Differential Roles of AT1 and AT2 Receptor Subtypes in Vascular Trophic and Phenotypic Changes in Response to Stimulation With Angiotensin II
The aim of this study was to investigate the roles of angiotensin II (Ang II) receptor subtypes 1 (AT1) and 2 (AT2) in producing vascular wall hypertrophy and qualitative changes in smooth muscle cell gene expression. Wistar rats were treated for 23 days with osmotic minipumps containing solvent and either Ang II (120 ng·kg−1·min−1) or PD123319 (30 mg·kg−1·d−1), an AT2 receptor antagonist. In addition, rats receiving solvent and either Ang II or PD123319 were given losartan, an AT1 receptor antagonist, in the drinking water (10 mg·kg−1·d−1). Vascular wall hypertrophy and smooth muscle phenotype were characterized by morphometric analysis combined with immunohistochemistry. Ang II–induced hypertension was associated with the development of medial hypertrophy of the aorta and coronary arteries accompanied by reversion of vascular smooth muscle cells (VSMCs) toward an immature phenotype, as shown by the expression of cellular fibronectin and nonmuscle myosin. Losartan treatment, which restored normal arterial pressure, prevented all these changes. PD123319 treatment, which had no effect on blood pressure, prevented only vascular hypertrophy, with no effect on VSMC phenotype. Administration of only losartan to normal rats reproduced the Ang II–induced vascular hypertrophy, with no effect on VSMC phenotype. Taken together, these results suggest that (1) the trophic effect of Ang II on VSMCs is mediated via AT2 receptor subtypes and (2) changes in VSMC phenotypes are triggered mainly through AT1 receptor subtypes.
- Received March 7, 1996.
- Revision received June 17, 1996.
Arterial hypertension induces vascular remodeling, which affects large and small arteries.1 2 3 The hypertrophy of the media results from hypertrophy and/or hyperplasia of VSMCs and is dependent on the cause of hypertension and arterial size.2 Medial hypertrophy is associated with changes in gene expression of VSMCs leading to an immature phenotype characterized by accumulation of NM myosin heavy chain and c-FN.4 Both growth and phenotypic changes of VSMCs may be due to paracrine/autocrine growth factors acting through specific receptors.5 6 Ang II, the active biological peptide of the renin-angiotensin system, has potent vasoconstrictor actions and is directly involved in vascular7 and cardiac8 remodeling. Depending on the in vitro experimental conditions used,9 10 11 Ang II produces hypertrophy and/or hyperplasia in isolated VSMCs. Two benzylimidazole derivatives, losartan and PD123319, are specific antagonists for Ang II receptors AT1 and AT2, respectively.12 Both AT1 and AT2 receptor subtypes contain a seven-transmembrane domain, but only the former is coupled to G protein. AT1 receptor has been identified in several tissues and is considered to be the major mediator of the Ang II–induced effects in the cardiovascular, renal, and central nervous systems.13 In adult rabbit and rat vessels, it is the predominant receptor.14 15 The AT2 receptor is found in adrenal medulla, uterus, and the developing fetus and to a lesser extent in heart and arteries of adult mammals.16 The percentages of AT1 and AT2 receptors vary depending on the cell type or tissues considered: in rat aorta, 60% of the receptors are AT1, whereas in total rat heart, the percentage of AT1 reaches 90%17 of the angiotensin receptors, but the cardiomyocytes expressed exclusively AT1 subtype,18 whereas the fibroblast expressed AT2 receptor subtype as well.19 In pathophysiological situations such as cardiac hypertrophy secondary to stenosis of ascending aorta, AT2 receptor expression increases, whereas the AT1 subtype is downregulated.15 Moreover, the ratio of the concentrations of AT1 to AT2 receptors is reversed in experimentally induced vascular response to injury20 or in wound healing.21 Recent studies showed that overexpression of AT2 receptors inhibits neointimal formation after vascular injury.22 Although it has been suggested that AT2 receptor activation is involved in the control of cell differentiation, proliferation, and apoptosis,20 21 22 23 24 the possible roles of AT2 receptor in vivo are poorly understood.
