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Ann Thorac Surg 1997;64:1770-1775
© 1997 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Hypertonicity Induces Injury to Cultured Human Endothelium: Attenuation by Glutamine

Alessandro Parolari, MD, Roberto Sala, MD, PhD, Carlo Antona, MD, Ovidio Bussolati, MD, PhD, Francesco Alamanni, MD, Paola Mezzadri, BSc, Valeria Dall'Asta, BSc, Gian C. Gazzola, MD, Paolo Biglioli, MD

Department of Cardiac Surgery, Centro Cardiologico, Fondazione I. Monzino, IRCCS, University of Milan, Milan, and Institute of General Pathology, University of Parma, Parma, Italy

Accepted for publication June 21, 1997.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Although most preservation solutions as well as some cardioplegic solutions used for organ storage and transplantation are hypertonic, the effects of extracellular hypertonicity on endothelium are not well established. Aims of this study were to evaluate the response of cultured human saphenous vein endothelial cells to extracellular hypertonicity and to investigate the role of the amino acid glutamine in preventing endothelial damage in vitro.

Methods. Eight distinct strains of human saphenous vein endothelial cells were studied. Hypertonic (350 and 400 mosm/kg) media were obtained by supplementing culture medium with sucrose. Cell viability was assessed in the absence or the presence of glutamine through the determination of cell number and protein content of the cultures. Confocal microscopy of cells loaded with the fluorescent dye calcein was also performed.

Results. Exposure of human saphenous vein endothelial cells to hypertonic media without glutamine caused significant cell loss within 30 minutes. Cell loss progressed steadily during incubation and after 6 hours reached 50% at 350 mosm/kg and 65% at 400 mosm/kg. In the presence of 2 mmol/L glutamine, endothelial damage was completely prevented at 350 mosm/kg and significantly lessened at 400 mosm/kg compared with glutamine-free media. Confocal microscopy showed that most hypertonicity-treated cells exhibited the typical features of an apoptotic death and confirmed the osmoprotective effect of glutamine.

Conclusions. These results indicate that the supplementation of hypertonic storage solutions with glutamine might exert a partial osmoprotective effect and suggest that the relationship between endothelial damage and tonicity of storage and cardioplegic solutions should be carefully investigated.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Endothelial dysfunction has been proposed as a mechanism for the impaired vasomotor function observed after successful cardiac operation and heart transplant procedures [1]. Much experimental evidence suggests that endothelial damage is related to some feature of the preservation and cardioplegic protocols currently used in clinical practice [1–5]. In this context, it is surprising that the effects of extracellular hypertonicity on the endothelium are not well established, although many of the cardioplegic solutions and organ storage solutions are hypertonic [6–11]. Moreover, damage from hypertonicity is commonly observed in a variety of cell models and is vigorously counteracted by specific mechanisms in mammalian cells [12].

The present study investigates the cytoprotective effects of L-glutamine during hypertonicity-induced stress of cultured human greater saphenous vein endothelial cells (HSVECs). This amino acid is defined as being "osmoprotective," as other mesenchymal models need glutamine to properly counteract the effects of such stress [13]. To assess the effect of glutamine, endothelial damage referable specifically to incubation under hypertonic conditions was carefully documented in terms of cell number and protein content of HSVEC cultures and directly visualized with confocal microscopy.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Cell Culture
The endothelial cell strains were obtained from venous remnants from 8 men (age range, 50 to 65 years) who underwent elective coronary artery bypass grafting. The cultures were established according to the method of Jaffe and colleagues [14], with minor modifications. Briefly, after isolation with 0.48% dispase solution (0.5 U/mg) (Dispase II; Boehringer-Mannheim Italia, Milan, Italy), HSVECs were incubated in complete growth medium consisting of M199 supplemented with 20% fetal bovine serum, heparin (90 U/mL), endothelial cell growth supplement (50 µg/mL), and glutamine (2 mmol/L). Cultures of the HSVECs were routinely grown in the same medium on plastic ware coated with 2 µg/cm2 of collagen (Collagen S; Boehringer Mannheim Italia) under the following conditions: pH 7.4; atmosphere, 5% carbon dioxide in air; and temperature, 37°C. Culture medium was refreshed every 2 days. Cultures were homogeneously positive for endothelial markers (Fig 1Go) and were used between the third and sixth passage in vitro.



