2.1. Composition, Structure and Three-Dimensional Organization of the Extracellular Matrix in the Interstitial Spaces

The composition and organization of the extracellular matrix determine the mechanical properties of the interstitium such as its strength, elasticity, and hydration [14,39,41,42,88,166,221]. The interstitium is composed mainly of collagen types I, III, and V, elastin, and glycosaminoglycans (mucopolysaccharides, such as hyaluronate and proteoglycans) which are mechanically entangled and cross-linked to form a gel-like reticulum reminiscent of a brush-pile in terms of its three-dimensional organization (Figure 1.1). These large polymeric molecules are synthesized by fibroblasts and released into the interstitial space. Fibroblasts also release a variety of enzymes that continuously degrade matrix components, such that complete turnover of the extracellular matrix occurs every 50 days [88]. The rate of synthesis is influenced by local conditions and hormonal factors (e.g., thyroid hormone).

Collagen represents a major structural component of interstitial spaces, functioning as a scaffold for support of the interstitial space and surrounding parenchymal cells (Figure 1.1). This extracellular matrix protein is formed into rod-like fibrils that are comprised of parallel linear arrays, which are then organized into bundles measuring several micrometers in diameter that exhibit tensile strengths that are approximately one-sixth of that measured for milled steel [39]. This high tensile strength is related to the formation of covalent intermolecular linkages produced by the oxidative deamination of specific lysine or hydroxylysine residues by the enzyme lysyl oxidase, a process that occurs extracellularly. Several structurally and genetically distinct collagens have been identified and collagen fibers found in the extracellular matrices of various tissues vary widely in terms of diameter, distribution, and relative contents of hydroxylysine, hydroxyproline, and glycosylated hydroxylysine residues.

Elastin is another major structural component of the extracellular matrix and is one of the most hydrophobic of all known proteins. Elastin-associated microfibrils are highly complex structures that appear as solid, branching and unbranching, fine and thick, rod-like fibers, can occur as concentric sheets, or can be arranged in three-dimensional meshworks. These microfibrils are found in tissues that undergo repetitive distension or passive lengthening movements and appear to confer elasticity to these structures.

While collagen fibrils and elastin provide much of the structural framework for tissues, the glycosaminoglycan constituents of the extracellular matrix play a major role in tissue hydration. These mucopolysaccharides are linear chains of disaccharide units that carry anionic charge sites. In the interstitial spaces, glycosaminoglycans exist as three-dimensional random coils that interact to produce a continuous network of intermeshing, entangled reticular structures that entrap water and resist compression by electrostatic repulsion of neighboring anionic sites and elastic recoil of the mechanically intertwined coils (Figure 1.1). Thus, the high fixed charge density of glycosaminoglycans establishes interstitial volume.

Proteoglycans form macromolecular assemblies that consist of a protein core that is long and rod-like, to which are attached numerous sulfated mucopolysaccharides by covalent bonds, yielding a bottle-brush configuration to the ensemble. The terminal ends of the proteoglycan cores attach by hydrogen bonds to form enormous aggregates of bottle-brush and random coil structures that become entangled in the randomly distributed collagen array in the tissue matrix to produce elastic, three-dimensional reticulum (Figure 1.1).

The physicochemical properties of the extracellular matrix are dynamic and apparently derive in large part from the behavior of the glycosaminoglycan molecules, of which hyaluronate is of principle importance. Thus, it is not surprising that the three-dimensional reticular structure of the extracellular matrix provides mechanical support for the tissues and provides a sponge-like continuum for containment of water and solutes. The gel-like properties of the interstitium limit the availability of free water for fluid flow, although rivulets of free fluid exist within the space. The flow of this free fluid in the interstitium, which is derived from capillary filtration, drives protein transport from the blood to parenchymal and interstitial cells (e.g., fibroblasts, dendritic cells, adipocytes, and inflammatory cells such as extravasated white blood cells and mast cells) because proteins are too large to readily diffuse the distances between capillaries. Dynamic stresses related to fluid flow in the interstitium also bestow a signaling function that serves as an important morphoregulator in tissue development, maintenance, and remodeling, as well as providing cues that allow interstitial cells to monitor the state of their surroundings, establish microenvironments, and guide immune cells towards draining lymphatic vessels (see below).

