Effects of bone marrow transplantation on the cardiovascular ... - Nature

4 downloads 115 Views 380KB Size Report
associated with disseminated metastatic hemangiosarcoma. Tissue samples for light microscopy were collected without perfusion to remove donor-derived ...
Bone Marrow Transplantation (2000) 25, 1289–1297  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt

Effects of bone marrow transplantation on the cardiovascular abnormalities in canine mucopolysaccharidosis VII C Sammarco2, M Weil1, C Just1, S Weimelt1, C Hasson1, T O’Malley1, SM Evans2, P Wang2, ML Casal2, J Wolfe1,2 and M Haskins1,2 Departments of 1Pathobiology and 2Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

Summary: The genetic mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting from defective catabolism of glycosaminoglycans (GAGs). Echocardiographic abnormalities in dogs with MPS type VII (Sly syndrome, ␤-glucuronidase deficiency) included mitral valve thickening and insufficiency, large aortic dimensions in both the long and short axes, and thickened aortic valves. Grossly, at post mortem examination, there was nodular thickening of the mitral valve, a prominent ductus diverticulum, and a dilated aorta with thickened walls. Histologically, cytoplasmic vacuolation was seen in cells of the mitral valves, coronary arteries, and aorta. By electron microscopy, the cells of the mitral valve were packed with electron-lucent cytoplasmic vacuoles. The mean residual activity of ␤-glucuronidase in the aorta and myocardium was ⬍1% of normal, the mean hexosaminidase A activity ⬎2.5 times normal, and the mean GAG concentrations more than twice normal. In three MPS VII dogs that received heterologous BMT at 6 weeks of age, the echocardiographic abnormalities were improved, and the histopathologic and ultrastructural pathology was reduced. In the aorta and myocardium, the mean ␤-glucuronidase activity of the BMT group was 4.5% and 11% of normal, respectively, and the hexosaminidase A activity and GAG concentrations were normalized. Bone Marrow Transplantation (2000) 25, 1289–1297. Keywords: bone marrow transplantation; cardiovascular; canine; lysosomal storage; mucopolysaccharidosis

Lysosomes function in the degradation of complex substrates via the step-wise activity of several hydrolases, each step requiring the action of the previous enzyme. If one step in the process fails, further degradation ceases resulting in a lysosomal storage disease.1 Lysosomal storage diseases are classified by the primary substrates that accumulate and are defined by the individual enzyme that is deficient in activity. The genetic mucopolysaccharidoses (MPS) are a Correspondence: Dr M Haskins, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6051, USA Received 29 December 1999; accepted 20 March 2000

family of lysosomal storage diseases resulting from defective catabolism of several glycosaminoglycans (GAGs). Depending on the particular enzyme which is deficient in activity, the MPS syndromes are defined MPS I to VII, with several subgroups. In humans, clinical features may include cardiac murmurs, dysostosis multiplex, hepatosplenomegaly, hypertelorism, facial dysmorphia, corneal clouding, and mental retardation.2 In animals, naturally occurring MPS syndromes have been described in cats, dogs, goats, mice and rats (for review see Ref. 3) and emus.4 MPS type VII (Sly syndrome, ␤-glucuronidase deficiency) was reported in mixed-breed dogs5–8 and a colony was established at the University of Pennsylvania, School of Veterinary Medicine. All of the affected dogs are descendants of a single carrier female and were therefore homozygous for a single mutant allele, an arginine to histidine amino acid substitution at position 166.8 Typical features of the syndrome in the dog included dysostosis multiplex, diffuse corneal clouding, and growth retardation. Affected dogs excreted chondroitin 4- and 6-sulfates and dermatan sulfate in urine. Cardiac abnormalities were variable from having no clinical signs of heart disease by 2 years of age, to murmurs consistent with mitral insufficiency, to signs of congestive heart failure at 10 days of age. During the normal production and trafficking of most lysosomal enzymes, post-translational modification results in the addition of a mannose-6-phosphate moiety that can be recognized by integral membrane glycoprotein receptors, sorting the enzymes to the lysosome. In addition, a proportion of the modified enzyme may also be released from the cell via a secretory pathway. Secreted enzyme can ultimately reach a lysosome in another cell because the receptor is also present in the plasma membrane of most cells.9–11 Thus, secreted enzymes that connect with this receptor can be internalized and transferred to a lysosome. This ‘cross correction’ pathway provides the rationale for BMT therapy for lysosomal storage diseases. The strategy relies on adequate secretion of the normal hydrolase from normal donor bone marrow-derived cells, and the receptormediated uptake of the normal enzyme by diseased host cells. This principle of cross correction was first demonstrated using cultured fibroblasts from human MPS patients even before the enzymes and receptors had been characterized.12 Fortunately, relatively little enzyme needs to reach the lysosome to decrease substrate storage and ameliorate disease.13

BMT in canine MPS C Sammarco et al

1290

Materials and methods

Chimerism was evaluated in the two male dogs receiving male marrow by measuring serum GUSB activity in the recipient and donor. Both had levels that varied from 30 to 90% of donor activity, which was similar to that seen in M258 shown to be a full lymphocyte chimera by karyotyping.

