Dystrophin-deficient myofibers are vulnerable to mast cell granule-induced necrosis

Neuromusc. Disord., Vol. 4, No. 4, pp. 325-333, 1994 Elsevier Science Ltd Printed in Great Britain

Pergamon

09~r-Sg~94)EOOlS-4 DYSTROPHIN-DEFICIENT MAST

CELL

MYOFIBERS

ARE

GRANULE-INDUCED

VULNERABLE

TO

NECROSIS

J. RAFAEL M. GOROSPE,* MICHAEL THARP,t TOSHIO DEMITSU'~ a n d ERIC P. HOFFMAN*~ *Departments of Molecular Genetics and Biochemistry,Human Genetics, and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.; tDepartment of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.

Abstraet--Duchenne muscular dystrophy is the most common inherited lethal X-linked disorder of mankind and is caused by dystrophin deficiency. The steps involved in the dystrophin-deficiency-induced cascade which lead to myofiber necrosis, progressive muscle wasting in humans and dogs and prominent .muscle hypertrophy in mice and cats are obscure. Dystrophin is an intracellular component of the membrane cytoskeleton and its absence would be expected to cause necrosis 9f isolated myofibers (cell autonomous defect). However, all dystrophin-deficient muscles characteristically show simultaneous degeneration of large groups of muscle fibers (grouped necrosis). This implies that cell death may be mediated by extracellular, non-cell autonomous factors which occur as a secondary consequence of dystrophin deficiency. We have proposed a model where tissue pathology may be mediated by infiltrating mast cells (Gorospe et al., J Neurol Sci 1994). Here we show that intramuscular injections of purified mast cell granules induce widespread myofiber necrosis in dystrophindeficient mdx mice, but not in normal mice. These data support the hypothesis that dystrophin acts as a plasma membrane stabilizer and that its deficiency renders myofibers more susceptible to damage from mast cell proteases. Moreover, our results support the hypothesis that mast cell degranulation may be a trigger for myofiber death in dystrophin-deficient muscle. Key words: Dystrophin, rndx, mast cells, Duchenne dystrophy.

INTRODUCTION

Dystrophin is a large (427 kDa) protein which resembles spectrin [1]. It is a component of the membrane cytoskeleton and is associated with a series of proteins, some of which bind the extraceUular matrix [2, 3]. Intracellularly, it is associated with actin filaments [4] and is colocalized with vinculin [5, 6]. Thus, the complete membrane cytoskeleton may link the intracellular myofibrils to the extracellular matrix, imparting stability during the forceful contractions of muscle fibers. Genetically determined dystrophin deficiency, as in Duchenne muscular dystrophy (DMD), results in the secondary loss of many of the dystrophin-associated proteins [7]. The consequences of dystrophin deficiency at the cellular level is probably functionally analogous to spherocytosis in erythrocytes (i.e. :~Authorto whom correspondence should be addressed at: Room W1211 Biomedical Science Tower, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.

spectrin deficiency): loss of the membrane cytoskeleton of erythrocytes has been shown to cause fragility of the plasma membrane under conditions of torsional stress [8, 9]. Similarly, considerable evidence exists for an unstable plasma membrane in dystrophin-deficient muscle fibers: muscle contraction induces membrane damage in m d x mice [10, 11], cultured m d x myogenic cells are osmotically fragile [12], and dystrophin-deficient neurons are more susceptible to death from hypoxia [13] (a physiological insult which is thought to mediate cell death via increased membrane flux and consequent calcium influx). There is also ample evidence for overt leakiness of dystrophin-deficient muscle fibers at all ages in all species: all show striking elevations of serum creatine kinase [14], and serum albumin is seen inside viable dystrophindeficient fibers of female D M D carriers but not adjacent dystrophin-positive fibers [15]. Most myofibers, however, appear capable of maintaining homeostasis and preserving their integrity permitting good to excellent muscle function in spite of the unstable membrane. It appears that 325

