Venous hypertension plays an important role in the development of microangiopathy



by M. das Graças Coelho de Souza,
C. E. Virgini-Magalhães, B. Senra Barros,
C. L. Lascasas Porto, E. Bouskela, Brazil

Eliete BOUSKELA, MD, PhD

Eliete BOUSKELA1, MD, PhD
Maria DAS GRAÇAS COELHO DE SOUZA1, MD, PhD
Carlos Eduardo VIRGINIMAGALHÃES1,2, MD, PhD
Bernardo SENRA BARROS1,2, MD, PhD
Carmen Lucia LASCASAS PORTO1,3, MD, PhD
1 Laboratory for Clinical and Experimental Research on Vascular Biology
2 Discipline of Vascular Surgery Department of Surgery
3 Discipline of Angiology Department of Internal Medicine Biomedical Center State University of Rio de Janeiro Rio de Janeiro, BRAZIL

Venous hemodynamic changes leading to venous hypertension are important in the development of microangiopathy. To understand microangiopathy in chronic venous disease (CVD), one needs to know about microcirculation: how it functions and how it can be studied. Here we discuss the concept of the microcirculatory unit, which is composed of small arteries and arterioles, capillaries, venules, and lymphatics. Small arteries and arterioles are responsible for the control of blood flow to organs and tissues via the modulation of contraction or relaxation of the vascular wall. Capillaries, the smallest vessels of the cardiovascular system, vary in number according to the metabolic activity of the tissue. Venules are capacitance vessels with well-developed elastic walls. Lymphatics are histologically similar to veins and responsible for lymph transport. Analysis of microcirculatory changes in CVD is challenging because of the lack of practical tools. The orthogonal polarization spectral (OPS) imaging technique is fifteen years old and seems to be suitable to study these patients. The equipment (Cytoscan or similar) has a small handheld probe that can be noninvasively applied to the internal perimalleolar region. Using OPS imaging and comparing CVD patients to healthy controls, we found that capillary morphology (percentage of abnormal capillaries per field) and capillary diameter were significantly different from C2 onwards. The largest diameter of the capillary bulk and of the dermal papilla also increased with progression of CVD and was significantly different from C3 to C5. Functional capillary density (number of capillaries with flowing erythrocytes per unit of tissue area) decreased significantly from C4 to C5.

The main role of the microcirculation is to provide energy and nutrients to cells and to remove the waste products resulting from metabolism.1 The microcirculation represents the smallest functional unit of the cardiovascular system, where the interaction between blood and tissue creates the necessary environment for cellular function. The main components making up the microcirculatory unit (Figure 1, page 214) are arterioles, capillaries, venules, and lymphatic capillaries. Each component has a different and specialized function and behavior; however, in all of them, the endothelium has an important function, as under physiological conditions, it assures local tissue homeostasis. Capillaries are very thin structures with a wall consisting of one layer of endothelial cells. As a whole, the human body has approximately 10 billion capillaries, with a total estimated area of 500 to 700 m2 (around 1/8 of a football field). In fact, it is rare that any functional cell of our body is more than 20 to 30 mm away from a capillary. Capillary diameter ranges from 4 to 9 mm, which is just sufficient to allow the passage of water, electrolytes, and circulating blood cells. Capillaries can be referred to as arteriolar, middle, or venular, according to their proximity to arterioles or to venules.2

Figure 1. Schematic representation of the microcirculatory unit.

There is marked heterogeneity in microvascular endothelium, depending not only on the tissue but also on the organ and vascular segment, as well as vascular branching. Pronounced differences in the vascular wall structure may be observed. For example, in arterioles, the number of smooth muscle cells tends to decrease with the diminution of the arteriolar diameter. Capillary wall structure is different in that it consists of a single layer of endothelial cells that are superimposed on a basal membrane. Furthermore, several wellcontrolled studies of organ models have shown that venules can be several times more permeable to water than the arteriolar part of the capillary. Moreover, it is well-known that venules are more susceptible to inflammatory agonists, which can elicit a marked increase in water and macromolecular permeability.

Microcirculatory unit

• Arterioles
Small arteries and arterioles are mainly responsible for the control of blood flow to organs and tissues via variations in contraction and relaxation of the vascular wall. Changes in the degree of contraction of the circular smooth muscle layer of these small vessels allow the regulation of blood flow to tissues and control of mean arterial pressure. The diameter of resistance vessels (arterioles) is determined by intravascular pressure. In special situations, for example, when there is a generalized sympathetic discharge, the contractile activity of vascular smooth muscle cells in small arterioles could completely close the lumen. This phenomenon differs in magnitude in different regions, favoring the distribution of blood to organs such as the brain and heart. Arterioles are responsible for the biggest resistance to blood flow in the vascular system, and they play a fundamental role in the control of mean arterial pressure. Ten to one hundred capillaries originate from successive ramifications in each arteriole, and the mean arteriolar diameter ranges from 8 to 50 mm. In some tissues, it is possible to find metarterioles, intermediary between arterioles and venules, that could form a non-nutritional deviation of blood flow, from arterioles directly to venules.3

• Precapillary sphincter
The precapillary sphincter is where the last smooth muscle cell is located before the capillary itself. When metabolism in the tissue increases, for example, during physical exercise, a larger number of capillaries need to be perfused. Thus, in this case, precapillary sphincters would be predominantly open to allow blood to enter the capillaries.