To gain insights into the Ang II transduction mechanisms involved in the vascular remodeling, we have analyzed the cardiovascular effects of a long-lasting inhibition of the Ang II receptors AT1 and AT2 with losartan and/or PD123319 in rats perfused with or without Ang II. The smooth muscle cell phenotype was characterized by a differential expression of NM myosin and the extracellular matrix protein c-FN, both of which are developmentally regulated25 26 27 and considered to be reliable markers of smooth muscle cell differentiation in vivo and in vitro.4 28 29 30 The arterial smooth muscle cell phenotype and the degree of vascular hypertrophy were analyzed by a morphometric analysis combined with immunolabeling of cardiac and aortic sections with antibodies directed against extracellular matrix, smooth muscle, and nonmuscle proteins.
A total of 53 male normotensive Wistar rats weighing 300±20 g were used in this study. An osmotic minipump (Alzet, model 2ML4) was implanted subcutaneously in the back of the neck of rats anesthetized with sodium pentobarbital (50 mg/kg IP). Pumps were filled with solvent vehicle NaOH (pH 9) (n=11), Ang II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, acetate salt, Sigma Chemical Co) (n=12), or PD123319 (30 mg·kg−1·d−1, a dose previously shown to result in an effective AT2 blockade31 ) (n=6) in solvent solution (1 mg/mL). Minipumps were left in place for 22 days and infused at a constant rate of 2.5 μL/h, producing an Ang II infusion of 120 ng·kg−1·min−1. Rats infused with Ang II, PD123319, or solvent solution received losartan (10 mg·kg−1·d−1) or its vehicle (n=6) for 22 days by daily gavage.
At the end of the treatment period, SBP was measured by the tail-cuff method (BP recorder 8006, W+W Electronic). The rats were then anesthetized with sodium pentobarbital (50 mg/kg IP) and killed. The hearts, arrested in diastole (with a saturated solution of KCl), were quickly removed, weighed, and cut transversely at the equator of the ventricles. After the upper parts of the heart and the ascending aorta had been mounted, they were frozen in isopentane precooled with liquid nitrogen. The blocks were kept at −70°C until use.
Extracellular matrix protein was immunolabeled with either monoclonal antibodies directed against the extradomain A sequence of c-FN (Biohit)32 or polyclonal antibodies against total FN (Chemicon) that recognize both plasmatic FN and c-FN.33 Cytocontractile protein immunolabelings were performed with monoclonal antibodies directed against human SM α-actin (Dako)34 and NM myosin (NMG2). These latter antibodies are directed against a myosin epitope specific for nonmuscle cell types.35
Serial ventricular and aortic cryosections (5 μm) were labeled by use of a double immunolabeling technique previously described.4 Briefly, sections were incubated overnight at 4°C with monoclonal antibodies directed against either c-FN, SM α-actin, or NM myosin and diluted (1/100, 1/50, and 1/250, respectively) in PBS (in mmol/L: NaCl 150, KCl 2.5, and phosphate buffer 10; pH 7.2) containing 2% BSA. Sections were rinsed two times at room temperature in PBS and incubated for 30 minutes at 37°C with rabbit IgGs directed against total FN at a dilution of 1/100 in PBS/2% BSA. After two washings in PBS, sections were incubated for 30 minutes at room temperature with biotinylated anti-mouse IgGs (1/200 dilution in PBS+2% rat serum, Vector Laboratories). Sections were washed again and incubated with anti-rabbit IgGs conjugated to FITC (Amersham) and then with a streptavidin–Texas Red complex (1/50 dilution in PBS, Amersham). Sections were mounted in aqueous medium (Fluoprep, Biomerieux). Fluorescence was observed with a Leitz microscope equipped with epifluorescence optics (Leica).
