Adrenaline and noradrenaline as possible chemical mediators in the pathogenesis of arteriosclerosis.

We studied the relationships between adrenaline and noradrenaline and factors associated with arteriosclerosis to determine whether catecholamines contribute to the atherogenetic process. We investigated the effects of adrenaline and noradrenaline on cultures of vessel wall cells from rats and analyzed plasma catecholamine levels in humans exposed to atherogenic risk factors, undergoing hemodialysis treatment or following myocardial infarction or stroke. I. Cultured endothelial and smooth muscle cells from vessel walls exhibited enhanced proliferation when exposed to adrenaline or noradrenaline. This indicates that catecholamines trigger the activation of vascular wall cells in vitro. Such activation, the unspecific mesenchymal reaction, is the predominant characteristic change in early atherogenesis. II. In individuals subjected to the atherogenic risk factors smoking, essential hypertension and mental stress, plasma adrenaline concentrations were statistically significantly elevated. Mental stress also caused significantly elevated plasma noradrenaline levels. Plasma noradrenaline concentrations were also elevated in smoking and hypertensive individuals when compared with certain controls, but the differences failed to be statistically significant. III. In dialysis patients, plasma adrenaline and noradrenaline concentrations showed a positive correlation with the activity of the sclerotic process; i.e., plasma catecholamine concentrations increased with the severity of the disease. IV. Patients with persisting arteriosclerotic vascular disease, i.e., patients who had had a myocardial infarction or stroke, had significantly elevated plasma adrenaline and/or noradrenaline levels as late as one year after the event. The results of our investigations suggest that adrenaline and noradrenaline may act as chemical mediators during atherogenesis in man, thus contributing to the development and subsequent complications of arteriosclerosis.

A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis.

Cultured smooth muscle cells (SMC) from rat aorta and endothelial cells (EC) from pig aorta were used to study the effect of the catecholamines epinephrine and norepinephrine on cell proliferation. Both stimulated growth of SMC and EC when added to the culture medium. Besides epinephrine and norepinephrine, dopamine and some of their metabolites also stimulated proliferation of cultured endothelial cells. Smooth muscle cells originating from rats being exposed to atherosclerotic risk factors, like diabetes, hypertension and balloon-injury, exhibited an increased susceptibility to these catecholamines compared to SMC from control animals. In comparison to normotensive control animals a 10-fold elevated plasma concentration of epinephrine was found in hypertensive rats. In man plasma epinephrine and norepinephrine concentration was determined in a healthy control group and in patients suffering from diabetes mellitus and coronary artery disease. Plasma epinephrine and norepinephrine levels were similar in patients suffering from diabetes mellitus compared to the control group. But in patients with coronary artery disease significantly higher plasma concentrations for epinephrine (p less than 0.001) and norepinephrine (p less than 0.01) were observed. These data support the hypothesis that catecholamines may play a role in the development and subsequent complications of atherosclerosis.

[A single, rapid, selective and quantitative determination of adrenaline and noradrenaline in the plasma by a combination of solvent extraction, HPLC separation and electrochemical detection]. / Einfache, schnelle, selektive und quantitative Bestimmung von Adrenalin und Noradrenalin im Plasma durch Kombination von Flüssigextraktion, HPLC-Trennung und elektrochemischer Detektion.

A very simple and rapid solvent extraction system for the selective and quantitative isolation of epinephrine and norepinephrine from plasma is described. The extraction system makes use of the complex formation in alkaline medium between diphenylborate and the diol group of the catecholamines in combination with ion-pair formation. The extraction procedure, in conjunction with HPLC-separation and electrochemical detection, allows the quantitative determination of epinephrine and norepinephrine from plasma. This method is a specific as the commonly used adsorption on alumina, but has a much better recovery and even greater precision, especially for the quantitative determination of epinephrine.

Connective tissue proteins on the injured endothelium of the rat aorta.

Type V collagen (TVC), fibronectin (FN), and laminin (LAM) were detected on the endothelial surface of mechanically injured rat aortas with the help of monospecific antisera and protein A - gold conjugates, carbon film surface replicas, and conventional embedding techniques. Deendothelialized tracks were produced in the thoracic aorta, and the presence of the connective tissue proteins on the luminal surface of the endothelium was studied. The changes in the distribution of the proteins during repair of the endothelial surface was followed for up to 6 days after injury. From 1 to 3 days after injury small numbers of gold particles, indicating the presence of TVC, were found between the adherent platelets on the freshly deendothelialized subendothelial matrix and in higher amounts on cell debris and collagen fibers. On the sixth day after injury, however, the amount of TVC between the sparsely distributed platelets on the deendothelialized areas was significantly higher than it was previously. FN and LAM were readily detectable on the subendothelial matrix and on the damaged marginal endothelial cells. These proteins were especially obvious on both margins of the tracks even from the first day after treatment. FN was found also in connection with fibrin precipitations as well as on the surface of some platelets and monocytes. The amount of FN and LAM present on the damaged area decreased slightly up to the sixth day. Monocytes and leukocytes adhered mostly at the margin of the wound area in the vicinity of the lesions on the endothelium. FN and LAM were often detectable under and around these adherent cells. Little of the connective tissue proteins was found on the uninjured and on the regenerated endothelial cells. The results showed subtle transitory changes in the surface pattern of the subendothelial connective tissue matrix of the injured intima. The adhesion of blood-borne cells may have been induced by FN and LAM on the endothelial surface near the lesions, and later partly prevented by increasing amounts of TVC on the surface.

