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Study Title:

Multiple Functions of the Endothelial Cells

Study Abstract

2.1. HETEROGENEITY OF THE ENDOTHELIAL CELLS
The first hint of endothelial cell heterogeneity, a structural heterogeneity, was obtained following electron microscopy observations where differences in intercellular junctions led to the classification of continuous endothelium, fenestrated endothelium and discontinuous endothelium [92]. Continuous endothelium is found in most arteries, veins and capillaries of the brain, skin, lung, heart and muscle. Endothelial cells are coupled by tight junctions and anchored to a continuous basal membrane. Fenestrated endothelium is also associated with a continuous basal membrane and is characterized by the presence of transcellular 50–60 nm wide pores, which are sealed by a 5- to 6-nm-thick diaphragm. This is observed in tissues with an elevated trans-endothelial transport or an increased filtration role, such as endocrine and exocrine glands, gastrointestinal tract, choroid plexus, kidney glomeruli and subpopulations of renal tubules. The discontinuous endothelium is associated with a poorly structured basal membrane and is characterized by the presence of large 100- to 200-nm-wide fenestrations without diaphragm. This occurs in sinusoidal vascular beds in the liver, predominantly, but also in the spleen and bone marrow [20,1250].

Structural heterogeneity of the endothelial cells also includes various cellular shapes, various amounts of structural component of the endocytic pathway, such as clathrin-coated pits or the transcytosis pathway such as of caveolae, various levels of expression of the predominant types of intercellular junctions, tight junctions, adherens junctions or gap junctions, various compositions of the glycocalyx and the associated endothelial surface layer (a stationery layer much thicker than the glycolalix that excludes red blood cells), etc. [20,1250,1427].

The endothelial cells are involved in different tasks, which are performed either by all the endothelial cells in general or predominantly by endothelial cells in specific subsets of organs or vascular beds. For instance, endothelial cells in general regulate hemostasis. However, endothelium-derived products that, on the one hand, maintain blood fluidity and, on the other hand, are involved in the coagulation cascade and the formation of blood clot, are unevenly distributed over the vascular tree. Similar observations could be made for other important functions of the endothelium such as permeability, leukocyte trafficking, regulation of vascular tone, angiogenesis, immunity, etc. [20,21,430]. Even contiguous endothelial cells can exhibit differential signaling and functional properties. For instance, “pacemaker cells” show unique ability to generate spontaneously intracellular calcium events that are transmitted to the neighboring non-pacemaker cells inducing inter-endothelial calcium waves [1768].

This heterogeneity in endothelial cells is linked to both intrinsic, i.e., genetic factor, and extrinsic factors, i.e., environmental causes such as location, soluble mediators, cell to cell contact, cell–matrix interactions, pH, pO2, mechanical forces (shear stress, physical constraints), etc. [1250]. Endothelial cells and hematopoietic cells arise from the mesoderm by the differentiation of the same precursor cells, the hemangioblast [248]. Hemangioblasts give rise to angioblasts or endothelial progenitor cells, which in turn differentiate into endothelial cells of arteries, veins and capillaries, each cell type being identified by a specific expression pattern of gene markers. Epigenetic-induced heritable changes, histone methylation and acetylation, occur in the early phase of differentiation, while micro-environmental changes, which are not transmitted during mitosis, occur predominantly in the late phase of differentiation, i.e., organ-specific differentiation of the endothelial cells [20,21,1466]. However, other cell lineages, such as adipose lineage cells or neural stem cells, can trans-differentiate into endothelial cells [1226,1718].

The remarkable plasticity of the endothelial cells could be seen as its principal property. One can make the assumption that each one of the trillion endothelial cells included in our body is phenotypically distinct since, like a chameleon, each one has to sense and adapt to the needs of the various neighboring cells and to many different microenvironments [20,21].

