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The modulation of transcapillary transport of macromolecules
Molec. AspectsMed. Vol. 10, pp. 273-280, 1988
0098-2997/88 $0.00 + .50 Copyright© 1988 Pergamon Press plc.
Printed in Great Britain. All rights reserved.
THE MODULATION OF TRANSCAPILLARY TRANSPORT OF MACROMOLECULES Ion
Baciu
and
Liviu
Grosu
Department of Physiology, Medical and Pharmaceutical Institute, 3400 Cluj-Napoca, Romania
Research over the last decades (Palade, 1953, 1960; Moore and Ruska, 1957; Palade and Bruns, 1968; Baciu, 1974; Palade et al., Simionescu et al., 1973, 1981) has shown that cytoplasmic vesicles make up the main pathway for macromolecules to pass through the capillary wall. More recently, by perfusion experiments of a single capillary and measurement of the minimum osmotic reflection coefficients (Clough and Michel, 1981; Curry and Michel, 1980; Mason et al., 1977), it has been shown that vesicular transport accounts for a major but variable part of the dissipative transport of macromolecules. However, other pathways such as intracellular junctions or vesicular channels (Simionescu et al., 1975) which are hydraulically conductive and which can be the support of either a diffusive (dissipative) or a convective transport, cannot be excluded (Renkin and Curry, 1982). It has also been shown that the transport through vesicles is not energetically dependent (it also takes place in the presence of metabolic inhibitors), and is also not a membrane consuming process (Casley-Smith, 1969; Allison and Davies, 1974; Simionescu, 1981; Williams and Wagner, 1981).
i.
The Modulation of Transport Through Vesicles
In systems of isolated and perfused capillaries, the vesicular uptake was found to be insensitive to many physical factors and vasoactive substances and hormones, except for histamine and serotonin, which depress it markedly, and prolactin which gives rise to a dose-dependent inhibition (Wagner and Mathews, 1980). Calcium and magnesium ions, on the other hand, stimulate the uptake, whilst calcium may also stimulate a rapid efflux of the tracer from the preloaded endothelial cells (Williams and Wagner, 1981). These modulatory effects on vesicular functions are probably the results of a direct action upon the endothelial membrane, because cellular metabolism is not involved. The modulation may be, actually, a regulation of fusion of the membranes of vesicles with the luminal and albuminal cytoplasmic membranes. Thus, the chemical environment which exists in blood and tissue fluids may regulate the flux of materials transported by vesicles through the capillary walls. Finally it is worth noting that studies of Buonassisi and Colburn (1980) and Simionescu et al. (1981, 1982) have revealed, at the surface of endothelial cells, the presence of receptors for hormones and the existence of different microdomains; it could be that such specific formations contribute to the binding and preferential transport of certain molecules.
273
274 2.
I Baciu and L Grosu The Global Modulation of Transcapillary Transport
The modulation of transcapillary transport may be accomplished not only by influencing the vesicular activity, but also may be exerted on other pathways for convective or diffusive transport.
2.1.
The effect of plasma proteins
The effect of plasma proteins upon capillary permeability, mentioned some 60 years ago by Krough and Harrop (1921) and studied by Drinker (1927) and later by Danielly (1940), was recently evaluated by Mason et al. (1977) on the elegant experimental model of isolated and perfused capillary from frog mesentery. They showed that the removal of plasma proteins from the perfusion liquid induces a five fold increase of the filtration coefficient, even if the colloidosmotic pressure is maintained with dextran; the presence of only 0.1% bovine serum albumin in the perfusion medium is sufficient to maintain permeability within normal levels. Since above 0.1% this effect of the protein was independent of concentration, it has been concluded that the phenomenon is caused by a direct adsorption of proteins onto the capillary surface. Clough and Michel (1981) have shown that, at least partially, the effect is also explained by an influence of proteins on the vesicular transport of macromolecules. Thus, in the absence of circulatory albumin, the number of luminal vesicles containing ferritin was almost two times higher than in the presence of 1% albumin; this was explained by a direct interaction of albumin with the capillary wall, more exactly with the glycocalyx which lines the endothelial cells. This led Curry and Michel (1980) to formulate the theory of the 'fibre matrix', according to which the permeability properties of unfenestrated continuous endothelium reside in the glycocalyx, a network of glycoproteins (with a diameter of 0.6 nm which occupies less than 8% of the network volume) that lines the endothelial surface and the intercellular junctions, the channels of fused vesicles or the caveoles (attached vesicles). Spaces between the fibre can provide resistance to movement of both small and large solute molecules. By picking up circulating proteins, the network tightens its mesh and thus makes the underlying vesicles less accessible to other water-soluble molecules. Schneeberger and Hamelin (1984) have recently shown that depletion of protein results in an increase of permeability to ferritin and that the intensification of ferritin transport is associated with the loss of adsorbed albumin and IgG from the glycocalyx.
2.2.
