Evidence for the presence of early vascular lesions in newborn Watanabe heritable hyperlipidemic (WHHL) rabbits

Atherosclerosis, 101 (1993) 11-24 0 1993 Elsevier Scientific Publishers

17 Ireland, Ltd. All rights reserved. 0021-9150/93/%06.00

Printed and Published in Ireland ATHERO 05033

Evidence for the presence of early vascular lesions in newborn Watanabe heritable hyperlipidemic (WHHL) rabbits G. Aliev”, A. Mironova, R. Cirillob, A. Mironov Jr.a, E. Gorelovaa and M. Prosdocimib

“Department of Electron Microscopy, AS. Bubnov Ivanovo State Medical Institute, Engelsa Av., 8 153000 Ivanovo (Russia) and bDepartment of Vascular Biology, FIDIA Research Laboratories. Via Ponte della Fabbrica. 3/A 35031 Abano Terme (PD) (Italy)

(Received 24 August, 1992) (Revised, received 28 January, 1993) (Accepted 10 February, 1993)

We have investigated the morphology of the aortic wall of newborn New Zealand White (NZW) (n = 10) and newborn Watanabe heritable hyperlipidemic (WHHL) (n = 10) rabbits. In both strains, lipid levels (cholesterol and triglycerides) were elevated above the concentrations expected. This was particularly evident in WHHL. The morphology of the aortas of NZW rabbits suggested an intensive biosynthetic and bioenergetic activity of endothelium. This was most evident in areas where blood flow underwent division. No major abnormalities were noted in the endothelium or subendothelium. In newborn WHHL rabbits, leucocyte adhesion (usually monocytes) to endothelium and migration into the subendothelium was apparent, particularly on the aortic arch and around areas of dividing blood flow in the thoracic aorta. Tuberous raised structures were present in low numbers and distributed randomly on the aortic wall. Endothelial cells had elevated nuclear zones projecting into the vessel lumen. At regions of blood flow division, endothelium was polygonal in shape and silver staining of cell borders was more intense. Fatty streaks were present at blood flow divisions and micro-plaque was seen. Transmission electron microscopy of fatty streak-like areas showed the presence of up to two layers of smooth muscle cells and in some areas, lipidladen macrophages were seen. The presence of atherosclerotic lesions in newborn WHHL rabbits suggests that the process may commence in utero. Key words: Atherosclerosis;

Embryogenesis; WHHL rabbits; Aortic lesions

Introduction Correspondence to: Dr. Gjumrakch Aliev, Department of Vas-

cular Biology, FIDIA Research Laboratories, Via Ponte della Fabbrica, 3/a, 35031 Abano Tenne (PD), Italy. Fax: 049/810927.

Atherosclerosis is conventionally associated the process of aging except in situations where genetic hyperlipidemia exists [ 1,2]. The factors which promote development of atherosclerowith

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sis in man include: turbulent blood flow, hypertension, metabolic disturbances, immune responses, exogenous toxins, such as nicotine, and obturation of adventitial lymphatic vessels [3]. In hypercholesterolemia, atherosclerotic lesions have been demonstrated in children and teenagers [ 1,4]. Furthermore, a clear relationship exists between advancing age and atherosclerosis. Rabbits with heritable hypercholesterolemia, related to LDL receptor deficiency, develop lesions which resemble atherosclerosis in man [5,6]. During embryogenesis there is evidence that developing hemodynamic tensions may promote vascular reorganisation and changes in the mechanical properties of the large arteries [7]. It is therefore conceivable that hemodynamic tension could initiate atherosclerotic lesions at these early stages. We have studied the morphology of the proximal regions of the aortic wall in newborn Watanabe heritable hyperlipidemic (WHHL) rabbits to examine the possibility that atherosclerosis may be present at birth. A group of newborn/New Zealand white rabbits was also included in the study. Materials and Metbods Our colony of WHHL rabbit, raised from animals kindly provided by Prof. Y. Watanabe (Kobe University, Japan) has been previously described R&11]. For the present study, pregnant normal and WHHL female received an ad libitum daily supply of a diet containing. 13.5% crude protein, 2.5% crude fat, 21.5% crude fiber and 1.200 Cal/g of metabolic energy (Diet 2RB15, Mucedola srl, Milan, Italy). The experiments were performed using 10 Watanabe heritable hyperlipidemic (WHHL) rabbits (1 day old) and 10 New Zealand White (NZW) newborn rabbits as controls. The rabbits were anesthetised (pentobarbital 30 mg/kg i.p.) and the left ventricle of the heart was cannulated using a probe with an external collar to prevent its ejection from the ventricle during the subsequent process of perfusion at 60 mmHg. Blood samples were collected from the left ventricle for measurement of plasma triglycerides, HDL and total cholesterol. The perfusion fluid consisted of medium 199 (Sigma Co., St. Louis, MO) containing 0.6% (w/v)

