Regulation of microvascular permeability by ... - Wiley Online Library

J. Anat. (2002) 200, pp581–597

REVIEW Blackwell Science, Ltd

Regulation of microvascular permeability by vascular endothelial growth factors* D. O. Bates,1 N. J. Hillman,2 B. Williams,2 C. R. Neal1 and T. M. Pocock1 1 2

Microvascular Research Laboratories, Department of Physiology, University of Bristol, Bristol BS2 8EJ, UK Cardiovascular Research Institute, University of Leicester, Leicester LE2 7LX, UK

Abstract Generation of new blood vessels from pre-existing vasculature (angiogenesis) is accompanied in almost all states by increased vascular permeability. This is true in physiological as well as pathological angiogenesis, but is more marked during disease states. Physiological angiogenesis occurs during tissue growth and repair in adult tissues, as well as during development. Pathological angiogenesis is seen in a wide variety of diseases, which include all the major causes of mortality in the west: heart disease, cancer, stroke, vascular disease and diabetes. Angiogenesis is regulated by vascular growth factors, particularly the vascular endothelial growth factor family of proteins (VEGF). These act on two specific receptors in the vascular system (VEGF-R1 and 2) to stimulate new vessel growth. VEGFs also directly stimulate increased vascular permeability to water and large-molecular-weight proteins. We have shown that VEGFs increase vascular permeability in mesenteric microvessels by stimulation of tyrosine autophosphorylation of VEGF-R2 on endothelial cells, and subsequent activation of phospholipase C (PLC). This in turn causes increased production of diacylglycerol (DAG) that results in influx of calcium across the plasma membrane through store-independent cation channels. We have proposed that this influx is through DAG-mediated TRP channels. It is not known how this results in increased vascular permeability in endothelial cells in vivo. It has been shown, however, that VEGF can stimulate formation of a variety of pathways through the endothelial cell, including transcellular gaps, vesiculovacuolar organelle formation, and fenestrations. A hypothesis is outlined that suggests that these all may be part of the same process. Key words calcium; endothelium; hydraulic conductivity; transcellular gaps; TRPC channels; vascular permeability; VEGF; VVO.

Vascular permeability and angiogenesis Vascular permeability is increased in angiogenesis Generation of new blood vessels from pre-existing vasculature (angiogenesis) is accompanied in almost all states by increases in vascular permeability. This

Correspondence Dr David Bates, Microvascular Research Laboratories, Department of Physiology, The Preclinical Veterinary School, The University of Bristol, Southwell Street, Bristol BS2 8EJ, UK. Tel. +44 (0)117 9289818; fax: +44 (0)117 9288151; e-mail: [email protected] *From a paper presented at an Anatomical Society of Great Britain and Ireland symposium on the modulation of endothelial cell permeability, Royal Holloway College, UK, January 2002 Accepted for publication 29 April 2002 © Anatomical Society of Great Britain and Ireland 2002

was shown in physiological angiogenesis in the 1940s using large-molecular-weight dyes in wound healing (Abell, 1946), as well as in angiogenesis in the developing tail of immature amphibians (Clark & Clark, 1935). A more detailed ultrastructural study in 1963 showed that large-molecular-weight tracers such as colloidal carbon leaked out of growing capillaries in wound healing models (Schoefl, 1963). Increased vascular permeability during angiogenesis is now recognized as a cardinal feature of pathological angiogenesis. Although it is more marked during disease states, as capillaries grow and form new vessels in physiological systems there is also a regulated increase in vascular permeability to both solutes and water (Spanel-Borowski & Mayerhofer, 1987; Dejana et al. 2001). Since physiological angiogenesis can occur

582 VEGF and vascular permeability, D.O. Bates et al.

throughout adult life as well as during both prenatal and postnatal development (Hudlicka et al. 1992), the regulation of vascular permeability during angiogenesis is of interest to aid our understanding of normal physiological function. Angiogenesis occurs during muscle remodelling after exercise-induced training (Pearce et al. 2000), during fat deposition (Crandall et al. 1997), hair growth (Yano et al. 2001) and wound repair, as well as during the female reproductive cycle in the endometrium and the developing follicle (Hudlicka, 1984). Most studies, however, have concentrated on pathological angiogenesis, since this is apparent in a wide variety of diseases, including all the major causes of mortality in the western world: heart disease (Mack et al. 1998), cancer (Brown et al. 1993), stroke (Van Bruggen et al. 1999), peripheral vascular disease (Couffinhal et al. 1997) and diabetes (Aiello & Wong, 2000). Angiogenesis is regulated by a variety of growth factors and inhibitors, mainly of the vascular endothelial growth factor (VEGF), angiopoietin (Ang) and ephrin (Eph) families (Ferrara, 2001). These molecules co-ordinate physiological angiogenesis to produce viable patent mature vessels through which flow is regulated and vascular permeability is low. In pathological angiogenesis, production of vascular growth factors such as VEGF often becomes up-regulated (Brown et al. 1993) and a vasculature develops in which blood flow is abnormally regulated and the relationship between metabolic demand and flow appears to be disturbed (Dvorak et al. 1999).

Angiogenic pathologies are associated with oedema In pathological angiogenesis novel vasculature is characterized by weak, friable vessels that are inherently leaky, and often bleed (Schoefl, 1963). The tissue itself may become oedematous as a result of this increase in permeability, and this effect is compounded by unregulated flow through these vessels. This increased vascular permeability appears to have a minor effect in physiological angiogenesis, but causes considerable damage in pathologies. The abnormal vasculature results in cerebral oedema in glioblastoma multiforme, ascites and pleural effusions in liver metastasis and lung cancer, respectively (Vaquero et al. 2000; Xu et al. 2000; Yano et al. 2000). It may contribute to increased lipid deposition in atherosclerosis, substrate formation in neointimal hyperplasia (Celletti et al. 2001), and

neuropathy and retinopathy in diabetes (Aiello & Wong, 2000).

