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Endothelin-1 (ET-1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET-1-mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho-Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST-1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). ET-1 treatment of human VSMCs resulted in an increase in phospho-Smad2L level. The TGF-β receptor antagonist, SB431542 and the mixed ET

Cardiovascular disease is one of the major contributors to global disease burden. Atherosclerosis is an inflammatory process that involves the accumulation of lipids and fibrous elements in the large arteries, forming an atherosclerotic plaque. Rupture of unstable plaques leads to thrombosis that triggers life-threatening complications such as myocardial infarction. Current diagnostic methods are invasive as they require insertion of a catheter into the coronary artery. Molecular imaging techniques, such as magnetic resonance imaging, have been developed to image atherosclerotic plaques and thrombosis due to its high spatial resolution and safety. The sensitivity of magnetic resonance imaging can be improved with contrast agents, such as iron oxide nanoparticles. This review presents the most recent advances in atherosclerosis, thrombosis, and myocardial infarction molecular imaging using iron oxide-based nanoparticles. While some studies have shown their effectiveness, many are yet to undertake comprehensive testing of biocompatibility. There are still potential hazards to address and complications to diagnosis, therefore strategies for overcoming these challenges are required.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are new oral drugs for the therapy of patients with type 2 diabetes mellitus (T2DM). Research in the past decade has shown that drugs of the SGLT2i class, such as empagliflozin, canagliflozin, and dapagliflozin, have pleiotropic effects in preventing cardiovascular diseases beyond their favorable impact on hyperglycemia. Of clinical relevance, recent landmark cardiovascular outcome trials have demonstrated that SGLT2i reduce major adverse cardiovascular events, hospitalization for heart failure, and cardiovascular death in T2DM patients with/without cardiovascular diseases (including atherosclerotic cardiovascular diseases and various types of heart failure). The major pharmacological action of SGLT2i is through inhibiting glucose re-absorption in the kidney and thus promoting glucose excretion. Studies in experimental models of atherosclerosis have shown that SGLT2i ameliorate the progression of atherosclerosis by mechanisms including inhibition of vascular inflammation, reduction in oxidative stress, reversing endothelial dysfunction, reducing foam cell formation and preventing platelet activation. Here, we summarize the anti-atherosclerotic actions and mechanisms of action of SGLT2i, with an aim to emphasize the clinical utility of this class of agents in preventing the insidious cardiovascular complications accompanying diabetes.

[Image: see text] Toll-like receptors (TLRs) are dominant components of the innate immune system. Activated by both pathogen-associated molecular patterns and damage-associated molecular patterns, TLRs underpin the pathology of numerous inflammation related diseases that include not only immune diseases, but also cardiovascular disease (CVD), diabetes, obesity, and cancers. Growing evidence has demonstrated that TLRs are involved in multiple cardiovascular pathophysiologies, such as atherosclerosis and hypertension. Specifically, a trial called the Canakinumab Anti-inflammatory Thrombosis Outcomes Study showed the use of an antibody that neutralizes interleukin-1β, reduces the recurrence of cardiovascular events, demonstrating inflammation as a therapeutic target and also the research value of targeting the TLR system in CVD. In this review, we provide an update of the interplay between TLR signaling, inflammatory mediators, and atherothrombosis, with an aim to identify new therapeutic targets for atherothrombotic CVD.

Transforming growth factor (TGF)-β signalling commences with the engagement of TGF-β ligand to cell surface TGF-β receptors (TGFBR) stimulating Smad2 carboxyl-terminal phosphorylation (phospho-Smad2C) and downstream biological responses. In several cell models, G protein-coupled receptors (GPCRs) transactivate the TGF-β receptors type-1 (TGFBR1) leading to phospho-Smad2C, however, we have recently published that in keratinocytes thrombin did not transactivate the TGFBR1. The bulk of TGFBRs reside in the cytosol and in response to protein kinase B (Akt phosphorylation) can translocate to the cell surface increasing the cell's responsiveness to TGF-β. In this study, we investigate the role of Akt in GPCR transactivation of the TGFBR1. We demonstrate that angiotensin II and thrombin do not phosphorylate Smad2C in human vascular smooth muscle cells and in keratinocytes respectively. We used Akt agonist, SC79 to sensitise the cells to Akt and observed that Ang II and thrombin phosphorylate Smad2C via Akt/AS160-dependent pathways. We show that SC79 rapidly translocates TGFBRs to the cell surface thus increasing the cell's response to the GPCR agonist. These findings highlight novel mechanistic insight for the role of Akt in GPCR transactivation of the TGFBR1.

