Novel protective properties of IGFBP-3 result in enhanced pericyte ensheathment, reduced microglial activation, increased microglial apoptosis, and neuronal protection after ischemic retinal injury.

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GFP(+) hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity.

Cell-based therapies for diabetic retinopathy.

Autologous endothelial progenitor cell (EPC) populations represent a novel treatment for therapeutic revascularization and vascular repair for diabetic patients with complications including diabetic retinopathy. Current therapies are applicable to late-stage disease and carry significant side effects, whereas cell-based therapy may provide an alternative by repairing areas of vasodegeneration and reversing ischemia. However, EPCs from diabetic patients with vascular complications are dysfunctional. Moreover, the diabetic environment poses its own challenges and complicates the use of autologous EPCs. Before EPCs become the ideal "cell therapy," the optimal EPC must be determined, any functional dysfunction must be corrected prior to use, and the diabetic milieu will require modification to accept the EPCs. This review describes the rationale for harnessing the vascular reparative properties of EPCs with emphasis on the molecular and phenotypic nature of healthy EPCs, how diabetes alters them, and novel strategies to improve dysfunctional EPCs.

Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation.

RATIONALE: Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury. OBJECTIVE: To examine the mechanism of IGFBP-3-mediated repair following vascular injury. METHODS AND RESULTS: We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3-expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3-mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway. CONCLUSION: These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

EPCs and pathological angiogenesis: when good cells go bad.

Bone-marrow-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated pathological neovascularization, and recent studies have begun to recognize the biological significance of this contribution. This review will discuss the ability of EPCs to contribute to neovascularization in both physiological and pathological conditions. Circulating EPCs were originally identified in 1997 by Asahara as CD34(+) VEGFR2(+) mononuclear cells. These cells differentiated into an endothelial phenotype, expressed endothelial markers, and incorporated into neovessels at sites of ischemia (Asahara et al., 1997). EPCs provide both instructive (release of pro-angiogenic cytokines) and structural (vessel incorporation and stabilization) functions that contribute to the initiation of neo-angiogenesis. EPC populations can be characterized based on surface markers of freshly isolated cells, or they can be described by their in vitro characteristics once placed in culture. However, a major stumbling block to progress in the field has been the lack of consensus among investigators as to the optimal characterization of EPCs. This review intends to address the role of both EPC classes and evaluate how they interact in the setting of pathological angiogenesis. Since the EPCs may be responsible for turning on the "angiogenic switch," strategies have been employed to keep this switch in the "off" position for diseases like cancer, retinopathy, and wet AMD. The expectation is that EPCs will evolve into clinically useful prognostic and predictive tools in cancer and in ocular diseases associated with pathological neovascularization and that targeting this cell type is a key to successful management of patients suffering from diseases associated with pathological neovascularization.

Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model.

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow-derived CD34+ cells and vascular wall-derived endothelial colony-forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model.

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair.

Studies with an orally bioavailable alpha V integrin antagonist in animal models of ocular vasculopathy: retinal neovascularization in mice and retinal vascular permeability in diabetic rats.

The alpha(V) integrins are key receptors involved in mediating cell migration and angiogenesis. In age-related macular degeneration (AMD) and diabetic retinopathy, angiogenesis plays a critical role in the loss of vision. These ocular vasculopathies might be treatable with a suitable alpha(V) antagonist, and an oral drug would offer a distinct advantage over current therapies. (3,S,beta,S)-1,2,3,4-Tetrahydro-beta-[[1-[1-oxo-3-(1,5,6,7-tetrahydro-1,8-naphthyridin-2-yl)propyl]-4-piperidinyl]methyl]-3-quinolinepropanoic acid (JNJ-26076713) is a potent, orally bioavailable, nonpeptide alpha(V) antagonist derived from the arginine-glycine-asparagine binding motif in the matrix protein ligands (e.g., vitronectin). This compound inhibits alpha(V)beta(3) and alpha(V)beta(5) binding to vitronectin in the low nanomolar range, it has excellent selectivity over integrins alpha(IIb)beta(3) and alpha(5)beta(1), and it prevents adhesion to human, rat, and mouse endothelial cells. JNJ-26076713 blocks cell migration induced by vascular endothelial growth factor, fibroblast growth factor (FGF), and serum, and angiogenesis induced by FGF in the chick chorioallantoic membrane model. JNJ-26076713 is the first alpha(V) antagonist reported to inhibit retinal neovascularization in an oxygen-induced model of retinopathy of prematurity after oral administration. In diabetic rats, orally administered JNJ-26076713 markedly inhibits retinal vascular permeability, a key early event in diabetic macular edema and AMD. Given this profile, JNJ-26076713 represents a potential therapeutic candidate for the treatment of age-related macular degeneration, macular edema, and proliferative diabetic retinopathy.

Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes.

Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.

Increased Indoleamine 2,3-Dioxygenase and Quinolinic Acid Expression in Microglia and Müller Cells of Diabetic Human and Rodent Retina.

Purpose: We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D. Methods: Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels). Results: Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39-/Iba-1+ bone marrow-derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls. Conclusions: Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.

The dipeptide Arg-Gln inhibits retinal neovascularization in the mouse model of oxygen-induced retinopathy.

PURPOSE: Premature infants undergoing intensive care are highly vulnerable to amino acid deprivation. Supplementation of glutamine or arginine has resulted in beneficial effects in human neonates. This study was conducted to examine the effect of the dipeptide arginyl-glutamine (Arg-Gln) on vascular endothelial cell growth factor (VEGF) levels in primary human retinal pigment epithelial (hRPE) cell cultures and on inhibition of neovascularization in the oxygen-induced retinopathy (OIR) model. METHODS: The effects of Arg-Gln on VEGF levels were measured in supernates from hRPE cells by using ELISAs. For in vivo studies, mouse pups received twice-daily intraperitoneal injections of Arg-Gln, a control dipeptide (Ala-Gly) or were not injected. Retinal flatmounts from one cohort were prepared and retinal vessel morphology examined. The contralateral eyes were embedded, sectioned, and stained to count preretinal neovascular nuclei. RNA was isolated from retinas of selected animals and was used to quantify VEGF mRNA by real-time RT-PCR. RESULTS: Treatment of hRPE cells with Arg-Gln decreased VEGF levels in a dose-dependent manner. In the OIR model, Arg-Gln at 5 g/kg per day reduced preretinal neovascularization by 82%+/-7% (P<0.005), when compared with the control dipeptide Ala-Gly, and reduced VEGF mRNA by 64%+/-9% (P<0.001). CONCLUSIONS: Arg-Gln dramatically inhibited retinal neovascularization in the OIR model. This effect was associated with a reduction in retinal VEGF mRNA levels. Similarly the dipeptide reduced VEGF expression in hRPE cells, a cell type likely to respond to retinal hypoxia by expressing VEGF. Arg-Gln appears to be safe and, with future studies in human infants, may prove beneficial in the prevention of ROP.

Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells.

PURPOSE: To evaluate whether transfection of human retinal endothelial cells (HRECs) with plasmids expressing ribozymes designed to specifically cleave the mRNA and reduce expression of either vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1), or VEGF receptor-2 (VEGFR-2), or insulin-like growth factor-I receptor (IGF-IR) modulates occludin expression in high glucose-treated cells. METHODS: Hammerhead ribozymes that specifically cleave the human VEGFR-1, VEGFR-2, and IGF-IR mRNAs were developed and tested in vitro to determine ribozyme kinetics and cleavage specificity. HRECs grown in normal (5.5 mM) and high (25 mM) glucose medium were transfected with plasmids expressing VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes. VEGF and IGF-I levels were measured in conditioned medium of HREC exposed to high glucose conditions, and the effect of varying glucose concentration on VEGFR-1 and VEGFR-2 phosphorylation was examined. The amount of the tight junction protein occludin was determined by western analysis, and the protein was localized by immunohistochemistry. RESULTS: Exposure of HRECs to high glucose resulted in increased VEGF and IGF-I expression as well as VEGFR-2 but not VEGFR-1 phosphorylation. Immunocytochemistry and western analysis revealed that HRECs exposed to high glucose had reduced occludin staining and protein expression, respectively. Transfection of HRECs exposed to high glucose with either VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes prevented the downregulation of occludin protein expression. CONCLUSIONS: Our studies support that activation of VEGFR-1, VEGFR-2, and IGF-IR by high glucose contributes to disruption of tight junctions by decreasing occludin expression and may be important in the pathogenesis of blood-retinal barrier dysfunction in diabetic retinopathy.

