Chapter 162. Endothelium in Inflammation and Angiogenesis

The blood vascular system is a continuous series of hollow tubes that carry blood from the heart to the tissues and back again. Blood exits the heart through the aorta that gives rise to a series of diverging, progressively narrower muscular arteries and arterioles, delivering blood to all organs of the body. The terminal arterioles arborize into an interconnecting network of microvessels, mostly capillaries that nourish and cleanse the peripheral tissues. The capillary network empties into a series of converging venules and progressively larger veins that ultimately return the blood to the heart. All segments of this vascular system are lined by a one-cell-thick layer of epithelium-like cells called endothelium (Fig. 162-1). All vascular endothelial cells (ECs) share common features and functions, and hence may be collectively described as one cell type. However, ECs from one segment of the vascular system may differ in significant ways from the ECs at other anatomic sites. Blood vessel ECs differ from lymphatic ECs, which are not discussed in this chapter.

Blood vessel ECs must perform three important constitutive functions. First, vascular ECs must maintain homeostasis of the blood, which consists of roughly equal volumes of fluid (plasma) and cellular elements (erythrocytes, leukocytes, and platelets). The plasma contains the proteins of the coagulation system. In addition, both leukocytes and platelets have membrane receptors that trigger cellular activation on contact with the extravascular environment. The EC lining prevents intravascular activation both of the coagulation system and of these responsive cellular elements, yet keeps both systems poised respond to wounding or infection.

Second, ECs must separate circulating blood from the tissues. The barrier formed by ECs permits limited passage of fluid and solutes, allowing the blood to nourish the tissues while displaying “permselectivity” for macromolecules and presenting a virtually impenetrable barrier for blood cells (except at specialized sites of high permeability and leukocyte trafficking). In other words, vascular ECs regulate rather than prevent interactions between blood and tissues.

Third, ECs must regulate local blood flow. Because ECs are arranged in alignment with flow, their contraction opens gaps between cells, enhancing permeability without altering flow (see Fig. 162-1). However, ECs indirectly regulate flow by acting on smooth muscle cells (SMCs), which are oriented almost parallel to the vessel's circumferential or radial axis.

ECs do so principally by the generation of SMC relaxants [e.g., nitric oxide (NO), prostaglandin E2 or prostaglandin I2 (PGI2), and endothelium-derived hyperpolarizing factor] or contractants (e.g., endothelin) or by catalyzing the generation of vasoconstrictors from plasma protein-derived mediators (e.g., angiotensin II through the action of EC-expressed angiotensin-converting enzyme, which also inactivates bradykinin, a plasma protein-derived agonist that acts on ECs to generate NO.

In addition to these constitutive functions, ECs may be activated to induce additional functions, a topic discussed later in the chapter.

Approximately three decades ago Irwin Braverman described the organization of the vascular network of human skin, which he summarized in 1989.1 The human dermal vasculature consists of two interconnected systems—(1) a superficial and (2) a deep vascular plexus—with additional vascular networks surrounding sweat glands and hair follicles2,3 (Fig. 162-2). The superficial vascular plexus (SVP) is comprised of paired arterioles and venules that form an interconnected network of vessels coursing on a plane parallel to and just beneath the epidermal surface.3 Capillaries arise from the arterioles, extend upward within the papillary dermis at sites between the epidermal rete ridges, and then loop back down to the venules forming arcade-like structures. The basement membrane of the arterioles appears homogeneous by electron microscopy whereas that of the venules is multilayered. In normal skin, most of the capillary loop is invested with a basement membrane that resembles that of the arteriole, acquiring a venule-like investiture only at the level of the deepest rete just proximal to the anastomosis with the venule of the SVP.2 There are no ultrastructural differences between capillary loops at different sites of the skin. Inflammatory leukocytes extravasate through the venule-like portions of the capillary and the venule proper but only rarely infiltrate through the arteriole-like portions of the capillary or the arteriole itself.

The arterioles and venules of the SVP are connected by short, straight vessels to the arterioles and venules of a deeper planar network of anatomizing vessels called the deep vascular plexus (DVP) (see Fig. 162-2). The plane of the DVP is parallel to that of the SVP and courses above the boundary between the reticular dermis and the underlying subcutis (see Fig. 162-2). The DVP is fed through penetrating vessels from the subcutis. The DVP is drained by valve-containing veins into the subcutaneous fat. The vessel wall in the SVP consists of discontinuous layers of elastic fibers and SMCs. The DVP contains arterioles and venules of larger caliber consisting of three layers: (1) an intima, (2) a media, and (3) an adventitia. Capillary networks connecting the arterioles and venules of the DVP provide nourishment for the adnexal structures within the reticular dermis. The venules of the DVP serve as portals of entry for leukocytes associated with inflammation of the adnexa or for inflammation within the reticular dermis itself, as occurs in erysipelas.

Dermal microvessels are surrounded by flat adventitial cells, ultrastructurally most closely resembling a fibroblast, called veil cells.1 Veil cells are external to the wall, demarcating the vessel from the surrounding dermis. These cells lack leukocyte markers and are negative for HLA-DR and CD1a expression. They express glycoprotein Ib α, von Willebrand factor (vWF) receptor, and factor XIIIa, a coagulation transglutaminase, and are sometimes called XIIIa-positive dendrocytes. Factor XIIIa-positive dendrocyte rarefaction is found in the classic type of Ehlers–Danlos syndrome, but the significance of this finding is not clear. They should be distinguished from dermal dendritic cells, a population of specialized antigen-presenting leukocytes resident within the dermis.

Dermal microvessels, especially capillaries, contain, in addition to ECs, a population of contractile mesenchymal cells called pericytes (PCs). Unlike veil cells, PCs are an integral component of the vessel wall. They extend long cytoplasmic processes over the abluminal surface of the ECs and make interdigitating contacts. The coverage of ECs by PCs varies considerably among different microvessel types. Communicating gap junctions, tight junctions, and adhesion plaques are present at these points of contact.

Because PCs are rich in contractile proteins and have complex interdigitations with ECs in postcapillary venules, it is assumed that they are responsible, at least in part, for gap formation in response to, for example, histamine or bradykinin (although cultured ECs are capable of contraction in the absence of PCs). Moreover, interaction between PCs and ECs is important for the maturation, remodeling, and maintenance of the vascular system via the secretion of growth factors or modulation of the extracellular matrix. There is also evidence that PCs are involved in the transport across the blood–brain barrier and the regulation of vascular permeability.

General Morphology

ECs usually appear as flattened epithelium-like cells, with a thickness typically less than 10 μm and a surface area covering up to 1,000 μm2 (see Fig. 162-1). In large vessels, ECs have an elongated shape, especially in regions of laminar flow, and have their long axis oriented in the direction of blood flow. ECs located in regions of disturbed flow (i.e., at branch points of the arterial tree) are often polygonal rather than elongated. Cultured ECs are typically polygonal but elongate when flowing medium is passed over them for periods of 24 hours or more.4 Shear stress, the force imparted by viscous drag of flowing liquid, is responsible for inducing a cytoskeletal rearrangement and the change of shape from polygonal to elongated. Interestingly, the microtubular organizing center of ECs, located near the nucleus, is always oriented toward the heart (i.e., upstream of the nucleus in arteries and downstream of the nucleus in veins) regardless of the direction of blood flow.5 Moreover, ECs have an apical/luminal polarization, integrin receptors for matrix macromolecules are concentrated on the basal/abluminal surface, and adhesion molecules for leukocytes are almost exclusively displayed on their apical surface, which faces the lumen of the blood vessel.6