Toll-like receptors (TLRs) recognise pathogen‑associated molecular patterns, which allow the detection of microbial infection by host cells. Bacterial derived toxin lipopolysaccharide activates TLR4 and leads to the activation of the Smad2 transcription factor. The phosphorylation of the Smad2 transcription factor is the result of the activation of the transforming growth factor-β receptor 1 (TGFBR1). Therefore, we sought to investigate LPS via TLR4 mediated Smad2C phosphorylation dependent on the (trans)activation of the TGFBR1. The invitro used human aortic vascular smooth muscle cells (HA-VSMCs) to assess the implications of TLR4 (trans)activation of the TGFBR1 in vascular pathophysiology. We show that LPS mediated Smad2 carboxy-terminal phosphorylation is inhibited in the presence of TGFBR1 inhibitor SB431542 in HA-VSMCs. Treatment with MyD88 and TRIF pathways antagonists does not affect LPS mediated phosphorylation of Smad2C; however, LPS mediated Smad2 phosphorylation was inhibited in the presence of MMP inhibitor, GM6001 and unaffected in the presence of ROCK inhibitor Y27632. LPS via transactivation of the TGFBR1 stimulates PAI-1 mRNA expression. TLRs are first in line to respond to exogenous invading substances and endogenous molecules; our findings characterise a novel signalling pathway in the context of cell biology. Identifying TLR transactivation of the TGFBR1 may provide future insight into the detrimental implications of pathogens in pathophysiology.

The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1β monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.

Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-β receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4 h whereas transactivation via TGFBR1 is delayed for 120 min but also persists for at least 4 h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

Atherosclerosis, the principal cause of cardiovascular death worldwide, is a pathological disease characterized by fibro-proliferation, chronic inflammation, lipid accumulation, and immune disorder in the vessel wall. As the atheromatous plaques develop into advanced stage, the vulnerable plaques are prone to rupture, which causes acute cardiovascular events, including ischemic stroke and myocardial infarction. Emerging evidence has suggested that atherosclerosis is also an epigenetic disease with the interplay of multiple epigenetic mechanisms. The epigenetic basis of atherosclerosis has transformed our knowledge of epigenetics from an important biological phenomenon to a burgeoning field of cardiovascular research. Here, we provide a systematic and up-to-date overview of the current knowledge of three distinct but interrelated epigenetic processes (including DNA methylation, histone methylation/acetylation, and non-coding RNAs), in atherosclerotic plaque development and instability. Mechanistic and conceptual advances in understanding the biological roles of various epigenetic modifiers in regulating gene expression and functions of endothelial cells (vascular homeostasis, leukocyte adhesion, endothelial-mesenchymal transition, angiogenesis, and mechanotransduction), smooth muscle cells (proliferation, migration, inflammation, hypertrophy, and phenotypic switch), and macrophages (differentiation, inflammation, foam cell formation, and polarization) are discussed. The inherently dynamic nature and reversibility of epigenetic regulation, enables the possibility of epigenetic therapy by targeting epigenetic “writers”, “readers”, and “erasers”. Several Food Drug Administration-approved small-molecule epigenetic drugs show promise in pre-clinical studies for the treatment of atherosclerosis. Finally, we discuss potential therapeutic implications and challenges for future research involving cardiovascular epigenetics, with an aim to provide a translational perspective for identifying novel biomarkers of atherosclerosis, and transforming precision cardiovascular research and disease therapy in modern era of epigenetics.

Cardiovascular and metabolic diseases remain the leading cause of morbidity and mortality worldwide. Endothelial dysfunction is a key player in the initiation and progression of cardiovascular and metabolic diseases. Current evidence suggests that the anti-diabetic drug metformin improves insulin resistance and protects against endothelial dysfunction in the vasculature. Hereby, we provide a timely review on the protective effects and molecular mechanisms of metformin in preventing endothelial dysfunction and cardiovascular and metabolic diseases.

The endothelium is the single-cell monolayer that lines the entire vasculature. The endothelium has a barrier function to separate blood from organs and tissues but also has an increasingly appreciated role in anti-coagulation, vascular senescence, endocrine secretion, suppression of inflammation and beyond. In modern times, endothelial cells have been identified as the source of major endocrine and vaso-regulatory factors principally the dissolved lipophilic vosodilating gas, nitric oxide and the potent vascular constricting G protein receptor agonists, the peptide endothelin. The role of the endothelium can be conveniently conceptualized. Continued investigations of the mechanism of endothelial dysfunction will lead to novel therapies for cardiovascular disease. In this review, we discuss the impact of endothelial dysfunction on cardiovascular disease and assess the clinical relevance of endothelial dysfunction.

Curcumin is a natural compound that has been widely used as a food additive and medicine in Asian countries. Over several decades, diverse biological effects of curcumin have been elucidated, such as anti-inflammatory and anti-oxidative activities. Monocyte chemoattractant protein-1 (MCP-1) is a key inflammatory marker during the development of atherosclerosis, and curcumin blocks MCP-1 expression stimulated by various ligands. Hence, we studied the action of curcumin on lysophosphatidic acid (LPA) mediated MCP-1 expression and explored the specific underlying mechanisms. In human vascular smooth muscle cells, LPA induces Rho-associated protein kinase (ROCK) dependent transforming growth factor receptor (TGFBR1) transactivation, leading to glycosaminoglycan chain elongation. We found that LPA also signals via the TGFBR1 transactivation pathway to regulate MCP-1 expression. Curcumin blocks LPA mediated TGFBR1 transactivation and subsequent MCP-1 expression by blocking the ROCK signalling. In the vasculature, ROCK signalling regulates smooth muscle cell contraction, inflammatory cell recruitment, endothelial dysfunction and vascular remodelling. Therefore, curcumin as a ROCK signalling inhibitor has the potential to prevent atherogenesis via multiple ways.

Type 2 diabetes mellitus (T2DM) is closely associated with cardiovascular diseases (CVD), including atherosclerosis, hypertension and heart failure. Some anti-diabetic medications are linked with an increased risk of weight gain or hypoglycemia which may reduce the efficacy of the intended anti-hyperglycemic effects of these therapies. The recently developed receptor agonists for glucagon-like peptide-1 (GLP-1RAs), stimulate insulin secretion and reduce glycated hemoglobin levels without having side effects such as weight gain and hypoglycemia. In addition, GLP1-RAs demonstrate numerous cardiovascular protective effects in subjects with or without diabetes. There have been several cardiovascular outcomes trials (CVOTs) involving GLP-1RAs, which have supported the overall cardiovascular benefits of these drugs. GLP1-RAs lower plasma lipid levels and lower blood pressure (BP), both of which contribute to a reduction of atherosclerosis and reduced CVD. GLP-1R is expressed in multiple cardiovascular cell types such as monocyte/macrophages, smooth muscle cells, endothelial cells, and cardiomyocytes. Recent studies have indicated that the protective properties against endothelial dysfunction, anti-inflammatory effects on macrophages and the anti-proliferative action on smooth muscle cells may contribute to atheroprotection through GLP-1R signaling. In the present review, we describe the cardiovascular effects and underlying molecular mechanisms of action of GLP-1RAs in CVOTs, animal models and cultured cells, and address how these findings have transformed our understanding of the pharmacotherapy of T2DM and the prevention of CVD.

[Image: see text] Atherosclerosis begins in the vessel wall with the retention of low density lipoproteins to modified proteoglycans with hyperelongated glycosaminoglycan (GAG) chains. Bacterial infections produce endotoxins such as lipopolysaccharide that exacerbate the outcome of atherosclerosis by generating a heightened state of inflammation. Lipopolysaccharide (LPS) via its toll-like receptor (TLR) is well-known for its role in mediating an inflammatory response in the body. Emerging evidence demonstrates that TLRs are involved in regulating vascular functions. In this study we sought to investigate the role of LPS in proteoglycan modification and GAG chain elongation, and we hypothesize that LPS will signal via Smad2 dependent pathways to regulate GAG chain elongation. The in vitro model used human aortic vascular smooth muscle cells. GAG gene expression was assessed by quantitative real-time polymerase chain reaction. Western blotting was performed using whole-cell protein lysates to assess the signaling pathway. LPS via TLR4 stimulates the expression of GAG synthesizing enzymes to an equal extent to traditional cardiovascular agonists. LPS phosphorylates the Smad2 linker region via TAK-1/MAPK dependent pathways which correlated with genes associated with GAG chain initiation and elongation. The well-characterized role of LPS in inflammation and our data on GAG gene expression demonstrates that GAG chain elongation is the earliest marker of the inflammatory cascade in atherosclerosis development.

Atherosclerosis commences with the trapping of low density lipoproteins (LDLs) in blood vessels by modified proteoglycans (PGs) with hyperelongated glycosaminoglycan (GAG) chains. GAG chain synthesis and growth factor mediated hyperelongation regulates the composition and size of PGs in a manner that would cause low density lipoprotein (LDLs) retention in vessel wall. Galactosaminoglycans are a class of GAGs, commonly observed on PGs. Multiple enzymes are involved in galactosaminoglycan biosynthesis. Galactosaminoglycan synthesis is regulated by various signalling pathways which are amenable to pharmacological manipulation to treat atherosclerosis. Receptor mediated signalling pathways including protein tyrosine kinase receptors (PTKRs), serine/threonine kinase receptors (S/TKRs) and G-protein coupled receptors (GPCRs) pathways regulate galactosaminoglycan synthesizing enzyme expression. Increased expression of these enzymes modify galactosaminoglycan chain structure by making them hyperelongated. This review focuses on the signalling pathways regulating the expression of genes involved in galactosaminoglycan synthesis and modification. Furthermore, there are multiple other processes for inhibiting the interactions between LDL and galactosaminoglycans such as peptide mimetics of ApoB100 and anti-galactosaminoglycan antibodies and the therapeutic potential of these strategies is also addressed.

Transforming Growth Factor (TGF) β is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that leads to increased binding of atherogenic lipoproteins in the wall of blood vessels. TGF-β signalling pathway components leading to the modification of GAG chains on biglcyan are therefore potential targets for the treatment of atherosclerosis. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signalling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have previously shown that flavopiridol, a well known cyclin dependent kinase (CDK) inhibitor inhibited biglycan synthesis. We have used flavopiridol to study the role of linker region phosphorylation in the TGF-β-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS PAGE to assess proteoglycan synthesis; RT PCR to assess gene expression and chromatin immunoprecipitation (ChIP) to assess the binding of phosphorylated Smads to the promoter region of GAG synthesis genes. Flavopiridol blocked TGF-β-stimulated synthesis of mRNA for the GAG synthesizing enzymes, chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1), and xylosyltransferase-1 (XT-1); as well as biglycan core protein mRNA and protein expression. The incorporation of radiosulfate into proteoglycans stimulated by TGF-β as well as GAG hyperelongation were also blocked by flavopiridol. Flavopiridol blocked TGF-β-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of polyphosphorylated Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-β and this response was blocked by flavopiridol demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential target(s) for the development of a therapeutic agent to prevent atherosclerosis.

