Blood Vessels: the Endothelium

Blood vessels: the endothelium

Peter Takizawa Department of Cell Biology • Structure of blood vessels

• Endothelium and structures that control permeability

• Increasing endothelial permeability and leukocyte transmigration

• Angiogenesis Blood vessels comprise three functional layers.

Most blood vessels consist of three layers. The outer layer (adventitia) is mostly connective tissue: collagen fibers, some elastic fibers. Larger vessels contain blood vessels within the adventitia. The middle layer (media) contains mix of smooth muscle and elastic fibers. This components of this layer vary the most. Elastic arteries such as the aorta contain more elastic fibers to generate stretch and recoil. Smaller muscular arteries contain more smooth muscle to control the distribution of blood. The inner layer is called the endothelium is composed of a simple squamous epithelium. This is functionally the most interesting and important. Blood vessels comprise three functional layers.

As one progresses through circulatory system, the walls of arteries become smaller. As vessels become smaller in diameter, size of connective tissue layer and smooth muscle decrease. Also, the function of walls changes: Large, elastic -> recoil and maintain pressure. Muscular -> control distribution of blood. Capillaries contain only endothelial cells with occasional support cell. Endothelium one continuous layer of cells in all vessels. Endothelia cells are polarized with one surface, lumenal, facing the lumen of blood vessels and the opposite surface, adluminal, facing the basement membrane. Endothelial cells perform a variety of structural and biochemical functions. • Permeability

• Immune response

• Angiogenesis

• Modulate vascular resistance

• Antihaemostatic and haemostatic control

• Enzymatic action on plasma proteins

Endothelial cells only cell to directly contact blood -> perform a variety of functions. 1. Regulate permeability of vessels -> control passage of proteins and small molecules. 2. Regulate immune response -> entry site into tissue for leukocytes. 3. Initiate growth of new blood vessels. 4. Detect and respond to changes in blood pressure and ﬂow -> signal to underlying smooth muscle. 5. Regulate clotting. 5.1. Inhibit platelet aggregation. 5.2. Secrete Von Willebrand part of clotting cascade. 6. Enzymes that modify plasma proteins. 6.1. Angiotensin converting enzyme -> increase blood pressure. Endothelial cells restrict the diffusion of protein between blood and tissues.

Protein Protein

One of the most important functions of the endothelial cells is to prevent diffusion of proteins from the blood into the surrounding tissue. In most of the circulatory system, endothelial cells form the only cellular layer that separates blood from the tissue. Blood is under hydrostatic pressure that pushes water out of the vessel. Blood vessels are leaky to most ions, so there is no difference in osmotic pressure between blood and surrounding tissue. To counter the hydrostatic pressure, blood retains a high concentration of protein to generate oncotic pressure that draws water into the vessel. Maintaining oncotic pressure requires keeping a high protein concentration within the vessel and thus endothelial cells must prevent diffusion of proteins out of the vessel. Loss of protein out of vessels would decrease oncotic pressure, allowing fluid to leak into surrounding tissue -> edema. Albumin generates ~ 70% of oncontic pressure. But, some proteins must exit the blood to reach the surrounding tissue. These could be antibodies or proteins that escort hormones or lipids. So endothelial cells need a mechanism to get specific proteins out of the blood. In addition, one response to infection is inflammation where fluid is leaked into surrounding tissue. Endothelial cells restrict the diffusion of protein between blood and tissues.

Protein Protein

One of the most important functions of the endothelial cells is to prevent diffusion of proteins from the blood into the surrounding tissue. In most of the circulatory system, endothelial cells form the only cellular layer that separates blood from the tissue. Blood is under hydrostatic pressure that pushes water out of the vessel. Blood vessels are leaky to most ions, so there is no difference in osmotic pressure between blood and surrounding tissue. To counter the hydrostatic pressure, blood retains a high concentration of protein to generate oncotic pressure that draws water into the vessel. Maintaining oncotic pressure requires keeping a high protein concentration within the vessel and thus endothelial cells must prevent diffusion of proteins out of the vessel. Loss of protein out of vessels would decrease oncotic pressure, allowing fluid to leak into surrounding tissue -> edema. Albumin generates ~ 70% of oncontic pressure. But, some proteins must exit the blood to reach the surrounding tissue. These could be antibodies or proteins that escort hormones or lipids. So endothelial cells need a mechanism to get specific proteins out of the blood. In addition, one response to infection is inflammation where fluid is leaked into surrounding tissue. Endothelial cells regulate passage of immune cells into surrounding tissue.