The role of fibrinolysis in capillary permeability
Systematic research on the role of coagulation and fibrinolysis in capillary permeability was started in our Institute in 1960, based on the hypothesis of the existence of an endovascular film of fibrin (Duguid, 1949; Copley, 1964). In the first experiments, carried out on dogs with the thoracic duct cannulated for lymph collection, we noticed that i.v. administration of Evans Blue (T 1824) resulted in an impressive increase of transcapillary transport and an activation of fibrinolysis. At the same time plasma kinins increased progressively and reached a maximum in 15-30 min (Baciu et al., 1967; Baciu and Grosu, 1970). The administration of thrombin to modify the coagulation and fibrinolytic activities, induced a significant reduction of the transcapillary transport of the plasma proteins with radical alterations of the coagulant and fibrinolytic reactions.
Transcapillary Transport
275
The data obtained in this experimental model, although striking, are difficult to interpret, owing to its complexity. On one hand, the i.v. administration of the macromolecular dyestuff per se resulted in activation of coagulation, fibrinolytic and kinin forming systems; on the other hand, thrombin also has a complex effect on these systems. We tried, therefore, in subsequent experiments to simplify the experimental model by using another macromolecular marker, the i.v. administration of which does not induce an activation of the above mentioned systems and some more specific modifiers of the fibrinolytic activity. The experiments were performed on rats and the macromolecular marker was radioiodinated serum albumin (RISA), the i.v. administration of which does not give rise to significant changes in coagulation, or fibrinolysis. For altering the activity of the fibrinolytic system we used streptokinase as a fibrinolytic activator, and aprotinin (Trasylol, TR) and g-aminocaproic acid (EACA) as fibrinolytic inhibitors. The transcapillary transport of macromolecules was estimated by determining the transcapillary escape rate of RISA. We showed that EACA has a clear-cut inhibitory effect on transcapillary transport of RISA, aprotinin has no effect. With streptokinase, although the slope of the regression line is lower than that in control animals, the initial value of radioactivity (extrapolated zero value) is below the other, suggesting an increased transport in the very first minutes (Fig. i) (Don, 1979).
100zx-x-x-x--x--x--x--x'-x-×-x--x] EA C A
60
-.m
"':-- " . o ~ j
40
C
\SK
20
'5 1"0 26 Fig. i.
3'0
4"0
5'0
60
min
Transcapillary escape rate of RISA (regression lines calculated from variations of blood RISA against time) in animals treated with activators (streptokinase, SK) and inhibitors (g-amino-caproic acid, EACA, and aprotinin, TR) of fibrinolysis and in control animals (C).
We compared these results with those obtained in another experimental model, which allowed the exploration of a more homogenous capillary area, the dog hind paw. One or more prenodal popliteal lymphatics were cannulated for lymph collection, and transcapillary transport was measured by determining the lymph to plasma concentration ratio of RISA. RISA was administrated immediately after the inhibitory substances, but 40 min before streptokinase to allow a certain plateau in the curve of RISA disappearance from plasma in the moment of streptokinase administration. In this model the data indicated that EACA and aprotinin have no
276
I. Baciu and L. Grosu
I -"@- -
I
-o
SK
A 13-
l l f
-@ n.-
~,.---,
TI~
~.~~CA
C
I'0 30 50 ?0 90 110 min Fig. 2.
Alteration of transcapillary transport of RISA after administration of streptokinase. Lymph to plasma ratio of RISA concentration [L]/[P] in animals treated with streptokinase (SK), aprotinin (TR), E-amino-caproic acid (EACA) and in control animals (C). The differences in the curves C, EACA, TR and SK (before the arrow), are not significant; they reflect a variability in the reactivity of various animals.
significant effect on transcapillary transport of RISA, while streptokinase administration produced a very marked activation (Fig. 2) (Don, 1979). Finaly, we tried other conditions associated with an activation of fibrinolysis, such as hypoxia, to see whether this is correlated with an increase in transport of macromolecules. The experiments were performed on dogs with general hypoxia and cephalic ischaemic hypoxia. It was found that both general hypoxic hypoxia and cephalic ischaemic hypoxia increase capillary permeability for macromolecules during the first 15 min (Fig. 3). The findings suggest that the increase in capillary permeability induced by hypoxia is achieved by neurohumoral mechanisms (Baciu et al., 1970). Actually, Kowarzyk et al. (1962) have shown that cephalic ischaemic hypoxia results in activation of fibrinolysis, which may explain the increase of permeability. With regard to the mechanism by which the activation of fibrinolysis induces an enhanced transcapillary transport of macromolecules, we must mention that subsequent research by Vermylen and Chamone (1979) could not confirm the attractive hypothesis of an endovascular film of fibrin continuously renewed by coagulation and fibrinolysis processes. In contrast, Witte (1981) found in rats injected i.v. with fibrinogen labelled with fluorescein isothiocyanate that 5-15 min after injection, fibrinogen accumulates on the endothelial luminal face in the form of points, bands or as a network. Such accumulations occur mainly at the level of venules and especially at interendothelial junctions. This author also observed the appearance of labelled fibrinogen in the perivascular tissue. It is worth mentioning that a polypeptidic fraction derived from factor VIII behaves similarly to fibrinogen. This fraction has an important inhibitory action on the capillary permeability to plasma proteins (Witte, 1984). In the light of recent research we can thus imagine the endothelial cells as
Transcapillary Transport
ml/rain.