Type 2 diabetes mellitus is a complex and heterogeneous disorder which in its most common manifestation arises from insulin resistance and later insulin insufficiency. Type 2 diabetes is characterised by impaired insulin sensitivity and diagnosed as hyperglycaemia. Because of its cardiovascular consequences, Type 2 diabetes represents one of the world's leading causes of mortality and morbidity. Drug discovery and development are required to produce better ways to prevent, treat and manage diabetes and its complications. Diabetes is a human, not an animal disease, so animals do not get Type 2 diabetes. However there are animal models which are variously suitable for the investigation of new agents for the treatment of Type 2 diabetes. In this Review we have examined the various models that are available for the study of natural products with a focus on models (genetic, nutritional and spontaneous) for the metabolic abnormities of diabetes. These models are also relevant to the investigation of Western medicines for the treatment of diabetes. A suitable experimental model plays an important role in drug discovery for translational studies leading to increased understanding of the molecular basis and management of diabetes.

directly or as a source of small chemical entities for the treatment of diabetes and its associated cardiovascular disease. Thus, what is required is animal models which reproduce the pathophysiology of diabetes and cardiovascular with high fidelity and which thus can be used to test or screen natural products. Such a comprehensive model does not exist in a single animal model. Furthermore, the pathophysiology of diabetes is more complex than can be reproduced comprehensively in an animal model and with respect to cardiovascular disease the pathology of disease in animal models does not reflect the development of atherosclerotic plaques in human arteries. This review addresses these issues and suggests that different animal models are required to separately address effects on hyperglycaemia and atherosclerosis. It is further addressed that the therapeutic environment for the development of medicines for the treatment of diabetes and cardiovascular disease has changed considerably in the last year. Successful clinical trials have demonstrated the benefits in terms of reduced cardiovascular events and reduced deaths from the use of sodium glucose cotransport 2 inhibitors and glucagon like peptide agonists and this was achieved with a reasonable degree of safety. These results set a new benchmark for the development of new drugs in this area. A sophisticated approach to the evaluation of natural products for the treatment of cardiometabolic disease will expedite the discovery and development of new medicines in an area that has an exploding global population of people with diabetes.

The discovery and extension of G-protein-coupled receptor (GPCR) transactivation-dependent signalling has enormously broadened the GPCR signalling paradigm. GPCRs can transactivate protein tyrosine kinase receptors (PTKRs) and serine/threonine kinase receptors (S/TKRs), notably the epidermal growth factor receptor (EGFR) and transforming growth factor-β type 1 receptor (TGFBR1), respectively. Initial comprehensive mechanistic studies suggest that these two transactivation pathways are distinct. Currently, there is a focus on GPCR inhibitors as drug targets, and they have proven to be efficacious in vascular diseases. With the broadening of GPCR transactivation signalling, it is therefore important from a therapeutic perspective to find a common transactivation pathway of EGFR and TGFBR1 that can be targeted to inhibit complex pathologies activated by the combined action of these receptors. Reactive oxygen species (ROS) are highly reactive molecules and they act as second messengers, thus modulating cellular signal transduction pathways. ROS are involved in different mechanisms of GPCR transactivation of EGFR. However, the role of ROS in GPCR transactivation of TGFBR1 has not yet been studied. In this review, we will discuss the involvement of ROS in GPCR transactivation-dependent signalling.

Transforming growth factor (TGF)-β signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-β signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-β receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-β can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-β signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-β signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.

Lysophosphatidic acid (LPA) via transactivation dependent signalling pathways contributes to a plethora of physiological and pathophysiological responses. In the vasculature, hyperelongation of glycosaminoglycan (GAG) chains on proteoglycans leads to lipid retention in the intima resulting in the early pathogenesis of atherosclerosis. Therefore, we investigated and defined the contribution of transactivation dependent signalling in LPA mediated GAG chain hyperelongation in human vascular smooth muscle cells (VSMCs). LPA acting via the LPA receptor 5 (LPAR5) transactivates the TGFBR1 to stimulate the mRNA expression of GAG initiation and elongation genes xylosyltransferase-1 (XYLT1) and chondroitin 6-sulfotransferase-1 (CHST3), respectively. We found that LPA stimulates ROS and Akt signalling in VSMCs, however they are not associated in LPAR5 transactivation of the TGFBR1. We observed that LPA via ROCK dependent pathways transactivates the TGFBR1 to stimulate genes associated with GAG chain elongation. We demonstrate that GPCR transactivation of the TGFBR1 occurs via a universal biochemical mechanism and the identified effectors represent potential therapeutic targets to inhibit pathophysiological effects of GPCR transactivation of the TGFBR1.

Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (Gi, Gs, G12/13 and Gq); Gq is further subdivided into four classes. Among them Gαq and Gαq/11 isoforms are most crucial and ubiquitously expressed; these isoforms are almost 88% similar at their amino acid sequence but may exhibit functional divergences. However, uncertainties often arise about Gαq and Gαq/11 inhibitors, these G proteins might also have suitability to the invention of novel-specific inhibitors for each isoforms. YM-254890 and UBO-QIC are discovered as potent inhibitors of Gαq functions and also investigated in thrombin protease-activated receptor (PAR)-1 inhibitors and platelet aggregation inhibition. The most likely G protein involved in PAR-1 stimulates responses is one of the Gαq family isoforms. In this review, we highlight the molecular structures and pharmacological responses of Gαq family which may reflect the biochemical and molecular role of Gαq and Gαq/11. The advanced understanding of Gαq and Gαq/11 role in GPCR signalling may shed light on our understanding on cell biology, cellular physiology and pathophysiology and also lead to the development of novel therapeutic agents for a number of diseases.

Classically, receptor-mediated signalling was conceived as a linear process involving one agonist, a variety of potential targets within a receptor family (e.g. α- and β-adrenoceptors) and a second messenger (e.g. cAMP)-triggered response. If distinct responses were stimulated by the same receptor in different tissues (e.g. lipolysis in adipocytes vs. increased beating rate in the heart caused by adrenaline), the differences were attributed to different second messenger targets in the different tissues. It is now realized that an individual receptor can couple to multiple effectors (different G proteins and different β-arrestins), even in the same cell, to drive very distinct responses. Furthermore, tailored agonists can mould the receptor conformation to activate one signal pathway versus another by a process termed 'biased signalling'. Complicating issues further, we now know that activating one receptor can rapidly trigger the local release of agonists for a second receptor via a process termed 'transactivation'. Thus, the end response can represent a cooperative signalling process involving two or more receptors linked by transactivation. This overview, with a focus on the GPCR, protease-activated receptor-1, integrates both of these processes to predict the complex array of responses that can arise when biased receptor signalling also involves the receptor transactivation process. The therapeutic implications of this signalling matrix are also briefly discussed. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

A major feature of early age-related macular degeneration (AMD) is the thickening of Bruch's membrane in the retina and an alteration in its composition with increased lipid deposition. In certain pathological conditions proteoglycans are responsible for lipid retention in tissues. Growth factors are known to increase the length of glycosaminoglycan chains and this can lead to a large increase in the interaction between proteoglycans and lipids. Using choroidal endothelial cells, we investigated the effects of a number of AMD relevant growth factors TGFβ, thrombin, PDGF, IGF and VEGF on proteoglycan synthesis. Cells were characterized as of endothelial origin using the specific cell markers endothelial nitric oxide synthesis and von Willebrand factor and imaged using confocal microscopy. Cells were treated with growth factors in the presence and absence of the appropriate inhibitors and were radiolabeled with [35S]-SO4. Proteoglycans were isolated by ion exchange chromatography and sized using SDS-PAGE. Radiosulfate incorporation was determined by the cetylpyridinium chloride (CPC) precipitation technique. To measure cellular glycosaminoglycan synthesizing capacity we added xyloside and assessed the xyloside-GAGs by SDS-PAGE. TGFβ, thrombin, PDGF & IGF dose-dependently stimulated radiosulfate incorporation and GAG elongation as well as xyloside-GAG synthesis, however VEGF treatment did not stimulate any changes in proteoglycan synthesis. VEGF did not increase pAKT but caused a large increase in pERK relative to the response to PDGF. Thus, AMD relevant agonists cause glycosaminoglycan hyperelongation of proteoglycans synthesised and secreted by retinal choroidal endothelial cells. The absence of a response to VEGF is intriguing and identifies proteoglycans as a novel potential target in AMD. Future studies will examine the relevance of these changes to enhanced lipid binding and the development of AMD.

G protein-coupled receptors (GPCR) are one of the most important targets for therapeutics due to their abundance and diversity. The G protein-coupled receptor for thrombin can transactivate protein tyrosine kinase receptors (PTKR) and we have recently established that it can also transactivate serine/threonine kinase receptors (S/TKR). A comprehensive knowledge of the signalling pathways that GPCR transactivation elicits is necessary to fully understand the implications of both GPCR activation and the impact of target drugs. Here, we demonstrate that thrombin elicits dual transactivation-dependent signalling pathways to stimulate mRNA expression of glycosaminoglycan synthesizing enzymes chondroitin 4-O-sulfotransferase 1 and chondroitin sulfate synthase 1. The PTKR mediated response involves matrix metalloproteinases and the phosphorylation of the MAP kinase Erk. The S/TKR mediated response differs markedly and involves the phosphorylation of Smad2 carboxy terminal serine residues and does not involve matrix metalloproteinases. This work shows that all of the thrombin mediated signalling to glycosaminoglycan synthesizing enzyme gene expression occurs via transactivation-dependent pathways and does not involve transactivation-independent signalling. These findings highlight the complexity of thrombin-mediated transactivation signalling and the broader implications of GPCR targeted therapeutics.

