Epoxyeicosatrienoic acid(s) (EET) have variable hemodynamic, anti-inflammatory, and growth regulatory effects, and inhibitors of their regulatory enzyme, soluble epoxide hydrolase (sEH), can mimic these effects. For this reason, sEH inhibitors are being studied as potential pharmaceuticals for the treatment of hypertension, atherosclerosis, and inflammatory diseases. We now show that a highly selective urea-based sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) attenuates human aortic vascular smooth muscle (HVSM) cell proliferation independently of any effect on sEH. CDU also inhibits endothelial cells when stimulated with basic fibroblast growth factor or serum. In addition, we demonstrate that EET, as well as several newer generation sEH inhibitors and a urea-based weak sEH inhibitor, do not affect proliferation in HVSM cells. Structure-activity relationships demonstrate that the addition of an acid group to the dodecyl carbon chain, changing the cyclohexyl group to an adamantyl group, and shortening the carbon chain to two carbons all abolish the antiproliferative effect. Our finding that a highly selective urea-based inhibitor of sEH can alter biology independently of its putative target enzyme suggests that there may be other useful properties of this class of compounds unrelated to their influence on epoxyeicosanoids. In addition, our results show that caution should be used when attempting to infer conclusions of EET biology based solely on the effects these inhibitors in tissue culture models, especially when used at micromolar concentrations.

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-alpha, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF-alpha mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73 beta, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-alpha, when added to these cells in the presence of serum-containing media, increases p73 beta expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH2-terminally deleted p73 plasmid results in markedly decreased TNF-alpha-induced apoptosis. Thus p73 beta is likely a mediator of the apoptotic effect of TNF-alpha in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies.

The intracellular localization of signaling proteins is critical in directing their interactions with both upstream and downstream signaling cascade components. While initially described as a cyclin kinase inhibitor, p21Waf1/Cip1 has since been shown to have bimodal effects on cell cycle progression and cell proliferation, and evidence is emerging that intracellular localization of this protein plays a role in directing its signaling properties by dictating its interactions with downstream molecules. Since we have previously demonstrated a pro-apoptotic and cell cycle inhibitory effect of p21 attenuation after transfection of antisense p21 oligodeoxynucleotides (ODN) in several cell lines, we asked whether cytosolic p21 mediates a positive effect on vascular smooth muscle (VSM) cell cycle transit. We now show that transfection of a nuclear-localization signal deficient (ΔNLS) p21 construct into VSM cells results in increased cytosolic levels of p21 and causes increased cell cycle transit as measured by [3H]thymidine incorporation. Thus, at least in VSM cells, cytosolic localization of p21 is a means by which this signaling protein transmits pro-mitogenic signals to the proteins responsible for G1/S transition. Furthermore, compartmentalization of p21 may help explain the biphasic nature of p21 in a variety of cell types and may lead to therapeutic advances directed at modulating pathologic cell growth in vascular diseases and cancer.

Abnormal vascular smooth muscle (VSM) cell proliferation contributes to the development of atherosclerosis and its associated disorders, including angioplasty restenosis. The tumor-suppressor protein p53 has been linked to the development of atherosclerotic lesions, and its homolog, p73, is proving to have contrasting functions in a variety of tissues. As an outgrowth of our previous finding that p73 is increased in serum-stimulated VSM cells and human atherosclerotic tissue, we examined p73 overexpression in VSM cells to elucidate causality of p73 expression with growth response. Overexpression of p73 results in decreased cell cycle transit and is accompanied by apoptosis. The apoptotic changes in p73 overexpressing VSM cells are independent of p53 and are associated with a decrease in levels of p21 waf1/cip1 . In conjunction with our previous data finding that p73 is increased in serum-stimulated VSM cells, this work suggests a role for p73 in vascular proliferative diseases.

Atherosclerosis, in its myriad incarnations the foremost killer disease in the industrialized world, is characterized by aberrant proliferation of vascular smooth muscle (VSM) cells in part as a result of the recruitment of inflammatory cells to the blood vessel wall. The epoxyeicosatrienoic acids are synthesized from arachidonic acid in a reaction catalyzed by the cytochrome P450 system and are vasoactive substances. Metabolism of these compounds by epoxide hydrolases results in the formation of compounds that affect the vasculature in a pleiotropic manner. As an outgrowth of our observations that urea inhibitors of the soluble epoxide hydrolase (sEH) reduce blood pressure in spontaneously hypertensive rats as well as the findings of other investigators that these compounds possess antiinflammatory actions, we have examined the effect of sEH inhibitors on VSM cell proliferation. We now show that the sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) inhibits human VSM cell proliferation in a dose-dependent manner and is associated with a decrease in the level of cyclin D1. In addition, cis-epoxyeicosatrienoic acid mimics the growth-suppressive activity of CDU; there is no evidence of cellular toxicity or apoptosis in CDU-treated cells when incubated with 20 microM CDU for up to 48 h. These results, in light of the antiinflammatory and antihypertensive properties of these compounds that have been demonstrated already, suggest that the urea class of sEH inhibitors may be useful for therapy for diseases such as hypertension and atherosclerosis characterized by exuberant VSM cell proliferation and vascular inflammation.

