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

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

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT3 receptor antagonists

PURPOSE: The ferret cisplatin emesis model has been used for ~30 years and enabled identification of clinically used anti-emetics. We provide an objective assessment of this model including efficacy of 5-HT(3) receptor antagonists to assess its translational validity. METHODS: A systematic review identified available evidence and was used to perform meta-analyses. RESULTS: Of 182 potentially relevant publications, 115 reported cisplatin-induced emesis in ferrets and 68 were included in the analysis. The majority (n = 53) used a 10 mg kg(−1) dose to induce acute emesis, which peaked after 2 h. More recent studies (n = 11) also used 5 mg kg(−1), which induced a biphasic response peaking at 12 h and 48 h. Overall, 5-HT(3) receptor antagonists reduced cisplatin (5 mg kg(−1)) emesis by 68% (45–91%) during the acute phase (day 1) and by 67% (48–86%) and 53% (38–68%, all P < 0.001), during the delayed phase (days 2, 3). In an analysis focused on the acute phase, the efficacy of ondansetron was dependent on the dosage and observation period but not on the dose of cisplatin. CONCLUSION: Our analysis enabled novel findings to be extracted from the literature including factors which may impact on the applicability of preclinical results to humans. It reveals that the efficacy of ondansetron is similar against low and high doses of cisplatin. Additionally, we showed that 5-HT(3) receptor antagonists have a similar efficacy during acute and delayed emesis, which provides a novel insight into the pharmacology of delayed emesis in the ferret

Animal models for the evaluation of the cardiometabolic therapeutic potential of traditional Chinese and natural product medicines

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

Animal Models for the Evaluation of the Cardiometabolic Therapeutic Potential of Traditional Chinese and Natural Product Medicines

There is considerable interest in the potential for Traditional Chinese Medicines and other natural products to be used 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

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

The Transforming Growth Factor ß (TGF-ß) superfamily of ligands regulate a diverse set of cellular functions. TGF-ß induces its biological effects through type I and type II transmembrane receptors which 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 R-Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Smad2/3 and co-Smad4 have overall similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. 2. Smad linker region phosphorylation appears to have an important role in the regulation of Smad activity and function. MAP kinase family, CDK 2, CDK4 and calcium-calmodulin dependent (CAM) kinase are the main kinases that phosphorylate sites in the linker region. The role of linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAP-kinase and other kinases. In some instances linker region phosphorylation regulates inhibition of nuclear translocation of Smads. 3. In this review we describe TGF-ß signalling through Smad2/3 and the importance of linker region role in regulation and expression of genes induced by TGF-ß super family ligands in the context of vascular smooth muscle

Therapeutic implications of endothelin and thrombin G-protein-coupled receptor transactivation of tyrosine and serine/threonine kinase cell surface receptors

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)-. 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

Assessing the role of Gaq/11 iin cellular responses: An analysis of investigative tools

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

Thrombin promotes PAI-1 expression and migration in keratinocytes via ERK dependent Smad linker region phosphorylation

Keratinocyte proliferation and migration is essential during re-epithelialisation for the restoration of the epithelial barrier during skin wound healing. Numerous growth factors are involved in the stimulation of keratinocyte proliferation and migration. The signalling pathways that drive these processes during wound healing are not well defined. This study investigated thrombin-mediated signalling in keratinocytes. The thrombin receptor, protease-activated receptor 1 (PAR-1) is a seven transmembrane G-protein coupled receptor that is known to transactivate the epidermal growth factor receptor (EGFR). Immortalized human keratinocytes (HaCaT cells) were treated with thrombin and selective inhibitors to EGFR and MAP kinases. Whole cell lysates were separated on SDS-PAGE and analysed by Western blot using antibodies against transcription factor Smad2. Quantitative real-time polymerase chain reaction was used to measure the mRNA expression of PAI-1 while scratch wound assays were used to measure keratinocyte migration. Western blot data showed that thrombin mediates PAR-1 transactivation of EGFR and the downstream phosphorylation of the transcription factor Smad2 linker (Smad2L) region. ERK1/2 inhibition by UO126 caused a decrease in Smad2L phosphorylation while the p38 inhibitor SB202190 and JNK inhibitor SP600125 did not. Smad2L Ser250 was specifically phosphorylated by this thrombin mediated pathway while Ser245 and Ser255 were not. Thrombin increased PAI-1 mRNA expression and keratinocyte migration and this was reduced when either EGFR or ERK1/2 were blocked. Taken together these results show that thrombin mediated mRNA expression of PAI-1 in keratinocytes and migration occurs via EGFR transactivation and involves signalling intermediates ERK1/2 and Smad2 and may be a key pathway in skin wound healing

Integrating the GPCR transactivation-dependent and biased signalling paradigms in the context of PAR1 signalling

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 &#039;biased signalling&#039;. 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 &#039;transactivation&#039;. 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

Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors

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