Majid Sheykhzade

Pulmonary exposure to carbon black nanoparticles and vascular effects

BackgroundExposure to small size particulates is regarded as a risk factor for cardiovascular diseases.MethodsWe exposed young and aged apolipoprotein E knockout mice (apoE-/- ) to carbon black (Printex 90, 14 nm) by intratracheal instillation, with different dosing and timing, and measured vasomotor function, progression of atherosclerotic plaques, and VCAM-1, ICAM-1, and 3-nitrotyrosine in blood vessels. The mRNA expression of VCAM-1, ICAM-1, HO-1, and MCP-1 was examined in lung tissue.ResultsYoung apoE-/- mice exposed to two consecutive 0.5 mg/kg doses of carbon black exhibited lower acetylcholine-induced vasorelaxation in aorta segments mounted in myographs, whereas single doses of 0.05-2.7 mg/kg produced no such effects. The phenylephrine-dependent vasocontraction response was shifted toward a lower responsiveness in the mice exposed once to a low dose for 24 hours. No effects were seen on the progression of atherosclerotic plaques in the aged apoE-/- mice or on the expression of VCAM-1 and ICAM-1 and the presence of 3-nitrotyrosine in the vascular tissue of either young or aged apoE-/- mice. The expression of MCP-1 mRNA was increased in the lungs of young apoE-/- mice exposed to 0.9-2.7 mg/kg carbon black for 24 hours and of aged apoE-/- mice exposed to two consecutive 0.5 mg/kg doses of carbon black seven and five weeks prior to sacrifice.ConclusionExposure to nano-sized carbon black particles is associated with modest vasomotor impairment, which is associated neither with nitrosative stress nor with any obvious increases in the expression of cell adhesion proteins on endothelial cells or in plaque progression. Evidence of pulmonary inflammation was observed, but only in animals exposed to higher doses.

Modest effect on plaque progression and vasodilatory function in atherosclerosis-prone mice exposed to nanosized TiO 2

BackgroundThere is growing evidence that exposure to small size particulate matter increases the risk of developing cardiovascular disease.MethodsWe investigated plaque progression and vasodilatory function in apolipoprotein E knockout (ApoE-/-) mice exposed to TiO2. ApoE-/- mice were intratracheally instilled (0.5 mg/kg bodyweight) with rutile fine TiO2 (fTiO2, 288 nm), photocatalytic 92/8 anatase/rutile TiO2 (pTiO2, 12 nm), or rutile nano TiO2 (nTiO2, 21.6 nm) at 26 and 2 hours before measurement of vasodilatory function in aorta segments mounted in myographs. The progression of atherosclerotic plaques in aorta was assessed in mice exposed to nanosized TiO2 (0.5 mg/kg bodyweight) once a week for 4 weeks. We measured mRNA levels of Mcp-1, Mip-2, Vcam-1, Icam-1 and Vegf in lung tissue to assess pulmonary inflammation and vascular function. TiO2-induced alterations in nitric oxide (NO) production were assessed in human umbilical vein endothelial cells (HUVECs).ResultsThe exposure to nTiO2 was associated with a modest increase in plaque progression in aorta, whereas there were unaltered vasodilatory function and expression levels of Mcp-1, Mip-2, Vcam-1, Icam-1 and Vegf in lung tissue. The ApoE-/- mice exposed to fine and photocatalytic TiO2 had unaltered vasodilatory function and lung tissue inflammatory gene expression. The unaltered NO-dependent vasodilatory function was supported by observations in HUVECs where the NO production was only increased by exposure to nTiO2.ConclusionRepeated exposure to nanosized TiO2 particles was associated with modest plaque progression in ApoE-/- mice. There were no associations between the pulmonary TiO2 exposure and inflammation or vasodilatory dysfunction.

