Hypertensive arterial disease and atherogenesis. Part. 1. Intimal changes in the old, spontaneously hypertensive rat (SHR).

The histological, ultrastructural and permeability aspects of the intima in 60 70-week-old spontaneously hypertensive (SHR) and Wistar Kyoto normotensive (WK) rats were studied and compared. The intima of aorta, coronary and renal arteries was unequally thickened owing to the smooth muscle cell (SMC) migration and proliferation, blood cell immigration and endothelial cell activation. The present work describes intimal changes that may act as potential atherogenic factors, i.e. hyper-reactivity of endothelial cells; decreased thinness of endothelial cell periphery; reduced intercellular junction pathways; increased quantity of basement lamina and glycosaminoglycan subendothelial material; platelet and monocyte-macrophage infiltration; widened fenestrae in the internal elastic lamina, and smooth muscle cell migration and proliferation. These changes might possibly be able to reduce the atheroresistance of this species by reducing the barrier function, increasing the trapping effect and stimulating smooth muscle cell proliferation and fibrogenesis. They are believed to promote the development of arterial lipidosis when hyperlipemia is an added risk factor.

Tunica media changes in the spontaneously hypertensive rat (SHR).

The histological, ultrastructural, morphometrical and histochemical aspects of the arterial media were studied in young and aged SHR, and compared to normotensive Wistar Kyoto rats. The diffuse thickening was the most characteristic feature of the hypertensive media. It seems due to three processes: Early generalized hypertrophy of smooth muscle cells (smc); connective matrix neogenesis and smc proliferation, more evident in peripheral vasculature. The present paper discusses the following hypertensive tunica media changes in relation to the atherosclerotic process: the decrease in lipolytic esterase and cholinesterase activities; the activation of some lysosomal enzymes; the increase in collagen, glycosaminoglycan and elastin content; the increased media thickness; the modified smc behavior (migration, secretion, proliferation). These alterations might positively influence arterial susceptibility to atherosclerosis through reduced smc lipolytic activity; slowed transmural diffusion; perturbed efflux and aggravated media hypoxia.

Thrombomodulin, a functional surface protein on human keratinocytes, is regulated by retinoic acid.

Thrombomodulin, a major anticoagulant proteoglycan of the endothelial cell membrane, is a thrombin receptor that acts as a cofactor for protein C activation. It has previously been shown that thrombomodulin, present in human epidermis and in lysates of cultured keratinocytes, is implicated in cellular differentiation during mouse fetal development. The role of retinoic acid in keratinocyte differentiation prompted us to study retinoic acid regulation of thrombomodulin expression in primary cultures of keratinocytes isolated from adult human skin, grown at low (undifferentiated keratinocytes) and normal calcium levels (differentiated keratinocytes). Thrombomodulin antigen levels and total and surface activities were measured in cultures without and with retinoic acid. Thrombomodulin mRNA visualized by in situ hybridization was quantified by computer-based image analysis. Functional thrombomodulin was expressed on the surface and in the cytoplasm of cultured human keratinocytes regardless of the calcium concentration. In contrast, retinoic acid induced significant increases in the total antigen level and in surface and intracellular thrombomodulin activities only in keratinocytes grown in a low-calcium medium. In these undifferentiated keratinocytes, quantification of mRNA transcripts showed a threefold increase after retinoic acid stimulation. Thus, functional thrombomodulin is a human keratinocyte surface protein whose expression is controlled through the keratinocyte differentiation program and is modulated in vitro by retinoic acid.

Shear stress modulates tumour cell adhesion to the endothelium.

The adhesion of breast adenocarcinoma cells (MDA-MB-231) to human umbilical vein endothelial cells (HUVEC) was studied in whole blood and under varying flow conditions. This study was done on HUVEC either kept under static conditions or pre-conditioned in flow for 2 hours at a shear stress of 5 or 13 dyn/cm(2). Coverslips coated by HUVEC were placed in a parallel plate perfusion chamber and perfused at a shear rate of 300 or 1500 sec(-1) with heparin-anticoagulated blood containing 111In labelled MDA-MB-231 cells. We report here the optimal conditions for studying the adhesion of MDA-MB-231 to endothelial cells under shear constraints corresponding to those observed into small and medium sized arteries.

