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Table of Contents
Vol. 39, No. 5, 2002
Issue release date: September–October 2002
Section title: Review
J Vasc Res 2002;39:375–390
(DOI:10.1159/000064521)

Transendothelial Transport: The Vesicle Controversy

Rippe B. · Rosengren B.-I. · Carlsson O. · Venturoli D.
Department of Nephrology, University Hospital, Lund, Sweden
email Corresponding Author

Abstract

The relative contribution of transcytosis vs. large pore transport to the passage of macromolecules across microvascular endothelia has been a controversial issue for nearly half a century. To separate transcytosis from ‘porous’ transport, the transcytosis inhibitors N-ethylmaleimide (NEM) and filipin have been tested in in situ or ex vivo perfused organs with highly conflicting results. In continually weighed isolated perfused organs, where measurements of pre- and post-capillary resistances, capillary pressure and capillary filtration coefficients can be repeatedly performed, high doses of NEM and filipin increased the bulk transport of macromolecules from blood to tissue, despite producing vasoconstriction. By contrast, in in situ perfused organs, marked reductions in the tissue uptake of albumin tracer have been observed after NEM and filipin. When tissue cooling has been employed as a means of inhibiting (active) transcytosis, results have invariably shown a low cooling sensitivity of albumin transport, compatible with passive transendothelial passage of albumin. This observation is further strengthened by the commonly observed dependence of albumin transport upon the capillary pressure and the rate of transcapillary convection. For low-density lipoprotein (LDL), a cooling-sensitive, non-selective transport component has been discovered, which may be represented by filtration through paracellular gaps, lateral diffusion through transendothelial channels formed by fused vesicles, or by transcytosis. From a physiological standpoint there is little evidence supporting active transendothelial transport of most plasma macromolecules. This seems to be supported by studies on caveolin-1-deficient mice lacking plasmalemmal vesicles (caveolae), in which there are no obvious abnormalities in the transendothelial transport of albumin, immunoglobulins or lipoproteins. Nevertheless, specific transport in peripheral capillaries of several hormones and other specific substances, similar to that existing across the blood-brain barrier, still remains as a possibility.

© 2002 S. Karger AG, Basel


  

Key Words

  • Capillary permeability
  • Transcytosis
  • Transport
  • Macromolecules
  • Pores
  • Caveolae

 Introduction

The exchange barrier between plasma and tissue, i.e. the walls of the capillaries and post-capillary venules, is very highly permeable to small solutes and water, yet nearly impermeable to macromolecules. This relative impermeability of microvascular walls to large solutes is a prerequisite for the maintenance of a fluid equilibrium between plasma and interstitium according to the classical Starling principle. Still, macromolecules do cross microvascular walls to provide the tissues with antibodies, protein-bound hormones, opsonins, cytokines and chemokines and a number of other high-molecular-weight substances that need to percolate through the interstitial space.

The endothelial pathway involved in macromolecular transport is conceivably additional to that (or those) mainly available to small hydrophilic solutes. For over 4 decades there have been two partly conflicting hypotheses for this pathway. One view is that macromolecules are shuttled back and forth by plasmalemmal vesicles across the capillary endothelium by ‘transcytosis’. The other view favours a completely passive transport mode, namely convective protein transport across large pores, located either paracellularly or transcellularly, i.e. ‘porous transport’ (fig. 1). The relative contribution of large pores vs. vesicular transport may vary from organ to organ and also with respect to the size and nature of the particular macromolecule and/or the need for specific transport. The role of transcytosis vs. porous transport has been extremely controversial over the years, and several previous excellent reviews have been written on the ‘vesicle controversy’ [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20]. The aim of the present overview is to summarize the latest developments in the field based on investigations using classical physiological techniques and on recent studies on the cell biology of the capillary endothelium.

FIG01

Fig. 1. Sketch of a section of a continuous capillary endothelium showing three tentative transcapillary exchange routes for macromolecules: 1 refers to (widened) interendothelial clefts; 2 to transcytosis (vesicular transport); 3 to potential channels formed by fusion of plasmalemmal vesicles. BM = basal membrane; N = nucleus of endothelial cell. From Rippe and Haraldsson [4].

 

 Protein Transport through Large Pores

In the early 1950s, Pappenheimer et al. [21], based on osmotic transient experiments utilizing a number of small solutes as molecular probes, formulated the so-called ‘pore theory’ of capillary permeability. It predicts the diffusion across capillary walls of small hydrophilic solutes through water-filled channels or pores of radius ∼40 Å. In continuous endothelia, such as those in skin, skeletal muscle, connective tissue, peritoneum and the brain, these small pores are assumed to be morphologically represented by structures in the clefts between endothelial cells [11, 22]. In 1956, Grotte [23] presented data based on experiments investigating the sieving of infused high-molecular-weight dextran fractions between plasma and lymph in the dog, strongly suggesting a dual (bimodal) size selectivity of the barrier partitioning blood from lymph Grotte observed that large-size dextrans appeared in canine leg lymph in a concentration that decreased rapidly as a function of increasing molecular size for molecules ≤40–45 Å in radius. Larger dextrans still appeared in the lymph, but their lymph to plasma concentration ratios were only moderately affected by increases in molecular size. Furthermore, small plastic spheres (methylmetacrylate) of an average radius of 400 Å (range 350–450 Å) did not appear in the leg lymph at all. Grotte therefore suggested that a few large pores, or ‘capillary leaks’, with a radius of 116–350 Å, and representing only one part per 30,000 of the small-pores, accounted for the transcapillary passage of plasma proteins. Such a bimodal capillary selectivity concerning molecular size has subsequently been amply demonstrated in a great number of tissues and organs using lymphatic protein flux analyses [5, 7, 8, 14]. On average, the small-pore and large-pore radii are entirely consistent with the parameters estimated by Grotte. In skeletal muscle, skin or in the peritoneum (in man), for example, the small-pore radius is usually estimated to be of the order of 45 Å, and the large-pore radius usually of the order of 250 Å (fig. 2). Large pores usually account for 2–8% of the total capillary membrane filtration coefficient, despite the fact that large pores contribute only by a fraction of a percent (∼0.2–0.5%) to the total diffusion (pore) area available to small solutes. Furthermore, whereas diffusion is the major mode of transport in small pores, large pore transport is more or less totally convection dominated in nature, for solutes of the size of albumin and larger [5, 24]. In continuous (non-fenestrated) endothelia a third porous pathway exclusive for water, aquaporin-1, accounts for a few percent of the total ultrafiltration coefficient (hydraulic conductance) [5, 11]. This pathway is of importance in conditions when large crystalloid disequilibria are created across the blood-tissue barrier, such as in peritoneal dialysis [17].

