GBP2 inhibits pathological angiogenesis in the retina via the AKT/mTOR/VEGFA axis.

Pathological retinal angiogenesis is not only the hallmark of retinopathies, but also a major cause of blindness. Guanylate binding protein 2 (GBP2) has been reported to be associated with retinal diseases such as diabetic retinopathy and hypoxic retinopathy. However, GBP2-mediated pathological retinal angiogenesis remains largely unknown. The present study aimed to investigate the role of GBP2 in pathological retinal angiogenesis and its underlying molecular mechanism. In this study, we established oxygen-induced retinopathy (OIR) mice model for in vivo study and hypoxia-induced angiogenesis in ARPE-19 cells for in vitro study. We demonstrated that GBP2 expression was markedly downregulated in the retina of mice with OIR and ARPE-19 cells treated with hypoxia, which was associated with pathological retinal angiogenesis. The regulatory mechanism of GBP2 in ARPE-19 cells was studied by GBP2 silencing and overexpression. The regulatory mechanism of GBP2 in the retina was investigated by overexpressing GBP2 in the retina of OIR mice. Mechanistically, GBP2 downregulated the expression and secretion of vascular endothelial growth factor (VEGFA) in ARPE-19 cells and retina of OIR mice. Interestingly, overexpression of GBP2 significantly inhibited neovascularization in OIR mice, conditioned medium of GBP2 overexpressing ARPE-19 cells inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, we confirmed that GBP2 downregulated VEGFA expression and angiogenesis by inhibiting the AKT/mTOR signaling pathway. Taken together, we concluded that GBP2 inhibited pathological retinal angiogenesis via the AKT/mTOR/VEGFA axis, thereby suggesting that GBP2 may be a therapeutic target for pathological retinal angiogenesis.

The Anti-Diabetic Drug Metformin Suppresses Pathological Retinal Angiogenesis via Blocking the mTORC1 Signaling Pathway in Mice (Metformin Suppresses Pathological Angiogenesis).

PURPOSE: Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). METHODS: OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. RESULTS: Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. CONCLUSIONS: These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis.

Porphyromonas gingivalis lipopolysaccharide affects the angiogenic function of endothelial progenitor cells via Akt/FoxO1 signaling.

AIMS: Endothelial progenitor cells (EPCs) function as the angiogenic switch of many physiological and pathological conditions. We aimed to investigate the effects of Porphyromonas gingivalis lipopolysaccharide on the angiogenic capacity of EPCs and delineate the underlying mechanisms. MATERIALS AND METHODS: EPCs were isolated from human umbilical blood. CCK-8 assay was undertaken to analyze the cell viability. The migration and tube formation capacity were assessed by wound healing and tube formation, respectively. The protein expression of Akt/p-Akt, endothelial nitric oxide synthase (eNOS)/p-eNOS, and Forkhead box O1 (FoxO1)/p-FoxO1 was determined by Western blot. The intracellular localization of FoxO1 was evaluated by immunofluorescent staining. RESULTS: P. gingivalis LPS at 10 µg/ml significantly increased the viability (10.9 ± 2.9%), migration (16.3 ± 3.1%), and tube formation (38.6 ± 5.5%) of EPCs, along with increased phosphorylation of Akt, eNOS, and FoxO1. Mechanistically, Akt inhibition by specific inhibitor wortmannin and FoxO1 forced expression by adenovirus transfection in EPCs markedly attenuated the P. gingivalis LPS-induced eNOS activation, tube formation, and migration. Moreover, P. gingivalis LPS-induced phosphorylation and nuclear exclusion of FoxO1 were blunted by Akt inhibition. CONCLUSIONS: The present study suggests that P. gingivalis LPS could affect the angiogenic function of EPCs through the Akt/FoxO1 signaling. The current findings may shed light on the clinical association of periodontitis with aberrant angiogenesis seen in atherosclerotic plaque rupture.

