Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor.

The goal of this study was to investigate whether insulin-like growth factor binding protein-3 receptor (IGFBP-3 receptor) is required for IGFBP-3 to inhibit retinal endothelial cell (REC) apoptosis. REC were grown in normal glucose (5 mM) or high glucose medium (25 mM) for 3 days. Once cells reached confluence, they were transfected with an endothelial- specific IGFBP-3 plasmid DNA (non-IGF binding; IGFBP-3 NB) at 1 µg/ml for 24 h. Cell proteins were extracted and analyzed for IGFBP-3 receptor expression by Western blotting or use in coimmunoprecipitation or co-localization experiments for detection of IGFBP-3 and IGFBP-3 receptor binding. REC were also transfected with or without IGFBP-3 receptor siRNA before IGFBP-3NB plasmid DNA transfection. Cell lysates were processed for a cell death ELISA, a cleaved caspase 3 ELISA, and Western blotting to measure key pro- and anti-apoptotic markers: Bcl-xL, Bax, Cytochrome C and Akt. The IGFBP-3 receptor is present on REC. Overexpression of IGFBP-3 in REC significantly increased protein levels of IGFBP-3 receptor (p < 0.05). Significant increases in cell death were found in cells transfected with IGFBP-3 receptor siRNA versus not treated samples (p < 0.05). Data suggest that IGFBP-3 inhibits retinal endothelial cell death through activation of an IGFBP-3 receptor in a hyperglycemic environment. This is the first demonstration of the involvement of IGFBP-3 receptor in inhibition of REC cell death. Future studies will investigate the mechanism by which IGFBP-3 receptor may inhibit retinal endothelial cell death.

Insulin-like growth factor binding protein-3 inhibits monocyte adhesion to retinal endothelial cells in high glucose conditions.

PURPOSE: Insulin-like growth factor binding protein-3 (IGFBP-3) is cytoprotective in the retina. The goal of this study was to investigate whether IGFBP-3 inhibits monocyte-endothelial cell adhesion associated with hyperglycemia. METHODS: Human retinal vascular endothelial cells (RECs) were grown in normal (5 mM), medium (15 mM), or high glucose medium (25 mM) for 72 h. After 48 h, cells were transfected with endothelial-cell-specific, non-IGF binding IGFBP-3 plasmid DNA (IGFBP-3NB) at 1 µg/ml for 24 h. Cells were serum starved for 16 h and treated with tumor necrosis factor-alpha (TNF-α; 10 ng/ml) for 4 h. Cell proteins were extracted and analyzed for intercellular adhesion molecule-1 (ICAM-1) expression with enzyme-linked immunosorbent assay. Additional RECs were plated onto attachment factor-coated slides, grown to 90% confluence in high glucose medium, and transfected with IGFBP-3 NB plasmid DNA or ICAM-1 small interfering RNA before treatment with or without TNF-α (10 ng/ml) for 4 h. Slides were then mounted in a parallel-plate flow chamber and subjected to a continuous flow of U937 human monocytes (10(5)/ml) in culture medium at shear stresses of 2 dynes/cm(2), with continual exposure to TNF-α. RESULTS: In high ambient glucose, overexpression of IGFBP-3 in RECs significantly decreased ICAM-1 expression when compared to the TNF-α-treated samples, whereas TNF-α increased monocyte-endothelial cell adhesion. IGFBP-3 significantly decreased monocyte adhesion to RECs in the high glucose condition. RECs transfected with ICAM-1 siRNA also had a decreased number of monocytes attached compared with the scrambled siRNA control. CONCLUSIONS: Data suggest that IGFBP-3 reduces monocyte-endothelial cell adhesion through decreased ICAM-1 levels in a hyperglycemic environment. This is the first demonstration of the role of IGFBP-3 in inhibiting monocyte-endothelial cell adhesion.

Sporadic late onset nemaline myopathy responsive to IVIg and immunotherapy.

Sporadic late onset nemaline myopathy (SLONM) is a progressive myopathy of indeterminate etiology and poor outcome. If associated with a monoclonal gammopathy, SLONM carries a more unfavorable prognosis. Immunotherapy was unsuccessful. We report two HIV-negative SLONM/monoclonal gammopathy patients who improved following intravenous immunoglobulin (IVIg) treatment alone or in combination with immunosuppressant agents. This favorable response to treatment suggests that a dysimmune mechanism is operative in some SLONM individuals. We suggest that IVIg deserves initial consideration for SLONM therapy.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.

BACKGROUND: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. OBJECTIVES: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. METHODS: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. RESULTS: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD106). Coronary MaVEC released significantly less EMP than MiVEC. CONCLUSION: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium.

IGFBP-3 and TNF-α regulate retinal endothelial cell apoptosis.

