Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice
Introduction
Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to α-l-iduronidase (IDUA; EC 3.2.1.76) deficiency that results in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfate [1]. The severe (Hurler syndrome), intermediate (Hurler–Scheie syndrome), and mild (Scheie syndrome) forms of MPS I can all result in fragmented elastin fibers in the ascending aorta and cardiac valves in humans [2], [3], which can result in aortic insufficiency due to reduced structural integrity of the valve. Dog [4], [5] and mouse [3], [6], [7] models of MPS I also have elastin fragmentation of the ascending aorta and heart valves, which can result in aortic dilatation in addition to aortic insufficiency.
Elastic fibers are complex structures that consist primarily of tropoelastin monomers organized into a highly crosslinked polymer in a process that involves elastin binding protein (EBP), components of extracellular matrix microfibrils, and crosslinking enzymes [8]. Abnormalities in any of the molecules or assembly steps could result in the fragmented elastic fibers seen in MPS I tissues. Indeed, EBP levels were low and elastin biogenesis was reduced in fibroblasts from patients with MPS I, and it was proposed that defects in elastin assembly resulted in fragmented elastic fibers in the aorta [9].
An alternative explanation for elastin fragmentation is that MPS I results in activation of elastin-degrading proteins such as metalloproteinases (MMPs), cathepsins, or neutrophil elastase [10], [11]. The metal-dependent MMP-2, 7, 8, 9, 10, 12, and 14 can degrade elastin and/or play a role in aortic aneurisms [12], [13], [14], [15]. Furthermore, MMP-9 or MMP-12 deficiency reduced elastin fragmentation within atherosclerotic lesions in hypercholesterolemic mice [14] or reduced CaCl2-induced aneurism formation [12], [16]. In addition, deficiency of MMP-9, but not MMP-12, reduced aneurism formation after elastase perfusion [13]. MMPs are activated proteolytically by urokinase plasminogen activator (uPA), or without cleavage by SIBLINGs (small integrin-binding ligand N-linked glycoproteins) such as osteopontin [17]. uPA deficiency reduced aneurism formation [10] while osteopontin is upregulated in aortic aneurisms [15]. Tissue inhibitor of metalloproteinases (TIMP) 1 binds and inactivates MMPs, while TIMP-2 protects MMPs from inactivation by TIMP-1; TIMP-1 overexpression [18] and TIMP-2 [19] deficiency reduced aneurism formation in mice.
Cathepsins are lysosomal cysteine proteases that can degrade elastin and are upregulated in aneurisms [11]. Although most are rapidly inactivated at the neutral pH found in the extracellular space, cathepsin S maintains 64% of its peak activity at pH 7.5 [20]. Cathepsin S RNA is increased in atherosclerotic lesions in mice [21] and its deficiency reduced elastin fiber fragmentation in hypercholesterolemic mice [22]. Cystatin C is a cathepsin inhibitor, and its mRNA is reduced in human aortic aneurisms, and its deficiency increases elastin fragmentation in hypercholesterolemic mice [11].
The goal of this study was to determine the pathogenesis of elastic fiber fragmentation in the ascending aorta of MPS I mice. We demonstrate here that elastin structure and function are normal in young mice, suggesting that MPS I does not adversely affect elastin synthesis and assembly in vivo. However, increases in mRNA and enzyme activity for the elastolytic proteases MMP-12 and cathepsin S correlated with progressive elastin fragmentation and aortic dilatation in older mice, suggesting that the mechanism underlying aortic disease in MPS I is enhanced elastin degradation.
Section snippets
Materials and methods
Reagents were from Sigma-Aldrich Chemical (St. Louis, MO) unless otherwise stated.
Effect of age and gene therapy on aorta dilatation and elastin fragmentation
The time course of development of ascending aortic dilatation and elastin fragmentation in MPS I mice was evaluated by studying the mechanical properties and pathology of aortas at different ages at a point halfway between the sinotubular junction and the innominate artery (Fig. 1). The outer diameters of ascending aortas from MPS I mice were modestly dilated at 1.5 and 3 months when 75 mm of Hg of pressure was applied at 1.6 ± 0.1 and 1.7 ± 0.2 mm [error bars represent standard deviation (SD)],
Discussion
The etiology of elastin fragmentation in the aorta and heart in MPS I is important because it is a major cause of morbidity, and is relatively refractory to treatments. For example, aortic insufficiency developed de novo or worsened in 89% of patients at 12 years after performing hematopoietic stem cell transplantation at ∼2 years [30] and in 60% of patients at 4–6 years after starting enzyme replacement therapy [31], [32]. Although pathological data were not available, progressive
Sources of funding
This work was supported by the Ryan Foundation, the National MPS Society, and the National Institutes of Health (DK66448). Histology was supported by P30 DK52574 and real-time PCR was supported by DK20579 awarded to Clay Semenkovich.
Acknowledgments
We thank Alexander Hinek for the anti-EBP antibody, Xu Zhang for performing immunostaining, Elizabeth Neufeld for MPS I mice, Robert Thompson and John Curci for helpful discussions, and Clay Semenkovich and Trey Coleman for help with real-time PCR.
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