Shedding of the endothelial glycocalyx in arterioles ......Shedding of proteoglycans and GAGs from cultured ECs, or their analogs, has been studied in response to a broad spectrum

doi:10.1152/ajpheart.00803.2011 301:H2235-H2245, 2011. First published 16 September 2011;Am J Physiol Heart Circ Physiol

Herbert H. Lipowsky, Lujia Gao and Ann Lescanichemodynamics during inflammationcapillaries, and venules and its effect on capillary Shedding of the endothelial glycocalyx in arterioles,

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Shedding of the endothelial glycocalyx in arterioles, capillaries, and venulesand its effect on capillary hemodynamics during inflammation

Herbert H. Lipowsky, Lujia Gao, and Ann LescanicDepartment of Bioengineering, The Pennsylvania State University, University Park, Pennsylvania

Submitted 10 August 2011; accepted in final form 13 September 2011

Lipowsky HH, Gao L, Lescanic A. Shedding of the endothelialglycocalyx in arterioles, capillaries, and venules and its effect oncapillary hemodynamics during inflammation. Am J Physiol HeartCirc Physiol 301: H2235–H2245, 2011. First published September 16,2011; doi:10.1152/ajpheart.00803.2011.—The endothelial glycocalyxhas been identified as a barrier to transvascular exchange of fluid,macromolecules, and leukocyte-endothelium [endothelial cell (EC)]adhesion during the inflammatory process. Shedding of glycans andstructural changes of the glycocalyx have been shown to occur inresponse to several agonists. To elucidate the effects of glycanshedding on microvascular hemodynamics and capillary resistance toflow, glycan shedding in microvessels in mesentery (rat) was inducedby superfusion with 10�7 M fMLP. Shedding was quantified byreductions of fluorescently labeled lectin (BS-1) bound to the EC andreductions in thickness of the barrier to infiltration of 70-kDa dextranon the EC surface. Red cell velocities (two-slit technique), pressuredrops (dual servo-null method), and capillary hematocrit (direct cellcounting) were measured in parallel experiments. The results indicatethat fMLP caused shedding of glycans in all microvessels withreductions in thickness of the barrier to 70-kDa dextran of 110, 80,and 123 nm, in arterioles, capillaries, and venules, respectively.Intravascular volumetric flows fell proportionately in all three divi-sions in response to rapid obstruction of venules by white blood cell(WBC)-EC adhesion, and capillary resistance to flow rose 18% due todiminished deformability of activated WBCs. Capillary resistance fellsignificantly 26% over a 30-min period, as glycans were shed from theEC surface to increase effective capillary diameter, whereas capillaryhematocrit and anatomic diameter remained invariant. This decreasein capillary resistance mitigates the increase in resistance due todiminished WBC deformability, and hence these concurrent rheologi-cal events may be of equal importance in affecting capillary flowduring the inflammatory process.

glycosaminoglycans; glycocalyx thickness; lectin binding; f-Met-Leu-Phe; resistance to flow

THE COATING OF PROTEINS AND polysaccharides on the vascularendothelium has received considerable interest in light of itsrole as a barrier to transvascular exchange and blood-endothe-lial interactions. Adsorbed proteins on the endothelial cell (EC)luminal surface, first postulated in studies of capillary perme-ability by Danielli (12), and a layer of polysaccharides boundto transmembrane and membrane-linked proteins (the glyco-calyx), first noted with electron microscopy by Luft (40), servea multifaceted role in vascular homeostasis (48, 52). Thisendothelial surface layer is an important barrier to transvascu-lar exchange of water and solutes (1, 24) and sieving ofplasma-borne macromolecules (29, 58, 60). The endothelialsurface layer also provides binding sites for antithrombin III,tissue factor pathway inhibitors, lipoprotein lipase, vascular

endothelial growth factor, fibroblast growth factor, and extra-cellular superoxide dismutase (52), serves as a barrier toleukocyte-endothelium adhesion (11, 42–44), and acts as ashear stress sensor and regulator of mechanotransduction (17).Experimental manipulations of the structure of the glycocalyxby perfusion of the microvasculature with heparinase haverevealed decreases in intravascular resistance to flow (50).Increases in capillary hematocrit (HCAP) have been attributedto removal of the glycocalyx in response to perfusion withheparinase (13) and hyaluronidase (7) and the presence ofreactive oxygen species derived from oxidized low-densitylipoprotein (LDL) (10). Thus the structure of the glycocalyx isa prime determinant of numerous physiological and hemody-namic processes, and maintenance of its stability may precludethe onset of many pathological disturbances (5).

Studies by electron microscopy have suggested that theglycocalyx is a porous layer composed of a matrix of mole-cules arranged in a regular pattern (55). The most prominentcomponents of the glycocalyx are the glycosaminoglycans(GAGs): heparan sulfate (HS), chondroitin sulfate (CS), andhyaluronan. The principal proteins on the EC surface that bindHS and CS to form the proteoglycans are the transmembranesyndecans and the membrane-bound glypicans (48). The mostprevalent proteoglycan, syndecan-1, is one of four members ofthis family of proteoglycans and is an integral membraneprotein composed of an intracellular domain, a transmembranedomain, and an extracellular core (53). Under normal physio-logical conditions, the structure of the glycocalyx layer isstable, with a molecular composition that represents a dynamicbalance between biosynthesis of new glycans and shear-depen-dent removal of existing constituents. Fluid shear stressesacting on the EC surface may affect the structure of theglycocalyx by either disrupting its molecular constituents, oractivation of proteases and lyases synthesized by the endothe-lium (4, 43).

Shedding of proteoglycans and GAGs from cultured ECs, ortheir analogs, has been studied in response to a broad spectrumof agonists (9, 16, 18, 31, 45–47). Shedding of HS proteogly-cans (namely the ectodomain of syndecans 1–4) has beenshown to occur in response to endotoxin (9), serine and/orcystein proteinases (30), complement activation (46), thrombinand growth factors (56), and activation of protein tyrosinekinase by phorbol esters (16). Using hydroxamic acid inhibi-tors of matrix metalloproteinases, it has been shown thatproteolytic cleavage of the syndecan ectodomain results fromthe concurrent action of multiple intracellular pathways thatactivate cell surface metalloproteinases (16).

