Nitric oxide inhibits Oct-1 DNA binding activity in cultured vascular smooth muscle cells

In this paper, the authors model the nonmonotonic relation between body mass index (BMI) (weight (kg)/height2 (m2)) and mortality in 13,242 black and white participants in the NHANES I Epidemiologic Follow-up Study in order to estimate the BMI at which minimum mortality occurs. The BMI of minimum mortality was 27.1 for black men (95% confidence interval (CI) 24.8-29.4), 26.8 for black women (95% CI 24.7-28.9), 24.8 for white men (95% CI 23.8-25.9), and 24.3 for white women (95% CI 23.3-25.4). Each confidence interval included the group average. Analyses conducted by smoking status and after exclusion of persons with baseline illness and persons who died during the first 4 years of follow-up led to virtually identical estimates. The authors determined the range of values over which risk of all-cause mortality would increase no more than 20% in comparison with the minimum. This interval was nine BMI units wide, and it included 70% of the population. These results were confirmed by parallel analyses using quantiles. The model used allowed the estimation of parameters in the BMI-mortality relation. The resulting empirical findings from each of four race/sex groups, which are representative of the US population, demonstrate a wide range of BMIs consistent with minimum mortality and do not suggest that the optimal BMI is at the lower end of the distribution for any subgroup.     

Retinoic acid and triiodothyronine stimulate ADP-ribosyl cyclase activity in rat vascular smooth muscle cells

Cyclic ADP-ribose (cADPR) is a nucleotide synthesized from beta-NAD- that can trigger or facilitate Ca2+-release through ryanodine-channels. We investigated the synthesis of cADPR (ADPR-cyclase activity) in cultured vascular smooth muscle cells (VSMC) from rat aorta in response to incubation with all-trans-retinoic acid (RA), 3,3',5'-triiodothyronine (T3), cortisol, beta-estradiol and 1-dehydrotestosterone. Only RA and T3 caused concentration-dependent (10(-9)-10(-6) M) stimulation of ADPR-cyclase activity in VSMC. Maximum stimulatory responses to RA (+100%) and T3 (+40%) were additive and the stimulatory effects of both hormones on ADPR-cyclase were due to an increase in Vmax without changes in the apparent Km. These observations indicate that in VSMC synthesis of cADPR can be upregulated by RA and T3. We propose that some of the actions of RA on VSMC such as enhancement of contractile competence, differentiation, and anti-proliferative effects might be elicited, at least in part, via upregulation of the cADPR/Ca2+-release signaling system.     

N-alpha-tosyl-L-lysine chloromethylketone prevents expression of iNOS in vascular smooth muscle by blocking activation of NF-kappa B

OBJECTIVE: To demonstrate that an agreement approach to applying equations on the basis of clinical and exercise test variables is an accurate, self-calibrating, and cost-efficient method for predicting severe coronary artery disease in clinical populations. DESIGN: Retrospective analysis of consecutive patients with complete data from exercise testing and coronary angiography referred for evaluation of possible coronary artery disease. After developing an equation in a training set, this equation and two other equations developed by other investigators were validated in a test set. The study was performed at two university-affiliated Veteran's Affairs medical centers. PATIENTS: 1080 consecutive men studied between 1985 and 1995 who had coronary angiography within 3 months of the treadmill test. The population was randomly divided into a training set of 701 patients and a test set of 379 patients. Patients with previous coronary artery bypass surgery, valvular heart disease, marked degrees of resting ST depression, and left bundle branch block were excluded. MEASUREMENTS: Recording of clinical and exercise test data along with visual interpretation of the electrocardiogram recordings on standardized forms and abstraction of visually interpreted angiographic data from clinical catheterization reports. RESULTS: Simple clinical and exercise test variables improved the standard application of exercise-induced ST criteria for predicting severe coronary artery disease. By setting probability thresholds for severe disease of <20% and >40% for the three prediction equations, the agreement approach divided the test set into three groups: low risk (patients with all three equations predicting <21% probability of severe coronary disease), no agreement, and high risk (all three equations with >39% probability) for severe coronary artery disease. Because the patients in the no agreement group would be sent for further testing and would eventually be correctly classified, the sensitivity of the agreement approach was 89% and the specificity was 96%. The agreement approach appeared to be unaffected by disease prevalence, missing data, variable definitions, or even angiographic criteria. CONCLUSIONS: Requiring diagnosis of severe coronary disease to be dependent on agreement between these three equations has made them likely to function in all clinical populations. The agreement approach should be an efficient method for the evaluation of populations with varying prevalence of coronary artery disease, limiting the use of more expensive noninvasive and invasive testing to patients with a higher probability of left main or triple-vessel coronary artery disease. This approach provides a strategy that can be applied by inputting the results of basic clinical assessment into a programmable calculator or a computer to assist the practitioner in deciding when further evaluation is appropriate, thus assuring patients access to subspecialty care.     