Is this increase in permeability due to vascular growth factor production? In physiological angiogenesis vascular growth factors are up-regulated in response to increased metabolic demand, particularly hypoxia (Mukhopadhyay et al. 1995). In pathological states, the same growth factors are overexpressed (Damert et al. 1997), but despite significant understanding of the mechanisms underlying regulation of transcription and translation of these factors (Sandner et al. 1997; Wenger et al. 1997), the underlying mechanisms in pathology are still not well understood. The functions of these growth factors, and the mechanisms through which they exert their actions, are also the subject of significant interest, and these metabolic pathways are currently being elucidated. The discovery in 1989 of vascular endothelial growth factor (VEGF) has radically enhanced our understanding of the changes in fluid balance and blood flow that occur during angiogenesis (Bates et al. 1999). The principal effect of developing a new vascular supply is to increase the delivery of nutrients to and removal of waste products from a deprived tissue. Vascular growth factors could do this through three main mechanisms of action. They could (1) increase blood flow to the tissue by acting as a vasodilator, (2) reduce the distance of the cells in the tissue from the nearest blood vessel, by stimulating angiogenesis and (3) increase the permeability of the vessels to water, small solutes and macromolecules (Michel, 1984; Taylor & Granger, 1984; Levick, 1991). VEGF is a unique molecule in that it is upregulated in all known endogenous physiological and pathological forms of angiogenesis, can stimulate angiogenesis directly (Ferrara & Bunting, 1996), is a potent vasodilator (Ku et al. 1993) and is able to increase vascular permeability (Bates & Curry, 1996). Although all three of these properties were claimed for VEGF when, or immediately after, it was discovered the mechanisms through which it acts to exert these effects are only now being elucidated. This review will address two aspects of those mechanisms – the signal transduction pathways through which VEGF stimulates increased permeability (Ferrara & Bunting, 1996; Bates et al. 1999; Neufeld et al. 1999) and the endothelial cell changes that are a result of those transduction pathways. © Anatomical Society of Great Britain and Ireland 2002

VEGF and vascular permeability, D.O. Bates et al. 583

Vascular permeability What is VEGF? Vascular endothelial growth factor (VEGF-A, hereafter termed VEGF) was independently described by four different groups using a variety of assays. As early as 1983, Senger et al. described a factor secreted by hepatocarcinoma cell lines (Senger et al. 1983) that increased dye extravasation into the skin of guinea-pigs. Although this was further characterized in 1986 (Senger et al. 1986), the protein structure and amino acid sequence were not described until 1990 (Senger et al. 1990). They termed this peptide vascular permeability factor since it stimulated the production of ascites, and increased interstitial accumulation of intravenously injected dye, Evans blue. At the same time, Criscuolo et al. (1988) had partially purified a protein from gliomas that also increased Evans blue dye extravasation. Furthermore, they subsequently showed that this factor increased intracellular calcium concentration [Ca2+]i in cultured endothelial cells (Criscuolo et al. 1989). In June 1989 Ferrara and Henzel published the partial amino acid sequence of a peptide purified from pituitary folliculostellate cells (Ferrara & Henzel, 1989) that stimulated mitosis in cultured endothelial cells. They termed this vascular endothelial growth factor, or VEGF. A fourth group also identified the peptide secreted by the hepatocarcinoma cell line, and in November of that year showed that it stimulated angiogenesis and was a specific and potent mitogen for endothelial cells (Connolly et al. 1989). In December 1989 both groups described the complete complementary DNA sequences encoding VEGF and VPF (Keck et al. 1989; Leung et al. 1989), and these turned out to be identical. The sequence described a protein that had some homology to the platelet-derived growth factor family of polypeptides. Subsequently, the protein was also purified using an angiogenesis assay, and termed vasculotropin, since it potently stimulated endothelial cell migration (Favard et al. 1991). This peptide is now referred to as VEGF. The most widely studied form of vascular endothelial growth factor (VEGF165 ) is a 38-kDa homodimeric peptide. It is one of a family of VEGFs that now include VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF) (Ferrara, 2001). The mRNA encoding VEGF is differentially spliced to form four common and three rare isoforms that vary from 121 to 206 amino acids in humans (one less in rodents) from eight exons (Houck et al. © Anatomical Society of Great Britain and Ireland 2002