Redox-responsive nanoparticles enhance radiation therapy by altering multifaceted radio-resistance mechanisms in human castration-resistant prostate cancer cells and xenografts.

INTRODUCTION: Radiation therapy (RT) is a major modality for the treatment of prostate cancer (PCa), especially castration-resistant PCa (CRPC). However, hypoxia, often seen in PCa tumors, leads to radiation-resistance. This work investigates the effect of a novel oxygen-generating polymer-lipid manganese dioxide nanoparticle (PLMDs) on improving RT outcomes in CRPC xenograft models by modulating the tumor microenvironment (TME) both before and after RT. MATERIALS AND METHODS: Human PC3 and DU145 PCa cells were used to investigate clonogenic inhibition and DNA repair pathways in vitro. Tumor hypoxia and post-RT angiogenesis were evaluated in a PC3-bearing SCID mouse model. PC3 and DU145 xenografts were used to study the efficacy of PLMD in combination with single or fractionated RT. RESULTS: PLMD plus RT significantly inhibited clonogenic potential, increased DNA double-strand breaks, and reduced DNA damage repair in hypoxic PC3 and DU145 cells as compared to RT alone. PLMD significantly reduced hypoxia-positive areas, hypoxia induced factor 1α (HIF-1α) expression, and protein carbonyl levels (a measure of oxidative stress). Application of PLMD with RT decreased RT-induced angiogenic biomarkers by up to 3-fold. Treatment of the human CRPC xenografts with PLMD plus RT (single or fractionated doses) significantly prolonged median survival of the host compared to RT alone resulting in up to a 40% curative rate. CONCLUSION: PLMD treatment modulated TME and sensitized hypoxic human CRPC cells to RT thus enhancing the efficacy of RT. These results confirmed the potential of PLMD as an adjuvant to RT for the treatment of hypoxic CRPC.

Exclusive surgical approach for facial arteriovenous malformations in precarious conditions.

Arteriovenous malformations (AVMs) are high-flow vascular lesions that does not regress spontaneously. They are located in the cranio-facial region in 50% of cases. Most of the time, the management of these lesions is a combination of surgery and vascular embolization. However, when the conditions are precarious, even without access to embolization, it's possible to treat some of those lesions with safety. We report four cases of patients suffering from cranio-facial AVM, treated exclusively by surgery during humanitarian missions.

VEGF-Targeted Multispectral Optoacoustic Tomography and Fluorescence Molecular Imaging in Human Carotid Atherosclerotic Plaques.

Vulnerable atherosclerotic carotid plaques are prone to rupture, resulting in ischemic strokes. In contrast to radiological imaging techniques, molecular imaging techniques have the potential to assess plaque vulnerability by visualizing diseases-specific biomarkers. A risk factor for rupture is intra-plaque neovascularization, which is characterized by overexpression of vascular endothelial growth factor-A (VEGF-A). Here, we study if administration of bevacizumab-800CW, a near-infrared tracer targeting VEGF-A, is safe and if molecular assessment of atherosclerotic carotid plaques in vivo is possible using multispectral optoacoustic tomography (MSOT). Healthy volunteers and patients with symptomatic carotid artery stenosis scheduled for carotid artery endarterectomy were imaged with MSOT. Secondly, patients were imaged two days after intravenous administration of 4.5 bevacizumab-800CW. Ex vivo fluorescence molecular imaging of the surgically removed plaque specimen was performed and correlated with histopathology. In this first-in-human MSOT and fluorescence molecular imaging study, we show that administration of 4.5 mg bevacizumab-800CW appeared to be safe in five patients and accumulated in the carotid atherosclerotic plaque. Although we could visualize the carotid bifurcation area in all subjects using MSOT, bevacizumab-800CW-resolved signal could not be detected with MSOT in the patients. Future studies should evaluate tracer safety, higher doses of bevacizumab-800CW or develop dedicated contrast agents for carotid atherosclerotic plaque assessment using MSOT.

