) o atrofia geográfica no neo vascular (seca). A pesar de que a solo el 20% de los pacientes se les diagnostica la forma húmeda, esta es la responsable del 90% de los casos de pérdida de la visión y su evolución es más aguda. TECNOLOGÍA: Brolucizumab es un fragmento variable humanizado de cadena única inhibidor de factor de crecimiento endotelial A. Un fragmento variable de cadena única es un agente de unión autónomo que compromete sólo los dominios variables del anticuerpo monoclonal responsables de unirse a su receptor. Al ser un fragmento de cadena único es pequeño (peso molecular 26 kDa) y le falta la porción cristalizable de su dominio proveyéndole la probable ventaja de mayor biodisponibilidad e inmunogenicidad reducida. Se encuentra indicado como primera línea en el tratamiento de la degeneración macular neo vascular asociada a la edad. OBJETIVO: El objetivo del presente informe es evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura del uso de brolucizumab en pacientes com degeneración macular neo vascular asociada a la edad. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas, en buscadores genéricos de internet, y financiadores de salud. Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias (ETS), evaluaciones económicas, guías de práctica clínica (GPC) y políticas de cobertura de diferentes sistemas de salud. RESULTADOS: Se incluyeron dos ECAs, una RS, dos estudios observacionales, una GPC, una evaluación económica, cuatro evaluaciones de tecnologías sanitarias y 15 informes de políticas de cobertura de brolucizumab en degeneración macular neo vascular asociada a la edad. CONCLUSIONES: Evidencia de moderada calidad sugiere que brolucizumab presenta un beneficio considerable em pacientes con degeneración macular neo vascular asociada a la edad en relación a la mejora en la agudeza visual y modificaciones anatómicas (reducción del grosor de la mácula y neo vascularización coroidea) al igual que el ranibizumab o aflibercept. En términos de seguridad la evidencia sugiere que brolucizumab presenta una mayor tasa de eventos adversos serios no oculares (eventos trombo embólicos arteriales: infarto de miocardio no fatal, accidente cerebrovascular no fatal y muerte por causa vascular) y oculares (endoftalmitis), aunque los mismos fueron poco frecuentes y de manera no significativa, en comparación con ranibizumab y aflibercept. Evidencia de moderada calidad sugiere que brolucizumab presenta una eficacia menor que bevacizumab fraccionado/fuera de prospecto en pacientes con degeneración macular neo vascular asociada a la edad, dada la mayor tasa significativa de eventos adversos serios no oculares (eventos trombo embólicos arteriales: infarto de miocardio no fatal, accidente cerebrovascular no fatal y muerte por causa vascular), aunque los mismos fueron poco frecuentes. La mayoría de las guías de práctica clínica desarrolladas para esta patología identificadas son de fecha de publicación previa a la autorización de comercialización de brolucizumab por las principales aseguradoras europeas y latinoamericanas. Agencias financiadoras públicas de Canadá, Reino Unido y algunas instituciones aseguradoras privadas de Estados Unidos cubren esta tecnología. Algunas evaluaciones de tecnologías sanitarias y económicas europeas (Alemania y EUnetHTA) mostraron que brolucizumab no fue costo efectivo dado que no se pudo demostrar el beneficio agregado de esta tecnología en relación con los comparadores. La agencia canadiense de evaluación de tecnologías sanitarias consideró que se debe realizar una reducción de precio significativa en caso de contarse con bevacizumab fraccionado/fuera de prospecto como opción terapéutica. En otros países, aunque no se encuentra aprobado para esta indicación, el uso de bevacizumab fraccionado/fuera de prospecto es ampliamente utilizado. Los financiadores latinoamericanos relevados no mencionan esta tecnología. No existen evaluaciones económicas que estudien el impacto de esta droga em Argentina, sin embargo, dado el frecuente uso de bevacizumab fraccionado/fuera de prospecto, se debe considerar que el costo de brolucizumab sería considerablemente mayor al mismo.

DEXAMETHASONE IMPLANT FOR DIABETIC MACULAR EDEMA IN NAIVE COMPARED WITH REFRACTORY EYES: The International Retina Group Real-Life 24-Month Multicenter Study. The IRGREL-DEX Study.

