Mechanisms of angiotensin II-induced expression of B2 kinin receptors

Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B(2) kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10(-9)-10(-6) M)- and time (0-24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10(-7) M. ANG II (10(-7) M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT(1) receptor antagonist) and/or PD-123319 (AT(2) receptor antagonist) at 10 microM for 30 min, followed by ANG II (10(-7) M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT(1A) receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44(mapk) (PD-98059) and/or an inhibitor of p38(mapk) (SB-202190), followed by ANG II (10(-7) M) for 24 h. Selective inhibition of the p42/p44(mapk) pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.     

Creatinine metabolite,HMH (5-hydroxy-1-methylhydantoin; NZ-419), modulates bradykinin-induced changes in vascular smooth muscle cells

Abstract

A creatinine metabolite, 5-hydroxy-1-methylhydantoin (HMH: NZ-419), a hydroxyl radical scavenger, has previously been shown to confer renoprotection by inhibiting the progression of chronic kidney disease in rats. In the current study, we demonstrate that HMH modulates the effects of glucose and bradykinin (BK) in vascular smooth muscle cell (VSMC). HMH a novel anti-oxidant drug completely suppressed the expression of B2-kinin receptors (B2KR) in response to high glucose (25?mM) stimulation in VSMC and was also shown to attenuate the effects of BK on VSMC remodeling. HMH inhibited the BK-induced increase in MAPK phosphorylation and attenuated the increase in connective tissue growth factor (CTGF) protein levels in VSMC. These findings suggest that HMH may confer vascular protection against high glucose concentrations and BK-stimulation to ameliorate vascular injury and remodeling through its anti-oxidant properties.     

High glucose enhances IL-1beta-induced cyclooxygenase-2 expression in rat vascular smooth muscle cells

The changes in vascular prostaglandin production are implicated in the derangement of vascular reactivity in diabetes. However, the mechanism of altered prostaglandin (PG) production in diabetes is largely unknown. In this study, we investigated the effect of high glucose on IL-1beta-induced PG production and the possible underlying mechanism in cultured vascular smooth muscle cell (VSMC). High glucose evoked an augmentation of IL-1beta-induced PG synthesis in a dose dependent manner and enhanced cyclooxygenase (COX) activity, which reached to maximum at 8-12 hours after stimulation. Western blot analysis supported the activity data. Protein kinase C (PKC) inhibitors, H-7 and chelerythrine, significantly inhibited the enhancement of IL-1beta-induced COX-2 expression by high glucose. The activation of PKC by PMA resulted in marked increase of PG production in low glucose group, whilst this was not the case in high glucose group. Furthermore, glucose-enhancing effect was significantly suppressed by zopolrestat, an aldose reductase inhibitor, and sodium pyruvate. These results suggest that the augmenting effect of high glucose on IL-1beta-induced PG production and COX-2 expression is, at least in part, due to increased glucose metabolism via sorbitol pathway following PKC activation.     

Glucose upregulates plasminogen activator inhibitor-1 gene expression in vascular smooth muscle cells

We investigated the effects of high concentrations of glucose on plasminogen activator inhibitor-1 (PAI-1) gene expression in cultured rat vascular smooth muscle cells (VSMC). In response to a high glucose concentration (27.5 mM), PAI-1 mRNA increased within 2 h, peaked at 4 h, remained elevated for another 4 h, then decreased to basal levels at 24 h. On the other hand, mannose at the same concentration (22.5 mM mannose plus 5.5 mM glucose) as an osmotic control had little effect on PAI-1 mRNA expression. The expression of PAI-1 mRNA that was also increased by H(2)O(2), angiotensin II, or phorbol myristate acetate, was reversed by the MAPK kinase (MEK) inhibitor PD98059 or the specific protein kinase C (PKC) inhibitor GF109203X. High glucose appeared to activate MAPK and PKC in VSMC judging from Elk-1 and AP-1 activation, respectively. PD98059 inhibited and GF109203X prevented subsequent PAI-1 induction by glucose. These results suggest that glucose at high concentrations induces PAI-1 gene expression in VSMC at least partially via MAPK and PKC activation. This direct effect of glucose might have important implications for the increased plasma concentrations of PAI-1 and possibly atherosclerosis that are associated with diabetes.     

Sodium Tanshinone IIA Silate Inhibits High Glucose-Induced Vascular Smooth Muscle Cell Proliferation and Migration through Activation of AMP-Activated Protein Kinase

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G₀/G₁ phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Role of the kinin B1 receptor in insulin homeostasis and pancreatic islet function

Kinins are potent vasoactive peptides generated in blood and tissues by the kallikrein serine proteases. Two distinct kinin receptors have been described, one constitutive (subtype B2) and one inducible (subtype B1), and many physiological functions have been attributed to these receptors, including glucose homeostasis and control of vascular permeability. In this study we show that mice lacking the kinin B1 receptor (B1-/- mice) have lower fasting plasma glucose concentrations but exhibit higher glycemia after feeding when compared to wild-type mice. B1-/- mice also present pancreas abnormalities, characterized by fewer pancreatic islets and lower insulin content, which leads to hypoinsulinemia and reduced insulin release after a glucose load. Nevertheless, an insulin tolerance test indicated higher sensitivity in B1-/- mice. In line with this phenotype, pancreatic vascular permeability was shown to be reduced in B1 receptor-ablated mice. The B1 agonist desArg9bradykinin injected intravenously can induce the release of insulin into serum, and this effect was not observed in the B1-/- mice or in isolated islets. Our data demonstrate the importance of the kinin B1 receptor in the control of pancreatic vascular homeostasis and insulin release, highlighting a new role for this receptor in the pathogenesis of diabetes and related diseases.     

