Biocompatibility and structural characterization of glycosaminoglycans isolated from heads of silver-banded whiting (Sillago argentifasciata Martin & Montalban 1935).

Glycosaminoglycans (GAGs) were extracted from heads of silver-banded whiting (SBW) fish and subjected to preliminary biocompatibility testing per ISO 10993: intracutaneous irritation, maximization sensitization, systemic toxicity, and cytotoxicity. When the GAG solution was injected intradermally, the observed irritation was within ISO limits and comparable to a marketed control. There was no evidence of sensitization, systemic toxicity, or cellular toxicity on the test organisms treated with the GAG mixture from SBW fish heads. Fractionation by size-exclusion chromatography has shown three distinct fractions: F1 as low molecular weight hyaluronic acid (190 kDa), F2 (82 kDa) and F3 (64 kDa), both as chondroitin sulfates. Structural characterization by 1D and 2D nuclear magnetic resonance spectroscopy and disaccharide analysis have shown sulfation ratios at positions C4:C6 of the F2 and F3 fractions respectively as 70:20% and 50:30%, and the balance of non-sulfated and 4,6-di-sulfated units. The preliminary results here suggest that GAG-based extracts from SBW fish heads are suitable alternative products to be used in soft tissue augmentation, although further long-term biocompatibility studies are still required.

Effects of carbon monoxide (CO) delivery by a CO donor or hemoglobin on vascular hypoxia inducible factor 1α and mitochondrial respiration.

We examined carbon monoxide (CO) delivery by carbon monoxide-releasing molecule 2 (CORM-2) or hemoglobin (Hb) on cellular oxygen sensing and mitochondrial respiration in bovine aortic endothelial cells (BAECs). CORM-2 reduced hypoxia-inducible factor-1α (HIF-1α) and endothelin-1 (ET-1) expression in normoxic and hypoxic cells, but while Hb alone significantly reduced HIF-1α stabilization in hypoxic cells, CO delivered by Hb (Hb-CO) had no effect on HIF-1α stabilization. CO dose-dependently increased basal oxygen consumption and reduced overall mitochondrial respiratory capacity. Hb-CO increased basal oxygen consumption but did not alter respiratory capacity. Together, CO reduced ET-1, and, at low doses, had no effect on endothelial mitochondria oxygen consumption. CO ligation to Hb may be developed further as non-vasoactive oxygen therapeutic without compromising mitochondrial function.

Epigallocatechin gallate induces expression of heme oxygenase-1 in endothelial cells via p38 MAPK and Nrf-2 that suppresses proinflammatory actions of TNF-α.

Epigallocatechin gallate (EGCG), the major polyphenol in green tea, acutely stimulates production of nitric oxide (NO) from vascular endothelium to reduce hypertension and improve endothelial dysfunction in spontaneously hypertensive rats. Herein, we explored additional mechanisms whereby EGCG may mediate beneficial cardiovascular actions. When compared with vehicle-treated controls, EGCG treatment (2.5 µM, 8 h) of human aortic endothelial cells (HAEC) caused a ~three-fold increase in heme oxygenase-1 (HO-1) mRNA and protein with comparable increases in HO-1 activity. This was unaffected by pretreatment of cells with wortmannin, LY294002, PD98059 or L-NAME (PI 3-kinase, MEK and NO synthase inhibitors, respectively). Pretreatment of HAEC with SB203580 (p38 MAPK inhibitor) or siRNA knockdown of p38 MAPK completely blocked EGCG-stimulated induction of HO-1. EGCG treatment also inhibited tumor-necrosis-factor-α-stimulated expression of vascular cell adhesion molecule (VCAM)-1 and decreased adhesion of monocytes to HAEC. siRNA knockdown of HO-1, p38 MAPK or Nrf-2 blocked these inhibitory actions of EGCG. In HAEC transiently transfected with a human HO-1 promoter luciferase reporter (or an isolated Nrf-2 responsive region), luciferase activity increased in response to EGCG. This was inhibitable by SB203580 pretreatment. EGCG-stimulated expression of HO-1 and Nrf-2 was blocked by siRNA knockdown of Nrf-2 or p38 MAPK. Finally, liver from mice chronically treated with EGCG had increased HO-1 and decreased VCAM-1 expression. Thus, in vascular endothelium, EGCG requires p38 MAPK to increase expression of Nrf-2 that drives expression of HO-1, resulting in increased HO-1 activity. Increased HO-1 expression may underlie anti-inflammatory actions of EGCG in vascular endothelium that may help mediate beneficial cardiovascular actions of green tea.