326

J. R. M. GOROSPE e t

A 1. H&E: mdx(4wks) uninjected

a/

B1. H&E: mdx(adult) uninjected

A2. Avidin-FITC: mdx(4wks) uninjected B2. Avidin-FITC: mdx(adult) uninjected Fig. 1. Grouped necrosis and mast cell infiltration in dystrophin-deficient mdx mice. Shown are parallel sections stained for histopathology (H&E) and mast cells (avidin-FITC). Panel A shows mdx muscle at the initial stage of large-scale grouped necrosis (4 weeks)(panel AI). There are a large numbers of mast cells in the area of grouped necrosis (panel A2). After the 4week necrotic period, mdx muscle successfullyregenerates, showing less frequent regions of grouped necrosis(panel B). Even in old mdx mice, mast cell infiltration is associated with regions of grouped necrosis. the characteristic finding of necrosis is not immediately consequent to this fragility and leakage [14]. On average, 1.5% of the fibers are usually necrotic in a 5-10 Nn thick crosssection and since the process is segmental, a considerably greater proportion of fibers are affected. Moreover, in those instances when necrosis can be seen histologically, it often occurs as "grouped necrosis" with neighboring fibers dying and regenerating synchronously [16]. Thus, the pathophysiological connections between dystrophin deficiency, necrosis, loss of muscle tissue, weakness, and death in D M D are undoubtedly complex and multifactorial. Grouped degeneration and regeneration has long been recognized as a consistent histopathological finding in Duchenne muscular dystrophy ( D M D ) [16]. More recently, grouped necrosis has been found to be a characteristic feature of dystrophin deficiency in dog, cat, and mouse muscle [14, 17]. The m d x mouse shows the most dramatic grouped necrosis: the muscle is histolo-

gically normal until 3-4 weeks post-partum, when groups of hundreds of neighboring fibers can be seen synchronously degenerating (Fig. 1, panels A l and A2). Such grouped necrosis is unusual for a typical " m y o p a t h y " where a myofiber-intrinsic (cell autonomous) defect characteristically results in segmental necrosis of single fibers in isolation from neighboring fibers. This paradoxical finding has led some investigators to propose that the histopathology in Duchenne dystrophy may be caused by a primary vascular defect: grouped necrosis is more characteristic of a vascular disease (ischemia), where muscle fibers within a vascular bed are subject to simultaneous cell death [18-20]. The identification of dystrophin and delineation of its expression pattern have not clarified the pathogenesis of the histopathology. As a step towards investigating the mechanisms leading to progressive histological fibrosis and muscle wasting in Duchenne dystrophy, we recently conducted a systematic study of mast

Dystrophin-deficientMyofibersand Mast Cell Granule-induced Necrosis cells in dystrophin-deficient muscle [21]. We have shown a correlation between mast cell number and localization with histopathological and disease progression in Duchenne muscular dystrophy and its dog and mouse models [21]. The mast cell numbers in D M D are elevated in the endomysiun; in most other myopathies, mast cells are limited to the perimysiun [21]. We also found evidence for substantial mast cell proliferation and degranulation in areas of grouped necrosis in both young and old mice (Fig. 1). While this was an incidental finding, this led us to hypothesize that mast cell degranulation may mediate myofiber death: the activation and degranulation of mast cells adjacent to dystrophin-deficient fibers would locally release proteases (chymase, tryptase, carboxypeptidase) which in turn could exacerbate the pre-existing membrane defect. This cascade of events may cause proteolysis of the myofiber sarcolemma, and may provide an explanation for the grouped necrosis seen in dystrophin-deficient muscle. Here we report the results of experiments designed to test this hypothesis. MATERIALS AND METHODS

