Immune-induced vascular connective tissue alterations in rabbits chronically immunized with bovine serum albumin: morphological and morphometric studies on normal and injured thoracic aorta.

The effect of persistent immunostimulation on normal and mechanically injured thoracic aorta was investigated histologically, histochemically and morphometrically. In the uninjured vessel wall no alterations suggestive of acute inflammation were observed following immunization, in accordance with previous biochemical studies. When mechanically elicited vascular injury and repair processes were induced in chronic immunostimulated rabbits, the neo-intimal aortic smooth muscle cell nuclear volume fraction of the vessel wall was significantly repressed, indicating, that the proliferative response to injury was inhibited. Further, the neo-intimal volume fraction of the vessel wall was reduced, suggesting impaired matrix neoformation. A highly significant linear correlation existed between the biochemically estimated DNA concentration and the nuclear volume fraction of smooth muscle cells in the vessel wall (r = 0.6275, P = 5 X 10(-5). Thus, the present study confirms previous biochemical observations, that the early processes of vascular inflammation and repair, i.e. smooth muscle cell proliferation and matrix accumulation, is inhibited following persistent immunostimulation. In addition to describing the histological correlates to the biochemical findings, important regional differences were quantified.     

Immune-induced vascular connective tissue alterations in rabbits chronically immunized with bovine serum albumin: biochemical studies on collagen, glycosaminoglycans, RNA, and DNA in normal and injured aorta

Repeated intravenous injections of bovine serum albumin in rabbits caused a significant reduction in the aortic in vivo biosynthesis of chondroitin-4,6-sulfate, whereas no changes were observed in the synthesis of other glycosaminoglycans nor in the content of collagen. This contrasts the biochemical changes generally seen in acute vascular injury. When experimentally elicited vascular injury and repair processes were induced in chronically immunized rabbits, the proliferative response was greatly inhibited, as reflected by a significant diminution of the DNA amount. Also, the vascular connective tissue matrix repair was restrained: the aortic content of collagen and the collagen type III/I ratio was repressed, and the in vivo biosynthesis of sulfated glycosaminoglycans was markedly reduced. All immunized rabbits developed antibodies to bovine albumin, but in only a few were circulating immune complexes detected. The inhibitory effect of persistent immunostimulation on the nonspecific processes of repair in vascular connective tissue may be of significance as to chronicity of vasculitis, as well as inflammation and repair in general, in inflammatory connective tissue diseases.     

Vascular smooth muscle cell in atherosclerosis

Vascular smooth muscle cells (VSMCs) exhibit phenotypic and functional plasticity in order to respond to vascular injury. In case of the vessel damage, VSMCs are able to switch from the quiescent ‘contractile’ phenotype to the ‘proinflammatory’ phenotype. This change is accompanied by decrease in expression of smooth muscle (SM)‐specific markers responsible for SM contraction and production of proinflammatory mediators that modulate induction of proliferation and chemotaxis. Indeed, activated VSMCs could efficiently proliferate and migrate contributing to the vascular wall repair. However, in chronic inflammation that occurs in atherosclerosis, arterial VSMCs become aberrantly regulated and this leads to increased VSMC dedifferentiation and extracellular matrix formation in plaque areas. Proatherosclerotic switch in VSMC phenotype is a complex and multistep mechanism that may be induced by a variety of proinflammatory stimuli and hemodynamic alterations. Disturbances in hemodynamic forces could initiate the proinflammatory switch in VSMC phenotype even in pre‐clinical stages of atherosclerosis. Proinflammatory signals play a crucial role in further dedifferentiation of VSMCs in affected vessels and propagation of pathological vascular remodelling.     

The aortic intima. II. Repair of the aortic lining after mechanical denudation.