GENERAL HYPOXIA rnl/min
277
CEPHALIC HYPOXIA
't '1
n"n llI nn ° NNINN NHnHn g~ 71
g~
i mp/min 8 Vmin
i-
]
i rnp/min t
150 180 210 240 2?0 30(3 0 15 60 g0 min
A Fig. 3.
'
150 180 210 240 270 300 0 30 50 90 120rain
B
The effects of general hypoxic hypoxia (left) and cephalic ischaemic hypoxia (right) on capillary permeability for macromolecules. Top: lymphatic rate. Middle: • • , the concentration of protein in plasma; , the concentration of protein in lymph; x i x , lymph to plasma protein concentration ratio. Hatched bars: protein clearance. Bottom: • - - • , the concentration of RISA in plasma; ..... , the concentration of RISA in lymph; x x , lymph to plasma RISA concentration ratio.
being surrounded by the environment of glycoproteins of the glycocalyx on which plasma proteins (albumins, globulins), fibrinogen and factor VIII are adsorbed. Consequently, the activation of fibrinolysis by means of a vascular activator, which is secreted, especially at the level of venules (Warren, 1964), could result in an increase in permeability. This could be due to the enlargement of the fibre network's mesh of glycocalyx as a result of the removal of fibrinogen and fibrin molecules. Secondly, the activation of fibrinolysis, both by direct action of plasmin and by factor VIII, results in the activation of prekallikrein (the Fletcher factor) to kallikrein, with consecutive release of bradykinin, which in turn causes an increase in capillary permeability (see below). Actually, we have noticed an increase in the concentration of kinins in plasma, in parallel with increasing rates of fibrinolysis and the activation of transcapillary transport. Moreover, preferred microdomains for transport of macromolecules could exist, for example the venular zone, where, as mentioned above, fibrinogen and factor VIII are preferentially accumulated, the vascular activator of plasminogen is synthesized, stored and released (Warre, 1964); the venular zone is also the zone with the
278
I. Baciu and L. Grosu
largest density of receptors for histamine, especially those of the }{2 type (Simionescu et al., 1982). In the same zone, histamine and bradykinin produce a contraction of endothelial cells with the enlargement of interendothelial space, favoured by the unusually lax organization of intercellular contacts (Majno et al., 1969). 2.3.
Other modulations factors
It is well known that histamine, bradykinin and serotonin increase capillary permeability. The electronmicroscopical studies of Majno et ai.~(1969) showed that the increase is produced by contraction of endothelial cells and the consequent enlargement of intercellular junctions. Venules with a diameter between 20-30 ~m make up, in this respect, the most reactive part of microcirculation. More recently the effect has been demonstrated on capillary permeability of prostaglandins (PGE I, PGE2) and leukotrienes C 4 and D 4 (Fantone et al., 1980; Ueno et al., 1981). Kallikrein, a purified fragment of active factor XII and the C 3 fraction of complement also increase capillary permeability. However, it is very likely that in these latter cases, the effect is mediated by liberation of kinins, owing to the complex interaction between plasma protease systems (Imamura et al., 1984). We may conclude from the material presented above that, despite the recent remarkable progress made in the knowledge of modulatory mechanisms of the transcapillary transport of macromolecules, our knowledge is still limited and continued efforts are necessary for its broadening. References Allison, A. C. and P. Davies (1974). Hechanisms of endocytosis and exocytosis. Symp. Soc. exp. Biol. 28, 419-446. Baciu, I. (1971). 44, 757-770.
Permeabilitatia capiliara pentru macromolecule.
Clujul Med.
Baciu, I., L. Grosu, M. Dorofteiu, F. Don, M. Zirbo, E. Enescu and N. Timar (1967). Efectele activarii procesului fibrinoplastic ?i fibrionolitic asupra permeabilita~ii capilare. Fiziol. norm. patol. 13, 11-29. Baciu, I. and L. Grosu (1970). Kininele plasmatice ~n reglarea circula~iei. In: Polipeptide endogene biologic active (I. Baciu, ed.), I.M.F. Cluj-Napoca. Baciu, I., L. Grosu, A. Olteanu and T. Pavel (1970).
Clujul Med. 43, 691-699.
Buonassisi, V. and P. Colburn (1980). Hormones and surface receptors in vascular endothelium. In: Advances in Microcirculation. Vascular Endothelium and Basement Nembranes (B. M. Altura, ed.), Vol 9, Karger, Basel. Casley-Smith, J. R. (1969). Endocytosis: The different energy requirement for the uptake of particles by small and large vesicles into peritoneal macrophages. J. Microsc. 90, 15-30. Clough, G. and C. C. Hichel (1981). The role of vesicles in the transport of ferritin through frog endothelium. J. Physiol., Lond. 315, 127-142. Copley, A. L. (1964). Capillary permeability venous fragility and the significance of fibrin as a physiologic cement of the blood vessel wall. In: 2nd European Conference on Microcirculation Pavia, 1962, Karger, Basel.
