Chapter 4
Cells of the Vascular System
Omaida Velazquez, Bo Wang
The effort to define the cell types in the vascular system is an ongoing process. The dynamic nature of cells makes this a challenging task. Cells of the vascular system constantly change their properties even during the postnatal period. Certain cell types with high plasticity undergo significant phenotypic and functional change in response to external stimuli. Currently, we have been able to define five main cell types that reside in the vascular wall. These are endothelial cells, vascular smooth muscle cells, pericytes, fibroblasts, and vessel-residing stem cells/progenitor cells. Nonnative cell types such as macrophages, neutrophils, and circulating stem cells can also be found in the blood vessel wall. Their migration into the vessel wall from the blood or distal sites is usually a response to different pathophysiologic conditions.
Traditionally, different cell types are defined by means of microscopy and differentiated from one another on the basis of their morphology and location. Modern technology relies more on molecular biological markers, which allow us to appreciate the dynamic nature of defining a certain cell type. Cells of the same type express different cell markers during different stages to reflect the ongoing changes of the intracellular processes. On the other hand, there can be a vast overlap among different cell types as to the cell markers they express, making it more difficult to differentiate them. Despite our confusion and the limits of technology, there is a fast-growing body of knowledge about the molecular pathways by which different cell types interact with one another as well as the signaling involved in inducing certain changes in cell properties. In this chapter, we discuss the five native cell types found in the blood vessels, their interactions with one another, and the roles they play in some pathophysiologic processes.
Endothelial Cells
Endothelial cells (ECs) are the innermost cellular component of the vessels. They form a cellular layer called endothelium that extends continuously throughout the entire vascular system. Throughout the endothelium layer, ECs display vast structural and functional heterogeneity depending on the organ they reside in and the specific function related to that organ.
The endothelial cells are supported by a layer of acellular components called basal lamina. The basal lamina is a specially organized extracellular matrix. The nature of the basal lamina and its components are complex and dynamic in nature. It borders the basolateral surface of the endothelium, providing attachment and support as well as regulating behaviors of the ECs such as migration and cell division. The interaction between the endothelial cells and their acellular environment is highly intricate. In vitro studies have shown that cultures of ECs on different acellular components, including collagens, fibronectins, and reconstructed basement membranes, show different rates of production of gap junctions, actin messenger RNA, and protein, cell proliferation, and membrane specialization.1–3
Endothelial Cells as a Transporting System and Selective Barrier
The endothelial cells are considered the most active part of the vascular system. They come in direct contact with the blood and sense the signals that are delivered through blood flow. These signals can be physical or chemical. In response to these signals, the cells carry out a complex intracellular and intercellular downstream process. Such a response is crucial in processes such as cardiovascular homeostasis, vasomotor regulation, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and innate and adaptive immunity.4
Endothelial permeability serves an important role in regulating the substance exchange between luminal vessel and extraluminal environment. There are several ways signals or substances can be exchanged—paracellular transport, endocytosis, transcytosis, and fenestration (Fig. 4-1).
Figure 4-1 Different types of endothelium and permeability. A, Continuous fenestrated endothelium is usually found in secretory tissue, such as endocrine glands, renal glomerulus, and gastrointestinal (GI) mucosa. This type of cell has large fenestrations that are covered by a selective diaphragm, which allow certain particles to pass by fairly easily. Continuous fenestrated endothelium demonstrates greater permeability to water and small solutes than to albumin and larger macromolecules. The diaphragms of the fenestrae act as molecular filters. B, Continuous nonfenestrated endothelium is the most selective type of endothelium. It represents a tight barrier between the lumen and interstitial tissue. It is commonly found in tissue such as heart, skin, and lung. Substances can pass through the endothelium through either formed caveolae or special intracellular channels. In continuous nonfenestrated endothelium, water and small solutes pass between endothelial cells, whereas larger solutes pass through such cells either via transendothelial channels or by transcytosis, the latter process being mediated primarily by caveolae (see Fig. 4-2). C, Discontinuous, or sinusoidal, endothelium can be found inside the liver. Discontinuous endothelium is characterized by fenestrae (without diaphragms), gaps, and poorly organized basement membrane. (Adapted from Boron WF, et al: Medical physiology, Philadelphia, 2002, WB Saunders.)
