Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II.

The concentration of high density lipoproteins (HDL) is inversely related to the risk of atherosclerosis. The two major protein components of HDL are apolipoprotein (apo) A-I and apoA-II. To study the role of apoA-II in lipoprotein metabolism and atherosclerosis, we have developed three lines of C57BL/6 transgenic mice expressing human apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed that human apoA-II mRNA was present only in the liver of transgenic mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma of transgenic mice. After 3 months on a regular chow, the plasma concentrations of human apoA-II were 21 +/- 4 mg/dl in the 25.3 line, 51 +/- 6 mg/dl in the 21.5 line, and 74 +/- 4 mg/dl in the 11.1 line. The concentration of cholesterol in plasma was significantly lower in transgenic mice than in control mice because of a decrease in HDL cholesterol that was greatest in the line that expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the plasma concentration of mouse apoA-I (32 +/- 2, 56 +/- 9, 91 +/- 7, and 111 +/- 2 mg/dl for lines 11.1, 21.5, 25.3, and control mice, respectively) that was inversely correlated with the amount of human apoA-II expressed. Additional changes in plasma lipid/lipoprotein profile noted in line 11.1 that expressed the highest level of human apoA-II include elevated triglyceride, increased proportion of total plasma, and HDL free cholesterol and a marked (>10-fold) reduction in mouse apoA-II. Total endogenous plasma lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a level directly correlated with the degree of increased plasma human apoA-II in the transgenic lines. LCAT activity toward exogenous substrate was, however, only slightly decreased. The biochemical changes in the 11.1 line, which is markedly deficient in plasma apoA-I, an activator for LCAT, are reminiscent of those in patients with partial LCAT deficiency. Feeding the transgenic mice a high fat, high cholesterol diet maintained the mouse apoA-I concentration at a normal level (69 +/- 14 mg/dl in line 11.1 compared with 71 +/- 6 mg/dl in nontransgenic controls) and prevented the appearance of HDL deficiency. All this happened in the presence of a persistently high plasma human apoA-II (96 +/- 14 mg/dl). Paradoxical HDL elevation by high fat diets has been observed in humans and is reproduced in human apoA-II overexpressing transgenic mice but not in control mice. Finally, HDL size and morphology varied substantially in the three transgenic lines, indicating the importance of apoA-II concentration in the modulation of HDL formation. The LCAT and HDL deficiencies observed in this study indicate that apoA-II plays a dynamic role in the regulation of plasma HDL metabolism.

Prenatal development of the rat sinuatrial node.

In order to study the origin and mode of differentiation of the cells which make up the sinuatrial node, samples of the sinuatrial junction of rat embryos of different ages were studied by transmission electron microscopy. From a seemingly morphologically homogeneous cell population at 11 days, an ultrastructural differentiation occurs from day 12. So, one could see: a) irregular-shaped cells with dark nucleus and medium-sized contractile apparatus which we have identified as ordinary ("working") atrial myocardiocytes and, b) pale cells with a clear spheroidal nucleus and cytoplasm containing few organelles and fine myofibrils which we have classified as nodal cells. Numerous undifferentiated cells of intermediate morphology appear intermingled with nodal and ordinary cells. Throughout development, nodal and ordinary cells progressively enhanced their mutual differences. Ordinary myocardiocytes become increasingly rich in myofibrils and mitochondria, and nodal cells contain scanty organelles and fine myofibrils, whereas undifferentiated cells are few at every stage. At the end of prenatal life, the sinuatrial node shows numerous unmyelinated axons of immature aspect but direct contacts between nodal cells and nerve fibers are not seen. The images we obtained suggest that the sinuatrial node must not be taken as an embryological remnant. Nodal cells are recognized from the 12th day of embryonic life as a particular form of differentiation of the cells which make up the sinuatrial region at the preceeding stages.

Presence or absence of glycogen-beta particles in sinoatrial node cells.

The sinoatrial node is formed by specialized cells, the main ultrastructural differences of which, as compared with ordinary atrial myocardium, are a pale cytoplasm and sparse myofibrils. Moreover, nodal cells have been described to contain large amounts of glycogen particles in their pale cytosol; these glycogen inclusions are often used as the main criterion for identifying nodal cells. Nevertheless, the presence of glycogen cytosolic inclusions has been discussed by several authors. This paradox was solved by the description of some undesirable effects of uranyl acetate when used en bloc. To prove the presence of glycogen granules in nodal cells and the effects of uranyl acetate en bloc, we performed an ultrastructural study of the sinoatrial node in rats of different ages using different staining techniques. Our results do not reveal any beta-particles in nodal cells in tissues processed by either general or glycogen-specific methods. Uranyl acetate staining did not cause any change of appearance in the nodal or ordinary myocardium. From these results, one could conclude that, on the one hand, sinoatrial nodal cells do not show deposits of beta-particles of glycogen which can be detected with ultrastructural techniques, and, on the other hand, that uranyl acetate does not cause any morphological artifacts.