Guido Majno

Chronic Inflammation: Links with Angiogenesis and Wound Healing

Late one night in the early days of electron microscopy I found myself staring at an electron micrograph (not mine) in the basement of Rockefeller University. “It is so beautiful,” I mumbled, “that it has to be true.” “What did you say?” asked a voice with a strong Romanian accent. George Palade did not let me get away with it. Since then I have been very careful in evaluating the scientific importance of beautiful photographs, although I must confess that occasionally I am still tempted to repeat that controversial statement. A collection of microphotographs that qualify as truly beautiful illustrates a paper in this issue of The American Journal of Pathology. 1 It is my privilege to comment on this excellent study by Thurston and colleagues at the University of California at San Francisco. Oddly enough, there are few microscopic studies of vessels in chronic inflammation and even fewer of vessels in infection. Yet this should be a productive field because some bacteria are directly angiogenic; the vascular component of the wart-like growth called verruga peruana, caused by Bartonella bacilliformis, is truly angiomatous. 2,3 In drafting this commentary I decided to let the paper by Thurston et al speak largely for itself. The fact that it links chronic inflammation with angiogenesis gives me the opportunity to broaden the discussion to include fibroblasts and wound healing because, as will be apparent below, these processes are more closely linked than is generally understood. The link is provided by circulating stem cells.

Vascular labelling with monastral blue B

Vascular labelling is an established technique of experimental pathology whereby leaky vessels can be identified in vivo. A suspension of a suitable colloidal pigment is injected intravenously; the pigment is then trapped in the wall of the leaky vessels. The colloidal preparation of carbon black, which has been used for many years for this purpose, is no longer commercially available. This communication introduces a substitute: Monastral blue B which gives beautiful preparations in whole mounts, is readily visible in paraffin and plastic embedded histologic sections, has a distinctive appearance in electron micrographs, and is nontoxic in the required dosage.

Cellular breakdown within the arterial wall: An ultrastructural study of the coronary artery in young and aging rats

The coronary arteries were studied by electron microscopy in normal rats weighing 65 to 535 gm; fixation was accomplished by perfusion for 20 min at 110–130 mm Hg. In rats of all ages (but especially in the oldest) the arterial wall contained deposits of abnormal intercellular material, consisting of granules, vesicles, myelin figures and other debris. These deposits were present in the intima and media, but rare in the adventitia; there was suggestive evidence that medial cells phagocytized some of the material. The adventitia was characterized by 1–4 layers of cells with extremely thin protoplasmic expansions wrapped around the vessel (“veil cells”) and containing lysosome-like bodies as well as phagosomes. These findings, taken as a whole, suggest the following sequence of events. During normal life, the media produces cellular debris, by cell death as well as by fragmentation of cellular processes; part of these debris are phagocytized by smooth muscle cells, part diffuse outward to the adventitia where they are taken up by specialized cells (the “veil cells”). However, due to relative inadequacy of phagocytosis in the media, the debris continue to accumulate and form intercellular deposits that increase with age. It is possible that this natural phenomenon may by itself set a maximal limit to the life-span of the arterial wall.

Endothelium 1977: A Review

This paper is conceived as a short update of the review published in 1965 (1). It is based on a set of 2,476 references assembled through a MEDLARS search limited to the single word endothelium (January 1966 – June 1977; a few references were added up to the end of 1977). Much to our regret, we were obliged to omit from our search the word capillary which would have yielded an unmanageable number of additional papers. About 5% of the MEDLARS titles referred to corneal endothelium and were eliminated. This decision, which was based on limitations of space, may well have discarded relevant data: vascular and corneal endothelium are both mesodermal structures, and although the corneal endothelium has some attributes which appear to be distinctive, such as a water pump (2), other properties are similar (3). In limiting the number of references to about 500, arbitrary choices had to be made; papers obviously belonging under the headings of inflammation, atherosclerosis, or thrombosis were not included and will be found in other portions of this volume. Some of the inevitable gaps will be filled by the bibliography of the papers quoted herein, by other reviews (4–15), and by the proceedings of the A. Benzon Symposium of 1969 (16).

Quantitation of oil red O staining of the aorta in hypercholesterolemic rats

A technique is described which provides morphologic and quantitative data on the amount of oil red O (ORO) staining in thoracic aortas of rats fed a high cholesterol diet. Samples are stained with ORO, the dye is extracted, and the concentration of ORO in the extract is measured colorimetrically. Wistar rats fed ad libitum either standard chow (control group: n = 15) or chow supplemented with 4% cholesterol, 1% cholic acid, and 0.5% thiouracil (CCT group: n = 23) were maintained on these diets for 1, 3, 6, 9, or 12 months. Plasma cholesterol levels averaged overall 87 and 737 mg/dl for the control and CCT groups, respectively. Animals were killed under anesthesia by perfusion fixation with formalin or glutaraldehyde, and samples of thoracic aorta were stained with ORO. After microscopic study en face and measurement of surface area, the ORO was extracted in chloroform-methanol (2:1). Concentrations of ORO (microM) were determined from a standard curve and expressed as microM/mm2 of aorta. Aortas of CCT animals showed progressive diet- and time-dependent increases in the amount of ORO staining compared to controls. We conclude that this method yields reliable quantitative data applicable to studying atherosclerosis in small animals.

