Hyperplastic vascular smooth muscle cells of the intrarenal arteries in angiotensin II type 1a receptor null mutant mice

Kidney International, Vol. 60 (2001), pp. 722–731

Hyperplastic vascular smooth muscle cells of the intrarenal arteries in angiotensin II type 1a receptor null mutant mice SACHIKO INOKUCHI, KENJIRO KIMURA, TAKESHI SUGAYA, KOITI INOKUCHI, KAZUO MURAKAMI, and TATSUO SAKAI Department of Anatomy, Juntendo University, School of Medicine, Tokyo; Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo; Institute of Applied Biochemistry, University of Tsukuba, Ibaragi; and Lead Generation Research Laboratories, Tanabe Seiyaku Co., Ltd., Kashima, Osaka; Third Department of Internal Medicine, Nippon Medical School, Tokyo; Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaragi; and Department of Anatomy, Juntendo University, School of Medicine, Tokyo, Japan

Hyperplastic vascular smooth muscle cells of the intrarenal arteries in angiotensin II type 1a receptor null mutant mice. Background. Angiotensin II (Ang II), which contracts vascular smooth muscle cells (VSMCs), has been reported to regulate VSMC growth. Recently formed transgenic mice without angiotensinogen or Ang II receptors showed vascular alterations. However, it is still unclear how their VSMCs alter. We explored the role of Ang II via the Ang II type 1a receptor (AT1a) in VSMCs in vivo using AT1a null mutant mice. Methods. We analyzed the ultrastructure of the intrarenal arteries in AT1a null mutant mice that were homozygous for a targeted disruption of AT1a receptor gene using light and electron microscopy. Results. The structural changes of the intrarenal arteries in AT1a null mutant mice showed the wall thickening, which in the interlobar, arcuate, and proximal interlobular arteries consisted of two additional populations of VSMCs, on the luminal and abluminal sides of the media. The luminal overpopulation of smooth muscle cells (SMCs) was arranged in a longitudinal direction separated by increased interposed elastic laminae. The abluminal overpopulation of SMCs ran in circumferential directions separated from the main population. The cytological structure of VSMCs in AT1a null mutant mice was smaller in size, contained more organelles for protein synthesis and secretion than in control mice, and had poorly developed contractile apparatus. Conclusions. The lack of AT1a signaling causes structural abnormalities in the renal vascular system and transforms the phenotype of VSMCs into cell proliferation, induces the escape of VSMCs from the circular mechanical integrity, and results in increased synthesis of extracellular matrices.

Angiotensin II (Ang II), the major effector of the renin-angiotensin system, is a potent constrictor of vascular smooth muscle cells (VSMCs) and modulates VSMC growth [1–3]. Ang II acts on two pharmacologically and structurally distinct receptors: Ang II type 1 and type 2 receptors (AT1 and AT2). AT1, the dominant receptor of the vascular effects of Ang II [4], was shown to consist of two isoforms in rodents, AT1a and AT1b [5]. In most cardiovascular tissue, AT1a is dominant over AT1b [6, 7]. Although specific pharmacologic blockers of the angiotensin receptors have been used extensively to examine the roles of each receptor, a more complete blockade of a receptor can be gained by targeted gene deletion. Angiotensinogen null mutant mice show hypotension and vascular hypertrophy in the vascular system [8–10]. Similar lesions are seen in angiotensin-converting enzyme null mutant mice [11, 12] and AT1 null mutant mice [13]. These findings suggest that the absence of Ang II via AT1 is critical to the pathogenesis of the phenotype. On the other hand, it is not clear whether the arterial wall in AT1a null mutant mice is altered, although AT1a null mutant mice exhibit hypotension, mesangial expansion [14, 15], abnormality in mesangial matrix formation [15], marked hypertrophy of the juxtaglomerular apparatus, and proximal expansion of reninproducing cells along the afferent arterioles [14]. Recently, there have been some interesting observations with regard to VSMC growth effects of Ang II. Geisterfer, Peach, and Owens reported that Ang II induces VSMC hypertrophy, but not hyperplasia, in confluent quiescent cells in a defined serum-free medium [1]. Furthermore, Gibbons and coworkers suggested that Ang II via transforming growth factor-␤1 inhibits smooth muscle cell proliferation and via platelet-derived growth factor stimulates smooth muscle cell proliferation [16, 17]. In the angiotensinogen- and angiotensin receptor-null