Paracellular Transport
Molecules can move from blood to extraluminal environment through the paracellular (between cells) space between endothelial cells. This space is regulated by certain types of junctions. Two types of junctions are recognized at this space, tight junctions and adherent junctions.
Adherent junction, or zona adherens, connects the ECs on their sides to form a continuous cellular layer. Tight junction, or zona occludens, forms barriers between the lumen and extraluminal environment by tightly sealing the gaps between adjacent cells. The density of distribution for tight junctions varies in different parts of the vascular system. In large arteries, the tight junctions are well formed to prevent the leaking of luminal content. Toward the distal circulation, the number of tight junction decreases drastically in order to accommodate the function of substance exchange between the vascular beds and the tissues they supply. The brain’s vascular system has particularly rigid tight junctions, which protect neural tissue from fluctuations in blood composition. Adherent junctions are less common and exist primarily in large arteries. Their principal role is to permit inter–endothelial cell communication via movement of ions, metabolites, and regulatory factors.5
Fenestrations
Substances can also directly pass in and out of the ECs through fenestrations, or pores, on the cells. This special pathway is especially developed in secretory organs such as exocrine and endocrine glands, gastric and intestinal mucosa, choroid plexus, glomeruli, and renal tubules. Fenestrae are transcellular pores approximately 70 nm in diameter that extend through the entire thickness of the endothelial layer.4 Fenestrations usually have a thin layer of diaphragm formed by glycoproteins across their opening. This diaphragm provides a selective filter based on size for the fenestrations. As a fenestration gets larger, the endothelium becomes discontinuous. This type of endothelium exists especially in sinusoidal vascular beds, most notably the liver. These fenestrations are about 100 to 200 nm in diameter and have no diaphragm. Their underlying basement membrane is poorly formed (see Fig. 4-1).
Endocytosis
Another important pathway for transduction of information from the luminal space is through endocytosis. ECs possess clathrin-coated pits, clathrin-coated vesicles, multivesicular bodies, and lysosomes, which represent the structural components of the endocytotic pathway.6 The endocytosis can happen either via receptor-dependent or receptor-independent mechanisms. The receptor-dependent pathway is responsible for uptake of low-density lipoprotein (LDL), transferrin, albumin, ceruloplasmin, and advanced glycosylation end products. Liver sinusoidal ECs demonstrate particularly high rates of clathrin-mediated endocytosis.4
Transcytosis
Transcytosis is a means of transcellular transfer of molecules across the endothelium. This process is mediated by caveolae and vesiculo-vascular organelles (VVOs). The number of caveolae is highest in continuous nonfenestrated endothelium, particularly in heart, lung, and skeletal muscle.7 When caveolae are bounded to the cell membrane, it opens toward the luminal or abluminal side. After picking up the target particles, it forms a free vesicle inside the cytoplasm. The particle is transported inside the vesicle and is released when it fuses with the cell membrane on the opposite side of the cell. The density of caveolae is far more prominent in capillaries than other parts of the vascular system, reflecting the highly active molecular trafficking in the microcirculation. VVOs are observed mainly in the venular endothelium. They are aggregations of smaller vesicles. VVOs, which contain caveoline-1, are thought to arise from the fusion of individual caveolae (Fig. 4-2).
Figure 4-2 Transcytosis transportation of caveolae and vesiculo-vascular organelles (VVOs). Left, Caveolae are the major apparatus for transcytosis. They contain clathrin inside the vesicles that carrier protein for particles. When vesicles fuse with the luminal side of the cell, the clathrin are exposed to the luminal side to pick up molecules; afterwards, the vesicles are reformed, internalizing the molecules from the luminal side. Caveolae are particularly prevalent in capillaries of heart and skeletal muscle and rare in the blood-brain barrier. After being internalized, the molecules move to either lysosomes or endosomes or are released into the abluminal side of the cell. Right, The VVO is another way of transcytosis through endothelium. This form of transport is predominant on the postcapillary venules, which are much larger than caveolae. Many consider venules to be formed by the fusion of many smaller caveolae. Their number can greatly increase in response to inflammation, which result in leaky endothelium that causes loss of water and small molecules to the interstitium. (Adapted from Boron WF, et al: Medical physiology, Philadelphia, 2002, WB Saunders.)