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2.2. HEMOSTASIS, THROMBOSIS AND FIBRINOLYSIS
In the heart, arteries, capillaries and veins and under normal conditions, the flowing blood is in contact with healthy endothelial cells and remains fluid. The luminal surface of quiescent endothelial cells is anticoagulant and non-thrombogenic, the platelets and leukocytes do not adhere to it and the coagulation system remains inactivated. By contrast, the macromolecules of the basal lamina, synthesized by the endothelial cells, are strongly thrombogenic, and activated endothelial cells promote thrombus formation. The endothelial cells, therefore, regulate the equilibrium between thrombosis, hemostasis and thrombo-resistance.

2.2.1. Hemostasis
Hemostasis processes are classified as primary, mainly involving platelets, and secondary, predominantly related to fibrin formation or blood coagulation, although the two processes strongly interact.

When the continuity of the endothelium is disrupted, the subendothelial matrix and the collagen fibers are exposed. Circulating platelets adhere to these structures and start the hemostatic process. This platelet recruitment depends on several integrin receptors that mediate the adhesion to extracellular matrix-associated fibronectin and laminin. However, vascular wall-associated von Willebrand factor (vWF) appears to play a major role in initial platelet recruitment and thrombus formation, especially when the blood flow is elevated (high shear rate). vWF, a multimeric protein, is predominantly synthesized by the endothelial cells and is stored in one of their characteristic ultrastructural features, the Weibel–Palade bodies [728]. vWF is released in the circulation, where it stabilizes factor VIII (FVIII) and is associated with collagen VI in the subendothelium. At sites of vascular injury, vWF acts as a bridge between the tissues and the platelet via the GPIb/IX/V glycoprotein receptor. Circulating vWF binds to the exposed collagen and participates in platelet–endothelial cell interactions [48,1609]. Additionally, endothelial cells synthesize the lipid mediator platelet-activating factor (PAF) that promotes the activation of platelets and their adhesion to the endothelial cells [1203]. These platelet–endothelial cell interactions are at the interface between hemostasis and inflammation (Figure 2).

FIGURE 2. Pivotal role of the endothelial cells in hemostasis, thrombosis and fibrinolysis.
FIGURE 2
Pivotal role of the endothelial cells in hemostasis, thrombosis and fibrinolysis. Upper panel: In normal conditions, endothelial cells show an anti-thrombotic phenotype [degradation of the aggregating agent ADP with ectonucleotidases; prevention of primary (more...)

The secondary recruitment of platelets and their aggregation involve morphological changes and degranulation of the activated platelets. The release of ADP, ATP, thromboxane A2, serotonin, vWF and fibrinogen further activates neighboring platelets, amplifies the aggregation process and produces vascular smooth muscle contraction facilitating the formation of the hemostatic plug. Healthy endothelial cells, in response to most of these mediators, would normally release prostacyclin and NO in order to limit the aggregation and a potential thrombosis. However, in some circumstances, endothelial cells can also contribute to the amplification phenomenon. In response to ADP, ATP or serotonin, endothelial cells can release arachidonic acid metabolites such as thromboxane A2, and, in response to thrombin, following the activation of protease-activated receptors (PAR), they release vWF, and the expression of tissue factor (TF) is up-regulated [1203,434] (Figure 2).

The initiation of the coagulation cascade is primarily mediated by TF. In healthy blood vessels, TF is located in the extracellular matrix underlying the endothelial cells, in the adventitia and in a number of cells surrounding blood vessels and in subcutaneous tissues. Exposure of TF to blood and the subsequent binding to FVIIa initiates coagulation. The complex TF–FVIIa converts circulating FIX and FX into active enzymes, FIXa and FXa, FIXa further enhancing the activation of FX. FXa catalyzes the generation of thrombin (FIIa). Thrombin is a key enzyme since it cleaves fibrinogen into fibrin. Fibrin polymerizes into a fibrin network, which with the aggregating platelets form the blood clot. Furthermore, thrombin is responsible for the propagation phase of coagulation by activating FV, FVIII, FXI and FXIII (Figure 3). Additionally, by interacting with its platelet receptor (PAR-1), it causes further platelet activation [48,1609]. During platelet activation, the adhesion molecule P-selectin is exposed at the surface of the membrane and allows the formation of conjugates between activated platelets and leukocytes.

