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Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:487-496
Published online before print January 6, 2005, doi: 10.1161/01.ATV.0000155325.41507.e0
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:487.)
© 2005 American Heart Association, Inc.

Brief Reviews

Proatherosclerotic Mechanisms Involving Protein Kinase C in Diabetes and Insulin Resistance

Christian Rask-Madsen; George L. King

From Joslin Diabetes Center, Harvard Medical School, Boston, Mass.

Correspondence to George L. King, MD, Joslin Diabetes Center, Harvard Medical School, One Joslin Place, Room 4504, Boston, MA 02120. E-mail george.king{at}joslin.harvard.edu

Series Editor: Richard A. Cohen
Diabetic Vascular Disease: Pathophysiological Mechanisms in the Diabetic Milieu and Therapeutic Implications
ATVB In Focus

Previous Brief Reviews in this Series:

•Naka Y, Bucciarelli LG, Wendt T, Lee LK, Rong LL, Ramasamy R, Yan SF, Schmidt AM. RAGE axis: animal models and novel insights into the vascular complications of diabetes. 2004;24:1342–1349.
•Natarajan R, Nadler JL. Lipid inflammatory mediators in diabetic vascular disease. 2004;24:1542–1548.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowConclusions
 down arrowReferences
 
In diabetes and insulin resistance, activation of protein kinase C (PKC) in vascular cells may be a key link between elevated plasma and tissue concentrations of glucose and nonesterified fatty acids and abnormal vascular cell signaling. Initial studies of PKC activation in diabetes focused on microvascular complications, but increasing evidence supports that PKC plays a role in several mechanisms promoting atherosclerosis. This review explains how PKC is thought to be activated in diabetes and insulin resistance through de novo synthesis of diacylglycerol. Furthermore, the review summarizes studies that implicate PKC in promoting proatherogenic mechanisms or inhibiting antiatherogenic mechanisms, including studies of endothelial dysfunction; gene induction and activation of vascular NAD(P)H oxidase; endothelial nitric oxide synthase expression and function; endothelin-1 expression; growth, migration, and apoptosis of vascular smooth muscle cells; induction of adhesion molecules; and oxidized low-density lipoprotein uptake by monocyte-derived macrophages.

In diabetes and insulin resistance, activation of PKC in vascular cells may be a key link between elevated plasma and tissue concentrations of glucose and nonesterified fatty acids and abnormal vascular cell signaling. This review summarizes studies that implicate PKC in promoting proatherogenic mechanisms or inhibiting antiatherogenic mechanisms.


Key Words: pathophysiology • cell signaling/signal transduction • gene regulation • type 2 diabetes • mechanism of atherosclerosis/growth factors • type 1 diabetes • endothelium/vascular type/nitric oxide


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowConclusions
 down arrowReferences
 
Abnormal signaling in vascular cells in diabetes and nondiabetic insulin resistance causes functional abnormalities long before the development of morphological manifestations of atherosclerosis. Activation of protein kinase C (PKC) by high concentrations of glucose and nonesterified fatty acids may have a key role in transducing abnormal metabolism into altered vascular cell signaling. As a result of increased intracellular concentrations of the lipid diacylglycerol (DAG), an intermediary in glucose and fat metabolism, PKC is chronically activated in diabetes and nondiabetic insulin resistance. Initially, most studies of PKC activation in diabetes focused on its significance for microvascular complications.1 However, increasing evidence supports that PKC is involved in many mechanisms promoting atherosclerosis. Such evidence is discussed in this review and is summarized in the Table and shown in schematic form in Figure 1.


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  		Overview of PKC Isoform–Specific Role in Proatherogenic Mechanisms in Arteries and Arterial Cells



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  		Figure 1. Schematic and simplified representation of proposed PKC-dependent mechanisms thought to promote atherogenesis and affect vascular remodeling. Please see text for details. VSMC indicates vascular smooth muscle cells; PKC, protein kinase C; LDL, low-density lipoprotein; LOX-1, receptor for oxidized LDL; NO, nitric oxide; ET-1, endothelin-1; O2–, superoxide.

PKC is an enzyme that phosphorylates protein substrates on serine (Ser) or threonine (Thr) residues. Of 10 known mammalian PKC isoforms, 8 are activated by DAG. PKC activation occurs through ligand activation of G-protein–coupled receptors, resulting in hydrolysis of a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), by phospholipase C. The products of this reaction are DAG and the PIP2 head group, inositol 1,4,5-triphosphate (IP3). IP3 contributes to PKC activation by a separate mechanism, through opening of IP3-gated Ca2+ transport channels in the endoplasmic reticulum membrane, thereby increasing [Ca2+]i. This is the classical mechanism for PKC activation seen, for example, during {alpha}-adrenergic stimulation,2 angiotensin II stimulation,3 and endothelin-1 (ET-1) stimulation.4 An alternative mechanism, producing a more sustained increase in DAG concentration, is through a pathway that begins with hydrolysis of phosphatidylcholine by phospholipase D.5 Much less is known about the chronic activation of PKC through synthesis of DAG from glucose or lipid metabolites in diabetes and insulin resistance (see next section).

PKC is ubiquitously expressed, but different tissues have specialized distribution of isoforms regulated by transcription of separate genes for each isoform. Similarities exist among protein kinase A, protein kinase B (Akt), and PKC, not only by their structural homology but also by resemblance in regulation of enzyme activity by phosphorylation.6 PKC can bind several activators; not all are described here. PKC isoforms are grouped according to whether they contain domains that bind Ca2+ or DAG, both of which positively regulate kinase activity. Conventional PKC (isoforms {alpha}, ß1, ß2, and {gamma}) binds both activators, novel PKC ({delta}, {epsilon}, {eta}, {theta}) binds DAG, but not Ca2+, and atypical PKC ({zeta}, {iota}) binds neither (the mouse and rat homologue of human PKC{iota} is named PKC{lambda}).