Animals

Pathology

Affected and control dogs were raised in the animal colony of the School of Veterinary Medicine, University of Pennsylvania, under NIH and USDA guidelines for the care and use of animals in research. The animals were housed with ad libitum food and water, 12-h light cycles, at 21°C, with 12–15 air changes per h. Physical examinations, electrocardiograms, and echocardiograms were performed on 13 untreated MPS VII dogs, and three MPS VII dogs treated by heterologous BMT (ages in Table 1). Electrocardiography was performed with the dogs in right lateral recumbency. Echocardiography was used to measure cardiac dimensions, and to evaluate valvular structure and function using 2D, M-mode, and Doppler (spectral and color flow) with a Hewlett-Packard 1000 echocardiograph. Echocardiograms were obtained using 5 MHz and 3.5 MHz transducers, with the dogs in left and right lateral recumbency. All examinations were performed blinded by one examiner who did not know which dogs had been treated by BMT. A subjective score was assigned for mitral valve and aortic valve thickness, mitral regurgitation, and aortic size.

Euthanasia was performed on all dogs using concentrated sodium pentobarbital in accordance with the American Veterinary Medical Association guidelines, except for the oldest BMT dog which died at 6 years of age of cardiac arrest associated with disseminated metastatic hemangiosarcoma. Tissue samples for light microscopy were collected without perfusion to remove donor-derived peripheral blood cells from within the vasculature, fixed in buffered 10% formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Specimens for electron microscopy were immersion fixed in 2.5% cacodylate-buffered gluteraldehyde, minced into 0.5–1.0 mm cubes, and stored overnight at 4°C. The tissues were post-fixed in osmium tetroxide, dehydrated through a graded series of alcohols, and embedded in Spurr’s low viscosity embedding medium.15 Embedded tissue blocks were sectioned to 70 nm on a Sorvall MT2-B ultramicrotome (Dupont/Sorvall, Newton, CT, USA) stained with lead citrate-uranyl acetate,16,17 and examined with a Zeiss EM109 transmission electron microscope (Gottingen, Germany).

This report describes the cardiovascular features of 13 dogs with MPS VII and results of BMT therapy in three affected dogs.

␤-Glucuronidase and hexosaminidase assays BMT Three 6-week-old MPS VII dogs received 700 cGy whole body irradiation in a single dose administered using a Picker Orthovoltage unit (Picker International, Cleveland, OH, USA), 250 kVp, and were transplanted with 3.4–3.9 ⫻ 108 nucleated bone marrow cells/kg of body weight from a mixed lymphocyte-reactivity-matched sibling donor.14 Two of the donors (for M258 and M335) were heterozygous for MPS VII. Prior to irradiation and for 3 days post irradiation, the dogs were given neomycin p.o. As long as the peripheral blood leukocyte count was below 1000 cells/␮l, dogs treated with BMT were given l-glutamine p.o. (30 mg twice a day), broad spectrum antibiotics (M258: cefoxitin sulfate, amikacin sulfate, and cefodroxil; M335: also trimethoprim sulfate and ciprofloxicin; M539: only trimethoprim sulfate and clavamox), and housed in a laminar flow, hepa-filtered room. Engraftment was apparent by 11–14 days post transplantation when the peripheral white blood cell count exceeded 1000/␮l. Cyclosporin was administered at 25 mg/kg once a day, p.o. for 3 weeks, and then reduced by half every 3 weeks over 2 months. One dog (M258) developed signs of graft-versus-host disease and was treated with prednisolone (2.2 mg/kg/once a day), and the lesions regressed. None of the untransplanted MPS VII dogs received medication other than routine vaccinations. Chimerism was evaluated in female M258, which received male donor marrow, by karyotype evaluation of lymphocytes at 1.5 months, 11 months, 2 years, and 3.5 years post transplantation (80 cells counted, all were XY). Bone Marrow Transplantation

␤-Glucuronidase (GUSB)18 and hexosaminidase A19 activities were measured using fluorescence assays. Frozen aorta and myocardium (2 × 2 × 3 mm) from four normal (ages 0.13, 0.3, 3, and 3.8 years), four MPS VII untreated, and the three BMT-treated dogs were ground in 1.0 ml 0.2% triton-X-100, 0.9% saline using a motor-driven pestle designed to fit into a conical 1.5 ml microcentrifuge tube. Samples were then frozen at −80°C for 30 min and thawed in a 37°C water bath. Cellular debris was pelleted in a microcentrifuge (12 000 g, 2 min). A standard curve was prepared from a 0.2 mm stock solution of 4-methylumbelliferone (M1381, Sigma, St Louis, MO, USA). ␤-Glucuronidase substrate, 10 mm 4-methylumbelliferone-␤-d-glucuronide (M5664, Sigma) was prepared in 0.1 m sodium acetate buffer, pH 4.8. hexosaminidase A substrate, 10 mm 4-methylumbelliferone-N-acetyl-␤-d-glucosaminide (M2133, Sigma), was prepared in 88.2 mm citric acid-55.9 mm disodium phosphate buffer, pH 4.4. Reaction mixtures contained 0.1 ml of sample supernatant and 0.1 ml of substrate in a final volume of 0.2 ml and were incubated at 37°C for 1 h. The reactions were stopped by the addition of 2.0 ml 0.32 m glycine, 0.2 m sodium carbonate, pH 10.5. Fluorescence was measured with a Hitachi model F-1200 fluorescence spectrophotometer (excitation wavelength 365 nm, emission wavelength 450 nm) (Hitachi, San Jose, CA, USA). Protein content was determined (BioRad Protein Assay kit, BioRad Laboratories, Hercules, CA, USA). Activity was expressed as nanomoles of 4-methylumbelliferone released per h per milligram protein. Results are

