Prolonged inhibition of nitric oxide synthesis in Yoshida hyperlipidemic rat: Aorta functional and structural properties

life

Sciences,Vol.

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copyright o 1997 Elsevier scicncc Inc. Printed in the USA. All rights reremd m442Q5/!37-s17.oo

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PII SOO24-3205(96)00674-l

PROLONGED INHlBITION OF NTTRIC OXIDE SYNTHESIS IN YOSHIDA HYPERLIPIDEMIC RAT: AORTA FUNCTIONAL AND STRUCTURAL PROPERTIES A. Chinellatoa, E. Ragazzia’, L. Pandolfoa, G. Froldia, L. Caparrottaa, B. Amoreb and S. SartorelW aDepartment of Pharmacology, University of Padova, Large Meneghetti 2, I-35 131 Padova, Italy bDepartment of Biomedical Sciences, University of Padova, Via Trieste 75, I-35 131 Padova, Italy cC.N.R. Unit for Muscle Biology and Physiopathology, Padova, Italy (Received in final form January l3.1997)

Summary To test whether inhibition of nitric oxide synthesis, associated with high levels of plasmatic lipids, can induce atherosclerotic lesions and phenotypic changes in smooth muscle cell composition in the aortic wall of an atherosclerotic-resistant species such as the rat, an inbred strain of hyperlipidemic Pittsburgh Yoshida rat was subjected to prolonged treatment (2 months) with the nitric oxidasynthase inhibitor L%itro-arginine-methyl ester or with L-arginine. The two types of in viva treatments were not able to modii in vitro aortic endothelium-mediated relaxation induced by acetylcholine or calcium-ionophore A-23187, the endotheliumindependent sodium nitrite relaxation and the contractile response to serotonin. Histology and lipid infiltration of vascular specimens showed that L%iitro-argininemethyl ester in viw treatment did not induce any significant change in the aortic wall. Monoclonal antibodies to myosin isoforms and immunofluorescence procedures revealed the presence of an immature smooth muscle cell subpopulation in aortic specimens from saline-treated Pittsburgh Yoshida rats, whose expansion has been related in other species to atherogenesis. This peculiar cell phenotype disappeared in our animal model after prolonged L%ritro-arginine-methyl ester treatment. These data indicate that, despite interference with endothelium-mediated nitric oxide synthesis, atherosclerosis does not develop in this animal model and tbrnish for the first time a biological justification for atherogenesis resistance of rat, i.e., the lack of activation of an immature aortic smooth muscle cell population which in atherosclerosis-prone species is involved in lesion formation. Key Wonlr: endothelium, vasc&r smooth muscle, nitric oxide, my&n isoforms, hyperlipidemia, Pittsburgh Yoshida rat

Vascular endothelium has a unique role in controlling changes of blood flow and wall stress occurring in experimental intimal thickening and atherogenesis (discussed in (1) ). In the atherosclerotic plaque, endothelial cell-produced nitric oxide (NO) -dependent relaxation is ‘Correspondence to: Eugenio Bagazzi, M.D., Department of Pharmacology, University of Padova, Largo E. Meneghetti, 2 - 35 131 Padova (Italy). E-mail: [email protected] Phone: +39-49-8275092. Fax: +39-49-8275093