Functional effects of the KCNQ modulators retigabine and XE991 in the rat urinary bladder

The anticonvulsant retigabine has previously been reported to inhibit bladder overactivity in rats in vivo but the mechanism and site of action are not known. In the present study we investigated the effect of retigabine in isolated rat bladder tissue. Bladders from Sprague-Dawley rats were cut transversally into rings and mounted on an isometric myograph. The average tension, the amplitude and frequency of bladder muscle twitches were measured. The bladder tissue was stimulated with carbachol, KCl (5, 10 and 60mM), and by electric field stimulation. Dose-response curves were obtained with increasing concentrations of the KCNQ((2-5)) selective positive modulator, retigabine or with the KCNQ((1-5)) negative modulator XE991. Retigabine experiments were repeated in the presence of 10 microM XE991. Retigabine reduced both the contractility and the overall tonus of bladder tissue independent of the mode of stimulation with EC(50) values ranging from 3.3 microM (20mM KCl) to 8.3 microM (0.2 microM carbachol). In support of a KCNQ-specific effect, retigabine had only weak effects after 60mM KCl pre treatment and all retigabine effects could be reversed by XE991. XE991 increased both the amplitude and mean tension of the bladder but was more potent at increasing the number rather than the size of the stimulated twitches. In conclusion, this study demonstrates an efficacious KCNQ dependent effect of retigabine and XE991 on rat bladder contractility.

Carbon black nanoparticles and vascular dysfunction in cultured endothelial cells and artery segments

Exposure to small size particulates is regarded as a risk factor for cardiovascular disease. We investigated effects of exposure to nanosized carbon black (CB) in human umbilical vein endothelial cells (HUVECs) and segments of arteries from rodents. The CB exposure was associated with increased surface expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in HUVECs at 100μg/ml. CB exposure was also associated with increased reactive oxygen species production and damage to the cell membranes in the form of increased lactate dehydrogenase leakage, whereas it did not alter the mitochondrial enzyme activity (WST-1) or the nitric oxide level in HUVECs. Incubation of aorta segments with 10μg/ml of CB increased the endothelial-dependent vasorelaxation, induced by acetylcholine, and shifted the endothelium-independent vasorelaxation, induced by sodium nitroprusside, towards a decreased sensitivity. In mesenteric arteries, the exposure to 10μg/ml was associated with a reduced pressure-diameter relationship. Incubation with 100μg/ml CB significantly decreased both acetylcholine and sodium nitroprusside responses as well as decreased the receptor-dependent vasoconstriction caused by phenylephrine. In conclusion, nanosized CB exposure activates endothelial cells and generates oxidative stress, which is associated with vasomotor dysfunction.

Characterization of calcitonin gene-related peptide(CGRP) receptors in intramural coronary arteries from male and female Sprague Dawley rats

In this study we characterized the CGRP‐receptor subtype by Schild‐plot analysis using the C‐terminal fragment, human‐αCGRP(8–37), a putative competitive CGRP1‐receptor selective antagonist. In addition, the effect of rat‐αCGRP was compared with that of homologous peptides rat‐βCGRP, rat‐amylin, rat‐adrenomedullin and [Cys(Acm)2,7]‐human‐αCGRP, a putative selective CGRP2‐receptor agonist, in the left coronary arteries of 3 months old male and female Sprague Dawley rats. Isolated rings from the distal, intramural part of the left anterior descending (LAD) coronary artery in both groups of rats were mounted on a double wire‐myograph. The arteries were then stretched to their optimal lumen diameter for active tension development and precontracted with 10−5 m prostaglandin F2α (PGF2α), after which agonists were added to the organ bath in a cumulative manner. Rat‐αCGRP induced endothelium‐independent relaxations in male and female Sprague‐Dawley rats. Rat‐βCGRP concentration‐response relations (10−11–10−7 m) were similar to those of rat‐αCGRP in either sex. The maximal relaxations induced by rat‐amylin and rat‐adrenomedullin, both at 10−6 m, were significantly (P<0.05) lower than those induced by rat‐α‐ and rat‐βCGRP. In contrast, the selective CGRP2‐receptor agonist [Cys(Acm)2,7]‐human‐αCGRP failed to induce significant relaxations at the highest concentration used (10−7 m) in the coronary arteries of male and female rats. The C‐terminal fragment, human‐αCGRP(8–37) blocked concentration‐dependently (10−7–10−6 m) the rat‐αCGRP‐induced relaxation in 10−5 m PGF2α‐precontracted coronary arteries. The slopes of the regression lines of the Schild‐plots for both male and female rats were not significantly (P>0.05) different from unity and the pA2 values for human‐αCGRP(8–37) were 6.93 and 6.98 in arteries from male and female rats, respectively. There was no significant (P>0.05) difference in estimated pKB values for human‐αCGRP(8–37) between male (6.99±0.10, n=13) and female (6.95±0.08, n=13) rats. The concentration‐response relationships for rat‐α‐ and rat‐βCGRP were similar in male and female Sprague Dawley rats. The predominant CGRP receptor subtype in small intramural coronary arteries appeared to belong to the CGRP1‐receptor subtype in both sexes.

