Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro

Nicotine is a major component of cigarette smoke and has been postulated to play an important role in atherogenesis and malignancy. Endothelial cell growth may be regulated by nicotine, yet operative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine (10(-14)-10(-4) M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in the presence and absence of hydroxyurea (an inhibitor of scheduled DNA synthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF and PDECGF, respectively). Nicotine significantly stimulated endothelial cell DNA synthesis and proliferation at concentrations lower than those obtained in blood after smoking (<10(-8) M). The stimulatory effects of nicotine were enhanced by serum (0.5%) and PDECGF and were blocked by the nicotinic-receptor antagonist hexamethonium. The response to nicotine was bimodal because cytotoxicity was observed at higher concentrations (>10(-6) M). This study has implications for understanding cellular mechanisms of nicotine action. The results may be important in tumor angiogenesis, atherogenesis, and vascular dysfunction in smokers.     

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS: Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION: When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.     

Effects of amide and amine plasma-treated ePTFE vascular grafts on endothelial cell lining in an artificial circulatory system

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities and an early death of the vitamin A-deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. We demonstrate that all-trans-, 9-cis-, 4-oxo-, and didehydroretinoic acids, and didehydroretinol and all-trans-retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor beta2 in the vitamin A-deficient quail embryo. The expression of RARbeta2 is at the same level and at the same sites where it is expressed in the normal embryo. Retinoids provided to the vitamin A-deficient embryo up to the 5-somite stage of development, but not later, completely rescue embryonic development, suggesting the 5-somite stage as a critical retinoid-sensitive time point during early avian embryogenesis. Retinoid receptors RARalpha, RARgamma, and RXRalpha are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A-deficient quail embryo, while the expression of RXRgamma is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARalpha and RARbeta. Our studies provide strong evidence for a narrow retinoid-requiring developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors is essential for a subsequent normal embryonic development.     

Shear stress gradients remodel endothelial monolayers in vitro via a cell proliferation-migration-loss cycle

Wall shear stress has been implicated in the genesis of atherosclerosis because a strong correlation exists between the location of developing arterial lesions and regions where particular gradients in stress occur. Studying the behavior of endothelial cells in such regions may contribute to our understanding of the disease etiology. We report the detailed migratory history of endothelial cells subjected to large shear stress gradients caused by a surface protuberance in an in vitro model system. The history of cell migration, cell division, and cell loss from the surface was continuously monitored in confluent human umbilical vein endothelial cell monolayers for 48 hours after the onset of flow. Individual cells were tracked using time-lapse video microscopy. In contrast to a uniform laminar flow field in which cells were observed to continually rearrange their relative position with no net migration, in a disturbed flow field there was a net migration directed away from the region of high shear gradient. This organized migration pattern under disturbed flow conditions was accompanied by more than a twofold increase in cell motility. In addition, cell division increased in the vicinity of the flow separation (maximum shear stress gradient of 34 dyne/cm2 per mm) whereas cell loss was increased upstream and downstream in the regions where the shear gradient diminishes. These data suggest a steady cell proliferation-migration-loss cycle and indicate that local shear stress gradient may play a key role in the morphological remodeling of the vascular endothelium in vivo.     

Chronic immobilization stress reduces sodium intake and renal excretion in rats

The influence of chronic exposure to immobilization (IMO) on sodium appetite as well as sodium and potassium renal excretion in adult male Wistar rats was studied. The animals were individually housed and all variables under observation were measured in metabolic cages the first, seventh, and thirteenth days once the experiment had started. Half of the rats had access to water, and the remainder of the rats had access to both water and saline solution (1.5% NaCl). IMO reduced the intake of saline solution. Renal water, sodium, and potassium excretion in those IMO rats having access to saline were lower than in control rats. The effects of IMO were very similar during all observation days; therefore no evidence of adaptation to repeated stress was found. The present data indicate the following: (i) IMO stress reduced sodium appetite, probably as a secondary effect to the deficit in sodium renal excretion; (ii) IMO caused antidiuresis and antikaliuresis, only in those rats taking saline solution; (iii) no adaptation to repeated IMO stress was found in any of the tested variables. The reduction of sodium appetite observed in stressed rats might be a homeostatic mechanism to maintain sodium balance after impairment of renal sodium excretion caused by stress.     

