Histology, Blood Vascular System

Issues of Concern

Pericytes are fundamental cells for the health, formation, and function of blood vessels. The latter was discovered in the 19th century by Rouget, but Zimmerman renamed them pericytes by Zimmermann. Depending on their location, they take on different roles and importance; in the brain and retina, they become fundamental to build the blood-retinal barrier and the blood-brain barrier. In the kidneys, they influence the functionality of the glomeruli for filtration; in the liver vessels, they are essential for the remodeling of the extracellular matrix. Recent evidence demonstrates their role in vessel immune response and phagocytic activity. The blood vessel must always be considered different, depending on the anatomical area and the demand of the tissue for the same blood vessel.

Structure

The structure of the vessels of the vascular system is highly specialized to suit their individual functions and anatomic relationships. These differential changes appear in three shared layers of the vessel wall.

The tunica externa, or tunica adventitia, is the outermost layer of the vessel wall. Responsible for vessel integrity and resisting physical wall strain. The externa houses the vasa vasorum and the nevi vasorum, which are vessels and nerves, respectively that supply the cells of the wall of the vessel itself. These colloquially called the “vessels of the vessel” or “nerves of the vessel.” Collagen is fundamental in the externa and facilitates anchoring of the vessel to nearby structures and tissue. 

The tunica media is the middle layer of the vessel wall. The tunica media is generally thicker on the arterial side of the vascular system and contains transversely arranged smooth muscle cells that are capable of changing the caliber of the vessel lumen. The media’s thickness varies widely between vessel types, being quite thick in some arteries to virtually non-functioning in some veins. The tunica media also contains elastic lamellae and is enveloped in the external elastic membrane, a layer of elastic fibers that separates the media from the externa.

The tunica intima is the innermost layer of the vessel wall that has exposure to, and interacts with, the contents of the lumen. The tunica intima is primarily defined by a single layer of simple squamous endothelial cells that is the innermost layer of any vessel. The endothelium gets supported by a basal lamina, also known as the extracellular matrix. Sometimes the tunica intima is surrounded by the subendothelial layer, which can be composed of smooth muscle, collagen, and elastic fibers and can vary in thickness. The tunica interna gets enveloped in the internal elastic membrane, a layer of elastic fibers that separates the interna from the media. It is thickest in the arterial system, specifically the muscular arteries and thin throughout the entire venous system.[1]

While these three common layers are present across the many different vessels of the vascular system, they have differential accentuations or reductions to their tunics to accommodate their role in the vascular system; this is so-called segmental differentiation. The structure of the gross vascular system begins on the arterial system at the outlets of the heart. Blood then flows proximally to distally stepwise through these vessels:

Large arteries, also known as elastic arteries, examples of large arteries include the aorta and the pulmonary arteries. The tunica intima of the large arteries is quite thick with predominant smooth muscle in the subendothelial layer of the tunica intima. Smooth muscle is the majority cell type and is responsible for the secretion of ground substance, housing macrophages, and caliber changes. The adventitia of the large arteries is comparatively thin. Generally, it constitutes less than half of the total vessel wall width. The adventitia houses both the intrinsic vessels (vasa vasorum) and nerves (nervi vasorum) that supply the vessel itself. The vasa vasorum allows oxygen, nutrients, and waste to move from the lumen to the cells of the vessel wall. The flow of the vasa vasorum progresses into a parallel network on the large veins that follow large arteries.

Muscular arteries, also known as medium arteries, are classically defined by a thick layer of transverse or spiraling smooth muscle in the tunica media. The subendothelial layer of muscular arteries is so thin it can be hard to visualize on histology, which leads to the appearance of the media abutting the endothelial lining

Small arteries and arterioles contain one or two layers of smooth muscle cells, which are essential in increasing peripheral resistance. The external elastic membrane is absent in arterioles. Arterioles flow into capillaries.

Capillaries have the smallest vessel diameter, which ranges from 4 to 10 microns. Capillaries have the most reduced vessel wall, comprised of endothelium surrounded by connective tissue. There is a basement membrane that houses pericytes. Pericytes are essential neurovascular signaling cells and respond to blood pressure changes. Capillaries are also highly specialized vessels, with different tunica features dependent on anatomic relationships and function. There are three types of capillaries with highly specialized functions. They are anatomically isolated to respective regions of the body.[2]

The first type of capillary is continuous capillaries. They have an uninterrupted endothelium and a continuous basal lamina. Continuous capillaries perform transcytosis that brings large molecules from the lumen toward the surrounding connective tissue and vice versa. They are present in all three muscle types, connective tissue, skin, lung, and central nervous system.

