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The role of arterial smooth muscle in vasorelaxation
Biochemical and Biophysical Research Communications 377 (2008) 504–507
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c
The role of arterial smooth muscle in vasorelaxation Igor B. Buchwalow a,*, Sona Cacanyiova b, Joachim Neumann c, Vera E. Samoilova a, Werner Boecker a, Frantisek Kristek b a
Gerhard Domagk Institute of Pathology, University of Muenster, 48149 Muenster, Germany Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic c Institute for Pharmacology und Toxicology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany b
a r t i c l e
i n f o
Article history: Received 29 September 2008 Available online 16 October 2008 Keywords: Vascular smooth muscle cells NO synthase Vascular relaxation Endothelium-derived relaxing factor
a b s t r a c t The concept of [Gren_OpEdothliumDervRaxingFctorGe_Cls][RdOpenendothelium-derived relaxing factor Red_Clos](EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. [Gren_OpHowvrGen_Cls][RdOpeHowever, Red_Clos]it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomic al integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts suf ficient to account for the vaso relaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle. © 2008 Elsevier Inc. All rights reserved.
The concept of the obligatory role of the endothelium in the vasorelaxation was drawn from the experiments with endothe lium-deprived blood vessels. Rubbing off the endothelial layer was reported to render blood vessels insensitive to ACh [1]. It was concluded, that the endothelial cells when stimulated by ACh, released a nonprostanoid, diffusible factor (later termed EDRF for endothelium-derived relaxing factor) that acted on the subjacent VSMC to produce relaxation. The biologic al activity of EDRF was later explained through NO release by endothelial cells [2]. To the time of those studies, VSMC were regarded as passive recipients of NO from the endothelium. Later it was, however, found that VSMC in various blood vessels do express all three NOS iso forms depending on the blood vessel type [3,4]. Moreover, aspects of the anatomical integrity of the organ (blood vessel) subjected to experiments with rubbing the blood vessel intimal surface were neglected. It should be taken in consideration that the destruction of the vascular wall integrity increases the concentration of vascu lar superoxides [5,6] that, in turn, impair vasodilatory responses to exogenous and endogenous nitrovasodilators [7]. Known as NO scavengers, superoxides drastically reduce NO bioactivity and NO bioavailability [8–10], whereas the intact endothelium pro tects VSMC from the superoxide attack [11,12]. In addition to NO * Corresponding author. Fax: +49 251 8355460. E-mail address: buchwalo@uni-muenster.de (I.B. Buchwalow) 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.019
s cavenging, superoxides can also directly exert a vasoconstrictor action [13,14]. We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer led to a vascular dysfunction and rendered blood vessels insensi tive to vasodilators as a consequence of oxidative stress. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scav engers restored vasodilatory responses to ACh in endotheliumdeprived blood vessels. Materials and methods Animals. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Insti tutes of Health (NIH Publication No. 85-23, revised 1996) and was performed in accordance with the guidelines of the Institutional Animal Care Committee, Institute of Normal and Pathological Phys iology, Bratislava. Male Wistar rats (350–450 g; n = 11) were used. Immunohistochemistry. Tissue probes of the thoracic aorta, mesenteric artery, and pulmonary artery were fixed in buffered 4% formaldehyde and routinely embedded in paraf fi n. 4-lm sec tions of the paraf fi n blocks were dewaxed in xylene, rehydrated in graded alcohols, pre-treated for antigen retrieval, and immunore acted with primary antibodies recognizing NOS1, NOS2 and NOS3
I.B. Buchwalow et al. / Biochemical and Biophysical Research Communications 377 (2008) 504–507