Activation of PKC in Diabetes and High-Glucose Conditions
High-glucose concentrations or diabetes cause increased activity of membrane-associated PKC and is associated with increased intracellular DAG concentrations in several tissues and cell culture systems, for example, in renal glomeruli from diabetic animals7 and in cultured aortic endothelial cells and vascular smooth muscle cells (VSMCs).8 Radioactive labeling studies have established that increased DAG mass occurs in such conditions, not through hydrolysis of PIP2, but partly by de novo synthesis from glucose,8 probably via glyceraldehyde 3-phosphate and phosphatidic acid or from nonesterified fatty acids.1 De novo synthesis of DAG and increased DAG mass are manifest 2 to 3 days after exposure of vascular cells to high-glucose concentrations. In the aorta of diabetic dogs, increased DAG levels are sustained for several years.9

High-glucose concentrations can likely cause increased DAG concentrations by de novo synthesis through several different metabolic pathways. One proposed mechanism is that increased synthesis of DAG is caused by inhibition of the glycolytic enzyme GAPDH, perhaps through poly(ADP-ribosyl)ation during high-glucose concentrations.10 This will cause build-up of the glycolytic intermediate dihydroxyacetone phosphate, which can be used for DAG synthesis after reduction to glycerol-3-phosphate.11

The PKC isoforms activated by diabetes depend on the tissue or cell type. In the aorta, membrane-associated PKCß2 was found to be increased in diabetic rats.12 PKCß2 and PKC{delta} activation has been demonstrated in aortic VSMCs grown in high-glucose concentrations,13 whereas other isoforms have been observed in the retina (PKC{alpha}, ß1, ß2, and {epsilon})14 and renal glomeruli of diabetic rats (PKC{alpha} and ß1).15 The introduction of ruboxistaurin (LY333531), a PKC inhibitor specific for the ß isoforms, showed improvement of glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats,16 supporting that activation of PKCß1 or ß2 is important for vascular dysfunction in several organs in diabetes. Even though PKCß isoforms are expressed at low levels in vascular cells, activation of PKCß in diabetes leads to an increase in membrane-associated general PKC activity.12

Interpreting Studies of Isoform-Specific PKC Function
Isoform-specific characteristics of PKC are important for drawing conclusions from studies of PKC-mediated effects. Phorbol esters are DAG analogues that only activate conventional and novel PKC, not atypical PKC that lack a DAG binding domain. The most widely used phorbol ester is 12-myristate 13-acetate (PMA) (synonymous with 12-O-tetradecanoylphorbol 13-acetate or TPA). Prolonged activation (6 to 12 hours or longer) by phorbol ester leads to downregulation of the expression of conventional and novel PKC,5 albeit differently for individual isoforms and in different cell types.

Phosphorylation of PKC in its activation loop is critical for kinase activity but is not sufficient for activation.6 Generally, PKC translocation (detected as protein content or as activity) from the cytosol to the plasma membrane or to internal membranes is the only reliable measure of PKC activation.

Only few PKC inhibitors possess useful specificity toward a single isoform. Ruboxistaurin inhibits the 2 splice variants of PKCß, ß1 and ß2, with an IC50 {approx}50-times lower than that for other PKC isoforms.16 Its general specificity, for PKC as opposed to other kinases, is favorable. Even though it was recently reported that ruboxistaurin inhibits 3-phosphoinositide-dependent kinase-1 (PDK1),17 it does so with an IC50 of 750 nM in kinase assays using purified enzyme,17 whereas PKC inhibition in vivo is observed at plasma concentrations of 19 nmol/L or lower.16 As another example, rottlerein inhibits PKC{delta} with an IC50 {approx}10-times lower than for PKC{alpha} and ß in vitro.18 Other inhibitors mentioned repeatedly in this review are GF109203X and calphostin C, both of which show selectivity toward conventional and novel PKC.

In general, a more specific approach than pharmacological inhibitors is targeting an individual PKC isoform with dominant-negative techniques (eg, expression of kinase-dead mutants or treatment with oligonucleotides), RNA interference, or targeted gene deletion in animals; reports have been published of mice with deletion of the genes for PKC{alpha}, ß, {gamma}, and {delta}, and they are all viable. In addition, isoform-specific activation or inhibition of PKC translocation can be achieved by peptides representing domains in PKC adaptor proteins.19

Endothelium-Dependent Vasomotor Function
In diabetes, endothelial vasodilator function is compromised because of changed production of vasodilator and vasoconstrictor substances, particularly nitric oxide (NO), prostacyclin,20 cyclooxygenase-dependent vasoconstrictors,20–22 and ET-1.23 Dysfunction of endothelium-dependent vasodilation in diabetes characterizes a proatherosclerotic state. It likely also plays a causal role in atherogenesis because the underlying changes in vascular intracellular signal transduction and in secretion of endothelium-derived vasomotive factors predispose to atherosclerosis. Through such mechanisms, PKC may mediate proatherosclerotic processes by causing increased production of reactive oxygen species (ROS),24,25 decreased NO production, or increased ET-1 production.26

The role of PKC activation in endothelial dysfunction in diabetes has been demonstrated both in animal models and in humans in vivo. In one study of aorta isolated from rabbit, high-glucose concentrations decreased endothelium-dependent vasodilation.27 PMA produced a similar effect, whereas inhibitors of PKC prevented this phenomenon.27 In aorta of rats with streptozotocin-induced diabetes, PKC activity in the membrane fraction was increased because of upregulation of the PKCß2 isoform.12 Endothelial dysfunction by hyperglycemia in healthy humans has been prevented by pretreatment with the PKCß inhibitor ruboxistaurin.28

Vascular NAD(P)H Oxidase Activation and Induction
PKC regulation of neutrophil NADPH oxidase was suggested 2 decades ago when it was shown to be stimulated by phorbol ester.30 It is possible that neutrophils or monocytes contribute to vascular oxidative stress. Thus, increased NADPH oxidase subunit expression in monocytes was demonstrated in diabetic patients and was partly normalized after lowering of plasma glucose.31 Vascular homologues of neutrophil NADPH oxidase are sources of a major part of ROS production in VSMC and endothelial cells during physiological and pathological conditions.29 However, this section focuses on regulation of vascular NAD(P)H oxidase.

In VSMC, angiotensin II stimulates vascular NAD(P)H oxidase.32 Angiotensin II is involved in the development of cardiovascular disease in people with and without diabetes, perhaps through mechanisms independent of hypertension.33 A very rapid angiotensin II-stimulated activation of NAD(P)H oxidase involving PKC may cause redox activation of c-Src, leading to a more sustained activation of NAD(P)H oxidase by mobilizing a PKC-independent pathway.34 Angiotensin II also increases NAD(P)H oxidase gene expression. Vascular cells express several catalytic subunit isoforms, including nox2 (synonymous with gp91phox, the catalytic subunit of neutrophil NADPH oxidase; the suffix stands for phagocyte oxidase).29 In rats, subcutaneous infusion of angiotensin II for 7 days increased PKC activity in aorta and increased ROS production from the aortic lamina media;35 gp91phox and p22phox were increased by 7-fold and 3-fold, respectively. Continuous coinfusion of the isoform-nonselective PKC inhibitor chelerythrine decreased superoxide production and prevented approximately half the increase in NAD(P)H oxidase subunit expression.35 Angiotensin II infusion caused hypertension in these animals.35 Even though increased intra-arterial pressure was shown to increase p47phox Ser phosphorylation (which precedes p47phox membrane translocation and NAD(P)H oxidase activation29) and superoxide production in a PKC-dependent manner,36 the VSMC culture studies described suggest that angiotensin II may stimulate activation of NAD(P)H oxidase independent of increased blood pressure.