In vivo, the endothelial glycocalyx has been shown to beshed in response to inflammation (25, 43), hyperglycemia (64),endotoxemia and septic shock (26), presence of oxidized LDL(10), TNF-� (8), atrial natriuretic peptide (6), abnormal blood

Address for reprint requests and other correspondence: H. H. Lipowsky,Dept. of Bioengineering, Penn State Univ., 205 Hallowell Bldg., Univ. Park,PA 16802 (e-mail: [email protected]).

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shear stress (20, 21), ischemia-reperfusion injury (43), andduring bypass surgery (51, 57). These observations have leadto the hypothesis of an underlying connection between integ-rity of the glycocalyx and vascular homeostasis (43, 64).Shedding of the glycocalyx in response to cytokines andchemoattractants has been shown to occur in all three principaldivisions of the microvasculature. It has been shown in arteri-oles, in response to TNF-� (25); capillaries, in response toTNF-� (25) and oxidized LDL (10); and venules, in responseto TNF-� (25) and fMLP (43). These studies were performedin multiple microvascular networks and species: cremastermuscle (10, 25, 43) and skin flap (7) of the hamster, ormesentery of the rat (43). There has also been limited quanti-zation of basic hemodynamic effects of the specific agonistduring the shedding process and consistent measurements ofstructural alterations of the glycocalyx, as, for example, interms of its thickness. Thus the present studies were under-taken to systematically quantitate agonist-induced shedding ofglycans in arteriolar, capillary, and venular divisions of themesentery of the rat. Parallel studies were performed to eval-uate changes in hemodynamic parameters [red cell velocity(VRBC)], in the three network divisions, and alterations in theresistance to flow in capillaries, derived from measurement ofpressure drops (�P) across the capillary network and changesin HCAP.

To this end, four separate protocols were employed to studythe glycocalyx and hemodynamics in the mesentery (rat),before and following superfusion with fMLP: 1) fMLP-in-duced shedding of glycans from arterioles, capillaries, andvenules was characterized by staining the microvessel endo-thelium with a fluorescently labeled lectin to quantify reduc-tions in bound lectin; 2) thickness of the glycocalyx andnetwork hemodynamics was evaluated from measurements ofthe exclusion of 70-kDa fluorescent dextran (Dx70) and VRBC,respectively; 3) HCAP were measured by direct cell counting;and 4) pressure gradients and red cell velocities were simulta-neously measured in the true capillaries to compute changes inthe resistance to flow. Taken together, these data provide aunified view of the shedding of the glycocalyx and hemody-namics during a well-defined model of the inflammatory pro-cess.

MATERIALS AND METHODS

Animal preparation. All animal studies conformed to the GuidingPrinciples in the Care and Use of Animals established by the Amer-ican Physiological Society, and all protocols have been approved bythe Institutional Animal Care and Use Committee of The Pennsylva-nia State University.

Male Wistar rats, weighing 250–400 g, were anesthetized withInactin (120 mg/kg ip), tracheostomized, and allowed to breathe underspontaneous respiration. The right jugular vein and its paired carotidartery were cannulated with polyethylene tubing (PE-50). Supplemen-tal anesthetic was administered via the jugular catheter, as needed, tomaintain a surgical plane of anesthesia. The carotid catheter wasconnected to a strain-gauge pressure transducer to monitor centralarterial pressure, which averaged a nominal 125 mmHg. Core tem-perature was monitored by a rectal probe and was maintained between36 and 37°C with the aid of a heating pad.

Intravital microscopy. The intestinal mesentery was exteriorizedthrough a midline abdominal incision and placed on a glass pedestalto permit viewing under bright-field microscopy by either trans- orincident illumination. The tissue was superfused with HEPES-buff-

ered Ringer solution (pH � 7.4) at a temperature of 37.0°C, with orwithout the chemoattractant fMLP at a concentration of 10�7 M.Fluorescence microscopy was performed under incident laser illumi-nation at a wavelength of 488 nm with a dichroic mirror and filtersappropriate for fluorescein excitation and emission spectra. Intensityof the laser illumination was controlled with an acousto-opticaltunable filter to modulate its intensity and turn it on and off. Tominimize photobleaching of the fluorophore, the tissue was excitedduring digitization of the video scenes for 0.5-s periods. Visualrecordings of the mesentery were made with a Yokogawa CSU-10,spinning disk confocal microscope (Solamere Technology, Salt LakeCity, UT) using an XR/MEGA-10 intensified charge-coupled devicecamera (Stanford Photonics, Palo Alto, CA). Output of the camerawas digitized and saved to computer disk in tagged image format, witheach image being 1,024 � 1,024 pixels in area with a depth inintensity of 10 bits (1,024). Bright-field images were made withtungsten illumination under transmitted light. Fluorescence micros-copy images were acquired using a Zeiss �20/0.50 numerical aperture(NA) water immersion objective, and spanned 170 � 170 �m in thefocal plane. For measurements of microvascular hemodynamics, themicroscope configuration was changed to view the tissue with a longworking distance Leitz UM20/0.33 NA metallurgical objective, withan effective magnification/NA of �13/0.22 NA (without the use of theglass hemispheres). An analog silicon target video camera was used toguide placement of the velocimeter photodetector and record mi-crovessel diameter.

Measurement of endothelial surface glycan concentration andglycocalyx thickness. To obtain an index of the glycan concentrationon the EC surface, the lectin Bandeira Simplicifolia (BS-1, Sigma, St.Louis, MO) was labeled with Alexa Fluor 488 (Invitrogen, Carlsbad,CA) and infused intravenously via the jugular indwelling catheter.The concentration of labeled lectin averaged 1.56 � 0.17 (SD) mg/mlin PBS with a molar ratio of fluorophore to protein equal to 17.5 � 3.9(SD) mg/ml, for 10 samples. A single bolus of labeled BS-1 wasadministered intravenously at a dose of 1 ml/kg and allowed toequilibrate for 20 min before intensity measurements on the ECsurface were made.

To quantify the thickness of the EC glycocalyx, the method ofHenry and Duling (24) was implemented as described previously (19).In brief, a bolus of 0.1% (in 0.15-ml PBS) of fluorescein isothiocya-nate labeled Dx70 (Sigma) was given intravenously (jugular vein),and fluorescence was allowed to reach a steady-state level, as ob-served in individual microvessels under incident fluorescence micros-copy. The radial profile of fluorescence intensity at the EC surface wasdigitized and fit by a sigmoidal curve using a least-squares method.The location of the inflection point in the radially decreasing dyeintensity curve was taken as the outer edge of the glycocalyx. Thesurface of the EC was taken as the outer edge of the dark refractiveband present near the wall in each microvessel under bright-fieldtrans-illumination. The spatial difference between the inflection pointand surface of the EC was taken as the thickness of the glycocalyx. Allimage processing and measurements were done using ImageJ (Na-tional Institutes of Health, Bethesda, MD).

