An important factor in the development of vascular wall alterations is degradation of the elastic fiber major protein–elastin. Elastin peptides derived from this degradation are present in the circulating blood and they are a stimulus for increased production of anti-elastin antibodies (AEAb). The aim of the present study was to examine the possible association between serum elastin AEAb and the development of diabetic vascular complications. Levels of AEAb (IgG, IgM and IgA) were determined by ELISA in sera of 28 children with Type 1 (insulin-dependent) diabetes mellitus (mean age 11.6±2.8 years, diabetes duration 5.1±2.5 years). None of the children had clinical or laboratory evidence of vascular complications. The children were followed over a period of 7 years, and 24 healthy children of similar age and sex served as a control group. During the study, four diabetics developed retinopathy, six microalbuminuria and two both retinopathy and microalbuminuria. Anti-elastin IgG showed correlation with diabetes duration (r=.48, P=.0007), HbA1c (r=.28, P=.05), triglycerides (r=.28, P=.05) and antibodies to advanced glycation endproducts (AGE) (r=.41, P=.005). Anti-elastin IgM correlated with HbA1c (r=.26, P=.038) and IgA with retinopathy (r=.32, P=.017). Our results suggest an association between the level of anti-elastin IgA antibodies and the development of diabetic retinopathy.
The alpha-thrombin peptide, TP508, accelerates the healing of full-thickness wounds in both normal and ischemic skin. In wounds treated with TP508, a pattern of increased vascularization is consistently observed both grossly and microscopically when compared to wounds treated with saline. One possible mechanism by which the peptide accelerates wound healing is by promoting revascularization of granulation tissue at the injured site. To evaluate the angiogenic potential of TP508, the peptide was tested in the chick embryo chorioallantoic membrane (CAM), where it increased the density and size of CAM blood vessels relative to controls. Additionally, TP508 stimulated chemokinesis and chemotaxis in a dose-dependent fashion in cultured human aortic and human microvascular endothelial cells. Taken together, these in vivo and in vitro data support an angiogenic role for TP508 in wound healing. A working model is presented to explain how this 23-amino-acid peptide, which lacks proteolytic activity, is generated during wound healing and contributes to the nonproteolytic functions associated with alpha-thrombin during tissue repair.
During the development of complicated multicellular organisms, the robust formation of specific cell-cell connections (cell matching) is required for the generation of precise tissue structures. Mismatches or misconnections can lead to various diseases. Diverse mechanical cues, including differential adhesion and temporally varying cell contractility, are involved in regulating the process of cell-cell recognition and contact formation. Cells often start the process of cell matching through contact via filopodia protrusions, mediated by specific adhesion interactions at the cell surface. These adhesion interactions give rise to differential mechanical signals that can be further perceived by the cells. In conjunction with contractions generated by the actomyosin networks within the cells, this differentially coded adhesion information can be translated to reposition and sort cells. Here, we review the role of these different cell matching components and suggest how these mechanical factors cooperate with each other to facilitate specificity in cell-cell contact formation.
This study sought to determine whether vascular endothelial growth factor (VEGF)-induced permeabilisation of pulmonary endothelium to macromolecules could be related to a permissive role for neutrophil-derived VEGF in neutrophil transmigration. Treatment of human pulmonary artery endothelial cell (HPAEC) monolayers with 1, 10 or 100 ng/ml VEGF for 15 min or 1, 10 ng/ml for 90 min significantly increased endothelial permeability to trypan blue-labelled albumin (TB-BSA). These increases were correlated with changes in the cellular distribution of F-actin, as visualised by rhodamine-phalloidin staining: increased stress fibre formation, cellular elongation and formation of intercellular gaps after 15 min; at 90 min, there was also evidence of microspike formation and extension of spindle processes from the cell surface. Treatment of human neutrophil suspensions with 200 nM phorbol myristyl acetate (PMA), n-formyl-methionyl leucylphenylalanine (fMLP, 10 nM), interleukin-8 (IL-8, 10 nM) (but not with leukotriene B(4) (LTB(4)) 100 nM), for 30 min caused significant extracellular release of neutrophil VEGF stores. A permissive role for neutrophil-derived VEGF in facilitating migration across HPAEC monolayers was assessed in experiments using a functional blocking antihuman VEGF antibody. In the presence of this antibody (10 microg/ml), neutrophil migration in response to fMLP (10 nM), IL-8 (10 nM) or LTB(4) (100 nM) was not significantly different to that in the absence of antibody. We conclude that neutrophil-derived VEGF does not play a functional role in facilitating neutrophil migration across pulmonary vascular endothelium, despite its ability to induce cytoskeletal changes and enhance endothelial macromolecular permeability.
