Blood Vessels

Macrovasculature

Large vessels are composed of three layers: intima, media, and adventitia. The intima comprises the endothelium and the subendothelium. The endothelial cells of large vessels contain distinct rod-shaped secretory organelles, called Weibel-Palade bodies, which contain von Willebrand factor (vWF) and other proteins, such as P-selectin and angiopoietin-2, that contribute to inflammation, angiogenesis, and tissue repair. The abluminal face of the endothelium rests on a basement membrane, which supports the endothelial cell and can act as a secondary barrier against the extravasation of blood. The subendothelial matrix contains occasional smooth muscle cells and scattered macrophages. Both smooth muscle cells and endothelial cells contribute to the extracellular matrix (ECM) of the intima, along with ECM components such as elastin and collagen. In large vessels, the media is separated from the intima by a layer of elastin, the internal elastic lamina. Diseases associated with mutations in elastin include supravalvular aortic stenosis, Williams syndrome, and autosomal dominant cutis laxa .

The medial layer is composed primarily of concentric layers of smooth muscle cells and their secreted matrix, which is a complex mix of glycoproteins and proteoglycans. This layer is responsible for the structural integrity of the wall and for maintaining vascular tone. Mutations of the fibrillin-1 gene, a microfilament protein in elastic fibers, result in the disruption of the media in Marfan syndrome. Defects of type III collagen can cause aortic rupture in patients with Ehlers-Danlos syndrome type IV. An attenuated band of elastic fibers, the external elastic lamina, separates the adventitia from the media.

The adventitia is composed of loose connective tissue, and the outer portion of the media contains an ECM scaffold containing fibroblasts, small nerves, progenitor cells, lymphatic vessels, and nutritive blood vessels, the vasa vasorum . The adventitia has been recognized as a dynamic environment important in the growth, disease, and repair of the artery. The external limit of the adventitial layer is loosely defined and becomes contiguous with the surrounding connective tissue of the organ.

Microvasculature

Capillaries and postcapillary venules are composed of two main cell types: endothelial cells and pericytes. The two cell types interact to contribute to basement membrane formation, maintenance, and remodeling. Long pericyte processes extend over the abluminal surface of each endothelial cell, and reciprocal extensions of the endothelial cell make contact with the pericyte. Pericytes and endothelial cells form specialized junctions with each other at distinct points in the basement membrane. These adherens junctions connect the cytoskeleton of pericytes and endothelial cells, mediating contact inhibition through contractile forces. Gap junctions between the cytoplasm of pericytes and endothelial cells enable communication through the passage of metabolites and ionic currents. Like endothelial cells, pericytes are also heterogeneous and display a variety of functions including : (1) contractile activity, which regulates blood flow; (2) multipotential capabilities resulting in differentiation to adipocytes, osteoblasts, phagocytes, and smooth muscle cells; and (3) regulation of capillary growth. In animal models and human disease (e.g., diabetic microangiopathy, hemangiomata), a lack of pericytes is associated with microaneurysms and disordered microvasculature.

High Endothelial Venules

Lymphocyte migration into secondary lymphoid sites, such as lymph nodes, Peyer’s patches, and chronically inflamed nonlymphoid tissues, occurs at specialized postcapillary venules called high endothelial venules (HEVs). The endothelial cells of these venules exhibit a plump morphology, display intense biosynthetic activity, and are encircled by a continuous thick basal lamina formed from ECM components produced by surrounding pericyte-like cells called fibroblastic reticular cells . HEV endothelial cells express tissue-specific adhesion molecules and secrete a thick adhesion molecule-containing glycocalyx. These HEV adhesion molecules are referred to as addressins and comprise the L-selectin ligands mucosal addressin cellular adhesion molecule 1 (MAdCAM-1), glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1), endomucin, and nepmucin. Other HEV receptors implicated in lymphocyte trafficking include α4β7 integrin, CD34, the chemokine receptor DARC (Duffy antigen receptor for chemokines) and the antiadhesive matrix protein Hevin. In addition to recruitment of immune cells, HEV can also influence the outcome of the immune response.