About the effect of risk factors on the structure and the proliferation of arterial wall cells. Part 1: Changes in structure and proliferation of cultivated aortic smooth muscle cells from hypertensive-diabetic rats.

In cultivated aortic smooth muscle cells (SMC) from normal or hypertensive-diabetic rats respectively, two types of cells, small SMC and large ones, were observed. The differences between the surface areas, form factors and nucleus-plasma relations of the small and the large cells, measured by morphometric methods are evident. The ratio of small to large cells originating from the normal group was 87%: 13%, and of those originating from the hypertensive-diabetic group 76%: 24%. The percentages of polynuclear cells from the normal group were 3.9% and from the hypertensive-diabetic group 6.1%. The small cells from hypertensive-diabetic rats proliferated faster (3H-thymidine labeling index 23.7%) than those from the control rats (3H-thymidine labeling index 12.9%). Their large cells showed on the contrary a trend to slower proliferation.

About the effect of risk factors on the structure and the proliferation of arterial wall cells. Part 2: Changes in structure and proliferation of cultivated aortic endothelial cells from hypertensive minipigs.

Cultured aortic endothelial cells (EC) from normotensive and hypertensive minipigs were studied in the second passage. Cytological measurements performed on confluent cultures of the same age in the second subculture revealed significant smaller surface areas and circumferences of EC and of their nuclei in the hypertensive group compared to those in the normotensive control group. The percentage of polynuclear EC was higher in cultures from hypertensive animals than in cultures from normotensive ones. EC derived from hypertensive minipigs differed from those of normotensive animals by a higher rate of proliferation as shown by 3H-thymidine labeling of the cells and by comparison of growth curves.

[Pathogenesis of coronary sclerosis]. / Pathogenese der Koronarsklerose.

It should first be pointed out that all arterial wall cells are mesenchymal cells, and that arteriosclerosis is a reactive disease of the arterial wall. A huge amount of different pathological factors is capable of inducing a reaction of arterial wall cells in the context of the "unspecific mesenchymal reaction". The reaction of the arterial wall cells to the influence of these factors is a biological process occurring as a complicated event of induction and reaction. The primary, necessary and fundamental pathological event in the process of atherogenesis is an alteration in the metabolism of the arterial wall cells induced by pathological risk factors. The direct result of these pathological alterations in the metabolism of the arterial wall cells are changes in structure and function of the entire arterial wall and the individual arterial wall cells (intimal lesion, hyalinosis, fibrosis, lipidosis, fibrinoses and thrombosis).

Cultivated human arterial smooth muscle cells. The effect of donor age, blood pressure, diabetes and smoking on in vitro cell growth.

The growth capacity and morphology of cultured human smooth muscle cells obtained from omental arteries from 153 patients were studied. The cells exhibited a very slow growth rate compared to rat or minipig SMC's, but they too developed cultures in a typical hill-and-valley pattern and possessed bundles of myofilaments. Two groups of smooth muscle cell cultures, slowly proliferating ones and rapidly proliferating ones were compared in their relation to donor age, blood pressure, nicotine abuse, diabetes mellitus and carcinoma. In the case of blood pressure and carcinoma significant differences indicated that those smooth muscle cells obtained from patients with high blood pressure or carcinoma revealed lower growth capacities. In the second subculture the proliferation of smooth muscle cells was found to be inversely proportional to donor age (4 to 78 years).

Special characteristics of cultured smooth muscle cell subtypes of hypertensive and diabetic rats.

In rat smooth muscle cell cultures two types of cells were detected. The majority of cells were small and spindle-shaped, 15% of which were labeled with H3-thymidine and displayed a nucleus/plasma relation of 1:15. Thirteen percent of the smooth muscle cell population was large and partly polynuclear. The H3-thymidine labeling was 1.3% in these rounder cells, and the nucleus/plasma ratio was 1:44. The surface area and cell shape of both types of cells were quantified morphometrically. Diabetes was induced in a group of animals by streptozotocin and hypertension by cellophane perinephritis. Data on smooth muscle cells (SMC) in culture obtained from diabetic and hypertensive rats differed from those of the controls. The percentage of small cells was significantly decreased, while the H3-thymidine index was increased in both diabetic and hypertensive rats. The number of large and polynuclear cells increased relative to the small cells, and they also displayed a higher proliferation rate as well as a change in their nucleus/plasma ratio. Another group of diabetic animals was treated with acetylsalicylic acid prior to SMC cultivation. Acetylsalicylic acid prevented the majority of changes found in cultured SMC of diabetic rats.

[Physiology and physiopathology of aging]. / Einiges zur Physiologie und Pathophysiologie des Alterns.