Vasomotor Regulation
The endothelial cell is an important source of vasoactive agents. Each agent has its specific receptor on vascular smooth muscle cells. Binding on these receptors results in downstream molecular signaling, which eventually leads to a change in Ca2+ concentration. The end result is either vasoconstriction or vasodilation. Vasoactive agents can be divided into vasodilators and vasoconstrictors on the basis of their effects (Table 4-1).
Nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (prostaglandin I2 [PGI2]) are three vasodilators secreted by endothelial cells. The three vasodilators work in an integrated manner to maintain the health of the vasculature. Each mediator possesses the capacity to interact with components of the synthesis/activation processes for the other mediators.
The distribution of vasodilators in the circulating system varies in the body. NO and PGI2 responses are primarily found in conduit vessels, whereas EDHF predominates in resistance arteries.8,9 Moreover, it has been found that NO tonically inhibits EDHF responses. There is a compensatory increase in EDHF when NO is deficient or inhibited. In this way, the vasolidatory capacity of the vessel is maintained.10,11 PGI2 and NO interact in a similar manner, although this activity is often evident only after inhibition of nitric oxide synthase (NOS).12
Nitric Oxide
Nitric oxide, originally named endothelium-derived relaxing factor, is a potent vasodilator. It is produced by the enzyme NOS. Currently, three isoforms of oxide synthase have been identified on the basis of their location. Endothelial nitric oxide synthase (eNOS or NOS III) is found only in the endothelial cells. It is a constitutive form of NOS that is present all the time (Fig. 4-3).
Figure 4-3 Dimer form of endothelial nitric oxide synthase (eNOS). For production of nitric oxide (NO), nitric oxide synthase (NOS) must exist in the dimer form. The monomer-formed molecule has two domains, the reductase domain and the oxygenase domain. Electrons (e−) are donated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the reductase domain and transferred through flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and Fe (iron) to the oxygenase domain. With the help of coenzyme tetrahydrobiopterin (BH4), arginine and O2 are catalyzed into NO and citrulline. H, Hydrogen; NADP, nicotinamide adenine dinucleotide phosphate.
The secretion of NO is under the control of several different mechanisms. Neuronal signal acetylcholine, inflammatory signal bradykinin, and increased shear stress can all increase the activity of eNOS. Other factors, such as substance P, thrombin, adenine nucleotides, and [Ca2+] (calcium ion concentration, can also affect eNOS. These molecules all affect the activity of eNOS through a Ca2+-and calmodulin (CaM)–dependent pathway. The molecular signals can trigger the entry of Ca2+, which binds to cytosolic CaM and then stimulates NOS. The activity of eNOS also requires cofactor tetrahydrobiopterin and NADPH (reduced nicotinamide adenine dinucleotide phosphate). Endothelial NOS catalyzes the formation of NO and L-citrulline from L-arginine. It is a lipophilic gas that can diffuse only a few cell lengths during its very short half-life. This feature limits the effect of NO on a local level.13
The receptor for NO inside the vascular smooth muscle cell (VSMC) is a soluble guanyl cyclase. Binding of NO activates the conversion from guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG). PKG phosphorylates myosin light chain kinase (MLCK), inhibiting its activity. This inhibition leads to a decrease in the activity of myosin light chain (MLC), whose activity depends on MLCK phosphorylation. The result is decreased interaction between myosin and actin, which leads to vasodilation.14,15
There are other pathways by which the NO can exert its effect on the VSMCs. In some blood vessels, cGMP can increase the level of cyclic adenosine monophosphate (cAMP) by preventing its degradation through inhibition of phosphodiesterase activity. An increase in cAMP results in muscle relaxation. NO may also induce relaxation by direct activation of Ca2+-dependent potassium (K+) channels in VSMCs.16 In addition, NO can function as a direct antagonist for the vasoconstrictors, including catecholamines, angiotensin II, and endothelin-1 (ET-1).17
In 1998, Furchgott Ignarro, and Murad shared the Nobel Prize for Physiology for their discovery of the NO signaling pathway. Today in medicine, NO donor drugs play an unparalleled role in the management of coronary artery disease and hypertensive emergency.