Interferon gamma up-regulates a novel protein in vascular smooth muscle cells

PURPOSE: By means of the technique of messenger RNA (mRNA) differential display, we previously isolated a partial DNA clone found to be down-regulated at the polytetrafluoroethylene (PTFE) hyperplastic arterial anastomosis compared with the normal artery. The partial DNA gene sequence was found to be homologous with interferon gamma up-regulated protein (IGUP) first found in human psoriatic keratinocytes. We cloned the entire IGUP gene from human vascular smooth muscle cells (VSMCs) to determine its regulation by gamma interferon (gamma-IFN) and other cytokines in cultured human VSMCs. METHODS: By means of polymerase chain reaction, the IGUP gene was amplified from a QUICK-Clone complementary DNA human aorta kit using 5' and 3' oligonucleotide primers to the known IGUP sequence. Immunohistocytochemistry studies compared normal artery and distal anastomotic IH. Human VSMCs were stimulated with 1000 U/mL of gamma-IFN, 5 ng/mL of platelet-derived growth factor BB (PDGF-BB), 3. 2 ng/mL basic fibroblast growth factor, 3.3 ng/mL transforming growth factor beta(TGF-beta), 10 ng/mL of vascular endothelial growth factor, and 10% fetal bovine serum (FBS) for zero, 24, 48 and 72 hours. Western blot analysis of lysates of the stimulated VSMCs was performed to determine up-regulation of IGUP. RESULTS: DNA sequencing confirmed the cloning of the entire coding region of the IGUP gene with 100% homology to the known IGUP DNA sequence. There was strong expression of IGUP in quiescent VSMCs and marked reduction of expression of IGUP in proliferating smooth muscle cells. gamma-IFN was the only cytokine, of the cytokines evaluated, to up-regulate production of IGUP in VSMCs. CONCLUSION: IGUP is a novel protein in VSMCs found to be down-regulated in areas of anastomotic IH, as compared with a normal artery. We have now shown IGUP to be up-regulated only by gamma-IFN in human VSMCs. IGUP may, therefore, be the intermediary for the known gamma-IFN inhibition of human VSMC proliferation.     

Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.     

Mitogen-activated signaling in cultured airway smooth muscle cells

Airway hyperresponsiveness and excess smooth muscle mass coexist in patients with asthma and bronchopulmonary dysplasia. This increase in airway smooth muscle mass, which in part relates to smooth muscle proliferation, may increase bronchoconstrictor-induced airway narrowing, even in the absence of excessive force generation. Thus, there is need for a precise understanding of the events involved in airway smooth muscle mitogenesis. This review examines the inflammatory substances and growth factors that induce airway smooth muscle proliferation, and the signaling pathways that may be involved in the transduction of these extracellular signals to the cell nucleus. Also discussed are various antimitogenic substances and potential mechanisms underlying the inhibition of cell proliferation. Central to the discussion are the extracellular signal regulated kinases (ERKs), serine/threonine kinases of the mitogen-activated protein kinase (MAP kinase) superfamily, which upon activation, translocate from the cytoplasm to the nucleus after mitogenic stimulation. Insight gained from studies of cultured airway smooth muscle growth and mitogen-activated signaling may shed light on parallel mechanisms that may operate in asthma and in bronchopulmonary dysplasia, and may lead to therapeutic interventions against airway remodeling.     

