We studied the metabolic behavior of exogenous and endogenous cholesterol carrying lipoproteins in vivo.
Normal and WHHL-rabbits were given intravenous injection of chylomicrons labeled either in the lipid portion with [3H] cholesterol or in the protein portion with [¹²⁵I] iodine. All radiolabeled components were removed from plasma at comparable rates and the bulk of the radioactivity was found in the liver in both genotypes. It suggests that liverr has a specific chylomicron remnants receptor.
Normal and WHHL-rabbits were given intravenous injection of ¹²⁵I-LDL and VLDL. The FCR for LDL in WHHL-rabbit was about one third the normal rate. That could not account for 20-fold increase of LDL in this rabbit. When ¹²⁵I-VLDL was injected to normal rabbit, it was converted to IDL and more than 90% of IDL was taken up by the liver rapidly. On the contrary, once VLDL was converted to IDL in WHHL-rabbit, it failed to enter the liver for catabolism, subsequently converted to LDL. These data suggest that endogenous lipoproteins require LDL receptor for their catabolism, and decreasing amounts of hepatic LDL receptors lead not only to impaired catabolism but to overproduction of LDL by the shunt mechanism.

View full abstract

The Watanabe Heritable Hyperlipidemic (WHHL) rabbit has been reported to be an animal model for familial hypercholesterolemia (FH) because both WHHL rabbits and FH patients show spontaneous hyperlipidemia due to low density lipoprotein (LDL) -receptor deficiency. Rabbits fed on cholesterol-rich chow have been reported to show suppressed level of LDL-receptor. We wished to clarify the resemblances and differences between WHHL rabbits and FH patients by comparing their lipoprotein profiles.
We obtained blood samples after overnight fasting from 16 normal (TC<230 mg/dl, TG<150 mg/dl) human (“NH”), from 15 patients with heterozygous FH, from 8 normal Japanese white rabbits (“NR”) on standard rabbit chow, from 4 Japanese white rabbits fed a 1% cholesterol chow (“CR”), and 8 WHHL rabbits fed on standard chow. We enzymatically measured lipid levels of serum and lipoprotein fractions (VLDL, very low density (d<1.006) lipoprotein; IDL, intermediate density (1.006<d<1.019) lipoprotein; LDL, low density (1.019<d<1.063) lipoprotein; HDL, high density (d>1.063) lipoprotein) . Serum apolipoprotein levels were measured by SRID method. Rabbit VLDL was washed and delipidated by aceton-ethanol and ether. ApoVLDL was electrophoresed in polyacrylamide gels containing 8M urea and 2% ampholine (pH 4-6) . The protein was fixed, stained, and destained, and the gels were scanned at 550 nm. We checked the isoprotein pattern of rabbit apoVLDL by measuring areas under each peaks of scanning chart.
Levels of VLDL-, IDL-, and LDL-TC elevated in FH patients compared with NH subjects, by 1.8, 1.0, and 2.6 times, respectively. WHHL rabbits showed increased levels of these lipoprotein TC compared with NR rabbits, by 26.2, 15.0, and 18.4 times, respectively. CR rabbits also showed elevated levels of lipoprotein TC by 13.4-20.0 times. The triglyceride (TG) levels of VLDL, IDL, and LDL fractions were higher in WHHL rabbits than in NR rabbits, by 8.0-12.8 times. Whereas, the TG levels of CR rabbits and FH patients showed no increase compared with normal controls. The TC/TG ratios of VLDL and IDL fractions were slightly increased in WHHL rabbits to the same degree as in FH patients. The TC/TG ratio of LDL fraction was normal. In CR rabbits. TC/TG ratio of VLDL, IDL, and LDL fraction elevated markedly by above 20 times.
The serum levels of apolipoprotein B (apoB) and apolipoprotein E (apoE) in FH patients were significantly higher than in NH subjects, by 2.0 and 1.5 times, respectively. WHHL rabbits showed higher apoB (1, 420±224 mg/dl) and apoE (52±9mg/dl) levels than NR rabbits (100±27 mg/dl and undetectable level, respectively) .
WHHL rabbits showed very high VLDL-TC level and resembled human type 3 hyperlipidemia, which was known to show elevated level of VLDL and abnormal isoprotein pattern of apoE. Therefore, we checked isoprotein pattern of apoVLDL of WHHL rabbits. There were no changes in isoprotein pattern of apoVLDL in WHHL rabbits compared with NR rabbits. There were some changes of isoprotein pattern in CR rabbits.
We summarized: 1) WHHL rabbits and FH patients showed two common characteristics; increases of slightly TC-rich VLDL and IDL and of LDL: elevated levels of apoB and apoE. 2) The increments of lipoprotein TC levels in WHHL rabbits were above 10 times, whereas those in FH patients were around twice. 3) All WHHL rabbits showed increased levels of VLDL, whereas a few number of patients with FH showed elevation of VLDL. 4) The CR rabbit was different from FH in showing elevation of markedly TC-rich lipoprotein and some changes in isoprotein pattern of apoVLDL. We conclude that the WHHL rabbit is an excellent animal model for FH.

