Section XI Extraskeletal (Ectopic) Calciﬁcation and Ossiﬁcation

Michael P. Whyte Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital and Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, Missouri

INTRODUCTION somewhat higher value because they have greater serum phosphate concentrations compared with adults. However, this is A significant number and variety of disorders cause extraskel- not well established.(5) etal deposition of calcium and phosphate (Table 1). In some, The material that comprises metastatic calcification may be mineral is precipitated as amorphous calcium phosphate or as amorphous calcium phosphate initially, but hydroxyapatite is crystals of hydroxyapatite; in others, osseous tissue is formed. deposited soon after.(2) The anatomic pattern of deposition The pathogenesis of ectopic mineralization is generally attrib- varies somewhat between hypercalcemia and hyperphos- uted to one of three mechanisms (Table 1). First, a supranormal phatemia, but occurs irrespective of the specific underlying “calcium-phosphate solubility product” in extracellular fluid condition or mechanism for the disturbed mineral homeostasis. can cause metastatic calcification. Second, mineral may be Additionally, there is a predilection for certain tissues. deposited as dystrophic calcification into metabolically im- Hypercalcemia is typically associated with mineral deposits paired or dead tissue despite normal serum levels of calcium in the kidneys, lungs, and fundus of the stomach. In these and phosphate. Third, ectopic ossification (or true bone forma- “acid-secreting” organs, a local alkaline milieu may account tion) occurs in a few disorders for which the pathogenesis is for the calcium deposition. In addition, the media of large becoming increasingly understood. arteries, elastic tissue of the endocardium (especially the left Discussed briefly in this introduction are these three mech- atrium), conjunctiva, and periarticular soft tissues are often anisms for extraskeletal calcification or ossification. Subse- affected. However, why these sites are predisposed is not well quently, there follows a description of disorders that illustrate understood. In the kidney, hypercalciuria may cause calcium each pathogenesis. phosphate casts to form within the tubule lumen, or calculi to develop in the calyces or pelvis. Furthermore, calcium phos- MECHANISMS FOR EXTRASKELETAL phate may precipitate in peritubular tissues. In the lung, calci- CALCIFICATION AND OSSIFICATION fication affects the alveolar walls and the pulmonary venous Calcium and inorganic phosphate are normally present in system. Well-established causes of metastatic calcification me- serum or extracellular fluid at concentrations that form a “meta- diated by hypercalcemia include the milk-alkali syndrome, stable” solution. That is, their levels are too low for spontane- hypervitaminosis D, sarcoidosis, and hyperparathyroidism (Ta- ous precipitation but sufficiently great to cause hydroxyapatite ble 1). [Ca10(PO4)6(OH)2] formation once crystal nucleation has be- gun.(1) In health, the presence of a variety of inhibitors of TABLE 1. DISORDERS ASSOCIATED WITH EXTRASKELETAL CALCIFICATION mineralization, such as inorganic pyrophosphate, helps to pre- OR OSSIFICATION vent ectopic calcification.(2) The pathogenesis of metastatic and dystrophic calcification A. Metastatic calcification at the cell level is partially understood. Both processes typi- I. Hypercalcemia cally involve mineral accumulation within matrix vesicles and a. Milk-alkali syndrome sometimes within mitochondria.(2) Conversely, the mecha- b. Sarcoidosis nisms which initiate ectopic ossification are less clear, but d. Hyperparathyroidism studies of progressive osseous heteroplasia (POH) identified e. Renal failure deactivating mutations in GNAS (which also causes pseudohy- II. Hyperphosphatemia poparathyroidism type IA).(3) Calcification and ossification a. Tumoral calcinosis within the vasculature is now being investigated intensely.(4) b. Hypoparathyroidism Metastatic calcification can occur from significant hypercal- c. Pseudohypoparathyroidism cemia or hyperphosphatemia (especially both) of any etiology d. Cell lysis after chemotherapy for leukemia (Table 1). In fact, therapy with phosphate supplements during e. Renal failure mild hypercalcemia or treatment with vitamin D or calcium B. Dystrophic calcification during mild hyperphosphatemia may trigger this problem. Min- I. Calcinosis (universalis or circumscripta) eral deposition can also occur ectopically from hyperphos- a. Childhood dermatomyositis phatemia despite concomitant hypocalcemia.(5) b. Scleroderma Direct precipitation of mineral occurs when the calcium– c. Systemic lupus erythematosis phosphate solubility product in extracellular fluid is exceeded. II. Post-traumatic A value of 75 (mg/dl ϫ mg/dl) is commonly taken as the limit C. Ectopic ossification that, if surpassed, causes mineral precipitation. However, the I. Myositis ossificans (post-traumatic) critical value for renal calcification is not precisely defined and a. Burns may vary with age.(5) In adults, some consider 70 to be the b. Surgery (joint replacement) maximal safe level for the kidney. Possibly, children tolerate a c. Neurologic injury II. Fibrodysplasia (myositis) ossificans progressiva (FOP) III. Progressive osseous heteroplasia (POH) IV. Osteoma cutis The author has reported no conflicts of interest.

Hyperphosphatemia of sufficient severity to cause metastatic lesions of calcinosis are small or medium-sized hard nodules calcification occurs in idiopathic hypoparathyroidism or that can cause muscle atrophy and contractures. Other etiolo- pseudohypoparathyroidism and with the massive cell lysis (re- gies for calcinosis include metastases or trauma that produce lease of cellular phosphate) that can follow chemotherapy for necrotic tissue. leukemia (Table 1). Renal insufficiency is commonly associ- Ectopic ossification is associated with two principal etiolo- ated with metastatic calcification—the mechanism may involve gies. It occurs sporadically with the fasciitis that follows neu- hyperphosphatemia, hypercalcemia, or both.(6) Of interest (but rological injury, surgery, burns or trauma, when it is called unexplained), ectopic calcification is more common in myositis ossificans. It also occurs as the major feature of a pseudohypoparathyroidism (type I) than in idiopathic hypopar- separate, heritable entity—fibrodysplasia (myositis) ossificans athyroidism despite comparable elevations in serum phosphate progressiva—where the pathogenesis is becoming understood. levels. Furthermore, the location of ectopic calcification in Some ascribe the ectopic bone formation in this latter, genetic pseudohypoparathyroidism and hypoparathyroidism (e.g., ce- disorder to be a muscle abnormality (myositis ossificans pro- rebral basal ganglion) is different from observations in hyper- gressiva), whereas others favor a connective tissue defect (fi- calcemia. With hyperphosphatemia, calcification of periarticu- brodysplasia ossificans progressiva). In all of these conditions, lar subcutaneous tissues is characteristic and may be related to osseous tissue is formed. The bone is lamellar, is actively tissue trauma from the movement of joints.(6) remodeled by osteoblasts and osteoclasts, has haversian sys- Dystrophic calcification occurs despite a normal serum tems, and sometimes contains marrow. Apparently, the injured calcium–phosphate solubility product.(7) Injured tissue of any or diseased tissue has the necessary inductive signals and kind is predisposed to this type of extraskeletal calcification. precursor cells to form cartilage and bone. Apparently, tissues can release material that has nucleating Described in the following chapters are tumoral calcinosis, properties. One classic example is the caseous lesion of tuber- dermatomyositis, fibrodysplasia ossificans progressiva (FOP), culosis. However, what local factor predisposes to the precip- and vascular diseases, which represent the principal examples itation of calcium salts is unknown. Indeed, several mecha- of each type of ectopic mineralization. nisms seem likely. It is clear that mineral precipitation into injured tissue is even more striking and more severe when REFERENCES either the calcium or phosphate level in extracellular fluid is also increased. The deposited mineral, as for metastatic calci- 1. Fawthrop FW, Russell RGG 1993 Ectopic calcification and ossification. fication, may be either amorphous calcium phosphate or crys- In: Nordin BEC, Need AG, Morris HA (eds.) Metabolic Bone and Stone Disease, 3rd ed. Churchill Livingstone, Edinburgh, UK, pp. 325–338. talline hydroxyapatite. 2. Anderson HC 1983 Calcific diseases: A concept. Arch Pathol Lab Med The term “calcinosis” refers to an important type of dystro- 107:341–348. phic calcification that commonly occurs in (or under) the skin 3. Eddy MC, Jan de Beur SM, Yandow SM, McAlister WH, Shore EM, from connective tissue disorders—particularly dermatomyosi- Kaplan FS, Whyte MP, Levine MA 2000 Deficiency of the ␣-subunit of tis, scleroderma, or systemic lupus erythematosus.(7) As the the stimulatory G protein and severe extraskeletal ossification. J Bone Miner Res 15:2074–2083. symptoms and the inflammatory process in the subcutaneous 4. Collett GD, Canfield AE 2005 Angiogenesis and pericytes in the initiation tissues from the acute connective tissue disease subside, pain- of ectopic calcification. Circ Res 96:930–938. ful masses of calcium phosphate appear under the skin. Calci- 5. Harrison HE, Harrison HC 1979 Disorders of Calcium and Phosphate nosis may involve a relatively localized area with small depos- Metabolism in Childhood and Adolescence. WB Saunders, Philadelphia, its in the skin and subcutaneous tissues, especially over the PA, USA. extensor aspects of the joints and the fingertips (calcinosis 6. Hamada J, Tamai K, Ono W, Saotome K 2006 Uremic tumoral calcinosis in hemodialysis patients: Clinicopathological findings and identification of circumscripta); or, it may be widespread and not only in the calcific deposits. J Rheumatol 33:119–126. skin and subcutaneous tissues, but deeper in periarticular re- 7. Boulman N, Slobodin G, Rozenbaum M, Rosner I 2005 Calcinosis in gions as well as areas of trauma (calcinosis universalis). The rheumatic diseases. Semin Arthritis Rheum 34:805–812.

Chapter 77. Tumoral Calcinosis

INTRODUCTION deposits.(5) The differential diagnosis includes periarticular metastatic calcification from hypercalcemia associated with Tumoral calcinosis, first described in 1899, is a heritable dis- (1) renal failure, milk-alkali syndrome, sarcoidosis, and vitamin D order that features periarticular metastatic calcification. Hy- intoxication. perphosphatemia is a pathogenetic factor in many patients.(2–4) Mineral deposition manifests as soft tissue masses around the major joints. Typically, the hips and shoulders are affected, CLINICAL PRESENTATION (5) although additional joints can be involved. Visceral calcifi- Most patients in North America with this disorder have cation does not occur, but segments of vasculature may contain black ancestry. About one third of cases are familial. Auto- somal recessive inheritance is usually described, although autosomal dominant transmission has also been re- The author has reported no conflicts of interest. ported.(1–6) There is no gender preference.

tion occurs along the endosteal surface of the diaphysis, perhaps from calcific myelitis.(7) This finding may be confused with osteomyelitis or a neoplasm.(11) When only calcific myelitis is present, CT and MRI are excellent tools for diagnosis.(11) Bone scanning, however, is the best method to detect and localize the calcified masses.(12) Periarticular masses that are radiologically indistinguishable from those of tumoral calcinosis occur in chronic renal failure when mineral homeostasis is poorly controlled.

