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Am J Physiol Heart Circ Physiol 293: H1721-H1728, 2007. First published June 8, 2007; doi:10.1152/ajpheart.01321.2006
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Validity of a patient-derived system of tissue-specific human endothelial cells: interleukin-6 as a surrogate marker in the coronary system

Karla Lehle,1,* Leoni A. Kunz-Schughart,2,3,* Peter Kuhn,1 Stephan Schreml,1 Dietrich E. Birnbaum,1 and Jürgen G. Preuner1

1Department of Cardiothoracic Surgery, and 2Institute of Pathology, University Hospital, Regensburg; and 3Medical Faculty Carl Gustav Carus, OncoRay Center for Radiation Research in Oncology, Dresden, Germany

Submitted 5 December 2006 ; accepted in final form 4 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
The aim of our study was to evaluate the relevance of tissue- and species-specific endothelial cells (EC) to study EC-dependent mechanisms in inflammatory-mediated tissue injury. We established an isolation protocol for highly purified EC (pEC) preparations of different origin and compared EC-specific inflammatory responses. Fluorescence-activated cell separation was used to obtain pEC cultures from different human arterial (coronary artery, internal thoracic artery) and venous (umbilical vein, saphenous vein) vessels. All pEC were analyzed for growth kinetics, morphology, release of cytokines/chemokines, and expression of E-selectin. For all different EC cultures, purities of ≥99% were reproducibly achieved. The EC isolation did not affect EC growth, morphology, and function. However, characterization of pEC from different vessel materials revealed an intrinsic, tissue-specific functional heterogeneity of EC cultures. Despite an arterial and venous difference in the secretion of IL-8 and monocyte chemoattractant protein-1, especially EC from coronary arteries produced significantly more IL-6 compared with other EC types, independent of age, gender, and disease of the cell donors. In contrast, the expression of E-selectin was not affected. We conclude that the proposed isolation protocol allows the generation of a pEC bank, enabling us to study tissue-specific aspects at the level of the endothelium.

purification; cell growth; fluorescence-activated cell separation; function

VASCULAR ENDOTHELIAL CELLS (EC) play a strategic role in the development of inflammatory-mediated tissue injury. Accordingly, endothelial activation and injury are associated with various pathological processes, including atherosclerosis, diabetes mellitus, and rejection in solid organ transplantation (4, 8, 22). Many vascular diseases are restricted to specific vessels (18, 19), and, consequently, EC may differ not only with respect to small vs. large vessels, but also between different tissues.

To date, in vitro research on the function of human EC has extensively been done by culturing human umbilical vein EC (HUVEC), a type of vessel that is rarely affected by the most common human vascular disorders (2). HUVEC differ from other EC in several functions that affect leukocyte migration (25) and thus may not be representative for adult endothelium. Taking into account that adult EC have been subject to prolonged exposure to cytokines, hormones, and other stimuli, there is an urgent need to develop a primary cell culture system for adult large-vessel endothelium. Cultures of EC from human saphenous veins (HSVEC), coronary arteries (HCAEC), or internal thoracic artery (HATEC) have previously been studied by several groups, but their usefulness has been limited by the problem of maintaining pure cultures for a reasonable period of time (26).

To gain a better understanding of the variety of EC properties, we examined highly pure cultures of macrovascular primary human EC from veins (HUVEC, HSVEC) and arteries (HCAEC, HATEC) and measured their anti-inflammatory responses under in vitro conditions. Our data demonstrate the functional heterogeneity of tissue- and nonvessel-type-specific EC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Tissue samples. EC from saphenous veins (HSVEC) and internal thoracic arteries (HATEC) were obtained from patients undergoing coronary artery bypass grafting. HCAEC were isolated from explanted hearts during orthotopic heart transplantations and HUVEC from umbilical cords. Informed consent was obtained from each patient, and the protocol for isolation of ECs was approved by the Institutional Review Boards of the Technical University of Munich and the University Hospital Regensburg, as well as by the local human ethics committee (no. 99/133). Patient characteristics of cell donors (except for HUVEC) are shown in Table 1.