Purification of mast cell granules Mast cell granules were purified from rats as previously described [22]. Briefly, rat peritoneal mast cells were washed from the abdominal cavities of exanguinated rats and mast cells counted using a hemocytometer. The mast cells were lysed and granules purified by centrifugation as described previously. The number of granules used was expressed on a per cell basis (mast cell equivalents [MCE]). The number of mast cells observed in large areas of endogenous grouped necrosis in mdx mouse was approximately 50 (see Fig. 1). We calculated that injection of 1 million mast cell equivalents was a dose similar to that seen in endogenous grouped necrosis by the following calculations. Five injection sites were done per gastrocnemius and with leakage from the muscle we calculated that we delivered approximately 10% of the solution (100,000 mast cell equivalents) per needle track. Assuming that the needle track goes the length of the gastrocnemius muscle (1 cm on the average), we calculated that there are 2000 tissue sections of 5 ~ thickness each per gastrocnemius, and thus 50 mast cell equivalents delivered per needle track per section. Thus, the 50 mast cell equivalents delivered by injection is similar to the

327

50 mast cells seen per section in endogenous grouped necrosis.

Mast cell granule injections Thirteen adult (1-2 yr old) C57BL/10J, and 13 adult ( 1-2 yr old) and three 4-week-old mdx were included in ur study. Ten of the C57BL/10J and 10 adult mdx mice were anesthetized prior to injection by methoxyflurane (Metofane) inhalation. A calculated number of mast cell equivalent (MCE) granules were resuspended in 25 /A of PBS and injected at multiple (4-5) sites into the whole length of the left gastrocnemius muscle using a 26G needle. Sham injection with PBS was done on the contralateral gastrocnemius. The rest of the mice were used as uninjected controls. Twenty-four hours after injection, mice were sacrificed by chloroform inhalation. The gastrocnemius muscles were then removed and flashfrozen in isopentane cooled in liquid nitrogen. Frozen cross-sections (4-6/an) were cut from the center of each muscle using an IEC microtome cryostat and thawed onto precleaned superfrost miscroscope slides (Fisher Scientific). Specimens prepared for H&E were left unfixed; those for fluorescence were fixed in cold acetone ( - 2 0 " C ) for 30 s then dried and rehydrated in 10% horse-serum PBS. H&E and avidin-FITC staining were performed as previously described [21].

Measurement of necrotic areas All observations were done with a Nikon FXA microscope. The gastrocnemii from 6 of 10 mast cell granule (MCG)-injected mdx, 5 of 10 PBSinjected mdx, 5 of 10 MCG-injected C57BL/10J, and 5 of 10 PBS-injected C57BL/10J showed observable injection sites (areas showing diffuse mast cell granules by avidin staining). Each muscle showing necrosis from injection (necrotic fibers and immediate areas affected by edema) was digitally photographed using a 2 × objective and a cooled-CCD camera (Photometrics). Images were contrast-stretched and the area of necrosis was interactively drawn to a bit plane using digital image analysis software (BDS Image). The number of pixels enclosed in each necrotic area and the actual area of necrosis were determined using a 1 mm 2 area as reference (1 mm 2 = 34,448 pixels). The areas of necrosis were averaged for each group and graphed as mean + S.E.M. Where indicated, p values were calculated by paired two-tailed Student's t-test.

328

J . R . M . GoRosPE

A1. H&E: C57(adult) unlnJected

al. H&E: C57(adult) PBS-inJected

et al.

C1. H&E: C57(adult) MCG-Injected

A2. Avldln-FITC: B2. Avldln-FITC: C2. Avidin-FITc: CST(adult) uninJected C57(adult) PBS-inJected C57(adult) MCG-inJected Fig. 2. Mast cell granule injection in normal C57BL/10J mice. Shown are serial sections from uninjected, PBS-injected, and mast cell granule (MCG)-injected normal C57BL/10J mice stained for histopathology (H&E; panels A I and B 1) and mast cell granules (avidin-FITC; panels A2 and B2). Uninjected mice show few, mostly perimysial, uniformly stained mast cells (panels A 1, A2). PBS-injected muscle shows limited necrosis immediately surrounding the needle track (panels B l, B2). The mast cell granule-injected muscle shows regions of necrosis surrounding the needle track similar to PBS-injected muscle (panel C1) despite the presence of granules by avidin-FITC staining (panel C2). Endogenous mast cells (brightly stained round cells) are invariably seen. RESULTS