FIGURE 3. Schematic representation of the coagulation cascade (solid arrow) and the fibrinolytic system (dotted arrow).
FIGURE 3
Schematic representation of the coagulation cascade (solid arrow) and the fibrinolytic system (dotted arrow).

Healthy endothelial cells do not express TF or show TF activity, although it can be induced in cultured endothelial cells by activated platelets, endotoxins, various cytokines, fibrin, thrombin or hypoxia. However, in vivo, it appears unlikely that the expression of TF by endothelial cells could contribute to the initiation phase of coagulation and, even under pathological conditions, there is no solid evidence so far for a contribution of endothelial TF in the formation of atherothrombosis [48,1203]. In contrast, endothelial cells may promote the propagation phase of coagulation. Once coagulation has been initiated, the activity of FXa is locally accelerated in neighboring endothelial cells by its binding on the endothelial cell surface to FVa, the expression of which is enhanced in endothelial cells after mechanical injury, with a concomitant decrease in the levels of thrombomodulin. These events concur to promote thrombin activation [48,1203].

Finally, if under normal conditions, the intact endothelium expresses low levels of adhesion molecules, upon activation, the expression of these molecules, such as intercellular adhesion molecule-I (ICAM-1), vascular cell adhesion molecule-I (VCAM-1), E-selectin and P-selectin, is enhanced. Platelets, leukocytes and even erythrocytes can bind to the endothelial cells and the stockpiling of these circulating cells can restrict blood flow. Furthermore, activated platelets provide a surface for the assembly of coagulation complexes [588] (Figure 2).

2.2.2. Blood Fluidity
Endothelial cells prevent adhesion, aggregation and activation of platelets and promote platelet de-aggregation by expressing 13-hydroxyoctadecadienoic acid (13-HODE) on their cell surface, by releasing prostacyclin and nitric oxide (NO), metabolizing ATP and ADP by membrane ectonucleotidases and preventing the action of thrombin, a potent aggregating agent [1039,1609]. Indeed, endothelium-derived NO and prostacyclin are not only potent vasodilators but also powerful inhibitors of platelet adhesion and aggregation. In fact, NO and prostacyclin, or in general, substances that activate guanylyl cyclase and adenylyl cyclase, show a remarkable synergistic interaction in inhibiting platelet aggregation [53,562,1068,1269–1271,1297,1298] (Figure 2).

Endothelial cells also act as a natural anticoagulant agent. They express at the cell surface the thrombin receptor thrombomodulin, which converts thrombin from a procoagulant to an anticoagulant enzyme. The thrombomodulin-bound thrombin activates protein C, a phenomenon facilitated by the neighboring presence of the endothelial protein C receptor. Activated protein C with the associated protein S act as a brake on the coagulation cascade by inactivating FVa and FVIIIa. Additionally, the endothelial cell surface is rich in heparin-like sulphated glycosaminoglycan molecules that bind and activate anti-thrombin, which is the main inhibitor of thrombin and FXa. Finally, the tissue factor pathway inhibitor (TFPI) inhibits the TF–FVIIa complex by forming a quaternary complex TFPI–TF–FVIIa–FXa [1039,1609] (Figure 2).

2.2.3. Fibrinolysis
When the blood clot is no longer required for hemostasis, the fibrinolytic system dissolves the clot and restores a patent blood vessel. The fibrinolytic system includes an inactive proenzyme, plasminogen, which is converted to the active plasmin by two different activators, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Plasmin degrades the fibrin component of the blood clot into soluble degradation products. t-PA is released by the endothelial cells constitutively and upon stimulation, for instance, by shear stress or by agonists such as thrombin or bradykinin. u-PA can also be synthesized and released but only by activated endothelial cells, for instance, in response to cytokines and growth factors [48,152,1203].

