Vascular Pathobiology: Atherosclerosis and Large Vessel Disease

Fatty streaks

Aortic fatty streaks are observed in early childhood and even in neonates; however, in the coronary artery, they appear at about 10 years of age. Fatty streaks are flat or slightly elevated and generally vary in size from 3 to 5 mm. They consist of many lipid-laden foam cells that contain cholesteryl esters and a variable amount of extracellular lipid . These cells, found in the intima, are mainly macrophages derived from blood monocytes, however, some are SMCs. Modified LDL ingested through the scavenger receptor-mediated pathway is a common mechanism to promote foam cell formation. Lesion transition is characterized by deep intimal extracellular deposition of cholesterol-rich vesicular lipid deposits as very early plaques begin to form . The presence of cholesterol crystals in the extracellular lipid pool may induce further foam cell formation and inflammation to promote fibroinflammatory lipid plaque formation. There is no fibrotic component to these flat fatty streak lesions. Women between the ages of 15 and 34 years have more extensive fatty streaks than men but do not end up with more raised plaques. Fatty streaks are reversible and are seen to the same extent in the aortas of the young when comparing populations that do and do not develop atherosclerosis in later life. At present, there is no marker that enables differentiation between those fatty streaks that will become fibroinflammatory lipid plaques and those that regress. Therefore it is likely that other factors interact with the fatty streak to promote progression toward a fibroinflammatory lipid plaque. For example, fatty streaks tend to progress to advanced lesions in areas of artery flow dividers and branch points probably related to altered hemodynamic forces.

Intimal thickening

Diffuse intimal thickening shows increased SMCs and extracellular matrix (ECM). Intimal thickening begins in the aorta during childhood and progresses throughout life, often present at flow dividers leading to eccentric intimal thickening. Excessive thickening leads to the development of the central necrotic core of the plaque since at beyond 300 μm thickness, diffusion of nutrients in the intima is disrupted leading to focal ischemia and necrosis at the intima–media border, resulting in the development of the fibroinflammatory lipid plaque.

Gelatinous lesions

Gelatinous lesions or elevations, soft translucent thickenings generally 2–4 mm in diameter, are characterized by edematous swelling of the intima that demonstrate reduced metachromasia in tissue sections stained with toluidine blue, compared with those in the normal vessel wall. The connective tissue is separated, and there is a decreased cellularity in the lesion relative to normal tissue. They contain little or no lipid and undergo transition to fibrous plaques . These lesions have not been extensively studied and are not referred to in many of the current theories of atherogenesis.

Mural microthrombi

The presence of rare fresh mural microthrombi has been described as a single platelet or an aggregate of platelets in contact with a normal endothelial cell. These microthrombi may contain fibrin. Very occasional microthrombi could be seen incorporated into the vessel wall. It is very unlikely that these rare findings result in pathobiologic effect because there is no evidence that the contact promoted functional changes in either platelet or endothelial cell.

Precursor lesions, endothelial dysfunction, lipoprotein metabolism, macrophage activation