Injection of saline (PBS) solution alone induced a small amount of myofiber necrosis along the path of the needle in both normal and mdx mice (Fig. 2, panel B). To determine the amount of mast cell granules which could induce a detectable but minimal change in histopathology in normal mice, a series of injections of decreasing mast cell granule concentrations were tested on C57BL/10J mice. It was found that mast cell granules isolated from ! million mast

cells induced a small region of necrosis approximately 3-fold greater than that seen with PBS injections. This number of mast cell granule equivalents is approximately equal to the number of mast cells seen in regions of grouped necrosis in mdx mice (see Materials and Methods). One million MCE were resuspended in 25 /21 and injected into each of 10 gastrocnemius muscles of both normal (C57BL/10J) and mdx adult mice. Mice were sacrified 24 h after injection, and muscles processed for parallel histopathology (H&E) and mast cell granule

Dystrophin-deficientMyofibersand Mast Cell Granule-inducedNecrosis fluorescence (avidin-FITC) [21]. We have previously shown the specificity of avidin-FITC staining for mast cell granules [21]. Uninjected normal mice showed no pathology, and the few mast cell granules present were located in intact mast cells in the perimysial connective tissue near blood vessels (Fig. 2, panels A I and A2). Uninjected 4-week-old and adult mdx muscle showed typical myopathic hystopathology with an increase in mast cell number compared to normal, as we recently reported (Fig. 1) [21]. PBS-injected normal and mdx muscle showed limited necrosis (0.37 .± 0.005 and 0.151 ± 0.040 mm% respectively) immediately surrounding the needle track (Fig. 2, panels BI and B2; Fig. 3, panels B1 and B2). Mast cell granule-injected normal mice showed regions of necrosis (0.167 ± 0.139 mm 2) surrounding the needle track which were approximately 3-fold greater than PBS-injected muscle and showed presence of mast cell granules by avidin staining (Fig. 2, panels C1 and C2). mdx mice showed grouped necrosis covering large areas (0.846 ± 0.387 mm 2) of the muscle (Fig. 3, panels C1, C2, D1, and D2; Fig. 4) approximately 5-fold larger than PBS-injected muscle. Mast cell granules induced necrosis in mdx mice about 5-fold greater than that seen in normal mice P < 0.007) (Fig. 4). FITC-avidin-positive material was seen only in intact mast cells in the muscle tissue or as isolated granules at injection sites. Autofluorescent lipofuscin granules stained dark orange and were easily distinguishable from bright green FITC-avidin-positive mast cell granules. DISCUSSION To determine if mast cell degranulation could play a direct role in the pathogenesis of grouped necrosis in dystrophin-deficient muscle, 'we conducted experiments using intramuscular injections of mast cell granules in both normal and mdx mice. We found that control intramuscular injections of PBS induced approximately 5fold more necrosis in mdx mice compared to agematched normal mice (P < 0.05). Intramuscular injection of mast cell granules in normal controls resulted in a 5-fold increase in necrosis compared to PBS-injected muscle. The most dramatic necrosis was seen in mdx mice injected with mast cell granules, where areas of myofiber degeneration were 5-fold increased compared to normal controls. These findings indicate that mdx muscle is