LABORATORY FINDINGS Serum calcium levels and alkaline phosphatase activity are usually normal. Hyperphosphatemia and increased serum calcitriol levels occur in some patients.(3,13) The TmP/GFR (phosphate transport maximum/glomerular filtration rate) may be supranormal, but renal function is otherwise unremarkable. Patients are in positive calcium/phosphate balance. Urinary studies reflect both the ongoing calcium and phosphate retention, and some patients are frankly hypocalciuric. The chalky fluid in lesions is predominantly hydroxyapatite.(14,15)

HISTOPATHOLOGY The masses of tumoral calcinosis are essentially foreign body granuloma reactions that form multilocular, cystic struc- FIG. 1. Tumoral calcinosis. Lobular, periarticular calcifications are (16) present at the right shoulder of this middle-aged man. tures. The early lesion may involve hemorrhage and histio- cyte accumulation.(16,17) There are ill-defined, perivascular, reactive-like, solid cell nests admixed with mononuclear and iron-loaded macrophages, or well-organized, variably-sized, Tumoral calcinosis often presents in childhood, but charac- fibrohistiocytic nodules embedded in a dense collagenous teristic masses have been discovered in infancy and in old age. stroma.(16) The cysts have tough connective tissue capsules, Hyperphosphatemic patients are usually black, have a positive and their fibrous walls contain numerous foreign body giant family history, manifest the disease before 20 years of age, and cells. Mature lesions are filled with calcareous material in a have multiple lesions.(3) viscous milky fluid. Occasionally, spicules of spongy bone and The soft tissue calcifications are typically painless and grow cartilage are found as well. at variable rates.(7) After 1 or 2 years, the masses may be the size of an orange or grapefruit and weigh 1 kg or more. Often ETIOLOGY AND PATHOGENESIS they are hard, lobulated, and firmly attached to deep fascia. Occasionally, the swellings infiltrate into muscles and ten- The genetic basis for tumoral calcinosis has recently been dons.(3) The major clinical complications are related to the revealed with deactivating mutations identified in the FGF23 tumors that occur around joints and the sequelae in skin, and GALNT3 genes.(18,19) The pathogenesis involves a defi- marrow, teeth, and blood vessels. Because the deposits are ciency of phosphaturia mediated by the kidney tubule cell. extracapsular, joint range of motion is not impaired unless the Increased renal reclamation of filtered phosphate becomes an tumors are particularly large. There can, however, be compres- important pathogenetic factor.(2,4) In hyperphosphatemic pa- sion of adjacent neural structures. The lesions can also ulcerate tients, enhanced kidney tubular reabsorption of phosphate oc- the skin and form a sinus tract that drains a chalky fluid; this curs independently of suppressed serum PTH levels.(3,13) De- complication may lead to infection. Other potential secondary ranged regulation of the renal 25-hydroxyvitamin D, problems include anemia, low-grade fever, regional lymphad- 1-hydroxylase causes increased calcitriol synthesis. Conse- enopathy, splenomegaly, and amyloidosis. Some patients have quently, dietary calcium absorption is enhanced, and serum characteristics of pseudoxanthoma elasticum (i.e., skin and PTH levels are suppressed.(3,13) vascular calcifications and angioid streaks in the retina). A The masses may begin as calcific bursitis but then grow into dental abnormality, featuring short bulbous tooth roots and adjacent fascial planes. Tissue damage with fat necrosis can be calcific deposits that often obliterate pulp chambers, is a hall- a pathogenetic factor.(15) mark.(6,8) Recurrent episodes of bone inflammation have been characterized.(9) This is a lifelong disorder. TREATMENT

RADIOGRAPHIC EXAMINATION Surgical removal of subcutaneous calcified masses may be helpful if they are painful, interfere with function, or are The tumors typically appear as large aggregations of irreg- cosmetically unacceptable. When tumor excision is complete, ular, densely calcified lobules that are confined to soft tissues recurrence seems unlikely.(20) (Fig. 1). Radiolucent fibrous septae account for the lobular Radiation therapy and cortisone treatment have not been appearance.(10) Occasionally, fluid layers are seen within the effective. Although it might seem that large masses of apatite masses. The joints per se are unaffected. Bone texture and crystals would be refractory to dissolution, success with alu- density are also unremarkable. minum hydroxide therapy (together with dietary phosphate and A “diaphysitis” has been recognized using radiographs, CT, calcium deprivation) has been reported.(2,21,22) Furthermore, or MRI in some cases of tumoral calcinosis. New bone forma- reduction of phosphate levels in extracellular fluid could help

© 2006 American Society for Bone and Mineral Research DERMATOMYOSITIS IN CHILDREN / 439 to prevent reformation of mineral deposits.(2) Preliminary stud- 12. Le Stanc E, Vilain D, Tainturier C 2004 Tumoral calcinosis appearances ies indicate that calcitonin therapy may also be efficacious by on skeletal scintigraphy. Clin Nucl Med 29:821–822. enhancing phosphaturia.(23) Acetazolamide, together with alu- 13. Lyles KW, Halsey DL, Friedman NE, Lobaugh B 1988 Correlations of (24) serum concentrations of 1,25-dihydroxyvitamin D, phosphorus, and para- minum hydroxide, seemed to be helpful for one patient. thyroid hormone in tumoral calcinosis. J Clin Endocrinol Metab 67:88– 92. 14. Boskey AL, Vigorita VJ, Sencer O, Stuchin SA, Lane JM 1983 Chemical, REFERENCES microscopic and ultrastructural characterization of mineral deposits in tumoral calcinosis. Clin Orthop 178:258–270. 1. McKusick-Nathans Institute for Genetic Medicine 2000 Online Mende- 15. Kindbolm L-G, Gunterberg B 1988 Tumoral calcinosis: An ultrastructural lian Inheritance in Man McKusick-Nathans Institute for Genetic Medi- analysis and consideration of pathogenesis. Acta Pathol Microbiol Immu- cine, Johns Hopkins University, Baltimore, MD, USA and National Cen- nol Scand 96:368–376. ter for Biotechnology Information, National Library of Medicine, 16. Pakasa NM, Kalengayi RM 1997 Tumoral calcinosis: A clinicopatholog- Bethesda, MD, USA. ical study of 111 cases with emphasis on the earliest changes. Histopa- 2. Yu X, White KE 2005 FGF23 and disorders of phosphate homeostasis. thology 31:18–24. Cytokine Growth Factor Rev 16:221–232. 17. Slavin RE, Wen J, Kumar WJ, Evans EB 1993 Familial tumoral calcino- 3. Prince MJ, Schaefer PC, Goldsmith RS, Chausmer AB 1982 Hyperphos- sis. A clinical, histopathologic, and ultrastructural study with an analysis phatemic tumoral calcinosis. Association with elevation of serum 1,25- of its calcifying process and pathogenesis. Am J Surg Pathol 17:788–802. dihydroxy-cholecalciferol concentrations. Ann Intern Med 96:586–591. 18. Chefetz I, Heller R, Galli-Tsinopoulou A, Richard G, Wollnik B, Indel- 4. Smack D, Norton SA, Fitzpatrick JE 1996 Proposal for a pathogenesis- man M, Koerber F, Topaz O, Bergman R, Sprecher E, Schoenau E 2005 based classification of tumoral calcinosis. Int J Dermatol 35:265–271. A novel homozygous missense mutation in FGF23 causes familial tu- 5. Martinez S 2002 Tumoral calcinosis: 12 years later. Semin Musculoskelet moral calcinosis associated with disseminated visceral calcification. Hum Radiol 6:331–339. Genet 118:261–266. 6. Lyles KW, Burkes EJ, Ellis GJ, Lucas KJ, Dolan EA, Drezner MK 1985 19. Ichikawa S, Lyles KW, Econs M 2005 A novel GALNT3 mutation in a Genetic transmission of tumoral calcinosis: Autosomal dominant with pseudoautosomal dominant form of tumoral calcinosis: Evidence that the variable clinical expressivity. J Clin Endocrinol Metab 60:1093–1096. disorder is autosomal recessive. J Clin Endocrinol Metab 90:2420–2423. 7. Narchi H 1997 Hyperostosis with hyperphosphatemia: Evidence of famil- 20. Noyez JF, Murphree SM, Chen K 1993 Tumoral calcinosis, a clinical ial occurrence and association with tumoral calcinosis. Pediatrics 99:745– report of eleven cases. Acta Orthop Belg 59:249–254. 748. 21. Davies M, Clements MR, Mawer EB, Freemont AJ 1987 Tumoral calci- 8. Burkes EJ Jr, Lyles KW, Dolan EA, Giammara B, Hanker J 1991 Dental nosis: Clinical and metabolic response to phosphorus deprivation. Q lesions in tumoral calcinosis. J Oral Pathol Med 20:222–227. J Med 242:493–503. 9. Blay P, Fernandez-Martinez JM, Diaz-Lopez B 2001 Vertebral involve- 22. Gregosiewicz A, Warda E 1989 Tumoral calcinosis: Successful medical ment in hyperphosphatemic tumoral calcinosis. Bone 28:316–318. treatment. J Bone Joint Surg Am 71:1244–1249. 10. Steinbach LS, Johnston JO, Tepper EF, Honda GD, Martel W 1995 23. Salvi A, Cerudelli B, Cimino A, Zuccato F, Giustina G 1983 Phosphaturic Tumoral calcinosis: Radiologic-pathologic correlation. Skeletal Radiol action of calcitonin in pseudotumoral calcinosis. Horm Metab Res 15:260. 24:573–578. 24. Yamaguchi T, Sugimoto T, Imai Y, Fukase M, Fujita T, Chihara K 1995 11. Martinez S, Vogler JB, Harrelson JM, Lyles KW 1990 Imaging of tumoral Successful treatment of hyperphosphatemic tumoral calcinosis with long- calcinosis: New observations. Radiology 174:215–222. term acetazolamide. Bone 16:247S–250S.

Chapter 78. Dermatomyositis in Children

INTRODUCTION 1–3 years after the disease onset and occurred in 25–50% of patients before intensive therapeutic regimens became avail- Dermatomyositis is a multisystem connective tissue disorder able for dermatomyositis. caused by small vessel vasculitis.(1,2) Acute and chronic, non- Calcinosis may predate the myopathy.(6) Mineral deposits de- suppurative inflammation involves especially the skin and striated velop over 1–3 years. In calcinosis universalis (see below), calci- muscles. Dystrophic calcification can follow episodes of inflam- (3–6) fication occurs throughout the subcutaneous tissues, but primarily mation and can be severely debilitating. in periarticular regions or in areas that are subject to trauma (Fig. 1). In calcinosis circumscripta, the deposits are more localized and CLINICAL PRESENTATION typically occur around joints. The ectopic mineralization can There are more female than male patients and two peak ages cause pain, ulcerate the skin, limit mobility, result in contractures, of incidence: childhood (5–15 years) and adulthood (50–60 and predispose to abscess formation. Although the dystrophic years). When the disorder manifests before age 16 years, it is calcification then typically remains stable, rarely some spontane- (1,2) called juvenile or childhood dermatomyositis.(1,2) The adult ous resolution is reported. Dystrophic calcification is rare in (3) form is associated with malignancy.(3) adults with dermatomyositis. In juvenile dermatomyositis, the patient’s sex and the age- of-onset of symptoms seem unrelated to the severity of any calcinosis, although increased time to diagnosis and treatment LABORATORY FINDINGS worsen this complication.(4) Calcification is generally noted Although hypercalcemia with hypercalciuria and hyperphos- phaturia may occur in juvenile dermatomyositis, parameters of mineral homeostasis are usually normal.(7) Elevated levels of The author has reported no conflicts of interest. ␥-carboxyglutamic acid have been found in the urine of af-

FIG. 1. Calcinosis universalis in childhood dermatomyositis. (A) Characteristic subcutaneous nodules are apparent in the left arm and anterior chest wall of this 15-year-old boy. (B) The nodules in this boy’s arm are composed of dense lobular calcifications. In addition, the muscles of the upper arm are encased in a characteristic calcified sheath. fected children—especially if there is calcinosis.(8) Hydroxy- A variety of mechanisms considered for the dystrophic calcifi- apatite comprises the nucleus of the calcinosis deposits, but cation include release of alkaline phosphatase or free fatty acids other factors (including cytokines and macrophages) are also from diseased muscle that, in turn, directly precipitate calcium or present.(9) first bind acid mucopolysaccharides. Increased urinary levels of ␥-carboxylated peptides suggest that calcium-binding proteins RADIOGRAPHIC FINDINGS may be responsible for the mineral deposition. In juvenile dermatomyositis, four types of dystrophic calcification occur.(10) TREATMENT 1. Superficial masses (small circumscribed nodules or High-dose prednisone therapy soon after the onset of plaques) within the skin symptoms seems to be important for minimizing the risk of 2. Deep, discrete, subcutaneous, nodular masses (Fig. 1) calcinosis and for ensuring good, functional recovery.(1,2,18,19) If the response is incomplete, consideration is near joints that can impair movement (calcinosis cir- given to additional immunosuppressive agents, including cumscripta) methotrexate and cyclosporine.(20) In a small clinical trial, 3. Deep, linear, sheet-like deposits within intramuscular warfarin treatment to decrease ␥-carboxylation was not as- fascial planes (calcinosis universalis) sociated with changes in calcium or phosphorus excretion or 4. Lacy reticular subcutaneous deposits that encase the in a reduction of calcinosis.(21) Phosphate-binding antacid torso to form a generalized “exoskeleton” therapy may reverse the mineral deposition.(22) Remarkable resolution of calcinosis can occur with probenecid therapy Children with severe disease refractory to medical therapy to improve renal handling of phosphate.(23) Positive re- seem especially prone to developing exoskeleton-like calcifi- sponses to alendronate(24) and increasingly positive re- cations. In turn, the exoskeleton is associated with severe sponses to diltiazem treatment are reported.(25) Troublesome calcinosis and poor physical function. Skeletal scintigraphy can calcium deposits can be removed surgically. be useful.(11) MRI is also helpful for diagnosis by showing muscle edema.(12) PROGNOSIS ETIOLOGY AND PATHOGENESIS The clinical course of dermatomyositis in children is vari- Juvenile dermatomyositis seems to be a form of complement- able. Some have long-term relapsing or persistent disease, mediated microangiopathy.(13) HLA-DQA1*051 may be a predis- whereas others recover. When recovery is incomplete, there posing factor.(14) The precise cause of the dystrophic calcification may be severe residual weakness, joint contractures, and cal- is unknown. However, immune deficiencies may predispose the cinosis. The calcinosis may be the principal cause of long-term (1–6,23) patient to this complication.(15) Calcinosis seems to occur in the disability. majority of long-term survivors and may reflect a scarring process. This hypothesis is supported by the observation that mineral deposition appears primarily in the muscles that were most se- REFERENCES verely affected during the disease’s acute phase. Electron microscopy shows that the calcification consists of hydroxyapatite crys- 1. Cassidy JT, Lindsley CB 2005 Juvenile dermatomyositis. In: Cassidy JT, (16) Petty RE (eds.). Textbook of Pediatric Rheumatology, 5th ed. WB Saun- tals, but other important factors and cells seem to be significant ders, Philadelphia, PA, USA, pp. 407–441. constituents.(9) ␮CT and X-ray diffraction reveal hydroxyapatite 2. Pachman LM 1995 Juvenile dermatomyositis. Pathophysiology and dis- with varied microstructures.(17) ease expression. Pediatr Clin North Am 42:1071–1098.