Hemodynamic measurements. To quantify changes in the resistanceto flow in response to shedding of the glycocalyx within individualcapillaries, VRBC and arteriovenous �P were measured. VRBC wasobtained using the two-slit photometric technique (61). The meanvelocity of cells plus plasma (VMEAN) was assumed to be proportionalto the centerline VRBC and given by VMEAN � VRBC/1.6 (39).Upstream-to-downstream �P in single capillaries was measured bythe dual servo-null technique (28). Finely drawn micropipettes(�5-�m tip diameter) were inserted into side branches of feedingarterioles (20- to 30-�m diameter) and draining venules (25- to40-�m diameter) to obtain �P in individual capillaries. The resistanceto flow was determined as R � �P/Q, where the volumetric flow Q �VMEAN � D2/4, and D � microvessel luminal diameter.

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Separate experiments were performed to measure HCAP duringapplication of fMLP by direct cell counting of RBCs in capillaries.Video recordings of capillary blood flow were made with a gatedcharge-coupled device camera (Optronics, Goleta, CA) at high shutterspeeds (1/4,000-s or 1/10,000-s exposure time) to obtain the number(N) of flowing red cells resident at any instant. HCAP was calculatedas HCAP � N � mcv/(D2 � L/4), where mcv is mean cell volume,and D and L are capillary diameter and length, respectively. A valueof mcv equal to 55 fl was used.

Statistics. Statistical analyses of trends in the data were performedusing SigmaStat (Systat, San Jose, CA) with either Student’s t-test forpaired measurements, or the Holm-Sidak method for ANOVA ofmultiple comparisons. Nonparametric tests were used when appropri-ate, as indicated.

RESULTS

Fluorescence intensity measurements. Measurement of flu-orescence intensity of fluorophores in living tissues under laserillumination necessitates correction for the uneven illuminationin the microscope field. To illustrate, shown in Fig. 1A is a plotof fluorescence intensity in the focal plane (x-y) of the micro-scope when focused on the midplane of a 100-�m deepmicrocuvette containing 0.01 mg/ml fluorescently labeledBS-1 lectin. The nonuniform laser excitation results in aparabolic distribution of emission intensity, I(x,y)uniform, forthis uniform distribution of fluorophores, with a range offluorescent intensities from 22 to 994 and a coefficient ofvariation equal to 17.2%. To correct for this artifact, eachimage scene was flattened by scaling the input image, I(x,y)in,

with the algorithm I(x,y)out � [I(x,y)in � I(x,y)0]/[I(x,y)uniform �I(x,y)0] � K, where I(x,y)0 is background intensity (no incidentillumination), and K is an arbitrary constant, typically taken as520. Application of this algorithm to the uniform field (Fig. 1A)resulted in the flat field of Fig. 1B, with an intensity range of332–649, and a coefficient of variation equal to 1.1%.

The linearity of I(x,y)out with varying fluorophore concen-trations was validated by regression of fluorescence intensityagainst serial dilutions of a known concentration of labeledBS-1. A representative regression of average fluorescenceintensity obtained over a 100 �m2 area against concentrationsof BS-1 ranging from 10 to 175 �g/ml is illustrated in Fig. 1C.Intensity was linear with concentration with a regression co-efficient of r2 � 0.998 and a root-mean-square error of 6.5%.An illustrative video scene of a capillary image corrected byflattening the field and subtracting off the background intensity(obtained in the avascular tissue space) is shown in Fig. 1D.Twenty minutes following infusion of the BS-1, the capillarywalls were intensely stained by the fluorophore, which reachedsteady-state levels. The intensity of the fluorescence was non-uniform because of the shallow depth of field of the objective,estimated to be on the order of 3 �m. The noncircular crosssection of microvessels resulted in one wall being in sharpfocus at the apex of its curvature, while the other wallobliquely traversed the focal plane, which resulted in a lessintense image. Variations in intensity also arose from the entireplane of the vessel being oblique to the focal plane, as illus-trated by the microvessel running horizontally in the image

Fig. 1. Schema of image processing for fluores-cence intensity measurements. A: spatial distribu-tion of fluorescence intensity from a microcuvettecontaining a 1% solution of the lectin BS-1 con-jugated with Alexa Fluor 488. Within the micro-scope focal plane (x–y � 1,024 � 1,024 pixels),emission intensity averaged 691.4 (maximumvalue � 1,024), with a coefficient of variation of17.2%. B: flattening the field by postprocessingreduced this nonuniformity to 1.1%. C: represen-tative calibration of emission intensity vs. BS-1concentration for corrected images yielded a lin-ear regression with r2 � 0.998 and a root-mean-square error of 6.5%. D: representative processedimage of a capillary stained with BS-1 (intrave-nous). The surface of the endothelium within thefocal plane stained brightly (left wall above),whereas portions of the vessel out of focus stainedless intensely, as indicated by the right wall andthe entire capillary at the top of the picture.

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field at the top. This vessel appeared to be in a focal planeabove the vertically running capillary and, as a result, fluo-resced much less intensely compared with the capillary. Thesethree-dimensional variations in intensity necessitated multiplesampling at different focal plane positions to obtain an averageintensity for a specific microvessel.