This article reports the ultrastructure of the aortic lining during the repair of mechanically denuded aortic intima in the rat. Three main features were observed: a) Although platelets form a pavement on the exposed components of the aortic intima, platelet thrombi do not form on the denuded surface. b) During the first weeks after injury, a temporary false endothelial lining is formed by modified intimal smooth muscle cells. While the modified smooth muscle cells do not constitute a continuous cell layer, they are like true endothelial cells in that platelets do not adhere to the cell membrane of either cell type. c) A continuous layer of true endothelial cells is formed within 2 months after the original injury. Even after reestablishment of a continuous endothelium, however, abnormalities persist in the form of incompletely formed intercellular junctions. This abnormal endothelium is associated with areas of intimal smooth muscle cell proliferation. These observations are compatible with two alternative interpretations of the role of endothelial injury in the intimal proliferation seen following injury to the vessel wall: a) persistent defects in the endothelium may result in proliferation of underlying arterial smooth muscle cells or b) the proliferation, in converse, may in some manner delay the healing process of the overlying endothelium.     

Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture

Intimal thickening, the accumulation of cells and extracellular matrix within the inner vessel wall, is a physiological response to mechanical injury, increased wall stress, or chemical insult (e.g., atherosclerosis). If excessive, it can lead to the obstruction of blood flow and tissue ischemia. Together with expansive or constrictive remodeling, the extent of intimal expansion determines final lumen size and vessel wall thickness. Plaque rupture represents a failure of intimal remodeling, where the fibrous cap overlying an atheromatous core of lipid undergoes catastrophic mechanical breakdown. Plaque rupture promotes coronary thrombosis and myocardial infarction, the most prevalent cause of premature death in advanced societies. The matrix metalloproteinases (MMPs) can act together to degrade the major components of the vascular extracellular matrix. All cells present in the normal and diseased blood vessel wall upregulate and activate MMPs in a multistep fashion driven in part by soluble cytokines and cell-cell interactions. Activation of MMP proforms requires other MMPs or other classes of protease. MMP activation contributes to intimal growth and vessel wall remodeling in response to injury, most notably by promoting migration of vascular smooth muscle cells. A broader spectrum and/or higher level of MMP activation, especially associated with inflammation, could contribute to pathological matrix destruction and plaque rupture. Inhibiting the activity of specific MMPs or preventing their upregulation could ameliorate intimal thickening and prevent myocardial infarction.     

The role of shear stress in the pathogenesis of atherosclerosis

Although the pathobiology of atherosclerosis is a complex multifactorial process, blood flow-induced shear stress has emerged as an essential feature of atherogenesis. This fluid drag force acting on the vessel wall is mechanotransduced into a biochemical signal that results in changes in vascular behavior. Maintenance of a physiologic, laminar shear stress is known to be crucial for normal vascular functioning, which includes the regulation of vascular caliber as well as inhibition of proliferation, thrombosis and inflammation of the vessel wall. Thus, shear stress is atheroprotective. It is also recognized that disturbed or oscillatory flows near arterial bifurcations, branch ostia and curvatures are associated with atheroma formation. Additionally, vascular endothelium has been shown to have different behavioral responses to altered flow patterns both at the molecular and cellular levels and these reactions are proposed to promote atherosclerosis in synergy with other well-defined systemic risk factors. Nonlaminar flow promotes changes to endothelial gene expression, cytoskeletal arrangement, wound repair, leukocyte adhesion as well as to the vasoreactive, oxidative and inflammatory states of the artery wall. Disturbed shear stress also influences the site selectivity of atherosclerotic plaque formation as well as its associated vessel wall remodeling, which can affect plaque vulnerability, stent restenosis and smooth muscle cell intimal hyperplasia in venous bypass grafts. Thus, shear stress is critically important in regulating the atheroprotective, normal physiology as well as the pathobiology and dysfunction of the vessel wall through complex molecular mechanisms that promote atherogenesis.     

Extracellular matrix remodeling in the vascular wall.