3. Jayalakshmi SS, Borgohain R, Mohandas S 2000 Dystrophic calcification tions in childhood dermatomyositis association with the development of in adult dermatomyositis: Neuroimage. Neurol India 48:407. calcinosis, raised IgE concentrations and granulocyte chemotactic defect. 4. Pachman LM, Hayford JR, Chung A, Daugherty CA, Pallansch MA, Fink Ann Rheum Dis 51:378–383. CW, Gewanter HL, Jerath R, Lang BA, Sinacore J, Szer IS, Dyer AR, 16. Landis WJ 1995 The strength of a calcified tissue depends in part on the Hochberg MC 1998 Juvenile dermatomyositis at diagnosis: Clinical char- molecular structure and organization of its constituent mineral crystals in acteristics of 79 children. J Rheumatol 25:1198–1204. their organic matrix. Bone 116:533–544. 5. Fisler RE, Liang MG, Fuhlbrigge RC, Yalcindag A, Sundel RP 2002 17. Stock S, Ignatiev K, Lee P, Abbott K, Pachman L 2004 Pathological Aggressive management of juvenile dermatomyositis results in improved calcification in juvenile dermatomyositis (JDM): MicroCT and srynchro- outcome and decreased incidence of calcinosis. J Am Acad Dermatol tron x-ray diffraction reveal hydroxyapatite with varied microstructures. 47:505–511. Connect Tissue Res 45:248–256. 6. Wananukul S, Pongprasit P, Wattanakrai P 1997 Calcinosis cutis present- 18. Bowyer SL, Blane CE, Sullivan DB, Cassidy JT 1983 Childhood der- ing years before other clinical manifestations of juvenile dermatomyositis: matomyositis: Factors predicting functional outcome and development of Report of two cases. Australas J Derm 38:202–205. dystrophic calcification. J Pediatr 103:882–888. 7. Perez MD, Abrams SA, Koenning G, Stuff JE, O’Brien KO, Ellis KJ 1994 19. DeSilva TN, Kress DW 1998 Management of collagen vascular diseases Mineral metabolism in children with dermatomyositis. J Rheumatol 21: in childhood. Dermatol Clin 6:579–592. 2364–2369. 20. Reiff A, Rawlings DJ, Shaham B, Franke E, Richardson L, Szer IS, 8. Lian JB, Pachman LM, Gundberg CM, Partridge REH, Maryjowski MC Bernstein BH 1997 Preliminary evidence for cyclosporin A as an alter- 1982 Gamma-carboxyglutamate excretion and calcinosis in juvenile der- native in the treatment of recalcitrant juvenile rheumatoid arthritis and matomyositis. Arthritis Rheum 25:1094–1100. juvenile dermatomyositis. J Rheumatol 24:2436–2443. 9. Mukamel M, Horev G, Mimouni M 2001 New insight into calcinosis of 21. Moore SE, Jump AA, Smiley JD 1986 Effect of warfarin sodium juvenile dermatomyositis: A study of composition and treatment. J Pediatr therapy on excretion of 4-carboxy-l-glutamic acid in scleroderma, 138:763–766. dermatomyositis, and myositis ossificans progressiva. Arthritis 10. Blane CE, White SJ, Braunstein EM, Bowyer SL, Sullivan DB 1984 Rheum 29:344–351. Patterns of calcification in childhood dermatomyositis. Am J Roentgenol 22. Wang W-J, Lo W-L, Wong CK 1988 Calcinosis cutis-juvenile dermato- 142:397–400. myositis: Remarkable response to aluminum hydroxide therapy. Arch 11. Bar-Sever Z, Mukamel M, Harel L, Hardoff R 2000 Scintigraphic eval- Dermatol 124:1721–1722. uation of calcinosis in juvenile dermatomyositis with Tc-99m MDP. Clin 23. Harel L, Harel G, Korenreich L, Straussberg R, Amir J 2001 Treatment of Nucl Med 25:1013–1016. calcinosis in juvenile dermatomyositis with probenecid: The role of phos- 12. Samson C, Soulen RL, Gursel E 2000 Milk of calcium fluid collections in phorus metabolism in the development of calcifications. J Rheumatol juvenile dermatomyositis: MR characteristics. Pediatr Radiol 30:28–29. 28:1129–1132. 13. Kissel JT, Mendell JR, Rammohan KW 1986 Microvascular deposition of 24. Ambler GR, Chaitow J, Rogers M, McDonald DW, Ouvrier RA 2005 complement membrane attack complex in dermatomyositis. N Engl J Med Rapid improvement of calcinosis in juvenile dermatomyositis with alen- 314:329–334. dronate therapy. J Rheumatol 32:1837–1839. 14. Reed AM, Pachman LM, Hayford J, Ober C 1998 Immunogenetic studies 25. Ichiki Y, Akiyama T, Shimozawa N, Suzuki Y, Kondo N, Kitajima Y in families of children with juvenile dermatomyositis. J Rheumatol 25: 2001 An extremely severe case of cutaneous calcinosis with juvenile 1000–1002. dermatomyositis, and successful treatment with diltiazem. Br J Dermatol 15. Moore EC, Cohen F, Douglas SD, Gutta V 1992 Staphylococcal infec- 144:894–897.

Chapter 79. Vascular Calciﬁcation

Dwight A. Towler1 and Linda L. Demer2 1Department of Medicine, Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri; and 2Department of Medicine, Division of Cardiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California

INTRODUCTION cell lineages control vascular mineral metabolism, entrained to morphogenetic, metabolic, inflammatory, and mechanical de- The details of tissue calcium homeostasis in the skeleton are mands experienced by any particular vascular segment. This beginning to emerge. Interactions between a functional trium- chapter provides a very brief overview of vascular calcifica- virate of endothelial, mesenchymal, and hematopoietic cell tion, organized into histoanatomic categories that highlight lineages control bone formation and bone resorption— known or probable differences in pathobiology, and thus may entrained to morphogenetic, metabolic, inflammatory, and me- potentially guide future development of effective pharmaco- chanical demands placed on the skeleton. However, with ad- therapeutic approaches. vancing age, vascular inflammation, hypertension, and certain dysmetabolic states (diabetes, dyslipidemia, uremia, hyperphosphatemia), calcium accumulates to a substantial extent in ATHEROSCLEROTIC CALCIFICATION another venue—the arterial macrovasculature.(1,2) Mechanistic The most common form of vascular calcification is athero- studies of vascular calcification and vascular calcium metabo- sclerotic calcification (Table 1), in which hydroxyapatite min- lism significantly lag behind those of skeletal mineral physiol- eral forms inside intimal plaque in association with lipid de- ogy. Recent data show that “osteogenic” and “chondrogenic” posits and monocyte–macrophage infiltration. Until recently, mechanisms resembling those of craniofacial or endochondral atherosclerotic vascular calcification was considered an un- bone formation control vascular mineral deposition.(1,2) As in common, passive, degenerative, inevitable process of aging. bone, cells of endothelial, mesenchymal, and hematopoietic Textbook examples seldom included calcification, and pathol- ogists classified calcified plaque as a category Vb lesion, giving the impression that it is limited to end-stage disease Dr. Towler is a consultant for Eli Lilly. Dr. Demer has reported no processes.(3) Selection bias may have occurred as a result of conflicts of interest. investigator avoidance of significantly calcified plaques, be-

TABLE 1. HISTOANATOMIC TYPES OF VASCULAR CALCIFICATION nucleated and located along the internal elastic laminae between the endothelial and medial layers. Proteases are gener- Atherosclerotic intimal calcification (type Vb atherosclerotic plaque) ated in atherosclerosis and degrade elastin, apparently opening Medial artery calcification of diabetes and chronic kidney disease sites of lipid and calcium binding. Normally the extracellular Elastocalcinotic medial artery calcification (Marfan’s syndrome, matrix of arteries includes collagen type III and collagen type pseudoxanthoma elasticum) I, but greater collagen I is produced in atherosclerosis. Loss of Cardiac valve calcification (native and bioprosthetic) elastin with upregulation of type I collagen may promote Calcific uremic arteriolopathy (“cutaneous calciphylaxis”) osteogenic phenotypic differentiation by oxylipids. Cardiac annulus calcification Post-infarct myocardial calcification Ectopic Bone in Atherosclerosis. Although it is well known Pericardial calcification that calcium deposits in the artery wall are generally composed Soft tissue calciphylaxis including vessels (acute hyperphosphatemia and of hydroxyapatite mineral, it is little known that ϳ15% of renal failure) calcified plaques contain fully formed lamellar bone.(6) As Calcifying primary cardiac tumors Virchow noted in 1863, some cases of vascular calcification are Portal vein calcification not mere calcification, but “ossification with real plates of Pelvic vein pleboliths bone.”(7) A prerequisite for bone formation within arteries is angiogenic invasion, driven by vascular endothelial growth factor, as in endochondral vascularization, which provides a cause these plaques can damage both scalpels and microtomes scaffold for bone tissue development. Sites of atherosclerotic and require prolonged decalcification or plastic embedding. ossification frequently include marrow spaces with fat tissue However, with growing use of cardiac gated electron beam CT and hematopoietic cells, as well as multinucleated cells that are scanning (EBCT), calcium deposits have been found in ϳ20% positive for osteoclast markers.(8) Usually, the bone tissue is of young adults, 60% of middle-aged adults, and 90% of the associated with and appears to arise from calcified matrix. The elderly.(4) mature bone tissue does not usually appear until vascular invasion of the deposit. It is remarkable to note that, in its Pathobiology of Atherosclerotic Calcification mature stage, atherosclerotic calcification actually contains Inflammatory Milieu of Atherosclerotic Lesion. Atheroscle- vessels within bone structures that are, themselves, within a rosis is an inflammatory vascular disease, which might more vessel. Thus, angiogenesis and biology of the vasa vasorum accurately be termed “atheroscleritis.” The process is initiated play important roles in the formation of advanced atheroscle- by a breach in the endothelial barrier or by high serum lipids, rotic calcification. resulting in accumulation of lipoprotein particles in the subendothelial space between the endothelial monolayer and the Molecular Mechanisms, Vascular Stem Cells, and Relation- underlying internal elastic lamina (Fig. 1).(1,2) When the re- ships to Bone Formation. As with skeletal bone formation tained lipoproteins undergo non-enzymatic oxidation, they and remodeling, vascular mineralization is subject to both generate bioactive phospholipids and oxysterols, which induce positive and negative regulation. Pathological calcification in chemotactic influx of smooth muscle cells from the vessel wall arteries and valves shares many features with normal skeletal as well as monocytes and T lymphocytes from the circula- mineralization. Matrix vesicles are found in human and animal tion.(1,2) These inflammatory lipids can also induce osteogenic atherosclerotic calcification,(9) along with bone regulatory pro- differentiation and mineralization of vascular smooth muscle teins, such as osteopontin, osteocalcin, osteonectin, matrix Gla cells.(5) Earliest sites of atherosclerotic calcification are lipid protein (MGP), and bone sialoprotein.(2) Human and animal

FIG. 1. Arterial calcification. In arterial cross-section, three layers—intima, media, and adventitia—are present from the lumen outward. In atherosclerosis, eccentric, subintimal atheroma formation, cholesterol deposition with inflammation, fibrosis, compromise of the internal elastic lamina, apoptic body formation, and calcium deposition herald formation of the type Vb calcified atherosclerotic plaque.(3) Atherosclerosis deforms the lumen and potentially provides a focus for thrombosis and acute occlusion. In medial artery calcification, calcium deposition is concentric, compromising vascular compliance without lumen deformation. Low-grade adventitial inflammation, elastinolysis, and vascular smooth muscle cell matrix vesicle formation drive concentric disease processes. Of note, vascular calcification is only one component of the pathobiology that contributes to reduced vascular compliance.(68) Myofibroblast proliferation, vascular monocyte–macrophage infiltration, and microvessel formation (angiogenesis) are key components of osteogenic vascular calcification responses in macrovascular settings.(1,2,6) CaPO4, apatitic calcium phosphate deposition.

