Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. II. Uptake and cytotoxicity of ultraviolet-treated LDL on lymphoid cell lines.

The 'cytotoxicity' of ultraviolet-treated low-density lipoproteins (LDL) has been investigated using cultured lymphoid cell lines from normal subjects and from a patient with receptor-negative familial hypercholesterolemia. The ultraviolet-treated LDL were taken up by control lymphoblasts through the classical apo B/E-receptor pathway, while they were slowly taken up by receptor-negative lymphoblasts by non-specific endocytosis. These LDL were found highly 'cytotoxic' on normal lymphoblasts as demonstrated by Trypan blue dye uptake, [3H]thymidine incorporation, lactate dehydrogenase release and by electron microscopy. The 'cytotoxicity' increased progressively with the concentration of ultraviolet-treated LDL in the culture medium and with the incubation time. In contrast, lymphoblasts from familial hypercholesterolemia were not sensitive to low doses of ultraviolet-treated LDL (up to 150 micrograms apo-B/ml). The comparison of cells from normals and familial hypercholesterolemia showed that the 'cytotoxic' effect occurred subsequently to the LDL uptake, either receptor-mediated or receptor-independent. Experiments combining short-time (5 h) pulse with ultraviolet-treated LDL (labelled with [3H]cholesteryl oleyl ether) and a relatively long-chase period (72 h) showed: (1) a relationship between the delay for the appearance of the 'cytotoxicity' and the amount of ultraviolet-treated LDL taken up by the cells; and (2) the existence of a minimal dose (threshold dose) for triggering the 'cytotoxic' effect. The use of 'hybrid' LDL, prepared by partial delipidation of non-treated LDL and reconstitution by re-incorporating the neutral lipid fractions isolated from ultraviolet-treated LDL, demonstrated that the 'cytotoxic' effect is mainly mediated by triacylglycerols and cholesteryl esters. Scanning electron microscopy showed that the most prominent morphological change resulting from the uptake of ultraviolet-treated LDL was the early blebbing of plasma membranes.

Healing of full-thickness cutaneous wounds in the pig. I. Immunohistochemical study of epidermo-dermal junction regeneration.

In order to determine the kinetics of epidermo-dermal junction (EDJ) regeneration during would healing, we studied the regeneration of five EDJ components during reepidermization. Cutaneous wounds (50-mm length, 2-mm width, and 5-mm depth) were produced on the flank area of two pigs and left unsutured. Daily biopsies from day 1 to day 20 were studied by light microscopy on paraffin-embedded sections and by indirect immunofluorescence on cryostat sections using human sera to bullous pemphigoid antigen (BPA) with specificity previously confirmed by indirect immuno-electron microscopy, rabbit antisera to type IV collagen (Coll IV) and to fibronectin, and the monoclonal antibodies (MoAb) 4C 12-8 to laminin and NP-76 to type VII collagen (Coll VII). Histologically, reepidermization started from day 1 and progressed unidirectionally and exclusively from the wound edges. Up to day 9, the distal tips of the neo-epidermal tongues generally extended between the crust and the granulation tissue (GT). They fused on day 10, restoring epidermal continuity. For each EDJ component, the date of appearance (emergence), the spreading under the neo-epidermis tongue (expression), and the morphologic aspect of the labeling were studied. BPA and Coll IV were detected from day 1 to day 20 and found to be expressed all along the neo-EDJ. Fibronectin and laminin were detected from day 1, were present in the proximal and median zones of the neo-EDJ before day 7, up to the distal tip from day 7 to day 9 and were all along the neo-EDJ from day 10 to day 20. Coll VII was only detected from day 3. It was present in the proximal zone on day 3 and day 4, in the proximal and median zones on day 5 and day 6, than all along the neo-EDJ from day 7 to day 20. From day 10, all the labeling characteristics of the five components were found to be similar in the neo-EDJ and in the normal EDJ. With regard to the neo-epidermis progression, we found a synchronism of emergence and expression for BPA and Coll IV, a synchronism of emergence but a delay of expression for fibronectin and laminin and lastly, a delay of emergence and expression for Coll VII. We concluded that BPA and Coll IV could constitute the framework on which the neo-EDJ is progressively built by adjunction of the other components, restitution being obtained just after epidermal continuity is restored.