In vitro and in vivo calcification of vascular bioprostheses

Efficacy of different chemical treatments on calcification of vascular graft in vitro and in vivo was studied. Culture medium-filled rat aortas were separately treated in 0.2% glutaraldehyde and epoxy compound, and photooxidized in 0.01% methylene blue for a shorter period (group 1). Another group of rat aortas were separately treated in the same chemicals for a longer period (group 2). All fresh and treated aortas of both groups were cultured for 21 days in an organ culture medium and implanted (except for group 1) in weanling rats for five months. Histology and immunohistochemistry revealed that differently treated aortas of group 1 grow and calcify, and the smooth muscle cells between elastin fibers are the primary site of calcium deposition. In contrast, differently treated aortas of group 2 neither grew, nor did calcify in the medium except the epoxy compound cross-linked aorta of group 2 which did not grow but did calcify. Untreated aorta did not calcify. All fresh and differently treated aortic homografts calcified severely in rats. Our whole arterial segment-calcification system would be useful for analyzing the molecular and cellular mechanisms of both bioprosthetic and atherosclerotic calcification of vascular graft. New anticalcification technique is the only hope for better outcome of future vascular bioprostheses.     

Gliding bacterial adjuvant stimulates feline cytokines in vitro and antigen-specific IgG in vivo

Gliding bacterial adjuvant (GBA) has been previously characterized as a potent immune modulator, stimulating the growth of murine B lymphocytes, inducing murine NK cell activity, and promoting the release of several murine cytokines. Based on these studies and our interest in potentiating the effectiveness of feline vaccines, GBA was tested for its ability to stimulate feline T cells in vitro and act as a vaccine adjuvant in vivo. In vitro, GBA stimulated feline PBLs to proliferate and release interferon (IFN) and IL-2. Unlike IFN, the release of IL-2 appeared to be unaffected by prior depletion of macrophages, indicating GBA directly stimulated feline T cells. In vivo GBA was co-administered with Keyhole Limpet Hemacyanin (KLH) and the anti-KLH antibody response was compared to cats receiving KLH emulsified in complete Freund's adjuvant (CFA). Fourteen days after the third immunization and continuing for a 30-day observation period, KLH-specific IgG titers in cats receiving GBA were significantly higher than those given CFA. However, when cats were subsequently boosted with KLH alone, those cats receiving CFA demonstrated significantly higher antibody titers throughout a second 30-day observation period. The anti-KLH antibody memory response was greatly enhanced when GBA was emulsified with incomplete Freunds adjuvant (IFA) prior to injection. Serum titers of cats given KLH in an oil-based GBA preparation were significantly higher than cats receiving KLH adjuvanted with IFA or CFA, an effect which persisted 38 days after boosting with KLH alone. Finally, GBA significantly enhanced the feline humoral response to a recombinant protein of Dirofilaria immitis, the causative agent of feline heartworm. Serum titers of cats inoculated with recombinant antigen in GBA were significantly greater than cats given recombinant antigen adjuvanted with Titermax, alum, or NAGO. These studies indicate that GBA induces T cell proliferation and the release of IL-2 and IFN in vitro and can be used to enhance the recall antibody response to both a T cell dependent antigen and an immunogen derived from Dirofilaria immitis.     

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.     

Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo

A replication-defective adenovirus 5 vector carrying the beta-galactosidase reporter gene was tested for its efficiency for gene delivery to vascular endothelial cells in various situations. Both porcine and human primary vascular endothelial cell cultures were very efficiently infected (>90%) at adenovirus concentrations of 10(10) pfu/ml or higher. Cultured rat fibroblasts and keratinocytes were even more readily infected, with >90% infection with adenovirus titers of 10(8) pfu/ml or higher. However, nondividing vascular endothelium in situ was very poorly transduced. Pieces of aorta from adult pigs, sheep, rabbit and rat, and pieces of human umbilical artery and vein were studied in organ culture. These showed only occasional positive vascular endothelial cells when exposed to the adenovirus vector at concentrations up to 5x10(11) pfu/ml. Kidney perfusion studies in rats and pigs gave similar results. The only exception to the above findings was in very young (3-4 day old) piglets, which showed excellent (>90%) infection of vascular endothelium with the adenovirus vector at titers of 10(10) pfu/ml. Our data suggest that adenovirus vectors will not be of value for gene delivery to uninjured vascular endothelium in situ, and are therefore unsuited for ex vivo genetic manipulation of vascular endothelium in organs for transplantation.     

Direct physical factors and PGI2 and TXA2 secretions by a human endothelial cell line: in vitro investigation of pressure and shear stress applied independently or in synergy

The direct effect of two types of mechanical stress was measured through the prostacyclin (PGI2) and thromboxane A2 (TXA2) secretions by a confluent monolayer of cells from the EA.hy926 line. Eight values of constant pressure were applied in the gas phase above the culture medium, around atmospheric pressure taken as a control (0 mm Hg), from -500 to +760 mm Hg. Three amplitudes of sinewave modulated pressure (+/- 40; +/- 80; +/- 160 mm Hg) were explored at a frequency of 1 Hz. Modulated pressure (+/- 40 mm Hg) was also applied synergetically to a shear stress generated under steady state conditions by a rectilinear laminar motion of the medium. The cells remained adherent and exhibited unchanged morphology and viability. Constant pressure or depressure increased both PGI2 and TXA2 release but to an extent depending on the pressure value. Under pressure, the PGI2/TXA2 ratio was unchanged, but was higher under depressure, compared to the control. Pressure modulation strongly stimulated the secretion of PGI2 but had no effect on TXA2. Modulation strongly increased the PGI2/TXA2 ratio to a similar extent for the three amplitudes. Pressure-shear synergy enhanced secretion of PGI2 markedly more than shear stress alone, but the level reached was similar to the one induced by pressure modulation. No cumulative effect on the secretion of PGI2 was observed, whereas TXA2 synthesis undergoes a more than cumulative effect. The PGI2/TXA2 ratio remained unchanged under shear alone or under combined shear-pressure modulation but was higher with the modulated pressure alone. These results demonstrate that pressure has an outstanding effect on secretion that may be origin to local disturbances of the vascular system, thus inducing pathologies such as thrombosis or atherosclerosis.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

目的 研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子(VEGF)及其受体( VEGFR)的影响。方法酶联免疫吸附法检测非细胞毒性浓度(5、10、20 ng/ml)青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果培养24、48 h后,无青蒿琥酯作用的SHI-1细胞培养上清液VEGF质量浓度分别为( 980.3±2.2)、(982.4±2.3) pg/ml,VEGFR-1表达率分别为(5.40±3.11)％和(4.45±2.85)％,VEGFR-2表达率分别为(13.90.± 2.26)％和（13.95±1.96）％。5、10、20 ng/ml青蒿琥酯作用24h后,SHI-1细胞培养上清液VEGF质量浓度分别为(234.6±1.8)、(114.9±1.6)、(108.8±1.5) pg/ml,作用48 h后分别为(62.3±1.7)、(60.9±1.6)、(32.7±1.7) pg/ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜ 0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义(均P＜ 0.05)。5、10、20 ng/ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为(4.30±2.21)％、(4.20±1.37)％和(3.90±1.86)％,作用48 h后分别为(3.80±2.87)％、(3.60±1.73)％和(3.00±1.82)％,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义(均P＞ 0.05);作用24h后,SHI-1细胞VEGFR-2阳性率分别为(4.40±1.15)％、(3.10±0.68)％和(1.10±0.72)％,作用48 h后分别为(3.00±1.68)％、(2.20±0.93)％和(0.60±0.92)％,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜ 0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞ 0.05）。结论SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

