Intimal healing. The pattern of reendothelialization and intimal thickening.

Studies were undertaken to investigate further the basis of intimal proliferation and the identity of the surface lining cell in rabbit aortas subjected to extensive deendothelialization. Endothelial cells were selectively removed by passage of an inflated balloon catheter through the arterial lumen. The healing response was evaluated at intervals up to 36 weeks by several techniques: 1) permeability to Evans blue, 2) reappearance of endothelial cells as indicated by the specific marker, adsorbed goat anti-rabbit tissue factor-horseradish peroxidase, 3) planimetric measurements of intimal thickness, and 4) electron microscopy. The results indicate that endothelial cell recovery progressed slowly and that it extended only from areas spared denudation. The regions not covered by endothelial cells were lined by cells of smooth muscle cell origin. Such areas were permeable to Evans blue-protein complex, and their luminal smooth muscle cells were associated with connective tissue-like material at their luminal surface; this material apparently acted as a base for platelet accumulation. The present findings indicate that the lumen of the extensively denuded vessel is lined by either endothelial or smooth muscle cells and that intimal healing is related to restoration of endothelial cell cover. In addition, intimal thickening reached a maximum well before reendothelialization was complete.     

Morphogenesis of intimal thickening in nonspecific aortoarteritis

Hyperplastic intima in nonspecific aortoarteritis was investigated immunomorphologically and by means of electronmicroscopic autoradiography. The cellular composition of the involved vascular wall was studied by the method of alcohol-alkaline dissociation. The prevalence of elongated cells with side processes and stellate cells in thickened intima was shown. These cells are characterized by essentially intensive RNA synthesis and, according to the electron microscopic data, can be regarded as pericytes. A significant amount of capillaries and precapillaries in thickened intima was visualized. Distribution of laminin and type IV collagen coincided with localization of the basal membrane of the vasa vasorum and with that of microvessels growing in the intima. Considered together, these data suggest that pericytes that surround a capillary/precapillary network may play a significant role in the morphogenesis of vascular transformation in nonspecific aortoarteritis intimal thickening.     

The mechanism of intimal thickening in arteriosclerosis

The limitations of current theories of the mechanism of intimal thickening in arteriosclerosis are briefly reviewed. The suggestion is advanced that this change is due to intimal oedema, arising from failure of the lymphatic system of the surrounding tissue to remove adequately, protein-containing fluid filtering outwards through the vascular endothelium. It is postulated that such local environmental influences account for the variability in the degree of arterial degeneration seen in different arteries, and in different portions of the same artery.     

Hyaluronic acid accumulation and endothelial cell detachment in intimal thickening of the vessel wall. The normal and genetically defective ductus arteriosus.

The closing ductus arteriosus (DA) was studied as a model for the development of intimal thickening of vessel walls using ultrastructural and immunohistochemical techniques. The material consisted of DA from neonatal dogs of three types: normal beagles, DA-defective pups from a line of mixed poodles with a genetic defect in the closure of the DA leading to persistent ductus arteriosus (PDA line), and normal litter-mates of DA-defective pups in the PDA line. The DA of the normal litter-mates of DA-defective pups did not differ from those of normal beagles. In the DA of normal beagles and normal PDA-line pups, closure is preceded by intimal thickening characterized by formation of a widened subendothelial region (SR), detachment of endothelial cells, invagination of endothelial cells, and migration of smooth muscle cells into the SR. It was observed that immediately before and after endothelial cell detachment, there was an increase in hyaluronic acid (HA) in the SR and inner media. In the DA-defective pups, the increase in hyaluronic acid failed to occur and there was no intimal thickening. The SR failed to expand, endothelium remained attached to the internal elastic membrane, and there was no invagination of endothelium or migration of smooth muscle cells. It is hypothesized that the increased synthesis of HA is an important early event leading to intimal thickening in the normal DA and perhaps to abnormal intimal thickening of other vessels. By its hygroscopic properties, HA may be directly involved in the formation of a wide SR, inducing endothelial cell detachment and favoring smooth muscle cell migration. In affected pups of the PDA line, there is a genetically-determined "block" in the normal process of intimal thickening at or before the initiation of increased HA synthesis.     

