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. 2011 Aug 22;589(Pt 21):5181–5192. doi: 10.1113/jphysiol.2011.209262

Excessive erythrocytosis compromises the blood–endothelium interface in erythropoietin-overexpressing mice

Vincent Richter 1, Michele D Savery 2, Max Gassmann 3, Oliver Baum 4, Edward R Damiano 2, Axel R Pries 1,5
PMCID: PMC3225673  PMID: 21859826

Non-technical summary

Elevated systemic haematocrit (Hct) increases cardiovascular risk, such as stroke and myocardial infarction. One possible pathophysiological mechanism could be a disturbance of the blood–endothelium interface. It has been shown that blood interacts with the endothelial surface via a gel-like layer (the ‘glycocalyx’, or ‘endothelial surface layer’– ESL) that modulates various biological processes, including inflammation, permeability and atherosclerosis. However, the consequences of an elevated Hct on the functional properties of this interface are incompletely understood. In a transgenic mouse (tg6) model exhibiting systemic Hct levels of about 0.85 the glycocalyx/ESL was nearly abolished. The corresponding increase in vessel diameter had only minor effects on peripheral flow resistance. This suggests that the pathological effects of elevated Hct may relate more strongly to the biological corollaries of a reduced ESL thickness and alterations of the blood–endothelium interface than to an increased flow resistance.

Abstract

Abstract

Elevated systemic haematocrit (Hct) increases risk of cardiovascular disorders, such as stroke and myocardial infarction. One possible pathophysiological mechanism could be a disturbance of the blood–endothelium interface. It has been shown that blood interacts with the endothelial surface via a thick hydrated macromolecular layer (the ‘glycocalyx’, or ‘endothelial surface layer’– ESL), modulating various biological processes, including inflammation, permeability and atherosclerosis. However, the consequences of elevated Hct on the functional properties of this interface are incompletely understood. Thus, we combined intravital microscopy of an erythropoietin overexpressing transgenic mouse line (tg6) with excessive erythrocytosis (Hct 0.85), microviscometric analysis of haemodynamics, and a flow simulation model to assess the effects of elevated Hct on glycocalyx/ESL thickness and flow resistance. We show that the glycocalyx/ESL is nearly abolished in tg6 mice (thickness: wild-type control: 0.52 μm; tg6: 0.13 μm; P < 0.001). However, the corresponding reduction in network flow resistance contributes <20% to the maintenance of total peripheral resistance observed in tg6 mice. This suggests that the pathological effects of elevated Hct in these mice, and possibly also in polycythaemic humans, may relate to biological corollaries of a reduced ESL thickness and the consequent alteration in the blood–endothelium interface, rather than to an increase of flow resistance.

Introduction

Many long-term studies on human cohorts have provided evidence that an elevated systemic haematocrit (Hct) predisposes the cardiovascular system to ischaemic disease. Corresponding events such as myocardial infarction, angina pectoris, stroke and intermittent claudication indicate impaired cardiac, cerebral and peripheral perfusion, respectively (Harrison et al. 1981; Gagnon et al. 1994; Wannamethee et al. 1994; Brown et al. 2001). Polycythaemia vera is a clinical condition that is typically associated with an overproduction of red blood cells (RBCs). Patients afflicted with this myeloproliferative disorder are also at increased risk for developing arterial and venous thromboembolic complications (Spivak, 2002). The precise pathomechanism for this predisposition is imperfectly understood. It is generally supposed that the increased cardiovascular risk has its origins in pathological alterations in cardiovascular and microvascular haemodynamics, arising from the exponential rise in viscosity and the associated increase in blood pressure (Goubali et al. 1995), spontaneous platelet aggregation (Lowe & Forbes, 1985), thrombosis (Assaf et al. 2011), and impaired left-ventricular function (Schunkert et al. 2000).

In addition, endothelial dysfunction has been shown to be present in polycythaemia vera and to be correlated with the extent and prognosis of patients at risk of thrombotic disease (Suwaidi et al. 2000; Neunteufl et al. 2001). However, beyond its effects on haemodynamics, the possibility that elevated haematocrit may lead to pathological alterations of the blood–endothelium interface (Skretteberg et al. 2010) and that these alterations could be involved in the pathogenesis of vascular disease in polycythaemia, has not been investigated.

A transgenic mouse line overexpressing the human erythropoietin cDNA (tg6) allows investigation of the possible pathophysiological consequences of excessive erythrocytosis (Ruschitzka et al. 2000; Vogel & Gassmann, 2011). Due to the antiapoptotic effect of erythropoietin on erythroid progenitor cells (Jelkmann, 2011), the systemic Hct in these mice reaches a level of 0.80 to 0.90 within the first 2 months of life (Wagner et al. 2001). Surprisingly, these animals are viable and show similar levels of mean arterial pressure and cardiac output in rest to their wild-type counterparts, and correspondingly similar total peripheral resistance (Wagner et al. 2001). Furthermore, no thromboembolic complications have been reported in these mice (Ruschitzka et al. 2000). Apparently, their cardiovascular and microvascular systems are able to adjust to the substantial increase in systemic Hct.