Morphology and Quantitative Analysis
The aorta and large coronary artery media were analyzed in serial sections labeled with antibodies directed against SM α-actin, c-FN, or NM myosin by use of a video-imaging microscopy approach previously described.4 Video images from a low-light-level camera (C-2400, Hamamatsu) were transmitted to a computer (Macintosh IIfx) equipped with an image analysis program (Optilab, Graphtek). This software permitted a real-time storage of images in digitally calibrated formats for later processing. All quantitative analyses were performed in a blinded fashion. Data are reported as the mean of quantification of three serial nonconsecutive sections.
Thickness of the Aorta and Myocardial Coronary Artery Media
The specific location of SM α-actin in the media of aorta and coronary arteries was used to define the internal and external medial perimeters and to evaluate the medial and lumen surface area. The size of the aorta and coronary vessels was evaluated with low magnification (×40 and ×100, respectively). All the coronary arteries were examined throughout the two ventricles, and only those found to have a transverse orientation were further analyzed.
Quantitative Analysis of c-FN and NM Myosin Labeling
Coronary arteries were examined throughout the two ventricles, and only those with lumen diameter >35 μm were considered. The percentage of coronary arteries exhibiting positive staining for c-FN and/or NM myosin in the media was determined. Quantitative analysis of aortic labeling with antibodies directed against c-FN or NM myosin was performed with a video-imaging microscopy technique as previously described.4 36 Briefly, the aortic surface occupied by c-FN or NM myosin was expressed as percentage of the aortic media surface area determined by the internal and external lamina.
Data are expressed as mean±SEM. Statistical significance was assessed by ANOVA followed by Bonferroni's test. P<.05 was considered significant.
Index of Hypertension and Hypertrophy
The increase in rat SBP induced by Ang II infusion (P<.01 versus control) was completely prevented by the losartan treatment, whereas PD123319 was without effect (Fig 1A⇓). Treatment of normotensive rats with either losartan or PD123319 separately had no effect on SBP. SBP was not significantly lower when both drugs were administered simultaneously.
Since body weight was similar in all the groups, the ratio of heart weight to body weight was used as an index of cardiac hypertrophy (Fig 1B⇑). The Ang II–induced cardiac hypertrophy (P<.01 versus control) was totally prevented by treatment with losartan but not by treatment with PD123319. PD123319 treatment of the Wistar rats induced a significant cardiac hypertrophy (P<.05 versus control). Treatment with either losartan alone or the combination of losartan and PD123319 had no effect on the ratio of heart weight to body weight.
Ang II–Induced Aortic Medial Hypertrophy and Changes in VSMC Phenotype
Ang II–Induced Aortic Medial Hypertrophy
SM α-actin immunolabeling outlined the aortic media thickness and revealed a marked increase in the Ang II–treated groups (+27±4%, P<.01 versus control) (Figs 2⇓ and 3). Both losartan and PD123319 treatment of Ang II–treated animals reduced the extent of hypertrophy, but PD123319 treatment appeared to be more effective (Fig 2⇓). When only losartan was administered to normotensive rats, an increase in aortic media thickness was observed (+10±2%, P<.05 versus control). Although PD123319 administration to rats had no effect on the aortic medial thickening, the combination of the two drugs decreased the medial thickness by 25±9% (P<.05 versus control).