Transcapillary Transport
279
Curry, F. E. and C. C. Michel (1980). A fiber matrix model of capillary permeability. Microvasc. Res. 20, 96-99. Danielly, J. F. (1940). Capillary permeability and oedema in perfused frog. J. Physiol. 98, i09-129. Don Felicia (1979). Influenta procesului fibrinolitic si fibrino plastic asupra microcircula~iei. Teza de doctorat I.M.F. Cluj-Napoca. Don Felicia, L. Grosu, T. Pavel, M. Cucuianu, M. Grigerosik and I. Baciu (1987). Influence of some inhibitors and activators of fibrinolysis on transcapillary transport of macromolecules. Rev. roum. Morphol. Embryol. Physiol. Physiol. (in press). Drinker, C. K. (1927). The permeability and diameter of the capillaries in the web of the brown frog when perfused with solutions containing pituitary extract and horse serum. J. Physiol. 63, 249-269. Duguid, J. B. (1949).
Pathogenesis of atherosclerosis.
Lancet ~, 925.
Fantone, C. J., S. L. Kunkel, P. A. Ward and R. B. Zurier (1980). Suppression by prostaglandin E l of vascular permeability induced by vasoactive inflammatory mediators. J. Immunol. 125, 2591-2596. Imamura, T., T. Yamamoto and T. Kambara (1984). Guinea pig plasma kallikrein as a vascular permeability enhancement factor. A. J. Pathol. 115, 92-101. Kowarzyk, H., I. Baciu, C. Opri§iu and M. Kotschy (1962). Sur quelques mecanismes d'activation de la fibrinolyse. Arch. Immunol. ThOr. Exp. Krogh, A. and G. A. Harrop (1921). J. Physiol. 54, LXXV-CXXVI.
I0, 527.
Some observation on stasis and oedema.
Majno, G., S. M. Shea and M. Leventhal (1969). Endothelial contraction induced by histamine-like mediators. An electron microscopic study. J. Cell. Biol. 42, 647-672. Mason, J. C., F. E. Curry and C. C. Michel (1977). The effects of proteins upon filtration coefficients of individually perfused frog mesenteric capillaries. Microvasc. Res. 13, 185-202. Moore, D. H. and H. Ruska (1957). The fine structure of capillaries and small arteries in muscle. Anat. Rec. 136, 254. Palade, G. E. (1953). 24, 1424.
Fine Structure of blood capillaries.
J. Appl. Physiol.
Palade, G. E. (1960). Transport in quanta across the endothelium of blood capillaries. Anat. Rec. 136, 254. Palade, G. E. and R. R. Bruns (1968). Structural modulation of plasmalemal vesicles. J. Cell. Biol. 37, 633-653. Palade, G. E., M. Simionescu and N. Simionescu (1979). Structural aspects of the permeability of the microvascular endothelium. Acta Physiol. Scand. suppl. 463, 11-32.
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Renkin, E. M. and F. E. Curry (1982). Endothelial permeability: modulations. Ann. N. Y. Acad. Sci. 401, 248-258.
Pathways and
Schneeberger, E. E. and M. Hamelin (1984). Interaction of serum proteins with lung endothelial glycocalyx: its effect on endothelial permeability. Am. J. Physiol. 247, H206-H217. Simionescu, M., N. Simionescu and G. E. Palade (1973). Permeability of muscle capillaries to exogenous myoglobin. J. Cell. Biol. 57, 424-452. Simionescu, M., N. Simionescu and G. E. Palade (1975). Permeability of muscle capillaries to small hemepeptides. Evidence for the existence of patent transendothelial channels. J. Cell. Biol. 64, 586-607. Simionescu, N. (1981). Transcitosis and traffic of membranes in the endothelial cell. In: International Cell Biology (H. G. Schweizer, ed.), Springer Verlag, Berlin, Heidelberg. Simionescu, N., C. Neltianu, F. Antohe and M. Simionescu (1982). Endothelial cell receptors for histamine. Ann. N.Y. Acad. Sci. 401, 132-148. Ueno, A., K. Tanaka, M. Katori, M. Hayashi and K. Arai (1981). Species difference in increased vascular permeability by synthetic leucotriene C 4 and D 4. Prostaglandins 2__1, 637-647. Vermylen, I. G. and D. A. F. Chamone (1979). The role of the fibrinolytic system in thromboembolism. Prog. Cardiovasc. Dis. 21, 255-266. Wagner, R. C. and M. A. Matthews (1980). Vesicular ingestion by isolated capillaries: Effects of hormones and vasoactive substances. Microvasc. Res. 19, 200. Warren. B. A. (1964). Bull. 2_.9_0,213.