1991; Tischer et al. 1991; Poltorak et al. 1997; Jingjing et al. 1999; Whittle et al. 1999). The VEGF molecule consists of a number of different functional domains coded by those exons (Fig. 1). Exon 1 encodes the first 23 moieties of a 26 amino acid signal sequence. Exon 2 contains the N terminus of the protein, and exon 3 the dimerization domain (Potgens et al. 1994). VEGF family members bind to three known receptors – VEGF-R1 (previously called flt-1) (De Vries et al. 1992), VEGF-R2 (flk-1 in the mouse, kinase insert domain receptor, KDR in humans) (Terman et al. 1992) and neuropilin (Soker et al. 1998). Exon 3 encodes the VEGF-R1 binding domain, exon 4 the VEGF-R2 binding domain (Keyt et al. 1996a), and the terminal part of exon 7 the neuropilin binding domain (Soker et al. 1996). Differential splicing from exon 5 to exon 8 generates the different isoforms of VEGF. Exon 6 (spliced out in the most common forms, VEGF121 and VEGF165) and exon 7 provide heparin binding affinity. A plasmin cleavage site towards the end of exon 5 means that when VEGF is digested with plasmin a 110 amino acid peptide is formed (VEGF110) which loses the ability to stimulate mitosis, indicating that the mitotic ability must reside in carboxyl terminus of VEGF. There are four cystine bonds in the longer common forms of VEGF (VEGF165 and VEGF189) that help to provide the VEGF dimers with their distinctive shape. In vitro studies have shown that VEGF acts as a homodimer that binds to the three different receptors, and that it can stimulate endothelial mitosis, migration, increased permeability of endothelial monolayers and even that it can stimulate tube formation in vitro (Iwasaka et al. 1996). Many of the signalling pathways stimulated by VEGF in vitro have been determined. In fact VEGF appears to be able to stimulate almost all known signalling pathways in endothelial cells in culture. In a recent review, 46 different signalling molecules were identified as having been activated by VEGF stimulation of endothelial cells in culture (Zachary & Gliki, 2001). To name a few, VEGF has been shown to stimulate phospholipase C and A (PLC, PLA) (WheelerJones et al. 1997; Wu et al. 2000), Thromboxane A2 (Nie et al. 2000), Protein kinase B (Akt), and PI3 Kinase (Gerber et al. 1998), ras (Guo et al. 1995), rho (Gingras et al. 2000), mitogen-activated protein kinase (MAPK, or ERK) and its kinase (MEK) (Parenti et al. 1998), focal adhesion kinase (FAK) (Wu et al. 2000), pyk-2 (Liu et al. 1997), fos (Seetharam et al. 1995), [Ca2+]i (Brock et al. 1991), nitric oxide (Parenti et al. 1998), and protein


Fig. 1 Exon structure of the VEGF mRNA. A. Structure of the mRNA encoding VEGF206. The amino acid sequence corresponding to the mRNA sequence is given underneath the graphical representation. The functional domains are also shown. B. Differential splicing results in different isoforms with differing properties. Splicing is specific to particular tissues (e.g. VEGF183 is only found in the iris). Coloured bars are coding regions.

kinase C (PKC) (Xia et al. 1996). What is more, inhibition of most if not all of these abolishes at least one effect (often mitosis) and sometimes many effects of VEGF on endothelial cells in vitro (Zachary & Gliki, 2001). Recent studies show that it is often difficult to reproduce these findings in vivo (Bates et al. 2001; Pocock & Bates, 2001b, 2002).

What is vascular permeability? Although VEGF was originally purified as a permeability factor (Senger et al. 1990), the assays used to screen for activity, although useful for purifying a vasoactive factor, were seriously flawed as a method for determining the effect of a factor on vascular permeability (Miles & Miles, 1952). All the methods used determined the uptake of a tracer into the tissue. This tracer was usually a high-molecular-weight dye such as Evans blue albumin (Senger et al. 1990), although occasionally radioactive tracers such as 125I-albumin were used (Collins et al. 1993). The parameters measured in these assays were therefore assessments of the amount of

tracer solute in the tissue. It has been well established that the rate of tracer movement into the tissue depends on a variety of factors including blood flow, capillary pressure, surface area available for exchange and heterogeneity of vascular supply as well as permeability (Curry, 1984; Taylor & Granger, 1984; Levick, 1991; Michel, 1996; Bates et al. 1999). All of these factors have been shown to be affected by VEGFs (Bates & Curry, 1996; Ku et al. 1993; Wu et al. 1996; Bates et al. 1999), and therefore this type of assay is particularly unsuitable for the determination of the permeability, or the mechanism of the permeability-enhancing effect of VEGF (for review see Bates et al. 1999). In order to determine the mechanisms through which VEGF acts to increase vascular permeability, some of these signal transduction pathways have been investigated in vivo using a model that measures vascular permeability independently of the other haemodynamic factors, and investigates the cellular changes that occur in endothelial cells lining blood vessel walls during this change in permeability. A modification of the Landis Michel technique has been © Anatomical Society of Great Britain and Ireland 2002


Fig. 2 Measurement of hydraulic conductivity, reflection coefficient and compliance in single capillaries. A. The frog is anaesthetized and an incision made in the skin and body wall and the ileum draped over a pillar. Three micropipettes – a restraining rod, an occluder and a cannulation pipette – are used. B. Schematic diagram of the mesentery during measurement. C. Schematic side view of a vessel during perfusion before (solid lines) and during (dashed lines) occlusion.

used for measurement of vascular permeability in the mesentery of anaesthetized animals (usually frogs or rats) (Michel et al. 1974). A detailed description of this technique has been given many times before and the reader is referred to a number of excellent reviews of how this technique is used (Landis & Pappenheimer, 1963; Curry et al. 1983; Michel, 1984; Levick, 1991; Curry, 1992). Briefly, the measurement of fluid flux (Jv) across a single capillary is measured by cannulating the vessel with a glass micropipette and perfusing the vessel at a known pressure (Pc ). The permeability to water (Lp ) is measured by occluding the vessel some distance downstream from the site of cannulation (Fig. 2). The fluid flow across the capillary wall is calculated from the velocity of marker cells recorded on videotape © Anatomical Society of Great Britain and Ireland 2002

