Meihua Ju

Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: Direct correlation with clinical retinopathy of prematurity

Retinopathy of prematurity is a blinding disease, initiated by lack of retinal vascular growth after premature birth. We show that lack of insulin-like growth factor I (IGF-I) in knockout mice prevents normal retinal vascular growth, despite the presence of vascular endothelial growth factor, important to vessel development. In vitro, low levels of IGF-I prevent vascular endothelial growth factor-induced activation of protein kinase B (Akt), a kinase critical for endothelial cell survival. Our results from studies in premature infants suggest that if the IGF-I level is sufficient after birth, normal vessel development occurs and retinopathy of prematurity does not develop. When IGF-I is persistently low, vessels cease to grow, maturing avascular retina becomes hypoxic and vascular endothelial growth factor accumulates in the vitreous. As IGF-I increases to a critical level, retinal neovascularization is triggered. These data indicate that serum IGF-I levels in premature infants can predict which infants will develop retinopathy of prematurity and further suggests that early restoration of IGF-I in premature infants to normal levels could prevent this disease.

Nonvascular role for VEGF: VEGFR-1, 2 activity is critical for neural retinal development

The purpose of this study was to evaluate the function of extravascular vascular endothelial growth factor (VEGF) receptors in developing neural retina. VEGF is routinely described as a vascular endothelial cell‐specific mitogen, and VEGF receptor 1 (VEGFR‐1) and VEGF receptor 2 (VEGFR‐2) are described as endothelial cell specific, but there is evidence that these VEGF receptors are found outside the vasculature in neural tissue. The developing eye presents a unique opportunity to examine the function of VEGF in neural tissue alone. The peripheral retina is normally avascular at birth and becomes vascularized over the first 2 wk after birth. We localized VEGFR‐1 and ‐2 mRNA and protein to extravascular neuronal tissue during early retinal development. Avascular cornea also expresses these receptors. Inhibition of VEGFR‐1 and ‐2 in vivo with a specific small‐molecule tyrosine kinase antagonist, SU5416, inhibits development of the nonvascularized immature retina, resulting in cell loss in the inner retina, including the inner nuclear layer containing Muller cells and the ganglion cell layer containing astrocytes. Isolated retinal Muller cells express both VEGF receptors. VEGF stimulation activates MAPK, which is abrogated with inhibition of the receptors. We conclude that VEGFR‐ 1 and ‐2 are necessary for normal neural retinal development independent of vascular development.

Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity.

Oxygen administration to immature neonates suppresses VEGF-A expression in the retina, resulting in the catastrophic vessel loss that initiates retinopathy of prematurity. To investigate the mechanisms responsible for survival of blood vessels in the developing retina, we characterized two VEGF-A receptors, VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). Surprisingly, these two VEGF-A receptors differed markedly during normal retinal development in mice. At 5 days postpartum (P5), VEGFR-1 protein was colocalized with retinal vessels, whereas VEGFR-2 was detected only in the neural retina. Real-time RT-PCR identified a 60-fold induction of VEGFR-1 mRNA in retina from P3 (early vascularization) to P26 (fully vascularized), and no significant change in VEGFR-2 mRNA expression. Placental growth factor-1 (PlGF-1), which exclusively binds VEGFR-1, decreased hyperoxia-induced retinal vaso-obliteration from 22.2% to 5.1%, whereas VEGF-E, which exclusively binds VEGFR-2, had no effect on blood vessel survival. Importantly, under the same conditions, PlGF-1 did not increase vasoproliferation during (a). normal vessel growth, (b). revascularization following hyperoxia-induced ischemia, or (c). the vasoproliferative phase, indicating a selective function supporting blood vessel survival. We conclude that VEGFR-1 is critical in maintaining the vasculature of the neonatal retina, and that activation of VEGFR-1 by PlGF-1 is a selective strategy for preventing oxygen-induced retinal ischemia without provoking retinal neovascularization.

Transforming growth factor β1 induction of vascular endothelial growth factor receptor 1: Mechanism of pericyte-induced vascular survival in vivo

Degeneration of vessels precedes and precipitates the devastating ischemia of many diseases, including retinopathy of prematurity and diabetic retinopathy. Ischemia then leads to proliferative retinopathy and blindness. Understanding the mechanisms of blood vessel degeneration is critical to prevention of these diseases. Vessel loss is associated with oxygen-induced suppression of vascular endothelial growth factor (VEGF) and with pericyte (vascular smooth muscle cell) dropout. The molecular mechanism of pericyte protection of the vasculature is unknown. We show that transforming growth factor β1 (TGF-β1)-expressing pericytes are specifically found on vessels resistant to oxygen-induced loss. TGF-β1 potently induces VEGF receptor 1 (VEGFR-1) expression in endothelial cells and thereby prevents oxygen-induced vessel loss in vivo. Vessel survival is further stimulated with a VEGFR-1-specific ligand, placental growth factor 1. TGF-β1 induction of VEGFR-1 in endothelial cells explains pericyte protection of vessels and the selective vulnerability of neonatal vessels to oxygen. These results implicate induction and activation of VEGFR-1 as critical targets to prevent vessel loss.

