Transforming growth factor-ß receptors: versatile mechanisms of ligand activation.

Transforming growth factor-ß (TGF-ß) signaling is initiated by activation of transmembrane TGF-ß receptors (TGFBR), which deploys Smad2/3 transcription factors to control cellular responses. Failure or dysregulation in the TGF-ß signaling pathways leads to pathological conditions. TGF-ß signaling is regulated at different levels along the pathways and begins with the liberation of TGF-ß ligand from its latent form. The mechanisms of TGFBR activation display selectivity to cell types, agonists, and TGF-ß isoforms, enabling precise control of TGF-ß signals. In addition, the cell surface compartments used to release active TGF-ß are surprisingly vibrant, using thrombospondins, integrins, matrix metalloproteinases and reactive oxygen species. The scope of TGFBR activation is further unfolded with the discovery of TGFBR activation initiated by other signaling pathways. The unique combination of mechanisms works in series to trigger TGFBR activation, which can be explored as therapeutic targets. This comprehensive review provides valuable insights into the diverse mechanisms underpinning TGFBR activation, shedding light on potential avenues for therapeutic exploration.

Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies.

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 1ß 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.

Lipopolysaccharide acting via toll-like receptor 4 transactivates the TGF-ß receptor in vascular smooth muscle cells.

Toll-like receptors (TLRs) recognise pathogen­associated 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-ß 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.

Endothelin-1 mediated glycosaminoglycan synthesizing gene expression involves NOX-dependent transactivation of the transforming growth factor-ß receptor.

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor (TGF) ß 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-ß. 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.

Targeted Molecular Imaging of Cardiovascular Diseases by Iron Oxide Nanoparticles.

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.

Endothelin-1 dependent expression of GAG genes involves NOX and p38 mediated Smad linker region phosphorylation.

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-ß 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.

Non-Mouse Models of Atherosclerosis: Approaches to Exploring the Translational Potential of New Therapies.

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.

Gαq Is the Specific Mediator of PAR-1 Transactivation of Kinase Receptors in Vascular Smooth Muscle Cells.

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-ß 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.

Smad linker region phosphorylation is a signalling pathway in its own right and not only a modulator of canonical TGF-ß signalling.

Transforming growth factor (TGF)-ß signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-ß signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-ß receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-ß 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-ß 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-ß signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.

Curcumin Inhibits Lysophosphatidic Acid Mediated MCP-1 Expression via Blocking ROCK Signalling.

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.

Artemisinin inhibits glycosaminoglycan chain synthesizing gene expression but not proliferation of human vascular smooth muscle cells.

Pleotropic growth factor, transforming growth factor (TGF)-ß drives the modification and elongation of glycosaminoglycan (GAG) chains on proteoglycans. Hyperelongated GAG chains bind and trap lipoproteins in the intima leading to the formation of atherosclerotic plaques. We have identified that phosphorylation of Smad2 linker region drives GAG chain modification. The identification of an inhibitor of Smad2 linker region phosphorylation and GAG chain modification signifies a potential therapeutic for cardiovascular diseases. Artemisinin renowned for its potent anti-malarial effects possesses a broad range of biological effects. Our aim was to characterise the anti-atherogenic role of artemisinin in vascular smooth muscle cells (VSMCs). We demonstrate that TGF-ß mediated Smad2 linker region phosphorylation and GAG chain elongation was attenuated by artemisinin; however, we observed no effect on VSMC proliferation. Our data demonstrates the potential for artemisinin to be developed as a therapy to inhibit the development of atherosclerosis by prevention of lipid deposition in the vessel wall without affecting the proliferation of VSMCs.

Flavopiridol Inhibits TGF-ß-Stimulated Biglycan Synthesis by Blocking Linker Region Phosphorylation and Nuclear Translocation of Smad2.

Transforming growth factor-ß (TGF-ß) 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 increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-ß-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-ß-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-ß-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-ß-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-ß, 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 therapeutic target(s) for the development of an agent to prevent atherosclerosis.