(Transduction Laboratories, KY; and Santa Cruz Biotechnology, CA) as described by us earlier [3,4,15]. Bound rabbit primary antibod ies were detected using DAKO EnVision-HRP system and NovaRed substrate kit (Vector Labor atories), counterstained with hema toxylin and mounted with an aqueous mounting medium GelTol (Immunotech, Marseille). The exclusion of the primary antibody from the immunohistochemic al reaction, substitution of primary antibodies with the rabbit IgG at the same final concentration, or preabsorption of primary antibodies with corresponding control peptides resulted in lack of immunostaining. Visualization and image processing. Immunostained sections were examined on a Zeiss microscope “Axio Imager Z1”. Micros copy images were captured using AxioC am 12-bit camera and Axi oVision single channel image processing (Carl Zeiss Vision GmbH). Images shown are representative of at least three independent experiments which gave similar results. Functional in vitro study. Rats were anaesthetized with diethyl ether, decapit ated and exsanguinated. The thoracic aorta, mesenteric artery, and pulmonary artery were immediately removed, cleaned of adhering fat and connective tissue, and cut into 2–4 mm wide rings. Endothelial cells were removed by gently rubbing the intimal surface with cotton-covered wire. The rings were vertically fixed between two stainless steel triangles in 20 ml incubation organ bath with Krebs solution, and bubbled with a 95% O2 and 5% CO2 gas mixture. The vessel segments were allowed to equilibrate for 1 h at a resting tension of 1 g and the changes of isometric tension were recorded as described previously [16]. Krebs solution containing 80 mM KCl was prepared by replacing NaCl with equimolar KCl and after an equili bration period the rings were stimul ated until a sustained response was obtained, in order to test their contractile capacity. The pres ence of functional endothelium was assessed in all preparations by determining the ability of ACh (10¡5 M) to induce relaxation of rings pre-contracted with phenylephrine. For relaxation studies, the rings were pre-contracted with maximum concentration of phen ylephrine (10¡5 M) and cumulative concentration–response curves for ACh (10¡10 to 3 £ 10¡5 M) were obtained. After washout the rings of aorta were preincubated with N-acetylcysteine (10¡4 M; 20 min) or tempol (3 £ 10¡3 M; 20 min). The rings of mesenteric artery and pulmonary artery with tempol only, and the relaxant responses to ACh were determined. Relaxation was expressed as a percentage of phenylephrine-induced contraction. Statistical analysis. Data are given as means ± SEM. For the statis tical evalua tion of differences between groups, one-way analysis of variance (ANOVA) was used and followed by Bonferroni’s post hoc test. The differences of means were considered as significant at P value <0.05.
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To gain evidence for the role of arterial smooth muscle in the regulation of vascular tension and to elucidate the role of superox ides in impairing vasodilatory responses, we examined ACh-induced vasorelaxation in endothelium-deprived blood vessels like in the experiment of Furchgott and Zawadzki [1] but in the presence of superoxide scavengers—tempol and N-acetyl-l-cysteine (NAC). In thoracic aorta rings with intact endothelium, cumulative addition of ACh (10¡10 to 3 £ 10¡5 M) produced concentrationdependent relaxation. The maximum relaxation was 85.59 ± 4.69% (Fig. 2A). In rings with denuded endothelium, ACh-induced relax ation was held back with the maximum relaxation 15.85 ± 4.31% (p < 0.01). Pre-treatment of denuded rings with NAC (10¡4 M) significantly restored ACh-induced relaxation to the level of 33.75 ± 7.25% (p < 0.05) (Fig. 2A). Tempol (3 £ 10¡3 M), a superox ide dismutase mimetic, also reversed the ACh-mediated relax ations in endothelium-deprived aortic ring preparations with the
Results and discussion Thoracic aorta rings, mesenteric artery rings, and pulmonary artery rings from intact and denudated blood vessels of rat were first subjected to morphological and immunohistochem ical control to confirm the absence of the endothelial layer in endothelium-deprived blood vessels and to demonstrate NOS expression in blood vessels under study. We found all three NOS isoforms expressed not only in the intima but also in media of the blood vessels under study. As an example, Fig. 1 shows strong expression of NOS3 in the thoracic aorta (Fig. 1A), mesenteric artery (Fig. 1B), and pulmonary artery (Fig. 1C), in both inti mal and medial cells. Inserts in this layout (Fig. 1) demonstrate the complete removal of the endothelial layer after denudation. The NOS expression by cells in the media of blood vessels was also confirmed by us earlier with Western blotting showing the presence of characteristic immunoreactive protein bands for NOS in the porcine carotid artery and rat aorta devoid of endo thelium [3,4].
Fig. 1. Expression of NOS3 in (A) thoracic aorta, (B) mesenteric artery, and (C) pulmonary artery, in both intimal and medial cells. Inserts demonstrate the com plete removal of the endothelial layer after denudation. Fifty micrometer scale bar for entire layout.