Assessment of Tumor Angiogenesis by Expression of CD 105 in Ameloblastoma, Odontogenic Keratocyst and Central Giant Cell Lesion.

BACKGROUND: Angiogenesis is critical for tumor growth and reflects the aggressive behavior of invasive odontogenic lesions [like Ameloblastoma (AM), Odontogenic Keratocyst (OKC) and Central giant cell lesion (CGCL)]. Mean vascular density (MVD) shows the angiogenic potential and CD105 is an ideal endothelial biomarker due to its specificity to new blood vessels for MVD detection. The aim of the study was to compare the MVD (angiogenic potential) among AM, OKC and CGCL in comparison to Pyogenic Granuloma (PG) using CD105 biomarker. METHODS: Sixty-four primary cases of odontogenic invasive tumors (AM, OKC and CGCL) and PG, diagnosed clinically and histologically were included in the study, with 16 samples in each group. Tissue samples of peripheral AM, Peripheral GCL of jaws, malignant AM, and specimen with insufficient tissue were excluded. Tissue sections were embedded, processed and stained using Hematoxylin and Eosin (H and E). Immunohistochemistry was performed using antibodies against CD105, with positive brown cytoplasmic staining in the endothelial cells of neo-vasculature. Distinct countable, positively stained endothelial cell or clusters were evaluated under light microscope for identification of MVD. ANOVA and t-test were applied for statistical analysis of data. RESULTS: Highest MVD was displayed in CGCL (32.99±0.77) and the minimum was observed in OKC (7.21± 0.75) respectively. CGCL showed significantly higher MVD to AM, OKC and PG lesions (p <0.05). AM (8.07± 0.36) and Odontogenic Keratocyst (7.21± 0.75) showed comparable MVD, which was lower than PG (14.7± 0.96) and CGCL vascular density (p < 0.01) respectively. CONCLUSION: CGCL was most aggressive, with highest MVD among the investigated odontogenic lesions (OKC, AM and PG). The proliferative aggressive behavior of Odontogenic Keratocyst is comparable to AM due to comparable mean vascular density.
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Novel Markers of Angiogenesis in the Setting of Cognitive Impairment and Dementia.

BACKGROUND: Aberrant angiogenesis may play a role in the development of Alzheimer's disease and related dementia. OBJECTIVE: To explore the relationship between angiogenesis activity and evidence of neurodegeneration among older adults. METHODS: Cross-sectional study of 49 older adults clinically characterized as cognitively normal, mild cognitive impairment, or early Alzheimer's disease. In addition to neuroimaging, we completed assays on peripheral blood, including: vascular endothelial growth factor, tumor necrosis factor, fibroblast growth factor, and amyloid-ß peptide 40. We used advanced polychromatic flow cytometry to phenotype circulating mononuclear cells to assess angiogenesis activity. RESULTS: Although we documented differences in cognitive performance, structural changes on neuroimaging, and burden of amyloid and tau on positron emission tomography, angiogenesis activity did not vary by group. Interestingly, VEGF levels were shown to be increased among subjects with mild cognitive impairment. In ANCOVA models controlling for age, sex, intracranial volume, and monocyte subpopulations, angiogenesis activity was correlated with increased white matter hyperintensities. CONCLUSION: We demonstrate a significant association between angiogenesis activity and cerebrovascular disease. To better understand the potential of angiogenesis as an intervention target, longitudinal studies are needed.

Atorvastatin increases oxidative stress and inhibits cell migration of oral squamous cell carcinoma in vitro.