Differential roles of hyperglycemia and hypoinsulinemia in diabetes induced retinal cell death: evidence for retinal insulin resistance.

Diabetes pathology derives from the combination of hyperglycemia and hypoinsulinemia or insulin resistance leading to diabetic complications including diabetic neuropathy, nephropathy and retinopathy. Diabetic retinopathy is characterized by numerous retinal defects affecting the vasculature and the neuro-retina, but the relative contributions of the loss of retinal insulin signaling and hyperglycemia have never been directly compared. In this study we tested the hypothesis that increased retinal insulin signaling and glycemic normalization would exert differential effects on retinal cell survival and retinal physiology during diabetes. We have demonstrated in this study that both subconjunctival insulin administration and systemic glycemic reduction using the sodium-glucose linked transporter inhibitor phloridzin affected the regulation of retinal cell survival in diabetic rats. Both treatments partially restored the retinal insulin signaling without increasing plasma insulin levels. Retinal transcriptomic and histological analysis also clearly demonstrated that local administration of insulin and systemic glycemia normalization use different pathways to counteract the effects of diabetes on the retina. While local insulin primarily affected inflammation-associated pathways, systemic glycemic control affected pathways involved in the regulation of cell signaling and metabolism. These results suggest that hyperglycemia induces resistance to growth factor action in the retina and clearly demonstrate that both restoration of glycemic control and retinal insulin signaling can act through different pathways to both normalize diabetes-induced retinal abnormality and prevent vision loss.

Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94.

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe(+4)) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl(2)). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.

Green tea polyphenol epigallocatechin gallate reduces endothelin-1 expression and secretion in vascular endothelial cells: roles for AMP-activated protein kinase, Akt, and FOXO1.

Epigallocatechin gallate (EGCG), a green tea polyphenol, promotes vasodilation by phosphatidylinositol 3-kinase-dependent activation of Akt and endothelial nitric oxide synthase to stimulate production of nitric oxide. Reduction in endothelin-1 (ET-1) synthesis may also increase bioavailability of nitric oxide. We hypothesized that the phosphatidylinositol 3-kinase-dependent transcription factor FOXO1 may mediate effects of EGCG to regulate expression of ET-1 in endothelial cells. EGCG treatment (10 microm, 8 h) of human aortic endothelial cells reduced expression of ET-1 mRNA, protein, and ET-1 secretion. We identified a putative FOXO binding domain in the human ET-1 promoter 51 bp upstream from the transcription start site. Trans-activation of a human ET-1 (hET-1) promoter luciferase reporter was enhanced by coexpression of a constitutively nuclear FOXO1 mutant, whereas expression of a mutant FOXO1 with disrupted DNA binding domain did not trans-activate the hET-1 promoter. Disrupting the hET-1 putative FOXO binding domain by site-directed mutagenesis ablated promoter activity in response to overexpression of wild-type FOXO1. EGCG stimulated time-dependent phosphorylation of Akt (S(473)), FOXO1 (at Akt phosphorylation site T(24)), and AMP-activated protein kinase alpha (AMPK alpha) (T(172)). EGCG-induced nuclear exclusion of FOXO1, FOXO1 binding to the hET-1 promoter, and reduction of ET-1 expression was partially inhibited by the AMPK inhibitor Compound C. Basal ET-1 protein expression was enhanced by short interfering RNA knock-down of Akt and reduced by short interfering RNA knock-down of FOXO1 or adenovirus-mediated expression of dominant-negative Foxo1. We conclude that EGCG decreases ET-1 expression and secretion from endothelial cells, in part, via Akt- and AMPK-stimulated FOXO1 regulation of the ET-1 promoter. These findings may be relevant to beneficial cardiovascular actions of green tea.

Dehydroepiandrosterone stimulates phosphorylation of FoxO1 in vascular endothelial cells via phosphatidylinositol 3-kinase- and protein kinase A-dependent signaling pathways to regulate ET-1 synthesis and secretion.