Effect of pressure on cultured smooth muscle cells

Recent studies indicate that smooth muscle cell (SMC) growth and morphology can be modulated by repetitive strain. However, there is very little known about the influence of pressure, without an associated cell stretch, on SMC phenotype. To study this, cultured bovine aortic SMC were grown on a rigid surface and placed in a custom-designed plexiglass pressure chamber with a carefully regulated 5% CO2/air environment. SMC were exposed to either atmospheric, 105 mm Hg or 120/90 mm Hg pressure at a frequency of 60 cycles/min (0.5 s systole, 0.5 s diastole). SMC number was determined on days 1, 3, 5, 7 and 9. SMC exposed to pressure were more elongated and displayed a significant increase in cell number by day 5 which persisted until day 9. Lactate dehydrogenase (LDH) in the conditioned media, an index of cytotoxicity, was not different between the groups at each time point. There was also no difference in pH or pCO2 of the media of SMC in any group. This is the first report of the effects of increased static and pulsatile pressure on SMC in vitro and indicates an increased proliferative rate. We hypothesize that the systemic pressure that the blood vessel is exposed to in vivo may have a significant regulatory influence on the phenotype of the smooth muscle cells which may affect the SMC response to injury or stimuli.     

Vascular smooth muscle nitric oxide synthase anomalies in Dahl/Rapp salt-sensitive rats

Salt-sensitive hypertension in the Dahl/Rapp rat (S strain) is prevented by L-arginine. Based on the observations that dexamethasone prevented the antihypertensive effect of L-arginine in these animals and the suggestion that a locus in or near an inducible nitric oxide synthase (NOS) gene on chromosome 10 cosegregated with hypertension in some F2 crosses that utilized the S rat, the present study explored the hypothesis that the vascular smooth muscle isoform of inducible NOS (NOS2) was abnormal in S rats. Primary cultures of aortic smooth muscle cells from S rats demonstrated impaired inducible NO production, which improved with increased L-arginine in the medium. Sequence analysis identified a single T-->C transversion that produced an amino acid substitution (S714P) between the FAD and FMN binding sites and a restriction fragment length polymorphism. This restriction fragment length polymorphism was present only in S rats. The mutation of NOS2 and the role of this enzyme in the pathogenesis of salt-sensitive hypertension in the Dahl/Rapp rat require further investigation.     

Ethanol inhibits mitogen activated protein kinase activity and growth of vascular smooth muscle cells in vitro

The intrathecal (i.t.) injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 (65 and 650 pmol) and endothelin-3 (650 pmol) enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases (between 1.4- and 2.2-fold) of vascular permeability were observed in other tissues (trachea, kidney, ears, skin of hind paws and back skin) with endothelin-1. Endothelin-1 (650 pmol) caused a similar increase (27-fold) in lung vascular permeability when injected at T-2, although the response was significantly less (P < 0.05) if injected at the L-4 (15-fold) spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 (650 pmol) did not produce lung oedema and the lung vascular permeability was increased by only 1.4-1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 (cyclo[D-Trp, D-Asp, L-Pro, D-Val, L-Leu]), a selective endothelin ET(A) receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 (650 pmol). Whereas i.v. treatment with phentolamine (2 mg/kg) or pentolinium (25 mg/kg + 50 mg/kg per h x 15 min) abolished the lung vascular permeability changes evoked by endothelin-1 (650) pmol), atropine (1 mg/kg), NG-nitro-L-arginine (50 mg/kg) or indomethacin (5 mg/kg) had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats (125 mg/kg, 10 days earlier) and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine (100 mg/kg, 24-48 h earlier). All these treatments except atropine and NG-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by alpha-adrenoceptors following the release of catecholamines in response to the activation of endothelin ET(A) receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism.     

Oxidized collagen stimulates proliferation of vascular smooth muscle cells

We have examined the use of presurgical morphine-midazolam combination in 80 children aged 2-10 y undergoing repair of hypospadias. They were allocated randomly, in a double-blind study, to receive one of four morphine-midazolam combination doses (n = 20 each); (group I: 75 microg/kg each) [corrected] (group II: 75 microg/kg [corrected] morphine, 50 microg/kg [corrected] midazolam); (group III: 50 microg/kg [corrected] morphine, 75 microg/kg [corrected] midazolam); (group IV: 50 microg/kg [corrected] each). Drugs were given after induction of anesthesia and before the start of surgery. Observational scoring system, using crying, movement, agitation, posture and localization of pain as scoring criteria, was used to assess the children during their stay in the recovery room together with their sedative and/or analgesic requirement. Pre-surgical morphine-midazolam administration produced stable hemodynamic variables with satisfactory postoperative analgesia suggesting 75 microg/kg [corrected] dose of both morphine and midazolam as upper permissible dose, and 50 microg/kg [corrected] each as lower effective dose.