Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required for Proper Elastic Fiber Formation and Inactivation of Endothelin-1
Background— Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with β-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome.
Methods and Results— We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin.
Conclusions— Our results provide the first evidence that CathA acts in vivo as an endothelin-1–inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.
Received August 8, 2007; accepted February 8, 2008.
Cathepsin A (protective protein; CathA) is a ubiquitously expressed multifunctional enzyme, with deamidase, esterase, and carboxypeptidase activities and a preference for substrates with hydrophobic amino acid residues at the P1′ position.1,2 Association with CathA, as part of the lysosomal multienzyme complex (LMC), is essential for stabilization of lysosomal β-galactosidase (GAL), as well as for activation of the lysosomal sialidase Neu1.3–5 CathA, Neu1, and an alternatively spliced variant of GAL can also be translocated to the cell surface of arterial smooth muscle cells as subunits of elastin receptor.6 In humans, inherited defects in the CathA gene result in the secondary deficiency of Neu1 and GAL and cause a lysosomal storage disorder, galactosialidosis, characterized by macular cherry-red spots, corneal clouding, skeletal dysplasia, hepatosplenomegaly, growth retardation, and neurological deterioration.7
Clinical Perspective p 1981
Although the enzymatic activity of CathA is not necessary for its function in the LMC, it has been conserved throughout evolution, which suggests that CathA may have a dual biological function. In vitro, CathA can hydrolyze a number of regulatory peptides, including angiotensin (Ang) I and endothelin-1 (ET-1).8–11 The potent vasoconstrictive peptide ET-1 is produced by proteolytic cleavage of inactive propeptide by the metalloendopeptidase endothelin-converting enzyme.12 The highest level of ET-1 is found in the endothelium, which also contains the highest level of CathA. Coincidentally, the endothelial cells of CathA knockout mice and galactosialidosis patients exhibit extensive cytoplasmic vacuolization.7,13
Studies of cultured fibroblasts from galactosialidosis patients demonstrated that these cells lack endothelin-degrading capacity, whereas brain autopsies showed high ET-1–specific immunoreactivity.14,15 Because in tissues and in the circulation, ET-1 is also cleaved by an abundant neutral endopeptidase, NEP,16 in vivo experiments are required to understand whether CathA is a nonredundant ET-1–degrading enzyme. The previously described CathA-knockout mice are not suitable for physiological and behavioral studies because they develop progressive and diffuse edema, tremor, and ataxia due to secondary Neu1 deficiency.13
To explore the physiological role of CathA, we generated a transgenic mouse model in which the normal CathA was replaced with a catalytically inactive enzyme that forms LMC and activates Neu1. Our studies indicate that CathA plays a nonredundant role in the regulation of blood pressure through inactivation of ET-1 and that as a component of the elastin-binding protein complex, it participates in the biogenesis of elastic fibers.
A 5.4-kb fragment of the mouse CathA (Ctsa) gene containing exons 2 to 15 was amplified by an Expand long-template polymerase chain reaction (PCR) system (Roche, Basel, Switzerland) with genomic DNA of 129/Sv mice and the primers 5′-CCC TAA AGT TCC TAG GAG GG CATG-3′ (I2F) and 5′-TTA GCG GAG GAC TCC TC TCT GCTGA-3′ (I15R) and cloned with a TOPO XL PCR kit (Invitrogen, Carlsbad, Calif). The fragment was inserted in pBluescript vector (Stratagene, La Jolla, Calif) in front of the thymidine kinase gene. The c.571AGC→GCA (p.S190A) mutation was generated by PCR mutagenesis with the primers 5′-TGG AGT CGC AGA ACG ACC CAA AGA ACA GC-3′ (E3F), 5′-GCA TAT GCC TCT CCT GTC AGG AAA AGT TTG TTG-3′ (E6R), 5′-CAG GAG AGG CAT ATG CTG GCA TCT ACA TCC (E6F), and 5′-CAC ACT CTG