Specialized Organelles of Endothelial Cells

The ultrastructure of a postcapillary venule is shown in Fig. 162-3. ECs have a number of characteristic organelles. The best described are Weibel–Palade bodies (WPBs), fenestrae, and the caveolar system. WPBs are elongated, membrane-bound organelles that display longitudinal striations by electron microscopy. WPBs are distributed randomly throughout the cytoplasm in vascular ECs of most vertebrates. WPBs constitutively contain vWF (also called factor VIII-related antigen), P-selectin (CD62P),7 lysosomal membrane-associated glycoprotein 3 (or CD63),8 1,3-fucosyltransferase VI,9 interleukin 8 (IL-8),10 and angiopoietin 2 (Ang-2).11 The longitudinal striations within the WPBs are formed by highly polymerized forms of vWF, reaching sizes of up to 20,000 kDa. On release, vWF polymers may attach to the basement membrane and stabilize platelet adhesion to the subendothelial matrix, particularly in high-velocity arterial flow. Although vWF is commonly used as an EC marker (see Section “Molecular Markers of Endothelial Cells”), it is also found in megakaryocytes, which appear to lack a physiologic mechanism of release, synthesizing vWF only for storage within the α granules of their platelet progeny. P-selectin is rapidly translocated to the cell surface as the WPB fuses with the plasma membrane, where it mediates leukocyte adhesion to ECs (see Section “Leukocyte Adhesion”). CD63 is a 53-kDa lysosomal membrane glycoprotein belonging to the tetraspanin superfamily; its function in the WPB is unknown.

Blood vascular ECs usually form a continuous monolayer, and most transendothelial transport takes place in the junctions between cells. One important exception is in the fenestrae of capillaries in lymph nodes, capillaries in the intestinal mucosa, renal glomerular ECs, and hepatic and bone marrow sinusoids. Fenestrae, uniquely found in ECs, are specialized annular patches of attenuated cytoplasm and membrane, about 175 nm in diameter, characterized by an increased concentration of anionic lipids.12 They group into structures resembling sieve plates that occupy 6% to 8% of the surface capillaries. Fenestrated capillaries are much more permeable to water and small-molecular-size solutes than those of the continuous type. The anionic charge probably precludes the transfenestral passage of anionic plasma proteins. Various factors, such as pressure, alcohol, serotonin, nicotine, and infection, change the number and diameter of fenestrae, and ECs of the continuous type may become transiently fenestrated.

All ECs appear to contain numerous vesicles subjacent to the plasmalemma, both luminal and abluminal. On careful examination, most of these vesicles turned out to have narrow tubular connections to the plasma membrane. Such flask-shaped invaginations are called caveolae, literally “little caves,” and are part of the surface membrane, either as a single caveola or as more complex racemose invaginations. In situ, they may account for 50% or more of the plasma membrane surface area of ECs. Caveolae arise from a late Golgi compartment and represent a stable membrane unit built up around caveolins, cholesterol, and glycosphingolipids. ECs are rich in caveolins 1 and 2. Caveolins bind to cholesterol and are required for caveolae formation; ECs in caveolin 1 knockout mice fail to form caveolae. Expression of caveolin 1 in some cell types normally lacking caveolae can cause their formation, but cultured ECs normally retain expression of caveolins yet lose their caveolae, with cholesterol and glycosphingolipids dispersing into submicroscopic lipid rafts on the now simplified plasma membrane, implying a key role for an as yet unidentified factor.

Caveolae can mediate vesicular transport, internalize certain plasma membrane proteins and provide a platform for the assembly of signaling complexes at the surface of the cell. Caveolins interact with a variety of downstream signaling molecules, including Src-family of tyrosine kinases, p42/44 mitogen-activated protein kinase, and endothelial NO synthase (eNOS). They hold these signal transducers in the inactive conformation until activation by an appropriate stimulus. For example, the amount of caveolin is inversely related to NO production by eNOS.13 Mice lacking caveolin 1 have impaired NO and calcium signaling in the cardiovascular system, which causes aberrations in EC-dependent relaxation, contractility, and maintenance of myogenic tone.14

Caveolin 1 is associated with a number of growth factor receptors, including the epidermal growth factor receptor, the high affinity nerve growth factor receptor (TrkA), the insulin receptor, the platelet-derived growth factor receptor and transforming growth factor receptors I and II.15,16 In addition, cytokine receptors like the tumor necrosis factor (TNF) receptor 1 (CD120a) and interferon receptors also localize to caveolae. For these receptors, the invaginated structure of caveolae provides an optimal environment for receptor function, forming a more stable signaling complex and allowing for cross talk with other receptors.

The lipid constituents of caveolae are the same as those of lipid rafts, but rafts are regions without membrane indentations and they are flat. A key limitation in a clear distinction between rafts and caveolae is due to methodical limitations to clearly separate them. Moreover, rafts and caveolae should be considered as dynamic entities that form and dissipate in response to various stimuli, and both may facilitate transport of entities to other cellular regions and across the cell.

Another organelle that is present in venular endothelium is so called vesicular–vacuolar organelle, grapelike structures of interconnected vesicles that extend throughout the cytoplasm. They show a wide variation in size and most vesicles do not contain caveolin. Consistent with this finding, they are present in normal size and numbers in caveolin null mice.17 These organelles appear to be critically involved in acute vascular hyperpermeability, which typically involves the postcapillary venules, where these vesicular–vacuolar organelles are typically situated.

Endothelial Junctions

Neighboring ECs connect to each other by forming interdigitating protrusions connected by junctions. Junctions possess different degrees of complexity along the vascular tree, reflecting different permeability for proteins and cells. Intercellular tight (occluding) junctions constitute the anatomic basis for the tightly regulated interfaces of the blood–testis and blood–brain barriers.18 They consist of a continuous belt-like meshwork of six anastomosing junctional strands, which prevent the transport of macromolecules into the tissue. Arterial ECs also possess tight junctions19; however, they consist of only one or two junctional strands, and tight junctions are difficult to find in postcapillary venules. Tight junctions contain a complex of transmembrane (junctional adhesion molecule-1, occludin, and claudins) and cytoplasmic (zonula occludens-1 and -2, cingulin, AF-6, and 7H6) proteins linked to the actin cytoskeleton. The claudin multigene family encodes tetraspan membrane proteins that are crucial structural and functional components of tight junctions. In mammals, the claudin family consists of 24 members, which exhibit complex tissue-specific patterns of expression. Their extracellular loops interact with each other to seal the cellular sheet and regulate paracellular transport between the luminal and basolateral spaces. The transmembrane protein occludin is linked to ZO-1, a cytoplasmic protein that interacts with the cytoskeleton and appears involved in opening and closing of the junctions. Junctional adhesion molecules, called JAMs, members of the immunoglobulin superfamily, form homophilic adhesion sites in tight junctions and play a role in the generation of cell polarity, keeping luminal proteins from diffusing to the abluminal cell surface and vice versa.

Adherens junctions are found in all vascular ECs (see Fig. 162-3). They are multimeric protein structures that mediate homotypic cell adhesion. In endothelial cells, they are distributed along the cleft between neighboring cells and are frequently intermingled with tight junctions. During embryonic development, this type of junction promotes adhesion of identical cells and is important for organization and separation of tissues. In the adult, they maintain tissue homeostasis and contribute to paracellular permeability. Moreover, they confer cell-to-cell signals such as contact inhibition of cell growth, or resistance to apoptosis. They also regulate cell shape and polarity. Endothelial adherens junctions are principally organized by VE-cadherin [also called cadherin 5 or vascular endothelial cadherin (CD 144)]. VE-cadherin-negative ECs derived by gene targeting fail to organize in vessel-like structures, blocking VE-cadherin interactions with monoclonal antibodies inhibits vessel formation.20,21 Whereas VE-cadherin appears critical for initial formation of blood vessels, for maintenance and remodeling of blood vessels, a receptor-type phosphotyrosine phosphatase (RPTP) called VE-PTP appears essential. VE-PTP binds to VE-cadherin via the extracellular domain and supports adhesive functions of VE-cadherin.22 Intracellularly, VE-cadherin is linked to a large cytosolic protein complex. This complex contains catenins, β-catenin, p120ctn, plakoglobin (also called γ-catenin), and α-catenin. Catenins β-catenin, p120ctn and plakoglobin contain homologous Armadillo repeats, whereas α-catenin is homologous to vinculin, which is another actin-binding protein. Moreover, this complex contains src, fyn and src homology-containing phosphatase (SHP)-1, and RPTP-μ and may associate with neighboring transmembrane receptors such as vascular endothelial growth factor receptor (VEGF-R)-2, angiopoietin receptor 1 (also known as Tie-2) and protease activated receptors (PAR)-3 and -6. Thus, adherens junctions integrate signals from neighboring cells and neighboring transmembrane receptor molecules and link these signals to the cytoskeleton and through focal adhesion kinase with integrin receptors to the basal matrix. This signaling pathway also functions retrograde from integrin receptors to adherens junctions. This network gets further input through RhoGTPases signaling to Rho kinase (ROCK) and leading to contraction of the cytoskeleton.17,23–29

