Birth weight and cardiac function assessed by echocardiography in adolescence: Avon Longitudinal Study of Parents and Children.

OBJECTIVE: Maternal hemodynamics in pregnancy is associated with fetal growth and birth weight, which in turn are associated with offspring cardiovascular disease later in life. The aim of this study was to quantify the extent to which birth weight is associated with cardiac structure and function in adolescence. METHODS: A subset of offspring (n = 1964; 55% female) of the Avon Longitudinal Study of Parents and Children were examined with echocardiography at a mean age of 17.7 (SD, 0.3) years. The associations of birth-weight Z-score for sex and gestational age with cardiac structure (assessed by relative wall thickness, left ventricular mass index (LVMI) and left atrial diameter index), systolic function (assessed by ejection fraction and left ventricular wall velocity) and diastolic function (assessed by early/late mitral inflow velocity (E/A) and early mitral inflow velocity/mitral annular early diastolic velocity (E/e')) were evaluated. Linear regression models were adjusted for several potential confounders, including maternal prepregnancy body mass index, age, level of education and smoking during pregnancy. RESULTS: Higher birth-weight Z-score was associated with lower E/A (mean difference, -0.024; 95% CI, -0.043 to -0.005) and E/e' (mean difference, -0.05; 95% CI, -0.10 to -0.001) and higher LVMI (mean difference, 0.38 g/m2.7 ; 95% CI, 0.09 to 0.67). There was no or inconsistent evidence of associations of birth-weight Z-score with relative wall thickness, left atrial diameter and measurements of systolic function. Further analyses suggested that the association between birth-weight Z-score and LVMI was driven mainly by an association observed in participants born small-for-gestational age and it did not persist when risk factors in adolescence were accounted for. CONCLUSIONS: Higher birth weight adjusted for sex and gestational age was associated with differences in measures of diastolic function in adolescence, but the observed associations were small. It remains to be determined the extent to which these associations translate into increased susceptibility to cardiovascular disease later in life. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Arterial waveform parameters in a large, population-based sample of adults: relationships with ethnicity and lifestyle factors.

Little is known about how aortic waveform parameters vary with ethnicity and lifestyle factors. We investigated these issues in a large, population-based sample. We carried out a cross-sectional analysis of 4798 men and women, aged 50-84 years from Auckland, New Zealand. Participants were 3961 European, 321 Pacific, 266 Maori and 250 South Asian people. We assessed modifiable lifestyle factors via questionnaires, and measured body mass index (BMI) and brachial blood pressure (BP). Suprasystolic oscillometry was used to derive aortic pressure, from which several haemodynamic parameters were calculated. Heavy alcohol consumption and BMI were positively related to most waveform parameters. Current smokers had higher levels of aortic augmentation index than non-smokers (difference=3.7%, P<0.0001). Aortic waveform parameters, controlling for demographics, antihypertensives, diabetes and cardiovascular disease (CVD), were higher in non-Europeans than in Europeans. Further adjustment for brachial BP or lifestyle factors (particularly BMI) reduced many differences but several remained. Despite even further adjustment for mean arterial pressure, pulse rate, height and total:high-density lipoprotein cholesterol, compared with Europeans, South Asians had higher levels of all measured aortic waveform parameters (for example, for backward pressure amplitude: ß=1.5 mm Hg; P<0.0001), whereas Pacific people had 9% higher loge (excess pressure integral) (P<0.0001). In conclusion, aortic waveform parameters varied with ethnicity in line with the greater prevalence of CVD among non-white populations. Generally, this was true even after accounting for brachial BP, suggesting that waveform parameters may have increased usefulness in capturing ethnic variations in cardiovascular risk. Heavy alcohol consumption, smoking and especially BMI may partially contribute to elevated levels of these parameters.

Ethnic differences in cross-sectional associations between impaired glucose regulation, identified by oral glucose tolerance test or HbA1c values, and cardiovascular disease in a cohort of European and South Asian origin.