View full abstract

The interactions between platelets and vascular walls have special significance for atherosclerotic heart disease. The aims of this study were to determine whether atherosclerosis is associated with altered vascular reactivity to aggregating platelets and to elucidate the responsible underlying mechanisms. Isolated helical strips of aortae and coronary arteries from age-matched control and Watanabe Heritable Hyperlipidemic rabbits (WHHL rabbits) (average age=10 months for aortic study and 22 months for coronary study) were mounted for isometric tension recording in oxygenated buffer. All the segments of aortae and coronary arteries from WHHL rabbits showed atherosclerotic lesions macroscopically. Cumulative additions of suspended platelets (10⁴-3×10⁶/μl) activated with thrombin into the organ bath resulted in platelet count-related aortic contractions. The maximum serotonin (5HT) and thromboxane (TX) B₂ concentration in the bath solution averaged 148 ng/ml and 24 ng/ml. Contractile responses of atherosclerotic aortae to aggregating platelets were markedly augmented as compared to controls. An incubation of platelets with aspirin, which completely inhibited TX A₂ synthesis without any alterations of 5HT release, did not attenuate hyperreactivity of atherosclerotic aortae compared to controls. Prior exposure to phentolamine or diphenhydramine also did not alter contractile responses to aggregating platelets. The treatment of aortic strips with methysergide inhibited responses to aggregating platelets in both atherosclerotic and control groups, indicating a serotonergic mechanism of its hyperreactivity. To further elucidate its responsible mechanism, contractile responses to exogenously administered 5HT, histamine and TX A₂ were examined in aortic strips from control and WHHL rabbits. Responsiveness of atherosclerotic aortae to 5HT was significantly augmented compared to controls. There were no differences in the responses to histamine between two groups. Atherosclerotic aortae exhibited impaired responses to TX A₂ prepared by incubation of PG H₂ with calf platelet microsomes or ST A₂, a stable TX-like analogue, as compared to controls. These results also can help to explain the mechanisms responsible for hyperreactivity of atherosclerotic aortae to aggregating platelets.
In addition, atherosclerotic coronary arteries exhibited a markedly enhanced reactivity to aggregating platelets and 5HT.

View full abstract

It is well known that cholesterol ester accumulate in atherosclerotic arterial wall. In this paper, two monoclonal antibodies against lipid laden cells (FCR1a/201F) and extracellular matrix where lipid deposited (EMRIa/212D) are discussed.
FCRIa/201F recognized lipid laden cells from both Watanabe-Heritable Hyperlipidemic rabbit and human atherosclerotic aorta. The cells probably both macrophages and modified smooth muscle cells are transformed to lipid laden cells during accumulation of cholesterol ester. EMRIa/212D recognized extracellular matrix where lipid deposited. Antigenic substances against this antibody must be glycoprotein, the epitope of which is sialic acid.
These evidences obtained from experimental atherosclerotis (WHHL rabbit) may help understanding how lipid accumulate in arterial wall in human patient.