FIG02

Fig. 2. Semi-logarithmic plot of transperitoneal unidirectional clearances (in man) of solutes ranging in size from urea to IgM, using heteroporous theory for transmembrane transport. Solid line is simulated for a small-pore radius (rS) of 47 Å, a large-pore radius (rL) of 300 Å and setting the unrestricted pore area over unit diffusion path length (A0/Δ×) at 46,000 cm, when the total blood-to-peritoneal filtration rate was set at 1 ml·min–1·1.73 m–2 of body surface area. Reproduced from Rippe and Stelin [108].

 

 Transcytosis Concept

Ever since the discovery of the so-called ‘plasmalemmal vesicles’ by routine electron microscopy of endothelial cells [25], such plasmalemmal vesicles have been postulated to take part in the bulk transport of large (and even small) solutes across the endothelial barrier by transcytosis. Indeed, in routine electron microscopy, where fixation was performed at various times after injection of electron-dense macromolecular tracers, tracers have been observed to enter plasmalemmal vesicles and to later appear in intracellular vesicular profiles, and subsequently, in the interstitial space. These studies no doubt give the impression that transcytosis may be a quantitatively important transfer mode for macromolecules. However, the interpretation can also be that the transfer of large solutes to the interstitium has occurred through nearby large pores or through pathways established by random confluences of plasmalemmal vesicles, forming transcellular channels. Thus, the appearance of tracer within abluminal vesicles or membrane invaginations may be a secondary phenomenon, due to ‘back-filling’ of the vesicles by solute. Contrary to the back-filling hypothesis, dynamic electron microscopic studies of capillaries in the rete mirabile of the eel, using terbium as an electron-dense macromolecular tracer, have demonstrated the presence of abluminal patches of intravascularly given tracer at a considerable distance away from the intercellular junctions [26]. Such patches, bounded by pericytes, thereby hindering them, diffuse to the interstitium, regularly showed even higher tracer concentrations than plasmalemmal vesicles. This suggests that the tracer may have used a transcellular pathway. Such a transcellular pathway could be represented by channels formed by very transiently fused plasmalemmal vesicles. Recently, Schnitzer et al. [18], by the use of very specific antibodies against membrane proteins of vesicular origin (TX 3.833), demonstrated that transcytosis in the classical sense is actually possible in endothelium, i.e. that domains of the plasmalemma can indeed move from one side of the endothelial cell to the opposite. This, however, gives no clue as to the rate or magnitude of membrane traffic, i.e. the quantitative importance of transcytosis.

Two principally distinct transcytotic processes have been identified: receptor mediated transcytosis (involving binding of the macromolecule to a membrane receptor), originally thought to be primarily carried out by so-called ‘clathrin-coated vesicles’ (see below), and fluid phase transcytosis, by which a quantum of plasma or interstitial fluid may be ‘engulfed’ into a caveola, without the need for macromolecules to specifically interact with a receptor. A special variant of receptor-mediated transcytosis is ‘adsorptive transcytosis’, implying some kind of non-specific adsorptive mechanism to account for the interaction of the macromolecule with the plasmalemma. Receptor-mediated transcytosis, whether occurring by caveolae (see below) or clathrin-coated vesicles, is thought to occur for a number of plasma molecules including insulin [27], albumin [28, 29, 30, 31, 32, 33] (via albondin [33], formerly known as gp60 [30]), transferrin [34], ceruloplasmin [35], HDL [36], LDL [37] and orosomucoid (α1-acidic glycoprotein) [38].

 

 Vesicle Controversy. Pros and Cons


 General Features of Endothelial Transcytosis

During the last decade, much has been learnt about the cell biology of transcytosis [11, 18, 19, 20, 39, 40]. When a vesicle is formed, a ‘coat’ is apparently used as a device to sculpt the vesicle out of the plasmalemma. Coats are spherical protein shells consisting of polymeric proteins and of assembly (AFR) proteins. In endocytic vesicles, i.e. vesicles which fuse with lysosomes, the coat is thick, electron-dense and made up by clathrin. These so-called ‘clathrin-coated’ vesicles (or coated pits), have diameters up to 100 nm. By contrast, in the slightly smaller ‘caveolae’ (∼70 nm in diameter), which are smooth, flask-shaped plasmalemmal invaginations from the endothelial surface, rich in cholesterol, the structural coat protein is caveolin (caveolin-1 and 2). Caveolae are present not only on the surface of endothelial cells, but also in fibroblasts, adipocytes or cardiomyocytes. It is generally believed that caveolae are responsible for various important cellular processes ranging from signalling and mechano-transduction to cholesterol trafficking, cell growth and various transport processes, including transcytosis, endocytosis and ‘potocytosis’ [18, 19]. Potocytosis was originally described as a process of fusion and fission events occurring in between static invaginations of vesicular profiles and/or clusters of vesicles, which bypassed the lysosomes. The fusion/fission model was first described by Clough and Michel [41], and later further elaborated upon by Mineo et al. [19] and Andersson et al. [42] (see below).

The mechanisms responsible for vesicular transport are schematically outlined in figure 3 [39]. These processes must be energy dependent. First, the formation of the vesicle (vesicle budding) is triggered by GTP-dependent and ATP-dependent proteins (and possibly by Mg2+) that initiate the polymerization of caveolin-1 and -2 [43]. Somehow, the polymerization of caveolin results in the formation of a vesicle in an all-or-none fashion [18]. In the next step, some vesicles may undergo fission to be released from the plasma membrane, which is mediated by the GTPase, dynamin, at the neck of the vesicle [44, 45]. After detachment, the vesicle may now be able to pass through the cell, conceivably guided by cytosolic tubules or fibres and aided forward by hitherto unknown mechanisms. The transcytotic vesicle movement is likely to require energy, since the viscosity of the cytosol is considerably higher than that in free medium [46]. Furthermore, the cell interior is crowded with macromolecular structures, which, in case the vesicle is not guided and aided through the cytoplasm, would further impede the transcytotic process [46]. The vesicle and its target membrane have specific identifiers called ‘SNAP receptors’, or SNAREs (see below), which can bind to each other, eventually docking the vesicle to the target membrane, usually after minutes of transcellular passage [47]. A NEM-sensitive fusion protein, N-ethylmaleimide-sensitive factor (NSF), and soluble NSF attachment protein (SNAP) bind to the docking receptor complex, thereby disrupting the complex and initiating fusion, which occurs when NSF hydrolyzes ATP. The targeting receptors located on the vesicles are denoted v-SNAREs (cf. VAMP or synaptobrevin), which recognize and dock with their cognate t-SNAREs (cf. syntaxin) on the target membrane.