One compensatory mechanism for the resistance effects associated with elevated systemic Hct in tg6 mice could be increased vessel diameter due to augmented synthesis of endothelial nitric oxide (Ruschitzka et al. 2000). Another possibility is a reduction in the thickness of the endothelial surface layer (ESL), or glycocalyx (Pries et al. 2000) (schaematically illustrated in Fig. 1B). This blood–endothelium interface has recently been the subject of increasing attention for its roles in vascular physiology and pathophysiology. Various experimental approaches have been taken to estimate the thickness and composition of this gel-like layer (Pries et al. 2000; Reitsma et al. 2007). The estimated thickness, tESL, of the glycocalyx/ESL in mammals varies between 0.4 and 1.2 μm, depending on species, location within the vasculature, and the method used to make the estimate. Since plasma flow through the ESL is retarded, the ESL effectively reduces the vascular cross-section available to free-flowing plasma, and directly affects microvascular flow resistance (Pries et al. 1994; Pries & Secomb, 2005).

Figure 1. Measurements and concept of microvascular blood flow.

Figure 1

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A, photomicrograph of a venule (45.5 μm in diameter). White encircled dots represent the dual image of a microsphere. Arrowheads denote the opposing sides of the inner vessel wall. The x/y/z-coordinates of the vessel section and the time signal superimpose the video signal at its upper edge. B, schematic illustration of the fluid layers adjacent to the luminal aspect of endothelial cells (ECs). While actual flow conditions (left-hand side) suggest a continuous reduction of local haematocrit towards the ESL–vessel lumen interface, the idealized two-phase flow model (right-hand side) assumes an instantaneous transition between regions of maximum Hct (RBC-core) and regions of zero Hct (cell-free layer, CFL). The endothelial surface layer (ESL) is considered consistently as a distinct, nearly quiescent fluid layer.

In addition to its influence on microvascular haemodynamics, the blood–endothelium interface also affects a number of relevant microvascular mechanisms pertaining to the normal functioning of the vascular bed. These range from fluid exchange to inflammatory processes (Vink & Duling, 2000; Potter et al. 2009). In this context, a reduction in ESL thickness, though beneficial from a haemodynamic perspective, may be detrimental to vascular health. It is evident from experimental observations that the vessel walls in tg6 mice are extremely thin and fragile. Microscopic investigations have revealed signs of impaired vessel integrity, progressive endothelial inflammation and multiple organ degeneration in aged tg6 mice (Heinicke et al. 2006; Ogunshola et al. 2006). This, ultimately, may contribute to the substantial reduction in mean life span of tg6 mice compared to wild-type control littermates, i.e. 7.4 versus 26.7 months (Wagner et al. 2001). Thusfar, however, no study has reported either on the possible mechanisms of the putative endothelial degeneration associated with excessive erythrocytosis or on the possible effects of Hct changes on the integrity of the blood–endothelium interface.

The objective of this study is to assess the haemodynamic and haemorheological consequences of strong elevations in systemic Hct on the microcirculation, with emphasis placed on the possible effects on the blood–endothelium interface, or ESL. Intravital microscopy, microparticle image velocimetry (μ-PIV), microviscometric analysis (Damiano et al. 2004), and a haemodynamic network flow simulation were employed to study microvascular haemodynamics and the ESL in postcapillary venules in the cremaster muscle of tg6 and wild-type mice. In order to discriminate between long-term adaptation to elevated haematocrit and sudden haemodynamic changes to an acute reduction in haematocrit, data were collected before and after systemic haemodilution in both tg6 and wild-type mice.

Methods

Ethical approval

All procedures were performed after approval by university and governmental committees on animal care (Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit Berlin, Tierversuchsantrag G239/02), and comply with the ethical policies and regulations as outlined in Drummond (2009). A total of 44 mice were used in the completion of this study.

Intravital microscopy

Male tg6 mice (tg, n = 8) and male C57Bl/6 wild-type (WT, n = 8) control littermates, aged 10–20 weeks, were used in the following groups: tg85 and wt46, with the subscripts referring to the mean systemic Hct (expressed as a percentage) of each group. Blood flow was examined by means of intravital microscopy (Ernst Leitz, Wetzlar, Germany) in a total of 32 (diameter, D, 40.7 ± 12.2 μm) and 37 (D 34.5 ± 8.3 μm) cremaster muscle venules in the tg85 and wt46 groups, respectively. Data was collected according to the μ-PIV method, as described in Smith et al. (2003), and the cross-sectional velocity distribution of each vessel was extracted from the circulating fluorescent micro-particles (D 0.5 ± 0.016 μm, FluoSpheres, Molecular Probes, Leiden, The Netherlands) injected via a venous catheter and visualized with intermittent stroboscopic flash illumination (Fig. 1A). Haemodilution of the same animals with hydroxyethyl starch (H) or plasma (p) gave rise to the following groups: tgH46, tgp53, wtH30, and wtp24 with a total of 10, 16, 15 and 19 vessels, respectively. The HES and plasma groups of the two strains were collected into a single group, denoted by tg50 (D 42.2 ± 12.6 μm) and wt27 (D 35.3 ± 8.6 μm), whenever no statistically significant differences were found.