Not only must specific proteins exit the blood but immune cells must also cross the endothelium to enter the surrounding tissue to fight infections. Given the significantly larger size of cells compared to proteins, this would seem to be a near impossible challenge. Endothelial cells play two important roles in this process. First, they attract immune cells to the sites of infection. Second, they allow immune cells to pass across and enter the surrounding tissue. Structures that control endothelial permeability Small molecules and ions diffuse across endothelia but proteins are restricted.

Sugars s

Small Protein Large Protein

The permeability of endothelium displays two phases. The ﬁrst is a size-selective phase in which the permeability of molecules is directly related to their size. This holds for small molecules like sugars. Larger molecules, such as proteins, show permeability that is not size restrictive. That is, all molecules above a certain size ~ 2 nm are equally permeable. This includes most proteins. Some vessels highly restrictive even to small molecules -> brain. Primary components that regulate permeability of endothelia.

• Structure of endothelium

• Basement membrane

• Glycocalyx

• Intercellular junctions Continuous endothelium most restrictive to diffusion of protein.

There are four structural components that help control passage of molecules, proteins and cells out of the blood. The ﬁrst is the structure of the endothelial cell. This is an example of a continuous endothelial cell in which the plasma membrane completely encloses the lumen of the vessel. The only gap is where neighboring cells meet and these are sealed via junctional complexes. These are the most restrictive in regards to diffusion of molecules and proteins. Again note the thinness of the cytoplasm. Fenestrated endothelium contains small pores that facilitate diffusion and retain basement membrane.

A second type of endothelium, called fenestrated, contains small holes that allow diffusion of molecules and small proteins. These holes are about 25 nm in diameter that is large enough to accommodate even the largest of proteins. However, several structures limit diffusion of protein. One is the diaphragm across the hole and the other is the basement membrane. Found in regions where high exchange of material between tissue and vessels: intestine and kidney. The fenestrated endothelia facilitates diffusion of smaller molecules. Fenestrated endothelium contains small pores that facilitate diffusion and retain basement membrane.

Basement Membrane

Endothelial Cell

A second type of endothelium, called fenestrated, contains small holes that allow diffusion of molecules and small proteins. These holes are about 25 nm in diameter that is large enough to accommodate even the largest of proteins. However, several structures limit diffusion of protein. One is the diaphragm across the hole and the other is the basement membrane. Found in regions where high exchange of material between tissue and vessels: intestine and kidney. The fenestrated endothelia facilitates diffusion of smaller molecules. Discontinuous endothelium has larger pores and lacks basement membrane.

Lumen Endothelial Cell

The third type of endothelial cells is called discontinuous. Similar to fenestrated, discontinuous endothelia have gaps, but these gaps tend to be larger than in fenestrated endothelia and lack the diaphragm across the gaps that is seen in fenestrated endothelia. Another critical difference is that discontinuous endothelial lack a basement membrane. Discontinuous endothelia are found primarily in the liver and spleen where large macromolecules must easily cross the endothelium. Basement membrane is a negatively-charged barrier on the basal surface of endothelial cells.

Similar to other epithelia, endothelial cells rest on a basement membrane. The basement membrane of endothelium performs similar functions as the basement membrane of other epithelium: structural support. Endothelial cells attach to the basement membrane via integrins. In addition, the basement membrane also inhibits diffusion of protein because it is strongly negatively charged. The basement membrane contains a lot of proteoglycans and the sugar groups on the proteins are all negatively charged. Albumin is negatively charged. The thickness of the basement membrane varies. Capillaries in kidney thick basement membrane to prevent loss of protein into urine. Glycocalyx restricts diffusion of large, negatively charged molecules.