Enzyme-histochemical changes in arteries of genetically hypertensive rats (SHR, SP-SHR).

To determine the effect of the duration and severity of hypertension on arterial wall metabolism 28 enzyme activities and several macromolecular complexes were histochemically studied in normotensive (WK), moderately (SHR) and strongly hypertensive (SP-SHR) rats at various ages. The results indicate that the abnormalities of 5' nucleotidase, acid esterase, cholinesterase and Alk.P. appeared in prehypertensive 4 w.old SHR. The posthypertensive changes, fluctuating in relation to the duration of hypertension, concerned: the pentose pathway, Krebs cycle and glycolosis -linked dehydrogenases; lysosomal enzymes; glycogen-phosphorylase and MAO; glycosaminoglycan and glycoprotein content. The structural and metabolic response presented several local and regional differences. The metabolic changes were greater in the aorta than in the caudal and femoral arteries. The comparison between SHR and SP-SHR indicates that the blood pressure (BP) at 170 mm Hg seems well tolerated during a long period of time. Severe lesions such as degeneration and failure of lipolytic activity in aortic smooth muscle cells (SMC), notable and early (8 mo.) in SP-SHR with 240 mm Hg were less intense and appeared later (13 mo.) in SHR with 190 mm Hg. The level of hypertension, rather than its duration, appears as a determining factor of posthypertensive vascular damage.

Thrombomodulin is synthesized by human mesangial cells.

Thrombomodulin (TM), an endothelial receptor for thrombin, endowed with a powerful anticoagulant activity, plays an important role in the antithrombogenicity of the vascular endothelium. Its presence within the human renal glomerulus is already known but was thought to be only endothelial. We looked for TM expression in human mesangial cells (MC), both in situ, in freshly prepared glomeruli, and in primary culture. Both fresh and cultured MC were strongly reactive for TM by immunocytochemical methods. Total TM antigen measured on MC lysates and surface TM activity on MC were 0.292 +/- 0.075 ng/mg of cellular proteins and 1.20 +/- 0.02 pmole of activated protein C/min/mg of cellular proteins, respectively. As shown by the presence of numerous transcripts detected by in situ hybridization, TM was shown to be synthesized by MC in vivo and in culture. The synthesis of active TM by both endothelial and mesangial cells within the renal glomerulus stresses the importance of its role in maintaining renal hemostatic equilibrium, and sheds some light on the conflicting reports of TM over- and underexpression in glomerulopathies to open a new field for investigation.

[Morphogenesis of central and peripheral arteriopathies in the hypertensive rat. 2. Histochemical aspects]. / Morphogénèse de l'artériopathie centrale et périphérique du rat spontanément hypertendu. 2. Aspects histochimiques.

Twenty-seven enzyme activities and five macromolecular complexes in central and peripheral arteries of the spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) 4, 20, 30 and 60 week-old rats were comparatively studied. Some of the four-week-old SHR (prehypertensive stage) showed several enzyme-cytochemical differences in comparison with WKY of the same age. In the adult SHR (20 to 60 weeks), the metabolic behaviour of different arterial segments was not uniform. Thus, the arteriopathy of elastic-type arteries was reflected by early and permanent activation of 5' nucleotidase and by the early increase (between 5 and 30 weeks) and later decrease (60th week) of esterase-and cholinesterase-activity. Peripheral arteriopathy was evidenced by hyperactivity and/or hypoactivity of glycogen-synthetase, glycogen-phosphorylase and monamine oxidase (in muscular-type arteries) and by early (5th week) and long lasting (60th week) hyperactivity of alcaline phosphatase (in arterioles). Some parameters were modified throughout the whole vasculature: increase in RNA, glycosaminoglycans, and glycoproteins, heightened lysosomal and dehydrogenase enzyme activities. Certain reported chemical modifications are discussed in connection with atherogenesis (carboxylic esterases and glycosaminoglycans) and peripheral tensiogenesis (5' nucleotidase, alcaline phosphatase, L-amino-peptidase, MAO).