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markedly impaired and this loss of finction is due to endothelial injury or to inactivation of NO (2). NO released at sites of vascular injury (3) has been demonstrated to attenuate the development of experimental intimal thickening (4-7), possibly by inhibiting smooth muscle and endothelial cell proliferation (8, 9). In addition, NO is a potent inhibitor of platelet aggregation, acting synergistically with prostacyclin, and is also able to decrease the adhesion of monocytes to the endothelial layer. Thrombus formation and leukocyte inilltration are crucial events in the atherosclerotic lesion development (10) and the reduction of their impact on the arterial wall could account for the reported anti-atherogenetic effect of L-arginine (the NO precursor) supplementation to cholesterol-fed rabbits (11). While rat is extensively used as a model of mechanically-induced endothelial lesion which gives rise to a typical intimal thickening, this species is generally considered to be atherosclerotic lesion-resistant to hyperlipidemic diets (12, 13). Despite alteration in the structure and function of endothelium in 6-month-old inbred hyperlipidemic Pittsburgh Yoshida @‘OS) rat, the animals do not develop atherosclerotic lesions, even with age or after a cholesterol-enriched diet (14, 15). We have reasoned that if the endothelium dysfunction plays a role in inducing a true atherosclerotic lesion in the presence of a high plasmatic lipid level, the in v&o prolonged reduction of NO endogenous synthesis in YOS rats, by using the NO-synthase inhibitor L%itro-arginine-methyl ester (L-NAME), would be instrumental to obtain such a lesion. We have also investigated on the feasibility to reverse the previously identified endotheliopathy (14) in this rat strain by prolonged in vivo treatment with L-arginine. Potential phenotypical changes in the pattern of smooth muscle cell (SMC) differentiation, which might accompany proliferation/migration of these cells from the medial layer into the putatively formed neointima, have been assessed by monoclonal antibodies specific for myosin isoforms (16).

Methods Pittsburgh Yoshida (YOS) male hyperlipidemic 5-month-old rats (450 f 1Ogof body weight) and Wistar normolipidemic male rats (480 f 15 g) of the same age were obtained liom Charles giver Italia (Calco, Italy). The age of 5 months was preferred over other ages for the in viva treatnaent,she2 6month-old YOS rats already displayed diminishedendothelialfunction (14). YOS rats were treated in viva for two months with L-NAME, Garginine or saline (NaClO9?A, as control). The animalsas&red to L-NAME treatment received daily via i.p. the drug dissolved in saline (in 0.5 ml volume) at the initial dose of 30 mg/kg for the Grst month; this dose was selected in order to reduce toxic efI&s, since prehminary data showed lethal etlbcts after 10 days of treatment with 100 m@kg/die of L-NAME (unpublished data). Afterwards rats were given a dose of 70 mg/kg for the second month in order to assure greater inhibitory eIl?ecton NO-synthesis. Another group of rata received daily i.p. L-rug&e 100 m@g (in 0.5 ml volume). The control group was treated i.p. with 0.5 ml of saline for the same twomonth period. All the animalswere maintainedat a 14L:1ODlight cycle and mceived a statAnd low celoty diet (4RP21, Mucedola, Settimo Milanese, Italy) and water ad libitum.Animalswere used and maintained in accordance with the recommendations in the Guide for the Care and Use of Laboratory Anin&, published by the US National Institutes of Health (NII-I Publication No. 8523, revised 1985) and the Animal Care Advisory Committee of the Italian Ministry of Public Health (DL 116/92). Seventy-two hours after the last treatment (this lag time was left to prevent acute e&cur due to the presence of the administered drugs) the animals were sacrificed for the in vim studies. Systolic blood pressure was measured before and during the treatment period by the tail-cufI?method. Heart rate was measmed simultaneously with blood pressure. Blood samples were obtained from tail vein for serum determmations.

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Functional e.xperiments A&r deep anaesthesiawith inhaled methoxytlurane, the descending thoracic aorta was removed, cleaned of adjacent tissue and cut into transverse rings approximately 3 mm wide. Each aortic preparation consisted of a chain of 4 rings, tied by silk thread, suspended in 30-ml tissue baths containing a solution of the following composition (mmol/l): NaCl 116.0; KC13.2; CaCl2 1.2; MgClz 1.2; NaH$04 1.2; NaHC@ 22.0; glucose 10.1; ascorbic acid 1.1, and equilibrated with a 95% 02 - 5% C@ gas mixture, pH 7.4, at 37 “C. Isometric force was recorded by means of transducers (Type DYO, Basile, Italy) connected to a chart recorder (Unirecord, Basile, Italy). The aortic chains were held at a resting force of 15 mN and allowed to equilibrate at optimal length for 60-90 min before experimentation was started, the bulTerbeing changed every 15 min Before experiments were started, all aortic preparations were tested with the approximate EC% of norepinephrine (0.03 @vi),washed and equilibratedfor at least 60 min. This procedure was found to increase and stabii any subsequent contra&e response to norepinephrine. One aortic preparation was used to determine the cmmdative concentration-response curve of norepinephrine in order to calculate the EC& of the contractile agonist for each rat. For relaxation studies, the vasodilator drugs were cumulativelyadded to aortic rings precontracted with E&I norepinephrine at the steady state. The drugs were separately tested at diffierentlevels of thoracic aorta. At the end of the experiment, ECW,norepinephrine was added to check the stabiity of contraction The spontaneous relaxation following the contraction to the last addition of EC50 norepinephrine was subtracted from the relaxation caused by the vasodilator agonists to calculate the net effect of the agonists. IN vitro experiments were performed by usii thoracic aorta preparations from untreated normolipidemicWistar rats (Charles giver Italia, Calco, Italy).

