Electrostatically Cross-Linked Bioinks for Jetting-Based Bioprinting of 3D Cell Cultures.

It has been acknowledged that thousands of drugs that passed two-dimensional (2D) cell culture models and animal studies often fail when entering human clinical trials. Despite the significant development of three-dimensional (3D) models, developing a high-throughput model that can be reproducible on a scale remains challenging. One of the main challenges is precise cell deposition and the formation of a controllable number of spheroids to achieve more reproducible results for drug discovery and treatment applications. Furthermore, when transitioning from manually generated structures to 3D bioprinted structures, the choice of material is limited due to restrictions on materials that are applicable with bioprinters. Herein, we have shown the capability of a fast-cross-linking bioink that can be used to create a single spheroid with varying diameters (660, 1100, and 1340 µm) in a high-throughput manner using a commercialized drop-on-demand bioprinter. Throughout this work, we evaluate the physical properties of printable ink with and without cells, printing optimization, cytocompatibility, cell sedimentation, and homogeneity in ink during the printing process. This work showcases the importance of ink characterization to determine printability and precise cell deposition. The knowledge gained from this work will accelerate the development of next-generation inks compatible with a drop-on-demand 3D bioprinter for various applications such as precision models to mimic diseases, toxicity tests, and the drug development process.

Determinants of self-monitoring of blood glucose in patients with Type 1 diabetes: a multi-centre study in Brazil.

AIM: The aim of this study was to determine the relationship between the daily frequency of self-monitoring of blood glucose and glycaemic control, demographic and socio-economic status in patients with Type 1 diabetes under routine clinical care in Brazil. METHODS: This was a cross-sectional, multi-centre study conducted between December 2008 and December 2010 in 28 public clinics in 20 Brazilian cities. The data were obtained from 3176 patients, aged 22 ± 11.8 years, of whom 56.3% were female and 57.4% were Caucasian. The mean time since diabetes diagnosis was 11.7 ± 8.1 years. RESULTS: The prevalence of self-monitoring of blood glucose was 88.5%. There was a significant increase in self-monitoring frequency associated with female gender, lower ages, more intensive diabetes management and higher socio-economic status. A correlation between HbA(1c) levels and the daily frequency of self-monitoring was observed (r(s) = -0.13; P = 0.001). The mean HbA1c levels were related to the daily frequency of self-monitoring (P < 0.001) without additional benefit to patients who performed self-monitoring more than four times daily (9.2, 11.2, 10.2,15.2 and 15% for one, two, three, four, five or more self-monitoring tests daily, respectively; P < 0.0001). CONCLUSIONS: The majority of our patients (88.5%) performed three or more self-monitoring tests daily, with more frequent testing reported by females, younger patients, those on intensive insulin regimens and of higher socio-economic status. No additional benefit was found in patients who performed self-monitoring more than four times daily. The diabetes care team must improve patients' education regarding self-monitoring of blood glucose and its benefits.

3D Bioprintable Hydrogel with Tunable Stiffness for Exploring Cells Encapsulated in Matrices of Differing Stiffnesses.

In vitro cell models have undergone a shift from 2D models on glass slides to 3D models that better reflect the native 3D microenvironment. 3D bioprinting promises to progress the field by allowing the high-throughput production of reproducible cell-laden structures with high fidelity. The current stiffness range of printable matrices surrounding the cells that mimic the extracellular matrix environment remains limited. The work presented herein aims to expand the range of stiffnesses by utilizing a four-armed polyethylene glycol with maleimide-functionalized arms. The complementary cross-linkers comprised a matrix metalloprotease-degradable peptide and a four-armed thiolated polymer which were adjusted in ratio to tune the stiffness. The modularity of this system allows for a simple method of controlling stiffness and the addition of biological motifs. The application of this system in drop-on-demand printing is validated using MCF-7 cells, which were monitored for viability and proliferation. This study shows the potential of this system for the high-throughput investigation of the effects of stiffness and biological motif compositions in relation to cell behaviors.

Impact of a multidimensional infection control strategy on central line-associated bloodstream infection rates in pediatric intensive care units of five developing countries: findings of the International Nosocomial Infection Control Consortium (INICC).