ECs form gap junctions, demonstrated by transfer of microinjected small-molecular-weight tracers between adjacent ECs. In ECs, the relevant gap junctional proteins are connexin-43, connexin-37, and connexin-40. Interestingly, ECs also may form heterogap junctions with PCs or SMCs in the vessel wall and sometimes with adherent leukocytes.

Endothelial Cell–Matrix Interactions

ECs rest on a basal lamina, which is formed by EC itself and by surrounding cells. It consists of laminins, collagens, fibronectin, nidogen (entactin), and heparan sulfate proteoglycans. Interaction with these matrix molecules is essential for maintaining normal endothelial function. For example, the laminin A chain peptide supports endothelial branching and formation of new capillaries.30 Furthermore, a 20-kDa C-terminal fragment of collagen XVIII specifically inhibits endothelial proliferation and angiogenesis,31 which suggests that collagen XVIII is also a positive regulator of vessel growth. On the other hand, EC–collagen V interactions inhibit endothelial attachment and growth. ECs interact with these components of the basal lamina by numerous transmembrane adhesion molecules. The complexities of these interactions have only been partially characterized. Members of the integrin family are particularly important for this function. For example, α6β1-, α2β1-, α5β1-, and αVβ3-receptor complexes are important for endothelial attachment to, and migration on, laminin, collagen, and tenascin, and therefore serve important roles in angiogenesis. In addition, integrin binding to matrix proteins also has a multitude of intracellular effects on the organization of the endothelial actin skeleton and on signaling processes within the cell.3 Endothelial-cell growth factors have been shown to differentially upregulate the biosynthesis of α2, α5, β1, and β3 integrins, serving the different needs of endothelium during growth, spreading, and new vessel formation. Moreover, integrins show certain tissue-and vessel-restricted expression; for example, α1β1 is found only in small blood vessels and capillaries but not in large vessel ECs. Matrix proteins such as thrombospondin 1, may also serve as inhibitors of angiogenesis.33

Molecular Markers of Endothelial Cells

ECs display a number of molecular markers that result in a characteristic profile detectable with antibodies, lectins, and other ligands (Table 162-1). In addition to containing molecules expressed on all endothelium, the skin vasculature shows several site-specific characteristics. Postcapillary venules of the SVP are CD36− and thus distinct from capillaries of the DVP and from capillaries of other tissues, which are CD36+.34 CD36 antigen is a cellular receptor for thrombospondin, collagen, low-density lipoproteins, long-chain fatty acids, and platelet-agglutinating protein p37.35 Moreover, CD36 functions as one of the receptors that mediate the adhesion of Plasmodium falciparum-infected erythrocytes to microvascular EC. The functional consequences of the disparate CD36 expression on SVP and DVP endothelium are unknown. Microvessels are best characterized by expression of the pathologische anatomie Leiden-endothelium (PAL-E) antigen (which is identical with plasmalemmal vesicle 1 or fenestrated endothelial-linked structure protein),36 N-cadherin, and CXC chemokine receptor 4 (CXCR4), and by their surrounding by smooth muscle actin-positive cells (which may be SMCs or PCs). Blood vascular ECs normally lack the lymphatic EC markers lymphatic vessel endothelial-1 [LYVE-1, (a hyaluronan receptor) and podoplanin], but may express these molecules when inflamed.37,38 ECs express receptors for binding members of the VEGF family. Blood endothelial cells express VEGFR-2 and VEGFR-1, whereas lymphatic vessel endothelial cells express VEGFR-2 and VEGFR-3.39–42 Neuropilins, another group of VEGF-binding membrane-bound receptors, are also differentially expressed in various types of vasculature. Whereas neuropilin-1 is strongly expressed in arterial endothelial cells, neuropilin-2 is expressed in veins and visceral lymphatic vessels.43 Another important signaling system is the angiopoietin/Tie receptor system (see Section “Regulation of Angiogenesis and Vasculogenesis”).

Postcapillary venules of the skin express toll-like receptor (TLR) family members and CD32 molecules (FcγRIIa).44 CD32 binds complexed immunoglobulin G and is thought to function in the elicitation of type III (immune complex-mediated) hypersensitivity as well as in immune complex clearing. ECs of postcapillary venules express significantly higher levels of the histamine H1 receptor than other ECs. Histamine produces increased albumin leakage and induces rolling of leukocytes, mediated in part by P-selectin expressed on the EC surface. PAR 2 expression at this site also plays a role in inflammatory reactions. PAR 2-activating peptides increase leukocyte rolling and adhesion to postcapillary venules. Finally, postcapillary vessels of the skin in situ constitutively express major histocompatibility complex (MHC) class I and II molecules at their luminal surfaces, which implies that ECs may directly participate in the elicitation of antigen-driven immune responses. As discussed later (see Section “Role of Endothelium in Adaptive Immunity”), skin microvessels in cell culture appear able to process and present antigen in an MHC-restricted manner to T cells.45

Innate Immunity

Innate immunity, also called natural or native immunity, comprises the host's defense systems against microbes that are independent of T and B lymphocytes and their receptors for specific antigens (see Chapter 10). The innate immune system is much older in evolutionary terms than the T- and B-cell-specific immune system (often called adaptive immunity to distinguish it from innate immunity). The humoral component primarily involves the complement system. The primary cellular effectors of innate immunity in humans are neutrophils, mononuclear phagocytes, and natural killer cells, but ECs also participate.

Endothelial cells represent the initial target in many different types of infection.46 For example, exposure to lipopolysaccharide, a major cell-wall constituent of Gram-negative bacteria, results in endothelial activation through a receptor complex consisting of TLR-4, CD14, and MD2. Then, recruitment of the adaptor protein myeloid differentiation factor (MyD) 88 to the ligand-activated TLR-4 receptor initiates an MyD88-dependent pathway that culminates in the early activation of nuclear factor-κ B and the mitogen-activated protein kinases. In parallel, an MyD88-independent pathway using an alternative adaptor protein called TRIF results in a late-phase activation of nuclear factor-κ B as well as generation of interferons.47,48 ECs also express TLR-2, which serves as a signaling receptor for Gram-positive cell-wall components, and intracellular TLRs that respond to various types of nucleic acids typically encountered during infections by viruses. Stimulation of TLR 2/6 by bacterial lipopeptides also may promote angiogenesis and results in the secretion of GM-CSF.49 Certain vascular beds express the macrophage mannose receptor (CD206), a receptor for oligosaccharides on the cell wall of bacteria, yeasts, and parasites. Other receptors of the innate immune system include a variety of lectin-type molecules, such as mannose-binding protein or galectins that recognize bacterial carbohydrates. Mannose-binding protein may stimulate the alternative pathway of complement activation.