© 2006 American Society for Bone and Mineral Research VASCULAR CALCIFICATION / 443 calcific arterial lesions also contain osteogenic regulatory and TABLE 2. EVIDENCE LINKING HYPERLIPIDEMIA WITH CORONARY AND transcription factors such as BMP2, Msx2, Runx2, Osterix, and VALVULAR CALCIFICATION Wnts.(1,2,10) Current evidence indicates that, in vascular cells, Coronary calcification correlates with Pohle et al. 2001 BMP2 signaling initiates ectopic osteoblastic differentiation.(2) LDL—independently of age In addition to bone, atherosclerotic lesions may contain Cardiac valve calcification progression Mohler et al. 2001 cartilage tissue, amorphous calcification, marrow-like tissue, correlates with LDL Pohle et al. 2001 and adipose tissue. The origin of all these tissues is not clear, High-fat diet induces matrix vesicles Hsu et al. 2001 but the possibilities are intriguing. One consideration is that Coronary calcification correlates inversely Kuller et al. 1999 (11) there are resident mesenchymal stem cells in the artery wall. with HDL and positively with LDL The smooth muscle cells of the tunica media contain hetero- Lipid-lowering treatment inhibits coronary Williams et al. 1998 geneous subpopulations. Some, including aortic myofibro- calcification in monkeys and in humans Callister et al. 1998 blasts, bovine aortic calcifying vascular cells (CVCs), and Hyperlipidemia induces and lipid-lowering Rajamannan et al. 2003, microvascular pericytes, are now known to have multilineage inhibits, aortic valve calcification 2005 potential in vitro, generating osteogenic, chondrogenic, leiomyogenic, marrow stromal, and adipogenic lineages under regulation of BMP2 and Wnt signaling. It is also possible that the multipotential cells in the artery wall arise from the adven- warfarin-induced vascular calcification in rats,(21) but without titial layer.(10,12) Adventitial cells, in turn, may originate in the atherosclerosis (see medial artery calcification below). bone as marrow stromal cells and immigrate to the atherosclerotic plaque through the circulation entering through the ad- Role of Hyperlipidemia and Inflammatory Lipids in Athero- ventitial vasa vasorum. Recent evidence that atherosclerotic sclerotic Vascular Calcification. Atherosclerosis and athero- plaques derive in part from progenitor cells in the adventitia sclerotic vascular calcification associate epidemiologically and circulation. However, the relative contributions of regional with hyperlipidemia (Table 2). In young adults with homozy- vascular mesenchymal progenitors versus circulating marrow- gous familial hypercholesterolemia, atherosclerotic coronary derived mesenchymal progenitors has yet to be determined. calcification is essentially universal, and its severity correlates with the severity and duration of the hypercholesterolemia as In Vitro Models of Vascular Calcification. A variety of cells measured in cholesterol-years.(22) In coronary artery disease harvested from the artery wall, with the exception of endothe- patients, progression of coronary calcification correlates with lial cells, produce hydroxyapatite mineral in vitro.(13,14) Uns- the severity of hyperlipidemia.(23) Conversely, when patients elected medial smooth muscle cells, in the same manner as successfully lower their cholesterol levels with lipid-lowering osteoblastic cells, mineralize the film of extracellular matrix agents, the rate of progression of coronary calcification is overlying cellular monolayers in the presence of exogenous reduced.(24) This epidemiological evidence together with the phosphate donors. Occasionally, these cultures will produce a close physical relationship between vascular calcification and 3D cellular aggregate containing mineral. Such mineralized atherosclerotic lesions strongly suggest that the two are mech- aggregates are produced in cultures of bovine microvascular anistically related. Lipid may directly contribute to hydroxy- pericytes, human aortic smooth muscle cells, and bovine aortic apatite mineral proliferation. At the ultrastructural level, hy- smooth muscle cells. The spatial frequency of nodules is in- droxyapatite mineral crystals from atherosclerotic lesions are creased several-fold in about one third of single-cell derived physically associated with microcrystals of cholesterol.(25) Bos- subcultures of bovine aortic smooth muscle cells (SMCs) key and Posner(26) showed that phospholipids form complexes termed CVCs.(14) The nodules range widely in size—from with calcium and phosphate in mineral initiation, and the about 100 to 1500 mm in diameter—and contain irregularly phospholipids in matrix vesicles may be crucial in nidus for- shaped hydroxyapatite mineral deposits within their core. The mation. One of the earliest sites of calcification in atheroscle- regular spacing of the nodules seems to be mediated by a rosis is in the elastin layer, where dietary lipids incorporate into reaction–diffusion process involving the morphogens BMP2 the molecular structure.(27) While it is generally assumed that and one of its inhibitors, matrix GLA protein (MGP).(15) Bo- the calcium deposits follow from inflammatory effects of the vine pericytes, retinal microvascular smooth muscle cells, re- cholesterol deposits, some data suggest that calcium hydroxy- quire several weeks to produce calcified nodules; bovine apatite crystals trigger the inflammatory reaction in atheroscle- cloned CVCs and human SMCs require about 10–14 days. The rosis rather than vice versa(28); such responses have the tremen- rate is affected by exogenous ascorbic acid, presumably caused dous potential to fuel rapid vascular disease progression by the changes in extracellular type I collagen production. through procalcific “feedforward” mechanisms. In vitro, inter- leukin-6 (IL-6), TNF-␣, 25-hydroxycholesterol, TGF-␤,fi- In Vivo Models of Atherosclerotic Calcification. Mice defi- bronectin and collagen I, and inflammatory lipoproteins/ phospholipids induce osteoblastic differentiation in vascular cient in apolipoprotein E develop spontaneous vascular calci- (5,29) (16) smooth muscle cells. In vivo, mice with hyperlipidemia fication, primarily in the form of cartilaginous metaplasia. (16) Interestingly, these mice also have increased BMD, presum- develop both atherosclerosis and vascular calcification. Ap- ably related to deficiencies in vitamin K delivery. Most se- optosis occurs in atherosclerotic plaques, producing apoptotic verely affected are the great vessels of the heart, especially the bodies that nucleate mineral deposition through mechanisms similar to those used by the geometrically smaller matrix brachiocephalic (innominate) artery.(16) Mice deficient in the vesicle.(30) low-density lipoprotein receptor (LDLR) develop hyperlipidemia and vascular calcification when exposed to a high-fat Clinical Issues in Atherosclerotic Calcification diabetogenic diets.(17) Mice expressing the human LPA gene develop calcified aortic lesions.(18) Certain strains of mice are Clinical Significance. The correlation between the degree of more predisposed to develop spontaneous vascular calcification calcification and atherosclerosis is strong enough that the “cal- through endochondral calcific metaplasia.(19) Atherosclerotic cium score” is a reliable clinical marker for coronary artery calcification can be induced by vitamin D and calcium supple- disease, predicting cardiovascular events independently and ments in hyperlipidemic rabbits.(20) Vitamin D also enhances more accurately than some conventional risk factors.(31) As

© 2006 American Society for Bone and Mineral Research 444 /CHAPTER 79 might be expected, the degree of calcification correlates quan- supplies coronary and systemic circulation, and hence can titatively with the volume of atherosclerotic plaque burden. rapidly threaten hemodynamic stability when severe. Calcifi- Clinical consequences of vascular calcification primarily stem cation is primarily on the aortic face of the valve, the layer from perturbed endothelial antithrombotic function and me- known as the fibrosa. The two other layers, the spongiosa and chanical rigidity of the aortic arch and cardiac valves. Nor- ventricularis, which face the ventricle, are generally spared.(36) mally highly resilient and rich in elastin, these structures de- The primary cell type, known as the valvular interstitial cell, is velop high flow impedance once calcified, resulting in intermediate between fibroblasts and vascular smooth muscle hypertension, left ventricular hypertrophy, heart failure, aortic cells. Valve interstitial cell myofibroblasts resemble the aortic stenosis, coronary ischemia, complications of cardiovascular adventitial myofibroblasts that contribute to medial artery cal- surgery and procedures, as well as possible acute coronary cification.(1,2) syndrome and myocardial infarction. Aortic recoil is required for maintaining diastolic aortic pressure, which, in turn, is Pathobiology of Cardiac Valve Calcification required for coronary perfusion. Calcified aortas lack recoil, resulting in attenuated diastolic perfusion, high pulse pressure, Native Cardiac Valve Calcification. For years, cardiac valvu- and coronary insufficiency. It remains controversial whether lar stenosis had been attributed solely to mechanical “wear and calcified plaques convey mechanical stability or instability to tear.” Indeed, the endothelium on the two faces are heteroge- atherosclerotic lesions. Although most plaques that rupture are neous, both in development and in adult valves, which have calcified, and numerous calcium deposits increase the risk of reduced expression of calcification inhibitors on the high- rupture,(32) ϳ80% of significantly narrowed plaques are calci- pressure, aortic face of the leaflets.(37) However, recent evi- fied.(33) Real-time imaging during human angioplasty and en- dence converges to support the concept that most aortic calcific gineering considerations suggest that solid mechanical failure valvular stenosis is atherosclerotic. Many of the same molec- stresses are concentrated at the edges of calcium deposits.(2) ular and cellular processes driving atherosclerotic calcification have now been shown to enhance valvular calcification. Ath- Inhibition of Atherosclerotic Calcification. If lipids promote erosclerotic and coronary risk factors also convey risk for vascular calcification, an obvious approach to inhibition calcific aortic stenosis. As with atherosclerotic calcification, would be lipid-lowering agents. Patients who successfully most of these factors can be categorized as inflammatory or lower their cholesterol levels significantly reduce progres- oxidative stressors. In early lesions, increases in subendothelial sion of their coronary calcification.(24) Atherogenic effects thicknesses on the aortic face, with myofibroblast intracellular of oxidized lipids are also blocked by high-density lipopro- lipid accumulation, expansion of the valve interstitial valve tein (HDL) and in vitro vascular calcification. In an entirely fibrosus, diffuse stippled calcium deposition, and monocyte– different mechanism, osteopontin seems to inhibit mineral- macrophage infiltration histologically evident.(36) Of note, ization both at the physico-chemical level of stearic inhibi- whether valvular calcification arises in response to atheroscle- tion of crystal growth, as well as at the level of cellular rotic stimuli or the hemodynamic stresses such as those expe- genetic regulation.(34) An in vivo model for regression of rienced with bicuspid aortic valves, inflammatory T-cell infil- ectopic calcification has been developed using an allograft trates are observed during the later stages of disease of glutaraldehyde-fixed cardiac valvular tissue implanted progression. As in atherosclerotic calcification, mature lamellar subcutaneously in mice. Using this model, Steitz et al.(35) or endochondral bone tissue is found in ϳ15% of stenotic showed that osteopontin blocks ectopic calcification; in ad- cardiac valves.(6) Calcified aortic valves express many of the dition, osteopontin promotes calcium egress by inducing same osteogenic processes as atherosclerotic calcification, in- invading monocyte/macrophage carbonic anhydrase II, thus cluding osteopontin at the mRNA and protein level, BMP2, acidifying the extracellular matrix and enhancing calcium RANKL, tenascin C, osteocalcin, and alkaline phosphatase mobilization. Given that atherosclerotic lesions are already activity.(2) rich in osteoclast progenitors in the form of diapedetic monocytes, it is conceivable that existing atherosclerotic Bioprosthetic Cardiac Valve Calcification. Cardiac valves calcification could be reversed by local induction of oste- can be replaced by mechanical or biological tissue protheses. oclastic resorption—essentially by eliciting osteoporosis in The biological prostheses are usually fashioned from devital- the artery wall. Indeed, osteoclast-like cells have been iden- ized, glutaraldehyde treated, allograft or xenograft (porcine, tified histopathologically in calcified atherosclerotic le- bovine) valve or pericardium. Glutaraldehyde treatment is be- sions.(8) These multinucleated cells stain positively for tar- lieved to reduce immunogenicity. Bioprosthetic valves have trate resistant acid phosphatase and cathepsin K, but it the advantage of not requiring long-term anticoagulation; how- remains to be established whether they are bona fide oste- ever, the greatest concern in these valves is ultimate mechan- oclasts. The net effects of augmenting “vascular osteoclast” ical failure because of mineralization. The observations that activity on vascular health and integrity is as yet unknown. cell-free bioprosthetic valves and vascular matrix can mineralize in vitro with inorganic phosphate supplementation(38) has CARDIAC VALVE CALCIFICATION generated some confusion about whether vascular calcification is “cell-regulated.” A clarifying point is that even normal, Cardiac valve leaflets are remarkably thin and pliable, yet physiological calcification occurs outside of cells, as in carti- strong and inelastic, consisting of two layers of interstitial cell lage calcification that occurs after apoptotic death of hypertro- myofibroblasts surrounded on either side by endothelial mono- phic chondrocytes. Interestingly, the valve fixation procedure layers.(36) Valvular sclerosis (fibrosis) is a common occurrence does not usually remove lipids; however, when lipids are during hypertension, inflammation, diabetes, dyslipidemia, and removed experimentally, ex vivo mineralization is reduced.(39) advanced age, and can occur in the absence of narrowing of the Thus, the cellular regulation occurs at the level of removing valvular opening. Once the scarring becomes sufficiently ad- PPi-like mineralization inhibitors and generating matrix:lipid vanced to narrow the orifice (i.e., stenotic), it is usually calci- complexes that nucleate mineral deposition. As such, valve fied. Hence, a common disorder is calcific cardiac valve ste- calcification occurs through cell-regulated mechanisms similar nosis. Stenosis is most clinically apparent in calcific aortic to those directing skeletal mineralization. Of note, vascular and sclerosis because the aortic valve is in a high pressure system, valvular cells require no more, and possibly less, exogenous