• Capillaries
Capillaries are the smallest vessels of the cardiovascular system, and their number varies according to the metabolic activity of the tissue: where there is higher metabolism, there are more capillaries, and vice versa. On the other hand, they occupy the highest cross-sectional area of the vascular system with only 5% of the circulating blood; inside the capillaries, blood flows with a velocity of approximately 0.3 to 1.2 mm/s under resting conditions, but this could increase several times during physical exercise.

The capillary wall is only one-endothelial-cell thick, the capillary is very thin and relatively short, and the blood flows with a low velocity; thus, it is an ideal structure for exchange between blood and tissue. However, there are different types of capillaries, depending on the organ or tissue, and these are classified as continuous (brain), fenestrated (kidney), or sinusoid (liver) capillaries: (i) Continuous. These are present in skeletal muscle, lung, adipose and conjunctive tissues, and in the nervous system. They are made up of one to three endothelial cells forming a circumference supported by the basal membrane. (ii) Fenestrated. These are present in tissues such as kidney tubules and glomerulus, exocrine glands, and intestinal mucosa. They have holes roughly 50-60 mm in diameter between endothelial cells and are more permeable to water and small hydrophilic solutes than continuous capillaries. (iii) Sinusoid. These are present in the bone marrow, liver, and spleen, and have intercellular gaps of approximately 100 nm, making these organs permeable to plasma proteins.

Capillaries do not actively control their diameter, as there are no smooth muscle cells in the capillary wall; passive changes in diameter occur by alterations of pre- or postcapillary resistance.2 The thin capillary wall resists high internal pressures without disruption.

Capillary blood flow is normally nutritional, but it could also be non-nutritional. Nutritional flow occurs when there is an exchange of gases and solutes. In certain tissues, such as the skin, one can observe an arteriovenous functional deviation that could be morphological or physiological. The morphological deviation occurs when there is a direct connection between arterioles and venules, ie, there is no passage through capillaries. The physiological deviation is characterized by an increase in blood flow through open capillaries. In tissues that have metarterioles, the functional deviation occurs during low metabolic activity. When there is an increase in the metabolic activity, the precapillary vessels dilate and the blood passes through metarterioles and becomes available for capillary perfusion.

In spite of capillaries having been traditionally considered to be mainly responsible for tissue oxygenation, recent data suggest that their primary role is the extraction of catabolite products from the tissues. The functional capillary density (number of capillaries with flowing red blood cells per unit of tissue area) varies according to the metabolic needs of each tissue. In the brain and myocardium, we find higher functional capillary density than in the skeletal muscle. In these organs, oxygen consumption is high and constant.

• Venules
Venules are capacitance vessels with well-developed elastic walls. Vein compliance is around 24 times higher and its diameter 3 times bigger than the corresponding artery.

Figure 2. Leukocyte-endothelium interaction.

The role of venules is to collect blood from capillaries and bring it back to the heart. Returning to the heart, capillary blood passes through venules, then from venules to bigger veins that decrease in number and change in vascular wall composition. The cross-sectional area is reduced and the velocity of the blood passing through these bigger veins increases. Venules and veins are the reservoir of blood in the vascular system. Owing to their high compliance and low resistance, they can store 60% of the total blood volume. Between 15% and 30% of the circulating blood volume can be easily compensated by the adaptation capacity of these vascular components.

• Lymphatics
The network of lymphatic capillaries converges to transition into lymphatic vessels and later on, lymphatic trunks. Lymphatic vessels are histologically similar to veins: the lumen is formed by a layer of endothelial cells, and the thinner vessels are covered by a discontinuous layer of smooth muscle; this layer of smooth muscle becomes continuous in vessels closer to lymphatic trunks.

In lymphatic vessels, the pressure oscillates between 1 and 2 mm Hg, similar to what is observed in the adjacent subcutaneous tissue. Lymphatic smooth muscle cells can elevate this pressure to 5-10 mm Hg during their rhythmic contraction. This contraction is synchronized in segments between valves and tends to push the lymph toward the thoracic duct. Lymphatic vessels have valves that restrict the movement of the lymph that proceeds toward the thoracic duct. Some tissues do not have the lymphatic system, such as bone marrow and cartilage. In other tissues, such as the dermis and the genitourinary, respiratory, and gastrointestinal tracts, lymphatic vessels are numerous.

Proteins that eventually exit the vascular system through the microcirculation are removed from the interstitial space by lymphatic capillaries through the lymph, formed by the difference between capillary filtration and reabsorption. As a whole, 2 to 4 L of lymph is formed every day. Lymph composition is similar to blood plasma, except in the quantity of proteins, which may be half that found in plasma.

Endothelium-leukocyte interaction

Interactions between the endothelium and leukocytes, as well as increases in fluid and protein filtration, are restricted almost exclusively to postcapillary venules (mean internal diameter is between 9 and 16 mm). The nature and magnitude of these adhesion interactions between leukocytes and endothelial cells are determined by a variety of factors, including the expression of adhesion molecules on leukocytes and/or endothelial cells, products of leukocyte (superoxide, among others) and endothelial cell (nitric oxide, among others) activation, and physical forces originating from the movement of blood close to the vessel wall (Figure 2). Evidence pointing to leukocytes as mediators of tissue injury in different diseases is accumulating rapidly.

Microangiopathy in CVD

The exact role of the microcirculation in the physiopathology of chronic venous disease (CVD) is still not completely defined, and it has only recently been subjected to systematic investigation. 4 In spite of all the progress in genetics and molecular biology, the impact of these new tools is also small. It is still possible to encounter professionals that think that the physiopathology in patients with varicose veins in the lower limbs is due only to mechanical alterations caused by reflux and venous hypertension seen on vascular echography, even though CVD has long been considered an inflammatory pathology.

Figure 3. (Left panel) Microangiopathy observed in chronic venous disease and (right panel) measured microcirculatory parameters. Abbreviations: CD, capillary limb diameter; CM, capillary morphology; DCB, diameter of the capillary bulk; DDP, diameter of the dermal papilla.

Although clinical and experimental studies have yet to completely elucidate the physiopathology of CVD,5 it is well-accepted that venous hemodynamic alterations leading to venous hypertension play an important role in the development of the observed microangiopathy.6-8 Elevated ambulatory pressure manifests not only in the macrocirculation with the development of varicose veins, but also in the capillaries, causing chronic damage and, finally, disruption of the microcirculation. Cutaneous capillaries become progressively enlarged and tortuous, forming “true” skeins described in the literature as glomerulus-like capillaries9-11,12 (Figure 3). The endothelial cells themselves become enlarged, with bigger interendothelial pores making the capillary lumen irregular.11,13 These alterations provoke an increase in microvascular permeability, with extravasation of plasma, blood cells, and macromolecules, such as fibrinogen. In the interstitium, fibrinogen is activated, forming a barrier involving the capillaries and limiting the exchange of nutrients,14 though there is no consensus about this point.13,15 The persistence of venous stasis and hypertension results in chronic inflammation of the capillary bed and surrounding tissues16 and in edema.4 The reduction in the number of capillaries leads to trophic disorders and leg ulceration.17

Hemodynamic forces, such as venous hypertension, circulatory stasis, and alterations in shear stress (acting on the vascular wall as a result of the tangential force produced by blood flow), seem to have an important role in the activation of the inflammatory cascade, promoting adverse reactions in the vascular wall, venous valves, and skin.18,19 As a consequence of venous hypertension, there is extravasation of blood fluid from the vessels, mainly from postcapillary venules. Plasma extravasation is responsible for the increase in the lymphatic content and edema, as well as the increase in viscosity and in the amount of red-blood-cell aggregates, leading to a decrease in red blood cells in the microcirculation. 20

Alterations in shear stress as a result of abnormal blood flow lead to changes in morphology, function, and gene expression in endothelial cells.21 When blood flow is pulsatile or laminar, the shear stress is normal, and factors that reduce inflammation, thrombus formation, and free radicals—such as nitric oxide, tissue plasminogen activator (tPA), thrombomodulin, and prostacyclin (PGI2)—are actively liberated. On the other hand, if the shear stress is zero or very low—as a result of whirl or even reverse blood flow—free radicals and proinflammatory and prothrombotic (eg, plasminogen activator inhibitor [PAI]-1, von Willebrand factor, monocyte chemoattractant protein [MCP]-1, angiotensin II, and endothelin-1) mediators are liberated.19,20

CVD is also accompanied by an increase in leukocyte infiltration in the affected leg.22 Leukocytes infiltrate the microcirculation by being trapped in the capillaries or by adhering to venular endothelium.23 Trapping of neutrophils in the microcirculation reduces capillary perfusion, increases free radical formation, and induces the liberation of proteolytic enzymes. Adhesion of leukocytes in postcapillary venules or in bigger veins could be facilitated by the expression of selectins (P- and L-selectin), integrins, and members of the immunoglobulin superfamily, such as intercellular adhesion molecule (ICAM)-1.23

In addition to the acute inflammatory process, where granulocytes infiltrate the venular and venous walls, there is also an infiltration of T and B lymphocytes. Monocytes/macrophages also infiltrate the venous valves and could be involved in their destruction. There is evidence for the involvement of ICAM-1, vascular adhesion molecule (VCAM)-1, L-selectin, E-selectin, and integrins in this process.23

Activation of leukocytes is characterized by synthesis and liberation of several inflammatory mediators; these include leukotrienes, prostaglandins, bradykinin, free radicals, and cytokines, such as tumor necrosis factor (TNF)-a and interleukin (IL)-6, which regulate and perpetuate the inflammatory reaction by autocrine and paracrine mechanisms.23

CVD of the lower limbs is a common public health problem worldwide that has had a highly negative impact on quality of life due to leg ulceration, pain, and sick leave. CVD is a multifactorialdiseasewith many clinical presentations and is increasing worldwide. Although the CEAP (C for clinical evaluation, E for etiology, A for anatomic findings, and P for pathophysiology) classification was revised in 2004,24 with enhancement of the pathophysiologic analysis, we still lack a large microcirculatory study. Methods, such as laser Doppler fluxometry, videocapillaroscopy, plethysmography, and fluorescence videomicroscopy have been used to visualize the microcirculation (directly or indirectly); however, the latest technique is orthogonal polarization spectral (OPS) imaging. OPS is based on intravital microscopy with incident polarized light that produces reflected depolarized light from hemoglobin. Cytoscan implements the OPS technique and was described for the first time in 1999.25 Cytoscan and its followers, Microscan and Cytocam, can be used for noninvasive studies of all tissue surfaces without the use of fluorescent dyes, and OPS imaging has been validated in comparison with conventional videocapillaroscopy and intravital microscopy.26 Nowadays, it is possible to quantify microangiopathic changes related to CVD with this equipment. It is important to evaluate the perimalleolar area in these patients as it is a gaiter zone where stasis ulcers usually appear.4

Quantification of microangiopathy in CVD: our experience

According to the concept that venous microangiopathy resulting from venous hypertension is one of the first signals of CVD, quantification of microcirculatory parameters can be used to monitor disease severity.27,28 In our laboratory, we have investigated several microcirculatory parameters in patients with CVD. We found that morphological alterations of the microcirculation that are characteristic of this disease increase according to its evolution, but they are already present in the C2 class of disease. The method to observe the cutaneous microcirculation in patients with CVD through OPS imaging was also developed in our laboratory.29 Reproducibility of studied parameters showed <20% variability, which is acceptable due to normal expected variation of the measurements.29 Functional capillary density, the number of capillaries with flowing red blood cells per unit of tissue area (mm2), is similar for C1 and C2 patients, but starting with C3 there is a gradual reduction in the number of perfused cutaneous capillaries leading to capillary rarefaction in more advanced stages of the disease. On the other hand, capillary limb diameter (mm) and capillary morphology (the percentage of abnormal capillaries in the total number of capillaries observed in each field) followed the progression of the venous disease, being significantly different from healthy subjects already at C2. Diameters of the capillary bulk (mm)—measuring the size of the skein in which the hairpin (capillary found in healthy subjects) was transformed—and of the dermal papilla (mm, to quantify the onset of edema)—the smallest functional skin unit—also increased with the progression of the disease and were significantly different from healthy subjects from C3 to C5.29,30

Capillary morphology seems to be a good parameter for evaluation of CVD patients, because already at C2, it was significantly different from controls. In healthy subjects, only 3.6% of cutaneous capillaries in the lower limb showed morphologic alterations; in patients with CVD, one can observe a gradual substitution of hairpin capillaries with glomerulus-like ones (Figure 3).

The increaseddiameter of the capillary may represent a change in shear stress and consequently elicit endothelial cell activation. The diameter of the capillary bulk also increased gradually, according to the evolution of the disease. The dermal papilla tended to increase when the capillary inside it became tortuous. It is possible that the initial increase was due to changes in capillary-tissue exchange and the appearance of a preclinical edema.10 The decrease in functional capillary density can lead to tissue ischemia and, subsequently, cutaneous ulceration.

In conclusion, we have found that capillary morphology and capillary diameter differs significantly from those in healthy subjects from C2 onwards. Diameter of the capillary bulk and diameter of the dermal papilla also increases with disease progression and is significantly different from those in healthy subjects from C3 to C5. Functional capillary density decreases significantly compared with healthy subjects from C4 to C5.29 Thus, our experience supports the idea that quantification of microangiopathy in chronic venous disease could facilitate monitoring of disease severity.

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