Proceedings of the Third International Symposium on Retinopathy of Prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California).

The Third International Symposium on Retinopathy of Prematurity (ROP) was convened with the aim of cross fertilizing the horizons of basic and clinical scientists with an interest in the pathogenesis and management of infants with ROP. Ten speakers in the clinical sciences and ten speakers in the basic sciences were recruited on the basis of their research to provide state of the art talks. The meeting was held November 9, 2003 immediately prior to the American Academy of Ophthalmology meeting; scholarships were provided for outreach to developing countries and young investigators. This review contain the summaries of the 20 platform presentations prepared by the authors and the abstracts of presented posters. Each author was asked to encapsulate the current state of understanding, identify areas of controversy, and make recommendations for future research. The basic science presentations included insights into the development of the human retinal vasculature, animal models for ROP, growth factors that affect normal development and ROP, and promising new therapeutic approaches to treating ROP like VEGF targeting, inhibition of proteases, stem cells, ribozymes to silence genes, and gene therapy to deliver antiangiogenic agents. The clinical presentations included new insights into oxygen management, updates on the CRYO-ROP and ETROP studies, visual function in childhood following ROP, the neural retina in ROP, screening for ROP, management of stage 3 and 4 ROP, ROP in the third world, and the complications of ROP in adult life. The meeting resulted in a penetrating exchange between clinicians and basic scientists, which provided great insights for conference attendees. The effect of preterm delivery on the normal cross-talk of neuroretinal and retinal vascular development is a fertile ground for discovering new understanding of the processes involved both in normal development and in retinal neovascular disorders. The meeting also suggested promising potential therapeutic interventions on the horizon for ROP.

Promoting vascular repair in the retina: can stem/progenitor cells help?

Since its first epidemic in the 1940s, retinopathy of prematurity (ROP) has been a challenging illness in neonatology. Higher than physiological oxygen levels impede the development of the immature retinal neuropil and vasculature. Current treatment regimens include cryotherapy, laser photocoagulation, and anti-VEGF agents. Unfortunately, none of these approaches can rescue the normal retinal vasculature, and each has significant safety concerns. The limitations of these approaches have led to new efforts to understand the pathological characteristics in each phase of ROP and to find a safer and more effective therapeutic approach. In the era of stem cell biology and with the need for new treatments for ROP, this review discusses the possible future use of unique populations of proangiogenic cells for therapeutic revascularization of the preterm retina.

Focal adhesion kinase overexpression induces enhanced pathological retinal angiogenesis.

PURPOSE: Focal adhesion kinase (FAK) is involved in processes integral to angiogenesis, such as cell growth, survival, and migration. FAK is activated by angiogenic growth factors, such as insulin-like growth factor (IGF)-I, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The study was conducted to determine whether overexpression of FAK or FAK-related nonkinase (FRNK), an inhibitor of FAK, could influence human retinal endothelial cell (HREC) migration and in vivo angiogenesis. METHODS: Migration in response to a combination of growth factors was examined in transfected HRECs overexpressing FAK or FRNK. The effect of FAK or FRNK overexpression on preretinal neovascularization was examined in a mouse model of oxygen-induced retinopathy. RESULTS: Overexpression of FAK in HRECs resulted in a 102% +/- 13% increase (P = 1.4 x 10(-4)) in cell migration, whereas overexpression of FRNK resulted in a 20% +/- 8% decrease (P = 0.01). Overexpression of FAK in mouse eyes led to formation of numerous large vascular tufts resembling glomeruli and a 57% +/- 7% increase in preretinal neovascularization (P = 3 x 10(-9)), whereas FRNK resulted in a 55% +/- 15% reduction (P = 5 x 10(-5)). CONCLUSIONS: Modulating the FAK/FRNK system may provide a novel approach to inhibiting pathologic retinal angiogenesis.

Expression of IGFBP-3 by human retinal endothelial cell cultures: IGFBP-3 involvement in growth inhibition and apoptosis.