329

more susceptible to damage from mast cell granules. This effect is most likely mediated through mast cell proteases which are at high concentration in granules: mast cell proteases may damage the already compromised myofiber membranes o f m d x muscle. Although we used rat mast cell granules, it is unlikely that the grouped necrosis was immune-mediated because the 24-h time frame of the experiment precludes the mounting of an immune response. Alternatively, the mast cell granules could trigger macrophage infiltration of the muscle and the macrophages could in turn cause the grouped necrosis. Previous studies have shown that the infiltrating mononuclear cells in D M D are predominantly macrophages and CD4 + helper/inducer T-cells and that the cells were activated [23]. It will be important to determine the cause/effect relationship of macrophages, T-cells, and mast cells in dystrophin-deficiency-induced muscle damage. Independent of the precise primary cause of the myofiber death, it is clear that mdx myofibers are considerably more vulnerable to this insult than normal muscle. We are currently conducting in vitro experiments on dystrophin-positive and dystrophin-negative muscle cultures to demonstrate a direct effect of mast cell proteases on myogenic cells. A number of characteristic findings in D M D could be rationalized by implicating mast cell mediators in their pathogenesis. Mast cells express considerable amounts of phospholipase [24] and dystrophin-deficient humans show high levels of this enzyme [25]. Thus, the damage to dystrophin-deficient fibers may be mediated both through mast cell-derived proteolysis ofextracellular matrix and membrane proteins and through direct digestion of membrane phospholipids. Normal muscle, with its intact membrane cytoskeleton, is probably more resistant to mast cell mediated damage, a hypothesis supported by the findings in this study. Chymase, another abundant mediator expressed by human connective tissue-type mast cells, is also known to convert angiotensin I to angiotensin II, a potent local vasoconstrictor [26, 27], and it also inactivates the vasodilating peptides bradykinin and kallidin [28]. Hence, mast cell degranulation could cause the vasculogenic-like features of the disease: mast cell mediators could induce localized functional ischemia in dystrophin-deficient muscle exacerbating the documented pathological Ca 2+ influx [14]. It is also important to note that mast cell chymase itself is a modulator for mast cell

330

J.R.M.

GOROSPE et aL

A 1. H&E:

B1. H&E:

mdx(adult) uninjected

mdx(adult) PBS-injected

I

A2. Avidin-FITC:

B2. Avidin-FITC:

mdx(adult) uninjected

mdx(aduit) PBS-injected

Fig. 3.

Dystrophin-deficient Myofibers and Mast Cell Granule-induced Necrosis

C1. H&E: mdx(adult) MCG-injected

331

D1. H&E: mdx(adult) MCG-injected

C2. Avidin-FITC:

D2. Avidin-FITC:

mdx(adult) MCG-injected

mdx(adult) MCG-injected

Fig. 3. Mast cell granule injection in dystrophin-deficient mdx mice. Shown are serial sections from uninjected, PBS-injected, and two different mast cell granule (MCG)-injected adult mdx mice stained for histopathology (H&E; panels A 1, B 1, C 1, D 1) and mast cells (avidin-FITC; panels A2, B2, C2, D2). Uninjected mdx muscle show typical myopathic histopathology (panel A l) with a large number of mast cells as previously documented (panel A2) [21]. PBS-injected muscle shows limited necrosis immediately surrounding the needle track (panel B 1, B2). Mast cell granule-injected muscles (panels C 1, D l) show massive grouped necrosis covering large areas of the muscle extending beyond the area photographed. The injection sites clearly show mast cell granules by avidin-FITC staining (panels C2, D2). Endogenous mast cells are invariably seen as intensely stained, plump cells in the connective tissue space.

332

J . R . M . GOROSPE et al. Mast cell granule-induced

necrosis

g

force-, osmotic-, and ischemia-induced damage. We have presented a pathogenesis model for Duchenne muscular dystrophy where mast cells play a prominent role. A complete definition of the cascade of events initiated by dystrophin deficiency may open additional avenues towards rational therapies via modulation of the pathophysiology of the disease process.

[ ] C57BI_/10J

mrnd~

Acknowledgements--The work presented here was supported by the March of Dimes Birth Defects Foundation. This was presented in part at the 5th National Colloquium on Neuromuscular Disorders of the Association Francaise Contre les Myopathies (AFM) in Strasbourg, France in June 1993. We would like to acknowledge the assistance of Eric Voigt in the computer analysis of the figures.

=.