dextran (mol. wt. 6000 approx.) and heparin (10 I.U./ml). Perfusion took place for 60 s at 37°C after which glutaraldehyde (final cont. 2.5% (v/v) in medium 199, pH 7.4) was perfused for 5 min. Immediately afterwards the entire aorta was removed and fixed in fixative (as above) for 2 h at 4°C. Subsequently,) samples for transmission electron microscopy were excised and posttixed for at least a further 2 h. Sections were taken from the ascending and descending parts of aortic arch, from the initial and the terminal regions of the thoracic aorta, from the infrarenal parts and from the iliac bifurcation of the abdominal aorta. For scanning electron microscopy (SEM) analysis, postfixation was performed (after washing the tissues in medium 199) by incubation of the tissue in 1% (w/v) osmium tetroxide in water at 4°C for 1 h followed by washing (water) and transfer to 1% (w/v) tanninic acid for a further 30 min. After a further brief wash, samples were fixed in 1% osmium tetroxide for 30 min. Finally the samples were dehydrated in graded concentrations of ethanol and critical-point dried. The samples were then split along the longitudinal axis using a razor blade and sputtered with gold or platinum in an EICO-III sputtering machine. Microscopic studies were performed using a Hitachi S-570 or a Philips SEM-505 electron microscope operating at 20-30 kV. The endothelial cell boards were stained after glutaraldehyde fixation by perfusion with 5.5% (w/v) glucose for 1 min at 37°C followed by perfusion with: (a) 0.15% (w/v) AgN03 for 1 min; (b) 5.5% (w/v) glucose for 1 min; (c) 1% (w/v) NH4Br and 3% (w/v) CoBrt for 1 min; and (d) glucose 5.5% for 1 min [12]. Finally, the animals were perfused again with glutaraldehyde prior to removal of vessels. For transmission electron microscopy (TEM) the specimens were washed with medium 199, postfixed for 60 min, washed again in medium 199 and post-fixed for 60 min in 1% osmium tetroxide in cacodylate buffer (pH 7.4; 4°C). After washing, the samples were dried in graded concentrations of ethanol and finally embedded in epoxy resin. In the intercostal branches, samples were selected from regions where scanning electron microscopy revealed atherosclerotic lesions. Semithin sections for light microscopy were stained with 1% (w/v) toluidine blue and examined using a light microscope (Olympus AN-2). Thin sections

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were stained with 3% (w/v) uranyl acetate and Reynold’s lead citrate and observed using a Philips EM 400 operating at 80 kV. Statistical analysis

Data are means f S.E.M. Statistic differences were assessed by using the unpaired Student’s ttest. A p-value of less than 0.05 was considered significant. Results

TABLE 1 PLASMA LIPID CONCENTRATIONS IN NORMAL NEWBORN NZW AND IN NEWBORN WHHL RABBITS Serum lipid parameters (mg/dl)

NZW (n = 10)

WHHL (n = IO)

Total cholesterol Triglycerides HDL cholesterol

228.6 f 18.6 209.4 + 24.6 33.6 f 5.9

766.5 f 119..5* 652.2 f 138.1; 44.3 It 6.8

*P < 0.05 vs. NZW rabbits. All the data are expressed as mean * S.E.M.

Plasma lipids

Plasma lipid levels are shown in Table 1. In newborn NZW rabbits total cholesterol, triglycerides and HDL cholesterol were 228.6 i 18.6, 209.4 i 24.6 and 33.6 f 5.9 mg/dl, respectively. In newborn WHHL rabbits total cholesterol, triglycerides and HDL cholesterol were 766.5 f 119.5 (P < 0.05), 652.2 f 138.1 (P < 0.05) and 44.3 f 6.8, respectively. Electron microscopy observations