PURPOSE: Growth hormone (GH), insulin-like growth factor (IGF), and somatostatin (SST) modulate each other's actions. SST analogues have been successfully used to treat proliferative diabetic retinopathy (PDR) that is unresponsive to laser therapy and to retard the progression of severe nonproliferative retinopathy to PDR. In this study, the endogenous expression of IGF-binding protein (IGFBP)-3 was examined in human retinal endothelial cells (HRECs), the direct effects of IGFBP-3 on HRECs were evaluated, and the possible involvement of IGFBP-3 in mediating the growth inhibitory effects of SST receptor (SSTR) agonists in HRECs was assessed. METHODS: The cellular localization of IGFBP-3 was examined with anti-IGFBP-3 and fluorescein-conjugated goat anti-rabbit IgG. HRECs were exposed to varying concentrations of human recombinant IGFBP-3, and growth inhibition was evaluated by thiazolyl blue (MTT) conversion. Apoptosis was examined using fluorochrome-annexin V staining. Conditioned media (CM) from SSTR2 agonist (L779976)-treated or SSTR3 agonist (L796778)-treated HRECs were analyzed by ELISA for changes in expression of IGFBP-3. RESULTS: HREC immunostaining showed cell surface and cytoplasmic IGFBP-3. Exogenous IGFBP-3 induced a dose-dependent inhibition of HREC proliferation and staining with fluorochrome-annexin V showed numerous apoptotic HRECs. HRECs exposed to the SSTR2 or SSTR3 agonists expressed IGFBP-3 in a concentration-dependent manner. CONCLUSIONS: Cultured HRECs expressed endogenous IGFBP-3. Exogenous administration of IGFBP-3-induced growth inhibition and apoptosis, supporting a regulatory role for IGFBP-3 in endothelial cells. SSTR agonists mediate their growth-inhibitory effect, in part, by increasing expression of IGFBP-3.

Fibronectin fragments promote human retinal endothelial cell adhesion and proliferation and ERK activation through alpha5beta1 integrin and PI 3-kinase.

PURPOSE: Extracellular matrix degradation is associated with neovascularization in diabetic retinas. Fibronectin fragments (Fn-fs) are generated during vascular remodeling. The effects of cellular fibronectin (Fn) and selected Fn-fs on adhesion, proliferation, and signal transduction in human retinal endothelial cells (HRECs) were characterized. METHODS: Relative quantitative RT-PCR, flow cytometry, and immunocytochemistry determined integrin expression on HRECs. Adhesion was evaluated by coating plastic with Fn or Fn-fs of 45, 70, 110, or 120 kDa, and MTT conversion was used to measure proliferation and survival. Peptide inhibitors and blocking antibodies determined adhesive sites and integrins used for adhesion. Pharmacologic inhibitors and Western analyses were used to evaluate intracellular signaling. RESULTS: HRECs produced significant levels of alpha(2), alpha(3), alpha(5), alpha(v), beta(1), beta(3), and beta(5) integrin subunit mRNA. Flow cytometry of surface integrin expression revealed high levels of alpha(3), alpha(5), and beta(1) and lower levels of alpha(1), alpha(v), beta(3), and beta(5). These results were confirmed by immunocytochemistry. For adhesion to Fn and Fn-fs. the alpha(5)beta(1) integrin was essential. Pharmacologic inhibitors of PI 3-kinase blocked adhesion to Fn and Fn-fs, whereas the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 blocked phosphorylation. The 110- and 120-kDa Fn-fs showed a concentration-dependent increase in proliferation, whereas 500 ng of the 70 kDa Fn-f-induced proliferation. Addition of III1-C, a matrix assembly domain, increased the proliferative effect of these Fn-fs. CONCLUSIONS: Fn and its Fn-fs modulate HREC adhesion and proliferation through signal-transduction pathways involving coupling of the alpha(5)beta(1) integrin through PI 3-kinase. Mitogenic signals for endothelial cells from degraded extracellular matrix may contribute to the development of diabetic retinopathy.

Decreased expression of the insulin-like growth factor 1 receptor by ribozyme cleavage.

