Diseases of Medium-Sized and Small Vessels

Introduction

Vascular diseases extract a large toll on the human population, both in terms of mortality and quality of life. Vascular disease is in fact the leading cause of death in developed countries. Numerous pathologic conditions alter the structure and/or function of blood vessels. This chapter will cover the mechanisms of common disorders affecting medium-sized and small blood vessels, which are not discussed elsewhere in this book. However, the basic mechanisms of vascular activation discussed in this chapter have relevance to many conditions discussed elsewhere in the book, including thrombosis ( Chapter 15 ), angiogenesis ( Chapter 10 ), atherosclerosis ( Chapter 12 ), aneurysms ( Chapter 13 ), and hypertension ( Chapter 14 ). This chapter will only focus on several of the more common vascular diseases. Additional information on less common vascular pathologic conditions not covered in this chapter or elsewhere in this book can be found in the end-noted textbooks.

Normal Vessel Wall Structure

The primary components of normal blood vessels are endothelial cells, vascular smooth muscle cells, and extracellular matrix including collagen, elastin, and proteoglycans. Structurally, blood vessels are composed of three distinct concentric layers: intima, media, and adventitia. The intima refers to the inner portion of the vessel wall, and in normal vessels is composed primarily of a single layer of endothelium with its basement membrane and subendothelial connective tissue. In small and medium-sized muscular arteries, the intima is separated from the next layer, the media, by a discrete layer of elastic fibers called the internal elastic lamina. The media is composed primarily of vascular smooth muscle cells. In muscular arteries, the media is separated from the outer adventitial layer by a second discrete layer of elastic fibers called the external elastic lamina. In elastic arteries, such as the aorta and its proximal branches, rather than discrete internal and external elastic lamina, there is a mesh of elastic fibers throughout the media, between the smooth muscle cells.

The adventitia is composed primarily of collagen and fibroblasts. It is becoming clear that the adventitia may serve as a reservoir of multipotent stromal cells, or stem cells, that can facilitate vascular responses to injury by differentiating into fibroblasts, endothelial cells, and vascular smooth muscle cells. Large vessels and some medium-sized vessels contain capillaries and other small vessels in the adventitia. In the aorta, this vasa vasorum infiltrates into the outer third of the media, contributing to the blood supply of the vessel wall. In normal medium-sized and small vessels, the media is typically not vascularized. The adventitia may also contain nerves, which innervate the vessel to regulate vasoconstrictor tone.

Proceeding from the aorta distally, the arteries branch to give rise to progressively smaller vessels. The medium-sized distributing arteries carry blood to the individual organs, and the small intraparenchymal arteries carry blood within the organs themselves. Both small arteries and medium-sized arteries are generally referred to as medium-sized vessels. The small arteries continue to branch until giving rise to arterioles, which contain only one to two layers of smooth muscle in the media. The arterioles branch to eventually become capillaries, which are essentially endothelial tubes lacking a defined media. Capillaries are surrounded and supported by interspersed pericytes. The structure of capillaries and their combined high luminal surface area facilitate diffusion of oxygen and nutrients to the surrounding tissues. The capillaries coalesce to form post-capillary venules, which themselves join to form veins. In contrast to arteries, veins have thinner walls relative to the luminal diameter, and less compact smooth muscle in the media with more extracelluar matrix ( Figure 11.1 ). Medium-sized and large veins will also contain longitudinal bands of smooth muscle outside of the standard concentric layers of smooth muscle. Large veins will also contain valves on the luminal surface to facilitate unidirectional blood flow. Such venous valves are particularly important in the large veins of the legs.

