Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases.

AIMS: The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. METHODS AND RESULTS: We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands. CONCLUSION: Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.

Phosphatidylserine decarboxylase is critical for the maintenance of skeletal muscle mitochondrial integrity and muscle mass.

OBJECTIVE: Phosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo. METHODS: To determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry. RESULTS: PSD knockdown disrupted muscle phospholipid homeostasis leading to an ∼25% reduction in PtdEtn and an ∼45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content. CONCLUSIONS: The PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass.

In vivo cardiac glucose metabolism in the high-fat fed mouse: Comparison of euglycemic-hyperinsulinemic clamp derived measures of glucose uptake with a dynamic metabolomic flux profiling approach.

RATIONALE: Cardiac metabolism is thought to be altered in insulin resistance and type 2 diabetes (T2D). Our understanding of the regulation of cardiac substrate metabolism and insulin sensitivity has largely been derived from ex vivo preparations which are not subject to the same metabolic regulation as in the intact heart in vivo. Studies are therefore required to examine in vivo cardiac glucose metabolism under physiologically relevant conditions. OBJECTIVE: To determine the temporal pattern of the development of cardiac insulin resistance and to compare with dynamic approaches to interrogate cardiac glucose and intermediary metabolism in vivo. METHODS AND RESULTS: Studies were conducted to determine the evolution of cardiac insulin resistance in C57Bl/6 mice fed a high-fat diet (HFD) for between 1 and 16 weeks. Dynamic in vivo cardiac glucose metabolism was determined following oral administration of [U-(13)C] glucose. Hearts were collected after 15 and 60 min and flux profiling was determined by measuring (13)C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Cardiac insulin resistance, determined by euglycemic-hyperinsulinemic clamp, was evident after 3 weeks of HFD. Despite the presence of insulin resistance, in vivo cardiac glucose metabolism following oral glucose administration was not compromised in HFD mice. This contrasts our recent findings in skeletal muscle, where TCA cycle activity was reduced in mice fed a HFD. Similar to our report in muscle, glucose derived pyruvate entry into the TCA cycle in the heart was almost exclusively via pyruvate dehydrogenase, with pyruvate carboxylase mediated anaplerosis being negligible after oral glucose administration. CONCLUSIONS: Under experimental conditions which closely mimic the postprandial state, the insulin resistant mouse heart retains the ability to stimulate glucose metabolism.

The CDP-Ethanolamine Pathway Regulates Skeletal Muscle Diacylglycerol Content and Mitochondrial Biogenesis without Altering Insulin Sensitivity.

Accumulation of diacylglycerol (DG) in muscle is thought to cause insulin resistance. DG is a precursor for phospholipids, thus phospholipid synthesis could be involved in regulating muscle DG. Little is known about the interaction between phospholipid and DG in muscle; therefore, we examined whether disrupting muscle phospholipid synthesis, specifically phosphatidylethanolamine (PtdEtn), would influence muscle DG content and insulin sensitivity. Muscle PtdEtn synthesis was disrupted by deleting CTP:phosphoethanolamine cytidylyltransferase (ECT), the rate-limiting enzyme in the CDP-ethanolamine pathway, a major route for PtdEtn production. While PtdEtn was reduced in muscle-specific ECT knockout mice, intramyocellular and membrane-associated DG was markedly increased. Importantly, however, this was not associated with insulin resistance. Unexpectedly, mitochondrial biogenesis and muscle oxidative capacity were increased in muscle-specific ECT knockout mice and were accompanied by enhanced exercise performance. These findings highlight the importance of the CDP-ethanolamine pathway in regulating muscle DG content and challenge the DG-induced insulin resistance hypothesis.

Application of dynamic metabolomics to examine in vivo skeletal muscle glucose metabolism in the chronically high-fat fed mouse.

RATIONALE: Defects in muscle glucose metabolism are linked to type 2 diabetes. Mechanistic studies examining these defects rely on the use of high fat-fed rodent models and typically involve the determination of muscle glucose uptake under insulin-stimulated conditions. While insightful, they do not necessarily reflect the physiology of the postprandial state. In addition, most studies do not examine aspects of glucose metabolism beyond the uptake process. Here we present an approach to study rodent muscle glucose and intermediary metabolism under the dynamic and physiologically relevant setting of the oral glucose tolerance test (OGTT). METHODS AND RESULTS: In vivo muscle glucose and intermediary metabolism was investigated following oral administration of [U-(13)C] glucose. Quadriceps muscles were collected 15 and 60 min after glucose administration and metabolite flux profiling was determined by measuring (13)C mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates via gas chromatography-mass spectrometry. While no dietary effects were noted in the glycolytic pathway, muscle from mice fed a high fat diet (HFD) exhibited a reduction in labelling in TCA intermediates. Interestingly, this appeared to be independent of alterations in flux through pyruvate dehydrogenase. In addition, our findings suggest that TCA cycle anaplerosis is negligible in muscle during an OGTT. CONCLUSIONS: Under the dynamic physiologically relevant conditions of the OGTT, skeletal muscle from HFD fed mice exhibits alterations in glucose metabolism at the level of the TCA cycle.