C57Bl/6 wild-type mice were obtained from the ‘Forschungseinrichtung Experimentelle Medizin’ (FEM) of the Charité Berlin. Tg6 mice, bred by mating hemizygous males to C57Bl/6 females (Ruschitzka et al. 2000), were supplied by the Institute of Veterinary Physiology Zurich, Switzerland.

All animals were anesthetized with an intraperitoneal bolus injection of a combination of xylazine (10 μg (g body weight (bw))−1), ketamine (100 μg (g bw)−1), and atropine (0.1 μg (g bw)−1). Absence of limb withdrawal to paw pressure indicated adequacy of anaesthesia. After completion of the experimental investigation, animals were killed with an overdose of pentobarbital (250 mg (g bw)−1).

Microviscometric analysis

The μ-PIV data were analysed according to the microviscometric method recently developed by Damiano et al. (2004). This method employs a non-linear regression analysis to determine the velocity profile and corresponding ESL thickness that results in the minimum least-squares error in the fit to the μ-PIV data. This velocity profile is then used to predict various microvascular flow quantities, including the cross-sectional distributions of shear rate, shear stress and viscosity (Supplemental Fig. S1, red curves), as well as the volumetric flow rate, axial pressure gradient, and relative apparent viscosity. Collectively, microviscometric analysis consists of an extensive array of novel analytical tools that is capable of predicting local flow and stress within individual microvessels in vivo with an accuracy and detail that heretofore were not achievable using traditional methods for analysing flow parameters in the microcirculation. Microviscometric analysis couples mechanics with in vivo measurements of microparticle tracers to analyse vascular functional correlates of changes observed after haemodilution in tg6 and WT mice as well as differences observed between tg6 and WT mice. These functional correlates include distributions of shear rate, shear stress, velocity and viscosity over the vessel cross section of each microvessel analysed. Each of these distributions is a fundamental determinant of vascular function as it dictates flow throughout the microvessel and the micromechanical state of stress at the endothelial cell surface. These distributions are precisely determined in each microvessel that we observed and are functionally correlated across groups (and before and after haemodilution within the same group).

Microviscometric analysis has its foundations in continuum mechanics and assumes only that blood flow in microvessels >20 μm in diameter can be approximated by the dynamics of a continuous homogeneous fluid with spatially varying viscosity; no assumptions about the linearity or non-linearity of blood's constitutive behaviour are necessary. Support for this continuum approximation was provided by Cokelet in his studies using physiological concentrations of red blood cells suspended in plasma flowing at physiological shear rates in glass tubes as small as 20 μm in diameter (Cokelet 1999). Whereas empirical curve fitting to μ-PIV data acquired in vivo provides a quantitative, but purely descriptive, account of our observations, microviscometric analysis of those μ-PIV data details, in a quantitative and predictive manner, full-field distributions that satisfy the conservation principles of mass and linear momentum. The accuracy of microviscometric analysis has been substantiated in vivo and in glass tube validation studies in vitro (Smith et al. 2003; Long et al. 2004). Further details of the microviscometric analysis used here are provided in Supplemental Material, Methods.

Data on wall shear stress and viscosity for the tg85 group could not be obtained. At very high levels of systemic haematocrit, as in the tg6 mice, the analytical technique is not able to properly determine local shear stress and apparent viscosity. In order to determine these two quantities, the microviscometric method requires a known viscosity at least at one radial position. For normal or low haematocrit levels, this condition is met at the virtually cell-free fluid layer adjacent to the vessel wall where plasma viscosity is assumed to prevail. For very high haematocrit levels, however, an effectively cell-free layer of sufficient width is not observed. Under these conditions, viscosity of the near-wall fluid layers might substantially exceed plasma viscosity, and thus neither the radial shear stress distribution nor the apparent viscosity can be properly assessed.

A two-phase model of microvascular blood flow was used to estimate the width of a cell-free layer (CFL). As illustrated in Fig. 1B, this model assumes an instantaneous transition between a core region of maximum Hct and a marginal region of zero Hct, termed accordingly RBC-core and CFL, respectively. The step distribution assumed by the two-phase flow model is a simplification of the non-uniform radial Hct distribution that prevails in microvessels (and which is predicted using microviscometric analysis). The width of the CFL derived from the two-phase flow model is an indicator of the Hct reduction close to the vessel wall. The ESL is considered as a distinct, nearly quiescent fluid layer that excludes RBCs. This is in contrast to previous studies that use a two-phase approach (Maeda et al. 1996; Kim et al. 2007).

Flow simulation model

In addition, a haemodynamic flow simulation for an experimental vascular network in the rat mesentery with 546 vessel segments was used to calculate the overall flow resistance, Rnet (Pries et al. 1994). For the vessel lumen available for free fluid flow (D− 2tESL), a parametric description of apparent viscosity in vitro as a function of diameter and systemic Hct (the ‘in vitro viscosity law’) was used (Pries et al. 1992).

An expanded Methods section is presented in the Supplemental Material.

Results

Macrohaemodynamic parameters for each experimental group are given in Supplemental Table S1. Consistent with previous studies (Ruschitzka et al. 2000; Wagner et al. 2001; Vogel et al. 2003), no statistically significant difference in baseline MAP was detected between tg6 mice and WT mice. Upon haemodilution, MAP declined from 77 to 52 mmHg (P < 0.01) and from 69 to 60 mmHg (P > 0.05) in tg6 mice and WT mice, respectively. The difference in MAP between the two strains of mice after haemodilution was not statistically significant. On the other hand, tg6 mice exhibited a significantly higher heart rate (HR) than WT mice, both before (P < 0.05) and after haemodilution (P < 0.001). After haemodilution, HR increased from 342 to 525 beats min−1 in tg6 mice (P < 0.001) and from 261 to 330 beats min−1 in WT mice (P < 0.05). These relatively low HR levels (Ruschitzka et al. 2000; Schuler et al. 2010) may be due to the depressant cardiovascular effect of the anaesthetic regime employed.

As shown in Fig. 2A, average flow velocity (vmean) in tg6 mice was 2.6 times lower than in WT mice before haemodilution (P < 0.001), and nearly doubled in tg6 mice after haemodilution (P < 0.001). In WT mice, haemodilution led to only a minor increase in vmean which was not statistically significant (P > 0.05). Similarly, centreline flow velocity (vmax) was significantly lower (2.9 times) in tg6 mice as compared to WT mice (P < 0.001). Haemodilution resulted in a 96% increase of vmax in tg6 mice (P < 0.001), but no significant change in WT mice. Blood flow rate in tg6 mice was 52% lower compared with WT control values (P < 0.05), and increased to WT levels after haemodilution (Supplemental Table S2). This corroborates data reported for the tg6 brain where cerebral blood flow was 44% lower compared with WT values (Frietsch et al. 2007b).

Figure 2. Microhaemodynamic parameters in post-capillary cremaster muscle venules.

Figure 2

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A, average flow velocity. B, averaged velocity profiles for each group. Each profile is normalized to the radial position of the blood–ESL interface (vessel radius−tESL) and to the centerline velocity, vmax. The dotted curve indicates a velocity profile of parabolic shape. The box-plot (inset) shows the ratio of vmax to vmean as a metric for the degree of bluntness of the velocity profiles. A value of 2 corresponds to a perfectly parabolic shape of the velocity profile, while a smaller value corresponds to a rather blunt profile. C, dependence of the ratio vmax/vmean on systemic Hct in tg6 mice (black symbols) and WT mice (grey symbols), before (circles) and after haemodilution (rhombs). n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; r2, coefficient of determination.

The velocity profiles of the tg85 group exhibited the lowest ratio of vmax/vmean, and hence the greatest degree of bluntness, while groups with lower mean systemic Hct showed successively higher ratios (Fig. 2B). Thus, a negative correlation was observed between systemic Hct and bluntness factor in both tg6 and WT mice (Fig. 2C). However, despite similar Hct levels of tg6 mice after haemodilution and WT mice prior to haemodilution, bluntness of the velocity profiles in the former group remained significantly higher than in the latter (Fig. 2B).

In Fig. 3, median values of relative apparent viscosity, ηrel (symbols), estimated using microviscometric analysis, are plotted against the group-specific mean systemic Hct. These experimental data show remarkable consistency with theoretical predictions (dashed line), based on in vitro data for the given average vessel diameter (38 μm) and typical red cell volume of WT mice (45 fl) (Pries et al. 1994). The arrow in Fig. 3 indicates the expected ηrel from the aforementioned in vitro viscosity law for the respective systemic Hct level. This extrapolation predicts that the ηrel for a systemic Hct of 0.85 would be approximately 3.5-fold greater than for a Hct of 0.46.

Figure 3. Dependence of the relative apparent viscosity on systemic Hct.

Figure 3

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Symbols represent median values of the respective group as a whole, and were estimated using microviscometric analysis. The dashed curve is derived from the in vitro viscosity law (Pries et al. 1994), assuming the experimental mean vessel diameter (38 μm) and the typical red cell volume of WT mice (45 fl). The arrow indicates the expected relative apparent viscosity for tg6 mice, obtained by extrapolation of the non-linear fit to glass tube data (Pries et al. 1992) to a systemic Hct of 0.85.

Figure 4A shows the estimated tESL range for each experimental group plotted versus group-specific average systemic Hct. Baseline tESL in tg6 mice was 0.13 μm, which was significantly (P < 0.001) lower than in WT mice (0.52 μm). After haemodilution, tESL in tg6 mice increased significantly, attaining a median value of 0.42 μm (P < 0.01), which is not significantly different from the baseline tESL in WT mice. In contrast, haemodilution of WT mice led to only a slight increase in median layer thickness (0.52 to 0.62 μm), which did not reach significance. As indicated in Fig. 4A, tESL exhibits a consistent inverse linear relation to the systemic Hct level, suggesting an imaginary regression line that intersects the abscissa (i.e. tESL = 0 μm) close to Hct 1, and the ordinate (Hct = 0) at an tESL of ∼0.8 μm.

