ORIGINAL ARTICLES

Microvascular Changes in Early and Advanced Dermatomyositis: A Quantitative Study Alison M. Emslie-Smith, MB, PhD, and Andrew G. Engel, MD

In adult dermatomyositis 10 muscle specimens with no or minimal histological alterations were compared with 7 that showed typical alterations. Five specimens from patients with inclusion body myositis, 5 from patients with polymyositis, and 8 from normal subjects served as controls. Vascular endothelium, visualized with the lectin Ulex europaeus agglutinin I, and complement membrane attack complex were demonstrated in the same cryostat sections by paired immunofluorescence. Large randomly selected fields were analyzed to determine the number of capillaries per square millimeter of fiber area (capillary density), per 1,000-pn2 area of each muscle fiber (capillary index), and in 100 x 100pm grid squares. In dermatomyositis specimens with minimal structural alterations there was focal capillary depletion, the capillary density was significantly reduced, and the frequency distributions of the capillary index and grid count were shifted to the left. In advanced dermatomyositis specimens, the findings were similar but more severe. In both kinds of specimens, clusters of capillaries reacted for complement membrane attack complex. The 2 patients with the highest proportion of vessels positive for membrane attack complex had a fulminant and fatal course. In polymyositis and inclusion body myositis specimens, the capillaries had a normal overall density and none reacted for membrane attack complex. The findings imply that the capillaries are an early and specific target of the disease process in dermatomyositis. Emslie-Smith AM, Engel AG. Microvascular changes in early and advanced dermatomyositis: a quantitative study. Ann Neurol 1990;27:343-356

Several lines of evidence implicate the muscle vasculame in the pathogenesis of dermatomyositis (DM). As early as 1912, Batten described infiltration of vessel walls with lymphocytes, thickening of the vessel walls, and “in some cases actual obliteration of the vessels” [l]. These findings were further extended by Banker and Victor, who emphasized the central role of arteritis and phlebitis in muscle and other tissues in childhood D M [2]. Subsequently, in an ultrastructural study of childhood DM, Banker found that the major abnormalities in muscle were in the endothelial cells of capillaries, arterioles, and veins, and suggested that these were primary alterations 131. Subsequently, Carpenter and co-workers confirmed these observations, also demonstrated a decreased capillary density in childhood DM, and suggested that the damage to muscle fiber and capillaries was coextensive [4]. There is also evidence that immune mechanisms contribute to the vasculopathy of DM. “Immune complexes” ( 1 6 , IgM, and complement C3) have been detected in the walls of intramuscular venules and arterioles in children and adults with DM [ 5 ] , and the C5b-9 complement membrane attack complex (MAC)

has been immunolocalized in intramuscular arterioles and capillaries in 10 of 12 children and 5 of 19 adults with DM [b}. However, none of the conventional morphological o r immunocytochemical studies have considered the earliest alterations in muscle in adult DM. In adults with DM, we recently detected ultrastructurd abnormalities in endomysial capillaries in muscle specimens that showed minimal or no structural abnormahties in muscle fibers and contained few or no inflammatory cells. A proportion of the endothelial cells harbored microtubular inclusions, displayed small vacuoles, or were swollen [7].These findings implied that the microvasculature is an early and specific target of the disease process in muscle. If this were so, then one would also expect that capillary depletion would occur in the earliest phases of the disease. However, the samples examined in the electron microscope proved too small for obtaining reliable estimates of capillary density and distribution. Therefore, we tested the hypothesis that capillary loss is an early event in DM by determining the overall density and spatial distribution of capillaries in minimally involved muscles

From the Muscle Research Laboratory, Mayo Clinic, Rochester, MN.

Address correspondence to Dr Engel, Mayo Clinic, Rochester, M N 55905.

Received Jul 10, 1989, and in revised form Sep 22. Accepted for publication Sep 22, 1989.