© 2006 American Society for Bone and Mineral Research VASCULAR CALCIFICATION / 445 organic phosphate than skeletal-derived osteoblastic primary ficiencies in elastin-based extracellular matrix metabolism and cells or cell lines; some vascular cells require no supplemental elastocalcinosis (i.e., not through matrix vesicle formation organic phosphate to mineralize in vitro.(2) [vide infra]). While both types of medial artery calcification are sometimes denoted as Monckeberg’s medial calcific sclerosis, Models of Valve Calcification. Myofibroblastic valve intersti- the emerging differences in pathobiology are highlighted by tial cells can be harvested from human, canine, lapine, or ovine individual consideration below. aortic valves obtained at surgery.(2) Rajamannan et al.(40) developed a rabbit model for studying calcific valvuloplasty Pathobiology of Medial Artery Calcification elicited by diet-induced hyperlipidemia. As with mural vascular cells, these valvular cells incorporate calcium and deposit Medial Artery Calcification of Diabetes and Uremia. Medial hydroxyapatite mineral in their matrix. Osteogenic calcification artery calcification is characterized by the deposition of apatitic is enhanced by oxysterols, RANKL, and canonical Wnt sig- calcium phosphate in the tunica media of large vessels—with naling.(41) However, it is as yet unclear whether these models the notable absence of neointima formation. Medial artery fully recapitulate the pathobiology and pharmacology of estab- calcium deposition is nucleated by lipidaceous matrix vesicles lished human cardiac valve calcification. that arise from a minimum of two sources: (1) the apoptotic bodies of dying vascular smooth muscle cells (VSMCs) remi- niscent of hypertrophic chondrocyte mineralization and (2) the Clinical Issues in Cardiac Valve Calcification regulated extrusion of mineralizing matrix vesicles from viable Clinical Significance of Calcific Valvular Stenosis and Bio- VSMCs.(30) The latter process closely resembles the mineral- prosthetic Valve Calcification. Calcific aortic stenosis is the ization of membranous bone formation during craniofacial most frequent cardiac valve disorder in developed countries skeletogenesis. Importantly, Reynolds et al.(48) have convinc- and the primary valve disorder in the elderly. It confers high ingly shown that matrix vesicles can promote or inhibit calcium morbidity and mortality.(42) Valvular calcification can be diag- deposition, dependent on whether serum-derived inhibitors nosed by ultrasonic imaging (echocardiography), but the nar- such as fetuin are recruited into MGP-containing complexes. rowing of the orifice is ideally assessed by Doppler techniques. Serum fetuin is taken up by VSMCs and packaged into matrix Recent evidence suggests that valve calcification can be reli- vesicles that serve to inhibit calcium deposition. Besides in- ably quantified by EBCT. While bioprosthetic valves do not hibiting matrix vesicle nucleation, fetuin promotes VSMC require long-term anticoagulation, the life span of these im- “phagocytosis” of pro-osteogenic matrix vesicles; this high- plants is generally limited to about 10 years because of calci- lights the complexity of VSMC-regulated vesicle metabolism fication that results in stenosis and insufficiency. that controls the initiation and propagation of vascular calcification. Importantly, production of pro-osteogenic matrix vesi- Inhibition of Cardiac Valve Calcification. Several strategies cles entails the upregulation of bone alkaline phosphatase have been evaluated in preclinical and clinical models to inhibit (ALP), a key osteoblast ectoenzyme that promotes deposition cardiac valve calcification. Osteopontin, known to be inhibitory of calcified extracellular matrix. ALP (a.k.a. tissue nonspecific in vascular calcification, is also inhibitory in the in vivo model alkaline phosphatase) is of particular importance. Inorganic of valvular calcification developed by Steitz et al.(35) and co- pyrophosphate (PPi) is a cell-generated organic anion that localizes with mineralization in valves. Another inhibitor val- inhibits mineralization-and is a physiologically relevant sub- idated in vivo is pulsatile teriparatide, a PTH/PTH-related strate for ALP hydrolysis. Johnson and Terkeltaub(49) have peptide (PTHrP) receptor agonist and bone anabolic agent that elegantly shown that loss of extracellular PPi derived from (1) concomitantly inhibited cardiac valve calcification in LDL the extracellular enzyme NPP1 (ectonucleotide pyrophos- receptor null mice.(43) Etidronate, used to inhibit heterotopic phatase/phosphodiesterase I) or (2) the cellular PPi exporter bone formation after hip surgery, seems to inhibit progression ANK predisposes to massive arterial calcification in murine of aortic calcification in patients with end-stage renal disease models. Intriguingly, extracellular pyrophosphate is re- (ESRD).(44) Some evidence suggests that lipid lowering may quired to stabilize the myogenic phenotype of VSMCs; reduce valvular calcification. In the hyperlipidemic rabbit VSMCs incapable of generating a PPi-replete extracellular model, treatment to lower serum lipids levels reduced the milieu undergo phenotypic drift and begin to express mo- severity of calcification(40) through effects on the LDL receptor lecular markers of the chondrogenic lineage. Importantly, related protein, LRP5, and canonical Wnt signaling.(41) Aortic chondrogenic “trans-differentiation” and tissue mineraliza- valve calcification progresses more rapidly in subjects with tion are inhibited by treatment with nanomolar concentra- high LDL levels.(23) However, strategies aggressively focused tions of PPi.(49) Of note, in the setting of ESRD, circulating on LDL-cholesterol reduction with statin therapy seem insuf- PPi levels are reduced.(50) Thus, along with the prevalent ficient to prevent vascular calcification progression once the glucose intolerance, hyperphosphatemia, and fetuin defi- disease has been initiated.(45) ciency, reduction in PPi synergistically promotes the profound calcific vasculopathy that assails patients with ESRD.(1) Strategies that seek to restore serum PPi “tone” MEDIAL ARTERY CALCIFICATION using non-hydrolyzable bisphosphonate PPi analogs may in (MONCKEBERG’S MEDIAL CALCIFIC SCLEROSIS) fact inhibit progression of vascular calcification.(44) Medial artery calcification is a highly characteristic feature of diabetes and ESRD.(1) Although diabetes is the leading cause Molecular Mechanisms, Vascular Stem Cells, and Relation- of ESRD, diabetes is also an independent risk factor for vas- ships to Bone Formation. The molecular mechanisms that cular calcification(46); indeed, even in the presence of chronic regulate vascular calcification in diabetes are beginning to be renal insufficiency, the extent of medial artery calcification understood. High-fat diets that induce obesity, insulin-resistant increases with worsening glycemic control.(47) Uremic and diabetes, and dyslipidemia promote vascular calcification in diabetic medial artery calcification proceed through matrix male LDLR-deficient mice.(17) In this physiologically relevant vesicle–dependent mineralization processes.(30) Medial artery model of type II diabetes, the high fat Western diet—a stimulus calcification also occurs in the setting of Marfan’s syndrome for obesity and vascular matrix vesicle formation(51)—activates and pseudoxanthoma elasticum, characterized by primary de- an aortic adventitial BMP2-Msx2 signaling cascade. Cell cul-

© 2006 American Society for Bone and Mineral Research 446 /CHAPTER 79 ture studies have shown that Msx2 enhances osteogenic differ- dependent internalization.(58) Thus, if PTH1R signaling plays entiation (ALP induction, calcification) of aortic myofibro- important roles in promoting skeletal mineral accumulation blasts through Osterix-dependent signals. Analysis of while simultaneously limiting vascular calcium accumulation, conditioned media from Msx2-expressing 10T1/2 mesenchy- the accumulation of such antagonistic PTH fragments may mal cells revealed the elaboration of a pro-osteogenic signal contribute to the calcific vasculopathy of CKD. characteristic of a canonical Wnt ligand.(10) Canonical Wnts signal through the heteromeric LDLR-related protein receptors Elastocalcinotic Vascular Calcification: A Distinct Form of LRP5 and LRP6 to activate osteogenic gene expression Medial Artery Calcification. Recently, it has become apparent through nuclear ␤-catenin–dependent transcription. Similarly, that vascular calcification associated with primary alterations in Msx2-expressing cells express a factor that enhances nuclear elastin metabolism may in fact represent a unique entry point in accumulation of ␤-catenin and upregulates activity of a feedforward cycle of medial artery calcification.(59) Large ␤-catenin–dependent transcription driven by a T-cell transcrip- muscular arteries contain elastin as a major extracellular matrix tion factor/lymphoid enhancer binding factor (TCF/LEF) opti- constituent. Aberrant elastin organization and metabolism is mal promoter (TOP)-reporter construct.(10) Pro-osteogenic ac- characterized by aortic root dilatation, aneurysm formation, tivities of Msx2 were reversed by treatment with Dkk1, an and medial calcification and degeneration. This is perhaps most inhibitory ligand for LRP5 and LRP6 signaling. Similar results evident in Marfan’s syndrome, where deficiencies in fibrillin 1 were observed in vivo in studies of cytomegalovirus immediate (1) cause homeostatic failure in the microfibrillar array of the early promoter (CMV)-Msx2 transgenic mice,(10) a model pre- tunica adventitia to withstand physiological hemodynamic viously validated in studies of Msx2-dependent ectopic calvar- stress; and (2) result in disruption of the tunica media elastin ial bone formation. Aortic Wnt3a and Wnt7a were upregulated network, smooth muscle cell phenotypic modulation, metallo- by the Msx2 transgene, with concomitant suppression of aortic proteinase induction, and calcification as secondary events.(59) Dkk1. Immunohistochemistry showed Msx2 accumulation in Elastolytic calcification—unlike medial artery calcification of the aortic adventitia but induction of ALP in the tunica media. diabetes—is not initially associated with matrix vesicle forma- Calcium deposition coincides with ALP expression. Thus, a tion; instead, calcium phosphate deposition occurs in associa- working model has emerged in which a paracrine BMP2- tion with degenerating elastin fibrils of the tunica media. As Msx2-Wnt signaling cascade, initiated by the adventitial oxi- such, it is a form of medial artery calcification. While molec- dative stressors of type 2 diabetes, controls the osteogenic ular mechanisms are not understood, it is apparent that elasti- differentiation and mineralization of vascular progenitors nolytic matrix remodeling processes degrading vascular tro- through non-endochondral processes.(10) The vector of mural poelastin and elastin enhance vascular matrix calcium microvascular flow is concentric, with the vasa vasorum cours- deposition. During the progression of any form of medial ing from the tunica adventitia to the tunica media. The con- calcification, perturbations in elastin metabolism likely contrib- centric medial calcification of diabetes arises in part from the ute to vascular calcium load. Interestingly, elastin glycoxida- anatomic relationship between (1) the Msx2 expressing cells of tion products such as pentosidine accumulate in ESRD, in- the periaortic adventitia that elaborate a Wnt-laden osteogenic crease vessel stiffness, and enhance matrix calcium binding.(60) milieu(10) and (2) CVCs of Demer in the tunica media that However, matrix vesicles are clearly evident in medial calci- undergo osteogenic differentiation in response to signals or fication of ESRD, and as such progresses through mechanisms cells conveyed through the vasa vasorum.(11) The precise ori- overlapping those of diabetic medial artery calcification.(30,48) gins of the Msx2-expressing cells and CVCs are not known; Elastin-nucleated calcification also occurs in the setting of however, a ScaIϩ stem cell population has recently been pseudoxanthoma elasticum (PXE), arising from mutations in shown to reside within the aortic adventitia.(12) Whether these the ABCC6 gene that causes fragmentation of elastic lamina.(61) cells arise from circulating progenitors(52) or aortic Mechanisms are again unknown. Electron microscopy con- mesoangioblast-like cells is also unclear. firms deposition of calcium along thickened elastin fibers in the In uremia, a “perfect storm” of calcific vasculopathy occurs. absence of matrix vesicle formation. A murine model of Approximately 5% of Americans have impaired renal function, ABCC6 deficiency has been recently reported.(61) Detailed and three million patients have clinically relevant chronic kid- study of this model should provide further insights into the ney disease (CKD). In this common clinical setting, phosphate pathobiology of elastocalcinotic medial artery calcification. retention, secondary hyperparathyroidism, and the accumulation of PTH fragments that perturb normal calcium phosphate In Vivo Models of Medial Artery Calcification. Several mod- homeostasis drive tremendous vascular calcium loads.(53) The els of medial artery calcification have been developed. The best phosphate retention of CKD presents opportunity for appreciated models are those associated with vitamin D excess intervention(54)-but confounds simple interpretation of disease with warfarin ϩ menadione or hypervitaminosis D plus nico- pathophysiology and progression. Hyperphosphatemia stimu- tine administration.(62,63) These treatments result in an elasto- lates vascular matrix accumulation of procalcific VSMC matrix calcinotic medial artery calcification but may also suppress vesicles.(30) Consistent with this, Giachelli et al.(55) have pro- vascular PTHrP, a paracrine inhibitor of vascular osteogenic vided evidence that inhibition of the cellular phosphate trans- differentiation.(57) Other models include genetic osteoprote- porter, Pit-1, inhibits VSMC calcification and subsequent gerin (OPG) deficiency and high-fat diet administration to Ϫ Ϫ osteo-/chondrogenic differentiation. Moreover, Vyavahare et nephrectomized LDLR / mice(64) or C57Bl/6 mice possess- al.(39) showed that paracrine vascular PTHrP limits VSMC ing the CMV-Msx2 transgene.(10) With OPG deficiency, inti- calcification, consistent with results obtained with pulsatile mal and medial artery calcification arises,(65) potentially related PTH(1-34) administration in vivo(43); thus, the widespread use to unopposed actions of RANKL on vascular myofibro- of calcitriol to limit secondary and tertiary hyperparathyroid- blasts.(66) Pro-osteogenic Wnt signaling cascades are activated ism may exert unintended deleterious consequences on vascu- by aortic Msx2 gene expression, with calcification triggered by lar calcium load(56) through suppression of vascular PTHrP.(57) high-fat diabetogenic diet (vide supra).(10) Induction of chronic A proteolytic fragment of PTH, PTH(7-84), that accumulates in renal insufficiency, with attendant phosphate retention, pro- Ϫ Ϫ ESRD and functions to induce resistance to PTH binds the foundly accelerates calcification in LDLR / mice.(64) Side- PTH1R and does not elicit signaling cascades; instead it down- by-side comparisons have yet to be performed with these regulates cell surface expression by enhancing dynamin- models to clarify mechanistic similarities and differences.

