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Key features of chronic kidney disease (CKD) include tubulointerstitial inflammation and fibrosis. Protease activated receptor-2 (PAR2), a G-protein coupled receptor (GPCR) expressed by the kidney proximal tubular cells, induces potent proinflammatory responses in these cells. The hypothesis tested here was that PAR2 signalling can contribute to both inflammation and fibrosis in the kidney by transactivating known disease associated pathways. Using a primary cell culture model of human kidney tubular epithelial cells (HTEC), PAR2 activation induced a concentration dependent, PAR2 antagonist sensitive, secretion of TNF, CSF2, MMP-9, PAI-1 and CTGF. Transcription factors activated by the PAR2 agonist 2F, including NFκB, AP1 and Smad2, were critical for production of these cytokines. A TGF-β receptor-1 (TGF-βRI) kinase inhibitor, SB431542, and an EGFR kinase inhibitor, AG1478, ameliorated 2F induced secretion of TNF, CSF2, MMP-9, and PAI-1. Whilst an EGFR blocking antibody, cetuximab, blocked PAR2 induced EGFR and ERK phosphorylation, a TGF-βRII blocking antibody failed to influence PAR2 induced secretion of PAI-1. Notably simultaneous activation of TGF-βRII (TGF-β1) and PAR2 (2F) synergistically enhanced secretion of TNF (2.2-fold), CSF2 (4.4-fold), MMP-9 (15-fold), and PAI-1 (2.5-fold). In summary PAR2 activates critical inflammatory and fibrotic signalling pathways in human kidney tubular epithelial cells. Biased antagonists of PAR2 should be explored as a potential therapy for CKD. [ABSTRACT FROM AUTHOR]

Aims: G protein-coupled receptor (GPCR) transactivation of kinase receptors greatly expands the actions attributable to GPCRs. Thrombin, via its cognate GPCR, protease-activated receptor (PAR)-1, transactivates tyrosine and serine/threonine kinase receptors, specifically the epidermal growth factor receptor and transforming growth factor-β receptor, respectively. PAR-1 transactivation-dependent signalling leads to the modification of lipid-binding proteoglycans involved in the retention of lipids and the development of atherosclerosis. The mechanisms of GPCR transactivation of kinase receptors are distinct. We aimed to investigate the role of proximal G proteins in transactivation-dependent signalling. Main Methods: Using pharmacological and molecular approaches, we studied the role of the G⍺ subunits, G⍺q and G⍺11, in the context of PAR-1 transactivation-dependent signalling leading to proteoglycan modifications. Key Findings: Pan G⍺q subunit inhibitor UBO-QIC/FR900359 inhibited PAR-1 transactivation of kinase receptors and proteoglycans modification. The G⍺q/11 inhibitor YM254890 did not affect PAR-1 transactivation pathways. Molecular approaches revealed that of the two highly homogenous G⍺q members, G⍺q and G⍺11, only the G⍺q was involved in regulating PAR-1 mediated proteoglycan modification. Although G⍺q and G⍺11 share approximately 90% homology at the protein level, we show that the two isoforms exhibit different functional roles. Significance: Our findings may be extrapolated to other GPCRs involved in vascular pathology and highlight the need for novel pharmacological tools to assess the role of G proteins in GPCR signalling to expand the preeminent position of GPCRs in human therapeutics. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is the largest single cause of disease-related mortality worldwide and the major underlying pathology is atherosclerosis. Atherosclerosis develops as a complex process of vascular lipid deposition and retention by modified proteoglycans, endothelial dysfunction and unresolved chronic inflammation. There are a multitude of current therapeutic agents, most based on lowering plasma lipid levels, but, overall, they have a lower than optimum level of efficacy and many deaths continue to arise from cardiovascular disease world-wide. To identify and evaluate potential novel cardiovascular drugs, suitable animal models that reproduce human atherosclerosis with a high degree of fidelity are required as essential pre-clinical research tools. Commonly used animal models of atherosclerosis include mice (ApoE−/−, LDLR−/− mice and others), rabbits (WHHL rabbits and others), rats, pigs, hamster, zebrafish and non-human primates. Models based on various wild-type and genetically modified mice have been extensively reviewed but mice may not always be appropriate. Thus, here, we provide an overview of the advantages and shortcomings of various non-mouse animal models of atherosclerotic plaque formation, and plaque rupture, as well as commonly used interventional strategies. Taken together, the combinatorial selection of suitable animal models readily facilitates reproducible and rigorous translational research in discovering and validating novel anti-atherosclerotic drugs. [ABSTRACT FROM AUTHOR]

