Smooth muscle and the cardiovascular and lymphatic systems

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CHAPTER 6 Smooth muscle and the cardiovascular and lymphatic systems

The cardiovascular system carries blood from the heart to all parts of the body through a series of tubes, all but the smallest of which are muscular. The muscle in these tubes is of two types: smooth muscle is characteristic of the walls of blood vessels and cardiac muscle provides the walls of the heart chambers with their powerful contractile pumping action. The general characteristics and classification of muscle tissues are given on page 103. Smooth muscle also forms an important contractile element in the walls of many other organ systems of the body, e.g. the gastrointestinal tract.

SMOOTH MUSCLE

In smooth muscle tissue the contractile proteins actin and myosin are not organized into regular sarcomeres, visible as transverse striations, so the cytoplasm has a smooth (unstriated) appearance. Smooth muscle is also referred to as involuntary muscle, because its activity is neither initiated nor monitored consciously. It is more variable, in both form and function, than either striated or cardiac muscle, a reflection of its varied roles in different systems of the body.

Smooth muscle cells (fibres) are smaller than those of striated muscle. Their length can range from 15 μm in small blood vessels to 200 μm, and even to 500 μm or more in the uterus during pregnancy. The cells are spindle-shaped, tapering towards the ends from a central diameter of 3–8 μm (Fig. 6.1). The nucleus is single, located at the midpoint, and often twisted into a corkscrew shape by the contraction of the cell. Smooth muscle cells aggregate with their long axes parallel and staggered longitudinally, so that the wide central portion of one cell lies next to the tapered end of another. Such an arrangement achieves both close packing and a more efficient transfer of force from cell to cell. In transverse section, smooth muscle is seen as an array of circular or slightly polygonal profiles of very varied size, and nuclei are present only in the centres of the largest profiles (Fig. 6.2). This appearance contrasts markedly with that of skeletal muscle cells, which show a consistent diameter in cross-section and peripherally placed nuclei throughout their length.

Fig. 6.1 Smooth muscle cells (fibres) in transverse (above) and longitudinal (below) section in the human intestinal wall. Individual cells are spindle-shaped with a single central nucleus, aligned in parallel with neighbouring cells in a fasciculus.

Fig. 6.2 Electron micrograph showing smooth muscle fibres in transverse section, two at the level of their single central nucleus. In several places, the plasma membranes of adjacent cells are closely approximated at gap junctions (arrows).

Smooth muscle is typically found in the walls of tubular structures and hollow viscera. It regulates diameter (e.g. in blood vessels, and branches of the bronchial tree); propels liquids or solids (e.g. in the ureter, hepatic duct, and intestines); or expels the contents (e.g. in the urinary bladder and uterus). The actual arrangement of the cells varies with the tissue. The account that follows will therefore be concerned with the generic properties of smooth muscle. The more specialized morphologies of smooth muscle are described in the appropriate regional chapters.

Smooth muscle has no attachment structures equivalent to the fasciae, tendons and aponeuroses associated with skeletal muscle. There is a special arrangement for transmitting force from cell to cell and, where necessary, to other soft tissue structures. Cells are separated by a gap of 40–80 nm. Each cell is covered almost entirely by a prominent basal lamina which merges with a reticular layer consisting of a network of fine elastin, reticular fibres (collagen type III) and type I collagen fibres (Fig. 6.3). These elements bridge the gaps between adjacent cells and provide mechanical continuity throughout the fascicle. The cell attaches to components of this extracellular matrix at dense plaques (see Fig. 6.4A) where the basal lamina is thickened; cell–cell attachment occurs at intermediate junctions or desmosomes, formed of two adjacent dense plaques. At the boundaries of fascicles, the connective tissue fibres become interwoven with those of interfascicular septa, so that the contraction of different fascicles is communicated throughout the tissue and to neighbouring structures. The components of the reticular network, the ground substance and collagen and elastic fibres, are synthesized by the smooth muscle cells themselves, not by fibroblasts or other connective tissue cells, which are rarely found within fasciculi.

Fig. 6.3 A, Three-dimensional representation of smooth muscle cells. For clarity, some structural features have been separated for illustration in different cells. The spindle-shaped cells interdigitate with their long axes parallel; mechanical continuity between the cells is provided by a reticular layer of elastin and collagen fibres. The cytoskeletal framework consists of intermediate filament arrays (mainly longitudinal) and bundles of actin and myosin filaments (shown in separate cells) inserted into cytoplasmic dense bodies and submembraneous dense plaques to form a three-dimensional network. The sarcolemma contains anchoring desmosomes (adherens junctions), gap junctions and caveolae. B, The concertina-like change in shape of smooth muscle cells as they contract.

Fig. 6.4 Electron micrographs showing the characteristic features of smooth muscle cells. Vascular smooth muscle in human kidney tissue, showing a cytoplasm packed densely with microfilaments (actin and myosin), cytoplasmic dense bodies (arrows) and submembraneous dense plaques (arrowheads). A basal lamina encloses the cell. Inset shows four caveolae (C) (vesicular invaginations of the cell surface at high magnification. These are associated with receptors, enzymes and ion channels important in smooth muscle function.

(Inset courtesy of Professor Chun Y. Seow, University of British Columbia.)

In some blood vessels, notably those of the pulmonary circulation, and in the airways, and probably in other smooth muscle types, there is evidence for heterogeneity of cell phenotype. Some myofibroblast-like cells have a function that is more secretory than contractile. The secretory phenotype is often increased in disease (e.g. chronic severe asthma, pulmonary hypertension) and is associated with increased proliferation and remodeling, also the secretion of cytokines and other mediators. Many smooth muscles seem to exhibit considerable plasticity between these contractile and secretory phenotypes.

Discontinuities occur in the basal lamina between adjacent cells, and here the cell membranes approach to 2–4 nm of one another to form a gap junction (Fig. 6.2). These junctions are believed to be structurally similar to their counterparts in cardiac muscle. They provide a low-resistance pathway through which electrical excitation can pass between cells, producing a coordinated wave of contraction. The incidence of gap junctions varies with the anatomical site of the tissue: they appear to be more abundant in the type of smooth muscle which generates rhythmic (phasic) activity.

Although some smooth muscles can generate as much force per unit cross-sectional area as skeletal muscle, the force always develops much more slowly than in striated muscle. Smooth muscle can contract by more than 80%, a much greater range of shortening than the 30% or so to which striated muscle is limited. The significance of this property is illustrated by the urinary bladder, which is capable of emptying completely from an internal volume of 300 ml or more. Smooth muscles can maintain tension for long periods with very little expenditure of energy. Many smooth muscle structures are able to generate spontaneous contractions: examples are found in the walls of the intestines, ureter and uterine tube.

MICROSTRUCTURE OF SMOOTH MUSCLE AND THE CONTRACTILE MECHANISM

Although electron microscopy revealed the presence of filaments in smooth muscle some years ago, this observation alone provided little insight into their mode of function because of the lack of any obvious organization of the filaments. More recent work, using high-resolution immunocytochemistry, has revealed further details of the internal architecture of the cell and suggests a structural basis for contractile function. The model, which is illustrated in Fig. 6.3, depends on the mutual interaction of two systems of filaments, one forming the cytoskeleton and the other the contractile apparatus.

Excluding the perinuclear region, the cytoplasm of a smooth muscle cell effectively consists of two structural domains. The cytoskeleton forms a structural framework that maintains the spindle-like form of the cell and provides an internal scaffold with which other elements can interact. Its major structural component is the intermediate filament desmin, with the addition of vimentin (which may also be present alone) in vascular smooth muscle. The intermediate filaments are arranged mainly in longitudinal bundles, but some filaments interconnect the bundles with each other and with the sarcolemma to form a three-dimensional network. The bundles of intermediate filaments insert into focal, electron-dense bodies, approximately 0.1 μm in diameter, which are distributed uniformly throughout the cytoplasm and also attach to dense plaques underlying the plasma membrane (Fig. 6.3A). The cytoplasmic dense bodies and submembraneous dense plaques are equivalent to the Z-discs of striated muscle cells. They contain the actin-binding protein α-actinin and thus also anchor the actin filaments of the contractile apparatus. These form a lattice of obliquely arranged bundles throughout the cytoplasm, which transmit force to the plasma membrane and thus the basal lamina and extracellular matrix via dense plaques. These are associated with a highly structured arrangement of ancillary proteins, including vinculin and talin, which in turn attach to integrins that cross the membrane and provide attachment to components of the extracellular matrix. An analogous arrangement underlies cell–cell attachment at desmosomes, but here the attachment between dense plaques is provided by transmembrane cadherin glycoproteins and intracellular catenins instead of integrins and talin. Mechanical deformation of the cell may be linked to cell signalling mechanisms via focal adhesion kinase (FAK) and its substrate paxillin; phosphorylation of talin and paxillin may modulate the deformability of the smooth muscle cell. Other regulatory proteins also associate specifically with actin, such as caldesmon and calponin. The cytoskeleton is therefore not a passive structure, and can adapt dynamically to load, presumably therefore contributing to the low energy requirements of smooth muscle contraction. The ratio of actin to myosin is about eight times greater in smooth compared to striated muscle, reflecting the greater length of actin filaments in smooth muscle.