FIG03

Fig. 3. The figure illustrates the principal steps of the formation and shuttling of (coated) vesicles across a cell, as reproduced from Rothman and Wieland [39]. In case of endothelial vesicles the ‘coat proteins’ are thought to be represented by caveolins, mainly caveolin-1, which are integral membrane proteins. These proteins are thought to be retained in the membrane, and not shed, during the shuttling process. a Illustrates the principal steps of the vesicle formation (vesicle budding). Step (i): Assembly of caveolar constituents and subsequent polymerization of coat proteins (caveolin) in endothelial caveolae results in a mold for the plasmalemmal membrane, which will form a caveola. This process is dependent upon ATP, GTP and Mg2+ (not shown). Step (ii): Fission and detachment of the vesicle is dependent upon a GTPase, dynamin, forming a spring-like structure around the vesicle neck. Upon activation dynamin is thought to induce hydrolysis of GTP, which causes neck extension and fission of the caveola to form a free transport vesicle. Caveolae do not seem to loose their caveolin structure (unlike many so-called COPI-coated vesicles) during shuttling. b Targeting of vesicles occurs by the vesicle identifiers, named v-SNAREs, which can bind to their cognate t-SNAREs, present on the target membrane, resulting in the formation of a SNARE complex. The supposed vectorial transport across the cytosol of the caveola may require energy. c Vesicle fusion by NEM-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAPs) which bind to the SNAP-receptor (SNARE) complex and initiate fusion when NSF hydrolyses adenosine triphosphate (ATP). The latter step is thought to result in release of molecular cargo and incorporation of caveolar membrane into the target membrane. It should be noted, that for the sake of simplicity, a great number of intermediate steps and molecular mechanisms have been left out. Additional pieces of machinery and mechanistic details remain to be uncovered, or are found in other reviews [11, 18, 19, 20, 39, 40, 41, 42]. Reprinted with permission from Rothman and Wieland [39], copyright 1996 American Association for the Advancement of Science.

In summary, practically all the steps of the transcytotic mechanism, from the vesicle budding (and the polymerization of the coat proteins) and the vesicle detachment to the vesicle fusion and docking with the target membrane are apparently energy-dependent processes, requiring an intact cellular metabolic machinery to function. Furthermore, if receptors are involved, transcytosis would be saturable, i.e. it would follow Michaelis-Menten kinetics and not first-order kinetics. Also, NEM should be a tool to effectively inhibit transcytosis. Conceptually, vesicular transport should not be dependent upon capillary hydrostatic pressure, nor on the rate of convection across the endothelium.

In the following section we will look more closely at these general features of vesicular transport: (1) its active, energy-dependent nature, (2) its dissipative (non-convective) nature, (3) its sensitivity to chemical transcytosis inhibition or chemical fixation, and (4) morphological correlates to the passage of macromolecules across endothelia.

 Is Large Solute Transport Active or Passive? Effects of Temperature Reduction

Rippe et al. [48] investigated the effects of tissue cooling on transvascular albumin passage in the rat hindquarter microvascular bed during maximal arteriolar dilatation in experiments which were designed as paired, parallel perfusions of one test hindquarter at 14°C and one control at 36°C. As perfusate, oxygenated, heparinized horse serum or mixtures of horse serum and bovine serum albumin was used. Hindquarters were arranged for a gravimetric technique, allowing continuous monitoring of tissue weight, arterial and venous pressures and perfusate flow. Whereas the control animal had a rectal temperature of 36–37°C, the rectal temperature in the test animal could, as mentioned above, be reduced (via cooling of the perfusate) to 14–16°C. Radiolabelled serum albumin (125I-albumin, RISA) was added to the perfusate and perfusion was continued for 40–60 min, after which tracer-free perfusate was employed to effectively wash the hindquarters free from tracer (during 6–8 min). Muscle tissue samples were then taken and analysed with respect to content of fluid and RISA. Blood-to-tissue RISA clearance was calculated as the transvascular mass transfer of RISA per unit time and tissue weight divided by the plasma RISA concentration during the tracer perfusion.

At 14°C, compared to 36°C, the transcapillary RISA flux was reduced in due proportion to the increased viscosity, but not to the marked extent that the tissue metabolism was reduced. It was concluded that the transvascular albumin flux must be completely passive in nature. The low cooling sensitivity of transendothelial albumin transport was later confirmed by Rutledge [49] in experiments on single perfused frog mesenteric microvessels. Rutledge investigated the permeability of post-capillary frog venules to sodium fluorescein (a small solute), FITC-labelled albumin and fluorescently labelled low density lipoprotein (LDL) at ∼20°C and ∼5°C. Reducing the temperature lowered the permeability of sodium fluorescein by approximately 40%, i.e. in direct proportion to the estimated increase in viscosity (and to the reduction in absolute temperature), whereas the reduction in albumin clearance was approximately 50%. The latter reduction was not significantly different from the reduction in sodium fluorescein permeability. However, the reduction in LDL permeability was much larger. Furthermore, whereas there was a coupling between fluid flow and LDL transport at 20°C, this coupling was more or less completely abolished at ∼5°C. Rutledge’s data seem to indicate that approximately 50% of the LDL transport may be occurring through an active pathway that is completely inhibited at 5°C. Rutledge suggested that LDL may pass through the microvasculature by lateral diffusion in the lipid membranes of large transendothelial channels, conceivably formed by fusion of vesicles. Alternatively, 50% of LDL transport may be occurring through bulk filtration through large pores, while the remaining 50% may be dependent upon other transport mechanisms, possibly transcytosis or transport through actively formed (inflammatory) gaps (i.e. very large pores). The ratio of albumin clearance to that of LDL in Rutledge’s experiments was much higher than that found in vivo across the peritoneal membrane or in cutaneous suction blister fluid [50, 51]. The reason for this discrepancy is not clear, but the absence of plasma orosomucoid in the perfusate in Rutledge’s experiments may have markedly increased the capillary albumin clearance (∼3-fold) above its physiological level [52].

In recent experiments in the isolated perfused rat lung Rippe and Taylor [53] measured the RISA clearance and the RISA reflection coefficient (σalb) at 35°C and 22°C. Albumin clearance during isogravimetric conditions decreased by 27% by cooling the lung to 22°C, which is exactly equal to the increment in viscosity occurring for the temperature reduction imposed. Again, the results demonstrate that the cooling sensitivity of albumin clearance across continuous vascular walls is consistent with passive transport.