OBJECTIVE: This study aimed to evaluate the effect of atorvastatin treatment on reactive oxygen species (ROS) production and tumor angiogenesis in oral squamous cell carcinomas. MATERIAL AND METHODS: An HN13 cell line was treated with 1 µM, 5 µM, and 10 µM of atorvastatin. VEGF-A gene expression was evaluated by quantitative real time PCR. VEGF-A protein expression was quantified from total protein and conditioned media by ELISA. Cellular oxidative stress was measured using 2',7'-dichlorfluorescein-diacetate (DCFH-DA). Angiogenesis assay was performed using human umbilical vein endothelial cells (HUVEC). The effect of atorvastatin on cell migration was evaluated by wound healing assay. RESULTS: 5 µM and 10 µM of atorvastatin significantly increased VEGF-A gene expression in the HN13 cell line. Intracellular expression of the VEGF-A protein was higher in the cells treated with 5 µM and 10 µM than in the control cells. VEGF-A protein expression was also higher in the conditioned media from the atorvastatin-treated cells than in the media from the DMSO-treated cells. 5 µM and 10 µM of atorvastatin increased oxidative stress. Regarding angiogenesis assay, 5 µM of atorvastatin resulted in higher numbers of branch points, compared to the solvent. 10 µM of atorvastatin treatment resulted in significantly reduced cell migration. CONCLUSIONS: This study showed that atorvastatin increases the oxidative stress and angiogenesis in oral squamous cell carcinomas. The decrease of cell migration indicates atorvastatin's inhibitory effect in oral tumors. These results suggest that atorvastatin could increase the intracellular oxidative stress in these cells, leading to a toxic microenvironment and inhibiting their metastasis.

Overexpression of Antiangiogenic Vascular Endothelial Growth Factor Isoform and Splicing Regulatory Factors in Oral, Laryngeal and Pharyngeal Squamous Cell Carcinomas

Background: Overexpression of proangiogenic vascular endothelial growth factor A family VEGFAxxx is associated with tumor growth and metastasis. The role of the alternatively spliced antiangiogenic family VEGFAxxxb is poorly investigated in head and neck squamous cell carcinomas (HNSCCs). The antiangiogenic isoform binds to bevacizumab and its expression level could influence the treatment response and progression-free survival. In this study, the relative expression of VEGFAxxx and VEGFA165b isoforms and splicing regulatory factors genes was investigated in a series of HNSCCs. Methods: VEGFAxxx, VEGFA165b, SRSF6, SRSF5, SRSF1 and SRPK1 gene expression was quantified by quantitative real time PCR in 53 tissue samples obtained by surgery from HNSCC patients. Protein expression was evaluated by immunohistochemistry. Results: VEGFAxxx and VEGFA165b were overexpressed in HNSCCs. Elevated protein expression was also confirmed. However, VEGFA isoforms demonstrated differential expression according to anatomical sites. VEGFAxxx was overexpressed in pharyngeal tumors while the VEGFA165b isoform was up-regulated in oral tumors. The VEGFA165b isoform was also positively correlated with expression of the splicing regulatory genes SRSF1, SRSF6 and SRSF5. Conclusions: We concluded that VEGFAxxx and VEGFA165b isoforms are overexpressed in HNSCCs and the splicing regulatory factors SRSF1, SRSF6, SRSF5 and SRPK1 may contribute to alternative splicing of the VEGFA gene. The findings for the differential expression of the antiangiogenic isoform in HNSCCs could facilitate effective therapeutic strategies for the management of these tumors.

Difference in EGFR expression and mean vascular density in normal oral mucosa, oral epithelial dysplasia and oral squamous cell carcinoma

To evaluate the expression of the epidermal growth factor receptor (EGFR) and mean vascular density (MVD) in normal oral mucosa (NOM), oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Material and methods: Descriptive case study. Nineteen histological samples diagnosed with NOM, 18 diagnosed with OED, and 19 with OSCC, were analyzed with immunohistochemistry against EGFR and CD31. EGFR expression was evaluated by extent and intensity of its expression in normal, dysplastic and neoplastic epithelium. MVD was determined through the detection of blood vessels by antibodies against CD31. Results: Extension of EGFR expression was highest in OSCC followed by OED and lowest in NOM, resulting in significant different between the degrees of extension (p<0.001). Intensity of EGFR was similar in NOM, OED and OSCC, without differences in its expression (p=0.533). Differences in MVD were found between NOM and OSCC groups (p<0.01), and between OED and OSCC groups (p<0.01), with no differences between NOM and OED groups (p=0.91). MVD was 21.17 +/- 4.98 in NOM, 23.40 +/- 5.77 in OED and 33.92 +/- 8.39 in OSCC. Conclusion: EGFR is expressed in normal, dysplastic or neoplastic oral epithelium. However, the extent of its expression is greater as malignancy increases. MVD varies according to the diagnosis.