Simultaneous but not prior inhibition of VEGF165 enhances the efficacy of photodynamic therapy in multiple models of ocular neovascularization

PURPOSE To investigate the effect of the combined treatment of photodynamic therapy and specific VEGF165 inhibition with pegaptanib sodium (Macugen; Eyetech Pharmaceuticals, Lexington, MA) on ocular neovascularization. METHODS Photodynamic therapys (PDTs) effects on the integrity of pegaptanib sodium were analyzed by HPLC, a VEGF165-binding assay, and a VEGF165-induced tissue factor gene expression assay. The effects of mono- or combined treatment on vessel growth and regression were determined in a murine corneal neovascularization model. The effects of combined treatment on vessel growth were also determined in a murine choroidal neovascularization model. RESULTS PDT did not affect the chemical composition of pegaptanib sodium nor the efficacy of pegaptanib sodium in the inhibition of VEGF165 binding to Flt-1 and VEGF165-induced gene expression. In an animal model of effects on existing ocular neovascular lesions (corneal neovascularization), PDT monotherapy yielded an initial regression of these vessels, but there followed a rapid regrowth. In contrast, pegaptanib sodium monotherapy yielded little regression but potently abrogated further vessel growth. The combination of pegaptanib sodium and PDT resulted in the regression of the neovascular lesions, as observed with PDT alone, but also prevented significant vessel regrowth, leading to a significantly greater reduction in lesion size than did each monotherapy. In addition, there was a significantly greater effect of the combination of pegaptanib sodium and PDT on lesion size in choroidal neovascularization than with each monotherapy. Pretreatment with pegaptanib sodium appeared to decrease the efficacy of PDT-induced vessel regression in corneal neovascularization, and as such the enhanced efficacy over monotherapy when the agents were delivered simultaneously was not observed. CONCLUSIONS Although the combined simultaneous treatment of ocular neovascularization with PDT and pegaptanib sodium may provide a more effective approach for the regression and overall treatment of CNV associated with AMD, the order of addition of these treatments may play a role in achieving optimal efficacy.

SOCS3 in retinal neurons and glial cells suppresses VEGF signaling to prevent pathological neovascular growth

Enhancing SOCS3 activity in neurons and glial cells in the retina may prevent abnormal blood vessel formation in proliferative retinopathies. Halting blood vessel formation in the eye Diabetics and preterm infants are susceptible to vision loss or blindness because of abnormal blood vessel formation in the eye, a process called retinal neovascularization. Using a model of this disease process, Sun et al. found that mice that lacked a protein called SOCS3 in the neurons and glial cells of the eye had greater retinal neovascularization than did control mice. Their results suggest that SOCS3 prevents neurons and glial cells from releasing too much VEGF (a cytokine that promotes blood vessel formation) by inhibiting the transcription factor STAT3, which can trigger the production of VEGF. Neurons and glial cells in the retina contribute to neovascularization, or the formation of abnormal new blood vessels, in proliferative retinopathy, a condition that can lead to vision loss or blindness. We identified a mechanism by which suppressor of cytokine signaling 3 (SOCS3) in neurons and glial cells prevents neovascularization. We found that Socs3 expression was increased in the retinal ganglion cell and inner nuclear layers after oxygen-induced retinopathy. Mice with Socs3 deficiency in neuronal and glial cells had substantially reduced vaso-obliterated retinal areas and increased pathological retinal neovascularization in response to oxygen-induced retinopathy, suggesting that loss of neuronal/glial SOCS3 increased both retinal vascular regrowth and pathological neovascularization. Furthermore, retinal expression of Vegfa (which encodes vascular endothelial growth factor A) was higher in these mice than in Socs3 flox/flox controls, indicating that neuronal and glial SOCS3 suppressed Vegfa expression during pathological conditions. Lack of neuronal and glial SOCS3 resulted in greater phosphorylation and activation of STAT3, which led to increased expression of its gene target Vegfa, and increased endothelial cell proliferation. In summary, SOCS3 in neurons and glial cells inhibited the STAT3-mediated secretion of VEGF from these cells, which suppresses endothelial cell activation, resulting in decreased endothelial cell proliferation and angiogenesis. These results suggest that neuronal and glial cell SOCS3 limits pathological retinal angiogenesis by suppressing VEGF signaling.

Sirtuin1 Over-Expression Does Not Impact Retinal Vascular and Neuronal Degeneration in a Mouse Model of Oxygen-Induced Retinopathy

Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy.