Peter Gaehtgens

Resistance to blood flow in microvessels in vivo.

Resistance to blood flow through peripheral vascular beds strongly influences cardiovascular function and transport to tissue. For a given vascular architecture, flow resistance is determined by the rheological behavior of blood flowing through microvessels. A new approach for calculating the contribution of blood rheology to microvascular flow resistance is presented. Morphology (diameter and length), flow velocity, hematocrit, and topological position were determined for all vessel segments (up to 913) of terminal microcirculatory networks in the rat mesentery by intravital microscopy. Flow velocity and hematocrit were also predicted from mathematical flow simulations, in which the assumed dependence of flow resistance on diameter, hematocrit, and shear rate was optimized to minimize the deviation between measured and predicted values. For microvessels with diameters below approximately 40 microns, the resulting flow resistances are markedly higher and show a stronger dependence on hematocrit than previously estimated from measurements of blood flow in narrow glass tubes. For example, flow resistance in 10-microns microvessels at normal hematocrit is found to exceed that of a corresponding glass tube by a factor of approximately 4. In separate experiments, flow resistance of microvascular networks was estimated from direct measurements of total pressure drop and volume flow, at systemic hematocrits intentionally varied from 0.08 to 0.68. The results agree closely with predictions based on the above-optimized resistance but not with predictions based on glass-tube data. The unexpectedly high flow resistance in small microvessels may be related to interactions between blood components and the inner vessel surface that do not occur in smooth-walled tubes.

Blood flow in microvascular networks. Experiments and simulation.

A theoretical model has been developed to simulate blood flow through large microcirculatory networks. The model takes into account the dependence of apparent viscosity of blood on vessel diameter and hematocrit (the Fahraeus-Lindqvist effect), the reduction of intravascular hematocrit relative to the inflow hematocrit of a vessel (the Fahraeus effect), and the disproportionate distribution of red blood cells and plasma at arteriolar bifurcations (phase separation). The model was used to simulate flow in three microvascular networks in the rat mesentery with 436,583, and 913 vessel segments, respectively, using experimental data (length, diameter, and topological organization) obtained from the same networks. Measurements of hematocrit and flow direction in all vessel segments of these networks tested the validity of model results. These tests demonstrate that the prediction of parameters for individual vessel segments in large networks exhibits a high degree of uncertainty; for example, the squared coefficient of correlation between predicted and measured hematocrit of single vessel segments ranges only between 0.15 and 0.33. In contrast, the simulation of integrated characteristics of the network hemodynamics, such as the mean segment hematocrit or the distribution of blood flow velocities, is very precise. In addition, the following conclusions were derived from the comparison of predicted and measured values: 1) The low capillary hematocrits found in mesenteric microcirculatory networks as well as their heterogeneity can be explained on the basis of the Fahraeus effect and phase-separation phenomena. 2) The apparent viscosity of blood in vessels of the investigated tissue with diameters less than 15 microns is substantially higher than expected compared with measurements in glass tubes with the same diameter.

Biophysical aspects of blood flow in the microvasculature

The main function of the microvasculature is transport of materials. Water and solutes are carried by blood through the microvessels and exchanged, through vessel walls, with the surrounding tissues. This transport function is highly dependent on the architecture of the microvasculature and on the biophysical behavior of blood flowing through it. For example, the hydrodynamic resistance of a microvascular network, which determines the overall blood flow for a given perfusion pressure, depends on the number, size and arrangement of microvessels, the passive and active mechanisms governing their diameters, and on the apparent viscosity of blood flowing in them. Suspended elements in blood, especially red blood cells, strongly influence the apparent viscosity, which varies with several factors, including vessel diameter, hematocrit and blood flow velocity. The distribution of blood flows and red cell fluxes within a network, which influences the spatial pattern of mass transport, is determined by the mechanics of red cell motion in individual diverging bifurcations. Here, our current understanding of the biophysical processes governing blood flow in the microvasculature is reviewed, and some directions for future research are indicated.

Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules.

At the onset of the inflammatory process, leukocytes roll along venular but not arteriolar walls before they firmly attach and emigrate. To test whether differences in hydrodynamic flow conditions are responsible for the preferential occurrence of leukocyte rolling in venules, we varied wall shear rate, gamma w, between 30 and 2,000 sec-1 by selective micro-occlusion of side branches in venules and arterioles (diameter, 20-37 microns) of the exposed mesentery of anesthetized rats. In venules, 39% (range, 6-77%) of all passing leukocytes were found interacting with the endothelium (rolling), whereas this fraction was only 0.6% in arterioles. The fraction of rolling leukocytes in venules decreased from 49 +/- 13% at gamma w less than 100 sec-1 (N = 12) to 24 +/- 13% at gamma w greater than 400 sec-1 (N = 12). Mean leukocyte rolling velocity in venules increased with gamma w, but the most frequent rolling velocity class was 20-40 microns/sec at all shear rates. In arterioles, even prolonged (up to 90 minutes) conditions of reduced flow (gamma w less than 150 sec-1) did not induce leukocyte rolling. Radial distribution of freely flowing leukocytes not different in arterioles and venules. The data indicate that hemodynamic factors are not responsible for the difference of leukocyte adhesion between arterioles and venules. The venular endothelium appears to be specialized to support leukocyte adhesion during inflammation. This finding correlates with reports on preferential expression of various endothelial-leukocyte adhesion molecules on venular endothelial cells.

Red Cell Distribution at Microvascular Bifurcations

The distribution of red cell and blood volume flow was studied at 65 arteriolar bifurcations in the rat mesentery. Hematocrit and flow velocity were measured simultaneously in all three vessel segments constituting a bifurcation. Blood flow distribution was manipulated by irreversibly occluding downstream side branches of one of the daughter vessels. The dependence of fractional red cell volume flow on fractional blood flow was described using a three-parameter (X0, B, A) logit function. The critical volume flow fraction below which only plasma enters a downstream branch (X0), the nonlinearity of the relation between red cell and blood volume flow (B), and the asymmetry of that relation which is described by the parameter A decrease with increasing diameter of the vessel feeding the bifurcation. At diameters above 30 microns, phase separation is very limited. In addition, the nonlinearity parameter B decreases with decreasing hematocrit in the feeding vessel. The asymmetry parameter A strongly depends on the diameter ratio between the two daughter branches: For a given fractional blood flow, the smaller branch receives more red cells than the larger branch. Using a model for plasma skimming based on the assumption of a planar separating surface, the shape of the radial hematocrit profile in the feeding vessel has been calculated. The model predicts a decrease in local hematocrit from the vessel axis toward the wall with a distinct marginal zone free from cell centers. With increasing vessel diameter the hematocrit profile becomes more blunted while the width of the marginal zone increases.

Structural adaptation and stability of microvascular networks: theory and simulations