The second type of capillary is fenestrated capillaries. They have circular fenestrations in the capillary wall with a continuous basal lamina. Found in endocrine organs and locations of metabolic absorption. These include the kidney, pancreas, intestinal tract.

The third type of capillary is discontinuous capillaries. Discontinuous capillaries have a disrupted endothelium that is defined by large gaps in between cells, as well as large gaps in the basal lamina. Discontinuous Capillaries are present in the liver, the spleen, and the bone marrow — all sites of blood formation at one time or another.

Blood then begins its return to the heart through the venous system. It flows distally to proximally from begging by flowing from capillaries into Venules. Venules have a diameter of up to 0.1 mm and are directly continuous with the capillary bed. Despite their direct connection, veins contain a more sparse subendothelial elastic fibers compared to the proximal capillary. Venules also contain pericytes in the basement membrane, which work in cell signaling. As these venules start to develop an appreciable tunica media further upstream and become known as ‘muscular venules.’ The beginning of a tunica externa is also present in muscular venules in some instances. 

The flow then flows into small veins. Small veins have a diameter of 0.1mm to 1mm; this is the first class of veins that contain all three tunics that are appreciable on histology. Pericytes are usually no longer observed at this point. 

The next vessel in the venous network is medium veins. They have a diameter as great as 1 cm and constitute many of the known ‘named’ veins. The presence of valves classically defines medium veins. These one-way valves are particularly predominant in the lower extremities and block retrograde flow. Medium veins have a much thinner tunica media than is seen in parallel arteries. The adventitia is the thickest layer in medium veins and contains collagen and elastin fibers.

Finally, blood flow directs into one last vessel before arriving back to the heart, the Large Veins. Large veins have a diameter of over 1 cm and include prominent structures like the portal vein, inferior vena cava, and superior vena cava. Large veins lack a clear demarcation between the tunica intima and tunica media with no obvious internal elastic membrane, and like medium veins, they contain a thick tunica externa. The externa also contains longitudinal smooth muscle. However, these general guidelines for the tunics in veins. The distinct layers are not as clearly appreciated as in arteries and highly variable in different persons.[2][3][1]

Function

The function of any given vessel depends on its structure and anatomic locations. There are general functions observed in both arteries and veins.

Arteries are responsible for the perfusion of every tissue in the body, and Veins work to return blood and waste from tissue to the heart and excretory organs, respectively. The presence of one-way valves allows veins to pump blood against gravity despite their relative lack of ability to contract. The lack of smooth muscle in veins is also what allows them to hold around 70% of the total blood volume. This is the degree of distensibility known as high compliance.

In the vascular system, arterioles are so-called “resistance vessels” and essential in the production of peripheral resistance. By contracting one or two layers of smooth muscle in the tunica media, the large arteriole system can generate tremendous resistance in an expansive parallel network. This function at baseline is important in defending capillaries and venules against large pressure flows.

To understand the functional aspect of capillaries, you must first consider how massive the parallel network of capillaries is. Fenestrated capillaries, found in endocrine organs, the GI tract, and kidneys, are an important capillary subtype that performs filtration of luminal contents. Their filtration is selective and does not allow free blood flow like discontinuous capillaries. Pinocytotic vessels present in the endothelial cells allow for the uptake of luminal contents. The fenestrations and pinocytotic vessels can be appreciated on an electron microscope only. Discontinuous capillaries have large, irregularly spaced gaps that function for blood and large protein uptake in the liver, spleen, and bone marrow. This type of large molecule transport is not possible in other capillaries.[4][3]

Housed in the basement membrane of the smallest vessels in the body, both capillaries and venules are pericytes. Pericytes, also known as Rouget cells, are circumferentially arranged inside the microvascular wall and communicate via both direct contact to the endothelial cells and paracrine signaling. They are essential in regulating the maturation of the endothelial cells and are crucial components of the neurovascular unit. Pericytes aid in maintaining microvascular blood flow, clearing cellular debris, and maintaining the integrity of the blood-brain barrier.