NO has commonly been regarded as a vessel-protective agent since its discovery; however, over the past decade, it has emerged as a fundamental signaling molecule that regulates virtually every critical cellular function and as a potent mediator of cellular damage in a wide range of conditions. We have introduced here the important functions of NO in protecting the vessel’s health, including vasodilation, antithrombosis, and anti-inflammation; however, the “double-edged sword” nature of NO is increasingly emphasized in recent literature.18,19 An important mediator for NO toxicity is peroxynitrite. NO is an antioxidant and a salvager of reactive oxygen species (ROS). The two substances, NO and ROS, form peroxynitrite when they meet. Peroxynitrite interacts with lipids, DNA, and proteins via either direct oxidative reactions or indirect radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders.18
NADPH oxidases such as superoxide anion are major sources of ROS in the vessel wall. The short-lived superoxide anion can rapidly form hydrogen peroxide. For the NOS enzyme to function normally, it must remain in a dimer form. Hydrogen peroxide can disrupt this dimer form.20 When NOS becomes uncoupled, it becomes a major producer of superoxide anion. To reverse back to dimer form, this process of reduction relies on an important cofactor, tetrahydrobiopterin (BH4). Under certain conditions, such as ischemic insult, the production of ROS overwhelms the NOS-BH4 reduction system.21
Growing evidence has shown that NO has an important role in modulating angiogenesis, especially in neovascularization developing in vivo in response to ischemia.22–24 This role is specifically restricted to eNOS-derived NO. NO derived from eNOS is involved in mobilizing endothelial progenitor cells (EPCs) to the site of injury.23,24 This function is impaired in diabetic patients. In such patients, eNOS is downregulated and wound healing potential is impaired as a result of decreased EPC mobilization from bone marrow. It has been found that this adverse condition can be reversed by hyperoxic conditions, providing evidence of hyperbaric O2 healing potential on a cell molecular level.24
It is believed that although sustained low concentrations of NO are mainly vasoprotective and excessive cytotoxic formation of NO (peroxynitrite) mainly results from inducible NOS (iNOS) and neuronal nitric oxide synthase (nNOS). White cell adherence is an early event in the development of atherosclerosis; therefore, NO may protect against the onset of atherogenesis. Furthermore, NO has been shown to inhibit DNA synthesis, mitogenesis, and proliferation of VSMCs. The inhibition of platelet aggregation and adhesion protects smooth muscle from exposure to platelet-derived growth factor(s). Therefore, NO also prevents a later step in atherogenesis, fibrous plaque formation. On the basis of the combination of those effects, endothelial NO probably represents the most important antiatherogenic defense principle in the vasculature. Many risk factors, such as diabetes and smoking, can lead to excess production of superoxide and diminished NO, thus damaging the endothelium.19
Endothelium-Derived Hyperpolarizing Factor NO, PGI2, and EDHF are the three native vasodilators that have been identified. Their effect on smooth muscle converges on causing hyperpolarization of the smooth muscle cell. It is believed that endothelium-dependent relaxation of vascular smooth muscle is a result of endothelial cell hyperpolarization.10–12,25–35
Endothelial cell hyperpolarization occurs as a result of influx of extracellular K+ through the opening of calcium-activated potassium channels in response to the rise in intracellular calcium. This endothelial hyperpolarization is transmitted to the smooth muscle through the gap junctions (ion channels) or the flow of K+ from the endothelial cell into the myoendothelial space. Smooth muscle hyperpolarization is then induced by the activation of rectifying potassium channels (KIR) or Na+/K+ adenosine triphosphatase (ATPase).25–29
EDHF is released by endothelial cells under the stimulation of acetylcholine, bradykinin, substance P, and increased shear stress. EDHF can cause hyperpolarization of VSMCs through three different pathways. First, it is able to diffuse across endothelial cells and activate calcium-activated potassium channels of VSMCs to promote K+ efflux. Second, EDHF can act in an autocrine manner to facilitate the activation of KCa channels on endothelial cells to cause endothelial hyperpolarization. Last, EDHF can enhance gap junction communication and increase the transmission of endothelial hyperpolarization to VSMCs25–30 (Fig. 4-4).
