Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model.

AIMS/HYPOTHESIS: We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells. METHODS: Wild-type, gp130(-/-) and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp(+) chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood. RESULTS: BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b(+)/CD45(hi)/ CCR2(+)/Ly6C(hi) inflammatory monocytes. Diabetic gp130(-/-) mice were protected from development of diabetes-induced changes in their HSCs. CONCLUSIONS/INTERPRETATION: The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA.

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A2A, A2B, and A3) and is a potential mediator of angiogenesis through the A2B receptor. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A2B receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a Vmax of 515+/-125 pmol/min and a Kcat of 36.1+/-8.3 min(-1) (P< or =1x10(-5)). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B receptor mRNA levels by 45+/-4.8% (P=5.1x10(-5)). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3+/-1.2% (P=7x10(-4)). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5+/-8.2% (P=4.5x10(-5)). Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A2B receptor in retinal angiogenesis.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Fibronectin fragments modulate human retinal capillary cell proliferation and migration.

Capillary morphogenesis involves cell-cell and cell-matrix interactions. Proteases elaborated by capillary cells modify the extracellular matrix (ECM) to facilitate capillary tube formation. Previously, we detected the presence of fibronectin fragments (Fn-f) associated with the proform of matrix metalloprotease-2 (MMP-2) in conditioned medium of human retinal endothelial cells (HRECs). Association of this fragment to latent MMP-2 prevented autocatalytic activation of MMP-2, suggesting a modulatory role of Fn-f in MMP-2 activation. In this report, we examined the potential role of Fn-f on two processes involved in angiogenesis, proliferation and migration of vascular cells. The effects of Fn-f on proliferation were determined by DNA synthesis and cell counts. Their effects on migration were assessed using modified Boyden chambers. Seven Fn-f were tested on vascular cell migration and/or proliferation. Three Fn-f induced migration. Fn-f of 30-kDa and 120-kDa size positively affected proliferation of microvascular cells but not macrovascular cells. A 45-kDa gelatin binding fragment of Fn inhibited HREC proliferation but stimulated pericyte and smooth muscle cell proliferation. The potency of these fragments exceeded that of the known angiogenic growth factor, basic fibroblast growth factor (bFGF), on HREC migration. ECM components such as fibronectin may influence capillary morphogenesis by the generation of fragments that can modulate proliferation, migration, and protease activation. In the setting of diabetes, excess Fn is generated and is available for degradation. Thus, the production of Fn-f may be specifically relevant to the angiogenesis observed in proliferative diabetic retinopathy.

Matrix metalloproteinase expression in human retinal microvascular cells.

The degree of hyperglycemia correlates with the development of diabetic retinopathy. We investigated the effect of glucose on the expression of matrix metalloproteinase (MMP)-2 and MMP-9 (72-kDa and 92-kDa type IV collagenases, respectively) by human retinal microvascular endothelial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donors were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, conditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the proform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose exposure or the disease state of the donors. CM from nondiabetic HREC cultures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they were exposed to 5 mmol/l glucose. This same activity was seen in CM from HREC cultures of diabetic origin in the presence of both 5 and 30 mmol/l glucose. Western analysis confirmed the identity of the 65-kDa band as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-based reverse transcription-polymerase chain reaction revealed that nondiabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibronectin. After exposure to 5 or 30 mmol/l glucose, no changes were detected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TIMP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by extracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.

The efficacy of octreotide in the therapy of severe nonproliferative and early proliferative diabetic retinopathy: a randomized controlled study.

