Ultrastructural variability of macrophages in the wall of selected aorto-coronary bypass grafts

This study involved 50 patients (37 male (74%) and 13 women (26%) with the mean age of 63.4±9.2 years who underwent elective CABG procedures with the simultaneous application of RA and SV segments.

During CABG procedures, surplus segments of vessels used as aorto-coronary grafts and at least one centimeter in length were taken for ultrastructural studies. The arterial grafts were harvested pedicled with the surrounding tissues to avoid any accidental microinjuries. All vessels were obtained in the standard manner applying a full skin incision over the entire course of them. Moreover, measures such as avoidance of touching (‘no-touch method’), excessive manipulation and dilatation as well as high-energy electrocauthery were applied to minimize surgical trauma.

Vessels with macroscopic abnormalities such as signs of atherosclerosis (only in RA segments), inflammation, abnormally dilated or with iatrogenic injuries were not used both by surgeons as well as for ultrastructural examinations.

Following macroscopic inspection harvested vascular segments were divided into two parts, one for analysis in transmission electron microscopy (TEM) were immersed in phosphate-buffered 2.5% glutaraldehyde (0.05 M, pH 7.4) while the another one in freshly prepared Boiun solution for light microscopy. The further preparatory steps for microscopic examination were previously described in the details [18].

In immunohistochemical analysis the Dako REAL EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse, K5007 (Dako, Copenhagen, Denmark) and mouse monoclonal anti-CD68 antibodies in dilution 1:100 (M0814, Dako) were used. Additionally, to identify macrophages in the vascular wall an eliminating assay with the following specific antibodies for lymphocyte subpopulations such as anti-CD20cy (1:400 dilution, M0755, Dako), anti-CD3 (1:50 dilution, M7254, Dako), anti-CD8 (1:100 dilution, M7103, Dako) and anti-CD30 (1:40 dilution, M0751, Dako) was carried out [18]. The peroxidase reaction was developed using diaminobenzidine.

Transverse vascular sections were analyzed under a brightfield microscope - Olympus BX 50 (OLYMPUS Optical Europe, Germany) equipped with a Mirax-Midi scanner (Carl Zeiss Microimaging GmbH, Germany), coupled with a Panoramic Viewer, version 1.15.4, software (3DHISTECH Ltd., Budapest, Hungary). All of the analyses were performed blind using coded samples and included positive and negative controls. The negative controls consisted of sections incubated with nonimmune IgG1 (X0931, Dako) as well as sections in which the primary or secondary antibodies were omitted. In addition, serial sections were stained in subsequent experiments as positive controls and to determine the consistency of staining. The cells presenting expression of CD68 protein were counted first throughout the vessel wall then within the consecutive layers: the tunica intima, the tunica media and the tunica adventitia.

For transmission electron microscopy (TEM), vascular samples were prepared in steps described previously [16]. Ultrathin sections were mounted on mesh nickel grids, counterstained with uranyl acetate then lead citrate and eventually examined using a JEM-1010 (JEOL, Tokyo, Japan) TEM. Typical utrastructural features of macrophages were assessed separately in all layer of the vascular wall and special attention was paid to cell membrane and cytoplasmic organella with respect to their biological activity.

First, continuous variables were checked for normality with the use of the Shapiro-Wilk test. Those satisfying normal distribution criteria were presented as the means with standard deviations (sd). The remaining continuous data were expressed as medians with 25th to 75th percentiles and then compared by means of the nonparametric Mann-Witney U test. A p value below 0.05 was considered as statistically significant. Analysis was performed with the use of statistical software Statistica 10.0 for Windows (StatSoft, Inc., Tulsa, OK, USA).

The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee. The study protocol was approved by the Local Bioethical Committee (No 1201/08) and all patients expressed written consent to participate in this study.

Expression of CD68 protein was noted in all segments of the examined vascular segments although their number was in the wide range, in RA between 15 and 244 while 28 through 289 in SV. Median (25th; 75th percentile) of a total number of CD68+ cells, considered as macrophages, on the vascular transverse sections was smaller in RA (66 (43; 108)) than in SV (95 (67; 135)) (p<0.05) (Fig. 1).

FIGURE 1

Histological analysis revealed that not only the absolute number of CD68+ cells in the consecutive layers but also their percentage rate differed significantly between RA and SV. In the latter one, more macrophages were found in the inner strata of their vascular wall. In RA, markedly more CD68+ cells were found in the tunica adventitia. The rate of adventitial macrophages exceeded 70% in RA and approximately 50% in the venous segments. The detailed data are outlined in table 1 (see also Fig. 1).

Detailed analysis of microelectrophotography of cells equipped with ultrastructures typical for macrophages revealed marked differences between these located in the tunica intima or media and in the tunica adventitia (Fig. 2).

FIGURE 2

Macrophages in the tunica intima and tunica media were very similar with respect to their morphology and manifested features of pronounced metabolic and phagocytic activity (Figs 2a and 2b). Folded cellular membrane with irregular outline formed cytoplasmic protrusions with numerous caveolae and vesicles. Inside the cells, the cytoplasm was rich in organelles such as non-membraneous aggregation of electron-dense material and vesicles of varying electron density. Moreover, numerous lysosomes (both primary and mature) manifesting marked heterogeneity of size and shape as well as phagocytic vacuoles were also found. Centrally positioned large and euchromatic nuclei of irregular outline had one or two nucleoli inside.

Otherwise, the vast majority of macrophages in the tunica adventitia presented properties of unreactive residual cells filled with cytoplasm poor in cellular organelles with few heterogenous lysosomes and cisterns of rough endoplasmic reticulum, poorly developed Golgy apparatus and small mitochondria (Fig. 2c). Folded cellular membrane had only single caveolae-like protrusions. These cells were usually noted close to vasa vasorum and also in the layers of the tunica adventitia adjacent to the external elastic lamina.

In the subendothelial layer, macrophages were embedded between collagen fibers. Similarly to the arterial wall, the majority of cells detected as macrophages in the tunica intima and tunica media presented ultrastructure typical for biologically active cells such as numerous lysosomes, heterogenous phagocytic vesicles (Figs 3a and 3b). These aforementioned findings were irrespective of the age of venous segments donors.

FIGURE 3

In the tunica adventitia there were macrophages among fibroblasts and smooth muscle cells. CD68+ cells were found predominantly in the areas close to vasa vasorum. Cytoplasmic equipment suggested deprived metabolic and phagocytic activity. Only single lysosomes were seen in the cytosol adjacent to the centrally positioned nuclei. In the majority of cells no caveolae and vesicles linked to the cellular membrane were detected (Fig. 3c).