Transforming growth factor (TGF)-β1 mediates glycosaminoglycan (GAG) chain hyperelongation on secreted proteoglycans and these modifications are associated with increased lipid binding in the vessel wall and the development of atherosclerosis. In vascular smooth muscle cells (VSMCs), TGF-β1 regulated GAG elongation via extracellular signal-regulated kinase (ERK) and p38 as well as Smad2 linker region phosphorylation. In this study, our aim was to identify the TGF-β1 mediated signalling pathway involving reactive oxygen species (ROS) and Smad2 linker region phosphorylation that regulate the mRNA expression of GAG synthesizing enzymes, chondroitin 4-O-sulfotransferase 1 (CHST11) and chondroitin sulfate synthase 1 (CHSY1) which are the rate limiting enzymes involved in GAG chain elongation. Signalling molecules were assessed by western blotting, quantitative real-time PCR was used for analysis of gene expression and intracellular ROS level was measured by a fluorescence based assay. TGF-β1 induced ROS production in VSMCs. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) inhibitors, diphenyleneiodonium (DPI) and apocynin blocked TGF-β1 mediated Smad2 linker region phosphorylation. TGF-β1 treatment increased the mRNA levels of CHST11 and CHSY1. Pharmacological inhibition of Nox blocked TGF-β1 mediated mitogen activated protein kinases (MAPKs) phosphorylation and TGF-β1 stimulated CHST11 and CHSY1 mRNA expression. These findings demonstrated that TGF-β1 mediated expression of CHST11 and CHSY1 can occur via Nox-dependent pathways and Smad2 linker region phosphorylation.

Growth factors such as thrombin and transforming growth factor (TGF)-β facilitate glycosaminoglycan (GAG) chain hyperelongation on proteoglycans, a phenomenon that increases lipoprotein binding in the vessel wall and the development of atherosclerosis. TGF-β signals via canonical carboxy terminal phosphorylation of R-Smads and also non-canonical linker region phosphorylation of R-Smads. The G protein coupled receptor agonist, thrombin, can transactivate the TGF-β receptor leading to both canonical and non-canonical Smad signalling. Linker region phosphorylation drives the expression of genes for the synthesis of the proteoglycan, biglycan. Proteoglycan synthesis involves core protein synthesis, the initiation of GAG chains and the subsequent elongation of GAG chains. We have explored the relationship between the thrombin stimulated phosphorylation of individual serine and threonine sites in the linker region of Smad2 and the expression of GAG initiation xylosyltransferase-1 (XT-1) and GAG elongation chondroitin 4-sulfotransferase-1 (C4ST-1) and chondroitin synthase-1 (CHSY-1) genes. Thrombin stimulated the phosphorylation of all four target residues (Thr220, Ser245, Ser250 and Ser255 residues) with a similar temporal pattern - phosphorylation was maximal at 15 min (the earliest time point studied) and the level of the phospho-proteins declined thereafter over the following 4 h. Jnk, p38 and PI3K, selectively mediated the phosphorylation of the Thr220 residue whereas the serine residues were variously phosphorylated by multiple kinases. Thrombin stimulated the expression of all three genes - XT-1, C4ST-1 and CHSY-1. The three pathways mediating Thr220 phosphorylation were also involved in the expression of XT-1. The target pathways (excluding Jnk) were involved in the expression of the GAG elongation genes (C4ST-1 and CHSY-1). These findings support the contention that individual Smad linker region phosphorylation sites are linked to the expression of genes for the initiation and elongation of GAG chains on proteoglycans. The context of this work is that a specific inhibitor of GAG elongation represents a potential therapeutic agent for preventing GAG elongation and lipid binding and the results indicate that the specificity of the pathways is such that it might be therapeutically feasible to specifically target GAG elongation without interfering with other physiological processes with which proteoglycans are involved.

Objectives Atherosclerosis, a progressive condition characterised by the build-up of plaque due to the accumulation of low-density lipoprotein and fibrous substances in the damaged arteries, is the major underlying pathology of most cardiovascular diseases. Despite the evidence of the efficacy of the present treatments for atherosclerosis, the complex and poorly understood underlying mechanisms of atherosclerosis development and progression have prevented them from reaching their full potential. Novel alternative treatments like usage of nanomedicines and theranostics are gaining attention of the researchers worldwide. This review will briefly discuss the current medications for the disease and explore potential future developments based on theranostics nanomaterials that may help resolve atherosclerotic cardiovascular disease. Key findings Various drugs can slow the effects of atherosclerosis. They include hyperlipidaemia medications, anti-platelet drugs, hypertension and hyperglycaemia medications. Most of the theranostic agents developed for atherosclerosis have shown the feasibility of rapid and noninvasive diagnosis, as well as effective and specific treatment in animal models. However, there are still some limitation exist in their structure design, stability, targeting efficacy, toxicity and production, which should be optimized in order to develop clinically acceptable nanoparticle based theronostics for atherosclerosis. Summary Current medications for atherosclerosis and potential theranostic nanomaterials developed for the disease are discussed in the current review. Further investigations remain to be carried out to achieve clinical translation of theranostic agents for atherosclerosis.

