Arnold G. Herman

Differential formation of prostacyclin (PGX or PGI2) by layers of the arterial wall. An explanation for the anti-thrombotic properties of vascular endothelium.

Abstract Different layers of rabbit aorta were separated and classified by microscopic examination into intima, internal elastic lamina, media and adventitia. The ability of each layer to produce prostacyclin (PGI 2 ), a potent endogenous inhibitor of platelet aggregation was investigated. The effect of adding the different layers to platelet rich plasma was studied and correlated with the synthetic capacity to form prostacyclin. Observations were also made after inhibiting prostacyclin formation with 15-hydroperoxy arachidonic acid (15-HPAA). The ability of the arterial wall to generate prostacyclin is highest in the intimal surface and progressively decreases to the adventitial surface. The pro-aggregatory activity of the different layers increased from the internal elastic lamina to the adventitia. These observations may explain the long known anti-thrombotic properties of vascular endothelium and help in the detailed elucidation of the mechanism of formation of intravascular thrombi and haemostatic plugs.

Effect of hypercholesterolemia on vascular reactivity in the rabbit. I. Endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits.

We studied the effects of hypercholesterolemia on vascular responsiveness in different arteries isolated from rabbits: control groups of rabbits and groups receiving the atherogenic diet consisted of eight animals each. In the arteries, 16 weeks of cholesterol-rich (0.3%) diet evoked intimal lesions which were more pronounced than those noted after 8 weeks of hypercholesterolemia; the aortic arch was affected significantly more by the lesions than the abdominal aorta and the pulmonary artery. Segments of the arteries were mounted in organ chambers for isometric tension recording or for measurement of the endothelium-derived relaxant factor. Contractions caused by acetylcholine and prostaglandin F2α were not altered by the hypercholesterolemia; those evoked by serotonin were moderately augmented only in the aortic arch of hypercholesterolemic rabbits. As the degree of intimal lesion formation increased, the contractions to norepinephrine and clonidine were progressively inhibited. The endothelium-independent relaxations to nitroglycerin were inhibited in only the most severely affected arteries; the endothelium-dependent relaxations to acetylcholine and adenosine triphosphate were progressively inhibited as the degree of fatty streak formation augmented. Thus, in the aortic arch, the relaxations to 3 ± 10–6 m acetylcholine, expressed as percent of the initial contraction, decreased from 86.7 ± 3.3% in control tissues to 16.3 ± 8.6% in the 16-week hypercholesterolemic vessels; in the abdominal aortas these relaxations averaged 93.5 ± 2.2% in control vessels and 72.0 ± 6.9% in the hypercholesterolemic tissues. The acetylcholine-induced release of endothelium-derived relaxant factor from the abdominal aorta was not significantly affected by the hypercholesterolemia. We conclude from these studies that in arteries obtained from hypercholesterolemic rabbits: (1) the contractions caused by serotonergic mechanisms tend to be augmented, while those to α-adrenergic activation are decreased, (2) the endothelium-independent relaxations are modified only in the more severely affected arteries, and (3) the endothelium-dependent relaxations are progressively inhibited as the degree of fatty streak formation augments, probably because a step subsequent to the release of endothelium-derived relaxant factor is altered.

Apoptosis and Related Proteins in Different Stages of Human Atherosclerotic Plaques

BACKGROUND The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions. METHODS AND RESULTS Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (n=18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (P<0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8+/-3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles. CONCLUSIONS We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques.

Elevated Levels of Oxidative DNA Damage and DNA Repair Enzymes in Human Atherosclerotic Plaques

Background—The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated whether reactive oxygen species-induced oxidative damage to DNA occurs in human atherosclerotic plaques and whether this is accompanied by the upregulation of DNA repair mechanisms. Methods and Results—We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques contained 160±29 8-oxo-dG residues/105 dG versus 3±1 8-oxo-dG residues/105 dG in mammary arteries. Single-cell gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (>90%). This suggests that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohistochemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear antigen. Conclusions—Our findings provide evidence that oxidative DNA damage and repair increase significantly in human atherosclerotic plaques.

Phagocytosis of Apoptotic Cells by Macrophages Is Impaired in Atherosclerosis

Objective—Apoptotic cell death has been demonstrated in advanced human atherosclerotic plaques. Apoptotic cells (ACs) should be rapidly removed by macrophages, otherwise secondary necrosis occurs, which in turn elicits inflammatory responses and plaque progression. Therefore, we investigated the efficiency of phagocytosis of ACs by macrophages in atherosclerosis. Methods and Results—Human endarterectomy specimens and human tonsils were costained for CD68 (macrophages) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (apoptosis). Free and phagocytized ACs were counted in both tissues. The ratio of free versus phagocytized AC was 19-times higher in human atherosclerotic plaques as compared with human tonsils, indicating a severe defect in clearance of AC. Impaired phagocytosis of AC was also detected in plaques from cholesterol-fed rabbits and did not further change with plaque progression. In vitro experiments with J774 or peritoneal mouse macrophages showed that several factors caused impaired phagocytosis of AC including cytoplasmic overload of macrophages with indigestible material (beads), free radical attack, and competitive inhibition among oxidized red blood cells, oxidized low-density lipoprotein and ACs for the same receptor(s) on the macrophage. Conclusion—Our data demonstrate that phagocytosis of ACs is impaired in atherosclerotic plaques, which is at least partly attributed to oxidative stress and cytoplasmic saturation with indigestible material.

