Chances and limitations of isolated mouse heart models for investigating the endothelial glycocalyx 1

Abstract

BACKGROUND:

The endothelial glycocalyx plays a decisive role in maintaining vascular homeostasis. Previous animal models have mainly focused on in-vitro experiments or the isolated beating guinea pig heart. To further evaluate underlying mechanisms of up- and down regulation, knock-out animals seem to be a promising option.

OBJECTIVE:

Aim of the present study was to evaluate if an isolated mouse-heart model is suitable for glycocalyx research.

METHODS:

Isolated beating mouse hearts (C57/Bl6J) underwent warm, no-flow ischemia and successive reperfusion. Coronary effluent was analyzed by ELISA and Western blot for the glycocalyx core protein: syndecan-1. Hearts were prepared for either immunofluorescence or electron microscopy and lysed for Western blot analysis.

RESULTS:

An endothelial glycocalyx covering the total capillary circumference and syndecan-1 were detected by electron and immunofluorescence microscopy. Ischemia/reperfusion seriously deteriorated both findings. Confoundingly, syndecan-1 was not detectable either in the coronary effluent or in the lysates of blood-free hearts by ELISA or Western blot technique.

CONCLUSIONS:

Blood vessels of mouse hearts contain an endothelial glycocalyx comparable to that of other animals also with respect to its core protein syndecan-1. But, for studies including quantification of intravascular soluble glycocalyx constituents, the amount of syndecan-1 in mouse hearts seems to be too low.

Keywords

Endothelial glycocalyx isolated beating mouse heart syndecan-1 electron microscopy immunofluorescence

1 Introduction

The endothelial glycocalyx (EG) is an impressive structure clothing the total vascular system. Being anchored in and on the endothelial cells, it consists of membrane-bound proteoglycans, predominantly syndecan-1, which carry glycosamino glycane (GAG) side chains, chiefly heparan sulfate [1]. In various experimental models, the existence and dimension of the EG has been revealed by electron microscopy and staining of EG constituents using various techniques [2, 3]. Together with receptor-bound hyaluronan and intercalated plasma proteins, it is built up into the endothelial surface layer (ESL), the functionally active form in vivo. The ESL has been shown to have a thickness of around 1 μm, recognizing that the thickness is also depending on the fixation technique [4, 5]. Some authors measured even a thickness of 6 μm using a special rapid freezing method [3, 6]. Today there is growing evidence for the importance of this structure in maintaining vascular and extracellular hemostasis. In the past decade, awareness of the ESL has changed thinking in various areas of perioperative medicine, like rational perioperative infusion therapy and sobriety [7, 8]. The detrimental effects of ischemia/reperfusion, inflammation and various enzymes on this structure have meanwhile been confirmed in clinical trials showing that shedding of the EG correlated with extent and duration of ischemia/reperfusion and was an independent predictor of mortality in patients suffering from septic shock or trauma [9 –15].

Despite knowledge of the fact that the EG is deteriorated in various pathophysiological situations, the underlying mechanisms and pathways remain vague, precluding rational therapeutic approaches [16]. The utilization of genetically modified or knock-out animal models seems a promising approach in order to gain insights into the degradation and restoration of the EG. With this in mind, establishing an isolated beating mouse heart model is one possible strategy. This should, theoretically, allow for quantification of the dimensions and functions of the EG in situ in an intact vascular bed, and for quantification of shedding of the EG under various conditions. Aim of the present report is to establish feasibility and to present preliminary results of this organ model, perfused in a modified Langendorff setting foremost to facilitate a comparison to the commonly used guinea pig model.

2 Material and methods

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The study was approved by the officially installed independent ethics committee of the State of Bavaria (file No. 55.2-1-54-2532.3-73-12).

The ELISA test used for tracing syndecan-1 was of the type solid phase sandwich ELISA (murine sCD138 ELIA Kit, Diaclone, Besancon, France). The primary mouse specific antibody for Western blotting of syndecan-1 immunofluorescence was purchased from R&D Systems (AF3190, R&D, Minneapolis, USA).