Copyright 0 1990 by the American Neurological Association

343

of 10 adults. For comparison, we applied the same analysis to more severely involved muscle specimens from patients with adult DM, polymyositis (PM), and inclusion body myositis (IBM), and to normal muscles obtained from nonweak control subjects. T h e capillaries were visualized in cryostat sections by an immunofluorescence method. This enabled us to colocalize MAC in the same sections and evaluate the proportion and distribution of the MAC-positive vessels. Material and Methods Clinical Material Limb muscles were biopsied for diagnosis in all patients. A total of 15 adults, 12 women and 3 men, with DM were studied. Their ages ranged from 29 to 83 years. All had the typical rash of the disease. The serum level of creatine kinase was increased 1.5- to 18.0-fold in 8 of the 15 patients. Nine patients (DM1-DM3 and DM5-DM10) had minimal or no weakness; their muscle specimens were essentially normal, showing little or no inflammatory or structural change by conventional histochemical criteria. Four patients (DM12DM 15) had moderately severe and predominantly proximal muscle weakness and pathological alterations characteristic of DM {8]. One patient (DM11) had minimal proximal weakness; however, perifascicular muscle fiber injury and a sparse perimysial and perivascular inflammatory infiltrate were present. One patient (DM4) had severe weakness of the upper but no weakness of the lower limbs. A specimen of her clinically weak extensor carpi radialis showed advanced pathological alterations typical of severe DM, but the clinically uninvolved vastus lateralis had only a few regenerating fibers and no inflammatory cells. In one patient (DM8), most of the muscle specimen was normal, but a few fascicles at one edge of the specimen showed perifascicular alterations typical of DM; sparse inflammatory cells were confined to this area. In this patient, the involved and uninvolved portions of the specimen were analyzed separately. Thus, our material consisted of 10 muscle specimens showing no or minimal pathological alterations (for example, Fig 1A) and 7 specimens showing changes typical of DM (for example, Fig lB, C). These specimens will be referred to as early DM (eDM) and advanced DM (aDM) specimens. Fourteen patients with DM had electromyographic studies. All patients had myopathic motor unit potentials; fibrillation potentials were absent in 4 patients with mild disease (DM1, DM6, DM9, and DMIO). Disease control subjects consisted of 5 patients with PM and 5 with IBM. The clinical and pathological criteria for diagnosis have been published previously [9, lOJ. Normal control subjects consisted of 8 patients who had undergone muscle biopsy for diagnosis but were ultimately found to be free of muscle disease by combined clinical, electromyographic, and histological criteria. Ten of 15 patients with DM received no treatment prior to biopsy. Patient DM6 had had a short course of corticosteroids 3 months before biopsy. Patient DM5 received 60 mg/ day of prednisone for 7 days and Patient DM 15 received 80 mg/day for 3 weeks prior to biopsy. Patients DM4 and DM 12 received corticosteroids at varying doses for 5 months and 12 months, respectively. Patients DM4, DM12, and D M l 5

344 Annals of Neurology Vol 27 No 4 April 1990

all continued to deteriorate clinically while receiving treatment. None of the patients with PM were treated prior to biopsy. Only 1 of the 5 patients with IBM was treated with prednisone, 30 mg on alternate days, until a few months before biopsy.

Estimation of Capillaly Density and Distribution in Muscle In pilot studies, we evaluated different methods for measuring the capillary density. Capillary counts in semithin resin sections, employed in previous studies {4, 11, 121, proved unsatisfactory because muscle fibers shrink during fixation, dehydration, and embedding, and the extent of shrinkage varies within and between specimens. A correction formula for fiber shrinkage during preparatory procedures is available 1137 but is unreliable when there is abnormal variation of fiber size. Further, we and others (121 found that collapsed or degenerating capillaries are difficult to distinguish from fibroblasts or mononuclear cells in semithin resin sections. Electron microscopic analysis accurately identified capillaries, but the sample area proved to be too small for reliable quantification, and shrinkage of muscle fibers remained a problem. Cryostat sections of unfixed muscle preserve the diameters of muscle fibers in the native state, but special markers are required for capillary identification. Many animal studies have relied on alkaline phosphatase as an endothelial marker (141, but this enzyme is not consistently present in human capillary endothelium. Muscle capillaries also can be visualized by staining their basal lamina with the periodic acid-Schiff method after diastase digestion {15], but this method is unsuitable in myopathies where capillary basal lamina can persist after the endothelial cells have disappeared, or where there is extensive endomysial fibrosis. Factor VIII-related antigen (161 and the lectin Ulex europaeus agglutinin I (UEA-I) are commonly used as endothelial markers { 17- 191. We found that both markers were equally expressed in capillaries in normal muscle, but UEA-I was expressed more strongly and consistently by capillaries in DM muscle. Therefore, we chose the UEA-I method to visualize the microvasculature. UEA-I and MAC were detected by paired immunofluorescence in the same cryostat sections (Fig 2).

