Herbert H. Lipowsky

Leukocyte-endothelium adhesion: microhemodynamics in mesentery of the cat.

The effects of leukocyte-endothelium adhesion on microhemodynamics were studied in cat mesentery under control conditions and following tissue suffusion with the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (FMLP). The results indicate that under normophysiological conditions there is little or no leukocyte-endothelium adhesion in arterioles and venules. Tissue suffusion with FMLP significantly increases the number of adhering leukocytes in venules, but not in arterioles. Analysis of the number of adhering leukocytes (in venules) as a function of wall shear rate suggested that increased adhesion of leukocytes was primarily due to elevated adhesive forces and not the result of decreases in dispersal forces, i.e., wall shear stress. From measurements of upstream to downstream pressure drop, red cell velocity, and microvessel hematocrit in 16 unbranched venules, no significant changes in diameter (mean of 39.9 +/- 7.8 (SD) micron), intravascular pressure gradient (0.59 +/- 0.40 X 10(-2) cm H2O/micron), nor microvessel hematocrit (31.0 +/- 9.8%) occurred in response to FMLP. There were significant decreases in upstream pressure (8%) and estimated bulk flow (28%) as well as significant increases in the number of adhering leukocytes, from 1.5 +/- 2.8 to 11.4 +/- 8.3 cells/100 micron, and vessel resistance (81%). Changes in hemodynamics were found to be more pronounced in venules with small diameters. The observed response to FMLP suggests that changes in hemodynamics during leukocyte-endothelium adhesion can be accounted for by a decrease in the effective diameter due to obstruction of the lumen by WBCs, and that adhesive interactions between WBCs and endothelium are a major determinant of blood flow resistance in the microcirculation.

In vivo measurements of “apparent viscosity” and microvessel hematocrit in the mesentery of the cat ☆

Abstract The arteriovenous (A-V) distribution of microvessel hematocrit (Hmicro) was determined throughout successive microvascular divisions in cat mesentery from in vivo measurements of optical density. In vitro correlations of optical density and tube hematocrit in small-bore glass tubes permitted the computation of in vivo values of Hmicro for luminal diameters ranging from 20 to 70 μm. For smaller-size vessels, Hmicro was determined by microocclusion and red cell counting. The results demonstrate a monotonic fall in the ratio of H micro H systemic from 0.80 in the 70-μm arterioles to a minimum of 0.21 in the immediate postcapillaries (10 μm diameter) followed by a subsequent monotonic rise to 0.95 in the 70-μm venules. Conservation of red cell flux throughout the mesenteric network was partially demonstrated upon applying previously established in vitro relationships between discharge and tube hematocrits, the resulting disparity being attributed to the rheological behavior of blood and possible A-V shunting of red cells. Simultaneous measurements of pressure drop and red cell velocity in unbranched arterioles during systemic hemodilution facilitated a comparison of in vivo and in vitro (cone-plate viscometer) apparent viscosities (η). No significant differences between the two approaches were found for arteriole diameters ranging from 24 to 47 μm in the absence of leukocyte-endothelium adhesion, with 0

The distribution of blood rheological parameters in the microvasculature of cat mesentery.

In vivo studies of the rheological behavior of blood in the microcirculation were conducted by direct in situ measurements in cat mesentery. Upstream to downstream pressure drops were measured in unbranched arterioles, capillaries, and venules, with diameters from 7 to 58 fim. Simultaneous measurements of red cell velocity and vessel geometry facilitated computation of bulk velocity, pressure gradient, apparent viscosity, wall shear stress, and resistance. Arteriovenous distributions of these parameters revealed the following. Maximum pressure gradient (0.015 cm H2O/;im) occurs in the true capillaries (7 μm in diameter); intravascular wall shear stress averaged 47.1 dynes/cm2 in arterioles and 29.0 dynes/cm2 in venules. Extreme values as great as 200 dynes/cm2 were observed in a few shunting arterioles. Apparent viscosity averaged 3.59 cP in arterioles, 5.15 cP in venules, and 4.22 cP overall. Intravascular resistance per unit length of microvessel varied with luminal diameter as a power law function with exponents of −4.04 for arterioles, −3.94 for venules, and −3.99 for all vessels combined. This apparent maintenance of Poiseuilles law is attributed to the opposing processes of hematocrit reduction and decreasing shear rate as blood is dispersed in successive arteriolar segments, and the converse action of these processes in the venous confluences which lessen the extent of network variations in apparent viscosity. Reductions in bulk velocity from the normal flow state to below 0.5 mm/sec resulted in increases in apparent viscosity by a factor of 2 to 10, which are attributed primarily to obstruction of the lumen by leukocyte-endothelium adhesion.