Figure 4-4 Hyperpolarization of smooth muscle cells induced by endothelium-derived hyperpolarizing factor (EDHF). EDHF causes smooth muscle cell relaxation by inducing hyperpolarization in the cell. The EDHF is released by stimulation of the acetylcholine, bradykinin, substance P receptor on the endothelial surface. Mechanical stress from blood flow can also lead to the release of EDHF. There are several mechanisms by which EDHF can cause hyperpolarization of smooth muscle cells. First, EDHF can diffuse through endothelial cells and bind the calcium-activated potassium channels of vascular smooth muscle cells (VSMCs), leading to potassium efflux. Second, endothelial cell hyperpolarization occurs as a result of efflux of extracellular K+ through the opening of calcium-activated potassium channels in response to the rise in intracellular calcium. This endothelial hyperpolarization can be transmitted to the smooth muscle cells through the gap junctions. This process in turn induces smooth muscle hyperpolarization via the activation of rectifying potassium channel (KIR) or Na+/K+ pump (adenosine triphosphatase [ATPase]). Third, pumping of K+ into the myoendothelial space can also directly lead to the hyperpolarization of VSMCs. KCa , calcium-sensitive potassium channel.
EDHF has been identified in coronary, peripheral, skin and venous vessels. It is defined as a group of molecules that carry out their vasodilatory function in the same manner rather than as a single type of molecule. This group of molecules includes hydrogen peroxide, epoxyeicosatrienoic acids (EEAs), anandamide, and C-natriuretic peptide.9
Prostacyclin
PGI2 is a product of arachidonic acid catalyzed by cyclooxygenase (COX). It is a potent vasodilator in arteries and vein, as well as an important platelet aggregation inhibitor. COX-1 is the constitutive form of the enzyme expressed in vascular endothelium that is thought to contribute to the maintenance of vascular homeostasis. COX-3, on the other hand, is an inducible form that is thought to be related to pathogenesis.31
PGI2 binds to specific receptors on smooth muscle cells. These are G protein–coupled adenyl cyclases that can increase cAMP levels, which in turn activate cAMP-dependent protein kinase (PKA). Like PKG, PKA phosphorylates MLCK, inhibiting MLCK activity, decreasing the interaction between myosin and actin, and leading to vasodilation.32 This mechanism of vasodilation is seen in NO-induced VSMC relaxation, as previously described. The difference is that NO activity depends on the activation of guanyl cyclase and cGMP rather than adenyl cyclase and cAMP.
Endothelin-1
ET-1 is the most potent vasoconstrictor. It is also the major isoform of endothelin found in humans. It is produced by ECs at a low physiologic level and inhibited by NO, PGI2, heparin, natriuretic peptides, and high levels of shear stress.33 It is converted from the pro-polypeptides by the membrane-bound ET-converting enzyme. ET-1 carries out its effects through two types of membrane–G protein–coupled receptors, ETA and ETB.34 The ET receptors can be found on VSMCs, adventitial fibroblasts, and endothelial cells. They are also expressed in kidney, liver, lung, and skin and are involved in a wide range of biological activities.