Identification of Terminal Lytic Complement Components in the Microvasculature In other pilot studies we compared the distribution of MAC and complement C9 in normal and pathological muscles.

b

Fig 1 . (A) Muscle specimen without structural alterations in muscle fibers or inflammatory cells from a patient with dermatomyositis. (B) and (C) Specimens from patients with dermatomyositis with structural changes typical of the disease. Compare with corresponding panels in Figure 2, which demonstrate UEA-I and MAC localization in the same specimens. In panel B, note rarefaction and vacuolar change in numerous fibers.Fibers marked with an asterisk correspond to MAC-positivefibers in Figure 28. In panel C , note atrophy and other structural changes in muscle fibers concentrated at the bottom edge ofthe fascicle. (A, hematoxylin-eosin, x 21 0; B, trichrome, X 180; C , hematoxylin-eosin, x 210.)

Emslie-Smith and Engel: Microvasculature in Dermatomyositis

345

Both markers were detected in necrotic muscle fibers, as previously described [207, and all DM capillaries that were MAC-positive were also C9-positive. However, some capillaries in normal muscles that were MAC-negative proved to be C9-positive, indicating that C9 reactivity is not specific for capillary injury. Therefore only MAC was used as a marker for capillary injury.

lmmunoreagents PRIMARY REAGENTS. Unlabeled UEA-I and fluorescein isothiocyanate (F1TC)-labeled UEA-I were obtained from Vector Laboratories (Burlingame, CA). The polyclonal rabbit antibody against human MAC neoantigenic determinants has been previously characterized [201. A monoclonal antibody to human complement C9 (a gift from Dr Peter Taylor, Molecular Diagnostics Inc, West Haven, CT) and FITCconjugated goat antibody to human factor VIII-related antigen (Atlantic Antibodies, Scarborough, ME) were used in pilot experiments. Heat-inactivated normal mouse serum and normal rabbit serum were used in control experiments. SECONDARY REAGENTS. Goat anti-UEA-I, affinity-purified biotinylated horse anti-mouse IgG, and rhodamineavidin D were from Vector Laboratories. Affinity-purified biotinylated swine anti-goat IgG and biotinylated swine antirabbit IgG antibodies were from Boehringer Mannheim Biochemicals (Indianapolis, IN) and Dako Corporation (Santa Barbara, CA), respectively. IMMUNOCYTOCHEMICAL PROCEDURES. All immunoreagents used in fluorescence localizations were diluted in phosphate-buffered saline (PBS) containing 2% bovine serum albumin and 10% heat-inactivated serum from the species in which the second antibody was raised (solution A). All rinses were made in three changes of PBS over a 30minute period. Sections were incubated in humid chambers and, except where otherwise indicated, all incubation steps were carried out at 25°C. All studies were performed on 6-pm-thick serial cryostat sections, mounted on polylysine-coated coverslips. The first section in each series was stained with trichrome for histological correlation. All immunoreacted sections were fixed with ice-cold acetone for 5 minutes and air dried. To reduce nonspecific binding, all sections were preincubated with solution A for 15 minutes. In every case, control sections were treated with a matching concentration of nonimmune immunoglobulin, instead of the first antibody, or were processed without the first antibody. For paired immunofluorescence localization of MAC and UEA-I binding, the following steps were carried out: rabbit anti-MAC (2.5 pdrnl, preabsorbed with solution A containing 2.5% heat-inactivated human AB serum) overnight at 4°C; UEA-I (2.5 pg/ml) for 60 minutes; rinse; goat antiUEA-I ( 5 pg/ml) plus biotinylated swine anti-rabbit Ig (1.75 pg/ml) for 30 minutes; rinse; and FITC-labeled swine antigoat I g G (4.75 pg/ml) plus rhodamine avidin D (0.5 pg/ml) for 30 minutes. After a final rinse, all sections were mounted in a glycerol-based mounting medium containing 1 mg/ml p phen ylenediamine.

