Ann Thorac Surg 2003;75:1145-1152
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Multi-photon microscopic evaluation of saphenous vein endothelium and its preservation with a new solution, GALA
Hemant S. Thatte, PhDa,
Kunda S. Biswas, MDa,
Samer F. Najjar, MDa,
Vladimir Birjiniuk, MDa,
Michael D. Crittenden, MDa,
Thomas Michel, MD, PhDb,
Shukri F. Khuri, MDa*
a Department of Surgery, Boston, MA, USA
b Cardiology Division, VA Boston Healthcare System, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
Accepted for publication October 24, 2002.
* Address reprint requests to Dr Khuri, Department of Surgery (112), VA Boston Healthcare System, 1400 V. F. W. Parkway, West Roxbury, MA 02132, USA
e-mail: shukri.khuri{at}med.va.gov
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Abstract
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BACKGROUND: Injury to endothelium can compromise the patency of bypass grafts harvested during coronary artery bypass graft (CABG) surgery. Maintaining structural and functional viability of endothelium in grafts may lead to improved long-term patency. The information gained from the application of multi-photon microscopy in transmission and epifluorescence mode was used to assess the structural and functional integrity of human saphenous vein segments stored in multiple preservation solutions, and to design a superior storage solution.
METHODS: Multi-photon microscopy was used to image deep within saphenous vein tissue harvested from patients undergoing CABG for analysis of endothelial structure and function. Endothelial cell structural viability, calcium mobilization, and nitric oxide generation were determined using specific fluorescence markers.
RESULTS: Within 60 minutes of harvest and storage in standard preservation solutions, calcium mobilization and nitric oxide generation were markedly diminished with more than 90% of endothelial cells no longer viable in the vein. In contrast, veins could be stored for 24 hours without substantial loss in cell viability in a newly formulated heparinized physiologic buffered salt solution containing glutathione, ascorbic acid, and L-arginine (GALA).
CONCLUSIONS: Standard solutions in clinical use today led to a profound decline in saphenous vein endothelial cell viability, whereas the newly designed physiologic salt solution (GALA) maintained endothelial function and structural viability for up to 24 hours. The improvements seen from using GALA as a vessel storage medium may lead to greater long-term vein graft patency following CABG surgery.
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Introduction
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The saphenous vein represents the most commonly used venous conduit for coronary artery bypass graft (CABG) surgery and other peripheral artery bypass procedures. However, the long-term patency of the saphenous vein graft is limited, with an occlusion rate of 15% to 26% in the first year [1]. Pathologic changes in the saphenous vein related to graft occlusion are well documented [2, 3]. Endothelial cell damage following vein harvest results in platelet activation and may be an important element in early graft occlusion. Therefore, the integrity of the endothelium is essential for successful short and long-term patency [2, 3]. Short-term storage of free vascular grafts is routine in CABG operations, where one to two hours may elapse between the vein harvest and reperfusion [2–4]. This interval may affect both the structure and function of the graft, depending upon the composition and temperature of the storage solution and the duration of tissue ischemia before vein reimplantation [2–8].
Until recently the intracellular events transpiring within endothelial cells in living vessels could not be directly evaluated because of the inability of conventional fluorescence microscopy to image deep into the tissues. With the recent advent of fluorescent multi-photon microscopy [9], it is now possible to overcome these limitations. Excitation of a fluorescent molecule by the simultaneous absorption of two (near) infrared photons of longer wavelength facilitates deeper penetration into tissues [10, 11]. Optical sectioning and construction of three-dimensional images has become possible without dissecting the tissue. Multi-photon excitation localized to a limited volume at the focal plane minimizes photo-damage and out-of-plane fluorescence and results in high-contrast, well-resolved images from deep within tissues [10, 11].
In this study, we utilized multi-photon imaging techniques in real time to characterize the endothelial cell viability in saphenous veins harvested from patients undergoing CABG surgery. We evaluated the comparative efficacy of a variety of storage solutions in preserving the structure and function of endothelium, including a newly formulated solution (GALA), which we hypothesized would provide optimum endothelial cell protection before CABG surgery. This newly designed solution was found to maintain the vascular structure and key endothelial cell regulatory pathways of calcium mobilization and nitric oxide generation for extended periods of time.