GGT CTT TGT TGTC-3′ (E8R); the mutagenic nucleotide sequence is underlined. The fragments were linked by overlap amplification with primers E3F and E8R and subcloned with PstI sites. A neomycin (Neo)-resistant cassette flanked by loxP sites was inserted into the NheI site in intron 7. The targeting construct (Figure 1A) linearized with NotI digestion was electroporated into R1 embryonic stem (ES) cells. G418- and ganciclovir-resistant ES cells were screened for homologous recombination by PCR (Figure 1A) with the allele-specific primers 5′-TCC CGG AGA TGT GCG CCA TC-3′ (I1F) and 5′-CGG GGC TGC TAA AGC GCA T-3′ (NeoR) and Southern blot (Figure 1B). Targeted ES clones were microinjected into C57BL/6J blastocysts and implanted into pseudopregnant female mice. Male chimeras were mated with C57BL/6J females, and offspring were genotyped by Southern blot as described above. The heterozygous mice were bred to produce homozygous CathAS190A-Neo mice or mated with FVB/N-TgN-EIIa-Cre strain (The Jackson Laboratory, Bar Harbor, Me) to remove the Neo cassette. Absence of the Neo gene was confirmed by PCR amplification of genomic DNA with the E3F and E8R primers followed by NdeI digestion (Figure 1C). All mice were bred and maintained in the Canadian Council on Animal Care–accredited animal facilities of the CHU Ste Justine Research Center. Approval for the experiments was granted by the animal care and use committees of the CHU Ste Justine Research Center and CHUM. CathA mRNA levels in tissues were studied by Northern blotting with a full-length [α-32P] dCTP-labeled mouse CathA cDNA as a probe. The expression of CathA protein was studied by Western blotting with polyclonal rabbit anti-CathA antibodies.17
Purification of LMC
Total glycoproteins purified from 40 g of combined mouse liver and kidney tissues with affinity chromatography on concanavalin A-Sepharose3 were dialysed against 20 mmol/L sodium acetate buffer, pH 4.75, which contained 0.15 mol/L NaCl, concentrated to ≈65 mg of protein per milliliter, applied on an FPLC Superose 6 column (GE Healthcare, Baie d’Urfe, Canada), and eluted with the same buffer at a flow rate of 0.4 mL/min. Thirty 0.5-mL fractions were collected and analyzed for sialidase, GAL, and carboxypeptidase activities. The molecular masses of the eluted proteins were determined with the calibration curve obtained with protein molecular weight standards (GE Healthcare).
Sialidase, β-galactosidase, and β-hexosaminidase activities were assayed with the corresponding fluorogenic 4-methylumbelliferyl glycoside substrates as described previously.3 Carboxypeptidase activity of CathA was measured with CBZ-Phe-Leu as a substrate17 or by the same procedure with 0.1 mmol/L ET-1 as a substrate.
Light Microscopy of Mouse Tissues
At 1, 4, and 8 months of age, mice were anesthetized with sodium pentobarbital and euthanized by exsanguination. Lungs, spleen, brain, testis, and liver were fixed by immersion in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer for 48 hours at 4°C. The tissues were embedded in paraffin and Epon (Hexion Specialty Chemicals, Columbus, Ohio), cut, and viewed by light microscopy.
Detection of Elastic Fibers in Mouse Skin, Lungs, and Aorta
Histological sections of skin, lungs, and aortas derived from 17-week–old mice were stained with Movat’s pentachrome,18 which shows elastin as black. The distribution of black-stained material entirely overlapped with the immunostaining by the specific anti-elastin antibodies performed on the parallel sections. At least 5 mice were studied for each phenotype.
Isolation and Culture of Primary Neural Cells From Cerebella and Cerebra
Primary neural cell cultures were prepared from wild-type and CathAS190A mice aged 1 to 2 days. Pooled cerebra from 6 to 7 brains were passed through a nylon mesh with a 40-μm pore size (BD Falcon, BD Biosciences, Mississauga, Canada) in Hank’s balanced salt solution (Invitrogen). The dissociated cells were cultured in DMEM, supplemented with 10% FCS, 5 μg/mL insulin, and antibiotics.