346 Annals of Neurology Vol 27 N o 4 April 1990

Data Analysis The sections were examined with a Zeiss photomicroscope equipped with phase optics and with epifluorescence for selective FITC and rhodamine detection. One or more randomly selected fascicles, comprising 0.408 to 1.800 x lo6 pm2 of muscle fiber area from DM samples and 0.575 to 1.620 x lo6 pm2 from other samples, were analyzed. All regions of the areas chosen for analysis were photographed at an original magnification of 63x under phase, FITC, and rhodamine optics. Each photograph was further enlarged with a projector to a final magnification of 444 x . The fiber outlines were then traced onto a transparent sheet and UEA-I-positive vascular profiles and any MAC-positive vascular profiles or MAC-positive necrotic fibers were marked with different colors (see Fig 2C). The sheets were then spliced together to form a montage of the entire sample area. The following were determined in each sample area: (1) the total number of muscle fibers; (2) the area of each fiber, using the formula (3.14 x D x d)/4, where D and d, respectively, are the largest and smallest caliper diameters of the fiber [21, 221; (3) the number of capillaries immediately adjacent to each fiber; (4) the total number of capillaries; (5) the number of capillaries in 100 x 100-pm grid squares superimposed on the entire sample area (grid count); (6) the number of MAC-positive, lectin-positive capillaries; and (7) the number of MAC-positive, lectin-negative capillaries. The following values were derived from these measurements: (1) the number of capillaries per square millimeter of muscle fiber area (capillary density); (2) for each muscle fiber, the number of capillaries per 1000 pm2 of fiber area (capillary index); ( 3 ) the frequency distribution of the capiIlary index; (4) the frequency distribution of capillaries per grid square; (5) the proportion of lectin-positive capillaries reacting for MAC; and (6) the proportion of all MAC-positive capillaries that were lectin-negative. ~

~~

Fig 2. Paired immunofEuorescencelocalization of UEA-I and MAC in dermatomyositis muscle without structural alterations in muscle fibers or infEammatoorycells (A) and demratomyositis muscle with advanced structural changes (B).Panel A is a higher power view of a part of the fascicle depicted in Figure IA. Panel B shows the same field as Figure I B in an adjacent section. Panel C is a tracing of an immunoreacted section adjacent to that shown in Figure I C with a superimposed 100 x I O O - p n grid. Green, yellow, and red capillaries indicate MAC-negative kin-positive, MAC-positive lectin-positive, and MAC-positive lectin-negative vessels, respectively. In panel A , note capillaty with MAC reactivity exterior to lectin reactivity (arrowheads) and MAC-positive lectin-negative capillaries (arrows). There are abnormal variations in the intensity of the green lectin reaction in diflmnt capillaries.In panel B, most capillaries display yellow dual fEuorescencefor lectin and MAC; few capillaries react only for MAC (arrowhead). Note two necrotic musclefibers showing intense red MAC reaction. Both fibers have adjacent capillaries in the plane of sectioning. The fiekdshown in panel C represents only a small part of the sampled area. Here note seueral vessels, including a venule (arrowhead), reacting for both lectin and MAC, and two capillaries reactingfor MAC only. (A, x 290; B, x 180; C, x 230.)

Emslie-Smith and Engel: Microvasculature in Dermatomyositis

347

Table I . Quantitative Analysis

of Muscle Capillaries”

Sample Type

Samples (no.)

Capillary Densityb

Capillary Index‘

Normal control Early dermatomyositis Advanced dermatomyositis Polymyositis Inclusion body myositis

8 10 7

393 f 28.8 275 ? 43.7d 217 2 23.gd*‘ 47.2 372 473 ? 111.5

1.005 f 0.041 0.738 ? 0.096d 0.575 ? O.15SdSf 1.067 k 0.158 1.175 2 0.267

*

5 5

“Values given are the mean +. SD. bNumber of capillaries per mm2 muscle fiber area Capillary density of each sample is shown in Figure 3 . ‘The capillary index for a given muscle fiber is the number of capillaries per l,OOO-pmz fiber area One hundred thirty-three to 651 capillary indices were determined in the individual samples. For each disease group, the table indicates the overall mean rt SD of the sample means in that group. The sample means are shown in Figure 4. dSignificantlydifferent from normal control values by Student’s two-tailed t test at p < 0.001. ‘significantly different from early derrnatomyositis values by Student‘s two-tailed I test at p < 0.01. ‘Significantly different from early derrnatomyositis values by Student‘s two-tailed t test at p < 0.02.

7001

Capillary density

N \ b .L

0

++

600{

1.81

Capillary indices

1.6

9

0

+

OO1

I

5

1

N

+

1.01

0.8.

+

*

+

* -4-

4

a yzoO~ 0

\

B (u

0.6-

4

b

4 4

If

loo1

04

8

N

I

eDM

I

aDM

I

IBM

I

PM

0.41 I

0.21

G G 0 N eDM aDM IBM PM

Fig 3 . Scatter diagram of capillary densities in muscle specimens from normal controls (N), dermutomyositis with no or minimal pathological changes (eDM), dermatomyositis with advanced changes (aDM), inclusion body myositis (IBM), andpolymyositis (PM).