Quantifying lectins bound to the endothelium. Fluorescenceintensity of the bound BS-1 was monitored in arterioles,capillaries, and venules during infusion of the BS-1, during a20-min period of its binding to the endothelium (EC) and for30 min following onset of superfusion of the tissue with fMLP.Illustrated in Fig. 2 are representative images of an arteriolarand venular bifurcation during this sequence of events. Toreduce the influence of random noise in the intensifiedimage, each image was averaged in real time over 16successive frames (taken at 30 frames/s) and thus representsthe average intensity acquired in a 0.53-s period. Initialstudies of the accumulation of BS-1 on the EC revealed thata steady-state level of staining was achieved within 20 minof circulation of the lectin. Hence, all experiments includeda 20-min stabilization period before onset of the fMLP.Following superfusion with fMLP, the fluorescence intensitysteadily declined, although visual evidence is difficult to dis-cern compared with analysis of the digitized intensity. Whiteblood cells (WBCs) also took up the lectin stain, as shown forWBCs briefly sequestered in the arterioles and their prolongedand firm adhesion in the venules. Also noted is the occurrenceof a slight arteriolar vasoconstriction due to the fMLP withinthe first 5 min and its subsequent lessening over a 30-minperiod. Prior studies have shown this response to have aninsignificant effect on arteriolar resistance (28). Both the timecourse of vasoconstriction and WBC firm adhesion to the ECwere consistent with prior observations of the effects of fMLP(28). Firm adhesion of WBCs in the postcapillary venules

reached a maximum within a 15- to 30-min period followingonset of superfusion with fMLP.

The accumulation of fluorescently labeled BS-1 on the ECwall was quantified as illustrated in Fig. 3 by a three-stepprocedure. First, a measurement path was digitally traced byeye along the curvature of the microvessel wall (Fig. 3A) andtypically ranged from 30 to 100 �m in length. In this example,the measurement path was 240 pixels in length (the equivalentof 40.3 �m), and in other cases ranged from 100 to 900 pixels.A radial line, normal to the measurement path, was thendelineated by prescribing a measurement line of sufficientlength to span the entire microvessel width and beyond. Acustom macro in ImageJ was then executed to sample imageintensity in each pixel along the measurement line. The loca-tion of the measurement line was then moved to the next pixelalong the measurement path, and a second set of radial inten-sities was digitized. This process was repeated along the entiremeasurement path (in this case, 240 times), and an averageradial intensity distribution was computed. The image formedby the matrix of radial measurement lines is shown in Fig. 3B,and the average intensity is given in panel C. As shown, thewall delimited by the measurement path became straightenedout, and the peak intensity at the wall corresponded to the peakin the radial profile of Fig. 3C. In cases in which both wallswere in focus in the same image, this process was repeated bydrawing a new measurement path line along the opposite wall,and its peak intensity was determined. The peak intensity at thewall was taken as the average of the values for the two walls.If the opposite wall was not in focus, the intensity was eithertaken as that of the one wall, or another digitized scene wasused in which the wall was brought into sharp focus.

Peak wall intensity measurements in 14 arterioles, 50 cap-illaries, and 17 venules are presented in Fig. 4. Also shown areseparate control (sham) experiments, where BS-1 accumula-

Fig. 2. Representative staining of arterioles andvenules in response to a 20-min accumulation ofBS-1 conjugated with Alexa Fluor 488. Nominalrelative times (t) of image acquisition are indi-cated. At t � 0, superfusion of the tissue with10�7 M fMLP was begun. The direction of redcell flow is indicated by the arrows. The inter-mittent adhesion of a white blood cell (WBC)can be seen in arteriole branches, and WBC firmadhesion is strikingly evident in venules follow-ing onset of fMLP.

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tion and fluorescence intensity were monitored during a similartime period without fMLP. The abscissa values for time cor-respond to the average acquisition time of the images followingonset of superfusion of fMLP or controls, which varied byapproximately �5–10 min while scanning multiple tissue re-gions. Baseline values of intensity before onset of fMLP aregiven in Table 1. As shown there, capillary baseline intensitywas significantly greater than those for either arterioles orvenules, P � 0.02 (ANOVA). However, a regression analysisof intensity vs. microvessel diameter revealed an insignificantcorrelation: r2 � 0.026, P � 0.260. This behavior suggests thatthe greater capillary intensity, and hence uptake of lectin, maybe due to factors other than optical (e.g., depth of field) orgeometric (e.g., departure from a circular lumen) parameters,as noted in the DISCUSSION.

Under control conditions, no statistically significant varia-tion of intensity was observed throughout the 30-min period foreach class of microvessel (ANOVA, P � 0.876). However,with fMLP superfusion, the intensity in all microvessels fellsignificantly during the first 5 min and exhibited a significantdecline over the 30-min superfusion period, ANOVA, P �0.05. This behavior suggests that the shedding of glycans inresponse to fMLP was not unique to the venular wall, butcommon to arterioles, capillaries, and venules.

Thickness changes of the glycocalyx. To characterize changesin thickness of the glycocalyx attendant to glycan shedding inresponse to fMLP, following 20 min superfusion with fMLPfluorescently labeled Dx70 was infused systemically and al-lowed to equilibrate for 10 min, following which thicknessmeasurements were made, as described in MATERIALS AND

METHODS. Thicknesses for 12 arterioles, 24 capillaries, and 12venules are shown in Fig. 5. Diameters of these microvesselsaveraged 18.0 � 3.3 (SD) �m for the arterioles, 8.2 � 2.0 (SD)�m for the capillaries, and 20.9 � 8.3 (SD) �m for the venules.Thickness of the glycocalyx before application of fMLP variedsignificantly among the three orders of microvessels, P �0.001, ANOVA. Statistically, thickness in the capillaries (348nm) was significantly less than that in arterioles (551 nm) orvenules (638 nm), P � 0.025, ANOVA. The arterioles were

not significantly different from the venules, P � 0.050. Pairedmeasurements of thickness before and following fMLP withinindividual microvessels fell significantly in all three divisions,with ratios of post- to pre-fMLP thickness of 0.74 � 0.16, 0.88 �0.51, and 0.87 � 0.46 (SD), for arterioles, capillaries, andvenules, respectively. The maximum fall occurred in arterioles(26%), followed by venules (13%) and capillaries (12%), withall decreases being statistically significant, P � 0.05.

Hemodynamic measurements and hematocrit. To assess theimpact of alterations in the thickness of the glycocalyx oncapillary hemodynamics, two sets of measurements weremade: 1) VRBC and Q values in arterioles, capillaries, andvenules; and 2) �P, VRBC, and resistance to flow, before andfollowing application of fMLP. Baseline hemodynamic valuesfor the measured (VRBC, �P, and diameter) and calculatedparameters [wall shear rate (̇), Q, and resistance] are given forthe two experiments in Table 1. The effect of fMLP on thedistribution of flows throughout the hierarchy of arterioles,capillaries, and venules is illustrated in Fig. 6A. With applica-tion of fMLP, Q fell �18% in arterioles, which was statisti-cally insignificant, P � 0.690. Small yet insignificant decreasesin flow also occurred in the capillaries (20%, P � 0.337) andvenules (3%, P � 0.941). To assess the effects of fMLP on theredistribution of flow throughout the mesenteric network, theratio of capillary (QCAP) and venular flows (QVEN) to theirrespective feeding arteriolar flows (QART) is given in Fig. 6B.In general, QCAP and QVEN changes were proportional toQART, as evidenced by the constancy of their fractions ofQART. The ratio of QCAP to QART after fMLP to its controlvalue was insignificantly different from 1.0, P � 0.375. Asimilar invariance of QVEN-to-QART ratio, QVEN/QART, wasevident, P � 0.195. Hence, it appeared that QCAP were notdisproportionately affected by changes in the glycocalyx of thesmaller capillary lumen.