AIMS: We contrasted impaired glucose regulation (prediabetes) prevalence, defined according to oral glucose tolerance test or HbA1c values, and studied cross-sectional associations between prediabetes and subclinical/clinical cardiovascular disease (CVD) in a cohort of European and South Asian origin. METHODS: For 682 European and 520 South Asian men and women, aged 58-85 years, glycaemic status was determined by oral glucose tolerance test or HbA1c thresholds. Questionnaires, record review, coronary artery calcification scores and cerebral magnetic resonance imaging established clinical plus subclinical coronary heart and cerebrovascular disease. RESULTS: Prediabetes was more prevalent in South Asian participants when defined by HbA1c rather than by oral glucose tolerance test criteria. Accounting for age, sex, smoking, systolic blood pressure, triglycerides and waist-hip ratio, prediabetes was associated with coronary heart disease and cerebrovascular disease in European participants, most obviously when defined by HbA1c rather than by oral glucose tolerance test [odds ratios for HbA1c -defined prediabetes 1.60 (95% CI 1.07, 2.39) for coronary heart disease and 1.57 (95% CI 1.00, 2.51) for cerebrovascular disease]. By contrast, non-significant associations were present between oral glucose tolerance test-defined prediabetes only and coronary heart disease [odds ratio 1.41 (95% CI 0.84, 2.36)] and HbA1c -defined prediabetes only and cerebrovascular disease [odds ratio 1.39 (95% CI 0.69, 2.78)] in South Asian participants. Prediabetes defined by HbA1c or oral glucose tolerance test criteria was associated with cardiovascular disease (defined as coronary heart and/or cerebrovascular disease) in Europeans [odds ratio 1.95 (95% CI 1.31, 2.91) for HbA1c prediabetes criteria] but not in South Asian participants [odds ratio 1.00 (95% CI 0.62, 2.66); ethnicity interaction P = 0.04]. CONCLUSIONS: Prediabetes appeared to be less associated with cardiovascular disease in the South Asian than in the European group. These findings have implications for screening, and early cardiovascular prevention strategies in South Asian populations.

KV 7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries.

BACKGROUND AND PURPOSE: Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca(2+) concentration ([Ca(2+)](i)) by opening of K channels and release of H2S. EXPERIMENTAL APPROACH: Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca(2+)](i), and the expression of voltage-gated K channels K(V)7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K(Ca)1.1) was examined. Voltage clamp assessed the role of K(V)7 channels in hypoxia. KEY RESULTS: Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca(2+)](i) in PGF(2α) (10 µM)-contracted arteries whereas no fall in [Ca(2+)](i) was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K(Ca)1.1 inhibited hypoxia-induced dilatation in PGF2α -contracted arteries; this inhibition was more marked in the presence of the K(v)7 channel blockers, XE991 and linopirdine, while a K(V)7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H2S- and adenosine-induced vasodilatation. PCR revealed the expression of K(V)7.1, K(V)7.4, K(V)7.5 and K(Ca)1.1 channels, and K(Ca)1.1, K(V)7.4 and K(V)7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions. CONCLUSION: The K(V)7.4 and K(V)7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K(V)7 channels in this vasodilatation. Activation of these K(V)7 channels may be induced by H2S and adenosine.

The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression.

Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.

Platelet and leukocyte activation, atherosclerosis and inflammation in European and South Asian men.