Fig 4. Scatter diagram of mean capillary indices in muscle specimens from normul controls (N), dermatomyositis with no or minimalpathological changes (eDM), dermatomyositis with advanced changes (aDM), inclusion body myositis (IBM), and polymyositis (PM).

Differences between distributions were compared by Smdent’s two-tailed t test and by the rank-sum test. Differences between proportions were evaluated by chi-square comparison.

Results N o m l Controls

samples, only rare fibers (0.13%) were without adjacent capillaries and none of the 104-pm2grid squares were devoid of capillaries. The frequency distributions of the pooled capillary indices (Fig 5) and grid counts (Fig 6) were close to gaussian. Figure 7A shows the localization of capillaries in normal muscle. None of the normal capillaries were immunoreactive for MAC.

The mean capillary density (number per square millimeter of muscle fiber area) was close to 400 (range, 348-435) (Table 1 and Fig 3). The mean capillary index was 1.0, with a relative standard error of only 4% (Table 1 and Fig 4). This index was essentially independent of age, sex, or mean fiber diameter. In all

Earb Dermutomyositis In 9 of 10 eDM samples the capillary density fell below the normal range; in one sample (DM2), the density was at the lower limit of normal (see Fig 3). The mean capillary density was 70% of the normal control

348 Annals of Neurology Vol 27 No 4 April 1990

Control

I

I

PM

-. -.

29

1

.

.

.

.

.

.

.

.

.

,

.

.

r U r U m m 3 - 3 -

a DM

111

.

.

- - r w r u m r l J - J -

Capi 11a r y Index

Capi 11a r y Index Fig 5 . FwquenLy histograms of capillary indices in muscle specimens from normal controls (N), &rt,wtomyositis with no or minimal structural changes (eDM), dermatomyositis with advanced changes (aDM), inclusion body myositis (IBM), and polymyositis (PM).

value, and the difference between the two means was highly significant (see Table 1). The mean capillary index was also significantly reduced (see Table 1 and Fig 4), and the frequency distribution of the index was shifted to the left. About 10% (range, 2.1-23.0s) of the fibers had no adjacent capillaries; that is, they had a capillary index of 0 (see Fig 5). The frequency distribution of the grid counts was also shifted to the left, and close to 10% (range, 2.1-19.0%) of the 104-pm2grid squares had no capillaries (see Fig 6). Figure 7B illustrates the focal capillary dropout in eDM muscle. Nine of 10 eDM specimens contained MAC-positive vessels in the sample area (Table 2). In 8 samples, 0.8 to 9.3% of the lectin-positive capillaries were

MAC-positive (Figs 2A and 8), and these vessels characteristically occurred in small groups or clusters. In patient DM10, 62.6% of the lectin-positive capillaries were MAC-positive. This patient had no weakness at the time of the biopsy but subsequently died of fulminant disease. In Patient DM7, the randomly selected sample area had no MAC-positive vessels, but two other areas contained clusters of MAC-positive vessels (see Fig 2A). In 8 of 10 eDM samples a few small venules or arterioles also reacted for MAC (see Fig 8). Finally, in 7 of 10 eDM samples there were MACpositive, lectin-negative profiles in the endomysium that resembled capillaries in their size, shape, and position within the fascicles. These vessels accounted for 5 to 74% of all MAC-positive capillary profiles (see Table 2). Often, MAC-positive lectin-positive and MAC-positive lectin-negative vessels occurred in the same region (see Fig 2A).

Emslie-Smith and Engel: Microvasculature in Dermatomyositis 349

I

eDM

-l

N

U

J-

1L PM

Lo

E

m

3-

Lo

3-

ul

D

!?

m

F?

aDM

Cap1 11 aries/lO 4 pm2 Fig 6. Frequency histograms of capillary grid counts in muscle specimens from normal controls (N), dewtomyositis with no or minimal pathological changes (eDM), demtatomyositis with advanced changes (aDM), inclusion body myositis (IBM), and polymyositis (PM). Each grid square corresponds to an area of 104 pm?

Advanced Dermutomyositis

In all 7 aDM samples, the capillary loss was more marked than in the eDM samples (Fig 9). Th'1s was reflected by significantly greater decreases of capillary density and index in the aDM than in the eDM samples (see Table 1 and Figs 3-6). It was of particular interest to note that 32% of the fibers (range, 7.454.4%) had a capillary index of 0 (see Fig 5), and about 21% (range, 10.5-34.6s) of the 104-km2grid squares contained no capillaries (see Fig 6). No consistent correlation was observed between zones of capillary depletion and perifascicular atrophy.

