Insulinlike Growth Factor-Binding Protein-1 Improves Vascular Endothelial Repair in Male Mice in the Setting of Insulin Resistance.

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.

Linking energy sensing to suppression of JAK-STAT signalling: A potential route for repurposing AMPK activators?

Exaggerated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling is key to the pathogenesis of pro-inflammatory disorders, such as rheumatoid arthritis and cardiovascular diseases. Mutational activation of JAKs is also responsible for several haematological malignancies, including myeloproliferative neoplasms and acute lymphoblastic leukaemia. Accumulating evidence links adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), an energy sensor and regulator of organismal and cellular metabolism, with the suppression of immune and inflammatory processes. Recent studies have shown that activation of AMPK can limit JAK-STAT-dependent signalling pathways via several mechanisms. These novel findings support AMPK activation as a strategy for management of an array of disorders characterised by hyper-activation of the JAK-STAT pathway. This review discusses the pivotal role of JAK-STAT signalling in a range of disorders and how both established clinically used and novel AMPK activators might be used to treat these conditions.

Progressive Development of Aberrant Smooth Muscle Cell Phenotype in Abdominal Aortic Aneurysm Disease.

Abdominal aortic aneurysm (AAA) is a silent, progressive disease with a high mortality and an increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA (≥5 cm) and cells cultured from a porcine carotid artery (PCA) model of early and end-stage aneurysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of the differentiation marker miR-145 (2.2-fold, p < 0.001) and the senescence marker SIRT-1 (1.3-fold, p < 0.05), features not recapitulated in aneurysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, p < 0.001, and 1.8-fold, p < 0.01, respectively, vs. control cells) and displayed an aberrant nuclear morphology. Human AAA-SMC exhibited higher levels of the DNA damage marker γH2AX (3.9-fold, p < 0.01, vs. control SMC). These features did not correlate with patients' chronological age and are therefore potential markers for pathological premature vascular aging. Early-stage PCA-SMC (control and aneurysmal) were indistinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at the end stage of the disease. Refinement of a porcine bioreactor model would facilitate the study of temporal modulation of SMC behaviour during aneurysm development and potentially identify therapeutic targets to limit AAA progression.

Mapping the methylation status of the miR-145 promoter in saphenous vein smooth muscle cells from individuals with type 2 diabetes.

Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus-smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus-smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs -112 and -106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus-smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR-145 observed in type 2 diabetes mellitus-smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.

Vascular Smooth Muscle as a Target for Novel Therapeutics.

Cardiovascular disease is the principal cause of death in patients with type 2 diabetes (T2DM). Exposure of the vasculature to metabolic disturbances leaves a persistent imprint on vascular walls, and specifically on smooth muscle cells (SMC) that favours their dysfunction and potentially underlies macrovascular complications of T2DM. Current diabetes therapies and continued development of newer treatments has led to the ability to achieve more efficient glycaemic control. There is also some evidence to suggest that some of these treatments may exert favourable pleiotropic effects, some of which may be at the level of SMC. However, emerging interest in epigenetic markers as determinants of vascular disease, and a putative link with diabetes, opens the possibility for new avenues to develop robust and specific new therapies. These will likely need to target cell-specific epigenetic changes such as effectors of DNA histone modifications that promote or inhibit gene transcription, and/or microRNAs capable of regulating entire cellular pathways through target gene repression. The growing epidemic of T2DM worldwide, and its attendant cardiovascular mortality, dictates a need for novel therapies and personalised approaches to ameliorate vascular complications in this vulnerable population.

Aberrant phenotype in human endothelial cells of diabetic origin: implications for saphenous vein graft failure?

Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate) to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30%) and angiogenesis (~40%) compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp.), effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.

Elevated expression levels of miR-143/5 in saphenous vein smooth muscle cells from patients with Type 2 diabetes drive persistent changes in phenotype and function.

Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFß) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.

