Flow modulates endothelial regulation of smooth muscle cell proliferation: a new model

BACKGROUND: With a co-culture model, we have previously demonstrated that endothelial cells (ECs) exert regulatory control over smooth muscle cell (SMC) behavior. ECs appeared to stimulate SMC proliferation in static culture. This study was performed to test the hypothesis that the EC stimulation of SMC proliferation was effected by shear stress. METHODS: Bovine SMCs were cultured on a thin semipermeable membrane either alone or opposite ECs in co-culture (SMC/EC). A novel parallel-plate flow device was developed and used for exposing the EC side of the co-culture to shear stress. EC and SMC proliferation rates were determined after 24 hours' exposure to 0, 1, or 10 dynes/cm2 of shear stress. RESULTS: SMC proliferation decreased significantly from 362 +/- 65 cpm/microgram DNA (control, mean +/- SEM) to 68 +/- 43 cpm/microgram (1 dyne/cm2) and 99 +/- 18 cpm/microgram (10 dynes/cm2)(P < .05). EC proliferation after flow decreased as compared with no-flow controls 71 +/- 15 cpm/micrograms DNA (control, mean +/- SEM) to 29 +/- 5 cpm/microgram (1 dyne/cm2) and 21 +/- 4 cpm/microgram (10 dynes/cm2)(P < .05). CONCLUSIONS: In a model designed to study SMC/EC interactions in a flow environment, it was seen that EC exposure to shear stress alters the growth characteristics of SMCs. This suggests that hemodynamic mechanical forces may be sufficient to alter the EC regulation of SMC behavior.     

Mechanical trauma in leukocytes

Mechanical and surface traumas in cardiopulmonary bypass circuits alter the function and morphology of human leukocytes. The effect of controlled in vitro shear stess (0 to 2,000 dynes/cm2, 2 to 10 min, 37 degrees C) on electronic cell count, morphology, adhesiveness, and phosphatase cytochemical staining was studied on whole blood from normal donors. Electronic cell counts droppped significantly after shear stress exposure (25% at 600 dynes/cm2 for 10 min). The frequency of disrupted leukocytes in blood smears increased with shear stress above 150 dynes/cm2, and aggregates of the disrupted cells appeared after exposure to higher shear stresses (450 dynes/cm2, 10 min). Cytochemical staining of the alkaline phosphatase in the granules of intact neutrophils was significantly reduced by the application of shear stress (150 dynes/cm2 for 10 min, or greater), but staining of acid phosphatase-containing granules was almost unaffected. Increased cell retention in columns of nylon fibers suggests that increased leukocyte adhesiveness results from exposure to shear stress. Thus exposure to shear stress may alter or disrupt leukocyte morphology and function at values somewhat lower than the 1,500 dynes/cm2 for 2 min which is required to hemolyze erythrocytes.     

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.     

In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts

PURPOSE: To perform an in vitro evaluation of electrostatic endothelial cell transplantation of human umbilical vein endothelial cells (HUVEC) onto segments of 4 mm internal diameter expanded polytetrafluoroethylene (ePTFE) vascular prostheses. METHODS: This evaluation consisted of exposing vascular graft segments that had been subjected to either electrostatic or gravitation transplantation with HUVEC to a physiologic shear stress (15 dynes/cm2) under steady flow conditions within a flow loop system. Biochemical assays were performed on freshly transplanted grafts by means of radioimmunoassay for prostacyclin and thromboxane A2. RESULTS: There was a 30% loss of HUVEC after 30 minutes of shear stress exposure from the grafts subjected to gravitational transplantation with no additional significant (alpha = 0.05) loss after 120 minutes. Grafts subjected to electrostatic transplantation had no significant (alpha = 0.05) loss of HUVEC during exposure to physiologic shear stress. Furthermore, after 120 minutes of shear-stress exposure, the grafts subjected to electrostatic transplantation (78,420 +/- 6274 HUVEC/cm2) retained 2.3 times more HUVEC than the counterparts subjected to gravitational transplantation (34,427 +/- 4637 HUVEC/cm2). The biochemical assay results indicated no significant (alpha = 0.05) production of prostacyclin or thromboxane A2 regardless of the method of cell transplantation. CONCLUSIONS: (1) The electrostatic transplantation technique was superior to the gravitational transplantation technique in terms of cellular retention when the ePTFE grafts were exposed to physiologic shear stress. (2) Production of prostacyclin and thromboxane A2 did not differ between transplanted HUVEC subjected to gravitational or electrostatic procedures.     