Smooth muscle myosin filaments are 1.5–2 μm long, somewhat longer than those of striated muscle. Although smooth muscle cells contain less myosin, the longer filaments are capable of generating considerable force. The myosin filaments of smooth muscle are also assembled differently, such that their head regions lie symmetrically on either side of a ribbon-like filament, rather than imposing a bipolar organization on the filament. Actin filaments, to which they bind, can thus slide along the whole length of the myosin filament during contraction. This difference underpins the ability of smooth muscle to undergo much greater changes in length than striated muscle. Actin–myosin filament sliding generates tension which transmits to focal regions of the plasma membrane, changing the cell to a shorter, more rounded shape (Fig. 6.3B) and often deforming the nucleus to a corkscrew-like profile.

Caveolae, cup-like invaginations of the plasma membrane with a resemblance to endocytotic vesicles, are a characteristic feature of smooth muscle cells, and may form up to 30% of the membrane (Fig. 6.4). They are associated with many receptors, ion channels and kinases, and the peripheral sarcoplasmic reticulum and may thus act as sites for highly localized signalling pathways. They may also act as specialized pinocytotic structures involved in fluid and electrolyte transport into the cell. Other organelles (mitochondria, ribosomes etc) are largely confined to the filament-free perinuclear cytoplasm, although in some smooth muscle types, including vascular smooth muscle, peripheral mitochondria, sarcoplasmic reticulum and sarcolemma seem to form signalling microdomains. Recent studies using mitochondria-specific staining of such smooth muscle cells often show mitochondria forming a reticular network within the cytosol, which would be consistent with a cell signalling function, especially that concerned with intracellular calcium homeostasis.

VASCULAR SUPPLY

The blood supply of smooth muscle is less extensive than that of striated muscle. Where the tissue is not too densely packed, afferent and efferent vessels gain access via connective tissue septa, and capillaries run in the connective tissue between small fascicles. However, unlike striated muscle, capillaries are not found in relation to individual cells.

INNERVATION

Smooth muscle may contract in response to nervous or hormonal stimulation, or electrical depolarization transferred from neighbouring cells. Some muscles receive a dense innervation to all cells; these are often referred to as multi-unit smooth muscles, and most blood vessels are of this type. Such innervation can precisely define contractile activity, e.g. in the iris, specific nervous control can produce either pupillary constriction or dilation. Other muscles are more sparsely innervated. They tend to display myogenic activity, initiated spontaneously or in response to stretch, which may be markedly influenced by hormones. In these muscles, which include those in the walls of the gastrointestinal tract, urinary bladder, ureter, uterus and uterine tube, innervation tends to exert a more global influence on the rate and force of intrinsically generated contractions. These muscles have been referred to as unitary smooth muscles. The terms multi-unit and unitary smooth muscles are widely used, but in practice such distinctions are better regarded as the extremes of a continuous spectrum.

Smooth muscles are innervated by unmyelinated axons whose cell bodies are located in autonomic ganglia, either in the sympathetic chain or, in the case of parasympathetic fibres, closer to the point of innervation (Fig. 6.5). They ramify extensively, spreading over a large area of the muscle and sending branches into the muscle fasciculi. The terminal portion of each axonal branch is beaded, and consists of expanded portions, varicosities, packed with vesicles and mitochondria, separated by thin, intervaricose portions. Each varicosity is regarded as a transmitter release site, and, in the functional sense, is therefore a nerve ending. In this way the axonal arborization of a single autonomic neurone bears a very large number of nerve endings (up to tens of thousands), as opposed to a maximum of a few hundred in somatic motor neurones. The neuromuscular terminals of autonomic efferents are considered in more detail on page 62.

Fig. 6.5 Confocal fluorescence micrograph illustrating the innervation of airway smooth muscle in the developing human lung. Smooth muscle cells are arranged circumferentially and are labelled for actin (red); nerves and ganglia form a loose network around the smooth muscle, separated from it by up to 40 μm. Nervous tissue is labelled for PGP 9.5 (green).

Reproduced with permission from Sparrow MP, Weichselbaum M, McCray PB Jr 1999 Development of the innervation and airway smooth muscle in human fetal lung. Am J Respir Cell Mol Biol 20: 550–60.

The neuromuscular junctions in smooth muscles do not show the consistent appearance seen in skeletal muscles. The neurotransmitter diffuses across a gap that can vary from 10 to 100 nm: even separations up to 1 μm may still allow neuromuscular transmission to take place, although more slowly. The nerve ending is packed with vesicles, but the adjacent area of the muscle cell is not structurally differentiated from that of non-junctional regions – there is no distinct synapse.

Intramuscular afferent nerves are the peripheral processes of small sensory neurones in the dorsal root ganglia. Since they are unmyelinated, contain axonal vesicles and have a beaded appearance, they are difficult to distinguish from efferent fibres, except by differential staining for neurotransmitters.

EXCITATION–CONTRACTION COUPLING IN SMOOTH MUSCLE

Excitation–contraction coupling in smooth muscle may be electromechanical or pharmacomechanical. Electromechanical coupling involves depolarization of the cell membrane by an action potential, and may be generated when a membrane receptor, usually linked with an ion channel, is occupied by a neurotransmitter, hormone or other blood-borne substance. It is most commonly seen in unitary smooth muscles such as those of the viscera, with transmission of electrical excitation from cell to cell via gap junctions. In some types of smooth muscle, depolarization may be the consequence of other stimuli, such as cooling, stretching, and even light.

Pharmacomechanical coupling is a receptor-mediated and G-protein coupled process, which can activate constriction via several pathways. These include triggering the formation of inositol trisphosphate, which acts as a signal for intracellular calcium release from the sarcoplasmic reticulum, activation of voltage-independent calcium channels in the sarcolemma, and depolarization causing activation of voltage-dependent calcium channels. In addition, some receptors couple to kinases that modulate contraction in a calcium independent fashion, either via myosin phosphatase (see below), or via the actin cytoskeleton. Different types of smooth muscle use these pathways to differing extents.

The regulation of contraction of smooth muscle is however largely calcium-dependent. In the cytoplasm, calcium binds to calmodulin. The complex so formed regulates the activity of myosin light chain kinase, which phosphorylates myosin regulatory light chains and initiates the myosin-actin ATPase cycle (p. 111). The enzymatic activation process is therefore inherently slow. Unphosphorylated myosin II of smooth muscle cannot initiate actin binding, although it can maintain contraction, with little energy expenditure. Myosin phosphatase dephosphorylates myosin light chains, and thus promotes relaxation. Inhibition of the phosphatase, for example by Rho kinase, increases phosphorylation for any level of calcium (i.e. increases calcium sensitivity). This is now believed to be a very important component of the response to many constrictor agonists.

DEVELOPMENT

It was thought that all smooth muscle cells developed in situ exclusively from the splanchnopleuric mesenchyme in the walls of the anlagen of the viscera and around the endothelium of blood vessels. However, recent experimental studies have traced the progeny of cells proliferating from the epithelial plate of the somite and have identified endothelial and tunica media smooth muscle cells arising from individual somites (Scaal & Christ 2004). The origin of the smooth muscle of the iris is still unclear. This region of the eye develops from the optic cup, and so the smooth muscle which arises there is derived either from the neurectoderm of the original optic cup or from the neural crest mesenchyme which later invades the iris.

Following a period of proliferation, clusters of myoblasts become elongated in the same orientation. Dense bodies, associated with actin and cytoskeletal filaments, appear in the cytoplasm, and the surface membrane starts to acquire its specialized features, i.e. caveolae, adherens junctions and gap junctions. Cytoskeletal filaments extend to insert into the submembranous dense plaques and cytoplasmic dense bodies. Thick filaments are seen a few days after the first appearance of thin filaments and intermediate filaments, and from this time the cells are able to contract. During development, dense bodies increase in number and further elements of the cytoskeleton are added. In addition to synthesizing the cytoskeleton and contractile apparatus, the differentiating cells express and secrete components of the extracellular matrix.

In a developing smooth muscle all the cells express characteristics of the same stage of differentiation, and there are no successive waves of differentiation. From its earliest appearance to maturity, a smooth muscle increases several hundredfold in mass, partly by a 2- to 4-fold increase in the size of individual cells, but mainly by a very large increase in cell number. Growth occurs by division of cells in every part of the muscle, not just at its surface or ends. Mitosis occurs in cells in which differentiation is already well advanced, as evidenced by the presence of myofilaments and membrane specializations. Mitotic smooth muscle cells may be found at any stage of life, but their numbers peak before birth, at a time that differs for different muscles; they are rare in the adult unless the tissue is stimulated to hypertrophy (as in the pregnant uterus) or to repair. The ability of mature cells to undergo mitosis therefore differs between the three major types of muscle: skeletal muscle cells cannot divide at all after differentiation; cardiac muscle cells can divide, but only before birth; and smooth muscle cells appear to remain capable of division throughout life.