VIP/PACAP receptors in cerebral arteries of rat: Characterization, localization and relation to intracellular calcium

BACKGROUNDnVasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP)-containing nerves surround cerebral blood vessels. The peptides have potent vasodilator properties via smooth muscle cell receptors and activation of adenylate cyclase. The purpose of this study was to describe the effects of two putative VIP/PACAP receptor antagonists and the distribution of the receptor protein in rat brain vessels.nnnMETHODSnThe vascular effects of VIP, PACAP-27 and PACAP-38 were investigated in segments of rat middle cerebral artery (MCA) by pressurized arteriography, and in a wire myograph. The antagonistic responses to PACAP6-38 and PG99-465 were evaluated. In addition, the receptor subtypes for VIP and PACAP (VPAC1, VPAC2 and PAC1) were visualized in the rat middle cerebral artery by immunohistochemistry and Western blotting.nnnRESULTSnIn the perfusion model, abluminal but not luminal VIP, PACAP-27 and PACAP-38 caused concentration-dependent relaxations of the MCA (27.1±0.2%, 25.2±0.4% and 0.3±0.1%, respectively). In the wire myograph, there was no significant difference in potency of the peptides in the MCA. In both systems, PACAP6-38 and PG99-465 inhibited the VIP induced relaxation. Western blot showed the presence of the receptor proteins in cerebral vasculature and immunohistochemistry showed that all three receptors are present and located in the cytoplasm of smooth muscle cells.nnnCONCLUSIONnIn both systems, the two blockers antagonized the relaxant VIP effect; the potency order of agonists and the immunohistochemistry suggest the presence of the dilatory VPAC1 and VPAC2 receptors on the smooth muscle cells.

Modest vasomotor dysfunction induced by low doses of C60 fullerenes in apolipoprotein E knockout mice with different degree of atherosclerosis

BackgroundExposure to small size particulate matter in urban air is regarded as a risk factor for cardiovascular effects, whereas there is little information about the impact on the cardiovascular system by exposure to pure carbonaceous materials in the nano-size range. C60 fullerenes are nano-sized particles that are expected to have a widespread use, including cosmetics and medicines.MethodsWe investigated the association between intraperitoneal injection of pristine C60 fullerenes and vasomotor dysfunction in the aorta of 11–13 and 40–42 weeks old apolipoprotein E knockout mice (apoE-/-) with different degree of atherosclerosis.ResultsThe aged apoE-/-mice had lower endothelium-dependent vasorelaxation elicited by acetylcholine in aorta segments mounted in myographs and the phenylephrine-dependent vasoconstriction response was increased. One hour after an intraperitoneal injection of 0.05 or 0.5 mg/kg of C60 fullerenes, the young apoE-/- mice had slightly reduced maximal endothelium-dependent vasorelaxation. A similar tendency was observed in the old apoE-/- mice. Hampered endothelium-independent vasorelaxation was also observed as slightly increased EC50 of sodium nitroprusside-induced vasorelaxation response in young apoE-/- mice.ConclusionTreatment with C60 fullerenes affected mainly the response to vasorelaxation in young apoE-/- mice, whereas the vasomotor dysfunction in old apoE-/- mice with more advanced atherosclerosis was less affected by acute C60 fullerene treatment. These findings represent an important step in the hazard characterization of C60 fullerenes by showing that intraperitoneal administration is associated with a moderate decrease in the vascular function of mice with atherosclerosis.