Application of the “two-slit” photometric technique to the measurement of microvascular volumetric flow rates☆

Abstract In vitro studies of red cell suspensions flowing through glass tubes were performed to provide additional details on the empirical relationship between red cell velocity measured by the “twoslit” photometric technique along the vessel centerline, V , and the mean velocity of cells plus plasma, V mean . Small bore glass tubes (17- to 60-μm internal diameter) were used to simulate a blanket application of this method to the microcirculation. The previously established ratio, V V mean = 1.6 , was found to be valid within 5 to 10% in these tubes and for velocities within the physiological range. For tube diameters decreasing from 60 to 17 μm, V V mean was found to increase slightly but was still within 10% of the 1.6 ratio. Analysis of earlier in vivo studies of the single-file motion of red cells in 6- to 10-μm capillaries, suggests that below 10 μm, V V mean should approach a value on the order of 1.3, or 19% below the 1.6 factor.

Microvascular Rheology and Hemodynamics

The goal of elucidating the biophysical and physiological basis of pressure–flow relations in the microcirculation has been a recurring theme since the first observations of capillary blood flow in living tissues. At the birth of the Microcirculatory Society, seminal observations on the heterogeneous distribution of blood cells in the microvasculature and the rheological properties of blood in small bore tubes raised many questions on the viscous properties of blood flow in the microcirculation that captured the attention of the Societys membership. It is now recognized that blood viscosity in small bore tubes may fall dramatically as shear rates are increased, and increase dramatically with elevations in hematocrit. These relationships are strongly affected by blood cell deformability and concentration, red cell aggregation, and white cell interactions with the red cells and endothelium. Increasing strength of red cell aggregation may result in sequestration of clumps of red cells with either reductions or increases in microvascular hematocrit dependent upon network topography. During red cell aggregation, resistance to flow may thus decrease with hematocrit reduction or increase due to redistribution of red cells. Blood cell adhesion to the microvessel wall may initiate flow reductions, as, for example, in the case of red cell adhesion to the endothelium in sickle cell disease, or leukocyte adhesion in inflammation. The endothelial glycocalyx has been shown to result from a balance of the biosynthesis of new glycans, and the enzymatic or shear‐dependent alterations in its composition. Flow‐dependent reductions in the endothelial surface layer may thus affect the resistance to flow and/or the adhesion of red cells and/or leukocytes to the endothelium. Thus, future studies aimed at the molecular rheology of the endothelial surface layer may provide new insights into determinants of the resistance to flow.

Capillary recruitment in response to tissue hypoxia and its dependence on red blood cell deformability