BACKGROUND: Increased platelet activation occurs in ischemic heart disease (IHD), but increased platelet activation is also seen in cerebrovascular atherosclerosis and peripheral artery disease. It is not clear therefore whether platelet activation is an indicator of IHD or a marker of generalized atherosclerosis and inflammation. South Asian subjects are at high risk of IHD, but little is known regarding differences in platelet and leukocyte function between European and South Asian subjects. METHODS: Fifty-four male subjects (age 49-79 years) had coronary artery calcification measured by multislice computed tomography (CT), aortic atherosclerosis assessed by measurement of carotid-femoral pulse wave velocity (aortic PWV), and femoral and carotid atherosclerosis measured by B-mode ultrasound. Platelet and leukocyte activation was assessed by flow cytometry of platelet-monocyte complexes (PMC), platelet expression of PAC-1 binding site and CD62P, and expression of L-selectin on leukocytes. RESULTS: Elevated circulating PMC correlated significantly with elevated aortic PWV and PMC were higher in subjects with femoral plaques. In contrast PMC did not differ by increasing coronary artery calcification category or presence of carotid plaques. Higher numbers of PMC were independently related to elevated levels of C-reactive protein (CRP), higher aortic PWV, hypertension and smoking in a multivariate model. Markers of platelet and leukocyte activation did not differ significantly by ethnicity. CONCLUSIONS: Increased PMC are related to the extent of aortic and femoral atherosclerosis rather than coronary or carotid atherosclerosis. The association between elevated CRP and increased PMC suggests that inflammation in relation to generalized atherosclerosis may play an important role in PMC activation.

Carotid angioplasty in a pulsatile flow model: factors affecting embolic potential.

OBJECTIVES: Carotid endoluminal intervention is an alternative to surgery but carries a risk of embolic stroke even with distal protection devices. We investigated the clinical features and degree of stenosis related to number and size of emboli during carotid angioplasty. DESIGN: An experimental ex vivo study. MATERIALS: An ex vivo pulsatile flow model was used in which temperature, velocity, flow, pressure and viscosity characteristics were designed to simulate the carotid circulation. METHODS: Carotid endarterectomy specimens excised as intact cylinders (n = 28) were subjected to a standardised angioplasty procedure using radiological guidance. Emboli collected in filters placed distally were counted and sized using microscopy. RESULTS: Median number of emboli during angioplasty was 133 (range 15-1331). Median size of the largest embolus was 700 microns (range 75-2400). Severity of stenosis correlated with increased maximum size (r = 0.55, p = 0.012). Statin therapy >4 weeks pre-operatively was associated with reduced emboli number and size (54 (range 15-748) vs 247 (range 37-1331) [p = 0.023] and 400 microm (range 75-2400) vs 1300 microm (range 600-2200) [p = 0.022]). CONCLUSIONS: In this model a wide range of emboli number and size were produced. Number and size of embolic particles were highest in patients with high-grade stenoses not receiving statin therapy.

Effect of tumour necrosis factor-alpha and interleukin 1beta on endothelium-dependent relaxation in rat mesenteric resistance arteries in vitro.

1. Pre-eclampsia is associated with elevated proinflammatory cytokine levels and endothelial dysfunction. This study examined the effect of two cytokines, tumour necrosis factor-alpha (TNF) and interleukin-1beta (IL-1) on endothelium-dependent relaxation in response to acetylcholine (ACH), bradykinin (BK) and histamine (HIS) in rat mesenteric small arteries in vitro. 2. Rat mesenteric arteries were mounted in an isometric myograph. Tone was induced with phenylephrine (PE) or a depolarizing solution containing 80 mM KCl (K(80)). Relaxation was measured in response to ACH, BK, HIS and sodium nitroprusside (SNP), an endothelium-independent relaxant. Inhibition of NO synthase by a combination of N(omega)-monomethyl-L-arginine (L-NMMA) and N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly inhibited relaxation in response to ACH and BK. Addition of an inhibitor of cyclooxygenase, indomethacin, had no additional effect when added to L-NMMA and L-NAME. Inhibition of endothelium-derived hyperpolarizing factor (EDHF) by K(80) partially reduced responses to ACH and BK. Inhibition of HIS-induced relaxation was more marked with K(80). L-NMMA and L-NAME largely abolished the remaining relaxation to ACH, BK and HIS in arteries contracted with K(80). 3. Preincubation with TNF for 30 min caused an inhibition of relaxation in response to ACH and BK in arteries contracted with PE. Responses to HIS and SNP were not affected by TNF under these conditions. TNF also inhibited ACH-induced relaxation in arteries contracted with K(80). IL-1 had no effect on responses to ACH and the combination of TNF and IL-1 was not more effective than TNF alone. 4. The inhibitory effect of TNF on ACH-induced relaxation was abolished by coincubation with superoxide dismutase (SOD) and was not seen if NO synthase was inhibited by L-NMMA and L-NAME. 5. TNF inhibits the NO-dependent component of endothelium-dependent relaxation in response to ACH and BK, but does not inhibit the EDHF-dependent component. This effect may be attributable to the ability of TNF to increase levels of superoxide anions (O(2)(-)) and the ability of O(2)(-) to inactivate NO. This mechanism could contribute to the endothelial dysfunction seen in situations where TNF is elevated, such as pre-eclampsia.