OBJECTIVE: The pilot study examined the ability of octreotide to retard progression of diabetic retinopathy (DR) and delay the need for panretinal photocoagulation (PRP) in patients with advanced stages of retinal disease. RESEARCH DESIGN AND METHODS: Patients with severe nonproliferative DR (NPDR) or early non-high-risk proliferative DR (PDR) were randomly assigned to conventional diabetes management (control group, 12 patients) or to treatment with maximally tolerated doses of octreotide (200-5,000 microg/day subcutaneously; 11 patients). Ocular changes in each eye were assessed at a minimum of every 3 months for 15 months or until disease progressed to high-risk PDR requiring laser surgery. Endocrine assessments occurred at 3-month intervals during the study RESULTS: Only 1 of 22 eyes from patients treated with octreotide reached high-risk PDR requiring PRP, compared with control patients, in whom 9 of 24 eyes required PRP. The decreased incidence of progression requiring laser surgery was statistically significant if events were considered independently (P < 0.006). The incidence of ocular disease progression was only 27% in patients treated with octreotide compared with 42% in patients with conventional diabetes management. This treatment effect on whether the retina worsened approached statistical significance using repeated measures analysis (P = 0.0605). Endocrine management was similar between treatment groups. Thyroxine replacement therapy was administered to maintain a euthyroid state for all octreotide-treated patients and 7 of 12 control patients. CONCLUSIONS: Our results suggest that octreotide treatment in euthyroid patients may retard progression of advanced DR and may delay the time to laser surgery.

Separation of the insulin-like growth factor-binding proteins in plasma and purification of the larger molecular weight species.

Methods were developed for purification of the high mol wt (150K) insulin-like growth factor (IGF)-binding protein and its acid stable (70K) component from human plasma. High mol wt IGF-binding protein was highly purified by chromatography of Cohn IV-1 fraction of human plasma on Concanavalin A-Sepharose followed by chromatography on IGF-I-Sepharose. The acid-stable component of the high mol wt IGF-binding protein was purified to near homogeneity by chromatography of Cohn IV-1 fraction of human plasma on Con-A Sepharose, followed by chromatography on Sephadex G-50 at pH 2.5 and subsequent chromatography on IGF-I-Sepharose. Both fractions obtained after IGF-I-Sepharose chromatography were capable of binding [125I]IGF-I and gave a single protein band of 79,000 mol wt, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Coomassie blue staining), suggesting that the large mol wt species may be a dimer of identical or iso-mol wt subunits. Three minor contaminants of less than 5% each were detected upon subsequent silver staining. These methods represent important tools that should aid in furthering our understanding of the role of the IGF carrier proteins in the actions of IGF-I and IGF-II.

Changes in insulin-like growth factors I and II and their binding protein after a single intramuscular injection of growth hormone.

The concentrations of insulin-like growth factors I and II (IGF-I and IGF-II) and their binding proteins in serum were measured in 10 GH-deficient patients before and after a single 6-IU injection of GH. Serum IGF-I concentrations were initially low, increased significantly by 8 and 24 h, and decreased to pretreatment levels 48 and 72 h after GH administration. Serum IGF-II concentrations also were low initially and did not increase by 8 and 24 h, but were, however, significantly higher 48 and 72 h after GH administration. In GH-deficient patients before GH administration, binding of IGF-I or IGF-II to serum proteins was restricted primarily to proteins of 50K mol wt. Little or no binding to proteins of 150,000 mol wt was found. By 8 and 24 h after GH injection, IGF-I, but not IGF-II, bound primarily to a protein(s) of 150K mol wt, as in normal subjects. IGF-II remained bound to a 50K mol wt protein. By 48 and 72 h after administering GH, however, the binding pattern was reversed, and IGF-II, but not IGF-I, bound predominantly to a protein(s) of 150K mol wt. Our data demonstrate both a temporal dissociation in the responses of IGF-I and IGF-II to GH and a similar temporal dissociation in the binding of IGF-I and IGF-II to the large mol wt (150K) binding protein. This dissociation, particularly the latter, may provide a means for better characterization of protein fractions in binding IGF, particularly in terms of specificity.

Elevated levels of plasma prorenin (inactive renin) in diabetic and nondiabetic patients with autonomic dysfunction.

We measured plasma inactive renin (prorenin) levels in 46 diabetic patients, 4 nondiabetic patients with idiopathic autonomic dysfunction, and 115 normal subjects. Plasma inactive renin levels were normal in the diabetic patients who had no complications (n = 6) and in those with microvascular disease (n = 8) who did not have coexistent autonomic dysfunction. Plasma inactive renin was either grossly elevated or in the upper limit of the normal range in diabetic patients with autonomic dysfunction (n = 18). No correlation was found between plasma inactive renin and glycemic control, as measured by hemoglobin A1c. High plasma inactive renin levels were also found in the 4 nondiabetic patients with idiopathic autonomic dysfunction. These data suggest that increased plasma inactive renin levels in diabetic patients are a consequence of coexistent autonomic dysfunction. This finding is consistent with other evidence that suggests autonomic regulation of the processing of prorenin to renin within the kidney.