Recently, Rosengren et al. [54] examined the clearance of some macromolecules, i.e. of albumin, IgG, IgM and of LDL, in peritoneal microvessels in a rat model of peritoneal dialysis in vivo, reducing the rat body temperature from 37°C to 19°C. 51Cr-EDTA and glucose were used as markers of paracellular transport. At 37°C the relationships between albumin, IgG, IgM and LDL clearances across the rat peritoneum were entirely in accordance with measurements performed during clinical peritoneal dialysis in man, consistent with convective protein transport through large pores (of radius ∼270 Å) [50]. During cooling, the clearances of all markers were markedly reduced. Assuming that IgG, LDL and IgM are all convected through large pores, cooling would reduce transport by the viscosity factor (in this case to 65% of control) and by the concomitant fall in mean arterial pressure (in this case to 45% of control) occurring during cooling. The combined effect of viscosity changes and hemodynamic effect would lead to a reduction of macromolecular clearance through large pores by approximately 70% (to 0.65 × 0.45 = 0.30 of control). This was, more or less, exactly the fall in IgG and albumin clearance observed. However, the clearances of LDL and IgM fell slightly, but significantly, more (i.e. by 80%). Also, small solute clearances fell dramatically (by 75–80%) due to the combined effect of the viscosity increments and of the (marked) blood flow reductions (assessed by laser Doppler flowmetry), and conceivably, also by derecruitment of effective vascular surface area occurring at low temperature.

The data may be interpreted to indicate that the large pore radius was moderately reduced during cooling, or alternatively, that a portion (maximum 50%) of the LDL (and IgM) transport may have occurred through an active, non-size-selective and temperature dependent pathway, whereas this pathway may have contributed only 10–15% to the albumin and IgG transport, and progressively less to the transport of solutes smaller than albumin. Such a non-size-selective pathway, existing at 37°C, but not at 19°C, may be represented by actively formed ‘intercellular gaps’ at 37°C (see below), absent at 19°C, or alternatively, by other mechanisms, such as transcytosis. Essentially larger solutes than IgM and LDL have to be investigated with respect to their transcapillary transport and temperature sensitivity to really settle whether a non-size-selective, active mechanism is involved in transendothelial transport of very large macromolecules.

 Is There a Coupling of Protein Transport to the Rate of Transcapillary Filtration?

In practically all previous lymphatic protein flux analysis a coupling of large solute transport to transcapillary volume flow has been demonstrated. This seems to hold even for macromolecules as large as fibrinogen, IgM and LDL [7, 8, 14, 49]. In the study by Rippe et al. [48] mentioned above, there was, indeed, a clear-cut coupling of albumin transfer to transcapillary filtration rate, the coupling coefficient being of the order of 0.06–0.08, which corresponds to an albumin reflection coefficient (σalb) of 0.92–0.94 (fig. 4). Furthermore, somewhat surprisingly, the clearance (Cl) of RISA during isogravimetric conditions, proved to be dependent upon the plasma (perfusate) colloid osmotic pressure (πp). Since ‘clearance’ represents the rate of mass transfer divided by the plasma (tracer) concentration, it should be completely independent of the plasma protein level. However, the dependence of Cl upon πp can be understood if the concept of ‘volume recirculation’ in heteroporous membranes is introduced. If there are large pores (gaps) in the microvascular wall, there will always be a bulk filtration of plasma across large pores along the local capillary to interstitial hydrostatic pressure gradient here, regardless of the direction and magnitude of the volume flow occurring across the small (protein-sieving) pores. The reason is that the effective colloid osmotic pressure gradient across large pores is extremely low and does not balance out the normal blood-tissue hydrostatic pressure gradient. By contrast, across small pores there is a balance between hydrostatic and colloid osmotic pressure gradients according to the Starling principle. During complete isogravimetry (no net filtration) this balance will actually be directed from tissue to plasma, completely balancing out the continuous filtration of protein-rich fluid occurring through the large pores. Thus, during isogravimetric conditions, there is a recirculation of fluid from large to small pores; plasma is leaving the microvasculature through large pores, but plasma water is being returned to the circulation via the small pores. Thus, protein is transferred to the tissue by convection, even though the net convection is zero. Such an isogravimetric situation, during which volume recirculation occurs, may prevail for up to 1 h in muscle, but cannot be maintained during steady-state conditions.

FIG04

Fig. 4. Clearance of albumin to rat hindquarter muscles in perfused, maximally vasodilated rat hindlimbs plotted against filtration rate at 36°C (broken line), and during tissue cooling at 13–15°C (solid line). Each data point represents a mean value of three duplicate determinations per experiment. There was a significant coupling of albumin clearance to fluid filtration in both situations, the parallel lines, however, differed significantly from each other (p < 0.001). Note that at zero filtration rate, the reduction in albumin clearance during cooling was in proportion to the increased plasma viscosity at 13–15°C compared to that of 37°C. Reproduced from Rippe et al. [48].

The importance of the volume recirculation phenomenon is that it demonstrates that a large portion of the transport of macromolecules, at or near isovolumetric conditions, may occur by convection through large pores in addition to diffusion through small pores. Failure to take volume recirculation into account in modelling heteroporous transport has led to large errors in the evaluation of the role of convection vs. other mechanisms (diffusion and transcytosis) in the transcapillary passage of macromolecules. If a heteroporous term (i.e. the volume recirculation term) is not included in the transport equations, a large ‘residual’ and default ‘non-convective’ fraction of transport will appear. Such a residual, non-convective transport fraction was obtained by Renkin et al.55], based on their lymphatic protein flux data from the dog’s paw and suggested to be represented by ‘vesicular transport’. By contrast, utilizing heteroporous transport theory in their calculations, Rippe and Haraldsson [5, 24] demonstrated that passive mechanisms (mainly convection through large pores) could fully account for the transvascular clearance of the proteins investigated by Renkin et al. [55] for the dog’s paw. Indeed, reevaluating their data, Renkin [14] and Curry [56] demonstrated full consistence of IgG and fibrinogen/α2-macroglobulin transport with passive convective transport through large pores [14, 56]. However, a fraction of the transport of albumin was still not fitting with a passive two-pore model. The discrepancy between the analysis of Rippe and Haraldsson and that of Renkin is based on different interpretations of the magnitude of the capillary filtration coefficient (LpS). Rippe and Haraldsson used the ‘capillary wall’ LpS value, consistent with the calculated diffusive parameters (A0/ΔX), whereas in the analysis by Renkin [14] and by Curry [56], the regression coefficient of the steady-state lymph flow vs. the calculated capillary pressure was used as the filtration coefficient. Rippe and Haraldsson [24] argued that, due to Starling force adjustments occurring as a consequence of the increases in fluid filtration, this steady-state value must be much less than the LpS. It was also argued that, since volume recirculation is a membrane event, the higher LpS value should be used in the calculations, whereas the net blood to lymph transport would be described by the much lower blood to lymph ‘filtration coefficient’.