The initiation of intimal thickening in human arteries

To obtain more detailed information about the relationship of intimal thickening to defects in the elastin structure of the arterial wall, the internal elastic lamina and subsequent elastin formation in the intima was studied by very oblique sectioning of paraffin sections of the arterial wall, and by scanning electron microscopy of formic acid digested preparations. Comparison was made in the same subject between the internal thoracic artery (a vessel showing only slight intimal thickening) and the anterior descending coronary (which usually develops advanced intimal thickening). There was no evidence of penetration of the normal fenestrations of the internal elastic lamina by medial smooth muscle cells. This took place through major defects of this lamina and resulted in a change from transverse to longitudinal orientation of these cells and the accompanying elastin fibers of the intima. A further condensation of elastin (greater for the internal thoracic) occurred in the intima subjacent to the endothelial cells.     

Apoptosis participates in cellularity regulation during rat aortic intimal thickening.

Intimal thickening induced after endothelial denudation of rat aorta is though to be due to migration and proliferation of smooth muscle cells (SMC). When the reendothelialization is achieved, intimal thickening shows an important decrease in cellularity. Using in situ end labeling of fragmented DNA and electron microscopy, we show that this remodeling is accompanied by apoptosis of SMC. The number of apoptotic SMC becomes important 15 days after endothelial injury and reaches a maximum at 20 days; at 45 days the intimal thickening is reendothelialized and no more apoptotic SMC are detected. Apoptotic SMC show nuclear and cytoplasmic condensation as well as cytoplasmic vacuolization. Our results indicate that apoptosis is an important mechanism in the regulation of intimal thickening evolution.     

Relation between arterial intimal thickening and the vasa-vasorum

Summary The histopathological data presented support a new concept for the origin of the cells which cause intimal thickening of arteries. Arterial segments, isolated between ligatures, when examined by intra-vascular contrast techniques, showed penetration of vasa-vasora and formation of intra-arterial granulation tissue which produced myointimal thickening. Transitional forms between pericytes and myointimal cells were found. In autoradiographic studies on the incorporation of 3H-thymidine, DNA synthesis was first seen in the adventitia, fundamentally in the vasa-vasora pericytes, and in the adjacent media, and later in the intimal thickening. In arterial segments between ligatures typical intimal thickening was produced when intra-arterial granulation tissue was formed and the ligatures were removed thus restoring the circulation. These results were not produced when the arterial segment was sectioned lengthways between ligatures. It is suggested that this intimal thickening originates in cells from the vasa-vasora, in particular from pericytes.     

Hemodynamic parameters and early intimal thickening in branching blood vessels

Intimal thickening due to atherosclerotic lesions or intimal hyperplasia in medium to large blood vessels is a major contributor to heart disease, the leading cause of death in the Western World. Balloon angioplasty with stenting, bypass surgery, and endarterectomy (with or without patch reconstruction) are some of the techniques currently applied to occluded blood vessels. On the basis of the preponderance of clinical evidence that disturbed flow patterns play a key role in the onset and progression of atherosclerosis and intimal hyperplasia, it is of interest to analyze suitable hemodynamic wall parameters that indicate susceptible sites of intimal thickening and/or favorable conditions for thrombi formation. These parameters, based on the wall shear stress, wall pressure, or particle deposition, are applied to interpret experimental/clinical observations of intimal thickening. Utilizing the parameters as "indicator" functions, internal branching blood vessel geometries are analyzed and possibly altered for different purposes: early detection of possibly highly stenosed vessel segments, prediction of future disease progression, and vessel redesign to potentially improve long-term patency rates. At the present time, the focus is on the identification of susceptible sites in branching blood vessels and their subsequent redesign, employing hemodynamic wall parameters. Specifically, the time-averaged wall shear stress (WSS), its spatial gradient (WSSG), the oscillatory shear index (OSI), and the wall shear stress angle gradient (WSSAG) are compared with experimental data for an aortoceliac junction. Then, the OSI, wall particle density (WPD), and WSSAG are segmentally averaged for different carotid artery bifurcations and compared with clinical data of intimal thickening. The third branching blood vessel under consideration is the graft-to-vein anastomosis of a vascular access graft. Suggested redesigns reduce several hemodynamic parameters (i.e., the WSSG, WSSAG, and normal pressure gradient [NPG]), thereby reducing the likelihood of restenosis, especially near the critical toe region.     

A morphometric study of arterial intimal thickening in kidneys of dialyzed patients.