Management of a large diffuse maxillofacial arteriovenous malformation previously treated with ligation of ipsilateral arterial supply.

A 32 year-old male presented with a pulsatile facial mass with palpable thrill and audible bruit. Imaging revealed a very large diffuse left-sided facial arteriovenous malformation with extensive bilateral supply, as well as a previously ligated left external carotid artery. Endovascular treatment was required to control associated hemorrhagic events as well as for palliation and was delivered via the contralateral and ipsilateral collateral supply because of ligation of the direct route to the nidus. In addition, the patient received intravenous bevacizumab and intraarterial bleomycin therapy. Under such circumstances, endovascular embolization remains often the only option when emergent therapy for massive haemorrhage is required. Collaboration and treatment planning with head and neck surgery is imperative and should be performed from the onset, avoiding disastrous ligation of arterial feeders.

Diabetes-Induced Jagged1 Overexpression in Endothelial Cells Causes Retinal Capillary Regression in a Murine Model of Diabetes Mellitus: Insights Into Diabetic Retinopathy.

BACKGROUND: Several mechanisms have been proposed to account for diabetes-induced microvasculopathy (DMV). Although Notch signaling was reported to be affected by glucose metabolism in endothelial cells during developmental angiogenesis, it has not been investigated in vascular remodeling of adult capillaries in relation to diabetes mellitus. METHODS: We induced diabetes mellitus in 8-week-old adult mice by intravenously administering streptozotocin. After 6 weeks, we harvested organs, including retina, heart, and skeletal muscle, and evaluated the capillaries with immunofluorescence and confocal microscopy. We modulated endothelial Notch signaling using chemical inhibitors in wild-type mice or transgenic mice, inducing conditional knockout of Jagged1 or Mib1. RESULTS: DMV was characterized by capillary remodeling, regression, and decreased density. Notch ligand Jagged1, but not δ-like ligand 4, was markedly increased in endothelial cells of diabetic mice. Using endothelium-specific Jagged1 knockdown mice, we found that blocking Jagged1 prevented DMV even under diabetic conditions. Furthermore, in the inducible endothelium-specific Jagged1 knockdown mice, blocking Jagged1 even at 4 weeks after the establishment of DMV could reverse it, leading to normalization of retinal vasculature. A search for downstream signals revealed that diabetes mellitus decreased the nuclear localization of Notch1 intracellular domain and reduced the expression of VE-cadherin and N-cadherin in endothelial cells. Chemical Notch inhibition phenocopied DMV in normal mice. CONCLUSIONS: Our findings indicate that diabetes mellitus induces Jagged1 overexpression and suppresses Notch signaling in endothelial cells, leading to DMV in adult mice. We conclude that dysregulated intercellular Notch signaling may be a novel mechanism of DMV.

Microvascular density and immunohistochemical expression of VEGF, VEGFR-1 and VEGFR-2 in benign prostatic hyperplasia, high-grade prostate intraepithelial neoplasia and prostate cancer.