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I.B. Buchwalow et al. / Biochemical and Biophysical Research Communications 377 (2008) 504–507
Fig. 2. (A) Effect of NAC (10¡4 M) on the concentration–response curves to ACh in thoracic aorta endothelium-intact (E+) and endothelium-denuded (E¡) rings. Tissues were exposed for 20 min to NAC before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. *p < 0.05; **p < 0.01; with respect to E+; +p < 0.05; ++ p < 0.01 with respect to E¡. (B) Effect of tempol (3 £ 10¡3 M) on the concentration–response curves to ACh in thoracic aorta endothelium-intact (E+) and endotheliumdenuded (E¡) rings. Tissues were exposed for 20 min to tempol before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. *p < 0.05; ** p < 0.01; with respect to E+; +p < 0.05; ++p < 0.01 with respect to E¡.
aximum relaxation of 34.58 ± 6.65% (p < 0.05), whereas pre-treat m ment of thoracic aorta intact rings with tempol but insignificantly inhibited ACh-induced relaxat ion (Fig. 2B). Cumulative addition of ACh (10¡9 to 3 £ 10¡5 M) relaxed phen ylephrine-pre-contracted intact mesenteric artery rings with a maximum relaxat ion of 74.0 ± 8.04% (Fig. 3A). Compared to intact mesenteric artery rings, endothelial denudation resulted in a signif icant depression of ACh-induced relaxation (20.2 ± 3.23%, p < 0.01), but pre-treatment of endothelium-denuded rings with tempol (3 £ 10¡3 M) restored ACh-induced relaxation up to 51.6 ± 6.2% (p < 0.01). Important, tempol pre-treatment of intact mesenteric artery rings did not affect the vasorelaxatory response to ACh. In pulmonary artery rings with intact endothelium, ACh-induced relaxation amounted to 89.9 ± 4.12% (Fig. 3). Like in intact mes enteric artery rings, ACh-induced relaxation was not affected by tempol pre-treatment. After endothelial denudation, ACh-induced relaxation was held back, and the maximum relaxation decreased to 20.0 ± 7.76% (p < 0.01). However, tempol (3 £ 10¡3 M) pre-treat ment of endothelium-deprived rings restored ACh-induced relax ation to the level of 57.4 ± 8.81% (p < 0.01). ACh-induced relaxation recovery in de-endothelia lized blood vessels by antioxidants was more pronounced in the mesenteric and pulmonary artery, the latter having also the strongest expres sion of NOS in the media. Additionally, blood vessels of the elas tic type like aorta have a lower ratio of VSMC compared to blood vessels of the muscular type [3]. This explains a lower response to protective action of superoxides in denuded aorta compared with mesenteric and pulmonary artery. Restoration of vasodi latory responses to ACh in endothelium-deprived blood vessels under protection against oxidative stress challenges the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle. The concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle implies, that NO generated by NOS in the endothelium diffuses to the underlying VSMC to produce relaxation [1,2]. However, the corner stone of this concept—inabil
ity of the smooth muscle to express NOS by themselves—appeared later as imaginary. With the advent of more powerful immunohis tochemical techniques increasing the antigen detectability, it turned out that VSMC in various blood vessels express all three NOS iso forms depending on the blood vessel type [3,4]. Further evidence for NOS expression in VSMC can also be drawn from more recent publications [17–19]. There are some other facts that are also inconsistent with the concept of the obligatory role of endothelial cells in the relaxa tion of arterial smooth muscle. Along with the oxidative stress, endothelial denudation can also impair K+-induced vasorelaxation (background-K+ channel activation) [20] as well as myoendothelial gap junctional communications in VSMC [21], which play a major role in endothelium-derived hyperpolarizing factor (EDHF)-med iated relaxations. It has been proposed that EDHF, whose chemi cal nature is as yet unknown, contributes to microvascular dilation more than NO does [22]. The major target of NO in VSMC is soluble guanylyl cyclase (sGC), which catalyzes the conversion of GTP to cGMP with subsequent activation of cGMP-dependent protein kinases or altered func tion of phosphodiesterases triggering the cell-specific physiologic response, vasodilation [23]. A mathematical model of the spatialtemporal gradient of the NO diffusion in a homogenous biological medium shows that the NO concentration on the border of vascu lar smooth muscle layer is less than the equilibrium dissociation constant of sGC. It means, that the diffusion of the NO produced in endothelium is insuf ficient to cause a relaxation of vascular smooth muscles in medium- and large-size blood vessels [24,25]. Taken together these considerations imply that the EDRF con cept derived from the experiments with de-endothelialized blood vessels is not free of assumptions and oversimplifications. With this study we have demonstrated that the endothelial denudation leads to a vascular dysfunction and renders blood vessels insensitive to vasodilators as a consequence of oxidative stress. Restoration of vasodilatory responses in endothelium-deprived blood vessels by antioxidants is in compliance with reports that superoxides
I.B. Buchwalow et al. / Biochemical and Biophysical Research Communications 377 (2008) 504–507
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Fig. 3. (A) Effect of tempol (3 £ 10¡3 M) on the concentration–response curves to ACh in mesenteric artery endothelium-intact (E+) and endothelium-denuded (E¡) rings. Tissues were exposed for 20 min to tempol before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. **p < 0.01; with respect to E+; + p < 0.05; ++p < 0.01 with respect to E¡. (B) Effect of tempol (3 £ 10¡3 M) on the concentration–response curves to ACh in pulmonary artery endothelium-intact (E+) and endo thelium-denuded (E¡) rings. Tissues were exposed for 20 min to tempol before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. ** p < 0.01; with respect to E+; +p<0.05; ++p < 0.01 with respect to E¡.