2.1 Heart preparation

Male C57BL/6J mice 12 weeks of age were anaesthetized with a body-weight (bw) adapted intraperitoneal injection of xylazin (10 mg/kg bw) and ketamin (120 mg/kg bw). The animals were fixed supine on an underlayer and oxygen was administered. Upon reaching a sure level of analgesia, laparotomy followed by a diaphragm incision and a lateral double-sided thoracotomy was performed. Simultaneously, the heart was cooled down with 1–4°C cold saline solution until cardiac arrest to avoid ischemic damage. The hearts were then generally (see Study Conditions) perfused within 3 min via an aortic perfusion cannula (inner diameter 0.55 mm) inserted above the aortic valve but distal to the coronary arteries. This early perfusion served to prevent hypoxic insufficiency and coronary occlusion before the perfusion experiment propper was started. The perfusate was a modified crystalloid Krebs-Henseleit-buffer (containing, besides all common plasma electrolytes, 95% O₂, 5% CO₂, pyruvate, glucose and insulin). To achieve physiological heart rates, norepinephrin (0.1 μmolar) was administered via a side port. Hearts were then perfused with a constant pressure of 100 cm H₂O during the experiments, except for protocols invoking no-flow ischemia as inidicated [17]. The upper caval veins, pulmonary veins and lungs were ligated before the truncus pulmonalis was cannulated with an especially designed cannula (inner diameter 0.75 mm, outer diameter 1.22 mm). This served to collect the coronary effluent. Last steps of the preparation were ligating the inferior caval vein and then removing the heart from the thorax. The spontaneously beating hearts were kept in a special temperature chamber to maintain a surface temperature of 38±0.5°C. Hearts generally underwent a total equilibration phase of 15 minutes to ensure steady state conditions before starting each experiment, unless otherwise stated.

All operational steps were performed with a stereo magnifying glass (Zeiss Stemi DV4 Spot, Zeiss, Jena, Germany).

2.2 Study conditions

Removal of the heart immediately after anaesthesia (without aortic canulation): group A

Removal of the heart after short perfusion with crystalloid buffer (blood free): group B

Removal of the heart after 20 min warm ischemia and 15 min reperfusion: group C

Removal of the heart after 45 min crystalloid perfusion: group D

The study protocols and the subsequent analyses are outlined in Fig. 1.

Fig.1

Experimental protocols. Group A: direct removal of the heart after thoracotomy; Group B: cannulation of the aorta and brief perfusion to flush out intracoronary blood. Group C: after 15 min of equilibration, 20 min warm (37°C), no-flow ischemia followed by 15 min reperfusion. Group D: after 15 min equilibration, 35 min perfusion before further processing. WB = Western blot, IF = immunofluorescence staining, EM = electron microscopy, ELISA = enzyme linked immunosorbent assay.

2.3 Determination of syndecan-1 in the effluent by ELISA

Total effluent was saved and frozen for assessing shedding of syndecan-1 (CD-138). Total sample volume (average volume about 25 ml) was concentrated to about 250 μl with a 3 kDa cutoff ultrafilter (Millipore, Billerica, MA, USA). Syndecan-1 concentrations were determined using an enzyme-linked immunosorbent assay (Diaclone Research, Besancon, France), as described by the manufacturer [18, 19].

2.4 Cell extracts for Western blot analysis and immunoblotting of syndecan-1

Protein lysates were prepared and protein content quantified from hearts of groups A, B, C and D and effluents from groups C and D, as described previously [20]. In short, tissues were homogenised in ice-cold cell lysis buffer (Cell Signaling Technology, Frankfurt am Main, Germany) using ceramic beads (Peqlab, Erlangen, Germany) and shaking in a CapMix (3M ESPE, Seefeld, Germany). Lysates were then subjected to Western blot analysis as described elsewhere [20]. Briefly, lysates or effluates were mixed with hot sample buffer (0.25M Tris pH 6.8, 8% SDS, 40% glycerol, 0.02% bromophenol blue, 400 mM β-mercaptoethanol) and loaded onto a 10% SDS-PAGE. Proteins were blotted onto nitrocellulose membranes (Peqlab, Erlangen, Germany) using wet blotting. Membranes were blocked 1 h at room temperature in 5% milk in TBS (tris buffered saline) supplemented with 0.1% tween, followed by incubation with primary antibody against mouse CD-138 (syndecan-1) (1:1000) over night. After washing 3 times in TBS supplemented with 0.1% tween, membranes were incubated with secondary, horse-radish peroxidase labelled antibody (1:10000) (Calbiochem, Darmstadt, Germany) for 1 h at room temperature. Upon application of enhanced chemiluminiscence solution (1M Tris pH8.5, 248 mM luminol, 91 mM p-coumaric acid, 0.2 % H₂O₂), membranes were developed using a chemiluminiscence detection system with a CCD camera (Hamamatsu, Germany). human β-actin was detected as a control for the loading of equal amounts of protein.