Vascular Cell Activation

Vascular cells respond to injury and other stimuli by undergoing phenotypic changes referred to as activation. The endothelium plays important roles in vascular responses, as this cell sits at the border between the luminal blood and the vessel wall. Endothelial cells are critical for maintaining a non-thrombogenic surface in normal circumstances and for regulating vasodilation and vasoconstriction. Endothelial cells are responsive to the shear stress generated by the blood flowing across its surface. Under normal non-activating conditions of laminar shear stress, endothelial cells align with the direction of the blood flow, and primarily secrete agents that prevent coagulation and foster vasodilation, such as nitric oxide and prostacyclin. However, at branch points in the vasculature, the steady laminar flow of blood is disrupted resulting in an altered and abnormal pattern of shear stress on the endothelium. In these sites of disturbed flow, endothelial cells lose their orderly alignment with the flow of blood and secrete increased amounts of agents that promote coagulation and/or vasoconstriction such as endothelin, von Willebrand factor, and superoxide. Endothelial cells are in fact the predominant source of reactive oxygen species in the normal vessel wall. Such endothelial activation is also termed endothelial dysfunction, since the endothelial cells become dysfunctional in facilitating vasodilation and preventing coagulation. The superoxide that is generated will inactivate nitric oxide. Nitric oxide would otherwise promote vasodilation and inhibit platelet aggregation.

The activated endothelium also secretes growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), stromal cell-derived factor 1 (SDF-1), and another reactive oxygen species hydrogen peroxide. Hydrogen peroxide is rapidly formed from superoxide by the action of superoxide dismutase. These growth factors activate the underlying vascular smooth muscle cells. The activated endothelial cells also play important roles in regulating inflammatory responses in the vessel wall by expressing cell adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P-selectin and E-selectin, which bind circulating leukocytes to facilitate their entry into the vessel wall. Endothelial cells also produce inflammatory mediators, such as interleukins (IL) including IL-1β, IL-6, and IL-8, and also tumor necrosis factor α (TNF-α), monocyte colony stimulating factor (M-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), and monocyte chemotactic protein 1 (MCP-1), which attract inflammatory cells and modulate their functions.

Activation of endothelial cells is orchestrated by multiple transcription factors. Endothelial responses to the shear stresses imparted by the flow of blood over the luminal surface are mediated primarily by two transcription factors, Kruppel-like factor 2 (KLF2) and nuclear factor erythroid-2-related factor-2 (Nrf2). Steady laminar shear stress, as is present in non-branching segments of the vasculature, stimulates expression of KLF2 and Nrf2, which help to maintain the endothelial cells in a relatively quiescent non-activated state. KLF2 inhibits induction of adhesion molecules by cytokines such as IL-1β, thus limiting the binding of inflammatory leukocytes to the endothelial cell surface. KLF2 also promotes the expression of nitric oxide synthase, which generates nitric oxide. As mentioned above, nitric oxide promotes vasodilation and prevents coagulation. Nrf2 promotes the expression of several genes that combat oxidative stress. With disturbed non-laminar flow, the expression of KLF2 and Nrf2 is decreased in endothelial cells, allowing the cells to acquire an activated phenotype, characterized by enhanced surface adhesion molecule expression, decreased nitric oxide generation, and increased oxidative stress. These changes promote increased coagulation, vasoconstriction, and entry of inflammatory cells into the vessel wall.

In addition to disturbed laminar flow, endothelial cells are directly activated by other stimuli including infection, mechanical injury, radiation therapy, and cytokines released from inflammatory cells and other activated vascular cells. Cytokines such as TNF-α, IL-1, and IL-4 stimulate endothelial cells to express adhesion molecules for inflammatory cells, such as VCAM-1 and ICAM-1. Many of the cytokine-stimulated effects on endothelial cells are mediated by the activation of the transcription factor NF-κB. In endothelial cells, NF-κB promotes the expression of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. NF-κB also stimulates the expression of cytokines and immune modulators such as GM-CSF, M-CSF, MCP-1, IL-1, IL-6, and TNF-α. In addition to being activated by cytokines such as TNF-α, NF-κB is also activated by toll-like receptors. Toll-like receptors are pattern recognition receptors that bind universal danger signals, such as structural components present on bacterial and viral surfaces.