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Material and methods
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Saphenous veins
Saphenous vein segments (inner diameter 0.05 to 0.2 mm, outer diameter 0.2 to 1.0 mm) were obtained before distension or any other manipulation from male patients of age 67.13 ± 9.78 (mean ± SD) years undergoing CABG surgery at the VA Boston Healthcare System, according to an approved Human Studies Subcommittee protocol. The vein segments were placed in the preservation solutions described below at 21°C and processed within 30 minutes. Excess adipose and adventitia were gently excised. Five different preservation solutions were used for the ex vivo preservation of explanted saphenous veins. The segments from the saphenous veins were stored from 1 to 24 hours at 21°C in one of the following: HLS (heparin, 40 U/ml; 0.25% lidocaine; 0.9% sodium chloride), a commonly used storage solution; AHB, autologous heparinized (40 U/ml) blood, also routinely used in the operating rooms; TCM, tissue culture medium (1:1 MEM [minimum essential medium], RPMI 1640 medium), a solution that is a standard for in vitro tissue culture and used in the laboratories to provide optimum conditions for cell growth in tissue propagation; HBSS, Hank’s balanced salt solution, a representative physiologic salt solution (HBSS consists of, all in gm/L, 0.14 calcium chloride, 0.4 potassium chloride, 0.06 potassium phosphate [monobasic], 0.1 magnesium chloride [hexahydrate], 0.1 magnesium sulfate [heptahydrate], 0.8 sodium chloride, 0.35 sodium bicarbonate, 0.05 sodium phosphate [dibasic; hepatahydrate] and 1 D-Glucose); GALA, an easily prepared, newly formulated storage solution, which we hypothesized will preserve the endothelial structural and functional viability of the surgical conduits during storage for an extended period of time. Hank’s balanced salt solution was modified by adding 0.09 gm/L of reduced glutathione (1000 µmol/L final concentration) and 0.31 gm/L of L-ascorbic acid (500 µmol/L final concentration) as antioxidants and reducing agents, with 0.15 gm/L of L-arginine (500 µmol/L final concentration) as a substrate for endothelial nitric oxide synthase (eNOS). Heparin (50 U/ml) was added as an anticoagulant and the pH was adjusted to 7.4 using 8.4% sodium bicarbonate solution. This new solution is referred to as the GALA solution (glutathione, ascorbic acid, L-arginine). The saphenous vein graft segments were collected from 9 different patients. Each segment was divided into five sections and each section was separately stored in one of the five preservative solutions described above.
Cell viability assay
Structural and functional viability of the endothelial cells was assessed with a fluorescence-based Live-Dead assay (calcein AM/ethidium homodimer; Molecular Probes [Eugene, OR]) [15, 16]. Vein segments were stored in one of the storage solutions as indicated and then incubated with calcein AM and ethidium homodimer dyes (10 µmol/L, final concentration) in 1.5-ml HBSS, pH 7.4, for 30 minutes at 21°C. After incubation, segments were washed three times with the preservation solution, mounted on the multi-photon microscope stage in an imaging chamber, and imaged as described below. A viability score was assigned to each experiment as follows: a score of 4+ = 76% to 100% viability, 3+ = 51% to 75% viability, 2+ = 26% to 50% viability, and 1+ = 
25% viability based on green and red fluorescence observed.
Determination of intracellular calcium mobilization and nitric oxide generation
Calcium mobilization in the endothelial cells of the saphenous veins was measured using calcium-sensitive calcium orange dye (Molecular Probes, Inc., Eugene, OR). Nitric oxide production in the segments was determined using the nitric oxide specific indicator dye DAF-2DA (5, 12, 13 [Calbiochem, La Jolla, CA]). Vein segments were incubated for 60 minutes at 37°C with calcium orange and DAF in HBSS (10 and 15 µmol/L final concentration, respectively). Under these incubation conditions the fluorescence dyes label endothelial cells and not the smooth muscle cells. After washing with HBSS to remove excess dye, the segments were imaged as described below. Calcium mobilization and eNOS activity were stimulated by adding bradykinin in HBSS (final concentration of 10 µmol/L), to the imaging chamber. Changes in calcium orange and DAF fluorescence were recorded in real time for 10 minutes at 21°C after bradykinin stimulation. To measure the specificity of eNOS activity, vein segments were preincubated for 30 minutes at 37°C with 100 µmol/L of eNOS inhibitor Nw-nitro-L-arginine (L-NNA) in 1.5 ml of HBSS, before incubation with DAF [5, 12, 13]. Resting calcium levels and basal activity of eNOS were measured in the absence of bradykinin stimulation.