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  		Table 1. Demographic data and operative details

 
EC preparation and primary culturing. For evaluation of optimal isolation, culture conditions, and functional analysis, EC were prepared according to Jaffe et al. (14). Briefly, after tissue/vessels were thoroughly rinsed to remove blood components, EC were detached by incubation with 0.05% collagenase A (Roche, Basel, Switzerland), washed several times with HEPES, and resuspended/cultivated in an endothelial medium (EGM-kit; Promocell, Heidelberg, Germany) containing 30% pooled human 0+ serum [cell culture medium with serum (CMS)]. Confluent cultures were dissociated and transferred to 0.1% gelatine-coated tissue culture surfaces (Merck, Darmstadt, Germany). EC stock cultures were stored in liquid nitrogen (in CMS containing 10% DMSO). Morphological characteristics (cobblestone pattern) were monitored by phase-contrast microscopy (Leica DMRBE, Bensheim, Germany).

EC purification. To evaluate the impact of fluorescence-activated cell separation (FACS) technique on cell growth and function, EC were isolated from a mixture of primary cultures of EC (HSVEC, HCAEC, HUVEC) and fibroblasts (prepared from human aorta following the same protocol as described above). Uncontaminated, pure cultures of EC (pEC) and fibroblasts were mixed in defined proportions and were also used as pure controls. Purity and recovery were quantified by flow cytometry using CD31 labeling. Dissociated cells (2 x 106) were washed with PBS/0.5% BSA/2 mM EDTA and labeled with a primary phycoerythrin (PE)-conjugated monoclonal antibody to human CD31 (20 µg/ml, 30 min, 37°C; Ancell, Läufelfingen, Switzerland). For FACS, labeled cells were then transferred into supplemented PBS [(0.5 – 1) x 106 cells/ml], and FACSseparation of the CD31-positive cell population was performed on a FACStarPLUS (Becton Dickinson, San José, CA) with a laser excitation of 200 mW at 488 nm. The sort windows were defined in the dot-plot diagrams of forward scatter vs. 90 side scatter signals and forward scatter vs. FL-2 (CD31-PE), both on a logarithmic scale. For each experiment, CD31-positive cells were sorted into 5-ml tubes containing 1.5-ml CMS. Cell suspensions were then pelleted by centrifugation and resuspended in CMS, and 5 x 104 cells were seeded in 1) 10-cm2 chamber slides for immunofluorescent characterization; and 2) six-well tissue culture trays for functional analyses. To demonstrate the influence of FACSorting procedure on EC growth and function, the release of IL-6 and the surface expression of E-selection on labeled and sorted cells of the same culture in the same set of experiment (see below) were analyzed.

Phenotypic and functional reanalysis of purified EC. CD31-positive cells in original and FACsorted cell fractions, as well as recultured detached cells, were distinguished by flow cytometry (FACSCalibur, Becton Dickinson). Samples were stained with the PE-conjugated mouse-anti-human CD31-antibody, as described above. Cells were routinely washed twice, spun down, and resuspended in 300 µl PBS/0.2% BSA. The data were analyzed using CellQuest (Becton Dickinson) and winMDI2.8 (Scripps Research Institute, LaJolla, CA). Unlabeled pure cells (EC and fibroblasts) and cells labeled with an isotype-matched control antibody (mouse IgG1, 20 µg/ml, Ancell) were used as a control (as shown in Fig. 1B).


Figure 1
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  		Fig. 1. Rationale of endothelial cell (EC) purification. A: labeling with FITC-conjugated anti-human CD31 antibodies reveals the proportion of EC in pure EC or mixed cultures in a confluent monolayer. B: pure EC (top) and mixed cultures with a content of 5% fibroblasts (F) (bottom) were analyzed for their CD31 content [dot plot vs. forward scatter (FSC)]. The expression of CD62E (= E-selectin) of the EC population was quantified by flow cytometry (isotype control, solid trace with open histogram; basal expression, dashed trace with open histogram; and TNF-induced expression after 4 h, solid trace with shaded histogram). C: the basal IL-6 release per cell of mixed cultures (n = 3) (the level of contamination was defined by the proportion of CD31-negative cells) was determined in the supernatant of confluent monolayers. PE, phycoerythrin.