induced by destruction of the vascular wall anatomical integrity [5–7] reduce the NO bioactivity as a relaxing factor. These findings challenge the concept of the obligatory role of endothelial cells in the relaxat ion of arterial smooth muscle. Acknowledgment This study was supported in part by a Grant VEGA 2/6139/28. References [1] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288 (1980) 373– 376. [2] R.M. Palmer, A.G. Ferrige, S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327 (1987) 524–526. [3] I.B. Buchwalow, T. Podzuweit, W. Bocker, V.E. Samoilova, S. Thomas, M. Well ner, H.A. Baba, H. Robenek, J. Schnekenburger, M.M. Lerch, Vascular smooth muscle and nitric oxide synthase, FASEB J. 16 (2002) 500–508. [4] I.B. Buchwalow, T. Podzuweit, V.W. Samoilova, M. Wellner, H. Haller, S. Grote, S. Aleth, W. Boecker, W. Schmitz, J. Neumann, An in situ evidence for auto crine function of NO in the vasculature, Nitric Oxide—Biol. Chem. 10 (2004) 203–212. [5] L.C.P. Azevedo, M.D. Pedro, L.C. Souza, H.P. de Souza, M. Janiszewski, P.L. da Luz, F.R.M. Laurindo, Oxidative stress as a signaling mechanism of the vascu lar response to injury: the redox hypothesis of restenosis, Cardiovasc. Res. 47 (2000) 436–445. [6] S.K. Ozel, M. Yuksel, G. Haklar, C.U. Durakbasa, T.E. Dagli, A.O. Aktan, Nitric oxide and endothelin relationship in intestinal ischemia/reperfusion injury (II), Prostaglandins Leukot. Essent. Fatty Acids 64 (2001) 253–257. [7] T. Heitzer, U. Wenzel, U. Hink, D. Krollner, M. Skatchkov, R.A.K. Stahl, R. Macharzina, J.H. Brasen, T. Meinertz, T. Munzel, Increased NAD(P)H oxidasemediated superoxide production in renovascular hypertension: evidence for an involvement of protein kinase C, Kidney Int. 55 (1999) 252–260. [8] I.B. Buchwalow, E. Shagdarsuren, J.K. Park, W. Schulze, J. Slezak, F.C. Luft, H. Haller, Biochemical and physical factors involved in reducing nitric oxide bio activity in hypertensive heart and kidney, Circulation 98 (1998) 118. [9] C. Napoli, L.J. Ignarro, Nitric oxide and atherosclerosis, Nitric Oxide 5 (2001) 88–97. [10] W. Zhao, S.A. Swanson, J. Ye, X. Li, J.M. Shelton, W. Zhang, G.D. Thomas, Reac tive oxygen species impair sympathetic vasoregulation in skeletal muscle in angiotensin II-dependent hypertension, Hypertension 48 (2006) 637–643.