PURPOSE: To analyze the impact of the International Nosocomial Infection Control Consortium (INICC) multidimensional infection control strategy including a practice bundle to reduce the rates of central line-associated bloodstream infection (CLAB) in patients hospitalized in pediatric intensive care units (PICUs) of hospitals, which are members of the INICC, from nine cities of five developing countries: Colombia, India, Mexico, Philippines, and Turkey. METHODS: CLAB rates were determined by means of a prospective surveillance study conducted on 1,986 patients hospitalized in nine PICUs, over a period of 12,774 bed-days. The study was divided into two phases. During Phase 1 (baseline period), active surveillance was performed without the implementation of the multi-faceted approach. CLAB rates obtained in Phase 1 were compared with CLAB rates obtained in Phase 2 (intervention period), after implementation of the INICC multidimensional infection control program. RESULTS: During Phase 1, 1,029 central line (CL) days were recorded, and during Phase 2, after implementing the CL care bundle and interventions, we recorded 3,861 CL days. The CLAB rate was 10.7 per 1,000 CL days in Phase 1, and in Phase 2, the CLAB rate decreased to 5.2 per 1,000 CL days (relative risk [RR] 0.48, 95% confidence interval [CI] 0.29-0.94, P = 0.02), showing a reduction of 52% in the CLAB rate. CONCLUSIONS: This study shows that the implementation of a multidimensional infection control strategy was associated with a significant reduction in the CLAB rates in the PICUs of developing countries.

A high-throughput 3D bioprinted cancer cell migration and invasion model with versatile and broad biological applicability.

Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.

Unraveling the effects on lithium-ion cathode performance by cation doping M-Li2CuO2 solid solution samples (M = Mn, Fe and Ni).

Cation doping is one of the most dynamic strategies to enhance the electrochemical properties of cathode materials for lithium-ion batteries. Nevertheless, the maximum partial substitution capacity depends on the solubility of each metal ion, and so the formation of impurities is a very common consequence. Thus, the correlation between electrochemical performance and the doping effect is frequently unknown. In this study, the effect of the partial substitution of copper by manganese, iron or nickel in Li2CuO2 is evaluated, as well as the effect on the electrochemical performance of the modified Li2CuO2 samples as lithium ion battery cathode materials. XRD characterization confirmed single phase formation for all samples, and the incorporation of the transition metal in the Li2CuO2 structure was evaluated by XRD profile fitting, EPR and 7Li-NMR. The results showed modifications in intra- and inter-chain interactions, associated with the variations in the Cu-O-Cu bond angle and changes in magnetic order, due to the presence of the doping transition metal. Among all samples, only manganese partial substitution reveals a drastic improvement in the electrochemical stability during the charge/discharge processes even at potentials higher than 3.9 V. It was corroborated that the higher stability is attributed to (i) the increase in the superexchange interactions between the copper sites and manganese, directly modifying lithium diffusivity and electronic conductivity, both inferred from dynamic thermogravimetric analysis for CO2 sorption and conductivity tests, respectively and (ii) the lower propensity to enable O2 evolution during several charge cycles. These results are totally attributed to manganese cation partial substitution, which has a huge impact on the utilization of copper-based materials in real applications.

Electronic density distribution of Mn-N bonds by a tuning effect through partial replacement of Mn by Co or Ni in a sodium-rich hexacyanoferrate and its influence on the stability as a cathode for Na-ion batteries.

This study evaluates the effect of equimolar substitution of manganese by cobalt or nickel in hexacyanoferrate (HCF) open frameworks as electrode materials for Na-ion batteries. As the stability of Mn-N bonds is crucial to obtain long term stability and cyclability of manganese (Mn-HCF), the samples were analyzed thoroughly using several spectroscopic and structural methods. The XPS and infrared experiments reveal that the charge density around Fe is modulated by the presence of Co or Ni, which is associated with their high polarizing power, leading to decreased cell distortion as revealed by XRD. The Rietveld refinement demonstrated that the octahedra built by 3d metals and the cyanide nitrogen were distorted with the axial bond distances being larger than the equatorial distances. This octahedral distortion promotes the spin behavior of 3/2 for Mn2+ confirmed by magnetic experiments; the arising of this spin state is attributed to d orbital splitting determined by UV-Vis experiments. Therefore, the changes upon Mn substitution are related to the modification of the covalent character of Mn-N bonds, modulated by the effect of the Ni and Co polarizing power. All these properties improve the electrochemical stability of the Ni or Co substituted materials as Na-ion batteries, leading to higher capacity retention even at higher C-rates (5C) and good capacity recovery, in comparison with those obtained for Mn-HCF.