ET-1 binding to ETA receptors on VSMCs leads to cellular contraction. Abnormal vasoconstriction, vasospasm, and vascular hypertrophy all share the feature of abnormal ET receptor expression.35 In recent years, ET-1 has been identified as a major factor in endothelial dysfunction of various cardiovascular diseases, including coronary artery disease, peripheral artery disease, and stroke due to hypertension. Elevated blood and tissue levels of ET-1 have been identified in relation to the pathology of these diseases.36–39
At low concentrations, ET-1 can induces a paradoxical vasodilatation through activation of endothelial ETB receptors coupled with the release of NO, PGI2, and EDHF. Activation of ETB receptor–coupled G proteins results in an increase in phospholipase C (PLC) activity, generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol, and eventual release of Ca2+ from intracellular storage. This causes an increase in activity of the Ca2+/calmodulin-dependent NOS enzyme, which allows liberated NO to diffuse into the SMC layer, initiate production of cGMP, and develop the cellular relaxation response. ETA receptors are coupled to Gq/11, G12/13, and Gi heterotrimer G protein subunits, which link to inhibition of phospholipase C, RhoA–guanosine triphosphatase, and adenylyl cyclase, respectively.35
Endothelium-Derived Constricting Factor
Endothelium-derived constricting factor is a poorly defined term. Most literature uses it as a common name for the group of peptides that are produced by endothelium and can induce vasoconstriction.40 Endothelin-1 is the best-described member of the group. A few other peptides have been defined as members of the group. COX appears to be at least partially involved in their activities40,41 Superoxide anions and endoperoxides have been included as members of this group.41
Angiotensin II
Angiotensin II (Ang II) is a product of the renin-angiotensin-aldosterone axis. It is produced mainly in the lung and kidney endothelial cells as a systemic response to low blood osmolality and low pressure that is initiated in the kidney. It is produced by angiotensin-converting enzyme (ACE) in the endothelium as a product of angiotensin I. Angiotensin I is a product of angiotensinogen, which is produced in the liver. The process is catalyzed by the kidney-secreted enzyme renin. Angiotensin II is a potent vasoconstrictor for the vascular system. However, its special role in the regulation of renal perfusion has always been the main emphasis. Its effect on renal perfusion is mediated through the regulation of arteriole smooth muscle tone. Under high concentrations of angiotensin II, the efferent arterioles constrict more than the afferent arterioles, retaining the salt in circulation and increasing the colloid osmotic pressure in the blood. On the other hand, angiotensin II is also able to interact with specific receptors in the kidney to increase the uptake of Na+ and stimulate the thirst sensation in the hypothalamus. Finally, it stimulates the production of aldosterone in the adrenal cortex, which also promotes Na+ reabsorption in the collecting tubules and ducts.
The exact mechanism of how angiotensin II causes vascular constriction is still under study. It has been determined that the AT1B receptor, a subtype of angiotensin II type 1 (AT1) receptors, predominantly mediates contractions induced by angiotensin II.42 Binding of AT1 receptors leads to activation of the G protein Gq, which in turn activates phospholipase C, which hydrolyzes phosphatidylinositol-4,5-bisphosphate to generate inositol-1,4,5-trisphosphate and diacylglycerol. Inositol-1,4,5-trisphosphate activates the intracellular release of Ca2+, and extracellular Ca2+ can also enter the cell through Ca2+ channels located on the cell membranes. Ca2+-calmodulin–dependent MLCK can turn on MLC phosphorylation and vasoconstriction. Angiotensin II also activates phospholipase D, which converts phosphatidylcholine into choline and phosphatidic acid; the latter is converted to diacylglycerol rapidly. This pathway is thought to play a major role in the activation of protein kinase C in the sustained phase of angiotensin II–induced contraction.43
Hemostasis versus Antithrombosis/Fibrinolysis
The endothelial cells produce both anticoagulant factors and procoagulant factors. This makes them crucial in maintaining the balance between the two processes. The anticoagulant factors ECs produce include tissue factor pathway inhibitor (TFPI), heparin, thrombomodulin (TM), endothelial protein C receptor (EPCR), tissue-type plasminogen activator (t-PA), ecto-adenosine diphosphatase (ADPase), prostacyclin, and NO. The procoagulant, factors are plasminogen activator inhibitor-1 (PAI-1), von Willebrand factor (vWF), and protease activated receptors.4
Other than endothelium-derived factor, the coagulation cascade involves circulating platelet from bone marrow as well as clotting factors, fibrinogen, and the anticoagulants proteins C and S and antithrombin III, which are produced by the liver. These factors are been produced at a fairly constant rate and distributed evenly throughout the circulation. On the other hand, the production of endothelium-derived factors can vary among different vascular beds. This feature allows them to create a site-specific coagulating environment. For example, thrombomodulin is highly expressed in all vessel types and calibers in all organs, whereas EPCR is expressed predominantly in large arteries. Although von Willebrand factor is predominantly found on the venous side of the circulation, t-PA expression in the endothelium is restricted to arteries of the pulmonary system and central nervous system44–46 (Figs. 4-5 and 4-6).