During the early stages of development, smooth muscle expresses embryonic and non-muscle isoforms of myosin. The proportions of these isoforms decrease progressively. Initially, SM-1 is the dominant or exclusive smooth muscle heavy chain isoform: the SM-2 isoform becomes more established later. For a review of the development of vascular smooth muscle see Owens, Kumar and Wamhoff (2004).

THE CARDIOVASCULAR AND LYMPHATIC SYSTEMS

GENERAL ORGANIZATION

Cells of peripheral blood, suspended in plasma, circulate through the body in the blood vascular system. Interstitial fluid from peripheral tissues returns to the blood vascular system via the lymphatic system, which also provides a channel for the migration of leukocytes and the absorption of certain nutrients from the gut.

The cardiovascular system carries nutrients, oxygen, hormones, etc. throughout the body and the blood redistributes and disperses heat. As a consequence of the hydrostatic pressure, the system also has mechanical effects, such as maintaining tissue turgidity and counteracting the effects of gravity. Blood circulates within a fast, high capacity system made up of the heart, which is the central pump and main motor of the system; arteries, which lead away from the heart and carry the blood to the peripheral parts of the body; and veins, which return the blood to the heart. The heart can be thought of as a pair of muscular pumps, one feeding a minor loop (pulmonary circulation), which serves the lungs and oxygenates the blood, the other feeding a major loop (systemic circulation), which serves the rest of the body. With limited exceptions, each loop is a closed system of tubes, so that blood per se does not usually leave the circulation.

From the centre to the periphery, the vascular tree shows three main modifications. The arteries increase in number by repeated bifurcation and by sending out side branches, in both the systemic and the pulmonary circulation. For example, the aorta, which carries blood from the heart to the systemic circulation, gives rise to about 4 × 10⁶ arterioles and four times as many capillaries. The arteries also decrease in diameter, although not to the same extent as their increase in number, so that a hypothetical cross-section of all the vessels at a given distance will increase in total area with increasing distance from the heart. At its emergence from the heart, the aorta of an adult man has an outer diameter of approximately 30 mm (cross-sectional area of nearly 7 cm²). The diameter decreases along the arterial tree until it is as little as 10 μm in arterioles (each with a cross-sectional area of about 80 μm²). However, given the enormous number of arterioles, the total crosssectional area at this level is approximately 150 cm², more than 200 times that of the aorta. As a result, blood flow is faster near the heart than at the periphery.

The walls of arteries decrease in thickness towards the periphery, although this is not as substantial as the reduction in vessel diameter. Consequently, in the smallest arteries (arterioles), the thickness of the wall represents about half the outer radius of the vessel, whereas in a large vessel it represents between one-fifteenth and one-fifth, e.g. in the thoracic aorta the radius is approximately 17 mm and the wall thickness 1.1 mm.

Venules, which return blood from the periphery, converge on each other forming a progressively smaller number of veins of increasingly large size. As with arteries, the hypothetical total cross-sectional area of all veins at a given level reduces nearer to the heart. Eventually, only the two largest veins, the superior and inferior venae cavae, open into the heart from the systemic circulation. A similar pattern is found in the pulmonary circulation, but here the vascular loop is shorter and has fewer branch points, and consequently, the number of vessels is smaller than in the systemic circulation. The total end-to-end length of the vascular network in a typical adult is twice the circumference of the earth.

Large arteries, such as the thoracic aorta, subclavian, axillary, femoral and popliteal arteries, lie close to a single vein which drains the same territory as that supplied by the artery. Other arteries are usually flanked by two veins, satellite veins (venae comitantes), which lie on either side of the artery, and have numerous cross-connections: the whole is enclosed in a single connective tissue sheath. The artery and the two satellite veins are often associated with a nerve, and when they are surrounded by a common connective tissue sheath they form a neurovascular bundle.

The close association between the larger arteries and veins in the limbs allows the counterflow exchange of heat to take place. This mechanism promotes heat transfer from arterial to venous blood, and thus helps to preserve body heat. Counterflow heat exchange systems are found in certain organs, e.g. in the testis, where the pampiniform plexus of veins surrounds the testicular artery (this arrangement not only conserves body heat, but also maintains the temperature of the testis below average body temperature). Counterflow ion exchange mechanisms are found in the microcirculation, as in the arterial and venous sinusoids of the vasa recta in the renal medulla. Here, countercurrent exchange retains sodium ions at a high concentration in the medullary interstitium, and efferent venous blood transfers sodium ions to the afferent arterial supply.

Arteries and veins are named primarily according to their anatomical position. In functional terms, three main classes of vessel are described: resistance vessels (arteries, but mainly arterioles), exchange vessels (capillaries, sinusoids and small venules) and capacitance vessels (veins). Structurally, arteries can also be divided into elastic and muscular types. Although muscle cells and elastic tissue are present in all arteries, the relative amount of elastic material is greatest in the largest vessels, whereas the relative amount of smooth muscle increases progressively towards the smallest arteries.

Arteries may also be subdivided into conducting and distributing, as well as resistance, vessels. The large conducting arteries which arise from the heart, together with their main branches, are characterized by the predominantly elastic properties of their walls. Distributing vessels are smaller arteries supplying the individual organs, and their wall is characterized by a well-developed muscular component. Resistance vessels are mainly arterioles. Small and muscular, they provide the main source of the peripheral resistance to blood flow, and they cause a marked drop in the pressure of blood which flows into the capillary beds within tissues.

Capillaries, sinusoids and small (postcapillary) venules are collectively termed exchange vessels. Their walls allow exchange between blood and the interstitial tissue fluid which surrounds all cells: this is the essential function of a circulatory system. Arterioles, capillaries and venules constitute the microvascular bed, the structural basis of the microcirculation.

Larger venules and veins form an extensive, but variable, large-volume, low-pressure system of vessels conveying blood back to the heart. The high capacitance of these vessels is due to the distensibility (compliance) of their walls, so that the content of blood is high even at low pressures. This part of the vascular bed contains the greatest proportion of blood, reflecting the large relative volume of veins.

Blood from the abdominal part of the digestive tube (with the exception of the lower part of the anal canal), and from the spleen, pancreas and gallbladder, drains to the liver via the portal vein. The portal vein ramifies within the substance of the liver like an artery and ends in the hepatic sinusoids. These drain into the hepatic veins which in turn drain into the inferior vena cava. Blood supplying the abdominal organs thus passes through two sets of capillaries before it returns to the heart. The first provides the organs with oxygenated blood, and the second carries deoxygenated blood, rich in absorption products from the intestine, through the liver parenchyma. A venous portal circulation also connects the median eminence and infundibulum of the hypothalamus with the adenohypophysis. In essence, a venous portal system is a capillary network that lies between two veins, instead of between an artery and a vein, which is the more usual arrangement in the circulation. A capillary network may also be interposed between two arteries, e.g. in the renal glomeruli, where the glomerular capillary bed lies between afferent and efferent arterioles. This maintains a relatively high pressure system, which is important for renal filtration.

A parallel circulatory system in the body is provided by the lymphatic vessels and lymph nodes. Lymphatic vessels originate in peripheral tissues as blind-ended endothelial tubes which collect excess fluid from the interstitial spaces between cells and conduct it as lymph. Lymph is returned to the blood vascular system via lymphatic vessels which converge on the large veins in the root of the neck.

The development of blood vessels is described on pages, 206–208.

General features of vessel walls

Blood vessels, irrespective of size, and with the exception of capillaries and venules, have walls consisting of three concentric layers (tunicae) (see Fig. 6.7). The intima (tunica intima), is the innermost layer. Its main component, the endothelium, lines the entire vascular tree, including the heart, and the lymphatic vessels. The media (tunica media) is made of muscle tissue, elastic fibres and collagen. While it is by far the thickest layer in arteries, the media is absent in capillaries and is comparatively thin in veins. The adventitia (tunica adventitia) is the outer coat of the vessel, and consists of connective tissue, nerves and vessel capillaries (vasa vasorum). It links the vessels to the surrounding tissues. Vessels differ in the relative thicknesses and detailed compositions of their layers and, in the smallest vessels, the number of layers represented.

Fig. 6.7 The principal structural features of the larger blood vessels as seen in a muscular artery.

Large elastic arteries

The aorta and its largest branches (brachiocephalic, common carotid, subclavian and common iliac arteries) are large elastic arteries which conduct blood to the medium-sized distributing arteries.

The intima is made of an endothelium, resting on a basal lamina, and a subendothelial connective tissue layer. The endothelial cells are flat, elongated and polygonal in outline, with their long axes parallel to the direction of blood flow (see Fig. 6.17). The subendothelial layer is well developed, contains elastic fibres and type I collagen fibrils, fibroblasts and small, smooth muscle-like myointimal cells. The latter accumulate lipid with age and in an extreme form, this feature contributes to atherosclerotic changes in the intima. Thickening of the intima progresses with age and is more marked in the distal than in the proximal segment of the aorta.