II 0 0

LApx — —

NSA NSA NSA

NSA NSA NSA NSA NSA NSA NSA NSA NSA NSA NSA NSA NSA 150 160 140

120 140 120 160 160 140 160 140 120 140 130 140 160 23 20 23

26 36 20 22 31 24 23 22 30 19 32 29 27 11 7 14

17 23 11 15 20 14 15 13 18 11 19 18 14

LVIDs mm

52 65 39

35 36 45 32 35 42 35 41 40 42 41 38 48

FS%

8 7 7

7 7 7 7.5 5 5 5 6 5 4 5 7 5

IVS mm

8 7 7

7 7 7 7.5 5 5 5 6 5 4 5 7 5

PW mm

22 17 27

27 31 26 17 20 29 21 19 26 23 22 21 26

LA mm

15 16 15

21 25 16 16 23 21 20 21 26 20 24 20 24

AO mm

1.5 1.1 1.8

1.3 1.2 1.6 1.1 0.9 1.4 1.1 0.9 1.0 1.2 0.9 1.1 1.1

L/A Rt

26 24 25

28 35 32 25 35 25 24 28 34 27 23 24 30

LA Lng mm

14 13 17

19 22 17 16 19 20 19 20 24 20 20 22 22

Ao Lng mm

2 1 0

2 4 2 2 3 3 3 3 4 2 1 1 3

MR

2 1 1

1 2 2 2 2 2 2 3 3 2 2 2 3

MV Th

0 0 2

4 4 2 2 4 4 4 4 4 4 3 3 4

Ao sub

0 0 0

0 2 2 1 2 2 2 3* 2 2 2 2 2

AoV Th

Echocardiographic evaluation of untreated and BMT-treated MPS VII dogs. Wt = weight in kilograms; MRMR = MR murmur; LOC = location; RHTM = rhythm; HR = heart rate; LVIDd = left ventricular internal dimension in diastole; LVIDs = left ventricular internal dimension in systole; FS% = fractional shortening percent; IVS = interventricular septum; PW = posterior wall; LA = left atrium in the short axis; AO = aortic size short axis; Ao Lng = aortic size in long axis; L/A Rt = LA to AO ratio; LA Lng = LA in the long axis; MR = mitral regurgitation; MV Th = mitral valve thickness (subjective, 0.4); Ao sub = overall aorta size (subjective, 0–4); AoV Th = aortic valve thickness (subjective, 0–4); Apx, Hf = apex, high frequency; Lapx = left apex; L&R Apx = left & right apex; NSA = normal sinus arrhythmia; * = moderate aortic insufficiency.

1.3 5.5 3.3

— Apx, Hf LApx — LApx LApx LApx L&R L&R LApx — — LApx

LVIDd mm

M F M

0 III II 0 II III II III III, II I 0 0 IV

HR

BMT-treated M439 8 M258 5.5 M335 7

0.8 0.8 0.7 0.8 1.1 1.1 0.4 0.3 0.4 0.3 0.6 0.6 1.3

RHTM

F M F F F M M F M F M M M

LOC

7.1 9.5 8 7.4 8 7 5 5 6.4 4 7.2 6.8 8

MRMR

Untreated M599 M604 M614 M628 M646 M669 M727 M737 M751 M785 M756 M723 M714

Age (yr)

Wt (kg)

Animal No.

Sex

Clinical measurements in MPS VII dogs with and without bone marrow transplants

Table 1

BMT in canine MPS C Sammarco et al

1291

Bone Marrow Transplantation

BMT in canine MPS C Sammarco et al

1292

presented as means ⫾ 1 standard deviation and were evaluated by analysis of variance and Tukey post-hoc analysis.

BMT-treated animal), had echocardiographic evidence of mitral regurgitation; however, the mitral regurgitation score was generally higher in the untreated group.

GAG assay Frozen aorta and myocardium from four normal, four MPS VII untreated (as above), and the three BMT-treated dogs was dried in a speed vac at room temperature to obtain a dry weight of 10–30 mg. The dried tissues were extracted, and the sulfated GAGs precipitated in a high salt (0.4 m guanidine hydrochloride), low pH (pH 1.75), buffer containing 0.25% Triton X-100 and alcian blue.20 Unbound dye was removed by washing with dimethylsulfoxide, the pellets solubilized (in 4.0 m guanidine hydrochloride in 33% propanol), and the reaction read at 600 nm on a Genesys 5 spectrophotometer (Spectronic Instruments, Rochester, NY, USA). Protein content was determined (BioRad Protein Assay kit, BioRad Laboratories) and the final values expressed as ␮g GAG/mg protein. Results are presented as means ⫾ 1 standard deviation and were evaluated by analysis of variance and Tukey post-hoc analysis. Results Physical examination Affected animals were identified soon after birth by low serum ␤-glucuronidase activity. Facial dysmorphia, chest deformity, growth retardation, and corneal clouding were all evident by 6 to 8 weeks of age. Body weight was similar in the treated and untreated groups (Table 1). The treated group’s mean age was 3.6 years while the untreated group was younger (mean 0.8 years) because untreated MPS VII dogs could not stand by 6 months of age and euthanasia was performed when their quality of life diminished. In the 13 untreated MPS VII dogs, nine (70%) had cardiac murmurs, one grade I/V, three grade II/V, four grade III/V, and one grade IV/V. All were band-shaped systolic murmurs. Of the three BMT-treated dogs, one had a grade II/V bandshaped, systolic cardiac murmur over the left apex. Electrocardiography All dogs had normal ECG findings. Of the untreated MPS VII dogs, two (M599 and M604) had pronounced Ta waves, but P wave size and morphology were normal.