Figure 4. Dimensions of the near-wall fluid layers.

Figure 4

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Estimated thickness of the ESL (A) and estimated width of the CFL (B) as a function of systemic haematocrit. Each symbol represents the mean ± SEM of the respective group as a whole. The arrows in panel A denote the corresponding median values (see also Table 1), which are referred to in Fig 6.

Table 1.

Cardiovascular and microvascular flow parameters of tg6 mice and WT mice both before and after isovolaemic haemodilution

tg85 tg50 wt46 wt27
MAP (mmHg) 77 ± 5 52 ± 3 69 ± 2 60 ± 7
HR (beats min−1) 342 ± 21 525 ± 37 261 ± 18 330 ± 21
Centreline velocity (μm s−1) 1182 ± 99 2317 ± 234 3422 ± 425 3856 ± 503
Average flow velocity (μm s−1) 786 ± 66 1464 ± 144 2054 ± 262 2149 ± 284
vmax/vmean 1.51 ± 0.02 1.58 ± 0.03 1.68 ± 0.02 1.81 ± 0.01
Pressure gradient (103 dyn cm−3) −9.0 ± 1.1 −12.2 ± 1.5 −17.7 ± 1.8 −15.0 ± 2.0
Flow rate (nl s−1) 1.1 ± 0.1 2.3 ± 0.4 2.3 ± 0.4 2.2 ± 0.3
Mean shear rate (s−1) 63 ± 6 115 ± 12 203 ± 22 222 ± 25
Interfacial shear rate (s−1) 646 ± 63 956 ± 111 1212 ± 130 1012 ± 126
Interfacial shear stress (dyn cm−2) — 12.0 ± 7.2 15.5 ± 9.9 12.9 ± 9.4
Relative apparent viscosity — 2.50 ± 0.1 2.06 ± 0.1 1.6 ± 0.1
ESL thickness (μm) 0.15 ± 0.03 [0.13] 0.45 ± 0.06 [0.42] 0.47 ± 0.04 [0.52] 0.58 ± 0.04 [0.62]
CFL thickness (μm) 1.1 ± 0.1 [0.9] 1.7 ± 0.2 [1.3] 1.8 ± 0.2 [1.6] 2.8 ± 0.3 [2.4]
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Interfacial shear rate and interfacial shear stress refer to the blood–ESL interface, at the luminal ESL surface. Values are expressed as means ± SEM. Figures in square brackets represent median values.

Similar to the findings associated with the ESL, the width of the CFL is also inversely related to systemic Hct (Fig. 4B). However, the relationship does not seem to be linear by nature. CFL width may approach a minimum value of about 0.8 μm as Hct is getting close to 1, and tends to match vessel radius as Hct approaches 0.

Discussion

In numerous population studies, chronically elevated haematocrit is accompanied by an increase in total peripheral resistance (TPR) resulting in decreased cardiac output and/or increased MAP (Goubali et al. 1995; Schunkert et al. 2000). Similar results have also been found in animal studies (Richardson & Guyton, 1959; Lipowsky & Firrell, 1986). The deteriorated haemodynamic conditions, accompanied by reduced microvascular perfusion, and oxygen supply deficiency, are generally supposed to be the main risk factor for pathological or fatal events in the cardiovasculature and microvasculature at elevated Hct. In addition, altered haemorheology may contribute to the increased risk, e.g. via disturbed microvascular blood distribution and platelet aggregation (Fig. 5, left).

Figure 5. Impact of elevated Hct on cardiovascular risk, possible role of compensatory mechanisms and alterations of blood–endothelium interface.

Figure 5

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Left, conventional concept. Increased Hct leads to increased viscosity (right branch) which causes increased total peripheral resistance (TPR) and thus increased mean arterial pressure (MAP), reduced blood flow, and reduced oxygen supply. In addition, the modified microrheological properties of the blood (left branch) may lead to increased heterogeneity of local perfusion. Both effects could contribute to the reported increase in cardiovascular risk. Right, present concept. In chronic haematocrit elevation in tg6 mice, a number of compensatory mechanisms are activated. The reported increases in cell flexibility and cell volume counteract the increase in effective blood viscosity in microvessels (right branch). Similarly, the increase in total intravascular volume also reduces TPR at a given viscosity. The reduction of glycocalyx/ESL thickness reduces TPR, but this effect is relatively limited (∼18%). These compensatory mechanisms taken together restore TPR to levels similar to wild-type controls, despite the excessive increase in Hct. However, the increased red cell concentration also compromises the blood–endothelium interface and reduces the ESL thickness (middle branch). This may have strong biological effects on the blood–endothelium interface contributing to the known cardiovascular risks associated with polycythaemia.