In NZW rabbits, the luminal surface of the aortic wall and, in particular, the aortic arch had a random relief and a characteristic ‘ebb and lee’ pattern. Other regions of the aortic endothelium were relatively flat and were more markedly spindle shaped. Some slightly raised areas were seen below regions of vessel branching (Fig. 1A). No other significant peculiarities were observed. TEM analysis of endothelial cells from newborn NZW rabbits showed a well developed granular endoplasmatic reticulum and a large number of mitochondria, indicating that a probable active state of protein synthesis, was present in the cell cytoplasm. The luminal surface of the aortic wall in newborn WHHL rabbits was characterized by the presence of a high number of adherent leucocytes, possibly monocytes. Some evidence of subendothelial migration was also noted (Fig. 1B). Below regions of blood flow division, internal bulges were noted (Fig. lC), occasionally tuberous in shape and randomly spread on the vessel wall (Fig. 1D). Occasionally, ovoid structures were seen (Fig. 1D). Endothelial cells had a prominent nuclear zone projecting into the vessel lumen (Figs. lE,F). At regions of blood flow division, endothe-

lial cells had a polygonal shape and a moresintense silver-like staining of the borders between cells was seen (Fig. 1G). The endothelium, particularly in the aortic arch, in the thoracic aortic ostia and abdominal aortic bifurcations of newborn WHHL rabbits, contained small polygonal or irregular cell forms with a sharply raised nuclear zone. Adherent leucocytes and fibrin threads were noted on the surface of every specimen from WHHL rabbit aorta (Fig. 1H). Raised areas, perpendicular to the axis of the vessel, were found on the lateral side of thoracic aorta (Fig. 11) and aortic arch (Figs. 2A,B). Endothelial cells of these raised areas were slightly smaller than elsewhere. Raised ovoid shapes were rare in the thoracic aorta and only occurred in areas of blood flow division (Fig. 2C), in particular at the intercostal branches in each animal. Endothelial cells Ibecame more distinctly spindle shaped in the more distal regions of the aorta which were examined, suggesting that regeneration was occurring (FCg. 2D). The endothelium covering these areas was flat and elongated and may have an increased microvilli expression suggesting the possibility of mitosis [ 13,141 (Fig. 2E). In zones surrounding the raised areas an increased number of surface-adherent cells were seen (Fig. 2F). Using transmission electron microscopy, dense concentrations of mitochondria and ribosomes were seen (Fig. 3C). Sections of raised areas (aortic arch and thoracic ostia - Figs. 3A,B) showed an underlying presence of l-2 layers of smooth muscle cells (Fig. 3D), or occasionally lipid-laden macrophages (Fig. 3E).

Fig. 1. Morphological characterization of the luminal surface of segments from the aorta of newborn NZW and WHHL rabbits. (A) Thoracic aorta. Intercostal branch (NZW) showing intimal creases (t 1).Some adherent erythrocytes (I) (SEM x 250). (B) Aortic arch of newborn WHHL rabbits showing irregular orientation of endothelium which have many microvilh (1). Some adherent leucocytes over endothelial margins (t 1). Some fibrin (t t t) (SEM x 820). (C) Aortic arch of newborn WHHL rabbits. Early plaque-like structures (*); some adherent leucocytes which may be migrating under endothelium (1) (SEM x 1200). (D) Thoracic aorta (WHHL) in the region of intercostal branch. Early raised structure protuding sharply into the vessel lumen (*) (SEM x 1200). (E) Aortic arch showing irregular pattern of endothelium and polygonal endothelium with adhesion and migration of leucocytes (1). Spontaneous de-endothelization areas (*) (SEM x350). (F) As above under a larger magnification showing leucocytes in margin the deendothelization area (1) (SEM X 1700). (G) Silver staining of WHHL thoracic aorta in the intercostal branch area. Note the association of leucocytes with silver-stained areas (1) (SEM-silver staining x 680). (H) Thoracic aorta (WHHL) showing leucocyte migration into the vessel wall (*) @EM x 5000). (I) Fatty-streak in the thoracic aorta of newborn WHHL rabbits (*). Endothelium has polygonal pattern (SEM x 700).

Fig. 2. Changes in endothelial morphology in the aortic wall of newborn WHHL rabbits. (A-B) Thoracic aorta. Evidence for tuberous wave structures at the branches of the intercostal arteries (*) (SEM, A x 80; B x 200). (C) Thoracic aorta showing fatty cells (t t) migrating towards vessel wall (SEM x 3540). (D) Thoracic aorta showing presence of a large number of endothelial cells with microvilli on their surface suggesting active cell division (SEM x 800). (E) Thoracic aorta. Terminal parts of intercostal branches showing endothelium which may be in division (*) (SEM x 2000). (F) Adhesion and migration of leucocytes in the thoracic aorta (1). Endothelium have sharply protruding nuclear zones (*) (SEM x 1890).

