Microalgae as biological treatment for municipal wastewater - effects on the sludge handling in a treatment plant.

A mix of microalgae and bacteria was cultivated on pre-sedimented municipal wastewater in a continuous operated microalgae-activated sludge process. The excess material from the process was co-digested with primary sludge in mesophilic and thermophilic conditions in semi-continuous mode (5 L digesters). Two reference digesters (5 L digesters) fed with waste-activated sludge (WAS) and primary sludge were operated in parallel. The methane yield was slightly reduced (≈10%) when the microalgal-bacterial substrate was used in place of the WAS in thermophilic conditions, but remained approximately similar in mesophilic conditions. The uptake of heavy metals was higher with the microalgal-bacterial substrate in comparison to the WAS, which resulted in higher levels of heavy metals in the digestates. The addition of microalgal-bacterial substrate enhanced the dewaterability in thermophilic conditions. Finally, excess heat can be recovered in both mesophilic and thermophilic conditions.

Anaerobic co-digestion of sludge and microalgae grown in municipal wastewater - a feasibility study.

In this study a natural mix of microalgae grown in wastewater of municipal character was co-digested with sewage sludge in mesophilic conditions, in both batch and semi-continuous modes. The semi-continuous experiment was divided into two periods with OLR1 (organic loading rate) of 2.4 kg volatile solids (VS) m-3 d-1 and HRT1 (hydraulic retention time) of 15 days, and OLR2 of 3.5 kg VS m-3 d-1 and HRT2 of 10 days, respectively. Results showed stable conditions during both periods. The methane yield was reduced when adding microalgae (from 200 ± 25 NmL CH4 g VSin-1, to 168 ± 22 NmL CH4 g VSin-1) but VS reduction was also decreased by 51%. This low digestibility was confirmed in the anaerobic batch test. However, adding microalgae improved the dewaterability of the digested sludge. The high heavy metals content in the microalgae resulted in a high heavy metals content in the digestate, making it more difficult to reuse the digestate as fertilizer on arable land. The heavy metals are thought to originate from the flue gas used as a CO2 source during the microalgae cultivation. Therefore the implementation of CO2 mitigation via algal cultivation requires careful consideration regarding the source of the CO2-rich gas.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

Measurement of cerebral microvascular compliance in a model of atherosclerosis with optical coherence tomography.

Optical coherence tomography (OCT) has recently been used to produce 3D angiography of microvasculature and blood flow maps of large vessels in the rodent brain in-vivo. However, use of this optical method for the study of cerebrovascular disease has not been fully explored. Recent developments in neurodegenerative diseases has linked common cardiovascular risk factors to neurodegenerative risk factors hinting at a vascular hypothesis for the development of the latter. Tools for studying cerebral blood flow and the myogenic tone of cerebral vasculature have thus far been either highly invasive or required ex-vivo preparations therefore not preserving the delicate in-vivo conditions. We propose a novel technique for reconstructing the flow profile over a single cardiac cycle in order to evaluate flow pulsatility and vessel compliance. A vascular model is used to simulate changes in vascular compliance and interpret OCT results. Comparison between atherosclerotic and wild type mice show a trend towards increased compliance in the smaller arterioles of the brain (diameter < 80µm) in the disease model. These results are consistent with previously published ex-vivo work confirming the ability of OCT to investigate vascular dysfunction.

Performance evaluation of adding ethanol production into an existing combined heat and power plant.

In this paper, the configuration and performance of a polygeneration system are studied by modelling the integration of a lignocellulosic wood-to-ethanol process with an existing combined heat and power (CHP) plant. Data from actual plants are applied to validate the simulation models. The integrated polygeneration system reaches a total efficiency of 50%, meeting the heating load in the district heating system. Excess heat from the ethanol production plant supplies 7.9 MW to the district heating system, accounting for 17.5% of the heat supply at full heating load. The simulation results show that the production of ethanol from woody biomass is more efficient when integrated with a CHP plant compared to a stand-alone production plant. The total biomass consumption is reduced by 13.9% while producing the same amounts of heat, electricity and ethanol fuel as in the stand-alone configurations. The results showed that another feature of the integrated polygeneration system is the longer annual operating period compared to existing cogeneration. Thus, the renewable electricity production is increased by 2.7% per year.

Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis.

BACKGROUND AND OBJECTIVES: Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury. METHODS: Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe. RESULTS: Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow=1.7%+/-0.6% vs. 34%+/-12.6%, P=0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P=0.0001), and contained four-fold more inflammatory cells (P=0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P=0.0024 and P=0.001, respectively). CONCLUSIONS: In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.

Regression of aortic valve stenosis by ApoA-I mimetic peptide infusions in rabbits.

BACKGROUND AND PURPOSE: Aortic valve stenosis (AVS) is the most common valvular heart disease, and standard curative therapy remains open heart surgical valve replacement. The aim of our experimental study was to determine if apolipoprotein A-I (ApoA-I) mimetic peptide infusions could induce regression of AVS. EXPERIMENTAL APPROACH: Fifteen New Zealand White male rabbits received a cholesterol-enriched diet and vitamin D(2) until significant AVS was detected by echocardiography. The enriched diet was then stopped to mimic cholesterol-lowering therapy and animals were allocated randomly to receive saline (control group, n=8) or an ApoA-I mimetic peptide (treated group, n=7), three times per week for 2 weeks. Serial echocardiograms and post mortem valve histology were performed. KEY RESULTS: Aortic valve area increased significantly by 25% in the treated group after 14 days of treatment (P=0.012). Likewise, aortic valve thickness decreased by 21% in the treated group, whereas it was unchanged in controls (P=0.0006). Histological analysis revealed that the extent of lesions at the base of valve leaflets and sinuses of Valsalva was smaller in the treated group compared with controls (P=0.032). The treatment also reduced calcification, as revealed by the loss of the positive relationship observed in the control group (r=0.87, P=0.004) between calcification area and aortic valve thickness. CONCLUSIONS AND IMPLICATIONS: Infusions of ApoA-I mimetic peptide lead to regression of experimental AVS. These positive results justify the further testing of high-density lipoprotein (HDL)-based therapies in patients with valvular aortic stenosis. Regression of aortic stenosis, if achieved safely, could transform the clinical treatment of this disease.

Chronic heart rate reduction by ivabradine prevents endothelial dysfunction in dyslipidaemic mice.

BACKGROUND AND PURPOSE: High resting heart rate is a predictor for total and cardiovascular mortality independent of other risk factors in patients with coronary artery disease. We tested the hypothesis that a reduction of resting heart rate with the cardiac pacemaker I(f) current inhibitor ivabradine prevents the endothelial dysfunction associated with dyslipidaemia. EXPERIMENTAL APPROACH: Three-month-old dyslipidaemic (DL) male mice expressing the human ApoB-100 were assigned or not (DL, n=16), to treatment for 3 months with ivabradine (10 mg kg(-1) d(-1), n=17). Wild-type C57Bl/6 mice (WT, n=15) were used as controls. Heart rate was measured at 3, 4.5 and 6 months. Dilatation to acetylcholine (ACh) of isolated cerebral and renal arteries was investigated at 6 months. KEY RESULTS: Heart rate remained stable in anaesthetized WT mice, increased (25%, P<0.05) with age in DL mice but was limited (11%, P<0.05) by ivabradine. At 6 months, left ventricular maximal pressure was similar in all groups. The minimal and end-diastolic left ventricular pressures were increased (P<0.05) in DL (10.2+/-1.0 and 18.7+/-1.4 mm Hg) compared to WT (-0.4+/-0.7 and 6.3+/-1.0 mm Hg) and reduced (P<0.05) by ivabradine (4.2+/-1.3 and 11.5+/-1.5 mm Hg). ACh-induced maximal dilatation was impaired (P<0.05) in renal and cerebral arteries isolated from DL compared to WT (56+/-7 versus 83+/-3% in renal arteries; 22+/-2 versus 42+/-2% in cerebral arteries). Ivabradine completely prevented (P<0.05) this dysfunction in renal and cerebral arteries. CONCLUSIONS AND IMPLICATIONS: Selective heart rate reduction with ivabradine limits cardiac dysfunction and prevents the renovascular and cerebrovascular endothelial dysfunction associated with dyslipidaemia.