Multi-photon imaging
Imaging and semiquantitative fluorescence measurements were done with a BioRad MRC 1024ES multiphoton imaging system (BioRad, Hercules, CA) coupled with a mode-locked titanium:sapphire laser (Spectra-Physics, Mountain View, CA) operating at 82 MHz repetition frequency, 80-fs pulse duration with a wavelength tuned to 820 nm, in transmission and epifluorescence mode. A Zeiss Axiovert S100 inverted microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a high quality water immersion 40x/1.2 NA, C-apochroma objective was used to image the segments and quantitate fluorescence. The 512 x 512 pixel images were collected in direct detection configuration at a pixel resolution of 0.484 µm with a Kalman 5 collection filter. The lumen and endothelial cell layer were identified by XYZ scanning and imaged, generally at depths of 50 to 200 µm in longitudinal vein segments depending on the size of the vein [5, 14], and at a depth of 100 µm from the site of excision in 10-mm cross sections of the veins.
Quantitative analysis of calcium and nitric oxide
Calcium mobilization and nitric oxide generation [5, 12, 13] were measured by recording changes in calcium orange and DAF fluorescence before and after bradykinin treatment in real time. Typically three to six specific regions were drawn along the endothelial region of the lumen identified by XYZ scanning in a field of view at 400x magnification using image processing software (MetaMorph Imaging Series [Universal Imaging Corp., West Chester, PA]). The change in fluorescence intensity integrated over all pixels within each boundary was monitored over time and quantitated separately using MetaMorph in red (calcium) and green (nitric oxide) fluorescence channels, respectively. Because of the variable size and shape of the boundaries and vein sizes, and in order to eliminate effects due to variation in fluorescence dye loading, the fluorescence intensity from each image was normalized by values determined from a reference image recorded before bradykinin treatment [5, 12, 14–16]. The data represents an average of blinded experiments performed on different days and expressed as mean ± standard error of the mean (SEM; n = 9).
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Results
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Effects of preservation solutions on endothelial cell viability in explanted saphenous vein
Vein segments stored in heparinized lidocaine saline (HLS), autologous heparinized blood (AHB) or tissue culture medium (TCM) exhibited a red fluorescence pattern in the lumenal region within 1 hour of storage indicative of extensive cell membrane damage and compromised viability of endothelial cells (Fig 1, Table 1).
In contrast, the endothelial cells were structurally intact
(green fluorescence) after 1 hour of storage in Hank’s balanced salt solution (HBSS; Fig 1), but lost cellular integrity during extended storage (Table 1). Similarly, smooth muscle cell architecture of the vessels also degenerated completely during prolonged storage in HBSS (Fig 1) and other storage solutions (not shown).
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Fig 1. This figure illustrates changes in structural integrity of vein segments stored in different preservation solutions. Vein segments were stored in heparinized lidocaine saline (HLS), autologous heparinized blood (AHB), tissue culture medium (TCM) for 60 minutes; and in Hank’s balanced salt solution (HBSS) and the newly designed glutathione, ascorbic acid, and L-arginine (GALA) preservation solution for a total of 24 hours. Green fluorescence indicates cell viability; red fluorescence indicates compromised cells. Extensive cell membrane damage and compromised endothelial cell integrity in the vessels was observed after short-term storage in HLS, AHB, and TCM. Cell viability was well preserved in veins during short-term storage in HBSS but resulted in cell death upon extended storage. In contrast, endothelial cells remained viable in vessels preserved in GALA solution throughout the extended time of storage. Representative image, at x400 magnification, n = 9. (EC = endothelial cells.)
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The protective effect of GALA solution on endothelial structural integrity was compared to other storage solutions. Vein segments were stored in GALA for 5 hours at 21°C, then for 19 hours at 4°C for a total storage time of 24 hours. In contrast to other solutions, segments stored in GALA solution exhibited a robust green fluorescence indicative of structural viability of the endothelium (Fig 1). Dead cells (red fluorescence) were rarely observed during 24 hours of preservation. Similarly, smooth muscle cell architecture and viability of the vessels were well maintained during extended storage in GALA (Fig 1, Table 1). Transmitted light images of cross sections of the saphenous vein stored for 60 minutes in HLS, AHB and GALA are illustrated in Figure 2.