CholesteroI and Pi@ceri&

akterm&ations

Cholesterol and triglycerides in serum were determined by using enzymanc-colorimetric assay kits (oxida.&peroxidase method, MenariniDiagnostic Division, Florence, Italy), leading to the formation of quinoneimine dye, revealed at 500 ran, according to the manr&ctu&s instructions. Aortic arch was used to estimate total tissue cholesterol. At& saponitlcation with 8.9 M KOH and ethanol (1: 10) at 37’ C for 3 h, extra&ion of cholesterol was carried out in n-hexane and tlnally determined with the above mentioned oxidase@oxidase assay kit.

Histoiqqy, histochemistry and immunojluorescence

Specimens from the aortic arch and the descending aorta of normolipidemic Wistar rats, hyperlipidemic L-NAME- or L-arginine-treated YOS rats as well as YOS rats treated with saline were filled with O.C.T. (Miles, Elkhart) immediately frozen in liquid nitrogen and stored at -8O’C until use. Five urn cryosections were cut using a Leitx 1720 cryostat and collected onto gelatincoated glass coverslips. The indiiect immunofluorescence procedure described in (17) with antimyosin antibodies was used. Bat non-immune IgG were included in the second incubation with IgG anti-mouse IgG coupled with rhodamine isothiocyanate (Dako, Dakopatts a/s, Glostrup, Denmark) to lower the background staining. BF-48 monoclonal antibody specific for smooth muscle-type SMI (204 kDa) and SM2 (200 kDa) myosin heavy chain (MyHC) isoforms was used (18) along with monoclonal NM-G2 specific for non-muscle (NM) 196 kDa MyHC (19, 20). Control experiments were those of (17).

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l OO-_. ACh, log [M] Fig. 1 Acetylcholine-induced relaxation after norepinephrine precontraction of aorta preparation from normolipidemic Wistar and thoracic hyperlipidemic YOS rats. Experiments were performed in the absence or presence of L-NAME (0.1 mM, preincubated for 20 minutes). Results show mean with SEM (indicated by vertical bars), n=4-5. Where no error bar is shown, SEM is within the sii of the symbol. ** p
L%itro-arginine-methyl ester &-NAME), Latginine, acetylchohne bromide (ACh), sodium nitrite, A-23 187, serotonin cmatink sulfate complex and norepinephrinebitartrate were purcha& f?om Sigma-Aldrich S.r.1. (Milano, Italy). AU drugs were dissolved daily in saline solution (0% NaCl). Aliquots were added in the tissue bath solution to obtain the final desired concentration. Stotisticul ondysis

Data were expressed as means f SEM. m between means were compared by Student’s t test for unpaired data. Signilkance was accepted at the 0.05 level of probabii (two tailed).

Results

Eficacy of L.-NM

in vi&o

Before the in viw treatment of YOS rats with GNAME, a prehmky study was conducted in aortic preparations from normolipidemic Wistar rats, in order to evaluate the ability of GNAME to

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inhibit in the rat species the relaxant efkct of endotheliumdependent agents, such as acetylcholine. The same experiment was performed also on YOS rats in order to verity the activity of L-NAME in this hyperlipidemicstrain. As presented in figure 1, after 20 minutes of L.-NAME(0.1 mM) incubation, the relaxation induced by ah the concentrations of acetylcholine completely disappeared in both animal groups. Body weight, bloodpressure and heart rate There was no signifkmt difkence in the body mass among the experimental groups, examined during the experimental time. Figure 2 shows the blood pressure and heart rate determined in YOS rats atkr the period of in viw treatment. Mean values of systolic blood pressure did not vary significantly among the three treatment groups, although a slight increase was observed after the tmatment period. Heart rate did not change signiticantlyin saline- and L-arginin~treated animals,but a signilicantreduction was found in L NAME-treated rats.