Assessment of Elastin and Other Extracellular Matrix Components Produced by Cultured Skin Fibroblasts
Skin fibroblasts isolated by collagenase digestion were cultured in DMEM, supplemented with 10% FCS and antibiotics as described previously.6 Seven-day-old confluent cultures were fixed in 100% cold methanol. The deposition of ECM components was then detected with antibodies to tropoelastin, collagen I, and fibronectin as described previously.19
Measurement of Insoluble Elastin Produced by Cultured Cells
Skin fibroblasts were grown to confluence in 10-cm cell culture dishes in quadruplicates. A total of 20 μCi of [3H]-valine was added to each dish along with fresh media at day 4. After 72 hours, levels of insoluble elastin were measured as described previously.6,19
Blood Pressure Measurements by Radiotelemetry
Male CathAS190A-Neo mice and appropriate littermate controls were implanted with TA11PA-C10 radiotelemetry sensors (Data Sciences International, St Paul, Minn) in the left carotid artery for direct measurement of arterial pressure and heart rate as described previously.20,21 Immobilization stress was performed by placing the mice in a transparent restraining plastic holder routinely used for tail-cuff measurement of blood pressure.22 At least 6 mice were studied for each genotype.
Measurement of ET-1 Degradation Rate in Mouse Blood and Tissues
CathAS190A mice and their wild-type siblings (5 mice per group) with a body weight of 40 to 45 g that had been anesthetized with urethane (1.5 g/kg) were injected intravenously with a solution of ET-1 peptide (Bachem Bioscience, King of Prussia, Pa) in saline at a dose of 10 nmol/kg body weight. After 15 minutes, blood was collected through cardiac puncture and immediately centrifuged to separate plasma. Lungs and liver were dissected and frozen in liquid nitrogen.
For peptide extraction, tissues (200 mg) were homogenized in 1 mol/L CH3COOH/20 mmol/L HCl. Plasma was supplemented with concentrated CH3COOH until the final concentration of 1 mol/L. After they were boiled for 10 minutes, samples were centrifuged at 20 000g for 10 minutes, and supernatant was applied to a Sep-Pac C18 column (Waters, Milford, Mass). Columns were washed with 3 volumes of 0.1% TFA in water, and peptides were eluted with 60% acetonitrile/0.1% TFA and lyophilized. Samples were reconstituted in 0.1% TFA in DMSO for ELISA analysis or 10% acetonitrile/0.1% TFA for tandem mass spectrometry analysis. Quantitative assay of ET-1 by ELISA was performed with a kit from IBL (Toronto, Canada).
Measurement of ET-1 With Multiple-Reaction Monitoring Tandem Mass Spectrometry
Samples were analyzed in duplicate with an 1100 Series nanoflow liquid chromatography system (Agilent Technologies, Santa Clara, Calif) and a 4000 QTrap mass spectrometer (Applied Biosystems, Foster City, Calif). The peptides were enriched on a Zorbax 300SB-C18 trap column (Agilent Technologies) and separated by reverse-phase chromatography on a PicoFrit column (New Objective, Woburn, Mass) packed with Biobasic C18. Acquired spectra were analyzed by Analyst software (Applied Biosystems).
Statistical analysis has been performed with 2-tailed t test, Welch’s modification of 2-tailed unpaired t test (Table), and ANOVA or repeated-measures ANOVA tests.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Generation of Mice With Deficient CathA Activity
To define the physiological role of CathA activity, we generated a mouse strain with the targeted point mutation c.571AGC>GCA in the Ctsa gene, which replaced the nucleophile of the CathA active site, Ser190, with Ala (Figure 1). R1 ES cells were electroporated with the targeting vector that contained the above mutation in exon 6 and a PGK-Neo cassette inserted in intron 7. Targeted ES cells were injected into C57BL/6 embryos and transfected to pseudopregnant females. The chimeras thus obtained were bred with C57BL/6 mice to obtain germline transmission (CathAS190A-Neo strain). Mice heterozygous for CathAS190A-Neo (Figure 1C, D) were crossed with mice carrying a Cre-expressing transgene23 to excise the PGK-Neo cassette. Littermates were genotyped for the presence of the PGK-Neo gene and CathAS190A allele (Figure 1E) and mated with each other to obtain animals homozygous for the CathAS190A allele.