 
Recultured cells were grown to confluence on chamber slides, fixed in acetone/methanol (1:1) for 10 min at –20°C, and labeled using anti-human CD31 (IgG1, clone JC/70A, Dako Diagnostika, Hamburg, Germany) and a FITC-conjugated goat anti-mouse IgG antibody (Amersham Pharmacia Biotech, Freiburg, Germany). Purity of cultures was monitored by microscopic imaging.

Functional analyses were performed to demonstrate potential effects of the sorting procedure. Here, pEC were used as 1) untreated control cells, 2) antibody-labeled nonsorted cells (labeling control), and 3) FACsorted cells (= CD31-positive fraction after FACSorting of mixed cultures) (n = 3 each for HSVEC, HUVEC, and HCAEC). All samples were inoculated into six-well plates (5.0 x 103 cells/cm2) and cultured for 5 days, and basal as well as TNF-induced (10 ng/ml, 4 h) cytokine release were determined in addition to the expression of E-selectin. IL-6 content in the supernatant was assayed using commercial ELISA kits (Coulter Immunotech, Krefeld, Germany). For flow cytometric analysis of cell surface-bound E-selectin expression, EC were double-labeled with anti-human CD62E-FITC antibody (Dianova) and anti-human CD31-PE.

In a separate set of experiments, functional interference of the contaminating cells with EC in the confluent monolayer was verified by analysis of 1) TNF-induced expression of E-selectin using CD62E-FITC labeling, and 2) basal release of IL-6 into the supernatant (as described above).

Functional analysis of EC of different origin. Purified macrovascular arterial (pHATEC n = 8, pHCAEC n = 9) and venous (pHUVEC n = 6, pHSVEC n = 8) EC (>98% CD31-positive cells) were grown to confluence under identical culture conditions (see above). In brief, EC (5.0 x 103 cells/cm2) were seeded on either 6-, 24-, or 96-well gelatine-coated culture plates and grown to confluence (5–6 days) without additional nutrient supply. Every 24 h, supernatants were collected from a six-well plate and routinely monitored with the blood-gas analyzer (Radiometer), and the content of lactate dehydrogenase (Roche Diagnostics, Basel, Switzerland) was documented parallel to the accumulated IL-6 concentrations. In the corresponding wells, membrane-intact cells were counted, and mean cell volumes were recorded using the CASY1 system following enzymatic detachment. Cell surface expression of E-selectin was measured by flow cytometry after cell dissociation at day 5 in culture (24-well plate), with and without TNF-induction (10 ng/ml, 4 h). For the analysis of basal vs. TNF-induced release of proinflammatory cytokines (IL-6) and chemokines [IL-8, monocyte chemoattractant protein (MCP)-1, and CXC chemokine ligand-10 (CXCL-10) formerly known as IFN-{gamma}-inducible protein-10], EC were routinely grown to confluence in 96-well culture plates, treated with or without 10 ng/ml TNF for 20 h. Supernatants were collected, and the content of IL-8, MCP-1, CXCL-10, and transforming growth factor (TGF)-beta2 was analyzed using commercially available ELISA kits (Bender MedSystems, Burlingham, CA). In another set of experiments, we compared basal and TNF-induced production of IL-6 (see above) from HCAEC isolated from coronary arteries of explanted hearts after transplantation (n = 3) or obtained from Clonetics (San Diego, CA) and Promocell (Heidelberg, Germany) (n = 3).