High concentrations of glucose and nonesterified fatty acids can also increase superoxide production from NAD(P)H oxidase in cultures of aortic endothelial cells and vascular smooth muscle cells.24 Rats with streptozotocin-induced diabetes were shown to have a 7-fold increase of gp91phox mRNA expression and increased activity of NAD(P)H oxidase in the aorta.25 These abnormalities could be partly normalized, without changes in plasma glucose, after treatment of rats with the PKC inhibitor N-benzoyl-staurosporine (synonymous with CGP4125118 a PKC inhibitor with selectivity toward conventional PKC isoforms).25 Induction of several NAD(P)H oxidase subunits have also been shown in endothelial cell culture exposed to constant or intermittent high-glucose concentrations, and such upregulation was inhibited by LY379196, a specific PKCß inhibitor.37 Evidence for increased activity of vascular NAD(P)H oxidase in Otsuka Long-Evans Tokushima Fatty rats, a model of insulin resistance, has also been published.38 In mammary arteries and saphenous veins from diabetic patients undergoing coronary artery bypass surgery, superoxide production was doubled compared with vessels from nondiabetic controls, likely because of increased endothelial expression of NAD(P)H oxidase subunits.39

Only limited information is available about which PKC isoform is involved in neutrophil NADPH oxidase activation. In isolated neutrophils, PKC{zeta} inhibition prevented oxidant production,40 and in monocytes superoxide production caused by incubation in high-glucose concentrations was dependent on PKC{alpha}.41 Activation of conventional or novel PKC as mediators of vascular NAD(P)H oxidase in endothelial and VSMC culture is suggested by the temporal association of increasing DAG concentrations and PKC activation as well as the fact that increased superoxide production can be recapitulated by stimulation with PMA.24 This is consistent with data from renal glomeruli from rats with streptozotocin-induced diabetes, in which ruboxistaurine treatment prevented increased membrane-associated p47phox and p67phox without changing total p47phox or p67phox mRNA or protein expression.42 In contrast, p47phox membrane translocation stimulated by tumor necrosis factor-{alpha} (TNF-{alpha}) in cultured endothelial cells was inhibited by antisense oligonucleotides or kinase-dead mutants targeting PKC{zeta}, but not by PKC{alpha}, {theta}, or {epsilon} oligonucleotides or prolonged PMA stimulation.43

Endothelial Nitric Oxide Synthase Expression and Dysfunction
The abnormal metabolic milieu of diabetes and insulin resistance results in the PKC-dependent dysregulation of many endothelium-derived substances that are primarily known as vasodilators or vasoconstrictors. For example, in aortic endothelial cells, PKC activation may lead to increased expression of cyclooxygenase, decreased production of the vasodilator prostacyclin, and increased production of the vasoconstrictor thromboxane A2.22 However, in the next 2 sections, the focus will be on regulation of NO and ET-1, for which extensive evidence exists for an implication of PKC. A schematic representation of endothelial NO synthase (eNOS) regulation by PKC is shown in Figure 2.



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  		Figure 2. Overview of PKC-dependent activation and induction of eNOS and vascular NAD(P)H oxidase. Please see text for details. IRS-1 indicates insulin receptor substrate-1; eNOS, endothelial nitric oxide synthase.

NO mediates endothelium-dependent vasodilation and inhibits many proatherogenic mechanisms. It has been reported that eNOS expression is decreased in aortic endothelial cells cultured during high-glucose concentrations.44 This is controversial, however. During very similar culture conditions, it was demonstrated that eNOS expression was upregulated, albeit insufficiently to compensate for decreased NO bioavailability because of increased production of superoxide, which breaks down NO.45 Decreased NO production during the latter conditions can be restored by calphostin C, an inhibitor of conventional and novel PKC.22 The relevance of this finding in cell culture was supported by studies of rats with streptozotocin-induced diabetes.25 These studies pointed to eNOS as a source of vascular superoxide production that was decreased by NG-nitro-L-arginine (L-NNA), an inhibitor of eNOS.25 Treatment of the diabetic rats with a PKC inhibitor decreased eNOS-derived superoxide production, prevented eNOS induction, and restored endothelium-dependent vasorelaxation of aortic rings.25

There are several mechanisms that may explain why eNOS synthesizes superoxide rather than NO, a situation termed "uncoupling" of eNOS. First, PKC may cause phosphorylation of eNOS at Thr495 in the calmodulin-binding domain of the enzyme, perhaps by direct phosphorylation of this site.46,47 Dephosphorylation of Thr495 during eNOS activation may be important for coupling electron flux in eNOS to NO production,48 and a relative increase in Thr495 phosphorylation may uncouple oxygen reduction from L-arginine oxidation so that eNOS produces superoxide. Second, superoxide, from vascular NAD(P)H oxidase or other sources, may react with NO and form peroxynitrite, which potently oxidizes tetrahydrobiopterin, causing uncoupling of eNOS and further eNOS superoxide production.49,50 Third, peroxynitrite may release Zn2+ from the Zn2+–thiolate complex, breaking the eNOS homodimer and uncoupling eNOS.51 Each mechanism is thus a positive feedback cycle, in which superoxide production causes even more superoxide production. Although PKC activation decreases NO production in cell culture,47,52 it remains to be directly demonstrated that this occurs by uncoupling eNOS.

