C. C. Michel

Microvascular fluid exchange and the revised Starling principle

Microvascular fluid exchange (flow J(v)) underlies plasma/interstitial fluid (ISF) balance and oedematous swelling. The traditional form of Starlings principle has to be modified in light of insights into the role of ISF pressures and the recognition of the glycocalyx as the semipermeable layer of endothelium. Sum-of-forces evidence and direct observations show that microvascular absorption is transient in most tissues; slight filtration prevails in the steady state, even in venules. This is due in part to the inverse relation between filtration rate and ISF plasma protein concentration; ISF colloid osmotic pressure (COP) rises as J(v) falls. In some specialized regions (e.g. kidney, intestinal mucosa), fluid absorption is sustained by local epithelial secretions, which flush interstitial plasma proteins into the lymphatic system. The low rate of filtration and lymph formation in most tissues can be explained by standing plasma protein gradients within the intercellular cleft of continuous capillaries (glycocalyx model) and around fenestrations. Narrow breaks in the junctional strands of the cleft create high local outward fluid velocities, which cause a disequilibrium between the subglycocalyx space COP and ISF COP. Recent experiments confirm that the effect of ISF COP on J(v) is much less than predicted by the conventional Starling principle, in agreement with modern models. Using a two-pore system model, we also explore how relatively small increases in large pore numbers dramatically increase J(v) during acute inflammation.

Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. An immortalised rat brain endothelial cell line (GPNT) was used to investigate the mechanisms underlying dexamethasone-induced decrease in paracellular permeability. Following treatment with 1 microM dexamethasone there was a decrease in transmonolayer paracellular permeability mainly to sucrose, fluorescein and dextrans of up to 20 KDa. According to pore theory, these differences in permeability were consistent with a decrease in the number of pores between brain endothelial cells. This effect was accompanied by a concentration of filamentous actin and cortactin to the cell periphery. Concomitantly, the continuity of the tight junctional protein ZO-1 at the cell borders was improved and was associated with an increase in both ZO-1 and occludin expression. By contrast, the expression and distribution of adherens junctional proteins such as beta-catenin and p100/p120 remained unchanged. These observations suggest that glucocorticoids induce a more differentiated BBB phenotype in cultured brain endothelial cells through modification of tight junction structure.

Pathways through the intercellular clefts of frog mesenteric capillaries.

1. The three‐dimensional ultrastructure of endothelial intercellular clefts of frog mesenteric capillaries of known hydraulic permeability (Lp) has been investigated in the absence and presence of lanthanum ions as tracers of extracellular solute. 2. Experiments were carried out on the exposed mesenteries of pithed frogs and Lp of a chosen microvessel perfused with a Ringer solution containing serum albumin (10‐40 mg ml‐1) was determined. In some experiments the mesentery was fixed in situ with 2.5% glutaraldehyde immediately after Lp had been measured. In other experiments, measurement of Lp was followed by brief microperfusion (10‐20 s) with a second Ringer solution containing 1% lanthanum nitrate as a tracer before in situ fixation of the tissue. The tissue was prepared for electron microscopy using standard techniques. The perfused capillary was identified in the block and serial transverse sections were cut along its length over regions where Lp had been measured. 3. In six capillaries where the tissues were fixed immediately after measurement of Lp, Lp had a mean value (+/‐ S.E.M.) of 4 (+/‐ 0.5) x 10(‐7) cm s‐1 (cmH2O)‐1. Serial (30‐40 nm) sections of these vessels revealed that a single short narrow region of the intercellular clefts ran almost continuously from section to section. Additional tight regions were regularly seen, but they usually extended for relatively few sections. In 13.36 microns of reconstructed cleft, there were three interruptions of the tight region of 0.14, 0.14 and 0.17 microns respectively. In the region of these discontinuities, the wide region was uninterrupted from luminal to abluminal surface. 4. Examination of the tight junction on a tilting stage revealed that the outer leaflets of the adjacent cells were not fused, but separated by a gap of mean width (+/‐ S.E.M.) 2.3 (+/‐ 0.1) nm. 5. In four capillaries perfused with lanthanum nitrate before fixation, mean Lp (+/‐ S.E.M.) was 6.5 (+/‐ 0.02) x 10(‐7) cm s‐1 (cmH2O)‐1. Segments of intercellular clefts, totalling 23.56 microns in length, were reconstructed from serial sections and throughout these, electron‐dense deposits of lanthanum were observed to fill the luminal parts of the intercellular clefts up to the tight region. Lanthanum deposits filled the entire cleft to the abluminal surface at eleven sites, which accounted for a length of 2.52 microns out of the 23.56 microns. Only five of these regions were delimited within a continuous series of sections and their mean length (+/‐ S.E.M.) was 0.16 (+/‐ 0.063) microns.(ABSTRACT TRUNCATED AT 400 WORDS)