A theoretical model was developed to simulate long-term changes of vessel diameters during structural adaptation of microvascular networks in response to tissue needs. The diameter of each vascular segment was assumed to change with time in response to four local stimuli: endothelial wall shear stress (τw), intravascular pressure (P), a flow-dependent metabolic stimulus (M), and a stimulus conducted from distal to proximal segments along vascular walls (C). Increases in τw, M, or C or decreases in P were assumed to stimulate diameter increases. Hemodynamic quantities were estimated using a mathematical model of network flow. Simulations were continued until equilibrium states were reached in which the stimuli were in balance. Predictions were compared with data from intravital microscopy of the rat mesentery, including topological position, diameter, length, and flow velocity for each segment of complete networks. Stable equilibrium states, with realistic distributions of velocities and diameters, were achieved only when all four stimuli were included. According to the model, responses to τw and P ensure that diameters are smaller in peripheral than in proximal segments and are larger in venules than in corresponding arterioles, whereas M prevents collapse of networks to single pathways and C suppresses generation of large proximal shunts.A theoretical model was developed to simulate long-term changes of vessel diameters during structural adaptation of microvascular networks in response to tissue needs. The diameter of each vascular segment was assumed to change with time in response to four local stimuli: endothelial wall shear stress (tauw), intravascular pressure (P), a flow-dependent metabolic stimulus (M), and a stimulus conducted from distal to proximal segments along vascular walls (C). Increases in tauw, M, or C or decreases in P were assumed to stimulate diameter increases. Hemodynamic quantities were estimated using a mathematical model of network flow. Simulations were continued until equilibrium states were reached in which the stimuli were in balance. Predictions were compared with data from intravital microscopy of the rat mesentery, including topological position, diameter, length, and flow velocity for each segment of complete networks. Stable equilibrium states, with realistic distributions of velocities and diameters, were achieved only when all four stimuli were included. According to the model, responses to tauw and P ensure that diameters are smaller in peripheral than in proximal segments and are larger in venules than in corresponding arterioles, whereas M prevents collapse of networks to single pathways and C suppresses generation of large proximal shunts.

Angiotensin II–Induced Leukocyte Adhesion on Human Coronary Endothelial Cells Is Mediated by E-Selectin

Clinical data suggest a link between the activation of the renin-angiotensin system and cardiovascular ischemic events. Leukocyte accumulation in the vessel wall is a hallmark of early atherosclerosis and plaque progression. E-Selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules participating in mediating interactions between leukocytes and endothelial cells and have been found to be expressed in athero-sclerotic plaques. We investigated whether angiotensin II, the effector of the renin-angiotensin system, influences the endothelial expression of E-selectin, VCAM-1, and ICAM-1. In coronary endothelial cells derived from explanted human hearts, angiotensin II (10(-11) to 10(-5) mol/L) induced a concentration-dependent increase in E-selectin expression. The effect was measured by cell ELISA and duplex reverse-transcription polymerase chain reaction (RT-PCR) and reached its maximum at 10(-7) mol/L. Angiotensin II induced only a small increase in E-selectin expression in cardiac microvascular endothelial cells. VCAM-1 and ICAM-1 were not affected by angiotensin II stimulation. In addition, the effect of angiotensin II-induced E-selectin expression on leukocyte adhesion was quantified under flow conditions. Angiotensin II (10(-7) mol/L) increased leukocyte adhesion significantly to 67% of the maximal effect by tumor necrosis factor-alpha at a wall shear stress of 2 dyne/cm2. This adhesion was found to be E-selectin dependent, as demonstrated by blocking antibodies. The AT1-receptor antagonist DUP 753 significantly reduced E-selectin-dependent adhesion, whereas the AT2-receptor antagonist PD 123177 had no inhibitory effect. In addition, only AT1-receptor, but not AT2-receptor, mRNA could be detected by RT-PCR in coronary endothelial cells. Therefore, it is suggested that AT1 receptors mediate the effects of angiotensin II on E-selectin expression and leukocyte adhesion on coronary endothelial cells.