Oxidized low density lipoproteins elicit DNA fragmentation of cultured lymphoblastoid cells.

Lymphoblastoid cell lines continuously pulsed with mildly oxidized low density lipoproteins, exhibited a significant increase of DNA fragmentation induced by oxidized LDL internalized by cells. DNA fragmentation was associated with an increasing number of morphologically characteristic apoptotic cells simultaneously with the increase of cytotoxicity indexes, and the activation of the poly(ADP-ribose) polymerase, a nuclear enzyme stimulated by DNA strand breaks. The potential involvement of these biochemical and morphological changes in atherogenesis is discussed.

A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells.

Bovine aortic endothelial cells (BAEC) pulsed for 5 h with mildly oxidized low density lipoproteins (LDL), exhibited a broad, sustained and high peak of [Ca2+]i occurring several hours after the end of the pulse and reaching very high [Ca2+]i values (around 2500-3000 nmol/l) and a concomitant drop of cytosolic pH (around 0.2-0.3 pH units) without any loss of cell viability. When BAEC were continuously pulsed with oxidized LDL, the peak of [Ca2+]i was more sustained than in short pulse experiments and was associated with irreversible morphological changes usually associated with cytotoxic events (blebbing) and with a marked loss of viability. The potential involvement of these biochemical and morphological changes in atherogenesis are discussed.

Protective effect of 17 beta-estradiol against the cytotoxicity of minimally oxidized LDL to cultured bovine aortic endothelial cells.

The ability of 17 beta-estradiol, progesterone, testosterone and cholesterol in preventing the cytotoxicity of oxidized LDL to cultured aortic bovine endothelial cells (BAEC) was tested and compared. The lipid peroxidation of LDL, promoted either by UV-C radiation, copper ions or cultured human lymphoblastoid cells, was inhibited in a dose-dependent manner by 17 beta-estradiol (IC50 were evaluated at around 50 +/- 10 mumol/l with UV on copper and 6 +/- 2 mumol/l with cells), whereas exogenous cholesterol, progesterone or testosterone were completely inactive under the range of concentrations tested (up to 100 mumol/l). Subsequently, this antioxidant effect of 17 beta-estradiol preventing LDL oxidation protected 'indirectly' BAEC against the cytotoxicity of oxidized LDL. 17 beta-Estradiol was also able to protect 'directly' BAEC against the cytotoxic effect of oxidized LDL (with an IC50 around 0.5 +/- 0.1 mumol/l), whereas the other steroids tested were almost completely inactive. This direct protective effect resulted from an increased resistance of BAEC against the cytotoxic effect of oxidized LDL as shown by pre-incubation of BAEC with 17 beta-estradiol. The protective effect of 17 beta-estradiol was present for 2-3 days. In conclusion, 17 beta-estradiol exhibited an antioxidant activity and was effective in protecting BAEC against the cytotoxicity of oxidized LDL by acting at two separate sites: (i) outside the cells, by inhibiting the LDL oxidation; (ii) inside the cells by increasing the cellular resistance against the cytotoxic effect of oxidized LDL. The potential relevance of these results in relation to prevention of atherogenesis is discussed.

Hypothesis on microangiopathy of cutaneous capillaries.

Microangiopathy is a more or less pronounced PAS deposit-located exterior to the endothelial cells of the lymphatics and the basal membrane of the capillaries. This lesion, found in various normal and pathological states, has generated numerous pathogenic hypotheses. The presence of microangiopathy in 5 groups of 50 subjects representing five different clinical conditions, subjects over 60 years old or less than 40, diabetics, latent diabetics or patients with severe coronary heart disease, together with microscopic and/or ultrastructural lesions of the connective tissue (fibroblasts, collagen and elastic fibers, ground substance) has enabled us to propose a pathogenic hypothesis applicable to any microangiopathy. The initial change, hereditary or acquired, would be fibroblastic or interstitial. It would be characterized by the accumulation of glycoproteins, proteoglyacans and soluble collagen in the interstitium. Incomplete drainage of these macromolecules would occur around the blood and lymphatic capillaries and manifest itself by a PAS deposit, the hallmark of the microangiopathy.