Advanced glycation end products increase retinal vascular endothelial growth factor expression.

Advanced glycation end products (AGEs) are linked with the development of diabetic retinopathy; however, the pathogenic mechanisms are poorly defined. Vascular endothelial growth factor (VEGF) levels are increased in ischemic and nonischemic diabetic retina, and VEGF is required for the development of retinal and iris neovascularization. Moreover, VEGF alone can induce much of the concomitant pathology of diabetic retinopathy. In this study, we found that AGEs increased VEGF mRNA levels in the ganglion, inner nuclear, and retinal pigment epithelial (RPE) cell layers of the rat retina. In vitro, AGEs increased VEGF mRNA and secreted protein in human RPE and bovine vascular smooth muscle cells. The AGE-induced increases in VEGF expression were dose- and time-dependent, inhibited by antioxidants, and additive with hypoxia. Use of an anti-VEGF antibody blocked the capillary endothelial cell proliferation induced by the conditioned media of AGE-treated cells. AGEs may participate in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo.

Elevated vascular endothelial growth factor (VEGF) levels are required for ocular and tumor angiogenesis in animal models. Ischemic hypoxia is strongly correlated with increased VEGF expression in these systems and is considered a physiologically relevant stimulus. Because ischemic hypoxia is often followed by reperfusion and reactive oxygen intermediate (ROI) generation, we examined the potential role of ROI in the control of VEGF gene expression. Human retinal pigment epithelial cells exposed to superoxide or hydrogen peroxide rapidly increased VEGF mRNA levels. Superoxide-associated mRNA increases were dose dependent, blocked by antioxidants, and associated with elevated VEGF protein levels in conditioned media. Increases in VEGF mRNA levels were also observed in cultured human melanoma and rat glioblastoma cells with superoxide or hydrogen peroxide. Cycloheximide prevented the ROI-associated increases in VEGF mRNA. Transcriptional inhibition with actinomycin D revealed an inducible increase in VEGF mRNA half-life, but nuclear run-on experiments showed no increase in VEGF transcriptional rate. Reoxygenation of human retinal pigment epithelial cells in vitro and ocular reperfusion in vivo increased retinal VEGF mRNA levels. Antioxidants prevented the reperfusion-associated VEGF mRNA increases in retina. We conclude that ROIs increase VEGF gene expression in vitro and during the reperfusion of ischemic retina in vivo. The ROI-associated increases are mediated largely through increases in VEGF mRNA stability.

A strategy to discover circulating angiogenesis inhibitors generated by human tumors.

The phenomenon of inhibition of tumor growth by tumor mass has been studied in many experimental animal systems and has been observed in several clinical scenarios. Not until the recent discovery of angiostatin, a circulating angiogenesis inhibitor generated in the presence of a murine Lewis lung tumor, has a satisfactory mechanism been proposed to explain this phenomenon. Thus far, no other animal or human tumors are known to generate angiostatin. In this study, we utilized a mouse corneal neovascularization model to detect circulating inhibitors of angiogenesis generated by three human tumors grown in immunodeficient mice: (a) the PC-3 human prostate carcinoma; (b) the CCL188 human colon carcinoma; and (c) the UBC urinary bladder carcinoma. Mice bearing these three primary tumors demonstrated significant inhibition of angiogenesis in the cornea induced by a pellet containing basic fibroblast growth factor. Corneas of mice bearing s.c. prostate and colon carcinomas showed significant inhibition of vessel length, clock-hours of neovascularization, and vessel density. However, corneas of mice bearing s.c. bladder carcinomas demonstrated significant inhibition of vessel density only. Three colon carcinomas (clone A, CX-1, and MIP101), the MDA-MB-435S breast carcinoma, the MM-AN melanoma, and the JE-3 choriocarcinoma did not significantly inhibit corneal neovascularization.