Figure 4-5 Inhibition of the thrombin pathway. Thrombin activation is a key step in the process of coagulation. Thrombin binding to a cell surface thrombin receptor (TR) can induce various pro-coagulation factors. Intact endothelium has various mechanisms to counteract coagulation. One of them is through the expression of heparan sulfate proteoglycans on endothelial cell surfaces. Heparan sulfate allows bound antithrombin III (AT III) to inhibit thrombin molecules generated by the coagulation cascade. Endothelial cells also synthesize and display thrombomodulin (TM). When thrombomodulin and thrombin complex (TM-thrombin complex), they work together to activate protein C, an enzyme that, when activated, destroys clotting factors and inhibits coagulation. NF-κB, Nuclear factor-κB; PAI-1, plasminogen activator inhibitor-1.
Figure 4-6 Anticoagulation pathways. A, Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are important factors in the process of platelet activation and thrombin release. Intact endothelial cells have ATP/ADPase that can convert ATP and ADP to adenosine monophosphate (AMP). B, Tissue factor pathway inhibitor (TFPI) is an inhibitor of the extrinsic pathway. Tissue factor exposure activation of factor VII, which in turn activates factor X, is a critical step in the extrinsic coagulation pathway. TFPI can bind to factors VII and X at the same time and deactivate the process of the extrinsic pathway. C, Platelet attachment to endothelium and activation are facilitated by von Willebrand factor (vWF). The endothelial cells can sequester vWF in Weibel-Palade bodies (WPBs) to prevent its exposure to platelet; furthermore, the constitutive form of nitric oxide synthase, extrinsic nitric oxide synthase (eNOS), also secretes nitric oxide (NO), which further inhibits platelet activation.
This site-specific distribution of clotting factors gives rise to site-specific thrombotic phenotypes. In the arterial system, platelet activation is based on the formation of thrombi, whereas in veins, fibrin is the main player in the formation of thrombi.46 Deficiency of specific factors is related to a thrombotic event that involves particular types of vessels or organs. For example, factor V Leiden is associated with increased risk of venous thrombosis but not of arterial myocardial infarction and stroke.47 Organ-specific thrombosis is observed in mice models with low levels of thrombomodulin, which increased fibrin deposition is found in the lung, heart, spleen, and liver but not in brain and kidney.48
The intact endothelium is an antithrombotic surface. It expresses heparin sulfate and chondroitin sulfate to prevent platelet binding. Endothelial cells also prevent the activation of platelets through the activity of ADPase, PGI2, and NO. The process of coagulation is initiated by the binding of factor VIIa with tissue factor complex. The binding is blocked by the tissue factor pathway inhibitor on the endothelial luminal surface. The action of thrombin, ATP, and ADP is a key component of the amplification of platelet aggregation signal. Heparin sulfate proteoglycans allows the binding of antithrombin III, which in turn inhibits thrombin molecule. ADPase blocks the platelet aggregation by metabolizing ADP to AMP and adenosine. The endothelium blocks coagulating factors from accessing the smooth muscle cells. Upon injury to the endothelium, the smooth muscle cells are exposed to circulating procoagulating factors, and thus the injured surface becomes an active site for neutrophil, monocyte, and platelet adhesion.49
NO, PGI2, and prostaglandin D2 are strong anticoagulative regulators able to interfere with platelet adhesion, activation, aggregation, secretion, and shape changes.50 PGI2 binds to specific receptors present on platelets that are linked to adenylate cyclase. Receptor activation leads to accumulation of intracellular cAMP in the platelet cytoplasm and downregulation of aggregation pathways.51 Thromboxane A2 (TxA2) and PGI2 usually plays each other’s counterparts in the process of coagulation. TxA2 is produced by platelet COX-1 and thromboxane synthase, and PGI2 is synthesized in vascular endothelial cells predominantly by the action of COX-2.51 PGI2 is continuously released into the circulation by the lungs to counter platelet aggregation from the release of TxA2. The PGI2/TxA2 ratio has been observed to be important; manipulation of this ratio with small doses of aspirin has beneficial effects similar to those of antithrombotic therapy.52 Endothelium-derived NO diffuses through the platelet membrane to bind its receptor. NO receptor is a soluble guanyl cyclase, like in the VSMCs. Binding to the receptor induces an elevation in cGMP and activation of cGMP-dependent kinases. This, in turn, inhibits intracellular Ca2+ increase, cytoskeletal rearrangements, integrin activation, and dense granule secretion, so the aggregation signal of platelet is blocked.53