Interestingly enough, the chronically elevated systemic Hct in tg6 mice, which is nearly twice the value found in WT mice (0.85 versus 0.46), is accompanied neither by a decrease in cardiac output nor by an increase in MAP (Ruschitzka et al. 2000; Wagner et al. 2001; Vogel et al. 2003). This indicates that TPR (= MAP/cardiac output) is unchanged in tg6 mice relative to WT mice, suggesting the presence of effective haemodynamic mechanisms compensating the strongly elevated systemic Hct. Also, oxygen supply of both the brain and the skin in tg6 mice is not lowered relative to WT mice (Frietsch et al. 2007a,b;). In accordance with this, red cell flux in post-capillary venules, an indicator of the potential for oxygen delivery, seems to be maintained in tg6 mice as shown in Supplemental Fig. S3. A recent study (Schuler et al. 2010) has demonstrated that the optimal systemic Hct (that corresponding to maximal O2 flux) during maximal exercise in tg6 mice is higher (0.68) than in their WT siblings (0.58), further indicating that the tg6 mice are able to adapt to this excessive erythrocytosis in haemodynamic terms.

Several mechanisms that may reduce TPR below the actually anticipated level have been identified, including NO-induced increase in vessel diameter (Ruschitzka et al. 2000) and increased erythrocyte flexibility (Vogel et al. 2003; Bogdanova et al. 2007).

A largely overlooked area that might be involved in the pathogenesis of vascular disease in polycythaemia is the alteration of the blood–endothelium interface (Fig. 5, right). A growing body of evidence indicates that the blood–endothelium interface comprises a ∼0.5 μm thick layer (endothelial surface layer, ESL or glycocalyx) (Pries et al. 2000; Smith et al. 2003), which is only present in vivo (Potter & Damiano, 2008) and not available for free fluid flow. Our study demonstrates that the excessive Hct in tg6 mice is associated with a severely decreased thickness of this layer (tESL) in post-capillary cremaster muscle venules (0.13 μm in tg6 mice versus 0.52 μm in WT mice, P < 0.001). The latter control value is consistent with reference values for tESL in WT mice given in the literature (Vink & Duling, 1996; Damiano et al. 2004; Savery & Damiano, 2008).

In recent years, various conditions resulting in ESL damage, degradation or reduction have been identified, including the presence of reactive oxygen species (ROS) (Rubio-Gayosso et al. 2006) cytokines (Mulivor & Lipowsky, 2004; Potter & Damiano, 2008), oxidized low-density lipoproteins (Vink et al. 2000; Constantinescu et al. 2001), and hyperglycaemia (Nieuwdorp et al. 2006). Given the elevated eNOS levels in the endothelial cells of tg6 mice (Ruschitzka et al. 2000), the formation of ROS associated with NO formation could result in biochemical degradation of the ESL, as previously demonstrated for ischaemia–reperfusion experiments (Rubio-Gayosso et al. 2006).

Increased levels of haematocrit will lead to widening of the red cell column and augmented radial forces generated by red cell interaction. Thus, a mechanical compression of the ESL by flowing red cells would be the most likely cause of the reported ESL thinning in these mice. In the present study, the thinning of the ESL was rapidly reversible upon haemodilution to physiological levels, suggesting a dynamic mechanical recovery of the layer. Comparison of the cell-free layer (CFL) in tg6 with that in WT mice provides further support for this hypothesis (Fig. 4B). The mean CFL width in tg6 mice (∼1 μm) estimated using the two-phase fluid model was found to be less by approximately 50% than in WT controls (∼2 μm). Haemodilution led to a significant increase in CFL width in both tg6 and WT mice. The analytical methods used here thus suggest a dynamic rehydration of a mechanically compressed layer (rather than molecular reconstitution of a degraded layer). It has recently been shown that it can take up to 1 week for the ESL to fully recover after enzymatic degradation (Potter et al. 2009). Therefore, it seems unlikely that endothelial protein synthesis is involved in this rapid ESL thickening, as μ-PIV data were collected within 60 min following haemodilution.

A decrease in tESL results in an increase in effective vessel diameter. Due to the strong inverse dependence of flow resistance on vessel radius, even a slight increase in vessel diameter can substantially decrease TPR. In light of this, we assessed the impact of tESL on flow resistance using a microvascular network model. Reduction in tESL in all vessels by the difference in the corresponding median values between tg6 mice and WT mice (i.e. by 0.4 μm), results in an ∼18% decrease of network flow resistance, Rnet, at a Hct of 0.85 (Fig. 6).

Figure 6. Overall flow resistance of a microvascular network.

Figure 6

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Dependence of flow resistance in an experimental microvascular network on ESL thickness (I–III) and systemic haematocrit (0.27, 0.46 and 0.85). In addition, four other factors are considered. A, bulk viscosity is assumed for all vessels independent of vessel diameter. B, reduction of apparent viscosity with declining vessel diameter according to Fåhraeus–Lindqvist effect is taken into account. C, effect of increased red cell volume, MCV, is included (WT 45 fl vs. tg6 57 fl). D, impact of increased cell flexibility on bulk viscosity according to Vogel et al. (2003) is integrated. Values are normalized to the flow resistance obtained for WT mice under spontaneous conditions (ESL thickness II, systemic Hct 0.46, MCV 45 fl, normal cell flexibility). Each column in light grey represents the predicted flow resistance of the considered experimental group (tg85, wt46, wt27) if the assumed parameters are applied to it. Categories of ESL thickness (I–III) correspond to the group-specific median values rounded to one decimal place.