Nelore females along a new Brazilian agricultural frontier: hematological and clinical-biochemical approaches / [Abordagens hematológicas e bioquímicas clínicas de fêmeas Nelore na nova fronteira agrícola brasileira]

The Nelore breed is the second largest bovine breed in the world and has actively participated in the expansion of new Brazilian agricultural frontiers. In this context, the purpose of this study was to determine the hematological and biochemical reference intervals of healthy Nelore matrices raised under an extensive regime without supplementation along southwest of Piauí state. Blood samples were collected from fifty-five multiparous female of the Nelore breed. Biochemical and hematological parameters were analyzed using a parametric statistical method with 95% CI of reference limits. The average values of red blood cells, hemoglobin as well as hematimetric indices showed reference ranges similar to reference standards. The hematocrit as well as granulocytes and agranulocytes presented alterations typical of animals raised in environments with higher temperatures. Mineral, enzymatic, protein and metabolic profiles were similar to other bovine breeds but with a narrower range of values. However, lower mean values were observed for levels of ionized calcium, total protein and urea. Nelore females present slightly different biochemical and hematological profiles from other breeds, which might result from the environmental and nutritional management applied and the natural deficiency of nitrogen, phosphorus and calcium in the region's pastures.(AU)

Nelore é a segunda maior raça bovina do mundo e tem participado ativamente da expansão das novas fronteiras agrícolas brasileiras. Nesse contexto, o objetivo do presente estudo foi determinar os intervalos de referência hematológicos e bioquímicos de matrizes Nelore criadas em regime extensivo sem suplementação, ao longo do sudoeste do estado do Piauí. Amostras de sangue foram coletadas de 55 fêmeas multíparas da raça Nelore. Os parâmetros bioquímicos e hematológicos foram analisados por método estatístico paramétrico com IC 95% para os limites de referência. Os valores médios de hemácias, hemoglobina e índices hematimétricos apresentaram intervalos de referência semelhantes aos padrões de referência. Tanto o hematócrito quanto os granulócitos e os agranulócitos apresentaram alterações típicas de animais criados em ambientes com temperaturas mais elevadas. Os perfis mineral, enzimático, proteico e metabólico foram semelhantes aos de outras raças bovinas, mas com uma faixa de valores mais estreita. No entanto, valores médios mais baixos foram observados para os níveis de cálcio ionizado, proteína total e ureia. Fêmeas Nelore apresentam perfis bioquímicos e hematológicos ligeiramente diferentes de outras raças, o que pode resultar dos manejos ambiental e nutricional aplicados e da deficiência natural de nitrogênio, fósforo e cálcio nas pastagens da região.(AU)

Regulation of vascular endothelial growth factor expression by insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) levels are correlated with retinal ischemia-associated intraocular neovascularization in humans. Since VEGF is required for iris and retinal neovascularization in animal models of retinal ischemia, we tested whether IGF-I could act as an indirect angiogenic factor by increasing VEGF gene expression. IGF-I increased retinal pigment epithelial (RPE) cell VEGF mRNA in a concentration-dependent manner with an EC50 of 7 nmol/1 (53.6 ng/ml). RPE and bovine smooth muscle cells exposed to 50 nmol/l (383 ng/m1) IGF-I achieved peak VEGF mRNA expression within 2 h. IGF-I-treated RPE cells increased VEGF protein levels in conditioned media and stimulated capillary endothelial cell proliferation. Blockade of the IGF-I receptor with a neutralizing antibody abrogated the VEGF increases in RPE cells. Further, hypoxia-mediated and IGF-I-mediated increases in VEGF mRNA and protein levels were additive in RPE cells, and the hypoxia-induced VEGF increases were independent of endogenous IGF-I. VEGF promoter activity was enhanced by IGF-I in RPE cells, but VEGF transcript half-life was unaltered. In summary, the supplementation of RPE and smooth muscle cell cultures with IGF-I at 5-100 nmol/l increased VEGF mRNA and secreted protein levels. The VEGF increases in RPE cells occurred primarily through enhanced transcription of the VEGF gene and via the IGF-I receptor. Elevated IGF-I levels may promote neovascularization through increased retinal VEGF gene expression.