A more recently discovered component of the permeability barrier is glycocalyx. Glycocalyx forms a brush like structure on the apical surface of endothelial cells. Glycocalyx can extend several hundred nanometers into the lumen of the vessels. Glycocalyx was ﬁrst thought to primarily prevent red blood cells from sticking to the surface of endothelium. Recent work has shown that glycocalyx is a critical component that prevents diffusion of protein across endothelium. Enzymatic removal of glycocalyx leads to edema in several tissues Glycocalyx is composed of a proteoglycans and GAGs. which gives glycocalyx a negative charge. The measure space between proteoglycans is 20 nm suggesting that glycocalyx functions in part like a sieve to restrict passage of larger proteins and macromolecules. Junctions between cells is the only site for diffusion across continuous endothelium.

The ﬁnal structure that restricts diffusion of molecules across the endothelium is the junctional complex between endothelial cells. Where neighboring endothelial cells meet the junctional complexes prevent paracellular diffusion of material. Junctional complexes restrict paracellular diffusion between endothelial cells.

The junctional complexes are similar to those of other epithelial cells. They contain tight junctions and adherins junctions. The tight junctions contain claudins that restrict the passage of small molecules and ions. The type of claudin expressed determines the restrictiveness of the junction. Most endothelial cells allow passage of ions and small molecules but endothelial cells in the brain express claudin 5 that make that endothelium extremely restrictive to passage of small molecules. Endothelial cells are also held together by adherins junctions. These interactions are mediated by a special type of cadherin called VE-cadherin by function similar to cadherins in other epithelia. Cadherins critical for preventing ﬂuid loss from vessels. Injecting antibodies against extracellular domain of cadherins disrupts connections between endothelial cells leading to edema. Transcytosis of protein across endothelial cells

Although endothelia usually restrict the difusion of protein, there are times when they must allow protein to move from blood into surrounding tissue. For example, endothelia are highly restrictive to passage of albumin to maintain oncotic pressure, but albumin is also a carrier for hormones and lipids must pass across endothelia to make available those hormones and lipids to cells. How does endothelia allow one type of albumin to pass while restricting the other. Transcytosis mediates movement of proteins across endothelial cells.

This image reveals the process of transcytosis. The black dots represent labeled albumin that was injected into the blood vessel. The albumin is being endocytosed from the lumen and transported across the cytoplasm of the endothelial cell and then released on the adluminal side of the endothelial cell. The inset image shows the junction between two endothelial cells and illustrates how the tight junction prevents diffusion of albumin. Note how the albumin accumulates in the endocytic vesicles. Importantly, notice that the albumin is stuck on the surface of the membrane in the endocytic vesicle, but internally the albumin is released from the membrane. Receptor-mediated endocytosis transports speciﬁc proteins across endothelium.

Albumin is passed across endothelial cells via receptor-mediated endocytosis. Mentioned that maintaining albumin in blood is important for oncotic pressure, so why does endothelium transport albumin from blood into surrounding tissue? Albumin is a carrier protein for several important lipids and steroid hormones, so endothelium must transport albumin conjugated to lipids into tissues. To accomplish this, endothelial cells express a receptor that binds more tightly to albumin-lipid than unbound albumin. Unlike endocytosis in other cells that utilize clathrin, endothelial cells employ a membrane proteins called caveolin. Caveolin clusters the receptors and triggers endocytosis. These vesicles are called caveolae but are similar to endosomes. Within the caveolae the afﬁnity between the receptor and albumin is reduced, possibly due to lowering of pH, releasing the albumin. The caveolae fuse with the plasma membrane on the adluminal side, releasing the albumin into the surrounding tissue. Endothelial express several different receptors for proteins and macromolecules in blood and all use the same mechanism to transport material across endothelial cells. Receptor-mediated endocytosis transports speciﬁc proteins across endothelium.

Occasionally, endothelial cells loosen junctions to allow ﬂuid and protein to leak into surrounding tissue. Inﬂammatory molecules and immune cells activate signaling pathways that disrupt junctions.