Both the CathAS190A-Neo and CathAS190A mice were vital and fertile, grew normally, and had a normal lifespan, but they had almost zero CathA activity measured with CBZ-PheLeu (Table) or ET-1 (not shown) in kidney, livers, and lungs, consistent with the presence of the Ser190Ala point mutation.24 CathAS190A mice, however, had normal sialidase activity, whereas in tissues of CathAS190A-Neo mice, the sialidase activity was reduced to ≈10% (Table). CathA mRNA levels were reduced dramatically in tissues of CathAS190A-Neo mice, consistent with the previously reported hypomorphic effect of the Neo gene,25,26 whereas CathAS190A mice expressed normal CathA mRNA levels (Figure 2A). Both Western blotting and immunohistochemistry showed normal levels of CathA protein in the tissues of CathAS190A mice (Figure 2B and 2C).
To confirm that the inactive S190A CathA mutant is capable of forming a complex with Neu1 and GAL, we performed a gel-filtration analysis of concentrated glycoprotein extracts from the combined kidney and liver tissues of the wild-type, CathAS190A, and CathAS190A-Neo animals. In the tissue extracts of the wild-type mice, both an ≈1200-kDa LMC and a 120-kDa CathA homodimer6,17 were detected by the presence of enzymatic activity peaks that eluted from the column with correspondingly appropriate retention times (Figure 2D, peaks I and II). Similar profiles for GAL and Neu1 activities were also observed in the fractions obtained by gel filtration of the tissue extracts of CathAS190A mice, but no carboxypeptidase activity was detected (Figure 2D), although CathA protein was present in the expected amount according to Western blot analysis (Figure 2D, inset). Both CathA activity and protein were absent from the fractions collected after gel filtration of the tissue extracts of CathAS190A-Neo mice (not shown).
CathAS190A Mice Have Pathological Changes in Elastin-Rich Tissues
Pathological examination of CathAS190A mice performed at the age of 1 and 8 months did not reveal any gross changes in the visceral organs. Similarly, microscopic investigation of tissue sections (Figure 3A) showed normal morphology in most tissues, including those with high expression of CathA activity, such as kidney, liver, testis, and brain. However, light microscopic histochemical examination demonstrated pathological changes in elastin-rich tissues such as skin, arteries, and lungs (Figure 3B). The dermis of CathAS190A mice showed a significant decrease in elastic fibers as detected by the pentachrome Movat method (Figure 3B) and by immunohistochemistry with specific anti-elastin antibodies (Figure 3C and online-only Data Supplement Figure I). The elastic arteries of CathAS190A mice exhibited a peculiar reduction of elastic fibers in the tunica adventitia. The deficiency in elastic fibers was also noted within the alveolar septae of the lung in CathAS190A mice and coincided with an apparent enlargement of the alveolar diameter, which resembled an initial stage of the emphysema. Finally, cultured skin fibroblasts of CathAS190A mice also showed impaired deposition of insoluble elastin, as measured by [3H]-valine incorporation, and a discriminatory reduction in the number of elastic fibers, as assessed by quantitative immunohistochemistry. Of note, cultured skin fibroblasts of CathAS190A mice produced normal levels of fibronectin and collagen (Figure 3D).
Changes in Cardiovascular Function in CathA-Deficient Mice
To test the hypothesis that CathA, as the ET-1–degrading enzyme, is involved in hemodynamic regulation, we measured heart rate, blood pressure (BP), natriuresis, and diuresis in CathAS190A and control mice by radiotelemetry and metabolic cages. BP and heart rate were measured at rest (over a 10-day period), after the stress caused by 30 minutes of immobilization or after intravenous injection of ET-1 (0.1 nmol per kg of body weight). The same measurements were performed after the mice were challenged with a high-salt diet (8% NaCl for 3 weeks).