Endothelium of Bone Marrow Sinuses

BM sinus endothelial cells are flat with loose interdigitated junctions and discontinuous basal lamina and are programmed to regulate the egress of hematopoietic cells. For example, if a red blood cell (RBC) that still is nucleated begins to enter the circulation, the body of the cell is allowed to cross and is released as a reticulocyte, while the nucleus is retained extravascularly. Adventitial reticular cells (similar to pericytes) also play an important role in controlling hematopoietic cell egress, and CXCL12 (SDF-1)–abundant reticular cells may also participate in hematopoietic stem cell maintenance. Most quiescent hematopoietic stem cells in the mouse are closely associated with marrow sinusoids, although it is possible that some are also present in a periarteriolar location. CXCL12 is highly expressed in endothelial cells, and interactions with chemokine receptor CXC-chemokine receptor (CXCR)4 are essential for stem cell homing, mobilization, and transendothelial migration into the BM. The CXCR4 antagonist, Pleraxifor, is used to mobilize stem cells for autologous transplantation in patients with lymphoma or myeloma. Vascular cell adhesion molecule 1 (VCAM-1) expressed on BM endothelial cells appears to be the major BM addressin for hematopoietic progenitor cells expressing VLA-4 (α4β1 integrin). Endothelial selectins have also been implicated in promoting hematopoietic stem and progenitor cell homing to the BM. Blockade or genetic targeting of E-selectin inhibits niche-mediated pro-survival signaling of acute myeloid leukemia (AML) blasts and synergizes with chemotherapy (see Chapter 60 ). Clinical trials with the small-molecule E-selectin mimetic Uproleselan (GMI-1271) are under way in AML. Factors such as CD44 and matrix metalloproteinases (MMPs) and mechanical factors such as cytoskeletal rearrangement are also key to the homing process related to the endothelium.

Endothelium as a Barrier

The microvessels act to exchange material brought to tissues through circulation. The movement of lipophilic and low-molecular-weight hydrophilic substances between blood and tissue is virtually unimpeded (except for the brain), but the vessels are selectively permeable to macromolecules. This semi-selective barrier is necessary to maintain the fluid balance between intravascular and extravascular compartments, yet antibodies, hormones, cytokines, and other molecules must have access to the interstitial space to exert their function.

The movement of macromolecules across the vessel wall is governed by (1) hydrostatic and oncotic pressure gradients; (2) physicochemical properties of the molecule, such as size, shape, and charge; and (3) properties of the barrier. The barrier of the vessel wall is formed by the cellular components, endothelial cells, and pericytes, as well as by the charge and compactness of the matrix components, glycocalyx, and basement membrane. Macromolecules can pass either directly through the endothelial cell (transcellular path) or between adjacent endothelial cells (paracellular path).

Although water mainly moves across endothelium paracellularly, a significant proportion (≤ 40%) uses the transcellular route and water-transporting membrane channels, the aquaporins. Macromolecular transport into cells can proceed by receptor-mediated systems, such as clathrin-coated pits, in which the molecules usually are targeted to the lysosome but may be transported through the cell. Caveolae are 50 to 100 nm membrane invaginations that are abundant in capillary endothelial cells, and participate in transcytosis, as well as in translocation of glycosylphosphatidylinositol-linked proteins into the cytoplasm and in transmembrane signaling. The known leakiness of tumor microvasculature led to the identification of a structure designated the vesiculovacuolar organelle (VVO). These organelles are clusters of interconnecting uncoated vesicles and vacuoles that span the entire thickness of the vascular endothelium, thereby providing a transendothelial connection between the vascular lumen and the extravascular space. Their function is enhanced by vascular endothelial growth factor (VEGF), which is known to increase the permeability of vessels.