[11] S.L. Linas, J.E. Repine, Endothelial cells protect vascular smooth muscle cells from H2O2 attack, Am. J. Physiol—Renal Physiol. 41 (1997) F767–F773. [12] M. Walia, L. Sormaz, S.E. Samson, R.M.K.W. Lee, A.K. Grover, Effects of hydro gen peroxide on pig coronary artery endothelium, Eur. J. Pharmacol. 400 (2000) 249–253. [13] A. Just, C.L. Whitten, W.J. Arendshorst, Reactive oxygen species participate in acute renal vasoconstrictor responses induced by ETA and ETB receptors, Am. J. Physiol—Renal Physiol. 294 (2008) F719–F728. [14] M.Y. Lee, K.K. Griendling, Redox signaling, vascular function, and hyperten sion, Antioxid. Redox Signal. 10 (2008) 1045–1059. [15] I.B. Buchwalow, E.A. Minin, V.E. Samoilova, W. Boecker, M. Wellner, W. Schmitz, J. Neumann, K. Punkt, Compartmentaliz ation of NO signaling cascade in skeletal muscles, Biochem. Biophys. Res. Commun. 330 (2005) 615–621. [16] J. Torok, F. Kristek, M. Mokrasova, Endothelium-dependent relaxation in rabbit aorta after cold-storage, Eur. J. Pharmacol.—Environ. Toxicol. Pharmacol. Sect. 228 (1993) 313–319. [17] V.P. Ekshyyan, V.Y. Heb ert, A. Khandelwal, T.R. Dugas, Resveratrol inhibits rat aortic vascular smooth muscle cell proliferat ion via estrogen recep tor dependent nitric oxide production, J. Cardiovasc. Pharmacol. 50 (2007) 83–93. [18] A.F. El-Yazbi, W.J. Cho, J. Cena, R. Schulz, E.E. Daniel, Smooth muscle NOS, co-localized with caveolin-1, modulates contraction in mouse small intestine, J. Cell Mol. Med. 12 (2008) 1404–1415. [19] J.M. Tadie, P. Henno, I. Leroy, C. Danel, E. Naline, C. Faisy, M. Riquet, M. Levy, D. Israel-Biet, C. Delclaux, Role of nitric oxide synthase/arginase balance in bron chial reactivity in patients with chronic obstructive pulmonary disease, Am. J. Physiol. Lung Cell. Mol. Physiol. 294 (2008) L489–L497. [20] D. Harris, P.E.M. Martin, W.H. Evans, D.A. Kendall, T.M. Grif fi th, M.D. Randall, Role of gap junctions in endothelium-derived hyperpolarizing factor responses + and mechanisms of K relaxation, Eur. J. Pharmacol. 402 (2000) 119–128. [21] H. Ding, C.R. Triggle, Novel endothelium-derived relaxing factors—identifica tion of factors and cellular targets, J. Pharmacol. Toxicol. Methods 44 (2000) 441–452. [22] H. Miura, Y.P. Liu, D.D. Gutterman, Human coronary arteriol ar dilation to bradykinin depends on membrane hyperpolarization—contribution of nitric oxide and Ca2+-activated K+ channels, Circulation 99 (1999) 3132–3138. [23] M.A. Moro, R.J. Russell, S. Cellek, I. Lizasoain, Y. Su, V.M. Darley-Usmar, M.W. Radomski, S. Moncada, cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the solub le guanylyl cyclase, PNAS 93 (1996) 1480. [24] I.P. Seraya, Y.R. Nartsissov, Theor etical approach to description of time-depen dent nitric oxide effects in the vasculature, Mol. Biol. Rep. 29 (2002) 151–155. [25] I.P. Seraya, Y.R. Nartsissov, G.C. Brown, Mathem atical modeling of nonstation ary spatial nitric oxide gradients in vascular smooth muscles, Biofizika 48 (2003) 91–96.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c
The role of arterial smooth muscle in vasorelaxation Igor B. Buchwalow a,*, Sona Cacanyiova b, Joachim Neumann c, Vera E. Samoilova a, Werner Boecker a, Frantisek Kristek b a
Gerhard Domagk Institute of Pathology, University of Muenster, 48149 Muenster, Germany Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic c Institute for Pharmacology und Toxicology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany b
a r t i c l e
i n f o
Article history: Received 29 September 2008 Available online 16 October 2008 Keywords: Vascular smooth muscle cells NO synthase Vascular relaxation Endothelium-derived relaxing factor
a b s t r a c t The concept of [Gren_OpEdothliumDervRaxingFctorGe_Cls][RdOpenendothelium-derived relaxing factor Red_Clos](EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. [Gren_OpHowvrGen_Cls][RdOpeHowever, Red_Clos]it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomic al integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts suf ficient to account for the vaso relaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle. © 2008 Elsevier Inc. All rights reserved.
