Endothelial Insulin Resistance Exacerbates Experimental Periodontitis.

Epidemiological studies suggest that the severity of periodontitis is higher in people with diabetes than in healthy individuals. Insulin resistance might play a crucial role in the pathogenesis of multiple diabetic complications and is reportedly induced in the gingiva of rodents with type 2 diabetes; however, the molecular mechanisms underlying the pathogenesis of diabetes-related periodontitis remain unclear. Therefore, we aimed to investigate whether endothelial insulin resistance in the gingiva may contribute to the pathogenesis of periodontitis as well as elucidate its underlying molecular mechanisms. We demonstrated that insulin treatment downregulated lipopolysaccharide (LPS)-induced or tumor necrosis factor α (TNFα)-induced VCAM1 expression in endothelial cells (ECs) via the PI3K/Akt activating pathway, resulting in reduced cellular adhesion between ECs and leukocytes. Hyperglycemia-induced selective insulin resistance in ECs diminished the effect of insulin on LPS- or TNFα-stimulated VCAM1 expression. Vascular endothelial cell-specific insulin receptor knockout (VEIRKO) mice exhibited selective inhibition of the PI3K/Akt pathway in the gingiva and advanced experimental periodontitis-induced alveolar bone loss via upregulation of Vcam1, Tnfα, Mcp-1, Rankl, and neutrophil migration into the gingiva compared with that in the wild-type (WT) mice despite being free from diabetes. We also observed that insulin-mediated activation of FoxO1, a downstream target of Akt, was suppressed in the gingiva of VEIRKO and high-fat diet (HFD)-fed mice, hyperglycemia-treated ECs, and primary ECs from VEIRKO. Further analysis using ECs transfected with intact and mutated FoxO1, with mutations at 3 insulin-mediated phosphorylation sites (T24A, S256D, S316A), suggested that insulin-mediated regulation of VCAM1 expression and cellular adhesion of ECs with leukocytes was attenuated by mutated FoxO1 overexpression. These results suggest that insulin resistance in ECs may contribute to the progression of periodontitis via dysregulated VCAM1 expression and cellular adhesion with leukocytes, resulting from reduced activation of the PI3K/Akt/FoxO1 axis.

A monoclonal antibody (3G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes.

The identification of microvascular pericytes in vitro relies principally on morphological characteristics and growth dynamics, as there is a paucity of immunochemical markers for these cells. Consequently, an attempt was made to identify mAb reagents that would aid in both the rapid identification and enrichment of retinal capillary pericytes in vascular cell cultures. A panel of mAbs raised by xenogeneic immunization of mice with various tissues was screened for immunoreactivity with dissociated cultures of bovine retinal capillary pericytes. Two antibodies from the panel (3G5 and HISL-8) were seen to react with pericytes by indirect immunofluorescence. The mAb 3G5 was selected for further study. mAb 3G5 did not react with dissociated cultures of smooth muscle cells, endothelial cells, or retinal pigmented endothelial cells. The pericyte 3G5 antigen was insensitive to the action of trypsin; therefore, mAb 3G5 was used to selectively purify pericytes from trypsinized mixed retinal cell cultures by flow cytometry. 3G5+ pericytes (representing 8% of cells in a mixed retinal cell culture) were enriched at least nine-fold to represent greater than 70% of cells. The mAb 3G5 stained retinal capillaries in vivo with a fluorescence distribution consistent with pericyte staining. The 3G5 antigen of cultured pericytes was found to be a glycolipid of mobility intermediate between ganglioside markers GM1 and GM2.

Receptor-mediated vectorial transcytosis of epidermal growth factor by Madin-Darby canine kidney cells.

Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose-dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier.

Receptor-mediated transport of insulin across endothelial cells.

Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.

Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor.

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

Characterization and partial purification of an endothelial cell growth factor from human platelets.