During inflammation, binding of neutrophils to the endothelium results in the generation of oxidants that can induce endothelial cell injury and increase permeability. Upon adhesion of neutrophils to the endothelium, leukocyte CD18 (β2 integrin)-mediated signals trigger the release of the neutrophil cationic protein called heparin-binding protein/CAP37/azurocidin , which in turn induces formation of gaps between endothelial cells and macromolecular efflux. Thrombin, a crucial hemostatic enzyme and inflammatory mediator, can increase endothelial permeability by several mechanisms resulting from activation of its receptor on endothelial cells. Both an increase in transcellular vesicular and paracellular permeability play a role. The contraction and retraction of endothelial cells are accompanied by “loosening” of intercellular junctions and focal integrin contacts with the ECM. An increase in paracellular permeability results from posttranslational modification of components of junctional proteins such as claudins and VE-cadherin. Thrombin may also alter the repulsive effect of the negatively charged glycocalyx. Pericyte contractility has been hypothesized as an additional mechanism for increasing permeability in inflammatory states.

Endothelium as a Non-Thrombogenic Surface

The molecular mechanisms of hemostasis and thrombosis are addressed in Chapters 121 and 125 . This section places the endothelium in the context of these processes, as shown in Fig. 122.1 . Normal unperturbed endothelium presents a non-thrombogenic surface to the circulation by inhibiting platelet aggregation, preventing the activation and propagation of coagulation, and enhancing fibrinolysis. Conversely, when inflamed and injured or under conditions of disturbed flow, the endothelium may become thrombogenic.

When near endothelial cells, platelets become unresponsive to agonists. This inhibition of platelet aggregation is accomplished by secretion of prostacyclin (prostaglandin I ₂ [PGI ₂ ]) and NO and by surface expression of an ecto-adenosine phosphatase (ADPase)/CD39/nucleoside triphosphate diphosphohydrolase (NTPDase-1). Prostacyclin is synthesized mainly by vascular endothelial and smooth muscle cells as a product of arachidonic acid metabolism. It inhibits platelet activation, secretion, and aggregation, as well as monocyte interaction with endothelial cells. It also causes vascular smooth muscle cell relaxation. NO also has a wide range of functions, including inhibition of platelet adhesion, activation, and aggregation. Most of the NO released from endothelial cells is elaborated abluminally, where it acts on the smooth muscle cell to cause vasodilation. However, some NO may enter the lumen and diffuse into platelets. Prostacyclin and NO can act synergistically to reverse platelet aggregation. The released platelet agonist, ADP, can be inactivated by endothelial membrane-associated CD39. Metabolism of ATP and ADP to adenosine monophosphate (AMP) by CD39 eliminates platelet recruitment and returns platelets to their resting state. Adenosine, which is generated by hydrolysis of AMP by ecto-5′-nucleotidase, inhibits platelet aggregation and promotes vasodilation. ATP/ADP can stimulate purinoreceptors on endothelial cells, resulting in the synthesis and release of PGI ₂ and NO.

Endothelial cells use three main pathways to inhibit thrombin generation and limit coagulation (see Chapters 121 and 125 ) :

• 1.

  Antithrombin system: Heparan sulfate proteoglycans are secreted onto the luminal surface of endothelial cells and into the subendothelium. Heparan sulfates are capable of binding and activating antithrombin (AT), thereby accelerating inactivation of several procoagulant serine proteases, including thrombin, factor Xa, and factor IXa.

• 2.

  Protein C system: Thrombomodulin on the surface of endothelial cells binds thrombin. This coupling inhibits the coagulant properties of thrombin and increases its affinity for protein C, which it cleaves and activates. Activation of protein C by the thrombin–thrombomodulin complex is augmented by its binding to the endothelial cell protein C receptor (EPCR). Protein S, synthesized primarily by the endothelial cell, acts as a cofactor for activated protein C (APC). Independent of protein C, free protein S can inhibit the prothrombinase and intrinsic tenase complexes and interact directly with factors Va and VIIIa.