Endothelial t-PA plays a major in role in the fibrinolysis process but its activity is heavily regulated. The major plasma inhibitor of t-PA (and u-PA), plasminogen activator inhibitor type-I (PAI-1), is also a protein constitutively secreted by the endothelial cells or upon stimulation by thrombin (Figures 2 and 3). Increased PAI-1 activity is considered as an independent risk factor for cardiovascular diseases [48,1137]. Mice deficient in PAI-1 exhibit a mild hyperfibrinolytic state and resistance to venous thrombosis, while transgenic mice that overexpress PAI-1 develop venous thrombosis occlusions [196,396].

2.2.4. Endothelial Dysfunction
Under normal conditions, endothelial cells become activated according to environmental needs, i.e., stopping the hemorrhage and repairing injured vascular tissue. However, endothelial dysfunction, a term that encompasses multiple potential defects of the endothelial cells, may tip the balance toward thrombosis and contribute to various pathological states such as atherothrombosis, arterial thrombosis (stroke, visceral and peripheral artery occlusive diseases), venous thrombosis, intravascular coagulation and thrombotic microangiopathies. In 1856, Virchow published his now famous triad of factors that lead to the development of thrombosis: blood hypercoagulability, stasis and vessel wall damage. While Virchow originally referred to venous thrombosis, the concept can be revisited and also applied to arterial thrombosis [110,250,950].

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2.3. VASCULAR PERMEABILITY
The endothelium forms a semi-permeable barrier between the blood and the surrounding tissues. The control of solute and macromolecule transfer, into and out of the blood, across the blood vessel wall is another major function of the endothelium. Permeability could be separated into basal permeability, which occurs at the level of capillaries, the major site of exchange in the vascular bed, and the induced permeability associated with inflammation, which predominantly involves post-capillary venules. While fluids and small solutes move passively across the barrier via a paracellular route, macromolecules use either transcellular or paracellular pathways [20,1051].

2.3.1. Transcellular Pathway
The transcellular transport involves membrane-attached and cytosolic caveolae that migrate across the capillary endothelial cells and shuttle macromolecules from the blood to the interstitium. Transcellular transport of macromolecules may involve receptor-dependent (for instance, albumin binding to GP60 protein) or -independent mechanisms (fluid phase transcytosis).

Caveolae are cholesterol-rich and glycosphingolipid-rich membrane microdomains coated on the cytoplasmic surface with the 22-kDa protein, caveolin-1 [1308]. They are characteristic structural components of endothelial cells and can constitute up to 15% of their volume. Numerous signaling molecules, such as G-proteins, kinases, eNOS or ion channels, are associated with caveolin-1 [4,507,941]. Thus, caveolin-1, by regulating protein–protein interactions, concentrates signaling molecules and optimizes their functions [1051].

The key-signaling event regulating transcytosis is the Src-dependent tyrosine phosphorylation of caveolin-1, which initiates the induction of the fission of the membrane-attached vesicle. Then the endocytosis, the trans-migration of the vesicle across the endothelium, the fusion of the vesicle to the basolateral membrane and finally exocytosis in the interstitial space can occur. However, the molecular events that determine whether a vesicle will be addressed to the basolateral membrane, completing the transcytosis process, or to any other intracellular compartment, are currently unknown [1051]. In caveolin-1 knock-out mice, the characteristic flask-shape caveolae structure is no longer present and the albumin transport across the endothelial cells is deficient [344,1372]. Interestingly, the disruption of caveolin-1 gene is not lethal, indicating that alternative mechanisms, such as an increase in junctional permeability and the presence of caveolin-independent vesicles, may partially compensate the deletion of caveolin-1 [812,1373].

When compared to continuous endothelium, the permeability in fenestrated endothelium to water and small solutes is enhanced but the transcellular transport of macromolecules is similar. In discontinuous sinusoidal endothelium small solutes, water and macromolecules diffuse through the inter-endothelial gaps. These endothelial cells are enriched with clathrin-coated pits that can contribute to transcytosis [20].