Atherosclerotic plaques initially develop as intimal precursor lesions at sites which human and animal studies have shown are predisposed to lesion formation. Mild endothelial dysfunction with mild altered permeability and/or accumulation of intimal SMCs is present at these sites. The former often results in fatty streak formations while the latter occurs as an intimal SMC mass, often located at arterial branch points. Endothelial cell injury may occur in a variety of ways including by altered hemodynamic shear stress, viral or microbial agents, immune mechanisms, systemic inflammatory agents, cytokines, chemokines, and the formation of ROS. Some recent observations suggest that in coronavirus disease 2019 (COVID-19) the virus may induce significant endothelial injury and dysfunction inducing thrombosis and thromboembolic events . At present it is too early to know whether this pandemic disease may predispose to atherosclerosis in survivors of the acute condition. The injuries noted above lead to further disruption of the integrity of the endothelial barrier leading to increased endothelial permeability . This can occur as follows: VE-cadherin mediates adherens junctions between ECs and extracellular homophilic binding. Intracellularly, it is stabilized through binding to the actin cytoskeleton mediated by α- and β-catenin. Sphingosine-1 phosphate 1 (S1P1) maintains EC integrity while S1P2 disrupts adherens junctions promoting paracellular permeability. Increased permeability allows lipoproteins to penetrate the vessel wall. Lipoproteins are lipid–protein complexes composed of cholesterol, triacylglycerol, phospholipids, and an apoprotein molecule, which normally permits the complex to circulate in the blood stream. These lipoproteins may be modified through oxidization and acetylation or other enzymatic processes. Oxidized LDLs are formed once LDL particles accumulate in the vessel wall and are oxidized by toxic oxygen species produced and released by macrophages as well as by NADPH oxidase (NOX) and secretory phospholipase A2. Lectin-like oxidized LDL (LDX-1) is the receptor that binds oxLDL to endothelial cells and initiates signaling. Once in the intima, oxLDL taken up by macrophage scavenger receptors induces the macrophage proinflammatory phenotype and activates macrophages to secrete chemokines, cytokines, and proteolytic enzymes. At this stage, additional are macrophages recruited to enter the wall. Normally, cholesterol enters the cell through the LDL receptor (LDLR), a family of endocytic receptors that are important in cholesterol homeostasis. Cholesterol acts to inhibit receptor recycling and HMGCoA reductase, an enzyme important in intracellular cholesterol synthesis. LDLR expression is regulated in part by proprotein converted subtilism/kexin type 9, which may be a valuable therapeutic target for atherosclerosis when its activity is inhibited . Modified LDL undergoing oxidation or acetylation becomes a high affinity ligand for a family of scavenger receptors on the cell surface which internalize LDL through pathways that are not inhibited by intracellular cholesterol. Thus influx is poorly regulated and although it may be initially considered a protective response, eventually pathways to metabolize modified lipoproteins are overwhelmed and there is a buildup of excessive free cholesterol which is toxic to the cell and leads to cell death. This sets the stage for plaque development.

ECM alterations

With more severe endothelial injury, lipids accumulate at a faster rate. The ECM present in the intima, including proteoglycans, fibronectin (FN), collagens, and elastin, all bind lipids with high affinity, which sequesters lipid in this layer. Once ingested by macrophages, oxLDL enhances the secretion of proteoglycans including biglycan and versican which promote high affinity binding of extracellular lipid. Decorin, a small leucine-rich proteoglycan, does not promote binding.

Inflammation

Monocyte recruitment to the plaque is mediated by monocyte chemotactic protein-1 (MCP-1), macrophage colony stimulating factor (M-CSF), and other cytokines. M-CSF promotes differentiation of monocytes to macrophages. Upregulation of cell adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) occurs in endothelial cells (ECs). Macrophage accumulation is enhanced by macrophage inhibitory factor and its receptors CXCR2 and CXCR4, the former secreted by EC, SMC, and macrophages upon stimulation by oxLDL . The macrophages and intimal SMCs accumulate modified lipids via scavenger receptors, especially CD36, leading to the development of foam cells. Activated macrophages release growth factors and cytokines, including tumor necrosis factor (TNF), which promotes further entry of macrophages and more T and B lymphocytes to enhance adaptive immunity. The macrophages may undergo cell death due to the excessive intracellular lipid. Cholesterol crystals also promote inflammation as they accumulate in the plaque. These crystals activate the caspase-1-activating NLRP3 inflammasome which results in secretion of IL-1β family of cytokines which in turn activate leukocytes .

Besides innate immunity, adaptive immunity also plays a role, although less well defined. For example, autoantibodies against oxLDL are candidate autoantigens that activate T cells. It is the oxidation of LDL in the vessel wall that generates these immunogenic epitopes called oxidation-specific epitopes which are target pattern recognition receptors making them immunogenic .

