2. Origin and proliferation
- Blood properties
- Defense / immune
At the beginning of the XIX century, von Reckingausen observed that blood vessels were covered by a sheet of cells. This one-cell thick layer is made up of endothelial cells that line the internal surface of blood and lymphatic vessels. In humans, it is estimated that the total endothelial layer surface is about 35 m2 and that there are around 1 to 1,6 103 endothelial cells. The total length of arteries, veins, and capillaries is about 90000 km (more than traveling twice around the world). Endothelial cells are flat and connected between each other by cell junctions. Their main function is as intermediaries between the blood and the other tissues, and contribute to blood properties, both in normal and pathology tissues. They are also important for the exchange of immune cells between the blood and tissues.
Endothelial cells are very flattened cells, so much that their nucleus is the taller structure of the cell, even if the nucleus is flattened as well. The cellular shape is adapted to the duct the endothelial cell is lining (Figure 1, 2, and 3). In the more narrow capillaries, endothelial cells may extend their cytoplasm all the perimeter of the blood vessel, so that the duct is a row of endothelial cells. In larger ducts, like arteries and veins, many endothelial cells are needed for covering the total perimeter of the duct.
Perinuclear cytoplasm contains most mitochondria and other organelles, such as Golgi apparatus, whereas periphery cytoplasm are thinner and has few organelles, although endoplasmic reticulum can be found close to the plasma membrane. Endothelial cells have many vesicles, most of them are endocytic vesicles.
In some regions, such as liver, kidney cortex, and endocrine glands, endothelial cells of capillaries show pores or small passages, known as fenestratrions. They are pathways of about 60 to 70 nm in diameter, with a narrow passage of about 5 nm, that directly communicate the blood and lymph with the other tissues. Capillaries having these types of endothelial cells are known as fenestrated capillaries. Pores are distributed in groups and their density depends on the type of endothelium.
Endothelial cells are separated from the surronding tissue by a layer of extracellular matrix called basal lamina. Endothelial cell contribute to this layer with the proteins laminin, fibronectin, and collagen type II, IV and V.
2. Source and proliferation
Vasculogenesis is the formation of new endothelial cells in tissues with no preexisting blood vessels. It happens in the embryo. In adults, and also in embryos, new blood vessels, and therefore new endothelial cells, are generated from already present blood vessels. This process is known as angiogenesis. New endothelial cells are produced by branching or dividing blood and lymphatic vessels. Angiogenesis is common in growing tissues or those under heavy remodeling, like the uterus in mammal females, or in pathologies like tumors, inflammation and wounds.
First endothelial cells of mammals are generated after gastrulation during the embryo development. They differentiate from cells called angioblasts, form groups, and get organized in short blood vessels. This process first happens in the vitelin sac of the embryo. These initial blood vessels grow and connect between each other to form a network. Later, they recruit fibroblasts and muscle cells. This initial network undergoes a continuous remodeling process during the next developmental stages. In embryos, all endothelial cells are initially similar, but they get later differentiated in vein, artery, capillary and lymphatic vessel endothelium. Several molecules like FGF2, BMP4, IHH and VEGF induce separated differentiation pathways. Notch inducer appears to be very important because it leads to artery endothelium, whereas its inhibition leads to vein endothelium. Lymphatic endothelium is formed from the cardinal vein endothelium and needs the expression of COUPF-II and SOX18. In addition, endothelium is differentiated accordingly to the organ it is located in. For example, endothelium forms a tightly sealed layer in the brain to form the blood-brain barrier, whereas it is fenestrated of loosely organized in the liver to favor the exchange of molecules with the blood.
As endothelial cells are getting organized in artery and vein ducts, some endothelial cells give rise to hematopoietic cells by a differentiation process known as endothelial-hematopoietic transition (EHT). This process also happens in some adult tissues like during the formation of placenta and other tissues during pregnancy.