The cause of induction of eNOS expression in streptozotocin-induced diabetes25 is unclear. In endothelial cells cultured in 5.5 mmol/L glucose, the PKC inhibitors staurosporine,53 chelerythrine,53 and GF109203X54 increased eNOS expression. PMA treatment for 24 hours, expected to downregulate PKC, decreased eNOS expression in endothelial cell culture.53 In another study, the same effect was present after PMA treatment with durations between 6 and 20 hours,55 consistent with PKC downregulation (although PKC expression was not measured at these time points, except for PKC{alpha} and {epsilon}). Ruboxistaurine (LY333531)—used at a dose in which it specifically inhibits PKCß—also increased eNOS mRNA.54 Thus, it seems that PKC activation in itself likely tends to downregulate eNOS expression. However, PKC inhibition was found to prevent eNOS upregulation in aortic endothelial cells cultured in high-glucose concentrations.22 One possible explanation is that PKC activation during high-glucose conditions increases eNOS transcription and eNOS mRNA half-life through promotion of oxidative stress.56

To further complicate this issue, it is controversial how eNOS expression is changed in nondiabetic insulin resistance. In the Zucker fatty rat, a model of nondiabetic obesity-associated insulin resistance, eNOS mRNA was found to be decreased to 29% of control levels in microvessels isolated from fat.54 In contrast, eNOS protein was increased 1.7-fold in brain arteries in another study of nondiabetic Zucker fatty rats.57 Thus, it is possible that eNOS expression is regulated differently in large and small vessels or in different tissues. eNOS expression is unchanged in skin biopsy samples from patients with insulin resistance or diabetes,58 but we are not aware of any published data on endothelial eNOS expression in human conduit vessels from such patients.

ET-1 Expression
ET-1 is primarily produced by endothelial cells and is one of the most potent vasoconstrictors known.23 Plasma concentrations of ET-1 are increased in patients with type 2 diabetes complicated with atherosclerosis, compared with nondiabetic patients with atherosclerosis and compared with healthy control subjects.59 Infusion of an ETA receptor blocker increased forearm blood flow in patients with type 2 diabetes, but not in healthy control subjects, indicating that ET-1 has a basal vasoconstrictor effect only in diabetes.60

A likely causal role for ET-1 in the development of atherosclerosis was established with a study of apolipoprotein E (apoE) knockout mice treated with an ETA receptor antagonist for 30 weeks. This intervention did not change blood pressure or plasma lipids but reduced ET-1 protein expression, increased NO-dependent vasorelaxation, and decreased atherosclerotic lesion area in the aorta.61

In microvascular cells, induction of ET-1 caused by high-glucose concentration is associated by increased membrane-associated PKCß and {delta} and can be blocked by GF109203X,62 as well as an inhibitor of the upstream activator of Erk. The nuclear factors downstream of Erk activation, fos and jun, are responsible for induction of ET-1.63 PKC also mediates other stimuli for ET-1 induction. Thus, in heart endothelial cells, PKC mediated angiotensin II-stimulated induction of ET-1 expression because this effect could be blocked by calphostin C.64 Furthermore, oleic acid increased ET-1 expression in human aortic endothelial cells, and this could be blocked by inhibition of PKC or nuclear factor {kappa}B (NF{kappa}B). Oleic acid also increased membrane-associated PKC{alpha} and ß2 and increased NF{kappa}B binding activity, and the latter was prevented by treatment with a PKC inhibitor.65 Finally, ET-1 increases its own expression through a PKC-dependent mechanism.66

Vascular Insulin Resistance
Insulin stimulates skeletal muscle blood flow in healthy, lean individuals,67 but this effect is blunted in people with obesity67 or type 2 diabetes.68 Insulin-stimulated vasodilation is dependent on NO.69–71 Systemic or extravascular effects may contribute to insulin-stimulated vasodilation,72 for example, through platelet release of vasodilatory factors mediated by platelet-derived NO.73 However, at least part of the vasodilatory effect of insulin is likely caused by stimulation of endothelial-derived NO as it occurs in isolated vascular rings, and only when the endothelium is intact;69,74 and because insulin-stimulated NO production is dependent on insulin signaling in endothelial cell culture, including activation of the insulin receptor, insulin receptor substrate (IRS), 1-phosphatidylinositol 3-kinase (PI3K), PDK1, Akt, and ultimately eNOS Ser1177 phosphorylation.75–77 Insulin also induces eNOS gene expression through the PI3K signaling pathway.54 In mice with conditional deletion of the insulin receptor gene only in endothelial cells, eNOS expression in aorta is decreased by 62%.78 Thus, insulin-stimulated regulation of endothelium-derived NO may be a factor necessary for vascular homeostasis that is absent in insulin resistance.

Although Ser/Thr phosphorylation of the insulin receptor with ensuing inhibition of insulin receptor autophosphorylation can inhibit insulin signaling, attempts to explain diabetes-related inhibition of PI3K–Akt signaling have given most attention to IRS modification. In endothelial cell culture, PMA inhibits IRS-2–associated PI3K activity,54 and in VSMC, PMA or PKC{alpha} overexpression inhibits IRS-1 tyrosine phosphorylation,79 necessary for binding PI3K. Mutations of IRS-1 are sufficient to alter insulin-stimulated eNOS function, demonstrated by elegant experiments in human umbilical vein endothelial cells with a common single nucleotide polymorphism in IRS-1. Cultures with this polymorphism had decreased insulin-stimulated activation and gene expression of eNOS through IRS-1, PI3K, and Akt.77 However, studies of insulin signaling compromised by PKC-mediated IRS modification in vascular cells are sparse. Currently, the most definite evidence for PKC-mediated inhibition of IRS comes from studies of insulin action in skeletal muscle of PKC{theta} knockout mice and shows that PKC{theta} activation during hyperlipidemia is necessary for inhibition of skeletal muscle IRS-1 tyrosine phosphorylation and insulin-stimulated glucose uptake.80 Although PKC{theta} can directly phosphorylate IRS-1,81 it may also activate other kinases,82 and the mechanism may include downregulation of IRS-1 expression.82 However, the PKC isoform involved may well be dependent on species, because lipid infusion in humans activate skeletal muscle PKCß1, ß2, and {delta}, but not PKC{epsilon}, {theta}, or {zeta}.83

In insulin-resistant states, impairment of vascular insulin signaling is selective for insulin-stimulated PI3K pathways, leaving insulin-stimulated mitogen-activated protein kinase (MAPK) signaling unaffected.84 It is possible that insulin-stimulated ET-1 expression85 is preserved in insulin resistance, because ET-1 expression is dependent on nuclear factors fos/jun downstream of MAPK signaling.63

Proliferation and Apoptosis of VSMCs
The MAPK Erk1/2 is a major regulator of growth and proliferation. In VSMCs, Erk1/2 activation and proliferation stimulated by oxidized low-density lipoprotein (oxLDL),86 glycated serum albumin,87 oleic acid,88 and angiotensin II88 is dependent on PKC. Angiotensin II and high-glucose concentrations may stimulate VSMC growth by PKC-dependent induction of transforming growth factor-ß.89,90 Other MAPK pathways are activated in diabetes. In rats with streptozotocin-induced diabetes, p38 MAPK activation in aorta was increased compared with control rats.13 p38 activation was also found in VSMC cultured in 16.5 mmol/L glucose, in which overexpression of PKC{delta}, but not PKCß1, increased p38 activation.13