Platelets have been shown to affect the growth of vascular endothelial cells. This report describes the characterization and partial purification from human platelets of a novel growth factor which can stimulate human endothelial cells to synthesize DNA and grow. Platelets were lysed by sonication and the particulate fraction removed by ultracentrifugation at 100,000 g. The supernatant of the platelet lysate stimulated the incorporation of [3H]thymidine into DNA of endothelial cells by 20-fold and caused a threefold increase of cell number in 2 d in culture. Gel filtration on Sephacryl S-200 and dialysis with exclusion membranes resulted in a 50-fold purification of this growth-promoting substance. Two peaks of endothelial-growth factor (ENDO-GF) were observed with apparent molecular weights of 65,000 and 135,000. Further characterization showed that ENDO-GF differed from platelet-derived growth factor since it was very heat labile and more potent in stimulating growth in endothelial cells than in fibroblasts. The isolation of an ENDO-GF from platelets suggests that platelets may have a role in the growth and healing processes of human endothelium.

Identification of the mechanism for the inhibition of Na+,K(+)-adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A2.

Inhibition of Na+,K(+)-ATPase activity by hyperglycemia could be an important etiological factor of chronic complications in diabetic patients. The biochemical mechanism underlying hyperglycemia's inhibitory effects has been thought to involve the alteration of the protein kinase C (PKC) pathway since agonists of PKC can normalize hyperglycemia-induced inhibition of Na+,K(+)-ATPase activity. Paradoxically, elevated glucose levels and diabetes have been shown to increase PKC activities in vascular cells. The present study tested the hypothesis that the inhibition of Na+,K(+)-ATPase activity is mediated by the sequential activation of PKC and cytosolic phospholipase A2 (cPLA2). In cultured rat vascular smooth muscle cells (VSMC), increasing glucose levels in the medium from 5.5 to 22 mM elevated cPLA2 activity and increased [3H]arachidonic acid release and PGE2 production by 2.3-, 1.7- and 2-fold, respectively. Similar increases in cPLA2 activity were also induced by elevated glucose levels in human VSMC and rat capillary endothelial cells. The activation of cPLA2 was mediated by PKC since the increases in cPLA2 phosphorylation and enzymatic activity were inhibited by the PKC inhibitor GFX. In contrast, elevation of glucose levels decreased Na+,K(+)-ATPase activity as measured by ouabain-sensitive 86Rb uptake by twofold in rat VSMC. Surprisingly, both PMA, a PKC agonist, and GFX, a PKC inhibitor, were able to prevent glucose-induced decreases in 86Rb uptake. Further, the PLA2 inhibitor AACOCF3 abolished both glucose-induced activation of cPLA2 and the decrease in 86Rb uptake. These data indicated that hyperglycemia is inhibiting Na+,K(+)-ATPase activity by the sequential activation of PKC and cPLA2, resulting in the liberation of arachidonic acid and increased the production of PGE2, which are known inhibitors of Na+,K(+)-ATPase.

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

Angiotensin II (AII)- and Arg8-vasopressin (AVP)-regulated gene expression in vascular cells has been reported to contribute to vascular homeostasis and hypertrophy. In this report, AVP-induced expression of plasminogen activator inhibitor (PAI)-2 mRNA in rat microvessel endothelial (RME) cells was identified using differential mRNA display. Further characterization of vasoactive peptide effects on PAI expression revealed that AII stimulated a 44.8 +/- 25.2-fold and a 12.4 +/- 3.2-fold increase in PAI-2 mRNA in RME cells and rat aortic smooth muscle cells (RASMC), respectively. AII also stimulated a 10- and 48-fold increase in PAI-1 mRNA in RME cells and RASMC, respectively. These AII effects were inhibited by either Sar1, Ile8-angiotensin or the AT1 antagonist DuP 735, but were not significantly altered in the presence of the AT2 antagonist PD123319. AII stimulation of RASMC and RME cells also significantly increased both PAI-1 protein and PAI activity released to the culture medium. Inhibition of protein kinase C completely blocked PMA-stimulated induction of PAI-2 mRNA in both cell types and inhibited the AII-stimulated increase in RASMC by 98.6 +/- 2.8%. In contrast, protein kinase C inhibition only partially decreased the AII-stimulated PAI-2 expression in RME cells by 68.8 +/- 11.1%, suggesting that a protein kinase C-independent mechanism contributes to a 6.9 +/- 1.5-fold AII induction of PAI-2 expression in endothelial cells. AII and PMA also stimulated protein tyrosine phosphorylation in RME cells, and the tyrosine kinase inhibitor genistein partially blocked their induction of PAI-2 mRNA. These findings suggest that AII may regulate plasminogen activation in the vasculature by inducing both PAI-1 and PAI-2 expression.