Activation of ETB receptors regulates the abundance of ET-1 mRNA in vascular endothelial cells.

BACKGROUND AND PURPOSE: The factors that influence the cellular levels of endothelin-1 (ET-1) include transcription, mRNA localization, stability and translation, post-translational maturation of preproET-1 and degradation of ET-1. We investigated the regulation of ET-1 mRNA abundance by extracellular ET-1 in porcine aortic endothelial cells (PAECs). EXPERIMENTAL APPROACH: Passsage one cultures of PAECs were incubated in starving medium in the presence or absence of ET-1 and antagonists or pharmacological inhibitors. PreproET-1 mRNA, endothelin-1 promoter activity, Erk and p38 MAPK activation were determined. KEY RESULTS: Exogenous ET-1 reduced cellular ET-1 mRNA content: a reduction of 10 000-fold was observed after 4 h. ET-1 simultaneously reduced the stability of ET-1 mRNA and increased the loading of RNA Polymerase II at the endothelin-1 promoter. In the absence of exogenous ET-1, the ETB-selective antagonist, BQ788, increased ET-1 mRNA. An ETA-selective antagonist had no effect. ET-1 mRNA returned to control levels within 24 h. Whereas activation of p38 MAPK induced by ET-1 peaked at 30 min and returned to control levels within 90 min, Erk1/2 remained active after 4 h of stimulation. Inhibition of p38 MAPK prevented the ET-1-induced decrease in ET-1 mRNA. In contrast, Erk1/2 inhibition increased ET-1 mRNA. Similarly, inhibition of receptor internalization increased ET-1 mRNA in the presence or absence of exogenous ET-1. CONCLUSIONS AND IMPLICATIONS: These results suggest that extracellular ET-1 regulates the abundance of ET-1 mRNA in PAECs, in an ETB receptor-dependent manner, by modulating both mRNA stability and transcription via mechanisms involving receptor endocytosis and both ERK and p38 MAPK pathways.

Inhaled but not intravenous milrinone prevents pulmonary endothelial dysfunction after cardiopulmonary bypass.

OBJECTIVE: Cardiopulmonary bypass triggers a systemic inflammatory response that alters pulmonary endothelial function, which can contribute to pulmonary hypertension. Milrinone is a type III phosphodiesterase inhibitor. The objective of this study was to compare the effects of inhaled and intravenous milrinone on the pulmonary endothelium-dependent relaxations and hemodynamic and oxygenation parameters after cardiopulmonary bypass in a porcine model. METHODS: Five groups of Landrace swine were compared: (1) control group, no cardiopulmonary bypass; (2) bypass group, 90 minutes of normothermic bypass and 60 minutes of reperfusion; (3) inhaled milrinone group, bypass preceded by a 1.8-mg bolus of inhaled milrinone followed by a continuous milrinone nebulization; (4) intravenous milrinone group, bypass preceded by 2 mg of intravenous milrinone; and (5) inhaled NaCl group, bypass preceded by inhaled saline solution. After sacrifice, pulmonary arterial endothelium-dependent relaxations to acetylcholine and bradykinin were studied in organ chambers. RESULTS: Inhaled milrinone caused less hypotension ( P < .05), a lesser decrease in peripheral vascular resistances ( P < .01), and a lower heart rate ( P < .05) than intravenous milrinone. Inhaled milrinone prevented the alterations in relaxations of pulmonary arteries to acetylcholine caused by cardiopulmonary bypass, and relaxations to bradykinin were improved in the inhaled milrinone group ( P < .05) compared with the cardiopulmonary bypass and control groups. CONCLUSIONS: Inhaled milrinone prevents the occurrence of the pulmonary endothelial dysfunction seen after cardiopulmonary bypass. The hemodynamic and oxygenation profiles obtained with inhaled milrinone are safer than with intravenous milrinone. These strategies might be useful in preventing pulmonary hypertension after cardiac surgery.