The concept of the obligatory role of the endothelium in the vasorelaxation was drawn from the experiments with endothe lium-deprived blood vessels. Rubbing off the endothelial layer was reported to render blood vessels insensitive to ACh [1]. It was concluded, that the endothelial cells when stimulated by ACh, released a nonprostanoid, diffusible factor (later termed EDRF for endothelium-derived relaxing factor) that acted on the subjacent VSMC to produce relaxation. The biologic al activity of EDRF was later explained through NO release by endothelial cells [2]. To the time of those studies, VSMC were regarded as passive recipients of NO from the endothelium. Later it was, however, found that VSMC in various blood vessels do express all three NOS iso forms depending on the blood vessel type [3,4]. Moreover, aspects of the anatomical integrity of the organ (blood vessel) subjected to experiments with rubbing the blood vessel intimal surface were neglected. It should be taken in consideration that the destruction of the vascular wall integrity increases the concentration of vascu lar superoxides [5,6] that, in turn, impair vasodilatory responses to exogenous and endogenous nitrovasodilators [7]. Known as NO scavengers, superoxides drastically reduce NO bioactivity and NO bioavailability [8–10], whereas the intact endothelium pro tects VSMC from the superoxide attack [11,12]. In addition to NO * Corresponding author. Fax: +49 251 8355460. E-mail address: buchwalo@uni-muenster.de (I.B. Buchwalow) 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.019
s cavenging, superoxides can also directly exert a vasoconstrictor action [13,14]. We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer led to a vascular dysfunction and rendered blood vessels insensi tive to vasodilators as a consequence of oxidative stress. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scav engers restored vasodilatory responses to ACh in endotheliumdeprived blood vessels. Materials and methods Animals. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Insti tutes of Health (NIH Publication No. 85-23, revised 1996) and was performed in accordance with the guidelines of the Institutional Animal Care Committee, Institute of Normal and Pathological Phys iology, Bratislava. Male Wistar rats (350–450 g; n = 11) were used. Immunohistochemistry. Tissue probes of the thoracic aorta, mesenteric artery, and pulmonary artery were fixed in buffered 4% formaldehyde and routinely embedded in paraf fi n. 4-lm sec tions of the paraf fi n blocks were dewaxed in xylene, rehydrated in graded alcohols, pre-treated for antigen retrieval, and immunore acted with primary antibodies recognizing NOS1, NOS2 and NOS3
I.B. Buchwalow et al. / Biochemical and Biophysical Research Communications 377 (2008) 504–507
(Transduction Laboratories, KY; and Santa Cruz Biotechnology, CA) as described by us earlier [3,4,15]. Bound rabbit primary antibod ies were detected using DAKO EnVision-HRP system and NovaRed substrate kit (Vector Labor atories), counterstained with hema toxylin and mounted with an aqueous mounting medium GelTol (Immunotech, Marseille). The exclusion of the primary antibody from the immunohistochemic al reaction, substitution of primary antibodies with the rabbit IgG at the same final concentration, or preabsorption of primary antibodies with corresponding control peptides resulted in lack of immunostaining. Visualization and image processing. Immunostained sections were examined on a Zeiss microscope “Axio Imager Z1”. Micros copy images were captured using AxioC am 12-bit camera and Axi oVision single channel image processing (Carl Zeiss Vision GmbH). Images shown are representative of at least three independent experiments which gave similar results. Functional in vitro study. Rats were anaesthetized with diethyl ether, decapit ated and exsanguinated. The thoracic aorta, mesenteric artery, and pulmonary artery were immediately removed, cleaned of adhering fat and connective tissue, and cut into 2–4 mm wide rings. Endothelial cells were removed by gently rubbing the intimal surface with cotton-covered wire. The rings were vertically fixed between two stainless steel triangles in 20 ml incubation organ bath with Krebs solution, and bubbled with a 95% O2 and 5% CO2 gas mixture. The vessel segments were allowed to equilibrate for 1 h at a resting tension of 1 g and the changes of isometric tension were recorded as described previously [16]. Krebs solution containing 80 mM KCl was prepared by replacing NaCl with equimolar KCl and after an equili bration period the rings were stimul ated until a sustained response was obtained, in order to test their contractile capacity. The pres ence of functional endothelium was assessed in all preparations by determining the ability of ACh (10¡5 M) to induce relaxation of rings pre-contracted with phenylephrine. For relaxation studies, the rings were pre-contracted with maximum concentration of phen ylephrine (10¡5 M) and cumulative concentration–response curves for ACh (10¡10 to 3 £ 10¡5 M) were obtained. After washout the rings of aorta were preincubated with N-acetylcysteine (10¡4 M; 20 min) or tempol (3 £ 10¡3 M; 20 min). The rings of mesenteric artery and pulmonary artery with tempol only, and the relaxant responses to ACh were determined. Relaxation was expressed as a percentage of phenylephrine-induced contraction. Statistical analysis. Data are given as means ± SEM. For the statis tical evalua tion of differences between groups, one-way analysis of variance (ANOVA) was used and followed by Bonferroni’s post hoc test. The differences of means were considered as significant at P value <0.05.