• 3.

  Tissue factor pathway inhibitor (TFPI): TFPI is a Kunitz-type serine protease inhibitor that modulates TF-initiated coagulation. TFPI binds to and directly inhibits the tissue factor (TF)–factor VIIa–factor Xa complex. It is mainly produced by and bound to endothelial cells, likely to surface glycosaminoglycans. There is also a plasma pool bound to low-density lipoprotein.

If coagulation occurs despite the many anticoagulant mechanisms, endothelial cells also provide proteins to promote fibrinolysis. Endothelium is a major source of tissue type plasminogen activator (t-PA). Approximately 40% of t-PA is bound to its inhibitor, PAI-1, which is also secreted by endothelial cells. Stresses such as exercise, acidosis, hypoxia, shear forces, increased venous pressure, and thrombin cause the release of t-PA, which converts plasminogen to plasmin, the key effector of fibrinolysis. Receptors for plasminogen and t-PA are present on the endothelial cell surface, enabling localized fibrinolytic activity.

Although intact endothelium is necessary to maintain blood in a fluid state and inhibit coagulation under normal conditions, injured endothelium can rapidly downregulate its anticoagulant functions and become procoagulant even without the overt vascular damage that occurs with trauma, surgery, or viral infection. Tissue injury or vascular pathology also leads to exposure of the underlying matrix, which is procoagulant by virtue of its binding to, and activation of platelets. Apoptotic endothelial cells become procoagulant through exposure of phosphatidylserine on their surfaces and increased thrombin formation. These effects coupled with platelet adhesion to the injured endothelium contribute to thrombosis in diverse diseases.

Even without endothelial death, perturbation of the vascular lining by inflammatory mediators converts the endothelium from a non-thrombogenic to a procoagulant surface because of downregulation of anticoagulant properties as well as induction of a procoagulant phenotype. Acute inflammation is associated with increased release of vWF, platelet-activating factor (PAF), and fibronectin, all of which may potentiate thrombus formation. Tumor necrosis factor (TNF), IL-1, and lipopolysaccharide increase endothelial cell expression of PAI-1 and decrease t-PA expression, thereby attenuating fibrinolysis, and downregulate thrombomodulin. Although they induce TF expression by cultured endothelial cells, evidence that they induce TF expression by endothelial cells in vivo is limited. Instead , circulating microparticles (also known as extracellular vesicles) generated by leukocytes, vascular or cancer cells have been shown to be a source of blood-borne TF and to contribute to coagulation. Although most microparticles are derived from platelets, RBCs, and monocytes, endothelial-derived microparticles may be an important source of circulating TF under conditions of massive activation.

Endothelial Control of Vascular Tone

Vascular tone is orchestrated primarily by a balance between endothelium-derived vasodilators (NO, PGI ₂ , and various endothelium-derived hyperpolarizing factors [EDHFs]) and vasoconstrictors (endothelin-1 [ET-1], thromboxane [TXA ₂ ] and superoxide). In addition to inhibiting platelet aggregation, NO and PGI ₂ serve as vasodilators. NO is produced by the conversion of l -arginine to l -citrulline by NO synthase (NOS). Three NOS genes exist, of which an inducible form (iNOS/NOS2) and a constitutively active endothelial form (eNOS/NOS-3) are expressed in endothelial cells. NOS2 may be responsible for the uncontrolled vasodilation seen in septic shock. NO release through NOS3 activation, mainly by unidirectional laminar flow-induced shear stress, is crucial for maintaining basal vasodilation. The action of NO on platelets (antiaggregatory), endothelial cells, and smooth muscle cells (relaxation) is caused by activation of guanylyl cyclase and formation of cyclic guanosine 3′,5′-cyclic monophosphate. Whereas NO is unstable, the formation of S -nitrosothiols in the presence of oxygen and thiols provides a stable reservoir of NO. Deoxygenation is accompanied by an allosteric change in S -nitrosohemoglobin that releases the NO group, thereby relaxing blood vessels and enabling sufficient blood flow to meet local oxygen requirements. Hemoglobin is a scavenger of NO, which may account for the vasoconstriction observed with the administration of cell-free, hemoglobin-based, RBC substitutes.