[Behaviour of the aortic lipoidosis in rats twenty months after the withdrawal of an hyperlipidic diet (author's transl)]. / Devenir de la lipoïdose aortique du rat vingt mois après l'arrêt d'un régime hyperlipémiant.

48 rats were placed on an hyperlipidic diet (cholesterol, cholic acid, cholin, propylthiouracil), 23 were sacrificed at various intervals from the fourth up to the twelve month of the experiment. In the remaining 25 rats, the atherogenic regimen was replaced by a normal one. These animals were sacrificed one to twenty months after the withdrawal of the experimental diet. Aortae of experimental and control animals were studied by means of histological and histochemical technics. All the animals developed hypercholesterolemia together with intima and media lipoidosis. None demonstrated any aortic cell proliferation. The only metabolic change of the smooth muscle cell was a progressive decrease in 5' nucleotidase, acid esterase and cholinesterase activities. The return to a normal diet involved the reversion of the serum cholesterol level to normal values and the disappearance of intima lipoidosis. The reduced enzymatic activities in the media returned to normal levels around the six month. The surfaces of the extracellular sudanophilic areas decreased. However, twenty months after the withdrawal of the atherogenic diet, some lipids still persisted at the edges of the elastic fibres.

[Behaviour of the aortic lipoidosis in rats twenty months after stopping of an hyperlipidic diet (author's transl)]. / Devenir de la lipoïdose aortique du rat vingt mois après l'arrêt d'un régime hyperlipémiant.

48 rats were placed on an hyperlipidic diet (cholesterol, cholic acid, cholin, propylthiouracil), 23 were sacrificed at various intervals from the fourth up to the twelve month of the experiment. In the remaining 25 rats, the atherogenic regimen was replaced by a normal one. These animals were sacrificed one to twenty months after stopping of the experimental diet. Aortae of experimental and control animals were studied by means of histological and histochemical technics. All the animals developed hypercholesterolemia together with intima and media lipoidosis. None demonstrated any aortic cell proliferation. The only metabolic change of the smooth muscle cell was a progressive decrease in 5' nucleotidase, acid esterase and cholinesterase activities. The return to a normal diet involved the reversion of the serum cholesterol level to normal values and the disappearance of intima lipoidosis. The reduced enzymatic activities in the media returned to normal levels around the sixth month. The surfaces of the extracellular sudanophilic areas decreased. However, twenty months after stopping of the atherogenic diet, some lipids still persisted at the edges of the elastic fibres.

Activation of the 92 kDa type IV collagenase by tissue kallikrein.

Type IV collagenases are secreted as latent 92 and 72 kDa proenzymes which are then activated extracellularly. The mechanisms by which they are activated in vivo are not clear. We have studied the activation of porcine endothelial cell type IV collagenases by tissue and plasma kallikrein, and found that tissue kallikrein was a very efficient activator of the 92 kDa type IV collagenase. Enzyme cleavage was observed at concentrations of tissue kallikrein as low as 0.1 microgram/ml. Plasma kallikrein had no effect. By comparison, plasmin, which has been proposed to be the physiological activator of interstitial collagenase and stromelysin, and elastase were much less effective, and high concentrations (plasmin at 100-200 micrograms/ml and elastase at 20 micrograms/ml) were required to cause only a limited cleavage which was not associated with an increase in activity, as observed by the gelatin-gel lysis assay. In addition tissue kallikrein was found by immunohistochemistry to be present in the extracellular matrix of the intima of porcine aortic vessel wall. These findings suggest that tissue kallikrein can be a potential activator of the 92 kDa type IV collagenase in vivo.