Under some conditions, p21(Waf1/Cip1) plays an assembly factor role for the cyclins and cyclin-dependent kinases, and recent reports demonstrate that p21 can act as an anti-apoptotic protein. Thus, it is logical to exploit this function of p21 as an anti-cancer target. We have performed a pilot study showing that daily subcutaneous injection of a phosphorothioate antisense p21 oligodeoxynucleotide, which we have previously shown to attenuate p21 levels in vitro, into nude mice who have been implanted with highly metastatic breast cancer cells results in inhibition of tumor growth and angiogenesis. Inhibition of in vitro endothelial capillary formation confirms that these oligodeoxynucleotides have a direct effect upon tumor angiogenesis. The attractiveness of our novel approach to breast cancer therapy, which capitalizes on the anti-apoptotic function of p21, derives from the ease of transfection of antisense oligodeoxynucleotides as well as the observations that p21(-/-) mice do not develop spontaneous tumors, making techniques exploiting the assembly factor and anti-apoptotic role of p21 worthy of further study against breast cancer.

The thiazolidenediones (TZDs) are commonly used to treat hyperglycemia in type 2 diabetes. Diabetes is associated with macrovascular disease, leading to accelerated atherosclerosis caused by aberrant vascular smooth muscle (VSM) cell proliferation. Although VSM cell proliferation is inhibited by the TZDs, the mechanism of this effect has not been established. Because of reports that the cyclin kinase inhibitors (CKIs) p21 Waf1/Cip1 and p27 Kip1 can exhibit both growth-inhibitory and growth-permissive effects in VSM cells, we asked whether alterations in these cell cycle regulatory proteins are the mechanism by which the TZDs inhibit VSM cell growth. We show that platelet-derived growth factor-BB increases p21 and p27 and that this increase is attenuated by TZDs. Surprisingly, when VSM cells were transfected with antisense oligodeoxynucleotides to p21 and p27, inhibition of DNA synthesis by TZDs occurred to the same degree as in control cells. Furthermore, the TZDs have inhibitory effects on cyclin D1 and cyclin E levels, suggesting another mechanism by which these drugs decrease VSM cell growth. These data suggest that the TZD-mediated reduction in CKI levels is not the sole mechanism for their antiproliferative effects. The observed decrease in levels of the G1 cyclins by the TZDs suggests a possible mechanism of VSM cell growth inhibition.

p73 is a newly described homologue of the tumour suppressor p53 that was cloned serendipitously and subsequently shown to possess considerable homology in the most evolutionarily conserved p53 domains. Yet despite the fact that p53 and p73 have extensive structural similarities, their functions are proving to be quite different. We now show that p73 is a growth-regulated protein in the vasculature, being markedly increased in cultured vascular smooth muscle (VSM) cells stimulated with 10% serum, with no significant change in p73 mRNA levels. Stability of p73 is increased after serum stimulation and, probably contributing to this increase in p73 stability, the c-Abl oncogene protein displays a higher molecular weight species and is probably phosphorylated and activated in serum-stimulated VSM cells. The serum-mediated induction of p73 is not altered when the cells are incubated with inhibitors of the MAP/ERK pathway or tyrosine kinases, and is not stimulated by PDGF-BB, demonstrating that the mechanism of the increase in p73 does not involve this classical receptor tyrosine kinase growth factor signalling cascade. p73 is markedly increased in plaque tissue taken from atherosclerotic human carotid arteries, but not in comparable intimal scrapings from normal human arteries. Our data indicate that the tumour suppressor homologue p73 probably plays a role in VSM cell cycle progression, being mediated by a specific, as yet unidentified, serum component, and identifies a new function for this protein as being important in the pathogenesis of human atherosclerosis as well as other vascular diseases.

The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet-derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation.