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  		Fig 1. . Characterization of human saphenous vein endothelial cells. The cells were seeded on collagen-coated coverslips, grown for 5 days, and treated as follows: (A) fixation in 3.5% formaldehyde in phosphate-buffered saline solution (PBS) for 10 minutes and immersion in methanol for 5 minutes at -20°C; primary antibody, polyclonal rabbit anti-human von Willebrand factor (DAKO, Milan, Italy), and secondary antibody, fluorescein-conjugated mouse anti-rabbit immunoglobulin G (DAKO); (B) fixation in 3.5% formaldehyde in PBS for 10 minutes; primary antibody, monoclonal mouse anti-human endothelial cell (CD31; DAKO), and secondary antibody, fluorescein-conjugated sheep anti-mouse IgG (DAKO); and (C) incubation for 4 hours at 37°C in complete growth medium supplemented with fluorescent acetylated low-density lipoprotein (Biogenesis, Ltd, England), washing in PBS, and fixation in 3% formaldehyde for 20 minutes. (A and C, x400 before 42% reduction; B, x800 before 42% reduction.)

 
Experimental Studies
SERIES I EXPERIMENTS.
The first series of experiments (pilot study) was designed to investigate specifically the effects of hypertonicity on the viability of the endothelial cells. To this purpose, cells were incubated in glutamine-free M199 (M3769; Sigma, Milan, Italy) supplemented with 20% fetal bovine serum, heparin sodium (90 U/mL), and endothelial cell growth supplement (50 µg/mL). The osmolality of this medium, called Experimental Isotonic M199 (EI-M199), was found to be 280 mosm/kg. Hypertonic media (EH-M199) were obtained by supplementing EI-M199 with sucrose, a metabolically inert, membrane-impermeant sugar widely used in studies on the effects of stress from hypertonicity [15], to obtain osmolalities of 350 and 400 mosm/kg. Before medium supplementation, serum was treated with 0.1 U/mL of asparaginase (Boehringer-Mannheim) to hydrolyze serum glutamine.

SERIES II EXPERIMENTS.
The second series of experiments was undertaken to see whether glutamine modifies the effects of incubation under hypertonic conditions on endothelial cell viability. To this purpose, cells were incubated for specified times in EI-M199 or EH-M199 or in the same media supplemented with glutamine at a concentration of 2 mmol/L, the concentration normally used for the growth of endothelial cells in vitro.

Cell loss was assessed either by determining cell number using a ZM cell counter after trypsinization of the cultures or by determining protein content of the cultures using a modified Lowry procedure [16]. To this purpose, cell monolayers were washed three times in ice-cold MgCl2 AND DISSOLVED IN SODIUM DEOXYCHOLATE (0.5% IN 1 N NAOH). FOR THESE EXPERIMENTS, HSVECS WERE SEEDED INTO 2-CM2 wells of disposable 24-well trays (nunc as, roskilde, denmark) and grown for 3 to 5 days in complete growth medium, which was renewed 24 hours before the experiment.

Confocal Microscopy
The viability of the endothelial cultures was also assessed with confocal laser scanning microscopy of cells preloaded with calcein acetoxy-methylester (Molecular Probes Inc, Eugene, OR). This dye is retained in the intracellular compartment as long as the plasma membrane is intact; however, the dye leaks rapidly from cells with compromised membranes, even in the presence of residual intracellular esterase activity [17].

Confocal studies were carried out on a Multiprobe 2001 system (Molecular Dynamics, Inc, Sunnyvale, CA) equipped with an argon laser and based on a Nikon inverted microscope (Diaphoto TMD; Nikon Instruments SPA, Florence, Italy). The HSVECs, seeded on a coverslip at a density of 5 x 104 cells/cm2 3 days before the experiment, were placed in a thermostated flow chamber [18], which allows repeated medium substitutions and prolonged periods of observation. At the beginning of the experiment cells were incubated in normal complete growth medium supplemented with 2 µmol/L calcein-acetoxy-methylester. After stabilization of the signal, usually reached within 30 minutes, cells underwent experimental manipulations, with laser intensity, photomultiplier setting, and gain kept constant throughout the experiment. At the designated times, cultures were observed through a 40x objective with a pinhole set at 100 µm (excitation wavelength of 488 nm employing a primary beamsplitter at 510 nm and a final barrier filter 510 long-pass). The same field was followed throughout the experiment. Image processing was performed on a Silicon Graphics Personal Iris workstation (Image Space software). Intensities were expressed on a scale from black (zero) to white (maximal intensity, 255); images were rendered in a 256 RGB pseudocolors scale from black (zero), through different hues of blue, yellow, and red, to white (255).