Statistics. Data are given as means ± SD. A paired t-test was used to compare responses between treated and untreated cells of the same origin. Wilcoxon signed-rank test was used to statistically verify differences between different EC types (HSVEC vs. HCAEC, HSVEC vs. HATEC, HATEC vs. HCAEC). A P value ≤0.05 was considered to be statistically significant, and P ≤ 0.01 was considered highly significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Rationale of EC purification. EC cultures from umbilical or saphenous vein preparations were rarely contaminated by fibroblasts. However, plating of cell suspensions after tissue dispersion of heart vessels (coronary artery, aorta, pulmonary artery) or the internal thoracic artery usually resulted in mixed adherent cell cultures. Labeling with a primary FITC-conjugated monoclonal anti-human CD31 antibody revealed the proportion of EC in these mixed cultures (Fig. 1A). The contaminating fibroblasts usually grew more rapidly than the EC population. A proportion of only 5% fibroblasts at seeding led to a final contamination of 18.1 ± 7.5% in the EC culture at confluence. Prolongation of cultivation time or additional transfer then resulted in a complete overgrowth of the EC monolayers. Due to the observation that contaminating cell populations such as fibroblasts are likely to affect EC function, activity, and differentiation (16), we analyzed the inflammatory response of EC in the presence of rising concentrations of fibroblasts. Thus fibroblasts were negative for E-selectin, whereas 100% of the CD31-positive cells expressed E-selectin (CD62E) on their cell surfaces (Fig. 1B). The mean fluorescence intensity of this cell population was not affected by rising concentrations of fibroblasts. Even at highest concentrations of 70% fibroblasts and 30% EC, the mean fluorescence intensity of E-selectin remained unchanged. Flow cytometry allows single-cell analysis and exclusion of the contaminating cells, while other functional parameters, such as the release of paracrine factors into the supernatant, cannot be easily discriminated for EC and fibroblasts. The activation of EC accompanied by IL-6 release, for example, interfered with the IL-6 production of contaminating fibroblasts, as indicated in Fig. 1C. Therefore, evaluation of functional heterogeneity of different EC types required EC purification. We, therefore, tested various purification strategies. FACSorting was finally used based on the validation method, described in detail in the following section.

Recovery, purity, and functionality of purified EC cultures. Since FACSorting technology allows the separation of single cells, purity of EC following FACS separation using anti-CD31-PE antibodies was always ≥99% for EC/fibroblast mixtures or contaminated primary cultures, as verified by immediate reanalysis. The loss of EC never exceeded 20% of the original EC fraction. Accordingly, FACS separation was a feasible technique for purifying EC from large-vessel preparations for recultivation.

Inoculation of pEC (untreated control, labeling control, and respective FACS-sorted EC) in six-well plates yielded confluent monolayers consisting of (3.7 ± 0.5) x 104 EC/cm2 with the typical cobble-stone structure after 3–4 days in culture. There was no significant difference in the final cell concentration of HSVEC, HUVEC, and HCAEC (data not shown). Labeling and sorting procedures affected neither cell growth kinetics nor the spontaneous and TNF-induced release of IL-6 (Fig. 2, top). FACS also did not affect E-selectin expression on the EC surface (Fig. 2, bottom). Unstimulated EC were negative for E-selectin and peaked at 4 h of stimulation. The applied sorting procedure was suitable to sufficiently regrow purified EC from organ-specific tissues, such as heart vessels or the internal thoracic artery, for functional analyses. In addition to that, the results presented in Fig. 2 for only a limited number of EC preparations (n = 3 for each EC type) indicated large interindividual variations, with EC derived from various sources, but systematic differences in some basic cellular characteristics, e.g., IL-6 release, could be observed. This phenomenon was studied in more detail.


Figure 2
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  		Fig. 2. Labeling and FACSorting procedures affected neither spontaneous and TNF-stimulated release of IL-6 (top), nor the expression of E-selectin (bottom) by different EC in regrown confluent monolayer cultures. Untreated control, labeling control, and FACSorted pure cultures of human umbilical vein EC (HUVEC; black bars), human saphenous vein EC (HSVEC; light gray bars), and human coronary artery EC (HCAEC; dark gray bars) (n = 3 for each origin) were grown to confluence and incubated without (left) and with (right) 10 ng/ml TNF (4 h). The concentration of IL-6 was determined in the culture supernatant. The surface expression of E-selectin was analyzed using fluorescence-activated cell separation (FACS) [mean fluorescence intensity (MFI)].