Fig. 6.17 Scanning electron micrograph of the luminal surface of the basilar artery. The tightly packed endothelial cells are elongated in the direction of blood flow.

(By courtesy of Masoud Alian, University College, London.)

A prominent internal elastic lamina, sometimes split, lies between intima and media. This lamina is smooth, measures about 1 μm in thickness, and, with the elastic lamellae of the media, is stretched under the effect of systolic pressure, recoiling elastically in diastole. Elastic arteries thus have the effect of sustaining blood flow despite the pulsatile cardiac output. They also smooth out the cyclical pressure wave. The media has a markedly layered structure, in which fenestrated layers of elastin (elastic lamellae) alternate with interlamellar smooth muscle cells (Fig. 6.6), collagen and fine elastic fibres. The arrangement is very regular, such that each elastic lamella and adjacent interlamellar zone is regarded as a ‘lamellar unit’ of the media. In the human aorta there are approximately 52 lamellar units, measuring about 11 μm in thickness. Number and thickness of lamellar units increases during postnatal development, from 40 at birth.

Fig. 6.6 Elastic artery (human aorta), stained for elastic fibres. The dense staining of the internal elastic lamina is seen close to the luminal surface (top); elastic lamellae fill the tunica media and merge with the external elastic lamina at its junction with the collagenous adventitia (red fibres, below). Compare with Fig. 6.20. Elastic van Gieson technique.

The adventitia is well developed. In addition to collagen and elastic fibres, it contains flattened fibroblasts with extremely long, thin processes, macrophages and mast cells, nerve bundles and lymphatic vessels. The vasa vasorum are usually confined to the adventitia.

Muscular arteries

Muscular arteries are characterized by the predominance of smooth muscle in the media (Fig. 6.8). The intima consists of an endothelium, similar to that of elastic arteries, which rests on a basal lamina and subendothelial connective tissue. The internal elastic lamina (Fig. 6.7, Fig. 6.8) is a distinct, thin layer, sometimes duplicated and occasionally absent. It is thrown into wavy folds as a result of contraction of smooth muscle in the media. Some 75% of the mass of the media consists of smooth muscle cells which run spirally or circumferentially around the vessel wall. The relative amount of extracellular matrix is therefore less than in large arteries, however, fine elastic fibres which run mainly parallel to the muscle cells are present. An external elastic lamina, composed of sheets of elastic fibres, forms a less compact layer than the internal lamina, and separates the media from the adventitia in the larger muscular arteries. The adventitia is made of fibroelastic connective tissue, and can be as thick as the media in the smaller arteries. The inner part of the adventitia contains more elastic than collagen fibres.

Fig. 6.8 The wall of a human muscular artery. The intima (I) forms the innermost layer, lined by an endothelium (arrowhead) and separated from the middle muscular layer, the media (M), by an internal elastic lamina (short arrow). A more diffuse external elastic lamina (long arrow) divides the media from the outermost collagenous adventitia (A), within which lie the vasa vasorum (V).

Arterioles

In arterioles (Fig. 6.9, Fig. 6.10) the endothelial cells are smaller than in large arteries, but their nuclear region is thicker and often projects markedly into the lumen. The nuclei are elongated and orientated parallel to the vessel length, as is the long axis of the cell. The basal surface of the endothelium contacts a basal lamina, but an internal elastic lamina is either absent or is highly fenestrated and traversed by the cytoplasmic processes of muscle cells or endothelial cells.

Fig. 6.9 A small arteriole (A) and accompanying venule (V) in loose connective tissue (human). Note the relative thicknesses of the vessel walls, in comparison with the diameters of their lumens.

Fig. 6.10 Electron micrograph of a small arteriole in the epineurium of a peripheral nerve. The vessel lumen contains erythrocytes and is lined by endothelial cells (with nuclei, N, projecting into the lumen); note the electron-lucent internal elastic lamina (pale, wavy line, arrowed), the media containing densely filamentous smooth muscle cells (M) and the connective tissue of the adventitia (A) merging with that of the epineurium (E).

The muscle cells are larger in cytoplasmic volume than those of large arteries and they form a layer one or two cells thick. They are arranged circumferentially and are tightly wound around the endothelium. In the smallest arterioles each cell makes several turns, producing extensive apposition between parts of the same cell. Their contractility controls the flow of blood into the capillary bed, and they act functionally as precapillary sphincters. Constriction of the vessel lumen is thought to be under myogenic, rather than neurogenic, control and is responsive to local vasoactive and metabolic factors. Arteriolar adventitia is very thin.

Arterioles are usually densely innervated by sympathetic fibres, via small bundles of varicose axons packed with transmitter vesicles, mostly of the adrenergic type. The distance between axolemma and muscle cell membrane can be as little as 50–100 nm and the gap is occupied only by a basal lamina. Autonomic neuromuscular junctions are very common in arterioles.

Capillaries

The capillary wall (Fig. 6.11) is formed by an endothelium and its basal lamina, plus a few isolated pericytes. Capillaries are the vessels closest to the tissue they supply and their wall is a minimal barrier between blood and the surrounding tissues. Capillary structure varies in different locations. Capillaries measure 4–8 μm in diameter (much more in the case of sinusoids) and are hundreds of microns long. Their lumen is just large enough to admit the passage of single blood cells, usually with considerable deformation. However, the true bottleneck of the circulatory system occurs at the level of the arterioles, where muscle contraction can obliterate the lumen.

Fig. 6.11 Electron micrograph of a capillary in a human muscle biopsy specimen. An endothelial cell with its nucleus in the plane of section forms adherens junctions (long arrows) with a second cell, or an extension of itself. The cytoplasm contains numerous transcytotic vesicles (short arrows). A basal lamina surrounds the capillary.

Typically a single endothelial cell forms the wall of a capillary, so that the junctional complex occurs between extensions of the same cell. The endothelial cells of some capillaries have fenestrations, or pores, through their cytoplasm. Fenestrations are approximately circular, 50–100 nm in diameter, and at their edge the luminal and abluminal membranes of the endothelial cell come into contact with each other. The fenestration itself is usually occupied by a thin electron-dense diaphragm of unknown molecular composition. Fenestrated capillaries occur in renal glomeruli, where they lack a diaphragm, in intestinal mucosae, and in endocrine and exocrine glands. Fenestrations are almost invariably present in capillaries which lie close to an epithelium, including the skin.

Capillaries without fenestrations, such as those in the brain, striated and smooth muscles, lung and connective tissues, are known as continuous capillaries. Capillary permeability varies greatly among tissues, and this can be correlated partly with the type of endothelium. Where efficient barriers to diffusion of large molecules occur, e.g. brain, thymic cortex and testis, continuous capillary endothelial cells are joined by tight junctions.

Sinusoids

Sinusoids are expanded capillaries (Fig. 6.12), and are large and irregular in shape. They have true discontinuities in their walls, allowing intimate contact between blood and the parenchyma. The discontinuities are formed by gaps between endothelial cells, which are also fenestrated, such that the sinusoidal lining, and sometimes also the basal lamina, is incomplete. Sinusoids occur in large numbers in the liver (where a basal lamina is completely absent), spleen, bone marrow, adenohypophysis (Fig. 6.12) and suprarenal medulla.

Fig. 6.12 Expanded sinusoids (S) typical of endocrine glands and certain other tissues, are seen here containing erythrocytes (orange) in the adenohypophysis. Endocrine cells stain blue, reddish-brown or are poorly stained in this trichrome preparation.

Venules

When two or more capillaries converge, the resulting vessel is larger (10–30 μm) and is known as a venule (postcapillary venule). Venules (Fig. 6.9) are essentially tubes of flat, oval or polygonal endothelial cells surrounded by basal lamina and, in the larger vessels, by a delicate adventitia of a few fibroblasts and collagen fibres mainly running longitudinally. Pericytes (see Fig. 6.21) support the walls of these venules.

Fig. 6.21 Scanning electron micrograph of capillary (C) and pericytes (P) supporting the vessel wall.

(By courtesy of T. Fujiwara and Y. Uehara, Department of Anatomy, Ehime University School of Medicine, Japan.)

Postcapillary venules are sites of leukocyte migration. In venules of mucosa-associated lymphoid tissue (MALT), particularly of the gut and bronchi, and in the lymph nodes and thymus, endothelial cells are taller and have intercellular junctions through which lymphocytes and other blood components can readily pass. These are known as high endothelial venules (HEV) (see Figs 6.18, 6.19). Elsewhere, venules are believed to be a major site where migration of neutrophils, macrophages and other leukocytes into extravascular spaces occurs, and where neutrophils may temporarily attach, forming marginated pools.

Fig. 6.18 A high endothelial venule in a human lymph node, sectioned longitudinally, lined by cuboidal endothelium (arrow). Erythrocytes and leukocytes (mainly lymphocytes and neutrophils) are seen in the lumen.