INTRODUCTION: The aim of our study was to determine and compare angiogenesis in benign prostatic hyperplasia (BPH), high-grade prostate intraepithelial neoplasia (HGPIN) and prostate cancer (Pca). Moreover, we evaluated its role as a prognostic factor for Pca. MATERIAL AND METHODS: We examined 39, 12 and 51 samples of BPH, HGPIN and Pca, respectively. Immunohistochemical methods were applied in order to evaluate the expression of VEGF and its receptors (VEGFR-1, VEGFR-2), while microvascular density (MVD) was determined using CD105. In Pca samples, we recorded stage, differentiation, perineural invasion, adjuvant radiotherapy and their correlation with angiogenesis. RESULTS: 225 The expression of VEGF, VEGFR-1 and VEGFR-2 was significantly higher in Pca than compared to BPH (p <0.001, p <0.001 and p <0.001, respectively) and HGPIN (p <0.001, p <0.001 and p = 0.04, respectively), while there was no difference between BPH and HGPIN. MVD was higher in Pca compared to BPH (p <0.001) and HGPIN (p <0.01), while there was no difference between BPH and HGPIN. VEGF expression and MVD were significantly greater in Pca samples with poor differentiation (p = 0.044 and p = 0.038, respectively) and perineural invasion (p <0.001 and p = 0.019, respectively), while overexpression of VEGF was associated with advanced pathological stage (p = 0.047). CONCLUSIONS: Angiogenesis is more prominent in Pca than in BPH and HGPIN, while there is no difference between BPH and HGPIN. Pharmaceutical inhibition of angiogenesis could be a valuable therapeutic option for Pca in the near future.

Endothelial precursor cell-based therapy to target the pathologic angiogenesis and compensate tumor hypoxia.

Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected: MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.

CDKN2B Regulates TGFß Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels.

RATIONALE: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. OBJECTIVE: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. METHODS AND RESULTS: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor ß (TGFß) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFß1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFß activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFß1-induced-1, which is a TGFß-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. CONCLUSIONS: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFß signaling and hypoxic neovessel maturation.

Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

A platform of integrative studies from in vitro to in vivo experiments: towards drug development for ischemic retinopathy.

Pathologic angiogenesis induced by hypoxia is a hallmark of ischemic retinopathy including diabetic retinopathy and retinopathy of prematurity. These 2 diseases affect substantial number of working population and preterm babies, respectively, resulting in visual deterioration. It is essential for novel therapeutics for ischemic retinopathy to demonstrate the potency in reducing pathologic angiogenesis and the safety without definite toxicity on the retina and the whole body. In this review, we suggest a novel platform of integrative studies from in vitro to in vivo experiments on angiogenesis and toxicity with the aim of accelerating and facilitating the development of novel therapeutic agents for ischemic retinopathy. Robust in vitro and in vivo studies with bridging microfluidic and ex vivo systems help researchers to evaluate the efficacy and anticipate the toxicity of candidate drugs. We hope that novel therapeutic approach based on this platform will be developed in near future and reduce the incidence of vision loss from ischemic retinopathy.

Anti-angiogenic effect of bare titanium dioxide nanoparticles on pathologic neovascularization without unbearable toxicity.

Local application requires fewer nanoparticles than systemic delivery to achieve effective concentration. In this study, we investigated the potential toxicity and efficacy of bare titanium dioxide (TiO2) nanoparticles by local administration into the eye. Mono-disperse, 20nm-size TiO2 nanoparticles did not affect the viability of retinal constituent cells within certain range of concentrations (~1.30µg/mL). Furthermore, local delivery of TiO2 nanoparticles did not induce any significant toxicity at the level of gene expression and histologic integrity in the retina of C57BL/6 mice. Interestingly, at the low concentration (130ng/mL) without definite toxicity, these nanoparticles suppressed in vitro angiogenesis processes and in vivo retinal neovascularization in oxygen-induced retinopathy mice when they are administered intravitreally. Taken together, our results demonstrate that even TiO2 nanoparticles can be safely utilized for the treatment of retinal diseases at the adequate concentration levels, especially through local administration. FROM THE CLINICAL EDITOR: In this paper the local application of titanium dioxide is described as a local treatment for retinal diseases associated with neovascularization. While these nanoparticles have known systemic toxicity, this work demonstrates that when applied locally in a mouse model, they can be used without observable toxicity even in their native forms.