Fibrinolytic activity of vascular endothelium.
Br. Med.
Williams, S. K., M. A. Matthews and R. C. Wagner (1979). Metabolic studies on the micropinocytic process in endothelial cell. Microvasc. Res. 18, 175-184. Williams, S. K. and R. C. Wagner (1981). Divalent cation regulation of micropinocytosis in capillary endothelium. Microvasc. Res. 20. 134-143. Witte, S. (1981). The affinity of fluorescent labelled fibrinogen to the vessel wall as seen by vital microscopy. Biorheology 18, 578-588. Witte, S. (1984). The role of blood coagulation in capillary permeability. Vital microscopic contributions. Biorheology 21, 121-133.
0098-2997/88 $0.00 + .50 Copyright© 1988 Pergamon Press plc.
Printed in Great Britain. All rights reserved.
THE MODULATION OF TRANSCAPILLARY TRANSPORT OF MACROMOLECULES Ion
Baciu
and
Liviu
Grosu
Department of Physiology, Medical and Pharmaceutical Institute, 3400 Cluj-Napoca, Romania
Research over the last decades (Palade, 1953, 1960; Moore and Ruska, 1957; Palade and Bruns, 1968; Baciu, 1974; Palade et al., Simionescu et al., 1973, 1981) has shown that cytoplasmic vesicles make up the main pathway for macromolecules to pass through the capillary wall. More recently, by perfusion experiments of a single capillary and measurement of the minimum osmotic reflection coefficients (Clough and Michel, 1981; Curry and Michel, 1980; Mason et al., 1977), it has been shown that vesicular transport accounts for a major but variable part of the dissipative transport of macromolecules. However, other pathways such as intracellular junctions or vesicular channels (Simionescu et al., 1975) which are hydraulically conductive and which can be the support of either a diffusive (dissipative) or a convective transport, cannot be excluded (Renkin and Curry, 1982). It has also been shown that the transport through vesicles is not energetically dependent (it also takes place in the presence of metabolic inhibitors), and is also not a membrane consuming process (Casley-Smith, 1969; Allison and Davies, 1974; Simionescu, 1981; Williams and Wagner, 1981).
i.
The Modulation of Transport Through Vesicles
In systems of isolated and perfused capillaries, the vesicular uptake was found to be insensitive to many physical factors and vasoactive substances and hormones, except for histamine and serotonin, which depress it markedly, and prolactin which gives rise to a dose-dependent inhibition (Wagner and Mathews, 1980). Calcium and magnesium ions, on the other hand, stimulate the uptake, whilst calcium may also stimulate a rapid efflux of the tracer from the preloaded endothelial cells (Williams and Wagner, 1981). These modulatory effects on vesicular functions are probably the results of a direct action upon the endothelial membrane, because cellular metabolism is not involved. The modulation may be, actually, a regulation of fusion of the membranes of vesicles with the luminal and albuminal cytoplasmic membranes. Thus, the chemical environment which exists in blood and tissue fluids may regulate the flux of materials transported by vesicles through the capillary walls. Finally it is worth noting that studies of Buonassisi and Colburn (1980) and Simionescu et al. (1981, 1982) have revealed, at the surface of endothelial cells, the presence of receptors for hormones and the existence of different microdomains; it could be that such specific formations contribute to the binding and preferential transport of certain molecules.
273
274 2.
I Baciu and L Grosu The Global Modulation of Transcapillary Transport
The modulation of transcapillary transport may be accomplished not only by influencing the vesicular activity, but also may be exerted on other pathways for convective or diffusive transport.
2.1.
The effect of plasma proteins
The effect of plasma proteins upon capillary permeability, mentioned some 60 years ago by Krough and Harrop (1921) and studied by Drinker (1927) and later by Danielly (1940), was recently evaluated by Mason et al. (1977) on the elegant experimental model of isolated and perfused capillary from frog mesentery. They showed that the removal of plasma proteins from the perfusion liquid induces a five fold increase of the filtration coefficient, even if the colloidosmotic pressure is maintained with dextran; the presence of only 0.1% bovine serum albumin in the perfusion medium is sufficient to maintain permeability within normal levels. Since above 0.1% this effect of the protein was independent of concentration, it has been concluded that the phenomenon is caused by a direct adsorption of proteins onto the capillary surface. Clough and Michel (1981) have shown that, at least partially, the effect is also explained by an influence of proteins on the vesicular transport of macromolecules. Thus, in the absence of circulatory albumin, the number of luminal vesicles containing ferritin was almost two times higher than in the presence of 1% albumin; this was explained by a direct interaction of albumin with the capillary wall, more exactly with the glycocalyx which lines the endothelial cells. This led Curry and Michel (1980) to formulate the theory of the 'fibre matrix', according to which the permeability properties of unfenestrated continuous endothelium reside in the glycocalyx, a network of glycoproteins (with a diameter of 0.6 nm which occupies less than 8% of the network volume) that lines the endothelial surface and the intercellular junctions, the channels of fused vesicles or the caveoles (attached vesicles). Spaces between the fibre can provide resistance to movement of both small and large solute molecules. By picking up circulating proteins, the network tightens its mesh and thus makes the underlying vesicles less accessible to other water-soluble molecules. Schneeberger and Hamelin (1984) have recently shown that depletion of protein results in an increase of permeability to ferritin and that the intensification of ferritin transport is associated with the loss of adsorbed albumin and IgG from the glycocalyx.