Microvascular corrosion casting was used to assess the effects of thrombin and D609, a phospholipase C inhibitor, on the vascularity of the chick embryo chorioallantoic membrane (CAM). Discs containing vehicle, thrombin or D609 were placed on the CAM of fertilized white Leghorn eggs on Day 9 of gestation and vascularity was assessed on Day 11. Thrombin caused significant increases in the numbers (43%), diameters (5%) and lengths (17%), of both pre- and postcapillaries (first-order vessels by centripetal ordering). Conversely, D609 caused a decrease in the numbers (27%), lengths (12%) and diameters (8%) of first-order vessels. D609 decreased the total vascular volume of first- to third-order vessels by 32%, whereas thrombin increased vascular volume by 27%. Additionally, thrombin increased capillary plexus density by 6%, whereas D609 decreased capillary plexus density by 3%. These findings provide a quantitative assessment of changing vascularity in the chick CAM--a model assay system in the development of pro- and antiangiogenic agents.
This study tested whether (1) L-type Ca(2+) channel blockade and extracellular Ca(2+) removal prior to endothelin-1, as compared to during the endothelin-1 constriction, resulted in lesser inhibition, and (2) the reduced inhibition due to prior L-type Ca(2+) channel blockade resulted from enhanced non L-type Ca(2+)-channel-dependent constriction. Pretreatment of rabbit basilar artery in vitro with 1 microM verapamil, an L-type Ca(2+) channel blocker, inhibited 3, 10, 30, and 100 nM endothelin-1 constrictions to a lesser extent than verapamil addition during the plateau endothelin-1 constriction. Ni(2+) (0.03 and 0.1 mM), a nonselective cation channel blocker, relaxed the plateau endothelin-1 constrictions in vessels pretreated with verapamil to greater magnitudes than vessels unexposed to verapamil. Extracellular Ca(2+) removal prior to 10, 30, and 100 nM endothelin-1 also inhibited the endothelin-1 constrictions to smaller magnitudes than Ca(2+) removal during the plateau endothelin-1 constrictions. These results suggest that the reduced inhibition of the endothelin-1 constriction following pretreatment with L-type Ca(2+) channel blocker or Ca(2+)-free solution, as compared to addition of these agents during the endothelin-1 constriction, is the result of non L-type Ca(2+) channel opening and enhanced Ca(2+)-independent constriction, respectively.
Our earlier observations suggest that M3 muscarinic acetylcholine (ACh) receptors (mAChRs) are involved in Ca2+ signaling and regulation of c-fos gene expression in T lymphocytes. Here, we describe the effects of YM905, a novel M3 antagonist, on evoked Ca2+ signaling and c-fos gene expression in CEM human leukemic T cells. YM905 significantly inhibited increases in intracellular free Ca2+ evoked by 10 μM oxotremorine-M, an M1/M3 agonist (IC50=100 nM), and also inhibited 10 μM oxotremorine-M-induced upregulation of c-fos gene expression at 1 μM. These findings demonstrate that YM905 antagonizes the intracellular responses in T cells induced via mAChRs, possibly M3 receptors.
Peroxisome proliferator-activated receptor gamma (PPAR gamma) immunohistochemical expression was analyzed in 75 human bladder tumor specimens, where the expression of some angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PDECGF), and tumor progression markers, such as epidermal growth factor receptor (EGFr), p16, mutated p53, and normal pRB, were also analyzed. The results were then compared to the clinical and pathological characteristics of the disease. PPAR gamma was expressed more significantly in papillary tumors than in solid cancers, and its presence was associated with statistical significance to low incidence of tumor recurrence or progression. This significant association was observed also when PPAR gamma was expressed in the presence of PDECGF, which resulted, when considered alone, to an angiogenic factor typical of solid cancers and appeared related to poor prognosis. In the presence of bFGF, on the contrary, PPAR gamma expression no longer resulted to a significant association with low incidence of tumor recurrence or progression, suggesting a possible worsening role of this angiogenic factor, typical of papillary cancers, in its interaction with PPAR gamma.