Measurements of HCAP in response to fMLP are given inTable 2 for 31 capillaries studied in three animals with asystemic hematocrit (large vessel) averaging 43 � 3.6% (SD).Although there was a 22% decrease in HCAP and a 20%decrease in HCAP-to-systemic hematocrit ratio following

Fig. 3. Illustrative example of quantifying theintensity of BS-1 staining on the microvesselsurface. A: a measurement path was manuallytraced along the wall of a vessel (240 pixels inlength in this example), and a radial measure-ment line was automatically generated normal tothe beginning of the trace. A computer algorithmaveraged the radial intensity distribution at eachof the 240 pixels. The rasterized display of eachintensity distribution is shown in B, and theresultant profile of average intensity is shown inC. The effect of the measurement process was tostraighten out the wall delimited by the measure-ment path. The peak intensity was taken as ameasure of lectin accumulation and averagedwith the peak intensity obtained from an imagewith the contralateral wall in focus.

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fMLP, they were not significant, P � 0.077 and P � 0.052,respectively. For a subset of 21 capillaries, for which pairedmeasurements of HCAP were obtained, the post-to-pre-fMLPratio was not significantly different from 1.0, P � 0.801,Mann-Whitney rank-sum test.

To elucidate the hemodynamic significance of changes inglycocalyx thickness in the capillaries directly, resistance mea-surements were made in 18 capillaries (Fig. 7). VRBC and ̇ fellsignificantly by �7%, flow (QCAP) decreased by 5%, and �Prose by 5%, as evidenced by the ratio of their post- topre-fMLP values. These changes were not significant, as as-sessed by paired t-test. However, an 18% rise in resistance(R � �P/QCAP) was significant, P � 0.05. This slight rise inresistance may be due to activation of WBCs by the fMLP,which caused them to stiffen, thus increasing the resistance toflow as they entered the capillaries.

Presented in Fig. 8 is the ratio of post- to pre-capillaryresistance, RPOST/RPRE, for these 18 capillaries, plotted againstcapillary diameter (A) and the time duration of exposure tofMLP (B). Average baseline resistance (pre-fMLP) is given inTable 1. Individual anatomic capillary diameter remained in-variant with superfusion of fMLP and averaged 6.69 � 1.06(SD) �m before fMLP, and 6.74 � 1.12 (SD) �m duringfMLP, which were not significantly different, P � 0.663. Withincreasing diameter (Fig. 8A), RPOST/RPRE showed a slight(although insignificant decline, r2 � 0.112, P � 0.175), sug-

Fig. 4. Shedding of the endothelial glycocalyx in arterioles (n � 14; A),capillaries (n � 50; B), and venules (n � 17; C). The variation of fluorescenceintensity with time (�) following onset of superfusion of the mesentery with10�7 M fMLP is shown. Peak intensity of fluorescence emission at theendothelial surface was normalized with respect to baseline values before onsetof fMLP (Table 1). In each case, intensity fell significantly during the first 5–7min and during a subsequent 30-min period. Control (sham) experimentsconducted over a 30-min period (Ringer solution alone; Œ) showed no signif-icant decline in intensity for 21 arterioles, 39 capillaries, and 20 venules.Values are means � SE.

Table 1. Baseline measurements before superfusionwith fMLP

Arterioles Capillaries Venules

Fluorescence intensity measurements (Fig. 4)

No. of vessels 14 50 17Baseline fluorescence intensity 71.9 � 47.0 133.2 � 101.1 73.9 � 46.0Diameter before fMLP, �m 17.1 � 6.4 8.8 � 2.5 28.8 � 14.8No. of control (sham) vessels 21 39 20Diameter of control (sham)

vessels, �m 14.9 � 6.0 9.1 � 2.5 28.2 � 13.9

Hemodynamic baseline measurements (Fig. 6)

No. of vessels 14 49 16Diameter, �m 20.7 � 9.1 9.1 � 2.7 27.0 � 8.4VRBC, mm/s 3.3 � 2.1 2.2 � 1.8 1.4 � 0.9̇, s�1 831 � 525 1,266 � 1111 247 � 162Q, 10�4 mm3/s 10.8 � 14.6 1.03 � 1.20 6.2 � 7.0

�P and resistance measurements (Figs. 7 and 8)

No. of vessels 18Diameter, �m 6.7 � 1.1DP, cmH2O 24.1 � 2.8VRBC, mm/s 1.7 � 0.7̇, s�1 1,566 � 637Q, 10�4 mm3/s 0.6 � 0.4R, 105 cmH2O·mm�3·s�1 5.1 � 2.5

Values are means � SD. �P, pressure drop; VRBC, red cell velocity; ̇, shearrate; Q, volume flow; R, resistance.

Fig. 5. Changes in the thickness of the glycocalyx measured by the infiltrationof FITC labeled dextran 70 kDa into the endothelial surface layer. Thicknesswas taken as the distance between the edge of the dextran column and thesurface of the endothelium for 12 arterioles, 24 capillaries, and 17 venules.Thickness in the capillaries was significantly smaller compared with that ofarterioles or venules, P � 0.025, ANOVA. In all three divisions of themicrovasculature, thickness decreased significantly following superfusion with10�7 M fMLP. Values are means � SE. *P � 0.05.