2.2.
The role of fibrinolysis in capillary permeability
Systematic research on the role of coagulation and fibrinolysis in capillary permeability was started in our Institute in 1960, based on the hypothesis of the existence of an endovascular film of fibrin (Duguid, 1949; Copley, 1964). In the first experiments, carried out on dogs with the thoracic duct cannulated for lymph collection, we noticed that i.v. administration of Evans Blue (T 1824) resulted in an impressive increase of transcapillary transport and an activation of fibrinolysis. At the same time plasma kinins increased progressively and reached a maximum in 15-30 min (Baciu et al., 1967; Baciu and Grosu, 1970). The administration of thrombin to modify the coagulation and fibrinolytic activities, induced a significant reduction of the transcapillary transport of the plasma proteins with radical alterations of the coagulant and fibrinolytic reactions.
Transcapillary Transport
275
The data obtained in this experimental model, although striking, are difficult to interpret, owing to its complexity. On one hand, the i.v. administration of the macromolecular dyestuff per se resulted in activation of coagulation, fibrinolytic and kinin forming systems; on the other hand, thrombin also has a complex effect on these systems. We tried, therefore, in subsequent experiments to simplify the experimental model by using another macromolecular marker, the i.v. administration of which does not induce an activation of the above mentioned systems and some more specific modifiers of the fibrinolytic activity. The experiments were performed on rats and the macromolecular marker was radioiodinated serum albumin (RISA), the i.v. administration of which does not give rise to significant changes in coagulation, or fibrinolysis. For altering the activity of the fibrinolytic system we used streptokinase as a fibrinolytic activator, and aprotinin (Trasylol, TR) and g-aminocaproic acid (EACA) as fibrinolytic inhibitors. The transcapillary transport of macromolecules was estimated by determining the transcapillary escape rate of RISA. We showed that EACA has a clear-cut inhibitory effect on transcapillary transport of RISA, aprotinin has no effect. With streptokinase, although the slope of the regression line is lower than that in control animals, the initial value of radioactivity (extrapolated zero value) is below the other, suggesting an increased transport in the very first minutes (Fig. i) (Don, 1979).
100zx-x-x-x--x--x--x--x'-x-×-x--x] EA C A
60
-.m
"':-- " . o ~ j
40
C
\SK
20
'5 1"0 26 Fig. i.
3'0
4"0
5'0
60
min
Transcapillary escape rate of RISA (regression lines calculated from variations of blood RISA against time) in animals treated with activators (streptokinase, SK) and inhibitors (g-amino-caproic acid, EACA, and aprotinin, TR) of fibrinolysis and in control animals (C).
We compared these results with those obtained in another experimental model, which allowed the exploration of a more homogenous capillary area, the dog hind paw. One or more prenodal popliteal lymphatics were cannulated for lymph collection, and transcapillary transport was measured by determining the lymph to plasma concentration ratio of RISA. RISA was administrated immediately after the inhibitory substances, but 40 min before streptokinase to allow a certain plateau in the curve of RISA disappearance from plasma in the moment of streptokinase administration. In this model the data indicated that EACA and aprotinin have no
276
I. Baciu and L. Grosu
I -"@- -
I
-o
SK
A 13-
l l f
-@ n.-
~,.---,
TI~
~.~~CA
C
I'0 30 50 ?0 90 110 min Fig. 2.
Alteration of transcapillary transport of RISA after administration of streptokinase. Lymph to plasma ratio of RISA concentration [L]/[P] in animals treated with streptokinase (SK), aprotinin (TR), E-amino-caproic acid (EACA) and in control animals (C). The differences in the curves C, EACA, TR and SK (before the arrow), are not significant; they reflect a variability in the reactivity of various animals.