The responses to endothelial vasodilators and exogenous nitric oxide (NO) were characterized in intact isolated uterine vascular beds of nonpregnant, midpregnant and late-pregnant rats perfused with Kreb's buffer (37 degreesC, 5% CO2 in air, pH similar to 7.4) containing 2% dextran and indomethacin. Phenylephrine increased perfusion pressure in the vascular beds equally in all three groups. In the presence of phenylephrine, N-omega-nitro-L-arginine methyl ester (L-NAME) significantly augmented perfusion pressure in the order: nonpregnant < midpregnant < latepregnant uterine vascular bed. Acetylcholine and bradykinin-induced attenuation of perfusion pressure did not depend on gestational age. The decrease in perfusion pressure induced by acetylcholine was nonsignificantly attenuated by L-NAME in vascular beds from pregnant rats. The attenuation induced by bradykinin reached significant level in the vascular beds from midpregnant rats. The diethylamine (DEA)/ NO-induced decrease in perfusion pressure was not influenced by L-NAME in any group. These data demonstrate the augmentation of basal release of NO associated with progression of pregnancy, while the responses to endothelial vasodilators do not depend on gestational age and are not abolished by inhibition of NO synthase, suggesting involvement of nonprostanoid non-NO factor in the control of uterine circulation. (C) 2002 Elsevier Science Inc. All rights reserved.
Spiradoline, an arylacetamide kappa (κ) opioid receptor agonist, produced a potent tonic block of rat neuronal (EC50=34±5 μM) and heart (EC50=183±13 μM) sodium channels and also blocked IFMQ3 mutant neuronal sodium channels (EC50=130±34 μM) that lack fast inactivation when expressed in Xenopus oocytes. Spiradoline produced a hyperpolarizing shift in the voltage-dependence of sodium channel inactivation and exhibited a marked frequency-dependent component to blockade of sodium channels. The onset of open channel block of the IFMQ3 channel by spiradoline was best fit with a first-order blocking scheme, yielding an affinity constant of 116±33 μM. Thus, spiradoline blocks sodium channels by interacting with the major states of the channel which could result in local anesthetic action in nerves and antiarrhythmic action in the heart.
(1) The role of L-type Ca2+ channels in the relaxation to nitric oxide (NO)-mediated MaxiKCa channel activation was examined in guinea pig aorta. (2) Acetylcholine (ACh) produced an endothelium-dependent relaxation of guinea pig aorta precontracted with noradrenaline (NA), which was abolished by an NO synthase inhibitor, NG-nitro-l-arginine (l-NNA). (3) Both endothelium-dependent relaxation by ACh and endothelium-independent relaxation by an NO donor, (±)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR3), were strongly suppressed by a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), suggesting that increased intracellular cGMP plays the key role in both responses. (4) ACh- and NOR3-induced relaxations were significantly suppressed by iberiotoxin (IbTX), a selective blocker of MaxiKCa channels. (5) ACh- and NOR3-induced relaxations were greatly attenuated when arteries were precontracted with high KCl instead of NA, supporting the idea that K+ channel activation mediates the relaxant responses. (6) NOR3-induced relaxations were not affected by a L-type Ca2+ channel blocker, diltiazem. Furthermore, endothelium-independent relaxation by a KATP channel opener, (+)-7,8-dihydro-6,6-dimethyl-7-hyroxy-8-(2-oxo-1-piperidinyl)-6H-pyrano[2,3-f]benz-2,1,3-oxadiazole (NIP-121) was not affected by diltiazem and nicardipine. (7) These findings suggest that blockade of L-type Ca2+ channels is not a major mechanism responsible for the vascular relaxation due to NO-mediated MaxiKCa channel activation in guinea pig aorta.