Investigating inherent functional differences between human cardiac fibroblasts cultured from nondiabetic and Type 2 diabetic donors.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) promotes adverse myocardial remodeling and increased risk of heart failure; effects that can occur independently of hypertension or coronary artery disease. As cardiac fibroblasts (CFs) are key effectors of myocardial remodeling, we investigated whether inherent phenotypic differences exist in CF derived from T2DM donors compared with cells from nondiabetic (ND) donors. METHODS: Cell morphology (cell area), proliferation (cell counting over 7-day period), insulin signaling [phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) Western blotting], and mRNA expression of key remodeling genes [real-time reverse transcription-polymerase chain reaction (RT-PCR)] were compared in CF cultured from atrial tissue from 14 ND and 12 T2DM donors undergoing elective coronary artery bypass surgery. RESULTS: The major finding was that Type I collagen (COL1A1) mRNA levels were significantly elevated by twofold in cells derived from T2DM donors compared with those from ND donors; changes reflected at the protein level. T2DM cells had similar proliferation rates but a greater variation in cell size and a trend towards increased cell area compared with ND cells. Insulin-induced Akt and ERK phosphorylation were similar in the two cohorts of cells. CONCLUSION: CF from T2DM individuals possess an inherent profibrotic phenotype that may help to explain the augmented cardiac fibrosis observed in diabetic patients. MINI SUMMARY: We investigated whether inherent phenotypic differences exist between CF cultured from donors with or without Type 2 diabetes. Cell morphology, proliferation, insulin signaling, and gene expression were compared between multiple cell populations. The major finding was that Type I collagen levels were elevated in fibroblasts from diabetic donors, which may help explain the augmented cardiac fibrosis observed with diabetes.

Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: possible role of differential RhoA activity.

BACKGROUND/PURPOSE: Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. METHODS/MATERIALS: SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. RESULTS: IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P<0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P<0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P<0.05 and 43% reduction in activity, P<0.01). CONCLUSIONS: IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients. SUMMARY: The internal mammary artery (IMA) is the conduit of choice for bypass grafting and is generally successful in all patients, including those with type 2 diabetes (T2DM). By contrast, saphenous vein (SV) is inferior to IMA and furthermore patients with T2DM suffer strikingly poorer outcomes than their non-diabetic (ND) counterparts. We discovered that SV-SMC from T2DM patients exhibit altered persistent morphology and function compared to ND SV-SMC, with differential expression and activity of the small GTPase RhoA, yet ND and T2DM IMA-SMC were indistinguishable. These data offer an explanation for the superior patency of IMA grafting independent of the presence of diabetes.

Exploring smooth muscle phenotype and function in a bioreactor model of abdominal aortic aneurysm.

BACKGROUND: Vascular smooth muscle cells (SMC) are central to arterial structure and function yet their involvement in the progression of abdominal aortic aneurysm (AAA) disease is not well studied. The progressive and silent nature of AAA in man essentially restricts research to the use of "end-stage" tissue recovered during surgical repair. This study aimed to generate an ex vivo model of AAA using protease-treated porcine carotid arteries maintained in a novel bioreactor, and to compare the structural and functional changes in SMC cultured from the recovered vessels with those from human tissue acquired at elective surgical repair. METHODS: Freshly isolated porcine arteries were pretreated with collagenase and/or elastase before culturing under flow in a bioreactor for 12 days. Human end-stage aneurysmal tissue and saphenous veins from age-matched controls were collected from patients undergoing surgery. SMC were cultured and characterised (immunocytochemistry, measurement of spread cell area) and assessed functionally at the level of proliferation (cell-counting) and matrix-metalloproteinase (MMP) secretion (gelatin zymography). Cellular senescence was investigated using ß-galactosidase staining and apoptosis was quantified using a fluorescence-based caspase 3 assay. RESULTS: Co-expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain confirmed all cell populations as SMC. Porcine SMC harvested and cultivated after collagenase/elastase pretreatment displayed a prominent "rhomboid" morphology, increased spread area (32%, P < 0.01), impaired proliferation (47% reduction, P < 0.05), increased senescence (52%, P < 0.001), susceptibility to apoptosis and reduced MMP-2 secretion (60% decrease, P < 0.01) compared with SMC from vehicle, collagenase or elastase pre-treated vessels. Notably, these changes were comparable to those observed in human AAA SMC which were 2.4-fold larger than non-aneurysmal SMC (P < 0.001) and exhibited reduced proliferation (39% reduction, P < 0.001), greater apoptosis (4-fold increase, P < 0.001), and increased senescence (61%, P < 0.05). CONCLUSIONS: Combined collagenase/elastase exposure of porcine artery maintained in a bioreactor under flow conditions induced a SMC phenotype characteristic of those cultured from end-stage AAA specimens. This model has potential and versatility to examine temporal changes in SMC biology and to identify the molecular mechanisms leading to early aberrancies in SMC function. In the longer term this may inform new targets to maintain aortic SMC content and drive cells to a "reparative" phenotype at early stages of the disease.

Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVß3 and RhoA/ROCK-mediated mechanisms.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVß3-dependent, but transforming growth factor ß-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVß3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.

The vascular smooth muscle cell: a therapeutic target in Type 2 diabetes?