In addition to its antithrombotic role (above) endothelial PGI ₂ acts locally to counterbalance the vasoconstriction induced by thromboxane A ₂ (TXA ₂ ). Whereas PGI ₂ transduces a cellular signal by increasing the levels of cyclic AMP (cAMP), TXA ₂ signals via the phosphoinositol pathway and lowers cAMP levels (see Chapter 123 ). The synthesis of prostaglandins is catalyzed by the action of cyclooxygenases (COX-1 and COX-2) on arachidonic acid. Aspirin irreversibly inhibits COX in both platelets and endothelial cells (see Chapters 123 and 124 ). However, its clinical effect is mainly exerted on platelets, which cannot synthesize new COX. Therefore, TXA ₂ synthesis recovers only when new platelets enter the circulation, while endothelial COX restores PGI ₂ levels within a few hours. In addition, platelets encounter aspirin before it is deacetylated by the liver and diluted by the venous circulation. The balance between the activity of PGI ₂ and TXA ₂ is important for vessel wall homeostasis, as evidenced by the fact that selective COX-2 inhibitors, which decrease the production of PGI ₂ without affecting the production of TXA ₂ , increase the risk for cardiac events.

Endothelial cells release other relaxing factors (EDHFs), which act by increasing the membrane potential of smooth muscle cells. These factors comprise a variety of constitutive and inducible effectors including calcium-activated potassium channels, various cytochrome P450 and 15-lipoxygenase derived arachidonic acid metabolites, H ₂ O ₂ , and C-type natriuretic peptide.

Endothelin 1 (ET-1) is a 21-amino acid peptide released preferentially at the abluminal surface of endothelial cells that exhibits potent vasoconstrictor activity. Of the three known ETs, only ET-1 is produced by endothelial cells. At least two receptors (ET-A and ET-B) bind all three ETs. Whereas ET-A is abundantly expressed on smooth muscle cells, ET-B is predominantly expressed on endothelial cells. The vasoconstrictor activity of ET-1 is preferentially mediated by ET-A G-protein–coupled receptors on smooth muscle cells. Engagement of the ET-B receptor on endothelial cells by ET-3 may paradoxically cause transient vasodilation. ET-1 may contribute to pregnancy-induced hypertension, reperfusion injury after ischemia, and pulmonary arterial hypertension. The dual ET receptor antagonist bosentan, and ET-A antagonist ambrisentan have been approved for treatment of the latter disease.

Another regulator of vascular tone is the superoxide anion. The source of this free radical may be the endothelium itself or inflammatory cells that have been recruited to sites of injury or inflammation. Interaction of superoxide radicals with NO produces peroxynitrite and reduces the concentration of NO. Peroxynitrite can oxidize low-density lipoprotein (LDL) and deleteriously modify other proteins, thereby causing endothelial dysfunction. Increased production of superoxide inhibits the synthesis of PGI ₂ but not TXA ₂ .

The endothelium expresses angiotensin-converting enzyme (ACE) on its surface; this enzyme converts angiotensin I to angiotensin II, a potent vasoconstrictor. The interaction between ET, angiotensin II, and α-adrenergic agonists contributes to the pathogenesis of hypertension. An altered balance of the vasoactive substances described in this section has been proposed as a cause of endothelial dysfunction and the attendant vascular pathology observed in atherosclerosis, hypertension, and diabetes mellitus (see Chapter 142 ). Alteration of vascular function in these diseases may then perpetuate endothelial dysfunction and, consequently, worsen disease.