significant effect on transcapillary transport of RISA, while streptokinase administration produced a very marked activation (Fig. 2) (Don, 1979). Finaly, we tried other conditions associated with an activation of fibrinolysis, such as hypoxia, to see whether this is correlated with an increase in transport of macromolecules. The experiments were performed on dogs with general hypoxia and cephalic ischaemic hypoxia. It was found that both general hypoxic hypoxia and cephalic ischaemic hypoxia increase capillary permeability for macromolecules during the first 15 min (Fig. 3). The findings suggest that the increase in capillary permeability induced by hypoxia is achieved by neurohumoral mechanisms (Baciu et al., 1970). Actually, Kowarzyk et al. (1962) have shown that cephalic ischaemic hypoxia results in activation of fibrinolysis, which may explain the increase of permeability. With regard to the mechanism by which the activation of fibrinolysis induces an enhanced transcapillary transport of macromolecules, we must mention that subsequent research by Vermylen and Chamone (1979) could not confirm the attractive hypothesis of an endovascular film of fibrin continuously renewed by coagulation and fibrinolysis processes. In contrast, Witte (1981) found in rats injected i.v. with fibrinogen labelled with fluorescein isothiocyanate that 5-15 min after injection, fibrinogen accumulates on the endothelial luminal face in the form of points, bands or as a network. Such accumulations occur mainly at the level of venules and especially at interendothelial junctions. This author also observed the appearance of labelled fibrinogen in the perivascular tissue. It is worth mentioning that a polypeptidic fraction derived from factor VIII behaves similarly to fibrinogen. This fraction has an important inhibitory action on the capillary permeability to plasma proteins (Witte, 1984). In the light of recent research we can thus imagine the endothelial cells as
Transcapillary Transport
ml/rain.
GENERAL HYPOXIA rnl/min
277
CEPHALIC HYPOXIA
't '1
n"n llI nn ° NNINN NHnHn g~ 71
g~
i mp/min 8 Vmin
i-
]
i rnp/min t
150 180 210 240 2?0 30(3 0 15 60 g0 min
A Fig. 3.
'
150 180 210 240 270 300 0 30 50 90 120rain
B
The effects of general hypoxic hypoxia (left) and cephalic ischaemic hypoxia (right) on capillary permeability for macromolecules. Top: lymphatic rate. Middle: • • , the concentration of protein in plasma; , the concentration of protein in lymph; x i x , lymph to plasma protein concentration ratio. Hatched bars: protein clearance. Bottom: • - - • , the concentration of RISA in plasma; ..... , the concentration of RISA in lymph; x x , lymph to plasma RISA concentration ratio.
being surrounded by the environment of glycoproteins of the glycocalyx on which plasma proteins (albumins, globulins), fibrinogen and factor VIII are adsorbed. Consequently, the activation of fibrinolysis by means of a vascular activator, which is secreted, especially at the level of venules (Warren, 1964), could result in an increase in permeability. This could be due to the enlargement of the fibre network's mesh of glycocalyx as a result of the removal of fibrinogen and fibrin molecules. Secondly, the activation of fibrinolysis, both by direct action of plasmin and by factor VIII, results in the activation of prekallikrein (the Fletcher factor) to kallikrein, with consecutive release of bradykinin, which in turn causes an increase in capillary permeability (see below). Actually, we have noticed an increase in the concentration of kinins in plasma, in parallel with increasing rates of fibrinolysis and the activation of transcapillary transport. Moreover, preferred microdomains for transport of macromolecules could exist, for example the venular zone, where, as mentioned above, fibrinogen and factor VIII are preferentially accumulated, the vascular activator of plasminogen is synthesized, stored and released (Warre, 1964); the venular zone is also the zone with the
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largest density of receptors for histamine, especially those of the }{2 type (Simionescu et al., 1982). In the same zone, histamine and bradykinin produce a contraction of endothelial cells with the enlargement of interendothelial space, favoured by the unusually lax organization of intercellular contacts (Majno et al., 1969). 2.3.
Other modulations factors
It is well known that histamine, bradykinin and serotonin increase capillary permeability. The electronmicroscopical studies of Majno et ai.~(1969) showed that the increase is produced by contraction of endothelial cells and the consequent enlargement of intercellular junctions. Venules with a diameter between 20-30 ~m make up, in this respect, the most reactive part of microcirculation. More recently the effect has been demonstrated on capillary permeability of prostaglandins (PGE I, PGE2) and leukotrienes C 4 and D 4 (Fantone et al., 1980; Ueno et al., 1981). Kallikrein, a purified fragment of active factor XII and the C 3 fraction of complement also increase capillary permeability. However, it is very likely that in these latter cases, the effect is mediated by liberation of kinins, owing to the complex interaction between plasma protease systems (Imamura et al., 1984). We may conclude from the material presented above that, despite the recent remarkable progress made in the knowledge of modulatory mechanisms of the transcapillary transport of macromolecules, our knowledge is still limited and continued efforts are necessary for its broadening. References Allison, A. C. and P. Davies (1974). Hechanisms of endocytosis and exocytosis. Symp. Soc. exp. Biol. 28, 419-446. Baciu, I. (1971). 44, 757-770.
Permeabilitatia capiliara pentru macromolecule.
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Baciu, I., L. Grosu, M. Dorofteiu, F. Don, M. Zirbo, E. Enescu and N. Timar (1967). Efectele activarii procesului fibrinoplastic ?i fibrionolitic asupra permeabilita~ii capilare. Fiziol. norm. patol. 13, 11-29. Baciu, I. and L. Grosu (1970). Kininele plasmatice ~n reglarea circula~iei. In: Polipeptide endogene biologic active (I. Baciu, ed.), I.M.F. Cluj-Napoca. Baciu, I., L. Grosu, A. Olteanu and T. Pavel (1970).