PURPOSE: To investigate efficacy and safety of repeated dexamethasone (DEX) implants over 24 months, in diabetic macular edema (DME) eyes that were treatment naive compared with eyes refractory to anti-vascular endothelial growth factor treatment, in a real-life environment. METHODS: This multicenter international retrospective study assessed best-corrected visual acuity and central subfield thickness (CST) of naive and refractory eyes to anti-vascular endothelial growth factor injections treated with dexamethasone implants. Safety data (intraocular pressure rise and cataract surgery) were recorded. RESULTS: A total of 130 eyes from 125 patients were included. Baseline best-corrected visual acuity and CST were similar for naive (n = 71) and refractory eyes (n = 59). Both groups improved significantly in vision after 24 months (P < 0.001). However, naive eyes gained statistically significantly more vision than refractory eyes (+11.3 ± 10.0 vs. 7.3 ± 2.7 letters, P = 0.01) and were more likely to gain ≥10 letters (OR 3.31, 95% CI 1.19-9.24, P = 0.02). At 6, 12, and 24 months, CST was significantly decreased compared with baseline in both naive and refractory eyes; however, CST was higher in refractory eyes than in naive eyes (CST 279 ± 61 vs. 313 ± 125 µm, P = 0.10). CONCLUSION: Over a follow-up of 24 months, vision improved in diabetic macular edema eyes after treatment with dexamethasone implants, both in eyes that were treatment naive and eyes refractory to anti-vascular endothelial growth factor treatment; however, improvement was greater in naive eyes.

EGFL7 enhances surface expression of integrin α5ß1 to promote angiogenesis in malignant brain tumors.

Glioblastoma (GBM) is a typically lethal type of brain tumor with a median survival of 15 months postdiagnosis. This negative prognosis prompted the exploration of alternative treatment options. In particular, the reliance of GBM on angiogenesis triggered the development of anti-VEGF (vascular endothelial growth factor) blocking antibodies such as bevacizumab. Although its application in human GBM only increased progression-free periods but did not improve overall survival, physicians and researchers still utilize this treatment option due to the lack of adequate alternatives. In an attempt to improve the efficacy of anti-VEGF treatment, we explored the role of the egfl7 gene in malignant glioma. We found that the encoded extracellular matrix protein epidermal growth factor-like protein 7 (EGFL7) was secreted by glioma blood vessels but not glioma cells themselves, while no major role could be assigned to the parasitic miRNAs miR-126/126*. EGFL7 expression promoted glioma growth in experimental glioma models in vivo and stimulated tumor vascularization. Mechanistically, this was mediated by an upregulation of integrin α5ß1 on the cellular surface of endothelial cells, which enhanced fibronectin-induced angiogenic sprouting. Glioma blood vessels that formed in vivo were more mature as determined by pericyte and smooth muscle cell coverage. Furthermore, these vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent. EGFL7-inhibition using a specific blocking antibody reduced the vascularization of experimental gliomas and increased the life span of treated animals, in particular in combination with anti-VEGF and the chemotherapeutic agent temozolomide. Data allow for the conclusion that this combinatorial regimen may serve as a novel treatment option for GBM.

Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia.

Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.

Effect of Epiretinal Membranes on Antivascular Endothelial Growth Factor Treatment for Neovascular Age-Related Macular Degeneration.

PURPOSE: To evaluate the effect of epiretinal membranes (ERMs), detected with spectral-domain optical coherence tomography (SD-OCT), on the outcome of antivascular endothelial growth factor (VEGF) treatment for neovascular age-related macular degeneration (nAMD). METHODS: A total of 434 eyes with treatment-naive nAMD were retrospectively included and analyzed. All patients were administered an initial series of 3 monthly loading injections of ranibizumab or aflibercept, followed by further injections as required. The visual and anatomical outcomes were compared between the eyes with ERMs and those without. Features of ERMs at baseline assessed with SD-OCT were evaluated and correlated with visual outcomes. RESULTS: Sixty-eight eyes (15.7%) with nAMD presented ERMs at baseline. The mean best-corrected visual acuity (BCVA) of these eyes, expressed as the logarithm of the minimum angle of resolution, improved from 0.75 ± 0.48 (Snellen equivalent: 20/112) to 0.59 ± 0.44 (20/77) after 12 months of treatment (P = 0.021). Central foveal thickness also decreased from 381 ± 191 µm to 294 ± 167 µm (P < 0.001). Compared to the eyes without ERMs (366 eyes), the eyes with ERMs had a significantly thicker central fovea after treatment (P = 0.020). However, the intergroup differences in BCVA improvement were not significant. No significant association was found between visual outcome after treatment and ERM features on OCT at baseline. CONCLUSIONS: In eyes with nAMD, ERMs were infrequent. Central foveal thickness was significantly greater after anti-VEGF treatment in eyes with nAMD and ERMs. However, the presence of ERMs in eyes with nAMD did not affect visual outcome.