Leukocytes

In the absence of an inflammatory stimulus, neutrophils circulate freely and do not interact with the endothelium. This contrasts with continuous, low-level physiologic traffic of monocytes and lymphocytes across the vessel wall. Monocytes emigrate from the bloodstream to develop into tissue macrophages that may exhibit tissue- or organ-specific functions. To maintain immune surveillance of tissue, lymphocytes recirculate between blood and lymphatics, gaining entrance to the latter at the high endothelial venule of postcapillary venules in lymphoid tissue.

Intravital microscopic studies have established a sequence of events involved in leukocyte emigration at extravascular sites of inflammation. Under conditions of flow, leukocytes first tether to, and then roll along, the endothelium of postcapillary venules adjacent to the site of inflammation. Some of the rolling leukocytes are activated and adhere firmly. The adherent leukocytes migrate along the endothelial surface and diapedese between endothelial junctions to enter the extravascular tissue. These steps in emigration (i.e., tethering, rolling, activation, firm adhesion, and diapedesis) are also involved in lymphocyte emigration at HEVs. They result from the interaction between distinct leukocyte counterreceptors and endothelial receptors in an adhesion cascade ( Fig. 122.2 ). Rolling is observed only under flow conditions and is the consequence of shear forces acting on the leukocyte and adhesive interactions between selectin receptors and their glycoconjugate counter structures. It is initiated primarily by activation of the endothelium by extravascular stimuli such as bacterial-derived products or by endogenous mediators produced by the endothelium or cells in tissue. Early on, rolling is mediated by endothelial P-selectin, which is rapidly translocated from Weibel-Palade bodies to the luminal surface, and L-selectin on leukocyte microvilli. E-selectin is involved only at later time points because it is not constitutively expressed by endothelium but rather is induced over hours by de novo synthesis.

For leukocytes to circulate freely, their integrin receptors must be minimally adhesive, but they also must be able to rapidly increase binding at sites of inflammation. After tethering to endothelium by selectin interactions, leukocyte integrin receptors are activated by endothelial membrane-expressed PAF, endothelial membrane-bound chemokines, or locally secreted chemoattractants. Activation of leukocyte integrins involves changes in receptor affinity or affinity-independent receptor clustering, thereby promoting firm adhesion to endothelial ligands, which are members of the immunoglobulin gene superfamily (IgSF). These IgSF ligands are constitutively expressed (ICAM-1, ICAM-2), further upregulated (ICAM-1), or induced (VCAM-1) by inflammatory mediators. Subsequent leukocyte migration over the endothelium requires reversible adhesion caused by cyclic modulation of receptor avidity.

There are several caveats regarding the multistep model of initial selectin-mediated rolling and subsequent integrin-mediated firm adhesion. First, selectin-mediated rolling is not a prerequisite for emigration under conditions of reduced flow, as might occur at sites of inflammation. Second, the model was developed from observations in the systemic microcirculation where leukocyte emigration occurs in postcapillary venules under relatively low shear forces. However, selectins do not appear to play a major role in neutrophil emigration in the pulmonary microcirculation, where emigration occurs predominantly in capillaries, or in the liver microvasculature, where leukocytes emigrate primarily in sinusoids. Third, under some conditions, leukocytes can tether and roll via receptors other than selectins and α4 integrins (e.g., CD44 or VAP-1). Finally, several other adhesion pathways have been implicated in leukocyte adhesion to endothelium in vitro, and their roles in the adhesion cascade in vivo remain uncertain.

While migrating on the endothelial surface, some leukocytes encounter an intercellular junction, diapedese between endothelial cells, enter extravascular tissue, and then migrate to the sites of inflammatory or immune reaction. This process depends on leukocyte integrin interactions with endothelial Ig superfamily ligands and on several junctional proteins, including PECAM-1 (CD31), JAM-1, CD99, CD99L2, ESAM, PVR, and CD47. Diapedesis involves signaling by the leukocyte to the endothelial cell that triggers the opening of endothelial cell junctions. In some cases, leukocytes may emigrate directly through the body of an endothelial cell.