2.2. Solute Exclusion and Osmotic Amplification in the Extracellular Matrix

The entangled nature of the fibrils allows the extracellular matrix to behave as if it were perforated by pores approximately 200–250 angstroms in diameter. Thus, one important property of the interstitium relates to the ability of the gel reticulum to exclude solutes from portions of the available gel water (Figure 2.1) [41,42,88,280]. As a consequence, extravasated plasma proteins present in the interstitium are normally distributed in only a fraction of the interstitial volume because these large solute molecules cannot gain access to certain regions of the matrix meshwork (Figure 2.1). In other words, these large molecules are distributed into the matrix spaces that have dimensions larger than the solute (the accessible volume) and are excluded from microdomains with smaller dimension (the excluded volume). For a given large solute, the excluded volume varies inversely with the hydration state of the interstitium, which varies in accord with capillary filtration and the rate of lymphatic outflow. As fluid accumulates in the interstitium, the density of matrix fibers decreases, thereby increasing matrix porosity and reducing the fraction of tissue fluid from which the solute is excluded (Figure 2.1, left panel). On the other hand, tissue dehydration compacts the extracellular matrix and increases the excluded volume (Figure 2.1, right panel).

Figure 2.1

Solute exclusion in the extracellular matrix gel reticulum varies with hydration state. With increased tissue hydration (left panel), as occurs in edema, the effective pore radii of the gel reticulum increase (i.e., matrix density decreases), resulting (more...)

The functional significance of this exclusion phenomenon is related in part to its effect on oncotic pressure generated by plasma proteins distributed within the interstitial space. As fluid accumulates in the interstitium, tissue oncotic pressure falls, thereby reducing the balance of forces favoring capillary filtration and limiting edema formation. However, this reduction in tissue oncotic pressure is not simply due to dilution by the capillary filtrate, but also relates to the increased tissue water that becomes available for solute distribution on hydration that occurs secondary to the reduction in excluded volume on hydration. As a consequence, the exclusion phenomenon amplifies the oncotic buffering response to capillary filtration by further diluting the concentration of large solutes in the available gel water. The exclusion phenomenon also influences protein diffusion in the interstitial space because alterations in the distribution volume modify the effective surface area for, and the frictional resistance to, the diffusion of macromolecules (Figure 2.1). From the foregoing discussion, it should be apparent that exclusion properties for a given solute depend on its molecular size, the concentration of the various matrix elements, and hydration state of the interstitium. In addition, because both the permeating solutes and matrix elements are polyionic in nature, electrostatic interactions may influence the distribution spaces.

The fact that extracellular matrix molecules are mechanically entangled and cross-linked to yield a three-dimensional structure that behaves as if it were permeated by 200–250 angstrom diameter pores implies that migrating cells would encounter difficulty in passively traversing this space. Rather, it has been proposed that active mechanisms exist to promote changes in cell shape and amoeboid movement between the interstices of extracellular matrix molecules or involve protease-dependent degradation of extracellular matrix molecules to facilitate cell motility within this space. (This topic is briefly reviewed here because degradation of matrix components may contribute to enhanced capillary filtration and solute permeability in inflammation (see below)). For example, it has been recently shown that endothelial cells form vascular guidance tunnels during angiogenesis by a mechanism that is dependent on release of matrix type 1-metalloproteinase [50,51,52,113,248,249]. In effect, these migrating endothelial cells digest a route of passage through the extracellular matrix, thereby creating physical spaces in the interstitium that serve as conduit pathways for both assembly and remodeling of tubular structures as well as recruitment of other cell types, such as pericytes, that are required for this morphogenetic process. Whether such processes are involved in the migration of immune cells, fibroblasts, and metastasizing tumor cells is unclear. However, penetration and transit through basement membranes appears to correlate with sites of focal matrix metalloproteinase activity, involves integrin-dependent adhesion steps, and occurs in regions where some matrix proteins are more sparsely expressed [72,132,140,274]. Upon gaining access to the interstitial spaces, leukocytes migrate at much faster velocities than that noted for activated fibroblasts and tumor cells, implying mechanistic distinctions [189]. Activated fibroblasts and tumor cells employ pericellular proteolysis to migrate through interstitial extracellular matrix molecule barriers. Proteases bound to cell membranes co-cluster with specific integrins at contacts with substrates and cleave the extracellular matrix [72,140]. In contrast, migrating leukocytes move through the extracellular matrix in an amoeboid manner independent of protease activity and integrins, tracking along collagen fibers and squeezing through narrow spaces within the matrix reticulum [22,72,132,150,189,204]. More recent work has demonstrated differences in migratory behavior for monocytes versus neutrophils in the interstitial space, implying that individual leukocyte types may employ diverse processes to directionally navigate the extracellular space [132,140].