Key words: renal artery, hypertrophy, cell signaling, extracellular matrix, renin-angiotensin system, targeted gene deletion, intrarenal vascular structure. Received for publication December 27, 1999 and in revised form January 19, 2001 Accepted for publication March 12, 2001

 2001 by the International Society of Nephrology

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mutant mice, the ultrastructural characteristics of their VSMCs have not yet been reported. To examine the role of Ang II via the AT1a receptor for VSMCs growth, we ultrastructurally evaluated the intrarenal vascular structure and the characteristics of the renal VSMCs in AT1a null mutant mice. METHODS Homozygous AT1a gene-deficient mice (AT1a null mutant mice) generated from C57BL/6 mice by Sugaya et al were used [7]. In the mutant mice, the AT1a receptor gene was disrupted by replacement with the ␤-galactosidase gene. C57BL/6 mice of the wild type were used as controls. Four AT1a null mutant mice aged 27 weeks and four wild-type mice of the same age were used for morphological examination. The mice were anesthetized by intraperitoneal injection of a mixture of ketamine (Ketarar 50; Sankyo, Tokyo, Japan) and xyladine (Ceraktar, Bayer Japan, Tokyo, Japan). After opening the abdominal cavity, the abdominal aorta was cannulated with a retrograde catheter and perfused by a fixative for five minutes with a perfusion apparatus (Kosaka Laboratory, Tokyo, Japan) operated at a pressure of about 120 mm Hg for AT1a null mutant mice and 140 mm Hg for the control mice. We preliminarily confirmed that each perfusion pressure was the lowest possible pressure to perfuse each kind of mouse. Additionally, two AT1a null mutant mice aged nine weeks were used to study an earlier period. The fixative contained 2% glutaraldehyde in 0.1 mol/L phosphate buffer at pH 7.4. Small pieces of the kidney, liver, and small intestine were cut into sections of 400 ␮m thickness and processed by a cold-dehydration technique described elsewhere [18]. After brief treatment with 0.1% osmium tetroxide, the specimens were immersed in 1% extracts of oolong tea (OTE, Suntory, Tokyo, Japan) in an acetone buffer solution (0.05 mol/L maleate buffer, pH 6.0, containing 10% acetone), followed by 1% uranyl acetate in the same acetone buffer solution, and then they were dehydrated with a graded acetone series at 0⬚C to ⫺30⬚C and embedded in Epon 812. These procedures enabled detailed morphological observation of the extracellular matrix and intracellular fibrillar structures [18]. Semi-thin sections (500 nm) were stained with toluidine-blue and prepared for light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with a JEM-1200EX electron microscope (JEOL, Tokyo, Japan). To quantify the morphological changes in the intrarenal arteries, cross-sectional profiles of all the proximal interlobular (interlobular arteries in the deeper one third of the cortex), arcuate, and interlobar arteries were examined morphometrically. The measurement was performed for each mouse on four arterial profiles for interlobar arteries, 8 to 12 for

