Rapid transformation of [3H]cholesteryl ester in rat high-density lipoprotein: in vivo and in vitro studies.

[24,25-3H]Cholesteryl ester-labeled rat high-density and low-density lipoproteins were administered to recipient rats. Following death of the rats, a major portion of the radioactivity in administered [3H]cholesteryl ester-high-density lipoprotein rapidly appeared in less dense [3H]cholesteryl ester-lipoproteins and was isolated with the low-density lipoprotein fraction. The specific activity of the esterified cholesterol in the product lipoproteins found with the low-density lipoproteins exceeded that of the precursor high-density lipoproteins. In vitro, the addition of [3H]cholesteryl ester-high-density lipoprotein to plasma resulted in a five- to six-fold increase in radioactivity recovered in the low-density lipoprotein. These results demonstrate that, under a variety of experimental conditions, isolated high-density lipoprotein particles (both in vitro and in vivo) tend to become larger and less dense. Rapid changes in the density of lipoproteins labeled with [3H]cholesteryl ester must be considered when interpreting physiologic studies using this label.

Suppression of fibroblast proliferation in vitro and of myointimal hyperplasia in vivo by the triazolopyrimidine, Trapidil.

Platelet derived growth factor (PDGF) is a mitogen capable of stimulating the proliferation of both vascular smooth muscle cells (SMC) and fibroblasts in tissue culture. Recently, it was reported that this in vitro stimulatory effect is significantly reduced by Trapidil, a triazolopyrimidine. We confirm this, and also that similar inhibition occurs in vivo, in balloon catheter deendothelialized aorta of male, Sprague-Dawley rats treated with Trapidil. Two groups of rats, of 8 animals each, were treated daily with oral doses of either 45 mg or 90 mg Trapidil. A third group of 9 control animals were treated identically, but received only the diluent used to dissolve the drug. ADP or collagen-induced platelet aggregation, and endothelial cell regrowth were unaffected by Trapidil administration, but the degree of myointimal hyperplasia was significantly reduced in all animals receiving the drug. Thus, Trapidil seems to possess the selective ability to alter the SMC proliferative response which normally follows deendothelialization.

Selective arterial medial injury fails to produce intimal hyperplasia in experimental animals.

The hypothesis was tested, in rats and rabbits, that selective medial injury may lead to arterial intimal hyperplasia. Lesions were produced by passage of a micro-suture through the arterial wall without penetration to the lumen, or controls in which the suture did penetrate. Vessels were examined histologically at intervals up to 2 weeks after suturing. Only vessels with penetrating sutures developed intimal hyperplasia, suggesting that medial injury alone is insufficient for the production of arteriosclerosis.

Side-chain oxidation of lipoprotein-bound [24,25-3H]cholesterol in the rat: comparison of HDL and LDL and implications for bile acid synthesis.

The purpose of the study was to test the hypothesis that high density lipoprotein (HDL) cholesterol would be more easily oxidized in vivo than low density lipoprotein (LDL) cholesterol. Homologous plasma was incubated with [24,25-3H]cholesterol and fractionated by ultracentrifugation to obtain HDL and LDL each labeled with [3H] free sterol. HDL and LDL labeled with [24,25-3H]cholesteryl esters were prepared by ultracentrifugation of plasma from donor rats injected 24 hr previously with [24,25-3H]cholesterol in propylene glycol. These four labeled lipoproteins were administered to recipient rats. It was found that more tritium oxide (3H2O) was produced after the HDL doses than after the corresponding LDL doses, from 2--3-fold more when lipoprotein free cholesterol was labeled and from 2--6-fold more when lipoprotein cholesteryl esters were labeled. More 3H2O was produced from free cholesterol-labeled lipoproteins than from cholesteryl ester-labeled lipoproteins. Since oxidation of cholesterol is a measure of bile acid formation, it is concluded that under the conditions of the study HDL-cholesterol is a better precursor of bile acids than LDL-cholesterol.

The influence of the pituitary on arterial intimal proliferation in the rat.

The formation of arteriosclerotic fibromusculoelastic intimal thickening following arterial de-endothelialization is well documented. Recent findings, both in vitro and in vivo, suggest that platelets are a major participant in the pathogenesis of this lesion by releasing a mitogen to medial smooth muscle cells (SMC). This mitogen results in SMC migration to and proliferation within the intima. A similar mitogen has been described as originating in brain and pituitary tissue. We now report that, in hypophysectomized rats with normal platelet counts, intimal hyperplasia is markedly delayed; pair-fed intact controls normally develop lesions. It therefore appears that the pituitary gland plays a significant role in the experimental arteriosclerotic response.

The effect of thrombocytopenia on experimental arteriosclerotic lesion formation in rabbits. Smooth muscle cell proliferation and re-endothelialization.