Hypoxia Induces Multidrug Resistance via Enhancement of Epidermal Growth Factor-Like Domain 7 Expression in Non-Small Lung Cancer Cells.

Chemotherapy is widely used in non-small cell lung cancer (NSCLC) treatment, yet multidrug resistance (MDR) is a major chemotherapeutic obstacle in both resectable and advanced NSCLC. Epidermal growth factor-like domain 7 (EGFL7), also known as vascular endothelial stain, is an endothelial cell-derived secreted factor that regulates vascular tube formulation. The aim of this study was to investigate the potential relationships between EGFL7 and MDR in NSCLC cells. We first obtained the CDDP-based MDR phenotype cell line A549/CDDP by repeated exposure to a proper concentration of CDDP (cisplatin) from original A549 cells. These A549/CDDP cells, which maintained relative high levels of EGFL7 and P-glycoprotein (P-gp), were resistant to other chemotherapy drugs, such as carboplatin (CBP), paclitaxel (TAX), and gemcitabine (GEM) (p < 0.05). We also found that hypoxia significantly reduced the chemosensitivity of NSCLC cells, and hypoxia-induced MDR was mediated by P-gp and EGFL7 (p < 0.05). EGFL7 was veryy relevant to NSCLC cell MDR, and downregulation of EGFL7 could significantly increase the chemosensitivity of NSCLC cells (p < 0.05). Thus, our findings first indicate that hypoxia induced NSCLC cell MDR at least partly by enhancing the expression of EGFL7 protein. EGFL7 might be a feasible target for reversing hypoxia-mediated MDR in NSCLC cells and a promising biomarker for predicting the development of MDR in NSCLC patients on chemotherapy.

Randomized Phase II Trial of Parsatuzumab (Anti-EGFL7) or Placebo in Combination with FOLFOX and Bevacizumab for First-Line Metastatic Colorectal Cancer.

LESSONS LEARNED: These negative phase II results for parsatuzumab highlight the challenges of developing an agent intended to enhance the efficacy of vascular endothelial growth factor inhibition without the benefit of validated pharmacodynamic biomarkers or strong predictive biomarker hypotheses.Any further clinical development of anti-EGFL7 is likely to require new mechanistic insights and biomarker development for antiangiogenic agents. BACKGROUND: EGFL7 (epidermal growth factor-like domain 7) is a tumor-enriched vascular extracellular matrix protein that supports endothelial cell survival. This phase II trial evaluated the efficacy of parsatuzumab (also known as MEGF0444A), a humanized anti-EGFL7 IgG1 monoclonal antibody, in combination with modified FOLFOX6 (mFOLFOX6) (folinic acid, 5-fluorouracil, and oxaliplatin) bevacizumab in patients with previously untreated metastatic colorectal cancer (mCRC). METHODS: One-hundred twenty-seven patients were randomly assigned to parsatuzumab, 400 mg, or placebo, in combination with mFOLFOX6 plus bevacizumab, 5 mg/kg. Treatment cycles were repeated every 2 weeks until disease progression or unacceptable toxicity for a maximum of 24 months, with the exception of oxaliplatin, which was administered for up to 8 cycles. RESULTS: The progression-free survival (PFS) hazard ratio was 1.17 (95% confidence interval [CI], 0.71-1.93; p = .548). The median PFS was 12 months for the experimental arm versus 11.9 months for the control arm. The hazard ratio for overall survival was 0.97 (95% CI, 0.46-2.1; p = .943). The overall response rate was 59% in the parsatuzumab arm and 64% in the placebo arm. The adverse event profile was similar in both arms. CONCLUSIONS: There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first-line mCRC. The Oncologist 2017;22:375-e30.

Choroidal Thickness Changes After Intravitreal Antivascular Endothelial Growth Factor Therapy for Age-Related Macular Degeneration: Ranibizumab Versus Aflibercept.