Openings Through Endothelial Cells Associated with Increased Microvascular Permeability

Rapid increases in microvascular permeability are associated with the appearance of openings in microvascular endothelium, which are believed to develop between the endothelial cells of venules. Reconstruction of these openings, from electron micrographs of serial sections of the endothelium reveal that many pass through the periphery of the endothelial cells close to intact intercellular junctions. Transcellular pathways are the principal type of opening induced in microvascular endothelium by the ionophore A23187, by VEGF, and by high transmural pressures. Some mediators induce the fusion of vacuoles with the luminal and abluminal surfaces of the endothelium, and it is suggested that the transcellular openings may develop from vacuolar channels.

Steady-state fluid filtration at different capillary pressures in perfused frog mesenteric capillaries.

1. The theory of steady‐state filtration through capillary walls (Michel, 1984) has been developed and investigated in experiments on single capillaries of the frog mesentery perfused with Ringer solutions containing bovine serum albumin (BSA) and Ficoll 70. 2. In each experiment, the micro‐occlusion technique of Michel, Mason, Curry, Tooke & Hunter (1974) has been used to investigate the relation between fluid movements per unit area of capillary wall (Jv/A) and capillary pressure (Pc) under two sets of conditions in the same vessel. First, the relation has been determined following brief perfusions, where the difference in oncotic pressure across the capillary wall approximated to the perfusate oncotic pressure at all values of Pc. These results are referred to as the transient data. Secondly, the relation was investigated by estimating Jv/A at values of Pc which had been maintained constant during at least 2 min of perfusion prior to the measurement. Under these conditions the concentration of macromolecules in the pericapillary fluid was determined by the steady‐state composition of the filtrate passing through the capillary wall, and these results are referred to as steady‐state data. 3. In all fifteen capillaries investigated, the relationship between Jv/A and Pc was linear for the transient data and conspicuously non‐linear in the steady state. When Pc exceeded the oncotic pressure of the perfusate, steady‐state values for Jv/A lay slightly above but parallel to the transient values for the same vessel. At values of Pc less than the perfusate oncotic pressure, the transient data showed reabsorption of fluid from the tissues, but in the steady state either fluid movements were so small as to be undetected or slight filtration was observed. The steady‐state data followed the pattern predicted by theory. 4. The transient data were used to estimate the reflection coefficient of the capillary wall (sigma) to the macromolecular solute. In seven vessels, the mean sigma to BSA was 0.76 (S.E. of mean +/‐ 0.04) and in eight different vessels mean sigma to Ficoll 70 in the presence of BSA (10 mg ml‐1) was 0.98 (S.E. of mean +/‐ 0.05). The steady‐state data were consistent with the prediction that the oncotic pressure opposing high filtration rates approximates to sigma 2 pi c in the steady state, where pi c is the perfusate oncotic pressure.(ABSTRACT TRUNCATED AT 400 WORDS)

Openings in frog microvascular endothelium induced by high intravascular pressures.