Disrupt Junctions

Modify Kinases Cadherins

Increase Myosin Tension

Inﬂammatory agents bind receptors on surface of endothelial cells, triggering signaling cascades that activate kinases or increase intracellular calcium. Kinases phosphorylate cadherins to reduce the strength of the intercellular junctions. Calcium activates myosin by activating myosin light chain kinase (MLCK) which phosphorylates myosin light chain. Active myosin increases intracellular tension, generating gaps between neighboring endothelial cells. Cytoplasmic domain of VE-cadherin interacts with proteins that stabilize adhesion junctions.

Endocytosis Endocytosis

Similar to adhesion junctions in other epithelial, the interaction between cadherins is stabilized by association with actin filaments. Interactions between individual cadherins is weak and strong cell adhesion is generated through the interaction of multiple cadherin molecules confined to one region of the cell membrane. Attachment to actin filaments limits the diffusion of cadherins and increases overall affinity of interaction between cadherins in neighboring cells. The link between cadherins and actin filaments is mediated by beta and alpha catenin. The interaction between cadherin and p120 is also important for the adhesion between adjacent endothelial cells. p120 masks a dileucine motif that when exposed, triggers endocytosis of VE-cadherin. Phosphorylation of cadherins and catenins disrupts adhesion junctions.

Disruption of cadherins mediated by phosphorylation. Phosphorylation of beta-catenin causes it to dissociate from VE-cadherins. This disrupts the interaction between cadherins and actin ﬁlaments. The increased mobility of cadherins leads to weaker intercellular interactions. The cytosolic domain of VE-cadherins is also a target for kinases. Phosphorylation of VE-cadherins dissociates p120, exposing an endocytosis motif. In addition, phosphorylation recruits beta-arrestin which stimulates endocytosis. Endocytosis of cadherin reduces the amount on the cell membrane and decreases the strength of cell adhesion. Activation of myosin ﬁlaments increases tension of adhesion junctions.

A second mechanism to disrupt cadherins interactions is by increased cellular tension via actin and myosin ﬁlaments. Actin and myosin ﬁlaments assemble at junctional complexes. Activation of myosin generates tension on junctional complexes. Leads to separation between neighboring cells. Thrombin increase vascular permeability through tension on adhesion junctions.

A number of agents increase the permeability of endothelium, including thrombin, bradykinin and histamine. The images on the left show the results of treating endothelial cells with thrombin. The red is stain for junctional protein on the plasma membrane. Note how the stain is continuous around the endothelial cells and there are no gaps between cells. 5 minutes after thrombin, small gaps are observed in the junctional complexes. At 15 minutes, spaces are seen between cells. The results showed that addition of thrombin increased the tension within cells. The tension could be relieved by adding agents that depolymerize actin ﬁlaments, suggesting that tension was generated by an actin dependent process. Leukocyte transmigration across endothelium Leukocyte transmigration consist of three distinct steps.

Rolling Adhesion Emigration

Timothy Springer Lab, Harvard Medical School

Not only must endothelial cells allow passage of proteins but remarkably also whole cells. These are usually immune cells that need to enter a tissue to ﬁght infection. How do immune cells cross endothelium that is usually so restrictive to small molecules? Immune cells cross endothelium in three observable steps. First is rolling where immune cells roll along the surface of endothelial cells whereas red blood cells go ﬂying by. The second is adhesion where immune cells stick to the surface of endothelial cells and don’t move. Finally, immune cells are seen to squeeze across endothelium to enter the surrounding tissue. Selectins and integrins mediate rolling and adhesion of immune cells.

Selectins Integrins

Capture Rolling Arrest Transmigration

The ﬁrst step is mediated by a set of proteins called selectins. Endothelial cells express selectins on their plasma membrane in response to infection. The selectins on endothelial cells interact with selectins on immune cells to capture the immune cell to the surface of the endothelium. The interaction between selectins is weak so that immune cells don’t stay in one place but are pushed along the surface of the endothelium by the ﬂow of blood. Next, chemokines and chemotractants released by cells in tissue in response to infection trigger the expression of integrins on the surface of immune cells. These integrins bind proteins on the surface of endothelial cells. The interaction mediated by integrins is much stronger and locks the immune cell to the surface of endothelium. Finally, the immune cell squeezes between endothelial cells to enter the surrounding tissue. Leukocytes disrupt adhesion junctions to migrate across endothelium.