Prospective evaluation of percutaneous central venous silastic catheters in newborn infants with birth weights of 510 to 3,920 grams.

With improved neonatal survival, especially of very low birth weight infants, our efforts should be directed toward reduction of morbidity. Sick preterm infants require total parenteral nutrition for prolonged periods of time due to extreme prematurity and feeding intolerance. However, the use of surgically placed Broviac catheters has been associated with a high complication rate. A prospective study of 53 percutaneous central venous Silastic catheterizations for administration of total parenteral nutrition was performed in 45 newborn infants. At the time of catheter insertion, 37 babies weighted less than 1,500 g and 19 weighed less than 1,000 g. Percutaneous central venous catheters were placed successfully the first time in 50 of 55 attempts. In three babies, insertion was successful on second attempt. The catheters remained in place for 25.4 +/- 16.7 days ([mean +/- SD] range two to 80 days). In babies weighing less than 1,000 g, the catheters remained in place for a longer period of time (34.0 +/- 18.0; range 12 to 80 days). Sixty-six percent of the catheters were removed electively. There were four cases of bacteremia (7.5%), and the overall incidence of mechanical complications was 26.4%. We conclude that percutaneous central venous catheters can be used safely and effectively in newborn infants for prolonged administration of total parenteral nutrition, especially in neonates weighing less than 1,000 g.

Requirement for vascular endothelial growth factor in wound- and inflammation-related corneal neovascularization.

PURPOSE: Vascular endothelial growth factor (VEGF) is required for vascular development and for ischemia-related tumor, iris, and retinal neovascularization. The role of VEGF in inflammatory corneal neovascularization is unknown and was investigated in these studies. METHODS: A rat model was used in which removal of the corneal and limbal epithelium resulted in circumferential neovascularization. Corneal VEGF mRNA levels were quantified with ribonuclease protection assays, and VEGF protein was studied in situ using immunohistochemical analysis. Controlled-release pellets containing anti-VEGF antibodies were implanted into the corneal stroma and were used to determine the requirement for VEGF in corneal neovascularization. RESULTS: VEGF mRNA and protein were induced to high levels after corneal injury and were temporally and spatially correlated with inflammation and neovascularization. VEGF immunoreactivity was localized primarily to the inflammatory cells invading the wounded cornea. The specific inhibition of VEGF bioactivity with neutralizing antibodies potently suppressed corneal neovascularization. CONCLUSIONS: These data are the first to demonstrate that VEGF may be required for inflammatory neovascularization of the rat cornea and to identify VEGF as a functional endogenous corneal angiogenic factor.

Cloning and mRNA expression of vascular endothelial growth factor in ischemic retinas of Macaca fascicularis.

PURPOSE: To identify and isolate cDNAs for the alternatively spliced vascular endothelial growth factor (VEGF) mRNAs present in retina and to compare the relative levels of the splice variants and localization of VEGF mRNA in nonischemic and ischemic adult simian retinas. METHODS: Retinas of cynomolgous monkeys were made ischemic by laser occlusion of the main branch retinal veins. Reverse transcription-polymerase chain reaction was used to amplify the VEGF coding region of RNA from ischemic and control retinas, and amplification products were analyzed by agarose gel electrophoresis, Southern blot, and nucleotide sequencing. Analysis of VEGF mRNA expression was accomplished by in situ hybridization. RESULTS: Control and ischemic retinas produce mRNAs that correspond to the 121 and 165 amino acid diffusible isoforms of VEGF, and relatively low levels of VEGF189, the heparin-binding isoform. There was no significant difference in the levels of the alternatively spliced VEGF transcripts between control and ischemic retinas. Cloning and sequencing revealed that simVEGF cDNAs are 99% identical to human VEGFs and are predicted to encode proteins identical to their respective human homologues. In situ hybridization of nonischemic retinas revealed a low level of VEGF mRNA in retinal ganglion cells and in the inner nuclear layer. VEGF mRNA levels were elevated in ischemic retinas as early as 1 day after laser vein occlusion, when there was a small but reproducible increase in signal. The expression peaked at approximately 13 days, coincident with maximal iris neovascularization, and was significantly reduced by 28 days, when the iris vessels largely regressed. CONCLUSIONS: The elevation of simVEGF121 and VEGF165 in ischemic retinas is consistent with a role for diffusible, retina-derived angiogenic factors in the development of ocular neovascularization.