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arcuate arteries, and 20 to 30 for proximal interlobular arteries. Both inner and outer diameters were measured for each artery. In the case of vascular profiles of a slightly oval shape, the inner and outer diameters were represented by the smallest diameter. The outer diameter minus the inner diameter divided by two and the outer diameter minus the inner diameter divided by the outer diameter (designed as arterial wall thickness and wall thickness ratio, respectively) were calculated. A single average for each artery for each mouse was calculated and compared between the wild-type mice and AT1a null mutant mice. Digital images of light microscopy (Photograb-300; Fuji Photo Film Co., Ltd., Tokyo, Japan) and computer software for image analysis (KS400; Carl Zeiss, Oberkochen, Germany) were used. Morphometric data are presented as means ⫾ SD. Statistical analysis was performed using Student t test of an unpaired design. A P value of less than 0.05 was considered statistically significant. RESULTS Structural changes of the intrarenal arterial system in AT1a null mutant mice The intrarenal arterial system (IAS) of AT1a null mutant mice showed remarkable changes in the wall structure as well as in the width of the inner diameter compared with that of control mice (Fig. 1). On the other hand, the IAS branching pattern appeared unchanged, and its segments, including the interlobar, arcuate, and interlobular arteries, as well as the afferent and efferent arterioles could be identified easily. The most remarkable structural changes of IAS consisted of a thickening of the arterial wall in the proximal IAS, namely in the interlobar, arcuate, and proximal interlobular arteries (Fig. 1 b–d). In the distal IAS, namely in the distal interlobular arteries and afferent arterioles, a mixture of thickening and thinning of the wall was found, in addition to an increase in renin-containing granular cells of afferent arterioles and their appearance in the distal interlobular arteries. The proximal IAS of AT1a null mutant mice showed unique structural specializations in their thickened wall that were never seen in the control mice. The specializations consisted of two additional populations of VSMCs, on the luminal side (Fig. 1) and abluminal side of the media (Fig. 2), as well as interposition of elastic lamina mainly on the luminal side. The luminal overpopulation of smooth muscle cells (SMCs) was arranged strictly in a longitudinal direction and were separated from the main circumferential population of VSMCs by the interposed elastic lamina (Figs. 3b and 4b). The abluminal overpopulation of SMCs ran in circumferential directions, but were separated from the main population of SMCs by a moderate amount of extracellular matrices,

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Fig. 1. Light micrographs showing the thickening in the arcuate and interlobar artery of angiotensin II type 1a (AT1a) null mutant mouse. (a) The arcuate artery in the wild-type mouse has two layers of medial smooth muscle cells and is constant in width. (b) The arcuate artery in AT1a null mutant mouse reveals the thickening of the tunica media accompanied with luminal dilation compared with that in the wild-type mice. An additional overpopulation of smooth muscle cells (SMCs) appears on the luminal side (arrows). In the tunica media, medial SMCs proliferate among the irregular intercalated elastic lamina. (c) In the oblique section of the interlobar artery in AT1a null mutant mouse, the wall is focally thickened accompanied with small clusters of luminal overpopulation of SMCs (arrow). (d) An oblique section of the interlobar artery in AT1a null mutant mouse reveals that the luminal overpopulation of SMCs is perpendicular to that of medial SMCs (a–c ⫻450, d ⫻900).

so that they were not incorporated in the mechanical armor of the arterial wall provided by main circumferential VSMCs (Fig. 2). The thickening of the arterial wall of the proximal IAS in the AT1a null mutant mice was due to the presence of the two additional population of SMCs, since the thickness of the main circumferential layer of VSMCs of the AT1a null mutant mice was similar or smaller than that of the media of the comparable segments of IAS in the wild-type mice. The distal IAS of AT1a null mutant mice possessed an occasional abluminal overpopulation of VSMCs in addition to the main circumferential population, but no luminal overpopulation of VSMCs with a longitudinal arrangement. The wall thickness of distal IAS in AT1a null mutant mice was increased by the presence of an abluminal overpopulation of VSMCs and proximal expansion of renin-producing cells along the interlobular arter-

ies and afferent arterioles, compared with comparable segments in control mice, but was decreased or unchanged where the additional VSMCs were absent. Ultrastructure of VSMCs and extracellular matrices in AT1a null mutant mice At a light microscopic level, the luminal overpopulation of VSMCs was distinguished from the main circumferential VSMCs by their typical longitudinal orientation and by the frequent interposition of elastic lamina between these two populations of VSMCs (Fig. 1). Even where the two populations were not separated by elastic lamina, the difference of orientation of VSMCs allowed for a clear demarcation of these two populations. We did not find any VSMCs with intermediate oblique orientation in the IAS of AT1a null mutant mice. At the electron microscopic level, the VSMC popula-

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Fig. 2. Transmission electron micrographs showing the interlobular artery in AT1a null mutant mouse. (a) An additional population of vascular smooth muscle cell (SMCs) appears on the abluminal side of the media (arrow). The abluminal overpopulation of SMCs runs in circumferential directions. (b) The abluminal overpopulation of SMCs is separated from the main population of SMCs by moderate amounts of extracellular matrices (arrowheads). aSMC, abluminal overpopulation of SMCs (a ⫻2400; b ⫻15,200).