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gesting that the initial increase in resistance arose from sourcesother than diameter, such as the effect of fMLP on WBCdeformability. Although a steeper negative regression slopewas evident for the group of capillaries with diameters �8 �m,it too was not statistically significant, r2 � 0.210, P � 0.086.Given that shedding of the glycocalyx increased with the timeduration of exposure to fMLP (Fig. 4), the ratio of RPOST/RPRE

was examined as a function of time (Fig. 8B). These trendsoccurred during the time course of sampling the capillarynetwork to make the sequence of hemodynamic measurements(velocity, �P, etc.), and revealed a significant 26% decrease incapillary resistance over the 30-min duration of fMLP, r2 �0.257, P � 0.032. The average duration of exposure to fMLPfor each capillary during this period was 14.4 � 7.3 (SD) min.

DISCUSSION

fMLP and shedding of the glycocalyx. In the present studies,the loss of lectins (BS-1) bound to the surface of the endothe-lium has been shown to rapidly occur in response to topical

application of fMLP. In prior studies, it has been demonstratedfor glass surfaces to which CS has been covalently linked thatthe adhesion of BS-1-coated fluorescent microspheres couldnot be disrupted by shear stresses far in excess of physiologicallimits (100 dyn/cm2), with or without the presence of 10�7 MfMLP (38). Hence, it is presumed that the loss of bound lectinsshown herein signifies shedding of GAGs or glycosylatedproteins from the EC surface. This assumption is also sup-ported by prior studies that have correlated fMLP-inducedshedding of antibodies for HS and CS bound to the EC surface

Fig. 6. A: calculated volumetric flow rates (Q) measured before and 10–20 min following onset of superfusion of mesentery with 10�7 M fMLP for 14 arterioles(QART), 49 capillaries (QCAP), and 16 venules (QVEN). Baseline hemodynamic parameters [red blood cell velocity (VRBC), shear rate (̇), and Q] are given inTable 1. A small, yet insignificant, decrease in Q occurred in arterioles (P � 0.690), capillaries (P � 0.337), and venules (P � 0.941). B: effects of fMLP onchanges in capillary and venular flows relative to their paired feeding arterioles are indicated by the paired ratio of QCAP/QART and QVEN/QART. No changeoccurred in the ratio of these parameters following and before the application of fMLP for QCAP/QART (P � 0.375) and QVEN/QART (P � 0.195), thus suggestingthat significant changes in the resistance to flow did not occur between arterioles, capillaries, and venules. Values are means � SE.

Table 2. Capillary hematocrit measurements pre- andpost-fMLP superfusion

Parameter Pre-fMLP Post-fMLP

HCAP, % 28.1 � 13.6 21.9 � 10.7*HCAP/HSYS 0.64 � 0.29 0.51 � 0.26†N 31 30

Values are means � SD; N, no. of measurements. HCAP, capillary hematocrit;HSYS, systemic hematocrit; HCAP/HSYS, ratio of HCAP to HSYS. The 22% fall inHCAP and 20% fall in HCAP/HSYS with fMLP were not significant: *P � 0.077,†P � 0.052. For 21 paired measurements, the ratio of (HCAP/HSYS)Post-fMLP/(HCAP/HSYS)Pre-fMLP was not significantly different from 1.0 (P � 0.801, Mann-Whitney rank sum test), as HCAP fell slightly from 23.8 � 8.2 to 23.2 � 12.2%with fMLP.

Fig. 7. Post- to pre-fMLP ratios of hemodynamic parameters, VRBC, ̇, Q, andpressure drop (�P) for 18 capillaries measured over a 30-min period ofsuperfusion, with an average duration of 14 min. The corresponding post/preratio of the calculated resistance to flow (R � �P/Q) is also shown. Post- topre-fMLP ratios did not change significantly for VRBC (P � 0.169), ̇ (P �0.206 ), Q (P � 0.454), and �P (P � 0.081), whereas R increased significantlyby 18% (*P � 0.05), assessed by paired t-test. Baseline (pre-fMLP) parametersare given in Table 1. Values are means � SE.

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with shedding of the bound lectins Bandiera Simplicifolia(BS-1) and Lycopersican Esculentum (43).

The pattern of lectin binding to the EC surface amongarterioles, capillaries, and venules may be indicative of theavailability of binding sites, as evidenced by the greater inten-sity of fluorophore at the capillary level (Table 1). However,other factors may influence the delivery and uptake of lectin,which necessitate that agonist-induced changes in intensity benormalized to initial values. Although optical and geometricfactors were ruled out (see RESULTS), flow-dependent factorsmay prevail. For example, wall ̇ were typically greatest in thecapillaries, compared with the arterioles and venules (Table 1).However, the large spatial variation of capillary ̇ throughoutthe network precluded a statistically significant difference fromthe arterioles, and the substantially lesser ̇ in venules com-pared with capillaries was significant. Assuming that the de-

position and binding of lectin to the EC is convection (flow)limited, the accumulation of lectin over time would be propor-tional to the volumetric flux per unit of vessel surface area.That is, within a microvessel, the convective flux of lectin withconcentration c in a stream with Q is given by the product cQ,and the area available for its accumulation would be propor-tional to diameter (D). Thus, given that the flux per unit ofwall surface area is thus cQ/D for a tube of unit length, andthat for a Newtonian fluid, ̇ is given by 32Q/D3, the buildupof lectin may be proportional to ̇. While the greater accumu-lation of lectin in the capillaries is consistent with the higher ̇,the equivalence of accumulation between arterioles andvenules (Table 1), with markedly different ̇ along the wall(Table 1), suggests that other factors may play a role. It is thusconceivable that the increased deposition of lectin on thecapillary surface, compared with arterioles and venules, mayreflect an increased level of binding sites.

The use of fMLP as an agonist to induce shedding of theglycocalyx stems from its role as a well-studied model of theinflammatory process (3, 28), whereby it mimics a variety ofpeptides released from either bacterial or host cells during anacute inflammation. It was selected as the agonist for thepresent studies because of the rapidity of its action and its welldocumented effect on the mechanical and adhesive propertiesof WBCs and microvascular hemodynamics. As a stimulus forWBC activation, fMLP causes a stiffening of polymorphonu-clear leukocytes (15) and increased polymorphonuclear leuko-cyte adhesion in postcapillary venules, as a result of stimula-tion of G protein-coupled receptors (42), and thus significantlyincreases resistance to flow (28). Based on studies of therolling and adhesion of WBCs on either artificial surfacescoated with receptors for specific ligands (2, 35, 36), ormonolayers of cultured ECs (4, 27, 34), it has long been heldthat adhesiveness was influenced by regulation of the affinityand avidity of the integrin molecules on the WBC and EC (32,33, 41, 62). Similar in vivo studies of postcapillary venulesin the living animal (3, 28, 37) have supported this concept.The apparent thickness of the glycocalyx has been estimatedby the exclusion of erythrocytes and macromolecules (59) tobe on the order of 400 –500 nm, which significantly exceedsthe lengths of EC receptors involved in leukocyte (WBC)rolling on the EC (selectins) and firm adhesion to the EC(integrins). Studies of the lengths of these receptors haveshown a range from 20 nm for the �2-integrin ligands to30–40 nm for E- and P-selectins (54). Thus, in view of theconcurrent shedding of glycans and increased binding of anti-bodies to ICAM-1 on the EC in response to fMLP, it has beenpostulated that stimulated shedding of the glycocalyx mayrapidly expose receptors on the EC to facilitate WBC-ECadhesion (43).