How do immune cells squeeze between endothelial cells? They appear to activate myosin ﬁlaments within endothelial cells that generates contraction and weakening of the junctional complexes. This separation between neighboring endothelial cells allows immune cells to insert extensions of the plasma membrane between the cells. The immune cell expresses on its surface proteins that bind to the receptors that make up the junctional complexes of endothelial cells. These interactions likely guide the immune cell across the endothelial cell. After passing across the endothelial cells, the immune cells must still penetrate the basement membrane. Immune cells express a variety of proteases that degrade the structural components of the basement membrane and allow it to pass into the tissue. Angiogenesis Low oxygen and wounding initiate growth of new blood vessels.

Hypoxia triggers growth of new vessels. VEGF triggers morphological changes in endothelial tip cells.

High O2 Low O2

Endothelial cells are also responsible for initiating growth of new vessels. In response to low oxygen or wounding, cells within a tissue express several proteins, the most prominent being vascular-endothelial growth factor (VEGF), that trigger growth of new vessels. VEGF causes certain endothelial cells, called tip cells, to undergo several radical changes. First, the tip cells reverse polarity, so whereas the apical surface normally faces the lumen, VEGF causes the apical surface to face the surrounding tissue. Second, the junctional complexes between tip cells and neighboring endothelial cells are chnaged. Finally, the tip cell invades the surrounding tissue by extending ﬁlopodia. Only tip cells respond to VEGF because if all cells responded, the entire vessels would degenerate. Tip cells prevent neighboring endothelial cells from responding to VEGF. Tip cells follow guidance cues to connect to existing vessels.

Semaphorins

The crawling of the tip cell is guided by a set of signaling molecules called semaphorins that are expressed by the surrounding tissue. Semaphorins also guide the growth of axon during neural development. Following the tip cell are specialized endothelial cells called stalk cells that can divide to produce more endothelial cells and have the ability to form the lumen of the new vessel behind the tip cell. Eventually the tip cell will reach an existing vessels and integrate into the vessel to form a conduit between vessels. Tumor cells initiate angiogenesis to increase their blood supply.

VEGF VEGF

Tumor cells Stromal cells

VEGF also appears to play an important role in the growth of tumors. Due to high cell mass, tumors are hypoxic environments. Tumors activate production of VEGF to recruit blood vessels to the tumor. Anti-VEGF antibodies (Avastin) are a treatment to slow growth of tumors. Vascular repair and smooth muscle Smooth muscle cells perform contractile and synthesis functions in vessel walls.

The major function of smooth muscle in media is to control the tone of vessels and regulate the diameter of vessels. The media also contains connective tissue: collagen, elastic fibers. However, the media lacks fibroblasts, so smooth muscle synthesizes the protein components of the media. The media likely contains two populations of smooth muscle cells: contractile and fibroblast-like. Smooth muscle cells progenitors recruited to intima after damage.

Damage

Damage to endothelial layer stimulates recruitment of smooth muscle cell progenitors. These cells synthesize connective tissue molecules that help repair wound. These progenitors come from the adventia and media but also the blood. The progenitors are capable of dividing and one consequence is proliferation of smooth muscle in the intimal layer which causes bulging of intima and occlusion of the vessel. Smooth muscle proliferation can lead to occlusion of arteries after insertion of stents.

Angioplasty is one common treatment for occluded arteries. Stents are inserted to keep the artery open. The procedure, however, damages the endothelial layer of the vessel and often trigger proliferation of smooth muscle cells in the intima. Over time the growth of smooth muscle cells occludes the artery despite the presence of the stent. To prevent the growth of smooth muscle cells, stents were designed to release chemicals that inhibit the proliferation of smooth muscle cells. Take home points…

• Blood vessels consist of three structural and functional layers

• Permeability of endothelium is determine by type of endothelium, basement membrane, glycocalyx and intercellular junctions

• Transcytosis mediates transport of protein across endothelium

• Immune cells loosen junctions to increase permeability and mediate transmigration

• VEGF promotes growth of new blood vessels through activation of endothelial cells