2.3.2. Paracellular Pathway
In continuous endothelium, fenestrated or not, and under basal conditions, inter-endothelial junctions are impermeable to macromolecules. Tight junctions and adherens junctions, which interact with the endothelial actin cytoskeleton, establish a restrictive barrier to macromolecules. However, in a state of acute or chronic inflammation, ischemia–reperfusion, atherosclerosis, sepsis, diabetes, thermal injury, angiogenesis or tumor metastasis, mediators such as histamine, serotonin, thrombin, bradykinin, substance P, PAF, cytokines, growth factors such as vascular endothelial growth factor (VEGF) and reactive oxygen species induce endothelial cell retraction, which increases the intercellular space and subsequently the permeability to solute and plasma proteins. Aberrations of endothelial barrier function lead to an abnormal extravasation of fluid and macromolecules, resulting in edema and tissular dysfunction. In the course of inflammation, this is the first recognized step occurring predominantly not only at the level of post-capillary venules but also, for instance, in response to VEGF at the levels of arterioles and capillaries [412,413,424,1035,1595] (Figure 4). Edema develops when plasma extravasation exceeds its re-absorption and the capacity of the lymphatic system to remove fluids from the interstitial space.

FIGURE 4. Changes in vascular permeability.
FIGURE 4
Changes in vascular permeability. The cheek pouch microcirculatory bed of the anesthetized hamster is directly observed under microscope, and the number of vascular leakage sites is shown by the extravasation of fluorescein isothiocyanate (FITC-dextran, (more...)

The integrity of the endothelial barrier is determined not only by the adhesive forces that couple endothelial cells with each other and to the extracellular matrix but also by the shape of endothelial cell. Endothelial adherens junctions link neighboring cells and consist in transmembrane VE-cadherins molecules, which are associated in the intercellular space, while the cytoplasmic tail is linked to catenins, which in turn are linked to the actin cytoskeleton [300]. Actin–myosin activation regulates the contractile function of endothelial cells. This calcium-dependent process is activated by inflammatory mediators. The traction exerted can thus disrupt the adherens junctions, precipitating the retraction of the endothelial cells [1051,1340]. This is a dynamic and reversible process. In experimental models, proper endothelial permeability and barrier integrity are generally restored within minutes (bradykinin or histamine) or hours (VEGF) following the administration of the inducing agent [412,424].

Additionally, induced permeability can involve a different pathway and not be associated with gap formation. A transcellular pathway, implicating the endothelial vesiculo–vacuolo organelles, which upon stimulation by inflammatory mediators form trans-endothelial pores, could be responsible for the leakage of macromolecules [435].

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2.4. LEUKOCYTE TRAFFICKING
The interaction between leukocytes and the vascular endothelium is a physiological and pathophysiological process. These interactions contribute to the immune response, wound repair and thrombosis as well as to acute and chronic inflammation. The passage of leukocytes from the circulation to the surrounding tissue requires the adhesion of the leukocyte to the endothelial cell surface, a multi-step cascade, already described in detail in the late 19th century [266], involving the capture (or tethering), rolling and arrest of the leukocytes, followed by their transmigration (or diapedesis). Again, these steps, often associated with inflammation, take place predominantly but not exclusively in post-capillary venules, since they can be observed in large veins, capillaries and arterioles [20,905].

2.4.1. Capture and Rolling
Under normal conditions, leukocytes do not adhere to endothelial cells. Following activation, the leukocyte first binds to the endothelial surface and the rolling process starts when new bounds are formed before those established initially are disrupted. This activation process mostly depends on the expression of the selectin family of adhesion molecules, in both endothelial cells and leukocytes. Selectins are transmembrane type I glycoproteins that share a significant structural homology (>50%) and which were named after the cells where they were first discovered: E-selectin, L-selectin and P-selectin for endothelium, leukocyte and platelets. The selectin extracellular domain is involved in the binding of leukocytes while the cytoplasmic domain has signaling functions, for instance, via the MAP-kinases and ERK pathways [905].