The rising epidemic of T2DM (Type 2 diabetes mellitus) worldwide is of significant concern. The inherently silent nature of the disease in its early stages precludes early detection; hence cardiovascular disease is often established by the time diabetes is diagnosed. This increased cardiovascular risk leads to significant morbidity and mortality in these individuals. Progressive development of complications as a result of previous exposure to metabolic disturbances appears to leave a long-lasting impression on cells of the vasculature that is not easily reversed and is termed 'metabolic memory'. SMCs (smooth muscle cells) of blood vessel walls, through their inherent ability to switch between a contractile quiescent phenotype and an active secretory state, maintain vascular homoeostasis in health and development. This plasticity also confers SMCs with the essential capacity to adapt and remodel in pathological states. Emerging clinical and experimental studies propose that SMCs in diabetes may be functionally impaired and thus contribute to the increased incidence of macrovascular complications. Although this idea has general support, the underlying molecular mechanisms are currently unknown and hence are the subject of intense research. The aim of the present review is to explore and evaluate the current literature relating to the problem of vascular disease in T2DM and to discuss the critical role of SMCs in vascular remodelling. Possibilities for therapeutic strategies specifically at the level of T2DM SMCs, including recent novel advances in the areas of microRNAs and epigenetics, will be evaluated. Since restoring glucose control in diabetic patients has limited effect in ameliorating their cardiovascular risk, discovering alternative strategies that restrict or reverse disease progression is vital. Current research in this area will be discussed.

Lipoprotein(a): Cellular Effects and Molecular Mechanisms.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease (CVD). Indeed, individuals with plasma concentrations >20 mg/dL carry a 2-fold increased risk of developing CVD, accounting for ~25% of the population. Circulating levels of Lp(a) are remarkably resistant to common lipid lowering therapies, and there are currently no robust treatments available for reduction of Lp(a) apart from plasma apheresis, which is costly and labour intensive. The Lp(a) molecule is composed of two parts, an LDL/apoB-100 core and a unique glycoprotein, apolipoprotein(a) (apo(a)), both of which can interact with components of the coagulation cascade, inflammatory pathways, and cells of the blood vessel wall (smooth muscle cells (SMC) and endothelial cells (EC)). Therefore, it is of key importance to determine the molecular pathways by which Lp(a) exerts its influence on the vascular system in order to design therapeutics to target its cellular effects. This paper will summarise the role of Lp(a) in modulating cell behaviour in all aspects of the vascular system including platelets, monocytes, SMC, and EC.

Divergent effects of 17-ß-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-α-induced neointima formation.

Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α). TNF-α can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-α on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-α (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-α, an effect that was abolished by co-culture with E2. TNF-α increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-α alone. SV-EC migration was significantly impaired by TNF-α (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-α increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-α potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-α induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers.

AIM: The aim of the study was to determine the potential for K(V)1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. METHODS AND RESULTS: Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptase-polymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function. K(V)1.3 was unique among the K(V)1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of K(V)1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC(50) of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. CONCLUSION: K(V)1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events.

CCAAT/enhancer binding protein alpha, beta and delta gene variants: associations with obesity related phenotypes in the Leeds Family Study.

OBJECTIVE: To identify novel polymorphisms in the genes encoding the transcription factors CCAAT/enhancer binding protein alpha, beta and delta ( CEBPA, CEBPB, CEBPD) and investigate associations between polymorphisms and obesity-related phenotypes. METHODS: Denaturing high-performance liquid chromatography (HPLC) was used to screen for novel gene variants and polymorphisms were genotyped in stored DNA from participants of the Leeds Family Study (537 subjects from 89 families). Genotype and haplotype analyses were carried out in STATA and PBAT, respectively. RESULTS: Twenty-five polymorphisms were identified; 11 in CEBPA, 12 in CEBPB and 2 in CEBPD. Several allelic variants were associated at a nominal 5% level with waist-to-hip ratio (-919G>A in CEBPA, -412G>T and 646C>T in CEBPB), leptin (1558G>A in CEBPA, -1051A>G and 1383T>- in CEBPB) and adiponectin (1382G>T and 1903G>T in CEBPB). Effects of CEBPA and CEBPB allelic variants were independent, but variants within each gene were in linkage disequilibrium. Several associations were observed between other obesity-related traits and allelic variants in CEBPA and CEBPB, but not CEBPD. CONCLUSION: These findings suggest that common allelic variants in CEBPA and CEBPB could influence abdominal obesity and related metabolic abnormalities associated with type 2 diabetes and cardiovascular disease in healthy White Northern European families, although results require independent confirmation.

Short-term stimulation of calcium-permeable transient receptor potential canonical 5-containing channels by oxidized phospholipids.