Effects of inhaled versus intravenous vasodilators in experimental pulmonary hypertension

Inhaled nitric oxide (NO) causes selective pulmonary vasodilation and improves gas exchange in acute lung failure. In experimental pulmonary hypertension, we compared the influence of the aerosolized vasodilatory prostaglandins (PG) PGI2 and PGE1 on vascular tone and gas exchange to that of infused prostanoids (PGI2, PGE1) and inhaled NO. An increase of pulmonary artery pressure (Ppa) from 8 to approximately 34 mmHg was provoked by continuous infusion of U-46619 (thromboxane A2 (TxA2) analogue) in blood-free perfused rabbit lungs. This was accompanied by formation of moderate lung oedema and severe ventilation-perfusion (V'/Q') mismatch, with predominance of shunt flow (>50%, assessed by the multiple inert gas elimination technique). When standardized to reduce the Pps by approximately 10 mmHg, inhaled NO (200 ppm), aerosolized PGI2 (4 ng x kg(-1) x min(-1)) and nebulized PGE1 (8 ng x kg(-1) x min(-1)) all reduced both pre- and postcapillary vascular resistance, but did not affect formation of lung oedema. All inhalative agents improved the V'/Q' mismatch and reduced shunt flow, the rank order of this capacity being NO > PGI2 > PGE1. In contrast, lowering of Ppa by intravascular administration of PGI2 and PGE1 did not improve gas exchange. "Supratherapeutic" doses of inhaled vasodilators in control lungs (400 ppm NO, 30 ng x kg(-1) x min(-1) of PGI2 or PGE1) did not provoke vascular leakage or affect the physiological V'/Q' matching. We conclude that aerosolization of prostaglandins I2 and E1 is as effective as inhalation of nitric oxide in relieving pulmonary hypertension. When administered via this route instead of being infused intravascularly, the prostanoids are capable of improving ventilation-perfusion matching, suggesting selective vasodilation in well-ventilated lung areas.     

Influence of the estrous cycle on the norepinephrine-induced contraction of rat aorta: relationship to vascular prostanoids biosynthesis

Since ovarian sex steroids (estradiol and progesterone) may affect both blood pressure and prostanoids synthesis, and because prostaglandin-E2 (PGE2) and prostacyclin (PGI2) can modulate the vascular action of pressor hormones, we investigated the vascular reactivity to norepinephrine during the estrous cycle of the rat. In addition, we determined the vascular biosynthesis of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2) at different stages of the estrous cycle. Cumulative dose-response curves were obtained by a stepwise increase in the concentration of norepinephrine. The contraction of thoracic aortic rings induced by norepinephrine did not change significantly between estrus, metestrus and diestrus. However, aortic rings obtained on proestrus showed a significant reduction in the maximal contraction (Emax) induced by norepinephrine (p < 0.001). In addition, we found significant increases in vascular synthesis of PGE2 and PGI2 on proestrus (p < 0.001). These results indicate that vascular reactivity and vascular prostanoids synthesis are influenced by the hormonal changes occurring during the estrous cycle of normal female rats. It is possible that prostanoids generated locally may play an important role in the regulation of vasomotor tone in the systemic vascular bed throughout the estrous cycle.     

Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase

Physiological levels of shear stress reduce endothelial cell turnover and exert a potent antiatherosclerotic effect. Here we demonstrate that oxidative stress-induced apoptosis of human endothelial cells was inhibited by shear stress exposure (15 dynes/cm2). Incubation with H2O2 (200 mumol/L) for 18 hours induced apoptosis of human umbilical venous endothelial cells as demonstrated by an enzyme-linked immunosorbent assay specific for histone-associated DNA fragments and visual analysis of fluorescence-stained nuclei. Shear stress-mediated inhibition of apoptosis was partially prevented by pharmacological inhibition of glutathione (GSH) biosynthesis with buthionine sulfoximine (BSO) or nitric oxide (NO) synthase with NG-monomethyl-L-arginine (LNMA), whereas inhibition of catalase by aminotriazol did not affect the inhibitory action of shear stress. Combined inhibition of NO synthase and GSH biosynthesis completely reversed the protective effect of shear stress, suggesting that both NO synthase and the GSH redox cycle system are involved in the apoptosis-suppressing effect of shear stress. Similar results were obtained when apoptosis was stimulated by tumor necrosis factor alpha (TNF alpha). To gain further insights into the interference of shear stress with apoptosis signal transduction, we measured caspase-3-like activity, a cysteine protease that has been shown to play a predominant role in the cell death effector pathway. Indeed, shear stress prevented the activation of caspase-3-like activity induced by H202 or TNF alpha. The inhibitory effect of shear stress was prevented by LNMA and BSO, suggesting that the reduction of oxidative flux by shear stress prevents the activation of caspase-like proteases and thereby inhibits apoptotic cell death in human endothelial cells.     

Computer analysis of single neuron activity during conditioned feeding task

Human recombinant tumor necrosis factors alpha and beta (TNF-alpha and TNF-beta), at and above 1 ng/ml (approximately equal to 70 pM), caused a dose- and time-dependent enhancement of 45Ca release from neonatal mouse calvarial bones in vitro. In addition, TNF-alpha and TNF-beta (3-100 ng/ml) caused a dose-dependent stimulation of prostaglandin E2 (PGE2) formation in the calvarial bones. TNF-alpha also enhanced the biosynthesis of PGI2, as assessed by analysis of the stable breakdown product 6-keto-PGF1 alpha. The stimulatory actions of TNF-alpha and TNF-beta on PGE2 formation was maximal at 12 h. Indomethacin, flurbiprofen, and meclofenamic acid, three structurally unrelated nonsteroidal antiinflammatory drugs, abolished PGE2 biosynthesis induced by TNF-alpha and TNF-beta (100 ng/ml). The 45Ca release stimulated by TNF-alpha and TNF-beta (100 ng/ml), however, was only slightly reduced by indomethacin, flurbiprofen, and meclofenamic acid. The partial inhibitory effect of indomethacin on 45Ca release was seen over a wide range of TNF-alpha concentrations, without affecting the concentration producing half-maximal stimulatory response. TNF-alpha and TNF-beta (100 ng/ml) stimulated bone matrix breakdown, as assessed by analysis of the release of 3H from bone prelabeled with [3H]proline. Also, the stimulatory effect of TNF-alpha and TNF-beta on bone matrix degradation was partially reduced by indomethacin. Hydrocortisone (1 microM) and dexamethasone (0.1 microM) abolished TNF-alpha- and TNF-beta-induced production of PGE2. In contrast to the cyclooxygenase inhibitors, the corticosteroids did not affect the stimulatory action by the cytokines on 45Ca release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness

OBJECTIVE: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. METHODS: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm2 shear stress and then for an additional 3 days with 1 or 25 dyne/cm2 shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with beta-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. RESULTS: The pretreatment with 25 dyne/cm2 but not with 0 or 1 dyne/cm2 shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that beta-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained alpha-smooth muscle actin were present. The thickness of this neointima averaged 41 +/- 12 micrometer and 60 +/- 23 micrometer in endothelial cell-seeded grafts that were pretreated with 25 dyne/cm2 shear stress and 1 dyne/cm2 shear stress, respectively, and 158 +/- 46 micrometer in grafts that were not seeded with endothelial cells. CONCLUSION: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness.     

