Lipopolysaccharide exposure modulates the contractile and migratory phenotypes of vascular smooth muscle cells.

INTRODUCTION: Sepsis survivors are at higher risk for cardiovascular events. Lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) in sepsis. Activation of TLR4 modulates vascular smooth muscle cells (VSMCs) phenotype and contributes to cardiovascular changes after sepsis. AIM: Investigate changes in VSMCs phenotype caused by LPS-induced TLR4 activation. METHODS: Rat VSMCs were incubated with LPS. Two incubation conditions were used in cell contraction and migration assays: acute stimulation - LPS stimulus was initiated at the beginning of the assay and maintained throughout; and preconditioning - LPS stimulation was applied prior to the assay then discontinued. Nitric oxide (NO) production, mRNA expression of cytokines and phenotype markers, and interleukin (IL)-6 production were evaluated. KEY FINDINGS: LPS increased gene expression of IL-1ß, IL-6, TNFα and MCP-1 (p < .001), of secretory phenotype markers collagen and vimentin (p < .0479) and of the contractile marker smooth muscle 22α (SM22α) (p = .0067). LPS exposure increased IL-6 secretion after 24 and 48 h (p < .0001), and NO at 8 and 24 h (p < .0249) via inducible nitric oxide synthase (iNOS), as demonstrated by a decrease in NO after incubation with aminoguanidine. Acute stimulation with LPS reduced migration and contraction in a NO-dependent manner, while preconditioning with LPS increased both in an IL-6-dependent manner. SIGNIFICANCE: LPS affects VSMCs by modulating their secretory, contractile and migratory phenotypes. LPS acute stimulation of VSMCs promoted a NO-dependent reduction in migration and contraction, while preconditioning with LPS promoted IL-6-dependent increases in migration and contraction, evidencing that VSMCs can present phenotype modifications that persist after sepsis, thereby contributing to postsepsis cardiovascular events.

Trans-4-methoxy-ß-nitrostyrene relaxes rat thoracic aorta through a sGC-dependent pathway.

1-Nitro-2-phenylethene (NPe) induces a more potent vasorelaxant effect in rat aorta than its structural analog 1-nitro-2-phenylethane, but mediated through a different mechanism, independent of soluble guanylate cyclase (sGC) stimulation. We hypothesized that introducing an electron donor into the aromatic moiety might stabilize NPe, enhancing its potency and/or interaction with sGC. Therefore, trans-4-methoxy-ß-nitrostyrene (T4MN) was synthesized, and mechanisms underlying its vasorelaxant effects were studied in rat aortic ring preparations. In endothelium-intact preparations, T4MN fully relaxed contractions induced by phenylephrine (PHE) with a potency similar to that of its parent drug, NPe. This vasorelaxant effect that was unchanged by endothelium removal, pretreatment with L-NAME, indomethacin, or MDL-12,330A, but was significantly reduced by tetraethylammonium, 4-aminopyridine, methyl blue, or ODQ. Under Ca2+-free conditions, T4MN did not alter contractions evoked by caffeine, but significantly reduced, in an ODQ-preventable manner, those induced by either PHE or extracellular Ca2+ restoration following depletion of intracellular Ca2+ stores in thapsigargin-treated aortic preparations. Under the same conditions, T4MN also reduced contractions induced by protein kinase C activator phorbol-12,13-dibutyrate with a potency similar to that evoked by this nitroderivative against PHE-induced contractions. In conclusion, T4MN induces potent vasorelaxation in rat aorta by stimulating the sGC-cGMP pathway through a NO-independent mechanism. Introduction of a methoxy group into the aromatic moiety apparently stabilizes NPe, thereby enhancing its interaction with sGC.

Impaired vascular function in sepsis-surviving rats mediated by oxidative stress and Rho-Kinase pathway.

We investigated long-lasting changes in endothelial and vascular function in adult rat survivors of severe sepsis induced by cecal ligation and puncture (CLP) model. For this, male Wistar rats (200-350g) had their cecum punctured once (non-transfixing hole) with a 14-gauge needle. Performed in this way, a mortality rate around 30% was achieved in the first 72h. The survivors, together with age-matched control rats (not subjected to CLP), were maintained in our holding room for 60 days (S60 group) and had the descending thoracic aorta processed for functional, histological, biochemical or molecular analyses. Endothelium-intact aortic rings obtained from sepsis-surviving S60 group displayed increased angiotensin II-induced contraction, accompanied by decreased activity of the endogenous superoxide dismutase, augmented reactive oxygen species generation, and increased levels of tyrosine nitration compared with vessels from control group. The superoxide scavengers superoxide dismutase and tempol, and the antioxidant apocynin, were able to avoid this enhanced contractility to angiotensin II in aortic rings from the S60 group. In addition, aortic rings from the S60 group presented reduced sensitivity to Y-27632, a Rho-kinase (ROCK) inhibitor. Immunoblot analyses revealed augmented RhoA and ROCK II, and high levels of phosphorylation of myosin phosphatase target subunit 1 in vessels from S60 rats. In conclusion, aortic rings from sepsis-surviving rats display endothelial dysfunction mediated by the increased production of reactive oxygen species, which in turn reduces the bioavailability of nitric oxide and increases the formation of peroxynitrite, and enhances RhoA-ROCK-mediated calcium sensitization, leading to augmented contractile responses to angiotensin II. Notably, this is the first study demonstrating long-term dysfunction in the vasculature of sepsis-surviving rats, which take place or remain beyond the acute septic insult.