tion in IAS could be identified only with reference to orientation of the vascular axis. In arterial cross-sections, the luminal overpopulation of VSMCs exhibited crosssectional profiles, whereas the main population of VSMCs appeared in longitudinal sectional profiles (Fig. 3). In longitudinal sections of arteries, the sectional profiles of these two populations were reversed (Fig. 4). In oblique sections, the identification of VSMC population was difficult. The abluminal overpopulations of VSMCs were more

difficult to identify compared with the luminal ones. They were most clearly identifiable in cross-sections of arteries. In such typical sections, the abluminal overpopulation of VSMCs appeared as small islands attached from the outside to the circumferential layer of the main VSMC population (Fig. 2). The cytological structures of both of the additional VSMC populations were fundamentally similar to that of the main population of VSMCs in AT1a null mutant mice. The VSMCs in AT1a null mutant mice were different from VSMCs of wild-type

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Fig. 3. Transmission electron micrographs showing the interlobar artery in cross-cut profile. (a) Tunica media of the interlobar artery in the wild-type mouse is composed of three layers of medial smooth muscle cells (mSMCs) between inner elastic lamina (iEL) and outer elastic lamina. mSMCs in cross-cut profile are spindle shaped. (b) In AT1a null mutant mouse, mSMCs, which are composed of three to five layers, are irregular in arrangement and size. In the luminal overpopulations of smooth muscle cells (lSMC) that appear in the tunica media, the cells are irregular in size and cubic in shape like cobblestones, whereas the main mSMCs are spindle shaped as seen in the wildtype mice. The inner elastic lamina has become partially thickened. Irregular intercalated elastic fibers (arrow) accumulate among SMCs. EC, endothelial cell (a, b ⫻5000).

mice in that they were smaller in size and contained more organelles for protein synthesis and secretion, such as rough endoplasmic reticulum and Golgi apparatus (Fig. 5). The dense bodies on the cytoplasmic surface of cell membrane served as anchoring sites of actin filaments that were poorly developed in VSMCs of AT1a null mutant mice as compared with those of wild-type mice (Fig. 5, arrow). Thus, the VSMCs in AT1a null mutant mice still possessed a clearly contractile phenotype, but slightly differentiated toward a synthetic phenotype compared with VSMCs in wild-type mice. In the IAS of AT1a null mutant mice, elastin was increased in amount and exhibited an irregular arrangement. The elastin was found in both AT1a null mutant and wild-type mice as inner and outer elastic lamina on both sides of the media. In the proximal IAS of wildtype mice, the inner elastic lamina was conspicuous, and the outer elastic lamina was mesh-like in appearance,

whereas in the distal IAS, the inner elastic lamina was mesh-like, and the outer elastic lamina was absent. In the proximal IAS of AT1a null mutant mice, intercalated elastic lamina was encountered on the luminal part of media in addition to the inner and outer elastic lamina (Fig. 3). Furthermore, spots of elastin as well as other components of extracellular matrices such as collagen fibrils and amorphous substance were increased between the VSMCs of the proximal IAS in AT1a null mutant mice compared with wild-type mice (Fig. 5). In the distal IAS of AT1a null mutant mice, whether the amount of extracellular matrices was increased was unknown. However, VSMCs got smaller in size in the AT1a null mutant mice than in wild-type mice, and the intercellular space among VSMCs was extended. Nine-week-old AT1a null mutant mice also showed the same findings as the 27-week-old AT1a null mutant mice, namely, increased arterial wall thickness, an emer-

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Fig. 4. Longitudinal profiles of the interlobar artery. (a) Medial smooth muscle cells (mSMC) in the wild-type mouse are cubic in shape. The tunica media shows constant width. (b) In AT1a null mutant mouse, overpopulations of SMCs appear on the luminal side; these cells are spindle shaped, whereas the main mSMCs are cubic. Elastic components lie between endothelial cells and luminal overpopulation of SMCs, become thick and continue on to the inner elastic lamina (arrowheads). Abbreviations are: lSMCs, luminal overpopulation of SMCs; EC, endothelial cell; iEL, inner elastic lamina (⫻5000).