Transforming growth factor

G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

G protein coupled receptor (GPCR) signalling is mediated by transactivation independent and transactivation dependent pathways. GPCRs transactivate protein tyrosine kinase receptors (PTKRs) and protein serine/threonine kinase receptors (PS/TKR). Since the initial observations of transactivation dependent signalling, there has been an effort to understand the mechanisms behind this phenomena. GPCR signalling has evolved to include biased signalling. Biased signalling, whereby selected ligands can activate the same GPCR that can generate multiple signals, but drive only a unique response. To date, there has been no focus on the ability of biased agonists to activate the PTKR and PS/TKR transactivation pathways differentially. As such, this represents a novel direction for future research. This review will discuss the main mechanisms of GPCR mediated receptor transactivation and the pathways involved in intracellular responses.

G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the "triple membrane bypass" pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.

In the past three decades, endothelin and endothelin receptor antagonists have received great scientific and clinical interest, leading to the publication of more than 27,000 scientific articles since its discovery. The Thirteenth International Conference on Endothelin (ET-13) was held on September 8-11, 2013, at Tokyo Campus of the University of Tsukuba in Japan. Close to 300 scientists from 25 countries from around the world came to Tokyo to celebrate the anniversary of the discovery of the endothelin peptide discovered 25 years ago at the University of Tsukuba. This article summarizes some of the highlights of the conference, the anniversary celebration ceremony, and particularly the participation of next generation of endothelin researchers in endothelin science and the anniversary celebration. As a particular highlight, next generation endothelin researchers wrote a haiku (a traditional form of Japanese poetry originating from consisting of no more than three short verses and 27 on, or Japanese phonetic units) to describe the magic of endothelin science which they presented to the conference audience at the anniversary ceremony. The text of each haiku - both in its original language together with the English translation - is part of this article providing in an exemplary fashion how poetry can be bridged with science. Finally, we give an outlook towards the next 25 years of endothelin research.

Objectives Suramin is a polysulfonated naphthylurea with antiparasitic and potential antineoplastic activity. Suramin's pharmacological actions, which have not yet been fully elucidated, include antagonism of the action of platelet-derived growth factor (PDGF) at its receptor. We investigated the effects of suramin on PDGF-stimulated proteoglycan synthesis. Methods Human vascular smooth muscle cells (VSMCs) were incubated in the presence and absence of PDGF and suramin with [3H]thymidine or 35SO4 as radiolabels. Mitogenic response was determined by [3H]thymidine incorporation. PDGFβ receptor phosphorylation was assessed by western blotting. Proteoglycan size and glycosaminoglycan chain synthesis and size were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The Alphascreen phosphotyrosine assay kit was used to investigate PDGFβ receptor tyrosine kinase inhibition by suramin. Key findings Suramin decreased PDGF-stimulated proliferation, proteoglycan synthesis and GAG chain hyperelongation. Suramin also directly inhibited PDGFβ receptor kinase activity as well as PDGFβ receptor phosphorylation in intact VSMCs. Conclusions These data show that inhibition of PDGFβ receptor phosphorylation in intact cells is necessary to define a fully active PDGF antagonist. They also confirm that PDGFβ receptor kinase activity is necessary for PDGF-mediated atherogenic changes in proteoglycan synthesis and support efforts to develop PDGFβ receptor antagonists as potential anti-atherosclerotic agents.

Objective G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). Method The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. Key finding ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4–6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ETA and ETB receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. Conclusion In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis.

Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (G

Introduction Tyrosine kinase inhibitors were the first class of smart drugs being specifically designed to inhibit a disease causing target. There is a very important but unresolved question as whether or not the overall therapeutic role of an individual tinib results from an action at its primary target, a single most likely, tyrosine kinase, or from the combined or aggregate action at the multiple targets which each tinib addresses. Methods We selected a series of ten tinibs (gefitinib, sunitinib, lapatinib, erlotinib, imatinib, sorafenib, axitinib, vanitinib, bosutinib, dasatinib) with various known targets and investigated their activities in the inhibition of proteoglycan synthesis and GAG hyperelongation stimulated by a tyrosine kinase receptor agonist, platelet derived growth factor (PDGF) and for contrast, a serine/threonine kinase receptor agonist, TGF β and some downstream signalling pathways. Results The inhibitory activity varied from little to total inhibition. The actions of the tinibs were directed more towards inhibition of the tyrosine kinase, PDGF receptor signalling pathway compared to the TGF β. Conclusion There was no suggestion of any synergistic effect arising from inhibition of multiple kinases as the most potent compound, dasatinib, is known to inhibit the broadest spectrum of kinases.

Objectives: Atherosclerosis is a disease process involving the early deposition of lipids in the vessel wall trapped by modified proteoglycans and subsequently a chronic inflammatory process leading to the clinical events. MTX has been chosen to study the potential efficacy of an anti inflammatory agent in preventing atherosclerosis and secondary cardiovascular disease in the cardiovascular inflammation reduction trial (CIRT). Methods: We have investigated cell proliferation and growth factor stimulated proteoglycan synthesis in vascular smooth muscle (VSMC) to assess some of the direct effects of MTX. Experiments were conducted in cultured human VSMC. Proliferation was assessed by the gold standard technique of cell counting and proteoglycan synthesis by 35S radiosulafate incorporation and size analysis by SDS PAGE. Key findings: MTX had a concentration-dependent inhibitory effect on serum stimulated VSMC proliferation with a maximum and total inhibitory effect at 10 μM. Thrombin, platelet-derived growth factor and transforming growth factor beta stimulated proteoglycan synthesis and increased the size of the biglycan molecules but MTX (10 μM) had no effect on any of these responses. Conclusions: The outcome of a trial with MTX will reflect the potential of targeting inflammation for the prevention of atherosclerosis and it remains an interesting proposition to evaluate the effects of a "proteoglycan inhibitor" on atherosclerosis.