Myocardial production of prostaglandins: its role in atrial natriuretic peptide release

In recent years, considerable evidence has been accumulated on prostaglandins (PG) in modulating atrial natriuretic peptide (ANP) release. In the current study we investigated whether eicosanoids promote isoproterenol-induced ANP secretion from superfused rabbit sliced atria. Inclusion of the cyclooxygenase inhibitor indomethacin (10 mumol) to the superfusing medium abolished isoproterenol-induced ANP release. Next, PGE2, but not PGF2 alpha or PGI2 (10 mumol), increased ANP release. Furthermore, isoproterenol-induced PGE2 formation was fully attenuated by indomethacin. Dibutyl-cAMP (0.5 mmol) had no effect on PGE2 formation, and the protein kinase A (PKA) inhibitor H89 (20 mumol) did not alter isoproterenol-induced PGE2 formation. On the other hand, indomethacin led to a significant decrease in isoproterenol-induced cAMP production. In addition, PGE2 enhanced basal cAMP concentration in superfusates. Superfusion of sliced atria by forskolin (10 mumol) or by dibutyl-cAMP (0.5 mmol) produced a significant rise in ANP release. Finally, H89 was ineffective on basal ANP release but abolished the increase of ANP release in response to isoproterenol or to PGE2. We conclude that: the effect of isoproterenol on ANP release is sensitive to indomethacin and H89; PGE2, but not PGE2 alpha or PGI2, increases ANP release; isoproterenol promotes myocardial PGE2 formation independently of adenylate cyclase and PKA activation pathways; and PGE2-induced ANP release is mediated by cAMP production and subsequent PKA activation. These results suggest that isoproterenol-induced ANP release appears to be mediated at least partly by PGE2 with underlying cAMP formation and PKA activation.     

Effect of ganglioside (GM1) on memory in senescent rats

We investigated the effects of aerosolized prostacyclin (PGI2) administration on hemodynamics and pulmonary gas exchange in 8 patients with severe respiratory failure and acute pulmonary hypertension. Nebulization of epoprostenol (5 ng/kg body weight for 15 min) decreased mean pulmonary blood pressure from 41.2 +/- 6.7 mm Hg (mean +/- SD, before administration) to 36.1 +/- 6 mm Hg < or = 15 min (p < 0.05). The effect was reversed 10 min after discontinuation of PGI2 (40.9 +/- 6.3 mm Hg). Pulmonary vascular resistance index (339 +/- 138 dynes.s.cm-5.m2, before administration) was significantly (p < 0.05) reduced < or = 15 min (260 +/- 89 dynes.s.cm-5.m2) and increased again after discontinuation of PGI2 (341 +/- 142 dynes.s.cm-5.m2). The ratio of arterial oxygen to the fraction of inspired oxygen (PaO2/FiO2) increased from 119 +/- 34 mm Hg (before administration) to 163 +/- 76 mm Hg (15 min after initiation of administration p < 0.05) and was reduced after PGI2 discontinuation (116 +/- 35 mm Hg). Heart rate, mean blood pressure, central venous pressure, and pulmonary arterial wedge pressure remained unchanged, whereas cardiac index was slightly reduced. We assume that PGI2 aerosolization is a beneficial technique, applied with a ventilator nebulization system. The beneficial effect might be caused by selective pulmonary vasodilatation in well-ventilated areas of the lung.     