During cyclic stretch of VSMC cultures, apoptosis is mediated by PKC-dependent p38 activation.91 In fact, PKC{delta} has been shown to promote VSMC apoptosis and limit intimal neoplasia in a model of vascular remodeling in response to increased vascular pressure.92 Thus, allografting of vena cava to the carotid artery in mice resulted in marked neointimal hyperplasia in the vein graft after 8 weeks.92 However, vein grafts from PKC{delta} knockout mice showed exaggerated neointimal hyperplasia and decreased apoptosis compared with vein grafts from wild-type mice.92 Whether these findings are relevant for diabetes is unclear because VSMCs grown in high-glucose concentrations show decreased apoptosis.93 This antiapoptotic effect was prevented by prolonged incubation with PMA or treatment with calphostin C, suggesting that conventional or novel PKC were involved.93 When cultured in 5.5 mmol/L glucose, apoptosis of VSMCs as a result of serum starvation was prevented by overexpression of PKC{alpha} or ß2, but not PKC{delta}.93 Whether VSMC apoptosis is increased or decreased in diabetes remains to be resolved.

The contribution of VSMC apoptosis to clinical atherosclerosis is complex, because intimal neoplasia may promote early atherosclerosis but result in instability of advanced plaques.94 It is more readily accepted that endothelial apoptosis promotes atherosclerosis.95 In endothelial cell culture, high-glucose concentrations have been shown to increase apoptosis through activation of caspase-3 and p38.96

Induction of Adhesion Molecules
A critical early step in atherogenesis is adhesion of monocytes to endothelium, followed by migration of monocytes to the intima where they transdifferentiate into macrophages that take up lipids and become foam cells. This process is initiated by binding of monocyte integrins to endothelial adhesion molecules. Vascular cell adhesion molecule-1 (VCAM-1) was first associated with early atherogenesis as an inducible adhesion molecule in endothelium covering fatty streaks in hyperlipidemic rabbits.97 In rabbits, VCAM-1 and E-selectin expression in aortic endothelium is increased by diabetes and further upregulated by diet-induced hyperlipidemia.98 The clinical relevance of these observations is supported by many human studies. For example, in healthy men, the future risk of myocardial infarction is associated with plasma concentrations of soluble intercellular adhesion molecule-1 (ICAM-1),99 and in patients with type 2 diabetes, soluble VCAM-1 concentrations are independently associated with cardiovascular mortality.100

Cell culture studies have firmly established a role for PKC in mediating leukocyte–endothelial interaction. Adhesion of monocytes to the endothelium is dependent on PKC activation in monocytes101–103 and PKC is necessary for monocyte differentiation into macrophages.104 In endothelial cells cultured in high-glucose concentrations, neutrophil adherence and expression of ICAM-1, E-selectin, and P-selectin are increased, but this can be prevented by incubation with PKC inhibitors.105

High-glucose concentrations was shown to increase NF{kappa}B activation and VCAM-1 expression in endothelial cell culture, and these events were inhibited by a selective PKCß2 inhibitor.106 However, VCAM-1 induction stimulated by thrombin has been shown to be mediated by PKC{delta} and PKC{zeta} through binding of NF{kappa}B and GATA-2, respectively, to the VCAM-1 promotor.107 Both conventional and atypical PKC may be involved in regulation of ICAM-1 expression as well. In endothelial cells, ICAM-1 expression is induced by NF{kappa}B binding to the ICAM-1 promotor, which may be mediated by PKC{zeta} after TNF-{alpha} stimulation108 or PKC{delta} after thrombin stimulation.109 The pathways regulating ICAM-1 transcription during high-glucose concentrations have not yet been described.

Monocyte–Macrophage LDL Uptake and Foam Cell Formation
The earliest lesion in atherosclerosis consists of lipid accumulation in the lamina intima and formation of foam cells through uptake of modified LDL, including oxLDL, by monocyte-derived macrophages, dependent on macrophage scavenger receptors.110 The oxLDL increase induction of ICAM-1 in isolated porcine coronary arteries and leukocyte adhesiveness to endothelium,111 as well as growth of cultured macrophages.112 ICAM-1 induction111 and macrophage growth112 can be partly prevented with calphostin C and other PKC inhibitors.

In cultured macrophages, oxLDL was shown to also increase expression of scavenger receptor CD36, and this could be prevented by calphostin C.113 High-glucose concentrations caused induction of another scavenger receptor, LOX-1, in cultures of monocyte-derived macrophages.114 This increase in LOX-1 expression was associated with increased membrane location of PKCß2 and could be prevented by calphostin C or the PKCß-specific inhibitor LY379196.114 The study pointed to ROS production as an upstream event of PKCß2 activation and LOX-1 induction because both could be prevented by the antioxidant N-acetyl-1-cysteine.114 Thus, PKC signaling may be involved in foam cell formation by mediating several different effects of oxLDL.

Pharmacotherapy
The PKCß inhibitor ruboxistaurine is currently being tested in ongoing clinical trials with microvascular end points. In the future, clinical trials may determine whether PKC inhibition can prevent atherosclerosis. However, PKC activation may be modified by strategies other than with PKC inhibitors. For example, troglitazone115 and other PPAR{gamma} agonists116 inhibit high-glucose–induced PKC activation in VSMCs115 and endothelial cells,116 and this may be caused by activation of DAG kinase, resulting in decreased DAG concentrations.116


*    Conclusions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Conclusions
down arrowReferences
 
A large body of literature supports that vascular DAG accumulation and ensuing PKC activation in diabetes lead to endothelial dysfunction by causing activation of vascular NAD(P)H oxidase, dysfunction of eNOS, induction of ET-1, or by other mechanisms (Figure 1). The information about eNOS expression and dysfunction in diabetes and high-glucose conditions is contradictory and more studies are clearly needed. Vascular remodeling by VSMC proliferation and apoptosis is regulated by PKC, either through activation of the DAG–PKC pathway or as intermediary signaling, eg, after angiotensin II stimulation. Lastly, leukocyte adhesion, monocyte transdifferentiation, and macrophage growth leading to intimal foam cell formation are critical events in early atherogenesis, and all may be partly regulated through PKC activation. With regard to most of these mechanisms, the characterization of which PKC isoforms are involved is only partial at best.