Pathology Grossly, all animals had some degree of nodular thickening of the mitral valve. However that observed in the BMTtreated dogs was minimal. A prominent ductus diverticulum and dilated aorta with thickened wall was present in the untreated dogs. Histologically, in the untreated dogs the vascular smooth muscle cells of the aorta were enlarged and rounded with highly vacuolated cytoplasm (Figure 1B), while those of the treated dogs (Figure 1C–E) resembled normal aorta (Figure 1A). Similar lesions were seen in the left coronary artery (Figure 2B) compared to normal (Figure 2A). The BMT-treated dogs (Figure 2C–E) had less storage in the coronary artery, but the lesions were more prominent than those in the aorta. By electron microscopy, the cells of the mitral valve of the untreated dogs were packed with electron-lucent cytoplasmic vacuoles, some of which appeared to have coalesced (Figure 3B). In the treated dogs, the vacuolation in the cells of the mitral valve was dramatically reduced (Figure 3C–E), and was indistinguishable from normal (Figure 3A). Enzyme assays In four untreated affected dogs, the mean residual activity of ␤-glucuronidase in the aorta and myocardium was 0.7% and 0.2% of normal, respectively (Table 2). In the three BMT-treated MPS VII dogs, the mean ␤-glucuronidase activity in the aorta and myocardium was 5.4% and 11.3% of normal, respectively (Table 2). Tukey post-hoc analysis revealed significant differences in activity of a second lysosomal enzyme, hexosaminidase A, in the aorta and myocardium (Figure 4) between the normal and MPS VII dogs, and between the MPS VII and BMT-treated MPS VII dogs (aorta: F = 22.2, df = 2, P = 0.001; myocardium: F = 18.0, df = 2, P = 0.001), but no significant difference was seen between the BMT-treated MPS VII and normal dogs (aorta P = 0.987; myocardium P = 0.126). Thus, the secondary increase in activity of another lysosomal enzyme activity found in untreated MPS VII aorta and myocardium was normalized by BMT. GAG assay

Echocardiography (Table 1) Abnormalities that were seen in untreated MPS VII dogs included mitral valve thickening and regurgitation, larger aortic dimensions in both the long and short axes, and aortic valves that were subjectively thicker. No difference in left atrial size was apparent between the treated and the untreated groups. The treated group generally had smaller aortic (short and long axes) and end diastolic left ventricular internal dimensions vs the untreated group. The mitral valve and aortic valve thickness scores in the BMT-treated animals were generally lower than those in the untreated dogs. All dogs except one (M335, a Bone Marrow Transplantation

The mean concentration of GAGs in the aorta and myocardium, respectively, from four normal dogs was 62.4 ⫾ 19.2 and 3.4 ⫾ 1.6 ␮g/mg protein; from four affected dogs was 146.4 ⫾ 7.8 and 12.1 ⫾ 3.7; and from the three BMTtreated dogs was 56.5 ⫾ 4.5 and 5.6 ⫾ 0.7 (Figure 5). Statistical analysis of aortic and myocardial GAG concentrations revealed significant differences between normal and affected, and between BMT-treated and affected MPS VII dogs (aorta: F = 57.5, df = 2, P ⬍ 0.001; myocardium: F = 13.4, df = 2, P = 0.003). There were no significant differences between aortal and myocardial GAG concentrations between normal and BMT-treated MPS VII dogs

BMT in canine MPS C Sammarco et al

1293

Figure 1 Light micrographs of the aortic wall of a 3-month-old normal dog (A), a 16-month-old untreated affected MPS VII dog (B), and the BMTtreated MPS VII dogs (C, D, E; 19, 45, and 69 months old, respectively). In the affected dog, many of the cells are enlarged with highly vacuolated cytoplasm compared to normal. The cell hypertrophy and cytoplasmic vacuolation is not present in two of the BMT-treated dogs (C and E), and greatly reduced in the third (D). Bar = 64 ␮.

Figure 2 Light micrographs of the left coronary artery of the 3-month-old normal dog (A), 16-month-old untreated affected MPS VII dog (B), and the three BMT-treated MPS VII dogs (C, D, E). In the affected dog, the cells have prominent cytoplasmic vacuolation and the arterial wall is thicker than normal. The vessel wall thickness is reduced in the BMT-treated dogs, but cytoplasmic vacuolation remains conspicuous. Bar = 64 ␮.