In intact rats, under conditions where the rate of transcapillary fluid filtration can not be directly assessed, it seems that the coupling of protein flux to volume flow is much less than obtained in skin and muscle either using lymphatic protein flux analyses or tissue uptake techniques. In vivo assessments of net fluid flow and tracer albumin clearance in muscle and skin (in other organs) have yielded estimates of the reflection coefficient for albumin of the order of 0.98–0.995 [57, 58, 59] and not of the order of 0.9–0.95, as obtained in lymph flux analyses or tissue uptake techniques. In these in vivo studies there was thus virtually no coupling between protein clearance (Cl) and fluid transfer (Jv) across vascular walls. The reason for this discrepancy between lymphatic flux analyses and in vivo assessments of transvascular albumin permeability is unclear. Conceivably, if in lymphatic protein flux studies steady-state conditions are not achieved at elevated filtration rates, and proteins are still washing out from the interstitium, a false coupling between protein transfer and transcapillary filtration rate will occur [59]. Conversely, if exogenous macromolecules (such as dextran, ficoll or RISA) are studied in lymphatic protein flux analyses during non-steady-state conditions, the reverse may be true, namely that the coupling of Cl to Jv may be underestimated. This is then a consequence of the fact that the interstitium has not become fully equilibrated with the exogenous macromolecular tracer. In this context, it is worth pointing out that lymphatic protein flux analyses usually include one stage in which true steady-state conditions are prevailing, namely the resting (control) condition, where venous pressure has not been perturbed. In studies in which attempts have been made to achieve true steady-state during, for example, conditions of elevated venous pressures [55], two-pore analyses usually yield approximately the same pore parameters for control (steady-state) and elevated venous pressure conditions (where steady-state can be difficult to attain). This, at least indirectly, lends support to ‘porous’ transport, implying the presence of coupling between protein transfer and transcapillary volume flow [5, 24].

Tissue uptake studies using protein tracers in intact animals may be subject to experimental error, because they usually imply the assessment of the difference between two large quantities, namely the total amount of tracer present in the tissue (interstitium plus intravascular space) minus that present only in the intravascular compartment at the time of sacrifice [59, 60]. This is a statistically unfavourable situation, leading to both positive and negative values for the apparent extravasated protein space. Moreover, it has been argued that in intact animals, a significant portion of the tracer protein extravasated may have left the tissue via the lymphatics during the period of observation, when lymph drainage from the tissue has not been hindered [61, 62]. These conclusions have been mainly based on tissue uptake studies in rapidly exchanging tissues (lung and intestine), in which rather lengthy tracer accumulation periods have been allowed. However, if tissue uptake studies are limited to short tracer accumulation periods, especially in organs that are slowly exchanging (cf. muscle and skin), then the loss of tracer via the lymphatics is minimal [59, 63, 64]. In conclusion, tracer accumulation techniques are transient state methods, which requires brief, initial uptake measurements, whereas lymphatic protein flux analyses require steady-state conditions and measurements over relatively long intervals. The pros and cons of the two techniques were recently discussed at some length in an excellent review [59].

In the tissue uptake study of Renkin et al. [57] venous congestion in rats was produced by unilateral femoral vein ligation, which was supposed to immediately raise venous pressure (Pv) in the test hindlimb by 10 mm Hg with the contralateral limb serving as a control. Haraldsson et al. [65] assessed Pv, using the same maneuver, via a T-tube inserted into the rat femoral vein, and found that femoral vein ligation increased Pv from 6 to 13 mm Hg distal to the site of ligation. However, there was a gradual return of Pv towards control during the following 30 min, the Pv levelling off at ∼9.5 mm Hg. This gradual return of Pv towards control may be partly due to recruitment of collateral routes for venous return, reducing the effective capillary pressure elevation just to 3 mm Hg. Haraldsson et al. argued that the Pv elevation had actually been very low and that the apparent rate of fluid filtration in the congested leg had been much overestimated. A reason for this overestimation could have been a bias in the wet to dry-weight ratio, assessed to calculate the rate of oedema fluid accumulation, because the non-congested leg was used as a control. Since femoral vein ligation is likely to have caused sympathetic activation (because of a reduced venous return) and peripheral vasoconstriction, and hence, a reduced capillary pressure affecting the contralateral leg, the ‘control’ wet-to-dry-weight ratio may have been underestimated.

All in all, the technique of measuring albumin extravasation and the technique of estimating oedema formation by indirect methods in vivo may be subject to systematic errors. On the other hand, the studies of Rippe et al. in the isolated perfused hindquarter [48] as well as the studies of Rutledge et al. [49], showing a clear-cut coupling of large solute transport to transcapillary filtration rate may have overestimated the coupling of protein transport to volume flow due to the presence of undue large pores (gaps?) in their preparations caused by low-grade inflammation or relative ischemia. Such disturbances may follow from the ‘exposure’ of the tissue to partly unphysiological conditions.

The formation of inflammatory gaps (large pores) in the microvascular membrane is the result of an active, calcium-dependent process, which is readily inhibited by temperature reduction or by cyclic adenosine monophosphate (cAMP) agonists [11, 66, 67, 68, 69]. If actively formed inflammatory gaps had, indeed, been present in the mentioned preparations at normal temperatures, they would be very scant at low temperatures. This would have resulted in an uncoupling of Cl to Jv at low temperatures. However, in the isolated perfused rat hindquarters [48], cooling did not uncouple albumin transfer from fluid flux, although the RISA reflection coefficient (σalb) tended to become slightly higher in the cooled compared to the control animals. Anyway, it seems unlikely that unphysiological inflammatory gaps would have substantially affected the data obtained in the studies of Rutledge [49] and Rippe et al. [48].

In summary, based on available data, it seems safe to conclude that protein flux is, at least moderately, coupled to transcapillary volume flow. Also, when volume flow is zero or near zero, protein transport seems to be dependent upon the level of capillary pressure, because macromolecules will always leak through large pores (due to the presence of ‘volume recirculation’). This lends support to the concept of porous transport of macromolecules. It should, however, be noted that Moffit and Michel [70] have presented data strongly indicating that increases in intraluminal hydrostatic pressure can induce an increased labelling of endothelial cell vesicles by (exogenous) macromolecular tracers.

 Can Endothelial Transcytosis Be Inhibited Using Chemicals?