Clinical Issues in Medial Artery Calcification Zebboudj et al.(71) first showed that MGP forms an inhibitory complex with BMPs that precludes ALP induction; this bioac- Clinical Significance of Medial Artery Calcification. Epide- tivity is dependent on modification of MGP by Gla residues. miological studies have clearly shown that medial artery cal- Moreover, as shown by Shanahan et al.,(48) MGP–fetuin com- cification increases the risk of cardiovascular morbidity and (67) (53) plexes assembled by vascular smooth muscle cells form vesi- mortality in patients with diabetes and uremia. The excess cles that can actually inhibit vascular calcium deposition. Of risk for lower extremity amputation and cardiovascular mor- note, recent data suggest that undercarboxylated MGP is asso- tality may arise from a type of vascular “diastolic” dysfunction ciated with the risk of calcific vascular disease in patients with that arises with reduced vascular compliance of elastic arteries. normal renal function.(72) Thus, given the above data, we spec- During systole, potential energy is stored within large elastic ulate that MGP–fetuin deficiencies associated with weight loss arteries such as the aorta. Kinetic energy is subsequently re- and warfarin treatment in patients with CUA contributes to leased during the relaxation phase of the cardiac cycle, provid- pathogenesis. However, until a robust animal model of CUA is ing diastolic perfusion of the myocardium and sustained per- (68) developed, these notions are again speculative; as previously fusion of distal vascular beds. With vessel stiffening, noted,(70) the original calciphylaxis model of Selye that causes elevated pulse pressure and highly pulsatile flow kinetics in- skin necrosis in experimental animals does not recapitulate the teract with elevated systolic blood pressure and increased myo- histopathology of CUA. Thus, the use of the term “cutaneous cardial oxygen consumption to increase workload and decrease (68) calciphylaxis” to connote CUA should probably be discontin- distal tissue perfusion. Compromised elastic artery compli- ued. Future studies will no doubt address whether patients with ance is likely a major contributor to the increased risk for lower (67) CUA are particularly deficient in the formation of these novel extremity amputation of patients with type 2 diabetes. inhibitory MGP:fetuin vesicles.(48) Infusion of sodium thiosulfate has been used to treat severe calcific uremic arteriolopa- Inhibition or Regression of Medial Artery Calcification. thy,(73) but no randomized control trial of thiosulfate therapy in Very few studies have explored whether medial artery calcifi- any form of vascular calcification has been reported. cation is preventable or reversible. Price et al.(63) have shown that vitamin D plus warfarin-induced vascular calcification in MYOCARDIAL, PERICARDIAL, AND ANNULAR the rat is inhibited by treatment with OPG. Giachelli et al.(55) have provided evidence that inhibition of the phosphate trans- CALCIFICATION porter, Pit-1, inhibits smooth muscle cell calcification and Calcium deposits also develop in human myocardial and osteo-/chondrogenic differentiation. The phosphate binding pericardial tissue in a variety of conditions; these include resin sevelamer inhibits the endochondral vascular calcification myocardial infarction—especially with aneurysm formation, of apoE null mice.(69) Moreover, aggressive lipid-lowering pericarditis, and myocarditis. Myocardial dystrophic calcifica- therapy with statins suppresses cardiovascular calcification and tion is visible by chest X-ray in ϳ5–10% of patients who have associated canonical Wnt signaling in dyslipidemic rabbits.(41) survived 5ϩ years after a left ventricular infarct.(74) In mice, However, in human studies, only sevelamer has been unam- spontaneous calcification of the myocardial tissue, known as biguously shown to decrease progression of vascular calcifica- dystrophic cardiac calcinosis, can be induced by a high-fat diet tion.(54) A very recent study showed that administration of the in certain strains. Using intercrosses of resistant C57Bl/6J and endothelin receptor antagonist darusentan induced regression susceptible C3H/HeJ inbred mice, Ivandic et al.(75) identified a of elastocalcinotic medial calcification in rodents by upregula- major predisposing quantitative trait locus, Dyscalc1, on prox- tion of carbonic anhydrase(62); whether this exciting new strat- imal chromosome 7. Granulomatous diseases (tuberculosis, egy is effective in other preclinical models of vascular calcifi- histoplasmosis, sarcoidosis) were historically the common cation has yet to be determined. causes of pericardial calcification; with the incidence of tuberculosis in decline, granulomatous pericardial calcification has CALCIFIC UREMIC ARTERIOLOPATHY also declined.(76) Uremia, systemic lupus, and postviral, post- (CUTANEOUS CALCIPHYLAXIS) irradiation, or post-hemopericardium pericardial inflammation are the more common settings in which pericardial calcification A particularly severe and mercifully uncommon form of is seen today.(76) The stiffening of this tissue produces clini- vascular calcification is calcemic uremic arteriolopathy (CUA), cally significant hemodynamic abnormalities leading to restric- observed in the setting of ESRD. Unlike the highly common tive heart failure. The valve annulus, a fibrous ring embedded macrovascular medial artery and atherosclerotic calcification of in the myocardium surrounding each valve, often undergoes ESRD, CUA afflicts much smaller arteries, most notably the calcification through endochondral metaplasia. Cardiac annu- arterioles of the dermis.(70) Clinically, it presents as a vasculitis, lus calcification is commonly observed after middle age in with livido reticularis followed by cord-like dermal thickening women (mitral) or in the setting of uremia and aortic valve and subsequent “dry” cutaneous necrosis. The histopathology calcification. Rarely, primary cardiac tumors such as rhab- is medial arteriolar (100–600 micron diameter) calcification domyomas, endotheliomas, and myxomas can also calcify. with concomitant (1) endovascular, fibroproliferative neointi- mal constriction; (2) frequent small vessel thrombosis; and (3) VENOUS VASCULAR CALCIFICATION fat necrosis with panniculitis and acute inflammatory changes. Similar histopathology can occur in intestinal mesenteric arte- Calcification does occur in the venous vasculature; indeed, rioles, and contributes to poor clinical outcome.(70) The patho- calcified pelvic venous thromboliths are commonly observed biology of CUA is poorly understood. Antecedent hyperphos- on plain film, but have little known clinical consequence. phatemia and elevated calcium-phosphate product is prevalent However, venous vasculature exposed to elevated transmural but insufficient to explain the disease process. However, treat- pressures may become subject to “arterialization” and thus ment with warfarin before onset of CUA is observed in one half clinically relevant macrovascular calcification in certain dis- of the afflicted patients.(70) MGP is a highly important modu- ease settings. Calcification of saphenous vein grafts used for lator of BMP signaling and inhibitor of osteo/chondrogenic coronary bypass certainly represents a visible and relevant vascular calcification. BMPs are powerful bone morphogens example. However, orthotopic venous calcification has been that promote osteogenic differentiation and ALP induction. uniformly reported in another common clinical setting—portal