The extracellular matrix provides a structural framework essential for the functional properties of vessel walls. The three dimensional organization of the extracellular matrix molecules--elastin, collagens, proteoglycans and structural glycoproteins--synthesized during fetal development--is optimal for these functions. Early in life, the vessel wall is subjected to injury: lipid deposition, hypoxia, enzyme secretion and reactive oxygen species production during inflammatory processes, and the extracellular matrix molecules are hydrolyzed by proteases--matrix metalloproteinases, leukocyte elastase, etc. In uninjured arteries and veins, some proteases are constitutively expressed, but through the control of their activation and/or their inhibition by inhibitors, these proteases have a very low activity. During the occurrence of vascular pathologies--atherosclerosis, hypertension, varicosis, restenosis, etc.--the balance between proteases and their inhibitors is temporally destroyed through the induction of matrix metalloproteinase gene expression or the secretion of enzymes by inflammatory cells. Smooth muscle cells, the most numerous cells in vascular walls, have a high ability to respond to injury through their ability to synthesize extracellular matrix molecules and protease inhibitors. However, the three dimensional organization of the newly synthesized extracellular matrix is never functionally optimal. In some other pathologies--aneurysm--the injury overcomes the responsive capacity of smooth muscle cells and the quantity of extracellular matrix decreases. In conclusion, care should be taken to maintain the vascular extracellular matrix reserve and any therapeutic manipulation of the protease/inhibitor balance must be perfectly controlled, because an accumulation of abnormal extracellular matrix may have unforeseen adverse effects.     

The renin-angiotensin system and extracellular matrix

Summary A hallmark of vascular disease is the inappropriate proliferative and synthetic behaviour of vascular smooth muscle cells. This phenotypically immature behaviour arises as a consequence of the myocytes undergoing phenotypic conversion and/or clonal proliferation of a fetal type of smooth muscle cell preexisting in the vessel wall. De-differentiation and initiation of proliferation is not only induced by endothelial desquamation and acute exposure of smooth muscle cells to platelet-derived mitogens, but also occurs in the uninjured blood vessel. Therefore normal components of the blood vessel are implicit in the pathological process. These include vasoconstrictor peptides, growth factor peptides and extracellular matrix molecules. In vitro and in vivo experimentation has indicated that while some of these compounds individually are only mild stimulators of smooth muscle proliferative metabolism, they may act synergistically to induce robust responses. Here we discuss the effects of the vasoconstrictor peptide angiotensin II, which can be locally generated within the vessel wall itself, on the expression of extracellular matrix molecules in vitro and in vivo. We focus on the angiotensin II-modulated expression of extracellular matrix glycoproteins, e.g. thrombospondin, tenascin, fibronectin and laminin.Abbreviations VSMC vascular smooth muscle cell - ECM extracellular matrix - Ang II Angiotensin II - ACE angiotensin converting enzyme - PDGF platelet-derived growth factor - TGFß transforming growth factor     

Adventitial lymphatic vessels -- an important role in atherosclerosis

Arterial inflammation is a significant component of atherosclerotic disease-specific immune responses directed against autoantigens or pathogen-derived antigens in the vascular wall could initiate and/or maintain atherosclerotic processes. Atherosclerosis is now regarded as a chronic inflammatory disease. Developing in response to injury in the vessel wall, it is characterized by the infiltration of mononuclear lymphocytes into the intima, local expansion of vascular smooth muscle cells, and accumulation of extracellular matrix. A number of potential mechanisms have been implicated in the development of inflammatory reactions in the vascular system. Adventitia provides cells and molecules with the ability to influence neointimal formation and vascular remodeling implemented in part by vasa vasorum. We hypothesize that lymphatic vessels, existing in adventitia in the atherosclerotic artery, could drain local inflammatory cells and cytokines to the lymphatic nodes and lymphoid tissues where inflammatory cells can be sensitized and activated. Or, blood vessels may deliver sensitized inflammatory cells and cytokines to the inflammatory site of the vascular wall. Therefore, both lymphatic and blood vessels constitute a complete circle of immune response, whereby the inflammatory cells and cytokines are effectively delivered to tissues and their effects magnified. Under certain circumstances, this situation may lead to a vicious circle of inflammation such as in atherosclerosis, resulting in perpetuating intimal hyperplasia and vascular remodeling. Inhibition of lymphangiogenesis may interrupt this self-perpetuating vicious circle of inflammation in atherosclerosis and provide a new approach to the prevention and treatment of the disease.     