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 ETA and ETB receptor antagonist bosentan, inhibited ET‐1‐mediated phospho‐Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET‐1‐mediated phospho‐Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post‐ET‐1 treatment were increased compared to untreated controls (p < 0.01). The ET‐mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N‐acetyl‐L‐cysteine (NAC). ET‐1‐mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation‐dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor (TGF) β receptor (TGFBR1) stimulates glycosaminoglycan (GAG) elongation on proteoglycans. GPCR agonists thrombin and lysophosphatidic acid (LPA) via respective receptors transactivate the TGFBR1 via Rho/ROCK dependent pathways however mechanistic insight for ET-1 transactivation of the TGFBR1 remains unknown. NADPH oxidase (NOX) generates reactive oxygen species (ROS) and is a signalling entity implicated in the pathogenesis of many diseases including atherosclerosis. If implicated in this pathway, NOX/ROS would be a potential therapeutic target. In this study, we investigated the involvement of NOX in ET-1/ET receptor-mediated transactivation of TGFBR1 to stimulate mRNA expression of GAG chain synthesizing enzymes chondroitin 4–O–sulfotransferase 1 (C4ST-1) and chondroitin sulfate synthase 1 (ChSy-1). The invitro model used vascular smooth muscle cells that were treated with pharmacological antagonists in the presence and absence of ET-1 or TGF-β. Proteins and phosphoproteins isolated from treated cells were quantified by western blotting and quantitative real-time PCR was used to assess mRNA expression of GAG synthesizing enzymes. In the presence of diphenyliodonium (DPI) (NOX inhibitor), ET-1 stimulated phospho-Smad2C levels were inhibited. ET-1 mediated mRNA expression of GAG synthesizing enzymes C4ST-1 and ChSy-1 was also blocked by TGBFR1 antagonists, SB431542, broad spectrum ET receptor antagonist bosentan, DPI and ROS scavenger N-acetyl-l-cysteine. This work shows that NOX and ROS play an important role in ET-1 mediated transactivation of the TGFBR1 and downstream gene targets associated with GAG chain elongation. As ROS is involved in GPCR to protein tyrosine kinase receptor transactivation, the NOX/ROS axis presents as the first common biochemical target in all GPCR to kinase receptor transactivation signalling. [ABSTRACT FROM AUTHOR]

Atherosclerosis, the major underlying pathology of cardiovascular disease, commences with the binding and trapping of lipids on modified proteoglycans, with hyperelongated glycosaminoglycan chains. Transforming growth factor (TGF)‐β stimulates glycosaminoglycan elongation in vascular smooth muscle cells. We have recently shown that this TGF‐β signaling pathway involves reactive oxygen species (ROS). YY‐11 is a dodecapeptide derived from camel milk and it has antioxidant activity. We have investigated the role of YY‐11 in blocking ROS signaling and downstream atherogenic responses. YY‐11 inhibited TGF‐β stimulated ROS production and inhibited the expression of genes for glycosaminoglycan chain elongation as a component of an in vitro model of atherosclerosis. This study provides a biochemical mechanism for the role of camel milk as a potential nutritional product to contribute to the worldwide amelioration of cardiovascular disease. Practical applications: The identification of readily accessible foods with antioxidant properties would provide a convenient and cost‐effective approach community wide reducing oxidative stress induced pathologies such as atherosclerosis. We demonstrate that camel milk‐derived peptide is an antioxidant that can inhibit growth factor‐mediated proteoglycan modification in vitro. As proteoglycan modification is being recognized as one of the earliest atherogenic responses, these data support the notion of camel milk as a suitable nutritional product to contribute to the prevention of early stage of atherosclerosis development. [ABSTRACT FROM AUTHOR]