1. We have investigated the effects of microvascular pressures, high enough to rupture the vessel wall, upon the ultrastructure and permeability of mesenteric capillaries in pithed frogs. 2. The vessels were perfused via micropipettes with Ringer solutions containing bovine serum albumin (1 mg ml‐1) and a few human red cells. After a perfused section of vessel had been closed downstream, pressure (applied via the micropipette) was raised in a series of steps of 10 mmHg, each lasting approximately 10 s, until the vessel ruptured. Fluid filtration through the vessel wall prior to rupture was estimated from the movements of the red cells. 3. Seven vessels were fixed in glutaraldehyde immediately after rupture and prepared for electron microscopy. The electron micrographs revealed openings in the vessel walls and thirty‐six of these gaps were completely defined in runs of serial sections made on four of the vessels. Twenty‐nine of these gaps passed through the endothelial cells (transcellular) and seven were intercellular. 4. The pressure at which a vessel ruptured, Pb, was measured in twenty‐nine vessels and had a mean +/‐ S.E.M. value of 79.6 +/‐ 5.0 cmH2O. In ten of these vessels, which had a mean +/‐ S.E.M. Pb of 84.2 +/‐ 6.5 cmH2O, microvascular pressure was lowered immediately after the initial rupture and the vessel perfused at a pressure of 20 cmH2O. Pb was then remeasured and found to be 69.9 +/‐ 8.4 cmH2O, which was not significantly different from its initial value. 5. Hydraulic permeability (Lp) was measured in six vessels over the range of 15‐30 cmH2O before and 10 min after the vessel wall ruptured at high pressure. Mean values were 5.5 x 10(‐7) and 4.0 x 10(‐7) cms‐1 cmH2O‐1 and were not significantly different. 6. At pressures equal to and 10 mmHg below Pb, small short‐lived increases in filtration rate were observed. It is suggested that these may correspond to the increased permeability to fluid and macromolecules observed at high microvascular pressures in intact capillary beds.

Transcellular gaps in microvascular walls of frog and rat when permeability is increased by perfusion with the ionophore A23187.

1. The experiments described in this paper aimed to determine whether the gaps which develop in microvascular endothelium in association with increases in permeability are located in the intercellular clefts or are openings passing through the endothelial cells. 2. Hydraulic permeability (Lp) was estimated in frog mesenteric capillaries and single rat venules using a microperfusion‐micro‐occlusion technique before and during perfusion with solutions containing the ionophore A23187 at a concentration of 10 microM. When Lp was seen to have increased, the tissues were fixed in situ with 2.5% glutaraldehyde. 3. The increases in Lp varied considerably from vessel to vessel. In six frog vessels Lp increased from 2.6 +/‐ 0.9 x 10(‐7) to 266 +/‐ 159 x 10(‐7) cm s‐1 cmH2O‐1 and in three rat venules Lp rose from 0.94 +/‐ 0.09 x 10(‐7) to 16.4 +/‐ 4.9 x 10(‐7) cm s‐1 cmH2O‐1 (means +/‐ S.E.M.). 4. Forty openings or gaps were completely reconstructed from electron micrographs of serial ultrathin sections of the six frog vessels. Thirty‐nine of these gaps passed through the endothelial cells and did not communicate with the intercellular clefts; one was intercellular. Similarly, fifteen out of sixteen gaps reconstructed from electron micrographs of the rat venules were transcellular and clearly separated from the intercellular clefts. 5. The increased Lp and associated ultrastructural changes induced by A23187 were reversed by perfusion with ionophore‐free solutions.

Perfusion of Single Tumor Microvessels: Application to Vascular Permeability Measurement

Objective: To develop a new method for determining the relative importance of convection versus diffusion in macromolecular transport across tumor microvessel walls.