Under normal conditions, the coagulation cascade is self-regulated. For example, NO production is in part triggered by the action of platelet-activating factor (PAF) on the endothelium.54 Although the importance of eNOS-produced NO has been confirmed, the contribution of other NOS species to platelet inhibition is poorly understood, and data on the subject are conflicting.50 The role of platelet-derived NO in the negative feedback system of platelet aggregation has been challenged, because independent studies have not demonstrated NOS messenger RNA and NOS activities in isolated platelets.55,56 The endothelial cells can respond to vasoactive agents, thrombin, or tumor necrosis factor-α (TNF-α) within minutes with the increased release of NO and prostacyclin. At the same time, the endothelial cells also recruit vesicles of pre-formed Weibel-Palade bodies to their plasma membranes. Weibel-Palade bodies are vesicles containing von Willebrand factor, P-selectin, and angiopoietin-S, which are involved in platelet binding, leukocyte recruitment, and inflammation modulation, respectively. Even though proteins C and S are not produced by endothelium, their activities occur in an endothelium-dependent fashion that involves multiple endothelially produced factors. Protein C and cofactor S can inactivate factors Va and VIIIa to turn off coagulation. The activation of protein C is facilitated by thrombomodulin and EPCR. EPCR binds protein C with high affinity to the surface of endothelial cells adjacent to the thrombomodulin-thrombin complex. Thrombomodulin transcription factor is enhanced in endothelial cells by arterial shear forces, which involve the transcription factor KLF-2.57 Arterial shear forces induce the transcription factor KLF2 and suppress inflammatory activation, a property that is lost in atherosclerosis-prone areas with disturbed blood flow.58–60
The key to the antithrombotic property of the endothelium is its integrity. If the endothelium is damaged or functions abnormally in any way, it will present a situation in which the disturbed balance favors thrombogenesis. Such disturbance has been recognized in clinical situations of traumatic vascular damage, diabetes, smoking, hypertension, and various other clinical diseases.
Leukocyte Trafficking
Migration of leukocytes from blood to peripheral tissue is crucial for the normal inflammatory process and also has important implications in pathologic conditions. The process, which is controlled by the endothelium, is a multistep cascade involving attachment, rolling, arrest, and transmigration. This process takes place almost exclusively in postcapillary venules.4
Leukocyte rolling is mediated by the interaction among endothelial E-selectin, P-selectin, and their respective leukocyte ligands. E-selectin is expressed in activated endothelium, and P-selectin is expressed constitutively. After the leukocytes are slowed down at the endothelial surface, firm adhesion is achieved by the interaction of endothelial intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) with leukocyte integrins. Both adhesion molecules are induced by activation agonists of inflammatory signaling. Leukocytes can pass through the endothelium either paracellularly or transcellularly.61,62 This passage is facilitated by CD99 antigen, platelet endothelial cell adhesion molecule-1 (PECAM-1, also known as cluster of differentiation 31 [CD31]), and junctional adhesion molecules-1.63–65
Endothelial cells can be activated by endotoxin and inflammatory cytokines such as tumor necrosis factor-α αand interleukin-1. These factors all activate the classical nuclear factor-κB (NF-κB) pathway. Nuclear factor-κB induces the amplification of the genes required for inflammatory responses such as E-selectin, VCAM-1, ICAM-1, COX-2, PAI-1, and urokinase-type plasminogen activator (u-PA). NO can counteract this inflammatory activation. It can inhibit leukocyte adhesion to the vessel wall either by interfering with the ability of the leukocyte adhesion molecule CD11/CD18 (integrin) to form an adhesive bond with the endothelial cell surface or by suppressing CD11/CD18 expression on leukocytes.19 NO also plays a role in modulating or inhibit expression of VCAM, platelet endothelial cell adhesion molecule, and ICAM on the endothelial cell, thus inhibiting the adhesion and migration of leukocytes.58–60