TNF and IL-1 are the principal effector cytokines of the innate immune response to bacteria. In general, endothelial responses to TNF and IL-1 are similar and are considered together. The major isoform of IL-1 that is produced during innate immune responses is IL-1β, but IL-1α, the major isoform made by ECs, has indistinguishable actions, and we will simply refer to both isoforms as IL-1. At least five changes in the phenotype of ECs that promote inflammation, collectively called endothelial activation, have been described.

Synthesis of Vasodilators

TNF- or IL-1-treated ECs increase production of vasodilators, such as PGI2 and NO, that relax smooth muscle cell tension, thereby producing vasodilation and increasing tissue perfusion. PGI2, also called prostacyclin, is synthesized in ECs by a series of three sequential enzyme-catalyzed reactions.52 ECs express one isoform of prostaglandin H synthase, PGHS-1, also called cyclooxygenase 1, that is constitutively active. Treatment of ECs with TNF or IL-1 dramatically increases PGHS activity by inducing de novo expression of a second isoform, PGHS-2, which is also called cyclooxygenase 2. This response markedly increases the capacity to make prostaglandin (PG)H2 from arachidonic acid, and the increased PGH2 is subsequently converted into PGI2 (Fig. 162-4). Pharmacologic inhibition of the synthesis of PGH2 is thought to contribute to the anti-inflammatory actions of aspirin and nonsteroidal anti-inflammatory drugs. NO is produced by conversion of arginine to citrulline through eNOS (Fig. 162-5). TNF exhibits opposing effects on vasodilation. Initially, it enhances eNOS function and NO synthesis through activation of Akt and by increasing the synthesis of tetrahydrobiopterin, a limiting cofactor for eNOS function.53 At later times, TNF destabilizes eNOS mRNA and thereby inhibits NO production.

Leukocyte Adhesion

(Tables 162-2 and 162-3). TNF- or IL-1-treated ECs exhibit changes in the cell surface that favor the initial capture, rolling, and, ultimately, firm adhesion and spreading of leukocytes (Fig. 162-6). Adhesion is a necessary prelude to extravasation of leukocytes into the tissues. Capture and rolling are typically mediated by selectins expressed on the leukocyte or on the EC.54 There are three structurally related selectin proteins: (1) L-selectin (CD62L), (2) E-selectin (CD62E), and (3) P-selectin (CD62P). In the presence of flowing blood, the leukocyte is propelled in the direction of blood flow, rapidly breaking and reforming selectin-mediated attachments. This results in rolling of the leukocyte along the EC surface (see Fig. 162-6). Ligands involved in selectin-mediated capture may not be the same as those involved in selectin-mediated rolling; for example, neutrophils may switch from L-selectin to E-selectin ligand (ESL)-1 as the principal E-selectin ligand during these two phases of the interaction.55,56 Studies using blocking monoclonal antibodies or selectin knockout mice have confirmed that selectins are required for neutrophil rolling and subsequent recruitment in vivo. In mice, E- and P-selectin functions may be redundant (i.e., both molecules must be knocked out or inhibited for complete loss of rolling). Of note, whereas IL-1 and TNF upregulate both P- and E-selectin in mice, they induce only E- but not P-selectin in humans. In humans, E-selectin expression is largely regulated by de novo synthesis induced by cytokines whereas P-selectin is preformed and mobilized from WPBs to the cell surface in response to stimuli like thrombin, histamine, reactive oxygen species, and the like. Consequently, E- and P-selectin may not be redundant in humans. Selectin expression is rapid, within 5 min for P-selectin and 1 to 2 hours for E-selectin, but is often transient, peaking at 15 minutes for P-selectin and 4 to 6 hours for E-selectin, but may persist much longer on skin ECs. Another important mechanism initiating rolling of T lymphocytes on ECs is the interaction of the hyaluronan receptor CD44. CD44 is expressed on endothelial cells and, under inflammatory conditions, binds hyaluronan, which then binds CD44 expressed on activated T lymphocytes and macrophages.57

Firm adhesion of leukocytes to ECs is generally mediated by leukocyte integrins. Leukocytes use two different types of integrins to interact with ECs. The first of these are the CD18 or β2 integrins, including lymphocyte function-associated antigen-1 (αLβ2 or CD11a/CD18) and Mac-1 (αMβ2 or CD11b/CD18). The adhesion mediated by LFA-1 or Mac-1 recognition of intercellular adhesion molecule-1 (ICAM-1) is much stronger than that mediated by selectins, so that once these molecules have engaged ICAM-1, leukocytes no longer roll. In the case of neutrophils, integrin engagement results in a gradual slowing of rolling velocity. In contrast, lymphocytes abruptly convert from rolling to firm adhesion. Once adherent, leukocytes spread out and then slowly crawl along the EC surface until they reach a cell junction, where they undergo diapedesis through the vessel wall into the tissues (see Fig. 162-6). It is likely that LFA-1-mediated attachment, disengagement, and reattachment to ICAM-1 play a role in the spreading, crawling, and diapedesis of leukocytes. ICAM-1 is induced at slightly later times than E-selectin, but it remains elevated for days; it is initially (at 4 hours after treatment with TNF) upregulated diffusely on the luminal surface of the EC but then preferentially localizes to the intercellular junctions over the first few days.6 Studies with blocking antibodies and in knockout mice confirm that β2 integrins and ICAM-1 play a major role in leukocyte adhesion to ECs and in subsequent recruitment.

The second group of integrins on leukocytes that mediate attachment to ECs are the α4 integrins (CD49d), which may pair with either β1 (CD29) to form very late activation antigen-4 (VLA-4) (α4β1) or with β7 to form lymphocyte Peyer patch adhesion molecule-1 (LPAM-1) (integrin α4β7). The principal EC ligand for VLA-4 is vascular adhesion molecule 1 (VCAM-1). This immunoglobulin superfamily member is not expressed on resting ECs under normal circumstances but is upregulated by TNF or IL-1 with a time course that peaks at about 24 hours. VCAM-1 may be selectively upregulated by IL-4, a cytokine associated with certain types of adaptive immunity, and IL-4 can act synergistically with TNF or IL-1. Although VCAM-1 expression is not responsive to interferon-γ (IFN-γ) itself, IFN-γ intensifies TNF-induced VCAM-1 expression.60 Interestingly, VCAM-1 may support initial tethering and rolling of some leukocytes (e.g., of T cells but not of neutrophils), as well as firm adhesion, depending on whether the affinity/avidity of the leukocyte VLA-4 is increased by leukocyte activation. Antibody-blocking experiments confirm a role for these molecules in leukocyte recruitment in vivo.61 True VCAM-1 knockout mice are embryonic lethals due to failure of placentation. Some VCAM-1 “knockouts” were made by disrupting the first exon. These animals make a truncated VCAM-1 protein, lacking the first Ig domain but express it at reduced (“hypomorphic”) levels. These animals also confirm a role for VCAM-1 in leukocyte recruitment.

Leukocyte Activation

TNF- or IL-1-treated ECs promote the activation of tethered or rolling leukocytes, triggering the transition from selectin-mediated low-affinity attachment to integrin-mediated firm adhesion and the transition from spherical, immotile leukocytes to spreading, migrating forms. In some experiments, engagement of selectins appears to be adequate by itself to trigger some of these changes, but most experiments indicate the requirement for a distinct signal. One candidate activator is the lipid mediator platelet-activating factor (PAF), which can be displayed in the EC plasma membrane. PAF synthesis is initiated by cytosolic phospholipase A2 activation, which is the same reaction that provides arachidonic acid for PGI2 synthesis. It has been reported that IL-1β may promote PAF synthesis in ECs, but most studies emphasize the role of autacoids (e.g., histamine), rather than cytokines, as inducers of PAF synthesis.