Secretion of adipokines by human adipose tissue in vivo: partitioning between capillary and lymphatic transport

Peptides secreted by adipose tissue (adipokines) may enter blood via capillaries or lymph. The relative importance of these pathways for a given adipokine might influence its biological effects. Because this has not been studied in any species, we measured the concentrations of seven adipokines and eight nonsecreted proteins in afferent peripheral lymph and venous plasma from 12 healthy men. Data for nonsecreted proteins were used to derive indices of microvascular permeability, which in conjunction with the molecular radii of the adipokines were used to estimate the amounts leaving the tissue via capillaries. Transport rates via lymph were estimated from the lymph adipokine concentrations and lymph flow rates and total transport (secretion) as the sum of this and capillary transport. Concentrations of nonsecreted proteins were always lower in lymph than in plasma. With the exception of adiponectin, adipokine concentrations were always higher in lymph (P < 0.01). Leptin and MCP-1 were secreted at the highest rates (means: 43 μg/h or 2.7 nmol/h and 32 μg/h or 2.4 nmol/h, respectively). IL-6 and MCP-1 secretion rates varied greatly between subjects. The proportion of an adipokine transported via lymph was directly related to its molecular radius (r(s) = +0.94, P = 0.025, n = 6), increasing from 14 to 100% as the radius increased from 1.18 (IL-8) to 3.24 nm (TNFα). We conclude that the lymph/capillary partitioning of adipokines is a function of molecular size, which may affect both their regional and systemic effects in vivo. This finding may have implications for the physiology of peptides secreted by other tissues.

Quantitative comparisons of hydraulic permeability and endothelial intercellular cleft dimensions in single frog capillaries.

1. We have investigated the ultrastructure of the intercellular clefts of the walls of single capillaries and venules of the frog mesentery in which the hydraulic permeability (Lp) and the reflection coefficient of the vessel walls to serum albumin (sigma BSA) had been measured using the micro‐occlusion technique of Michel (1980). Our aim was to investigate whether the dimensions of the clefts were sufficient to accommodate the pathways through the vessel walls necessary to account for the measured permeability. 2. Lp was measured in seventeen individually perfused vessels. The walls of fourteen of these were relatively impermeable to macromolecules with a sigma to albumin greater than 0.66 (mean value 0.83, S.E.M. +/‐ 0.04). The Lp of these fourteen vessels ranged from 1.8 x 10(‐7) to 12.5 x 10(‐7) cm s‐1 cmH2O‐1 and had a mean value of 5.9 (S.E.M. +/‐ 0.85) x 10(‐7) cm s‐1 cmH2O‐1. 3. Cleft dimensions estimated from electron micrographs of 642 transversely sectioned endothelial cell junctions from the same seventeen vessels gave a value for the mean cleft width (W) of 0.0220 micron (S.E.M. +/‐ 0.0064 micron). The mean depth of the clefts from luminal to abluminal surface of the endothelium (delta x) was 0.395 micron (S.E.M. +/‐ 0.091 micron) with a range of 0.104‐1.70 micron. The cleft length per unit area of cell wall (L), calculated using the formulation of Bundgaard & Frøkjaer‐Jensen (1982), was 2064 (S.E.M. +/‐ 112) cm cm‐2. Measurements were also made of cleft dimensions from longitudinally sectioned junctions from five of the seventeen vessels. 4. The fraction of the surface area of capillary wall occupied by the clefts (Ap = LW) had a mean value of 0.0048 (+/‐ 0.00014) for all seventeen vessels with a range of 0.0030‐0.0074 when estimated from transverse sections. There was no correlation between the variation of Lp between different vessels and the variations of Ap. 5. Data from the fourteen vessels when sigma BSA was greater than 0.66 revealed a correlation between values of Lp and the reciprocal of delta x (r = 0.6675, P less than 0.01). No correlation was found between Lp and the mean thickness of the endothelial cells in the vicinity of the clefts. This is strong evidence for the intercellular cleft being the principal pathway for fluid movements. Variation in cleft depth appears to be a factor determining variation in permeability between different capillaries.(ABSTRACT TRUNCATED AT 400 WORDS)