Growth factor-stimulated DNA synthesis in a variety of cell lines has been shown to be decreased after overnight (or longer) treatment with the 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, the statins. Although this anti-mitogenic effect had been presumed to be the result of the impairment of Ras lipidation, a stable modification (T1/2 approximately 20 h), this study provides new data demonstrating that brief (approximately 1 h) pretreatment of rat vascular smooth muscle cells with 100 microM pravastatin before platelet-derived growth factor-BB (PDGF-BB) stimulation results in attenuation of DNA synthesis through a Ras-independent mechanism. PDGF-BB-stimulated PDGF-beta receptor tyrosine phosphorylation, Ras activity, and mitogen-activated protein/extracellular signal-regulated kinase activity are unaffected by from 10 min to 1 h of pravastatin incubation, while Raf activity is markedly increased after 1 h of pravastatin. Phosphatidylinositol-3 kinase activity and phosphorylation of its downstream effector Akt are decreased after 1 h pravastatin incubation. Rho is stabilized by pravastatin, and ADP-ribosylation of Rho by C3 exoenzyme decreases PDGF-stimulated phosphatidylinositol-3 kinase activity, mimicking the effect of pravastatin on this signaling protein. Levels of the cyclin-dependent kinase inhibitor p27Kip1 are increased when cells were preincubated with pravastatin for 1 h and then exposed to PDGF, and apoptosis is induced by pravastatin incubation times as short as 1 to 4 h. Thus, short-term, high-dose pravastatin inhibits vascular smooth muscle cell growth and induces apoptosis independently of Ras, likely by means of the drug's effect on p27Kip1, mediated by Rho and/or phosphatidylinositol-3 kinase. This work demonstrates for the first time that the statins may be therapeutically useful when applied for short periods of time such that potential toxicity of long-term statin use (such as chronic Ras inhibition) may be avoided, suggesting future therapeutic directions for statin research.

Iron(III) complexes with four pentaaza macrocylic ligands, [Fe(L)Cl-2](PF6), have been synthesized, where the ligand L is 1,4,7,10,13-pentaazacyclopentadecane (L-1), 2R,3R,8S,9S-dicyclohexano-1,4,7,10,13-pentaazacyclopentadecane L-2), 2R,3R,8R,9R-dicyclohexano-1,4,7,10,13-pentaazacyclopentadecane (L-3), or 2S,5R,8S,11R,14S-pentamethyl-1,4,7,10,13-pentaazacyclopentadecane (L-4), respectively. Conductivity measurements in acetonitrile are consistent with two chloro anions coordinated to iron(III). In acetonitrile solution, all four iron complexes exhibit a reversible or quasi-reversible redox couple in the experimental range -1.5 to +1.5 V vs EAg/Ag+, and the redox potentials for those four complexes are similar (from -0.15 to -0.19 V vs NHE). In aqueous solutions, the electrochemical properties of those complexes are different from these in acetonitrile solution; the redox peaks shift more than 0.5 V more positive. The complexes with L-1, L-2, and L-3 display a reversible redox at 0.35, 0.45, and 0.43 V vs NHE, respectively, while [Fe(L-4)Cl-2](PF6) shows a cathodic peak at 0.44 V and two anodic peaks at 0.31 and 0.14 V vs NHE, respectively. The base titration results reveal that two water molecules are coordinated to the iron(III) in these complexes, pK(a1) and pK(a2): 3.46(7) and 7.31(7) for [Fe(L-1)Cl-2](PF6); 3.7(1) and 7.50(2) for [Fe(L-2)Cl-2](PF6); 4.1(1) and 7.73(2) for [Fe(L-3)Cl-2](PF6); 3.6(7) and 7.4(2) for [Fe(L-4)Cl-2](PF6). The superoxide dismutase (SOD) activity, assessed by stopped-flow experiments, reveals that all four metal complexes catalyze the fast disproportionation of superoxide in aqueous solution: at pH = 7.8, the catalyic rate constants for the complexes were 0.81 x 10(7), 1.42 x 10(7), 1.41 x 10(7), and 0.29 x 10(7) M-1 s(-1) for [Fe(L-1)Cl-2](PF6), [Fe(L-2)Cl-2](PF6), [Fe(L-3)Cl-2](PF6), and [Fe(L-4)Cl-2](PF6), respectively. The crystal structure of [Fe(L-2)Cl-2](PF6) was determined. Crystal data: triclinic,; a = 7.367(2), b = 10.707(2), c = 17.632(2) Angstrom; alpha = 81.27(2), beta = 79.74(2), gamma = 83.38(2)degrees; R = 0.0465 for observed data (I > 2 sigma(I)). The iron is seven-coordinate with five nitrogen atoms from the pentaaza macrocylic ligand and trans-chloro anions in a pentagonal bipyramidal arrangement. The average bond lengths for Fe-N and Fe-Cl are 2.271 and 2.339 Angstrom, respectively.