Prevalence of HTLV infection in the Dominican Republic: association with neurological disease.

The presence of the human T-cell leukemia virus (HTLV) in Dominican blood donors and patients with tropical spastic paraparesis (TSP) was first detected in 1987. To define further the seroprevalence in the country, nearly 4,000 samples from high- and low-risk populations, as well as patients with neurological disease and with leukemia or lymphoma were tested for HTLV antibodies. A 1-2% seropositivity rate was found among the low-risk population, a 2-5% in the high-risk, and at least 87% in those with TSP. A few patients with malignancy also had antibodies to HTLV. An increase in seropositivity with age and a predominance of female seropositive individuals were found. Infectious virus was isolated from TSP patients, prostitutes, and family members of index patients. These data indicate the substantial level of HTLV infection in another Caribbean country and its relation to neurologic disease.

Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate.

OBJECTIVE: To determine whether the angiogenic peptide vascular endothelial growth factor (VEGF) is sufficient to produce iris neovascularization in a nonhuman primate (Macaca fascicularis). METHODS: Eight eyes of 4 animals were studied. The 165-amino acid isoform of human recombinant VEGF (VEGF165) was injected into the vitreous of 5 cynomolgus monkey eyes (doses ranging from 0.25-2.5 micrograms per injection). Equal amounts of inactivated human recombinant VEGF (2 eyes) or vehicle (1 eye) were injected into contralateral control eyes. Eyes were assessed by slitlamp biomicroscopy, tonometry, iris color photography, fluorescein angiography, histopathologic examination, and immunostaining with antibodies against proliferating cell nuclear antigen. RESULTS: All 5 bioactive VEGF-injected eyes developed neovascularization with dilated and tortuous iris vessels that leaked fluorescein. None of the 3 control eyes exhibited any iris vascular changes. Inflammation was absent in both treatment groups. A dose response to VEGF was observed in the single animal that received 2.5 micrograms and 0.25 microgram in the right and left eyes, respectively. Iris vessel endothelial cells were positive for proliferating cell nuclear antigen in the bioactive VEGF-injected eyes only. Injections of 1.25 micrograms of VEGF every 3 days during a 30-day period produced advanced iris neovascularization, ectropion uvea, and neovascular glaucoma. CONCLUSIONS: Intravitreal injections of recombinant human VEGF165 in amounts comparable with those measured in eyes with active neovascularization are sufficient to produce noninflammatory iris neovascularization in a nonhuman primate. Prolonged exposure to VEGF165 can produce ectropion uveae and neovascular glaucoma.

Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate.

OBJECTIVE: To determine if the angiogenic peptide vascular endothelial growth factor (VEGF) is required for retinal ischemia-associated iris neovascularization in a nonhuman primate. METHODS: Laser retinal vein occlusion was used to produce retinal ischemia in 16 eyes of eight animals (Macaca fascicularis). Eyes were randomized to treatment every other day with intravitreal injections of either a neutralizing anti-VEGF monoclonal antibody or a control monoclonal antibody of the same isotype. Serial iris fluorescein angiograms were assessed using a standardized grading system and masked readers. Retinal VEGF and placental growth factor expression were assessed by Northern blotting. The specificity of the antibodies was determined in capillary endothelial cell proliferation assays prior to intravitreal injection. RESULTS: Zero of eight eyes receiving the neutralizing anti-VEGF antibodies developed iris neovascularization. Five of eight control antibody-treated eyes developed iris neovascularization. The difference was statistically significant (P = .03). Intravitreal antibody injection did not impair the ability of the ischemic retina to increase VEGF messenger RNA expression. The anti-VEGF antibodies specifically inhibited VEGF-driven capillary endothelial cell proliferation in vitro. CONCLUSION: These data demonstrate that VEGF is required for iris neovascularization in an adult nonhuman primate eye. The inhibition of VEGF is a new potential therapeutic strategy for the treatment of ocular neovascularization.