This study was designed to investigate the mechanisms involved in fibromusculoelastic lesion formation produced by selective de-endothelialization by the intra-arterial balloon catheter technique in thrombocytopenic rabbits. Thrombocytopenia was induced and maintained for up to 30 days by daily injections fo highly specific sheep anti-rabbit platelet sera (APS). Evidence for re-endothelialization was obtained by i.v. Evans blue dye 30 min before sacrifice. Rabbits received daily injections of APS, which reduced the mean platelet count to 5,600/cm3; control animals received identically treated normal sheep sera on the same schedule, and had mean daily platelet counts of 363,000/cm3. Evaluation of intimal thickness was assessed by counting cell layers in semithin sections. Intimal thickening in aortae from rabbits treated with APS was strikingly suppressed, in contrast to those from normal sheep sera-treated animals which showed a mean intimal thickness of 18 cell layers within 28 days often after de-endothelialization. Re-endothelialization was not affected by APS treatment. These results indicate that the proliferation of smooth muscle cells is dramatically inhibited by reduction of platelets.

Intimal healing. The pattern of reendothelialization and intimal thickening.

Studies were undertaken to investigate further the basis of intimal proliferation and the identity of the surface lining cell in rabbit aortas subjected to extensive deendothelialization. Endothelial cells were selectively removed by passage of an inflated balloon catheter through the arterial lumen. The healing response was evaluated at intervals up to 36 weeks by several techniques: 1) permeability to Evans blue, 2) reappearance of endothelial cells as indicated by the specific marker, adsorbed goat anti-rabbit tissue factor-horseradish peroxidase, 3) planimetric measurements of intimal thickness, and 4) electron microscopy. The results indicate that endothelial cell recovery progressed slowly and that it extended only from areas spared denudation. The regions not covered by endothelial cells were lined by cells of smooth muscle cell origin. Such areas were permeable to Evans blue-protein complex, and their luminal smooth muscle cells were associated with connective tissue-like material at their luminal surface; this material apparently acted as a base for platelet accumulation. The present findings indicate that the lumen of the extensively denuded vessel is lined by either endothelial or smooth muscle cells and that intimal healing is related to restoration of endothelial cell cover. In addition, intimal thickening reached a maximum well before reendothelialization was complete.

Hydrocephalus: III. Reconstitution of the cerebral cortical mantle following ventricular shunting.

Adult cats with hydrocephalus were sacrificed at varying times following valveless ventricular shunting. This shunting resulted in a prompt reduction of ventricular size and rapid gross reconstitution of cortical mantle. Ultrastructurally it was evident that white matter edema persisted for many weeks, even in the presence of normal size ventricles. The areas most severely affected by the hydrocephalus such as the corpus callosum showed a paucity of myelinated fibers and their replacement by numerous reactive astrocytes. Reconstitution of the cortical mantle consists predominantly of a diminution of white matter edema and reactive astrocytosis. Clinical improvement undoubtly results from the functional improvement of remaining elements rather than from the replacement of lost elements. This scheme of hydrocephalus suggests that if hydrocephalus is relieved when only ependymal disruption and periventricular water accumulation have occurred, it may be reversible. The subsequent sequence of events consisting of axonal degeneration, myelin disruption and reactive astrocytosis may be less, if at all reversible.

Hydrocephalus: I. Histological and ultrastructural changes in the pre-shunted cortical mantle.

Hydrocephalus results in a reduction in the thickness of the cerebral cortical mantle which seemingly is restored by ventricular shunting. In order to determine the effect of hydrocephalus and its potential reversibility, experimental hydrocephalus was produced in adult cats by the introduction of kaolin or silicone into the basal cisterns followed by bilateral craniectomies. Microscopic and fine structural studies of these animals suggest the occurrence of significant focal axonal destruction. These studies suggest a sequence of events consisting of ventriculomegaly, disruption of the periventricular ependyma, periventricular edema, axonal destruction, secondary myelin disintegration and finally reactive astrocytosis. The damage to axons and meylin and resulting gliosis is irreversible.

Hydrocephalus: II. Cell number and size, and myelin content of the pre-shunted cerebral cortical mantle.

The number of cells in the brain and their size can be calculated by determining the total brain DNA, RNA and protein. Myelin can be determined biochemically. This analysis was applied to brains of a group of adult cats with craniectomies that were made severely hydrocephalilc by the introduction of kaolin or silicone into the basal cisterns. In severely hydrocephalic brains sectioned rostral to the area of kaolin inflammation there was no loss of dry weight. Wet weight increased slightly reflecting periventricular edema. There was a slight increase in total DNA, RNA and protein, presumably reflecting the increase in small inflammatory cells. There was a significant reduction in galactolipids reflecting myelin loss.

Reconstitution of the cerebral cortical mantle in shunt-corrected hydrocephalus.

The purpose of this study was to ascertain the sequence of events and the cellular constituents involved in reconstituting the cortical mantle after ventricular shunting. The subjects were severely hydrocephalic adult cats. After insertion of a shunt, the ventricular system rapidly returned to normal size and there was gross reconstitution of the cortical mantle. However, there still remained in the cortical mantle many of the histological changes seen before insertion of the shunt. The effect of hydrocephalus is mainly upon axons in the periventricular white matter. The axons become stretched and finally disrupted, resulting in disintegration of the surrounding myelin. In the absence of axons, remyelination cannot take place. It would seem, therefore, that prompt reversal of hydrocephalus is necessary in order to preserve the anatomical and functional integrity of the brain.