Age-related changes in the elastic tissue of the human thoracic aorta.

Thirty human aortas with varying degrees of atheroma graded macroscopically according to the WHO classification were taken at autopsy from subjects of different ages (24-86 years). Study by light microscopy showed aortas with an intact wall (4 subjects, 25-46 years) with a thin intima and regular elastic layers, and aortas with varying degrees of modification of the wall, where the intima was of varying thickness and the elastic fibers showed varying degrees of damage (moderate lesions: 5 subjects, 35-52 yrs; severe lesions: 21 subjects, 26-86 yrs). From each aorta, a 4-cm segment from the tunica media, free of atheromatous lesions, was defatted and subjected to successive treatment with EDTA-Tris, 6 M guanidine-HCl-Tris, 6 M guanidine-HCl-Tris-DTE and collagenase. The residues (EP residues) were subjected to amino acid (AA) analysis and transmission electron microscopy (TEM) study. In the young subject, the AA composition was similar to that of elastin and the TEM images were characteristic of this substance. In the aging subject, an increase in polar AA and a parallel decrease in apolar AA and crosslinks was noted. By TEM, the elastin was seen to be associated with abundant fibrillar material. Trypsin treatment of EP residues gave E residues, whose composition and TEM appearance were similar in all samples, corresponding to the standard composition of elastin and its classic appearance by electron microscopy. We suggest that the fibrillar material removed by trypsin is the morphological reflection of the chemical variations observed in the EP residues. These correspond to contamination of the elastin by a polar protein fraction. This contamination is closely correlated with age but not with the degree of atheroma. Thus the age-related chemical changes in elastin appear to be independent of the onset and evolution of atheromatous lesions. The 10-15 nm diameter of the contaminating fibrillar material suggests that may be the microfibrillar fraction of elastic tissue.

Identification of coronary thrombus after myocardial infarction by intracoronary ultrasound compared with histology of tissues sampled by atherectomy.

This study compares the ability of intracoronary ultrasound (ICUS) to identify thrombus by means of actual criteria, with the histologic studies of tissues removed by directional atherectomy in patients treated previously with thrombolytic therapy. Coronary angiography and intravascular ultrasound imaging were performed before atherectomy in 34 patients who had received intravenous thrombolytic therapy for acute myocardial infarction a mean of 6 days before. The lesion morphology and the percentage of stenosis were defined on the angiogram. The ultrasound characteristics of the narrowing were described as intraluminal thrombus, mural thrombus, mixed plaque, and dense plaque. Thirty patients were studied. Thrombus was suspected in 8 patients on angiography. By ICUS, the presence of thrombus was predicted in 21 patients. Histologic studies of excised tissues found thrombus in 20 of the 30 patients. When ICUS was compared with histology, the true-positive rate was 80% and the false-positive rate was 50%; the true-negative rate was 50% and the false-negative rate was 20%. The correlation between observers was high. These observations suggest that ICUS may be useful in identifying fresh thrombus. The findings of this study help to confirm the criteria for diagnosing intraluminal thrombus by ICUS imaging.

Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.

BACKGROUND: Graft arteriosclerosis is a major cause of death after allotransplantation of organs such as the heart or the kidney. Aortic allotransplantation in mice is a useful experimental model to study the mechanisms of this pathology. However, the conventional heterotopic aortic model is limited by a high morbidity and is technically difficult to perform. We developed a new simple method for aortic transplantation in mice. METHODS: The infrarenal aorta from the donor mouse was anastomosed to the recipient's aorta at the same position using a sleeve technique. Orthotopic aortic transplantation was performed in 45 mice, 5 isografts and 40 allografts. No immunosuppression was given, and the mice were killed at day 15 or 30. The graft was examined macroscopically, and several histologic sections were made. RESULTS: The overall survival rate was 78%. The incidence of thrombosis was low (4 cases) compared with previously published series. Histology of aortas revealed typical aspects of rejection in the allografts with a chronic picture at day 30. No significant lesion was observed in isografts. CONCLUSIONS: We have developed a model of orthotopic aortic transplantation in mice. This new model is easy to carry out and has a low incidence of thrombosis, probably because there is no size discrepancy between donor and recipient aortic segment.

Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.

Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.

Early alterations of actin in cultured human keratinocytes and fibroblasts exposed to long-wavelength radiations. Possible involvement in the UVA-induced perturbations of endocytotic processes.

Exposure of cultured MRC5 human fibroblasts or NCTC 2544 human keratinocytes to mild doses of ultraviolet A (UVA: 320-400 nm) radiations markedly decreased the actin reactivity with fluorescein-labeled phalloidin. This indicates a change in the degree of polymerization of actin and thus in the organization of actin filaments. Such a phenomenon might be involved in the previously reported UVA-induced inhibition of specific and nonspecific endocytotic processes.

Metabolic and anti-atherogenic effects of long-term benfluorex in dyslipidemic insulin-resistant sand rats (Psammomys obesus).

Benfluorex is a clinical lipid-lowering agent with antihyperglycemic properties. The effect of long-term oral treatment (10 mg/kg/day for 7.5 months) on carbohydrate and lipid metabolism and aortic morphology was investigated in 24 insulin-resistant sand rats receiving a standard laboratory diet supplemented with cholesterol (2%). Untreated controls (n=34) developed impaired glucose tolerance, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia and elevated plasma LDL- and VLDL-cholesterol, positively correlated with the proportion of the thoracic aorta displaying oil red O-positive atherosclerosis; ultrastructural examination showed intimal lipid deposits, foam cells, polymorph infiltrates and fibrosis. Benfluorex-treated animals showed significant decreases in glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and plasma LDL- and VLDL-cholesterol, with no evidence of aortic atheroma. The metabolic benefits of benfluorex may protect against the long-term development of atherosclerosis in the insulin-resistant dyslipidemic syndrome.

Dermis ageing.

On ageing major changes within connective components of the dermis can be seen: collagen looses its regular and fascicular appearance, while ground substance increases, elastic material decreases and the fibroblast cell population becomes "at rest". The resulting dermal ageing is different according to individuals and is related to genetic back ground and exposition to multiple aggressions. This review presents these changes of the superficial dermis and the role of fibroblasts in the conservation of the connective tissue integrity.

Histopathology of the vessels of the femoral heads in specimens of osteonecrosis, osteoarthritis and algodystrophy.

The authors studied by light microscopy the vessels of the femoral head and neck in 38 well preserved specimens from core biopsy. There were 17 cases of osteonecrosis (ON), 11 cases of osteoarthrosis (OA) and 10 cases of reflex sympathetic dystrophy, so called algodystrophy (AD). Mean age of the patients was respectively 44, 45 and 42 years. The sex ratio, M/F, was respectively 12/5, 6/5 and 9/1. Types of staining used were hematoxylin-eosin, Masson Trichrome, P.A.S. and Verhoeff. In counting, thick-walled and thin-walled vessels were distinguished. There was a significant reduction in the number of the thick- and thin-walled vessels, in the ON group, by comparison with the OA and AD groups. An increased number of thin-walled vessels in the AD group were also observed. Morphological study showed an abnormal frequency of fibrosis of the media in the arteries of the ON group, i.e. arteriosclerosis. These data were compared with the few other histopathological studies previously published. The authors recommend further studies in order to precise their frequency and their significance.

Solid-state studies on synthetic fragments and analogues of elastin.

A series of synthetic fragments and analogues of elastin have been investigated, in the solid state, by means of differential scanning calorimetry and thermally stimulated current. Most of the polypeptides were shown to possess both amorphous regions and segments of long-range order. Water, which interacts preferentially with the amorphous zones, behaves as plasticizer, i.e. facilitates the localized motions of polypeptide chains. The results obtained have been correlated with elastin elasticity, in particular as far as the fundamental destructuring role of water is concerned.

Phase transitions and chain dynamics, in the solid state, of a pentapeptide sequence of elastins.