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Buonassisi, V. and P. Colburn (1980). Hormones and surface receptors in vascular endothelium. In: Advances in Microcirculation. Vascular Endothelium and Basement Nembranes (B. M. Altura, ed.), Vol 9, Karger, Basel. Casley-Smith, J. R. (1969). Endocytosis: The different energy requirement for the uptake of particles by small and large vesicles into peritoneal macrophages. J. Microsc. 90, 15-30. Clough, G. and C. C. Hichel (1981). The role of vesicles in the transport of ferritin through frog endothelium. J. Physiol., Lond. 315, 127-142. Copley, A. L. (1964). Capillary permeability venous fragility and the significance of fibrin as a physiologic cement of the blood vessel wall. In: 2nd European Conference on Microcirculation Pavia, 1962, Karger, Basel.
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Curry, F. E. and C. C. Michel (1980). A fiber matrix model of capillary permeability. Microvasc. Res. 20, 96-99. Danielly, J. F. (1940). Capillary permeability and oedema in perfused frog. J. Physiol. 98, i09-129. Don Felicia (1979). Influenta procesului fibrinolitic si fibrino plastic asupra microcircula~iei. Teza de doctorat I.M.F. Cluj-Napoca. Don Felicia, L. Grosu, T. Pavel, M. Cucuianu, M. Grigerosik and I. Baciu (1987). Influence of some inhibitors and activators of fibrinolysis on transcapillary transport of macromolecules. Rev. roum. Morphol. Embryol. Physiol. Physiol. (in press). Drinker, C. K. (1927). The permeability and diameter of the capillaries in the web of the brown frog when perfused with solutions containing pituitary extract and horse serum. J. Physiol. 63, 249-269. Duguid, J. B. (1949).
Pathogenesis of atherosclerosis.
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Fantone, C. J., S. L. Kunkel, P. A. Ward and R. B. Zurier (1980). Suppression by prostaglandin E l of vascular permeability induced by vasoactive inflammatory mediators. J. Immunol. 125, 2591-2596. Imamura, T., T. Yamamoto and T. Kambara (1984). Guinea pig plasma kallikrein as a vascular permeability enhancement factor. A. J. Pathol. 115, 92-101. Kowarzyk, H., I. Baciu, C. Opri§iu and M. Kotschy (1962). Sur quelques mecanismes d'activation de la fibrinolyse. Arch. Immunol. ThOr. Exp. Krogh, A. and G. A. Harrop (1921). J. Physiol. 54, LXXV-CXXVI.
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Some observation on stasis and oedema.
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Fine Structure of blood capillaries.
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Renkin, E. M. and F. E. Curry (1982). Endothelial permeability: modulations. Ann. N. Y. Acad. Sci. 401, 248-258.
Pathways and
Schneeberger, E. E. and M. Hamelin (1984). Interaction of serum proteins with lung endothelial glycocalyx: its effect on endothelial permeability. Am. J. Physiol. 247, H206-H217. Simionescu, M., N. Simionescu and G. E. Palade (1973). Permeability of muscle capillaries to exogenous myoglobin. J. Cell. Biol. 57, 424-452. Simionescu, M., N. Simionescu and G. E. Palade (1975). Permeability of muscle capillaries to small hemepeptides. Evidence for the existence of patent transendothelial channels. J. Cell. Biol. 64, 586-607. Simionescu, N. (1981). Transcitosis and traffic of membranes in the endothelial cell. In: International Cell Biology (H. G. Schweizer, ed.), Springer Verlag, Berlin, Heidelberg. Simionescu, N., C. Neltianu, F. Antohe and M. Simionescu (1982). Endothelial cell receptors for histamine. Ann. N.Y. Acad. Sci. 401, 132-148. Ueno, A., K. Tanaka, M. Katori, M. Hayashi and K. Arai (1981). Species difference in increased vascular permeability by synthetic leucotriene C 4 and D 4. Prostaglandins 2__1, 637-647. Vermylen, I. G. and D. A. F. Chamone (1979). The role of the fibrinolytic system in thromboembolism. Prog. Cardiovasc. Dis. 21, 255-266. Wagner, R. C. and M. A. Matthews (1980). Vesicular ingestion by isolated capillaries: Effects of hormones and vasoactive substances. Microvasc. Res. 19, 200. Warren. B. A. (1964). Bull. 2_.9_0,213.
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Williams, S. K., M. A. Matthews and R. C. Wagner (1979). Metabolic studies on the micropinocytic process in endothelial cell. Microvasc. Res. 18, 175-184. Williams, S. K. and R. C. Wagner (1981). Divalent cation regulation of micropinocytosis in capillary endothelium. Microvasc. Res. 20. 134-143. Witte, S. (1981). The affinity of fluorescent labelled fibrinogen to the vessel wall as seen by vital microscopy. Biorheology 18, 578-588. Witte, S. (1984). The role of blood coagulation in capillary permeability. Vital microscopic contributions. Biorheology 21, 121-133.