Relaxations to acetylcholine and contractions to acetylcholine in the presence of the nitric oxide (NO) synthesis inhibitor (l-NG-nitroarginine methyl ester, l-NAME) were studied in aortic rings from rabbits fed either a control or a diet containing 0.5% cholesterol+14% coconut oil for 14 weeks and treated or not with atorvastatin (2.5 mg kg−1 day−1). Rings were incubated with the endothelin (ETA) receptor antagonist BQ123, and/or the thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist ifetroban. In rabbits, high cholesterol and triglyceride plasma levels were associated with intimal thickening and blunted acetylcholine-relaxation as compared with controls. By contrast, acetylcholine+l-NAME response was higher. Incubation with either ifetroban or BQ123 increased acetylcholine-relaxation in both diet groups and it reduced the constrictor response only in dyslipidemic rabbits. Removal of endothelium reduced acetylcholine+l-NAME contraction in dyslipidemic rabbits, although increased it in control animals. Atorvastatin treatment reduced plasma lipid levels and lesion size in dyslipidemic animals. Likewise, it prevented acetylcholine-relaxation reduction. In addition, atorvastatin reduced constrictor response in dyslipidemic rabbits but only in rings with endothelium. Incubation with either ifetroban or BQ123 did not further modify these responses in atorvastatin-treated animals in any group. These data suggest that ET and TXA2 availabilities seem to participate in the endothelial dysfunction associated with dyslipidemia. Atorvastatin treatment reduces intimal thickening and improves endothelial dysfunction in rabbits. This effect seems to be a consequence of its ability to act on ET and TXA2 systems.
Hyperthyroidism was induced by subcutaneous injections of L-thyroxine (T(4)) (500 mg/kg/day) for 3 days in order to study whether adrenergic and muscarinic receptor-mediated vascular responses alter at an early stage of the disease. T(4) treatment was sufficient to induce a significant degree of thyroid weight loss, tachycardia, cardiac hypertrophy, and an elevation in serum T(4) levels. The tension of aortic ring preparations isolated from rats was measured isometrically to investigate the influence of acute hyperthyroidism. The contractions induced by norepinephrine (NE) were significantly suppressed in aortic rings from rats treated with T(4) compared with control rats. N(G)-nitro-L-arginine (L-NOARG), an inhibitor of nitric oxide synthase (NOS), significantly enhanced NE-induced contraction in aortic rings from both control and T(4)-treated rats, and the enhancement was greater in rats treated with T(4) than control rats. The relaxations induced by either acetylcholine (ACh) or sodium nitroprusside (SNP) were also significantly enhanced by T(4) treatment. L-NOARG abolished the relaxation induced by ACh in aortic rings from both control and T(4)-treated rats. L-NOARG shifted SNP-induced relaxation curves of aortic rings from those of control rats to the left, but not with rats treated with T(4). T(4) treatment showed no influence on the amount of endothelial NOS (eNOS) protein. These results suggest that vascular responses alter at an early stage of hyperthyroidism and that it may be due to a modification in the NO system which is independent from the amount of eNOS protein.