In resting endothelial cells, P-selectin is stored in the Weibel–Palade bodies and following activation (inflammatory mediators, trauma) is rapidly expressed at the cell surface, preferentially in post-capillary venules. E-selectin is among the very few genes that is highly restricted to endothelial cells. Constitutively, this gene is very poorly expressed but, following activation, is up-regulated, also predominantly in the endothelial cell of post-capillary venules. E- and P-selectin mediate leukocyte adhesion and rolling at the site of inflammation. Mice deficient in either E-selectin or P-selectin show only a moderate phenotype, while in the double knockout model, leukocyte adhesion is severely impaired, indicating redundancy between the two pathways [156]. Different selectin ligands have been identified in leukocytes, but the most important for both P-selectin and E-selectin appears to be the carbohydrate-based P-selectin glycoprotein ligand-1 (PSGL-1) [646,905].

A distinct form of leukocyte trafficking takes place in secondary lymphoid organs. Specialized post-capillary venules, named high endothelial venules, are involved in non-inflammatory, constituous leukocyte homing and recirculation. In these vascular beds, leukocyte capture and rolling is mediated by the interaction between L-selectin, which is constitutively expressed in most leukocytes, and its endothelial ligand, peripheral node addressin [20,905].

The rolling of leukocytes on the endothelial surface establishes a close contact between the two cell types that will lead to firm adhesion and arrest, these two later steps involving a different family of adhesion molecules.

2.4.2. Firm Adhesion and Arrest
Slowly rolling leukocytes are activated by endothelial chemokines, leading to conformational changes in integrins, a superfamily of adhesion receptors expressed by the leukocytes. At least 25 αβ heterodimers have been identified so far, each with a different function depending on the cell type where they are expressed and the ligand they bind to. Integrins interact with their ligands on the endothelial cells belonging to the immunoglobulin family, such as platelet–endothelial cell adhesion molecule (PECAM-1), vascular cell adhesion molecule (VECAM-1), intercellular adhesion molecule (ICAM-1) or junctional adhesion molecule (JAM).

ICAM-1 (CD 54) is constitutively expressed in resting endothelial cells and mediates both rolling and firm adhesion of leukocytes, where it interacts preferentially with lymphocyte function-associated antigen (LFA-1) and macrophage differentiation antigen-1 (Mac-1). VCAM-1 (CD106) is very poorly expressed in resting endothelial cells but is rapidly induced by inflammatory mediators. VCAM-1 interacts preferentially with leukocyte very late antigen-4 (VLA-4) and also mediates rolling and adhesion, indicating overlapping functions between the two pathways [905].

2.4.3. Diapedesis
Trans-endothelial migration occurs preferentially via inter-endothelial junctions (paracellular pathway), although a transcellular route has also been observed [436]. The paracellular pathway of diapedesis is under the control of adhesion molecules, some of which are highly expressed in inter-endothelial junctions. Leukocyte integrins may play a role not only in migration through the endothelium but also in infiltration through the subendothelial basal membrane [905].

The migration through the inter-endothelial junctions must involve the rupture of the selectin bonds on the endothelial surface and the establishment of new bonds on the margin of the endothelial cells. The activation of leukocytes reduces selectin binding and favors the integrin-mediated associations. In endothelial cells, PECAM-1, CD99, JAM are preferentially located in the inter-endothelial junctions and contribute to paracellular migration. Similarly, ICAM-1 and VECAM-1 are highly expressed in the transmigratory cups, the sites involved in transcellular migration of leukocytes [194,905].

In conclusion, leukocyte trafficking is essential in physiology for the development of immune response in T cells (lymphoid organs) and for hemopoietic homeostasis (maintenance of circulating leukocyte numbers). However, dysregulation of this mechanism leads to numerous pathophysiological conditions associated with inflammatory state including atherosclerosis where recruitment of leukocytes in the vessel wall appears to be a crucial early step in the development of the disease [1033].