The chemokines constitute an important group of TNF- or IL-1β-induced leukocyte activators that are defined by the presence and number of amino acids between two N-terminal cysteine residues (i.e., CC, CXC, etc.) (see Chapter 12). Chemokines induce migration and/or activation of various types of leukocytes through interaction with a group of seven transmembrane G protein-coupled receptors. The CXC chemokines (such as IL-8) are particularly important for neutrophil activation, and CC chemokines (such as MCP-1) are important for monocyte activation. IL-8 is stored in WPBs and released on stimulation with histamine or thrombin. Both IL-8 and MCP-1 are synthesized and secreted by TNF- or IL-1β-treated ECs. Furthermore, ECs can display their secreted chemokines in a complex with cell surface proteoglycans in a manner that renders them bioavailable to tethered or rolling leukocytes. The bound chemokines displayed by ECs could be made by ECs or could be captured and displayed once made by tissue cells or leukocytes. In addition, there is a transmembrane protein form of a chemokine called fractalkine that has its chemokine domain presented at the top of a mucinlike stalk. Fractalkine can be induced in vascular ECs and functions as an adhesion molecule. Accumulating evidence suggests that fractalkine mediates vascular injury in glomerulonephritis, allograft rejection, and atherosclerotic disease.63–65 Chemokines may also play a role in activation and recruitment of specific lymphocyte subsets, as discussed later.

Extravasation of Leukocytes

The extravasation of leukocytes requires the preceding steps of tethering, rolling and adhesion followed by locomotion to junctions. Junctional proteins such as PECAM-1 (CD31), CD99 and junctional adhesion molecules (JAMs) contribute to leukocyte transmigration, and as noted earlier, activated ECs also concentrate adhesive ligands, such as E-selectin, ICAM-1, and VCAM-1, at these same junctions. The details of interaction between leukocytes and ECs during diapedesis in vivo are incompletely defined. The principal pathway for extravasation appears to be through the intercellular junctions (see Table 162-2). Antibodies to PECAM-1 and to CD99 inhibit this process. Of note, in PECAM-1 knockout mice, effects on leukocyte transmigration appear to be mouse strain specific. PECAM-1 at the border of endothelium forms homophilic interaction between PECAM-1 on leukocytes and triggers targeted recycling of membrane from a reticulum localized compartment close to the endothelial cell lateral border and is called lateral border recycling compartment. This compartment constitutively recycles and is mobilized to sites of junctions and surrounds the transmigrating leukocyte. This compartment also contains CD99 and JAM-1 but not VE-cadherin.66 JAM proteins interact with leukocytes by binding to integrins. The integrin LFA-1 mediates neutrophil and T-cell adhesion to JAM-A; the integrin α4β1 mediates leukocyte adhesion to JAM-B. JAM-2–α4β1 interactions appear to be dependent on interactions between JAM-B and JAM-B. JAM-B binds to CD11b/CD18 on neutrophils, and this interaction is thought to be important for neutrophil transendothelial migration. It has been shown that VE-cadherin gap formation is required for leukocyte transmigration and p120 is a critical intracellular mediator of this process through its regulation of VE-cadherin expression at junctions.67 In addition, VE-cadherin may interact with leukocytes through an interposed fragment of fibrin, which is formed through thrombin-induced fibrinogen activation followed by plasmin or urokinase digestion.68

In certain instances, leukocytes may extravasate through the EC body. The factors that determine whether leukocytes use the intercellular or transcellular pathway are unknown. The early stages of this process appear to involve phagocytosis of the leukocyte through membrane extensions enriched in ICAM-1 and VCAM-1. The transcellular diapedesis of monocytes and neutrophils also involves the lateral border recycling compartment within which PECAM-1, CD99 and JAM-A are the critically involved adhesion molecules. In addition, the rapid transmigration of T cells, but not other leukocyte subsets, appears to depend on the presence of venular levels of shear stress, which contribute by signaling to the T cell by “stretching” tethered adhesion molecules such as LFA-1. TNF- or IL-1-treated ECs secrete and activate matrix metalloproteinases (MMPs), which degrade the condensed region of connective tissue matrix, that is, the basement membrane that underlies the endothelial monolayer. ECs may also trigger leukocytes to synthesize or release MMPs, and the signals for this release may be cytokine-inducible molecules such as VCAM-1 or chemokines.69

T cells in the circulation are heterogeneous. Some have never encountered their cognate antigen and are said to be naïve. Memory T cells arise following encounters with antigen and may be divided into central memory and effector memory subpopulations. Effector memory CD45RO+ CCR7low L-selectinlow CD4+ T cells transmigrate across endothelial monolayers in flow chambers in the presence of the IFN-γ-inducible protein-10 (IP-10) (CXCL10) whereas naive T cells or central memory T cells do not, despite the expression of CXCR3 on the latter cell type. In vitro assays established that the recognition of endothelial MHC class II by T cells blocks the response to IP-10 but causes delayed transendothelial migration of effector memory, but not of naïve or central memory CD4(+) T cells. Fractalkine, induced by TNF or IL-1 on microvessel EC, also contributes to the TCR-initiated response.70 The TCR pathway also involves PECAM-1, which is not used by T cells responding to IP-10.

Vascular Leakage and Provisional Matrix

Extravasation of plasma proteins into the tissue is a hallmark of inflammation. Autacoids such as histamine allow this to occur rapidly and transiently by causing ECs to contract their actin cytoskeleton, opening gaps between cells. VEGF may function similarly but also appears to have its primary effects on increasing fluid transit through vesiculovacuolar organelles. In contrast, TNF- or IL-1-treated ECs permit the nonspecific extravasation of fluid and plasma proteins, commonly called vascular leak, only after a delay of several hours in a process that depends on new protein synthesis. The loss of permselectivity (vascular leak) is a hallmark of inflammation. The principal purpose of this response is to allow plasma proteins, such as fibronectin and fibrinogen, to deposit in the tissues, where they form a provisional matrix that can be used by motile leukocytes. This is important because circulating leukocytes typically lack receptors that allow them to interact with the normal extracellular matrix, composed largely of interstitial collagens.

Collectively, these five TNF- or IL-1-mediated responses of ECs promote the development of an inflammatory infiltrate initially comprised of neutrophils and later of mononuclear phagocytes. The infiltrating leukocytes serve to phagocytose the eliciting microbes and clean up damaged tissue. Infiltrating phagocytes also can cause tissue damage through the same mediators and enzymes they use to eradicate microbes. These mechanisms can and often do injure local ECs, which is manifested by EC retraction and/or denudation and often accompanied by intravascular thrombosis. Intravascular thrombi exacerbate tissue ischemia and injury. Fortunately, TNF- or IL-1-treated ECs become more resistant to injury through expression of protective gene products such as detoxifying agents for reactive oxygen intermediates like manganese-superoxide dismutase and hemoxygenase, protease inhibitors like plasminogen activator inhibitor-1 and tissue inhibitors of metalloproteinases, and cytoprotective genes such as the zinc finger protein ubiquitinase/de-ubiquitinase enzyme A20 and Bcl-related antiapoptotic genes such as A1. These protective responses allow ECs to survive many inflammatory reactions without triggering thrombosis and consequent tissue infarction.

The acquisition of cytoprotective features has been observed in the ECs of organ grafts that have evaded transplant rejection.71 However, these protective responses are not always adequate. Moreover, some other TNF- or IL-1-mediated endothelial responses, for example, induction of procoagulant proteins such as tissue factor coupled with loss of anticoagulant proteins such as thrombomodulin, may actually exacerbate tissue and vascular injury. This combination of neutrophil recruitment and alterations in the endothelial coagulation system may underlie the tissue injury seen in the local Shwartzman reaction or in Gram-negative septicemia.

In summary, ECs, through their responses to cytokines, can contribute to host defense reactions mediated by the innate immune system. However, these same effector mechanisms may lead to endothelial injury and exacerbate tissue damage.