Selective dual nuclear scanning in low-risk patients with chest pain to reliably identify and exclude acute coronary syndromes.

STUDY OBJECTIVE: We sought to determine the use in routine clinical practice of selective dual nuclear cardiac scanning (rest and stress) in low-risk patients with chest pain for identifying and excluding acute coronary syndromes (ACSs) during the initial emergency department evaluation. METHODS: A prospective observational study was conducted over 13 months in 1,775 low-risk patients with chest pain who had intermediate- and high-risk ACSs ruled out by means of our 2-hour protocol, which consists of automated serial 12-lead ECG monitoring in conjunction with baseline and 2-hour creatine kinase (CK) MB and troponin I (cTnI) measurements. At the completion of the 2-hour evaluation period, low-risk patients were stratified by means of physician judgment into 1 of 2 categories: category III, possible ACS; and category IV, probable non-ACS chest pain. Level III patients underwent immediate dual nuclear scanning (rest thallium and stress sestamibi scanning), and level IV patients were discharged directly from the ED unless another serious non-ACS medical condition was thought to exist. Rest and stress scans were interpreted by a board-certified radiologist contemporaneous with patient evaluation. All patients were followed up for 30-day ACS, which was defined as acute myocardial infarction, percutaneous transluminal coronary angioplasty/coronary artery bypass grafting, coronary arteriography revealing stenosis of the major coronary artery of 70% or greater not amenable to percutaneous transluminal coronary angioplasty/coronary artery bypass grafting, life-threatening complication, or cardiac death within 30 days of ED presentation. RESULTS: A total of 2,206 ED patients with chest pain were evaluated for ACS during the study period. Four hundred thirty-one patients were excluded for having 1 or more of the following findings: initial ECG diagnostic of injury; baseline CK-MB level, cTnI level, or both diagnostic of acute myocardial infarction; 2-hour DeltaCK-MB level of +1.5 ng/mL or greater; 2-hour DeltacTnI level of +0.2 ng/mL or greater; injury or new or evolving ischemia on serial 12-lead ECG monitoring; or clinical diagnosis of ACS. Of the 1,775 study patients, 805 (45.4%) underwent immediate dual nuclear scanning. A positive stress nuclear scan result was more sensitive (97.3% versus 71.2%, P <.0001) and specific (87.7% versus 72.6%, P <.0001) for 30-day ACS than a positive resting nuclear scan result. The protocol of selective dual nuclear scanning (ie, patients who did not undergo dual nuclear scanning were counted as having a negative test result) had a sensitivity and specificity for 30-day ACS of 93.4% and 94.7%, respectively (positive likelihood ratio 17.6; negative likelihood ratio 0.07). CONCLUSION: Stress nuclear scanning is more sensitive and specific than resting nuclear scanning for identification of ACS in low-risk patients with chest pain. A strategy of using selective dual nuclear scanning once high- and intermediate-risk ACS has been ruled out with our 2-hour evaluation both reliably identifies and reliably excludes 30-day ACS.