Statistical Analysis
Data are presented as the mean ± one standard deviation of the mean. One- and two-factorial analysis of variance tests, followed by the Duncan test when indicated, were used for statistical evaluation. In select cases, the percentage changes from baseline were calculated. Linear regression analysis was used when needed. A p value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Series I Experiments
The incubation of HSVECs in EH-M199 caused a significant progressive loss of cells from the population (Fig 2AGo). The effect was already detectable after 1 hour at both 350 and 400 mosm/kg (p < 0.05) and was highly significant (p < 0.01) at the last measurements (3 hours and 6 hours), when cell loss was significantly more marked at the higher osmolality. After a 6-hour incubation at 400 mosm/kg, more than 70% of the cells were lost. Comparable results were obtained for protein content of the cultures (Fig 2BGo). Note that for both types of measurements, no significant differences were detected among results obtained with control EI-M199 at all the times tested.



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  		Fig 2. . Effect of incubation under hypertonic conditions on cultures of human saphenous vein endothelial cells (HSVECs). Two sets of cells were treated in parallel for each strain of HSVECs. Cells were seeded into 24-well trays at a density of 25 x 103 cells/cm2 and grown for 5 days. At time 0, Experimental Isotonic M199 (280 mosm/kg, [squares]) or Experimental Hypertonic M199 (350 mosm/kg, [triangles pointing up], 400 mosm/kg, [triangles pointing down]) was substituted for the medium. (see Material and Methods section for description of medium composition). At the indicated times, (A) cell number and (B) cell protein content of the cultures were determined. Points are means of eight triplicate values each obtained for a distinct strain of HSVECs with standard deviation shown when greater than value of the point.

 
The analysis of the relationship between cell number and protein content of the cultures using linear regression analysis showed a highly significant direct relationship between these two variables (R2 = 0.932; p < 0.001) (data not shown). For this reason, the results of the series II experiments are expressed only in terms of cell protein content of the cultures.

Series II Experiments
In EI-M199, no significant differences in protein content were detected between HSVECs incubated in glutamine-supplemented versus unsupplemented media until 6 hours of incubation (Fig 3AGo). Cell protein content remained fairly stable during the entire incubation period under either condition.



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  		Fig 3. . Effect of glutamine on cultures of human saphenous vein endothelial cells (HSVECs) incubated under hypertonic conditions. Cells were seeded into 24-well trays at a density of 25 x 103 cells/cm2 and grown for 5 days. At time 0 (A) Experimental Isotonic M199 (280 mosm/kg) or Experimental Hypertonic M199 (at (B) 350 mosm/kg; or (C) 400 mosm/kg) in the presence (black circles) or the absence (white circles) of 2 mmol/L glutamine was substituted for the medium. At the indicated times, cell protein content of the cultures was determined (see Material and Methods section). Points are means of eight triplicate values each obtained for a distinct strain of HSVECs with standard deviation shown when greater than value of the point.

 
On the other hand, glutamine supplementation had dramatic effects on cell protein content of HSVEC cultures incubated in EH-M199. Protein content decreased significantly (p < 0.05 at all experimental times tested versus time 0) in unsupplemented media at both 350 and 400 mosm/kg (Fig 3B, 3CGoGo). In the presence of glutamine, however, the fall in cell protein content was completely prevented at 350 mosm/kg for an 1 hour with only a minor decrease of 7% after 6 hours of incubation; the corresponding decrease in unsupplemented 350 mosm/kg EH-M199 was 50% (p < 0.01 glutamine-supplemented versus glutamine-free media) (see Fig 3BGo). In HSVEC cultures exposed to 400 mosm/kg, a significant progressive fall in cell protein context was detected after only 30 minutes of incubation even in the presence of glutamine (see Fig 3CGo). However, the presence of glutamine significantly attenuated the loss of cell proteins (p < 0.01 glutamine-supplemented versus glutamine-free media at all the experimental times tested). Finally, a more marked (p < 0.05) decrement in cell protein content was demonstrated at 400 mosm/kg than at 350 mosm/kg at all times tested.

Confocal Microscopy
Figure 4Go shows confocal images of calcein-loaded HSVECs during incubation at 400 mosm/kg in the presence or the absence of 2 mmol/L glutamine. Control cells (see Fig 4A, 4DGoGo) were clearly labeled by the dye and exhibited a characteristic endothelial morphology; fluorescence was homogeneously distributed throughout the cytoplasm, with a zone of high signal roughly corresponding to the nucleus. After 1 hour of incubation in hypertonic medium, cell-associated fluorescence was higher both in the presence (see Fig 4BGo) and the absence of glutamine (see Fig 4EGo). However, whereas cell morphology was spared in glutamine-supplemented EH-M199 (see Fig 4BGo), gross alterations in cell shape and dimension were evident in glutamine-free EH-M199 (see Fig 4EGo), where cells appeared shrunken and surrounded by bulging blebs. After 3 hours of hypertonic treatment, cells incubated in the presence of glutamine (see Fig 4CGo) were fully comparable with control conditions. On the other hand, the aspect of hypertonicity-treated cultures incubated without glutamine (see Fig 4FGo) was severely modified; a significant number of cells were no longer detectable; the others had lost endothelial morphology and appeared as small, rounded elements with a low level of intracellular fluorescence.