A number of compounds may be used to inhibit transcytosis, via, for example, effects on the cellular metabolism, such as NaCN [71] or on the integrity of the microtubule system, such as colchicine, vinblastine, cytochalasin B and nocodazole [72, 73]. Furthermore, compounds that inhibit acidification of intracellular organelles, such as monensin, chloroquine, methylamine and ammonium chloride, also prevent endocytosis and delivery of macromolecules to lysosomes [74, 75]. However, all these compounds are highly toxic and may have a number of unspecific effects. Nevertheless, many of these agents have been demonstrated to inhibit vesicular uptake of various macromolecular tracers in cell cultures in vitro [71, 72, 73, 74, 75, 76]. For example, Hastings et al. [73] apparently demonstrated the existence of monensin and nocodazole inhibitable endocytotic pathways in cultured alveolar type II cells. However, when the quantitative alveolar clearance of 125I-labelled immunoglobulin G or 131I-labelled albumin was investigated in anesthetized rabbits, there was no significant nocodazole inhibition of this bulk protein transport from the alveoli to the plasma. The authors therefore concluded that endocytic pathways must be insufficient to account for the clearance of large quantities of serum proteins from normal alveoli to the blood. They further claimed that the mere existence of a pathway does not prove that it is quantitatively important. It should also be noted that, in general, the permeability of cultured endothelium or epithelium to macromolecules is usually increased by one or two orders of magnitude in comparison to permeabilities measured in in situ perfused organs or in vivo [5, 11, 71]. In an in vitro system of cultured endothelial cells from large arteries grown to confluence on a polycarbonate micropore filter, Shasby and Shasby [71] clearly found evidence of a very high interstitial to lumen transport of radiolabelled albumin, inhibitable by NaCN. However, subsequent investigators were not able to reproduce the findings of preferential interstitial to lumen transport using similar endothelial cell cultures [77]. Thus, it should be noted that endothelial cells in culture may show increased or otherwise abnormal permeability properties due to phenotypic drift after several passages, trypsin digestion and other treatments, so that they may differ highly significantly from their native state.

In perfused rat hindquarters Haraldsson and Johansson [78] were able to produce a light to moderate chemical fixation of the vasculature by a short-term (3 min) perfusion fixation (after preceding intravascular washout) of the rat hindquarter vasculature using glutaraldehyde. After washout of the fixative, the hindquarters could again be perfused with serum. In such fixed, but perfused, vascular beds the clearance of albumin was reduced, but only to the extent that the capillary hydraulic conductance (LpS) and the small solute diffusion capacities (PS for Cr-EDTA and cyanocobalamine) were altered. The permeability of the microvascular walls with regard to albumin transport was apparently not reduced in a system where vesicle movements were completely inhibited, but where the effectively perfused capillary surface area seem to have been reduced.

More specific approaches to inhibiting vesicular transport than the highly cytotoxic procedures mentioned above appeared to emerge in the early 1990s by the use of NEM and filipin. NEM is an alkylating agent that cross-links SH-groups. It has been considered to more or less specifically interact with the NEM-sensitive fusion protein (NSF), and thereby with the docking and fusion of vesicles with their target membranes. The other substance, thought to specifically interact with transcytosis, is filipin. This agent is a cholesterol scavenging compound, which is assumed to inhibit transcytosis by removing cholesterol from the plasmalemma, thereby causing disassembly of the vesicles.

Schnitzer et al. [79] found that short-term exposure of the in situ perfused rat lung vasculature to 0.5 mM NEM reduced the subsequent uptake of radiolabelled albumin (RISA) into the lung tissue by 40–50%. Similar findings were obtained using filipin in the same in situ lung model [80]. In another animal model, the in situ perfused murine heart, NEM also caused marked reductions in the tissue uptake of labelled small proteins and albumin in tissue samples compared to control after 5 min of local NEM infusion (1 mM) using tracer containing perfusate, preceded by 5 min of NEM (1 mM) infusion using phosphate buffered saline as perfusate [81, 82].

Carlsson et al. [83] and Rippe and Taylor [53] were able to investigate the impact of NEM and/or filipin on isolated perfused rat hindquarters and isolated perfused rat lungs, respectively, under ex vivo conditions, where hemodynamic parameters, including capillary hydrostatic pressure (Pc), pre- and post-capillary resistances and the capillary filtration coefficient (LpS) were continually assessed. In isolated perfused rat hindquarters 5–10 min of exposure to NEM in doses ≥0.3 mM produced marked increases in pre-capillary resistance, conceivably due to vasoconstriction, and furthermore, large increases in microvascular permeability. Thus, there were marked increases in the LpS and increases in the clearance RISA to muscle tissues [83] (fig. 5). These results thus strongly suggest that NEM, besides producing marked vasoconstriction, also causes damage to the microvascular endothelium. Also, in the isolated perfused rat lung (using serum and albumin in a balanced salt solution as perfusate), NEM (0.13 mM) produced large increases in microvascular permeability, with increases in clearance of RISA from blood to tissue, increases in LpS, and reductions in the apparent RISA reflection coefficient [53] (fig. 6). Similar results were obtained also for high doses of filipin in the isolated perfused rat lung model [53]. That filipin can cause cell membrane damage and permeabilization of the plasmalemma by producing circular lesions of large dimension (cf. large pores) is well established [80, 84] and has been recently reinvestigated using atomic force microscopy [85].

FIG05

Fig. 5. Effect of pretreatment of perfused rat hindquarters with 0.33 mM NEM for 5–10 min upon the peripheral vascular resistance (PRU) (a), the capillary filtration coefficient (here denoted CFC) (b), and the albumin clearance from the perfusate to three different muscles (c). a, b Left panels demonstrate the effects of vehicle alone, whereas right panels exhibit the effects of NEM challenge (black columns). c The effects of NEM exposure is indicated by the black columns. Despite marked precapillary vasoconstriction NEM caused large increases in the capillary filtration coefficient and in the clearance of albumin from blood to muscle tissues. Data from Carlsson et al. [83].

FIG06

Fig. 6. Effects of 4 min of NEM (0.13 mM) exposure (solid bars) on isogravimetric clearance (Cliso; ml·min–1·100 g–1), the filtration coefficient (LpS; ml·min–1·cm H2O–1·100 g–1), σ for albumin (dimensionless), and total lung vascular resistance (Rtot; cm H2O· ml–1·min·100 g) compared with control (open bars) in isolated perfused rat lungs. Statistically significant increases occurred in both Cliso and LpS (p < 0.05). Furthermore, there was a reduction in σalb (p < 0.05). Reproduced from Rippe and Taylor [53].