Ang II–Induced Changes in Aortic Smooth Muscle Cell Phenotype
In the control group, SM α-actin antibodies stained all the medial smooth muscle cells, and labeling was homogeneously distributed throughout the media (Fig 3C⇓). The immunolabeling of c-FN and NM myosin was restricted to the intimal layer (Fig 3A and 3B⇓⇓). This staining pattern was found in 75% of the normal rats; in the remaining 25%, the c-FN and NM myosin labelings were present in the inner two or three layers of the aorta. The blood vascular profile of each animal cannot be used to discriminate between those expressing or not expressing NM myosin or c-FN in the aorta. In the Ang II group, the distribution of SM α-actin within VSMCs was qualitatively similar to that of the control group (Fig 3F⇓). Comparison of SM α-actin and c-FN immunolabelings indicated that the intima size was independent of the level of pressure in each animal. In the majority of the Ang II–treated rats (70%), c-FN immunolabeling extended to cells in the inner half of the media (Fig 3D⇓). An increased number of NM myosin–positive cells throughout the aortic media was also evident (Fig 3E⇓). These cells were identified as VSMCs by the codistribution of NM myosin and SM α-actin within the same cells throughout the entire media. Such a homogeneous distribution of NM myosin in the medial layer was observed in 60% of the Ang II–treated animals. In all of the Ang II–treated animals, administration of losartan prevented both the aortic medial hypertrophy (Fig 3I⇓) and the expression of c-FN and NM myosin that would have been induced by the Ang II treatment (Fig 3G and 3H⇓⇓). PD123319 treatment had no effect on Ang II–induced qualitative changes in phenotype (Fig 3J and 3K⇓⇓), and the majority of the animals (>80%) expressed extensive NM myosin and c-FN in the aortic media. In normal rats, the treatment with PD123319 increased the expression of c-FN and NM myosin. The labelings were detected in the inner three layers of the media in almost all animals (85%) (Fig 3P and 3Q⇓⇓). Losartan alone or in combination with PD123319 treatment did not affect the expression of SM α-actin, c-FN, or NM myosin of VSMCs in the aortic media (Fig 3M and 3N⇓⇓); consequently, the percentage of animals expressing c-FN and/or NM myosin within the inner part of the media was <20%.
Morphometric analysis revealed that c-FN and NM myosin occupied 6±0.6% and 7±0.7%, respectively, of the medial area in the control group (Fig 4⇓), whereas in the Ang II–treated group, the two proteins occupied 17±0.3% and 16±0.2%, respectively, of the medial area (P<.01 versus control). Administration of losartan prevented the expression of c-FN induced by the Ang II treatment (P<.05 versus Ang II group). The decrease in NM myosin was not significant. Treatment of normal rats with PD123319 increased the expression of c-FN and NM myosin, but only the expression of NM myosin was statistically significant (P<.05 versus control).
Ang II–Induced Coronary Artery Medial Hypertrophy and Changes in VSMC Phenotype
Ang II–Induced Medial Myocardial Coronary Hypertrophy
The immunolabeling of coronary arteries with anti–SM α-actin antibodies had a qualitatively similar distribution among all of the groups, and it illustrated the differences in the media thickness (Fig 5⇓). To measure changes in media thickness, the coronary artery population was subdivided into four groups according to their lumen size (Fig 6⇓). In almost all the groups, Ang II induced a significant thickening of the media that was prevented by the addition of either losartan or PD123319. Administration of losartan to normotensive rats resulted in a significant medial thickening similar to that observed in Ang II–treated rats. Finally, treatments with PD123319 did not affect the cross-sectional coronary medial size.
Ang II–Induced Phenotypic Changes in VSMCs
In contrast to SM α-actin, NM myosin was undetectable in the media of coronary arteries in normal rats (Fig 5A⇑) but was markedly induced in those of Ang II–treated animals (Fig 5B⇑). The cells that stained with NM myosin antibodies were VSMCs, since they were located mainly in the coronary artery media and codistributed with SM α-actin labeling. c-FN immunostaining was restricted to the endothelium of the control coronary arteries, whereas total FN was present throughout the media of the coronary arteries and around coronary capillaries (Fig 7A and 7D⇓⇓). Ang II treatment induced an accumulation of total FN and c-FN in the coronary artery media (Fig 7E and 7B⇓⇓). The increased expression of both c-FN and NM myosin in the media indicates a shift of VSMC phenotype toward an immature type, as previously demonstrated in SHR-SP.4 Smooth muscle cells exhibiting such an immature phenotype were found in 69±6% of the coronary arteries of all the Ang II–treated animals (P<.01 versus control), whereas the percentage of positive arteries in the controls did not exceed 10±5%. Losartan but not PD123319 totally prevented the expression of c-FN and NM myosin in Ang II–treated rat smooth muscle cells, the immunolabeling pattern being similar to that of the control group in all the coronary arteries whatever their size and location. Administration of PD123319 alone induced a slight increase to 39±10% of the coronary arteries staining for c-FN and NM myosin. This shift in phenotype was prevented by the combined administration of PD123319 and losartan. Losartan alone was without effect on the phenotype of smooth muscle cells of coronary arteries in normotensive rats.