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To gain evidence for the role of arterial smooth muscle in the regulation of vascular tension and to elucidate the role of superox ides in impairing vasodilatory responses, we examined ACh-induced vasorelaxation in endothelium-deprived blood vessels like in the experiment of Furchgott and Zawadzki [1] but in the presence of superoxide scavengers—tempol and N-acetyl-l-cysteine (NAC). In thoracic aorta rings with intact endothelium, cumulative addition of ACh (10¡10 to 3 £ 10¡5 M) produced concentrationdependent relaxation. The maximum relaxation was 85.59 ± 4.69% (Fig. 2A). In rings with denuded endothelium, ACh-induced relax ation was held back with the maximum relaxation 15.85 ± 4.31% (p < 0.01). Pre-treatment of denuded rings with NAC (10¡4 M) significantly restored ACh-induced relaxation to the level of 33.75 ± 7.25% (p < 0.05) (Fig. 2A). Tempol (3 £ 10¡3 M), a superox ide dismutase mimetic, also reversed the ACh-mediated relax ations in endothelium-deprived aortic ring preparations with the
Results and discussion Thoracic aorta rings, mesenteric artery rings, and pulmonary artery rings from intact and denudated blood vessels of rat were first subjected to morphological and immunohistochem ical control to confirm the absence of the endothelial layer in endothelium-deprived blood vessels and to demonstrate NOS expression in blood vessels under study. We found all three NOS isoforms expressed not only in the intima but also in media of the blood vessels under study. As an example, Fig. 1 shows strong expression of NOS3 in the thoracic aorta (Fig. 1A), mesenteric artery (Fig. 1B), and pulmonary artery (Fig. 1C), in both inti mal and medial cells. Inserts in this layout (Fig. 1) demonstrate the complete removal of the endothelial layer after denudation. The NOS expression by cells in the media of blood vessels was also confirmed by us earlier with Western blotting showing the presence of characteristic immunoreactive protein bands for NOS in the porcine carotid artery and rat aorta devoid of endo thelium [3,4].
Fig. 1. Expression of NOS3 in (A) thoracic aorta, (B) mesenteric artery, and (C) pulmonary artery, in both intimal and medial cells. Inserts demonstrate the com plete removal of the endothelial layer after denudation. Fifty micrometer scale bar for entire layout.
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Fig. 2. (A) Effect of NAC (10¡4 M) on the concentration–response curves to ACh in thoracic aorta endothelium-intact (E+) and endothelium-denuded (E¡) rings. Tissues were exposed for 20 min to NAC before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. *p < 0.05; **p < 0.01; with respect to E+; +p < 0.05; ++ p < 0.01 with respect to E¡. (B) Effect of tempol (3 £ 10¡3 M) on the concentration–response curves to ACh in thoracic aorta endothelium-intact (E+) and endotheliumdenuded (E¡) rings. Tissues were exposed for 20 min to tempol before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. *p < 0.05; ** p < 0.01; with respect to E+; +p < 0.05; ++p < 0.01 with respect to E¡.