To further establish the action of NEM and filipin on transvascular large solute transport Rosengren et al.86] tested these tentative ‘transcytosis inhibitors’ in a rat peritoneal dialysis model in vivo. Either NEM or filipin were incubated intraperitoneally for 4 min before starting a peritoneal dialysis dwell, during which the transport of RISA and LDL from peritoneal microvessels to the dialysis fluid were assessed. In concentrations higher than 0.5 mM, NEM progressively increased the peritoneal RISA and LDL clearance, whereas filipin in doses up to 4 μg/l neither affected the transport of RISA, nor that of LDL. Furthermore, in recent experiments on the in-situ-perfused rat thoracic aorta exposed to 0.1 mM NEM for 10 min followed by perfusion fixation, Ogawa et al. [87] noted marked extravasation of horseradish peroxidase into the subendothelial space as an indication of an increased endothelial permeability. Transendothelial channels, suggested to be formed by fusion of caveolae, were found to be much more frequent after NEM exposure than in controls, suggesting that NEM may have induced de novo formation of caveolar channels.

These data from rat peritoneum in vivo, or from the in situ perfused thoracic aorta, or from ex vivo perfused rat hindquarters and rat lungs clearly indicate that NEM and filipin are unsuitable as transcytosis inhibitors. At moderate or low doses of these agents, the transvascular clearance of albumin (or LDL) remained totally unaffected; at high doses (0.1–0.5 mM for NEM), they caused increases in microvascular permeability.

Given the marked vasoconstriction that NEM produces, at least in muscle, the reduction of the tissue uptake of labelled small proteins and albumin in murine heart after 10 min of local (in situ) NEM exposure (1 mM), compared with control [81, 82], may be explained by marked vasoconstriction and derecruitment of microvessels. In lung, vasoconstriction will not primarily cause derecruitment of microvascular surface area [88], but still a vasoconstriction-induced reduction in the intravascular space may be the main reason for the reduction in albumin space, but not in inulin space, observed in the lung. The reason why a reduction in intravascular volume would affect the apparent tissue uptake of a macromolecule, but not that of smaller solute, may be the following: For a large solute, the major portion of the apparent tissue ‘space’ of tracer in a ‘tissue uptake study’ is made up by the intravascular space. Efficient intravascular washout is crucial in short-term experiments to completely rid the vasculature of tracer. If tracer washout is insufficient, a vasoconstriction-induced reduction of the intravascular space will markedly affect the apparent ‘tissue uptake’ of a macromolecule. However, for a small or an intermediate-size solute (such as inulin) the relative intravascular portion of the total tissue space during tracer accumulation (in a tissue uptake experiment) is usually rather small. Thus, the apparent tissue space for inulin would not be significantly affected by NEM-induced reductions in intravascular volume. This may be the reason why the apparent inulin space, in contrast to that of albumin, was not significantly affected by NEM in the in-situ-perfused rat lung [79].

In the study of Schnitzer et al. [79], it was, however, claimed that lung vascular resistance was largely unaffected by NEM, since the total perfusion pressure during NEM infusion was largely unchanged. Still NEM-induced vasoconstriction may be a reason for the decrease in apparent albumin flux in these studies. Utilizing the isolated perfused rat lung model, the pressure drop along the lung vasculature proper is usually rather small, of the order of 3–4 mm Hg, for a perfusion flow of ∼10 ml/g lung. This usually represents one third of the total pressure drop (9–10 mm Hg) along the entire perfusion circuit at this perfusate flow. For such a low total flow resistance, a rise in pulmonary vascular resistance by ∼25% will give an overall pressure increment of only ∼1 mm Hg in the circuit, which is readily overlooked. Small perfusion pressure changes may thus be masked in the perfused lung model, unless arterial and venous pressures are continuously and very carefully monitored.

In summary, it is apparent that ‘transcytosis inhibition’ using chemicals in vivo or in situ perfused organs has not resulted in reductions in transcapillary protein flux, other than related to vasoconstriction, and hence, derecruitment of capillary surface area. The ‘transcytosis inhibitor’ NEM, when tested in situ or in vivo, is indeed a very toxic agent, which actually causes increases in vascular permeability. Filipin used in high doses has been demonstrated to have similar effects on the lung. Thus, the use of chemical transcytosis inhibition has not been successful in testing vesicular transport as a pathway for the bulk transport of proteins across vascular endothelia in vivo.

 Morphological Correlates to Transendothelial Protein Transport

Using ultrathin (∼140 Å) serial sectioning electron microscopy of endothelial cells from various capillaries, it was demonstrated that free plasmalemmal vesicles actually are very rare [89, 90, 91, 92, 93, 94]. Virtually all of the apparently free vesicles were demonstrated to actually be parts of complex, racemose invaginations from the cell surfaces (fig. 7). This has been very convincingly shown, using both conventional chemical fixation and ultra-rapid fixation, employing slam freezing [94]. This puts the original ‘shuttle hypothesis’ in doubt and would favour alternative involvements of vesicles in transcellular transport. Clough and Michel [41], based on very careful experiments of ferritin uptake into vesicles at different distances from the luminal plasma membrane, suggested that a number of fusion and fission events between vesicles or vesicular invaginations may have accounted for the transfer of ferritin particles from plasma to interstitium. The conclusion is mainly based on the fact that the labelling of vesicles by plasma tracers was attenuated more or less in proportion to their distance from the luminal plasmalemma. According to this view vesicles are not shuttling a definite cargo from one surface of the endothelial cell to the other, but the vesicle content is progressively diluted on its passage through the endothelium.

FIG07

Fig. 7. Diagrammatic sketch of a three-dimensionally reconstructed segment of endothelium from frog muscle capillaries. Reproduced from Frøkjær-Jensen et al. [92].

Does true transcytosis (shuttling) occur in parallel with transient fusions of vesicles to form transcellular channels? Conceivably, if these processes occur in parallel, the most efficient way of transporting proteins is by way of patent channels (‘large pores’). Only one or two such channels per (1 mm of) capillary can be calculated to account for the total passage of plasma proteins from blood to tissue [5, 11]. If channels indeed are formed at random, they may remain open during tissue cooling or even during tissue fixation, thereby explaining the insensitivity to cooling or chemical fixation of albumin transfer obtained in previous studies. Furthermore, they would be responsible for the coupling of protein transfer to capillary pressure (and to transcapillary volume flow) so frequently observed previously [5]. It is also possible that caveolae may not be involved in transvascular protein transport at all. This view is actually supported by the recent finding that knock-out mice who have lost their endothelial caveolae due to deficiency of caveolin-1, show normal interstitial protein concentrations and also have normal concentrations of albumin in the cerebrospinal fluid [95].

It can be argued that large pores, whether represented by modified (widened) interendothelial gaps or by channels of fused vesicles, are too infrequent to ever be detected by morphological techniques. Furthermore, it could be speculated that ‘actively formed’ endothelial gaps, such as those in inflammation [68], may always arise to some extent even in the absence of (overt) inflammation. It is not known to what extent inflammatory gaps are also of transcellular nature, even though a majority seems to be paracellular. Transcellular pores may actually be formed from intracellular clusters of vesicles, such as the newly discovered vesicular vacuolar organelles [96], described to exist in tumour microvessels. Another intriguing hypothesis is, that widened intercellular spaces may occur during cell turnover, i.e. cell-death and replacement of endothelial cells, so that the integrity of the tight junctions may be transiently, but continuously, disrupted at random [97, 98]. This could also account for the normal leakage of macromolecules from plasma to tissue. Again, however, it should be pointed out that the chance of finding such a ‘large pore’ is so small that, by definition, such a finding would be dismissed as an artefact.