Pharmacological characterization of the mechanisms underlying the vascular effects of succinate.

We investigated the mechanisms underlying the vascular effects of succinate. Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats and C57BL/6 wild type (WT) or GPR91(-/-) mice. Nitrate/nitrite (NOx) was measured colorimetrically whereas 6-keto-prostaglandin F1α (stable product of prostacyclin) was measured by enzyme immunoassay (EIA). Phosphorylation of endothelial nitric oxide synthase (eNOS) was assessed by western immunoblotting. Functional assays revealed that the direct effect of succinate in the vasculature is biphasic. At lower concentrations succinate induced relaxation while at higher concentrations succinate induced vascular contraction. Succinate concentration dependently relaxed rat aortic rings with intact endothelium. Endothelial removal reduced, but not abolished succinate-induced relaxation. Similarly, succinate relaxed endothelium-intact and endothelium-denuded aortas isolated from both C57BL/6 and GPR91(-/-) mice. Pre-incubation of endothelium-intact, but not endothelium-denuded rat aortic rings with l-NAME, indomethacin and tetraethylammonium (TEA) reduced succinate-induced relaxation. In endothelium-intact rings, succinate-induced relaxation was attenuated by ODQ, haemoglobin, Rp-8-Br-Pet-cGMPS, thapsigargin, wortmannin and SC-560. Blockade of K(+) channels with 4-aminopyridine, apamin and charybdotoxin reduced succinate-induced relaxation. Succinate increased the concentration of NOx and 6-keto-prostaglandin F1α as well as eNOS phosphorylation at ser(1177) residue. CaCl2-induced contraction of endothelium-intact or endothelium-denuded aortas was not affected by succinate. The major finding of our study is that it first demonstrates a direct effect of succinate in the vasculature. Succinate displays a biphasic and concentration-dependent effect. The vascular relaxation induced by succinate is partially mediated by endothelial GPR91 receptors via the NO-cGMP pathway, a vasodilator cyclooxygenase (COX) product(s) and the opening of K(+) channels.

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

Apocynin decreases AGEs-induced stimulation of NF-κB protein expression in vascular smooth muscle cells from GK rats.

CONTEXT: Elevated oxidative stress plays a key role in diabetes-associated vascular disease. Excessive production of reactive oxygen species via advanced glycation end products (AGEs) activates peroxisome proliferator-activated receptor gamma (PPARγ) and the transcription factor nuclear factor-kB (NF-κB) in aortic vascular smooth muscle cells (VSMCs). Apocynin, a drug with an antioxidant effect, has also been proposed as a therapeutic agent for atherosclerotic disease. OBJECTIVES: This work investigates the effects of apocynin on the PPARγ and NF-κB protein expression evoked by AGEs in cultured VSMCs from Goto-Kakisaki (GK) rats, a non-obese insulin model of both insulin resistance and type 2 diabetes. MATERIALS AND METHODS: VSMCs, isolated from aortas of GK and non-diabetic rats, were cultured. The expression of proteins was evaluated by Western blot. The blood glucose concentration was measured with a blood glucose test meter. The diabetes of GK rats was controlled by blood glucose and insulin determinations (non-fasting values). The serum insulin concentration was determined by radioimmunoassay. RESULTS: In VSMCs from non-diabetic and GK rats, apocynin (1 and 10 µM) abolished the protein overexpression of NF-κB induced by glycated bovine serum albumin (AGEs-BSA) incubation. However, apocynin (1 and 10 µM) enhanced the expression of PPARγ protein in the presence of AGEs-BSA (100 µg/mL) in VSMCs from non-diabetic, but not from GK rats. CONCLUSION: These findings suggest that apocynin decreases the incidence of alterations in VSMCs induced by AGEs through the reduction of NF-κB and may represent an attractive therapeutic approach to treat diabetes mellitus.

ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel.