PBS-Inle©ted

MCG-Injected

Fig. 4. Quantitation of necrosis in injected muscles. Necrotic areas in gastrocnemii showing injection sites were quantitated using digital image analysis. Mast cell granules induced approximately 5-fold more necrosis in dystrophin-deficient (mdx) muscle compared to dystrophin-positive (C57BL/10J) controls (P < 0.007).

activation [29]. Thus, a cascade of mast cell degranulation and myofiber necrosis could be initiated by a single degranulation event. This cascade could rationalize the intense mast cell infiltration and degranulation seen in dystrophin-deficient muscle [21]. A recent finding which may have bearing on the pathophysiological cascades induced by dystrophin deficiency is the dramatic elevations of b F G F levels in the serum of D M D patients [30]. bFGF, presumably released from myofibers by dystrophin-deficiency-induced membrane leakage, would normally be expected to be quickly bound in an inactive state by heparin proteoglycans in the basement membrane. However, mast cell mediators are known to activate b F G F by releasing it from the basal lamina stores [31, 32]. The correlation of our mast cell data with serum b F G F data is quite suggestive: Duchenne dystrophy shows much higher numbers of mast cells in muscle and very high circulating levels of bFGF. Thus, there may be a causal link between dystrophin deficiency, mast cell accumulation and degranulation, activation of bFGF, progressive proliferation of connective tissue, and failure of muscle regeneration which is hypothesized to result in clinical weakness [14, 33]. Our results reinforce the prevailing hypothesis that dystrophin deficiency causes generalized membrane fragility: protease-induced damage can be added to the previously documented susceptibility of dystrophin-deficient muscle to

REFERENCES

1. 2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12. 13.

Koenig M, Monaco A P, Kunkel L M. The complete sequence ofdystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988; 53: 219-228. Ibraghimov-Beskrovnaya O, Ervasti J M, Leveille C J, Slaughter C A, Sernett S W, Campbell K P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 1992; 355: 69# 702. Hoffman E P and Wang J. Duchenne/Becker muscular dystrophy and the non-dystrophic myotonias: paradigms for loss-of-function and change-of-function of gene products. Arch Neurol 1993; 50: 1227-1237. Levine B A, Moir A J G, Patchell V B, Perry S V. Binding sites involved in the interaction of actin with the N-terminal region of dystrophin. FEBS Lett 1992; 28: 4448. Minetti C, Bentrame F, Marcenaro G, Bonilla E. Dystrophy at the plasma membrane of human muscle fibers shows a costameric localization. Neuromus Dis 1992; 2:99 109. MineUi C, Tanji K, Bonilla E. Immunologic study of vinculin in Duchenne muscular dystrophy. Neurology 1992; 42: 1751-1754. Ervasti J M, Ohlendieck K, Kahl S D, Gaver M G, Campbell K P. Deficiency ofa glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 1990; 345: 315-319. Eber S W, Armburst R, Schroter W. Variable clinical severity of hereditary spherocytosis: relation to erythrocytic spectrin concentration, osmotic fragility, and autohemolysis. J Pedia 1990; 117: 409-416. Whitfield C F, Follweiler J B, Loprasti-Morrow L, Miller B A. Deficiency of alpha-spectrin in burstforming units-erythroid in lethal hereditary spherocytosis. Blood 1991; 78: 3043-3051. Petrof B J, Shrager J B, Stedman H H, Kelly A M, Sweeney H L. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci USA 1993; 90: 3710-3714. Hutter O F, Burton F L, Bovell D L. Mechanical properties of normal and mdx mouse sarcolemma: bearing on function of dystrophin. J Muscle Res Cell Motil 1991; 12:585 589. Menke A, Jockusch H. Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse. Nature 1991; 349:69 71. Mehler M F, Haas K Z, Kessler J A, PK. Enhanced sensitivity of hippocampal pyramidal neurons from mdx mice to hypoxia-induced loss of synaptic transmission. Proc Natl Acad Sci USA 1992; 89:2461 2465.