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Fig. 2 Systolic blood pressure and heart rate, determined in the three experimental groups of YOS rats atIer the weatment period. Bars represent mean values 1 SEM, n=5. ** ~4.01 vs control group.

Serumand time lipid compaxition Figure 3 shows the serum lipid protile in YOS rata atIer two months of tm&ment. The rats treated with L-NAME presented a lower cholesterol level in serum if compared to the control YOS rats treated with saline @
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Cholesterol content in aortic tissue obtained &om aortic arch in YOS rats afkr the tmatment period (Figure 3) appeared to be increased in the group treated with GNOME and U&nine, although not signiiicantly,compared to the saline+treatedcontrol group.

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Fig. 3 Serum cholesterol and triglycerides and aortic cholesterol in the three groups a&x two months of treatment. Bars represent mean values f SEM, n=5. *** p
Adylcholine (10 nM - 10 pM) induced a concentration-dependent rekation on norepinephrine-precontracted.thoracic aorta rings of YOS nits f&m all the Watment groups WW 4 A). No di&rence was deteckxl among L-NAME or Garginine kxtment groups in comparison to ssline&eated rats.

Figure 4 B illustrates the concentration-response curves to the calcium-ionophore A-23187 (1 nM - 1 @VI)of norepinephrine-precontracted thoracic aorta rings obtained fiom the three groups of YOS rats. In both GNAME and L-arginine treated groups, A-23187 caused a concentration-dependent endothelium-mediatedrelaxation on precontracted aortas that was not d&rent i+omthe control group. Figure 4 C shows aortic rekation induced by sodium nitrite (1 w - 10 mM) on norepinephrine-precontracted thoracic aortas. No di&rence was observed among the three groups of YOS rats.

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Fig.4 In v&o relax& response to acetykholine (ACh, panel A), A23187 (panel B) watment groups of andNaN~(padC)ofthoracicaortaf?omthethree YOS rats. Results show mean with SEM indicated by vertical bars, n=5. Where no error bar is shown, SEZMis within the size of the symbol.

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Fig. 5 Contraction to serotonin in thoracic aorta from the three treatment groups of YOS rats. Data are expressed as mN and each point is mean f SEM, n=5. Aortic contractile response to serotonin In order to test the elEct of the in viva treatments on contractile response of the aorta, complete concentration-response curves of serotonin were obtained in the range 10 nM - 100 @i. No sign&ant difference of aortic contraction to the drug was observed among the preparations from the three treatment groups (Figure 5). Histology histochemistty and immunofluorescence In order to check the appearance of altered vascular smooth muscle phenotype atter the treatment of YOS rats with GNOME or Garginine, immunochemicalanalysis of aortic arch and distal thoracic aorta was. performed by using monoclonal isoform-spechic anti-myosin NM-G2 or BF48 antibodies (18-20). Specimens from aortic arch or from descending aorta gave a similar immunofluorescence pattern when examined with anti-myosin antibodies (data not shown). Figure 6 shows the results obtained with descending aorta fi-om normolipidemic Wistar rat reacted with BF48 and NM-G2 antibodies. All SMC are stained for SM myosin, whereas a number of SMC containing SM and NM myosin (immature-type SMC) (18, 20) are dispersed unevenly in the media. No intimal layer was visible at the magnification used. Figure 7 shows the immunofluorescence pattern obtain4 with aortic samples from YOS rats: saline- (A,B), L-NAME- (CD) and L-arginine- (E,F) treated animals. In all samples examined, BF48 stained all medid SMC, though immunofluorescence labeling was occasionally absent in limited medial regions of L-NAME-treated rats (C). With regard to NM-G2 antibodies, aortas from saline-treated YOS rats showed NM myosin-containing medial immature type SMC grouped close

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to the luminal surf& (B), whereas in both L-NAME- and L-arginine-treated YOS rats no hnmunoreactivity was found. As the NM-G2 positive cells appeared to be localized in the inner region of the aortic media, the possibility exists that these cells accumulated because of the lipid deposits in this area. To test this hypothesis, we compared serial sections, processed for Sudan black staining, with NM-G2 antibody (Fig. 8 A,B). The accumulation of NM-G2 positive cells does not correlate with the existence of sudanophylic deposits in the aortic wall.