Previous work from our laboratory and others has shown that, in some epithelia, the epithelial sodium channel (ENaC) increases its expression during wound healing. In these cases, inhibition of the channel determines a decrease in the healing rate, a result suggesting a role for ENaC in the overall healing process. To understand further this role of ENaC in epithelia, we explored the participation of ENaC in wound healing in four cultured epithelial cell lines selected on the basis of their different embryonic origins, function and modality of healing, i.e., by lamellipodial cell crawling or by actin cable formation. Three of the cell lines (bovine corneal endothelial cells, rabbit corneal epithelial cells and Madin-Darby canine kidney cells) exhibited an increase in ENaC expression and consequent membrane potential depolarization and an increase in cytosolic sodium and calcium, whereas one line (bovine aortal endothelial cells, BAEC) did not exhibit any of these changes. In all of the cell lines, however, ENaC inhibition determined a similar decrease in the rate of wound healing. In BAEC monolayers, the increase in ENaC activity produced plasma membrane depolarization, increased cytosolic sodium and calcium, and augmented the velocity of healing. These novel findings contribute to the idea that ENaC plays a critical role in wound healing in various epithelia, independently of the modality of healing and of any increase in the expression of the channel.

Angiotensin-converting enzyme 2 activation improves endothelial function.

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Brown spider (Loxosceles intermedia) venom triggers endothelial cells death by anoikis.

Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α5ß1 integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.

Variation of the origin of aortic arch branches: in relationship with plates of atheroma / Variación del origen de las ramas del arco aórtico: relación con placas de ateromas

The aortic arch branches variations have called the attention of several authors, who have handled studies and classifications, both human and in different animals. The common trunk, which is between the brachiocephalic trunk and the common left carotid artery, is the most common variation. We conducted a descriptive and randomized study of the presence of the trunk mentioned before, trying to establish the possible relationship between this variation and the distribution plates of atheroma. The lumen observation makes it possible to define and check the distribution of the ostium, among the common ostium and the ones with common trunks. Regarding the plates of atheroma, it was found that there is a slight prevalence in common trunks cases, with respect to the classics (no variety) or the ones who had common ostium. In all cases, the presence of a plaque in the distal aortic arch was certified near the left subclavian artery. The knowledge of the existence of the common trunk sets up an act of academic interest, as practice interventions and diagnostic imaging and clinical work, since the presence of the common trunk might be related to the prevalence of the plates of atheroma at the level of its origin.

Las variaciones de las ramas del arco aórtico han llamado la atención de diversos autores, quienes han realizados estudios y clasificaciones, tanto en humanos, como en diferentes animales. El tronco común, entre el tronco braquiocefálico y la arteria carótida común izquierda, es la variación más frecuente. Realizamos un estudio descriptivo y randomizado de la presencia del mencionado tronco, tratando de verificar la posible relación entre dicha variación y la distribución de placas de ateroma. La observación luminal permitió precisar, entre los casos de ostios comunes y aquellos con troncos comunes, y comprobar la distribución de los ostios. En cuanto a las placas de ateroma, se observó una leve prevalencia en los casos de troncos comunes respecto de los clásicos (sin variedad) o de los que presentaron ostios comunes. En todos los casos se verificó la presencia de una placa en el arco aórtico distal, inmediato a la arteria subclavia izquierda. El conocimiento de la existencia del tronco común, constituye un hecho de interés académico, como práctico en intervencionismo, diagnóstico por imagen y la clínica. La presencia del tronco común pareciera estar relacionada con cierta prevalencia de placas de ateroma a nivel de su origen.

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta.

AIM: To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. METHODS: The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-l-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2×10(-5) M) and methyl-ß-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 µM), in the presence and absence of DMB (2×10(-5) M). RESULTS: The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+) exchanger (NCX), and partially blunted by the caveolae disassembly. CONCLUSIONS: These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.

Isometric contraction increases endothelial nitric oxide synthase activity via a calmodulin antagonist-sensitive pathway in rat aorta.

In this work, the possibility that isometric contraction activates endothelial nitric oxide synthase (eNOS) in a calcium/calmodulin (Ca2+/CaM)-dependent manner was examined in rat thoracic aorta. Step-wise stable contractile responses (precontractions) to phenylephrine were obtained in endothelium-intact and endothelium-denuded aortic rings. The subsequent addition of the NO synthase inhibitor, N(G)nitro-l-arginine methyl ester (l-NAME), or the soluble guanylyl cyclase inhibitor, ODQ, further augmented precontractions in a concentration-dependent manner. The amplitude of l-NAME- and ODQ-induced increases in tone were dependent on the level of precontraction; the maximal increments for l-NAME and ODQ were observed in arteries precontracted with phenylephrine at 67% of its maximal effect. Likewise, in endothelium-intact non-contracted arteries, l-NAME and ODQ induced small but significant increases in tone. Neither l-NAME nor ODQ had any effect in endothelium-denuded preparations. In endothelium-intact aortic rings precontracted with high K+ solutions, l-NAME also elicited supplementary contractions dependent on precontraction level. The CaM antagonist, calmidazolium, inhibited in a concentration-dependent, noncompetitive, manner the effects of l-NAME on the tone of endothelium-intact phenylephrine-precontracted aortic rings. These results suggest that isometric contraction increases the activity of eNOS by means of the Ca2+/CaM complex in rat aorta.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex.