The sections were imaged at 100 µm from the site of excision. The endothelial monolayer in juxtaposition to the lumen showed a substantial amount of disruption in vein segments stored in HLS and AHB. Similarly, thinning and disruption of smooth muscle layers was also observed in these vein segments. In contrast, well-preserved endothelium as well as smooth muscle layers remained morphologically intact in vein segments stored in GALA, Figure 2.
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Fig 2. Transverse sections of the saphenous veins imaged using multiphoton microscopy in transmission mode. The segments were imaged using XYZ scanning  100-µm deep from the site of excision. The vessel lumen, endothelial cell layer, and smooth muscle layer are clearly visible. Endothelial and smooth muscle cell damage was observed in segments stored for 60 minutes in heparinized lidocaine saline (HLS) and autologous heparinized blood (AHB). Structural integrity of the vessel was well preserved in vessels stored in glutathione, ascorbic acid, and L-arginine (GALA). Representative image at x400 magnification, n = 9.
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Effect of preservation solutions on endothelial calcium mobilization and nitric oxide generation in stored saphenous veins
The functionality of the vein segment endothelium was determined by the increase in internal calcium concentration and subsequent production of nitric oxide, a key endothelium-derived mediator, as a marker for endothelial cell function [17–19]. A time-dependent two- to threefold increase in calcium and nitric oxide fluorescence was observed upon bradykinin stimulation (Fig 3).
As expected, the calcium signal (orange/red fluorescence) and the nitric oxide signal (green fluorescence) were found to colocalize (orange-yellow fluorescence) in the endothelium. The effect of the various preservation solutions on saphenous vein endothelial function is illustrated in Table 2
and Figure 4. The bradykinin-dependent calcium mobilization was impaired in vein segments stored for 60 minutes in HLS, AHB and TCM (Table 2). Correspondingly, activation of eNOS was also severely attenuated
during 60 minutes of storage in HLS, AHB and TCM (Fig 4). Initially, a robust bradykinin-mediated calcium mobilization (Table 2), activation of eNOS’ and generation of nitric oxide (Fig 4) was observed in segments stored in HBSS solution. As expected, production of nitric oxide was completely inhibited by treating the segments with the eNOS inhibitor, L-NNA (1.0 ± 0.012, mean ± SEM of normalized nitric oxide fluorescence, arbitrary units, n = 6) indicating specificity. However, a time-dependent decay in calcium mobilization (Table 2) and nitric oxide generation was observed in vessels stored in HBSS as shown in Figure 4, and as reported previously [20]. In contrast to all the storage solutions tested, endothelial cells remained robustly functional in veins stored in the GALA preservation solution for the total duration of the study (Table 2, Fig 4). The bradykinin-mediated mobilization of calcium (Table 2) and activation of eNOS lead to sustained synthesis and release of nitric oxide by the endothelial cells of the vessels during prolonged storage in GALA (Fig 4). These results clearly demonstrate that the GALA solution can preserve both the structure and function of endothelium in ex vivo stored veins. To demonstrate that the structural integrity of the vein was maintained during 24 hours of storage in GALA, the vein was labeled with live/dead assay reagents and imaged using multiphoton microscopy. The vessel was imaged at increasing depths along the Z axis throughout the intact longitudinal vein segment. As illustrated in Figure 5, living cells (green fluorescence) were clearly observed throughout all the regions of the vein, including media and intima, demonstrating structural viability of the GALA preserved vessel.
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Fig 3. Bradykinin mediated calcium mobilization and nitric oxide generation in saphenous veins stored in glutathione, ascorbic acid, and L-arginine. Segments (similar to those illustrated in Fig 2) were labeled with calcium orange and diaminofluorescein, stimulated with bradykinin and imaged using multiphoton microscopy. A robust two- to threefold increase in calcium (red) and nitric oxide (green) fluorescence signal in the endothelial region was observed 10 minutes after bradykinin stimulation, that found to colocalize (orange-yellow fluorescence). Representative image of nine independent experiments.