In summary, there have been indications that are strongly suggestive of the presence of transiently formed large pores of transcellular nature, i.e. at a distance away from the intraendothelial gaps [26]. There are also strong indications that plasmalemmal membrane can actually move from one side of the endothelial cell to the other [18]. From dynamic morphological studies, however, it is not possible to quantitatively assess the role of vesicular transport in the overall passage of macromolecules from blood to tissue. From a physiological standpoint, passive transport through porous pathways, either represented by paracellular large pores, preformed, or formed during cell-turnover or low-grade endothelial activation, or by fused plasmalemmal vesicles, seems to more or less exactly account for the observed functional behaviour of transendothelial macromolecular transport.

 

 Concluding Remarks

During the last decade there has been a very rapid and significant increase of the knowledge concerning the cell biology of transcytosis. In endothelium it is likely that caveolae have most of the molecular machinery present to be able to take part in endothelial transport [47]. Indeed, endo- and exocytosis play important roles in the cellular uptake or release of various proteins and polypeptides, as well as the insertion and deletion of proteins (e.g. receptors) in the plasma membrane. Examples are excretion of hormones from endocrine cells, such as insulin from the pancreatic β-cells [99, 100, 101], the reabsorption of proteins in renal proximal tubuli [102, 103], the (transient) insertion of aquaporin-2 water channels in the plasmalemma of the principal cells of renal collecting ducts [104, 105, 106] or the recruitment of dopaminergic receptors to the cell surface in proximal tubular cells induced by atrial natriuretic peptide [107], all of which are highly cooling-sensitive processes [100, 106]. Furthermore, it was recently demonstrated that caveolae are essential for endothelial (nitric oxide)-dependent vasodilatation and calcium signalling, based on experiments on caveolin-1-deficient mice. However, in these caveolae-lacking mice, there was no evidence of abnormal transendothelial transport of either albumin or lipoproteins [95].

It may thus be concluded that endocytosis and exocytosis in microvascular endothelia normally have a number of very important primary functions other than taking part in transcytosis. Endothelial caveolae are capable of importing molecules and delivering them to specific locations within the cell, exporting molecules to the extracellular space and also compartmentalizing a variety of signalling activities. In brief, caveolae represent a major route for the communication and interaction of intracellular compartments with the cell surface. Hence, their primary function is obviously not to act as transport carriers across the endothelium. Further studies are, however, needed to give a definite answer to the question whether transcytosis, in excess of large pore transport, be quantitatively involved in the transfer of proteins across the microvascular endothelium or not.

 

 Acknowledgments

This study was supported by the Swedish Medical Research Council (Grant No 08285) and by European Union contract FMRX-CT98–0219. The skillful typing of the manuscript by Kerstin Wihlborg is gratefully acknowledged.


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Author Contacts

Prof. Bengt Rippe
Department of Nephrology
University Hospital of Lund
S–211 85 Lund (Sweden)
Tel. +46 46 171247, Fax +46 46 2114356, E-Mail Bengt.Rippe@njur.lu.se

  

Article Information

Received: Received: January 11, 2002
Accepted after revision: June 19, 2002
Number of Print Pages : 16
Number of Figures : 7, Number of Tables : 0, Number of References : 108

  

Publication Details

Journal of Vascular Research
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964–1973) continued as Blood Vessels by J.A. Bevan (1974–1991)

Vol. 39, No. 5, Year 2002 (Cover Date: September-October 2002)

Journal Editor: M.J. Mulvany, Aarhus
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/journals/jvr


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

The relative contribution of transcytosis vs. large pore transport to the passage of macromolecules across microvascular endothelia has been a controversial issue for nearly half a century. To separate transcytosis from ‘porous’ transport, the transcytosis inhibitors N-ethylmaleimide (NEM) and filipin have been tested in in situ or ex vivo perfused organs with highly conflicting results. In continually weighed isolated perfused organs, where measurements of pre- and post-capillary resistances, capillary pressure and capillary filtration coefficients can be repeatedly performed, high doses of NEM and filipin increased the bulk transport of macromolecules from blood to tissue, despite producing vasoconstriction. By contrast, in in situ perfused organs, marked reductions in the tissue uptake of albumin tracer have been observed after NEM and filipin. When tissue cooling has been employed as a means of inhibiting (active) transcytosis, results have invariably shown a low cooling sensitivity of albumin transport, compatible with passive transendothelial passage of albumin. This observation is further strengthened by the commonly observed dependence of albumin transport upon the capillary pressure and the rate of transcapillary convection. For low-density lipoprotein (LDL), a cooling-sensitive, non-selective transport component has been discovered, which may be represented by filtration through paracellular gaps, lateral diffusion through transendothelial channels formed by fused vesicles, or by transcytosis. From a physiological standpoint there is little evidence supporting active transendothelial transport of most plasma macromolecules. This seems to be supported by studies on caveolin-1-deficient mice lacking plasmalemmal vesicles (caveolae), in which there are no obvious abnormalities in the transendothelial transport of albumin, immunoglobulins or lipoproteins. Nevertheless, specific transport in peripheral capillaries of several hormones and other specific substances, similar to that existing across the blood-brain barrier, still remains as a possibility.

© 2002 S. Karger AG, Basel


  

Author Contacts

Prof. Bengt Rippe
Department of Nephrology
University Hospital of Lund
S–211 85 Lund (Sweden)
Tel. +46 46 171247, Fax +46 46 2114356, E-Mail Bengt.Rippe@njur.lu.se

  

Article Information

Received: Received: January 11, 2002
Accepted after revision: June 19, 2002
Number of Print Pages : 16
Number of Figures : 7, Number of Tables : 0, Number of References : 108

  

Publication Details

Journal of Vascular Research
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964–1973) continued as Blood Vessels by J.A. Bevan (1974–1991)

Vol. 39, No. 5, Year 2002 (Cover Date: September-October 2002)

Journal Editor: M.J. Mulvany, Aarhus
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/journals/jvr


Article / Publication Details

First-Page Preview
Abstract of Review

Published online: 9/18/2002
Issue release date: September–October 2002

Number of Print Pages: 16
Number of Figures: 7
Number of Tables: 0

ISSN: 1018-1172 (Print)
eISSN: 1423-0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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