Overexpression of sphingosine kinase 1 in liver reduces triglyceride content in mice fed a low but not high-fat diet.

Hepatic insulin resistance is a major risk factor for the development of type 2 diabetes and is associated with the accumulation of lipids, including diacylglycerol (DAG), triacylglycerols (TAG) and ceramide. There is evidence that enzymes involved in ceramide or sphingolipid metabolism may have a role in regulating concentrations of glycerolipids such as DAG and TAG. Here we have investigated the role of sphingosine kinase (SphK) in regulating hepatic lipid levels. We show that mice on a high-fat high-sucrose diet (HFHS) displayed glucose intolerance, elevated liver TAG and DAG, and a reduction in total hepatic SphK activity. Reduced SphK activity correlated with downregulation of SphK1, but not SphK2 expression, and was not associated with altered ceramide levels. The role of SphK1 was further investigated by overexpressing this isoform in the liver of mice in vivo. On a low-fat diet (LFD) mice overexpressing liver SphK1, displayed reduced hepatic TAG synthesis and total TAG levels, but with no change to DAG or ceramide. These mice also exhibited no change in gluconeogenesis, glycogenolysis or glucose tolerance. Similarly, overexpression of SphK1 had no effect on the pattern of endogenous glucose production determined during a glucose tolerance test. Under HFHS conditions, normalization of liver SphK activity to levels observed in LFD controls did not alter hepatic TAG concentrations. Furthermore, DAG, ceramide and glucose tolerance were also unaffected. In conclusion, our data suggest that SphK1 plays an important role in regulating TAG metabolism under LFD conditions.

Transforming growth factor-ß signalling: role and consequences of Smad linker region phosphorylation.

Transforming growth factor-ß (TGF-ß) is a secreted homodimeric protein that plays an important role in regulating various cellular responses including cell proliferation and differentiation, extracellular matrix production, embryonic development and apoptosis. Disruption of the TGF-ß signalling pathway is associated with diverse disease states including cancer, renal and cardiac fibrosis and atherosclerosis. At the cell surface TGF-ß complex consists of two type I and two type II transmembrane receptors (TßRI and TßRII respectively) which have serine/threonine kinase activity. Upon TGF-ß engagement TßRII phosphorylates TßRI which in turn phosphorylates Smad2/3 on two serine residues at their C-terminus which enables binding to Smad4 to form heteromeric Smad complexes that enter the nucleus to initiate gene transcription including for extracellular matrix proteins. TGF-ß signalling is also known to activate other serine/threonine kinase signalling that results in the phosphorylation of the linker region of Smad2. The Smad linker region is defined as the domain which lies between the MH1 and MH2 domains of a Smad protein. Serine/threonine kinases that are known to phosphorylate the Smad linker region include mitogen-activated protein kinases, extracellular-signal regulated kinase, Jun N-terminal kinase and p38 kinase, the tyrosine kinase Src, phosphatidylinositol 3'-kinase, cyclin-dependent kinases, rho-associated protein kinase, calcium calmodulin-dependent kinase and glycogen synthase kinase-3. This review will cover the role of Smad linker region phosphorylation downstream of TGF-ß signalling in vascular cells. Key factors including the identification of the kinases that phosphorylate individual Smad residues, the upstream agents that activate these kinases, the cellular location of the phosphorylation event and the importance of the linker region in regulation and expression of genes induced by TGF-ß are covered.

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)-ß. 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.

Thrombin-mediated proteoglycan synthesis utilizes both protein-tyrosine kinase and serine/threonine kinase receptor transactivation in vascular smooth muscle cells.

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, ß-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-ß receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.

G protein coupled receptor transactivation: extending the paradigm to include serine/threonine kinase receptors.