Like endothelial cells, vascular smooth muscle cells also undergo activation. In the normal state, vascular smooth muscle cells are considered as displaying a contractile phenotype. The cells are rich in contractile apparatus proteins such as smooth muscle α-actin, smooth muscle myosin, SM-22α, smoothelin and h-caldesmon. The primary function of contractile vascular smooth muscle cells is to maintain blood pressure and normal vascular wall structure in the setting of pulsatile cyclic strain. Vascular smooth muscle cells are responsive to various stimuli including growth factors and cytokines released by the endothelium and infiltrating leukocytes, injured cells, and alterations in cyclic strain. Smooth muscle cells express receptors for endothelial-derived growth factors. These receptors include platelet-derived growth factor receptor and CXCR4, the receptor for SDF-1. Smooth cells respond to these stimuli by a phenotypic transition from the contractile state to a synthetic or activated state. The transition from the contractile to the synthetic phenotype is accompanied by a decrease in the expression of contractile proteins, and an increase in secretory vesicles. This phenotypic switch is accompanied by an increased production of extracellular matrix components, particularly proteoglycans such as versican and biglycan ( Figure 11.2 ). Activated smooth muscle cells also secrete increased amounts of matrix metalloproteinases (MMPs), which facilitate extracellular matrix remodeling. Activated vascular smooth muscle cells show increased expression of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear mRNA binding protein that facilitates mRNA processing and nuclear export. Activated smooth muscle cells may also acquire a pro-inflammatory phenotype, expressing inflammatory cytokines, surface adhesion molecules such as VCAM-1, and the transcription factor NF-κB. Activated smooth muscle cells may demonstrate increased rates of proliferation and have increased expression of nuclear proteins that facilitate cell division such as proliferating cell nuclear antigen (PCNA), which promotes DNA synthesis by DNA polymerase.

Key activators of vascular smooth muscle cells include growth factors such as platelet-derived growth factor, which engage surface receptor tyrosine kinases. In addition, other mediators, such as angiotensin-II, activate G-protein-coupled receptors. Cell surface receptor activation stimulates intracellular protein kinase signaling cascades, including activation of mitogen-activated protein (MAP) kinases. Hydrogen peroxide released from adjacent cells including endothelial cells also directly stimulates smooth muscle cells to proliferate, but the specifics of how this occurs are not completely understood. In contrast, transforming growth factor β (TGF-β) drives smooth muscle cells towards the contractile phenotype by activating a group of transcription factors known as Smads, which up-regulate the expression of contractile proteins.

Intimal Hyperplasia

Intimal hyperplasia refers to a process in which the intima becomes thickened due to the presence of vascular smooth muscle cells and proteoglycan-rich extracellular matrix located between the endothelium and the internal elastic lamina ( Figure 11.3 ). This pathologic change is also referred to as neointimal hyperplasia and intimal thickening, in different settings. Since this process is associated with an increase in the number of cells, it is by definition a form of hyperplasia. The formation of intimal hyperplasia is often linked with vascular cell activation. Numerous factors promote the formation of intimal hyperplasia such as vascular wall injury, aging and inflammation. Non-laminar shear stress, particularly at branch points in the vasculature, results in a mild form of intimal hyperplasia often referred to as intimal thickening. Marked increases in blood pressure can stimulate intimal hyperplasia, such as occurs in the small arteries of the lungs in the setting of pulmonary hypertension.

Intimal hyperplasia may be either eccentric or concentric. Eccentric intimal hyperplasia often develops at branch sites due to the eccentric nature of altered shear stress at these locations. Such eccentric intimal hyperplasia induced by disturbed flow often progresses to a more concentric form with time. Vascular wall inflammation and injury may also be eccentric and stimulate the formation of eccentric intimal hyperplasia. In contrast, circumferential or concentric vascular wall injury or inflammation stimulates the formation of concentric intimal hyperplasia as occurs frequently in giant cell arteritis (see below).

The origin of the intimal smooth muscle cells in intimal hyperplasia is a topic of great interest. Originally, it was largely assumed that these cells derived from medial smooth muscle cells that migrated from the media into the intima. While medial smooth muscle cells do appear to be a source for intimal smooth muscle cells in many circumstances, it is becoming clear that in some settings, other sources may contribute to intimal smooth muscle cells, including either resident or circulating stem cells. Models with which to study the development of intimal hyperplasia include animal models in which arterial flow is disturbed by ligating an artery, and animal models in which injury is induced to either the adventitia or to the endothelium, using either a wire or balloon. Intimal hyperplasia is also studied using ex vivo human arteries in organ culture. This later model indicates that all of the cells necessary for the formation of intimal hyperplasia in this setting are present within the normal human artery wall. In these model systems, experimental perturbations that inhibit or prevent vascular smooth muscle cell activation generally prevent the development of intimal hyperplasia.