Endothelial G protein beta-subunits trigger nitric oxide-but not endothelium-derived hyperpolarizing factor-dependent dilation in rabbit resistance arteries.

A single subtype of heterotrimeric G protein-coupled receptor controls both nitric oxide (NO) (sensitive to L-arginine analogues) and endothelium-derived hyperpolarizing factor (EDHF) (sensitive to high-external K(+) and apamine) production by the vascular endothelium leading to dilation. We hypothesized that alpha- and betagamma-subunits of the G protein serve as distinct intermediates to produce NO and EDHF. In pressurized resistance arteries, selective pinocytotic endothelial incorporation of specific antibodies (Abs) directed against alpha(q/11)-subunits abolished acetylcholine (Ach)-mediated dilation but failed to influence oxymetazoline (Oxy, alpha(2)-adrenergic receptor agonist)-induced dilation. In contrast, alpha(i1-2)-subunit Abs prevented Oxy- but not Ach-induced dilation. Thus, as expected, endothelial muscarinic and alpha(2)-adrenoceptors couple to G(q) protein and G(i) proteins, respectively. beta-subunit Abs reduced both Ach- and Oxy-induced dilation. The beta-subunit Abs abolished the nitro-L-arginine (L-NNA)-sensitive component but did not impair the high-external K(+)-sensitive component of the dilation induced by Ach and Oxy. Thus, G protein beta-subunits primarily accounted for NO production. Neutralization of Hsp90 and inhibition of the phospholipase C by U73122 (1 micromol/L) or intracellular Ca(2+) buffering with BAPTA-AM (10 micromol/L) sharply reduced NO-dependent but not K(+)-sensitive dilation. In conclusion, mobilization of the G protein beta-subunit is pivotal to NO-dependent dilation triggered through muscarinic and alpha(2)-adrenergic receptors. In contrast, receptor-operated EDHF-dependent dilation was insensitive to beta-subunit Abs. Although not directly activating the NO pathway, alpha-subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries.

Endothelium-derived endothelin-1 reduces cerebral artery sensitivity to nitric oxide by a protein kinase C-independent pathway.

BACKGROUND AND PURPOSE: Nitric oxide (NO) reduces endothelin-1 (ET-1) production and blunts ET-1 dependent vasoconstriction. The direct effects of smooth muscle ET(A) receptor stimulation on NO-mediated relaxation are unknown. We hypothesized that endothelium-derived ET-1 regulates vascular tone by reducing smooth muscle sensitivity to NO, possibly through activation of protein kinase C (PKC). METHODS: Rings of rabbit middle cerebral artery were mounted on microvessel myographs to measure isometric tension. Dose-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP; an NO donor) were obtained with or without ET-1 receptor blockade. Experiments were performed in the presence of indomethacin (10 micromol/L). Results are expressed as mean+/-SEM. RESULTS: In depolarized conditions (40 mmol/L KCl physiological solution), ACh-induced relaxation was entirely NO-dependent, as indicated by its suppression by N(omega)-nitro-L-arginine (P<0.05). Arterial sensitivity (pD(2)) to ACh (6.32+/-0.11, n=6) was increased (P<0.05) to 6.77+/-0.10 (n=6) by BQ123 (ET(A) receptor antagonist, 5 micromol/L) but not by BQ788 (ET(B) receptor antagonist, 5 micromol/L; 6.08+/-0.22, n=5). Consistent with this finding, blockade of ET(A) receptors increased (P<0.05) vascular sensitivity to SNP (6.95+/-0.10, n=8), whereas BQ788 had no influence on arterial sensitivity to SNP (6.17+/-0.07, n=7) compared with control (6.43+/-0.13, n=11). In denuded arteries, the sensitivity to SNP (7.10+/-0.08, n=8) was reduced by exogenous ET-1 (6.51+/-0.35, n=7, P<0.05). Chelerythrine, a PKC inhibitor, did not alter smooth muscle sensitivity to NO, whereas phorbol 12-myristate 13-acetate, a PKC activator, strongly increased it. CONCLUSIONS: Blockade of ET(A) but not ET(B) receptors sensitizes vascular smooth muscle to exogenous and endothelium-derived NO. This suggests that ET-1 regulates smooth muscle sensitivity to NO by a PKC-independent pathway. This represents an alternative pathway by which NO and ET-1 interact to regulate vascular tone.