2.5 Immunofluorescence staining

Following the perfusion-fixation of the mouse hearts from groups B, C and D with 4,5% formaldehyde, hearts were embedded in paraffin and cut into 4 μm thin sections. These were deparaffinized and transferred in cuvettes containing PBS (phosphate buffered saline). The cuvettes were exposed to strong light (Arcadia-OT2, Redhill, UK) for 72 h with a substantial portion of near-UV visible light. This was done to minimize autofluorescence prior to immunohistochemistry. After washing the tissue sections in PBS, they were blocked with donkey normal serum. The primary antibody (Anti-mouse CD138 = syndecan-1, AF3190, R&D, Minneapolis, USA) was then applied and the tissue sections incubated at room temperature for 2 hours, followed by overnight incubation at 4°C and another 30 minutes at room temperature. The tissue sections were then washed thoroughly with PBS for 15 minutes and then incubated with the secondary fluorescent antibody conjugated with Alexa Fluor 546 nm, (A11056, AF546 donkey anti goat, Life Technologies, Carlsbad, California, USA) for 1 hour. Another 15 minutes of washing with PBS was followed by the incubation of the tissue sections with DAPI for 5 minutes. The sections were mounted with Kaiser's glycerine jelly. Immunofluorescence pictures were taken with a mercur-vapour lamp microscope (Zeiss Axiophot, Carl Zeiss Jena, Jena, Germany) and a microscope camera (Zeiss AxicomHRc, Carl Zeiss Jena, Jena, Germany) The image files were then analysed both qualitatively and quantitatively using the Java based image processing software Image J (version 1.48c, National Institutes of Health, Bethesda, Massachusetts, USA).

2.6 Electron microscopy

Electron microscopy of hearts from groups B, C and D was performed upon fixation of the tissue by a method modified from that of Vogel et al. [21]. After the end of the actual experiment, hearts were perfused with 10 ml of a fixation solution consisting of 2.5% glutaraldehyde, 12% sucrose, 0,4 M sodium cacodylate buffer (pH 7,3) and 4% lanthanum(III) nitrate. Thereafter, hearts were diced into pieces of approximately 1mm³ each and immersed in the fixation solution for two hours. Following this step, the pieces remained overnight in a solution without glutaraldehyde before being washed in a solution consisting of 0,1 N sodium hydroxide, 12% sucrose and destilled water. For contrasting, material was given into a solution containing 1% osmium tetroxide and 2% lanthanum(III) nitrate in a final concentration. After this, pieces were dehydrated in an ethanol step gradient, starting with 30% ethanol and ending up with pure ethanol. Samples were embedded using an epoxy resin embedding medium (Araldite CY212, Serva, Heidelberg, Germany), solved in propylenoxyd and fixed at 60°C for 48 hours. Semi-thin-sectioning was performed, samples for electron microscopy were selected after staining with toluidine blue. Finally, microtomic sectioning was performed to get sections of about 70 nm thickness, which were fished onto copper grids and contrasted with uranyl acetate (Serva, Heidelberg, Germany) solved in 70% methanol (2 spade points uranyl acetate in 1 ml methanol). Pictures were obtained with an electron microscope (TEM Libra 120, Fa. Zeiss, Oberkochen, Germany).

2.7 Statistics

For unpaired data, comparisons were made with Kruskal-Wallis and Mann-Whitney-U tests, as appropriate. When indicated, post-hoc corrections for multiple measurements were performed with the Holm-Bonferroni method (IBM SPSS Statistics, Armonk, NY, USA).

3 Results

3.1 Enzyme linked immunosorbent assay (ELISA)

Measurement of syndecan-1 was performed by ELISA in the coronary effluent of hearts undergoing 45 min perfusion with a modified crystalloid Krebs-Henseleit buffer (group D) and hearts undergoing 20 min of warm no-flow ischemia followed by 15 min of reperfusion (group C). Total effluent (approx. 25 ml) was collected and concentrated to obtain a final sample volume of about 250 μl in each experimental condition. The standard curve of the ELISA was exactly as described by the manufacturer. However, all measured syndecan-1 concentrations of the concentrated effluents of both groups C and D were either below the zero point or not differentiable from the blank values (data not shown). Therefore, no differences were seen between the two groups with and without ischemic challenge.

3.2 Western blot

In an attempt to quantify syndecan-1 and its shedding in the mouse heart, Western blots of total heart lysates (groups A, B, C and D) and concentrated effluents (groups C and D) were performed. Syndecan-1 could only be detected in the lysate of the blood containing hearts (Fig. 2: panel A). The loading control indicates adequate amounts of protein in the heart lysates, whereas no protein bands at all were detectable in the effluent (Fig. 2: panel B).