Intimal hyperplasia occurs in several distinct vascular diseases, and its severity and clinical significance vary greatly depending on the context. Nearly all human coronary artery atherosclerosis develops in the setting of pre-existing intimal hyperplasia. In humans, intimal hyperplasia forms in the coronary arteries within the first decade of life, and a particular vessel’s propensity to develop intimal hyperplasia mirrors its propensity to develop atherosclerosis later in life. In this setting, the intimal hyperplasia usually develops first as eccentric lesions around branch points and then later progresses to concentric lesions. While this type of intimal hyperplasia is largely stimulated by disturbed flow, it is also enhanced by other risk factors for atherosclerosis including smoking and aging. However, in this setting the intimal hyperplasia is usually mild and does not progress to significantly occlude the arterial lumen. However, this intimal hyperplasia likely serves as a precursor for the development of atherosclerosis by facilitating entrapment of LDL in the vessel wall. In intimal hyperplasia the thickened intima contains large amounts of proteoglycans, particularly versican, biglycan, and perlecan. Proteoglycans are highly negatively charged and avidly bind and retain positively charged LDL particles in the intima.

In other settings, intimal hyperplasia is not self-limited, and does progress to significantly narrow the lumen of the artery. In these settings the stimulus is usually more intense, and the development of the intimal hyperplasia is more rapid, occurring in weeks rather than years. One common example of this occlusive type of intimal hyperplasia is that seen after vascular injury such as balloon angioplasty or after surgical removal of an atherosclerotic plaque, a procedure referred to as endarterectomy. These procedures directly injure the intimal surface of the artery, provoking an inflammatory response and a strong smooth muscle cell proliferative response that may cause restenosis of the vessel. For this reason, coronary artery angioplasty is often now followed by placement of drug-eluting stents that secrete antiproliferative agents, such as rapamycin and paclitaxel, to prevent restenosis from intimal hyperplasia. Intimal hyperplasia is also the primary mechanism of vascular occlusion in some forms of vasculitis, such as giant cell arteritis.

The specific setting of the intimal hyperplasia will also influence the composition of the hyperplastic intima. In the rapid severe forms of intimal hyperplasia there is often an exuberant amount of extracellular matrix produced, often markedly exceeding the amount of extracellular matrix deposited in the more mild chronic forms of intimal hyperplasia. In the rapid severe forms, the intimal smooth muscle cells are actively dividing and express proliferation markers such as PCNA and Ki-67. In the mild chronic forms of intimal hyperplasia, the smooth muscle cells mostly do not express proliferation markers and are more densely packed together, often resembling the media itself. However, in the mild chronic forms of intimal hyperplasia, the intimal smooth muscle cells still possess an activated phenotype compared with the medial smooth muscle cells, as evidenced by nuclear hnRNP-C expression and enhanced extracellular matrix production ( Figure 11.4 ).

The specific signaling pathways driving intimal hyperplasia depend on both the particular setting or model being studied and the species. For example, murine endothelial injury models not uncommonly show different results than are obtained with murine ligation models. In addition, many markers shown to be important in rodent models do not directly translate to human intimal hyperplasia. However, one model system that has been studied in some detail is the murine carotid artery ligation model. In this model, intimal hyperplasia is enhanced by infusion of the vascular smooth muscle cell activator angiotensin-II. In the setting of angiotensin-II infusion, the intimal hyperplasia is largely independent of the leukocyte attractant MCP-1, suggesting that in this model, infiltrating leukocytes may not be important for the development of intimal hyperplasia. However in this model, NF-κB, TNF-α, and IL-1 have all been shown to promote the formation of intimal hyperplasia, likely in part by mediating vascular cell activation. In contrast to the murine carotid ligation model, the infiltration of inflammatory cells appears to play a more definitive role in promoting intimal hyperplasia in murine endothelial injury models.