Influence of nitric oxide synthase inhibition and endothelin-1 receptor blockade on acetylcholine-induced coronary artery contraction in vitro in dilated and ischemic cardiomyopathies.

The normal dilatory response to acetylcholine (ACH) is reduced in coronary vessels from patients with dilated cardiomyopathy (DCM) and reversed to a contraction in patients with coronary artery disease (CAD) and ischemic cardiomyopathy (ICM). This study investigated the influence of nitric oxide synthase inhibition and endothelin (ET)-1 receptor blockade on the reactivity to ACH of coronary arteries isolated from patients with end-stage congestive heart failure (CHF) associated or not with CAD. Small (approximately 400 microm) epicardial right coronary arteries were isolated from explanted hearts of patients undergoing transplantation for DCM or ICM. Segments were mounted on a wire myograph to record changes in isometric tension. ACH (1 microM) dilated pre-contracted vessels from DCM hearts but contracted pre-contracted vessels from ICM hearts. In the absence of pre-contraction, ACH (10(-9)-3 x 10(-5) M) induced a small contraction of rings from DCM hearts and a larger contraction (p < 0.05) of rings from ICM hearts. N(omega)-nitro-L-arginine (L-NNA, 100 microM), a NO synthase inhibitor, increased (p < 0.05) sensitivity and maximal response to ACH of vessels from DCM hearts only. In the presence of L-NNA, blockade of ET(A) with BQ123 (1 microM) prevented the effects of L-NNA in DCM, whereas blockade of ET(A/B) receptors with bosentan (10 microM) only reduced vascular sensitivity to ACH without significantly reducing the maximal contraction to ACH in DCM. The antagonists had no effects in vessels from ICM hearts. ACH, however, induced similar contractions of vessels without endothelium in DCM and ICM. These results suggest that ACH induces a contraction by stimulating smooth muscle muscarinic receptors. In coronary arteries isolated from DCM hearts, the contraction is regulated by NO and ET-1, whereas these factors seem to have little influence in ICM. This suggests that endothelial muscarinic receptors are either not expressed or uncoupled in ICM hearts.

Crosstalk between endothelin and nitric oxide in the control of vascular tone.

Several lines of evidence indicate that nitric oxide (NO) impairs endothelin (ET) production/action in vitro. Acute pressor responses caused by the blockade of NO formation with arginine analogues in vivo are blunted by selective ET(A) or dual ET(A)/ET(B) receptor blockade whereas blockade of NO formation magnifies ET-induced constriction of various vascular territories. Given that ET receptor blockade has normally limited effects on mean arterial pressure, the reversal of pressor responses caused by the blockade of NO formation with ET receptor blockade most likely reflects a significant crosstalk between NO and ET. Suppression of NO formation also leads to significant increases in ET production caused by agents targeting the endothelium, such as acetylcholine and thrombin. In addition, the inhibitory effect of shear stress on endothelial cells ET production also involves NO as an intermediate.Paradoxically, chronic exposure to organic nitrates which causes nitrate tolerance leads to an augmented vascular ET content. An increased angiotensin II (AII) production is apparently pivotal in this process. This article reviews observations pointing to the importance of NO/ET interactions as a fundamental and common regulatory mechanism shared across species. As a consequence of this crosstalk between NO and ET, experimental strategies designed to assess endothelial NO-dependent activity by the blockade of NO formation may be mitigated by magnified ET-dependent influences.

Different contribution of endothelial nitric oxide in the relaxation of human coronary arteries of ischemic and dilated cardiomyopathic hearts.