(usually erythrocytes included in the perfusate at a low haematocrit), and the cross-sectional area (A) of the vessel (calculated from its diameter). The fluid flux per unit area (Jv /A) can be calculated since the length and diameter of the vessel are known. Since the permeability to water (Lp, or hydraulic conductivity) is by definition the fluid flux per unit pressure difference per unit area, it can be calculated from Starling’s equation by plotting measured fluid flux against the applied pressure (see Fig. 3). The effective oncotic pressure difference between tissue and plasma σ(πp − πi) equals the hydrostatic pressure difference between the capillary (Pc) and the interstitum (Pi), and is the point at which no flow occurs. Therefore the effective oncotic pressure can be


Fig. 3 The effect of VEGF on hydrau ic conductivity and reflection coefficient. The filtration rate per unit area measured across a single capillary is plotted against the pressure at which the measurements were made. The slope of the regression line gives the hydraulic conductivity (Lp) according to Starling’s equation. When filtration rate is theoretically zero (xintercept), the hydrostatic pressure in the vessel equals the effective oncotic pressure of the perfusate (σ∆π). In this figure measurements were made on the same vessel before (day 1, triangles) and 24 h after perfusion with VEGF (day 2, squares). Lp, was increased 12.8-fold, but σ∆π was not altered.

measured (Michel & Phillips, 1987). Since, at high filtration rates, this value is equal to the product of the perfusate oncotic pressure and the square of the reflection coefficient to the macromolecules that exert an osmotic pressure across the wall (σ2πp) then the oncotic reflection coefficient (σ) can also be calculated using this method: Jv /A = Lp[(Pc − Pi) − σ(πp − πi)] (Starling equation). One additional advantage of this technique is that the compliance of the capillary wall can also be measured. A small change in pressure (e.g. 10 cmH2O) will distend the vessel by a fraction of a nanometre. However, this will result in a measurable movement of the marker red cells along the vessel (Smaje et al. 1980), because the submicrometre change in radius results in a rapid volume flux that is proportional to the length of the vessel. The volume flux results in a red cell jump that can be measured on the video tape. Therefore, the change in diameter per unit change in pressure (compliance) can also be measured (Bates, 1998b). Additionally, after measurement of these parameters, the pipette cannulating the vessel can be removed, allowing the mesentery to be replaced in the animal. With appropriate mapping of the macro- and microvascular architecture the same vessel may be located on subsequent anaesthetization of the animal and exposure of the mesentery so that permeability and compliance may be measured a second time (Bates & Curry, 1996). This method for measurement of hydraulic conductivity, reflection coefficient and microvascular compliance, under conditions of controlled flow and haemodynamic forces, enables true mechanistic

experiments to be carried out to determine the signal transduction pathways through which VEGF acts (Bates & Curry, 1997; Bates et al. 2001; Hillman et al. 2001; Pocock & Bates, 2001b). In addition, after (or in fact during) the permeability measurement, the vessel may be glutaraldehyde fixed. Thereafter, the vessel and surrounding connective tissue can be dissected from the mesentery and the vessel embedded and sectioned for electron microscopy (Clough et al. 1988). Ultrathin serial sectioning and reconstruction allows both qualitative and quantitative measurement of the ultrastructural changes that are stimulated by VEGFs (Neal & Michel, 1997).

What is the effect of VEGF on vascular permeability? The effects of VEGF on all three parameters that are a true measurement of vascular permeability – hydraulic conductivity, reflection coefficient and the diffusive permeability – have all been investigated in intact vessels (Bates & Curry, 1996; Wu et al. 1996; Neal & Michel, 1997; Bates, 1998). Many of these parameters have also been measured in endothelial cell monolayers in culture (Hippenstiel et al. 1998; Lakshminarayanan et al. 2000). However, since cultured endothelial cells form much leakier monolayers than the vessel wall (Albelda et al. 1988), are already in a state of growth factor stimulation (usually being grown with growth factors), and are grown independently of pericytes and other surrounding vascular structures (or circulating factors including red cells and leucocytes), we have concentrated on in vivo experiments rather than their in vitro counterparts. © Anatomical Society of Great Britain and Ireland 2002


VEGF perfusion results in a bi-phasic increase in hydraulic conductivity measured using the Landis micro-occlusion technique (Michel et al. 1974) – an acute transient phase lasting a few minutes, and a subsequent chronic phase that is established for at least 24 h (Bates & Curry, 1996). The acute phase is characterized by a rapid, transient increase in Lp in mesenteric microvessels of the frog (Bates & Curry, 1996) and the rat (Neal & Michel, 1998a). This increase was, on average, seven-fold greater than control Lp in the frog, yet returned to control values during continuous perfusion with VEGF within 90 s. The response in the rat mesenteric capillaries was a smaller increase but with a similar time course. Interestingly, only a subset of vessels (about two-thirds) perfused with VEGF responded by increasing Lp (Bates & Curry, 1996). Additionally, a responsive vessel is not capable of giving a second response for at least 15 min afterwards (Bates & Curry, 1997). This transient increase has been extensively investigated to determine the signal transduction pathways responsible (see below). The chronic response to perfusion with VEGF has also been determined (Bates & Curry, 1996). The permeability of isolated perfused microvessels in situ was significantly increased 24 h after a 10-min perfusion with VEGF. The Lp of vessels that were exposed to a short bolus dose of VEGF was five times greater than on the previous day, and this increase correlates with the acute response (Bates, 1998b), suggesting that the chronic increase is associated with the acute increase. VEGF was still capable of increasing permeability, acutely and transiently. The chronic increase in permeability resolves over a further 48 h. The effect of VEGF on the diffusive permeability to albumin (Ps ) has been determined in isolated perfused rat coronary vessels (Wu et al. 1996). The rate of albumin flux across 50-µm-diameter coronary venules (dissected and mounted on glass pipettes, in the manner of a perfusion myograph) was measured under conditions in which haemodynamic factors were controlled (surface area measured, pressure differences controlled and defined interstitial and plasma tracer concentrations). Ps was shown to be transiently increased two- to threefold in these vessels, and again resolved over a period of a few minutes (Wu et al. 1996). Recently, these measurements were repeated in the frog mesenteric microcirculation by Fu et al. using perfusion of capillaries in situ, and the diffusive permeability to albumin was also increased two- to three-fold (Fu, 2001). Mathematical modelling of the changes in transvascular pathways © Anatomical Society of Great Britain and Ireland 2002

