Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar anti proliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.

Synthesis and cytotoxicity studies of new dimethylamino-functionalised and aryl-substituted titanocene anti-cancer agents

From the carbolithiation of 6-N,N-dimethylamino fulvene (3a) and different lithiated aryl species [p-N,N-dimethylanilinyl lithium, p-anisyl lithium and 4-lithio-benzo[1.3]dioxole (2a-c)], the corresponding lithium cyclopentadienide intermediates 4a-c were formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl4 resulting in dimethylamino-functionalised and aryl-substituted titanocenes 5a-c. When these titanocenes were tested against LLC-PK cells, the IC50 values obtained were of 54, 45 and 26 mu M for titanocenes 5a, b and c, respectively. The most cytotoxic titanocene in this paper, 5c is approximately 10 times less cytotoxic than cis-platin, which showed an IC50 value of 3.3 mu M, when tested on the LLC-PK cell line, but approximately 100 times better than titanocene dichloride. (C) 2007 Elsevier Masson SAS. All rights reserved.

The integration of lipid-sensing and anti-inflammatory effects: how the PPARs play a role in metabolic balance

The peroxisomal proliferating-activated receptors (PPARs) are lipid-sensing transcription factors that have a role in embryonic development, but are primarily known for modulating energy metabolism, lipid storage, and transport, as well as inflammation and wound healing. Currently, there is no consensus as to the overall combined function of PPARs and why they evolved. We hypothesize that the PPARs had to evolve to integrate lipid storage and burning with the ability to reduce oxidative stress, as energy storage is essential for survival and resistance to injury/infection, but the latter increases oxidative stress and may reduce median survival (functional longevity). In a sense, PPARs may be an evolutionary solution to something we call the 'hypoxia-lipid' conundrum, where the ability to store and burn fat is essential for survival, but is a 'double-edged sword', as fats are potentially highly toxic. Ways in which PPARs may reduce oxidative stress involve modulation of mitochondrial uncoupling protein (UCP) expression (thus reducing reactive oxygen species, ROS), optimising forkhead box class O factor (FOXO) activity (by improving whole body insulin sensitivity) and suppressing NFkB (at the transcriptional level). In light of this, we therefore postulate that inflammation-induced PPAR downregulation engenders many of the signs and symptoms of the metabolic syndrome, which shares many features with the acute phase response (APR) and is the opposite of the phenotype associated with calorie restriction and high FOXO activity. In genetically susceptible individuals (displaying the naturally mildly insulin resistant 'thrifty genotype'), suboptimal PPAR activity may follow an exaggerated but natural adipose tissue-related inflammatory signal induced by excessive calories and reduced physical activity, which normally couples energy storage with the ability to mount an immune response. This is further worsened when pancreatic decompensation occurs, resulting in gluco-oxidative stress and lipotoxicity, increased inflammatory insulin resistance and oxidative stress. Reactivating PPARs may restore a metabolic balance and help to adapt the phenotype to a modern lifestyle.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous alpha,beta-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Future innovations in anti-platelet therapies

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed.

The neurokinin 1 receptor: a potential new target for anti-platelet therapy?

The important role of platelets in the development of arterial thrombosis and cardiovascular disease is well established. Current treatments for arterial thrombosis include anti-platelet agents such as aspirin, thienopyridines and glycoprotein IIb-IIIa inhibitors. Despite these drugs being effective there remains a substantial unmet clinical demand for more effective therapeutic approaches, which may reflect the existence of alternative underlying regulatory mechanisms to those already targeted. Recent publications have demonstrated a key role for tachykinins in the positive feedback regulation of platelet aggregation and thrombus formation. The pro-thrombotic effects of tachykinins on platelets are mediated through the neurokinin 1 receptor, which may therefore offer a novel therapeutic drug target in the prevention and the treatment of arterial thrombosis.