Role of Endothelium in Adaptive Immunity

ECs of microvessels constitutively express MHC molecules. To date, the only well-established function of class I and class II MHC molecules is to present peptides to T cells. Vascular ECs not only express MHC molecules but also express the proteins involved in generating peptides and loading these peptides into nascent class I and class II molecules.72 The expression of class I genes (and the gene encoding the associated β2-microglobulin molecule), as well as the proteins involved in peptide generation and class I MHC molecule peptide loading, are all upregulated by cytokines, particularly by IFNs, and by TNF.73 Under basal culture conditions human ECs do not express class II MHC molecules or the proteins responsible for peptide loading of class II molecules, but these proteins are induced by IFN-γ. In humans, class II molecules are basally expressed on microvascular cells in vivo.73,74 It is unclear whether this “constitutive” expression depends on low levels of circulating IFN-γ or is truly constitutive.

The typical diameter of a capillary lumen is less than 10 μm, i.e., narrower than the diameter of a circulating lymphocyte. Consequently, circulating T cells are forced to encounter antigenic peptides complexed to both class I and class II MHC molecules displayed on the endothelial surface during the course of normal circulation. A key question is what effect this encounter has on the T cell. Studies of the consequences of T-cell recognition of antigen presented by ECs have largely used cell culture systems. Naive T cells ignore antigens presented by ECs whereas resting memory T cells or recently activated T blasts respond by cytokine gene expression and a limited degree of proliferation. This limitation correlates with the observation that human ECs are deficient in expressing certain costimulatory molecules, for example, CD80 and CD86, and primarily costimulate T cells with LFA-3 (CD58).76 Naive T cells poorly express CD2, the receptor for LFA-3. Another factor may be that naive T cells lack the expression of adhesion molecules necessary to attach firmly to ECs. In addition to constitutive expression of LFA-3, human ECs display inducible expression of ligands for inducible T-cell costimulator, CD137, and CD134 that are typically associated with the stimulation or reactivation of memory T cells, as well as expression of molecules that inhibit T-cell activation, such as ligands for PD-1.77–79

Several nonexclusive roles have been proposed for presentation of antigen by ECs if it were to occur in vivo. The first is the maintenance of immunologic memory. Long-lived memory T cells may require periodic low-affinity signaling to survive, and the expression of MHC molecules bearing self-peptides by ECs may provide such low-affinity signals as these cells pass through the microcirculation. Recent data indicate that human blood ECs are able to present self-antigens in vivo75, which may help sustain the viability of memory-cell populations. A second role might come into play when there is reinfection by a particular pathogen. In this case, ECs may present microbe-derived peptides in association with MHC molecules, providing an antigen-specific signal to circulating memory T cells that pathogen is present. This mechanism would contribute to the efficiency of immune surveillance. This is a potentially important function, because T cells specific for any one antigen, even among memory T cells expanded by prior encounter with antigen, are rare (less than 1 in 10,000–100,000). Presentation of antigen by ECs could markedly increase the likelihood that antigen is recognized in a timely manner and that memory T cells are recruited to sites of danger. A third possibility involves the postthymic induction and activation of CD4+25+forkhead box P3 regulatory T cells.80 It has been shown that, in mice, alloantigen presentation by vascular ECs to CD4+ T cells induces such regulatory T cells, which could be important for induction of tolerance. However, it must be noted that mouse ECs usually do not express class II MHC molecules in vivo and that cultured human T cells preferentially activate CD4+ effector cells rather than regulatory T cells.

T cells may differentiate into specialized cytokine-producing subsets. For CD4+ T cells, such responses may become polarized into T helper 1 (Th1, they produce IFN and lymphotoxin), or into T helper 2 (Th2, they produce IL-4 and IL-5). More recently, it has been appreciated that some effector CD4+ T cells may selectively secrete IL-17, a proinflammatory cytokine. IL-17 producing helper T (Th17) cells bind via LFA-1 to ICAM-1, which is expressed on EC. IL-17 has been identified as an important proinflammatory mediator in various diseases like atopic dermatitis or bronchial asthma. In various models, IL-17 was also able to directly disrupt the blood–brain barrier as a direct effect of Th17 cells on ECs. Disruption of blood–brain barrier is an important step for the development of central nervous system inflammation as observed in diseases like multiple sclerosis.81,82 On HUVEC, IL-17 induces expression of adhesion molecules like ICAM-1, VCAM-1, E-selectin83 but the potency of IL-17 is markedly less than that of TNF or IL-1. Another subclass of specialized T cells, regulatory T cells (Treg; CD4+/CD25+/FOXP3+) play an important role in inflammatory and neoplastic diseases.84,85 ECs of tumor vessels express a specific addressin signature to guide Tregs into the tumor.86

Cytokines released by Th1 or Th2 cells may differentially shape EC activation patterns. For example, IFN-γ combined with lymphotoxin causes prolonged expression of E-selectin, an important ligand for Th1 T cells. Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin, and migration of Th1 cells into inflamed skin is blocked by antibodies against P- and E-selectin.87 P-selectin glycoprotein ligand 1 appears to function as P- and E-selectin ligands for Th1 cells, which correlates with increased expression of the α3-fucosyltransferases in Th1 cells.87,88 In contrast, Th2-derived IL-4 increases VCAM-1 expression while suppressing E-selectin, a cell surface molecule that favors eosinophil and possibly also Th2-cell recruitment. In other words, T cell-derived cytokines may modify the innate EC responses in a way that biases leukocyte recruitment toward recruitment of specific effector cells. Finally, differential expression of chemokine receptors may also account for selective recruitment of T-cell subsets. For example, CCR5 and, to a lesser degree, CXCR3 are preferentially found on Th1 cells, whereas CCR4, CCR8, and, more controversially, CCR3 and CXCR4 are preferentially found on Th2 cells.

Skin Microvessels in Cutaneous Inflammation

It is clear that vascular ECs are important participants in the development of inflammatory responses and that ECs may be partially responsible for tissue-specific differences in inflammation. One mechanism by which vascular ECs may contribute to tissue-specific inflammatory patterns is through distinctive patterns of adhesion molecule expression, which results in selective recruitment and diapedesis of circulating leukocytes bearing the proper ligands. In the skin, E-selectin, VCAM-1, and ICAM-1 may be particularly important in this process, and they have been extensively evaluated in normal and disease states34,89–91 (see Table 162-3).

The induction of E-selectin appears to be particularly important in cutaneous inflammatory reactions. In addition to being readily inducible by cytokines and uniformly associated with leukocytic infiltration, E-selectin may play a role in the selective recruitment of lymphocytes to cutaneous sites of inflammation through binding to the cutaneous lymphocyte antigen (CLA).92,93 CLA is a carbohydrate moiety on the surface of T cells closely related to sialylated Lewis X, which has homology to the core structure of P-selectin glycoprotein-1.94 By using the identifying monoclonal antibody HECA-452, CLA has been found on 80% to 90% of skin infiltrating lymphocytes compared with 5% to 10% at other peripheral sites. CLA is also present on circulating precursor Langerhans cells and presumably plays a role in their eventual localization to the skin. The expression of E-selectin in cultured dermal microvascular ECs is relatively persistent compared to endothelium derived from other sources. This persistent expression of E-selectin in vivo has been proposed to be responsible for the preferential homing of CLA-positive lymphocytes to the skin. Work in a chimeric human mouse model of inflammation suggests that CLA-positive lymphocytes specifically migrate through the superficial, as opposed to the deep, vascular plexus.95 It is unlikely that E-selectin–CLA interactions alone are sufficient to account for the homing of lymphocytes to the skin. The transmigration of CLA-positive lymphocytes across activated ECs is dependent on interactions with VCAM-1 and ICAM-1, in addition to E-selectin.