suggest that this increase could be due to a 2.5-fold increase in the pore diameter through the vessel walls. Interestingly, a 2.5-fold increase in pore diameter would also result in a seven-fold change in hydraulic conductivity (Lp is dependent on the fourth power of the radius of a pore, whereas diffusive permeability is proportional to the square of the radius). It remains to be seen whether this correlates with the ultrastructural changes measured in these vessels (discussed later). The oncotic reflection coefficient (σ) to albumin has not been measured during acute perfusion with VEGF. However, σ measured during the chronic, sustained increase in hydraulic conductivity reported 24 h after exposure to VEGF (Bates, 1998b) was not significantly different from that before exposure to VEGF, despite a 6.8-fold mean increase in Lp (Bates, 1998b) (see Fig. 3) Vessels previously exposed to VEGF had a reflection coefficient of 0.87, compared to 0.84 before exposure. These findings show that vessels which are highly permeable to water may still retain selectivity to large-molecular-weight solutes. A significant oncotic pressure may therefore act across the vascular wall after stimulation by VEGF. This would limit the rate of fluid movement across the capillary wall and may help to prevent oedema having a significant impact in pathologies where VEGF is up-regulated and the vessels are chronically leaky, such as psoriasis (Bull et al. 1992; Detmar et al. 1994).

VEGF-stimulated signal transduction pathways Vascular endothelial growth factors form a family of angiogenic and lymphangiogenic growth factors. The most well studied forms, VEGF and VEGF-C, both stimulate endothelial cells through pathways that have been partially characterized both in vitro and in vivo. It is known which receptors are stimulated by which VEGFs and many of the signalling pathways have been identified for the different receptors. This review will concentrate on the current understanding of the pathways involved in VEGF-stimulated permeability in vivo, rather than in stimulating angiogenesis, endothelial cell migration, vasodilatation or migration.

The VEGF receptor responsible for increased permeability There are four known receptors for the VEGF family of cytokines, VEGF-R1, VEGF-R2 and neuropilin, as


described above, and VEGF-R3, also known as flt-4. VEGF-R3 is found predominantly on lymphatic endothelial cells, and there are few reports of VEGF-R3 expression on vascular endothelial cells in vivo. VEGF specifically activates VEGF-R1 and VEGF-R2 (Neufeld et al. 1999). Placental growth factor (PlGF) has been shown to specifically act on VEGF-R1 (Park et al. 1994), whereas VEGF-C is known to stimulate both VEGF-R2 and VEGF-R3 (Joukov et al. 1996). Recent studies have used this receptor-specific signalling to determine which VEGF receptors are responsible for the transduction of signals that result in increased vascular permeability (Hillman et al. 2001). VEGF is known to transiently and chronically increase hydraulic conductivity in the mesenteric microcirculation of rats and frogs. Frogs share a high degree of homology with humans in the amino acid sequence of their VEGF receptors. In fact, antibodies to human VEGF receptors specifically detect frog receptors, including those in mesenteric microvessels (Hillman et al. 2001). Further they can distinguish between VEGF-R2 and VEGF-R1. Interestingly, VEGF C, but not PlGF can stimulate both acute and chronic increases in hydraulic conductivity in a similar manner to VEGF (Hillman et al. 2001), suggesting that VEGFs increase permeability through stimulation of VEGF-R2 rather than VEGF-R1. This hypothesis is lent further weight by studies investigating the effect of VEGF receptor inhibitors on the VEGF induced increase in permeability. The development of VEGF-receptor inhibitors has been of significant interest for the development of novel treatments for angiogenesis-related diseases such as cancer, diabetic retinopathy and rheumatoid arthritis. A number of inhibitors are now commercially available from pharmaceutical companies that inhibit VEGF receptors. Although most of these inhibit both VEGF-R2 and VEGF-R1 [i.e. PTK787 from Novartis (Wood et al. 2000), or SU5416 from Sugen (Fong et al. 1999)], one of these compounds, ZM323881, has been developed by Astra Zeneca and is specific to VEGF-R2 (Whittles et al. 2001). A study of the effects of VEGF on hydraulic conductivity of frog mesenteric microvessels has shown that ZM323881 inhibits the acute permeability response to VEGF at doses much lower than that which inhibited VEGF-R1 (Whittles et al. unpubl. obs.). In fact the same study showed that ZM323881 inhibited VEGF-induced phosphorylation of VEGF-R2 in frog tissue, but did not affect VEGF-R1 phosphorylation. PTK787 also inhibited VEGF-stimulated permeability

(Pocock & Bates, 2001a), and tyrosine phosphorylation of both VEGF-R1 and VEGF-R2 (Whittles et al. 2001). These studies, combined with the PlGF and VEGF-C results show that VEGFs stimulate increased Lp through VEGF-R2 phosphorylation.