2.3. Compliance and Hydraulic Conductance in the Extracellular Matrix

The compliance characteristics of the interstitium vary with hydration state and are of great importance in determining the hydrostatic forces operating across the capillary and lymphatic walls and thus fluid movements across these endothelial cell membranes [14,87,88,141]. Under normal conditions, the volume conductance of the interstitium is very low because most of the interstitial fluid is immobilized in the tissue matrix. Tissue volume conductance is even lower under dehydrated conditions but increases dramatically with overhydration. From Figure 2.2, which depicts the compliance characteristics of the interstitial spaces, it is apparent that small changes in capillary filtration – and thus tissue volume – from normal levels produce very large increments in interstitial fluid pressure. However, there is an abrupt change in interstitial compliance when interstitial fluid volume increases by just 20% from its normal value of 25 ml/100g (i.e., to 30 ml/100g) (Figure 2.2). Although the mechanisms responsible for this abrupt shift to high compliance are uncertain, it is presumed to reflect a reversible disentanglement of hyaluronic acid chains and cross-link rupture [41,42,87,88]. In addition, fibroblasts, which form attachments with the extracellular matrix, relax in response to mediators released during inflammation, and may contribute to the increase in compliance with increasing hydration under such conditions (Figure 2.3) [215,279]. Since both hydraulic conductance and macromolecule exclusion of the interstitium are influenced by the degree of matrix hydration, an increase in interstitial fluid volume decreases excluded volume for proteins in this space and increases tissue hydraulic conductance, enhancing blood-to-lymph transport of fluid and macromolecules (Figures 2.1 and 2.2). Indeed, doubling interstitial fluid volume can increase its hydraulic conductance over a thousand fold.

Figure 2.2

Relationship between interstitial fluid volume, interstitial fluid pressure, and interstitial hydraulic conductance. As interstitial fluid volume increases from its normal level, interstitial fluid pressure rises very quickly, owing to the low compliance (more...)

Figure 2.3

Fibroblasts form attachments to collagen fibrils in the extracellular matrix by an α2β1-integrin-dependent mechanism, which confers cellular tension to the fibrous reticulum that restrains the hyaluronate/proteoglycan gel from taking up (more...)

The intestinal mucosal interstitium (and other organs with transporting epithelium) is rather distinctive in this regard in that its hydration state is not only influenced by capillary filtration, but also by net fluid absorption from the intestinal lumen. Indeed, when interstitial hydration occurs secondary to enhanced fluid absorption from the intestinal lumen, this may induce a larger change in interstitial compliance than produced by increased venous pressure (which increases capillary pressure and thus capillary filtration) because the congested microvasculature contributes to tissue stiffness in the latter case [87]. These considerations may provide an explanation for the efficient transfer of chylomicrons (lipoprotein particles that are secreted by intestinal epithelial cells after a meal and range in size from 750 to 6000 A in diameter) through the interstitium, which should offer substantial resistance to their movement, while en route to the central lacteals of mucosal villi. Although it has been suggested that interstitial chylomicron movement may also be facilitated by inhomogeneities in the mucosal interstitial gel, which represent non-endothelialized channels that form on matrix expansion secondary to fluid absorption, ultrastructural studies have failed to demonstrate such pathways [87].