Fig. 6 Micrographs showing the immunofluorescence patterns of descending aorta from normolipidemic Wistar rats with BF-48 (A) and NM-G2 (B) antibodies. Note that all medial SMC are stained by BF-48, whereas some of them are also labeled by NM-G2 (asterisks in B). A and B are serial cryosections. Bar, 50 pm.

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Fig. 7 Micrographs showing the immunofluoresce-nce patterns of descending aorta from hyperlipidemic control (A,B), L-NAME- (C,D) and L-a&nine (E,F) - treated YOS rat with BF-48 (A,C,E) and NM-G2 (B,D,F). Note that all (A,E) and the large majority (C; some cells are negative, double asterisk) of medial (m) SMC are stained with BF-48. NM-G2 positive cells are grouped in a medial region facing the lumen in A (asterisks), or completely absent in aortic samples ftom L-NAME- or L-arginiwtreated animals. a: Adventitia. Bar, 50 pm.

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Discussion

The present study indicates that prolonged tmatment of hyperlipidemic YOS rat with the NO-synthase inhibitor L-NAME neither substantially mod&s the endotheliumdependent relaxation properties of aortic tissue nor induces the formation of atherosclerotic lesions. Resistance to atherosclerosis in this rat strain is related to inabiity of expansion of immature type medial SMC population, which in other atherosclerosis-prone species is markedly increased. In vitro, L-NAME can inhibit endothelium-mediated relaxation induced by acetylcholine in aortic tissue from both normolipidemic and hyperlipidemic rats (see Fig. 1). In viva, however, saline-treated YOS rats show a marked endothelial dysltmction (endotheliopathy: (14) ) which is not attenuated, as shown here, by L-arginine administration nor mod&d by L-NAME supplementation. In fact, both endotheliumdependent (mediated by acetylcholine or calcium ionophore A-23 187) and endothelium-independent (mediated by sodium nitrite) relaxation as well as serotonin-stimulated contraction tests performed on aortic rings from L-NAME-treated YOS rats are similar to that of preparations from saline treated-animals or from L-arginine-treated rats. Impairment of NO-dependent relaxation (6,21) is directly related to development of intimal lesions (1). Reduced NO synthesis has been detected in vascular districts with high shear stress (22), where atherosclerosis is more likely to occur (22). Despite these correlations, prolonged interference with NO synthase has no efficacy in the animal model used in the present study. Even blood pressure in YOS rats treated with L-NAME (and also with L&nine) was not statistically diierent compared to saline-treated YOS rats. With regard to inducible blood pressure variations under L-NAME treatment, this rat strain behaves differently compared to the WistarKyoto rats used by other investigators (23,24). In t&t, chronic inhibition of NO synthesis increases blood pressure which can be reversed by a diuretic administration (24). As increased blood pressure is a well recognized risk factor for atherogenesis (25), the lack of lesions in YOS rats might be due to the inability of these animals to respond to L-NAME with a marked increase of hemodynamic parameters. Serum cholesterol was lower in L-NAME treated compared to control group of YOS rats, though the content in aortic tissue was similar in both groups. Navarro et al. (26) have found no changes in plasma cholesterol afler L-NAME treatment (oral administration of 5-30 mg/lOO ml for 5 weeks) of normolipidemic rats. YOS rat shows a marked hyperlipidemia mainly due to a lipoprotein overproduction (27). It remains to be elucidated whether chronic L-NAME treatment may influence lipoprotein turnover leading to a lowering of plasma cholesterol. The histological and histochemical examinations of aortic tissues from YOS rats treated with L-NAME indicate that no neointimal formation occurs. In addition, the rearrangement of NMG2 labelling antimyosin antibody observed in this animal group, compared to normolipidemic rats, is not related with histologically detectable stable lipid deposits in the media. We cannot exclude that lipoproteins can penetrate through the endothelium and accumulate in the subendothelial region of aortic wall, but it is likely that no retention of lipids and/or metabolic transformation occur (25). The presence of a unique SMC population in the media may be related to the absence of atherosclerotic lesions in this rat model. In several experimental systems (rat (28), rabbit (21) and human (29) ) the progressive structural and functional maturation achieved during development is characterized by a number of protein isoform switch&s, which in the case of myosin consists of a down-regulation of NM myosin expression (typical of immature type SMC cells) and up-regulation of smooth muscle adult-type SM2 isoform. In experimental models in which there is SMC prol%eration/migration, such as in exogenous- or endogeneous-hypercholesterolemic or renovascular hypertensive rabbits (16), the size of the SMC subpopulation with dual myosin content (SM+NM myosin) markedly increases (17).