Endothelium and “silver lines”

The significance of endothelial “silver lines” was studied by TEM in rat aortas after perfusion with glutaraldehyde followed by silver nitrate. Standard TEM technique proved unsatisfactory (coarse silver granules, imprecise localization, artefacts). Exposure of the silver-treated aortas to photographic fixer markedly improved the image of the deposits leaving fine, stable, uniform “residual granules” about 100 Å in diameter. Most of these granules were localized along the intercellular junctions; they also tended to pool in the basement membrane beneath each junction. This image suggests that the Ag+ ions pass through the junction, and react with its contents as well as with the basement membrane beyond it. A scheme is proposed to explain the reaction of Ag+ ions with anions and negatively charged radicals within the junction. It is concluded that the “silver lines” represent not only a histochemical effect, but also the visualization of a transendothelial electrolyte pathway.

Adriamycin impairs phagocytic function and induces morphologic alterations in human neutrophils

Normal human polymorphonuclear leukocytes (PMNL) were preincubated in vitro with methotrexate, 5‐fluorouracil, vincristine, cisplatin, Adriamycin (doxorubicin), and daunomycin for 15 hours before being tested in a phagocytic–bactericidal assay. Anthracycline‐treated PMNL were defective in phagocytosis and killing of the bacteria, in contrast to the other chemotherapeutic agents which allowed the PMNL to remain functional. The defect of Adriamycin‐treated PMNL resulted from decreased ingestion: 3 μg/ml Adriamycin inhibited by 50% of the uptake of Oil Red‐O particles. In this assay, the proportion of noningesting PMNL increased from ≦20% with 0.62 μg/ml to ≧90% with 10 μg/ml Adriamycin. Electron microscopy revealed that Adriamycin‐inactivated PMNL had rounded up, were depleted in glycogen, and had undergone profound nuclear changes. RNA and protein synthesis in PMNL were also affected. Adriamycin, besides producing neutropenia, may decrease the phagocytic function of circulating PMNL.

Cellular changes during hypertension: A quantitative study of the rat aorta☆

Using rats made hypertensive by aortic ligation or by the one kidney--one clip method, we searched the aorta for morphologic clues that could explain why hypertension aggravates atherosclerosis. Both atherosclerosis and hypertension are characterized by an increased migration of mononuclear cells into the aortic intima; we therefore quantitated this phenomenon and studied its time course. In the thoracic aorta of hypertensive rats intimal cells (emigrated mononuclear cells) increased up to 15 times 2 weeks after surgery and remained stationary thereafter. In both control and experimental rats, leukocyte emigration was heavier in the thoracic aorta than in the abdominal region. A two- to threefold increase in medial smooth muscle herniae into the intima (myointimal herniae) was also found at 8 weeks, indicating a smooth muscle cell dysfunction. Electron microscopic study of the intima showed that its thickening was due to blood-borne material and also to extracellular matrix synthesized by the endothelium. Heightened secretion reflects cell activation, a condition that (in the endothelium) leads also to leukocyte adhesion. These data suggest that, in renovascular hypertension, the aortic endothelium is in an activated state, possibly through a hormonal stimulus.

Venular endothelium in vitro: isolation and characterization.

SummaryThe structural and functional properties of the endothelium vary in relation to anatomic site and position along the vascular tree. Cultures of endothelial cells have been obtained so far from large arteries, large veins and capillaries, but not from venules. We now report techniques for culturing not only rat arterial and venous endothelium, but also a special method for obtaining and culturing venular endothelium. The technique is based on the principle of “vascular labeling,” whereby an insoluble pigment can be permanently deposited in the wall of thevenules, making them easily visible by light microscopy. The venules of a rat cremaster muscle are labeled with a local injection of histamine followed by Monastral blue B intravenously (i.v.); 24 hours later selected venules are isolated by microdissection and either enzymatically dispersed or placed directly into tissue culture wells. The wells are coated with fibronectin and laminin and supplemented with DMEM, 20% fetal calf serum, and endothelial cell growth factor. Polygonal and spindly endothelial cells begin as clusters, grow in sheets, and sometimes form tubes. The cells stain variably for Factor VIII-related antigen,Ulex Europeus I lectin, and non-muscle specific actin. They synthesize angiotensin-converting enzyme, but do not metabolize acetylated LDL. Ultrastructurally, they display pinocytic vesicles, microtendons, and tight junctions, but not Weibel-Palade bodies. We believe that this method will be important for studying the pathophysiology of venules, which are the preferential target of inflammatory mediators and the typical site of inflammatory cell diapedesis.

Atherosclerosis and Inflammation

It is probably safe to state that atherosclerosis is the sum of several different processes. Each one of the past and current theories of atherosclerosis emphasizes one of these processes. Thus, if we open a contemporary textbook of pathology (1), we find at least four theories: (1) lipid insudation, (b) structural or metabolic alteration of the arterial wall, (c) intimal stress or injury, (d) thrombogenesis or encrustation. To these we should add the two newer theories that emphasize a further aspect of the process, cell proliferation: the theory of Ross et al. (2, 3) which gives platelets the stimulating role, and the theory of Benditt (4, 5) in which the proliferation is considered akin to neoplasia.