Diabetic Vasculopathy

Diabetes mellitus refers to a group of disorders characterized by elevated serum glucose levels or hyperglycemia. Serum glucose levels are regulated by the hormone insulin, which stimulates the uptake of glucose by target tissues such as skeletal muscle, adipose tissue, and the liver. Hyperglycemia results from either impaired insulin secretion from the pancreas or defective insulin action in target tissues such as skeletal muscle. In classic type 1 diabetes mellitus, there is an immune-mediated destruction of the β-cells in the pancreas. These cells are responsible for secreting insulin into the blood. In the more common adult-onset type 2 diabetes mellitus, genetic predisposition and/or obesity act to render target tissue relatively resistant to the effects of insulin. In this disorder, the pancreatic β-cells initially respond with compensatory hyperplasia and overall increased insulin production, but eventually the β-cells undergo failure, resulting in a relative insulin deficiency in the setting of insulin resistance.

The vascular complications of diabetes include both macrovascular disease and microvascular disease. The macrovascular disease is essentially accentuated atherosclerosis, which most severely impacts the heart and the peripheral arteries in the legs. Diabetics are at markedly increased risk for both myocardial infarction and peripheral vascular occlusive disease (PVOD). Involvement of cerebral vessels put diabetics at increased risk for strokes. In addition to promoting routine atherosclerosis, diabetes also results in an arteriosclerosis of smaller intraparenchymal arteries. The microvascular disease of diabetes primarily affects capillaries and other small vessels in the kidneys, retina, and nerves resulting in the clinical disorders of diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy respectively.

While our understanding of the mechanisms underlying diabetic vascular disease continues to develop, most of the current evidence indicates that the complications are a result of hyperglycemia. Elevated blood glucose results in the formation of advanced glycation end products (AGEs). AGEs are created by the non-enzymatic reaction of protein amino groups with glucose-derived metabolites including glyoxal, methylglyoxal, and 3-deoxyglucosone. The vascular extracellular matrix is particularly prone to development of AGEs, which can result in crosslinking of matrix proteins with several deleterious consequences. Crosslinking of collagen renders the arteries stiffer and less elastic, altering shear stress and promoting endothelial activation and injury. AGE modification of the endothelial basement membrane impairs endothelial adhesion and promotes increased permeability. The increased permeability allows for increased serum proteins to enter into the intima. AGE-modified proteins are also relatively resistant to digestion by proteases. Since the amount of a protein at any given time is based on its rates of synthesis and degradation, this impaired degradation promotes an increase in the overall amount of vascular extracellular matrix in diabetes. The abundant AGE-modified extracellular matrix traps and retains serum proteins in the vessel wall. In large and medium-sized arteries, this relatively abundant cross-linked extracellular matrix promotes retention of LDL in the intima, spurring development of atherosclerosis. In smaller arteries, otherwise resistant to atherosclerosis, and in capillaries and other small vessels, the AGE-modified extracellular matrix binds other plasma proteins including albumin, which have entered the intima in relatively large amounts due to the increased endothelial permeability. The binding of plasma proteins to the extracellular matrix causes the vessel wall to thicken and to acquire a glassy or hyalinized appearance ( Figure 11.5 ). Such diabetic vasculopathy can cause severe stenosis of the vessel and end-organ ischemia. The diseased vessels are also more fragile and are prone to rupture. In some vascular beds such as the retina, diabetic vascuopathy results in the death of pericytes around the capillaries, and the formation of microaneurysms. In the retina, the activated endothelial cells show increased proliferation resulting in proliferative diabetic retinopathy.

In addition to the ECM, other extracellular proteins are modified by AGEs. Some of these modified proteins will bind to cellular surface receptors for AGEs, such as RAGE, on endothelial cells. Activation of RAGE results in endothelial activation, with increased secretion of reactive oxygen species, growth factors, and cytokines. These agents released from the endothelial cells stimulate vascular smooth muscle cell activation, promoting an increased production of extracellular matrix. Small vessels afflicted with diabetic vasculopathy contain increased amounts of collagen, fibronectin, and laminin. Activation of RAGE also leads to down-regulation of glyoxalase-1 (Glo1), an enzyme that metabolizes methylglyoxal. Thus, the interaction of AGE-modified proteins with RAGE fosters the generation of even more methylglyoxal and more AGEs, creating a vicious cycle leading to vascular compromise.