 
In vitro characteristics of different types of human macrovascular EC. Three groups of human macrovascular EC were available. HSVEC, HATEC, and HCAEC were derived from patients undergoing coronary artery bypass surgery or orthotopic heart transplantation. HUVEC were analyzed as controls. Three to five days after plating, all cell cultures reached confluence with quite similar mean cell densities in the monolayer (Fig. 3A). The integrity of the monolayer persisted over the next 2–3 days, with cell viabilities ranging from 85 to 95%. Due to identical growth kinetics, the accumulated IL-6 concentration in the supernatant of different cell types was compared. EC from coronary arteries produced significantly higher amounts of IL-6 than other arterial (HATEC) or venous (HUVEC, HSVEC) EC (Fig. 3B). However, there was no general correlation between cell doubling and cytokine accumulation, e.g., HATEC cultures with the highest cell numbers did not produce the highest levels of IL-6 (data not shown).


Figure 3
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  		Fig. 3. Primary cultures of human macrovascular EC show high interindividual variability in cell density of a confluent monolayer (A), as well as in the production of IL-6 in culture under identical growth conditions (B). HUVEC, HSVEC, human arteria thoracica interna EC (HATEC), and HCAEC were seeded at a density of 5 x 103 cells/cm2 and grown to confluence (5 days). Cell count per centimeter squared was determined, and supernatants were analyzed for accumulated IL-6 content. Each data point represents the mean ± SD of six to nine individual cultures per EC type. EC of different vascular beds were compared with each other: **P ≤ 0.05, ***P ≤ 0.01.

 
Since synthesis of IL-6 is a relevant prognostic marker in different chronic heart diseases (e.g., chronic heart failure, chronic rejection after heart transplantation), we also studied the inflammatory response of human patient-derived EC after stimulation with an exogenous stimulus (TNF). These experiments were performed in a 96-well system over 20 h. Due to the diverse experimental protocols, the absolute values (ng/ml) of Figs. 3B and 4 cannot be directly compared. However, the difference in basal IL-6 release was consistent for the different studied EC types, i.e., HCAEC produced significantly more IL-6 (Fig. 4). Incubation with TNF resulted in a reproducible stimulation of IL-6 release to maximum values of ~1 ng/ml (P ≤ 0.01). Under such stimulating conditions, IL-6 production of HCAEC, HATEC, and HSVEC did not differ (Fig. 4). To evaluate whether the basal release of IL-6 in coronary arteries and coronary artery-derived EC cultures was critically affected by the disease pattern, we compared HCAEC isolated from human recipient (diseased) and donor (nondiseased) hearts. There was no difference in either the basal or TNF-induced IL-6 release rates for the two HCAEC populations (diseased vs. nondiseased: basal, 377 ± 162 vs. 310 ± 169 pg/ml and TNF-induced, 1,371 ± 783 vs. 957 ± 685 pg/ml; n = 3, respectively).


Figure 4
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  		Fig. 4. HCAEC show a significantly higher basal release of IL-6 than other macrovascular EC in culture, while E-selectin expression does not differ. Basal and TNF-stimulated production of IL-6 and expression of E-selectin in four to nine individual confluent cultures from each EC type are documented. IL-6 release was measured in supernatants of cultures grown in 96-well plates over a time period of 20 h. Cell surface expression of E-selectin was determined by flow cytometry. EC of different origins were compared with each other: *P ≤ 0.05, **P ≤ 0.01.

 
To verify the heterogeneity of organ-specific EC, the release of additional proinflammatory proteins was measured. None of the EC types produced TGF-beta2 or CXCL-10. Under stimulating conditions, the concentration of CXCL-10 increased significantly. The stimulation was independent of the cell type. As shown in Table 2, arterial EC (especially HATEC) produced by a factor of two to three times more MCP-1 and twice as much IL-8 than venous EC. Independent of the cell type, stimulation with TNF for 20 h resulted in a significant increase of each protein (P ≤ 0.05). HUVEC produced the smallest amounts of IL-8 (both basal and TNF induced). HCAEC showed no exceptional position as documented for IL-6. Due to a high interindividual variability for the release rates of HCAEC, there are no statistically significant differences.