gence of an overpopulation of VSMCs, and increased elastic fibers. In addition, we compared the structure of arteries in the liver and small intestine in AT1a null mutant mice to those in wild-type mice. Interlobular arteries in the liver and small arteries in small intestine, which were muscular artery, had one or two layers of VSMCs, a mesh-like inner elastic lamina, and spotted outer elastic lamina in wild-type mice. Their fundamental structures in AT1a null mutant mice were similar to those in wildtype mice and did not show an overpopulation of VSMCs, which was found in the IAS. However, some alterations of VSMCs in the liver and small intestine in the AT1a null mutant mice were found, including increased extracellular matrices, developed intracellular organelles, and poorly developed dense bodies as well as in the intrarenal arteries. The comparison among arteries of the kidney, liver, and small intestine in AT1a null mutant mice revealed that overpopulation of VSMCs was specific to the intrarenal arteries and that ultrastructural intracellular alterations of VSMCs commonly appeared in muscular arteries.

Morphometric analysis The morphometric data clearly showed that the proximal IAS in AT1a null mutant mice was dilated, and the wall was thicker compared with the wild-type mice (Table 1). The inner diameter and wall thickness in the proximal IAS were significantly greater in AT1a null mutant mice than in wild-type mice. In the proximal interlobular arteries, the inner diameter and wall thickness in AT1a null mutant mice were each 1.4 and 1.6 times as large as in wild-type mice, while in the arcuate arteries they were 1.5 and 1.7 times and in the interlobar arteries 1.3 and 1.7 times as large, respectively. The wall thickness ratio in arcuate and proximal interlobular arteries was not significantly different between AT1a null mutant and wild-type mice, and the values were almost constant throughout the study. In the interlobar arteries, the wall thickness ratio of AT1a null mutant mice was increased compared with wild-type mice. DISCUSSION Since Ang II strongly contracts VSMCs and has been reported to regulate their growth, it is thought to be one

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Fig. 5. High-power magnification views of longitudinal profiles of the interlobar artery showing the phenotypic differences of the medial smooth muscle cells (mSMCs) between the wild-type and AT1a null mutant mouse. (a) The SMCs of the wild-type mouse have well-developed bundles of microfilaments (arrows). The extracellular spaces around the SMCs are narrow and regular in width. (b) In AT1a null mutant mouse, mSMCs have welldeveloped cytoplasmic organelles, including rough endoplasmic reticulum (rER) and Golgi apparatus (G). On the other hand, the microfilaments bundles, which characterize contractile SMCs, are poorly developed (arrows) compared with those of the wild-type mouse. The amount of the extracellular matrices around SMCs has increased (⫻12,500).

of several factors causing the structural changes of the vascular wall in hypertension or atherosclerosis both in human beings and experimental animals. The cellular mechanism underlying the vascular alteration has been analyzed with in vitro studies using cultured VSMCs and with transgenic mice. Studies on the vascular structure of transgenic mice, including null mutant mice of angiotensinogen, angiotensin-converting enzyme (ACE) and AT1, thus far has employed only light microscopy to show vascular wall thickening in intrarenal arteries [8–13]. Our present electron microscopic analysis provides the first observation, to our knowledge, on the structure of the arterial wall in these transgenic mice. It reveals the unexpected behavior of VSMCs after the shutdown of Ang II signaling via AT1a.

The present study demonstrates that the thickening of the vascular wall in AT1a null mutant mice is accompanied by an increased number of VSMCs and by an increased amount of extracellular matrices. The individual VSMCs in AT1a null mutant mice were smaller and had poor development of the contractile apparatus compared with those in the control mice. This is the first evidence showing that the vascular thickening after a shutdown of genes for the renin-angiotensin system is brought about by hyperplasia and not by hypertrophy of VSMCs. Furthermore, it shows that a part of the VSMCs escaped from the circular mechanical integrity and appears either as a luminal longitudinal overpopulation or as an abluminal overpopulation. Concerning the effect of Ang II in VSMC growth, it

Inokuchi et al: AT1a in smooth muscle cells Table 1. Morphometric analysis of proximal intrarenal arterial system in wild-type and AT1a null mutant mice

Proximal interlobular artery Inner diameter lm Wall thickness lm Wall thickness ratio Arcuate artery Inner diameter lm Wall thickness lm Wall thickness ratio Interlobar artery Inner diameter lm Wall thickness lm Wall thickness ratio

Wild-type (N ⫽ 4)