PURPOSE: We hypothesized that loss of insulin-like growth factor binding protein 3 (IGFBP-3) signaling would produce neuronal changes in the retina similar to early diabetes. METHODS: To understand better the role of IGFBP-3 in the retina, IGFBP-3 knockout (KO) mice were evaluated for neuronal, vascular, and functional changes compared to wild-type littermates. We also cultured retinal endothelial cells (REC) in normoglycemia or hyperglycemia to determine the interaction between IGFBP-3 and TNF-α, as data indicate that both proteins are regulated by ß-adrenergic receptors and respond antagonistically. We also treated some cells with Compound 49b, a novel ß-adrenergic receptor agonist we have reported previously to regulate IGFBP-3 and TNF-α. RESULTS: Electroretinogram analyses showed decreased B-wave and oscillatory potential amplitudes in the IGFBP-3 KO mice, corresponding to increased apoptosis. Retinal thickness and cell numbers in the ganglion cell layer were reduced in the IGFBP-3 KO mice. As expected, loss of IGFBP-3 was associated with increased TNF-α levels. When TNF-α and IGFBP-3 were applied to REC, they worked antagonistically, with IGFBP-3 inhibiting apoptosis and TNF-α promoting apoptosis. Due to their antagonistic nature, results suggest that apoptosis of REC may depend upon which protein (IGFBP-3 versus TNF-α) is active. CONCLUSIONS: Taken together, loss of IGFBP-3 signaling results in a phenotype similar to neuronal changes observed in diabetic retinopathy in the early phases, including increased TNF-α levels.

TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis.

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1(Ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IR(Tyr960), both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(Ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IR(Tyr960), both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.

Intra-ophthalmic artery chemotherapy triggers vascular toxicity through endothelial cell inflammation and leukostasis.

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

Compound 49b prevents diabetes-induced apoptosis through increased IGFBP-3 levels.

PURPOSE: To determine whether Compound 49b, a novel PKA-activating drug, can prevent diabetic-like changes in the rat retina through increased insulin-like growth factor binding protein-3 (IGFBP-3) levels. METHODS: For the cell culture studies, we used both human retinal endothelial cells (REC) and retinal Müller cells in either 5 mM (normal) or 25 mM (high) glucose. Cells were treated with 50 nM Compound 49b alone of following treatment with protein kinase A (PKA) siRNA or IGFBP-3 siRNA. Western blotting and ELISA analyses were done to verify PKA and IGFBP-3 knockdown, as well as to measure apoptotic markers. For animal studies, we used streptozotocin-treated rats after 2 and 8 months of diabetes. Some rats were treated topically with 1 mM Compound 49b. Analyses were done for retinal thickness, cell numbers in the ganglion cell layer, pericyte ghosts, and numbers of degenerate capillaries, as well as electroretinogram and heart morphology. RESULTS: Compound 49b requires active PKA and IGFBP-3 to prevent apoptosis of REC. Compound 49b significantly reduced the numbers of degenerate capillaries and pericyte ghosts, while preventing the decreased retinal thickness and loss of cells in the ganglion cell layer. Compound 49b maintained a normal electroretinogram, with no changes in blood pressure, intraocular pressure, or heart morphological changes. CONCLUSIONS: Topical Compound 49b is able to prevent diabetic-like changes in the rat retina, without producing systemic changes. Compound 49b is able to prevent REC apoptosis through increasing IGFBP-3 levels, which are reduced in response to hyperglycemia.

Fusion of HIV-1 envelope-expressing cells to human glomerular endothelial cells through an CXCR4-mediated mechanism.

A central question in the pathogenesis of HIV-associated thrombotic microangiopathic (HIV-TMA) lesions is whether the HIV-1 envelope glycoprotein (HIV-1 Env) can interact directly with human glomerular endothelial cells (HGECs) through specific HIV-1 co-receptors. The goal of this study was to determine whether cultured primary HGECs express significant levels of the major HIV-1 co-receptors CD4, CXCR4, and/or CCR5 to allow fusion interactions with HIV-1. The expression of CD4, CXCR-4 and CCR-5 was assessed in cultured HGECs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry using specific antibodies. The HIV-1 Env-mediated membrane fusion of target glomerular cells was evaluated by a fluorescent dye transfer-based cell-cell fusion microscopic method. HGECs express CXCR4 mRNA and protein as determined by RT-PCR and immunostaining with phycoerythrin-conjugated anti-CXCR4 Mab 12G5. CD4 and CCR5 were not detected in HGECs, either by RT-PCR or by surface immunostaining with specific antibodies. Incubation of HGECs with cells expressing a CD4-independent envelope strain (HIV-1IIIB-8x) and the CD4-dependent envelope strain (HIV-1IIIB) resulted in transfer of fluorescent dyes of approximately 20% after 8-16 h incubation at 37 degrees C. Incubation in the presence of inhibitors (C34, which blocks six-helix bundle formation, and AMD3100, which interacts with CXCR4) reduced dye transfer by 60%-80%, confirming that the dye transfer was specific with respect to gp120-gp41-mediated fusion. Cultured primary HGECs express CXCR4 but not CD4 or CCR5. The ability of HGECs to promote fusion by a CD4-independent HIV-1 envelope glycoprotein suggests that these cells may become a potential direct target of certain HIV-1 isolates.

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation.

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E+ EMP. Markers of apoptosis were negative. In TTP patients, CD62E+ and CD31+/CD42b- EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E+ EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31+/42b- to CD62E+ EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.