The effect of reduced red blood cell (RBC) deformability on microvessel recruitment attendant to a reduction in tissue PO2 was studied in rat cremaster muscle using indicator-dilution techniques. Transit times (TT) of fluorescently labeled RBCs (TTRBC) and plasma (TTPl) between functionally paired arterioles and venules were obtained from their dispersion throughout the microvascular network. Changes in PO2 were effected by superfusing the tissue with Ringer solution deoxygenated to different levels. Arteriolar blood flow (Q) was measured with the two-slit technique, and the vascular volume (V) occupied by RBCs and plasma was computed from the product of Q x TT during bolus infusions of rat and less deformable human RBCs to obtain VRBC and fluorescently labeled albumin to obtain VPl. Measurements of TTRBC and TTPl permitted computation of an average flow-weighted tissue (microvascular) hematocrit (HM) relative to systemic values (HS). During infusions of autologous rat RBCs, Q and total V increased threefold in response to hypoxia, whereas normalized RBC TT (TTRBC/TTPl) and normalized tissue hematocrit (HM/HS) did not show a significant trend, indicating an increase in the number of pathways through which the RBCs can traverse the network because of spatial recruitment of capillaries. In contrast, during infusions of human RBCs, TTRBC/TTPl and HM/HS decreased significantly in response to hypoxia. Although Q exhibited an increase similar to that during rat RBC infusions, VRBC exhibited a smaller increase compared with VPl, suggesting that reduced RBC deformability leads to a redistribution of RBCs through larger-diameter pathways within the network and exclusion of these RBCs from pathways normally recruited during hypoxia. Hence, reduced RBC deformability may adversely affect capillary recruitment and physiological mechanisms that ensure adequate delivery of oxygen to tissue.

Network analysis of microcirculation of cat mesentery

Abstract The modular configuration of the microcirculatory system in cat mesentery is subjected to a hydrodynamic network analysis assuming Poiseuillian dynamical behavior. Intravascular pressure, vessel pressure gradient, and wall shear stress are computed for an isolated module and presented as a function of vessel diameter, from arterial affluent to venous effluent. Computed and in vivo intravascular pressures show a marked disparity on the arterial side of the true capillaries and a fair agreement on the venous side. This is attributed to the effects of precapillary sphincter action and non-Newtonian rheological behavior. Computed pressure gradients based on a simple Poiseuillian relationship are approximately six times greater than those measured in vivo . By comparison of predicted and measured pressure gradients, the magnitude of maximum vessel wall shear stress is estimated to be on the order of 10 dyn/cm 2 .

Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes

The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10(-)(7)M fMLP. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 (D(Dx70)), and a smaller sublayer 173nm above the EC surface (D(173)), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D(Dx70) was twice that of D(173) suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D(Dx70) and D(173). However, heparinase decreased D(Dx70), and did not induce any significant change for the D(173). These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.

Intravital microscopy of capillary hemodynamics in sickle cell disease.

Direct intravital microscopic examinations were made in nailfold capillaries in subjects with homozygous sickle cell disease (HbSS red cells). In the resting state, capillary red cell (rbc) flux exhibited greater intermittence compared with normal subjects, which increased with painful crisis. In crisis-free HbSS subjects, capillary occlusion and red cell sequestration occurred in only 8.2% of all capillaries and diminished to 5.8% during crisis, possibly due to sequestration of less deformable rbcs in other organs. Velocities of rbcs (Vrbc) were measured by video techniques under resting conditions and during postocclusive reactive hyperemia (PORH) induced by a pressure cuff around the finger. Resting Vrbc was normal in crisis-free HbSS subjects, averaging 0.7 mm/s. In contrast, Vrbc was significantly elevated during crisis, to 0.98 mm/s, apparently due to compensatory arteriolar dilation. Crisis subjects exhibited a significantly depressed PORH with the ratio of peak red cell velocity to resting values reduced by 15% due to a loss of vasodilatory reserve, whereas crisis-free subjects exhibited a normal response. A 55% increase in the time to attain peak Vrbc was attributed to resistance increases, possibly resulting from red cell and leukocyte-to-endothelium adhesion during the induced ischemia.

Inhibition of Glycan Shedding and Leukocyte-Endothelial Adhesion in Postcapillary Venules by Suppression of Matrixmetalloprotease Activity with Doxycycline

Objective: The aims of this study were to examine the role of matrixmetalloproteinases (MMPs) in causing shedding of glycan components of the endothelial glycocalyx and delineate the efficacy of doxycycline as an inhibitor of white blood cell–endothelial cell (WBC‐EC) adhesion and glycan shedding in postcapillary venules.