Eur J Vasc Surg 8, 70-77 (1994)
Segmental Mediolytic Arteritis-- Electronmicroscopic and Immunohistochemical Study Tatu Juvonen ~, Olavi Riisiinen 2, Alli Reinil~i 2, Seppo Parkkila 3, Juha Nissinen ~, Matti I. Kairaluoma ~, Raija Sormunen 2 and Onni Niemel~i 4 Department of ~Surgery, ~-Pathology, 3Anatomy and 4Clinical Chemistry, Oulu University Hospital and University oJ Oulu, SF-90220 Oulu, Finland We examined specimens of human gastroepiploic artery aneurysm from a patient having several visceral aneurysms using electronmicroscopic and immunohistochemical techniques. The histopathological and ultrastructural findings confirmed the diagnosis of segmental mediolytic arteritis. Arterial smooth muscle cells from the gastroepiploic artery contained cytoplasmic vacuoles, media was thin and the internal elastic membrane showed distortion. X-ray microanalysis revealed calcium deposits in the medial extracellular space. Antigenic determinants of human immunoglobulins, fibrinogen, complement C3a and factor VIII were demonstrated in the injured artery wall, suggesting that immunocomplexes deposited in the artery wall may be associated with local injury. These findings support the role of autoimmune disorders in the pathogenesis of segmental mediolytic arteritis. Key Words: Segmental mediolytic arteritis; Visceral aneurysm; Arterial pathology; Immunohistochemistry.
Introduction Segmental mediolytic arteritis (SMA) is considered an extremely rare lesion of the splanchnic artery, which was first described b y Slavin and Gonzales in 1976.1.2 This disease has led to the formation of a n e u r y s m s and s p o n t a n e o u s intraabdominal h a e m o r r h a g e in most of the subsequently r e p o r t e d cases. 1-3 Since SMA is a rare entity its etiology has r e m a i n e d controversial. Slavin p r o p o s e d an autoimm u n e cause in his first report ~ but more recently concluded that SMA is a result of an inappropriate vasospastic response expressed in a splanchnic vascular bed u n d e r g o i n g vasoconstriction resulting from shock or severe hypoxemia. 2 The present s t u d y was u n d e r t a k e n to examine the role of a u t o i m m u n e m e c h a n i s m s in the pathogenesis of SMA using electron microscopic and i m m u n o histochemical methods.
Materials and Methods Patient Specimens of gastroepiploic artery a n e u r y s m s (Fig. 1)
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Fig. 1. Aneurysm of the gastroepiploic artery. w e r e obtained from a 73-year-old lady w h o und w e n t mitral valvular replacement in O u l u Univer~, Hospital. She had u n d e r g o n e surgery 3 years bef, admission for r u p t u r e of an omental artery ane ysm. In the follow-up arteriography several viscc a n e u r y s m s w e r e d e m o n s t r a t e d , one of those bein£ the gastroepiploic artery. 4 Control stainings were p e r f o r m e d for two g troepiploic artery specimens obtained from patie (male 67-year-old and female 70-year-old) w u n d e r w e n t total gastrectomy as a treatment for g tric cancer.
0950-821X/9ad010070+08$08.00/0(~) 1994W. B. Saunders Company Ltd.
Segmental Mediolytic Arteritis
Histological procedures The artery specimens were divided into 2 mm long segments that were frozen in liquid nitrogen and stored at -80°C or fixed in 4% neutral-buffered formaldehyde, dehydrated and embedded in paraffin. The following stains were employed for routine histology: hematoxylin-eosin, Verhoeff's elastica and van Kossa. For electron microscopy some segments of the specimens were also fixed in a glutaraldehydeformaldehyde mixture (1% glutaraldehyde, 4% formaldehyde, 0.1 M phosphate buffer, pH 7.4) and postfixed with osmium-tetroxide. Thin sections of 100 nm were stained with uranyl acetate-lead citrate and used for transmission electronmicroscopy. Unstained thin sections were used for X-ray microanalysis which was performed using a Jeol 100 CX transmission EM equipped with energy dispersive Xray spectrometer (Link, AN 10/25 S).
Immunohistochemistry Direct immunoyquorescence 5 ~m thick frozen sections of the specimens placed on clean object slides were air-dried for 30 min, rinsed in phosphate buffered saline (PBS) for 30 rain and incubated at room temperature with FITC-conjugated rabbit antibodies to human immunoglobulins (IgA, IgG and IgM), complement C3a and fibrinogen (Dakopatts, Glostrup, Denmark). The antibodies were diluted 1:5 in PBS. Then the sections were washed three times (10min each) in PBS and mounted in Shandon mounting liquid (Immu-Mount, Pittsburg, PA, U.S.A.). Sections were examined with fluorescence microscopy and photographed on black and white negative film (Kodak T Max p 3200).
washed in PBS for 30 min, and were finally examined and photographed as above. All incubations were carried out at room temperature.