High glucose abolishes the antiproliferative effect of 17beta-estradiol in human vascular smooth muscle cells

We examined effects of 17beta-estradiol (E(2)) on human vascular smooth muscle cell (VSMC) proliferation under normal (5 mmol/l) and high (25 mmol/l) glucose concentrations. Platelet-derived growth factor (PDGF) BB (20 ng/ml)-induced increases in DNA synthesis and proliferation were greater in high than normal glucose concentrations; the difference in DNA synthesis was abolished by a protein kinase C (PKC)-beta inhibitor, LY-379196 (30 nmol/l). Western blotting showed that PKC-beta(1) protein increased in cells exposed to high glucose, whereas PKC-alpha protein and total PKC activity remained unchanged, compared with normal glucose cultures. In normal glucose, E(2) (1-100 nmol/l) inhibited PDGF-induced DNA synthesis by 18-37% and cell proliferation by 16-22% in a concentration-dependent manner. The effects of E(2) were blocked by the estrogen receptor (ER) antagonist ICI-182780, indicating ER dependence. In high glucose, the inhibitory effect of E(2) on VSMC proliferation was abolished but was restored in the presence of the PKC-beta inhibitor LY-379196. Thus high glucose enhances human VSMC proliferation and attenuates the antiproliferative effect of E(2) in VSMC via activation of PKC-beta.     

Altered glucose homeostasis and hepatic function in obese mice deficient for both kinin receptor genes

The Kallikrein-Kinin System (KKS) has been implicated in several aspects of metabolism, including the regulation of glucose homeostasis and adiposity. Kinins and des-Arg-kinins are the major effectors of this system and promote their effects by binding to two different receptors, the kinin B2 and B1 receptors, respectively. To understand the influence of the KKS on the pathophysiology of obesity and type 2 diabetes (T2DM), we generated an animal model deficient for both kinin receptor genes and leptin (obB1B2KO). Six-month-old obB1B2KO mice showed increased blood glucose levels. Isolated islets of the transgenic animals were more responsive to glucose stimulation releasing greater amounts of insulin, mainly in 3-month-old mice, which was corroborated by elevated serum C-peptide concentrations. Furthermore, they presented hepatomegaly, pronounced steatosis, and increased levels of circulating transaminases. This mouse also demonstrated exacerbated gluconeogenesis during the pyruvate challenge test. The hepatic abnormalities were accompanied by changes in the gene expression of factors linked to glucose and lipid metabolisms in the liver. Thus, we conclude that kinin receptors are important for modulation of insulin secretion and for the preservation of normal glucose levels and hepatic functions in obese mice, suggesting a protective role of the KKS regarding complications associated with obesity and T2DM.     

Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF-beta and MAPK

Accumulation of extracellular matrix (ECM) is a hallmark feature of vascular disease. We have previously shown that hyperglycemia induces the expression of B(2)-kinin receptors in vascular smooth muscle cells (VSMC) and that bradykinin (BK) and hyperglycemia synergize to stimulate ECM production. The present study examined the cellular mechanisms through which BK contributes to VSMC fibrosis. VSMC treated with BK (10(-8) M) for 24 h significantly increased alpha(2)(I) collagen mRNA levels. In addition, BK produced a two- to threefold increase in alpha(2)(I) collagen promoter activity in VSMC transfected with a plasmid containing the alpha(2)(I) collagen promoter. Furthermore, treatment of VSMC with BK for 24 h produced a two- to threefold increase in the secretion rate of tissue inhibitor of metalloproteinase 1 (TIMP-1). The increase in alpha(2)(I) collagen mRNA levels and alpha(2)(I) collagen promoter activity, as well as TIMP-1 secretion, in response to BK were blocked by anti-transforming growth factor-beta (anti-TGF-beta) neutralizing antibodies. BK (10(-8) M) increased the endogenous production of TGF-beta1 mRNA and protein levels. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 inhibited the increase of alpha(2)(I) collagen promoter activity, TIMP-1 production, and TGF-beta1 protein levels observed in response to BK. These findings provide the first evidence that BK induces collagen type I and TIMP-1 production via autocrine activation of TGF-beta1 and implicate MAPK pathway as a key player in VSMC fibrosis in response of BK.     