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2.5. VASCULOGENESIS, ANGIOGENESIS AND ARTERIOGENESIS
Vasculogenesis refers to the initial events in vascular development in which endothelial cell precursors migrate, differentiate and assemble into a primitive vascular network. Additionally, after birth, post-natal vasculogenesis may occur following the recruitment of endothelial progenitor cells, derived from the bone marrow, which are incorporated into nascent vessels or which stimulate new vessel growth by releasing pro-angiogenic stimuli. Angiogenesis is the sprouting of new capillaries and blood vessels derived from pre-existing blood vessels. This is a fundamental process in reproduction, development and repair. Angiogenesis can also be pathological. Both physiological and pathological angiogenesis primarily involve the endothelial cells. Arteriogenesis is the stabilization of these newly formed blood vessels resulting from the association of pericytes or vascular smooth muscle cells, a critical step for the new vasculature to become stable, mature and functional [195,447,1555].

2.5.1. Vasculogenesis
During embryonic vasculogenesis, mesoderm-derived hemangioblasts migrate to the yolk sac where they form blood islands that give rise to endothelial and primitive blood cells. Blood islands fuse to form the extra-embryonic blood vessels with endothelial cells precursors lining spaces containing the hematopoietic progenitors. Within the embryo, similar endothelial precursors proliferate locally and interconnect in a loose meshwork that undergoes differentiation into a primary vascular plexus, which gives rise to the rest of the vasculature, forming with the pumping heart the first functioning organ system in the embryo [1466,1555].

Circulating endothelial progenitor cells can differentiate to mature endothelial cells and replace injured or senescent endothelial cells in order to restore the integrity of the endothelial lining. Additionally, endothelial progenitor cells could contribute to post-natal vasculogenesis as they have been shown to be home to sites of neovascularization. The mobilization and the recruitment of these bone marrow-derived endothelial progenitor cells to the sites of vasculogenesis involve similar mechanisms to those associated with angiogenesis (various growth factors, metalloproteinases, adhesion molecules and cytokines). However, to what extent and under which precise conditions endothelial progenitor cells contribute to vascular growth remains an open question. Besides bone marrow-derived endothelial progenitor cells, bone marrow-derived multipotent adult progenitor cells with angioblastic potency, tissue-specific stem cells and a subset of circulating endothelial cells, endothelial colony-forming cells, which show high proliferative potency, could play a similar vasculogenic role [447,1769].

2.5.2. Angiogenesis
When a primitive network has been formed through vasculogenesis, it expands and remodels into an organized vascular tree. This process involves angiogenesis, the sprouting of microvessels from pre-existing vessels. After birth, angiogenesis still continues to contribute to organ growth, and endothelial cells could play a master role in regulating the size of the various organs in the body [1555]. In the adult, angiogenesis is involved in the physiology of reproduction, in wound repair and in responses to stimuli such as hypoxia and inflammation. Numerous diseases are characterized or caused by abnormal or excessive angiogenesis or conversely by insufficient angiogenesis or vessel regression [447].

Endothelial cells are normally quiescent and are among the most genetically stable cells of the body as their turnover time is over hundreds of days. However, during angiogenesis, endothelial cells can proliferate rapidly with a turnover time of less than 5 days [465]. Angiogenesis is a complex phenomenon tightly regulated in a spatial and temporal manner. In response to angiogenic factors released by surrounding hypoxic tissues, endothelial cells are activated. They degrade the extracellular matrix, proliferate, migrate toward these angiogenic cues and establish tubular structures, the basis of the new blood vessels [384].