Avidin-biotin (AB) complex method The immunohistochemical staining was performed using the slightly modified AB Complex/HRP method of Dakopatts. In brief, the steps of the staining were as follows: the paraffin sections of the artery specimen were deparaffinised and rehydrated. Then the sections were treated with 3% H202 in methanol for 5 rain, rinsed in PBS and incubated with swine serum (Dakopatts) diluted 1:5 in PBS for 20 min. Incubation with primary antibodies was identical with that of the IIF-method. The sections were washed in PBS for 30 min and incubated at room temperature with biotinylated swine anti-rabbit immunoglobulins (Dakopatts) diluted 1:400 in PBS for 30min. After being rinsed in PBS the sections were treated with AB complex/HRP prepared according to the instructions of the manufacturer at room temperature for 30 min and rinsed in PBS. The antigenic sites were visualised using 3,3'-diaminobenzidine tetrahydrochloride and H202.
Confocal laser scanning microscopy The sections were also examined with a confocal laser scanning microscope (Leitz CLSM, Leica Laser technichs, Germany) using an air-cooled argon-krypton laser (75 mW output). The specimens were excited with a laser beam at a wavelength of 568 nm and the emission light was focused through a pinhole aperture. The full field of view was scanned in square image formats of 512 x 512 pixels and a built in software system was finally used to reconstruct the images obtained from the confocal sections.
Indirect immunoJluorescence (IIF) The IIF-method was performed according the ordinary Coon's method 5 with some modifications. 6 Frozen sections placed on clean object slides were airdried for 30 min, rinsed in PBS for 30 min and incubated with swine serum (Dakopatts) diluted 1:5 in PBS for 20min. Then the sections were incubated with primary antibodies to human IgA, IgG, IgM, factor VIII (Dakopatts) or with normal rabbit serum diluted 1:50 in PBS for 30 min. After washing in PBS for 30min, the sections were incubated with FITCconjugated swine anti-rabbit antibodies (Dakopatts) diluted 1:50 in PBS for 30 min. Then the sections were
On gross examination the excised artery specimen was unevenly thickened (Fig. 1). Histologically the intima showed diffuse thickening without any atherosclerotic changes (Fig. 2). The internal elastic lamina p a r t y disappeared and showed reduplication and distortion (Fig. 3). The medial layer was thin (Fig. 3) and showed patchy "lytic" degeneration of smooth muscle cells (Fig. 4). In addition, focal vascularisation was observed in media (Fig. 5). Both ABC-complex Eur J VascSurg Vol 8, January 1994
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Fig. 2. Diffuse intimal thickening of gastroepiploic artery. I, intima; M, media. Hematoxylin and eosin stain, x40.
Fig. 4. Vacuolar degeneration of medial smooth muscle cells (arrows). Note the preserved internal elastic lamina (arrowhead). Hematoxylin and eosin stain, x310.
Fig. 3. Reduplication and distortion of internal elastic lamina (arrow). Note the thick intima and thin media, and the lack of internal elastic lamina on the lower side. I, intima; M, media. Verhoeff's elastica stain, x 180.
Fig. 5. Slit-like vascular space inside the media. Note positive staining of endothelial cells for factor VIII (arrow head), PAP, x420.
a n d i m m u n o f l u o r e s c e n c e m e t h o d s s h o w e d positive s t a i n i n g for factor VIII in the e n d o t h e l i a l cells of n e o vessels (Figs 5 a n d 6). N o distinct h a e m o r r h a g e w a s f o u n d in the arterial wall or adventitia. A n i m m u n o -
histochemical staining using anti-human macrophage a n t i b o d i e s ( D a k o p a t t s ) s h o w e d the p r e s e n c e of s o m e m a c r o p h a g e s in t h e m e d i a l layer (data n o t s h o w n ) . In o n e of a p p r o x i m a t e l y 10 l i g h t m i c r o s c o p i c sections
Eur J Vasc Surg Vol 8, January 1994
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Fig. 7. Electron micrograph showing degenerating smooth muscle cell with fine granular material containing vacuole (arrowhead). Notice normal smooth muscle cell on the right (arrow), x48 000.
Fig. 6. CLSM also revealed distinct determinants for factor VIII in the endothelial cells while the subendothelial layer was negative. Voltage 656, Pinhole 128, Offset -40. Bar 10 p~m.
examined, focal calcium deposition was present in the media near the internal elastic membrane. On electron microscopy, medium dense homogenous material was found in the media associated with single degenerating smooth muscle cells (Fig. 7).