Kv7 Positive Modulators Reduce Detrusor Overactivity and Increase Bladder Capacity in Rats

The effects of the Kv 7 channel modulators retigabine (opener) and XE991 (blocker) on rat bladder function were investigated ex vivo and in vivo to assess the potential of Kv 7 openers for the treatment of overactive bladder. In organ bath studies, capsaicin-stimulated rat urinary bladder rings were exposed to retigabine and XE991 and the effect on tension and amplitude was evaluated. In anaesthetized rats, retigabine (0.01-1u2003mg/kg, i.v.) effects on bladder function, in which overactivity was induced by continuous infusion of 0.5% acetic acid, were assessed. The effect of retigabine (10u2003mg/kg, p.o.) on cystometric parameters was also measured in conscious rats with capsaicin-induced irritated bladders. Localization of Kv 7 subunits within bladder tissue was analysed by RT-qPCR and western blotting. In organ bath studies, retigabine robustly reduced capsaicin-induced contractility of bladder rings and this effect was blocked by XE991 confirming the specificity of action via Kv 7 channels. In anaesthetized rats with acetic acid-irritated bladders, retigabine markedly increased bladder capacity with no concomitant reduction in blood pressure. Retigabine also reduced bladder pressure and delayed voiding in conscious rats with capsaicin-irritated bladders. Kv 7.1, Kv 7.4 and Kv 7.5 subunit mRNA transcripts were detected in rat bladder. Western blot analysis confirmed that Kv 7.4 subunit protein was expressed in rat bladder. These results suggest that retigabine and other Kv 7 channel positive modulators may have beneficial effects on bladder overactivity partly via activation of Kv 7 channels expressed in bladder tissue.

Mechanism of CGRP-induced relaxation in rat intramural coronary arteries

This study investigates the mechanism of CGRP‐induced relaxation in intramural coronary arteries by determining the effect of CGRP on cytosolic Ca2+ concentration ([Ca2+]i) using FURA‐2 technique. CGRP concentration‐dependently (10u2003pMu2003–u2003100u2003nM) decreased the [Ca2+]i and tension of coronary arteries precontracted with either U46619 or BAY K 8644, and also of resting coronary arteries in PSS. In 36u2003mM K+‐depolarized arteries, CGRP reduced only the tension without affecting the [Ca2+]i. In 300u2003nM U46619‐ precontracted arteries, pretreatment with 10u2003μM thapsigargin significantly (P<0.05) attenuated the CGRP‐induced reduction in the tension (but not [Ca2+]i). In 300u2003nM U46619‐precontracted arteries, pretreatment with either 100u2003nM charybdotoxin or 100u2003nM iberiotoxin or 10u2003nM felodipine significantly (P<0.05) attenuated the CGRP‐induced reduction in both [Ca2+]i and tension. In contrast, 1u2003μM glibenclamide did not affect the CGRP‐induced responses in these coronary arteries. In resting coronary arteries, only pretreatment with the combination of 1u2003μM glibenclamide and 100u2003nM charybdotoxin attenuated the CGRP‐induced decrease in the [Ca2+]i and tension, suggesting a different mechanism of action for CGRP in resting coronary arteries. We conclude that CGRP relaxes precontracted rat coronary arteries via three mechanisms: (1) a decrease in [Ca2+]i by inhibiting the Ca2+ influx through membrane hyperpolarization mediated partly by activation of the large conductance Ca2+‐activated potassium channels, (2) a decrease in [Ca2+]i presumably by sequestrating cytosolic Ca2+ into thapsigargin‐sensitive Ca2+ storage sites and (3) a decrease in the Ca2+‐sensitivity of the contractile apparatus. In resting coronary arteries, however, there seems to be an interplay between different types of K+ channels.

Pulmonary exposure to particles from diesel exhaust, urban dust or single-walled carbon nanotubes and oxidatively damaged DNA and vascular function in apoE(-/-) mice.

Abstract This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. The apoE-/- mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal (i.t.) instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas i.t. instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta.