Differential Regulation of Permeability in the Microvasculature

Having described the general mechanisms through which the cytoskeleton regulates endothelial permeability, we will now outline specific characteristics intrinsic to microvascu-lar barrier function. Although the endothelium was initially regarded as relatively homogeneous throughout the vascula-ture, important phenotypic and functional differences are now appreciated at various sites along the vascular tree. These differences account for the tremendous physiologic variability observed in vascular function along various EC segments in different organ systems. Studies of endothelial heterogeneity typically divide ECs into two primary groups, macrovascular cells from large conduit vessels and microvascular cells from small arterioles, venules, and capillaries.

Isolation and culture of the microvasculature pose a particular challenge, in large part because it is not possible to cannulate the microvasculature. Isolation of microvascular ECs requires physical dissociation of ECs via homogenizing, mincing, or cutting the tissue into 3- to 5-mm3 pieces before incubation with proteolytic enzymes to release the microvascular ECs. However, these techniques often yield other cell types in addition to microvascular ECs (most frequently fibroblasts); therefore, purification is a key step in the isolation of microvascular ECs. Visual identification of cells based on morphologic characteristics followed by manual separation techniques was an early method of purification but is labor intensive and time consuming. Microvas-cular ECs can also be removed with specially coated glass beads that specifically adhere to ECs when added to the culture and then are removed using a magnet. Other techniques include fluorescence activated cell sorting (FACS) and gradient centrifugation to separate cells differentially by weight. Regardless of the technique employed, the possibility that other cell types are present in microvascular EC cultures needs to be considered when evaluating experimental results.

Multiple factors contribute to the differential barrier properties of microvascular and macrovascular EC (Figure 2), including cell-specific attributes such as surface protein expression, cell morphology, and permeability. Morphologic differences include a fourfold larger surface area for microvascular ECs relative to pulmonary artery ECs, while electron microscopy reveals tighter intercellular connections, fewer visible gaps, and increased focal adhesion sites in microvascular cells compared to macrovascular ECs. In addition, microvessel ECs contain a more abundant array of intercellular junctional complexes. All of these microvascu-lar EC characteristics combine to decrease basal permeability in these cells relative to large conduit ECs. Specifically, cultured microvascular ECs have tenfold higher barrier integrity than macrovascular ECs as measured by electrical resistance across monolayers. Even within each of these broad categories of macro- and microvascular ECs, significant variability is present. EC morphologic appearance, cell surface glycoproteins, cell-cell interactions, and protein and mRNA expression are all highly variable throughout the vasculature. The functional importance of this variability remains a highly active area of research.

Studies comparing the responses of various EC pheno-types to specific agonists have provided insight into differential barrier function along the vasculature. In general, microvascular ECs are more resistant to agonist-induced lung permeability. Cultured bovine and sheep pulmonary artery ECs have been shown to be more sensitive than microvascular ECs to barrier disruption by agents such as endotoxin or TNFa. However, this relative resistance of microvascular ECs is not universal to every model of agonist-induced permeability since thrombin produces more dramatic barrier disruption in microvascular ECs compared to pulmonary artery ECs. The mechanisms underlying this differential EC barrier regulation remain poorly understood, but it appears that Ca2+-dependent signaling events play a role. For example, macrovascular ECs are more sensitive to barrier disruption induced by store-operated Ca2+ entry, whereas microvascular ECs have increased levels of intra-cellular cAMP, a determinant of focal adhesion complex formation that contributes to barrier integrity. Differential adherens junction protein expression may also account for some of the variability in EC barrier function along the vascular tree. Microvascular ECs express significantly more VE-cadherin protein than macrovascular cells, an observation that appears to have functional importance for cell-cell integrity since the infusion of anti-VE-cadherin antibodies causes increased permeability primarily in alveolar capillaries. Finally, variability in the extracellular environment likely plays an important role in differential EC barrier function. Mechanical forces such as shear stress and cyclic stretch significantly affect EC cytoskeletal organization in ways that alter permeability (as discussed earlier) and are differentially distributed along the vasculature. For example, understanding the effects of shear stress on pulmonary EC barrier integrity must take into account regional differences in blood flow that affect the amount of shear applied

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Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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