The current paradigm of G protein coupled receptor signaling involves a classical pathway being the activation of phospholipase C and the generation of 1,4,5-inositol trisphosphate, signaling through ß-arrestin scaffold molecules and the transactivation of tyrosine kinase growth factor receptors. Transactivation greatly expands the range of signaling pathways and responses attributable to the receptor. Recently it has been revealed that G protein coupled receptor agonists can also transactivate the serine/threonine kinase cell surface receptor for transforming growth factor-ß (Alk5). This leads to the generation of carboxyl terminal phosphorylated Smad2 which is the immediate downstream product of the activated Alk5. Thus, the current paradigm of G protein coupled signaling can be expanded to include the transactivation of the serine kinase receptor Alk5. These insights expand the possibilities for outcomes of therapeutically targeting GPCRs where more substantive and prolonged actions such as the synthesis of extracellular matrix may be affected.

The paradigm of G protein receptor transactivation: a mechanistic definition and novel example.

Seven transmembrane G protein-coupled receptors are among the most common in biology and they transduce cellular signals from a plethora of hormones. As well as their own well-characterized signaling pathways, they can also transactivate tyrosine kinase growth factor receptors to greatly expand their own cellular repertoire of responses. Recent data in vascular smooth muscle cells have expanded the breadth of transactivation to include serine/threonine kinase receptors, specifically the receptor for transforming growth factor beta (TGF-beta). Stimulation with endothelin and thrombin leads to the rapid formation of C-terminal phosphorylated Smad2, which is the immediate product of activation of the TGF-beta receptor. Additionally, it appears that no definition of transactivation based on mechanism is available, so we have provided a definition involving temporal aspects and the absence of de novo protein synthesis. The phenomenon of transactivation is an important biochemical mechanism and potential drug target in multiple diseases.

Smad linker region phosphorylation in the regulation of extracellular matrix synthesis.

The canonical TGF-ß signalling pathway involves Smad transcription factors through direct serine phosphorylation of the carboxy termini, nuclear translocation and regulation of transcription by receptor-regulated (R)-Smad complexes. Smads can also be phosphorylated in the linker region most prominently by the action of mitogen-activated protein (MAP) kinases, which in turn have been activated by TGF-ß or a multitude of other growth factors and hormones. Linker region phosphorylation can prevent nuclear translocation of Smads and inhibit TGF-ß signalling, potentially leading to oncogenesis. However, some evidence has revealed that linker region phosphorylated Smads can be translocated to the nucleus where they regulate transcription particularly of the synthesis of extracellular matrix molecules. Matrix molecules such as collagen and proteoglycans are involved in diseases such a fibrosis and atherosclerosis, respectively, and the involvement of linker region phosphorylation may represent a new therapeutic target.

Transforming growth factor-ß regulation of proteoglycan synthesis in vascular smooth muscle: contribution to lipid binding and accelerated atherosclerosis in diabetes.

Atherosclerosis is accelerated in the setting of diabetes, but the factors driving this phenomenon remain elusive. Hyperglycemia leads to elevated levels of transforming growth factor (TGF)-ß and TGF-ß has been implicated as a factor in atherosclerosis. Given the established association between hyperglycemia and elevated TGF-ß, it is plausible that elevated TGF-ß levels in diabetes play a pathogenic role in the development of accelerated atherosclerosis. TGF-ß is a potent regulator of extracellular matrix synthesis, including many actions on proteoglycan synthesis that lead to increased binding to low-density lipoprotein and therefore potentially increased lipid retention in the vessel wall and accelerated atherosclerosis. TGF-ß signals through the canonical TGF-ß receptor I-mediated phosphorylation of Smad transcription factors and TGF-ß signaling is also known to involve, positively and negatively, interactions with the mitogen-activated protein kinase pathways. The focus of the present review is on the effects of TGF-ß on proteoglycan synthesis in vascular smooth muscle and particularly the signaling pathways through which TGF-ß exerts its effects, because those pathways may be therapeutic targets for the prevention of pathological modifications in the proteoglycan component of the vessel wall in the vascular diseases of diabetes.

Endothelin-1 stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by endothelin receptor transactivation of the transforming growth factor-[beta] type I receptor.

We utilized human vascular smooth muscle cells to address the question if a G-protein-coupled receptor, the endothelin (ET) receptor, could transactivate a serine/threonine kinase receptor, specifically the transforming growth factor (TGF)-[beta] receptor, T[beta]RI. Functionality of the interaction was addressed by studying endothelin-1-stimulated proteoglycan synthesis. Signaling molecules were assessed by Western blotting and proteoglycan synthesis by [35S]sulfate and 35S-met/cys incorporation and molecular size by SDS-PAGE. Endothelin-1 treatment led to a time- and concentration-dependent increase in cytosolic phosphoSmad2C, which was inhibited by the mixed endothelin receptor antagonist bosentan and the T[beta]RI antagonist SB431542. Endothelin-1 treatment led to a time-dependent increase in nuclear phosphoSmad2C. Endothelin-1-stimulated proteoglycan synthesis was partially inhibited (40%) by SB431542 and completely blocked by bosentan. The effect of endothelin-1 to stimulate an increase in glycosaminoglycan size on biglycan was also blocked in a concentration-dependent manner by SB431542. These data extend the current paradigm of G-protein coupled receptor signaling to include the transactivation of the serine kinase receptor for TGF-[beta] (T[beta]RI). This response should be considered in the context of response to endothelin-1, and the options for therapeutically targeting endothelin-1 are accordingly broadened to include downstream signaling otherwise associated with TGF-[beta] receptor activation.