Earlier studies of the role of vascular PKC activation in diabetes were primarily focused on microvascular dysfunction.1 Preliminary data show that PKCß/apolipoprotein E double knockout mice have decreased atherosclerosis in the aorta compared with apolipoprotein E knockout mice,117 confirming a role for PKCß in atherogenesis, even in the nondiabetic condition of hypercholesterolemia. Before deciding whether the role of PKC deserves to be tested in clinical studies of atherosclerosis, animal models will help evolve this field from the current suggestive evidence concerning proatherosclerotic signaling to studies of actual atherothrombosis.


*    Acknowledgments
 
Dr Rask-Madsen is supported by Danish Medical Research Council fellowship 22-01-0498 and the Danish Heart Foundation grant 01-2-2-79-22946. Dr King receives National Institutes of Health grant R01 DK53105 and R01 DK59725.

Received July 30, 2004; accepted December 27, 2004.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowConclusions
 *References
 

  1. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes. 1998; 47: 859–866.[Abstract]
  2. Feng X, Zhang J, Barak LS, Meyer T, Caron MG, Hannun YA. Visualization of dynamic trafficking of a protein kinase C betaII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization. J Biol Chem. 1998; 273: 10755–10762.[Abstract/Free Full Text]
  3. Dixon BS, Sharma RV, Dickerson T, Fortune J. Bradykinin and angiotensin II: activation of protein kinase C in arterial smooth muscle. Am J Physiol. 1994; 266 (5 Pt 1): C1406–C1420.
  4. Sugden PH. An overview of endothelin signaling in the cardiac myocyte. J Mol Cell Cardiol. 2003; 35: 871–886.[CrossRef][Medline] [Order article via Infotrieve]
  5. Liu WS, Heckman CA. The sevenfold way of PKC regulation. Cell Signal. 1998; 10: 529–542.[CrossRef][Medline] [Order article via Infotrieve]
  6. Newton AC. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J. 2003; 370 (Pt 2): 361–71.[CrossRef][Medline] [Order article via Infotrieve]
  7. Craven PA, DeRubertis FR. Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose. J Clin Invest. 1989; 83: 1667–1675.
  8. Inoguchi T, Xia P, Kunisaki M, Higashi S, Feener EP, King GL. Insulin’s effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues. Am J Physiol. 1994; 267 (3 Pt 1): E369–E379.
  9. Xia P, Inoguchi T, Kern TS, Engerman RL, Oates PJ, King GL. Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia. Diabetes. 1994; 43: 1122–1129.[Abstract]
  10. Du X, Matsumura T, Edelstein D, Rossetti L, Zsengeller Z, Szabo C, Brownlee M. Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Invest. 2003; 112: 1049–1057.[CrossRef][Medline] [Order article via Infotrieve]
  11. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001; 414: 813–820.[CrossRef][Medline] [Order article via Infotrieve]
  12. Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci U S A. 1992; 89: 11059–11063.[Abstract/Free Full Text]
  13. Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, Kuboki K, Meier M, Rhodes CJ, King GL. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest. 1999; 103: 185–195.[Medline] [Order article via Infotrieve]
  14. Shiba T, Inoguchi T, Sportsman JR, Heath WF, Bursell S, King GL. Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation. Am J Physiol. 1993; 265 (5 Pt 1): E783–E793.
  15. Koya D, Jirousek MR, Lin YW, Ishii H, Kuboki K, King GL. 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. J Clin Invest. 1997; 100: 115–126.[Medline] [Order article via Infotrieve]
  16. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science. 1996; 272: 728–731.[Abstract]
  17. Komander D, Kular GS, Schuttelkopf AW, Deak M, Prakash KR, Bain J, Elliott M, Garrido-Franco M, Kozikowski AP, Alessi DR, van Aalten DM. Interactions of LY333531 and other bisindolyl maleimide inhibitors with PDK1. Structure (Camb). 2004; 12: 215–226.
  18. Way KJ, Chou E, King GL. Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci. 2000; 21: 181–187.[CrossRef][Medline] [Order article via Infotrieve]
  19. Mackay K, Mochly-Rosen D. Localization, anchoring, and functions of protein kinase C isozymes in the heart. J Mol Cell Cardiol. 2001; 33: 1301–1307.[CrossRef][Medline] [Order article via Infotrieve]
  20. Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004; 11: 89–97.[CrossRef][Medline] [Order article via Infotrieve]
  21. Tesfamariam B, Brown ML, Deykin D, Cohen RA. Elevated glucose promotes generation of endothelium-derived vasoconstrictor prostanoids in rabbit aorta. J Clin Invest. 1990; 85: 929–932.
  22. Cosentino F, Eto M, De Paolis P, van der Loo B, Bachschmid M, Ullrich V, Kouroedov A, Delli Gatti C, Joch H, Volpe M, Luscher TF. High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species. Circulation. 2003; 107: 1017–1023.[Abstract/Free Full Text]
  23. Hopfner RL, Gopalakrishnan V. Endothelin: emerging role in diabetic vascular complications. Diabetologia. 1999; 42: 1383–1394.[CrossRef][Medline] [Order article via Infotrieve]
  24. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes. 2000; 49: 1939–1945.[Abstract]
  25. Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88: E14–E22.
  26. Dashwood MR, Tsui JC. Endothelin-1 and atherosclerosis: potential complications associated with endothelin-receptor blockade. Atherosclerosis. 2002; 160: 297–304.[CrossRef][Medline] [Order article via Infotrieve]
  27. Tesfamariam B, Brown ML, Cohen RA. Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C. J Clin Invest. 1991; 87: 1643–1648.
  28. Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Creager MA. Inhibition of protein kinase Cbeta prevents impaired endothelium- dependent vasodilation caused by hyperglycemia in humans. Circ Res. 2002; 90: 107–111.[Abstract/Free Full Text]
  29. Lassegue B, Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R277–R297.[Abstract/Free Full Text]
  30. Wolfson M, McPhail LC, Nasrallah VN, Snyderman R. Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme. J Immunol. 1985; 135: 2057–2062.[Abstract]