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  		Table 2. Cell type-specific release of MCP-1, IL-8, and CXCL-10 (= IP-10)

 
A general activation of EC in culture should be reflected by an upregulation of cellular E-selectin expression. We analyzed basal and TNF-stimulated E-selectin surface expression via flow cytometry. In contrast to IL-6, cell surface expression of E-selectin was independent of EC type and negligible under nonstimulating conditions. As expected, TNF significantly stimulated E-selectin expression (P ≤ 0.01), independent of the EC type (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLAIMER
 REFERENCES
 
The purpose of our study was to describe the in vitro heterogeneity of highly pure macrovascular EC from different vessel tissues to create an in vitro EC system for cardiovascular diseases. The analysis of the chemokine/cytokine repertoire of human EC from different organs could prove to be important for understanding coronary artery diseases. We demonstrated an enhanced IL-6 release by EC from coronary artery vessels compared with other EC types. This could be a relevant observation to explain tissue-specific responses to pathophysiological stimuli in cardiovascular research.

Considerations for EC purification.

It is well known that primary EC cultures are often overgrown by fibroblasts, smooth muscle cells, and pericardial and endocardial cells, which are characterized by a high proliferative activity in vitro and a significant expression of cytokines/chemokines (1, 3, 30). This problem was often discussed for preparations of endothelial progenitor cells (28), microvascular (20), and coronary artery EC (3, 10, 18). In our study, we could show that the proportion of non-EC in the monolayer of arterial vessel preparations increased exponentially. While the upregulation of TNF-induced expression of E-selectin was not affected, the release of IL-6 was increased to such an extent that it was not possible to attribute it to an EC-relevant response. Furthermore, fibroblasts are likely to affect EC function, activity, and differentiation, as shown earlier (16). Thus long-term culturing of EC requires elimination of contaminants. Different isolation protocols have been described in the literature (perfusion of whole organs, mincing tissues segments, cannulation of whole vessels). Enzymatic digestion (14) only results in EC cultures with high purities (≥98%), if vessels can be tightened throughout the preparation process, and is feasible for umbilical or saphenous veins but not for heart tissues. Due to further clinical applications of the explanted heart valves, we were obliged to prepare the coronary vessels inside the operating room, with the result that most of the vessels were leaky. Furthermore, we isolated macrovascular EC from calcified vessels, and our cell donors are, on average, 55–75 yr old. As far as our experience goes, each of the mentioned points impaired effectiveness of cell preparation and increased the risk for contamination. These nonperfusable tissues or parts of large vessels were frequently minced before protease digestion, with final purities of <85% (29), requiring further purification, e.g., using bead technology. In a preliminary unpublished study, we also used antibody-labeled bead technology (MACS, Miltenyi Biotech) to isolate EC from mixed cultures, resulting in purities ranging from 65 to 95%. However, the remaining fibroblasts overgrew our EC cultures, notably after transfer to the next passage or after long-term cultivation. Two-step bead sorting increased the purity, but, in parallel, >20% of the original EC disappeared. Magnetic bead separation affected neither morphology nor the expression of EC-specific markers, such as platelet endothelial cell adhesion molecule-1, von Willebrand factor, cytokine-induced E-selectin, VCAM-1, or ICAM-1, but further functional analyses could once again not be performed due to long-term overgrowth and potential cytokine/chemokine release by contaminating cell populations (5, 10).

In the present study, we benefit from single-cell separation of FACSorting technology with purities ≥99%. Similar FACS procedures have been described by several authors (6, 12, 23). However, they have not considered that the purification procedure itself may, in fact, affect the functional activity of EC. Indeed, both magnetic cell separation and FACS were shown to alter membrane physiology of cells, which was discussed as a sort-induced stress syndrome due to exposure to hydrodynamic forces or magnetic fields (27). Therefore, we confirmed the functional authenticity of FACS-separated, recultured EC before the analysis of tissue-specific characteristics. Using our protocol, neither cell growth nor spontaneous/TNF-induced cytokine/chemokine release was affected, as demonstrated in one set of experiments with treated cells of the same cultures. An activation of ECs due to antibody-labeling or FACSorting could be excluded, which is a crucial point for the analysis of inflammatory cell responses.