Amyloid Vasculopathy

The amyloidoses are a group of disorders characterized by the formation of a specific type of protein deposit in tissues. In amyloid deposits, there is a specific culprit protein that adopts an abnormal protein fold with an extended β-sheet conformation ( Figure 11.6 ). These long β-sheets will wrap around each other in groups of 3–5 to form large insoluble fibrils. Amyloid fibrils can be deposited in almost any tissue, but the vasculature is particularly susceptible.

Currently there are at least 27 distinct proteins that are known to form amyloid. The extended β-sheet structure of amyloid-fibrils allows Congo red dye to bind to the deposits in an orderly fashion, and in so doing enables the amyloid deposits to display green birefringence with plane-polarized light. In addition to the misfolded culprit protein, amyloid deposits also contain non-specific proteins including serum amyloid P, apolipoprotein E, and heparan sulfate proteoglycans. The proteoglycans in amyloid deposits result in these deposits staining with sulfated alcian blue. Serum amyloid P is often utilized as a general tissue immunohistochemical marker for all amyloid deposits and in nuclear medicine as a radiolabeled probe for assessing amyloid deposits throughout the whole body.

One mechanism leading to amyloidosis is the overproduction of a protein with a tendency to misfold and form these long β-sheet structures. Such proteins are referred to as being amyloidogenic. A common example of this process is the overproduction of immunoglobulin light chain by plasma cell neoplasms or myelomas. Plasma cell neoplasms are monoclonal and secrete a specific immunoglobulin. In some cases the light chain in the immunoglobulin is amyloidogenic, and the elevated levels of the amyloidogenic light chain released by the neoplasm result in amyloid deposition. A similar mechanism is responsible for amyloidosis due to serum amyloid A. Serum amyloid A is an amyloidogenic protein that is up-regulated during inflammation. Patients with long-standing inflammatory diseases, such as rheumatoid arthritis, have chronically elevated levels of serum amyloid A, which can lead to amyloid formation.

Another mechanism leading to the formation of amyloid deposits is the presence of a genetic alteration or polymorphism that enhances the amyloidogenic potential of a protein. Such genetic forms of amyloid typically display an autosomal dominant pattern of inheritance. A relatively common form of inherited amyloidosis results from mutations in the transthyretin gene. Over 80 such admyloidogenic transthyretin mutations are known to exist. In addition to transthyretin, mutations in other genes can also result in hereditary amyloidosis, including mutations in apolipoprotein A-I and apolipoprotein A-II.

In very rare cases, amyloid can result from being ‘infected’ by a small protein already in the extended β-sheet structure. Such infectious protein particles are referred to as prions. The deposition of the environmentally derived amyloid fibril in the tissue causes the exposed patient’s endogenous corresponding protein to misfold and deposit as amyloid along with the externally derived amyloid fibril. Such prion diseases are predominantly neurodegenerative disorders such as mad cow disease and Creutzfeldt-Jakob disease. However, it should be noted that the vast majority of amyloid-related diseases are not believed to be infectious.

In many circumstances it remains unknown as to why the amyloid deposits occur. One common example of this situation is the formation of amyloid due to wild-type transthyretin. This results in the relatively common disorder known as senile systemic amyloidosis, which as the name implies, is most often seen in older patients. This last type of amyloid is difficult to treat. While many forms of amyloid are essentially treated by reducing the level of the amyloidogenic protein, for senile systemic amyloidosis, efforts are focused at trying to prevent or reverse the misfolding of the protein.

Amyloid deposition in tissues such as the heart can lead to organ dysfunction, such as heart failure. Blood vessels are particularly susceptible to amyloid deposition ( Figure 11.7 ). Once deposited in blood vessels, amyloid can result in vascular obstruction and impaired vasoreactivity leading to end-organ ischemia. In addition, amyloid in small blood vessels can increase vascular fragility leading to hemorrhage. For example, in the brain cerebral amyloid angiopathy can result in hemorrhagic stroke, particularly in older patients.