Thrombogenesis of different cell types seeded on vascular grafts and studied under blood-flow conditions

BACKGROUND: Small-diameter vascular grafts tend to have an early and high occlusion rate. Cell seeding on the luminal surfaces of small-diameter vascular prostheses may provide an antithrombotic lining and improve both the short-term and the long-term patency rates. We studied the net results of procoagulant and anticoagulant properties of seeded grafts under blood-flow conditions, and we compared the different available types of donor cells. METHODS: Monolayers of liposuction-derived cultured human microvascular endothelial cells (MVECs), human adult endothelial cells (HAECs), human umbilical vein endothelial cells (HUVECs), and human mesothelial cells (MCs) that had been seeded on expanded polytetrafluoroethylene (ePTFE) grafts were perfused with marginally anticoagulated blood (20 U/mL low molecular weight heparin; shear rate, 400/s, 10 minutes) or with non-anticoagulated blood (shear rate, 100/s, 5 minutes). The thrombin and fibrin generation in time was studied with the measurement of the plasma levels of prothrombin fragment 1 and 2 (F 1+2) and of fibrinopeptide A (FPA). The plain ePTFE graft was taken as a control. RESULTS: When the seeded MCs were perfused with recirculating anticoagulated blood, a linear generation of F 1+2 in time was seen, with high levels of F 1+2 and FPA after 10 minutes (4.38 nmol/L and 362 ng/mL, respectively). Allopurinol was added, and the MCs generated less F 1+2 than the HAECs (0.7 nmol/L vs 1.86 nmol/L; P <.05). No fibrin formation was seen. The MVECs generated low amounts of F 1+2 (0.7 nmol/L; 10 minutes), and the HUVECs and the plain ePTFE graft generated the lowest amounts of F 1+2 (0.26 and 0.25 nmol/L, respectively). When the MCs were perfused with non-anticoagulated blood, high amounts of thrombin and fibrin were generated immediately and constantly and could not be decreased with allopurinol. The perfusion of the plain ePTFE graft showed a dramatic increase in F 1+2 and FPA levels towards the end of the experiments. The seeded HAECs, HUVECs, and MVECs inhibited this increase. These results were confirmed by means of scanning electron microscopy. CONCLUSION: Vascular prostheses that are seeded with cultured MCs are highly procoagulant. Standard ePTFE graft prostheses also initiate coagulation, which supports the idea of cell seeding. The endothelial cells, of which the MVECs are the most readily available, seem to preserve their anticoagulant properties after being seeded on vascular grafts.     

Effects of troglitazone and pioglitazone on cytokine-mediated endothelial cell proliferation in vitro

We examined whether troglitazone and pioglitazone, antidiabetic thiazolidinediones, would directly induce endothelial cell proliferation or influence cytokine-driven proliferation in vitro. Monolayers of Balb/c mouse aortic endothelial cells were treated with troglitazone or pioglitazone in the absence of fetal bovine serum. Endothelial cells also were exposed to varying concentrations of basic fibroblast growth factor (bFGF) or insulin with or without either thiazolidinedione. After 48 h, 3-[4,5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays were performed to quantitate endothelial cell proliferation by using the various treatment regimens. The data demonstrate that the antidiabetic thiazolidinediones troglitazone and pioglitazone negligibly affect direct endothelial cell proliferation in vitro. Furthermore, troglitazone and pioglitazone significantly inhibit bFGF-induced endothelial cell mitogenesis, whereas only high concentrations of troglitazone affect insulin-mediated proliferation.     