Gaq proteins: molecular pharmacology and therapeutic potential.

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.

The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier.

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-ß 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.

Peptidyl-prolyl isomerases: functionality and potential therapeutic targets in cardiovascular disease.

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.

Atherogenic, fibrotic and glucose utilising actions of glucokinase activators on vascular endothelium and smooth muscle.

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-dependent signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-ß stimulated proteoglycan synthesis was measured by 35S-SO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28-1675 on TGF-ß 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-ß stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity. CONCLUSIONS: GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase 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.

Mechanistic insight: Linking cardiovascular complications of inflammatory bowel disease.

Cardiovascular diseases (CVD) are the leading cause of mortality worldwide despite an aggressive reduction of traditional cardiovascular risk factors. Underlying inflammatory conditions such as inflammatory bowel disease (IBD) increase the risk of developing CVD. A broad understanding of the underlying pathophysiological processes between IBD and CVD is required to treat and prevent cardiovascular events in patients with IBD. This review highlights the commonality between IBD and CVD, including dysregulated immune response, genetics, environmental risk factors, altered gut microbiome, stress, endothelial dysfunction and abnormalities, to shed light on an essential area of modern medicine.

PAR2 activation on human tubular epithelial cells engages converging signaling pathways to induce an inflammatory and fibrotic milieu.

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-ß receptor-1 (TGF-ßRI) 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-ßRII blocking antibody failed to influence PAR2 induced secretion of PAI-1. Notably simultaneous activation of TGF-ßRII (TGF-ß1) 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.

Moscatilin inhibits vascular calcification by activating IL13RA2-dependent inhibition of STAT3 and attenuating the WNT3/ß-catenin signalling pathway.

INTRODUCTION: Vascular calcification, a devastating vascular complication accompanying atherosclerotic cardiovascular disease and chronic kidney disease, increases the incidence of adverse cardiovascular events and compromises the efficacy of vascular interventions. However, effective therapeutic drugs and treatments to delay or prevent vascular calcification are lacking. OBJECTIVES: This study was designed to test the therapeutic effects and mechanism of Moscatilin (also known as dendrophenol) from Dendrobium huoshanense (an eminent traditional Chinese medicine) in suppressing vascular calcification in vitro, ex vivo and in vivo. METHODS: Male C57BL/6J mice (25-week-old) were subjected to nicotine and vitamin D3 (VD3) treatment to induce vascular calcification. In vitro, we established the cellular model of osteogenesis of human aortic smooth muscle cells (HASMCs) under phosphate conditions. RESULTS: By utilizing an in-house drug screening strategy, we identified Moscatilin as a new naturally-occurring chemical entity to reduce HASMC calcium accumulation. The protective effects of Moscatilin against vascular calcification were verified in cultured HASMCs. Unbiased transcriptional profiling analysis and cellular thermal shift assay suggested that Moscatilin suppresses vascular calcification via binding to interleukin 13 receptor subunit A2 (IL13RA2) and augmenting its expression. Furthermore, IL13RA2 was reduced during HASMC osteogenesis, thus promoting the secretion of inflammatory factors via STAT3. We further validated the participation of Moscatilin-inhibited vascular calcification by the classical WNT/ß-catenin pathway, among which WNT3 played a key role in this process. Moscatilin mitigated the crosstalk between WNT3/ß-catenin and IL13RA2/STAT3 to reduce osteogenic differentiation of HASMCs. CONCLUSION: This study supports the potential of Moscatilin as a new naturally-occurring candidate drug for treating vascular calcification via regulating the IL13RA2/STAT3 and WNT3/ß-catenin signalling pathways.

Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle.

The transforming growth factor (TGF)-ß superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-ß 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-ß binds to the TGF-ß Type II receptor (TßRII) at the cell surface, recruiting the Type I receptor (TßRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TßRI, 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. 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. In the present review, we describe TGF-ß signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-ß superfamily ligands in the context of vascular smooth muscle.