The synthesis of [Ru(NO(2))L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO)L(bpy)(2)](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO(2))L(bpy)(2)](+) in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around -0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at -0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2))L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mumol L(-1) phenylephrine responded with relaxation in the presence of cis-[Ru(II)(NO(2))L(bpy)(2)](+). The potential of rat aorta cells to metabolize cis-[Ru(II)(NO(2))L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[Ru(II)(NO(2))L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[Ru(II)(NO(2))L(bpy)(2)](+) complex.

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle.

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.

New insights in endothelial and smooth muscle cell communication.

Based on immunohistochemical techniques against connexins and the intercellular flux of staining molecules, it has previously been shown that electrotonic communication occurs among endothelial and vascular smooth muscle cells, this due to the presence of myoendothelial gap junctions. The aim of this study was to evaluate the density of myoendothelial contacts in the left coronary and internal mammary arteries as well as in the left saphenous vein by means of electron microscopy, the distance between both cells participating in an myoendothelial contact with a semi-automatic image analysis system and the presence of homocellular and heterocellular gap junctions between endothelial and smooth muscle cells by using the immunohistochemical technique and confocal microscopy in thoracic aorta were also analyzed. The results are that all blood vessels studied present myoendothelial contacts, while density studies show that they are more abundant in the saphenous vein. The myoendothelial contact distance is constant and in no case the cytoplasmic processes reach the plasma membrane of the partner cell toward which they are advanced. Homocellular gap junctions were found between smooth muscle cells and between endothelial cells. Heterocellular gap junctions were absent, evidencing the possibility that signaling molecules between endothelial and smooth muscle cells may be transferred through plasma membranes as was once thought and not necessarily by electrotonic communication.

11,13-dihydro-dehydroleucodine, a derivative of dehydroleucodine with an inactivated alkylating function conserves the anti-proliferative activity in G2 but does not cause cytotoxicity.

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.

Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II.

The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.

Hiper-homocisteinemia causando aterogênese na aorta de coelhos: modelo experimental / Hyperhomocysteinemia causing atherogenesis in rabbits´aorta: an experimental model

Objetivo: Este estudo tem por objetivo avaliar os efeitos da homocisteinemia plasmática elevada na formação da placa aterosclerótica na aorta de coelhos. Material e método: Realizou-se estudo experimental comparativo em dois grupos homogêneos de coelhos durante 60 dias. Foram utilizados 20 coelhos da linhagem New Zealand divididos em dois grupos de 10 animais: grupo controle (C) e grupo metionina (M). Todos os animais receberam a mesma dieta sólida e 500 ml de água. Os animais do grupo M receberam 2 ml de uma solução de metionina na concentração de 200 mg/ml a cada 24 horas. Foram colhidas amostras de sangue para a dosagem de colesterol, triglicerídeos, HDL, LDL e homocisteína após 0, 30 e 60 dias. Os animais foram submetidos a eutanásia por dose letal de anestésico no 60° dia. A aorta torácica e a aorta abdominal foram retiradas para estudo anatomopatológico...

Vascular non-endothelial nitric oxide induced by swimming exercise stress in rats.

1. Herein, we report the effects of acute or chronic forced swimming on vascular responsiveness to angiotensin (Ang) II. 2. The possible involvement of locally produced substances, such as nitric oxide (NO) and prostanoids, in these effects were studied in rat thoracic aorta and superior mesenteric arteries. 3. Chronic, but not acute, swimming reduced the efficacy (maximal effect; Emax) of AngII in thoracic aorta and mesenteric arteries, either with intact or denuded endothelium. 4. The efficacy of AngII was reduced in the presence of indomethacin in mesenteric arteries, but not in the aorta, from either control or chronically stressed rats. 5. Treatment with NG-monomethyl-l-arginine reversed the effect of chronic stress on the response to AngII, suggesting that chronic stress may increase non-endothelial NO activity in both the aorta and mesenteric arteries. 6. The effects of acute and chronic stress on vascular reactivity were selective for AngII because no changes were observed on the effects of phenylephrine.