In vitro and in vivo biodurability of a compliant microporous vascular graft

We examined the extent to which program integrity (i.e., the degree to which programs were implemented as planned) was verified and promoted in evaluations of primary and early secondary prevention programs published between 1980 and 1994. Only 39 of 162 outcome studies featured specified procedures for the documentation of fidelity. Of these, only 13 considered variations in integrity in analyzing the effects of the program. Lowered adherence to protocol was often associated with poorer outcome. There was mixed evidence of dosage effects. The omission of integrity data, particularly measures of adherence, may compromise the internal validity of outcome studies in the prevention literature. We do not view procedures for integrity verification as inconsistent with the adaptation of interventions to the needs of receiving communities.     

Anti-inflammatory drugs and endothelial cell adhesion molecule expression in murine vascular beds

STATEMENT OF PROBLEM: There are discrepancies among researchers concerning the reliability and use of temporomandibular joint sounds. PURPOSE: This study examined the reliability of mandibular movements and sounds and determined the correlation between movements and sounds. MATERIAL AND METHODS: The mandibular movements of 35 subjects diagnosed with temporomandibular disorders were recorded with 2 CCD cameras, and sounds were recorded bilaterally with Panasonic electret condenser microphones in the ear canal. Subjects performed 3 movements, each repeated 5 times. RESULTS: Reliability of maximum movements across the 5 trials was good to excellent, with Intraclass Correlation Coefficients (ICC) between 0.76 and 0.91 for all movements except protrusion. Temporomandibular sound event counts were reliable for most movements, including vertical opening, protrusion, and right and left laterotrusion (ICCs between 0.41 and 0.81). Most subjects produced sound events either in 100% or in none of the trials. Reliability for sound events was better during protrusion (ICCs between 0.56 and 0.81) than vertical opening (ICCs 0.41 to 0.64). Subjects with sound events during vertical opening (followed by closing) were significantly more likely to have sound events during protrusion (followed immediately by vertical opening and closing) (P <.01). CONCLUSION: Temporomandibular sound events are generally reliable and warrant study regarding their use in classifying and diagnosing patients with temporomandibular disorders. Condylar translation, which occurs during both vertical opening and protrusion, appears to have a strong influence on the production of temporomandibular sound events.     

Characteristics of vascular wall cells subjected to dynamic cyclic strain and fluid shear conditions in vitro

Surfactant bolus instillation may be associated with a drop in blood pressure. Platelet-activating factor (PAF) has been found in surfactant preparations. The aim of this study was to evaluate rapid tracheal infusion of surfactant during 5 min as an alternative to bolus instillation and to examine whether a PAF receptor antagonist is able to prevent the decrease in blood pressure. METHODS: Surfactant deficiency was induced in 16 adult rabbits by lung lavages with saline. Six animals received a bolus of a porcine surfactant preparation (Curosurf (CS); 200 mg/kg), labeled with red microspheres to assess pulmonary distribution. In another 5 rabbits, the same amount of labelled CS was instilled by tracheal infusion within 5 min. A third group of 5 animals received 3 mg/kg body weight of the PAF antagonist WEB 2170 before CS bolus instillation. RESULTS: After CS bolus administration, mean PaO2 increased by 44.7 +/- 8.3 kPa (mean +/- SD) within 2 min and remained at this level. Mean arterial blood pressure dropped transiently by 2.3 +/- 2 kPa within 5 min. Pulmonary distribution of surfactant was even. After infusion, mean PaO2 rose by 22.4 +/- 16.3 kPa within 15 min. Blood pressure dropped by 1.8 +/- 1.1 kPa within 15 min. The distribution was extremely uneven. Blood pressure decreases also occurred after pretreatment with PAF receptor antagonist. CONCLUSION: Rapid tracheal infusion of surfactant results in poorer oxygenation, an inhomogeneous distribution and a similar decrease in blood pressure compared to the bolus instillation method. Blood pressure changes could not be prevented by a PAF receptor-specific antagonist.