PDGF beta-receptor kinase activity and ERK1/2 mediate glycosaminoglycan elongation on biglycan and increases binding to LDL.

The initiation of atherosclerosis involves the subendothelial retention of lipoproteins by proteoglycans (PGs). Structural characteristics of glycosaminoglycan (GAG) chains on PGs influence lipoprotein binding and are altered adversely by platelet-derived growth factor (PDGF). The signaling pathway for PDGF-mediated GAG elongation via the PDGF receptor (PDGFR) was investigated. In human vascular smooth muscle cells, PDGF significantly increased (35)S-sulfate incorporation into PGs and GAG chain size. PGs from PDGF-stimulated cells showed increased binding low-density lipoprotein (P < 0.001) in gel mobility shift assays. Knockdown of PDGFRbeta using small interfering RNA demonstrated that PDGF mediated changes in PGs via PDGFRbeta. GAG synthesis and hyperelongation was blocked by inhibition of receptor tyrosine kinase autophosphorylation site Tyr857 activity using Ki11502 or imatinib. Downstream signaling to GAG hyperelongation was mediated through ERK MAPK and not phosphatidylinositol-3 kinase or phospholipase Cgamma. In high-fat-fed apolipoprotein E(-/-) mice, inhibition of PDGFRbeta activity by imatinib reduced aortic total lipid staining area by 35% (P < 0.05). Inhibition of PDGFRbeta tyrosine kinase activity leads to inhibition of GAG synthesis on vascular PGs and aortic lipid area in vivo. PDGFRbeta and its signaling pathways are potential targets for novel therapeutic agents to prevent the earliest stages atherosclerosis.

Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type I receptor.

Growth factors modify the structure of the glycosaminoglycan (GAG) chains on biglycan leading to enhanced LDL binding. G-protein receptor-coupled agonists such as thrombin, signal changes the structure of proteoglycans produced by vascular smooth muscle cells (VSMCs). One component of classical G-protein-coupled receptor (GPCR) signaling invokes transactivation of protein tyrosine kinase receptors such as the epidermal growth factor receptor. Serine/threonine receptor growth factors such as transforming growth factor-(TGF)-beta are potent activators of proteoglycan synthesis. We have used the model of proteoglycan synthesis to demonstrate that the signaling paradigm of GPCR signaling can be extended to include the transactivation of serine/threonine receptor, specifically the TGF-beta type I receptor (TbetaRI) also known as activin-like kinase (ALK) V. Thrombin stimulated elongation of GAG chains and increased proteoglycan core protein expression and these responses were blocked by the TbetaRI antagonist, SB431542 and TbetaRI siRNA knockdown, as well as several protease-activated receptor (PAR)-1 antagonists. The canonical downstream response to TGF-beta is increased C-terminal phosphorylation of the transcription factor Smad2 generating phospho-Smad2C (phosphorylation of Smad2 C-terminal region). Thrombin stimulated increased phospho-Smad2C levels, and the response was blocked by SB431542 and JNJ5177094. The proteolytically inactive thrombin mimetic thrombin-receptor activating peptide also stimulated an increase in cytosolic phospho-Smad2C. Signaling pathways for growth factor regulated proteoglycan synthesis represent therapeutic targets for the prevention of atherosclerosis, but the novel finding of a GPCR-mediated transactivation of a serine/threonine growth factor receptor almost certainly has implications well beyond the synthesis of proteoglycans.

TGF-beta stimulates biglycan synthesis via p38 and ERK phosphorylation of the linker region of Smad2.

Transforming growth factor (TGF)-beta treatment of human vascular smooth-muscle cells increases the expression of biglycan and causes marked elongation of its glycosaminoglycan (GAG) chains. We investigated the role of MAP kinases and Smad transcription factors in this response. TGF-beta-stimulated phosphorylation of p38, ERK, and JNK as well as Smad2 at both its carboxy terminal (phospho-Smad2C) and in the linker region (phospho-Smad2L). Pharmacological inhibition of ERK and p38 blocked TGF-beta-mediated GAG elongation and expression of biglycan whereas inhibition of JNK had no effect. Inhibition of ERK and p38 but not JNK attenuated the effect of TGF-beta to increase phospho-Smad2L. High levels of phospho-Smad2L were detected in a nuclear fraction of TGF-beta treated cells. Thus, MAP kinase signaling through ERK and p38 and via phosphorylation of the linker region of Smad2 mediates the effects of TGF-beta on biglycan synthesis in vascular smooth-muscle cells.