Fig.2

Western blot (left panel) and loading control (right panel). A = Ladder, B = Heart lysate (blood containing, group A), C = Heart lysate (blood free, group B), D/E = ultraconcentrated effluent of control hearts (group D) and hearts undergoing ischemia/reperfusion (group C), F = Heart lysate after ischemia/reperfusion (group C). Syndecan-1 is only to be detected in the lysate of the blood containing hearts.

3.3 Electron microscopy

Tissue samples of all groups, randomly selected, were prepared for electron microscopic examination.

In isolated beating mice hearts undergoing crystalloid perfusion (group D), electron microscopic pictures revealed an endothelial glycocalyx in the coronary vessels that seemed to cover the entire endothelial cell surface on the luminal side (Fig. 3: A). This lining reached a thickness of up to approx. 150 nm (average over circumference 130 nm±20 nm) (Fig. 3: panels A–C). In contrast, samples of hearts undergoing ischemia reperfusion (group C) show a dramatic decrease in thickness of the endothelial glycocalyx layer and an only spurious coating of the endothelial cells (Fig. 3: panels D & E). The electron microscopic examinations thus clearly illustrate a shedding of the endothelial glycocalyx in mouse hearts subjected to warm, no-flow ischemia and reperfusion.

Fig.3

Electron microscopic pictures showing an intact (Panels A-C) and degraded (Panels D, E) endothelial glycocalyx. Panel. A: Overview of a mouse capillary presenting an endothelial glycocalyx in the total circumference (group D). Panels B and C: More detailed pictures of the endothelial glycocalyx of a capillary (B: group B; C: group D). Panels D and E: destroyed endothelial glycocalyx following 20 min of warm (37°C) no-flow ischemia and 15 min reperfusion (group C).

3.4 Immunofluorescence

Immunofluorescence pictures employing a mouse-specific antigen against syndecan-1 were taken for qualitative (Fig. 4) and semi-quantitative analysis (Fig. 5). Pictures were obtained from blood free hearts removed immediately after thoracotomy (group B), from control group hearts (group D) and from hearts subjected to 20 min warm (37°C), no-flow ischemia and 15 min reperfusion (group C). Blood-free crystalloid-buffer perfused control hearts revealed strong immunofluorescence covering the entire luminal surface of microvessels (Fig. 4: A & B). In contrast, only weak and discontinuous fluorescence was detectable after ischemia/reperfusion (Fig. 4: C & D). For statistical analysis of differences in syndecan-1, seven sections were taken from group B, eight sections from group C and eight slices from group D. Hypothesis of the present method is that more syndecan-1 results in more fluorescent material in the vessel wall. The mean grey values of the vascular wall were determined manually and the grey scale of the background was subtracted. The time lapse between the immunofluorescence staining and obtaining the picture was equal for all pictures. The settings of the microscope camera were identical for all images. Fluorescence did not differ significantly between hearts merely flushed free of blood (group B) and group D hearts perfused for 45 min with Krebs-Henseleit buffer D (Fig. 5). Pertinently, there was a significant difference in the amount of fluorescent material between hearts of the non-ischemic groups B and D, and mouse hearts stained after ischemia/reperfusion (Fig. 5).

Fig.4

Immunofluorescence staining of syndecan-1 in mouse capillaries using a primary antibody against CD138 (syndecan-1) and a fluorescent secondary antibody (AlexaFluor 546 nm). Panel A: a group B (blood free) heart shows syndecan-1 in the total circumference of the microvessel. Panel B: capillary of a heart following 15 min (equilibration) + 35 min perfusion with a crystalloid buffer. There is a fluorescent signal over the total vascular circumference. Panels C & D: capillaries of hearts after 20 min warm ischemia followed by 15 min reperfusion. There is only discontinuous fluorescent material over the vascular wall.

Fig.5

Semi quantitative Immunofluorescence. Group B: cannulation of the aorta and short perfusion until total removal of intracoronary blood. Group D: 15 min equilibration and 35 min perfusion before further processing. Group C: 15 min of equilibration, then 20 min warm (37°C), no-flow ischemia, followed by 15 min reperfusion.