View full abstract

The aim of this experiment was to study the effects of atherosclerosis on the cardiovascular hemodynamics in conscious condition and in response to hemorrhagic hypotension. Arterial pressure was monitored, via a catheter chronically inserted into the aortic arch, for six hours in seven normal and eight WHHL (Watanabe-Heritable Hyperlipidemic) conscious rabbits, sampled at one sec intervals using AD-converter and stored in a digital computer. Percentage frequency distribution curve, mean and standard deviation of the mean arterial pressure for six hours (21, 600 data points) were calculated for each string of pressure data. Mean and standard deviation were pooled for the seven normal rabbits and the eight WHHL rabbits, respectively. Mean and standard deviation of mean values were 117±10 mmHg in the WHHL rabbits, and 87±7 mmHg in the normal rabbits. They showed significant difference (p<0.05) . Mean and standard deviation of standard deviations were 9.5±1.8 mmHg in the WHHL rabbits and 6.5±1.4 mmHg in the normal rabbits. Difference in mean standard deviation between the normal and WHHL rabbits was significant (p<0.05) . Thus, the WHHL rabbits were slightly but significantly hypertensive and their systemic pressure was less controlled. This suggests that the pressure control capacity of the rapidly acting arterial pressure control system, i.e., the baroreflex system, deteriorates. In order to quantify the capacity of the rapidly acting arterial pressure control system, the open-loop gain (G) of the system was estimated in fifteen WHHL rabbits, aged 3 to 31 months. Twenty-five normal Japanese white rabbits, aged 6 to 30 months, were used as a control. After being anesthetized by intravenous injection of Nembutal, the rabbits were bled by 2 ml /kg body weight within 1 to 2 sec through a catheter inserted into the aortic arch. The arterial pressure change after this quick hemorrhage was monitored via a catheter placed in the aortic arch for more than 2 min. G was calculated as (ΔAP_I/ΔAP_s) -1, where dAP_I was the immediate fall and ΔAP_s the steady-state fall. The pulse pressure, ΔAP_I and ΔAP_s increased and G decreased with aging in the WHHL rabbit. These results indicate that the progress of atherosclerosis with aging in the WHHL rabbit decreases the vascular compliance and impairs the arterial pressure restoring function of the rapidly acting arterial pressure control system.

View full abstract

By means of a tensile testing instrument (MINEBEA, TOM 30J), tension-strain curves, stress-strain curves and stress relaxation curves were measured of circumferential strips excised from the proximal portion of the thoracic aorta in normal and WHHL rabbits aged from 8 to 30 months. Areas of atheromatous plaques on the intimal surface of the aorta in WHHL rabbits were measured using a area computing equipment (Planimex-25, Nihon Regulator) .
Tension-strain curves were concave toward the tension-axis, indicating a decrease in wall distensibility with increasing strain. The wall distensibility of atherosclerotic aortas was lower than that of normal aortas and progressively decreased with advancing age. A positive relationship was obtained between wall thickness and age with the correlation coefficient of 0.66 and also between wall thickness and area ratio of the atherosclerotic lesion with the correlation coefficient of 0.77. In the old age group of WHHL rabbits, aortic walls showed fairly high elastic moduli calculated from the stress-strain curves. In the young age group, however, elastic moduli of the aortic walls decreased with increasing wall thickness. These results led us to the following speculations. The decreased distensibility of the atherosclerotic wall may be attributed mainly to an increase in wall thickness caused by an intimal thickening. In advanced atherosclerotic aortas, wall stiffness explained by an increase in elastic modulus may be an additional factor of the increased distensibility.
Normal and atherosclerotic aortas exhibited some amount of stress relaxation curing the experimental period of five minutes, the degree of stress relaxation being smaller in the former than in the latter. There was a positive relationship between the degree of stress relaxation and wall thickness with the correlation coefficient of 0.58 for the aortas in WHHL rabbits aged 8 months. This may be mainly due to the increased viscoelasticity of the thickened intima associated with the proliferation of foam cells and the deposition of lipids.

View full abstract