Kirsten Riches

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.

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 KV1 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. KV1.3 was unique among the KV1 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 KV1.3 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 IC50 of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. Conclusion KV1.3 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.

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 Gi/o proteins but not on previously identified G protein-coupled receptors for oxidized phospholipids. Conclusion—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.

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 (TGFβ) 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.

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% O2) or hypoxia (1% O2) 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.

Hypoxic inhibition of human cardiac fibroblast invasion and MMP-2 activation may impair adaptive myocardial remodelling

Cardiac fibroblasts account for up to two-thirds of the total number of cells in the normal heart and are responsible for extracellular matrix homoeostasis. In vitro, type I collagen, the predominant myocardial collagen, stimulates proteolytic activation of constitutively secreted proMMP-2 (pro-matrix metalloproteinase-2). This occurs at the cell membrane and requires formation of a ternary complex with MT1-MMP (membrane-type-1 MMP) and TIMP-2 (tissue inhibitor of metalloproteinases-2). Following MI (myocardial infarction), normally quiescent fibroblasts initiate a wound healing response by transforming into a proliferative and invasive myofibroblast phenotype. Deprivation of oxygen to the myocardium is an inevitable consequence of MI; therefore this reparative event occurs under chronically hypoxic conditions. However, species and preparation variations can strongly influence fibroblast behaviour, which is an important consideration when selecting experimental models for provision of clinically useful information.

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.

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.

CCAAT/enhancer binding protein α, β and δ 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 α, β and δ ( 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.

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.