The Table⇓ summarizes the qualitative and quantitative changes in VSMC gene expression observed in the vasculature of the different animal groups. Ang II induced both a shift of the VSMC phenotype toward an immature type and a media thickening. The two processes were prevented by an AT1 receptor antagonist that normalized the blood pressure. The AT2 antagonist prevented the media thickening but had no effect on the VSMC phenotype. Interestingly, the treatment of control rats with AT1 antagonist induced the hypertrophy of the media without affecting phenotypic changes of VSMCs.
The present study analyzed the different roles of AT1 and AT2 receptors in the Ang II–dependent quantitative and qualitative changes that occur in rat heart and aorta during the development of hypertension.
In agreement with previous data,37 38 our findings indicate that Ang II–induced hypertension and associated cardiac hypertrophy are mediated mainly via the AT1 receptor subtype, since both processes were prevented by AT1 but not by AT2 antagonists. The Ang II–induced medial hypertrophy of both the aorta and the coronary arteries (Figs 2 and 6⇑⇑) was associated with specific qualitative changes in gene expression as detected by the increased expression of both c-FN and NM myosin, markers of an immature VSMC phenotype.4 39 40 Such a change in the medial VSMC pattern of gene expression in aorta and large coronary arteries of hypertensive rats occurs regardless of a genetic (SHR-SP) or induced (Goldblatt, chronic Ang II infusion rats) cause of the hypertension (References 4, 8, 39, 41, and this issue). The qualitative response of aortic and coronary VSMCs to Ang II was heterogeneous, as previously observed in other models of hypertension.4 29 Some medial cells maintained their adult contractile phenotype, whereas others exhibited an immature one, as evidenced by the heterogeneous distribution of either c-FN or NM myosin within the media. c-FN has also been observed in human diffuse intimal thickening, atherosclerotic fibrous plaque, experimental intimal thickening in rabbits and rats, and in migrating rat VSMCs in vitro.30 42 Changes in VSMC-matrix interactions are thought to be of major importance in controlling cell growth and vascular remodeling.5 26 On the other hand, NM myosin expression in VSMCs has been shown to control the adhesiveness and cytokinesis in vitro,43 its downregulation inhibiting VSMC proliferation.44 The presence of both c-FN and NM myosin in the media indicates that the process of vascular remodeling may still be active 3 weeks after the induction of hypertension by Ang II.
One of the important findings of the present study is the discrimination between the mechanisms involved in the qualitative and quantitative changes in VSMCs described above. The two processes are thought to be linked and to be triggered by mechanical and/or humoral factors.4 45
Several lines of evidence indicate that Ang II mediates the VSMC trophic effect via AT2 receptor subtype and independently of a pressure-dependent mechanism (Figs 2 and 6⇑⇑). First, the AT2 receptor blockade with PD123319 infusion in Ang II–treated rats had no significant effect on blood pressure but prevented the development of vascular hypertrophy of both aorta and coronary arteries. Second, the finding that in normotensive rats, treatment with losartan alone had no effect on blood pressure but induced a medial hypertrophy that was prevented by an additional treatment with PD123319 strongly suggests that the increase in systemic Ang II concentration, as a result of AT1 receptor blockade46 46A activates the AT2 receptors and, as a consequence, unmasks their trophic effect. The trophic effect of the AT2 receptor subtype appears to be specific to VSMCs and independent of the artery type (conductive as aorta or resistive as coronary artery). These results extend recent reports showing that AT2 receptors play a major role in the myointimal formation after arterial injury20 and that the expression of AT2 receptor is increased in hypertrophied left ventricle.15