In order to emigrate, endothelial cells must loosen their endothelial connections, a crucial determinant of vascular permeability, relieve peri-endothelial cell contact, a major factor in vessel stability and maturity and break down the basal lamina and the extracellular matrix, the scaffold of the blood vessel. Angiopoietin-2, the physiological antagonist of angiopoietin-1 is involved in the destabilization of the mature vessels [499]. Extracellular matrix degradation is mediated and regulated by several proteinase families, plasminogen activators and inhibitors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, chymases, heparanases, tryptases, cathepsins, kallikreins and their inhibitors. Under the action of proteinases, numerous growth factors with angiogenic properties are released from the matrix such as FGF-2, TGF-β, VEGF, IGF-1 and TNF-α. Conversely, and still in the spirit of a tightly regulated phenomenon, inhibitors of angiogenesis are also released from the matrix such as thrombospondin-1, canstatin, arrestin, tumstatin, angostatin, endostatin, etc., in order to switch off the angiogenic process. Once the physical barrier is disrupted and under the influence of the growth factors released from the matrix and the chemo-attractive substances produced by the environment, the endothelial cells proliferate and migrate, again a finely tuned process. Among the growth factors released, VEGF plays an essential role in this phenomenon with the additional contribution of other factors such angiopoietin-1, integrins and various chemokines. VEGF and angiopoietin-1 could also be involved in tube formation while ephrins play an important role in the guiding of the forming vessel toward its target. Finally, VEGF is also an essential survival factor for the endothelial cell-constituted new blood vessel. Depending on the origin of the neovascularization (physiological, pathological) or the vascular bed involved, the mechanisms leading to the angiogenic process can differ markedly [447,1480].

2.5.3. Arteriogenesis
In order to evolve into a mature and functional blood vessel, the endothelial tube must recruit smooth muscle cells (arteries, arterioles and veins) or pericytes (capillaries and venules) and generate extracellular matrix.

A considerable heterogeneity is observed in the origin of vascular smooth muscle cells both during embryologic development and in the adult vasculature. Indeed, vascular smooth muscle is a mosaic of tissue produced from at least seven origins in vertebrate embryos, neural crest, proepicardium, mesothelium, heart field, somites, mesoangioblasts and stem cell–progenitor cells. Pericytes and microvascular smooth muscle cells are derived from the same lineage as the smooth muscle cells that constitute the neighboring large arteries [972]. Adult blood vessels contain a distinct population of smooth muscle progenitor cells in the adventitia [680].

In adults, the recruitment of vascular smooth muscle cells is achieved by the division of pre-existing smooth muscle cells and the differentiation of various cell types, local smooth muscle progenitor cells, bone marrow-derived smooth muscle cells, circulating endothelial progenitor cells or fibroblasts. The PDGF family of growth factors plays a predominant role in the recruitment of vascular smooth muscle cells, in their division and in the stabilization of the newly formed blood vessels. Additionally, angiopoietin-1, PlGF and TGF-β contribute to the blood vessel stabilization and maturation processes [447].

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2.6. METABOLISM AND CATABOLISM
Endothelial cells are involved in intense metabolic activities with again a marked heterogeneity depending on the vascular bed, the size of the vessel or the type of vessel (arteries vs veins vs capillaries). Besides their involvement in hemostasis, endothelial cells can metabolize or conversely activate numerous circulating factors, with or without vasoactive properties, including polypeptide hormones, amines, nucleotides, lipoproteins, metabolites of arachidonic acid and reactive oxygen species. For instance, endothelial cells express monoamine oxidase (MAO) and cathecol-o-methyltransferase (COMT) [inactivating epinephrine and serotonin], angiotensin-converting enzyme (ACE) [activating angiotensin-II and degrading bradykinin, substance P or enkephalins], ACE-2 [activating Ang-(1–9) and Ang(1–7)], neutral endopeptidase (NEP) [inactivating natriuretic peptides, substance P, tachykinins, bradykinin, activating and inactivating endothelins], endothelin-converting enzymes (ECE) [producing endothelins, inactivating tachykinins, degrading amyloid peptides], dipeptidyl-peptidase IV (DPP-IV) [cleaving glucagon-like peptide-1(GLP-1), metabolizing neuropeptide Y (NPY-1)], cathepsins, extracellular superoxide dismutase, etc. [276,405,415,547,869,1105,1170,1254,1302,1572,1573,1701].