© 2006 American Society for Bone and Mineral Research 448 /CHAPTER 79 hypertension. Verma et al.(77) have recently identified that Kaplan FS 2001 Bone formation and inflammation in cardiac valves. ϳ11% of patients with cirrhosis have portal and mesenteric Circulation 103:1522–1528. venous calcification. The pathobiology and clinical conse- 7. Virchow R 1863 Cellular Pathology as Based Upon Physiological and Pathological Histology. Dover Publications, New York, NY, USA. quences have yet to be evaluated, but a detailed understanding 8. Hunt JL, Fairman R, Mitchell ME, Carpenter JP, Golden M, Khalapyan T, of how venous Wnt/LRP signaling and splanchnic venous Wolfe M, Neschis D, Milner R, Scoll B, Cusack A, Mohler ER III 2002 matrix remodeling responds to elevated transmural pressure Bone formation in carotid plaques: A clinicopathological study. Stroke promises to be fruitful. 33:1214–1219. 9. Tanimura A, McGregor DH, Anderson HC 1983 Matrix vesicles in atherosclerotic calcification. Proc Soc Exp Biol Med 172:173–177. SUMMARY 10. Shao JS, Cheng SL, Pingsterhaus JM, Charlton-Kachigian N, Loewy AP, Towler DA 2005 Msx2 promotes cardiovascular calcification by activat- The above pathogenetic mechanisms—reduced tissue pyro- ing paracrine Wnt signals. J Clin Invest 115:1210–1220. phosphate, elevated serum phosphate levels, enhanced vascular 11. Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer inflammation and oxylipid formation with reduced serum fe- LL 2003 Multilineage potential of cells from the artery wall. Circulation tuin, activated vascular BMP2-Msx2-Wnt signaling, and di- 108:2505–2510. minished vascular PTH/PTHrP receptor signaling–offer mul- 12. Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, Xu Q 2004 Abundant progenitor cells in the adventitia contribute to atherosclerosis of tiple potential therapeutic strategies. However, in humans, only vein grafts in ApoE-deficient mice. J Clin Invest 113:1258–1265. sevelamer has been unambiguously shown to decrease progres- 13. Schor AM, Allen TD, Canfield AE, Sloan P, Schor SL 1990 Pericytes sion of vascular calcification(54); clinical studies of bisphospho- derived from the retinal microvasculature undergo calcification in vitro. nates and statins have been either too small(44)or disappoint- J Cell Sci 97:449–461. ing.(45) There are a number of biological and epidemiologic 14. Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL links between the diseases of osteoporosis and atherosclerosis, 1993 Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest 91:1800–1809. suggesting common pathophysiological mechanisms and, thus, 15. Garfinkel A, Tintut Y, Petrasek D, Bostrom K, Demer LL 2004 Pattern (78) potential therapeutic linkage. Given that bone has vascular formation by vascular mesenchymal cells. Proc Natl Acad Sci USA channels, lipids deposits in subendothelial spaces of bone tis- 101:9247–9250. sue and resultant oxidative stress may contribute to both dis- 16. Rattazzi M, Bennett BJ, Bea F, Kirk EA, Ricks JL, Speer M, Schwartz orders.(79) Of note, maintaining bone anabolism is important to SM, Giachelli CM, Rosenfeld ME 2005 Calcification of advanced ath- diminish the risk of vascular calcification in ESRD; excessive erosclerotic lesions in the innominate arteries of ApoE-deficient mice: Potential role of chondrocyte-like cells. Arterioscler Thromb Vasc Biol reductions in serum PTH in hemodialysis patients results in 25:1420–1425. low-turnover osteoporosis and profound vascular calcifica- 17. Towler DA, Bidder M, Latifi T, Coleman T, Semenkovich CF 1998 tion.(80,81) Indeed, in murine models bone anabolic agents in- Diet-induced diabetes activates an osteogenic gene regulatory program in hibit vascular calcium deposition.(43,64) The pulsatile PTH re- the aortas of low density lipoprotein receptor-deficient mice. J Biol Chem sponses elicited by calcium receptor antagonists in chronic 273:30427–30434. renal insufficiency may thus help normalize both vascular and 18. Teivainen PA, Eliassen KA, Berg K, Torsdalen K, Svindland A 2004 (56) Atherogenesis and vascular calcification in mice expressing the human skeletal calcium homeostasis. However, the biological het- LPA gene. Pathophysiology 11:113–120. erogeneity of vascular calcification demonstrates that carefully 19. Qiao JH, Fishbein MC, Demer LL, Lusis AJ 1995 Genetic determination crafted, controlled and monitored translational research studies of cartilaginous metaplasia in mouse aorta. Arterioscler Thromb Vasc Biol are desperately needed to address the tremendous unmet clin- 15:2265–2272. ical need in this bone and mineral disease. 20. Demer LL 1991 Effect of calcification on in vivo mechanical response of rabbit arteries to balloon dilation. Circulation 83:2083–2093. 21. Price PA, Faus SA, Williamson MK 2000 Warfarin-induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb ACKNOWLEDGMENTS Vasc Biol 20:317–327. 22. Hoeg JM, Feuerstein IM, Tucker EE 1994 Detection and quantitation of The authors are supported by grants from the National In- calcific atherosclerosis by ultrafast computed tomography in children and stitutes of Health (D.A.T., L.L.D), the American Diabetes young adults with homozygous familial hypercholesterolemia. Arterio- Association (D.A.T.), and the American Heart Association scler Thromb 14:1066–1074. (L.L.D.). 23. 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Chapter 80. Fibrodysplasia (Myositis) Ossiﬁcans Progressiva

Frederick S. Kaplan, David L. Glaser, and Eileen M. Shore Division of Molecular Orthopaedics, Department of Orthopaedic Surgery, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

INTRODUCTION including intramuscular injections and influenza-like viral illnesses.(2,8,9) Swellings develop rapidly during the course of Fibrodysplasia ossificans progressiva (FOP) is a rare heritable several days. Typically, lesions affect the paraspinal muscles in disorder of connective tissue disease characterized by (1) con- the back or in the limb girdles and may persist for several genital malformations of the great toes and (2) recurrent epi- months.(10) Aponeuroses, fascia, tendons, ligaments, and con- sodes of painful soft-tissue swelling that lead to heterotopic nective tissue of voluntary muscles may be affected. Although ossification.(1,2) some swellings may regress spontaneously, most mature Post-traumatic myositis ossificans, a different disorder, also through an endochondral pathway, engendering true hetero- features heterotopic bone and cartilage formation within soft topic bone.(10) The episodes of induration recur with unpredict- tissues. Heterotopic ossification may also follow hip replace- able frequency. Some patients seem to have periods of quies- ment, spinal cord injury, and brain injury. cent disease. However, once ossification develops, it is FOP was first described in 1692; Ͼ600 cases have been permanent. reported.(1,2) This disorder is among the rarest of human afflic- Gradually, bony masses immobilize joints and cause con- tions, with an estimated incidence of one per two million live tractures and deformity, particularly in the neck and shoulders. births.(1,2) All races are affected.(2) Autosomal dominant trans- Ossification around the hips, typically present by the third mission with variable expressivity is established.(3) However, decade of life, often prevents ambulation.(6) Involvement of the reproductive fitness is low, and most cases are sporadic. Go- muscles of mastication (frequently the outcome of injection of nadal mosaicism has been described.(4) local anesthetic or overstretching of the jaw during dental procedures) can severely limit movement of the mandible and CLINICAL PRESENTATION ultimately impair nutrition.(11,12) Ankylosis of the spine and rib If the typical congenital skeletal malformations are recog- cage further restricts mobility and may imperil cardiopulmo- nized, FOP can be suspected at birth before soft tissue lesions nary function (Fig. 1).(1,2,6,13) Scoliosis is common and associ- occur.(1,2) The characteristic feature is short great toes, caused ated with heterotopic bone that asymmetrically connects the rib by malformation (hallux valgus) of the cartilaginous anlage of cage to the pelvis.(14) Hypokyphosis results from ossification of the first metatarsal and proximal phalanx (Fig. 1). In some the paravertebral musculature. Restrictive lung disease and cases, the thumbs also are strikingly short. Synostosis and predisposition to pneumonia may follow. However, the vocal hypoplasia of the phalanges is typical.(1,2) FOP is usually di- muscles, diaphragm, extraocular muscles, heart, and smooth agnosed when soft tissue swellings and radiographic evidence muscles are characteristically spared.(1) Although secondary of heterotopic ossification are first noted, although misdiagno- amenorrhea may develop, reproduction has occurred.(1–3) Hear- sis is common.(1,2) ing impairment (beginning in late childhood or adolescence) The severity of FOP differs significantly among patients,(3,5) manifests with increased frequency.(15) although most become immobilized and confined to a wheel- chair by the third decade of life.(1,2,6) Typically, episodes of soft tissue swelling begin during the first decade of life (Fig. 1),(7) RADIOLOGIC FEATURES although occasionally, the onset occurs as late as early adulthood. Skeletal anomalies and soft tissue ossification are the char- (16) Painful, tender, and rubbery soft tissue lesions appear spon- acteristic radiologic features of FOP. The principal malfor- taneously or may seem to be precipitated by minor trauma mations involve the great toe, although other anomalies of digits in the feet and hands may occur. Exostoses are frequent.(10) A remarkable feature of FOP is progressive fusion of cervical vertebrae that may be confused with Klippel-Feil The authors have reported no conflicts of interest. syndrome.(1,17) The femoral necks may be broad yet short.

However, the remainder of the skeleton is generally unremarkable.(16) Ectopic ossification in FOP progresses in several regular patterns or gradients (involvement is generally proximal before distal, axial before appendicular, cranial before caudal, and dorsal before ventral).(7) Paraspinal muscles are involved early in life, with subsequent spread to the shoulders and hips. The ankles, wrists, and jaw may be affected at later stages.(7) Radiographic and bone scan findings suggest normal modeling and remodeling of heterotopic bone.(18) Fractures are not increased and respond similarly in either the heterotopic or normotopic skeleton.(19) Bone scans are abnormal before ossification can be shown by conventional radiographs.(18) CT and MRI of early lesions have been described.(20)

LABORATORY FINDINGS Routine biochemical studies of mineral metabolism are usually normal, although alkaline phosphatase activity in serum may be increased, especially during disease “flare-ups,” (i.e., periods of active heterotopic bone formation).(1,2,21) Urinary basic fibroblast growth factor (FGF) levels may be elevated during disease flare-ups and coincide with the preosseous angiogenic fibroproliferative lesions.(22)

HISTOPATHOLOGY The earliest stage of FOP lesion formation consists of an intense aggregation of B and T lymphocytes in the perivascular spaces of otherwise normal-appearing skeletal muscle.(23) Sub- sequently, a nearly pure T cell occurs between edematous muscle fibers at the leading edge of an angiogenic fibroproliferative lesion, which is indistinguishable from aggressive juvenile fibromatosis.(23,24) Immunostaining with a monoclonal antibody against bone morphogenetic protein (BMP)-2/4 is intense in FOP lesions, but not in aggressive fibromatosis.(24) Mast cell infiltration is seen at all stages of FOP flare-ups.(25) Endochondral ossification is the major pathway for heterotopic bone formation.(10) Mature osseous lesions have haversian sys- tems and can contain hematopoietic tissue.

ETIOLOGY AND PATHOGENESIS Similarities between FOP and the effects of Drosophila decapentaplegic gene (BMP4 homolog) mutations have sug- gested involvement of the BMP signaling pathway in the pathogenesis of FOP (26). In fact, the BMP signaling pathway is highly dysregulated in FOP cells.(27–31) FOP cells overexpress BMP4, and are unable to appropriately upregulate the expression of multiple BMP antagonists, including Noggin and Gremlin, in response to a BMP challenge.(27,29,30) Additionally, FOP cells exhibit a defect in BMP receptor internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease.(31) Recently, BMP4 transgenic mice that develop an FOP-like phenotype have been described.(32) An initial genome-wide linkage analysis mapped FOP to 4q27-31; however, subsequent DNA sequence analysis of can- FIG. 1. Fibrodysplasia (myositis) ossificans progressiva. Characteristic fea- didate genes in this and other regions did not identify any tures of FOP are seen in early childhood. The presence of short malformed mutations.(33,34) With the discovery of additional pedigrees, a great toes at birth (A, arrows) heralds the later spontaneous appearance of the more conservative genome-wide linkage analysis excluded the preosseous soft tissue lesions on the neck and back (B, arrowheads) and should 4q27-31 region and identified linkage of FOP to 2q23-24, a provoke suspicion of FOP even before the transformation to heterotopic bone locus that includes the activin A type I receptor gene, ACVR1, (arrows). An inspection of the toes (C) will confirm the diagnosis and may a receptor for bone morphogenetic protein.(35) An identical alleviate the need for a lesional biopsy (trauma) that could exacerbate the heterozygous missense mutation (c.617GϾA; R206H) in the condition [from Kaplan FS and Smith RM 1997 Clinical vignette: Fibrodys- glycine-serine (GS) activation domain of ACVR1 was identi- plasia ossificans progressiva (FOP). J Bone Miner Res 12:855 with permission of the American Society for Bone and Mineral Research]. fied in all affected individuals with classic features of either

