Induction of macrophage elastase (MMP-12) gene expression by statins

The statins (including mevastatin and lovastatin) are a widely prescribed class of serum-cholesterol lowering drugs that function by inhibiting 3-hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity and cellular sterol synthesis. Statins are also widely being appreciated for their inhibitory effects upon inflammation, primarily mediated through direct regulation of inflammatory gene expression. Here we report that statins are also capable of increasing the expression of macrophage elastase (MMP-12). The induction of MMP-12 in mouse macrophages by statins is specific for HMG CoA reductase inhibition, rescued by mevalonate and not observed after inhibition of subsequent steps in the cholesterol biosynthetic pathway. Modulation of cholesterol metabolism may lead to changes in MMP-12 expression and subsequent impacts during physiological and pathophysiological states. We conclude that statins, in addition to their previously described anti-inflammatory properties, may promote the production of some proteinases from activated macrophages.     

Therapeutic levels of the hydroxmethylglutaryl-coenzyme A reductase inhibitor lovastatin activate ras signaling via phospholipase D2

Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 μM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target.     

Effects of cyclooxygenase inhibition on canine coronary artery blood flow and thrombosis

This study was designed to determine the effect of inhibitors of cyclooxygenase (COX)-1, COX-2, and the nonselective COX inhibitor naproxen on coronary vasoactivity and thrombogenicity under baseline and lipopolysaccharide (LPS)-induced inflammatory conditions. We hypothesize that endothelial COX-1 is the primary COX isoform in the canine normal coronary artery, which mediates arachidonic acid (AA)-induced vasodilatation. However, COX-2 can be induced and overexpressed by inflammatory mediators and becomes the major local COX isoform responsible for the production of antithrombotic prostaglandins during systemic inflammation. The interventions included the selective COX-1 inhibitor SC-560 (0.3 mg/kg iv), the selective COX-2 inhibitor nimesulide (5 mg/kg iv), or the nonselective COX inhibitor naproxen (3 mg/kg iv). The selective prostacyclin (IP) receptor antagonist RO-3244794 (RO) was used as an investigational tool to delineate the role of prostacyclin (PGI(2)) in modulating vascular reactivity. AA-induced vasodilatation of the left circumflex coronary artery was suppressed to a similar extent by each of the COX inhibitors and RO. The data suggest that AA-induced vasodilatation in the normal coronary artery is mediated by a single COX isoform, the constitutive endothelial COX-1, which is reported to be susceptible to COX-2 inhibitors. The effect of the COX inhibitors on thrombus formation was evaluated in a model of carotid artery thrombosis secondary to electrolytic-induced vessel wall injury. Pretreatment with LPS (0.5 mg/kg iv) induced a systemic inflammatory response and prolonged the time-to-occlusive thrombus formation, which was reduced in the LPS-treated animals by the administration of nimesulide. In contrast, neither SC-560 nor naproxen influenced the time to thrombosis in the animals pretreated with LPS. The data are of significance in view of reported adverse cardiovascular events observed in clinical trials involving the use of selective COX-2 inhibitors, thereby suggesting that the endothelial constitutive COX-1 and the inducible vascular COX-2 serve important functions in maintaining vascular homeostasis.     

Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductase

Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose‐ and time‐dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase‐8 and ‐9; BID cleavage, cytochrome C release and PARP cleavage. Statin‐sensitive cancers expressed high levels of HMG‐CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG‐CoA reductase since mevalonate pre‐incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG‐CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies.     