Thrombomodulin in the central nervous system.

Thrombomodulin (TM), a thrombin receptor, present on the endothelial surface of blood vessels is a major anticoagulant proteoglycan. In this work the presence of TM antigen in the human brain and in the whole central nervous system of calves was investigated by immunocytochemistry using specific antibodies against human and bovine TM. When TM antigen was well preserved by immediate fixation on frozen tissues, TM was found present in the brain vessels of all human surgical specimens examined as well as in the whole vasculature of the calf central nervous system (CNS), from the medulla to the cortex. Moreover, even without immediate fixation, TM antigen was always found on the surface of the cerebrospinal fluid (CSF) cavity: on the arachnoid and on the inner aspect of the dura mater in humans and in calves. This suggests that the proteolytic enzymes responsible for the cleavage of TM from the endothelium are not present in the CSF and in the meningeal cells. The results reported here emphasize the importance of TM in the CNS as protecting, by its antithrombogenicity, a free circulation of blood and CSF in normal and even more, in pathological situations in which the permeability of the meningeal barrier increases.

Human neutrophil elastase in temporal (giant cell) arteritis: plasma and immunohistochemical studies.

OBJECTIVE: Few enzymes are able to attack the internal elastic lamina, which is destroyed in temporal arteritis (TA). Because human neutrophil elastase (HNE) is one of these, its role in the pathogenesis of TA was examined in patients undergoing temporal artery biopsy for suspected TA. METHODS: Over a 6 month period, 33 patients undergoing temporal artery biopsy were prospectively included in the study. TA was diagnosed in 15 patients; 9 of them had positive temporal artery biopsy. The other 18 patients made up the non-TA group. Nineteen healthy age matched subjects (mean age 74 +/- 9 yrs) served as controls. Levels of plasma HNE bound to alpha1-antitrypsin (pHNE-alpha1AT) were measured by ELISA. The presence of HNE in the temporal artery wall of 7 TA and 7 non-TA patients was evaluated immunohistochemically. RESULTS: Age, neutrophil counts, and erythrocyte sedimentation rates were similar in TA and non-TA patients. The mean pHNE-alpha1AT concentration in the TA group (84 +/- 20 microg/l) was significantly higher (p < 0.001) than in the non-TA group (51 +/- 26 microg/l) or in healthy controls (52 +/- 23 microg/l). The diagnostic sensitivity of pHNE-alpha1AT > 50 microg/l was 100%. Immunohistochemistry detected no HNE within the temporal artery wall of any patient. CONCLUSION: High levels of pHNE-alpha1AT were associated with TA. Our preliminary results indicate this could be a diagnostic marker for TA. Further studies are needed to confirm its reliability. Because HNE was not detected locally, no conclusions can be drawn as to its pathogenic role in TA.

Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas.

Neonatal hemangioma is a common benign proliferation of unorganized structures containing stromal and capillary endothelial cells. We tested the hypothesis that such cell proliferation might result from the release by stromal cells of endothelial cell mitogens. Stromal cells cultured from biopsies of surgically removed life-threatening hemangiomas released an endothelial cell mitogen in vitro that was indistinguishable from vascular endothelial growth factor (VEGF) based on independent criteria such as affinity chromatography for heparin or anti-VEGF IgG and radioreceptor assay. A functional product of the KDR gene encoding a cognate VEGF receptor was also expressed by these stromal cells. Transient transfection with antisense oligonucleotides targeted on the translation initiation codon of KDR abolished its tyrosine phosphorylation and mitogenic response of neonatal hemangioma cells to VEGF, confirming the existence of an autocrine loop of proliferation. When grafted in nude mice, these stromal cells elicited an angiogenic response that was blocked by neutralizing anti-VEGF IgG. These results might provide a clue to the importance of stromal cells in the pathogeny of neonatal hemangiomas.