AT1a null mutant mice (N ⫽ 4)

43.65 ⫾ 2.77 3.54 ⫾ 0.25 0.14 ⫾ 0.01

62.78 ⫾ 3.79a 5.64 ⫾ 0.26a 0.15 ⫾ 0.01

86.54 ⫾ 5.75 5.79 ⫾ 0.37 0.12 ⫾ 0.01

125.75 ⫾ 9.57a 10.02 ⫾ 0.94a 0.14 ⫾ 0.01

216.89 ⫾ 29.45 12.24 ⫾ 0.91 0.10 ⫾ 0.01

272 ⫾ 9.37c 20.96 ⫾ 2.09a 0.13 ⫾ 0.01b

Values are expressed as mean ⫾ SD. a P ⬍ 0.001, b P ⬍ 0.01, c P ⬍ 0.05, significantly different values from wild-type mice, by the unpaired t test.

is still unclear which effect Ang II brings about in vivo: VSMC hypertrophy or proliferation. We showed VSMC hyperplasia in proximal IAS of AT1a null mutant mice, although the distal IAS showed hyperplasia of renincontaining granular cells due to stimulation of renin production by the lack of Ang II action via AT1a. Recent studies indicate that Ang II is a bifunctional VSMC growth modulator that promotes either hyperplasia or hypertrophy. The hypertrophic effect was brought about by active TGF-␤1, which exerts an antiproliferative influence on the growth stimulatory effects of Ang II [16]. Consequently, the net growth response of VSMCs to Ang II seems to be hypertrophy without hyperplasia [1, 16]. In the models of chronic hypertension such as the spontaneously hypertensive rat, changes in smooth muscle content are due to hypertrophy of pre-existing smooth muscle cells within the media of the blood vessel with little or no cellular proliferation [19, 20]. According to the view that the net growth response of VSMCs to Ang II in vivo is the result of hypertrophy, we can speculate that the lack of Ang II signaling via AT1a causes the hyperplasia of VSMCs in renal arteries. In addition to hyperplasia of VSMCs, VSMCs in AT1a null mutant mice contained an abundant rough endoplasmic reticulum and Golgi apparatus accompanied with an increased extracellular matrix around the VSMCs and poorly developed dense bodies. In general, SMCs consist of two distinct cell types, myofilament-rich (contractile) SMCs and rough endoplasmic reticulum-rich (synthetic) SMCs [21]. Synthetic or contractile phenotype can be interchanged in cultured smooth muscle cells by the effect of extracellular matrices, growth factors, and mechanical stresses [22, 23]. In cell culture, rough endoplasmic reticulum-rich SMCs can synthesize a wide variety of extracellular matrix components [21, 24]. These findings indicate that the VSMCs in AT1a null mutant mice were

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differentiated toward the synthetic phenotype. We therefore suggest that the lack of AT1a signaling results in the increased synthesis of extracellular matrix of VSMCs. The luminal overpopulation of VSMCs found in the proximal IAS in AT1a null mutant mice is frequently separated by conspicuous elastic lamina from both endothelial cells and the main population of SMCs. This location is comparable with that for intimal SMCs, which are found in both physiological and pathological conditions [25–29]. The physiological intimal SMCs have been found in normal aortas in young adult rat [25, 27] and in prenatal human aortas [28], and the pathological ones in atherosclerotic plaque [21]. The intimal SMCs are thought to have escaped from mechanical integrity of media possibly by increased mechanical stress in a physiological condition [29] or by various humoral factors, including Ang II in a pathological condition [21]. The luminal overpopulation of VSMCs in AT1a null mutant mice is also thought to have escaped from the medial integrity, but there is no direct information about whether their escape is caused by mechanical stress or by humoral factors. The younger, nine-week-old AT1a null mutant mice also showed the same findings as the 27-week-old AT1a null mutant mice. Since the effects of antihypertensive drugs such as hydralazine (a direct smooth muscle relaxant having a different mechanism from renin-angiotensin system–related antihypertensive drugs) do not influence smooth muscle cell growth [30], low blood pressure in AT1a null mutant mice does not seem to influence the appearance of luminal overpopulation of SMCs. These findings suggest that luminal overpopulation of SMCs in AT1a null mutant mice results from a lack of Ang II signaling via AT1a. The manner of VSMCs in AT1a null mutant mice is different from the results of in vitro culture studies. Several studies indicate that Ang II stimulates production of the extracellular matrix in cultured SMCs [21, 31], and that Ang II stimulates SMC migration via AT1 in a concentration-dependent manner in the cell culture [32, 33]. In spite of the block of Ang II signaling via AT1a, VSMCs in AT1a null mutant mice revealed the same manner of VSMC as stimulation of Ang II in cultured SMCs. Concerning this discrepancy, although we could not elucidate a clear reason, three possibilities are hypothesized. First, in vivo net action of Ang II to VSMCs might be different from that in cultured SMCs due to the effects of Ang II-related humoral factors or Ang II concentration. Second, in AT1a null mutant mice, other Ang II receptors might actively be at work instead of AT1a. Third, the reaction of VSMCs to Ang II via AT1a might represent a specific manner depending on the organ. Arteries in AT1a null mutant mice were structurally different in various parts of arteries in the kidney as well as in the arteries of various organs. The proximal IAS is characterized by both luminal and abluminal over-