The induction of E-selectin on cutaneous microvessels also appears to play a role in immediate hypersensitivity reactions in atopic individuals. These reactions are characterized by an immunoglobulin E-dependent biphasic response. Within minutes after exposure, the relevant antigen cross-links immunoglobulin E, which is bound to the high-affinity FcRϵ receptors on the surface of mast cells, and results in histamine release. Histamine-dependent stimulation of ECs leads to vasodilation and increased vascular permeability, which produces the classic wheal-and-flare response that typically resolves within 30 to 90 minutes. This reaction, known as the early-phase response, is not accompanied by endothelial E-selectin upregulation or inflammatory cell recruitment. The late-phase reaction begins 3 to 4 hours after antigen challenge and is characterized by the infiltration of eosinophils, neutrophils, and mononuclear cells into the inflamed area. Granulocytes are most prominent at 6 to 8 hours, and, by 24 to 48 hours, the cellular infiltrate consists largely of mononuclear cells. The expression of E-selectin, which indicates endothelial activation, is detectable at the onset of infiltration. Although this E-selectin induction may simply be due to cytokine release from infiltrating inflammatory cells, there is evidence that the necessary mediators are first released from skin-resident cells. In skin organ culture, the addition of antigen either before or during the culture period results in significant E-selectin expression without inflammatory infiltrate.96

VCAM-1 may play a role in the specific homing of leukocytes to the skin through binding to the integrin α4β7 on the surface of lymphocytes. This integrin is associated with the specific localization of lymphocytes to the gut through binding to mucosal addressin cell adhesion molecule 1 (Madcam-1) on ECs of intestinal microvessels.91 Another ligand for α4β7 includes fibronectin. α4β7 has been found to be expressed on a high percentage of intraepidermal lymphocytes, particularly in cutaneous T-cell lymphoma (CTCL) and, to a lesser degree, in spongiotic dermatitis.91 Interestingly, in CTCL, the expression of α4β7 strongly correlated with the expression of integrin αEβ7. αEβ7 is also found on a high percentage of gut (intraepithelial) and intraepidermal lymphocytes.

In chronic inflammatory conditions such as psoriasis and CTCL, the cutaneous microvascular endothelium develops a “high endothelial venule” morphology similar to that seen in peripheral lymph nodes. This is accompanied by the expression of peripheral lymph node addressin identified by the antibody MECA-79, which serves as an L-selectin ligand. This has led to the speculation that peripheral node addressins in chronic T cell-mediated skin diseases are responsible for sustained lymphocyte recruitment. Interestingly, there is MECA-79 reactivity in the perifollicular vessels in noninflamed skin.97 This constitutive expression in normal skin may serve a function in continuous lymphocyte recirculation.98

Chemokines also play a role in the selective homing of T cells to the skin. CCR4 is expressed at high levels by CLA-positive memory T cells. The CCR4 receptor TARC is constitutively expressed on ECs in cutaneous postcapillary venules. In vitro TARC triggers CLA-positive T cells that are rolling on E-selectin to firmly adhere ICAM-1. Another skin-associated chemokine, CCL27 (cutaneous T cell-attracting chemokine), is produced by keratinocytes and also preferentially attracts CLA-positive memory T cells.99

New Vessel Formation in the Skin

Definition of Angiogenesis and Vasculogenesis

In the healthy adult, blood vessels are stable structures with very slow turnover of ECs. In settings of chronic inflammation, tissue injury, or tumor growth, new vessels may be formed and existing vessels may undergo remodeling. Much of the process of vessel formation has been learned by the study of the formation of the vascular system during embryogenesis. In the embryo, ECs arise from angioblasts,100,101 which migrate from their site of origin (initially blood islands, later liver, and ultimately bone marrow) to peripheral tissues, where they form primitive blood vessels (vasculogenesis). Subsequently, new blood vessels arise from preexisting ones (angiogenesis). Mesenchymal cells are then recruited into the vessel wall, where they subsequently differentiate into smooth muscle cells and pericytes, as part of a process called vascular remodeling. New vessels can be formed in the adult as part of chronic inflammation, as part of the repair of injured tissues or during tumor growth. In the adult, new blood vessels develop through adult angiogenesis (sprouting from preexisting, mature blood vessels); it has been proposed that there may also be a contribution through recruitment of circulating endothelial progenitor cells, mesoangioblasts, and multipotent adult progenitor cells derived from the adult bone marrow (adult vasculogenesis).102,103 However, it is unclear that such cells give rise to stable ECs and many such observations have now been reinterpreted to imply a role for monocytes that express EC markers, promote angiogenesis, and then disappear as the new vessels become stable. Stem cells and endothelial progenitor cells have been identified in vessel walls as resident cells. Such cells are thought to reside in a niche, awaiting appropriate stimuli like vessel injury to take part in the repair of damaged and the formation of new blood vessels. Among such vascular wall progenitor cells are CD34+/CD31− cells that differentiate into endothelial cells.108 Other resident vascular wall progenitor cells are smooth muscle-cell progenitors and mesenchymal stromal cells,109 which may arise from PCs.

Regulation of Angiogenesis and Vasculogenesis

(Table 162-4). Molecules of the VEGF family, VEGF A through E, and their receptors, VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt-4), are required to initiate the formation of primitive vessels. Mice with a targeted disruption of VEGF or VEGFR-2 genes fail to develop a vasculature. VEGFR-1 knockout mice have immature leaky vessels. Disruption of VEGFR-3 results in the disorganization of large vessels and lymphatic hypoplasia. A missense mutation in VEGFR-3 occurs in primary human lymphedema.107,110,111 Fibroblast growth factors 1 and 2 (FGF-1 and FGF-2, also called acidic and basic FGF, respectively) mainly signal through FGF receptor I.112 Knockout mice die before vasculogenesis starts; thus, specific evaluation of FGF function in vascular development is not possible in these animals. Both FGF-1 and FGF-2 are potent EC mitogens but they also act on SMCs, PCs and fibroblasts. FGFs “synergize” with VEGF in stimulation of cultured EC mitogenesis and in bioassays of angiogenesis. In the adult, FGF-1 and FGF-2 are stored in the cytoplasm of a variety of cells, which is consistent with the fact that these proteins lack the signal sequences required for efficient secretion. Stored FGFs are released when cells are injured, acting as “wound hormones” to stimulate local angiogenesis and connective tissue growth.112 Other polypeptide factors have also been found to act on ECs in vivo and in vitro, including epidermal growth factor,113 heparin-binding EGF-like growth factor,114 and hepatocyte growth factor (HGF)/scatter factor (SF).115 For vascular remodeling and stabilization, tyrosine kinase receptors 1 (Tie-1) and 2 (Tie-2; Tek) are required. Tie-2 mediates the dialogue between mesenchymal cells and the endothelium of the immature blood vessel through binding to angiopoietins (Ang-1, Ang-2, murine Ang-3, and its human ortholog Ang-4). In humans, Tie-2 mutations cause venous malformations typified by enlarged vascular channels lacking smooth muscle cells. Ang-2 appears to be a competitive inhibitor of Ang-1, and Ang-2 overexpression results in a phenotype resembling an Ang-1 knockout animal.116 Ligands for Tie-1 are not known, but knockout of this receptor results in defective vascular maturation later in embryonic development.107,117 The eph family of receptor tyrosine kinases (ephA1 to ephA8 and ephB1 to ephB6) and their corresponding ephrin ligands (ephrin-A1 to ephrin-A5 and ephrin-B1 to ephrin-B3) are membrane bound and appear to mediate bidirectional cell-to-cell signaling. Mice lacking ephrin-B2 and its ephB4 receptor have lethal defects in early angiogenic remodeling.118 During vascular development ephrin-B2 marks the ECs of early arterial vessels and ephB4 marks the ECs of early veins.119

PDGF and TGF-β are secreted by ECs and promote migration of mesenchymal cells, matrix biosynthesis, and differentiation.120 PDGF and TGF-β act through receptors equipped with tyrosine and serine/threonine kinase activities. TGF-β also uses nonsignaling receptors that present TGF-β to the signaling receptor, for example, endoglin (CD105). A truncation mutation in the endoglin gene in humans causes a hereditary form of vascular malformations (hereditary hemorrhagic telangiectasia type I),121 and endoglin knockout mice die of defective vascular development.