The blood-brain barrier is another unique function of the vascular system. It insulates the parenchyma from increased edema in its confined space while also limiting chemical traffic. This strict barrier is due to the tight junctions present in endothelial cells as well as a denser basement membrane.[5][6]

Tissue Preparation

Histological visualization of blood vessels is most commonly with routine hematoxylin and eosin (H&E) staining. The vessel specimen gets fixed with a formalin solution for 24 to 48 hours, which preserves tissue structure. It then gets embedded in paraffin wax for easier sectioning. This process first involves dehydrating the specimen with alcohol changes, and further removing the alcohol with xylene. After dehydration, the specimen gets placed in a paraffin mold with enough paraffin wax to anchor the section. Once the specimen gets anchored in the paraffin, a cassette top is placed over the mold and filled with paraffin, which rapidly cools. Next, the vessel specimen is processed by a rotary microtome sectioning of the vessel into very thin slices for visualization. The sectioning is crucial in allowing light from the microscope to pass through. After the specimen is fixed and sectioned, it is de-waxed by rinsing through progressive changes of alcohol. The specimen is then rinsed in water to hydrate the cells for penetration by reagents. The specimen then gets stained with hematoxylin combined with the mordant aluminum salt in solution. At this point, the nuclei of the tunics will be stained a red color. The specimen is then rinsed in water, a process known as “bluing.” By exposure to a weakly alkaline solution, the hematoxylin converts to a dark blue color. The specimen gets checked to see if the nuclei have taken up the proper stain and show contrast. The tissue is then de-stained with weak acid alcohol and again rinsed with water for further “bluing.” The specimen then gets counterstained with either aqueous or alcoholic eosin Y, which shades non-nuclear elements pink. The specimen passes through several changes of alcohol to dehydrate it. This alcohol must be removed again via rinsing in xylene in which it is miscible. After the xylene rinse, the result is a transparent slide ready for light microscopy.[7]

Verhoeff stain is an option to visualize elastic tissue in the vessel wall. This process follows the same basic principles outlined above but differs as to which stains get used. Instead, the staining uses hematoxylin, iron (III) chloride, and iodine. The specimen is then counterstained with picric acid and fuschsin to stain collagen for contrast against the hematoxylin stained elastin.[8]

To prepare a large section of the vessel, the alternative method of preparing vessels is the en face preparation allows for a wide area of blood vessel surface visualization. This process is to analyze large vessels like the aorta and to visualize the organization of the endothelial cells that line the lumen. Using en face preparation, one can pinpoint specific locations of lesions and commonly combines with immunofluorescent staining.[9]

Histochemistry and Cytochemistry

CD31 is a platelet endothelial adhesion molecule that is used in immunofluorescence to identify endothelial cells on microscopy. This process is crucial in evaluating the degree of tumor angiogenesis, which may reflect the rapidity of growth. CD31 is also used to identify angiomas and angiosarcomas, two blood vessel tumors that maintain the adhesion molecule.[10]

Microscopy, Light

The structural organization of the vessel wall is easily appreciated with a light microscope, being observed most clearly in large vessels. The tunica intima appears as a single layer of simple squamous cells on light microscopy. The internal elastic membrane which surrounds the intima presents as a dark basophilic line that undulates close to the lumen. The tunica media is easily appreciated in some vessels as the cytoplasm of the abundant myofiber stains pink, while the nucleus stains blue and normally appears long and stretched to a prolated spheroid.[11] As expected, the smooth muscle lacks the arrangement of the actin and myosin, and there is no observable striations. The tunica externa is visible just outside the deeply basophilic staining external elastic membrane. The externa is easily visible in large vessels, like the inferior vena cava, where it is the thickest layer. Wavy bundles of eosinophilic collagen fibers are seen with scattered smooth muscle cells and nuclei interspersed.

Microscopy, Electron

On electron microscopy, it is possible to visualize vessel features that are unobservable by other means. Endothelial cells, which are difficult to visualize at lower magnifications, can be readily seen on an electron microscope as can their morphological changes to mechanical stress. These morphological change of the endothelial cells is known to occur as a response to shear stress. As the shear stress on a vessel increases, its length increases, and its width decreases.[2] The differential endothelial features of the capillary subtypes, continuous, fenestrated, and discontinuous is appreciated only on the electron microscope. At the high magnification of the electron microscope, pericytes, also become visible. These small cells are seen in capillaries and venules and are important in regulating endothelial maturation and neuroendocrine unit signaling.[12][13]