The rapid shedding of glycans in response to fMLP in allthree divisions of the microvasculature (Fig. 4) suggests that acascade of signaling events is initiated by EC activation, whichis independent of the presence of WBCs. The brief transit timeof WBCs in the arteriolar portion of the network (14) and alack of WBC-EC adhesion in arterioles suggest that the effectsof WBC activation are not evident until WBCs reach the truecapillaries. Furthermore, it was observed here that shedding oflectins occurred rapidly in venules in which no WBC adhesionoccurred, as has been indicated previously by shedding oflectin-coated microspheres (43). Prior studies of the time

Fig. 8. Post- to pre-fMLP ratio of capillary resistance (RPOST/RPRE) followingonset of superfusion. A: values obtained during a 30-min duration of superfu-sion regressed against capillary luminal diameter (D), revealed a regression ofRPOST/RPRE � 1.9 � 0.11D, with r2 � 0.112, which was not significant, P �0.175. (Also shown are 95% confidence intervals, dashed lines.) B: regressionRPOST/RPRE with time duration of superfusion (t) revealed a significantregression: RPOST/RPRE � 1.5 � 0.03t, with r2 � 0.257, P � 0.032. The rangeof times corresponded to the time taken to sample hemodynamic parametersduring the experiments. While the average value of RPOST/RPRE was elevated18% (Fig. 7) for the 14-min average duration of fMLP (presumably due todiminished WBC deformability), a significant decrease in resistance ratio overthe 30-min period lessened this effect, most likely from shedding of thecapillary glycocalyx (Fig. 4), which reduced the resistance to flow.

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course of changes in the composition of the glycocalyx inresponse to topical application of TNF-� (in hamster cremastervenules) revealed a similar length of time for changes incomposition of the glycocalyx, as evidenced by a 30% de-crease in thickness of the barrier to infiltration of Dx70 within20 min, as shown by Henry and Duling (25). In these latterstudies, it has also been suggested that TNF-� activates surfacebound proteases. This hypothesis is consistent with subsequentobservations that fMLP-induced shedding of bound lectins canbe inhibited by the matrix metalloproteinase inhibitors, ilomas-tat and doxycycline (44). The rapidity of shedding of constit-uents of the glycocalyx in response to TNF-� has also beendemonstrated by the rapid outflow of syndecan-1 and HS fromthe isolated guinea pig coronary circulation (8). Within 5 minof initiation of perfusion with cell-free buffer containingTNF-�, significant amounts of syndecan-1 and HS were foundin the effluent, which diminished with time, suggestive of adepletion of components shed from the endothelium.

Thickness of the glycocalyx. Using the exclusion of Dx70 asa measure of the thickness of the glycocalyx, changes inresponse to fMLP were found that are consistent with priormeasurements in arterioles, capillaries, and venules of hamstercremaster muscle (24, 25, 59, 60). Although the present mea-surements appear slightly larger, which may be due to eithermethodology or species differences, similar trends amongvascular divisions between the two species were found. In bothpreparations, the capillary Dx70 exclusion thickness wassmaller than that of either arterioles or venules. The order ofmagnitude reductions in Dx70 exclusion thickness of 100 nmin arterioles, capillaries, and venules found here (Fig. 5) isconsistent with reported venular responses, where the thicknessdecreased from 400 to 300 nm within 20-min exposure ofvenules to TNF-� (25). Given that resistance is inverselyproportional to the fourth power of diameter, a reduction incapillary diameter from 6.0 to 5.8 �m by the presence of asignificant glycocalyx would theoretically increase the re-sistance to flow by 15%. With larger vessels, for examplewith a 30-�m diameter, resistance to flow would be in-creased by only �3%.

Network hemodynamics and hematocrit. To determinewhether shedding of the glycocalyx in response to fMLPproduced flow redistribution effects due to changes in capillaryeffective diameter, the relative flow ratios between capillariesand arterioles were examined (Fig. 6). These results suggestedthat both QCAP and QVEN changed in proportion to QART, andthat shedding of the glycocalyx in the smaller diameter capil-laries did not measurably affect resistance across the capillarybed. Given the heterogeneity of capillary diameters in mesen-tery, shunting of flow through larger diameter thoroughfarechannels might have lessened the effect of the glycocalyx onthe ensemble of capillaries. Overall reductions in flow through-out the microvascular network with fMLP have been shownpreviously to principally result from obstruction of venules byWBC adhesion and a modest (although insignificant) arteriolarvasoconstriction (28). As shown therein, as few as 12 WBCsadhered to the endothelium per 100 �m of venular lengthwas sufficient to double the resistance to flow. The modestreductions in flow here reflect the substantially lesseramount of WBC-EC adhesion in rat mesentery comparedwith that in cat (28).

The small, yet insignificant, reductions in HCAP were con-sistent with prior observations of the effects of fMLP onarterioles and venules (28). As shown therein, no significantchange in arteriolar or venular hematocrit occurred with fMLP,as assessed by direct spectrophotometric methods. In contrast,HCAP was found to increase in response to degradation of theglycocalyx twofold with direct infusion of heparinase withmicropipettes (13) and systemic infusion of hyaluronidase (7).Systemic infusion of oxidized LDL increased it twofold (10),and sustained epifluorescence illumination increased it by 68%(59), presumably due to generation of reactive oxygen speciesin both studies. Thus the mechanisms of glycan shedding inthese studies might differ from those invoked by fMLP.