2.4. Fluid Flow in the Interstitium Modifies the Function of Tissue Cells

Capillary filtration drives fluid flow into the interstitium, which not only assists transport of nutrients through tissues, but also plays important roles in tissue morphogenesis and remodeling, regulation of vascular function, inflammation and lymphedema, formation of new lymph vessels, tumor biology and immune cell trafficking (Figure 2.4) [89,202,224,251,252,253]. The flow of fluid through the interstitium not only occurs through the three-dimensional ensemble of extracellular matrix molecules, but also around interstitial cells such as fibroblasts, extravasated immune cells, and adipocytes, as well as parenchymal cells comprising the tissue or organ (Figure 1.1). The flow of fluid derived from the capillary filtrate in the extravascular space occurs at a much slower velocity (0.1–4 μm/s) than capillary luminal flow, owing to the high resistance offered by the extracellular matrix [37,49]. However, this flow rate may increase 10-fold in edemagenic stress. It is important to note that interstitial flow occurs in all directions around the tissue cell-matrix interface, which may impart unique shear, pressure, and tethering force signals to tissue cells (Figure 1.1) [224]. Furthermore, interstitial fluid flow can also influence pericellular protein gradients, which may be particularly important for macromolecules that bind to matrix components, and are involved in directing cell migration (see below) [224].

Figure 2.4

Capillary filtration drives flow into the interstitium, which not only assists transport of nutrients through tissues, but also plays an important role in guiding the organization of a wide variety of cellular processes in the direction of interstitial (more...)

The shear stress induced by capillary filtrate flowing through the extracellular matrix (0.005 to 0.015 dyn/cm²) [224] is far smaller than that experienced in the capillary lumen, interendothelial clefts, or basal lamina, leaving it unclear as to whether such levels are sufficient to activate mechanosensors on tissue cells. Again, however, shear stress produced by interstitial flow may increase dramatically secondary to enhanced transcapillary filtration during edemagenic stress. Moreover, recent work indicates that the low shear stress levels normally present with interstitial flow can induce chemokine expression by dendritic cells embedded in an in vivo extracellular matrix construct [265]. Several other examples clearly indicate that interstitial flow exerts important effects on tissue function (Figure 2.4). In cartilage, interstitial flow has been shown to promote collagen and proteoglycan synthesis and increase chondrocyte metabolism [131,210]. Interestingly, interstitial flow induces proteoglycan deposition and matrix fiber compaction in the direction of flow and guides remodeling by enhancing the transport of tissue inhibitor of metalloproteinases-1 [69,77]. Interstitial fluid flow has also been suggested to direct the migration of endothelial and epithelial cells in wounded tissues [10,160,224] and appears to enhance blood and lymphatic capillary formation [26,96,97,185,235]. In regenerating skin, interstitial fluid channels form prior to the development of lymphatic capillaries, while lymphatic cell migration, expression of vascular endothelial cell growth factor-C, and lymphatic network organization occur in the direction of lymph flow [26]. These results suggest that lymphatic cells use the fluid channels for network development by a mechanism directed by interstitial flow.