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 Smad2 carboxy terminal phosphorylation dependent on the transactivation of the TGFBR1. The in vitro model used human aortic vascular smooth muscle cells to assess the implications of TLR4 transactivation of the TGFBR1 in vascular pathophysiology. We show that LPS-mediated Smad2 carboxy terminal phosphorylation is inhibited in the presence of TGFBR1 inhibitor, SB431542. Treatment with MyD88 and TRIF pathway antagonists does not affect LPS-mediated phosphorylation of Smad2 carboxy terminal; however, LPS-mediated Smad2 phosphorylation was inhibited in the presence of MMP inhibitor, GM6001, and unaffected in the presence of ROCK inhibitor Y27632 or ROS/NOX inhibitor DPI. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, G, G and G); G is further subdivided into four classes. Among them G and G 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 G and G 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 G 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 G family isoforms. In this review, we highlight the molecular structures and pharmacological responses of G family which may reflect the biochemical and molecular role of G and G. The advanced understanding of G and G 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. [ABSTRACT FROM AUTHOR]

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 (Tβ RI). 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 Tβ RI 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 n m treatment of ET-1 at 4-6 h. Tβ RI 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 Tβ RI. The transactivation-dependent response is dependent upon de novo protein synthesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peptidyl-prolyl cis/ trans isomerases ( PPIases) are a conserved group of enzymes that catalyse the conversion between cis and trans conformations of proline imidic peptide bonds. These enzymes play critical roles in regulatory mechanisms of cellular function and pathophysiology of disease. There are three different classes of PPIases and increasing interest in the development of specific PPIase inhibitors. Cyclosporine A, FK506, rapamycin and juglone are known PPIase inhibitors. Herein, we review recent advances in elucidating the role and regulation of the PPIase family in vascular disease. We focus on peptidyl-prolyl cis/ trans isomerase NIMA-interacting 1 (Pin1), an important member of the PPIase family that plays a role in cell cycle progression, gene expression, cell signalling and cell proliferation. In addition, Pin1 may be involved in atherosclerosis. The unique role of Pin1 as a molecular switch that impacts on multiple downstream pathways necessitates the evaluation of a highly specific Pin1 inhibitor to aid in potential therapeutic drug discovery. [ABSTRACT FROM AUTHOR]

Objectives Transforming 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. Methods Proteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues. Key findings TGF-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 ( PI3 K), while Ser255 was phosphorylated by the PI3 K/ 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. Conclusions TGF-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. [ABSTRACT FROM AUTHOR]

Background Pharmaceutical interventions for diabetes aim to control glycaemia and to prevent the development of complications, such as cardiovascular diseases. Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role. Glucokinase activity in the pancreas is critical in enhancing insulin release in response to hyperglycaemia. Glucokinase activators (GKAs) are novel agents for diabetes which act by enhancing the formation of glucose-6-phosphate leading to increased insulin production and subsequent suppression of blood glucose. Little, however, is known about the direct effects of GKAs on cardiovascular cells. Methods The effect of the GKAs RO28-1675 and Compound A on glucose utilisation in bovine aortic endothelial cells (BAEC) and rat MIN6 was observed by culturing the cells at high and low glucose concentration in the presence and absence of the GKAs and measuring glucose consumption. The effect of RO28-1675 at various concentrations on glucose-dependant signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-β stimulated proteoglycan synthesis was measured by 35SSO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28- 1675 at on TGF-β mediated Smad2C phosphorylation in BAEC was observed by measurement of pSmad2C levels. The direct actions of RO28-1675 on vascular reactivity were observed by measuring arteriole tone and lumen diameter. Results GKAs were demonstrated to increase glucose utilisation in pancreatic but not endothelial cells. Glucose-activated Smad2 phosphorylation was decreased in a dose-dependent fashion in the presence of RO28-1675. No effect of RO28-1675 was observed on TGF-β stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity. Conclusions GKA RO-28-1675 did not increase glucose consumption in endothelial cells indicating the absence of GK in those cells. No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs. If reflected in vivo, these drugs are unlikely to have their use compromised by direct cardiovascular toxicity. [ABSTRACT FROM AUTHOR]

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 [35 S]-sulfate incorporation into glycosaminoglycan chains and [35 S]-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. [ABSTRACT FROM AUTHOR]

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 35 SO4 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. [ABSTRACT FROM AUTHOR]

Objectives This 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 findings Multiple 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. Summary The 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

1. The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. 2. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. 3. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle. [ABSTRACT FROM AUTHOR]