American Journal ofPathology, Vol. 143, No. 1, July 1993 Copyright X American Societyfor Investigative Pathology Immunohistochemical Study of Intimal Microvessels in Coronary Atherosclerosis Yixia Zhang,* Walter J. Cliff,* Gutta 1. Schoefl,* over 1000 hearts, concluded that nobody with ab- and Grace Higginst normal vascularization of the coronary artery wall was From the Clinical Science Division,* John Curtin School of free from the risk of death from coronary artery dis- Medical Research, The Australian National University, ease. However, some workers consider that vessels Canberra, and the School ofPathology,t University ofNew invading the coronary intima are of no signifi- South Wales, Sydney, Australia cance12,13 and are a function of intimal thickness and not of atherosclerosis.14 Those who emphasize the importance of intimal Two hundred ninety-nine human coronary artery vascularization propose that the microvessels may paraffin-embedded tissue blocks were examined play two roles: one, of providing blood components to for intimal microvessel invasion by probing for "nourish" the growing plaques,2'10'15 and the other, of factor VIII-associated antigen with indirect im- causing intimal hemorrhages by rupturing. Repeated munofluorescence and high resolution confocal intimal hemorrhages may eventually lead to coronary microscopy. The results obtained confirm that in- artery thrombosis.11 As to the first hypothesis, no di- timal microvessels originate in the adventitia and rect evidence of plasma components leaking from in- show that the richness ofintimal microvessels is timal microvessels to nourish atherosclerotic plaques strongly positively correlated with intimal thick- has yet been obtained but newly formed vessels are ness and negatively correlated with relative lu- known to be highly permeable.16 In addition, newly men size. A number ofplasma constituents were formed small vessels are deficient in supporting con- examined in serial sections. Comparison of im- nective tissue and basement membrane and are frag- munofluorescence distribution patterns of these ile.16'17 The hemorrhage hypothesis generally pos- components with intimal microvessel distribution tulates that the ruptured microvessels arise directly patterns reveals that intimal microvessels leak from the arterial lumen.1 1 This is not in agreement with plasma albumin into artery waUs, exude fibrin- Barger's observations18 that intimal capillaries origi- ogen, and are associated with the build-up of nate more frequently from the adventitia than the lu- plasma ceUs within atherosclerotic lesions. men. The origin of intimal capillaries and their role in Therefore, intimal microvessels are demon- intimal hemorrhage are, therefore, still uncertain. stratedtoplay important roles in the development The endothelial cells of these thin-walled microves- of atherosclerosis. (AmJPathol 1993, 143.164- sels contain factor VIII-associated antigen (VIll.a.- 172) a.).19 Applying anti-factor VIII.a.a. antibody to coro- nary arteries will, therefore, reveal any microvessels present in their walls. The present work examines the Coronary arteries, as is the rule for most muscular intramural microvascular distribution patterns using arteries,1 normally have no vessels in their intima or the sensitive indirect immunofluorescence technique media. Intimal vascularization of human arteries was with anti-factor VIII.a.a. as the first antibody in con- first noticed and linked with atherosclerosis, and in- junction with high resolution confocal microscopy. timal thickening in general, by Koester2 in 1876. This Through analysis of the microvascular patterns, the observation has been confirmed by a large number origin of intimal microvessels and their relation to the of observers3-6 and has been beautifully demon- development of atherosclerosis can be examined. strated by perfusion of India ink7 and most recently of Comparison of microvessel distributions with those of silicone polymer into the walls of cleared coronary arteries with accompanying cinematography.8 Accepted for publication January 14, 1993. The microvessel invasion of has been em- plaques Address reprint requests to Dr. W. J. Cliff, John Curtin School of phasized as being crucial in the pathogenesis of Medical Research, The Australian National University, Canberra, atherosclerosis.9-11 Osborn,5 after the analysis of ACT 2601, Australia. 