(aorta P = 0.827; myocardium P = 0.21). Thus, the GAG concentrations were normalized by BMT in both aorta and myocardium. Discussion In this study of MPS VII dogs, the clinical and pathologic cardiovascular manifestations in a large series of untreated dogs was compared to three dogs receiving neonatal BMT,

one being followed for almost 6 years. The group of untreated animals was younger than those with BMT because untreated MPS VII dogs could not stand by 6 months of age and euthanasia was performed when their quality of life diminished. BMT-treated animals were able to stand and walk. Thus, euthanasia was performed to measure the effects of BMT on organ system pathology, not due to a diminished quality of life. Published normal echocardiographic values for dogs are limited. Regression equations for dogs of a mean weight Bone Marrow Transplantation

BMT in canine MPS C Sammarco et al

1294

Figure 3 Electron micrographs of the mitral valve of the 3-month-old normal dog (A), 26-month-old untreated affected MPS VII dog (B), and the three BMT-treated MPS VII dogs (C, D, E). The cell from the untreated MPS VII valve is highly distended with a large number of empty vacuoles that deform the nuclear outline. In all three BMT-treated dogs, the cells appear normal. Bar = 3.5 ␮.

Table 2

␤-Glucuronidase activity in aorta and myocardium

Animal

P90 P113 M784 M854 M714 M723 M756 M785 M258 M335 M539 a

Phenotype

normal normal normal normal affected affected affected affected affected affected affected

BMT

no no no no no no no no yes yes yes

Aorta

Myocardium

␤-Glucuronidase activitya

% of normalb

␤-Glucuronidase activitya

% of normalb

103.6 104.4 104.4 326.4 1.67 1.02 0.77 1.25 9.50 8.58 8.19

— — — — 1.0 0.7 0.5 0.7 5.5 5.3 5.3

64.8 70.8 83.5 274 0.36 0.26 0.27 0.37 30.4 4.1 7.2

— — — — 0.3 0.2 0.2 0.3 24.7 3.3 5.8

Mean activity in nmol/h/mg protein. The mean of P90, P113, M784, M854 aorta and myocardium: 159.7 and 123 nmol/h/mg protein, respectively.

b

of 6.8 kg are left atrium, long axis, 24.1 ⫾ 3.9; aorta, short axis, 14.1 ⫾ 1.1; and LVIDd, 27.1 ⫾ 4.5.21 The dogs with MPS VII could not be directly compared to these normal values because affected dogs had skeletal malformations and were of short stature, and muscle atrophy diminished body weight after they could no longer stand. Cardiovascular abnormalities seen by echocardiology in untreated MPS VII dogs included thickened mitral valves with regurgitation, larger aortic dimensions in both the long axis and the short axis, and thickened aortic valves. These values in the BMT-treated dogs suggest that the therapy reduced the echocardiographic abnormalities. In the untreated MPS VII dogs, the left ventricular end diastolic dimension and left atrial dimensions were within normal limits; there was no left ventricular hypertrophy. Bone Marrow Transplantation

The untreated dogs generally had larger aortic dimensions than a normal dog of this weight. However, as affected, untreated dogs are dwarfed by the skeletal lesions and have disuse atrophy of muscle and bone, body weight is not a reliable denominator. The aortic dimensions were only slightly increased above normal for the treated dogs, which remained mobile. At post-mortem examination, in untreated MPS VII dogs there was prominent nodular thickening of the mitral valve, with cytoplasmic vacuolation seen by light and electron microscopy similar to that reported in children, and other animals with MPS.22–25 The gross, microscopic, and ultrastructural abnormalities of the mitral valve, aorta, and coronary arteries were markedly improved in the dogs that had BMT.

BMT in canine MPS C Sammarco et al

1

GUSB to HEX A ratio

0.1

Aorta Myocardium

0.01

0.001

0.0001

0.00001

Normal n = 4

Affected n = 4

BMT n = 3

Figure 4 The ratios of the specific activity of ␤-glucuronidase to hexosaminidase A in aorta and myocardium. In lysosomal storage diseases, while the primary enzyme has decreased activity, other lysosomal enzymes have increased activity, represented here by hexosaminidase A. Successful therapy should both increase the deficient enzyme activity and reduce the secondary increase in the other enzymes. Bar = 1 standard deviation from the mean.

160

Aorta Myocardium

m g GAG/mg protein

120

80

40

0

Normal n = 4

Affected n = 4

BMT n = 3

Figure 5 Mean total glycosaminoglycan concentrations in the aorta and myocardium of four normal dogs, four untreated MPS VII dogs, and the three BMT-treated MPS VII dogs. Bar = 1 standard deviation from the mean.