Leukocyte recruitment is terminated by several mechanisms. Whereas E-selectin and P-selectin are removed from the endothelial cell surface by endocytosis, L-selectin is cleaved from leukocytes by a membrane protease. Decay of cytokine, chemokine, or chemoattractant generation leads to gradual resolution of endothelial adhesion molecule expression and integrin activation. Locally expressed mediators, such as NO, TGFβ, and Fas ligand, also inhibit further leukocyte adhesion to endothelium.

The adhesion molecules involved in leukocyte trafficking from the bloodstream to tissue have emerged as important therapeutic targets. Extensive preclinical studies showed that blockade of leukocyte or endothelial adhesion molecules was efficacious in diverse disease models, prompting the development of adhesion antagonists. In spite of the seemingly sound biology, clinical development of these agents was largely unsuccessful, and progressive multifocal leukoencephalopathy, a rare viral infection of the central nervous sytem (CNS), was observed in patients treated with the α4-integrin antagonist natalizumab. However, approval of two integrin antagonists: efalizumab for treatment of psoriasis and natalizumab for treatment of multiple sclerosis suggests a potential benefit for specific indications.

Platelets

Like neutrophils, non-activated platelets do not interact with unperturbed endothelium (see Chapter 124 ). After a vascular injury that produces endothelial denudation or retraction, platelets rapidly adhere to the exposed subendothelium. As discussed in greater detail in Chapters 123 and 124 , at high shear rates, this initial adhesion does not require platelet activation and involves platelet glycoprotein Ib/V/IX (GPIb–V–IX) binding to vWF in the subendothelial matrix and platelet glycoprotein VI (GPVI) binding to collagen in the injured arterial wall. Platelet activation occurs after adhesion, leading to platelet spreading mediated by integrin receptor binding to matrix components and aggregation mediated by fibrinogen binding to activated GPIIb/IIIa (αIIbβ3).

In the context of inflammation, platelets are the first cells to adhere to the endothelium in postcapillary venules, where they recruit neutrophils and direct them through the endothelial barrier while maintaining vascular integrity. This process is mediated through cell-cell interactions and the release of chemokines. Platelets can bind directly to activated endothelium in vivo via endothelial P-selectin and PECAM-1 and can roll on venular endothelium via interaction of platelet GPIbα with endothelial P-selectin. Platelets bind to HEVs in vivo via platelet P-selectin. In vitro, platelets adhere to intact endothelium via a platelet GPIIb/IIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, αVβ3 integrin, and GPIbα. Platelet adhesion to intact endothelium via these various pathways may contribute to thrombus formation in the circulation and may provide a link between thrombosis and inflammation in diseases such as atherosclerosis (see Chapter 142 ). Platelet-endothelial interactions also may contribute to the pathogenesis of thrombotic thrombocytopenic purpura (see Chapter 132 ). Normally, ultra-large vWF multimers remain attached to endothelium via P-selectin until they are cleaved by the plasma metalloproteinase ADAMTS-13. Failure of vWF multimer cleavage due to a deficiency of ADAMTS-13 may lead to spontaneous platelet adhesion to the endothelium and microvascular thrombosis in thrombotic thrombocytopenic purpura.