Dehydroepiandrosterone (DHEA) is an endogenous adrenal steroid hormone with controversial actions in humans. We previously reported that DHEA has opposing actions in endothelial cells to stimulate phosphatidylinositol (PI) 3-kinase/Akt/endothelial nitric-oxide synthase leading to increased production of nitric oxide while simultaneously stimulating MAPK-dependent secretion of the vasoconstrictor ET-1. In the present study we hypothesized that DHEA may stimulate PI 3-kinase-dependent phosphorylation of FoxO1 in endothelial cells to help regulate endothelial function. In bovine or human aortic endothelial cells (BAEC and HAEC), treatment with DHEA (100 nM) acutely enhanced phosphorylation of FoxO1. DHEA-stimulated phosphorylation of FoxO1 was inhibited by pretreatment of cells with wortmannin (PI 3-kinase inhibitor) or H89 (protein kinase A (PKA) inhibitor) but not ICI182780 (estrogen receptor blocker), or PD98059 (MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor). Small interfering RNA knockdown of PKA inhibited DHEA-stimulated phosphorylation of FoxO1. DHEA promoted nuclear exclusion of FoxO1 that was blocked by pretreatment of cells with wortmannin, H89, or by small interfering RNA knockdown of PKA. DHEA treatment of endothelial cells increased PKA activity and intracellular cAMP concentrations. Transfection of BAEC with a constitutively nuclear FoxO1 mutant transactivated a co-transfected ET-1 promoter luciferase reporter. Treatment of BAEC with DHEA inhibited transactivation of the ET-1 promoter reporter in cells overexpressing FoxO1. ET-1 promoter activity and secretion in response to DHEA treatment was augmented by PI 3-kinase blockade and inhibited by MAPK blockade. We conclude that DHEA stimulates phosphorylation of FoxO1 via PI 3-kinase- and PKA-dependent pathways in endothelial cells that negatively regulates ET-1 promoter activity and secretion. Balance between PI 3-kinase-dependent inhibition and MAPK-dependent stimulation of ET-1 secretion in response to DHEA may determine whether DHEA supplementation improves or worsens cardiovascular and metabolic function.

Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.

Diabetic retinopathy is characterized by early onset of neuronal cell death. We previously showed that insulin mediates a prosurvival pathway in retinal neurons and that normal retina expresses a highly active basal insulin receptor/Akt signaling pathway that is stable throughout feeding and fasting. Using the streptozotocin-induced diabetic rat model, we tested the hypothesis that diabetes diminishes basal retinal insulin receptor signaling concomitantly with increased diabetes-induced retinal apoptosis. The expression, phosphorylation status, and/or kinase activity of the insulin receptor and downstream signaling proteins were investigated in retinas of age-matched control, diabetic, and insulin-treated diabetic rats. Four weeks of diabetes reduced basal insulin receptor kinase, insulin receptor substrate (IRS)-1/2-associated phosphatidylinositol 3-kinase, and Akt kinase activity without altering insulin receptor or IRS-1/2 expression or tyrosine phosphorylation. After 12 weeks of diabetes, constitutive insulin receptor autophosphorylation and IRS-2 expression were reduced, without changes in p42/p44 mitogen-activated protein kinase or IRS-1. Sustained systemic insulin treatment of diabetic rats prevented loss of insulin receptor and Akt kinase activity, and acute intravitreal insulin administration restored insulin receptor kinase activity. Insulin treatment restored insulin receptor-beta autophosphorylation in rat retinas maintained ex vivo, demonstrating functional receptors and suggesting loss of ligand as a cause for reduced retinal insulin receptor/Akt pathway activity. These results demonstrate that diabetes progressively impairs the constitutive retinal insulin receptor signaling pathway through Akt and suggests that loss of this survival pathway may contribute to the initial stages of diabetic retinopathy.

The Ins2Akita mouse as a model of early retinal complications in diabetes.

PURPOSE: This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age. METHODS: Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity. RESULTS: The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P < 0.005), a modest increase in acellular capillaries after 36 weeks of hyperglycemia (P < 0.0008), and alterations in the morphology of astrocytes and microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P < 0.01). After 22 weeks of hyperglycemia, there was a 16.7% central and 27% peripheral reduction in the thickness of the inner plexiform layer, a 15.6% peripheral reduction in the thickness of the inner nuclear layer (P < 0.001), and a 23.4% reduction in the number of cell bodies in the retinal ganglion cell layer (P < 0.005). In vitro insulin receptor kinase activity was reduced (P < 0.05) after 12 weeks of hyperglycemia. CONCLUSIONS: The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.