Action of an HMG CoA reductase inhibitor, lovastatin, on apoptosis of untransformed and ts-SV40 transformed human smooth muscle cells derived from saphenous vein.

The effect of lovastatin, an inhibitor of 3-hydroxymethyl-3-glutaryl coenzyme A (HMG CoA) reductase, was examined on human vascular smooth muscle cells (HVSMC). Untransformed HVSMC were obtained from saphenous vein and in addition an SV-40 transformed immortalized cell line (HVTs-SM1) derived from saphenous vein smooth muscle was also used. HVTs-SM1 cell proliferation and DNA synthesis were measured, and cell cycle analysis was performed by flow cytometry. Apoptosis in both cell types was assessed by a combination of flow cytometry, terminal deoxynucleotidyl transferase (TUNEL) reagent-based immunocytochemistry, DAPI staining, and DNA agarose gel electrophoresis. Lovastatin had no effect on apoptosis of HVSMC over 96 h in serum-free conditions or after stimulation with platelet-derived growth factor (PDGF-BB), although PDGF-BB increased apoptosis in HVSMC, and this was prevented by lovastatin. In HVTs-SM1 cells lovastatin inhibited cell proliferation and DNA synthesis and induced apoptosis in a time- and concentration-dependent manner. The effects of lovastatin on cell proliferation, DNA synthesis, and apoptosis were prevented by coincubation with mevalonate and geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate. Lovastatin does not induce apoptosis in saphenous vein HVSMC in culture and inhibits PDGF-BB-induced DNA synthesis and apoptosis. In contrast, in SV40 transformed immortalized HVTs-SM1 cells, lovastatin induces apoptosis and inhibits cell proliferation and DNA synthesis. The pro-apoptotic effects of lovastatin in SV40 transformed HVTs-SM1 cells may be related to the enhanced rate of proliferation or deregulation of the cell cycle in this cell line.

p53, p21(WAF1/CIP1), and MDM2 involvement in the proliferation and apoptosis in an in vitro model of conditionally immortalized human vascular smooth muscle cells.

Using an in vitro model of a conditionally immortalized cell line, we have investigated how human vascular smooth muscle cells (VSMCs) are affected by the expression of simian virus 40 (SV40) large T antigen (LT antigen), which binds to cell cycle regulators such as the tumor suppressor protein p53. Cells were obtained after infection of saphenous vein-derived VSMCs with a nonreplicative retroviral vector containing a temperature-sensitive mutant of SV40 LT antigen and were shown to have maintained some characteristics and responses of VSMCs. Under permissive-temperature conditions (36 degrees C), the increased rate of cell proliferation was shown to be associated with expression of LT antigen and with LT antigen binding to and inactivation of p53. p53 inactivation failed to block apoptosis induced by serum withdrawal or UV irradiation. Downregulation of LT antigen expression at a nonpermissive temperature (39 degrees C) was shown to be associated with growth arrest, increased expression of the cell cycle inhibitor p21(WAF1/CIP1), increased murine double minute-2 promoter activity, and differential expression of murine double minute-2 gene products, suggesting that p53-induced transcription/transactivation may be involved in VSMC cycle control but not necessarily in apoptosis. The established SMC line HVTs-SM1 may be a useful model for study of the processes involved in myointimal hyperplasia and cellular aging, as well as for the study of cell cycle control in general.

Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60(c-src) kinase activity.