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The apparent SMC insensitivity to hyperlipidemia and endotheliumdysfunction in YOS rat treated with L-NAME might be due to the presence exclusively of an adult-type SMC population, demonstrated by the lack of NM myosin isoform. This peculiar SMC phenotype might be relatively incapable of LDL accumulation (30), and thus its expansion could be protective against atherogenesis. Alternatively, in YOS rat the inhibitor of NO-synthase L-NAME might have undergone blood (31) or systemic metabolism allowing the production of L-arginine which stimulates NO-synthase expression. This phenomenon may explain the similarity between arginine and L-NAME-treated YOS rat immunohistochemical aortic patterns. However, metabolism of LNAME in rat has been describedmainly to nitro-L-arginine, which should retain inhibitory effects on NO-synthase (32).

A Fig. 8 Micrographs showing the Sudan black staining (A) and the NM-G2 immunostaining (B) of serial cryosections from aorta of YOS rats treated with saline. While the adventitial layer (a) is strongly labeled with Sudan black, no staining is visible in the medial layer (m). NM-G2-positive medial SMC are grouped in the innermost layer (small arrowheads). Bar, 50 pm.

It is known that rat is the less suitable model in which to study the impact of hyperlipidemia on atherosclerotic lesion formation (12, 13), owing to its intrinsic genetic background which determines, among other consequences, only minimal thrombus formation and leukocyte inflltration in aortic tissue (1). In rodents, only genetic manipulations in mouse are able to convert the SMC from resistant to prone cellular elements with respect to fatty streak development (33). Thus, it is reasonable to assume that differences in the biology of aortic SMC, shown here, along with differences in lipoprotein profile/metabolism (12, 13, 32) and lipoprotein oxidation by endothelial cells (34) plus genetic background can account for the observed lack of susceptibiity to develop atherosclerosis in the rat. L-Arginine administration enhances NO production at sites of arterial injury with beneficial effects on neointimal thickening development, possibly by reducing the hyperplastic SMC growth (4, 6). Though a direct anti-proliferative effect of this aminoacid on SMC in vitro has been demonstrated (8), there are no conclusive experiments to validate its e5cacy in vivo. Further investigation of the effect of L-arginine treatment on myosin isoform expression in different

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pathological conditions leading to cell proliferation might clarify this point. Taken together all these data strongly suggest that the vascular biology of YOS rat is indeed different from other animal species and probably from other rat strains.

Acknowledgements

This work is dedicated to Professor Giuliana Fassina who planned and supervised with constructive suggestions a great part of the research project. She died on October 19, 1995. The work was supported by a special grant from MURST and the Biomedical Association for Vascular Research. We would like to thank Dr. Marco Prosdocimi (Fidia Research Laboratories, Abano Terme, Italy) for the in vivo treatment of the animals, Mr. Enrico Se& for excellent technical assistance and Dr. Cristiano Meggiato for performing many of the experiments as part of his research for his degree. We thank Ms. Marissa GallianiSanavio for her editorial assistance. References 1.

2. 3.

8. 9. 10. 11. 12. 13.

14.

15.

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