Capillary resistance to flow. To more precisely delineatechanges in flow resistance at the capillary level in response tofMLP, �P and flow measurements were made in capillaries.The invariance of capillary hemodynamic parameters (VRBC,volume flow, ̇, and �P) and resistance (Fig. 7) are consistentwith the relative changes in flows throughout arteriolar, capil-lary, and venular divisions of the network. The 18% increase incapillary resistance with fMLP is contrary to the 14–20%decreases in resistance found during enzymatic removal of theglycocalyx by perfusion of the microvascular network withheparinase by Pries et al. (50). This disparity could be due toseveral factors. Although the two studies examined similartissues (mesentery of the rat), the �P and flows measuredacross the microvascular networks were strikingly different. Inthe present study, QART on the order of 65 nl/min (1 nl/s inTable 1) were measured in 21-�m-diameter arterioles, com-pared with 740 nl/min in the experiments by Pries et al. forslightly larger vessels averaging 29 �m. �P measured hereinwere only 17.8 mmHg (24.1 cmH2O, Table 1) compared with 52mmHg by Pries et al. Thus the baseline resistance to flow measuredby Pries et al. was on the order of 0.06 � 105 cmH2O·mm�3·s�1

(calculated from Table 1 in Pries et al.), which was 1/100th thatmeasured here (5.1 � 105 cmH2O·mm�3·s�1, Table 1). This lowerresistance is suggestive of a greater number of parallel path-ways to carry off the stream. Comparison of the flows betweenthe two studies suggests that there might be 10 times as manypathways between arteriole and venule in the Pries studycompared with herein (i.e., 740 compared with 65 nl/min inarterioles). Prior studies by Pries et al. (49) have indeed showna greater degree of vascularity peculiar to the Wistar rats usedin their studies, compared with the strain of Wistar usedpresently and in previous studies from this laboratory. Hence,from a hemodynamic perspective, the current baseline condi-tions are reasonably consistent with those of Pries et al. (50).Another source of disparity between the two studies could alsobe the method for removal of the glycocalyx.

Using direct micropipette infusion of a mixture of heparinase,chondroitinase, and hyaluronidase in postcapillary venules (19), ithas been shown that the Dx70 exclusion thickness could bemaximally reduced in 40-�m-diameter venules from 463 to 52nm (�90%). In those studies, superfusion with 10�7 M fMLPcaused a reduction of Dx70 exclusion zone by 130 nm, whichis similar in magnitude to the reductions of 110, 80, and 123nm observed here in arterioles, capillaries, and venules, respec-tively (Fig. 5). For heparinase alone, the reductions in Dx70thickness were smaller than the mixture, from 463 to 233 nm,or �50% (19). Thus it appears unlikely that the resistance

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reductions measured by Pries et al. (50) in response to hepa-rinase were entirely from removal of the glycocalyx.

Previous studies on the effects of superfusion of the mesen-tery with fMLP showed a twofold increase in resistance due toobstruction of the venular lumen with firmly adhered WBCs. Inthe present studies, no firm adhesion of WBCs was observed inthe arterioles or capillaries, although a few transient seques-tration events were observed. It is conceivable that these eventsarose from stiffening of the WBCs as they become activated bythe fMLP, and hence their entry time into the capillaries wasprolonged, which served to raise capillary resistance. Thesmall, albeit insignificant, decrease in the resistance ratio ofpost- to pre-fMLP superfusion for the true capillaries withincreasing diameter (Fig. 8A) supports the hypothesis thatdiminished WBC deformability is largely responsible for theincrease in capillary resistance with fMLP. Less deformableWBCs will take longer to enter a smaller capillary and requirea greater �P. This effect is well known to depend on the ratioof cell to capillary diameter (15). The smaller the pore size, thegreater will be the �P required for the WBC to deform andenter a capillary. Hence, the increase in resistance with WBCstiffening will diminish as diameters become larger. Increasedcapillary plugging and resistance to flow in the spinotrapeziusmuscle has been shown in response to superfusion with fMLP,where a 20% increase in capillary resistance was attributed tofMLP activation (22, 23), which is similar to that found here.

A significant decrease in post- to pre-fMLP capillary resis-tance (RPOST/RPRE) was found with increased duration ofexposure to fMLP (Fig. 8B). Based on linear regression of thedata, this decrease amounted to 26% over the 30-min durationof superfusion with fMLP. Hence, the overall effect of super-fusing the mesentery with fMLP was to raise capillary resis-tance by diminishing WBC deformability, which was mitigatedin time by shedding of the glycocalyx to increase the effectivecapillary diameter. This level of resistance reduction is thusconsistent with that obtained by Pries et al. (50) by strippingoff portions of the glycocalyx with heparinase. In contrast,however, the experiments of Chappell et al. (8) revealed anincreased resistance to flow concurrent with HS and synde-can-1 shedding in response to TNF-�. These latter experimentswere done with cell-free solutions, and hence there were nocontributions to resistance by WBCs. It is likely that theincrease in resistance therein resulted from the observed in-crease in transvascular fluid exchange attendant to TNF-�which caused edema and compression of capillaries to increaseresistance. In the case of fMLP in the absence of WBCs,studies have revealed that no increase in transvascular fluidflux occurs, as assessed by direct measurement of capillarypermeability (63).

In summary, the present studies reveal that shedding of theendothelial glycocalyx rapidly occurs within all divisions ofthe rat mesentery network in response to the chemoattractantfMLP. A concurrent 18% increase in the resistance to flow atthe capillary level occurs due to diminished WBC deformabil-ity and the added �P required for WBCs to traverse thecapillary network. Shedding of the capillary glycocalyxattendant to endothelial activation by fMLP results in anincrease in capillary effective diameter and a reduction incapillary resistance that mitigates the effects of WBC stiff-ening. Thus these events contribute to the overall hemody-namic response during the inflammatory process and sug-

gest that concurrent shedding of the endothelial glycocalyxand diminished WBC deformability are equally importantrheological factors that affect QCAP.

GRANTS

This work was supported by National Heart, Lung, and Blood InstituteResearch Grant R01 HL-39286-20.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: H. H. L. conception and design of research; H. H. L.,L. G., and A. L. performed experiments; H. H. L., L. G., and A. L. analyzeddata; H. H. L. interpreted results of experiments; H. H. L. prepared figures;H. H. L. drafted manuscript; H. H. L. edited and revised manuscript; H. H. L.approved final version of manuscript.

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H2245SHEDDING OF THE GLYCOCALYX AND CAPILLARY HEMODYNAMICS

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