Differential regulation of protein kinase C and (Na,K)-adenosine triphosphatase activities by elevated glucose levels in retinal capillary endothelial cells.

Elevated cellular sorbitol levels resulting from conversion of increased glucose by aldose reductase might deplete cellular myoinositol content, which could then lower inositol phosphates (InsPs) and diacylglycerol levels, key regulators of protein kinase C (PKC). Secondary to altered PKC activity, other cellular enzymes such as (Na,K)-ATPase could be affected. To test this hypothesis we examined the association between PKC activity, (Na,K)-ATPase activity, and sorbitol, myoinositol, and InsP levels in cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy. Elevating glucose concentration in culture media from 100 to 400 mg/dl led to a 100% increase in sorbitol levels, which could be inhibited completely by sorbinil, an aldose reductase inhibitor. In contrast, no changes were observed in myoinositol or InsP levels. Subfractionated PKC activities showed a 100% increase in the membranous pool with a parallel decrease in the cytosolic fraction. Adding sorbinil did not affect PKC activity, whereas the PKC agonist, phorbol myristate acetate (PMA), stimulated translocation of PKC. Ouabain-inhibitable (Na,K)-ATPase activity was decreased 70% by elevated glucose levels. This decrease could be prevented by adding either PMA or sorbinil. Thus, in retinal capillary endothelial cells elevated glucose concentration can affect PKC and (Na,K)-ATPase activities, probably via different mechanisms.

Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulinlike growth factor) using antibodies to the insulin receptor.

Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the growth-promoting action of both peptides is mediated by the MSA receptor or other growth factors.

Receptors and growth-promoting effects of insulin and insulinlike growth factors on cells from bovine retinal capillaries and aorta.

It has been suggested that elevated levels of insulin or insulin-like growth factors (IGFs) play a role in the development of diabetic vascular complications. Previously, we have shown a differential response to insulin between vascular cells from retinal capillaries and large arteries with the former being much more insulin responsive. In the present study, we have characterized the receptors and the growth-promoting effect of insulinlike growth factor I (IGF-I) and multiplication-stimulating activity (MSA, an IGF-II) on endothelial cells and pericytes from calf retinal capillaries and on endothelial and smooth muscle cells from calf aorta. We found single and separate populations of high affinity receptors for IGF-I and MSA with respective affinity constants of 1 X 10(-9) M-1 and 10(-8) M-1 in all four cell types studied. Specific binding of IGF-I was between 7.2 and 7.9% per milligram of protein in endothelial cells and 9.1 and 10.4% in the vascular supporting cells. For 125I-MSA, retinal endothelial cells bound only 1.7-2.5%, whereas the aortic endothelial cells and the vascular supporting cells bound between 5.6 and 8.5% per milligram of protein. The specificity of the receptors for IGF-I and MSA differed, as insulin and MSA was able to compete with 125I-IGF-I for binding to the IGF-I receptors with 0.01-0.1, the potency of unlabeled IGF-I, whereas even 1 X 10(-6) M, insulin did not significantly compete with 125I-MSA for binding to the receptors for MSA. For growth-promoting effects, as measured by the incorporation of [3H]thymidine into DNA, confluent retinal endothelial cells responded to IGF-I and MSA by up to threefold increase in the rate of DNA synthesis, whereas confluent aortic endothelial cells did not respond at all. A similar differential of response to insulin between micro- and macrovascular endothelial cells was reported by us previously. In the retinal endothelium, insulin was more potent than IGF-I and IGF-I was more potent that MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In addition, insulin and IGF-I at 1 X 10(-6) and 1 X 10(-8) M, respectively, stimulated these cells to grow by doubling the number of cells as well. In all responsive tissues, the combination of insulin and IGFs were added together, no further increase in effect was seen. These data showed that vascular cells have insulin and IGF receptors, but have a differential response to these hormones. These differences in biological response between cells from retinal capillaries and large arteries could provide clues to understanding the pathogenesis of diabetic micro- and macroangiopathy.

Characterization of protein kinase C beta isoform activation on the gene expression of transforming growth factor-beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats.

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.

Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels.