The contribution of endothelial factors and mechanisms underlying decreased acetylcholine-induced relaxation and endothelial inhibitory action on phenylephrine-induced contraction were evaluated in aortas of two-kidney, one-clip hypertensive (2K-1C) and normotensive (2K) rats. Relaxation induced by acetylcholine in 2K-1C precontracted by phenylephrine was lower [Maximum Effect (ME): 71.33+/-3.36%; pD(2): 7.050+/-0.03] than in 2K (ME: 95.26+/-1.59%; pD(2): 7.31+/-0.07). This response was abolished by N(G)-nitro-L-arginine (L-NNA) in 2K-1C, but was only reduced in 2K (ME: 29.21+/-9.28%). Indomethacin had no effect in 2K-1C, and slightly attenuated acetylcholine-induced relaxation in 2K. The combination of L-NNA and indomethacin almost abolished acetylcholine-induced relaxation in 2K-1C, while in 2K, the inhibition (ME: 56.61+/-8.95%) was lower than the effect of L-NNA alone. During the KCl-induced precontraction, 2K and 2K-1C aortas showed similar acetylcholine-induced relaxation (43.50+/-5.64% vs. 41.60+/-4.36%), which was abolished by L-NNA. The levels of cGMP produced in response to acetylcholine were not different between 2K and 2K-1C. The sensitivity to sodium nitroprusside was lower in phenylephrine-precontracted aortas from 2K-1C than 2K, as showed by the pD(2) values (7.72+/-0.20 vs. 8.59+/-0.17), and this difference was abolished in aortas precontracted by KCl. The membrane potential was less negative in 2K-1C than in 2K (-41.57+/-1.19 vs. -51.00+/-1.13 mV) and hyperpolarization induced by acetylcholine was lower in 2K-1C than in 2K aortas (6.00+/-0.66 vs. 13.27+/-1.61 mV). Phenylephrine-induced contraction in aortas with endothelium was similar in both groups, and increased by the endothelium removal. This increase was lower in 2K-1C (from 1.32+/-0.06 to 1.90+/-0.21 g) than 2K (from 1.49+/-0.07 to 2.83+/-0.18 g). L-NNA and the endothelium removal had similar effect in 2K-1C (1.85+/-0.18 g) and were lower in 2K (2.18+/-0.20 g). Indomethacin decreased phenylephrine-induced contraction only in 2K. In conclusion, our major finding was a selective defect in smooth muscle membrane hyperpolarization, which could explain the decreased relaxation to acetylcholine and the attenuated inhibitory effect of endothelium on the contractile function in 2K-1C aortas.
The present study further examined the functional presence and the signal transduction mechanism(s) for adenosine A2A and A2B receptors through nitric oxide (NO) and the guanosine 3′, 5′-cyclic monophosphate (cGMP) pathway in cultured porcine coronary artery endothelial cells (PCAEC). The application of adenosine receptor agonists, NECA, CGS-21680 and CAD between 10−7 and 10−4 M, enhanced the production of NO (measured as nitrite) in a dose-dependent manner. On the basis of EC50 values, these agonists showed the following order of potency: NECA>CGS-21680>CAD. This order appears to be of the A2 adenosine receptor subtype. Similarly, the same concentrations of adenosine agonists evoked the production of cGMP in a dose-dependent manner, exhibiting a rank order that is similar to that of NO production. NO synthase inhibitor, N-nitro-l-arginine methylester (l-NAME, 10−5 M), inhibited the production of NO and cGMP, which was reversed by l-arginine (10−4 M). Selective A2A adenosine receptor antagonists, ZM-241385 and SCH-58261, at 10−7 M, significantly inhibited the effects of CGS-21680, but only partly inhibited the effect of NECA on NO and cGMP production. Along with the earlier molecular evidence from this laboratory [Am. J. Physiol. 279 (2000) H650], the present data further support the presence of both A2A and A2B receptors in PCAEC. These results further support that coronary endothelial cells express functional A2A and A2B adenosine receptors, leading to GMP production through the NO-synthase-linked mechanism. This is the first direct evidence where an A2B adenosine receptor has been linked to NO production in cultured endothelial cells and could play a role in coronary artery physiology and pathophysiology.
Bisphosphonates are used for the treatment of bone resorption, hypercalcemia, osteoporosis and Paget's disease. Etidronate, pamidronate and clodronate also inhibit the development of experimental atherosclerosis without altering serum lipid profile. Bisphosphonates inhibit the arterial calcification, lipid accumulation and fibrosis. They accumulate extensively in arterial walls and suppress macrophages in atheromatous lesions. In macrophage cultures, bisphosphonates inhibit the cellular accumulation and degradation of atherogenic LDL-cholesterol and foam cell formation. Further, they inhibit various enzymes involved in cell signal transduction and cholesterol biosynthesis. Recently, etidronate has been shown to inhibit the thickening of carotid arterial wall even in man.