Matrix glycoproteins such as fibronectin and laminin and receptors for matrix glycoproteins such as β1 and β3 integrins are also believed to play a role in vasculogenesis and angiogenesis.122 This seems intuitive, because neither ECs nor mesenchymal cells can survive unless they interact with matrix proteins via integrin receptors. Knockout of most of these molecules causes early embryonic death, which prevents a direct test of their role in vascular embryology. An exception is the β3-integrin knockout mouse, which shows normal development but enhanced tumor angiogenesis. Coagulation factors, like tissue factor or coagulation factor V, are implicated in vasculogenesis by observations of defective vascular development in knockout mice.123 It is thought that these defects are an indirect result of defective thrombin production, because knockout of the thrombin receptor PAR-1 produces similar phenotypes.

Cutaneous Angiogenesis and Vasculogenesis

Cutaneous angiogenesis and vasculogenesis occur in wound healing, inflammation, and tumorigenesis. New vessel formation in the skin differs in several ways from that in other organs. Skin harbors hair follicles, which, besides having a role in forming hair shafts, appear to play a role in new vessel formation. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located in the follicular bulge region. During the follicular growth cycle, they differentiate into the various cell types of the hair follicle and, in addition, can form a variety of epidermal cells. It has been shown that these nestin-positive cells can also develop into cutaneous blood vessels that originate from hair follicles and form a follicle-linked vascular network. The quantitative contribution of hair follicle-derived angiogenesis compared with conventional angiogenesis through local sprouting is unknown.

Skin endothelium is also unique by virtue of its close proximity to keratinocytes, which form the overlying epidermis coating. Keratinocytes have been shown to express a wide range of angiogenic factors, including members of the fibroblast or TGF protein family, platelet PDGF, or VEGF.124,125 Of note, not only ECs respond to VEGF, but also epithelial cancer cells possess receptors for and respond to VEGF.126 Under conditions of injury, hypoxia, or inflammation, keratinocytes produce and release these growth factors. Concomitantly, VEGFR-2 is upregulated on ECs, which augments endothelial responses to VEGF.127 Ang-1 and Ang-2 are also upregulated during injury, which is consistent with the role they are thought to play in the destabilization, followed by stabilization, of the vasculature. This allows a window for VEGF-mediated sprouting and elongation, followed by Ang-1-mediated remodeling.68 FGF-2 is released in cutaneous wounds and is derived from ECs and from infiltrating macrophages. Antibodies against FGF-2 inhibit wound angiogenesis nearly completely.128

Adhesion molecules are necessary for physiologic angiogenesis in the skin. The expression of integrin αVβ3 on the tips of growing capillaries appears to facilitate endothelial migration and to be required for wound healing. In an in-vivo model in which human skin placed on immunodeficient mice was wounded, blocking antibodies directed to CD31 and VE-cadherin reduced wound vascularization and healing.20 In a similar model, blocking antibodies directed against αVβ3 inhibited angiogenesis and wound healing.129 Administering αVβ3 inhibitors reduce granulation tissue formation and wound-induced angiogenesis, which suggests that αVβ3 is required for proper wound healing. In contrast, mice lacking β3 integrins show enhanced wound healing with complete reepithelialization several days earlier than do wild-type mice,130 apparently due to an increase in TGF-β1 and enhanced dermal fibroblast infiltration into wounds of β3-null mice. This suggests that the role of β3 integrins is complex and that these molecules may influence both angiogenic and antiangiogenic effects.

Angiogenesis is also necessary in skin tumorigenesis to maintain adequate nutritive supply. Skin tumors are known to produce angiogenic factors such as VEGF, FGF-2, EGF, and HGF/SF.131 Some of these growth factors play a role in the pathogenesis of both benign and malignant skin tumors. There is a significant vascular contribution to the growth and metastasis of cutaneous melanoma and increased number of vessels in primary melanomas compared with severely atypical nevi. This vascular proliferative response appears to be in part mediated by FGF-2, which is produced by melanoma cells. Antisense oligonucleotides directed against FGF-2 inhibit the proliferation of both primary and metastatic melanoma.132 HGF/SF and VEGF are also produced by melanoma cells and also contribute to vascularization. Peptide growth factors also appear to play a role in the pathogenesis of vascular tumors. The spindle cells of Kaposi sarcoma also produce large amounts of VEGF (see Chapter 128). When human ECs are cultured in the presence of HGF/SF, they acquire a Kaposi sarcoma cell-like morphology. In hemangiomas, high levels of FGF-2 and VEGF characterize the proliferative phase. However, there is also abundant FGF-2 expression in the involuting phase.

Vascular Remodeling in the Skin

Vascular remodeling plays a role in the pathogenesis of some inflammatory skin diseases, notably psoriasis (see Chapter 18). Psoriatic vessels become tortuous and elongated, and the normally well-defined boundary between capillary ECs resting on an arteriole-like homogenous basement membrane and ECs on a venule-like laminated basement membrane becomes unclear. The intrapapillary portions of capillaries acquire a venular phenotype.133 It is unknown whether this is due to metaplasia from arteriolar to a venular endothelium or to an elongation of the postcapillary venule. The first ultrastructurally visible alterations in eruptive guttate psoriasis lesions consist of gap formation within postcapillary venules, EC hypertrophy with a cuboidal shape similar to that seen in high endothelial venules, and compression of the vascular lumen, all of which precedes the invasion of inflammatory cells into the tissue.133 This morphologic alteration is accompanied by altered endothelial function. For example, there is increased blood flow in perilesional psoriatic skin even in the absence of any microscopically detectable changes.134 It has been suggested that these vascular alterations may, in part, be mediated by increased production of VEGF by keratinocytes. There is also increased expression of VEGFR-1 and VEGFR-2 on the papillary dermal microvascular ECs.135 VEGF appears to directly upregulate expression of ICAM-1, VCAM-1, and E-selectin. The VEGF-mediated vascular leak and adhesion molecule upregulation can be inhibited by concurrent overexpression of Ang-1. The role of angiogenesis in inflammatory processes has been further exemplified by the anti-inflammatory effects of angiogenesis inhibitors.136

The skin is prone to frequent trauma. Therefore, vascular remodeling, angiogenesis, and vasculogenesis in the skin are most often caused by wound healing processes. Wound healing requires a complex interplay between resident cells (endothelial cells, fibroblasts, and smooth muscle-like cells), soluble mediators, infiltrating leukocytes, and extracellular matrix molecules.137 The most important soluble factors in wound healing are members of the VEGF family of growth factors. VEGF acts as an EC mitogen, a regulator of vascular permeability, and a chemotactic agent.138 VEGF in the wound healing situation is mainly produced by stromal fibroblasts. In situations where the VEGF production cannot be appropriately adjusted to the increased demands, as is the case in elderly or diabetic patients, vessel formation is strongly reduced and wound healing impaired.139,140

The skin vascular system is unique in several respects. It is organized into functionally distinct vascular segments: the loops within the tips of the papillae, the SVP and the DVP, and the subcutaneous vascular plexus. These segments may individually or conjointly respond to exogenous or endogenous stimuli, thereby influencing skin disease expression. Due to their proximity to the epidermis, ECs of the SVP may directly respond to keratinocyte-derived factors like cutaneous T cell-attracting chemokine or VEGF, which results in an altered vascular architecture, phenotype, and function. Moreover, skin EC may be directly exposed to environmental antigens. In this context, the constitutive expression of HLA class II molecules by postcapillary venules implies that ECs play a role in antigen presentation, which may increase the likelihood that antigen is recognized in a timely manner to efficiently recruit memory T cells to sites of danger.