Vascular permeability, vascular hyperpermeability and angiogenesis

Surprisingly, there is not good agreement as to what is meant by the term “vascular permeability” and from this it follows that there is no consensus about how vascular permeability should be measured. Over the last half-century eminent physiologists including Pappenheimer, Landis, Starling, Renkin, Michel, Curry, Rippe, and Bates have investigated the mechanisms by which plasma and its solutes cross the vascular barrier [5, 9‐13]. They recognized that capillaries were the vascular segment involved in molecular exchange in normal tissues and that gases, water, and other small molecules crossed the capillary endothelial cell barrier freely whereas the passage of larger molecules such as plasma proteins was tightly restricted. Physiologists have commonly regarded capillary endothelium as a passive barrier, a thin, cellophane-like membrane that is punctuated by large numbers of small pores and lesser numbers of large pores. They postulated that the numerous small pores allowed the ready passage of small molecules and that the lesser number of large pores allowed limited extravasation of plasma proteins. With these assumptions in mind they developed elegant methods for investigating the flux of water and of plasma solutes across individual cannulated microvessels. They developed equations to calculate the three parameters that determine permeability, namely, hydraulic conductivity, reflection coefficient, and diffusion. Diffusion is the most important of these for the exchange of small molecules and is driven by the molecular concentration gradient across vascular endothelium as determined by the Fick equation: where J _s is the diffusion rate (e.g., ml/s) of a particular solute; D is the diffusion coefficient for that solute; A is surface area available for exchange; T is the thickness of the capillary; and C _v −C _i is the difference in solute concentration between the plasma and the interstitial fluid.

The value of D in the Fick equation depends heavily on molecular size; for example, the diffusion of albumin across the vasculature is estimated to be ∼1,000-fold less than that of water [11, 12]. As a result, filtration is much more important than diffusion for the flux of large molecules such as plasma proteins and is determined by the Starling equation:

where J _v is filtration rate (e.g., ml/s); L _P is hydraulic conductivity or the filtration coefficient, a property of the capillary wall and a measure of capillary permeability to water; A is surface area available for molecular exchange; P _v −P _i and π _v −π _i are, respectively, the hydrostatic and osmotic pressure differences between the plasma and the interstitium; and σ is the osmotic reflection or solvent-drag reflection coefficient. σ varies in different tissues from 0 to 1 and tissues such as skin with high values (e.g., 0.9) permit little plasma-protein escape. Further details concerning the diffusion and Starling equations can be found in standard textbooks of Physiology and in several excellent reviews [12‐16].

In contrast to physiologists, vascular biologists have used the term vascular permeability in a less restrictive sense. Rather than being concerned with the permeability of a single cannulated microvessel, they have sought to measure the net amount of a solute, typically a macromolecule such as plasma albumin, that has crossed a vascular bed and accumulated in the interstitium in response to a vascular permeabilizing agent or at a site of pathological angiogenesis. Generally speaking, the vessels involved are not of a single type, and the measurements made combine together all of the factors, both intrinsic properties of the blood vessels as well as extrinsic properties such as blood flow, that regulate extravasation.

To obtain this type of information, they have generally used the Miles assay or one of its variants [17‐19]. Typically, a dye such as Evan’s blue that binds noncovalently to albumin is injected intravenously and its accumulation is measured at some later time at a skin test site (Fig. 1), in a tumor, or in other tissues of interest. Permeability is defined as the amount of albumin–dye complex that is present at some time (often 30 min) after Evan’s blue injection. The intensity of local bluing observed visually provides sufficient information for some purposes. For example, local bluing in guinea pig skin was used to evaluate column fractions in the original purification of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) [19]. However, quantitative measurements can be made by extracting the dye from tissues and measuring it spectrophotometrically [20]. A limitation of the Miles assay, whether permeability is assessed visually or by quantitative measurement, is that it does not distinguish between dye that has extravasated from that present within the vasculature. However, serious error does not result when intravascular volumes are small relative to the amounts of dye that have leaked, for example, at skin test sites injected with column fractions containing different amounts of VPF/VEGF. Another limitation is that the Miles assay measures net accumulation of dye–albumin complex over a period of time and return of extravasated molecules to the circulation, either by way of capillaries or lymphatics, is not considered. Despite these limitations the Miles assay has provided much useful information.

However, in tumors and in other examples of pathological angiogenesis, the vasculature undergoes dramatic changes and is not comparable to that of adjacent normal control tissues. In such instances it is important to measure both the content of tracer within blood vessels and that which has extravasated. This can be accomplished using a dual isotope approach [20‐22]. Operationally, ¹²⁵I-albumin is injected i.v. into a mouse at time zero. After 25 min, a second i.v. injection is administered, this time of ¹³¹I-albumin. After 5 min, at time 30 min, blood is collected, animals are euthanized and tissues of interest are harvested, weighed, and subjected to gamma counting. The following equations are used to calculate intravascular plasma volume (V _p ) and the albumin leakage rate (LR): where A is total tissue radioactivity (μCi/g) of ¹²⁵I-albumin or ¹³¹I-albumin; C _p is concentration of radioactive tracer in plasma (μCi/μl); V _p is volume of plasma in tissue (μl/g); LR is the leakage rate from plasma into tissue expressed as μl/min-g and is equivalent to the permeability–surface area product; and t is time elapsed since injection of tracer (min).