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  		Fig 4. . Effect of incubation under hypertonic conditions on human saphenous vein endothelial cells loaded with calcein acetoxy-methylester, in presence or absence of glutamine (2 mmol/L) on. Single confocal planes of two representative fields are shown. (A, D) Control cells at time 0 incubated in Experimental Isotonic M199 in the presence of glutamine (E). Cells incubated for 1 hour in Experimental Hypertonic (EH) M199 at 400 mosm/kg in (B) presence or (E) absence of glutamine. Cells incubated for 3 hours in EH-M199 at 400 mosm/kg in (C) presence or (F) absence of glutamine. (All, x400 before 33% reduction.)

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Many authors have proposed the use of hypertonic cardioplegic and storage solutions to counteract the cell swelling and to reduce the myocardial edema that can follow cardiac and transplant operations. However, because stress from hypertonicity delays replication of human umbilical vein endothelial cells [19] and triggers apoptosis in vascular smooth muscle cells [20], we hypothesized that such stress can contribute to the endothelial dysfunction observed after exposure to preservation or storage solutions [1–5]. We chose cultures of HSVECs as an endothelial model to test this hypothesis because they are closer to human "adult" endothelium than more common endothelial models such as human umbilical vein cells. It is well known that there is some functional heterogeneity of endothelium derived from different vessels [21], and HSVECs have previously been used to evaluate the cell toxicity of some storage solutions [15, 22].

The effects of hypertonic cardioplegic solutions on endothelial cells have been reported by Carpentier and colleagues [23]. However, that study was not undertaken to establish the specific effect of hypertonicity, and consequently it is difficult to attribute the differences in cell death measured after incubation in solutions of markedly different composition exclusively to changes in extracellular osmolality. The experimental procedures selected here, on the other hand, were meant to discriminate the effect of tonicity on the endothelium and to eliminate, to the extent possible, the contribution of any other factor. For this reason, the cells were incubated at normothermia to avoid the deleterious effects of hypothermia on cultured endothelium [15]; experiments were performed in complete growth medium to conform as much as possible to physiologic conditions in vitro. However, because preservation and cardioplegic solutions are glutamine free [2–11] and because this amino acid can substantially alter the cell response to hypertonicity-induced stress [13], we decided to carry out our study with both glutamine-free and glutamine-supplemented media. Finally, incubation times ranged up to 6 hours, which is the usual range for cardioplegic protection or ideal graft storage.

Our study yielded two main conclusions. First, even a moderate increase in extracellular osmolality exerts significant toxic effects on HSVECs in vitro. This conclusion is supported by the results of confocal microscopy of calcein-loaded cells, which indicate that the accelerated cell death induced by stress from hypertonicity is associated with typical morphologic hallmarks of an apoptotic process [24]. In HSVECs, fully developed caused by hypertonicity damage requires several hours and involves most cells of the population. Even much smaller losses of endothelial cells in vivo could cause thrombotic phenomena and coronary artery spasm, which occurs in approximately 1% to 3% of patients undergoing coronary operations [1]; moreover, any endothelial damage may underlie increased graft vascular resistance after the use of storage solutions, a relatively common finding in both experimental and clinical studies [1]. We emphasize, however, that to assess the relevance of these findings in vivo, the study needs to be extended to models of native endothelium and to take into account important variables such as the temperature maintained during incubation and the composition of the extracellular medium [1–5, 15, 22].

The second conclusion is that medium supplementation with glutamine is able to reduce significantly the cell damage caused by incubation under hypertonic conditions. The cytoprotective effect of glutamine described here is consistent with previous findings pointing to an osmoregulatory role for this amino acid in human adult fibroblasts [13] and contributes additional evidence of the beneficial effects of glutamine supplementation recently described in an ischemia-reperfusion setting [25]. In this regard, the possibility that glutamine supplementation of preservation solutions and cardioplegic solutions exerts favorable effects on endothelium is of particular interest and deserves further investigation.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by a grant from Centro Cardiologico, Fondazione Monzino IRCCS, and by a grant from Fondazione Cassa di Risparmio di Parma.

The confocal microscope is at the Centro Interfacoltà Misure of the University of Parma.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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