A possible limitation in deriving this value may result from the uncertainty in the determination of individual vessel diameters while establishing the network database used. It was reported that this diameter determination is burdened by a relatively large error margin of about 0.5–1.0 μm (Pries et al. 1997a). However, this error will occur in a random fashion (influencing the distribution of flow between vessels) while the assumed reduction of ESL thickness leads to a systematic increase of luminal diameter in all vessels of the network (thus influencing overall flow resistance). Thus, the random diameter measurement error should not have a systematic effect on the estimated change in overall flow resistance even if the actual error of diameter determination is higher than the ESL thickness or its change due to high levels of haematocrit.

The estimated decrease in flow resistance due to ESL thinning is quantitatively similar to values reported for microinfusion of heparinase (Pries et al. 1997b), which is known to degrade the ESL. The haemodynamic network simulation was used to compare this effect with those of other factors, including the Fåhraeus–Lindqvist (FL) effect, cell volume, and cell flexibility (Fig. 6). Stepwise implementation of these factors leads to successive reduction in Rnet. The effect of the ESL on Rnet is relatively small, while the greatest reduction in Rnet results from the FL effect (Fig. 6, A→B). Thus, the resistance changes resulting from the modification in tESL are relatively minor.

The FL effect (Pries et al. 1992) takes into account the dynamic reduction of ηrel with decreasing tube diameter. While bulk viscosity prevails in large-diameter tubes (D≥ 1000 μm), values very close to plasma viscosity are achieved in small-diameter tubes in the capillary regime (D 5–7 μm). This diameter-dependent decrease in ηrel is especially strong at higher haematocrit levels, giving rise to the pronounced reduction in Rnet for conditions similar to those found in tg6 mice. The changes in ηrel described by the FL effect are dependent not only on absolute tube diameter, but more precisely, on the ratio of tube diameter to particle (i.e. RBC) diameter. Hence, the increase in mean corpuscular volume (MCV) in tg6 mice reported by Vogel et al. (2003) has the same effect as a reduced vessel diameter. Accordingly, an increase in MCV will generate an apparent rightward shift of the viscosity curve associated with a slight decrease of apparent viscosity (Fig. 6, B→C).

The increased cell deformability reported in tg6 mice (Vogel et al. 2003) could also account for a substantial fraction in the observed reduction in TPR. Since only in vitro data are available, an attempt was made to integrate the reported effect on bulk viscosity, i.e. a reduction of about 50%, into our in vivo flow simulation. This was done by halving the RBC-dependent component of relative apparent viscosity (ηrel− 1) according to the equation ηreladj = (ηrel− 1)/2 + 1. Since the RBC-dependent component of flow resistance is reduced in small vessels due to the FL effect, the resulting effect for a microvascular network is smaller than expected from the bulk viscosity data. A 38% reduction in Rnet, relative to that obtained with baseline cell deformability, is seen for a systemic Hct of 0.85 (Fig. 6, C→D).

Taking all of these factors into account, the estimated Rnet in tg6 mice is still approximately 40% greater than in WT mice. This would correspond to a substantially elevated TPR for identical network angioarchitecture. However, our histological examinations of the extensor digitorum longus (EDL) muscle in tg6 mice demonstrate a 1.6-fold higher capillary density (capillary sections per area) relative to WT mice (P < 0.001, Supplemental Fig. S2). A corresponding alteration of the total number capillaries perfused in parallel would suggest a reduction of overall flow resistance by about 38% (1/1.6 × 100), which may compensate for the aforementioned remaining discrepancy in Rnet for identical network architecture. In this reasoning it is assumed that the findings for individual vessels and vascular beds reflect more general systematic changes. This applies to the reduction of ESL thickness and other microrheological parameters determined in the cremaster muscle venules and the findings of increased capillarization in the EDL muscle of tg6 mice. While this assumption cannot be proven based on the available data, it is supported by the systemic changes in TPR addressed earlier and the reported 2-fold increase in total blood volume of tg6 mice (Vogel et al. 2003). An increased number of microvessels perfused in parallel in tg6 mice would also explain the apparent discrepancy between the predicted systemic RBC flux (cardiac output times systemic haematocrit), which is about doubled in tg6 mice relative to WT mice (Vogel et al. 2003), and the microvascular RBC flux, which is not significantly different from WT mice (present data, Supplemental Fig. S3).

While the haemodynamic effects of ESL thinning may be relatively small, the physiological implications of a change in the blood–endothelium interface due to ESL damage, degradation, or compression could be much more substantial. ESL thinning may evoke pathophysiological events on the microvascular level associated with endothelial dysfunction. Vascular pathologies related to an injury of the blood–endothelium interface include atherosclerosis (van den Berg et al. 2006), microangiopathy in diabetes (Nieuwdorp et al. 2006), tissue damage induced by ischaemia–reperfusion (Platts et al. 2003), leukocyte recruitment and inflammation (Smith et al. 2003; Mulivor & Lipowsky, 2004), and altered microvascular permeability (Henry & Duling, 2000; Vink & Duling, 2000) (Fig. 5, right).