  31. Avogaro A, Pagnin E, Calo L. Monocyte NADPH oxidase subunit p22(phox) and inducible hemeoxygenase-1 gene expressions are increased in type II diabetic patients: relationship with oxidative stress. J Clin Endocrinol Metab. 2003; 88: 1753–1759.[Abstract/Free Full Text]
  32. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.[Abstract/Free Full Text]
  33. Sleight P, Yusuf S, Pogue J, Tsuyuki R, Diaz R, Probstfield J. Blood-pressure reduction and cardiovascular risk in HOPE study. Lancet. 2001; 358: 2130–2131.[CrossRef][Medline] [Order article via Infotrieve]
  34. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.[Abstract/Free Full Text]
  35. Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Forstermann U, Meinertz T, Griendling K, Munzel T. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res. 2002; 90: E58–E65.
  36. Ungvari Z, Csiszar A, Huang A, Kaminski PM, Wolin MS, Koller A. High pressure induces superoxide production in isolated arteries via protein kinase C-dependent activation of NAD(P)H oxidase. Circulation. 2003; 108: 1253–1258. Epub July 21, 2003.[Abstract/Free Full Text]
  37. Quagliaro L, Piconi L, Assaloni R, Martinelli L, Motz E, Ceriello A. Intermittent high glucose enhances apoptosis related to oxidative stress in human umbilical vein endothelial cells: the role of protein kinase C and NAD(P)H-oxidase activation. Diabetes. 2003; 52: 2795–2804.[Abstract/Free Full Text]
  38. Kim YK, Lee MS, Son SM, Kim IJ, Lee WS, Rhim BY, Hong KW, Kim CD. Vascular NADH oxidase is involved in impaired endothelium-dependent vasodilation in OLETF rats, a model of type 2 diabetes. Diabetes. 2002; 51: 522–527.[Abstract/Free Full Text]
  39. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.[Abstract/Free Full Text]
  40. Dang PM, Fontayne A, Hakim J, El Benna J, Perianin A. Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst. J Immunol. 2001; 166: 1206–1213.[Abstract/Free Full Text]
  41. Venugopal SK, Devaraj S, Yang T, Jialal I. Alpha-tocopherol decreases superoxide anion release in human monocytes under hyperglycemic conditions via inhibition of protein kinase C-alpha. Diabetes. 2002; 51: 3049–3054.[Abstract/Free Full Text]
  42. Kitada M, Koya D, Sugimoto T, Isono M, Araki S, Kashiwagi A, Haneda M. Translocation of glomerular p47phox and p67phox by protein kinase C-beta activation is required for oxidative stress in diabetic nephropathy. Diabetes. 2003; 52: 2603–2614.[Abstract/Free Full Text]
  43. Frey RS, Rahman A, Kefer JC, Minshall RD, Malik AB. PKCzeta regulates TNF-alpha-induced activation of NADPH oxidase in endothelial cells. Circ Res. 2002; 90: 1012–1019.[Abstract/Free Full Text]
  44. Ding Y, Vaziri ND, Coulson R, Kamanna VS, Roh DD. Effects of simulated hyperglycemia, insulin, and glucagon on endothelial nitric oxide synthase expression. Am J Physiol Endocrinol Metab. 2000; 279: E11–E17.[Abstract/Free Full Text]
  45. Cosentino F, Hishikawa K, Katusic ZS, Luscher TF. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation. 1997; 96: 25–28.[Abstract/Free Full Text]
  46. Michell BJ, Chen Z, Tiganis T, Stapleton D, Katsis F, Power DA, Sim AT, Kemp BE. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem. 2001; 276: 17625–17628. Epub April 5, 2001.[Abstract/Free Full Text]
  47. Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: E68–E75.
  48. Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA, Jr, Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.[Abstract/Free Full Text]
  49. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001; 103: 1282–1288.[Abstract/Free Full Text]
  50. Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem. 2003; 278: 22546–22554. Epub April 10, 2003.[Abstract/Free Full Text]
  51. Zou MH, Shi C, Cohen RA. Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest. 2002; 109: 817–826.[CrossRef][Medline] [Order article via Infotrieve]
  52. Hirata K, Kuroda R, Sakoda T, Katayama M, Inoue N, Suematsu M, Kawashima S, Yokoyama M. Inhibition of endothelial nitric oxide synthase activity by protein kinase C. Hypertension. 1995; 25: 180–185.[Abstract/Free Full Text]
  53. Ohara Y, Sayegh HS, Yamin JJ, Harrison DG. Regulation of endothelial constitutive nitric oxide synthase by protein kinase C. Hypertension. 1995; 25: 415–420.[Abstract/Free Full Text]
  54. Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M, Yamauchi T, Feener EP, Herbert TP, Rhodes CJ, King GL. Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo: a specific vascular action of insulin. Circulation. 2000; 101: 676–681.[Abstract/Free Full Text]
  55. Li H, Oehrlein SA, Wallerath T, Ihrig-Biedert I, Wohlfart P, Ulshofer T, Jessen T, Herget T, Forstermann U, Kleinert H. Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene. Mol Pharmacol. 1998; 53: 630–637.[Abstract/Free Full Text]
  56. Drummond GR, Cai H, Davis ME, Ramasamy S, Harrison DG. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res. 2000; 86: 347–354.[Abstract/Free Full Text]
  57. Erdos B, Snipes JA, Miller AW, Busija DW. Cerebrovascular dysfunction in Zucker obese rats is mediated by oxidative stress and protein kinase C. Diabetes. 2004; 53: 1352–1359.[Abstract/Free Full Text]
  58. Szabo C, Zanchi A, Komjati K, Pacher P, Krolewski AS, Quist WC, LoGerfo FW, Horton ES, Veves A. Poly(ADP-Ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity. Circulation. 2002; 106: 2680–2686.[Abstract/Free Full Text]
  59. Perfetto F, Tarquini R, Tapparini L, Tarquini B. Influence of non-insulin-dependent diabetes mellitus on plasma endothelin-1 levels in patients with advanced atherosclerosis. J Diabetes Complications. 1998; 12: 187–192.[CrossRef][Medline] [Order article via Infotrieve]
  60. Cardillo C, Campia U, Bryant MB, Panza JA. Increased activity of endogenous endothelin in patients with type II diabetes mellitus. Circulation. 2002; 106: 1783–1787.[Abstract/Free Full Text]
  61. Barton M, Haudenschild CC, d’Uscio LV, Shaw S, Munter K, Luscher TF. Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 1998; 95: 14367–14372.[Abstract/Free Full Text]