Fig. 6.19 A high endothelial venule in transverse section in the human palatine tonsil. The lumen is completely filled by a neutrophil (N). Cuboidal endothelial cells (EC) line the vessel. Two lymphocytes (L) with heterochromatic nuclei are seen below, in transit within the wall of the vessel.

(By courtesy of Dr Marta Perry, Department of Anatomy, St Thomas’s Hospital Medical School, London.)

In general, the endothelium of venules has few tight junctions, and is relatively permeable. The intercellular junctions of venules are sensitive to inflammatory agents which increase their permeability to fluids and defensive cells, and facilitate leukocyte extravasation by diapedesis.

Venules do not acquire musculature until they are about 50 μm in outer diameter, when they are known as muscular venules. This distinction is important, because postcapillary venules, which lack muscle in their walls, are as permeable to solutes as capillaries and are thus part of the microcirculatory bed. At the level of the postcapillary venule the cross-sectional area of the vascular tree is at its maximum, and there is a dramatic fall in pressure (from 25 mmHg in the capillary to approximately 5 mmHg). Muscular venules converge to produce a series of veins of progressively larger diameter. Venules and veins are capacitance vessels, i.e. they have thin distensible walls which can hold a large volume of blood and accommodate luminal pressure changes.

Veins

Veins are characterized by a relatively thin wall in comparison to arteries of similar size and by a large capacitance. Wall thickness is not correlated exactly to the size of the vein, and varies in different regions, e.g. the wall is thicker in veins of the leg than it is in veins of a similar size in the arm.

The structural plan of the wall is similar to that of other vessels, except that the amount of muscle is considerably less than in arteries, while collagen and, in some veins, elastic fibres, predominate. In most veins, e.g. those of the limbs, the muscle is arranged approximately circularly. Longitudinal muscle is present in the iliac, brachiocephalic, portal and renal veins and in the superior and inferior venae cavae. Muscular tissue is absent in the maternal placental veins, dural venous sinuses and pial and retinal veins, veins of trabecular bone and the venous spaces of erectile tissue. These veins consist of endothelium supported by variable amounts of connective tissue. Distinction between the media and adventitial layers is often difficult, and a discrete internal elastic lamina is absent.

Tethering of some veins to connective tissue fasciae and other surrounding tissues may prevent collapse of the vessel even under negative pressure. Pressure within the venous system does not normally exceed 5 mmHg, and it decreases as the veins grow larger and fewer in number, approaching zero close to the heart. Because they contain only a small amount of muscle, veins have limited influence on blood flow. However, during a sudden fall in blood pressure, e.g. following a haemorrhage, elastic recoil and reflex constriction in veins compensate for the blood loss and tend to maintain venous return to the heart. Vasoconstriction in cutaneous veins in response to cooling is important in thermoregulation.

Most veins have valves to prevent reflux of blood (Fig. 6.13, Fig. 6.14). A valve is formed by an inward projection of the intima, strengthened by collagen and elastic fibres, and covered by endothelium which differs in orientation on its two surfaces. Surfaces facing the vessel wall have transversely arranged endothelial cells, whereas on the luminal surface of the valve, over which the main stream of blood flows, cells are arranged longitudinally in the direction of flow. Most commonly two, or occasionally three, valves lie opposite one another, sometimes only one is present. They are found in small veins or where tributaries join larger veins. The valves are semilunar (cusps) and attached by their convex edges to the venous wall. Their concave margins are directed with the flow and lie against the wall as long as flow is towards the heart. When blood flow reverses, the valves close and blood fills an expanded region of the wall, a sinus, on the cardiac side of the closed valve. This may give a ‘knotted’ (varicose) appearance to the distended veins, if these have many valves. In the limbs, especially the legs where venous return is against gravity, valves are of great importance to venous flow. Blood is moved towards the heart by the intermittent pressure produced by contractions of the surrounding muscles. Valves are absent in veins of the thorax and abdomen.

Fig. 6.13 The upper portions of the femoral and long saphenous veins laid open to show the valves. About two-thirds of the natural size.

Fig. 6.14 A valve in a human small vein, formed from flap-like extensions of the intima which close when pressure increases on the proximal side, preventing backflow.

VASCULAR SHUNTS AND ANASTOMOSES

Arteriovenous shunts and anastomoses

Communications between the arterial and venous systems are found in many regions of the body. In some parts of the microcirculation (e.g. mesentery), the capillary circulation can be bypassed by wider thoroughfare channels formed by metarterioles (Fig. 6.15A). These have similarities to both capillaries and the smallest arterioles, and have a discontinuous layer of smooth muscle in their walls. Metarterioles can deliver blood directly to venules or to a capillary bed, according to local demand and conditions. When functional demand is low, blood flow is largely limited to the bypass channel. Periodic opening and closing of different arterioles irrigates different parts of the capillary network. The number of capillaries in individual microvascular units and the size of their mesh determine the degree of vascularity of a tissue: the smallest meshes occur in the lungs and the choroid of the eye.

Fig. 6.15 A, A microcirculatory unit, showing a terminal arteriole, thoroughfare channels, capillaries and collecting venule. The distribution of smooth muscle cells and one of the precapillary sites where perfusion of the capillary bed is regulated are also shown. B, An arteriovenous anastomosis. Note the thick wall of the anastomotic channel composed of layers of modified smooth muscle cells.

Arteriovenous anastomoses (Fig. 6.15B) are direct connections between smaller arteries and veins. Connecting vessels may be straight or coiled, and often possess a thick muscular tunic. Under sympathetic control, the vessel is able to close completely, diverting blood into the capillary bed. When patent, the vessel carries blood from artery to vein, partially or completely excluding the capillary bed from the circulation. Simple arteriovenous anastomoses are widespread and occur notably in the skin of the nose, lips and ears, nasal and alimentary mucosae, erectile tissue, tongue, thyroid gland and sympathetic ganglia. In the newborn child, there are few arteriovenous anastomoses, but they develop rapidly during the early years. In old age they atrophy, sclerose and diminish in number. These factors may contribute to the less efficient temperature regulation which occurs at the two extremes of age.

In the skin of the hands and feet, especially in digital pads and nail beds (see Fig. 7.18), anastomoses form a large number of small units termed glomera. Each glomus organ has one or more afferent arteries, stemming from branches of cutaneous arteries which approach the surface. The afferent artery gives off a number of fine periglomeral branches and then immediately enlarges, makes a sinuous curve, and narrows again into a short funnel-shaped vein which opens at right angles into a collecting vein (Fig. 6.16).

Fig. 6.16 Digital arteriovenous anastomosis prepared by intravascular perfusion of stain in a full thickness specimen of skin, followed by clearance. The heavily stained, thick-walled, tortuous anastomotic channels (AC) contrast with the central arterial stem (A) and the thin-walled venous (V) outflow channels.

(By courtesy of the late RT Grant, GKT School of Medicine, London.)

Arterial anastomoses

Arteries can be joined to each other by an anastomosis, so that one can supply the territory of the other. An end-to-end anastomosis occurs when two arteries communicate directly, e.g. the uterine and ovarian arteries, the right and the left gastroepiploic arteries, the ulnar artery and the superficial palmar branch of the radial artery. Anastomosis by convergence occurs when two arteries converge and merge, as happens when the vertebral arteries form the basilar artery at the base of the brain. A transverse anastomosis occurs when a short arterial vessel links two large arteries transversely, e.g. the anastomoses between the two anterior cerebral arteries; the posterior tibial artery and the fibular artery; and the radial and ulnar arteries at the wrist.

The angiosome concept and vascular territories

An angiosome is a three-dimensional block of tissue (known as an anatomical territory) supplied by a source artery and its accompanying veins. It can be a composite of skin, underlying fascia, muscle and bone. These blocks of tissue form a complex three-dimensional jigsaw puzzle: some pieces have a predominantly cutaneous component while others are predominantly muscular. Each angiosome is made up of arteriosomes and venosomes and they are linked to neighbouring angiosomes by either simple anastomoses composed of similar calibre vessels or reduced calibre vessels termed choke vessels. The anastomoses between adjacent angiosomes can occur within the skin or within muscle. Some muscles are supplied by a single artery and its accompanying veins and therefore lie within one angiosome, while other muscles are supplied by more than one vessel and therefore cross more than one angiosome.

The clinical relevance of the angiosome concept is reflected in the potential connections of adjacent vascular territories. Should the source vessels for one angiosome become blocked or damaged, then that anatomical territory can be ‘rescued’ by receiving a blood supply from the immediately neighbouring angiosome via the connecting simple and choke vessel anastomoses. A detailed knowledge of angiosomes and vascular territories is essential for plastic and reconstructive surgeons when designing and surgically raising flaps of tissue which can reliably be moved from one part of the body to another without disrupting their blood supply.

FUNCTIONAL MICROSTRUCTURE OF VESSELS

Intima

The intimal lining of blood vessels consists of an endothelium, and a variable amount of subendothelial connective tissue, depending on the vessel.