The activity of ␤-glucuronidase (GUSB) in aorta and myocardium of two BMT-treated dogs was about 5% of normal, even in the dogs transplanted with a heterologous donor (with cells that have half-normal GUSB activity). This result may be higher than would have been found if the donor blood had been removed by perfusion, although the effect would be expected to be greater in the more vascular myocardium than aorta. In the myocardium of the 6year old BMT-treated dog, the GUSB activity was 25% of normal, which may have been caused by the foci of meta-

static hemangiosarcoma present throughout the myocardium. These lesions would be expected to contain more blood with donor white blood cells than myocardium. In lysosomal storage diseases, an increase in the activity of other lysosomal enzymes in tissues is a common finding. The activity of hexosaminidase A was elevated in MPS VII aorta and myocardium. Restoration of normal hexosaminidase A activity would be expected with effective therapy, which was seen in these tissues in the BMT-treated dogs. The GAG content of the aorta was also normalized, correlating with the improvements in gross, histologic, and ultrastructural pathology seen with BMT. It is important to note that the improvements in aortic pathology, GAG concentration, and hexosaminidase A activity were produced by restoration of only 5% of normal ␤-glucuronidase activity by BMT, which is consistent with similar observations in other organ systems in mice with MPS VII.13 More than 270 human patients with MPS have had results of cardiovascular system evaluations published.26–41 Of these patients, the majority did not have a specific enzyme diagnosis. However, the largest group with an identified diagnosis was MPS I (Hurler syndrome, alphal-iduronidase deficiency). The most common abnormality described was mitral valve insufficiency and, at post mortem, nodular thickening of the free margin of the valve leaflets. Other abnormalities observed in some patients included thickening of the tricuspid, aortic, and pulmonary valves, aortic stenosis, thickened aortic wall, thickened and stenotic coronary arteries, myocardial hypertrophy, thickening of the septal and posterior left ventricular wall, and cardiomegaly. ECG abnormalities were noted in a few patients with occasional premature ventricular contractions and right axis deviation without ventricular hypertrophy. In dogs with MPS I (Hurler syndrome, alpha-l-iduronidase deficiency), a wide P wave was noted on ECG, together with echocardiographic evidence of mitral valve thickening, enlarged aortic root diameter, and heart enlargement.42,43 Grossly, the dogs with MPS I had mitral valve nodules with some dogs having tricuspid, pulmonary, and aortic valve involvement, but no chamber enlargement. The aorta wall was thickened. Histologically, there was substantial storage in the valves, aorta, and the left coronary artery. Cardiovascular evaluations have been reported following BMT in children, dogs, and mice with MPS.36,39,41–43,44–46 In a 9.5-year-old MPS I child, the ventricular septal thickness and sigmoidal valve thickening were reduced.36 In another study, a series of 16 children from 3 to 108 months of age with MPS were followed post BMT.39 Of the 13 MPS I, one MPS IV (Morquio syndrome), and two MPS VI (Maroteaux–Lamy syndrome) patients examined after BMT, two of four cleared a restrictive left ventricle, one of two resolved left ventricular hypertrophy, and there was no change seen in the mitral valve dysplasia or aortic stenosis, each seen in two patients. Only one child (12 years old) with MPS VII has been studied following BMT.41 The mitral and aortic valve regurgitation seen in this patient was not changed 15 months following BMT. The cardiovascular effects of BMT performed at 5 months of age on three dogs with MPS I that were followed for 15 months were compared to three untreated dogs.42,43 The features that were unchanged by BMT included a wide

1295

Bone Marrow Transplantation

BMT in canine MPS C Sammarco et al

1296

P wave on electrocardiography, and the gross and histologic appearance of the mitral valve leaflets. The characteristics which improved with BMT in canine MPS I included aortic root diameters which were large by echocardiography and at post mortem in treated dogs, cytoplasmic vacuolation in the mitral valve, aorta, and coronary arteries, and GAG concentrations in mitral valves and myocardium. Several studies have examined the effects of BMT in MPS VII mice. In the first study, 10- to 18-week-old MPS VII mice were irradiated and given BMT. At 10 weeks post transplant, lysosomal distension was reduced in the endocardium and myocardial interstitial cells.44 In three animals examined, only a few cells with lysosomal storage were seen in the cardiac valves. Cytoplasmic storage in medial cells of the aorta was not improved. In another study, 1day-old mice were irradiated and given BMT. When 10 mice were examined at 10 weeks and six mice at 10 months, myocardial endomysial fibroblasts had a marked decrease in lysosomal distension, cardiac valve fibroblasts displayed a more variable appearance, and medial cells in the muscular arteries had persistent abundant lysosomal vacuolation.45 In a third study, five mice with MPS VII treated with BMT at 2 months of age had cardiovascular pathology reported 200 days post transplant.46 The GUSB activity was 53% of normal, the secondary elevation of ␤hexosaminidase activity was reduced almost to normal, and GAG concentrations in the myocardium were normalized. In addition to BMT, MPS VII mice have been treated with intravenous administration of purified GUSB, and with a viral vector transferring a GUSB cDNA. Both types of experiments provide a source of GUSB for cross correction, similar to what is provided by the normal bone marrow cells in BMT. In five mice treated in the neonatal period with six injections of purified recombinant mouse GUSB (enzyme replacement therapy), interstitial cells in the heart had less lysosomal storage than untreated MPS VII mice.47 However, GUSB activity was not detected histochemically. Injections of purified human GUSB administered to MPS VII dogs produced significant neutralizing antibodies, abrogating a therapeutic response (unpublished). Finally, in a gene transfer experiment using a single intravenous injection of an adeno-associated viral vector to transfer the human GUSB cDNA to 15 neonatal MPS VII mice, by 16 weeks of age the mice had 2000% of the normal GUSB activity in the heart with histochemical evidence of GUSB activity throughout the myocardium, and a reduction in the histopathologic evidence of cytoplasmic vacuolation in cardiac valves.48 A similar experiment is currently being evaluated in MPS VII dogs. From these experiments in mice and dogs, it appears that if GUSB enzyme activity can be provided to the cardiovascular system, be it by BMT, enzyme replacement therapy, or gene transfer, then the cardiovascular pathology of MPS VII can be reversed or prevented.