Insulin-like growth factor secretion by human B-lymphocytes: a comparison of cells from normal and pygmy subjects.

Freshly isolated human B-lymphocytes from eight subjects and Epstein-Barr virus-transformed human B-lymphocytes from seven subjects were examined for their capacity to secrete insulin-like growth factor-I (IGF-I) and IGF-II and for their capacity to respond to human GH. Similar studies were conducted with Epstein-Barr virus-transformed lymphocytes collected from six African pygmies. When transformed B-lymphocytes from normal stature subjects were cultured for 3 weeks in RPMI-1640 medium (6 x 10(3) cells/75-cm2 flask at seeding), significant amounts of IGF-I, but no IGF-II, were produced. GH (150 ng/mL) significantly increased for control cells the amount of IGF-I produced at each sampling interval compared to that by unstimulated cultures (P less than 0.05 at 1 week; P = 0.005 at 3 weeks). At 3 weeks, cell counts of cultures compared were 4.13 +/- 0.39 X 10(6)/mL for unstimulated cells and 4.23 +/- 0.87 X 10(6)/mL for GH-stimulated cells. IGF-I production at this time interval by unstimulated cells was 2.8 +/- 2.3 ng/mL, and that by GH-stimulated cells was 12.3 +/- 2.5 ng/mL (P = 0.005). Cell multiplication rates of control cultures were increased in 1 week by GH stimulation [GH stimulated, [16.7 +/- 22.0 X 10(4) cells, unstimulated, 5.73 +/- 4.1 X 10(4) cells; (mean +/- SD); n = 14; P less than 0.01]. Similar results occurred with GH studied at a lower concentration of 10 ng/mL for 3 weeks. Freshly isolated B-lymphocytes did not secrete IGF-I and II after 5 days of culture with GH. Cultures established from cells derived from pygmies produced significantly less IGF-I (4.24 +/- 2.62 ng/mL) when stimulated with 150 ng/mL GH than cultures of cells from normal stature subjects (12.3 +/- 2.5 ng/mL; 0.005 less than P less than 0.01). The cultures compared had a similar cell density. A similar significant difference in IGF-I secretion occurred between cultures of pygmy and control cells stimulated with 10 ng/mL GH. These data are consistent with previous in vivo studies in which pygmies failed to increase IGF-I and exhibit metabolic responses to exogenous GH.

Increased H2O2, vascular endothelial growth factor and receptors in the retina of the BBZ/Wor diabetic rat.

Hyperglycemia in diabetes induces increased levels of hydrogen peroxide (H2O2), a reactive oxygen species generated by reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nontoxic levels of H2O2 increase endothelial cell permeability. Using a model of non-insulin-dependent diabetes, the BBZ/Wor rat, we investigated retinal levels of H2O2, vascular endothelial growth factor (VEGF) and its receptors, VEGF-R1 and VEGF-R2 by transmission electron microscopy at sites of the blood-retinal barrier (BRB). H2O2 localization was done by the cerium NADH oxidase method, and extravasation of endogenous serum albumin was used to document disruption of the BRB. Higher levels of H2O2 were detected in blood vessels of diabetic (78.7 +/- 4.84%) as compared with vessels from nondiabetic rats (39.0 +/- 4.47%). VEGF immunoreactivity was statistically higher in the inner BRB (24.67 +/- 0.33 colloidal gold particles/63 microm2 vs. 21.52 +/- 0.43 colloidal gold particles/63 microm2, p = .0001) and outer BRB (42.56 +/- 0.45 colloidal gold particles/63 microm2 vs. 15.51 +/- 0.51 colloidal gold particles/63 microm2, p = .0001) of diabetic rats as compared with age matched nondiabetic control rats. VEGF-R1 immunoreactivity was significantly higher in diabetic retinas in both the inner BRB (21.66 +/- 0.75 colloidal gold particles/63 microm2 vs. 12.69 +/- 0.61 colloidal gold particles/63 microm2, p = .0001) and outer BRB (22.76 +/- 2.36 colloidal gold particles/63 microm2 vs. 8.53 +/- 2.67 colloidal gold particles/63 microm2, p = .0013). VEGF-R2 was statistically higher in the inner BRB (8.97 +/- 0.57 colloidal gold particles/63 microm2 versus 7.03 +/- 0.65 colloidal gold particles/63 microm2, p = .0419) but not in the outer BRB (29.42 +/- 1.25 colloidal gold particles/63 microm2 vs. 28.07 +/- 1.42 colloidal gold particles/63 microm2, p = .4889). H2O2 levels correlated with increased VEGF (correlation coefficient = 0.82, p = .001) in this model of nonproliferative diabetic retinopathy. These results support that hyperglycemia is one factor that induces retinal endothelial cells in vivo to increase H2O2 via NADH oxidase and stimulates increases in VEGF resulting in disruption of the BRB.