An underlying assumption of this method is that even in the case of highly leaky blood vessels only negligible amounts of ¹³¹I-albumin will have had time to extravasate at 5 min after injection. Therefore, the ¹³¹I-albumin value at 5 min provides a quantitative measure of intravascular volume whereas the ¹²⁵I-albumin value provides a measure of the sum of both intravascular and extravascular albumin. Extravasated albumin (i.e., the volume of plasma extravasated in 25 min) can then be determined by subtracting the 5 min value from the 30 min value. This method has the disadvantages of using a strong, short-lived gamma emitter (¹³¹I) and of not permitting visual inspection of tracer leakage as when Evan’s blue dye is used as tracer. To circumvent these limitations we recently modified the method by substituting Evan’s blue dye (hence plasma albumin) for the first tracer (30 min time point) and using ¹²⁵I-albumin for the second (5 min time point).

The assays described above measure permeability in living animals. However, a number of groups have used in vitro assays to measure the flux of small or large molecules across lawns of confluent endothelial cells cultured on membrane filters in transwell chambers [5, 10, 23‐26]. These assays are appealing in that they are relatively easy to perform and avoid the complexities of studies in living animals. However, in our view they suffer from severe limitations. Confluent-cultured endothelial cell monolayers, whether isolated from large or small vessels, are generally leakier than the normal blood vessel wall in vivo, perhaps because pericytes or smooth muscle cells that normally modify endothelial cell behavior are missing. Also, cultured endothelial cells generally have relatively few cytoplasmic vesicles and vacuoles, structures which are numerous in these same cells in vivo and provide the means by which solutes, and especially proteins, cross capillary and venular endothelium in vivo. Attempts to restore these vesicles have only been achieved in cultured endothelium under specialized conditions that are not easily amenable to permeability assays [27, 28]. Cultured endothelial cells are extremely flattened cells that do not resemble, for example, the cuboidal venular endothelium that is responsive to permeability agents such as VEGF-A or histamine in vivo. Finally, the kinetics of leakage in response to agents such as VEGF-A differ markedly in vivo and in cultured endothelium. In vivo, leakage in response to a single exposure to VEGF-A begins within a minute and is largely complete by ∼30 min. However, increased permeability develops much more slowly in cultured endothelium and often peaks over a period of hours, suggesting that the permeability observed may reflect, at least in part, a loosening of intercellular connections as endothelial cell are stimulated to migrate by VEGF-A. In sum, current in vitro assays do not mimic the basal vascular permeability or acute vascular hyperpermeability observed in vivo, but may provide a model for measuring the chronic vascular hyperpermeability characteristic of pathological angiogenesis as found in tumors, healing wounds, and chronic inflammation (see below).

As was already noted, low levels of vascular permeability to plasma proteins are essential for the health of normal tissues and these levels may vary considerably at different times in different organs and tissues in response to different physiological stimuli, e.g., exercise. However, it is important to distinguish between the basal permeability levels of normal tissues and the greatly increased levels of plasma protein extravasation that occur in pathology. These hyperpermeable states may be acute or chronic and differ from each other and from basal levels of permeability with respect to the vessels that leak, the composition of the extravasate, and the anatomic pathways that solutes follow in crossing vascular endothelium. Each of the three types of permeability will now be discussed in turn.

This review has shown that vascular permeability, far from being a single, well-defined entity, is instead an extremely complex process that in different settings, involves distinctly different types of blood vessels and makes use of different anatomic pathways. Further, whereas the fluid extravasating in BVH is a plasma filtrate consisting almost entirely of water and small solutes, that extravasating in AVH and CVH, is a protein-rich exudate. Agents such as VEGF-A have long been known to induce AVH and CVH, but, apart from hemodynamic factors, much less is known about the molecular events that are responsible for the normal permeability of BVP. Even less is known about the molecules that are involved in regulating permeability, though this is changing rapidly. In Table 1 we have listed as many of the published gene products of which we are aware that have been implicated in vascular permeability. Some of these molecules have long been known to have roles in permeability whereas others have only recently been recognized to have such a role. However, the signaling pathways by which even such well-studied molecules as eNOS and caveolin-1 act to induce permeability are poorly understood. Almost nothing is known about the molecular mechanisms that regulate such critical events as caveolar shuttling, the opening of VVO diaphragms, the formation of fenestrae, changes in endothelial cell junctions, etc. [61‐63]. We have attempted to catalog the molecules in Table 1, as far as is possible with existing knowledge, under the headings of BVP, AVH, and CVH. Some of these molecules are clearly involved in all three types of permeability whereas others apparently are not. The molecular mechanisms that govern each of the different types of permeability may well be different and are a subject for further research. As an example, Phung et al. found that, unlike the AVH induced by VEGF-A, the CVH found in mice over-expressing myr-Akt in vascular endothelium was not regulated by eNOS [64].

Finally, just as the angiogenic response induced by VEGF-A differs significantly in different mouse strains [65], it is likely that the permeability response, both basal and that induced by permeability factors, will also differ in mice with different genetic backgrounds, though this has not as yet been investigated in any systematic manner.