PURPOSE: To compare the changes in subfoveal choroidal thickness (CT) in eyes with neovascular age-related macular degeneration (nAMD) treated with intravitreal ranibizumab or aflibercept. METHODS: In this retrospective case series, the medical records of 28 patients with nAMD treated with at least 3 consecutive monthly injections of ranibizumab (0.5 mg/0.05 mL) or aflibercept (2 mg/0.05 mL) between December 2013 and June 2014 and who were followed up for at least 3 months were reviewed. Subfoveal choroidal thickness was measured using enhanced depth imaging optical coherence tomography. RESULTS: Choroidal thickness decreased over time in the aflibercept group, but was unchanged throughout the study in the ranibizumab group. At each time point, the decrease was significantly greater in aflibercept-treated eyes compared with ranibizumab-treated eyes (P<0.05). No significant change in best-corrected visual acuity (BCVA) was seen in either group during follow-up. There was no correlation between change in choroidal thickness and age, sex, duration of previous antivascular endothelial growth factor treatment, number of previous injections, spherical equivalent, baseline choroidal thickness, and the BCVA outcome in either group. CONCLUSIONS: Subfoveal choroidal thickness appeared to decrease significantly in eyes with nAMD during 3 months of aflibercept treatment. No corresponding decrease in choroidal thickness occurred in ranibizumab-treated eyes.

Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.

BACKGROUND: Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. MATERIALS AND METHODS: EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. RESULTS: After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. CONCLUSIONS: Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

Contrast-enhanced Ultrasound Imaging of Antiangiogenic Tumor Therapy.

BACKGROUND/AIM: Anti-angiogenic treatment is a promising strategy for cancer therapy and is currently evaluated in clinical trials. The aim of the present study was to further investigate the effects of an anti-angiogenic therapy, inhibiting vascular endothelial growth factor (VEGF) and endothelial growth factor (EGF) using a tyrosine kinase inhibitor for blocking tumor angiogenesis and tumor progression in vivo. MATERIALS AND METHODS: Experiments were performed using C57/Bl6 mice (25 ± 5 g of body weight (b.w.)) implanted with subcutaneous Lewis lung carcinoma (LLC-1). From day 7 till 21 after tumor cell implantation, animals (n=7 per group) were treated by monotherapy using ZD6474 (50 mg/kg b.w. per os (p.o.)) daily. A control group received only the solvent polysorbate 80. Using contrast enhanced ultrasound (CE-US) parameters of intra-tumoral microcirculation animals were examined 24 h after the last application of ZD6474. Moreover, subcutaneous tumor growth was measured over the whole therapy period. Finally, histological analyses were performed to analyze the functional vessel density in the tumor tissue. RESULTS: ZD6474 reduced tumor growth of LLC-1 in C57/Bl6 mice significantly. A significant difference of maximal signal intensity (ΔSImax) and area below the intensity time curve (AUC) after antiangiogenic therapy was recorded in the tumor center by CE-US. Vessel density after hematoxyline and eosin, as well as CD31, staining showed no significant difference in both groups. CONCLUSION: Anti-angiogenic effects can be quantitatively demonstrated using CE-US imaging, which represents the spreading of efficient vessels in the tumor tissue, especially in the tumor center.

Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.

Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.

Safety and efficacy of dexamethasone intravitreal implant (Ozurdex) for the treatment of persistent macular edema secondary to retinal vein occlusion in eyes previously treated with anti-vascular endothelial growth factors.

PURPOSE: To report our results concerning the safety and efficacy of repeated sustained-release dexamethasone 0.7 mg implants (Ozurdex, Allergan, Inc., Irvine, CA) in patients with persistent Macular Edema (ME) due to Retinal Vein Occlusion (RVO) previously treated with anti- VEGF injections. PATIENTS AND METHODS: Ten patients (5 males and 5 females/ 5 with CRVO and 5 with BRVO), all previously treated with at least 3 consecutive anti- VEGF injections and presented lack of anatomic improvement (CRT >250 µm in OCT) accompanied by lack of visual improvement (no change or deterioration of VA), received one or more Ozurdex injections (up to five). Ozurdex was administrated on an 'as needed' regimen and patients underwent a complete ophthalmological examination, including Best Corrected Visual Acuity (BCVA) measurements, biomicroscopy, tonometry, and Fourier Domain Optical Coherence Tomography (FD-OCT) on a monthly basis. Furthermore, mean time intervals for OZURDEX re-injection were estimated. RESULTS: In 9 out of 10 cases, the patients experienced improvement in BCVA after the Ozurdex implantation with reduction in CRT. The most frequently observed adverse events included IOP elevation (20% of cases), cataract progression (10%) and cataract formation (10%). No serious systemic or topical adverse events were observed in eyes undergoing repeated Ozurdex implantations. In one of the patients, it was observed that the implant was fractured into two pieces without affecting the efficacy of the implant or causing any side effects. CONCLUSION: Provided the complexity of molecular mechanisms involved in ΜΕ development and the effect of corticosteroids on many of these mechanisms, in our case series, Ozurdex appeared to be a safe and beneficial treatment option for persistent ME due to RVO, in patients with poor or complete lack of response after at least 3 consecutive monthly intravitreal anti-VEGF injections. The complication rate of cataract reported in our study is relatively high compared to previous reports. This might be attributed to the multiple injections, as the incidence of cataract increases with time. Regarding IOP elevation, there is no consensus among published clinical trials on the percentage of patients requiring IOP-lowering medications. Further studies with a greater patient population as well as a control group are required in order to confirm our findings.