350 Annals of Neurology Vol 27 No 4 April 1990

Capillaries/lO 4 urn2 Six of 7 aDM samples contained MAC-positive capillaries; in these samples 4.3 to 38.9% of the lectinpositive capillaries were MAC-positive, and MACpositive lectin-negative capillaries accounted for 9 to 74% of all MAC-positive capillaries (see Table 2 and Fig 2B, C). As in the eDM samples, the MAC-positive vessels occurred in clusters. The mean percentages of MAC-positive vessels in the aDM and eDM groups were not significantly different. However, the proportion of lectin-positive vessels reacting for MAC was significantly higher in the involved than in the uninvolved muscle in Patient DM4 and in the involved versus uninvolved region of the muscle studied in Patient DM8 (see Table 2). It was again of interest that the patient with the highest proportion of MACreactive lectin-positive capillaries (DM 15) had a fulminant and eventually fatal course. Four aDM samples also contained a few MAC-reactive arterioles or venules (see Fig 2C). Here the MAC deposits were not

Fig 7. Capillaries in n o m l muscle (A) and in dematomyositis muscle without structural change (B) visualized Sy the UEA-I mthod. Regions of Capillary depletion in panel B are marked Sy X. (A, x 250; B, X 170.)

associated with inflammatory cells or recognizable structural alteration (see Fig 1C). However, in the sample from Patient DM12, a small artery and adjacent arteriole and one other arteriole were clearly occluded. MAC reactivity was present in the mural elements of each of these vessels and in the occluding thrombi (Fig 10). Inclusion Body Myositis and Polymyositis In PM specimens, the mean capillary density and index were similar to those observed in normal control samples. In IBM specimens, these values were higher than those in normal control samples, but the differences were not significant (see Table 1, Figs 3 and 4). The higher IBM means were due to increased values in 2 patients (IBM1 and IBM5). A region of increased capillary density in Patient IBM5 is illustrated in Figure 11A. In both PM and IBM specimens, the capillary distribution showed greater than normal regional variation. A small proportion of the fibers, 2.4% in PM and 1.1% in IBM specimens, had a capillary index of 0 (see Fig 5). Many of these fibers, however, were highly atrophic and fell in grid squares that were not depleted

of capillaries. A small proportion of the grid squares, 3.1% in IBM and 1.6% in PM samples, had no capillaries (see Fig 6). There were two reasons for the empty grid squares: (1) in both diseases there was an uneven distribution of capillaries (Fig 11A, B); (2) large fibers, present more frequently in IBM than in PM specimens, occupied entire grid squares. Finally, the frequency distributions of the capillary index in PM and IBM specimens and of the grid count in IBM specimens were skewed to the right, consistent with focal hypervascularization (see Figs 5 and 6). None of PM or IBM vessels bound MAC. Other Observations In each disease, sections reacted with UEA-I showed a few microvessels with thick walls. This was particularly prominent in Patients PM2 (Fig 11B) and PM4 (Fig 12). In Patient PM4, this alteration was confined to a single fascicle.

Discussion Pathogenetic Implications for Demzatomyositis CAPILLARY DEPLETION. The present study demonstrates capillary depletion in adult DM muscles that are not weak on examination and lack the conventional pathologic criteria required for the diagnosis of DM. The fact that capillary depletion can precede structural changes in muscle fibers and the appearance of infiam-

Emslie-Smith and Engel: Microvasculature in Dermatomyositis 35 1

Table 2. Frequencies of CapiClaries Reactive for the Lectin UEA-I and for MAC

Patient No.

Early DM samples DM1 DM2 DM3 DM4, VL DM5 DM6 DM7" DM8, unaffected DEL DM9 DMlO Total Mean % 2 SD Advanced DM samples DM4, ECR DM8, affected DEL DM1 1 DM12 DM13 DM14 DMl5 Total Mean % ? SD

UEA-I+ (no.) 183 633 320 457 238 120 30 1 24 1 269 246 3,008

-

184 184 331 298 215 124 175 1,511

-

MAC+ (no.) 22 27 47 43 25 10 0

2 7 162 345

-

48 27 32 106 62 0

96 37 1

-

UEA-I+MAC+ (no.) 17 9 22 11 19 4 0 2 7 154 245

-

23 8 29 28 42 0 68 198

-

UEA-I-MAC" (no.) 5 18 25 32

6 6 0 0 0 8 100

UEA-I + MAC UEA-I+ (%) 9.3 1.4 6.9 2.4b 8.0 3.3 0.0 0.8' 2.6 62.6

(%I

-

-

9.7

25 19 3 78 20 0 28 173

12.5b 4.3' 8.8 9.4 19.5 0.0 38.9

2

18.8

2

31

&

30

&

29

52 70 9 74 32 0 29

-

13.3

UEA-I -MAC + MAC'

23 67 53 74 24 60 0 0 0 5

-

-

+

-

12.8

38

"Randomly selected sample area contained no MAC-positive vessels, but two other areas contained MAC-positive vessels. bProportions significantly different by chi-square comparison at p < 0.001. 'Proportions significantly different by chi-square comparison at p < 0.02.