The role of the signalling molecules – PLC and MAPK VEGF-R2 activation has been shown to result in stimulation of a variety of downstream signalling pathways including phospholipase C (Guo et al. 1995), calcium entry (Bates & Curry, 1997), nitric oxide synthase activation (Wu et al. 1996), raf activation, protein kinase C (Doanes et al. 1999), PI3 kinase, MAPK and MEK activation (Parenti et al. 1998), as discussed above. Although many of these pathways have been shown to be involved in increased vascular permeability, experiments to determine the signalling pathways through which VEGF increases permeability have only recently been completed and published (Bates et al. 2001; Pocock & Bates, 2001b). The first question is whether VEGF increases permeability through activation of MEK or PLC? As shown in Fig. 4(a,b), studies on frog mesenteric microvessels have shown that inhibition of MEK with PD98059 did not inhibit the increase in Lp brought about by VEGF (Bates et al. 2001), whereas PLC inhibition with U73122 did block the increase (Pocock & Bates, 2001b). These studies conclusively showed that Lp was increased through PLC activation. However, MEK inhibition did block the chronic increase in vascular compliance brought about by VEGF, despite having no effect on the chronic increase in Lp (Bates et al. 2001). It appears therefore that there are two different signalling pathways stimulated by VEGF that can reach their measurable endpoints (increased Lp and compliance) independently of each other. This indicates that the end results of VEGF stimulation (permeability and angiogenesis) may be stimulated or inhibited independently.

VEGF-induced calcium increases PLC is known to produce inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) from phosphoinositol 2 phosphate (PIP2). This results in release of calcium ions from intracellular stores, and subsequent calciuminduced calcium influx across the plasma membrane (Berridge, 1993). Increased intracellular calcium has © Anatomical Society of Great Britain and Ireland 2002


Fig. 4 The effect of various inhibitors on the VEGF mediated increase in permeability (Lp). A. MAPK (PD98059). B. PLC (U73122), C. Store release (Thapsigargin). D. Calcium influx (Nickel). Filled triangles are VEGF alone; open circles, VEGF + inhibitor; error bars are standard errors of the mean.

been shown to be stimulated by VEGF in frog mesenteric (Bates & Curry, 1997) and rat coronary (Wu et al. 1999) microvessels in vivo, as well as endothelial cells in vitro (Brock et al. 1991). This increase can be blocked by inhibition of calcium entry, so appears to be due to calcium influx (Bates & Curry, 1997; Pocock et al. 2000). Furthermore, inhibition of this calcium influx using either nickel ions or calcium channel inhibitors blocks the increase in Lp brought about by VEGF (see Fig. 4d and Bates & Curry, 1997). It has also been shown that this increase in [Ca2+]i is not inhibited by depletion of calcium stores with thapsigargin – an inhibitor of the calcium store ATPase (Pocock et al. 2000). Furthermore, as shown in Fig. 4(c), thapsigargin does not inhibit the VEGF-mediated increase in Lp. This is in stark contrast to other inflammatory mediators such as ATP, which stimulates calcium increases (He et al. 1990) that are dependent on store release (are inhibited by thapsigargin), and induces permeability effects that are also blocked by thapsigargin perfusion (Pocock et al. 2000). It appears therefore that VEGF stimulates a calcium influx that is independent of store release, and hence probably IP3 independent. This finding led to the © Anatomical Society of Great Britain and Ireland 2002

suggestion that VEGF could exert its effects through production of diacylglycerol by PLC.

DAG/OAG and the TRP channels DAG is known to stimulate protein kinase C (PKC) activation. If IP3 is not involved in the VEGF mediated stimulation of increased permeability, then DAG is a strong candidate, possibly by activation of PKC. VEGF is known to stimulate PKC in mesenteric microvessels in vivo (Mukhopadhyay et al. 1998). However, perfusion with an inhibitor of PKC (bisindolylmaleimide, BIM), did not inhibit the acute VEGF-mediated increase in Lp in frog mesenteric microvessels (Pocock & Bates, 2001b). There is one report that BIM inhibited the VEGF-mediated increase in solute permeability (Wu et al. 1999). In this investigation of the effects of BIM on the diffusive permeability to albumin, VEGF was perfused at 10-min intervals. However, we have demonstrated that following VEGF-induced increases in Lp, vessels must be washed out for at least 15 min before VEGF will evoke another response (Bates & Curry, 1997). Wu et al., however, neither repeated the experiments giving BIM


and VEGF first and then giving VEGF alone, nor demonstrate that giving VEGF alone on two successive occasions gave a comparable increase in permeability. Therefore, the inhibition described by Wu et al. is likely to be simple tachyphylaxis. If IP3 and PKC are not involved in the VEGF-mediated increase in Lp, then how might DAG stimulate increased permeability? DAG is broken down by the enzyme DAG lipase to arachidonic acid. It is possible that this results in increased prostacyclin production, as described for VEGF in vitro (Wheeler-Jones et al. 1997) and this in turn increases permeability. However, studies using the DAG lipase inhibitor RHC80267 have shown that the VEGF-mediated response is unaffected by this drug (Pocock & Bates, 2001b). In fact, RHC80267 actually stimulates a transient increase in permeability. It is therefore likely that DAG itself can increase permeability by stimulating calcium influx. Recently, it has been shown that the non-selective cation channels TRPC6 and TRPC3 are directly activated by diacylglycerol analogues such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) (Hofmann et al. 1999). Interestingly, perfusion of single vessels with OAG results in a transient and rapid increase in permeability, very reminiscent of the VEGF response, and also can stimulate calcium entry into endothelial cells in microvessels in vivo (Pocock & Bates, 2002). This suggests that DAG, once stimulated by VEGF, can stimulate calcium entry through non-selective cation channels independently of store release, possibly by acting directly on DAG-sensitive TRP channels such as TRPC6 or TRPC3.