Apoptosis in atherosclerosis: beneficial or detrimental?

Several groups have demonstrated apoptotic cell death in atherosclerotic plaques. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Both macrophages and smooth muscle cells undergo apoptosis in atherosclerotic plaques. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. Smooth muscle cell apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in early stages of atherosclerosis smooth muscle cells become susceptible to undergoing apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that smooth muscle cells may be killed by activated macrophages. The loss of the smooth muscle cells can be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by SMC. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies are removed. Apoptotic cells that are not scavenged in the plaque activate thrombin which could further induce intraplaque thrombosis. It can be concluded that apoptosis in the primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis. Recent data of our group indicate that apoptosis decreases after lipid lowering which could be important in our understanding of the cell biology of plaque stabilization.

Non-adrenergic non-cholinergic relaxation mediated by nitric oxide in the canine ileocolonic junction

The nature of the inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitter was studied in circular muscle strips of the canine terminal ileum and ileocolonic junction. Nitric oxide (NO) induced tetrodotoxin-resistant NANC relaxation, similar to that induced by electrical stimulation or acetylcholine (ACh). Incubation with the stereospecific inhibitors of NO biosynthesis, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), resulted in an increase of basal tension in the ileocolonic junction which was partly reversed by L-arginine but not by D-arginine. Moreover, L-NMMA and L-NNA, but not D-NMMA, concentration dependently inhibited the NANC relaxation in response to electrical stimulation and ACh, but not that in response to NO or nitroglycerin. This inhibitory effect was reversed by L-arginine but not by D-arginine. Hemoglobin reduced the NANC relaxation in response to electrical stimulation, ACh and nitroglycerin, and abolished the responses to NO. Our results suggest that NO or a NO releasing substance mediates the NANC relaxation in the canine terminal ileum and ileocolonic junction.

Phagocytosis and Macrophage Activation Associated With Hemorrhagic Microvessels in Human Atherosclerosis

Objective—Previously, we demonstrated that activated inducible NO synthase (iNOS)-expressing foam cells in human carotid plaques often produce autofluorescent (per)oxidized lipids (ceroid). Here, we investigate whether intraplaque microvessels can provide foam cells with lipids and trigger macrophage activation. Methods and Results—Microvessels (von Willebrand factor [vWf] immunoreactivity), activated macrophages (iNOS immunoreactivity), and ceroid were systematically mapped in longitudinal sections of 15 human carotid endarterectomy specimens. An unbiased hierarchical cluster analysis classified vascular regions into 2 categories. One type with normal vWf expression and without inflammatory cells was seen, and another type with cuboidal endothelial cells, perivascular vWf deposits, and iNOS and ceroid-containing foam cells was seen in 4 (27%) of 15 plaques. The perivascular foam cells frequently contained platelets (glycoprotein Ib&agr;) and erythrocytes (hemoglobin, iron), pointing to microhemorrhage/thrombosis and subsequent phagocytosis. Similar lipid-containing cells, expressing both ceroid and iNOS, were generated in atherosclerosis-free settings by incubating murine J774 macrophages with platelets or oxidized erythrocytes and also in vivo in organizing thrombi in normocholesterolemic rabbits. Conclusions—Focal intraplaque microhemorrhages initiate platelet and erythrocyte phagocytosis, leading to iron deposition, macrophage activation, ceroid production, and foam cell formation. Neovascularization, besides supplying plaques with leukocytes and lipoproteins, can thus promote focal plaque expansion when microvessels become thrombotic or rupture prone.

Cell Composition, Replication, and Apoptosis in Atherosclerotic Plaques After 6 Months of Cholesterol Withdrawal

Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8+/-0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.

Vascular endothelial dysfunction

Injury or activation of the endothelium changes its regulatory functions and results in abnormal endothelial cell function. Dysfunction of the endothelium has been defined as an imbalance between relaxing and contracting factors, between procoagulant and anticoagulant mediators or between growth-inhibiting and growth-promoting substances. The first part of this review describes endothelial dysfunction in hypercholesterolemia, and atherosclerosis in intimal thickening induced by perivascular and intravascular techniques. We focus on the implications of endothelial dysfunction in these conditions and the role of nitric oxide, endothelium-dependent hyperpolarization, endothelin, cytokines, adhesion molecules, growth factors, and thrombosis. In the second part, the endothial dysfunction in other diseases including hypertension, syndrome X, and diabetes is discussed. Finally, a short overview of therapeutic approaches of the dysfunctional endothelium is given.