Epidermal growth factor-like domain-containing protein 7 (EGFL7) enhances EGF receptor-AKT signaling, epithelial-mesenchymal transition, and metastasis of gastric cancer cells.

Epidermal growth factor-like domain-containing protein 7 (EGFL7) is upregulated in human epithelial tumors and so is a potential biomarker for malignancy. Indeed, previous studies have shown that high EGFL7 expression promotes infiltration and metastasis of gastric carcinoma. The epithelial-mesenchymal transition (EMT) initiates the metastatic cascade and endows cancer cells with invasive and migratory capacity; however, it is not known if EGFL7 promotes metastasis by triggering EMT. We found that EGFL7 was overexpressed in multiple human gastric cancer (GC) cell lines and that overexpression promoted cell invasion and migration as revealed by scratch wound and transwell migration assays. Conversely, shRNA-mediated EGFL7 knockdown reduced invasion and migration. Furthermore, EGFL7-overexpressing cells grew into larger tumors and were more likely to metastasize to the liver compared to underexpressing CG cells following subcutaneous injection in mice. EGFL7 overexpression protected GC cell lines against anoikis, providing a plausible mechanism for this enhanced metastatic capacity. In excised human gastric tumors, expression of EGFL7 was positively correlated with expression levels of the mesenchymal marker vimentin and the EMT-associated transcription repressor Snail, and negatively correlated with expression of the epithelial cell marker E-cadherin. In GC cell lines, EGFL7 knockdown reversed morphological signs of EMT and decreased both vimentin and Snail expression. In addition, EGFL7 overexpression promoted EGF receptor (EGFR) and protein kinase B (AKT) phospho-activation, effects markedly suppressed by the EGFR tyrosine kinase inhibitor AG1478. Moreover, AG1478 also reduced the elevated invasive and migratory capacity of GC cell lines overexpressing EGFL7. Collectively, these results strongly suggest that EGFL7 promotes metastasis by activating EMT through an EGFR-AKT-Snail signaling pathway. Disruption of EGFL7-EGFR-AKT-Snail signaling may a promising therapeutic strategy for gastric cancer.

Neurofilament Heavy polypeptide CpG island methylation associates with prognosis of renal cell carcinoma and prediction of antivascular endothelial growth factor therapy response.

Neurofilament Heavy polypeptid (NEFH) belongs to the group of type IV intermediate filament proteins. DNA methylation of the NEFH promoter and loss of expression have previously been shown to activate the AKT/ß-catenin pathway in tumor cells. When identifying hypermethylation of the NEFH CpG island (CGI) in renal cell cancer (RCC) we asked whether methylation could provide clinical or prognostic information for RCC and/or predict therapy response in patients with metastatic RCC (mRCC) undergoing antiangiogenic therapy. Relative methylation of the NEFH CGI was analyzed in 132 RCC samples and 83 paired normal tissues using quantitative methylation-specific PCR. Results were statistically compared with tumor histology, clinicopathological parameters, progression-free survival (PFS) as well as with overall survival (OS) in a subset of 18 mRCC patients following antiangiogenic therapy regimens. The NEFH CGI methylation demonstrated a tumor-specific increase (P < 0.001), association with advanced disease (P < 0.001), and distant metastasis (P = 0.005). Higher relative methylation was also significantly associated with a poor PFS (HR = 8.6, P < 0.001) independent from the covariates age, gender, diameter of tumors, state of advanced disease, and local and distant metastasis. Median OS following targeted therapy was 29.8 months for patients with low methylation versus 9.8 months for the group with high methylation (P = 0.028). We identified NEFH methylation as a candidate epigenetic marker for prognosis of RCC patients as well as prediction of anti-vascular endothelial growth factor-based therapy response.