Endothelium

The endothelium is a monolayer of flattened polygonal cells which extends continuously over the luminal surface of the entire vascular tree (Fig. 6.17, see Fig. 6.10). Its structure varies in different regions of the vascular bed.

The endothelium is a key component of the vessel wall, and subserves several major physiological roles. Endothelial cells are in contact with the bloodstream and thus influence blood flow. They regulate the diffusion of substances and migration of cells out of and into the circulating blood. In the brain, endothelial cells of small vessels actively transport substances, e.g. glucose, into the brain parenchyma. Endothelial cells participate in the formation of blood clots (by secreting clot-promoting factors – von Willebrand factor); in minimizing clot formation (by secreting prostacyclin and thrombomodulin); and in the process of clot dissolution or fibrinolysis (by secreting tissue plasminogen activator). They have selective phagocytic activity and are able to extract substances from the blood. For example, the endothelium of pulmonary vessels removes and inactivates several polypeptides, biogenic amines, bradykinin, prostaglandins and lipids from the circulation. Endothelial cells secrete both vasoconstrictive (thromboxane) and vasodilator (prostacyclin) prostaglandins, nitric oxide (NO, relaxing factor) and endothelin (a vasoconstrictor), which affect the tone of smooth muscle in vessel walls. They are sensitive to stretch (e.g. increased pressure) and the shear effect of blood flow, via stretch-sensitive ion channels in the cell membrane. Endothelial cells synthesize components of the basal lamina. They proliferate to provide new cells during the growth in size of a blood vessel, to replace damaged endothelial cells, and to provide solid cords of cells which develop into new blood vessels (angiogenesis). Angiogenesis, which may be stimulated by endothelial production of autocrine growth factors (see Ch. 1) in response to locally low oxygen tension, is important in wound healing, and in the growth of tumours. Endothelial cells are also active participants in, and regulators of, inflammatory processes (reviewed in Pober & Sessa, 2007).

Endothelial cells are thin but extend over a relatively large surface area. They are generally elongated in the direction of blood flow, especially in arteries (see Fig. 6.17). They usually adhere firmly to each other at their edges, so that the lining of the lumen presents no discontinuity, other than in sinusoids. The thickness of endothelial cells is maximal at the level of their nucleus, where it can reach 2–3 μm, and this part of the cell often bulges slightly into the lumen (see Fig. 6.10). Elsewhere, the endothelial cell is thinner and laminar: in capillaries, these portions of the cell often measure as little as 0.2 μm in thickness.

Transcytotic (pinocytotic) vesicles (see Fig. 6.11) are present in all endothelial cells, but are particularly numerous in exchange vessels; they include caveolae (see Fig. 6.4) typical of smooth muscle cells. They shuttle small amounts of extracellular fluid or blood plasma across the endothelial cytoplasm and thus facilitate the bulk exchange of dissolved gases, nutrients and metabolites between these compartments. In spite of the factors known to be released by endothelial cells, they do not have the morphological characteristics of secretory cells.

An organelle which characterizes endothelial cells is the Wiebel–Palade body, an elongated cytoplasmic vesicle, 0.2 × 2–3 μm in length, which contains regularly spaced tubular structures parallel to its long axis. Wiebel–Palade bodies store the adhesion molecule, P-selectin, and a large glycoprotein known as von Willebrand factor, which is released into the subendothelial connective tissue; it mediates the binding of platelets to the extracellular matrix and platelet aggregation, after vascular injury. Von Willebrand factor is also produced by megakaryocytes and is stored in platelets. Plasma von Willebrand factor binds factor VIII clotting protein, which is secreted into the bloodstream by hepatocytes. Hereditary deficiency or defective function of von Willebrand factor causes a clotting disorder of the same name.

Endothelial cells adhere to adjacent cells through the junctional complex, an area of apposition where adherent and tight junctions are found. They also communicate via gap junctions. Tight junctions are most marked in continuous capillaries. Cell contacts between endothelial cells and smooth muscle cells are common in arterioles, where the separation between endothelium and media is reduced and the inner elastic lamina is either very thin or absent.

Endothelial–leukocyte interactions

The luminal surface of endothelial cells does not normally support the adherence of leukocytes or platelets. However, many functions of human vascular endothelial cells are dynamic rather than fixed. Activated endothelial cells and the characteristic endothelium of high endothelial venules (HEV) of lymphoid tissues, are sites of leukocyte attachment and diapedesis (see below).

HEV (Fig. 6.18) are located within the T cell domains, between and around lymphoid follicles in all secondary lymphoid organs and tissues except the spleen. They are specialized venules of 7–30 μm diameter, which possess a conspicuous cuboidal endothelial lining. The luminal aspect of HEVs shows a cobblestone appearance. The endothelial cells rest on a basal lamina and are supported by pericytes and a small amount of connective tissue (Fig. 6.19). They are linked by discontinuous adhesive junctions at their apical and basal aspects: the junctions are circumnavigated by migrating lymphocytes. Ultrastructurally, the endothelial cells have the characteristics of metabolically active secretory cells. They contain large, rounded euchromatic nuclei with one or two nucleoli, prominent Golgi complexes, many mitochondria, ribosomes and pinocytotic vesicles. Typically, they also possess Wiebel–Palade bodies (see above).

Many of the adhesion molecules which mediate interactions between blood leukocytes and HEVs or cytokine-activated endothelium have been identified. They can be divided into three general families: selectins, integrins and the immunoglobulin supergene family. Selectins and integrins are expressed on leukocytes and mediate adhesion of circulating cells to the endothelium, which expresses selectins and members of the immunoglobulin supergene family. Regulated expression of these molecules by both cell types provides the means by which leukocytes recognize the vessel wall (leukocyte homing antigens and vascular addressins), adhere to it and subsequently leave the circulation.

The first step in this cascade is the loose binding or tethering of leukocytes, and is initiated via L-, P- or E-selectin (see below). This weak, reversible adhesion allows leukocytes to roll along the endothelial surface of a vessel lumen at low velocity, making and breaking contact, and sampling the endothelial cell surfaces. Recognition of chemokines (chemotactic signalling molecules) presented by the endothelium leads to ‘inside-out’ signalling and conversion of integrins at the leukocyte surface into actively adhesive configurations which bind strongly to their endothelial ligands, resulting in stable arrest. Finally, the leukocyte migrates through the vessel wall (diapedesis), passing between (paracellular migration) or across (transcellular migration) endothelial cells. It is now thought that transcellular migration is the preferred pathway and endothelial transcytotic vesicles (caveolae), intermediate filaments (vimentin) and F-actin are important in the creation of transient transcellular channels through which leukocytes pass. They then cross the basal lamina and migrate into the surrounding tissue by mechanisms which involve CD31 antigen and matrix metalloproteinases (see Ch. 2).

Cell adhesion molecules

Three members of the selectin family of adhesive proteins are currently recognized. They are L-selectin (also known as lymphocyte homing receptor), E-selectin and P-selectin. L-selectin is expressed on most leukocytes. Endothelial cells of HEVs in lymphoid organs express its oligosaccharide ligand, although other molecules such as mucins may be alternative ligands. Thus, L-selectin mediates homing of lymphocytes, especially to peripheral lymph nodes, but also promotes the accumulation of neutrophils and monocytes at sites of inflammation. E-selectin is an inducible adhesion molecule which mediates adhesion of leukocytes to inflammatory cytokine-activated endothelium, and is only transiently expressed on endothelium. P-selectin is rapidly mobilized from Wiebel–Palade bodies, where it is stored, to the endothelial surface after endothelial activation. It binds to ligands expressed on neutrophils, platelets, and monocytes and, like E-selectin, tethers leukocytes to endothelium at sites of inflammation. However, since P-selectin is quickly endocytosed by the endothelial cells, its expression is short-lived.

The integrins are a large family of molecules which mediate cell-to-cell adhesion as well as interactions of cells with extracellular matrix. Certain β-1 integrin heterodimers are expressed on lymphocytes 2–4 weeks after antigenic stimulation (very late antigens, VLA), and bind to the extracellular matrix. Additionally, VLA-4 present on resting lymphocytes (the expression of which increases after activation), monocytes and eosinophils, binds to the vascular cell adhesion molecule-1 (VCAM-1), the ligand on activated endothelium. In contrast to β-1 integrins, which many cells express, the expression of β-2 integrins is limited to white blood cells. Although the leukocyte integrins are not constitutively adhesive, they become highly adhesive after cell activation and therefore play a key role in the events required for cell migration. The endothelial ligands for one such β-2 integrin are the intercellular adhesion molecules-1 and -2 (ICAM-1 and ICAM-2), which belong to the immunoglobulin superfamily.

Three members of the large immunoglobulin superfamily of proteins are involved in leukocyte-endothelial adhesion, providing integrin counter-receptors on the endothelial cell membrane. ICAM-1 and ICAM-2 are constitutively expressed but upregulated by inflammatory cytokines. VCAM-1 is absent from resting endothelium but is induced by cytokines on activated endothelium and promotes extravasation of lymphocytes at sites of inflammation.