Vascular endothelial cells and renin-angiotensin system

The vascular renin-angiotensin system (RAS) is regulated independently from circulating RAS and plays a role in the local regulation of vascular tone, the modulation of sympathetic activity and vascular remodeling. Endothelial cells are a major source of angiotensin converting enzyme (ACE), which produces angiotensin II and degrades bradykinin, in normal arteries. Mechanical stress such as transmural pressure, stretch stress and shear stress appear to contribute to the regulation of endothelial ACE activity. In contrast, vessels with intimal proliferation such as atheromatous plaque and neointima following balloon injury show expression of ACE in smooth muscle cells and macrophages in the intimal lesions. Activation of ACE in intimal SMC may relate to a phenotypic change of SMC from the contracting type of the synthetic type. Activation of ACE in macrophages is also related to the transformation of macrophages from monocytes. Concerning the role of the activated RAS, elevated blood pressure and vascular tonus by angiotensin II are candidates of vascular injury and plaque rupture. Angiotensin II stimulates migration and proliferation of smooth muscle cells and production of extracellular matrix. Furthermore, angiotensin II increases oxidized-LDL which may be related to the forming of macrophages. These evidence suggest that activation of vascular RAS following endothelial dysfunction/injury play an important role in the pathogenesis of vascular remodeling and atherosclerosis.     

Reversal of indomethacin-induced inhibition of tubuloglomerular feedback by prostaglandin infusion

Experiments were performed in rats to study the effect of infusion of PGI2, PGE2, and PGF2 alpha on tubuloglomerular feedback responses (i.e. the change of SNGFR in response to a change of loop of Henle flow rate) in the presence and absence of simultaneous inhibition of endogenous PG synthesis with indomethacin. Infusion of PGI2 or PGE2 at rates that did not alter arterial blood pressure did not significantly modify the magnitude of feedback responses (PGI2 8.5 micrograms/hr, PGE2 85 micrograms/hr). Some inhibition of feedback responses was seen when PGI2 and PGE2 were administered at higher rates that were associated with a reduction of blood pressure (PGI2 20 micrograms/hr, PGE2 200 micrograms/hr). PGI2 (8.5 micrograms/hr) and PGE2 (85 micrograms/hr) largely prevented feedback inhibition induced by indomethacin. When given subsequent to indomethacin PGI2 and PGE2 restored feedback responsiveness almost to normal. In contrast, PGF2 alpha did not influence feedback inhibition caused by indomethacin. Infusion of PGI2 induced partial restoration of feedback responses in DOCA-salt treated animals in which the feedback system is virtually completely inactive. Our results indicate that availability of PGI2 or PGE2 is necessary for the normal operation of the tubuloglomerular feedback mechanism for control of nephron filtration rate.     

Living-related liver transplantation and neurological outcome in children with fulminant hepatic failure

In vivo, endothelial cells (ECs) are subjected to a complex mechanical environment composed of shear stress, pressure, and circumferential stretch. The aim of this study was to subject bovine aortic ECs to a pulsatile pressure oscillating from 70 to 130 mm Hg (mean of 100 mm Hg) in combination with pulsatile shear stresses from 0.1 to 6 dyne/cm2 (1 dyne/cm2=0.1 N/m2) with or without a cyclic circumferential stretch of 4% for 1, 4, and 24 hours. The effect of highly reversing oscillatory shear stress (range -3 to +3 dyne/cm2, mean of 0.3 dyne/cm2) typical of regions prone to the development of atherosclerotic plaques was also studied at 4 and 24 hours. Endothelin-1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) mRNA expression was time and mechanical force dependent. ET-1 mRNA was maximal at 4 hours and decreased to less than static culture expression at 24 hours, whereas ecNOS mRNA increased over time. Pressure combined with low shear stress upregulated ET-1 and ecNOS mRNA compared with static control. Additional increase in expression for both genes was observed under a combination of higher shear stress and pressure. A cyclic circumferential stretch of 4% did not induce a further increase in ET-1 and ecNOS mRNA at either low or high shear stress. Oscillatory shear stress with pressure induced a higher expression of ET-1 mRNA but lower expression of ecNOS mRNA compared with unidirectional shear stress and pressure. We have shown that the combination of pressure and oscillatory shear stress can downregulate ecNOS levels, as well as upregulate transient expression of ET-1, compared with unidirectional shear stress. These results provide a new insight into the exact role of mechanical forces in endothelial dysfunction in regions prone to the development of atherosclerosis.     