Early plaque growth

Besides prominent inflammation, the early plaque can grow in several ways involving all three vascular layers. Endothelial injury due to several factors including hypercholesterolemia, hyperglycemia, cigarette smoking, and viral infections leads to a reduction in the anticoagulant, antiplatelet, and fibrinolytic properties of the vessel wall, predisposing it to mural thrombosis. This stimulates the release of growth factors including fibroblast growth factor and platelet-derived growth factor (PDGF) and further accelerates SMC migration and proliferation into the intima and the secretion of matrix components . Small mural thrombi become organized and are incorporated into the plaque. Consequentially, the intima is thickened, its deeper layers are poorly nourished and undergo necrosis. Oxidized lipid and proteolytic enzymes released by macrophages cause additional tissue damage. Inefficient clearance of dead cells, termed efferocytosis, contributes to the formation of the necrotic core. The plaque is formed as a fibrous cap develops separating the central necrotic core from the vascular lumen. The fibrous cap is rich in SMCs, collagen, and elastin fibers and serves to protect the weakened necrotic core against plaque rupture. Angiogenesis promotes vascularization of the plaque with the vessels arising from the vasa vasorum of the adventitia and outer media. The plaque becomes heterogenous with respect to SMC localization, inflammatory cell infiltration, and matrix organization. Cell turnover does occur as plaques show low levels of SMC and macrophage proliferation and apoptosis.

There are processes that help to moderate the growth of the plaque. Important transcription factors such as Krüppel-like factor 2 (KLF2) and Krüppel-like factor 4 (KLF4) are antileukocyte adhesive and antithrombotic. KLF2 reduces the expression of proinflammatory cytokines, tissue factor (TF), and lipid expression in macrophages . This appears to limit macrophage activation and favor the macrophage phenotype M2, the repair phenotype, over the M1 proinflammatory phenotype.

Human and experimental in vivo and in vitro studies show that the adventitia is a dynamic microenvironment in which adventitial cells initiate and regulate important vascular functions in atherosclerosis . Although well away from the blood–wall interface where much pathology has been identified, the adventitia has a profound influence on the population of intimal and medial endothelial, macrophage, and SMC function . This regulates biologic processes including fibroblast and myofibroblast migration and proliferation, inflammation, immunity, stem cell activation and regulation, ECM remodeling, and angiogenesis. A debate exists as to whether the adventitia initiates disease or is just an important participant. Vascular injury and dysfunction of the PVAT which is loosely attached to the adventitia promote expansion of the vasa vasorum, activation of fibroblasts, differentiation of myofibroblasts, and activation of adipocytes which release adipokines.

Vessel wall remodeling

The artery wall adapts to the presence of an atherosclerotic plaque. As the plaque encroaches on the vessel lumen, the wall of the vessel undergoes remodeling lending to structural changes that dilate the artery wall in order to maintain normal lumen size. Hemodynamic shear stress is an important regulator through endothelial mechanotransduction. It is likely that cell proliferation, apoptosis, and both matrix synthesis and degradation are important factors in remodeling the artery wall. It is also likely that the plaque itself undergoes remodeling and that apoptosis and efferocytosis are important processes since factors associated with their regulation are found in plaques. Once a plaque encroaches on about 45% of the lumen, compensatory remodeling cannot maintain lumen size and it narrows leading to luminal stenosis.

Continual plaque growth

Plaques continue to grow in the adaptation stage by dynamic interactions involving SMCs, macrophages, lymphocytes, matrix synthesis, matrix degradation, and neovascularization. Surface thrombi may develop and be incorporated into the plaque. Mural thrombosis occurs as a result of disrupted blood flow around the plaque, at the site of its protrusion into the lumen. Hemorrhage arising from new, fragile vessels within the plaque may also increase plaque size. Plaque rupture may occur during this stage of mild to moderate plaque formation and either produce a sudden thrombotic clinical event or undergo a process of healing so that the thrombus does not occlude the lumen and becomes incorporated into the plaque.