Subendothelial connective tissue

The subendothelial connective tissue, also termed the lamina propria, is a thin but variable layer. It is largely absent in the smallest vessels, where the endothelium is supported instead by pericytes (see Fig. 6.21). It contains a typical fibrocollagenous extracellular matrix, a few fibroblasts and occasional smooth muscle cells. Endothelial von Willebrand factor concentrates in this layer and participates in the clotting process when the overlying endothelium is damaged.

Media

The media consists chiefly of concentric layers of circumferentially or helically arranged smooth muscle cells with variable amounts of elastin and collagen.

Smooth muscle

Smooth muscle forms most of the media of arteries (see Fig. 6.8) and arterioles. A thinner layer of smooth muscle is also found in venules and veins, with the exception of small segments of the pulmonary veins, where striated cardiac muscle is present in the portions nearest to the heart. Contraction of the smooth muscle in arteries and arterioles reduces the calibre of the vessel lumen, which reduces blood flow through the vessel and raises the pressure on the proximal side. This role is particularly effective in small resistance vessels where the wall is thick, relative to the diameter of the vessel. Smooth muscle can also alter the rigidity of the wall, without causing constriction (isometric contraction), and this affects the distensibility of the wall and propagation of the pulse.

The smooth muscle cells synthesize and secrete elastin, collagen and other extracellular components of the media which bear directly on the mechanical properties of the vessels. The mechanics of the musculature of the media are complex. Distensibility, strength, self-support, elasticity, rigidity, concentric constriction etc., are interrelated functions and are finely balanced in the different regions of the vascular bed.

In large arteries, where the blood pressure is high, the muscle cells are shorter (60–200 μm) and smaller in volume than in visceral muscle. In arterioles and veins, smooth muscle cells more closely resemble visceral muscle cells. The cells are packed with myofilaments and other elements of the cytoskeleton, including intermediate filaments. Vascular muscle cells have intermediate filaments of either vimentin alone or both vimentin and desmin; the intermediate filaments of visceral smooth muscle are exclusively of desmin. Intercellular junctions are mainly of the adhesive (adherens) type and provide mechanical coupling between the cells. In addition, there are gap (communicating) junctions which couple cells electrically. Junctions between muscle cells and the connective tissue matrix are particularly numerous, especially in arteries.

The muscle cells of the arterial media can be regarded as multifunctional mesenchymal cells. After damage to the endothelium, muscle cells migrate into the intima and proliferate, forming bundles of longitudinally oriented cells which reform the layer. In certain pathological conditions, muscle cells (and macrophages) undergo fatty degeneration and participate in the formation of atheromatous plaques.

Collagen and elastin

Components of the extracellular matrix are major constituents of vessel walls, and in large arteries and veins they make up more than half of the mass of the wall, mainly in the form of collagen and elastin. Other fibrous components such as fibronectin, and amorphous proteoglycans and glycosaminoglycans, are present in the interstitial space.

Elastin is found in all arteries and veins and is especially abundant in elastic arteries (see Fig. 6.6). Individual elastic fibres (0.1–1.0 μm in diameter) anastomose with each other to form net-like structures, which extend predominantly in a circumferential direction. More extensive fusion produces lamellae of elastic material, which though usually perforated and thus incomplete, separate the layers of muscle cells. A conspicuous elastic lamella, the internal elastic lamina, is seen in arteries, between intima and media. This is a tube of elastic material which allows the vessel to recoil after distension. When the intraluminal pressure falls below physiological limits (postmortem), the inner elastic lamina is compressed and it coils up into a regular corrugated shape (Fig. 6.20, see Figs 6.8, 6.10): in these conditions the lumen is much reduced but is not obliterated, and the profile of the artery remains circular. Fenestrations in the elastic lamina, which may also be split in thickness, allow materials to diffuse between intima and media. An outer elastic lamina, similar in appearance to, but markedly less well developed and less compact than the internal elastic lamina, lies at the outer aspect of the media at its boundary with the adventitia (see Fig. 6.8). These laminae are less evident in elastic arteries, where elastic fibres occupy much of the media (see Fig. 6.6).

Fig. 6.20 The wall of a muscular artery (a biopsied human temporal artery), stained for elastin, showing the internal elastic lamina (black wavy line nearest to the lumen, IEL) and more diffuse external elastic lamina (EEL). Fine, incomplete elastic lamellae are interspersed between smooth muscle cells of the tunica media between the IEL and EEL. Elastic van Gieson technique.

Collagen fibrils are found in all three vessel layers. Type III collagen (reticulin) occupies much of the interstitial space between the muscle cells of the media, and is also found in the intima. Collagen is abundant in the adventitia, where type I collagen fibres form large bundles which increase in size from the junction with the media to the outer limit of the vessel wall. In veins, collagen is the main component of the vessel wall, and accounts for more than half its mass.

In general terms, collagen and elastic fibres in the media run parallel to, or at a small angle to, the axes of the muscle cells, and they are therefore mainly circumferentially arranged. In contrast, the predominant arrangement of collagen fibres in the adventitia is longitudinal. This arrangement imposes constraints on length change in large vessels under pressure, e.g. in large arteries, in which the radial distension under the effect of the pulse far exceeds the longitudinal distension. The outer sheath of type I collagen in the adventitia therefore has a structurally supportive role. The more delicate type III collagen network of the media provides attachment to the muscle cells and its role is to transmit force around the circumference of the vessel. In a distended vessel, the elastic fibres store energy and, by recoiling, help to restore the resting length and calibre.

The extracellular material of the media, including collagen and elastin, is produced by the muscle cells. Its turnover is slow compared to that in other tissues. In the adventitia, collagen is synthesized and secreted by fibroblasts, as in other connective tissues. During postnatal development, while vessels increase in diameter and wall thickness, there is an increase in elastin and collagen content. Subsequent changes in vessel structure, seen during ageing, include an increase in the collagen-to-elastin ratio, with a reduction in vessel elasticity.

Cerebral capillaries are the site of the blood–brain barrier (p. 49). They are lined by endothelial cells which are joined by tight junctions. The endothelial cytoplasm contains a few pinocytotic vesicles. The cells are surrounded by a basal lamina (see Fig. 3.13): at points of contact with perivascular astrocytes the intervening basal lamina is formed by fusion of the endothelial and glial basal laminae. Pericytes, completely surrounded by basal lamina, are present around capillaries. Perivascular macrophages are attached to the outer walls of capillaries and to other vessels: they are phenotypically distinct from parenchymal microglia, which are also of monocytic origin. A thin layer of meningeal cells derived from the pia mater surrounds arterioles but disappears at the level of capillaries. For further descriptions of cerebral vessels, see Ch. 17.

LYMPHATIC VESSELS

Lymphatic capillaries form wide-meshed plexuses in the extracellular matrices of most tissues. They begin as dilated, blind-ended tubes with larger diameters and less regular cross-sectional appearances than those of blood capillaries. A basal lamina is incomplete or absent and they lack associated pericytes. The smaller lymphatic vessels are lined by endothelial cells, which have numerous transcytotic vesicles within their cytoplasm, and so resemble blood capillaries. However, unlike capillaries, their endothelium is generally quite permeable to much larger molecules: they are readily permeable to large colloidal proteins and particulate material such as cell debris and microorganisms, and also to cells. Permeability is facilitated by gaps between the endothelial cells, which lack tight junctions, and by pinocytosis.

Lymph is formed from interstitial fluid, which is derived from blood plasma via the microcirculation. Much of this fluid is returned to the venous system. Lymphatic vessels take up residual fluid (about one tenth) by passive diffusion and the transient negative pressures in their lumina which are generated intrinsically by contractile activity of smooth muscle in the largest lymphatic vessel walls, and extrinsically, by movements of other tissues (muscles, arteries) locally. The unidirectional flow of lymph is maintained by the presence of valves in the larger vessels (see Fig. 6.22). Lymphatic capillaries are prevented from collapsing by anchoring filaments which tether their walls to surrounding connective tissue structures and exert radial traction.

Fig. 6.22 A valve (V) in a lymphatic vessel (L), accompanying a small venule (Ven) and arteriole (A) in human connective tissue.

In most tissues, lymph is clear and colourless. In contrast, the lymph from the small intestine is dense and milky, reflecting the presence of lipid droplets (chylomicrons) derived from fat absorbed by the mucosal epithelium. The terminal lymphatic vessels in the mucosa of the small intestine are known as lacteals and the lymph as chyle. Lymphatic capillaries are not ubiquitous: they are not present in cornea, cartilage, thymus, the central or peripheral nervous system or bone marrow, and there are very few in the endomysium of skeletal muscles.

Lymphatic capillaries join into larger vessels which pass to local lymph nodes. Typically, lymph percolates through a series of nodes before reaching a major collecting duct. There are exceptions to this arrangement: the lymph vessels of the thyroid gland and oesophagus, and of the coronary and triangular ligaments of the liver, all drain directly to the thoracic duct without passing through lymph nodes. In the larger vessels, a thin external connective tissue coat supports the endothelium. The largest lymphatic vessels (200 μm) have three layers, like small veins, although their lumen is considerably larger than is the case in veins with a similar wall thickness. The tunica media contains smooth muscle cells, mostly arranged circumferentially. Elastic fibres are sparse in the tunica intima, but form an external elastic lamina in the tunica adventitia.