What does increased calcium do? Once increased intracellular calcium has been stimulated, the associated permeability increase occurs through the development of transvascular pathways for fluid and solute flux. This is likely to occur through calcium activation of nitric oxide synthase, since NOS inhibitors block both the acute (Wu et al. 1996) and the chronic increase in permeability brought about by VEGF (Bates, 1998b). The mechanism behind the link between NOS and the transvascular pathways is still very poorly understood, but may be through activation of guanylyl cyclase and production of cGMP (Wu et al. 1996). The transvascular pathways that occur have been the subject of much debate and this will be discussed in this section. In vessels exposed to VEGF, a number of ultrastructural features of endothelial cells forming these vessels

have been described. However, very few of these have been described in vessels in which the permeability has actually been measured. The major pathways for transvascular flux of fluid and solutes are the interendothelial cleft (Majno et al. 1969), fenestrations (Levick & Smaje, 1987; Neal et al. 1989), transcellular gaps (Neal & Michel, 1992) and vesiculo-vacuolar organelles (Dvorak et al. 1996).

The intercellular cleft The boundary between two endothelial cells is characterized by the formation of tight junctions, usually close to the luminal edge of the cells, and an overlapping cleft that may extend for some micrometres (Adamson et al. 1993). Fluid and small solutes are thought to pass through this cleft, which is relatively permeable to small polar solutes. Water passes through the cleft by bulk convection of fluid down the pressure gradient determined by the Starling pressures acting across the vascular wall (Michel & Curry, 1999). The tight junctions form a network of junctional strands that are overlapping and have occasional breaks in them (Adamson et al. 1993). Fluid and larger molecularweight solutes pass through these breaks. There is little or no evidence to suggest that these breaks become more or less frequent, or increase in size after treatment with VEGF. The permeability of the vessel wall will depend on the frequency and size of these breaks, as well as the length of the intercellular cleft (Weinbaum et al. 1992). It is possible that any or all three of these parameters are altered, although there is no direct evidence that VEGF specifically changes these. This lack of evidence is predominantly because the frequency or size of the breaks, or the length of the cleft has not been measured during the VEGFmediated permeability increase.

Transcellular gaps The presence of gaps in the vascular wall after stimulation with inflammatory cytokines such as histamine and bradykinin has been known for many years (Majno et al. 1969). However, it is relatively recently that the three-dimensional structure of these gaps has been elucidated in capillary walls after exposure to VEGF (Feng et al. 1997; Neal & Michel, 1997). Neal and Michel have shown that, in the frog mesenteric microvessels, most of these gaps are actually through endothelial © Anatomical Society of Great Britain and Ireland 2002


cells when the vessels are examined during the acute increase in permeability. Interestingly, in frog mesenteric microvessels serial section reconstruction of ultrathin electron microscopy sections showed the presence of large transendothelial gaps stimulated by VEGF. These gaps were not seen in rat mesenteric microvessels (Neal & Michel, 1998a). There are a number of differences, other than species, between the frog and rat microcirculations. Examples are: 1 The endogenous pressure in the capillaries [experiments were done at the same pressures (30 –40 cmH2O) in frog and rat, although the frog capillary pressure is normally 5–10 cmH2O, whereas the rat is 20– 30 cmH2O]. 2 The microvessels in rat are venular but capillaries in the frog. 3 The magnitude of the increase in permeability being greater in the frog experiments. Any or all of these differences may account for the different nature of the ultrastructural changes. There is strong evidence to show that these gaps are transient. If ferritin, used as a tracer, is perfused into the vessel the tracer is found not only immediately outside the gap trapped by the basement membrane but also on the abluminal side of intact endothelium. This suggests that the gaps had allowed ferritin out, but had subsequently closed (Neal & Michel, 2001). Interestingly, these transcellular gaps can also be seen in vessels with a chronically increased hydraulic conductivity, 24 h after perfusion with VEGF. Figure 6 shows a series of serial sections of a vessel perfused 24 h earlier with VEGF, and the permeability measured. In this vessel, the hydraulic conductivity was significantly increased 24 h after VEGF perfusion. It can be seen from the electron micrographs, and the associated three-dimensional reconstruction from line drawings of the membrane of the endothelial cells, that the gap in the cell passes through the endothelial cell. In this vessel two gaps were seen and reconstructed, from sampling of merely a 20-µm section of vessel. Further examination of vessels exposed to VEGF also demonstrated transcellular gaps, and their frequency was greater in vessels with increased hydraulic conductivity (Hillman et al. 2000).

Fenestrations One of the earliest noted effects of VEGF on the microcirculation was the description of fenestrations formed © Anatomical Society of Great Britain and Ireland 2002

Fig. 5 Diagram to show signalling pathways for increased vascular permeability. Numbers show the site of actions of the following inhibitors. (1) U73122, (2) RHC80267, (3) BIM, (4) PD98059.

in mesenteric vessels close to the injection site of VEGF (Roberts & Palade, 1995). This fenestrated endothelium has been reproduced in vitro in endothelial cell cultures (Esser et al. 1998), but was not reproduced in the vasculature of the eye in monkeys injected with VEGF (Hofman et al. 2000). Fenestrations have been described in single perfused microvessels exposed to VEGF in both frog and rat (Neal & Michel, 1998a).