We examined the role of endothelium-derived relaxing factors nitric oxide (NO), endothelium-derived hyperpolarising factor (EDHF), and prostaglandins (PGs) to P2Y1- and P2Y2-purinoceptor-induced vasodilation in isolated rat kidney. To do it, we analysed the renal response to ATP, 2-methylthio ATP, and UTP in rat renal vasculature under normal conditions and after the administration of: Nw-nitro-l-arginine (l-NAME), increased K+ concentration, indomethacin, and l-NAME and increased K+ together. Our results indicate that the vasodilator response to P2Y1- and P2Y2-purinoceptor activation in the isolated perfused kidney of rats is subserved by EDHF and NO.
In the present study, we observed that pentoxifylline (PTX) significantly augmented the nitric oxide (NO) production and the iNOS gene expression by interleukin-1β (IL-1β)-stimulated vascular smooth muscle cells (VSMCs). The enhancing effects of PTX on the IL-1β-induced NO production was associated with an increased intracellular cyclic AMP (cAMP) levels, and the synergistic effects of PTX on the IL-1β-induced NO production was blocked by cAMP-dependent protein kinase A (PKA) inhibitors. PKA inhibitors, KT5720 and H89, markedly decreased the augmented expression of iNOS gene whereas ODQ, a soluble guanylate cyclase inhibitor, did not affect the enhancing effect. In addition, the pretreatment with KT5720 or H89 abolished the increased translocation of the p65 subunit of NF-κB into the nucleus by PTX in the IL-1β-stimulated VSMCs. These results suggest that enhancing effects of PTX on the iNOS gene expression in the IL-1β-stimulated VSMCs is mediated predominantly through the activation of NF-κB via cAMP-dependent PKA pathway.
Tizanidine, an alpha-2 adrenergic agonist, is a centrally active muscle relaxant and a spasmolytic drug. The aim of our study was to investigate the activity of tizanidine on maximal electroshock seizures (MES) in mice. In the first part of the study, convulsive current 50 (CC 50) value to produce seizures was found. Then, tizanidine was given intraperitoneally (IP) at the doses of 0.5, 1, and 2 mg/kg, and orally (PO) at the doses of 5, 10, 20, 40 mg/kg. We found that tizanidine at the doses of 1 and 2 mg/kg IP and 40 mg/kg PO caused a significant protection against MES. In the second part of the study, after pretreatment with yohimbine, an alpha-2 adrenergic receptor blocker, at the dose of 2 mg/kg, anticonvulsant effect of tizanidine is diminished. We concluded that the mode of action of the anticonvulsant effect of tizanidine may be mediated by the central alpha-2 adrenergic receptors.
This study characterizes the reactivity of human chorionic plate vein in full-term (39.4±0.3 weeks of gestation) and preterm (34.4±0.6 weeks of gestation) pregnancy to endothelin-1 (ET-1) and attempts to characterize ET-1 receptor subtype, and the contribution of nitric oxide and cyclooxygenase products in these responses. In placental veins from full-term and preterm pregnant women, cumulative addition of ET-1 (10−10–10−6 M) caused marked and long-lasting concentration-dependent contractile responses. The mean EC50 and Emax values for ET-1-induced venoconstriction did not differ between the full-term and preterm pregnancy groups. In the veins from preterm placental preparations, the ETA receptor-selective antagonist cyclo(d-α-aspartyl-l-propyl-d-valyl-l-leucyl-d-tryptophyl (BQ123) reduced the ET-1-induced contraction by 28.6±2.4%, compared to a decline in tension of 51.2±4.2% in the full-term placental vessels. The ETB receptor-selective antagonist, N-[N-[N-[2,6-dimethyl-1piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl]-d-norleucinemonosodium (BQ788), did not influence ET-1-induced contraction in placental vein from both pregnancy groups in terms of maximal contraction and sensitivity. Pretreatment with the cyclooxygenase inhibitor, indomethacin (1 μM) and the nitric oxide synthase inhibitor Nw-nitro-l-arginine (NOLA, 100 μM) did not significantly affect either the EC50 or the maximum contraction to ET-1 in veins from normal full-term and preterm preparations. The results of this study suggest that there is no correlation between ET-1-induced vasoconstriction and gestational age and that this vasoconstriction is mediated predominantly via ETA receptor subtype in both groups of pregnant women, independent of NO and eicosanoids.