OBJECTIVE: To determine whether calcium-permeable channels are targets for the oxidized phospholipids: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC). METHODS AND RESULTS: Oxidized phospholipids are key factors in inflammation and associated diseases, including atherosclerosis; however, the initial reception mechanisms for cellular responses to the factors are poorly understood. Low micromolar concentrations of PGPC and POVPC evoked increases in intracellular calcium in human embryonic kidney 293 cells that overexpressed human transient receptor potential canonical 5 (TRPC5) but not human TRP melastatin (TRPM) 2 or 3. The results of electrophysiological experiments confirmed stimulation of TRPC5. To investigate relevance to endogenous channels, we studied proliferating vascular smooth muscle cells from patients undergoing coronary artery bypass surgery. PGPC and POVPC elicited calcium entry that was inhibited by anti-TRPC5 or anti-TRPC1 antibodies or dominant-negative mutant TRPC5. Calcium release did not occur. The effect was functionally relevant because it enhanced cell migration. The actions of PGPC and POVPC depended on G(i/o) proteins but not on previously identified G protein-coupled receptors for oxidized phospholipids. CONCLUSIONS: Stimulation of calcium-permeable TRPC5-containing channels may be an early event in cellular responses to oxidized phospholipids that couples to cell migration and requires an unidentified G protein-coupled receptor.

Inherent differences in morphology, proliferation, and migration in saphenous vein smooth muscle cells cultured from nondiabetic and Type 2 diabetic patients.

Individuals with Type 2 diabetes mellitus (T2DM) are at increased risk of saphenous vein (SV) graft stenosis following coronary artery bypass. Graft stenosis is caused by intimal hyperplasia, a pathology characterized by smooth muscle cell (SMC) proliferation and migration. We hypothesized that SV-SMC from T2DM patients were intrinsically more proliferative and migratory than those from nondiabetic individuals. SV-SMC were cultured from nondiabetic and T2DM patients. Cell morphology (light microscopy, immunocytochemistry), S100A4 expression (real-time RT-PCR, immunoblotting), proliferation (cell counting), migration (Boyden chamber assay), and cell signaling (immunoblotting with phosphorylation state-specific antibodies) were studied. SV-SMC from T2DM patients were morphologically distinct from nondiabetic patients and exhibited a predominantly rhomboid phenotype, accompanied by disrupted F-actin cytoskeleton, disorganized alpha-smooth muscle actin network, and increased focal adhesion formation. However, no differences were observed in expression of the calcium-binding protein S100A4, a marker of rhomboid SMC phenotype, between the two cell populations. T2DM cells were less proliferative in response to fetal calf serum than nondiabetic cells, but both populations had similar proliferative responses to insulin plus PDGF. Under high glucose concentration conditions in the presence of insulin, migration of diabetic SV-SMC was greater than nondiabetic cells. Glucose concentration did not affect SV-SMC proliferation. No differences in insulin or PDGF-induced phosphorylation of ERK-1/2 or components of the Akt pathway (Akt-Ser473, Akt-Thr308, and GSK-3beta) were apparent between the two populations. In conclusion, SV-SMC from T2DM patients differ from nondiabetic SV-SMC in that they exhibit a rhomboid phenotype and are more migratory, but less proliferative, in response to serum.

Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts.

Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O(2)) or hypoxia (1% O(2)) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay. In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P<0.001), paralleled by inhibition of myofibroblast invasion (P<0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P<0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.

The -1562C/T MMP-9 promoter polymorphism does not predict MMP-9 expression levels or invasive capacity in saphenous vein smooth muscle cells cultured from different patients.

Matrix metalloproteinase-9 (MMP-9) is an important regulator of vascular smooth muscle cell (SMC) invasion and proliferation. The T allele of the -1562C/T MMP-9 promoter polymorphism reportedly confers increased MMP-9 promoter activity, plasma MMP-9 levels and susceptibility to vascular pathologies. The aim of this study was to determine whether the MMP-9 -1562C/T polymorphism directly influences endogenous MMP-9 expression levels in saphenous vein (SV) SMC cultured from patients with different genotypes. Genotyping of 408 patients revealed -1562C/T genotype frequencies of 73.3% CC, 25.0% CT and 1.7% TT. Using a standardized, controlled protocol we investigated the effects of phorbol ester (TPA) and a physiological stimulus (PDGF+IL-1) on MMP-9 expression in cultured SV-SMC from 15 CC, 15 CT and 3 TT patients, and on PDGF+IL-1-induced SV-SMC invasion (Boyden chamber with Matrigel barrier). A strong correlation between MMP-9 mRNA levels (real-time RT-PCR) and MMP-9 protein secretion (gelatin zymography) was observed. However, no significant differences were observed in MMP-9 expression levels, or in SV-SMC invasion, between cells with different -1562C/T genotypes. Moreover, MMP-9 promoter activity of the C and T variants was similar. Our data challenge the functional nature of the -1562C/T polymorphism and its capacity to modulate MMP-9 expression levels and SV-SMC invasion, and hence susceptibility to vascular pathologies in vivo.