The larger lymphatic vessels differ from small veins in having many more valves (Fig. 6.22). The valves are semilunar, generally paired and composed of an extension of the intima. Their edges point in the direction of the current, and the vessel wall downstream is expanded into a sinus, which gives the vessels a beaded appearance when they are distended. Valves are important in preventing the backflow of lymph.

Deep lymphatic vessels usually accompany arteries or veins, and almost all reach either the thoracic duct or the right lymphatic duct, which usually join the left or right brachiocephalic veins respectively at the root of the neck.

The thoracic duct is structurally similar to a medium-sized vein, but the smooth muscle in its tunica media is more prominent. Most lymphatic vessels anastomose freely, and larger ones have their own plexiform vasa vasorum and accompanying nerve fibres. If their walls are acutely infected (lymphangitis) this vascular plexus becomes congested, marking the paths of superficial vessels by red lines, which are visible through the skin, and tender to the touch.

Lymphatic vessels repair easily and new vessels readily form after damage. They begin as solid cellular sprouts from the endothelial cells of persisting vessels and later become canalized.

CARDIAC MUSCLE

In cardiac muscle, as in skeletal muscle, the contractile proteins are organized structurally into sarcomeres which are aligned in register across the fibres, producing fine cross-striations that are visible in the light microscope. They both contain the same contractile proteins (although many are cardiac isoforms), which are assembled in a similar way, and the molecular basis for contraction, but not its regulation, is the same. Release of calcium into the sarcoplasm triggers contraction, which corresponds to cardiac systole, the pumping phase of the heart cycle. Reuptake of calcium produces relaxation, which corresponds to cardiac diastole, the filling phase of the cycle.

MICROSTRUCTURE

The myocardium, the muscular component of the heart, constitutes the bulk of its tissues. It consists predominantly of cardiac muscle cells, which are usually 120 μm long and 20–30 μm in diameter in a normal adult. Each cell has one or two large nuclei, occupying the central part of the cell, whereas skeletal muscle has multiple, peripherally placed nuclei. The cells are branched at their ends, and the branches of adjacent cells are so tightly associated that the light microscopic appearance is of a network of branching and anastomosing fibres (Fig. 6.23). Cells are bound together by elaborate junctional complexes, the intercalated discs (Fig. 6.23, Fig. 6.24, see Fig. 6.26).

Fig. 6.23 Cardiac muscle fibres (human heart), sectioned longitudinally. Faint fine cross-striations are just visible (arrow) and indicate the intracellular organization of sarcomeres. The pale transverse lines (some appear stepped) are intercalated discs (D). Endomysium (E) contains nuclei of endothelial cells and fibroblasts.

Fig. 6.24 Three-dimensional reconstruction of cardiac muscle cells in the region of an intercalated disc, a junctional complex between neighbouring cells. The interdigitating transverse parts of the intercalated disc form a fascia adherens, with numerous desmosomes; gap junctions are found in the longitudinal parts of the disc. The organization of the transverse tubules and sarcoplasmic reticulum is also shown.

Fig. 6.26 An intercalated disc in cardiac muscle, with several zones of electron dense fascia adherens (FA) and a gap junction (arrow).

(By courtesy of Professor Brenda Russell, Department of Physiology and Biophysics, University of Illinois at Chicago.)

Fine fibrocollagenous connective tissue is found between cardiac muscle fibres. Although this is equivalent to the endomysium of skeletal muscle, it is less regularly organized because of the complex three-dimensional geometry imposed by the branching cardiac cells. Numerous capillaries and some nerve fibres are found within this layer. Coarser connective tissue, equivalent to the perimysium of skeletal muscle, separates the larger bundles of muscle fibres, and is particularly well developed near the condensations of dense fibrous connective tissue that form the ‘skeleton’ of the heart (see Ch. 56). The ventricles of the heart are composed of spiralling layers of fibres which run in different directions. Consequently, microscope sections of ventricular muscle inevitably contain the profiles of cells cut in a variety of orientations. A linear arrangement of cardiac muscle fibres is found only in the papillary muscles and trabeculae carneae.

Electron micrographs of cardiac muscle cells in longitudinal section (Fig. 6.25) show that the myofibrils separate before they pass around the nucleus, leaving a zone that is occupied by organelles, including sarcoplasmic reticulum, Golgi complex, mitochondria, lipid droplets, and glycogen. At the light microscopic level, these zones appear in longitudinal sections as unstained areas at the poles of each nucleus. They often contain lipofuscin granules, which accumulate there in individuals over the age of 10; the reddish-brown pigment may be visible even in unstained longitudinal sections.

Fig. 6.25 Low power electron micrograph of cardiac muscle in longitudinal section, including the perinuclear zone of one of the fibres. Note the abundant large mitochondria (M) between myofibrils (My), and an intercalated disc (circled).

By courtesy of Professor Brenda Russell, Department of Physiology and Biophysics, University of Illinois at Chicago.)

The cross-striations of cardiac muscle are less conspicuous than those of skeletal muscle. This is because the contractile apparatus of cardiac muscle lies within a mitochondria-rich sarcoplasm. The myofibrils are less well delineated than they are in skeletal muscle: in transverse sections they often fuse into a continuous array of myofilaments, irregularly bounded by mitochondria and longitudinal elements of sarcoplasmic reticulum. The large mitochondria, with their closely spaced cristae, reflect the highly developed oxidative metabolism of cardiac tissue. The proportion of the cell volume occupied by mitochondria (approximately 35%) is even greater in cardiac muscle than it is in slow twitch skeletal muscle fibres. The high demand for oxygen is also reflected in high levels of myoglobin and an exceptionally rich network of capillaries around the fibres.

The force of contraction is transferred through the ends of the cardiac muscle cells via the junctional strength provided by the intercalated discs. As in skeletal muscle, force is also transmitted laterally to the sarcolemma and extracellular matrix via vinculin-containing elements which bridge between the Z-discs of peripheral myofibrils and the plasma membrane.

Atrial muscle cells are smaller than ventricular cells. The cytoplasm near the Golgi complexes at the poles of the nuclei exhibits dense membrane-bound granules, which contain the precursor of atrial natriuretic factor. This is a hormone whose action is to promote loss of sodium chloride and water in the kidneys, reducing body fluid volume and thereby lowering blood pressure. It is released in response to stretch of the atrial wall. The actions of atrial natriuretic factor are normally balanced by the opposing effects of aldosterone and antidiuretic hormone.

The sarcolemma of ventricular cardiac muscle cells invaginates to form T-tubules with a wider lumen than those of skeletal muscle; atrial muscle cells have few or no T-tubules. Unlike skeletal muscle, most T-tubules penetrate the sarcoplasm at the level of the Z-discs (Fig. 6.24). The T-tubules are interconnected at intervals by longitudinal branches to form a complex network. They probably serve a similar function in skeletal and cardiac muscle, i.e. to carry the wave of depolarization into the core of the cells. The actin-binding proteins, spectrin and dystrophin, are important components of the cardiac muscle cell cytoskeleton, which associate independently with the sarcolemma to provide mechanical support.

Intercalated discs

Intercalated discs are unique to cardiac muscle. In the light microscope they are seen as transverse lines crossing the tracts of cardiac cells (see Fig. 6.23). They may step irregularly within or between adjacent tracts, and may appear to jump to a new position as the plane of focus is altered. At the ultrastructural level these structures, which are complex junctions between the cardiac muscle cells, are seen to have transverse and lateral portions (see Fig. 6.24, Fig. 6.26). The transverse portions occur wherever myofibrils abut the end of the cell, and each takes the place of the last Z-disc. At this point, the actin filaments of the terminal sarcomere insert into a dense subsarcolemmal matrix which anchors them, together with other cytoplasmic elements such as intermediate filaments, to the plasma membrane. Prominent desmosomes, often with a dense line in the intercellular space, occur at intervals along each transverse portion. This junctional region is homologous with, and probably similar in composition to, the structure found on the cytoplasmic face of the myotendinous junction, and is a type of fascia adherens junction. It provides firm adhesion between cells, and a route for the transmission of contractile force from one cell to the next.

The lateral portions of the intercalated disc run parallel to the myofilaments, and the long axis of the cell, for a distance which corresponds to one or two sarcomeres before it turns again to form another transverse portion. It is therefore responsible for the stepwise progression of the intercalated disc which can be seen microscopically. The lateral portions contain gap junctions, which are responsible for the electrical coupling between adjacent cells (see Fig. 6.26). Conductance channels within these junctions enable the electrical impulse to propagate from one cell to the next, spreading excitation and contraction rapidly along the branching tracts of interconnected cells. In this way the activity of the individual cells of the heart is coordinated so that they function as if they were a syncytium.