Background. Glomerulonephritis is characterized by the accumulation of extracellular matrix protein within the glomerulus. This process, when allowed to proceed unimpeded, leads to glomerulosclerosis and eventually to cessation of glomerular filtration. There is evidence that protein kinase C (PKC) activation plays an important role in mediating at least some of the effects of TGF-beta in vascular smooth-muscle cells. The current study was undertaken to determine whether PKC activity is required for both TGF-beta and angiotensin II (Ang II) to induce mesangial cell matrix protein secretion.Methods. PKC was inhibited by two separate methods, and [H-3]thymidine incorporation was assessed in both the presence and the absence of PKC inhibition. Conditioned medium from cells stimulated with TGF-beta or Ang II was collected and analysed for secreted matrix proteins and sulphated proteins by SDS-polyacrylamide gel electrophoresis and western blotting.Results. Twenty-four-hour incubation of rat mesangial cells with phorbol-12-myristate-13-acetate (PMA) reduced total PKC activity to basal levels. Both TGF-beta and Ang II were mitogenic in mesangial cells, and chronic PMA pre-incubation inhibited this DNA synthesis. TGF-beta- and Ang-II-induced sulphated protein secretion into conditioned medium was markedly attenuated in PKC-downregulated cells. Secretion of the specific matrix proteins laminin and fibronectin by mesangial cells stimulated with either TGF-beta or Ang II was also diminished in PKC-downregulated cells and in cells pre-incubated with the specific PKC inhibitor, chelerythrine. There was no evidence of generalized cell toxicity or decreased non-specific protein synthesis caused by these PKC inhibitors.Conclusions, PKC is a key intermediary in the process by which TGF-beta and Ang II cause DNA synthesis and mesangial cell matrix protein production. Thus, PKC inhibitors deserve further study as potential therapeutic agents for a variety of glomerular diseases.

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.

Two new isomeric bis(trans-fused cyclohexano) substituted 1,4,7,10,13-pentaazacyclopentadecane ligands and their Mn(II) complexes, 3 and 14, have been synthesized, and their activity as superoxide dismutase (SOD) catalysts has been studied. Complex 3 is an excellent SOD catalyst with a second-order rate constant at pH 7.4 of 1.2 x 10(+8) M-1 s(-1). In contrast, the isomeric complex 4 has virtually no detectable catalytic SOD activity, implying the need to understand the effect that the position, number, and stereochemistry of substituents exert on the catalytic rate. The crystal structure of the complex 4 was determined and reveals that the Mn(II) ion is coordinated in a pentagonal bipyramid array of the dye nitrogens of the macrocyclic ligand and capped by two trans-chloro ligands. Crystal data for MnC18H37Cl2N5 are as follows: triclinic at 20 degrees C, space group P-1-C(i)2 (no. 2); a = 9.746(3) Angstrom, b = 12.631(6) Angstrom, c = 11.311(5) Angstrom; alpha = 73.14(4)degrees, beta = 76.39(3)degrees, gamma = 79.98(3)degrees, V = 1287(1) Angstrom(3), and Z = 2 (rho(calc) = 1.279 g/cm(3); mu(2) Mo K-alpha = 6.23 mm(-1)). Mechanistic studies with the complex 3 and the pentamethyl susbstituted complex, 5, including D2O rate studies, are reported and are consistent with the existence of two pathways for the rate-determining electron-transfer from Mn(II) to superoxide: (1) hydrogen atom transfer from a bound water on Mn(IP) to HO2. to yield a Mn(III) hydroxo intermediate and (2) the dissociative pathway in which superoxide anion binds to a vacant coordination site on Mn(II) followed by protonation/oxidation to yield a Mn(III)hydroperoxo species. Subsequent reduction of the intermediate Mn(III) with superoxide anion completes the catalytic cycle. Substituent effects on the rates and relative contribution of the two pathways to the overall rate of SOD activity is ascribed to the propensity of the ligand to fold around Mn(II) forming a pseudo-octahedral complex similar in geometry to the oxidized Mn(III) complex. Folding of the pentaaza macrocyclic ligand is confirmed as a relevant structural motif for this series of Mn(TI) complexes by the X-ray structure determination of the bis(nitrate) derivative of 1, [Mn(C10H25N5)NO3]NO3, which reveals a six-coordinate structure with a folded conformation of the macrocyclic ligand. Crystal data for [Mn(C10H25N5)NO3]NO3: orthorhombic at -100 degrees C, space group P2(1)2(1)2(1); a = 9.457(2) Angstrom, b = 12.758(2) Angstrom, c = 13.834(2) Angstrom, V = 1669.1(5) Angstrom(3), and Z = 4 (rho(calc) = 1.549 g/cm(3)).

The synthesis of a bis-cyclohexyl-fused pentaazamacrocycle via head-to-tail pseudopeptide cyclization chemistry is described. The bis-cyclohexyl-fused cyclic pseudopeptide 11 displays intramolecular hydrogen bonding which has been characterized by variable temperature NMR spectroscopy and X-Ray Crystallography.