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Fig 4. Vein segments were stored in preservative solutions for various time points, then incubated with diaminofluorescein (DAF) to measure nitric oxide generation. The integrated DAF fluorescence intensity in the endothelial region of each vein was quantitated using multi-photon microscopy after a 10-minute treatment with bradykinin (10 µmol/L) and was then normalized to the fluorescence intensity measured before the drug treatment (dashed line = 1). Each bar represents the mean ± SEM of independent experiments (n = 9). Nitric oxide generation was severely impaired in veins stored in heparinized lidocaine saline (shaded bars), autologous heparinized blood (black-spotted bars), and tissue culture medium (white-spotted bars). Nitric oxide production was maintained in vessels during short-term storage in Hank’s balanced salt solution (black bars); however, a temporal decrease in endothelial nitric oxide synthase (eNOS) activity was observed. In contrast, eNOS activation and nitric oxide generation was well preserved in vessels stored in glutathione, ascorbic acid, and L-arginine (GALA) solution (white bars). A sustained increase in nitric oxide production was observed during extended storage of the vessels in GALA.
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Comment
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The preservation of saphenous vein endothelial cells during the course of CABG surgery is essential for the long-term patency of the grafts. Endothelial cells are important mediators in regulating platelet function and in determining the coagulant and fibrinolytic pathways in the vessel wall. Endothelial injury can lead to acute alterations in these essential pathways, leading to thrombosis and stenosis [1–5]. Histochemical analyses have suggested that structural derangements in the harvested veins can be detected within two hours of storage [2–7]. In this study, we used multi-photon microscopy to demonstrate that endothelial cell viability is compromised within minutes of storage in standard preservation solutions. Current storage solutions, which vary from physiologic salt solutions to heparinized blood, may not represent a medium sufficient for endothelial or smooth muscle cell support [5–8]. For example, standard saline solutions lack an energy source, such as glucose, and have a low pH that may be injurious to the fragile endothelial cells [5, 8]. Moreover, the currently available solutions are deficient in free radical scavengers, antioxidants, and nitric oxide synthase substrate that might help sustain endothelial cell function during the storage period [5].
Endothelial injury may occur at the time of surgery as a result of graft preservation, graft insertion and turbulent blood flow, or with local stasis [5]. Defining the time and nature of this injury is of prime importance. Our ability to evaluate the structural viability of an intact vein segment and simultaneously measure calcium mobilization and nitric oxide production in real time in the same segment using multiphoton microscopy offers a new and powerful approach into the examination of endothelial cell structure and function of stored grafts [5, 14]. We were able to image to a depth of 200 µm into preserved intact saphenous vein segments because of the robust fluorescence signals of the calcein-ethidium homodimer dyes. Imaging was limited to a depth of 50 to 70 µm using nitric oxide dye due to degradation of the weak fluorescence signal by the thick tissue [11, 14]. Thus, signal decay was a limiting factor in using the intact vessel segments for functional studies. To compensate for this limitation, and to consistently use the same vessel for both structural and functional studies, calcium and nitric oxide assays were performed on cross sections of the intact vein segments that were used in structural evaluation.
We have demonstrated that the structural viability, the bradykinin-mediated increase in internal calcium concentration, and the activation of eNOS leading to nitric oxide generation in the endothelium, are lost in saphenous veins within minutes of storage in HLS, AHB, and TCM (Figs 1–4, Tables 1 and 2). Similarly, HBSS provides only short-term protection to the vein endothelium against structural and functional decay and does not protect against disruption-of-flow-induced apoptosis [21] in explanted vessels (manuscript in preparation). These results are in agreement with other published studies that have demonstrated that storage of vessels in HLS and AHB and other storage solutions leads to damaged endothelium and depressed vasomotor function, perhaps due to free radical damage, cessation of flow induced apoptosis, low pH, storage media composition, and a hostile environment [2–8, 17, 21, 22].