Acknowledgements Grateful appreciation is expressed to Dr Niels Pederson for referral of the propositus, Pat Miller-Wilson for assistance with irradiation, Donna Oakley and Mary Taylor for nursing expertise, Bone Marrow Transplantation

Drs Beth Callan, Urs Giger, and Robert Washabau for medical advice, Dr Kathryn Michel for nutrition advice, Drs Charles August and H Joachim Deeg for transplantation expertise, James Hayden of Biographics for the illustrations, Ulana Prociuk for cytogenetics, and Dr Carla Chieffo, Dr Anne Lannon, and a cadre of veterinary students for compassionate animal care. This work was supported by NIH grants DK54481, NS33526, DK25759, DK46637, DK42707, and RR02512.

References 1 Holtzman E. Storage disease. In: Siekevitz P (ed). Lysosomes. Plenum Press: New York, 1989, pp 344–356. 2 Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Disease. McGraw Hill: New York, 1995, pp 2465–2508. 3 Haskins ME, Giger U. Lysosomal storage diseases. In: Kaneko JJ, Harvey JW, Bruss ML (eds). Lysosomal Storage Diseases in Clinical Biochemistry of Domestic Animals. Academic Press. New York, 1997, pp 741–761. 4 Giger U, Shivaprasad HL, Wang P et al. Mucopolysaccharidosis IIIB (Sanfilippo B syndrome) in emus. Vet Pathol 1997; 34: 473. 5 Haskins ME, Desnick RJ, DiFerrante N et al. Beta-glucuronidase deficiency in a dog: a model of human mucopolysaccharidosis VII. Pediatr Res 1984; 18: 980–984. 6 Schuchman EH, Tordyan TK, Haskins ME, Desnick RJ. Characterization of the defective ␤-glucuronidase activity in canine mucopolysaccharidosis type VII. Enzyme 1989; 42: 174–180. 7 Sheridan O, Wortman J, Harvey C et al. Craniofacial abnormalities in animal models of mucopolysaccharidoses I, VI, and VII. J Craniofac Genet Dev Biol 1994; 14: 7–15. 8 Ray J, Bouvet A, DeSanto C et al. Cloning of canine ␤-glucuronidase: identification of a missense mutation in canine mucopolysaccharidosis type VII, and correction of mutant cells by retroviral vector-mediated gene transfer. Genomics 1998; 48: 248–253. 9 Kaplan A, Achord DT, Sly WS. Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts. Proc Natl Acad Sci USA 1977; 74: 2026–2030. 10 Distler J, Hieber V, Sahagian G, Schmickel R, Jourdian GW. Identification of mannose 6-phosphate in glycoproteins that inhibit the assimilation of beta-galactosidase by fibroblasts. Proc Natl Acad Sci USA 1979; 76: 4235–4239. 11 Natowicz MR, Chi MM, Lowry OH, Sly WS. Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts. Proc Natl Acad Sci USA 1979; 76: 4322–4326. 12 Neufeld EF, Fratantoni JC. Inborn errors of mucopolysaccharide metabolism. Science 1970; 169: 141–146. 13 Wolfe JH, Sands MS, Barker JE et al. Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer. Nature 1992; 360: 749–753. 14 Wolfe JH, Haskins ME, Zmijewski CM. Mixed lymphocyte reactivity in cats. Transplantation 1984; 37: 509–513. 15 Spurr AR. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 1969; 26: 31–43. 16 Reynolds E. The use of lead citrate at a high pH as an electronopague stain in electron microscopy. J Cell Biol 1963; 17: 209–212. 17 Sheehan DC, Hrapchak BB. Instrumentation. In: Theory and