Inflammation induces large increases in capillary filtration secondary to endothelial barrier dysfunction (increased permeability, reduced reflection coefficient) and elevated microvascular hydrostatic pressure induced by arteriolar vasodilatation. The associated increase in interstitial fluid flow, when coupled with hydrolytic cleavage of extracellular matrix components (which reduces the frictional resistance to solute movement), enhances the delivery of differentiation factors released from infiltrating inflammatory cells to fibroblasts, causing them to differentiate and remodel the extracellular matrix [224]. High interstitial flows characteristic of inflammatory states have also been shown to induce autocrine upregulation of transforming growth factor-b1 in fibroblasts seeded into three-dimensional collagen matrices, inducing their differentiation into myofibroblasts which then increase collagen production and alignment in a flow-directed manner [183,184,186]. These results suggest that enhanced capillary filtration associated with inflammation increases interstitial fluid flow, which in turn provides an early directional cue for rapid matrix repair. It has also been suggested that this may explain the development of tissue fibrosis induced by prolonged inflammation and why fibrosis can occur in the absence of inflammatory cells, as occurs with idiopathic pulmonary fibrosis [198,233,264].

Conditions characterized by abnormally low interstitial fluid flow may also induce dramatic changes in cell function. For example, lymphedema is a condition characterized by low interstitial flow that can result from either congenital lymphatic malformations (primary lymphedema) or from downstream lymphatic blockage that occurs with lymph node resection or compression due to tumor growth (secondary lymphedema). This results in excessive accumulation of fluid in the extravascular compartment in the absence of normal interstitial convection patterns, pathologic changes that cause chronic swelling of the interstitial compartment and precipitate inflammation and extensive remodeling of the extracellular matrix (fibrosis), adipocyte growth, and lipid accumulation [193,225]. These changes are exacerbated by the absence of normal immune trafficking in the affected tissue [224]. These findings underscore the importance of normal interstitial fluid flow in the maintenance of healthy tissue.

Interstitial fluid flow has also been shown to affect pericellular diffusion gradients for signaling molecules such as growth factors, including members of the VEGF, FGF, Wnt and TGF families, and immunosuppressive chemokines such as CCL19 and CCL21 (Figure 2.5) [71,211,224,265]. Many of these molecules, which are secreted by and can act upon the same cell, also bind strongly to elements of the extracellular matrix. Asymmetric distribution of the morphogen or chemokine by interstitial convective flow delivery, especially when maintained in pericellular proximity by matrix binding, creates both liquid and solid-phase gradients that provide the secreting cell with more nuanced and directed control of its microenvironment (Figure 2.5) [71,211,224,265]. Matrix binding allows establishment of an autologous gradient of morphogen or chemokine in the direction of interstitial flow that the cell can follow for directed movement using cues that these cells secrete. Although the directional bias in the distribution of these signaling molecules that is introduced by interstitial fluid flow is small owing to its low velocity, secretion of proteases from the same cell liberate the matrix-bound growth factor or morphogen, which amplifies the biasing effect of subtle interstitial flow and creates a stronger autocrine concentration gradient for directed motion (Figure 2.5) [71,211,224,265]. This phenomenon, whereby cells receive directional cues while at the same time being the source of these prompting signals, has been termed autologous chemotaxis [71,211,224,265]. Autologous chemotaxis has been demonstrated for VEGF directed capillary organization in vitro and more recently for leukocyte, dendritic cell, and tumor cell homing to draining lymphatics and lymph nodes [71,96,211,224,236,239,254,265].

Figure 2.5

In the presence of interstitial fluid flow, autologous pericellular gradients of self-secreted proteins (e.g., chemokines/morphogens) can develop in the direction of flow. The top panel depicts the chemotactic gradient development in the absence of interstitial (more...)

Interstitial fluid flow may also be exploited to enhance the targeted delivery of chemotherapeutic agents, especially given the promise of protein and antibody therapies, as well as synthetic drug carriers and delivery systems, including nanoparticles, liposomes and retroviruses, all of which encounter transport limitations owing to their large size [214,254]. The steric hindrance effects of the mechanically entangled and cross-linked extracellular matrix may be overcome by the use of ultra-small nanoparticles (25 nm) to activate the complement cascade, thereby generating signals to stimulate dendritic cells to trigger adaptive immune responses [214]. In addition, coincident introduction of enzymes such as collagenase or hyaluronidase to disrupt the extracellular matrix of tumors would be expected to facilitate the interstitial delivery of chemotherapeutic agents by enhanced convective flow and reduced molecular sieving.