Oxidation-responsiveness of nanomaterials for targeting inflammatory reactions

Oxidation is an almost ubiquitous feature of inflammatory reactions. We discuss the development of nanocarriers that respond to the presence of oxidants with profound physical reorganization, which could in perspective allow their use for delivering anti-inflammatory principles in an inflammation-responsive fashion. We also present a study demonstrating that the response of polysulfide nanoparticles has a bulk character, i.e., the odixation reactions happen homogeneously throughout the nanoparticles, and not interfacially.

The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury

Neuroinflammation plays an integral role in the progression of neurodegeneration. In this study we investigated the anti-inflammatory effects of different classes of flavonoids (flavanones, flavanols and anthocyanidins) in primary mixed glial cells. We found that the flavanones naringenin and hesperetin and the flavols (+)-catechin and (-)-epicatechin, but not the anthocyanidins cyanidin and pelargonidin, attenuated LPS/IFN-gamma-induced TNF-alpha production in glial cells. Naringenin also inhibited LPS/IFN-gamma-induced iNOS expression and nitric oxide production in glial cells, thus showing the strongest antiinflammatory activity among all flavonoids tested. Moreover, naringenin protected against inflammatory-induced neuronal death in a primary neuronal-glial co-culture system. Naringenin also inhibited LPS/IFN-gamma-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and downstream signal transducer and activator of transcription-1 (STAT-1) in LPS/IFN-gamma stimulated primary mixed glial cells. Taken together, our results suggest that naringenin may produce an anti-inflammatory effect in LPS/IFN-gamma stimulated glial cells that may be due to its interaction with p38 signalling cascades and the STAT-I trascription factor. (C) 2009 Elseiver Inc. All rights reserved.

Insights into dietary flavonoids as molecular templates for the design of anti-platelet drugs

Flavonoids are low-molecular weight, aromatic compounds derived from fruits, vegetables, and other plant components. The consumption of these phytochemicals has been reported to be associated with reduced cardiovascular disease (CVD) risk, attributed to their anti-inflammatory, anti-proliferative, and anti-thrombotic actions. Flavonoids exert these effects by a number of mechanisms which include attenuation of kinase activity mediated at the cell-receptor level and/or within cells, and are characterized as broad-spectrum kinase inhibitors. Therefore, flavonoid therapy for CVD is potentially complex; the use of these compounds as molecular templates for the design of selective and potent small-molecule inhibitors may be a simpler approach to treat this condition. Flavonoids as templates for drug design are, however, poorly exploited despite the development of analogues based on the flavonol, isoflavonone, and isoflavanone subgroups. Further exploitation of this family of compounds is warranted due to a structural diversity that presents great scope for creating novel kinase inhibitors. The use of computational methodologies to define the flavonoid pharmacophore together with biological investigations of their effects on kinase activity, in appropriate cellular systems, is the current approach to characterize key structural features that will inform drug design. This focussed review highlights the potential of flavonoids to guide the design of clinically safer, more selective, and potent small-molecule inhibitors of cell signalling, applicable to anti-platelet therapy.

Sesquiterpenoids lactones: benefits to plants and people

Sesquiterpenoids, and specifically sesquiterpene lactones from Asteraceae, may play a highly significant role in human health, both as part of a balanced diet and as pharmaceutical agents, due to their potential for the treatment of cardiovascular disease and cancer. This review highlights the role of sesquiterpene lactones endogenously in the plants that produce them, and explores mechanisms by which they interact in animal and human consumers of these plants. Several mechanisms are proposed for the reduction of inflammation and tumorigenesis at potentially achievable levels in humans. Plants can be classified by their specific array of produced sesquiterpene lactones, showing high levels of translational control. Studies of folk medicines implicate sesquiterpene lactones as the active ingredient in many treatments for other ailments such as diarrhea, burns, influenza, and neurodegradation. In addition to the anti-inflammatory response, sesquiterpene lactones have been found to sensitize tumor cells to conventional drug treatments. This review explores the varied ecological roles of sesquiterpenes in the plant producer, depending upon the plant and the compound. These include allelopathy with other plants, insects, and microbes, thereby causing behavioural or developmental modification to these secondary organisms to the benefit of the sesquiterpenoid producer. Some sesquiterpenoid lactones are antimicrobial, disrupting the cell wall of fungi and invasive bacteria, whereas others protect the plant from environmental stresses that would otherwise cause oxidative damage. Many of the compounds are effective due to their bitter flavor, which has obvious implications for human consumers. The implications of sesquiterpenoid lactone qualitiesfor future crop production are discussed.