Angiography of fluoresceinated anti-vascular endothelial growth factor antibody and dextrans in experimental choroidal neovascularization.

OBJECTIVE: To determine if anti-vascular endothelial growth factor antibody and a range of dextrans with varying diffusion radii and molecular weights are permeable through experimental choroidal neovascularization (CNV). METHODS: Choroidal neovascularization was induced in 10 cynomolgus monkey retinas by means of argon laser injury. Digital fundus fluorescein angiograms were performed with fluorescein sodium, fluoresceinated IgG antibodies (anti-vascular endothelial growth factor and a control antibody), and fluoresceinated dextrans with molecular weights of 4, 20, 40, 70 and 150 kd. The 40- and 70-kd dextrans straddle the effective diffusion radius of IgG. For each reagent, early and late angiograms were performed in a standardized fashion, with follow-up images obtained to monitor residual fluorescence. RESULTS: Perfusion of retinal vessels and choroidal vasculature was seen with all reagents. Fluorescein and 4- and 20-kd dextran leaked rapidly from the CNV within the first minute. Angiography with the use of 40-kd dextran and fluoresceinated antibody, either anti-vascular endothelial growth factor or control IgG, showed fluorescence within the CNV that increased during the first 1 to 5 hours, with mild leakage from the CNV. By 24 hours, fluorescence in the CNV was minimal, although in some cases persistent fluorescence in the surrounding tissue was evident up to 2 weeks. The 70-kd dextran showed fluorescence within the CNV and leakage in 1 of 3 eyes. The 150-kd dextran showed fluorescence within the CNV but did not demonstrate leakage. CONCLUSIONS: Fluoresceinated antibodies and dextran with smaller effective diffusion radii showed CNV perfusion and leakage. Dextrans with larger effective diffusion radii (70 kd and 150 kd) perfused into CNV but did not show leakage consistently. CLINICAL RELEVANCE: Determining the permeablity of antibodies and molecules of similar size through CNV can help ascertain the feasibility of using intravenously administered antibodies against angiogenic growth factors as a future treatment for choroidal neovascularization.

Systemic hyperoxia decreases vascular endothelial growth factor gene expression in ischemic primate retina.

OBJECTIVES: To determine whether systemic hyperoxia can reverse retinal hypoxia and decrease vascular endothelial growth factor (VEGF) gene expression in ischemic nonhuman primate retina. METHODS: Six eyes of 3 cynomolgus monkeys were studied. Retinal ischemia was induced via laser vein occlusion and confirmed with fluorescein angiography. Animals were randomly assigned to treatment with either 21% or 100% inhaled oxygen. Arterial PO2 was monitored while systemic acid-base status was maintained. An oxygen microelectrode on a micromanipulator was used to measure preretinal oxygen concentrations in ischemic and nonischemic retina in situ. RNA was isolated from fresh whole retinas, and VEGF messenger RNA levels were quantified with Northern blotting. RESULTS: The preretinal PO2 in ischemic retina was less than the PO2 in nonischemic retina in animals breathing 21% oxygen (intervascular zone PO2, 14.3+/-0.53 vs 21.8+/-0.55 mm Hg; P=.002). After 8 hours of systemic hyperoxia (arterial PO2, 512+/-18 mm Hg), the preretinal PO2 in ischemic retina increased to 166.2+/-15.6 mm Hg (21.8% oxygen) and retinal VEGF messenger RNA levels were reduced by an average of 55%. CONCLUSIONS: These data demonstrate that systemic hyperoxia can lower retinal VEGF gene expression and reoxygenate ischemic adult primate retina.