ObjectivesTransforming growth factor (TGF)-β regulates the function of vascular endothelial cells and may be involved in endothelial dysfunction. The canonical TGF-β pathway involves TGF-β receptor-mediated carboxy-terminal phosphorylation of Smad2; however, TGF-β signalling also activates numerous serine/threonine kinases that phosphorylate Smad2 in its linker region. The expression of phosphorylated Smad linker proteins were determined following TGF-β stimulation in the absence and presence of different serine/threonine kinase inhibitors in vascular endothelial cells.MethodsProteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues.Key findingsTGF-β mediated the phosphorylation of all four Smad2 linker region residues of interest. Erk and Jnk specifically phosphorylate Ser245 while all mitogen-activated protein kinases phosphorylate Ser250 and Ser255. Thr220 and Ser245 are phosphorylated by phosphoinositide 3 kinase (PI3K), while Ser255 was phosphorylated by the PI3K/Akt pathway. CDK and GSK-3 were shown to phosphorylate Thr220 and Ser245. TGF-β also mediated plasminogen activator inhibitor-1 gene expression that was attenuated by p38 and CDK inhibitors.ConclusionsTGF-β-mediated phosphorylation of individual serine/threonine sites in the linker region of Smad2 occurs in a highly specific manner by kinases. These phosphorylations provide an opportunity to further understand a therapeutically targeted and very specific signalling pathway in vascular endothelial cells.

Seven transmembrane G Protein Coupled Receptors (GPCRs) are one of the major classes of cell surface receptors and play a major role through agonists and antagonists in human therapeutics [1]. GPCRs are associated with a group of G proteins which consist of 3 subunits termed alpha, beta and gamma. G proteins may be classified according to their effector molecules of the alpha subunit, which in mammals falls into several subtypes, Gαs, Gαi, Gα12 and Gαq. The Gαq family consists of four subunits Gαq, Gα11, Gα14 and Gα15/16. In contrast to the protein kinase receptors which have intrinsic (kinase) enzymatic activity, GPCRs do not have enzymatic activity-enzymatic activity mediating signal transduction resides in the Gα proteins which have GTPase activity [2]. Gα proteins exist in the GTP bound form. Ligand initiated conformational changes in the GPCR causes the release of bound Gα proteins. This dissociation initiates the GTPase activity, hydrolyzing GTP to GDP which is released from the proteins and allows alterative interactions leading to downstream signal transduction.

ObjectivesThis review discusses the latest developments in G protein coupled receptor (GPCR) signalling related to the transactivation of cell surface protein kinase receptors and the therapeutic implications.Key findingsMultiple GPCRs have been known to transactivate protein tyrosine kinase receptors for almost two decades. More recently it has been discovered that GPCRs can also transactivate protein serine/threonine kinase receptors such as that for transforming growth factor (TGF)-β. Using the model of proteoglycan synthesis and glycosaminoglycan elongation in human vascular smooth muscle cells which is a component of an in vitro model of atherosclerosis, the dual tyrosine and serine/threonine kinase receptor transactivation pathways appear to account for all of the response to the agonists, endothelin and thrombin.SummaryThe broadening of the paradigm of GPCR receptor transactivation explains the broad range of activities of these receptors and also the efficacy of GPCR antagonists in cardiovascular therapeutics. Deciphering the mechanisms of transactivation with the aim of identifying a common therapeutic target remains the next challenge.

Objectives (S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)-β stimulates proteoglycan synthesis, leading to increased binding of low-density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)-[6]-gingerol on these TGF-β-mediated proteoglycan changes to explore its potential as an anti-atherosclerotic agent. Methods Purified (S)-[6]-gingerol was assessed for its effects on proteoglycan synthesis by [35S]-sulfate incorporation into glycosaminoglycan chains and [35S]-Met/Cys incorporation into proteoglycans and total proteins in human vascular smooth muscle cells. Biglycan level was assessed by real-time quantitative polymerase chain reactions and the effects of (S)-[6]-gingerol on TGF-β signalling by assessment of the phosphorylation of Smads and Akt by western blotting. Key findings (S)-[6]-Gingerol concentration-dependently inhibited TGF-β-stimulated proteoglycan core protein synthesis, and this was not secondary to inhibition of total protein synthesis. (S)-[6]-Gingerol inhibited biglycan mRNA expression. (S)-[6]-Gingerol did not inhibit TGF-β-stimulated glycosaminoglycan hyperelongation or phosphorylation of Smad 2, in either the carboxy terminal or linker region, or Akt phosphorylation. Conclusions The activity of (S)-[6]-gingerol to inhibit TGF-β-stimulated biglycan synthesis suggests a potential role for ginger in the prevention of atherosclerosis or other lipid-binding diseases. The signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-β responses.