The importance of endothelial calcium and nitric oxide in regulating vasomotor function has been documented [2–8, 17–19, 22]. Nitric oxide is known to inhibit smooth muscle cell proliferation, as well as platelet adhesion and activation [18, 19]. Therefore, a loss of endothelium-derived nitric oxide may contribute both to acute thrombosis and intimal proliferation in the graft [2–6]. Endothelial damage also reduces the production of prostacyclin, a powerful inhibitor of platelet aggregation, the loss of which may further promote platelet activation. Platelet activation may precipitate early thrombosis and lead to neointimal proliferation during the first few months following bypass surgery [2–6]. The loss in eNOS activity and in endothelial cells’ ability to generate nitric oxide which we observed in veins stored in standard solutions may adversely affect the vaso-reactivity and long-term patency of the grafts.
We hypothesized that the rapid loss of endothelial cell structural and functional integrity in saphenous vein stored in standard storage solutions can be avoided by developing a physiologic salt solution, GALA, that combines free radical scavengers and antioxidants (glutathione, ascorbic acid,) and nitric oxide synthase substrate (L-arginine). These components provide a favorable environment and cellular support during ex vivo storage. The protection of structure and function of the endothelium, which we observed in vessels stored over a prolonged period in GALA, confirms this hypothesis (Figs 1–5, Tables 1 and 2).
The three key ingredients added to the GALA were chosen because of their putative effect on endothelial cell function. The role of glutathione as a cellular reducing agent has been extensively investigated. Glutathione has been found to increase L-arginine transport in endothelial cells and may lead to the formation of biologically active S-nitrosoglutathione and to the stimulation of eNOS activity, nitric oxide generation, and coronary vasodilatation [20, 23]. Ascorbic acid is an antioxidant known to scavenge reactive oxygen species and thus demonstrate a sparing action on cellular glutathione and alpha tocopherol in the plasma membranes [20, 23]. Ascorbic acid also increases eNOS activity by preserving endothelium-derived nitric oxide bioactivity by possibly scavenging superoxide anions and preventing oxidative destruction of tetrahydrobiopterin, an eNOS cofactor [20, 23]. The presence of ascorbic acid in GALA may prevent the oxidation of this cofactor during vessel storage and help maintain eNOS function and nitric oxide generation in vascular endothelium [23]. Nitric oxide also reacts with thiols and can be trapped inside cells in the form of S-nitrosoglutathione and other nitrosothiols. These nitrosylated compounds can facilitate long-term availability of nitric oxide in the cell [20, 23, 24]. Therefore, ascorbic acid, by its reducing property, may assist sustained long-term release of nitric oxide from these compounds in vessels preserved in GALA, and thus help maintain the patency and tone of the vessels during storage. Additionally, ascorbic acid mediated reversal of endothelial dysfunction, reduced platelet activation and leukocyte adhesion, inhibition of smooth muscle cell proliferation and lipid peroxidation, and increased prostacyclin production have been demonstrated in numerous cardiovascular pathologies [2, 25]. L-arginine is a known substrate of nitric oxide synthase [19, 23] and has been shown to decrease neutrophil-endothelial cell interactions in inflamed vessels [23]. Therefore, the use of glutathione, ascorbic acid, and L-arginine may act synergistically to enhance the cell preservation properties of the GALA solution.
Preservation of endothelial viability in GALA-stored grafts may help counterbalance detrimental effects of vein graft arterialization [2, 5], the ill effects of which may be exacerbated when grafts with already damaged endothelium are used for implantation. We have completed preliminary studies in which GALA preserved grafts were exposed to arterial pressures in vitro, and the endothelial structure and function remained intact for extended periods of time. GALA also prevented disruption-of-flow induced apoptosis in these vessels (manuscript in preparation). It is well established that the improved patency of internal mammary artery grafts may be due to the fact that they are not placed in ex vivo storage and as such maintain their endothelial and smooth muscle cell functions [2–6]. The findings from this study prompt us to speculate that storage in GALA during conduit harvesting may render a long-term protective effect on the saphenous vein and may improve its long-term patency to a degree approaching to that of the internal mammary artery graft.
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Acknowledgments
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We gratefully acknowledge the expert technical assistance of Jin-Hwa Rhee, Thomas McGarry and Sofija Zagarins. We would like to thank Nancy Healey for editorial assistance and Aditi Thatte for her encouragement. This work was supported by National Institutes of Health grants (TM), a Veteran’s Affairs Merit Review Grant (SFK), and the Richard Warren Surgical Research and Educational Fund (HST, SFK).
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Abstract
Introduction