Vascular oxidant stress and inflammation in hyperhomocysteinemia

Elevated plasma levels of homocysteine are a metabolic risk factor for atherosclerotic vascular disease, as shown in numerous clinical studies that linked elevated homocysteine levels to de novo and recurrent cardiovascular events. High levels of homocysteine promote oxidant stress in vascular cells and tissue because of the formation of reactive oxygen species (ROS), which have been strongly implicated in the development of atherosclerosis. In particular, ROS have been shown to cause endothelial injury, dysfunction, and activation. Elevated homocysteine stimulates proinflammatory pathways in vascular cells, resulting in leukocyte recruitment to the vessel wall, mediated by the expression of adhesion molecules on endothelial cells and circulating monocytes and neutrophils, in the infiltration of leukocytes into the arterial wall mediated by increased secretion of chemokines, and in the differentiation of monocytes into cholesterol-scavenging macrophages. Furthermore, it stimulates the proliferation of vascular smooth muscle cells followed by the production of extracellular matrix. Many of these events involve redox-sensitive signaling events, which are promoted by elevated homocysteine, and result in the formation of atherosclerotic lesions. In this article, we review current knowledge about the role of homocysteine on oxidant stress-mediated vascular inflammation during the development of atherosclerosis.     

Serum stimulation, cell-cell interactions, and extracellular matrix independently influence smooth muscle cell phenotype in vitro.

Vascular injury profoundly alters the vessel wall microenvironment, and smooth muscle cells respond with cell cycle re-entry, loss of contractile elements, extracellular matrix remodeling, and altered signaling by endogenous growth factors and their receptors. Environmental cues include stimulation by exogenous mitogens and both cell-cell and cell-matrix interactions. Modeling this process in smooth muscle cells in vitro, these environmental determinants were varied independently and the phenotypic consequences assessed. Mitogenic stimulation with serum promoted the synthesis of collagen and fibronectin and the expression of fibroblast growth factor receptor-1 and suppressed the content of smooth muscle alpha-actin, myosin heavy chain, and basic fibroblast growth factor. Low cell density (reduced cell-cell contact) was also associated with enhanced extracellular matrix protein production, increased fibroblast growth factor receptor-1 expression, and reduced contractile protein and basic fibroblast growth factor content. The influence of serum stimulation and reduced cell-cell contact were independent and additive. Provision of a type I collagen matrix blunted the influence of serum and cell-cell contact on collagen synthesis but had minor effects on other measures of phenotype. Environmental factors thus independently influence smooth muscle cell phenotype, including endogenous growth factor expression and responsiveness, which can in turn influence the microenvironment of the vessel wall after injury.     

Autoimmune vasculitis resulting from in vitro immunization of lymphocytes to smooth muscle.

Lymphocytes sensitized in vitro to syngenic microvascular smooth muscle and transferred to syngeneic recipients produced in vivo microvessel vasculitis characterized by mononuclear cells which adhered to endothelium, infiltrated the vessel wall, and formed a perivascular cuff. A granulomatous type of vascular inflammation was seen in 20% of the affected recipients in which the vessel smooth muscle appeared to be preferentially attacked. These lesions bear a striking resemblance to certain human vasculitides, and the model provides an important means of studying vasculitis as well as general cellular autoimmune disease mechanisms.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter.

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Proliferative changes in the pulmonary arterial wall during short-term hyperoxic injury to the lung.

Injury to the lung during in vivo exposure to hyperoxia results in vascular restructuring and pulmonary hypertension. This study reports the pattern of cellular proliferation that occurs in proximal intrapulmonary arteries over time during vessel wall injury and adaptation to increased partial pressures of oxygen. Although the remodeling of the capillary bed has been emphasized particularly during oxygen injury to the lung, this report identifies significant proliferative changes within the vessel wall of proximal arterial segments isolated from rats exposed to 85% oxygen. An increased incorporation of 3H-thymidine by endothelial cells is the earliest and most dramatic vessel wall response. The labeling index of these cells is increased more than tenfold by the end of 7 days in hyperoxia. Proliferation of medial smooth muscle cells and adventitial fibroblasts is also significantly increased. The increased cell number within these compartments is noted especially for its contribution to the overall vessel wall hypertrophy observed in chronic hyperoxic pulmonary hypertension. This general proliferative response is accompanied by specific shifts in the relative percentages of different actin protein isoforms as identified by two-dimensional gel electrophoresis. Changes in the distribution of actin isoforms are discussed as potential markers of a phenotypic modulation among vascular smooth muscle cells that occurs during the progression of pulmonary vessel wall remodeling.     