MicroRNA-223 antagonizes angiogenesis by targeting ß1 integrin and preventing growth factor signaling in endothelial cells.

RATIONALE: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. OBJECTIVE: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. METHODS AND RESULTS: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223(-/y) animals were enhanced. MiR-223 overexpression had no effect on the growth factor-induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced phosphorylation of their receptors and activation of Akt. ß1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of ß1 integrin into miR-223-ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. CONCLUSIONS: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting ß1 integrin.

Análisis morfométrico del endotelio corneal tras ranibizumab intravítreo en la degeneración macular asociada a la edad / Morphometric analysis of corneal endothelium after intravitreal ranibizumab (Lucentis®) in age-related macular degeneration treatment

ObjetivoEvaluar el efecto de la inyección intravítrea de 0,5mg ranibizumab sobre el endotelio corneal en pacientes con degeneración macular asociada a la edad (DMAE).MétodoEstudio piloto de serie de casos, observacional y prospectivo con observador enmascarado. Veintiséis ojos (26 pacientes) con DMAE fueron evaluados. Todos los pacientes recibieron 1 inyección intravítrea mensual de 0,5mg de ranibizumab durante 3 meses consecutivos. El periodo de seguimiento fue de 6 meses. El endotelio corneal central fue evaluado antes, a los 7 días y a los 6 meses de la primera inyección de ranibizumab con microscopía especular. La densidad de las células endoteliales, coeficiente de variación del tamaño celular y porcentaje de hexagonalidad celular fueron analizados. También fue medido el espesor corneal central.ResultadosNo se encontraron diferencias estadísticamente significativas entre los valores preinyección y postinyección a los 7 días y 6 meses de la primera inyección de ranibizumab en las densidades de células endoteliales, coeficientes de variación del tamaño celular y porcentajes de hexagonalidad celular (p>0,5). Tampoco se encontraron modificaciones significativas en el espesor corneal central a lo largo del periodo de seguimiento (p>0,5).ConclusionesInyecciones intravítreas repetidas de 0,5mg ranibizumab no parecen causar cambios importantes sobre el endotelio corneal a los 6 meses de seguimiento(AU)

PurposeTo determine the effect of intravitreal injection of 0.5mg ranibizumab on the corneal endothelium in patients with age-related macular degeneration (AMD).MethodsObservational, prospective case series pilot study. Twenty-six eyes of 26 consecutive patients with AMD were evaluated. All participants received one monthly intravitreal injections of 0.5mg ranibizumab for three consecutive months. The follow-up period was 6 months. Central corneal specular microscopy was performed before injection and at 7 days and 6 months after the first intravitreal injection. The endothelial cell density, coefficient of variation of cell size, and percentage of hexagonal cells were analyzed and the central corneal thickness was measured.ResultsThere were no significant differences in the endothelial cell densities, coefficients of variation of cell size and percentages of hexagonal cells before injection and at 7 days and 6 months after the first intravitreal ranibizumab injection (P>0.5). There was also no significant difference in central corneal thickness measurements through the follow-up period (P>0.5).ConclusionsRepeated intravitreal injections of 0.5mg ranibizumab do not seem to cause substantial changes in the corneal endothelium(AU)

Drug discovery in ovarian cancer.

Drug discovery in the ovarian cancer arena has led to the activation of several important clinical trials. Many biologic agents have come down the pipeline and are being studied in phase II trials for recurrent disease. These agents include antivascular compounds that disrupt angiogenesis through a variety of mechanisms (e.g., prevention of ligand-binding to the vascular endothelial growth factor receptor-2 (VEGF-R2), high-affinity VEGF blockade, oral inhibitors of tyrosine kinases stimulated by VEGF, inhibition of alpha5beta1 integrin, neutralization of angioproteins, etc.). Other novel drugs include oral platinum compounds as well as those that antagonize the tumor proliferation genes in the Hedgehog pathway, and that target folic acid receptors which are expressed by ovarian cancer cells. In addition, studies are underway with oral agents that inhibit the tyrosine kinase activity associated with two oncogenes (epidermal growth factor receptor (EGFR) and HER-2/neu). Finally, emerging technologies in clinical trials include nanotechnology to enhance delivery of chemotherapy to ovarian tumors, drug resistance/sensitivity assays to guide therapy, and agents that mobilize and induce proliferation of hematopoetic progenitor cells to aid in red blood cell, white blood cell, and platelet recovery following chemotherapy. The relevant patents in drug discovery of ovarian cancer are discussed.