Functional heterogeneity of macrovascular EC from different origins.

The system of pEC cultures enables us to evaluate specific functions of atherosclerotic stressed EC. The present study mainly focused on the involvement of these EC in the increased release of IL-6 in different chronic heart diseases (e.g., chronic heart failure, chronic rejection after heart transplantation). Therefore, we compared EC cultures isolated from different macrovascular vessel materials, assuming vessel-specific functional variations. Since (embryonic) HUVEC has been widely applied as a system to study endothelial dysfunction in vitro, we analyzed HUVEC as a reference or internal control group.

In our study, all EC cultures grew to confluence and established the typical cobblestone pattern. However, confluent monolayers of macrovascular patient-derived EC differed in their basal release rates of cytokines and chemokines. EC from venous and arterial origin did not produce TGF-beta2 and CXCL-10. Under stimulating conditions, the release of CXCL-10 was independent of the EC type. However, arterial EC produced more MCP-1 and IL-8. HCAEC showed the highest release rates of IL-6 compared with other EC types. Our data on basal IL-6 release in HUVEC, HSVEC, and HATEC, ranging between 40 and 200 pg/ml over 20 h, confirms results described in previous literature (11, 15). Despite the high interindividual variability of different preparations, we were able to clearly show that HCAEC, while not differing in their capacity of maximum IL-6 production, release significantly higher amounts of IL-6 in contrast to other macrovascular EC under noninducing conditions. Hooper et al. (11) partly confirmed our data but did not discuss the enhanced IL-6 release of HCAEC at all, since they were mainly interested in activated protein C-induced EC activity. Our data indicate that basal and TNF-stimulated release of IL-6 is not altered by the disease state of the coronary arteries, since HCAEC from donors and recipients did not differ in IL-6 release. Therefore, the high basal IL-6 release in HCAEC as opposed to other coronary artery EC types implies intrinsic functional heterogeneity. It is tempting to speculate that this may be the basis for an increased or accelerated responsiveness of HCAEC to pathophysiological stimuli. The stress situation during extracorporal circulation or acute coronary artery syndrome, for example, might enhance transcardiac IL-6 gradients (7, 9), which is discussed to be primarily released from vascular endothelium but not the myocardium (17, 21).

The relevance of endothelium in controlling and fine-tuning inflammatory responses via modulation of cellular adhesion molecule expression is generally accepted. Invernici et al. (13) showed organ-specific differences in basal and TNF-induced expression of cell adhesion molecule in human fetal EC isolated from different organs. However, comparable variations could not be shown in our study with macrovascular EC from adult vessel materials, despite the divergent results on basal IL-6 production. In accordance with previous investigations using HUVEC and HSVEC (24, 31), HUVEC expressed higher levels of E-selectin on TNF-induction than HSVEC and arterial EC.

The efficient isolation and culture of human EC, as presented in our study, provides sufficient amounts of highly pure cultures to generate EC banks for functional studies. Due to their phenotypic and functional heterogeneity, it seems most relevant to establish and study tissue-specific EC, e.g., HCAEC should be applied to gain insight into the mechanism of systemic endothelial dysfunction associated with coronary artery diseases.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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DISCLAIMER
 REFERENCES
 
The project was supported by the Ministry of Nutrition, Health and Consumer Protection of the Government of Bavaria (STEMMAT-Project as part of the BayernAktiv Health Initiative).


   DISCLAIMER
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 DISCLAIMER
REFERENCES
 
All experiments comply with the current national laws, and the preparation protocol was approved by the local human ethics committee (no. 99/133).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the excellent technical assistance of A. Urbanek, S. Bergman, C. Leykauf, K. Bielenberg, K. Hollnberger, and M. Wondrak. Umbilical cords were kindly supplied by Drs. Niemeier and Gottschalk (Technical University Munich) and by Dr. Hofstaedter (Institute of Pathology, University of Regensburg).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Lehle, Dept. of Cardiothoracic Surgery, Univ. Hospital, Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany (e-mail: Karla.Lehle{at}klinik.uni-regensburg.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K. Lehle and L. A. Kunz-Schughart contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 

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