Angiogenesis is the formation of new blood vessels and endothelium by branching or dividing preexisting blood vessels. In adults, the proliferation rate of endothelial cells is very low. For example, in mammals, an endothelial cell may divide once per month or may wait several years. It means that angiogenesis is rare in tissues under normal physiology, excepting female reproductive organs. However, it can be activated in pathological processes like tissue damages and tumors. Some substances induce angiogenesis, such as endothelial growth factor (EGF), acidic EGF, fibroblast growth factor (FGF), transforming growth factor (TGF) and prostaglandins. Angiogenesis begins when angiogenic substances are released from surrounding tissues. The endothelial cell of a near blood vessel, usually from a venule, starts to migrate to the angiogenic signal source, dragging the other endothelial cells, which begins to proliferate and form a new vessel. These endothelial cells transiently lose their tight connections resulting in the uncovering of the basal membrane, which is quickly digested by enzymes released from the endothelial cells. Some endothelial cells go through the digested basal membrane while the proliferation goes on. All this process makes possible the elongation and branching of the new blood vessel until vasculogenesis signal disappears. The tips of the blood vessels fuse with other blood vessels to form closed circuits and allowing the flux of blood without dead endings.
Regions with endothelial cell progenitors have been found in the dorsal aorta and endocardium. Mesenchymal cells can be differentiated from endocardium endothelial cells and form the tricuspid valve and some fibroblasts of the heart, but not cardiomiocytes. Endothelial cells are so sensible to external signals that keeping the endothelium integrity is an active process, which is mediated by other signals like FGF. The active process is not just for integrity but to keep the cellular phenotype as well. When endothelial cells do not receive proper signals they die by apoptosis or become mesenchymal cells that may synthesize abundant extracellular matrix. The transition from endothelial to mesenchymal cell is behind some diseases like arteriosclerosis and fibrous miocarditis.
The idea that the endothelium is just a passive lining coat of lymphatic and blood vessels has to be changed. Endothelium functions are varied and essential for the organism. Indeed, it is a physical structure of cardiovascular and lymphatic ducts, but it also regulates the exchange of molecules between the lumen of the vessel and the surrounding tissues, and it influences the physiological properties of the blood. Besides a physical barrier, endothelial cells develop secretory, metabolic and immune functions. They change their physiological behavior influenced by molecules like growth factors, coagulants and anticoagulants, low density lipoproteins, nitric oxide, serotonin, enkephalin, and many others. Endothelial cells have receptors for all these substances.
Endothelial cells form a layer that usually acts as a barrier between the blood and the surrounding tissues. The cohesion between endothelial cells is fulfilled by cell junctions, such as tight junctions and adherent junctions. Gap junctions have also been observed, although their main function is the communication between adjoining cells. Endothelial cells can modulate these adhesions and change the permeability of the barrier, that may affect not only to molecules but also to cells crossing the endothelium. However, in some organs like liver, endothelial cells are much more loosely packaged and leave much free space so that endothelium can hardly been regarded as a barrier.
Transmission electron microscopy images show many vesicles in the cytoplasm of endothelial cells. They are thought to be involved in intracellular transport between the apical (facing the blood) and basolateral membranes (facing the basal lamina). This type of transport is known as transcytosis. It is interesting that transcytosis vesicles are more abundant in the endothelial cells of capillaries than in the those of larger blood vessels. It indicates that the endothelial cells in capillaries has a more intense exchange of molecules between blood and tissues, whereas in large diameter vessels they are mainly involved in conducting the blood. Some endothelial cells, known as fenestrated, have pores or very small ducts that directly connect the blood with the surrounding tissues, allowing some small size molecules to cross the endothelium without entering the cytoplasm of any endothelial cells (Figure 4). Finally, there are organs with very permeable endothelium. In the liver, sinusoids are blood vessels where endothelial cells leave free space between each other, and a function as a barrier hardly exists.