Neuron-specific protein F1/GAP-43 shows substrate specificity for the beta subtype of protein kinase C

We determined whether the beta or gamma protein kinase C (PKC) subtypes implicated in long-term potentiation (LTP) selectively regulates protein F1 phosphorylation. Purified bovine PKC subtypes and recombinant PKC subtypes activated by phosphatidylserine (PS) and calcium were tested for their relative ability to phosphorylate purified rat protein F1 (a.k.a. GAP-43). After equalizing enzyme activity against histone, the recombinant beta II PKC phosphorylated protein F1 to a 6 fold greater extent than the recombinant gamma PKC. Bovine beta I PKC phosphorylated protein F1 to a 3 fold greater extent than bovine gamma PKC. Even when PS was replaced by lipoxin B4, which can selectively increase gamma PKC activity, beta I PKC was still superior to gamma PKC in phosphorylating protein F1. Taken together with previous cellular studies of brain showing parallel levels of expression of beta PKC mRNA and protein F1 mRNA, the present results make it attractive to propose that beta PKC regulates protein F1 phosphorylation during the development of synaptic plasticity.     

Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

Glucose-induced changes in protein kinase C and nitric oxide are prevented by vitamin E

Changes in activity or expression of protein kinase C (PKC), reactive oxygen products, and nitric oxide (NO) may account for the alteration in cell behavior seen in diabetes. These changes have been proposed to be part of the pathophysiology of erectile dysfunction. We sought to ascertain if corpus cavernosal vascular smooth muscle cells (CCSMC) grown in a high glucose milieu exhibit changes in the activity and expression of PKC isoforms, NO, and reactive oxygen products and to find out if these changes are prevented by alpha-tocopherol. Rat CCSMC were grown in 5, 15, and 30 mM glucose concentrations for 3, 7, and 14 days. PKC isoform expression was assayed with isoform-specific antibodies. In CCSMCs grown in 30 mM glucose for 2-wk, PKC-beta(2)-isoform was upregulated (n = 4; P < 0.01), whereas the expression of alpha-, delta-, epsilon-, and beta(1)-isoforms was unchanged. NO as measured by nitrate-to-nitrite ratio was greatly diminished at 14 days in 30 mM (n = 4; P < 0.002) compared with 5 mM glucose. Reactive oxygen products were upregulated at 14 days when they were assayed by the fluorescent probe dichlorofluorescein diacetate bis(acetoxy-methyl) (DCFH-DA) (n = 5; P < 0.01). When these same cells were exposed to alpha-tocopherol for 14 days, there was a reduction of PKC-beta(2) (57.8%; P < 0.01; n = 4) and a reduction in reactive oxygen product formation (71.1%; P < 0.001; n = 4), along with an increase in nitrate-to-nitrite ratio (43.9%; P < 0.01, n = 4). These results suggest that there may be an interrelationship between PKC, NO, and reactive oxygen product formation in CCSMC exposed to a high glucose environment.     

Glucose down-regulation of cGMP-dependent protein kinase I expression in vascular smooth muscle cells involves NAD(P)H oxidase-derived reactive oxygen species

Reduced levels of cGMP-dependent protein kinase I (PKG-I) in vasculature have been shown to contribute to diabetic vascular dysfunctions. However, the underlying mechanisms remain unknown. In this report, using primary rat aortic smooth muscle cells (VSMC), we investigated the mechanisms of glucose-mediated regulation of PKG-I expression. Our data showed that high glucose (30 mM glucose) exposure significantly reduced PKG-I production (protein and mRNA levels) as well as PKG-I activity in cultured VSMC. Glucose-mediated decreases in PKG-I levels were inhibited by a superoxide scavenger (tempol) or NAD(P)H oxidase inhibitors (diphenylene iodonium or apocynin). High glucose exposure time-dependently increased superoxide production in VSMC, which was abolished by tempol or apocynin treatment, but not by other inhibitors of superoxide-producing enzymes (L-NAME, rotenone, or oxypurinol). Total protein levels and phosphorylated levels of p47phox (an NADPH oxidase subunit) were increased in VSMC after high glucose exposure. Transfection of cells with siRNA-p47phox abolished glucose-induced superoxide production and restored PKG-I protein levels in VSMC. Treatment of cells with PKC inhibitor prevented glucose-induced p47phox expression/phosphorylation and superoxide production and restored the PKG-I levels. Decreased PKG-I protein levels were also found in femoral arteries from diabetic mice, which were associated with the decreased DEA-NONOate-induced vasorelaxation. Taken together, the present results suggest that glucose-mediated down-regulation of PKG-I expression in VSMC occurs through PKC-dependent activation of NAD(P)H oxidase-derived superoxide production, contributing to diabetes-associated vessel dysfunctions.     

High glucose blocks the effects of estradiol on human vascular cell growth: differential interaction with estradiol and raloxifene

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E(2) on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E(2)- (but not RAL-) stimulated [3H] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E(2)-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E(2)-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E(2) (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E(2)-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E(2)-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E(2) on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E(2)'s anti-atherosclerotic properties. Since RAL's estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.     

Dual regulation of glycogen synthase kinase-3beta by the alpha1A-adrenergic receptor

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and G? 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.