The pathologies of diabetic micro- and macroangiopathy are different, suggesting that diabetes affects these two types of vascular tissue in a dissimilar manner. We have compared insulin receptors and the effects of insulin on cultured endothelium from calf retinal capillaries and aorta, and the vascular supporting cells, retinal pericytes, and aortic smooth muscle cells. 125I-insulin binds to high affinity insulin receptors on all four cell types. Receptor concentrations were similar except for aortic smooth muscle cells, which have 10-fold fewer receptors than the other cell types. Insulin at a concentration of 10 ng/ml stimulated [14C]glucose incorporation into glycogen in retinal endothelial cells and pericytes and aortic smooth muscle cells, but had no effect on aortic endothelium. Insulin over a concentration range of 10 ng/ml-10 microgram/ml, stimulated [3H]thymidine incorporation into the DNA of retinal pericytes, and endothelial cells and aortic smooth muscle cells but had no effect on aortic endothelial cells. These data suggested that a differential response to insulin may exist between endothelium of micro- and macrovasculature, and suggest that retinal capillary endothelium and retinal pericytes are both very insulin-sensitive tissues.

Identification of persistent defects in insulin receptor structure and function capillary endothelial cells from diabetic rats.

Insulin actions and receptors were studied in capillary endothelial cells cultured from diabetic BB rats and their nondiabetic colony mates. The endothelial cells from diabetic rats of 2 mo duration had persistent biological and biochemical defects in culture. Compared with normal rats, endothelial cells from diabetic rats grew 44% more slowly. Binding studies of insulin and insulin-like growth factor I (IGF-I) showed that cells from diabetic rats had 50% decrease of insulin receptor binding (nondiabetic: 4.6 +/- 0.7; diabetic: 2.6 +/- 0.4% per milligram protein, P less than 0.01), which was caused by a 50% decrease in the number of binding sites per milligram protein, whereas IGF-I binding was not changed. Insulin stimulation of 2-deoxy-glucose uptake and alpha-aminoisobutyric acid uptake were also severely impaired with a 80-90% decrease in maximal stimulation, in parallel with a 62% decrease in insulin-stimulated autophosphorylation (P less than 0.05). 125I-insulin cross-linking revealed an 140-kD alpha subunit of the insulin receptor similar to that in cells from nondiabetic rats, although bands at greater than 200 kD were also detected. The molecular weight of the insulin receptor beta subunit (by SDS-PAGE) was smaller in cells from diabetic than from normal rats (88-90 vs. 95 kD). Neuraminadase treatment of the partially purified insulin receptors decreased the molecular weight of the insulin receptors from nondiabetic rats to a greater degree than its diabetic counterpart. In contrast, Northern blot analysis of insulin receptor mRNAs using human cDNA probes revealed two species of 9.4 and 7.2 kb with no difference in mRNA abundance between cells from diabetic and nondiabetic rats. We conclude that the exposure of capillary endothelial cells to a diabetic milieu in vivo can cause specific and persistent changes in the insulin receptor and insulin action.

Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Primary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Gal beta 1-3GalNAc alpha R beta 1-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of O-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats.

Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca(2+)-sensitive PKC isoforms (alpha and betaII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms delta and epsilon. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cgamma (PLCgamma). This was demonstrated by parallel increases in PLCgamma tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF-induced PKC activation and endothelial cell proliferation. VEGF's mitogenic effect was inhibited by a PKC isoform beta-selective inhibitor, LY333531, in a concentration-dependent manner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-alpha protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCgamma and PKC pathway, involving predominately PKC-beta isoform activation in endothelial cells.

Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways.

Hyperglycemia can cause vascular dysfunctions by multiple factors including hyperosmolarity, oxidant formation, and protein kinase C (PKC) activation. We have characterized the effect of hyperglycemia on p38 mitogen-activated protein (p38) kinase activation, which can be induced by oxidants, hyperosmolarity, and proinflammatory cytokines, leading to apoptosis, cell growth, and gene regulation. Glucose at 16.5 mM increased p38 kinase activity in a time-dependent manner compared with 5.5 mM in rat aortic smooth muscle cells (SMC). Mannitol activated p38 kinase only at or greater than 22 mM. High glucose levels and a PKC agonist activated p38 kinase, and a PKC inhibitor, GF109203X, prevented its activation. However, p38 kinase activation by mannitol or tumor necrosis factor-alpha was not inhibited by GF109203X. Changes in PKC isoform distribution after exposure to 16.5 mM glucose in SMC suggested that both PKC-beta2 and PKC-delta isoforms were increased. Activities of p38 kinase in PKC-delta- but not PKC-beta1-overexpressed SMC were increased compared with control cells. Activation of p38 kinase was also observed and characterized in various vascular cells in culture and aorta from diabetic rats. Thus, moderate hyperglycemia can activate p38 kinase by a PKC-delta isoform-dependent pathway, but glucose at extremely elevated levels can also activate p38 kinase by hyperosmolarity via a PKC-independent pathway.

Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats.

Both insulin resistance and hyperinsulinemia have been reported to be independent risk factors for cardiovascular diseases. However, little is known regarding insulin signaling in the vascular tissues in insulin-resistant states. In this report, insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker (fa/fa) rats in both ex vivo and in vivo studies. Ex vivo, insulin-stimulated tyrosine phosphorylation of insulin receptor beta subunits (IRbeta) in the aorta and microvessels of obese rats was significantly decreased compared with lean rats, although the protein levels of IRbeta in the 2 groups were not different. Insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and their protein levels were decreased in the aorta of obese rats compared with lean rats. The association of p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats. In addition, insulin-stimulated serine phosphorylation of Akt, a downstream kinase of PI 3-kinase pathway, was also reduced significantly in isolated microvessels from obese rats compared with the lean rats. In euglycemic clamp studies, insulin infusion greatly increased tyrosine phosphorylation of IRbeta- and IRS-2-associated PI 3-kinase activity in the aorta of lean rats, but only slight increases were observed in obese rats. In contrast, insulin stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) equally in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. To our knowledge, these data provided the first direct measurements of insulin signaling in the vascular tissues, and documented a selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats.

In vivo phosphorylation of cardiac troponin I by protein kinase Cbeta2 decreases cardiomyocyte calcium responsiveness and contractility in transgenic mouse hearts.

Recently, it has been reported that the protein kinase C (PKC) beta isoform plays a critical role in the development of hypertrophy and heart failure. The purpose of the present study was to clarify the mechanism by which activation of PKCbeta led to depressed cardiac function. Thus, we used a PKCbeta2 overexpressing mouse, an animal model of heart failure, to examine mechanical properties and Ca2+ signals of isolated left ventricular cardiomyocytes. The percentage of shortening, rate of shortening, and rate of relengthening of cardiomyocytes were markedly reduced in PKCbeta2 overexpression mice compared to wild-type control mice, although the baseline level and amplitude of Ca2+ signals were similar. These findings suggested a decreased myofilament responsiveness to Ca2+ in transgenic hearts. Therefore, the incorporation of [32P] inorganic phosphate into cardiac myofibrillar proteins was studied in Langendorff-perfused hearts. There was a significant increase in the degree of phosphorylation of troponin I in PKCbeta2-overexpressing transgenic mice. The depressed cardiomyocyte function improved after the superfusion of a PKCbeta selective inhibitor. These findings indicate that in vivo PKCbeta2-mediated phosphorylation of troponin I may decrease myofilament Ca2+ responsiveness, and thus causes cardiomyocyte dysfunction. Since chronic and excess activation of PKCbeta2 plays a direct and contributory role in the progression of cardiac dysfunction, the PKCbeta selective inhibitor may provide a new therapeutic modality in the setting of heart failure.

The role of protein kinase C activation in diabetic nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease worldwide and an independent risk factor for all-cause and cardiovascular mortalities in diabetic patients. New insights into the molecular mechanisms that underlie the development and progression of microvascular complications of diabetes including nephropathy are emerging rapidly from experimental and clinical studies. Chronic hyperglycemia is a major initiator of diabetic microvascular complications. Activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway, enhanced polyol pathway, increased oxidative stress, and overproduction of advanced glycation end products have all been proposed as potential cellular mechanisms by which hyperglycemia induces diabetic vascular complications. The DAG-PKC pathway contributes to vascular function in many ways such as the regulation of endothelial permeability, vasoconstriction, extracellular matrix synthesis/turnover, cell growth, angiogenesis, cytokine activation, and leukocyte adhesion. We will briefly review the current knowledge base regarding the pathogenic role for the activation of DAG-PKC pathway in diabetic nephropathy and other microvascular complications of diabetes. The results from animal studies and key clinical studies investigating specific effects of the PKC isoforms on the renal and other vascular tissues to induce diabetic complications are also reviewed.