Release of prostaglandin I2-like activity from the rat aorta: effect of captopril, furosemide, and sodium

The effect of captopril, furosemide, indomethacin and intake of sodium on the production of PGI2-like material was studied in the rat aorta. Release of PGI2-like material from these vessels was estimated by its ability to inhibit ADP-induced platelet aggregation. Pretreatment with indomethacin (15 mg/kg/day) reduced the capacity of the aorta to release PGI2-like material. Pretreatment with captopril (10 mg/kg/day) had no effect. Intravenous furosemide (60 microgram/ml plasma volume) increased the capacity of the aorta to inhibit by 28% (p less than 0.25). The inhibitory capacity of aorta removed from rats on a low sodium diet did not differ from those on a high sodium diet. We conclude that the action of furosemide in reducing vascular tone may be related to stimulation of PGI2 synthesis in blood vessels whereas the effect of captopril and sodium in reducing vascular tone may involve a mechanism unrelated to PGI2 synthesis or may involve the synthesis of a prostaglandin other than PGI2.     

Expression and regulation of vascular endothelial growth factor in osteoblasts

Bone formation is linked closely to angiogenesis. Because prostaglandin E2 (PGE2) is a potent stimulator of bone formation, its effects were evaluated on vascular endothelial growth factor, a secreted endothelial cell-specific mitogen, and a potent angiogenic protein. Prostaglandin E2 increased vascular endothelial growth factor protein in conditioned media of osteoblastic RCT-3 cells within 3 hours. Prostaglandin E2 also increased the steady-state levels of vascular endothelial growth factor mRNA in a dose-dependent manner. The increased expression of vascular endothelial growth factor mRNA produced by PGE2 was rapid (maximal at 1 hour) and was enhanced by the protein synthesis inhibitor cycloheximide (5 micrograms/ml). The increase in vascular endothelial growth factor mRNA by PGE2 was inhibited strongly by pretreatment for 3 hours with dexamethasone (10(-7) M). Stimulation of vascular endothelial growth factor by PGE2 and its suppression by dexamethasone implicate the involvement of vascular endothelial growth factor in bone metabolism.     

Polymorphisms of the p53 gene in women with ovarian or endometrial carcinoma

The mechanisms underlying macrovascular complications in NIDDM are partially understood. In addition to increased prevalence and severity of systemic cardiovascular risk factors, local alterations of arterial wall and hemodynamics may play a role. Atherosclerotic lesions usually lie in regions of low wall shear stress. We therefore investigated the wall shear stress--that is, the frictional force acting tangentially to the endothelial surface--in the common carotid artery of diabetic and control subjects. Enrolled were 18 male NIDDM subjects and 18 age-matched control subjects. None of the participants were hypertensive, hyperlipidemic, or a cigarette smoker. Common carotid wall shear stress was calculated according to the following equation: blood viscosity x blood velocity/internal diameter. Blood viscosity was measured by use of a cone/plate viscometer. Blood velocity and internal diameter were measured by high-resolution echo-Doppler. Wall shear stress was significantly lower in NIDDM subjects than in control subjects (mean wall shear stress: 9.7 +/- 2.4 vs. 11.7 +/- 2.6 dynes/cm2, P < or = 0.005). Six diabetic participants had a plaque in one carotid tree and no lesions in the contralateral carotid. Among these subjects, mean wall shear stress was significantly lower in the side with lesion (8.1 +/- 1.6 vs. 10.5 +/- 2.4 dynes/cm2, P < or = 0.02). These findings suggest that diabetes is associated with a more atherosclerosis-prone carotid hemodynamic profile, which might represent an additional factor contributing to the increased prevalence and severity of carotid atherosclerosis in diabetic patients compared with general population.     

Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions

The objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti-4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.