Coronary artery disease and congestive heart failure (CHF) have been associated with a reduction in nitric oxide (NO) release or bioavailability from the vascular endothelium. The objectives of this study were to compare the role of NO in human coronary vessels isolated from nonischemic dilated (DCM) (n = 10) and ischemic (ICM) (n = 12) cardiomyopathic hearts. Segments were mounted on a wire myograph to record changes in isometric tension. All experiments were performed in the presence of indomethacin (10 microM). Contractions induced by angiotensin II (0.1 microM) or a depolarizing physiologic solution containing 40 mM KCl, were of similar amplitude in DCM and ICM. In vessels precontracted with angiotensin II, acetylcholine (1 microM) caused an endothelium-dependent relaxation of rings from DCM but a paradoxical contraction of rings from ICM; NO synthase inhibition with Nomega-nitro-L-arginine (L-NNA, 100 microM) did not affect acetylcholine-induced relaxation or contraction of DCM or ICM vessels, respectively. By contrast, substance P (0.1 microM) induced an endothelium-dependent relaxation in both groups of vessels; this relaxation was prevented (p < 0.05) by L-NNA in vessels from ICM hearts but only reduced (p < 0.05) by L-NNA in vessels from DCM hearts. In depolarized conditions, acetylcholine contracted (p < 0.05) whereas substance P induced a complete relaxation (p < 0.05) of vessels from both groups: substance P-induced relaxation was abolished (p < 0.05) by L-NNA. Our data suggest that in the presence of indomethacin, NO does not contribute to acetylcholine-induced relaxation of human epicardial coronary arteries isolated from DCM hearts. Furthermore, whereas NO and a secondary endothelium-derived relaxing factor sensitive to high K+ contribute to substance P-induced relaxation of rings from DCM hearts, only NO is involved in ICM hearts.

In vitro response of human and porcine vascular cells exposed to high dose-rate gamma-irradiation.

AIM: The objective of this study was to compare the in vitro response of human and pig endothelial cells, smooth muscle cells and fibroblasts exposed to conventional high dose-rate gamma-irradiation. MATERIALS AND METHODS: Clonogenic cell survival and growth responses were obtained after irradiation of plateau-phase cells with a 60Co source at a dose-rate of 1.5 Gy/min. DNA single-strand breaks were also evaluated using an alkaline filter elution technique. RESULTS: Overall, both the pig and human cell lines showed a similar response to conventional high dose-rate irradiation. Using clonogenic assays, the human aortic smooth muscle cell line was more sensitive than the fibroblast and endothelial cell lines, whereas the pig endothelial cell line was more sensitive than smooth muscle cells and fibroblasts. Shortly after irradiation (10 days) there was a temporary growth arrest, which was similar for endothelial, smooth muscle cells and fibroblasts with doses above 6 Gy. There was also a non-linear, dose-dependent growth delay up to 4 weeks after irradiation. This effect was also consistent between the different cell lines. Using alkaline filter elution, there was no significant difference in relative elution between endothelial cells, smooth muscle cells and fibroblasts, indicating similar DNA damage among the different cell lines. CONCLUSION: The in vitro response of human and pig endothelial cells, smooth muscle cells and fibroblasts exposed to high dose-rate irradiation appeared similar. The pig model seems well suited to evaluate the short- and long-term effects of ionizing radiation in the prevention of restenosis after vessel injury.

Role of ET(A) receptors in the regulation of vascular reactivity in rats with congestive heart failure.

Endothelium-derived nitric oxide (NO) and endothelin (ET)-1 interact to regulate vascular tone. In congestive heart failure (CHF), the release and/or the activity of both factors is affected. We hypothesized that the increased ET-1 production associated with CHF may result in a reduced smooth muscle sensitivity to NO. The aim of this study was to evaluate the effects of a chronic treatment with the ET(A)-receptor (ET receptor A) antagonist LU-135252 (LU) on cerebrovascular reactivity to sodium nitroprusside (SNP) in the rat infarct model of CHF. Rats were subjected to coronary artery ligation and were treated for 4 wk with placebo (n = 24) or LU (50 mg. kg(-1). day(-1), n = 29). CHF was associated with a decreased (P < 0.05) efficacy of SNP to induce relaxation of isolated middle cerebral arteries. Furthermore, neither NO synthase inhibition with N(omega)-nitro-L-arginine (L-NNA) nor endothelial denudation affected the efficacy of SNP. Thus the endothelium no longer influences smooth muscle sensitivity to SNP. LU treatment, however, normalized (P < 0.05) smooth muscle sensitivity to SNP. Sensitivity of ET-1-induced contraction was increased in CHF only in the presence of L-NNA, whereas contraction induced by ET(B) receptor (receptor B) stimulation was increased (P < 0.05) in endothelium-denuded vessels. LU treatment restored these changes in reactivity and revealed a significant endothelium-dependent ET(B)-mediated relaxation after NO synthase inhibition. In conclusion, CHF decreases and uncouples cerebrovascular smooth muscle sensitivity to SNP from endothelial regulation. The observation that chronic ET(A) blockade restored most of the changes associated with CHF suggests that activation of the ET-1 system importantly contributes to the alteration in vascular reactivity observed in experimental CHF.