VL

= vastus lateralis; DEL = deltoid; ECR = extensor carpi radialis; DM = dermatomyositis; UEA-I complement membrane attack complex.

matory cells implies that the capillaries are an early target of the disease process. We and others have found no capillary depletion in PM specimens C12, 231 or IBM specimens [ 2 4 ] . Conversely, another study has demonstrated T-cell-mediated cytotoxicity in PM and IBM, but not in DM [9]. These findings are strong evidence that the basic pathological process in DM is inherently different from that in PM or IBM and militate against considering DM and PM as closely related disorders. At first, it may seem surprising that capillary loss can occur in DM without clinical weakness or structural changes in the muscle fibers. Animal studies of the muscle microcirculation in vivo, however, have demonstrated that blood flow in most individual capillaries is intermittent at rest and during low-frequency stimulation 1251. Thus, only a proportion of the capillary bed is perfused at any one time, and there is a considerable circulatory reserve in muscle. Enlargement of the capillaries in DM, noted in a previous quantitative ultrastructural study C261, may be a response to increased blood flow through the remaining capillaries. As noted previously, 4 of the 15 patients with DM had been exposed to corticosteroids at various times before muscle biopsy. However, this is unlikely to 352 Annals of Neurology Vol 27 No 4 April 1990

=

Ukx europaeus agglutinin 1; MAC =

have affected the capillary density and distribution, because the values obtained in these patients were randomly distributed about the mean values.

MAC REACTIVITY. The fact that MAC-reactive capil-

laries were present in 10 of 10 eDM and 6 of 7 aDM muscle specimens implies that MAC is a participant in both the earliest and later stages of the disease. There was no significant difference between the proportion of MAC-reactive vessels in the two DM groups. However, the 2 patients with the highest proportion of MAC-reactive vessels (DM10, 6396, and DM15, 39%) had a fulminant and eventually fatal course. In our samples there were marked regional variations in the incidence of MAC-positive vessels within and between fascicles. Therefore, estimates of the incidence of MAC-positive vessels are subject to sampling error. Nevertheless, the proportion of adults with DM with MAC-reactive vessels in our series is significantly greater than that observed by Kissel and co-workers C61. The precise significance of MAC deposition in DM capillaries is still not known. In some capillaries, the MAC deposits surrounded rather than coincided with the lectin-binding endothelial cell surfaces (see Fig

Fig 8. Paired immunofluorescence localization of UEA-I (A) and MAC (B) in dermatomyositis muscle without structural change. Note MAC-positive Capillaries (arrowheads)and venule in paneZ B. Tiny subsarcolemmd white dots in panel B represent autofluorescent lipofuscin granules. ( x 250.)

2A). This could be consistent with immune complexes lying outside the vessel. In other capillaries, the MAC and lectin deposits coincided (see yellow capillary profiles in Fig 2B). This appearance could be consistent with activation of the classic complement pathway by antibodies bound to microvascular components, or activation of the alternative complement pathway by an altered cellular component. MAC deposits were also observed in profiles that resembled capillaries in size, shape, and position but did not bind UEA-I. These formations may represent necrotic endothelial cell remnants, or residual capillary basal lamina (capillary ghosts) associated with immune complexes. Ultrastructural immunolocalization studies will be required to establish the structural concomitants of MAC positivity in DM capillaries. The clustering of MAC-reactive vessels, also noted by Kissel and co-workers [b},represents an interesting but still unexplained feature of both eDM and aDM. The following possible causes could be considered: (1) ischemic necrosis of capillaries induced by spasm or occlusion of a feeding vessel upstream of the capillary bed, followed by MAC accumulation in necrotic endothelial cells; (2) a selective deposition of immune complexes favored by local blood flow conditions; and

(3) selective regional expression on endothelial cells of antigens recognized by complement-fixing antibodies, which would trigger the classical complement pathway, or of altered cellular components, which would trigger the alternative complement pathway. Observations in IBM and PM