Many cell types travel in the blood toward their target organs in the body. There, they cross the endothelium of the blood vessels, commonly at the level of post-capillary veins. It means that endothelial cells have to modify the cell junctions to let cells go through the endothelium layer. Leukocytes exit the blood vessel by recognizing and anchoring to specific molecules of the apical membrane of the endothelial cells. Selectins, integrins and immunoglobulins are responsible for the recognition and adhesion of leukocytes to the endothelial layer. Selectins begin the anchoring of leukocytes, which roll over the endothelial surface. This initial adhesion is weak and reversible.
Many leukocytes exit the blood during inflammatory processes to move to the affected tissues. Chemokins are leukocyte-attractive molecules that are released by damaged tissues and linked to the glycocalix of endothelial cells. Leukocytes rolling over the endothelial surface having chemokines are activated, cell-cell adhesion gets stronger, and leukocyte remains in the place. The activation of the leukocyte leads to the activation of its integrins, which recognize the immunoglobulins of the endothelial cell. These immunoglobulins are expressed in the endothelial cell membranes after the activation of the cell by chemokines. The cell-cell adhesion raises the calcium concentration in endothelial cells and leads to cell junctions disorganization and cytoplasm retraction. In this way, leukocytes can move to the border of endothelial cells and cross the endothelium. Adhesion molecules are also involved in this movement of leukocytes.
The endothelial functions are more complex than just control the molecules and cells going across the endothelium. Endothelial cells are also involved in blood pressure, coagulation, and some others blood properties.
Primitive circulatory system is thought to emerge 600 million years ago in invertebrates, but it lacked endothelium. Endothelial cells appeared 100 million years ago providing blood with a more laminar flux (not turbulent) and therefore a more efficient gas exchange.
Endothelial cells modulate the blood pressure by releasing substances that act on smooth muscle of blood vessels. They release nitric oxide (NO) and prostacyclin, which relax vascular smooth muscle. They also release endothelin and the platelet activator factor, both decreasing the blood vessel diameter. Nitric oxide is constitutively released and provides a proper muscle tone, inhibit platelet aggregation and leukocyte adhesion. Endothelin is a strong vasoconstrictor. How endothelial cells decide what molecule has to be released is not clear yet, but mechanorreceptors in their apical membranes that are able to feel the flux properties of the blood may be plausible.
Under normal conditions, endothelial cells release molecules into the blood that help to maintain a proper fluidity. They work at two levels: fluidity (anticoagulants) and preventing platelet aggregation (antithrombotics). Proteins C and S are important molecules affecting blood fluidity. C protein, forming a complex with S protein, inactivates coagulation factors VIIIa and Va. S protein is synthesized by endothelial cells. Furthermore, the endothelial glycocalix contains a glycosaminoglycan similar to heparin that is able to inactivate thrombin.
Related to their anticoagulant function, endothelial cells release nitric oxide and prostacyclin, both rising the cyclic AMP in platelets and making more difficult platelet aggregation. These two molecules are continuously released into the blood. Endothelial cells have ectonucleases in their apical membranes, which remove ATP and ADP, both strong promoters of platelet aggregation. In addition, endothelial cells release an activator molecule that transform plaminogen into plasmin, which favor removing thrombi.
All these molecular pathways may change when endothelial cells receive some signals or tissue are damaged, which lead to blood coagulation and platelet aggregation. In these circumstances, endothelial cells then become active participants of the coagulation and thrombosis.
Endothelial cells play a major role in the immune defense and are involved in two mechanisms: antigens presentation to T lymphocytes and recruiting immune cells. Together with macrophages, endothelial cells can present antigens to T lymphocytes because they constitutively express MHC-I (major histocompatibility complex) and may be induced to express MHC-II, both necessary for antigen presentation. Endothelial cells are able to activate immune memory, but not new T lymphocytes. There is bidirectional activation between endothelial cell and T lymphocytes, so that endothelial cell release molecules for attracting inflammatory cells and express adhesion molecules for anchoring blood leukocytes.
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