Dystrophin-deficient Myofibers and Mast Cell Granule-induced Necrosis 14. Hoffman E P, Gorospe J R M. The animal models of Duchenne muscular dystrophy: windows on the pathophysiological consequences of dystrophin deficiency. In: Morrow J, Mooseker M, eds. Current Topics in Membranes, Vol. 38. Academic Press: New York, 1991: 113-154. 15. Morandi L, Mora M, Gussoni E, Tedeschi S, Cornelio F. Dystrophin analysis in Duchenne and Becket muscular dystrophy carriers: correlation with intracellular calcium and albumin. Ann Neurol 1990; 28: 674-679. 16. Engel A G. Duchenne dystrophy. In: Engel A G and Banker B Q, Eds. Myology: Basic and Clinical. McGraw-Hill: New York, 1986, 1185 1240. 17. Coulton G R, Morgan J E, Partridge T A, Sloper J C. The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation. Neuropathol Appl Neurobiol 1988; 14: 53-70. 18. Engel W K. Integrative histochemical approach to the defect of Duchenne muscular dystrophy. In: L P Rowland, Ed Pathogenesis of Human Muscular Dystrophies. Excerpta Medica; Amsterdma, 1977: 277-309. 19. Mendel J R, Engel W K, Derrer E C. Duchenne muscular dystrophy: functional ischemia reproduces its characteristic lesions. Science 1971; 172:1143-1145. 20. Mendel J R, Engel W K, Derrer E C. Increased plasma enzyme concentrations in rats with functional ischemia of muscle provide a possible model of Duchenne muscular dystrophy. Nature 1972; 239: 522-524. 21. Gorospe J R M, Tharp M D, Hinckley J, Kornegay J N, Hoffman E P. A role for mast cells in the progression of Duchenne muscular dystrophy? Correlations in dystrophin-deficient humans, dogs, and mice. J Neurol Sci 1994; 122: 44-56. 22. Tharp M D, Kasper C, Theile D, Charley M R, Kennedy D A, Sullivan T J. Studies of connective tissue mast cell-mediated cytotoxicity. J Invest Dermato11989; 93: 423-428. 23. McDouall R M, Dunn M J, Dubowitz V. Nature of the

24.

25. 26.

27.

28.

29.

30.

31.

32. 33.

333

mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy. J Neurol Sci 1990; 99: 199-217. Murakami M, Kudo I, Suwa Y, Inoue K. Release of 14kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process. Eur J Biochem 1992; 209: 257-265. Tagesson C, Henriksson K G. Elevated phopholipase A2 in Duchenne muscle. Muscle Nerve 1984; 2: 260. Wintroub B U, Schechter N B, Lazarus G S, Kaemrfer C E, Schwartz L B. Angiotensin I conversion by human and rat chymotryptic proteinases. J Invest Dermatol 1984; 83: 33(~339. Reilly C F, Tewksbury D A, Schechter N B, Travis J. Rapid conversion of angiotensin I to angiotensin II by neutrophil and mast cell proteinases. J Biol Chem 1984; 257: 8619-8622. Reilly C F, Schechter N B, Travis J. Inactivation of bradykinin and kallidin by cathepsin G and mast cell chymase. Biochem Biophys Res Commun 1985; 127: 443~49. Goldstein S M, Wintroub B U. Mast cell proteases. In: Kaliner M A, Metcalfe D D, eds. The Mast Cell in Health and Disease. Marcel Dekker: New York, 1993: 343-380. D'Amore P A, Brown R, Ku P T, Hoffman E P, Watanabe H, Arahata K, Folkman J. Elevated levels of bFGF in the serum of patients with Duchenne muscular dystrophy. Ann Neurol 1994; 25: 362-365. Bashkin P, Doctrow S, Klagsbrun M, Svahn C M, Folkman J, Vlodavsky I. Basic fibroblast growth factor binds to subendithelial extracellular matric and is released by heparitinase and heparin-like molecules. Biochemistry 1989; 28: 1737-1743. D'Amore P A. Modes of FGF release in vivo and in vitro. Cancer Metastasis Rev 1990; 9: 227-238. Gorospe J R M and Hoffman E P. Duchenne muscular dystrophy. Curr Op Rheumatol 1992; 4: 794-800.