Thrombospondin deposition in rat carotid artery injury.

The balloon catheter injury model was used to determine the relative contributions of vascular smooth muscle cells (SMC) and platelets to thrombospondin (TSP) antigen deposition in the artery wall. Rat carotid arteries were denuded of endothelium, exposing the thrombogenic subendothelial extracellular matrix (ECM) to the circulation. Rats were killed after 1 hour, or 5, 10, or 20 days. Thrombospondin antigen deposition in the injured arteries was assessed using a specific polyclonal antiserum raised in rabbit against rat platelet TSP and a sensitive silver-enhanced immunogold staining method. Faint immunostaining for TSP antigen was detected, associated mostly with cells, in the media of the carotid artery of the nonoperated controls. One hour after balloon catheter injury, however, prominent cell-associated immunostaining was evident in the media; extracellular matrix staining was negligible. At this time, large foci of immunostaining were present on the lumenal surface of the vessel. Intimal proliferation was evident on most stained sections of tissue taken 5 days after balloon injury. Thrombospondin antigen immunostaining was markedly increased compared to nonoperated controls in all sections, regardless of the degree of intimal thickening. Thrombospondin immunostaining remained associated with cells in the neointima and media; extracellular matrix staining remained negligible. Ten days after endothelial injury, immunostaining for TSP antigen was detected in all layers of the artery, but was greater in the neointima and media. Reaction product was still associated only with cells. Thrombospondin antigen levels, as detected by this procedure, remained high in the injured tissue through 10 days of observation but appeared less prominent 20 days after injury. At this time extracellular matrix staining was obvious and cell-associated staining was reduced. These data support the hypotheses that thrombospondin (TSP) expression by vascular smooth muscle cells is an early response to injury and that the primary source of TSP antigen in injured artery is the vascular smooth muscle cells (SMC). These results support data derived from in vitro studies of TSP secretion.     

Responses of inner and outer muscle of the sheep carotid artery to injury.

1. Direct injury to the smooth muscle of the sheep carotid artery in vivo caused large, persistent and sharply limited annular contraction, even with tetrodotoxin 10(-5)M present to block nerves. 2. Surcose gap records from artery strips showed that mechanical injury caused slow, prolonged depolarization of the smooth muscle that spread for a few millimetres in a circular direction in relation to the intact artery wall with an apparent space constant of 1-26--3.49 mm. In the longitudinal direction, no depolarization was recorded 1 mm from the site of injury. No spikes were recorded more than 1 mm in either direction from the site of injury except when procaine, which facilitates electrical activity in the smooth muscle, was present. 3. When responses of inner and outer muscle were recorded separately, injury caused comparable contraction of both parts. 4. Clotted blood caused large contractions of intact artery strips; it contracted inner much more than outer muscle. 5. The main factors in the intact vessel's response to injury therefore seem to be inner and outer muscles' direct response to injury, reinforced by spread of depolarization round the vessel wall, and inner muscle's response to vasoconstrictor agents released by clotting blood.     

Role of blood platelets in thrombosis of the arteries and artificial surfaces

Blood platelets play an important role in thrombus formation in arteries and on artificial surfaces and in the development of atherosclerosis under hemodynamic conditions of high blood flow. This paper reviews the biological mechanisms implicated in the interactions of blood with vascular and artificial surfaces and, in particular, the role of endothelium injury, platelet activation pathways, the thrombogenic properties of the injured vessel wall and the interactions between blood cells (platelets, monocytes), growth factors and the endothelial and smooth muscle cells of the vessel wall.