On a normal diet, CathAS190A mice had significantly (P=0.004) higher day (Figure 4A) and night (not shown) levels of both diastolic BP (day 1, 99.5±2.4, day 2, 97.3±2.5, and day 3, 95.7±1.5 mm Hg) and systolic (day 1, 137.6±3.2, day 2, 133.9±2.9, and day 3, 132.1±1.9 mm Hg) BP than their wild-type siblings (day 1, 90.7±1.3, day 2, 87.3±0.6, and day 3, 87.4±1.3 mm Hg, and day 1, 124.0±1.8, day 2, 119.2±1.9, and day 3, 119.2±1.8 mm Hg, respectively), whereas heart rates were the same (not shown). CathAS190A mice also showed on average a lower increase in BP during immobilization stress, although the difference compared with the wild-type group did not reach statistical significance (Figure 4B). A significant (P=0.038) difference in systolic and diastolic BP between wild-type and CathAS190A mice was also detected for the mice challenged by a high-salt diet (Figure 4D) and between these groups in the immobilization stress experiment (Figure 4E). CathAS190A mice kept on a high-salt diet also consumed more water and produced more urine than their wild-type siblings (not shown) and showed a different response to the intravenous injections of ET-1. Although in wild-type mice, intravenous administration of ET-1 resulted in an ≈40% increase in BP that lasted for at least 50 minutes, BP in the CathAS190A mice remained mostly unchanged (Figure 4F).
CathA-Deficient Mice Have Decreased Degradation Rate of ET-1
Cultured embryonic brain cells of CathAS190A mice secreted a significantly higher level of ET-1 (Figure 5A); however, the comparisons of ET-1 levels in tissues of CathAS190A mice and their wild-type siblings measured with both ELISA (Figure 5B) and immunohistochemistry (not shown) were not conclusive. To determine whether the ET-1 degradation rate was different for the wild-type and CathA-deficient mice, we measured the concentration of the exogenous ET-1 in lungs and plasma 15 minutes after the intravenous injection (0.1 nmol per kg of body weight). Because antibody-based assays could not differentiate between the active full-length ET-1 peptide and the inactive peptide generated from ET-1 by CathA and lacking the C-terminal Trp residue, the ET-1 levels were assayed by multiple-reaction monitoring tandem mass spectrometry. The ET-1 concentration was determined by spiking the samples with known concentrations of ET-1 standard and comparing the mass spectrometry intensities to those of nonspiked samples. The data showed that 15 minutes after the intravenous ET-1 injection, its concentration in lungs (Figure 5C) or plasma (not shown) was on average 3-fold higher in CathAS190A mice than in their wild-type siblings, which suggests that in CathAS190A mice, the degradation rate of ET-1 is considerably reduced.
Despite the fact that ubiquitously expressed lysosomal carboxypeptidase, CathA, was described almost 7 decades ago,27 its biological function related to carboxypeptidase activity remained mainly unknown. Mice with targeted disruption of the Ctsa gene and human sialidosis patients with CathA mutations develop splenomegaly, skeletal abnormalities, and neuronal death.7,13 The present results show that these defects are caused by the secondary deficiency of Neu1 and not by the lack of CathA activity.
However, mutant mice in the present study that expressed normal levels of a catalytically inactive CathA protein showed a significant loss of elastic fibers in elastin-rich tissues, such as the skin and the tunica adventitia of elastic arteries. Also, the lungs, which are normally rich in elastic fibers, showed an unusual enlargement of the alveolar sacs and thinning of the alveolar septae. Previously, we have identified that CathA is a component of the elastin-binding protein complex, a nonintegrin cell-surface receptor expressed in all elastin-producing cells.6 The complex also contains Neu1 and the elastin-binding protein (EBP), an alternatively spliced product of the GAL gene.28 We also reported that cultured fibroblasts from galactosialidosis patients produce sufficient amounts of microfibrillar components and tropoelastin, which fail to assemble into elastic fibers.6 The present study constitutes the first in vivo demonstration that CathA activity in the elastin-binding protein complex is a prerequisite for the efficient assembly of elastic fibers.