BMT in canine MPS C Sammarco et al

18

19 20 21 22 23 24 25 26

27 28 29 30 31

32 33

Practice of Histotechnology. CV Mosby: St Louis, 1980, pp 17, 296, 298–299, 301. Wolfe JH, Sands MS. Murine mucopolysaccaridosis type VII: a model system for somatic gene therapy of the central nervous system. In: Lowenstein PR, Enquist LW (eds). Protocols for gene transfer in neuroscience: towards gene therapy of neurological disorders. John Wiley: Chichester, 1996, pp 265–274. Okada S, O’Brien JS. Tay–Sachs disease: generalized absence of a beta-d-N-acetylhexosaminidase component. Science 1969; 165: 698–700. Bjornsson S. Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue. Anal Biochem 1993; 210: 282–291. O’Grady MR, Bonagura JD, Powers JD, Herring DS. Quantitative cross-sectional echocardiography in the normal dog. Vet Radiol 1986; 27: 34–49. Haskins ME, Aguirre GD, Jezyk PF, Patterson DF. The pathology of feline arylsulfatase B-deficient mucopolysaccharidosis. Am J Pathol 1980; 101: 657–674. Haskins ME, Aguirre GD, Jezyk PF et al. The pathology of feline alpha-l-iduronidase deficient mucopolysaccharidosis. Am J Pathol 1983; 112: 27–36. Shull RM, Helman RG, Spellacy E et al. Morphologic and biochemical studies of canine mucopolysaccharidosis I. Am J Pathol 1984; 114: 487–495. Yoshida M, Ikadai H, Maekawa A et al. Pathological characteristics of mucopolysaccharidosis VI in the rat. J Comp Pathol 1993; 109: 141–153. Krovetz LJ, Lorincz AE, Schiebler GL. Cardiovascular manifestations of the Hurler sundrome: cardiovascular and angiocardiographic observations in 15 patients. Circulation 1965; 31: 132–141. Krovetz LJ, Schiebler GL. Cardiovascular manifestations of the genetic mucopolysaccharidoses. Birth Defects 1972; 8: 192–196. Schieken RM, Kerber RE, Ionasescu VV, Zellweger H. Cardiac manifestations of the mucopolysaccharidoses. Circulation 1975; 52: 700–705. Renteria VG, Ferrans VJ, Roberts WC. The heart in the Hurler syndrome: gross, histologic and ultrastructural observations in five necropsy cases. Am J Cardiol 1976; 38: 487–501. Wilson CS, Mankin HT, Pluth JR. Aortic stenosis and mucopolysaccharidosis. Annal Int Med 1980; 92: 496–498. Brosius FC, Roberts WC. Coronary artery disease in the Hurler syndrome. Qualitative and quantitative analysis of the extent of coronary narrowing at necropsy in six children. Am J Cardiol 1981; 47: 649–653. Johnson GL, Vine DL, Cottrill CM, Noonan JA. Echocardiographic mitral valve deformity in the mucopolysaccharidoses. Pediatrics 1981; 67: 401–406. Gross DM, Williams JC, Caprioli C et al. Echocardiographic

34 35 36 37 38

39 40 41

42 43 44

45 46

47 48

abnormalities in the mucopolysaccharide storage diseases. Am J Cardiol 1988; 61: 170–176. Nelson J, Shields MD, Mulholland HC. Cardiovascular studies in the mucopolysaccharidoses. J Med Genet 1990; 27: 94–100. John RM, Hunter D, Swanton RH. Echocardiographic abnormalities in type IV mucopolysaccharidosis. Arch Dis Child 1990; 65: 746–749. Vinallonga X, Sanz N, Balaguer A et al. Hypertrophic cardiomyopathy in mucopolysaccharidoses: regression after bone marrow transplantation. Pediatr Cardiol 1992; 13: 107–109. Braunlin EA, Hunter DW, Krivit W et al. Evaluation of coronary artery disease in the Hurler syndrome by angiography. Am J Cardiol 1992; 69: 1487–1489. DuCret RP, Weinberg EJ, Jackson CA et al. Resting T1-201 scintigraphy in the evaluation of coronary artery disease in children with Hurler syndrome. Clin Nucl Med 1994; 19: 975–978. Gatzoulis MA, Vellodi A, Redington AN. Cardiac involvement in mucopolysaccharidoses: effects of allogeneic bone marrow transplantation. Arch Dis Child 1995; 73: 259–260. Wippermann CF, Beck M, Schranz D et al. Mitral and aortic regurgitation in 84 patients with mucopolysaccharidoses. Euro J Pediatr 1995; 154: 98–101. Yamada Y, Kato K, Sukegawa S et al. Treatment of MPS VII (Sly disease) by allogeneic BMT in a female with homozygous A619V mutation. Bone Marrow Transplant 1998; 21: 629–634. Brieder MA, Schull RM, Constantopoulos G. Long-term effects of bone marrow transplantation in dogs with mucopolysaccharidosis I. Am J Pathol 1989; 134: 677–692. Gompf RE, Schull RM, Breider MA et al. Cardiovascular changes after bone marrow transplantation in dogs with mucopolysaccharidosis I. Am J Vet Res 1990; 51: 2054–2060. Birkenmeier EH, Barker JE, Vogler C et al. Increased life span and correction of metabolic defects in murine mucopolysaccharidosis type VII after syngeneic bone marrow transplantation. Blood 1991; 78: 3081–3092. Sands MS, Barker JE, Vogler C et al. Treatment of murine mucopolysaccharidosis type VII by syngeneic bone marrow transplantation in neonates. Lab Invest 1993; 68: 676–686. Poorthuis BJHM, Romme AE, Willemsen R, Wagemaker G. Bone marrow transplantation has a significant effect on enzyme levels and storage of glycosaminoglycans in tissues and in isolated hepatocyes of mycopolysaccharidosis type VII mice. Pediatr Res 1994; 36: 187–193. Sands MS, Vogler C, Kyle JW et al. Enzyme replacement therapy for murine mucopolysaccharidosis type VII. J Clin Invest 1994; 93: 2324–2331. Daly TM, Vogler C, Levy B et al. Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease. Proc Natl Acad Sci USA 1999; 96: 2296–2300.

1297

Bone Marrow Transplantation