Vesiculovacuolar organelles (VVOs) Probably the most striking ultrastructural change brought about by exposure to VEGF is the development of vesiculo-vacuolar organelles (VVO) and vacuolar channels in response to VEGF (Feng et al. 1996; Neal & Michel, 1998a). These channels have been shown to occur during both the acute and the chronic increase in permeability, as well as in tumour vessels. They consist of a series of small (50–100 nm) vesicles connected together by fenestrations, or a more amorphous structure that contains both vacuoles and vesicles, with fenestrations at each side of the cell (VVO) (Feng et al. 1999). Three-dimensional reconstruction of these VVOs show that the vesicles form a continuous chain from the luminal to the abluminal side of the vessel. One form of these VVOs is the vacuolar channel that essentially spans the endothelium in a single structure – a


Fig. 6 Serial section reconstruction of a transcellular gap. A. Ten consecutive 100nm-thick sections were examined under the electron microscope. A region of one endothelial cell of a vessel exposed to VEGF-C 24 h previously, and in which Lp was measured, is shown here. It can be seen that the gap is present in section S167 through S171, but not before or afterwards. The interendothelial junction was more than 1 µm to the left of this gap. B. A reconstruction of the gap formed by tracings of the cell membrane of the region shown.

multifenestrated vacuole (Neal & Michel, 1998a). These VVOs have been described in both acute and chronically increased permeability stimulated by VEGF and VEGF-C (see Fig. 7 and Hillman et al. 2000). These have been shown to occur in frog as well as rat mesenteric microvessels, in both capillaries (in frog) and venules (in frog and rat). Interestingly, VVOs, but not transendothelial gaps, were seen in rat vessels (Neal & Michel, 1998a), whereas both have been described in frog vessels (Neal & Michel, 1997; Hillman et al. 2000). The possible reasons for this difference are described above. In addition, VVOs were seen in VEGF-stimulated vessels only, were not apparent in vessels where the permeability had been increased by exposure to histamine, or the calcium ionophore A23187 (Neal & Michel, 1998b).

A unifying theory Dvorak has suggested that the development of transcellular gaps, VVOs, vacuoles and fenestrations may be all part of the same process, but at different times, and with differing degree of response (Feng et al. 1999). For instance, calcium-mediated fusion of vesicles to form a VVO with fenestrated diaphragms may be the initial ultrastructural event, followed by the breakdown of the diaphragms between the vesicles to result in the formation of a vacuole. If this structure – with fenestrations on either side, were to lose one fenestration, then the formation of a single fenestrated unit through the endothelial cell would occur (see Fig. 7). With continued, or more potent, stimulation, or with other concurrent events such as increased © Anatomical Society of Great Britain and Ireland 2002


frog vessels perfused at close to physiological pressure (15 cmH2O) produce a greater increase in Lp compared to when perfused at a higher pressure (30 cmH2O) (Neal & Bates, 2002). Therefore, the event that stimulates the breakdown of the fenestration, if indeed this hypothesis is correct, is not pressure but may be another factor such as shear stress.

Summary

Fig. 7 Vesiculovacuolar organelles in vessels with chronically increased permeability 24 h after perfusion with VEGF. (A). Ultrathin (40 nm) serial sections of a VVO, showing multiple vesicles connected together from the luminal to the abluminal side. B. Line drawings of the endothelial cells showing the inside of the vesicles connected together (shaded). Dotted lines are areas of indistinct membranes; solid lines are clearly visible membranes. C. Thin (100 nm) section through a VVO in an endothelial cell 24 h after exposure to VEGF. These VVOs were significantly more prevalent in vessels exposed to VEGF than those not exposed. L = lumen, V = vesicle, I = interstitium. Scale bar = 200 nm.

pressure, then this fenestration may be removed to result in a transcellular gap. A process of recovery would then be stimulated which would result in closing of the gap. We have hypothesized that the event that might stimulate the breakdown of the final endothelial fenestration may be the increased pressure (relative to normal pressure) in the frog compared to the rat vessels, which is why transcellular gaps are seen in frog capillaries, but not rat post-capillary venules. However, recent experiments have shown that in fact © Anatomical Society of Great Britain and Ireland 2002

VEGF is clearly a potent stimulator of endothelial cells. Activation by VEGF of its receptors leads to a multifaceted activation of downstream signalling pathways. Some of these, but not all, are involved in increasing microvascular permeability. In order to determine the signal transduction pathways through which VEGF can exert its effect on permeability, it is necessary to use methodology that can measure permeability changes directly in vivo, rather than inaccurate measurements of factors affected by permeability changes, such as dye extravasation. Using these specific technologies it has been possible to define some of the pathways responsible for the increase in permeability, and these experiments have shown that VEGF increases permeability acutely by acting on its receptor VEGF-R2 to stimulate PLC that results in DAG production. DAG directly stimulates calcium influx, through store-independent cation channels such as the TRP channels. This calcium influx then results in increased intracellular calcium that stimulates NOS to produce nitric oxide. This activates guanylyl cyclase and cGMP is produced. The link between cGMP and increased permeability is not known, but it may rest on increased vesicle fusion to form transcellular pathways through VVOs, fenestrations and ultimately transcellular gaps. These gaps are transient and close up within a few minutes of stimulation. Many of the mechanisms underlying the chronic increase in permeability are still waiting to be defined.

Acknowledgments Supported by the Wellcome Trust (050742, NJH, 58083 CRN), and the British Heart Foundation (PG97198, PG2000057 TMP, FS98023, BB200003, DOB).

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