Qualitative changes in VSMC gene expression of arteries have been related to the increased perfusion pressure and/or mediated by humoral factors such as Ang II.39 45 47 In the latter case, Ang II–induced expression of oncogenes and fibronectin is mediated mainly through the AT1 receptor subtype.48 Similarly, we show here that in Ang II–treated rats, losartan but not PD123319 prevented the qualitative changes in VSMC phenotype of both aorta and coronary arteries, indicating that AT1 receptor subtypes, directly or in combination with pressor mechanisms, are the main entrance to the pathway controlling VSMC phenotype. Moreover, the finding that the AT2 blockade in normotensive rats was associated with an increased NM myosin expression by the VSMCs suggests that AT2 blockade unmasks an AT1 receptor pathway, as proposed by Siragy and Carey.49
As a result, the qualitative and quantitative changes in the phenotype of the aorta and coronary arteries secondary to Ang II–induced hypertension appear to be triggered by two independent pathways: (1) Ang II induces, via the AT2 receptor, a medial hypertrophy of both aorta and coronary arteries independently of blood pressure elevation, and (2) VSMC phenotypic changes are controlled through AT1 receptor activation and/or blood pressure elevation.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1||=||angiotensin receptor type 1|
|AT2||=||angiotensin receptor type 2|
|NM myosin||=||nonmuscle myosin|
|SBP||=||systolic blood pressure|
|SHR-SP||=||stroke-prone spontaneously hypertensive rats|
|SM α-actin||=||smooth muscle α-actin|
|VSMC||=||vascular smooth muscle cell|
This study was supported by INSERM, CNRS, European Union (Biomed, BMH1CT 92-1171), and Fondation de France. The authors wish to thank Pr S. Winegrad for helpful discussions.
Owens GK. Control of hypertrophic versus hyperplastic growth of vascular smooth muscle cells. Am J Physiol. 1989;257:H1755-H1765.
Levy BI, Diurez M, Philipe M, Poitevin P, Michel JB. Effect of chronic dihydropyridine (Isradipine) on the large arterial walls of spontaneously hypertensive rats. Circulation. 1994;90:3024-3033.
Contard F, Sabri A, Glukhova M, Sartore S, Marotte F, Pomies JP, Schiavi P, Guez D, Samuel JL, Rappaport L. Arterial smooth muscle cell phenotype in stroke-prone spontaneously hypertensive rats. Hypertension. 1993;22:665-676.
Pauletto P, Sarzani R, Rappelli A, Pessina AC, Sartore S. Differentiation and growth of vascular smooth muscle cells in experimental hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Ltd; 1995:697-709.
Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995;75:487-517.
Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanisms of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension. 1991;18(suppl II):II-100-II-105.
Crawford DC, Chobanian AV, Brecher P. Angiotensin II induces fibronectin expression associated with cardiac fibrosis in the rat. Circ Res. 1994;74:727-739.
Geistefer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988;62:749-756.
Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension. 1989;13:305-314.
de Gasparo M, Husain A, Alexander W, Catt KJ, Chiu AT, Drew M, Goodfriend T, Harding JW, Inagami T, Timmermans PBMWM. Proposed update of angiotensin receptor nomenclature. Hypertension. 1995;25:924-927.
Lopez JJ, Lorell BH, Ingelfinger JR, Weinberg EO, Schunkert H, Diamant D, Tang SS. Distribution and function of cardiac angiotensin AT1- and AT2-receptor subtypes in hypertrophied rat hearts. Am J Physiol. 1994;36:H844-H852.
Zhuo J, Allen AM, Alcorn D, Aldred GP, MacGregor DP, Mendelsohn FAO. The distribution of angiotensin II receptors. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Ltd; 1995:1739-1762.
Lassègue B, Griendling KK, Alexandre W. In: Saavedra JM, Timmermans PBMWM, eds. Angiotensin II Receptors. New York, NY: Plenum Press; 1994:17-48.
Meggs LG, Coupet J, Huang H, Cheng W, Li P, Capasso JM, Homcy CJ, Anversa P. Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats. Circ Res. 1993;72:1149-1162.
Janiak P, Pillon A, Prost JF, Vilaine JP. Role of angiotensin subtype 2 receptor in neointima formation after vascular injury. Hypertension. 1992;20:737-745.
Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, Horiuchi M, Pratt RE, Dzau VJ. The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci U S A. 1995;92:10663-10667.
Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651-657.
Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. 1996;93:156-160.
Samuel JL, Farhadian F, Sabri A, Marotte F, Robert V, Rappaport L. Expression of fibronectin during rat fetal and postnatal development: an in situ hybridisation and immunohistochemical study. Cardiovasc Res. 1994;28:1653-1661.
Kuro-o M, Nagai R, Nakahara KI, Katoh H, Tsai RC, Tsuchimochi H, Yazaki H, Ohkubo A, Takaku F. cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis. J Biol Chem. 1991;266:3768-3773.
Sartore S, Scaneta M, Chiavegato A, Faggin E, Giuriato L, Pauletto P. Myosin isoform in smooth muscle cells during physiological and pathological vascular remodelling. J Vasc Res. 1994;31:661-681.
Glukhova MA, Frid MG, Shekhonin BV, Vasilevskaya TD, Grunwald J, Saginati M, Koteliansky VE. Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent. J Cell Biol. 1989;109:357-363.
Vartio T, Laitinen L, Narvanen O, Cutolo M, Thornell LE, Zardi L, Vartanen I. Differential expression of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. J Cell Sci. 1987;88:419-430.
Ruoslathi E, Hayman EG, Pierschbacher M, Engvall E. Fibronectin: purification, immunochemical properties, and biological activities. Methods Enzymol. 1982;82:803-831.
Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against α-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986;103:2787-2796.
Zanellato AMC, Borrione AC, Tonello M, Giuriato L, Scannapieco G, Pauletto P, Sartore S. Myosin isoform expression and smooth muscle cell heterogeneity in normal and atherosclerotic rabbit aorta. Arteriosclerosis. 1990;10:996-1009.
Bardy N, Karillon GJ, Merval R, Samuel JL, Tedgui A. Differential effects of pressure and flow on DNA and protein synthesis and on fibronectin expression by arteries in a novel organ culture system. Circ Res. 1995;77:684-694.
Pauletto P, Chiavegato A, Giuriato L, Scatena M, Faggin E, Grisenti A, Sarzani R, Paci MV, Fulgeri PD, Rapelli A, Pessina AC, Sartore S. Hyperplastic growth of aortic smooth muscle cells in renovascular hypertensive rabbit is characterized by the expansion of an immature cell phenotype. Circ Res. 1994;74:774-788.
Bauters C, Marotte F, Hamon M, Oliviero P, Farhadian F, Robert V, Samuel JL, Rappaport L. Accumulation of fetal fibronectin mRNA after balloon denudation of rabbit arteries. Circulation. 1995;92:904-911.
Seidel CL, Wallace CL, Dennison DK, Allen JC. Vascular non muscle myosin expression during cytokinesis, attachment, and hypertrophy. Am J Physiol. 1989;256:C793-C798.
Simons M, Rosenberg D. Antisense nonmuscle myosin heavy chain and oligonucleotides suppress smooth muscle cell proliferation in vitro. Circ Res. 1992;70:835-843.
Bardy N, Merval R, Samuel JL, Tedgui A. Effect of angiotensin II, pressure and flow on fibronectin expression in a novel organ culture system of perfused, pressurized rabbit aorta. Circulation. 1994;90(suppl I):I-515. Abstract.
Goldberg MR, Tanaka W, Barchowsky A, Bradstreet TE, McCrea J, Lo MW, McWilliams EJ, Bjornsson TD. Effects of losartan on blood pressure, plasma renin activity, and angiotensin II in volunteers. Hypertension. 1993;21:704-713.
Samuel JL, Barrieux A, Dufour S, Dubus I, Contard F, Koteliansky V, Farhadian F, Marotte F, Thiéry JP, Rappaport L. Accumulation of fetal fibronectin mRNAs during the development of rat cardiac hypertrophy induced by pressure overload. J Clin Invest. 1991;88:1737-1746.
Kim S, Kawamura M, Wanibuchi H, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Iwao H. Angiotensin II type 1 receptor blockade inhibits the expression of immediate-early genes and fibronectin in rat injured artery. Circulation. 1995;92:88-95.