4 Discussion

The importance of the endothelial glycocalyx for maintaining vascular homeostasis is well recognized [8, 16]. A large part of this physiological and pathophysiological knowledge was obtained from experiments using isolated beating guinea pig hearts in the Langendorff perfusion mode. An isolated organ model seemed ideal to gain fundamental knowledge about the real dimension, vascular distribution and functions of the endothelial glycocalyx in an intact vascular bed, as well as to ascertain its reaction to various stimuli like ischemia/reperfusion, tumor necrosis factor and natriuretic peptides [22, 23]. Furthermore, there were some protective strategies targeting the endothelial glycocalyx, which could be evaluated with this model, like pre-ischemic application of sevoflurane or various substances, which are physiologically found in the human blood such as hydrocortisone, antithrombin III and human albumin [24 –26]. However, the Langendorff model based on guinea pig hearts has various limitations, foremost a paucity of commercially available, species-specific antibodies and the lack of genetically modified strains. Implementing isolated beating mouse hearts promises new opportunities to gain mechanistic data and – owing to the availability of knock-out and gene hyperexpression animals – to elucidate more detailed mechanisms related to endothelial glycocalyx pathophysiology. Aim of the present study was to evaluate if the results derived from the guinea pig hearts principally could be transferred to the results of isolated beating mouse hearts.

In the past, quantification of glycocalyx shedding was performed mainly by analyzing levels of its constituents in the coronary effluent using specific ELISA kits. Even though providing conclusive results, the measured values from guinea pigs were at the lower limit of the standard concentration curve even after ischemia/reperfusion, i.e., in hearts undergoing massive shedding of the glycocalyx. With respect to an at least ten-fold lower heart weight in mice compared to guinea pigs, the potential hazard to realization of the mouse model becomes apparent: The total endothelial mass of the vascular bed of an about 100–150 mg mouse heart may well be too small to afford quantifiable amounts of the core protein syndecan-1 in the coronary effluent. Indeed, our ELISA tests failed to detect syndecan-1 in measurable amounts in effluents of any of the 4 groups of mouse hearts, even after concentration of effluents by a factor of about 100 (25 ml to 250 μl).

After this disappointing result, Western blots were performed with samples of tissue and effluent of different experimental conditions. Interestingly, a syndecan-1 band was neither detected in the blood free heart lysates, nor in the concentrated coronary effluents, although the loading control confirms sufficient amounts of protein for the lysated hearts. However, tissue of hearts not flushed of blood cells after removal did contain a syndecan-1 band in the Western blot (Fig. 2). Thus, extractable amounts of syndecan-1 in the heart of the mouse exist probably only on blood cells filling the coronary system. This conclusion is in accordance to the results of Sanderson and colleagues, who found syndecan expressed on circulating B cells [27].

Since syndecan-1 was not detectable in the coronary effluent, either by ELISA or Western blot, probably due to too little total protein available for shedding in hearts of the mouse, further efforts were focused on immunofluorescence and electron microscopic imaging.

Our group has demonstrated the existence of an endothelial glycocalyx in guinea pig hearts, and in both human veins and arteries [4, 28]. Previously, a glycocalyx was evidenced in the cerebral vasculature of the mouse by Vogel et al. [21]. As depicted in Fig. 3, we now demonstrate the existence of an endothelial glycocalyx also in mouse heart coronary vessels. Although the total height of the glycocalyx appears to be smaller (between 100–150 nm) than in guinea pig hearts (200–300 nm), the capillaries seem to be coated by an EG in their total circumference.

Additionally, immunofluorescence staining with a mouse specific anti-syndecan-1 antibody was performed. In Fig. 4 syndecan-1 is visualized on the endothelium of a capillary. In both, immediately fixated (group B) hearts and in control hearts after 45 min of crystalloid perfusion (group D), a prominent signal is detectable, whereas practically no syndecan-1 could be evidenced in hearts having undergone ischemia/reperfusion.

5 Perspectives

Taking all of these results into account, the present study demonstrates several important findings. First, there is an endothelial glycocalyx also in mouse hearts. Second, the endothelial glycocalyx of mice consists also of syndecan-1. Third, the amounts of syndecan-1 present in hearts of mice devoid of blood cells are too low for shedding to be quantified in the coronary effluent, given the sensitivity of ELISA tests commercially available today.

Footnotes

Acknowledgments

The present work was supported by a DFG (Deutsche Forschungsgemeinschaft)-grant awarded to Daniel Chappell (no. CH1019/2-1). The authors thank Gaby Groeger (Lab technician, Department of Anaesthesiology) for support in ELISA measurement, Beate Aschauer, Astrid Baltruschat, Sabine Tost and Ursula Fazekas (Lab technicians, Institute of Anatomy, Chair II, Ludwig-Maximilians University), for support in providing electron microscopic and immunofluorescence pictures of the glycocalyx and many years of close collaboration with experimental research on the endothelial glycocalyx.

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