Axitinib inhibits retinal and choroidal neovascularization in in vitro and in vivo models

AbstractAge-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy

Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses

PurposeIn conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its increased local concentration triggers transient exocytosis. In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles.MethodsCentral retina tissue from C57BL/6 mice was used. Laser scanning confocal microscopy (LSCM) experiments were conducted on fixed-frozen vertical sections. Primary antibodies were selected for their tissue/cellular specificity and ability to recognize single, multiple or all splice variants of selected isoforms. Electron microscopy (EM) and 3-D electron tomography (ET) studies used our standard procedures on thin- and thick-sectioned retinas, respectively. Calibrated fluo-3-Ca2+ imaging experiments of dark- and light-adapted rod and cone terminals in retinal slices were conducted.ResultsConfocal microscopy showed that mitochondria, ER, PMCA, and NCX1 exhibited distinct retinal lamination patterns and differential distribution in photoreceptor synapses. Antibodies for three distinct mitochondrial compartments differentially labeled retinal areas with high metabolic demand: rod and cone inner segments, previously undescribed cone juxtanuclear mitochondria and the two plexiform layers. Rod spherule membranes uniformly and intensely stained for PMCA, whereas the larger cone pedicles preferentially stained for NCX1 at their active zones and PMCA near their mitochondria. EM and ET revealed that mitochondria in rod spherules and cone pedicles differed markedly in their number, location, size, volume, and total cristae surface area, and cristae junction diameter. Rod spherules had one large ovoid mitochondrion located near its active zone, whereas cone pedicles averaged five medium-sized mitochondria clustered far from their active zones. Most spherules had one ribbon synapse, whereas pedicles contained numerous ribbon synapses. Fluo-3 imaging studies revealed that during darkness rod spherules maintained a lower [Ca2+] than cone pedicles, whereas during light adaptation pedicles rapidly lowered their [Ca2+] below that observed in spherules.ConclusionsThese findings indicate that ATP demand and mitochondrial ATP production are greater in cone pedicles than rod spherules. Rod spherules employ high affinity/low turnover PMCA and their mitochondrion to maintain a relatively low [Ca2+] in darkness, which increases their sensitivity and signal-to-noise ratio. In contrast, cone pedicles utilize low affinity/high turnover NCX to rapidly lower their high [Ca2+] during light adaptation, which increases their response kinetics. Spatiotemporal fluo-3-Ca2+ imaging results support our immunocytochemical results. The clustering of cone pedicle mitochondria likely provides increased protection from Ca2+ overload and permeability transition. In summary, these novel studies reveal that several integrated cellular and subcellular components interact to regulate ATP and Ca2+ dynamics in rod and cone synaptic terminals. These results should provide a greater understanding of in vivo photoreceptor synaptic terminal exocytosis/endocytosis, Ca2+ overload and therapies for retinal degenerations

Low-level human equivalent gestational lead exposure produces sex-specific motor and coordination abnormalities and late-onset obesity in year-old mice

Background. Low-level developmental lead exposure is linked to cognitive and neurological disorders in children. However, the long-term effects of gestational lead exposure (GLE) have received little attention. Objectives. Our goals were to establish a murine model of human equivalent GLE and to determine dose–response effects on body weight, motor functions, and dopamine neurochemistry in year-old offspring. Methods. We exposed female C57BL/6 mice to water containing 0, 27 (low), 55 (moderate), or 109 ppm (high) of lead from 2 weeks prior to mating, throughout gestation, and until postnatal day 10 (PN10). Maternal and litter measures, blood lead concentrations ([BPb]), and body weights were obtained throughout the experiment. Locomotor behavior in the absence and presence of amphetamine, running wheel activity, rotarod test, and dopamine utilization were examined in year-old mice. Results. Peak [BPb] were < 1, ≤ 10, 24–27, and 33–42 μg/dL in control, low-, moderate- and high-dose GLE groups at PN0–10, respectively. Year-old male but not female GLE mice exhibited late-onset obesity. Similarly, we observed male-specific decreased spontaneous motor activity, increased amphetamine-induced motor activity, and decreased rotarod performance in year-old GLE mice. Levels of dopamine and its major metabolite were altered in year-old male mice, although only forebrain utilization increased. GLE-induced alterations were consistently larger in low-dose GLE mice. Conclusions. Our novel results show that GLE produced permanent male-specific deficits. The nonmonotonic dose-dependent responses showed that low-level GLE produced the most adverse effects. These data reinforce the idea that lifetime measures of dose–response toxicant exposure should be a component of the neurotoxic risk assessment process

Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses.