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population of VSMCs, the distal IAS by abluminal overpopulation of VSMCs, and hyperplasia of renin-containing granular cells. The interlobular arteries in the liver and small arteries in small intestine are characterized by abundant cytoplasmic organelles and poorly developed actin filament bundles of VSMCs without an overpopulation of VSMCs. The overpopulation of VSMCs and hyperplasia of renin-containing granular cells were specifically found only in the IAS. Hyperplasia of renin-containing granular cells in the distal IAS seems to be caused by a negative feedback pathway owing to the lack of AT1a signaling. These structural differences are thought to represent different attitudes of reaction of VSMCs that are mediated by a similar signaling pathway. The alterations of AT1a null mutant mice reported in literature [13, 14] were not nearly as severe as that observed in angiotensinogen, ACE, or AT1 null mutant mice. In angiotensinogen null mutant mice, we found that the renal vasculature showed hyperplasia of VSMCs and increased extracellular matrix among VSMCs in considerably greater magnitude than in the AT1a null mutant mice (unpublished observation in collaboration with M. Kihara and S. Umemura; Department of Internal Medicine II, Yokohama City University School of Medicine, Kanagawa, Japan). On the other hand, AT1b [34] and AT2 [35, 36] null mutant mice showed no vascular abnormalities. These facts suggest that Ang II acts on VSMCs mainly through AT1a and that AT1b compensates for the lack of AT1a signaling on the VSMCs. The compensatory effect of AT1b might be supported by the physiological study that the blood pressure in hypotensive AT 1a null mutant mice is slightly increased by exogeneous Ang II [37]. The present observations also showed that perfusion fixation and electron microscopic observations are necessary for evaluation of vascular alterations. In vascular samples processed without perfusion fixation, the vascular lumina are frequently collapsed, and the inner elastic lamina is folded due to contraction of VSMCs postmortem. Therefore, values of the parameters of vascular alterations, including wall thickness, inner diameter, and wall thickness ratio, are variable depending on whether the mice are perfused. Perfusion fixation operated at optimum pressure prevents collapse of vascular lumina by counteracting the contracting force by VSMCs. Visualization of the major mechanical components of the vascular wall, namely, cytoskeleton and extracellular matrices, including elastic fibers, have been remarkably improved by the cold-dehydration technique for electron microscopy [18]. We conclude that the lack of AT1a signaling causes structural abnormalities in the renal vascular system and transforms the phenotype of VSMCs into cell proliferation, induces the escape of VSMCs from the circular

mechanical integrity, and results in increased synthesis of extracellular matrices. ACKNOWLEDGMENTS The supply of angiotensinogen-deficient mice from Dr. M. Kihara and Dr. S. Umemura (Department of Internal Medicine II, Yokohama City University School of Medicine, Japan) is appreciated. We thank Dr. I. Shirato for helpful advice and Dr. K. Koizumi, Mr. K. Igarashi, Mr. M. Yoshida, Mr. K. Sato, Mr. J. Nakamoto, and Ms. Y. Kojima for their skillful technical assistance. Reprint requests to Dr. Sachiko Inokuchi, Department of Anatomy, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: [email protected]

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