Insulin promotes rat retinal neuronal cell survival in a p70S6K-dependent manner.

The purpose of this study was to examine the role of the ribosomal protein S6 protein kinase (p70S6K), a protein synthesis regulator, in promoting retinal neuronal cell survival. Differentiated R28 rat retinal neuronal cells were used as an experimental model. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, and during the period of experimentation were exposed either to the absence or presence of 10 nm insulin. Insulin treatment induced p70S6K, mTOR, and Akt phosphorylation, effects that were completely prevented by the PI3K inhibitor, LY294002. Insulin-induced phosphorylation of p70S6K and mTOR was prevented by the mTOR inhibitor, rapamycin. Apoptosis, induced by serum deprivation and evaluated by Hoechst staining, was inhibited by insulin treatment in R28 cells, but not in L6 muscle cells. This effect of insulin was also largely prevented by rapamycin. Inhibition of p70S6K activity by exogenous expression of a dominant negative mutant of p70S6K prevented insulin-induced cell survival, whereas, overexpression of wild type p70S6K or expression of a rapamycin resistant form of the kinase enhanced the effect of insulin on survival. Enhanced cell survival under the latter condition was accompanied by increased p70S6K activity and phosphorylation. Rapamycin did not inhibit insulin induced p70S6K phosphorylation and activity in cells transfected with the rapamycin-resistant mutant. Together, these results suggest that p70S6K plays a key role in insulin stimulated retinal neuronal cell survival.

An eye on insulin.

Diabetic retinopathy, the most frequent complication of diabetes and leading cause of vision loss, involves vascular and neural damage in the retina. Insulin and IGF-1 signaling are now shown to contribute to retinal neovascularization, in part, by modulating the expression of various vascular mediators.

Characterization of insulin signaling in rat retina in vivo and ex vivo.

Insulin receptor (IR) signaling cascades have been studied in many tissues, but retinal insulin action has received little attention. Retinal IR signaling and activity were investigated in vivo in rats that were freely fed, fasted, or injected with insulin by phosphotyrosine immunoblotting and by measuring kinase activity. A retina explant system was utilized to investigate the IR signaling cascade, and immunohistochemistry was used to determine which retinal cell layers respond to insulin. Basal IR activity in the retina was equivalent to that in brain and significantly greater than that of liver, and it remained constant between freely fed and fasted rats. Furthermore, IR signaling increased in the retina after portal vein administration of supraphysiological doses of insulin. Ex vivo retinas responded to 10 nM insulin with IR beta-subunit (IRbeta) and IR substrate-2 (IRS-2) tyrosine phosphorylation and AktSer473 phosphorylation. The retina expresses mRNA for all three Akt isoforms as determined by in situ hybridization, and insulin specifically increases Akt-1 kinase activity. Phospho-AktSer473 immunoreactivity increases in retinal nuclear cell layers with insulin treatment. These results demonstrate that the retinal IR signaling cascade to Akt-1 possesses constitutive activity, and that exogenous insulin further stimulates this prosurvival pathway. These findings may have implications in understanding normal and dysfunctional retinal physiology.

Functions of insulin and insulin receptor signaling in retina: possible implications for diabetic retinopathy.

Insulin action regulates the metabolic functions of the classically insulin-responsive tissues: liver, adipose, and skeletal muscle. Evidence also suggests that insulin acts on neural tissue and can modulate neural metabolism, synapse activity, and feeding behaviors. Insulin receptors are expressed on both the vasculature and neurons of the retina, but their functions are not completely defined. Insulin action stimulates neuronal development, differentiation, growth, and survival, rather than stimulating nutrient metabolism, e.g., glucose uptake as in skeletal muscle. Insulin receptors from retinal neurons and blood vessels share many similar properties with insulin receptors from other peripheral tissues, and retinal neurons express numerous proteins that are attributed to the insulin signaling cascade as in other tissues. However, undefined neuron-specific signals downstream of the insulin receptor are likely to also exist. This review compares retinal insulin action to that of peripheral tissues, and demonstrates that the retina is an insulin-sensitive tissue. The review also addresses the hypothesis that dysfunctional insulin receptor signaling in the retina contributes to cell dysfunction and death in retinal diseases.