1. Tyrosine kinases have been proposed as regulators of voltage-operated calcium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage-operated calcium channel currents (I(Ba)) and pp60(c-src) kinase (c-src) activity in vitro. 2. I(Ba) was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage-clamp techniques. The activity of purified human c-src was measured in vitro using a non-radioactive assay. 3. Bath application of tyrphostin-23 and genistein (non-selective PTK inhibitors), bistyrphostin (a receptor-PTK-selective inhibitor) and PP1 (a src family-selective inhibitor) inhibited I(Ba) in a concentration-dependent manner over a range of test membrane potentials. Intracellular application of peptide-A, a peptide inhibitor of c-src also inhibited currents. Inhibitor potency series against I(Ba) was PP1 > genistein > tyrphostin 23 > bistyrphostin. 4. Tyrphostin-23, genistein, PP1, and peptide-A shifted the steady-state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no significant effects on I(Ba) activation calculated from current-voltage relationships. 5. The agents inhibited c-src activity in a concentration-dependent manner. The order of potency was PP1 > genistein > peptide-A > tyrphostin-23 > bistyrphostin. The IC(50) for inhibition of c-src activity was similar to the IC(50) for inhibition of I(Ba) in all cases. 6. Western blot analysis with a specific antibody to c-src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 7. A range of structurally dissimilar inhibitors of PTKs inhibit I(Ba) and c-src activity with similar potency. These data provide further evidence implicating endogenous c-src in the modulation of L-type calcium channels in vascular smooth muscle cells.

p53, p21(WAF1/CIP1), and MDM2 involvement in proliferation and apoptosis in an in vitro model of conditionally immortalized human vascular smooth muscle cells.

Using an in vitro model of a conditionally immortalized cell line, we investigated how human vascular smooth muscle cells (VSMCs) are affected by the expression of simian virus 40 (SV40) large T antigen (LT antigen), which binds to cell cycle regulators, such as the tumor suppressor protein p53. Cells were obtained after infection of saphenous vein-derived VSMCs with a nonreplicative retroviral vector containing a temperature-sensitive (ts) mutant of SV40 LT antigen and were shown to have maintained some characteristics and responses of VSMCs. Under permissive temperature conditions (36 degrees C), the increased rate of cell proliferation was shown to be associated with expression of LT antigen and with LT-antigen binding to and inactivation of p53. p53 inactivation failed to block apoptosis induced by serum withdrawal or by UV irradiation. Downregulation of LT-antigen expression at the nonpermissive temperature (39 degrees C) was shown to be associated with growth arrest, increased expression of the cell cycle inhibitor p21(WAF1/CIP1), increased MDM2-promoter activity, and differential expression of MDM2 gene products, suggesting that p53-induced transcription/transactivation may be involved in VSMC cell cycle control but not necessarily apoptosis. The established SMC line HVTs-SM1 may be a useful model for the study of processes involved in myointimal hyperplasia and cellular aging, as well as for the study of cell cycle control in general.

Regulation of phospholipase C-delta by GTP-binding proteins-rhoA as an inhibitory modulator.

The regulation of Phospholipase C (PLC)delta activity remains obscure. These studies show that PLCdelta1 activity is significantly enhanced by both guanosine thiotriphosphate (GTPgammaS) and Clostridium botulinum exoenzyme C3 (C3) but not by aluminium fluoride. C3 ADP ribosylated a 21-kDa protein in the PLCdelta1 preparation and Western blotting identified rhoA in these samples. RhoA acts as an inhibitory modulator of PLCdelta activity.

Platelet-derived growth factor beta-receptors can both promote and inhibit chemotaxis in human vascular smooth muscle cells.

The effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on migration was investigated in cultured human saphenous vein smooth muscle cells. The modified Boyden chamber technique yielded efficacies BB >> AB, AA = 0. However, the BB concentration-response relationship displayed a pronounced peak, occurring between 1 and 10 ng/mL, with no response above this range. Checkerboard analysis showed that the promotion of migration at low concentrations was chemotactic in nature but that the downturn was independent of gradient. Furthermore, at high concentrations BB was able to prevent chemotaxis induced by fetal calf serum and epidermal growth factor (EGF). Experiments using low concentrations of BB in combination with high concentrations of AA to saturate PDGF alpha-receptors in the presence and absence of a neutralizing antibody to alpha-receptors revealed that alpha-receptor activation induced partial inhibition of chemotaxis but this did not account for the inhibition of migration by high concentrations of BB. Despite possessing no significant chemotactic action itself, high concentrations of the AB isoform completely inhibited BB induced chemotaxis. Taken together these results suggest that the chemotactic signal induced by PDGF is dominated by PDGF beta-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [3H] thymidine incorporation) yielded saturable responses for the AB and BB isoforms, with similar efficacy and weak or no response for the AA isoform. Concentration-dependent patterns of tyrosine phosphorylation of certain proteins mirrored the form of the chemotactic response and suggest one possible underlying regulatory mechanism to account for the disparity between PDGF-induced chemotaxis and DNA synthesis.

Inhibition of proliferation by heparin and expression of p53 in cultured human vascular smooth muscle cells.

Human vascular smooth muscle cells (HVSMC) cultured from restenotic lesions are resistant to inhibition of proliferation by heparin. We examined if altered expression of p53 protein might be related to this phenomenon. HVSMC were cultured from saphenous vein and p53 protein levels examined using immunocytochemistry, and by immunoprecipitation with antibodies specific for wild-type or mutant conformations followed by SDS PAGE and immunoblotting. Inhibition of proliferation by heparin was measured in 14-day growth assays. Elevated levels of p53 were found in five out of 41 HVSMC strains. The accumulated p53 protein was not precipitated by a mutant conformation-specific anti-p53 antibody in any of the five strains. In one of the five positive strains there was concomitant human cytomegaloviral infection. No significant difference was found between efficacy of heparin in the p53-positive and -negative strains. Furthermore heparin had no detectable effect on p53 protein levels. Although aberrant levels of p53 are detectable in a minority of HVSMC strains, these data do not support a role for altered p53 expression in resistance to the growth inhibitory effects of heparin in these cells.

Activation of endogenous c-Src or a related tyrosine kinase by intracellular (pY)EEI peptide increases voltage-operated calcium channel currents in rabbit ear artery cells.

The effect of activation of endogenous c-Src tyrosine kinase by (pY)EEI peptide was examined on voltage-operated calcium channel (VOC) currents in arterial smooth muscle cells. In single rabbit ear artery cells intracellular application of (pY)EEI peptide increased calcium channel currents. Inactive, non-phosphorylated YEEI peptide had no effect on currents. Peptide-A, a 21 amino acid inhibitor of c-Src inhibited currents and prevented the effect of (pY)EEI peptide on calcium channel currents. These results indicate that activation of intrinsic c-Src increases VOC and support a role for c-Src in the regulation of VOC in vascular smooth muscle cells.

Effect of angiotension II on the expression of the early growth response gene c-fos and DNA synthesis in human vascular smooth muscle cells.

OBJECTIVES: The aims of this study were to characterize the angiotensin II receptor subtype present on vascular smooth muscle cells from human saphenous vein and to assess the effect of angiotensin II on the expression of the early growth response gene c-fos and on DNA synthesis. METHODS AND RESULTS: Using radioligand binding studies, we have defined the angiotensin II receptors present on these cells as being predominantly of the AT1 subtype. Angiotensin II increased peak intracellular calcium levels by 126 +/- 16 nmol/l (mean +/- SEM) in 17/49 cultures. Angiotensin II induced c-fos expression in a concentration-dependent manner only in cultures that exhibited an intracellular calcium transient in response to stimulation with angiotensin II. The induction of c-fos was inhibited by the selective AT1 antagonist losartan in accordance with the binding studies. Angiotensin II stimulated DNA synthesis with a maximal increase of 66.4% +/- 20.5% over serum-free levels at 1 nmol/l (mean +/- SEM, n = 6, P < 0.05). DNA synthesis declined with increasing angiotensin II concentration, falling to control values at 1 mumol/l, suggesting that a growth-inhibitory influence may counter-balance the stimulatory effect that is observed at lower concentrations. CONCLUSION: Vascular smooth muscle cells from human saphenous vein possess predominantly AT1 receptors and in response to angiotensin II show an induction of c-fos and a modest increase in DNA synthesis.