Increased NADH oxidase activity in the retina of the BBZ/Wor diabetic rat.

This morphological study demonstrates a role for endothelial cells in generating reactive oxygen species in early stages of retinopathy in the BBZ/Wor rat, an obese, noninsulin dependent model of diabetes. Hyperglycemia induced pseudohypoxia results in an imbalance in cytosolic NADH/NAD+. In the oxygen-rich environment of the retina, NADH oxidase generates superoxide radical which is dismutated to hydrogen peroxide. Localization of hydrogen peroxide by the cerium NADH oxidase enzyme activity cytochemical localization technique shows a statistically significant increase of peroxide localization in the central retina of diabetic rats as compared to age-matched, nondiabetic controls. Endothelial cell dysfunction, indicated by leakage of endogenous serum albumin, coincided with areas of NADH oxidase activity localization. In diabetic rats there are increased levels of fibronectin in areas of hydrogen peroxide localization. This in vivo, morphological study is the first demonstration of oxidative injury and endothelial cell dysfunction in the retina of a spontaneous, noninsulin dependent model of diabetes.

Radiotherapy for pituitary adenoma: long-term outcome and sequelae.

PURPOSE: To review outcome and treatment sequelae in patients treated with external beam radiotherapy for pituitary adenomas. METHODS AND MATERIALS: One hundred forty-one patients with pituitary adenomas received radiotherapy at the University of Florida and had 2-year minimum potential follow-up. One hundred twenty-one had newly diagnosed adenomas, and 20 had recurrent tumors. Newly diagnosed tumors were treated with surgery and radiotherapy (n = 98) or radiotherapy alone (n = 23). Patients with recurrent tumors received salvage treatment with surgery and radiotherapy (n = 10) or radiotherapy alone (n = 10). The impact of age, sex, presenting symptoms, tumor extent, surgery type, degree of resection, hormonal activity, primary or salvage therapy, and radiotherapy dose on tumor control was analyzed. Tumor control is defined by the absence of radiographic progression and stable or decreased hormone level (in hormonally active tumors) after treatment. Effect of therapy on vision, hormonal function, neurocognitive function, life satisfaction, and affective symptoms were examined. A Likert categorical scale survey was used for assessment of neurocognitive, life satisfaction, and affective symptom status. Survey results from the radiotherapy patients were compared with a control group treated with transsphenoidal surgery alone. Multivariate analysis used the forward step-wise sequence of chi squares for the log rank test. RESULTS: At 10 years, tumor control for the surgery and radiotherapy group (S + RT) was 95% and not statistically different (p = 0.58) than for patients treated with radiotherapy alone (RT) (90%). Patients with prolactin- and ACTH-secreting tumors had significantly worse tumor control, as did patients treated for recurrent tumors. Multivariate analysis for tumor control revealed that only young age was predictive of worse outcome (p = 0.0354). Visual function was either unaffected or improved in most patients, although four patients developed visual loss due to treatment. Hormonal function was affected adversely in 46 of the 93 patients for whom detailed hormonal information was available. Neurocognitive function evaluation revealed that patients in the S + RT group were more likely (p = 0.005) to report difficulty with memory than those in the RT-alone or S-alone groups. No significant difference in life satisfaction or affective symptoms was evident. CONCLUSIONS: Pituitary adenomas are well controlled by external beam radiotherapy, either alone or in combination with surgery. Visual symptoms often improve after treatment. Hormonal sequelae require medical intervention in many patients. Neurocognitive sequelae may be different among treatment groups.

Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.

Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.

Plasminogen activator production by human retinal endothelial cells of nondiabetic and diabetic origin.

The authors examined the effect of insulin-like growth factor 1 (IGF 1), epidermal growth factor (EGF), and acidic fibroblast growth factor (AFGF) on the synthesis by human retinal endothelial cell (HREC) of plasminogen activators (PA; tissue-type [t-PA] and urokinase-type [u-PA]) and plasminogen activator inhibitor (PAI). Immunologic and functional assays for t-PA, u-PA, and PA1 were conducted with cell lines derived from three diabetics and three nondiabetic controls. Confluent HREC of nondiabetic origin did not respond to IGF I (100 ng/ml) with any change of t-PA antigen in the medium (10.7 +/- 1.1 ng/ml unstimulated versus 10.1 +/- 0.8 ng/ml) stimulated, P = not significant). Likewise AFGF and EGF caused no significant change of t-PA levels. Both IGF I and EGF caused a significant increase of t-PA from HREC of diabetic origin (9.6 +/- 0.8 ng/ml unstimulated versus 16.6 +/- 1.9 ng/ml IGF I-stimulated, P less than 0.001, and 14.6 +/- 2.7 ng/ml EGF-stimulated P less than 0.005). Supplementation of AFGF had no effect on HREC of diabetic origin. In confluent cultures, only small quantities of u-PA were detected. After wounding confluent cultures, u-PA activity was associated with cells migrating from the wound edges. Functional PA activity was also measured by chromogenic assay. Results further supported a predominance of t-PA activity being produced by confluent HREC in culture. These results suggest that modulation of PA production by HREC is influenced by exposure to growth factors, by the state of confluency, and the origin of the cells (diabetic vesus nondiabetic).

Measurement of gene expression in human retinal microvessels by solution hybridization.

Changes in gene expression could play a central role in the phenotypic abnormalities of the retinal vascular cells observed in diabetic retinopathy and other retinal diseases. To measure gene expression in human retinal microvessels, a RNA-probe excess solution hybridization assay was used. Retinal microvessels were isolated from eyes obtained within 36 hr of death, and intact RNA was extracted by the guanidine method. Hybridization of poly(A)+ RNA northern blots revealed only the cytoskeletal beta-actin message; by using the more sensitive solution hybridization assay, the plasminogen activator-inhibitor 1 (PAI-1) and von Willebrand factor (vWF) mRNAs were quantified. The prevalence of these transcripts in the retinal microvessels was 0.04 x 10(6) copies/ng RNA for PAI-1 and 0.14 x 10(6) copies/ng RNA for vWF, much less than the prevalence in human umbilical vein endothelial cells (1.93 x 10(6) and 3.90 x 10(6), respectively). The PAI-1 mRNA levels in retinal microvessels isolated from five type II diabetic patients were significantly higher than those in vessels isolated from ten age-matched controls (0.06 x 10(6) versus 0.04 x 10(6) copies/ng RNA, P less than 0.05). The solution hybridization assay accurately measured low-abundance mRNAs in human retinal microvessels; determination of gene expression in these cells could aid in understanding the pathogenesis of important ophthalmologic diseases such as diabetic retinopathy.

Effects of epidermal growth factor, fibroblast growth factor, and transforming growth factor-beta on corneal cell chemotaxis.

The effects of recombinant basic fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta) on migration of human and bovine corneal cells were determined using checkerboard analysis in Boyden chambers. EGF, FGF, and TGF-beta each stimulated high levels of chemotactic migration. Each growth factor, however, induced a different dose-response pattern. Migration stimulated by FGF reached a plateau at a concentration between 100 and 200 ng/ml for endothelial, epithelial, and stromal fibroblasts. By contrast, chemotactic responses to EGF peaked between 10 and 50 ng/ml, then decreased at higher concentrations. TGF-beta also stimulated a peak in migration in all three corneal cells, but the peak of migration occurred at an approximately 1000-fold lower concentration (1 pg/ml) than for EGF. Checkerboard analysis demonstrated that FGF and EGF, but not TGF-beta, stimulated chemokinesis of bovine, stromal, and endothelial cells. These results demonstrate that FGF, EGF, and TGF-beta induce migration in pure populations of bovine and human corneal cells and support the concept that these growth factors may play key roles in corneal wound healing by regulating migration of corneal cells.

Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation.

PURPOSE: The nucleoside adenosine has been implicated in angiogenesis. A previous study demonstrated that activation of the A(2B) adenosine receptor (AdoR) increases cAMP accumulation, cell proliferation, and VEGF expression in human retinal endothelial cells (HRECs). In the present study, the role of this receptor was further characterized by examination of the effects of the selective A(2B) AdoR antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX) on AdoR-mediated HREC proliferation, capillary tube formation, and signal-transduction pathways. METHODS: HRECs were exposed to the adenosine analogue 5'-N-ethylcarboxamido-adenosine (NECA) in the absence or presence of AdoR antagonists. Migration was measured using Boyden chambers. Proliferation was assessed by counting cells. Western analysis was used to assess extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in cell lysates. The effect of AdoR activation on tube formation was studied using cells grown on a synthetic basement membrane matrix. RESULTS: NECA induced proliferation in a concentration-dependent manner that was inhibited by enprofylline and IPDX. NECA stimulated chemotaxis in a concentration-dependent manner that was also blocked by both A(2B) AdoR antagonists. NECA activated ERK and CREB in HRECs. Both A(2B) AdoR antagonists diminished activation of ERK by NECA exposure. ERK activation was also blocked by the ERK-mitogen-activated protein kinase (MAPK) inhibitor PD98059, but not by the protein kinase A (PKA) inhibitor H-89. CREB activation was blocked by H-89, but not by PD98059, suggesting that ERK activation is independent of PKA. NECA enhanced tube formation on the matrix, whereas both A(2B) AdoR antagonists attenuated this effect. CONCLUSIONS: The selective A(2B) AdoR antagonists, enprofylline and IPDX, inhibited NECA-stimulated proliferation, ERK activation, cell migration, and capillary tube formation. A(2B) AdoR inhibition may offer a way to inhibit retinal angiogenesis and provide a novel therapeutic approach to treatment of diseases associated with aberrant neovascularization, such as diabetic retinopathy and retinopathy of prematurity.

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Adenosine receptor antagonists and retinal neovascularization in vivo.

PURPOSE: The role of adenosine receptor (AdoR) antagonists in human retinal endothelial cell function in vitro has previously been determined. In this study, efficacy of AdoR antagonist administration in reducing retinal neovascularization was examined in a mouse pup model of oxygen-induced retinopathy. METHODS: A previously described model of oxygen-induced retinal neovascularization in newborn mouse pups was used to examine the effect of various AdoR antagonists on neovascularization. The nonselective AdoR antagonist xanthine amine congener (XAC), the A(2A)-selective antagonist ZM241385, the A(2B)-selective antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX), and the A(1)-selective antagonist cyclopentyl-1,3-dipropylxanthine (CPX) were used. After the hyperoxia exposure the animals received daily intraperitoneal injections of pharmacologically relevant doses of AdoR antagonists for 5 days. Control animals received vehicle (0.1% dimethyl sulfoxide [DMSO]) alone. The animals were then killed and perfused with fluorescein-dextran. Wholemounts of retinas from one eye were prepared and examined, whereas the retinas of the contralateral eye were embedded, sectioned, and stained for counting neovascular nuclei extending beyond the internal limiting membrane into the vitreous. RESULTS: Angiography of wholemount retinas showed reduction of neovascular tufts in animals treated with selective A(2B) AdoR antagonists. Quantification of the extraretinal neovascular nuclei showed that only animals treated with XAC, enprofylline, or IPDX showed a significant reduction in retinal neovascularization. By contrast, neither CPX nor ZM241385 had an effect on neovascularization. CONCLUSIONS: The A(2B)-selective AdoR antagonists inhibited oxygen-induced retinal neovascularization in vivo and may provide a basis for developing pharmacologic therapies for the treatment of proliferative retinopathies.