164 Microvessels in Coronary Atherosclerosis 165 AJPJuly 1993, Vol. 143, No. 1 plasma components such as albumin, fibrinogen, im- Control Tissue Sections munoglobulin y (IgG), a (IgA), and p (IgM) in serial sections, will establish whether blood components Human appendix (Woden Valley Hospital, Can- are entering the atherosclerotic plaques from the in- berra), tonsil (Woden Valley Hospital, Canberra), timal microvessels. Furthermore, the significance of and selected human coronary sections were cho- intimal neovascularization in the development of cor- sen as positive controls for IgG, IgA, IgM, CRP, fi- onary atherosclerosis will be analysed through relat- brinogen, albumin, and factor Vill.a.a. ing its presence and richness to intimal thickness and arterial stenosis. Antibodies Rabbit anti-human polyclonal antibodies were used Materials and Methods as the primary antibodies in the indirect immunoflu- orescence methods. Antibody specificity was Tissue Procurement, Fixation, and proved by absorption of the antibody with the corre- Embedding sponding pure antigen (Sigma Chemical Co, St. Louis, MO) at 4 C for 24 hours. After such absorp- Hearts were obtained at autopsy from the Pathology tion, the antibodies showed no immunoreactivity on Department of Royal Canberra Hospital and from human coronary arteries. The second antibody the School of Pathology, University of New South used was sheep anti-rabbit polyclonal antiserum Wales, Sydney. The coronary arteries were cannu- conjugated with fluorescein isothiocyanate (FITC) lated and perfused at 100 mm Hg pressure with (Silenus, Melbourne, Australia) diluted 1:100 with warm heparinized saline followed by 10% neutral phosphate-buffered saline (PBS) before use. Nor- buffered formalin (NBF) and then barium gelatin.20 mal rabbit serum (Dakopatts, Glostrup, Denmark) at The coronary arteries were clamped and fixation the same dilution as that of the tested antibodies continued by immersing the heart in NBF for at least was used as the negative control in the indirect im- 24 hours. They were then dissected free from the munofluorescence method. Normal sheep serum surface of the heart and made transparent by pro- was used to prevent nonspecific binding of the sec- longed soaking in two or three changes of glycer- ondary antibody and was separated by centrifuga- ine. The arteries were examined minutely under the tion from the blood of normal sheep maintained at dissecting microscope and numerous blocks were the John Curtin School of Medical Research, Aus- taken for histology of normal and diseased seg- tralian National University. ments. Sixty-eight cases were selected from sub- jects dying of acute myocardial infarction, other car- diovascular diseases, infections, neoplasms, Immunohistochemical Techniques alcoholism, and suicide-accident. Paraffin sections were deparaffinized, rehydrated, washed, and digested with 0.1% trypsin (Sigma) Coronary Artery Sections solution in Tris buffer (pH 7.8) for 2 to 3 hours at 37 C in a humidified chamber. Sections were then Two hundred ninety-nine paraffin-embedded blocks rinsed three times with distilled water and twice with of decalcified human coronary arteries from 68 pa- PBS for 5 minutes each. The final wash with PBS tients were selected according to histological ap- contained 0.5% Tween 20 (TPBS) to reduce non- pearances without knowledge of cause of death, specific staining. Sections were incubated for 20 sex, or age. The blocks were chosen to include a minutes at room temperature with 10% normal range from normals through the different aspects of sheep serum to block nonspecific binding. The se- coronary artery disease to provide examples of ma- rum was drawn off and the sections were then incu- jor pathological features such as fibrosis, inflamma- bated with the desired rabbit anti-human antibod- tory cell infiltration, intimal vascularization, plaque ies. Adjacent se'ctions were incubated with normal hemorrhage, and lipid deposition. Ten 4-p-thick se- rabbit serum for 30 minutes at room temperature in rial sections were cut from each block for the inves- the humidified chamber to serve as controls. After tigation of various components present in the coro- three 5-minute washes with TPBS, sections were in- nary arteries and for comparison of their distribution cubated with the FITC-conjugated second antibody patterns. The sections were mounted on poly-L- for 30 minutes at room temperature in the humid lysine coated slides and air-dried at 60 C for 1 chamber. Sections were then rinsed three times with hour.21 TPBS and mounted in PBS and glycerol (1:9) at pH 166 Zhang et al AJPJuly 1993, Vol. 143, No. 1 8.6 under glass coverslips. In general, the sections plasma components were graded on scales of 0 to were examined immediately but when this could not 3 in terms of their specific immunofluorescence in- be done, they were stored briefly in light-proof tensities and distributions in the wall. packets at 4 C until used. Fluorescent microscopy was performed using an MRC-500 Confocal Image System (Bio-Rad Micro- Statistical Methods science Division, Hemel Hempstead, England) with an argon ion laser operating