Fig. 3. Ultrastructural characterization of the aortic wall in newborn WHHL rabbits. (A) Fatty streak zone in aortic wall of thoracic aorta (*) (SEM x 2000). (B) Same areas in semithin section. See the wave characteristic of intima (1). (Toluidine blue staining; LM x 112). (C) Aortic arch. Evidence for synthetic phenotype endothelial cells. Accumulation of amorphous structures in the subendothelial layer (I). (TEM x 24 600). (D) Thoracic aorta. Sample selected from a fatty streak area showing synthetic type SMC in the subendothelial area (f = gold layer). (TEM x 20 600). Thin section samples were selected after SEM examination. (E) Aortic arch. Macrophage in subendothelium. Lipid accumulation (*) and myelin-like structures in the cytoplasm (**) (TEM x 21 000). EC = endothelial cell; SMC = smooth muscle cell; VL = vessel lumen.

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Discussion Elevated levels of cholesterol and triglyceride were seen in both groups of newborn rabbits. In newborn NZW rabbits, total cholesterol and triglyceride levels were approximately 5 and 2 times, respectively, higher than those observed in adults (approx. 3 months old). To our knowledge, no published paper reported similar observations and we think that these values may be normal in young animals. Furthermore, an intact vascular morphology of newborn NZW rabbits was seen despite high lipid levels. Newborn WHHL rabbits had very high values of total cholesterol and triglycerides. The values were higher than those expected in normal adult NZW rabbits or those observed in newborn NZW. We did not measure foetal levels of lipids or cholesterol but, since elevations take some time to occur, it is reasonable to suggest that elevated blood lipid levels existed before birth in the NZW strain and that these were greatly elevated in the WHHL rabbits. Experiments with diet-induced hypercholesterolemia have shown that stable elevation of cholesterol and triglyceride in serum does not occur rapidly [15-181. Careful morphological analysis has revealed that in normal vessels of NZW rabbits there is evidence for division and growth of endothelial cells as seen by the relatively random surface pattern, small square cells, prominent nuclei and the presence, at subcellular levels, of abdundant ribosomes and mitochondria. In the WHHL newborn subjected to the same observation as newborn NZW, we saw an increased adhesion of leucocytes, probably monocytes, specific wave-like structures and fatty streaks mainly associated with blood flow branches where some areas of turbulence or stasis could occur [7]. In another study [19], it has been shown that the initial stages in the development of atherosclerosis involve leucocyte adhesion, mural deposition of lipids and formation of fatty streaks. Our study has shown that in newborn animals with heritable hypercholesterolemia this process has already started. Since it can take 12 days of high cholesterol diet to induce these changes in adult NZW rabbits it is probable that the process of atherosclerosis in WHHL rabbits begins in utero.

Previously, it has been suggested that loss of endothelium is the most important initiating factor in atherosclerotic lesions [ 1,201. However, more recently, it has been suggested that an earlier parameter may be a morphological and functional change in endothelial cells [21-251. Consistent with the latter observations, in this study we have described lesions which occur in almost exactly the same regions of aorta in which others have recorded atherosclerotic injury in WHHL rabbits at 3 months and later [4-6,10,11,26-301. In rabbits and in man the development of atherosclerosis is most evident at areas of blood flow division usually under areas of low flow or at points of relative stagnation of blood [l l] and not in areas of high hemodynamic stress as previously suggested [ 111. Resistance to atherosclerosis may exist at points of bifurcation since smooth muscle in that region has the so-called synthetic phenotype which can transform to contractile cells after collagen synthesis [311. In summary, we have described the presence of early vascular lesions in newborn WHHL rabbits which were most evident in the proximal parts of the aorta and at places of blood flow division. Furthermore, these findings indicate that studies on prevention of lesion in WHHL rabbits should begin very early and suggest that, in selected patients with familial LDL receptor deficiehcy, an early treatment of dyslipidemia should be considered. Acknowledgments We are indebted to Mr. Edward J. Hornby for helpful comments, to Dr. Emanuela Salvatico for plasma lipid determination and to Fidia Animal Care staff for providing NWZ and WHHL newborn rabbits. References Ross, R., The pathogenesis of atherosclerosis. An update, N. Engl. J. Med., 20 (1986) 488. Ross, R., Mechanisms of atherosclerosis. A review, Adv. Nephrol., 19 (1990) 79. Constandinides, P., The role of arterial wall injury in atherogenesis, Zentralbl. Allg. Pathol. Anat., 5 (1989) 517. Buja, L.M., Clubb, F.J., Bilheimer, D.W. and Willerson,

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