This material could probably represent immunoglobulin deposits. In addition, focal electron dense granular material was present in the medial extracellular space among vesicle-like membranous structures (Fig. 8a) which seemed to be membrane remnants resulting from cell death. X-ray microanalysis showed the extracellular granular material to be calcium (Fig. 8b). The frozen artery sections were studied for h u m a n immunoglobulins IgA, IgG and IgM, comple1 0 0 s : R e m ~ i ni ng= Dead
10,9 > 20 cl;s
MEN I :SOR I Fig. 8. (a) Electron micrograph showing electron dense material (arrowheads) in extracellular space in the vicinity of vacuolar structures representing cell remnants, x23 000; (b) Energy/dispersive X-ray spectrum of the electron dense material showing peaks of calcium (arrow). Other peaks originate from preparation procedures and the supporting grid. Eur J Vasc Surg Vol 8, January 1994
D Fig. 9. Direct immunofluorescence. (a) Fine granular immunoreactive deposits are associated with degenerated and tortuous inte elastic lamina (IEL) and with external elastic lamina (EEL) when anti-human IgG antibodies were used. Note IEL also gives br autofluorescence, x500. (b) In anti-IgM staining numerous fine granular immunoglobulin deposits are seen in the thickened organised infima (zone between arrowheads), x500. (c) Fine granular IgM-material (arrowheads) associated with the degenen internal elastic lamina (IEL + arrow). (d) Fine granular IgM-material (arrowheads) associated with the degenerated external elastic lan (EEL) and to the endothelium of neovessels (NV) of the degenerated media (M), x 250. (e) Anti-C3 staining demonstrated fine gran immunoreactive material (arrowheads) associated with the internal elastic lamina (IEL) and in the walls of neovessels of the degenen media (M). (f) In the anti-fibrinogen staining two thin linear bands (arrowheads) were present in the old and new "endothelium" of thrombous area of the arteria (L = lumen), Eur J Vasc Surg Vol 8, January 1994
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m e n t C3, fibrinogen and factor VIII using direct a n d indirect immunofluorescence techniques. Anti-IgG antibodies revealed distinct immunoglobulin deposits attached to the degenerated internal elastic lamina (IEL) and the external elastic lamina (EEL) (Fig. 9a). In anti-IgM staining fine granular i m m u n o globulin deposits were present in the intima layer as a moderately a b u n d a n t zone (Fig. 9b). In addition, fine granular fluorescence for IgM was associated with the degenerated IEL, EEL and the endothelium of neovessels of the media (Fig. 9c and d). In anti-C3a staining a moderately fine granular immunoreactive material was associated with IEL. Furthermore, C3apositive fluorescence was present in the neovessel walls of the lytic media as a b u n d a n t deposits as in the anti-IgM staining (Fig. 9e). Fibrinogen was seen in the lyric media and it was also associated with IEL a n d EEL. The positive fluorescence was clearly granular and present in thin linear bands of "old a n d
Fig. 10. (a) Indirect anti-lgG staining with the avidin-biofinconjugated method (ABC-complex)shows IgG-immunoreactivematerial (arrows) associated with the degenerated internal elastic membrane. (b) Control staining is negative. The degenerated internal elastic membrane can be seen (arrows), x250.
n e w " endothelium (Fig. 9f). Anti-IgA staining and all control stainings were negative (data not shown), Indirect anti-IgG staining using ABC-complex m e t h o d also s h o w e d distinct immunoglobulin deposits associated with IEL while the control staining with normal rabbit serum was negative (Fig. 10a and b).
Fig. 11. Immunofluorescencestainings of IgG. (a) indirect immunofluorescence and IgM; (b) direct immunofluorescence deposits in the aneurysmatic tissue studied by CLSM.Note that the fluorescence of IgG is most distinct in the surface of the IEL (arrows). Anti-IgM staining showed positive fluorescence in the intima layer. The staining pattern was clearly granular. Voltages 664 (a) and 888 (b), pinhole 128, offset -127, Bars 10 ~m. Eur J Vasc Surg Vol 8, January 1994
T. Juvonen etaL
Using CLSM the positive fluorescent deposits for IgG were clearly present in the degenerated internal elastic membrane (Fig. 11a). The immunoglobulin deposits seemed to be located in the surface of the lamina. A distinct immunogenicity for anti-IgM was detected in the organised intima layer where the fluorescence pattern was clearly granular (Fig. 11b).