Inflammatory modulation of exercise salience: using hormesis to return to a healthy lifestyle

Most of the human population in the western world has access to unlimited calories and leads an increasingly sedentary lifestyle. The propensity to undertake voluntary exercise or indulge in spontaneous physical exercise, which might be termed "exercise salience", is drawing increased scientific attention. Despite its genetic aspects, this complex behaviour is clearly modulated by the environment and influenced by physiological states. Inflammation is often overlooked as one of these conditions even though it is known to induce a state of reduced mobility. Chronic subclinical inflammation is associated with the metabolic syndrome; a largely lifestyle-induced disease which can lead to decreased exercise salience. The result is a vicious cycle that increases oxidative stress and reduces metabolic flexibility and perpetuates the disease state. In contrast, hormetic stimuli can induce an anti-inflammatory phenotype, thereby enhancing exercise salience, leading to greater biological fitness and improved functional longevity. One general consequence of hormesis is upregulation of mitochondrial function and resistance to oxidative stress. Examples of hormetic factors include calorie restriction, extreme environmental temperatures, physical activity and polyphenols. The hormetic modulation of inflammation, and thus, exercise salience, may help to explain the highly heterogeneous expression of voluntary exercise behaviour and therefore body composition phenotypes of humans living in similar obesogenic environments.

Novel synthesised flavone derivatives provide significant insight into the structural features required for enhanced anti-proliferative activity

With many cancers showing resistance to current chemotherapies, the search for novel anti-cancer agents is attracting considerable attention. Natural flavonoids have been identified as useful leads in such programmes. However, since an in-depth understanding of the structural requirements for optimum activity is generally lacking, further research is required before the full potential of flavonoids as anti-proliferative agents can be realised. Herein a broad library of 76 methoxy and hydroxy flavones, and their 4-thio analogues, was constructed and their structure-activity relationships for anti-proliferative activity against the breast cancer cell lines MCF-7 (ER+ve), MCF-7/DX (ER+ve, anthracycline resistant) and MDA-MB-231 (ER-ve) were probed. Within this library, 42 compounds were novel, and all compounds were afforded in good yields and > 95% purity. The most promising lead compounds, specifically the novel hydroxy 4-thioflavones 15f and 16f, were further evaluated for their anti-proliferative activities against a broader range of cancer cell lines by the National Cancer Institute (NCI), USA and displayed significant growth inhibition profiles (e.g Compound-15f: MCF-7 (GI50 = 0.18 μM), T-47D (GI50 = 0.03 μM) and MDA-MB-468 (GI50 = 0.47 μM) and compound-16f: MCF-7 (GI50 = 1.46 μM), T-47D (GI50 = 1.27 μM) and MDA-MB-231 (GI50 = 1.81 μM). Overall, 15f and 16f exhibited 7-46 fold greater anti-proliferative potency than the natural flavone chrysin (2d). A systematic structure-activity relationship study against the breast cancer cell lines highlighted that free hydroxyl groups and the B-ring phenyl groups were essential for enhanced anti-proliferative activities. Substitution of the 4-C=O functionality with a 4-C=S functionality, and incorporation of electron withdrawing groups at C4’ of the B-ring phenyl, also enhanced activity. Molecular docking and mechanistic studies suggest that the anti-proliferative effects of flavones 15f and 16f are mediated via ER-independent cleavage of PARP and downregulation of GSK-3β for MCF-7 and MCF-7/DX cell lines. For the MDA-MB-231 cell line, restoration of the wild-type p53 DNA binding activity of mutant p53 tumour suppressor gene was indicated.