The effect of substrates and competitive inhibitors on the phosphatase-dependent activation of hepatic hydroxymethylglutaryl CoA reductase

Previous studies have demonstrated that the in vitro activation of microsomal hepatic hydroxymethylglutaryl (HMG) CoA reductase by dephosphorylation is inhibited by HMG CoA or NADPH, the substrates of HMG CoA reductase (13). In the present study the effect of three competitive inhibitors of HMG CoA reductase on the activation of HMG CoA reductase was investigated. Adenosine-2'-monophospho-5'-diphosphoribose, a competitive inhibitor for the NADPH binding site, blocked the phosphatase-mediated activation of HMG CoA reductase. By contrast, neither compactin nor mevinolin, competitive inhibitors for the HMG CoA binding site, altered the activation of HMG CoA reductase. Moreover, the HMG CoA-mediated inhibition of the activation of HMG CoA reductase was not blocked even by very high concentrations of either compactin or mevinolin. These observations suggest that HMG CoA can bind to two sites on HMG CoA reductase. One site of HMG CoA binding serves as a catalytic site and is competitively blocked by compactin or mevinolin, and the second binding site is an allosteric site to which only HMG CoA is capable of binding. The binding of HMG CoA to this second site inhibits the activation of HMG CoA reductase by phosphatases.     

Mevalonate-suppressive dietary isoprenoids for bone health

Osteoclastogenesis and osteoblastogenesis, the balancing acts for optimal bone health, are under the regulation of small guanosine triphosphate-binding proteins (GTPases) including Ras, Rac, Rho and Rab. The activities of GTPases require post-translational modification with mevalonate-derived prenyl pyrophosphates. Mevalonate deprivation induced by competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (e.g., statins) prevents the activation of GTPases, suppresses the expression of the receptor for activation of nuclear factor kappa B (NFκB) ligand (RANKL) and activation of NFκB and, consequently, inhibits osteoclast differentiation and induces osteoclast apoptosis. In contrast, statin-mediated inactivation of GTPases enhances alkaline phosphatase activity and the expression of bone morphogenetic protein-2, vascular epithelial growth factor, and osteocalcin in osteoblasts and induces osteoblast proliferation and differentiation. Animal studies show that statins inhibit bone resorption and increase bone formation. The anabolic effect of statins and other mevalonate pathway-suppressive pharmaceuticals resembles the anti-osteoclastogenic and bone-protective activities conferred by dietary isoprenoids, secondary products of plant mevalonate metabolism. The tocotrienols, vitamin E molecules with HMG CoA reductase-suppressive activity, induce mevalonate deprivation and concomitantly suppress the expression of RANKL and cyclooxygenase-2, the production of prostaglandin E2 and the activation of NFκB. Accordingly, tocotrienols inhibit osteoclast differentiation and induce osteoclast apoptosis, impacts reminiscent of those of statins. In vivo studies confirm the bone protective activity of tocotrienols at nontoxic doses. Blends of tocotrienols, statins and isoprenoids widely found in fruits, vegetables, grains, herbs, spices, and essential oils may synergistically suppress osteoclastogenesis while promoting osteoblastogenesis, offering a novel approach to bone health that warrants clinical studies.     

Effect of pitavastatin on apolipoprotein A-I production in HepG2 cell

There are few reports describing the mechanism of HDL-elevating action of HMG-CoA reductase inhibitors (statins). As it is considered that the key step of HDL production is the secretion of apolipoprotein A-I (apoA-I), we investigated the effect of statins on apoA-I synthesis and secretion by HepG2 cell to elucidate the mechanism of the action. Each statin induced apoA-I expression (mRNA and protein) dose-dependently: the rank order of the apoA-I induction pitavastatin (3 μM) > simvastatin (10 μM) > atorvastatin (50 μM). The induction of apoA-I by statins disappeared with addition of mevalonate, which indicates that the effect is HMG-CoA reductase inhibition-dependent. Based on HMG-CoA reductase inhibition, pitavastatin-induced apoA-I more efficiently than simvastatin and atorvastatin. Further study revealed that pitavastatin increased ABCA1 mRNA in HMG-CoA reductase-dependent manner and that Rho and Rho kinase inhibitor (C3T and Y27632) increased apoA-I production in the HepG2 cells. These results suggest that pitavastatin efficiently increases apoA-I in the culture medium of HepG2 cells by promoting apoA-I production through inhibition of HMG-CoA reductase and suppression of Rho activity and by protecting apoA-I from catabolism through ABCA1 induction and lipidation of apoA-I.     

Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells

AMP-activated protein kinase (AMPK) is a key energy sensor, known to regulate energy metabolism in diverse cell types. Hypoxia is encountered frequently in the microenvironments of inflammatory lesions and is a critical regulator of function in inflammatory cells. Energy deficiency is one of the consequences of hypoxia, but its potential role in modulating leucocyte function has received little attention. Using micropore chemotaxis assays to assess migratory responses of the monocyte-like cell line U937, it was found that the AMPK activators AICAR and phenformin rapidly reduced random migration (chemokinesis) as well as chemotaxis due to stromal cell-derived factor (SDF)1alpha. There was an approximate 50% reduction in both chemokinesis and chemotaxis following 30 min preincubation with both AICAR and phenformin (P < 0.01), and this continued with up to 24 h preincubation. The binding of SDF1alpha to its receptor CXCR4 was unaltered, suggesting AMPK was acting on downstream intracellular signalling pathways important in cell migration. As AMPK and statins are known to inhibit HMG CoA reductase, and both reduce cell migration, the effect of mevastatin on U937 cells was compared with AMPK activators. Mevastatin inhibited cell migration but required 24 h preincubation. As expected, the inhibitory effect of mevastatin was associated with altered subcellular localization of the Rho GTPases, RhoA and cdc42, indicating decreased prenylation of these molecules. Although the effect of AMPK activation was partially reversed by mevalonate, this was not associated with altered subcellular localization of Rho GTPases. The data suggest that activation of AMPK has a general effect on cell movement in U937 cells, and this is not due to inhibition of HMG CoA reductase. These are the first data to show an effect of AMPK on cell movement, and suggest a fundamental role for energy deficiency in regulating cellular behaviour.     

Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.     

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.     

The role of selective cyclooxygenase isoforms in human intestinal smooth muscle cell stimulated prostanoid formation and proliferation.

Intestinal smooth muscle plays a major role in the repair of injured intestine and contributes to the prostanoid pool during intestinal inflammatory states. Cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostanoids exists in two isoforms, COX-1 and COX-2. The purpose of this study was to determine the relative contributions of COX-1 and COX-2 in the production of prostanoids by human intestinal smooth muscle (HISM) cells when stimulated by interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS). Furthermore the effects of specific COX-1 and COX-2 inhibitors on the proliferation of smooth muscle cells was also evaluated. Confluent monolayer cultures of HISM cells were incubated with IL-1beta or LPS for 0-24h while control cells received medium alone. PGE2 and PGI2 as 6-keto-PGF1alpha and LTB4 were measured by a specific radioimmunoassay. COX enzymes were evaluated by Western immunoblotting. Unstimulated and stimulated cells were exposed to the specific COX-1 inhibitor valerylsalicylic acid (VSA) and the COX-2 inhibitors NS-398 and SC-58125. The effects of serum on proliferation were then evaluated in the presence of each of the specific COX inhibitors by incorporation of 3H-thymidine into DNA. IL-1beta and LPS increased both PGE2 and 6-keto-PGF1alpha in a dose dependent fashion with enhanced production detected two hours following exposure. Neither stimulus stimulated LTB4 release. Immunoblot analysis using isoform-specific antibodies showed that both COX-1 and COX-2 were present constitutively. Furthermore, COX-1 was upregulated by each inflammatory stimulus. In a separate set of experiments cells were pretreated with either the selective COX-1 inhibitor VSA or the selective COX-2 inhibitors NS-398 or SC-58125 prior to treatment with IL-1beta or LPS. The COX-1 and COX-2 inhibitors decreased both basal and IL-1beta and LPS stimulated prostanoid release. Spontaneous DNA synthesis was present and serum consistently increased proliferation. 3H-thymidine incorporation, stimulated by serum, was inhibited by both COX-1 and COX-2 inhibitors. This study suggests that the prostanoid response stimulated by proinflammatory agents of gut-derived smooth muscle cells appears to be mediated by both COX-1 and COX-2 enzymes. Proliferation of smooth muscles cells also appears to be influenced by both COX-1 and COX-2.     

Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 μM of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-α-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 μM), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.