Pathophysiology

The vast network of blood vessels is involved either directly or indirectly in many pathological disease states. The most common of which is atherosclerotic disease. This pathology begins with low-density lipoprotein (LDL) being taken up by the endothelial cells of vessels. As LDL begins to accumulate in the intima, macrophages work to digest the oxidized it. When the LDL is present in the intima in excessive amounts, the macrophages become overwhelmed and turn into foam cells. A ‘Fatty streak,’ composed of these foam cells, begins to form in the subendothelial space between within the tunica intima. The smooth muscle cells in the tunica media being to proliferate due to inflammatory cytokine signaling and secrete fibrous cap that entombs to fatty streak. The risk of such changes to the vascular wall includes thrombotic events, embolic events, and restriction of flow, which progresses in severe cases to occlusion of flow. In some instances, these fatty streaks can be so impressive that they are visible with the naked eye on a gross specimen.[14][15]

Cytokine response to stress or inflammation will cause separation of the endothelial lining of the vessel, which allows fluid extravasation into the surrounding tissue. These cytokines induce endothelial cell retraction, allowing otherwise impermeable large molecules, to pour out into the surrounding extravascular space. Similar changes within this functional array of hyperpermeability, angiogenic growth, and endothelial proliferation carries implications in angiomas and angiosarcomas.[16]

Diabetic neuropathy is thought to be secondary to sclerotic changes and increased resistance in the small blood vessels that supply the peripheral nerves, the nervi vasorum.[17] 

On histology, myocardial fibers are sometimes visible, extending into the pulmonary veins and vena cava walls. These continuations of myocardial tissue into the vessel wall are what are known as myocardial sleeves and are possible foci for the generation of atrial fibrillation.[18]

Clinical Significance

The vasculature system is associated with a broad spectrum of diseases due to its critical function and vast network throughout the body.

1. Atherosclerosis and fatty plaque deposition in vessel walls.

2. Arteriolosclerosis which involves hardening and the loss of the intrinsic elasticity of vessels. This condition most commonly relates to hypertension and diabetes mellitus.

3. Vasculitides which involves damage of blood vessels by the immune system

4. Connective tissue disorders, such as scleroderma which can affect the vessels.

Preliminary studies on the human model indicate that mobilized peripheral blood stem cells can become a treatment to treat lymphedema; further studies are necessary to endorse this therapeutic practice.[19]

During fetal growth, if the vessels that make up the placenta show alterations of vascular endothelial growth factor or VEGF, Angiopoietin/Tie2 signaling by the pericytes, they could cause serious placental diseases (pre-eclampsia). [20]

Thrombotic microangiopathy is a pathological clinical condition characterized by an alteration of the vascular wall that causes the formation of multiple small thrombi in the microcirculation, with functional disorders of the various organs and microangiopathic hemolytic anemia. Historically it is classified in primitive forms (such as thrombotic thrombocytopenic purpura and uremic-hemolytic syndrome) and secondary forms (from malignant hypertension, scleroderma, etc.), the latter much more rare, less severe and for which the therapy appears slightly different from primary forms. The disease is relatively rare, with an incidence of about six cases per million inhabitants per year. However, it remains a crucial diagnosis because the mortality of untreated patients is around 90%, mortality that is reduceable with the prompt execution of life-saving therapy (plasmapheresis). Early death occurs despite everything within 24 hours of presenting the disease, especially in women.[21]

Takayasu arteritis is an inflammatory pathology that affects the aorta, its branches, and pulmonary arteries. It occurs mainly in young women. Etiology is unknown. Vascular inflammation can cause arterial stenosis, occlusions, dilations, or aneurysms. Patients may have an asymmetric pulse or unequal blood pressure measurements between limbs, limb claudication, symptoms of decreased brain perfusion and hypertension or its complications. Diagnosis is via aortic arteriography or MRI angiography. Treatment involves the use of corticosteroids and other immunosuppressants, and in case of ischemia potentially dangerous for the organs, vascular interventions such as the by-pass.[22]

Familial hemorrhagic telangiectasia or Osler-Weber-Rendu syndrome is a condition characterized by malformations of the junctions between arteries and veins, which occur directly and without the "mediation" of the capillaries. The most common clinical manifestation is represented by spontaneous and recurrent bleeding of the nose, around 12 years of age. About 25% of patients also have bleeding from the gastrointestinal tract, typically from the age of 40. Sudden bleeding can also occur in various districts (e.g., brain or lungs), with serious consequences.[23]

Soft tissue sarcomas are rare tumors of which at least 50 subtypes are known. In adults, malignant cells are formed within the so-called soft tissues of the body and can be localized in practically all body areas. These tissues are muscles, connective tissues, blood or lymphatic vessels, nerves, ligaments, and adipose tissue. Only the biopsy allows you to find out which particular type of sarcoma it is and from which tissue it originated.