  62. Park JY, Takahara N, Gabriele A, Chou E, Naruse K, Suzuma K, Yamauchi T, Ha SW, Meier M, Rhodes CJ, King GL. Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation. Diabetes. 2000; 49: 1239–1248.[Abstract]
  63. Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshizumi M, Quertermous T. Regulation of endothelin-1 gene expression by Fos and Jun. J Biol Chem. 1991; 266: 19034–19039.[Abstract/Free Full Text]
  64. Chua BH, Chua CC, Diglio CA, Siu BB. Regulation of endothelin-1 mRNA by angiotensin II in rat heart endothelial cells. Biochim Biophys Acta. 1993; 1178: 201–206.[Medline] [Order article via Infotrieve]
  65. Park JY, Kim YM, Song HS, Park KY, Kim MS, Pak YK, Lee IK, Lee JD, Park SJ, Lee KU. Oleic acid induces endothelin-1 expression through activation of protein kinase C and NF-kappa B. Biochem Biophys Res Commun. 2003; 303: 891–895.[CrossRef][Medline] [Order article via Infotrieve]
  66. Saito S, Hirata Y, Imai T, Marumo F. Autocrine regulation of the endothelin-1 gene in rat endothelial cells. J Cardiovasc Pharmacol. 1995; 26 (Suppl 3): S84–S87.
  67. Laakso M, Edelman SV, Brechtel G, Baron AD. Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man. A novel mechanism for insulin resistance. J Clin Invest. 1990; 85: 1844–1852.
  68. Laakso M, Edelman SV, Brechtel G, Baron AD. Impaired insulin-mediated skeletal muscle blood flow in patients with NIDDM. Diabetes. 1992; 41: 1076–1083.[Abstract]
  69. Chen YL, Messina EJ. Dilation of isolated skeletal muscle arterioles by insulin is endothelium dependent and nitric oxide mediated. Am J Physiol. 1996; 270 (6 Pt 2): H2120–H2124.
  70. Steinberg HO, Brechtel G, Johnson A, Fineberg N, Baron AD. Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release. J Clin Invest. 1994; 94: 1172–1179.
  71. Duplain H, Burcelin R, Sartori C, Cook S, Egli M, Lepori M, Vollenweider P, Pedrazzini T, Nicod P, Thorens B, Scherrer U. Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation. 2001; 104: 342–345.[Abstract/Free Full Text]
  72. Cardillo C, Kilcoyne CM, Nambi SS, Cannon RO, III, Quon MJ, Panza JA. Vasodilator response to systemic but not to local hyperinsulinemia in the human forearm. Hypertension. 1998; 32: 740–745.[Abstract/Free Full Text]
  73. Randriamboavonjy V, Schrader J, Busse R, Fleming I. Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway. J Exp Med. 2004; 199: 347–356. Epub January 26, 2004.[Abstract/Free Full Text]
  74. Schroeder CA, Jr., Chen YL, Messina EJ. Inhibition of NO synthesis or endothelium removal reveals a vasoconstrictor effect of insulin on isolated arterioles. Am J Physiol. 1999; 276 (3 Pt 2): H815–H820.
  75. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation. 2000; 101: 1539–1545.[Abstract/Free Full Text]
  76. Montagnani M, Chen H, Barr VA, Quon MJ. Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179). J Biol Chem. 2001; 276: 30392–30398.[Abstract/Free Full Text]
  77. Federici M, Pandolfi A, De Filippis EA, Pellegrini G, Menghini R, Lauro D, Cardellini M, Romano M, Sesti G, Lauro R, Consoli A. G972R IRS-1 variant impairs insulin regulation of endothelial nitric oxide synthase in cultured human endothelial cells. Circulation. 2004; 109: 399–405.[Abstract/Free Full Text]
  78. Vicent D, Ilany J, Kondo T, Naruse K, Fisher SJ, Kisanuki YY, Bursell S, Yanagisawa M, King GL, Kahn CR. The role of endothelial insulin signaling in the regulation of vascular tone and insulin resistance. J Clin Invest. 2003; 111: 1373–1380.[CrossRef][Medline] [Order article via Infotrieve]
  79. Motley ED, Kabir SM, Gardner CD, Eguchi K, Frank GD, Kuroki T, Ohba M, Yamakawa T, Eguchi S. Lysophosphatidylcholine inhibits insulin-induced Akt activation through protein kinase C-alpha in vascular smooth muscle cells. Hypertension. 2002; 39 (2 Pt 2): 508–12.[Abstract/Free Full Text]
  80. Kim JK, Fillmore JJ, Sunshine MJ, Albrecht B, Higashimori T, Kim DW, Liu ZX, Soos TJ, Cline GW, O’Brien WR, Littman DR, Shulman GI. PKC-theta knockout mice are protected from fat-induced insulin resistance. J Clin Invest. 2004; 114: 823–827.[CrossRef][Medline] [Order article via Infotrieve]
  81. Li Y, Soos TJ, Li X, Wu J, DeGennaro M, Sun X, Littman DR, Birnbaum MJ, Polakiewicz RD. PKCAtheta inhibits insulin signaling by phosphorylating IRS1 at Ser1101. J Biol Chem. 2004; 16: 16.
  82. Gao Z, Zhang X, Zuberi A, Hwang D, Quon MJ, Lefevre M, Ye J. Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3–L1 adipocytes. Mol Endocrinol. 2004; 18: 2024–2034. Epub May 13, 2004.[Abstract/Free Full Text]
  83. Itani SI, Ruderman NB, Schmieder F, Boden G. Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha. Diabetes. 2002; 51: 2005–2011.[Abstract/Free Full Text]
  84. Jiang ZY, Lin YW, Clemont A, Feener EP, Hein KD, Igarashi M, Yamauchi T, White MF, King GL. Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats. J Clin Invest. 1999; 104: 447–457.[Medline] [Order article via Infotrieve]
  85. Oliver FJ, de la Rubia G, Feener EP, Lee ME, Loeken MR, Shiba T, Quertermous T, King GL. Stimulation of endothelin-1 gene expression by insulin in endothelial cells. J Biol Chem. 1991; 266: 23251–23256.[Abstract/Free Full Text]
  86. Yang CM, Chien CS, Hsiao LD, Pan SL, Wang CC, Chiu CT, Lin CC. Mitogenic effect of oxidized low-density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway. Br J Pharmacol. 2001; 132: 1531–1541.[CrossRef][Medline] [Order article via Infotrieve]
  87. Hattori Y, Kakishita H, Akimoto K, Matsumura M, Kasai K. Glycated serum albumin-induced vascular smooth muscle cell proliferation through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by protein kinase C. Biochem Biophys Res Commun. 2001; 281891–6.
  88. Greene EL, Lu G, Zhang D, Egan BM. Signaling events mediating the additive effects of oleic acid and angiotensin II on vascular smooth muscle cell migration. Hypertension. 2001; 37: 308–312.[Abstract/Free Full Text]
  89. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Inves