Differential scanning calorimetry (d.s.c.) and thermally stimulated current (t.s.c.) have been applied to the study of thermal transitions and dielectric relaxations of a pentapeptide sequence: Gly-Leu-Gly-Gly-Val of elastin. The manifestation of the glass transition has been observed by both techniques. The analysis of the fine structure of t.s.c. spectra reveals the existence of local order in the amorphous phase upon physical ageing. In the 'true' amorphous phase, cooperative motions of sequences of various length are observed. The corresponding activation parameters are characteristic of the 'structure' of the amorphous phase and might be used as reference for further studies.

An unusual dermal collagen disorder in a dog.

An adult dog developed a connective tissue disorder characterized by hyperextensibility of the skin on the head, neck and shoulders. Under light microscopy, the thickness of the dermis was reduced and the collagen bundles were fragmented. Hypodermic lesions appeared as fat necrosis and swelling of the wall of the blood vessels. Electron microscopically, the packing of collagen bundles and fibres in the dermis was highly disorganized and the rough endoplasmic reticulum of fibroblasts showed dilatation of the cisternae. Whereas the dermal lesions were non-specific, this case differed fundamentally from canine inherited collagen diseases in that the clinical features appeared in an old dog and the dermal lesions were only localized and were associated with hypodermal atrophy.

An atypical aortic atherosclerotic lesion in cynomolgus monkeys during hypercholesterolemia: a protection by smooth muscle cells against advanced lesions?

This study focuses on the fortuitous discovery of an atypical atherosclerotic lesion in four of 49 male adult cynomolgus monkeys (macacus fascicularis) which were maintained for a long time at a high level of hypercholesterolemia, and in seven of 19 female cynomolgus monkeys examined from the second to the 24th week of hypercholesterolemic diet: this lesion was in formation or already mature during this period of diet. This atypical lesion was formed by a collagen and elastic network surrounding synthetic smooth muscle cells without fibrofatty or fibrous plaques. Lipids were occasionally seen in the inner intima. The lesion appeared early (from the third week of diet). Once established, its morphology did not change. It became more extensive, but was not complicated by lipid overload in spite of prolonged, permanent hypercholesterolemia. This response to hypercholesterolemia is interesting because the activity of the smooth muscle cells differs from that observed in the classic lesion: they intervene earlier, their replication is very marked and rapid, their elastin secretion is greater and remains constant over time, and their phagocytic properties are reduced. This experimental study examines the installation and the maintenance of this lesion and raises the problem of its origin.

Simultaneous orthotopic transplantation of carotid and aorta in the rat by the sleeve technique.

Graft vascular disease (GVD) remains the major limitation to long-term survival after solid organ transplantation. Aortic or carotid allografts in rats have been shown to be useful models because similar changes to those observed in man develop within weeks. Both immunological and non-immunological factors influence the process of GVD and a method that could permit rapid multiple arterial allotransplantation in the rat would be of great value. We performed simultaneous orthotopic aortic and carotid allotransplantations in 25 rats. The vessels were anastomosed using a sleeve technique. No immunosuppression was given. The animals were killed at 15, 30, or 60 days and histological analyses of the grafts were performed. The overall survival rate was 80% and the incidence of technical failure was very low. The histopathological aspect revealed typical progressive GVD. In conclusion, we have developed a new model of simultaneous aortic and carotid transplantation in rats. This model, which incorporates a modification of the sleeve anastomosis, is rapid and yields an easy tool to investigate immunological and non-immunological processes driving GVD.

[D-Penicillamine effect on collagen synthesis and structural maintenance Red Sirius staining with polarized-light examination. The value of this staining in cases of collagen diseases (author's transl)]. / Appréciation de l'effet de la D-Pénicillamine sur la synthèse et le maintien de la structure collagène, grâce à la coloration histologique par le Rouge Sirius examinée en lumière polarisée. Discussion de la valeur de cette coloration en pathologie du collagène.

A severe systemic sclerodermia has been treated by D-Penicillamine. A pathological study before and after treatment showed important changes of the collagen structure. The soluble or foetal collagen fraction is enhanced. By the Red-Sirius staining with polarized light examination of the bundles of foetal collagen are green and bright. Then, this staining appears as a marker of collagen structures.