First of all some information will be given on history, methods and the actual situation of the research in aging. Aging ist not a disease but a physiologic process characterized by growth and maturity events which legally change the functional performances and the anatomical structures of the human beings in time. This aging trend depends on the metabolism of the living cells whose metabolism changes over the years. This will be demonstrated by the example of the connective tissue metabolism. Even if aging in itself is not a disease at all there still is the fact that morbidity, a multimorbidity, is certainly higher in old age than in the youth. These diseases which are almost continually existing with old age individually vary the length of life which is determined by genetic factors. The whole mode of procedure of the pathogenesis of a chronical disease will be treated on the basis of the most frequent disease in aging: arteriosclerosis.

Growth rate differences between arterial smooth muscle cells cultivated from rat impaired by short--or long--term hypertension respectively.

In former studies short-term renal hypertension (2 weeks) in rats led to a remarkable increase of arterial smooth muscle cells (ASMC) proliferation in cultures and subcultures, examined by cell counting and 3H-thymidine indices /flasks in cultures or subcultures of hypertensive rats. The activated cell growth was observed up to the 6th subcultures. The proliferation of ASMC cultivated from short-term hypertensive rats is suppressed or eliminated respectively by antirheumatic drugs up to the 6th subcultures. Long-term renal hypertension (4-6 weeks) in rats showed the same increase in ASMC proliferation rate up to the 2nd subcultures but in the higher passages e.g. in the 6th subcultures the ASMC growth increase was not existing anymore. The limited reduplication capacity of postmitotic cells, we assume, is the cause of this difference.

Effect of risk factors and antirheumatic drugs on the proliferation of aortic wall cells.

The proliferation of aortic smooth muscle cells (ASMC) of Wistar rats, impaired by risk factors such as arterial hypertension, diabetes mellitus, atherogenic diet and staphylolysin injections and of normal Wistar rats treated with antirheumatic drugs such as prednisolone and acetylsalicylic acid was investigated. The cells of these animals were cultivated, subcultivated, and in the 2nd subcultures the cell numbers/5 ml medium were counted by means of Coulter Counter, and the cells were incubated with [3H]thymidine and the percentage of labelling in 100 or 1000 counted cells was stated. The effect of risk factors such as LDL and staphylolysin and of antirheumatic drugs such as prednisolone, acetylsalicylic acid, D-penicillamine and chloroquine added to the 2nd subcultures of cultivated ASMC of normal minipigs was investigated by the same method. The proliferation of cultivated ASMC of rats impaired by risk factors was accelerated. The proliferation of cultivated ASMC of rats treated with antirheumatic drugs was inhibited. The proliferation of ASMC of minipigs in the 2nd subcultures was activated by addition of risk factors and inhibited by addition of antirheumatic drugs. Antirheumatic drugs given to the rats and added to the medium of the 2nd subcultures of ASMC of normal minipigs inhibit the acceleration of ASMC proliferation induced by simultaneously given risk factors. The proposal to augment up our arsenal of the hitherto existing preventive and therapeutical measures by the application of antirheumatic drugs based on the experimental models referred to is supported by the result of a limited prospective double-blind-study of a sample of 133 male patients after myocardial infarction. The most remarkable result that the acceleration of the ASMC proliferation, the real pathologic process of arteriosclerosis, is inhibited by the application of antirheumatic drugs, at exactly the same time as the acceleration of the fibroblast proliferation, the real pathologic process in rheumatic diseases--ASMC and fibroblast, both being mesenchymal cells--recommends the use of these drugs in the prevention and therapy of human arteriosclerosis. The surprising result of our in-vivo experiments, that the acceleration of the growth of the ASMC induced by risk factors and the inhibition of the growth induced by antirheumatic drugs persist in the subcultures, is explained by the "selection theory" that there are dissimilar kinds of ASMC in normal arteries and that they react differently.

[The effect of pathogenic factors and anti-rheumatic drugs on the proliferation of cultured human fibroblasts]. / Uber die Wirkung von Noxen und antirheumatischen Medikamenten auf die Proliferation von kultivierten menschlichen Fibroblasten.

1. Staphylolysin and low density lipoprotein (LDL) stimulate the proliferation of cultivated fetal human fibroblasts of the lung. 2. Antirheumatic drugs (prednisolone, acetylsalicylic acid, D-penicillamine and chloroquine) inhibit the proliferation of cultivated fetal human fibroblasts. 3. Antirheumatic drugs (prednisolone, acetylsalicylic acid, D-penicillamine and chloroquine), simultaneously added to the cluture eliminate the acceleration of the proliferation induced by staphylolysine or low density lipoprotein. 4. These results suggest, that in rheumatic diseases either pathogenic factors or antirheumatic drugs stimulate or inhibit, respectively, directly the cells of the connective tissue. Arterial wall cells, being mesenchymal cells, react in similar way on staphylolysine, low density lipoprotein and antirheumatic drugs. The similarity between the rheumatic and the arteriosclerotic process suggest to use antirheumatic drugs in the prevention and therapy of human arteriosclerosis.