Influence of postangioplasty beta-irradiation on endothelial function in porcine coronary arteries.

BACKGROUND: Postangioplasty (PTCA) intracoronary radiation therapy (ICRT) has been demonstrated to limit restenosis. The consequences of these procedures on coronary reactivity are unknown. METHODS AND RESULTS: Porcine coronary arteries were studied after PTCA immediately (n=5) and 6 weeks (n=5) after ICRT (n=5 and 5, respectively), after combined PTCA+ICRT (n=5 and 7, respectively), and after no intervention (n=11). A 3-cm-long source train of Sr/Y(90) was used in vivo to deliver 16 Gy at a depth of 2 mm from the source center, as used in clinical trials. Arterial rings were mounted on myographs to record isometric tension. After achieving steady-state contraction to depolarizing physiological solution containing 40 mmol/L KCl, measured baseline tension was significantly elevated immediately after all interventions. It returned to normal levels 6 weeks after PTCA and ICRT alone but was significantly reduced if combined. Active contractions induced by 40 mmol/L KCl were maintained after combined therapy both immediately after and at 6 weeks. In these depolarizing conditions, nitric oxide-dependent relaxation to substance P was trivial after PTCA+ICRT and reduced after ICRT, whereas in the presence of physiological solution and N(omega)-nitro-L-arginine, substance P-induced relaxation was reduced after PTCA and abolished after PTCA+ICRT 6 weeks after intervention. In rings without endothelium, the relaxation mediated by sodium nitroprusside (0.1 micromol/L) was reduced immediately after PTCA and at 6 weeks. CONCLUSIONS: PTCA+ICRT altered the passive mechanical properties of porcine coronary arterial wall. Furthermore, at 6 weeks, receptor-operated release of endothelium-derived nitric oxide and endothelium-derived hyperpolarizing factor was reduced by ICRT and PTCA alone, respectively, and was prevented by their combination.

Simultaneous measurement of ERK, p38, and JNK MAP kinase cascades in vascular smooth muscle cells.

Activation of the mitogen-activated protein kinase (MAP kinase) pathways in cultured porcine aortic vascular smooth muscle cells (VSMCs) was determined following a 5-min stimulation with endothelin-1 (ET-1), phorbol 12-myristate 13-acetate (PMA), H2O2, or sodium arsenite. Extracellular signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2) MAP kinase activation was assessed using anti-phospho-MAPK kinase antibodies. The activation of these kinase cascades was also determined by resolving lysates on Mono Q using a fast protein liquid chromatography (FPLC) system and measuring the phosphorylation of specific substrates ERK1, c-Jun, and hsp27. The substrates were subsequently resolved from each other and the [gamma-32P]ATP in the reaction mixture by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the incorporation of 32P was quantified by phosphor imaging. This technique revealed the presence of multiple peaks of activity phosphorylating ERK1 (5), c-Jun (7), and hsp27 (9). Differences in activation revealed by the chromatographic technique suggest that, although equivalent levels of activation may be detected by immunoblotting, the actual nature of the response differed depending upon the stimulus. Each stimulus that activated the MAP kinase cascades did not result in equivalent 'profile' of activation of kinase activities. These results suggest the presence of a mechanism of structural organization of the MAP kinase signaling molecules themselves resulting in the compartmentalization of responses with respect to the various cellular stimuli.