The ESL thinning that we observed allows platelets and leukocytes to come in closer proximity to the vessel wall, which provides those blood components with greater access to the endothelium, and may undermine the important barrier role of the ESL, making the vascular interface a more thrombogenic and proinflammatory surface than under conditions of normal haematocrit. Based on estimates of the stiffness of the ESL derived from several modelling studies (Damiano & Stace, 2002, 2005; Weinbaum et al. 2003), it is not surprising that de novo capture of leukocytes from the free stream is rare in post-capillary venules with an intact 500 nm-thick ESL (Smith et al. 2003). However, compression of the ESL to ∼100–200 nm would increase the likelihood that selectin molecules and their receptors on the endothelium come in close proximity frequently enough to allow elevated adhesive interactions, more capture events from the free stream, and more leukocyte rolling. Thus, the ESL thinning observed in tg6 mice could lead to a more chronically inflamed state for microvascular endothelium. Such inflammation could, in turn, lead to the release of cytokines, and to increased platelet aggregation and thrombogenicity. This concept is supported by findings showing that the increased incidence of leukocyte adhesion and thrombosis in polycythaemia vera is related to vascular damage and endothelial dysfunction (Neunteufl et al. 2001).

Secomb et al. (2002) showed that the blood–endothelium interface plays an important role in buffering excessive shear stress peaks in capillaries due to wall shape irregularities. Thus, the reduced tESL in tg6 mice may also lead to considerable variation in shear stress (especially if this reduction is due to ESL compression), giving rise to endothelial dysfunction as well as red cell damage. Consistent with such mechanisms, a reduced erythrocyte lifespan has been reported for tg6 mice (Bogdanova et al. 2007). Furthermore, histological studies on elderly tg6 mice (7.2 months of age) have found evidence of vascular degeneration and signs of latent chronic vascular inflammation in the liver, the kidney and the brain (Heinicke et al. 2006; Ogunshola et al. 2006). A chronically compromised microvasculature and reduced red cell survival in tg6 mice could have detrimental effects in terms of multiple organ degeneration and may be involved in the markedly reduced life span of these erythrocytic animals (Wagner et al. 2001).

In summary, our results show that in constitutively high haematocrit in transgenic mice, haemodynamic compensatory mechanisms may redress an increase in TPR that might otherwise accompany erythrocytosis. Hence, impaired haemodynamics is unlikely to play a significant role for the increased cardiovascular and microvascular risk observed in tg6 mice. However, the direct effects of polycythaemia on the blood–endothelium interface reported in this study may have corollaries going beyond merely haemodynamic considerations. Excessive erthrocytosis and the accompanying alterations in the interaction between blood components and the vascular wall may trigger pathophysiological mechanisms that lead to a variety of conditions directly impacting cardiovascular health and disease in humans with excessive erythrocytosis, such as in polcythaemia vera, high-altitude-training athletes, and blood doping. The reduction in tESL in tg6 mice is likely to have an adverse impact on microvascular barrier function in terms of both water and macromolecular permeability. Furthermore, a reduction in tESL, together with a reduced cell-free layer, inevitably brings platelets and leukocytes into close proximity with the vessel wall, which, in essence, undermines the role of the blood–endothelium interface making it a more prothrombogenic surface relative to control conditions. The fact that erthrocytosis may lead to chronic thinning of the ESL by a haemodynamic mechanism (i.e. compressive and spontaneously reversible) provides a novel pathophysiological concept which may have important implications for therapeutic intervention.

Acknowledgments

Partial support for this work was provided by the National Institutes of Health, grant R01 HL076499 (E.R.D.) and by a graduate fellowship from the Clare Boothe Luce Foundation (M.D.S.).

Glossary

Abbreviations

CFL

cell-free layer

ESL

endothelial surface layer

FL effect

Fåhraeus–Lindqvist effect

ηrel

relative apparent viscosity

Hct

haematocrit

HES

hydroxyethyl starch

HR

heart rate

MAP

mean arterial pressure

MCV

mean corpuscular volume

μ-PIV

microparticle image velocimetry

Rnet

network flow resistance

RBC

red blood cell

tESL

ESL thickness

TPR

total peripheral resistance

Author contributions

V.R. contributed to conception and design of the experiments, analysis and interpretation of the results, drafting of the article, and revising of the article critically for important intellectual content; M.D.S. contributed to analysis and interpretation of the results, and drafting of the article; M.G. generated the tg6 mouse line, contributed to drafting of the article, and helped revise the article; O.B. contributed to analysis and interpretation of the results, and drafting of the article; E.R.D. contributed to conception and design of the experiments, analysis and interpretation of the results, drafting of the article, and revision of the article critically for important intellectual content; and A.R.P. contributed to conception and design of the experiments, analysis and interpretation of the results, drafting of the article, and revision of the article critically for important intellectual content. All authors have approved the final version for publication. All experiments for his study were conducted in the laboratories of the Department of Physiology, Charité Berlin, Berlin, Germany.

Supplementary material

MATERIAL AND METHODS

Table S1

Table S2

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

tjp0589-5181-SD1.pdf (185.7KB, pdf)

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