© 2006 American Society for Bone and Mineral Research 452 /CHAPTER 80 sporadic or inherited FOP.(35) Protein modeling predicts desta- 2. Kaplan FS, Shore EM, Connor JM 2002 Fibrodysplasia ossificans pro- bilization of the GS domain, consistent with constitutive acti- gressiva. In: Royce PM, Steinmann B (eds.) Connective Tissue and Its vation of ACVR1 as the underlying cause of the ectopic chon- Heritable Disorders: Molecular, Genetic, and Medical Aspects, 2nd ed. (35) John Wiley & Sons, New York, NY, USA, pp. 827–840. drogenesis, osteogenesis, and joint fusions seen in FOP. 3. Delatycki M, Rogers JG 1998 The genetics of fibrodysplasia ossificans progressiva. Clin Orthop 346:15–18. TREATMENT 4. Janoff HB, Muenke M, Johnson LO, Rosenberg A, Shore EM, Okereke E, (1,2) Zasloff M, Kaplan FS 1996 Fibrodysplasia ossificans progressiva in two There is no established medical treatment for FOP. The half-sisters. Evidence for maternal mosaicism. Am J Med Genet 61:320– disorder’s rarity, variable severity, and fluctuating clinical 324. course pose substantial uncertainties when evaluating experi- 5. Janoff HB, Tabas JA, Shore EM, Muenke M, Dalinka MK, Schlesinger S, mental therapies. Binders of dietary calcium, radiotherapy, and Zasloff MA, Kaplan FS 1995 Mild expression of fibrodysplasia ossificans warfarin are ineffective.(1,2,36) Limited benefits have been re- progressiva: A report of 3 cases. J Rheumatology 22:976–978. ported using corticosteroids and disodium etidronate together 6. Rocke DM, Zasloff M, Peeper J, Cohen RB, Kaplan FS 1994 Age and joint-specific risk of initial heterotopic ossification in patients who have during flare-ups or using isotretinoin to prevent disease activa- fibrodysplasia ossificans progressiva. Clin Orthop 301:243–248. (37,38) tion. However, these impressions reflect uncontrolled 7. Cohen RB, Hahn GV, Tabas JA, Peeper J, Levitz CL, Sando A, Sando N, studies. Accordingly, medical intervention is currently support- Zasloff M, Kaplan FS 1993 The natural history of heterotopic ossification ive. Nevertheless, physical therapy to maintain joint mobility in patients who have fibrodysplasia ossificans progressiva. A study of 44 may be harmful by provoking or exacerbating lesions.(1,2) Sur- patients. J Bone Joint Surg Am 75:215–219. gical release of joint contractures is unsuccessful and risks 8. Lanchoney TF, Cohen RB, Rocke DM, Zasloff MA, Kaplan FS 1995 (1,2) Permanent heterotopic ossification at the injection site after diphtheria- new, trauma-induced heterotopic ossification. Removal of tetanus-pertussis immunizations in children who have fibrodysplasia os- FOP lesions is often followed by significant recurrence. Os- sificans progressiva. J Pediatr 126:762–764. teotomy of ectopic bone to mobilize a joint is uniformly coun- 9. Scarlett RF, Rocke DM, Kantanie S, Patel JB, Shore EM, Kaplan FS 2004 terproductive because additional heterotopic ossification devel- Influenza-like viral illnesses and flare-ups of fibrodysplasia ossificans ops at the operative site. Spinal bracing is ineffective, and progressiva (FOP). Clin Orthop Rel Res 423:275–279. surgical intervention is associated with numerous complica- 10. Kaplan FS, Tabas JA, Gannon FH, Finkel G, Hahn GV, Zasloff MA 1993 tions.(14) Dental therapy should preclude injection of local The histopathology of fibrodysplasia ossificans progressiva: An endo- (1,2,11,12) chondral process. J Bone Joint Surg Am 75:220–230. anesthetics and stretching of the jaw. In fact, newer 11. Luchetti W, Cohen RB, Hahn GV, Rocke DM, Helpin M, Zasloff M, dental techniques for focused administration of anesthetic are Kaplan FS 1996 Severe restriction in jaw movement after routine injection available. Guidelines for general anesthesia have been of local anesthetic in patients who have progressiva. Oral Surg Oral Med reported.(11) Intramuscular injections should be avoided.(8) Pre- Oral Pathol Oral Radiol Endod 81:21–25. vention of falls is crucial.(39) Measures against recurrent pul- 12. Janoff HB, Zasloff M, Kaplan FS 1996 Submandibular swelling in pa- monary infections and onset of cardiopulmonary complications tients with fibrodysplasia ossificans progressiva. Otolaryngol Head Neck Surg 114:599–604. of restrictive lung disease are important. More focused efforts 13. Kussmaul WG, Esmail AN, Sagar Y, Ross J, Gregory S, Kaplan FS 1998 based on inhibition of BMP signaling may offer hope for the Pulmonary and cardiac function in advanced fibrodysplasia ossificans future.(40) progressiva. Clin Orthop 346:104–109. 14. Shah PB, Zasloff MA, Drummond D, Kaplan FS 1994 Spinal deformity PROGNOSIS in patients who have fibrodysplasia ossificans progressiva. J Bone Joint Surg Am 76:1442–1450. Despite widespread heterotopic ossification and severe dis- 15. Levy CE, Lash AT, Janoff HB, Kaplan FS 1999 Conductive hearing loss ability, some patients live productive lives into the seventh in individuals with fibrodysplasia ossificans progressiva. Am J Audiol decade. Most, however, die earlier from pulmonary complica- 8:29–33. tions including pneumonia, secondary to restricted ventilation 16. Mahboubi S, Glaser DL, Shore EM, Kaplan FS 2001 Fibrodysplasia (1,2,13) ossificans progressiva (FOP). Pediatr Radiol 31:307–314. from chest wall involvement. 17. Schaffer AA, Kaplan FS, Tracy MR, O’Brien ML, Dormans JP, Shore EM, Harland RM, Kusumi K 2005 Developmental anomalies of the PROGRESSIVE OSSEOUS HETEROPLASIA cervical spin in patients with fibrodysplasia ossificans progressiva are distinctly different from those in patients with Klippel-Feil syndrome. Research on FOP led to the discovery of progressive osseous Spine 30:1379–1385. heteroplasia (POH), a distinct developmental disorder of hete- 18. Kaplan FS, Strear CM, Zasloff MA 1994 Radiographic and scintigraphic rotopic ossification.(41–43) Like FOP, POH is an autosomal features of modeling and remodeling in the heterotopic skeleton of pa- dominant genetic disorder of heterotopic ossification within tients who have fibrodysplasia ossificans progressiva. Clin Orthop 304: soft connective tissues. However, unlike in FOP, heterotopic 238–247. ossification in POH commonly occurs within the dermis and 19. Einhorn TA, Kaplan FS 1994 Traumatic fractures of heterotopic bone in forms by an intramembraneous, rather than an endochondral patients who have fibrodysplasia ossificans progressiva. Clin Orthop 308: (43) 173–177. pathway. Identification of two patients with POH-like fea- 20. Shirkhoda A, Armin A-R, Bis KG, Makris J, Irwin RB, Shetty AN 1995 tures who also had Albright hereditary osteodystrophy sug- MR imaging of myositis ossificans: Variable patterns at different stages. J gested the possibility of a genetic link between the two condi- Magn Reson Imaging 65:287–292. tions,(43,44) which was confirmed in a third patient with pure 21. Lutwak L 1964 Myositis ossificans progressiva: Mineral, metabolic, and POH.(45) These discoveries led to the rapid identification of radioactive calcium studies of the effects of hormones. Am J Med 37: paternally inherited inactivating mutations of the GNAS gene as 269–293. 22. Kaplan F, Sawyer J, Connors S, Keough K, Shore E, Gannon F, Glaser D, (46) ␣ the genetic cause of POH. Reduced expression of Gs , one Rocke D, Zasloff M, Folkman J 1998 Urinary basic fibroblast growth of several proteins encoded by GNAS, can induce an factor: A biochemical marker for preosseous fibroproliferative lesions in osteoblast-like phenotype in human mesenchymal stem patients with FOP. Clin Orthop 346:59–65. cells.(47) 23. Gannon FH, Valentine BA, Shore EM, Zasloff MA, Kaplan FS 1998 Acute lymphocytic infiltration in an extremely early lesion of fibrodysplasia ossificans progressiva. Clin Orthop 346:19–25. REFERENCES 24. Gannon F, Kaplan FS, Olmsted E, Finkel G, Zasloff M, Shore EM 1997 Differential immunostaining with bone morphogenetic protein (BMP) 2/4 1. Connor JM, Evans DAP 1982 Fibrodysplasia ossificans progressiva: The in early fibromatous lesions of fibrodysplasia ossificans progressiva and clinical features and natural history of 34 patients. J Bone Joint Surg Br aggressive juvenile fibromatosis. Hum Pathol 28:339–343. 64:76–83. 25. Gannon FH, Glaser D, Caron R, Thompson LD, Shore EM, Kaplan FS

2001 Mast cell involvement in fibrodysplasia ossificans progressiva. Hum on excretion of 4-carboxy-L-glutamic acid in scleroderma, dermatomyo- Pathol 32:842–848. sitis, and myositis ossificans progressiva. Arthritis Rheum 29:344–351. 26. Kaplan F, Tabas JA, Zasloff MA 1990 Fibrodysplasia ossificans progres- 37. Brantus J-F, Meunier PJ 1998 Effects of intravenous etidronate and oral siva: A clue from the fly? Calcif Tissue Int 47:117–125. corticosteroids in fibrodysplasia ossificans progressiva. Clin Orthop 346: 27. Shafritz AB, Shore EM, Gannon FH, Zasloff MA, Taub R, Muenke M, 117–120. Kaplan FS 1996 Dysregulation of bone morphogenetic protein 4 (BMP4) 38. Zasloff MA, Rocke DM, Crofford LJ, Hahn GV, Kaplan FS 1998 Treat- gene expression in fibrodysplasia ossificans progressiva. N Engl J Med ment of patients who have fibrodysplasia ossificans progressiva with 335:555–561. isotretinoin. Clin Orthop 346:121–129. 28. Lanchoney TF, Olmsted EA, Shore EM, Gannon FA, Rosen V, Zasloff 39. Glaser DM, Rocke DM, Kaplan FS 1998 Catastrophic falls in patients MA, Kaplan FS 1998 Characterization of bone morphogenetic protein 4 who have fibrodysplasia ossificans progressiva. Clin Orthop 346:110– receptors in fibrodysplasia ossificans progressiva. Clin Orthop 346:38– 116. 45. 40. Glaser DL Economides AN, Wang L, Liu X, Kimble RD, Fandl JP, 29. Olmsted EA, Kaplan FS, Shore EM 2003 Bone morphogenetic protein-4 Wilson JM, Stahl, N, Kaplan FS, Shore EM 2003 In vivo somatic regulation in fibrodysplasia ossificans progressiva. Clin Orthop 408:331– cell gene transfer or an engineered noggin mutein prevents BMP4- 343. induced heterotopic ossification. J Bone Joint Surg Am 85:2332– 30. Ahn J, Serrano de La Penã L, Shore EM, Kaplan FS 2003 Paresis of a 2342. bone morphogenetic protein antagonist response in a genetic disorder of 41. Kaplan FS, Craver R, MacEwen GD, Gannon FH, Finkel G, Hahn G, heterotopic skeletogenesis. J Bone Joint Surg Am 85:667–674. Tabas J, Gardner RJ, Zasloff MA 1994 Progressive osseous heteroplasia: 31. Serrano de la Penã L, Billings PC, Fiori JL, Ahn J, Kaplan FS, Shore EM A distinct developmental disorder of heterotopic ossification. J Bone Joint 2005 Fibrodysplasia ossificans progressiva (FOP), a disorder of ectopic Surg Am 76:425–436. osteogenesis, misregulates cell surface expression and trafficking of BM- PRIA. J Bone Miner Res 20:1168–1176. 42. Rosenfeld SR, Kaplan FS 1995 Progressive osseous heteroplasia in male 32. Kan L, Hu M, Gomes WA, Kessler JA 2004 Transgenic mice overex- patients. Clin Orthop 317:243–245. pressing BMP4 develop a fibrodysplasia ossificans progressiva (FOP)-like 43. Kaplan FS, Shore EM 2000 Progressive osseous heteroplasia. J Bone phenotype. Am J Pathol 165:1107–1115. Miner Res 15:2084–2094. 33. Feldman G, Li M, Martin S, Urbanek M, Urtizberea JA, Fardeau M, 44. Eddy MC, Jan De Beur SM, Yandow SM, McAlister WH, Shore EM, LeMerrer M, Connor JM, Triffitt J, Muenke M, Kaplan FS, Shore EM Kaplan FS, Whyte MP, Levine MA 2000 Deficiency of the alpha-subunit 2000 Fibrodysplasia ossificans progressiva (FOP), a heritable disorder of of the stimulatory G protein and severe extraskeletal ossification. J Bone severe heterotopic ossification, maps to human chromosome 4q27-31. Am Miner Res 15:2074–2083. J Human Genet 66:128–135. 45. Yeh GL, Mathur S, Wivel A, Li M, Gannon FH, Ulied A, Audi L, 34. Xu MQ, Feldman G, Le Merrer M, Shugart YY, Glaser DL, Urtizberea Olmstead EA, Kaplan FS, Shore EM 2000 GNAS1 mutation and Cbfa1 JA, Fardeau M, Connor JM, Triffitt J, Smith R, Shore EM, Kaplan FS misexpression in a child with severe congenital platelike osteoma cutis. 2005 Linkage exclusion and mutational analysis of the noggin gene in J Bone Miner Res 15:2063–2073. patients with fibrodysplasia ossificans progressiva. Clin Genet 58:291– 46. Shore EM, Ahn J, Jan de Beur S, Li M, Xu M, Gardner RJ, Zasloff MA, 298. Whyte MP, Levine MA, Kaplan FS 2002 Paternally-inherited inactivating 35. Shore EM, Xu M, Feldman GJ, Fenstermacher DA, The FOP Interna- mutations of the GNAS1 gene in progressive osseous heteroplasia. N Engl tional Research Consortium, Brown MA, Kaplan FS 2006 A recurrent J Med 346:99–106. mutation in the BMP type I receptor ACVR1 causes inherited and spo- 47. Leitman SA, Ding C, Cooke DW, Levine MA 2005 Reduction in Gs- radic fibrodysplasia ossificans progressiva. Nat Genet 38:525–527. alpha induces osteogenic differentiation in human mesenchymal stem 36. Moore SE, Jump AA, Smiley JD 1986 Effect of warfarin sodium therapy cells. Clin Orthop Rel Res 434:231–238.