Arterial intimal thickening is common in the end-stage kidneys of patients maintained on hemodialysis. We measured the intimal thickening in patients dialyzed for varying periods and in patients with the malignant phase of essential hypertension and with scleroderma-associated renal failure. The ratio of intimal area to medical area in intrarenal arteries was used as a measure of intimal thickening. In the dialysis groups, intimal thickening was relatively constant in arteries of all sizes and correlated with duration of dialysis, particularly in larger arteries. In the malignant hypertension and scleroderma groups, the intimal thickening was greatest in arteries less than 200 mu in diameter and least in those over 500 mu in diameter. There was much less intimal thickening in arteries of all sizes in kidneys of patients with end-stage polycystic disease than in other end-stage kidneys from patients with a similar diastolic blood pressure and similar duration of dialysis. We believe that the intimal thickening in dialyzed patients is probably a disuse type of change and may be related to reduction in the area of the renal microvascular bed.     

Cytoskeleton of rat aortic smooth muscle cells. Normal conditions and experimental intimal thickening

The organization of actin, vimentin, and desmin in smooth muscle cells of rat aortic media and intima under normal conditions and 15 or 75 days after endothelial injury has been studied by means of electron microscopy, indirect immunofluorescence, densitometric analysis of sodium dodecyl sulfate-polyacrylamide gels, isoelectric focusing, and bidimensional gels. In the normal aortic media, practically all smooth muscle cells contain vimentin, and about 50% of them contain, in addition, desmin; upon analysis of actin isotypes by bidimensional gels, smooth muscle cells show a predominance of alpha-actin, some beta-actin, and very little gamma-actin. Fifteen days after endothelial injury, cells that have migrated into the intima contain decreased amounts of actin and desmin and increased amounts of vimentin compared with normal medial smooth muscle cells. Moreover, beta-actin becomes the predominant actin isotype and significant amounts of gamma-actin appear, whereas alpha-actin decreases. Seventy-five days after endothelial injury, regenerated endothelial cells have repaired the injury. Intimal smooth muscle cells are less numerous than 15 days after injury, and the organization of their cytoskeletal elements has reverted almost to normal conditions. At both 15 and 75 days after endothelial injury, no significant changes of cytoskeletal elements are seen in the aortic media underlying intimal thickenings.     

Stenotic intimal thickening of the external iliac artery in competition cyclists

Twenty-three cases of an arterial disease that affects competition cyclists are reported. Patients complained of intermittent acute claudication appearing on one lower limb only at the time of a maximal strain while cycling. Doppler hemodynamic investigation on an ergometric bicycle revealed a collapse of the ankle systolic pressure. Arteriography showed a sinuous lengthening and moderate stenosis of the external iliac artery. Pathologic examination of the artery disclosed a stenotic intimal thickening due to moderately cellular loose connective tissue with a variable distribution of collagen and elastic fibers. The cells in the affected zone were readily labeled with anti-actin and anti-myosin antibodies, and electron microscopy revealed features of synthetic smooth muscle cells. The lesion observed differs from intimal fibrodysplasia and from artherosclerosis. Abnormal local hemodynamic conditions may lead to this type of lesion. Thus, stenotic intimal thickening of the external iliac artery appears to be a new arterial disease defined by clinical, arteriographic, and pathologic features.     

Experimental diffuse intimal thickening of the femoral arteries in the rabbit

Summary The temporary placing of a polyethylene cuff around the superficial femoral artery leads to a diffuse intimal thickening without thrombosis. The evolution of the phenomenon during 16 months has been studied by light and electron microscopy. The intimal thickening that developed in the first 2 months, consisted mainly of modified smooth muscle cells, with few leukocytes and some cells of the endothelial type adhering to the endothelium. In the intercellular space, there is a significant production of elastic and collagen material.The internal elastic lamina is interrupted over part of the vessel circumference for varying distances. The proliferation and dedifferentiation of the medial smooth muscle cells is seen until the 4th month. The adventitial reaction, characterized by fibroblast multiplication and the appearence of numerous histiocytes, disappears at about the 2nd month.Over time, the heterogeneous cellular population is no longer identified and the intimal thickening is composed, after the 6th month, of well differentiated smooth muscle cells. After the 9th month, the elastic structures have a tendency to become rarefied, while the fibrous tissue increases at the expense of the cellular components. Signs of damage are noted within the intimal and medial smooth muscle cells. These long-standing modifications are related to ageing.The origin of neointimal cells is discussed and possible participation of endothelial cells is suggested.The author are indebted to Mrs D. Tenza, J. Demeurie and N. Sebbagh for their skilful technical assistance.     

Aggregates of modified low-density lipoproteins induce lipid accumulation in intimal cells of the human aorta in vitro

Institute of Experimental Cardiology, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Smirnov.) Translated from Byulleten's Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 7, pp. 34–36, July, 1990.