PurposeIn conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its increased local concentration triggers transient exocytosis. In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles.MethodsCentral retina tissue from C57BL/6 mice was used. Laser scanning confocal microscopy (LSCM) experiments were conducted on fixed-frozen vertical sections. Primary antibodies were selected for their tissue/cellular specificity and ability to recognize single, multiple or all splice variants of selected isoforms. Electron microscopy (EM) and 3-D electron tomography (ET) studies used our standard procedures on thin- and thick-sectioned retinas, respectively. Calibrated fluo-3-Ca2+ imaging experiments of dark- and light-adapted rod and cone terminals in retinal slices were conducted.ResultsConfocal microscopy showed that mitochondria, ER, PMCA, and NCX1 exhibited distinct retinal lamination patterns and differential distribution in photoreceptor synapses. Antibodies for three distinct mitochondrial compartments differentially labeled retinal areas with high metabolic demand: rod and cone inner segments, previously undescribed cone juxtanuclear mitochondria and the two plexiform layers. Rod spherule membranes uniformly and intensely stained for PMCA, whereas the larger cone pedicles preferentially stained for NCX1 at their active zones and PMCA near their mitochondria. EM and ET revealed that mitochondria in rod spherules and cone pedicles differed markedly in their number, location, size, volume, and total cristae surface area, and cristae junction diameter. Rod spherules had one large ovoid mitochondrion located near its active zone, whereas cone pedicles averaged five medium-sized mitochondria clustered far from their active zones. Most spherules had one ribbon synapse, whereas pedicles contained numerous ribbon synapses. Fluo-3 imaging studies revealed that during darkness rod spherules maintained a lower [Ca2+] than cone pedicles, whereas during light adaptation pedicles rapidly lowered their [Ca2+] below that observed in spherules.ConclusionsThese findings indicate that ATP demand and mitochondrial ATP production are greater in cone pedicles than rod spherules. Rod spherules employ high affinity/low turnover PMCA and their mitochondrion to maintain a relatively low [Ca2+] in darkness, which increases their sensitivity and signal-to-noise ratio. In contrast, cone pedicles utilize low affinity/high turnover NCX to rapidly lower their high [Ca2+] during light adaptation, which increases their response kinetics. Spatiotemporal fluo-3-Ca2+ imaging results support our immunocytochemical results. The clustering of cone pedicle mitochondria likely provides increased protection from Ca2+ overload and permeability transition. In summary, these novel studies reveal that several integrated cellular and subcellular components interact to regulate ATP and Ca2+ dynamics in rod and cone synaptic terminals. These results should provide a greater understanding of in vivo photoreceptor synaptic terminal exocytosis/endocytosis, Ca2+ overload and therapies for retinal degenerations

Integrin αvβ8 on T&nbsp;cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy.

αvβ8 integrin, a key activator of transforming growth factor β (TGF-β), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvβ8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvβ8 is expressed at the highest levels in CD4+CD25+ T&nbsp;cells in tumors, and specific deletion of β8 from T&nbsp;cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T&nbsp;cells normally inhibited by TGF-β and involved in tumor cell killing, including granzyme B and interferon-γ. The in&nbsp;vitro cytotoxic effect of tumor CD8 T&nbsp;cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvβ8 integrin as a promising target for cancer immunotherapy

Integrin αvβ8 on T&nbsp;cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy.

αvβ8 integrin, a key activator of transforming growth factor β (TGF-β), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvβ8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvβ8 is expressed at the highest levels in CD4+CD25+ T&nbsp;cells in tumors, and specific deletion of β8 from T&nbsp;cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T&nbsp;cells normally inhibited by TGF-β and involved in tumor cell killing, including granzyme B and interferon-γ. The in&nbsp;vitro cytotoxic effect of tumor CD8 T&nbsp;cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvβ8 integrin as a promising target for cancer immunotherapy