Characterisation of Ki11502 as a potent inhibitor of PDGF beta receptor-mediated proteoglycan synthesis in vascular smooth muscle cells.

Platelet-derived growth factor (PDGF) receptor signalling is implicated in cardiovascular diseases such as atherosclerosis and restenosis. PDGF expression levels are elevated in atherosclerotic lesions and play a key role in migration and proliferation of vascular smooth muscle cells in the neointima. PDGF stimulates glycosaminoglycan elongation on vascular proteoglycans biglycan and decorin, a process implicated in the aetiology of atherosclerosis. We investigated the ability of the specific kinase inhibitor Ki11502 to inhibit PDGF beta receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. Ki11502 inhibited PDGF-mediated tyrosine phosphorylation of the PDGF beta receptor autophosphorylation site and at least six other receptor-associated proteins. Ki11502 also caused a concentration-dependent inhibition of PDGF-stimulated [(3)H]-thymidine incorporation. Total proteoglycan synthesis was assessed as incorporation of [(35)S]-sulfate. PDGF-induced a two-fold increase in [(35)S]-sulfate incorporation into proteoglycans secreted over 24h and was inhibited in a concentration-dependent manner by Ki11502. PDGF treatment resulted in a statistically significant (P<0.01) increase in total proteoglycan core protein secretion. Treatment of cells with Ki11502 (300 nM) had no effect on basal core protein secretion and completely abolished the PDGF-stimulated component. Analysis of isolated cleaved glycosaminoglycan chains by size-exclusion chromatography demonstrated that PDGF stimulated the synthesis and secretion of proteoglycans with elongated glycosaminoglycan chains and this effect was inhibited by Ki11502. Inhibition was also seen in the length of xyloside-glycosaminoglycan chains. The results demonstrate that Ki11502 is a potent and selective inhibitor of PDGF beta receptor phosphorylation, proliferation and proteoglycan synthesis in human vascular smooth muscle cells.

Growth factor-mediated hyper-elongation of glycosaminoglycan chains on biglycan requires transcription and translation.

The mechanism through which growth factors cause glycosaminoglycan (GAG) hyper-elongation is unclear. We have investigated the role of transcription and translation on the GAG hyper-elongation effect of platelet-derived growth factor (PDGF) in human vascular smooth muscle cells (VSMCs). To determine if the response involves specific signalling pathways or the process of GAG hyper-elongation we have also investigated the effects of epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta) and thrombin. We report that both actinomycin D and cycloheximide completely abolished the ability of PDGF to stimulate radiosulphate incorporation and GAG elongation into secreted proteoglycans, and to increase the size of xyloside GAGs. Blocking de novo protein synthesis completely prevented the action of all growth factors tested to elongate GAG chains. These results lay a foundation for further investigation into the genes and proteins implicated in this response.

p38 MAP kinase mediated proteoglycan synthesis as a target for the prevention of atherosclerosis.

The major underlying pathology of most cardiovascular disease is the chronic inflammatory disease of atherosclerosis. Type 2 diabetes, also recognised as an inflammatory condition, accelerates the development of atherosclerosis. Current therapies for atherosclerosis target risk factors such as elevated blood lipids and hypertension and are of strong but limited efficacy. The "response to retention" hypothesis states that atherosclerosis is initiated by the accumulation of lipids through binding to extracellular matrix, and this is specifically the glycosaminoglycan (GAG) chains on proteoglycans. Many vasoactive agonists stimulate changes in the structure of the GAGs which increase lipid binding and the relevant signalling pathways are a potential therapeutic target. It has recently been demonstrated that the actions of transforming growth factor beta on vascular smooth muscle proteoglycan synthesis involves signalling through p38 MAP kinase and inhibition of this pathway reduces binding of lipids. Inhibition of p38 MAP kinase will elicit a wide spread anti-inflammatory response which may alleviate some of the deleterious processes in cardiovascular tissues. This article explores the potential for the actions of p38 MAP kinase inhibitors directed at proteoglycan synthesis in vascular smooth muscle to contribute to the beneficial outcomes from targeting p38 MAP kinase for the prevention of cardiovascular disease.