aximum relaxation of 34.58 ± 6.65% (p < 0.05), whereas pre-treat m ment of thoracic aorta intact rings with tempol but insignificantly inhibited ACh-induced relaxat ion (Fig. 2B). Cumulative addition of ACh (10¡9 to 3 £ 10¡5 M) relaxed phen ylephrine-pre-contracted intact mesenteric artery rings with a maximum relaxat ion of 74.0 ± 8.04% (Fig. 3A). Compared to intact mesenteric artery rings, endothelial denudation resulted in a signif icant depression of ACh-induced relaxation (20.2 ± 3.23%, p < 0.01), but pre-treatment of endothelium-denuded rings with tempol (3 £ 10¡3 M) restored ACh-induced relaxation up to 51.6 ± 6.2% (p < 0.01). Important, tempol pre-treatment of intact mesenteric artery rings did not affect the vasorelaxatory response to ACh. In pulmonary artery rings with intact endothelium, ACh-induced relaxation amounted to 89.9 ± 4.12% (Fig. 3). Like in intact mes enteric artery rings, ACh-induced relaxation was not affected by tempol pre-treatment. After endothelial denudation, ACh-induced relaxation was held back, and the maximum relaxation decreased to 20.0 ± 7.76% (p < 0.01). However, tempol (3 £ 10¡3 M) pre-treat ment of endothelium-deprived rings restored ACh-induced relax ation to the level of 57.4 ± 8.81% (p < 0.01). ACh-induced relaxation recovery in de-endothelia lized blood vessels by antioxidants was more pronounced in the mesenteric and pulmonary artery, the latter having also the strongest expres sion of NOS in the media. Additionally, blood vessels of the elas tic type like aorta have a lower ratio of VSMC compared to blood vessels of the muscular type [3]. This explains a lower response to protective action of superoxides in denuded aorta compared with mesenteric and pulmonary artery. Restoration of vasodi latory responses to ACh in endothelium-deprived blood vessels under protection against oxidative stress challenges the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle. The concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle implies, that NO generated by NOS in the endothelium diffuses to the underlying VSMC to produce relaxation [1,2]. However, the corner stone of this concept—inabil
ity of the smooth muscle to express NOS by themselves—appeared later as imaginary. With the advent of more powerful immunohis tochemical techniques increasing the antigen detectability, it turned out that VSMC in various blood vessels express all three NOS iso forms depending on the blood vessel type [3,4]. Further evidence for NOS expression in VSMC can also be drawn from more recent publications [17–19]. There are some other facts that are also inconsistent with the concept of the obligatory role of endothelial cells in the relaxa tion of arterial smooth muscle. Along with the oxidative stress, endothelial denudation can also impair K+-induced vasorelaxation (background-K+ channel activation) [20] as well as myoendothelial gap junctional communications in VSMC [21], which play a major role in endothelium-derived hyperpolarizing factor (EDHF)-med iated relaxations. It has been proposed that EDHF, whose chemi cal nature is as yet unknown, contributes to microvascular dilation more than NO does [22]. The major target of NO in VSMC is soluble guanylyl cyclase (sGC), which catalyzes the conversion of GTP to cGMP with subsequent activation of cGMP-dependent protein kinases or altered func tion of phosphodiesterases triggering the cell-specific physiologic response, vasodilation [23]. A mathematical model of the spatialtemporal gradient of the NO diffusion in a homogenous biological medium shows that the NO concentration on the border of vascu lar smooth muscle layer is less than the equilibrium dissociation constant of sGC. It means, that the diffusion of the NO produced in endothelium is insuf ficient to cause a relaxation of vascular smooth muscles in medium- and large-size blood vessels [24,25]. Taken together these considerations imply that the EDRF con cept derived from the experiments with de-endothelialized blood vessels is not free of assumptions and oversimplifications. With this study we have demonstrated that the endothelial denudation leads to a vascular dysfunction and renders blood vessels insensitive to vasodilators as a consequence of oxidative stress. Restoration of vasodilatory responses in endothelium-deprived blood vessels by antioxidants is in compliance with reports that superoxides
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Fig. 3. (A) Effect of tempol (3 £ 10¡3 M) on the concentration–response curves to ACh in mesenteric artery endothelium-intact (E+) and endothelium-denuded (E¡) rings. Tissues were exposed for 20 min to tempol before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. **p < 0.01; with respect to E+; + p < 0.05; ++p < 0.01 with respect to E¡. (B) Effect of tempol (3 £ 10¡3 M) on the concentration–response curves to ACh in pulmonary artery endothelium-intact (E+) and endo thelium-denuded (E¡) rings. Tissues were exposed for 20 min to tempol before addition of phenylephrine. Data points are mean values and vertical lines represent SEM. ** p < 0.01; with respect to E+; +p<0.05; ++p < 0.01 with respect to E¡.
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