Microvascular blood flow resistance: role of endothelial surface layer

Observations of blood flow in microvascular networks have shown that the resistance to blood flow is about twice that expected from studies using narrow glass tubes. The goal of the present study was to test the hypothesis that a macromolecular layer (glycocalyx) lining the endothelial surface contributes to blood flow resistance. Changes in flow resistance in microvascular networks of the rat mesentery were observed with microinfusion of enzymes targeted at oligosaccharide side chains in the glycocalyx. Infusion of heparinase resulted in a sustained decrease in estimated flow resistance of 14-21%, hydrodynamically equivalent to a uniform increase of vessel diameter by approximately 1 micron. Infusion of neuraminidase led to accumulation of platelets on the endothelium and doubled flow resistance. Additional experiments in untreated vascular networks in which microvascular blood flow was reduced by partial microocclusion of the feeding arteriole showed a substantial increase of flow resistance at low flow rates (average capillary flow velocities < 100 diameters/s). These observations indicate that the glycocalyx has significant hemodynamic relevance that may increase at low flow rates, possibly because of a shear-dependent variation in glycocalyx thickness.Observations of blood flow in microvascular networks have shown that the resistance to blood flow is about twice that expected from studies using narrow glass tubes. The goal of the present study was to test the hypothesis that a macromolecular layer (glycocalyx) lining the endothelial surface contributes to blood flow resistance. Changes in flow resistance in microvascular networks of the rat mesentery were observed with microinfusion of enzymes targeted at oligosaccharide side chains in the glycocalyx. Infusion of heparinase resulted in a sustained decrease in estimated flow resistance of 14-21%, hydrodynamically equivalent to a uniform increase of vessel diameter by ∼1 μm. Infusion of neuraminidase led to accumulation of platelets on the endothelium and doubled flow resistance. Additional experiments in untreated vascular networks in which microvascular blood flow was reduced by partial microocclusion of the feeding arteriole showed a substantial increase of flow resistance at low flow rates (average capillary flow velocities < 100 diameters/s). These observations indicate that the glycocalyx has significant hemodynamic relevance that may increase at low flow rates, possibly because of a shear-dependent variation in glycocalyx thickness.

A role for β2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response

Growing evidence supports the idea that adhesion via β2 integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether β2 integrin‐mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the β2 integrin Mac‐1 (CD11b/CD18) by antibody cross‐linking was found to induce substantial de novo synthesis of IL‐8 mRNA as measured by semiquantitative RT‐PCR and Northern blotting technique, respectively. Induction of IL‐8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac‐1. Results were confirmed using PMN derived from CD18‐deficient mice, which were unable to produce MIP‐2 mRNA, a homologue of human IL‐8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP‐2 mRNA was observed when wild‐type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL‐8 and IL‐1β. In contrast, no TNF‐α or IL‐6 production was found, revealing that β2 integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.—Walzog, B., Weinmann, P., Jeblonski, F., Scharffetter‐Kochanek, K., Bommert, K., Gaehtgens, P. A role for β2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J. 13, 1855–1865 (1999)

Beta2 integrins (CD11/CD18) promote apoptosis of human neutrophils.

Apoptosis of human polymorphonuclear neutrophils (PMN) is thought to be critical for the control of the inflammatory process, but the mechanisms underlying its regulation in physiological settings are still incompletely understood. This study was undertaken to test the hypothesis that the β2 integrin (CD11/CD18) family of leukocyte adhesion molecules contributes to the control of activated PMN by up‐regulating apoptosis. Apoptosis of isolated human PMN was investigated by 1) analysis of DNA content, 2) detection of DNA degradation, 3) morphological studies, and 4) measurement of CD16 expression on the cell surface. We found that β2 integrins potentiated the tumor necrosis factor α (TNF‐α) ‐induced apoptosis within 4 and 8 h after stimulation. The effect required aggregation of the β2 integrin Mac‐1 (CD11b/CD18), which was induced by antibody cross‐linking, and was independent of Fc receptors. An enhancement of apoptosis was also observed after migration of PMN through an endothelial cell monolayer. TNF‐α‐induced apoptosis as well as potentiation by β2 integrins was prevented by inhibition of tyrosine kinases with herbimycin A or genistein. The present study provides a new model for the regulation of PMN apoptosis by a functional cross‐talk between β2 integrins and TNF‐α with a promoting role for the β2 integrins. This mechanism, which allows enhanced elimination of previously emigrated PMN, may be critical to abate local inflammatory processes in vivo.—Walzog, B., Jeblonski, F., Zakrzewicz, A., Gaehtgens, P. β2 integrins (CD11/CD18) promote apoptosis of human neutrophils. FASEB J. 11, 1177–1186 (1997)