The present study gives further support to the role of autoimmune factors underlying the extremely rare but distinctive arterial lesion called "segmental mediolytic arteritis" (SMA), which is confined to a small number of topographically related arteries within the abdomen or heart, large muscular arteries being the primary targets. 1,2 Earlier studies have indicated that SMA histologically represents a vascular lesion of a non-atherosclerotic and non-inflammatory nature. 1-3 The main morphologic characteristic of SMA is medial disappearance through an apparent lytic process. This is apparently initiated by the development of an interconnection maze of vacuoles in the smooth muscle cells. The disruptions of these vacuoles and cell membranes, with concomitant loss of their fluid contents, complete the lytic process. We suggest that calcium deposits near vacuoles, which were demonstrated in this study, indicate destruction of medial muscle cells. Transmural arterial lesions develop when the lesion is transmural and associated with the focal removal of the internal elastic lamina and intima. These transmural "gaps" may predispose towards sudden massive haemorrhage or the formation of aneurysms. In relation to the pathogenesis of SMA, Slavin et al. first suggested the autoimmune origin. 1 Since the histological features of autoimmune diseases traditionally include inflammatory reactions created by autoimmune complex precipitations between a selfantigen and antibodies, the non-inflammatory histological picture of SMA has led to the excluding of such possibilities as causative agents underlying SMA. Recently, Slavin et al. have suggested a vasospasm and consequent vascular injury as a mechanism for SMA. 2 This hypothesis implies an episode of hypoxia or shock some time before the above-mentioned pathological changes would occur. In previous reports, however, no attempts have been made to determine the occurrence of either immunoglobulin precipitants or complement in fresh samples in a prospective manner. A likely explanation for this is Eur J Vasc Surg Vol 8, January 1994
the fact that SMA is an extremely rare clinical condition and previous reports mainly concern autopsy material. Because multiple visceral aneurysms had previously been found in our patient, 4 it was possible to design a prospective immunohistochemical study with fresh histological samples. Immunofluorescence stainings with both conventional, microscopy and CLSM showed immunoglobulin deposits in the degenerated or fragmented elastic membranes and in the intima layer. A recent study of nonspecific abdominal aortic aneurysm disease could also demonstrate the presence of large amount of immunoglobulins in soluble extracts and, furthermore, an inflammatory filtrate located at the junction of the media and adventitia. 7 An immunologically mediated injury is also supported by the fact that antigen determinants for C3a were present together with IgG in the internal elastic membrane in the neovessel walls of the lytic media. The initial trigger for the autoimmune cascade remains unclear. The present findings could be explained by previous results indicating that certain elastin derived peptides are highly immunogenic and tissue damage could be initiated by interactions between microfibrillar proteins and such elastin peptides. 8 This subsequently leads to elastin degradation, further release of elastin-derived chemotactic peptides and tissue damage. 9-11 One aim of our future studies is to elucidate the possible role of these chemotactic peptides in the pathogenesis of SMA.
References 1 SLAvmRE, CONZALEs-VrrALEJC.Segmentalmediolyticarteritis. A clinical pathologic study. Lab Invest 1976; 35: 23-29. 2 SLAVlN RE, C ~ E R a ~ L, CARTWRIGHTJ. Segmental mediolytic arteritis. A clinicopathologic and ultrastructural study of two cases. Am J Surg Pathol 1989; 13: 558-568. 3 HERITZDM, JAGDIS, B, JOHNSTONKW, SmDERMANKW. Intraabdominal hemorrhage as a result of segmental mediolytic arteritis of an omental artery: Case report. J Vasc Surg 1990; 12: 561565. 4 JUVONENT, NIEMEL.~O, REINIL-x.A, NISSINENJ, KAIRALUOMAMI. Spontaneous intra-abdominal haemorrhage caused by segmental mediolytic arteritis in a patient with systemic lupus erythematosus--an underestimated entity of auto-immune origin? Eur J Vasc Surg 1994; 8: 96-100. 5 COON AH, CREECH HJt JONES RN, BERLINERE. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 1942; 4,5: 159-170. 6 R.~-S;4NENO, J.~,RVINENM, RINNE A. Localization of the human SH-protease inhibitor in the epidermis. Acta Histochem 1978; 63: 193-196. 7 BROPHYCM, REILLYJM, SMITH GJW, TILSON MD. The role of inflammation in nonspecific abdominal aortic aneurysm disease. Ann Vasc Surg 1991; 5: 229-233. 8 CLEAR¥ EG, GIBSON MA. Elastin-associated microfibrils and microfibrillar proteins. Int Rev Connect Tissue Res 1983; 10: 97209.
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9 WRENN DS, GRIFFIN GL, SENIOR RIM,MECHAMRP. Characterization of biologically active domains on elastin: identification of a m o n o d o n a l antibody to a cell recognition site. Biochemistry 1986; 25: 5172-5176. 10 SENIOR RM, GRIFFIN GL, MECHAM RP. Chemotactic activity of elastin-derived peptides. J Clin Invest 1980; 66: 859-862.
11 SENIORRM, GRIFFIN GL, MECHAM RP, WRENN DS, PRASADKU, URRY DW. Val-gly-val-ala-pro-gly, a repeating peptide in elastin, is chemotactic for fibroblast and monocytes. ] Cell Biol 1984; 99: 870-874.
Accepted 31 August 1993
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