Plaque complications

Several complications develop which destabilize the plaque and result in manifestations of serious clinical disease. These include surface erosion and ulceration, fissure formation, intraplaque hemorrhage, plaque rupture, mural and occlusive thrombosis, calcification, and aneurysm formation with or without artery wall rupture ( Fig. 7.8 ).

Advanced plaque growth

Processes described in early growth continue with emphasis on innate and acquired immune processes, further macrophage and lymphocyte accumulation, further lipid accumulation, SMC proliferation, ECM expansion by remodeling of matrix macromolecules, intraplaque hemorrhage, and incorporation of mural thrombi ( Fig. 7.9 ). The expansion of the central necrotic core may occur by continued macrophage activation by cytokines and by activation of NOD2 which promotes influx of macrophages and lymphocytes and accumulation of intracellular lipid by scavenger receptor-mediated uptake of oxLDL and by inhibition of HDL-mediated cholesterol efflux from macrophages, likely due to downregulation of the reverse cholesterol ATP-binding cassette transporter (ABCA1) . In the adventitia, fibroblasts, myofibroblasts, and inflammatory cells continue to become activated, and stem/progenitor cells differentiate into SMC myofibroblasts, and macrophage-like cells, osteocytes, and chondrocytes, which may populate the plaque and surrounding tissue . The PVAT also contributes to growth especially with activated adipocytes secreting adipokines . Advanced plaque growth leads to severe luminal stenosis. Severe luminal stenosis (greater than 75% in the coronary arteries) and/or superimposed thrombotic vascular occlusion lead to clinical disease, including CHD (angina pectoris, acute myocardial infarction, sudden death), stroke, and peripheral vascular disease.

Plaque rupture

The catastrophic event of plaque rupture with ensuing thrombosis and occlusion of the vascular lumen usually occurs in advanced plaques. However, angiographic studies suggest that even plaques causing less than 50% lumen stenosis may rupture. Therefore a plaque’s stability is not directly correlated with size or age of the plaque but with active biologic processes occurring within. Often there is clinical evidence of destabilization of the coronary artery plaque prior to rupture due to the occurrence of thromboatherosclerotic emboli or transient mural thrombi. However, rupture may produce the first sign or symptom of clinical disease. Plaque destabilization and rupture are important processes leading to ACS which includes ST-segment elevation myocardial infarction, non-ST-segment elevation myocardial infarction, and unstable angina . Plaque rupture results from disruption of the fibrous cap due to structural or functional changes within the cap or at its surface usually at the shoulder of the plaque. It is hypothesized that interactions of physical hemodynamic forces with proteolytic degradative processes associated with metalloproteinases in the fibrous cap lead to a weakening of the plaque, especially at the interface of normal artery and plaque. Plaque rupture occurs at sites of thinning of the fibrous cap, at sites of foam cell accumulation and lipid deposits in the cap, in association with areas of decreased smooth muscle cellularity in the cap, and near sites with microcalcifications. Conditions believed to cause plaque rupture include endothelial denudation with ulceration or fissure formation, hemodynamic shear stress at the plaque shoulder, the presence of a thin fibrous cap, apoptosis of SMCs within the cap, inflammatory activity at the interface between an area of lipid deposition and fibrous tissue, and the presence of matrix metalloproteinases (MMPs) released by activated macrophages, SMCs, and endothelial cells that digest ECM ( Table 7.3 ). In addition, another pathway to rupture is associated with hemorrhage from thin, newly formed vessels within the plaque. The hemorrhage may expand the plaque and rupture the fibrous cap from inside out. Once the plaque ruptures, the thrombogenic material in the plaque often triggers thrombosis in the lumen resulting in the formation of an occlusive thrombus. However, some ruptures heal without producing an acute clinical event. It is not known why some ruptures behave this way.