Red Blood Cells

RBCs normally do not bind to endothelium unless damaged by sickle cell disease or infection with malaria parasites. The binding of sickle cells to postcapillary endothelium with secondary trapping of poorly deformable cells is thought to be an important pathogenic factor in vasoocclusive events (see Chapter 42 ). Several interactions between sickle RBC receptors and endothelial ligands have been described, including α4β1/VCAM-1; α4β1/Lutheran blood group (basal cell adhesion molecule); and ICAM-4/αVβ3. The adhesive proteins thrombospondin (TSP-1) and vWF promote adhesion by serving as bridging factors between various sickle RBC and endothelial adhesion molecules. In addition to direct adhesion to endothelium, sickle RBC binding to adherent leukocytes appears to be an important mechanism for vascular occlusion in sickle cell disease. Further, microparticles generated primarily from RBC, as well as free Hb, heme and iron released during hemolysis can activate endothelial cells and neutrophils to increase adhesion, potentially accelerating vasoocclusion. Like leukocyte adhesion to endothelium in inflammatory and immune diseases, drugs targeting sickle RBC adhesive interactions with endothelial cells or leukocytes may prove useful in preventing or treating vaso-occlusive crises (see Chapter 43 ).

Among human malarial parasites, only Plasmodium falciparum modifies the surface of the RBCs such that asexual parasites and gametocytes can adhere to the vascular endothelium. The binding of trophozoite- and schizont-infected RBCs to endothelium not only allows the parasite to escape destruction in the spleen but also may contribute to the pathogenesis of cerebral malaria. Multiple endothelial adhesion receptors have been implicated in mediating cytoadherence of infected RBCs, including P-selectin, ICAM-1, VCAM-1, and CD36.

Vascular Endothelial Growth Factors

Vascular endothelial growth factor-A (VEGF-A), which is also known as VEGF or vascular permeability factor (VPF), is indispensable for vascular development. VEGF is the key member of a larger family of related polypeptides, including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGFR-F, and placental growth factor (PlGF). Upon dimerization, these factors bind to their tyrosine kinase receptors (RTKs/VEGFRs), including VEGFR1/Flt-1, VEGFR2/KDR/Flk-1, and VEGFR3/Flt-4, often in conjunction with their neuropilin co-receptors (NRP1, NRP2), as depicted in Fig. 122.3 . For instance, VEGF-A interacts with VEGFR2, VEGFR1, and VEGFR3, whereas PlGF is selective for VEGFR1. The distribution of different VEGFRs on vascular (VECs) and lymphatic (LECs) endothelial cell subsets, as well as among endothelial progenitor cells (EPCs), hematopoietic, myeloid, and certain tumor cells, defines the known biologic activities of the various VEGF ligands. The effects of VEGF include stimulation of endothelial mitogenesis, migration, survival, morphogenesis, and vascular permeability (e.g., through formation of intercellular gaps, vesiculo-vacuolar organelles or transcellular structures know as fenestrae ). The signaling activity of VEGFR2 is crucial for these processes, whereas VEGFR1 is often expressed as a soluble splice variant (sFlt-1) that neutralizes VEGF (serves as a VEGF “sink”), thereby inhibiting angiogenesis.

VEGF activity is also regulated by splicing of the corresponding mRNA, resulting in the generation of several protein isoforms, including VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 (designations based on the number of amino acids). Alternative splicing leads to the formation of a series of proangiogenic (VEGF-A _xxx a) and antiangiogenic (VEGF-A _xxx b) VEGF isoforms, the former being biologically predominant. VEGF-A isoforms differ in their cell association, solubility, and ability to bind heparinoids, or to interact with neuropilins, all of which define the formation of extracellular gradients and related biologic responses to these factors. In this regard, VEGF165 is an especially potent inducer of angiogenesis (see Fig. 122.3 ).

VEGF-C and VEGF-D stimulate the growth of lymphatics (lymphangiogenesis) via activation of VEGFR3, while VEGF-B and PlGF interact with VEGFR1 and are involved in vascular pathologies and inflammation.

Platelet-Derived Growth Factors

This family of VEGF-related growth factors consists of four members: PDGF-A, PDGF-B, PDGF-C, and PDGF-D, the homo- or heterodimers of which interact preferentially with one of the three known cellular RTKs, namely, PDGFRα, PDGFRβ, and PDGFRγ, each endowed with different cellular functions. For example, PDGF-BB is expressed by endothelial cells and mediates their capacity to attract mural cells harboring PDGFRβ.