The development of arterial hypertension, previously reported in galactosialidosis but not in sialidosis patients,29,30 is another clinical feature that could not be explained just on the basis of impaired lysosomal catabolism of sialylated glycoconjugates. We show that CathAS190A mice fed a normal and a high-salt diet had elevated BP compared with their wild-type siblings. The heart rate values were similar for both strains, which suggests that the observed difference in BP relates to a vascular effect. Because previous data showed that CathA can inactivate ET-1 in vitro,11 the elevated BP in CathA-deficient mice could be attributed to higher circulating levels of this peptide. Indeed, mass spectroscopy studies showed that these mice had a reduced degradation rate of ET-1 in blood and tissues.
Elevated ET-1 values have been observed previously in vascular and cardiovascular disorders such as acute myocardial infarction, congestive heart failure, ischemia, atherosclerosis, and hypercholesterolemia, as well as in patients with atrial and pulmonary hypertension.31 ET-1–deficient mice show abnormal manifestations in fetal development and hemodynamics,32 whereas overexpression of human ET-1 in mice caused vascular remodeling and endothelial dysfunction but no increase in BP.33,34 The latter result is contrary to the present findings, which demonstrated elevated basal BP levels in CathA–deficient mice. One possible explanation for the difference in BP between CathA-deficient mice with reduced capacity of ET-1 degradation and the transgenic mice stably secreting high levels of ET-1 could be the compensatory suppression of the renin-angiotensin system previously shown in men systemically infused with ET-1.35 In addition, CathA has a high activity against Ang I, converting it to Ang 1-9, which competes with Ang I in the reaction catalyzed by angiotensin-converting enzyme and potentiates bradykinin action on its B2 receptor.9,36,37 It is tempting to speculate, therefore, that the CathA-deficient mice would fail to generate this peptide and to release Ang II through the alternative stepwise pathway. Atrial natriuretic peptide is another regulator of BP that can be affected by the increased levels or reduced degradation rate of ET-1.38 Although in the present study, we did not directly measure levels of atrial natriuretic peptide, the increased urinary volume and water consumption in CathA-deficient mice challenged by a high-salt diet are consistent with higher levels of this hormone.
Together, the present results demonstrate that CathA, in addition to its known role in the lysosome, is an important factor that contributes to BP regulation and normal development of elastic fibers, a crucial component of the cardiovascular and respiratory systems.
We thank Marcos R. Di Falco for help in the tandem mass spectrometry analysis, Lucie Sedova and Michal Abrahamowicz for help with the statistical analysis, Raffaela Ballarano for help in preparation of the manuscript, and Professor Igor P. Ashmarin for helpful advice.
Sources of Funding
This work was supported in part by operating grants from the Canadian Institutes of Health Research to Dr Pshezhetsky (FRN 15079) and to Dr Tremblay (MOP 11463 and MOP 43859). Dr Seyrantepe was supported by postdoctoral fellowships from the Fonds de la recherche en santé du Québec and Fondation de l’Hôpital Sainte-Justine.
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Our studies in a pediatric genetic disorder, galactosialidosis (OMIM No. 256540), caused by an inherited defect of lysosomal carboxypeptidase (cathepsin) A/protective protein unexpectedly led to the hypothesis that cathepsin A may be involved in hemodynamic functions. Here, we demonstrate that gene-targeted mice in which the normal cathepsin A was replaced with an active site (Ser190Ala) mutant have a reduced degradation rate of the vasoconstrictor peptide endothelin-1 and significantly increased arterial blood pressure. Cathepsin A–deficient mice also display scarcity of elastic fibers in aortic adventitia, lungs, and skin. Our results provide the first evidence that cathepsin A acts in vivo as an endothelin-1–inactivating enzyme and has a crucial role in elastogenesis, revealing a new facet of cardiovascular biology pertinent to hypertension, vascular resilience, and the cardiovascular complications of lysosomal diseases.
The online-only Data Supplement, consisting of a Figure and an expanded Methods section, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.733212/DC1.