PURPOSE: Insulin-like growth factor (IGF)-1 and its receptor (IGF-1R) are associated with abnormal retinal neovascularization. Ribozymes were designed that selectively decreased the expression of the IGF-1R and these ribozymes were tested in angiogenesis models in vitro and in vivo. METHODS: Two hammerhead ribozymes were designed that cleave the human IGF-1R mRNA. The ribozymes were cloned into recombinant adeno-associated viral vectors (rAAV). The rAAV constructs were transfected into human retinal endothelial cells (HRECs). IGF-1R mRNA and protein levels were examined and the modified Boyden chamber assay used to examine ribozyme effects on cell migration. These constructs were injected intravitreally into mice to determine the effect of the ribozymes on retinal neovascularization in a mouse model of oxygen-induced retinopathy. RESULTS: Relative quantitative RT-PCR analysis showed that IGF-1R Rz1 reduced IGF-1R mRNA levels by 40% +/- 10% (P = 0.003), and Western blot analysis showed a 41% +/- 5% (P = 4.6 x 10(-5)) reduction of IGF-1R protein, confirming that this ribozyme reduces IGF-1R expression. IGF-1R Rz1 also reduced IGF-1-induced cell migration by 90% +/- 5% (P = 2.9 x 10(-9)) showing that IGF-1R Rz1 reduces IGF-1R function in HRECs. IGF-1R Rz1 also reduced the amount of preretinal neovascularization by 65% +/- 6% (P = 2.7 x 10(-5)), as measured by the average number of endothelial preretinal nuclei per section. CONCLUSIONS: These studies demonstrate that the IGF-1R ribozymes are effective at reducing the expression and function of the IGF-1R in vitro and in vivo. Therefore, the IGF-1R ribozymes are an effective method for studying the process of angiogenesis and may ultimately be effective as gene therapy tools for the reduction of pathologic retinal angiogenesis.

Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice.

PURPOSE: To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS: The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad). Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy. The ability of recombinant 16K hPRL expressed in E. coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC). RESULTS: 16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation. Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls. r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs. CONCLUSIONS: Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy. 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells. These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies.

An ocular view of the IGF-IGFBP system.

IGFs and their binding proteins have been shown to exhibit both protective and deleterious effects in ocular disease. Recent studies have characterized the expression patterns of different IGFBPs in retinal layers and within the vitreous. IGFBP-3 has roles in vascular protection stimulating proliferation, migration, and differentiation of vascular progenitor cells to sites of injury. IGFBP-3 increases pericyte ensheathment and shows anti-inflammatory effects by reducing microglia activation in diabetes. IGFBP-5 has recently been linked to mediating fibrosis in proliferative vitreoretinopathy but also reduces neovascularization. Thus, the regulatory balance between IGF and IGFBPs can have profound impact on target tissues. This review discusses recent findings of IGF and IGFBP expression in the eye with relevance to different retinopathies.

Per2 mutation recapitulates the vascular phenotype of diabetes in the retina and bone marrow.

In this study, we assessed whether Per2 clock gene-mutant mice exhibit a vascular phenotype similar to diabetes. Per2 (B6.129-Per2(tm1Drw)/J) or wild-type control mice 4 and 12 months of age were used. To evaluate diabetes-like phenotype in Per2 mutant mice, retina was quantified for mRNA expression, and degree of diabetic retinopathy was evaluated. Bone marrow neuropathy was studied by staining femurs for tyrosine hydroxylase (TH) and neurofilament 200 (NF-200). The rate of proliferation and quantification of bone marrow progenitor cells (BMPCs) was performed, and a threefold decrease in proliferation and 50% reduction in nitric oxide levels were observed in Per2 mutant mice. TH-positive nerve processes and NF-200 staining were reduced in Per2 mutant mice. Both retinal protein and mRNA expression of endothelial nitric oxide synthase were decreased by twofold. Other endothelial function genes (VEGFR2, VEGFR1) were downregulated (1.5-2-fold) in Per2 mutant retinas, whereas there was an upregulation of profibrotic pathway mediated by transforming growth factor-ß1. Our studies suggest that Per2 mutant mice recapitulate key aspects of diabetes without the metabolic abnormalities, including retinal vascular damage, neuronal loss in the bone marrow, and diminished BMPC function.