Although the mean capillary density in PM and IBM did not differ significantly from that of normal control values, the capillary density was noticeably increased in Patients IBM1 and IBMS. An increase in capillary density has been previously observed in IBM [24}. The frequency distributions of the capillary indices and grid counts revealed an excessive number of high and low values. This finding is explained partly by small hypervascularized and hypovascularized regions in the muscle samples (see Fig ll), and partly by the presence of very small and very large muscle fibers (see Results). An uneven distribution of muscle capillaries in IBM and PM also could stem from remodeling of the microvascular bed by cycles of capillary loss and proliferation accompanying cycles of muscle fiber injury and regeneration.

Methodological Considmations Visualization of capillaries with the UEA-I lectinbinding method in cryostat sections of muscle has obviated problems associated with previous studies [4, 11, 21, 22}. Our method can distinguish fibroblasts or mononuclear cells from small or collapsed capillaries,

Emslie-Smith and Engel: Microvasculature in Dermatomyositis 35 3

Fig 9. Capillary depletion in advanced dermutomyositis. The blood vessels are visualized 6v the UEA-I reaction. Panel A is printed with negative contrast, so that theJuorescent vessels appear bluck and the background is light. In panel B, the muscle j b w s are traced from a phase micrograph of the s a m field. Note muscle fibers without adjacent capillaries (asterisks). A w w heads in panel A indicate venuks or arterioles. v = perimysial venules. ( x 210.)

and can be combined with other immunolocalization procedures. Because the method can be applied to cryostat sections, the capillary density is not affected by shrinkage of tissue and must be close to that in the native state. This method should be generally applicable to quantitative studies of the microvasculature in other organs or tissues. Estimates of the capillary density provide information on the capillary supply over the entire sample area (see Fig 3). The analysis of capillaries falling in lo4pm2 grid squares provides information on the spatial distribution of capillaries over the entire sample area at an increased resolution. In DM, this revealed a high proportion of grid squares without capillaries, thus highlighting the focal nature of the capillary loss (see Fig 6). In PM and IBM’ it revealed areas Of hypovascdarization and hypervascularization that were masked when the was ‘Onsidered in isolation. A limitation of the grid count is that separation of individual fibers by edema, connective tissue, 354 Annals of Neurology Vol 27 No 4 April 1990

Fig 10. A small artery (large aterisk) and an adjacent arteriole (small asterisk) are occluded &y thrombi. The vessel uMllJand thrombi react intensebfor MAC. Fluorescence in adjacent venule represents autojuorescence ofelastica under rhodamine optics. ( X 250.)

Fig I I . Capillaries in inclusion body myositis (A)and poIymyositis (B) visualized b., the UEA-I method. In both panels the capillary distribution is uneven. In panel A, a band of connective tissue (x) separates two muscle fibers. On either side of the band, several capillaries are adjacent to each fiber. In panel B, many capillary profiles are abnormally large. The two largest profils in the right upper corner represent perimysial arterioles or venules. ( X 250.)

or artifact produces low values that may not reflect changes in muscle fiber perfusion. The capillary index is a measure of the number of capillaries supplying a 1,000-pm2 muscle fiber area, and is therefore independent of alterations in the interstitial space. Determination of this index for each muscle fiber in the sample area proved useful in defining the proportion of muscle fibers supported by a reduced number of capillaries or by no capillaries (see Fig 5). A drawback of this analysis, however, is that highly atrophic fibers in a given region may have an index of 0 even though capillary density in the same region is normal; and when an atrophic fiber abuts on a capillary, it produces a higher than normal capillary index. Despite limitations of the individual evaluation systems, the combined use of the capillary density, capillary index, and capillary grid count provides a powerful method for analyzing the density and distribution of capillaries in normal and diseased muscle.

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Fig 12. A cluster of large and thick-walled capillaries in Patient PMS.( X 390.)

Emslie-Smith and Engel: Microvasculature in Dermatomyositis 35 5

Supported by National Institutes of Health grant NS6277 and a Research Center Grant from the Muscular Dystrophy Association. Dr Emslie-Smith was the recipient of a postdoctoral research fellowship from the Muscular Dystrophy Association. The authors wish to thank Linda Murphy for expert technical assistance.

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Microvascular changes in early and advanced dermatomyositis: a quantitative study.

In adult dermatomyositis 10 muscle specimens with no or minimal histological alterations were compared with 7 that showed typical alterations. Five sp...
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