ISSN: 1524-4539 Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online

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DOI: 10.1161/CIRCULATIONAHA.107.733212 published online Apr 7, 2008; Circulation

Johanne Tremblay and Alexey V. Pshezhetsky Ernest, Yoshito Kadota, Maryssa Canuel, Kohji Itoh, Carlos R. Morales, Julie Lavoie,

Volkan Seyrantepe, Aleksander Hinek, Junzheng Peng, Michael Fedjaev, Sheila for Proper Elastic Fiber Formation and Inactivation of Endothelin-1

Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required

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Enzymatic Activity of Lysosomal Carboxypeptidase(Cathepsin) A Is Required for Proper Elastic Fiber

Formation and Inactivation of Endothelin-1Volkan Seyrantepe, PhD; Aleksander Hinek, MD, PhD; Junzheng Peng, PhD; Michael Fedjaev, PhD;

Sheila Ernest, PhD; Yoshito Kadota, PhD; Maryssa Canuel, MSc; Kohji Itoh, PhD;Carlos R. Morales, PhD; Julie Lavoie, PhD; Johanne Tremblay, PhD; Alexey V. Pshezhetsky, PhD

Background—Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomalmultienzyme complex along with �-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GALand Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactivesplice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA causegalactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomalstorage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, includinghypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest thatCathA 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 enzymecarrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalyticallyinactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiencyof 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 andstrongly confirm a crucial role of this enzyme in effective elastic fiber formation. (Circulation. 2008;117:1973-1981.)

Key Words: blood pressure � endothelin � enzymes � genes

Cathepsin A (protective protein; CathA) is a ubiquitouslyexpressed multifunctional enzyme, with deamidase, es-

terase, and carboxypeptidase activities and a preference forsubstrates with hydrophobic amino acid residues at the P1�position.1,2 Association with CathA, as part of the lysosomalmultienzyme complex (LMC), is essential for stabilization oflysosomal �-galactosidase (GAL), as well as for activation ofthe lysosomal sialidase Neu1.3–5 CathA, Neu1, and an alter-natively spliced variant of GAL can also be translocated tothe cell surface of arterial smooth muscle cells as subunits ofelastin receptor.6 In humans, inherited defects in the CathAgene result in the secondary deficiency of Neu1 and GAL andcause a lysosomal storage disorder, galactosialidosis, charac-terized by macular cherry-red spots, corneal clouding, skele-

tal dysplasia, hepatosplenomegaly, growth retardation, andneurological deterioration.7

Clinical Perspective p 1981

Although the enzymatic activity of CathA is not necessaryfor its function in the LMC, it has been conserved throughoutevolution, which suggests that CathA may have a dualbiological function. In vitro, CathA can hydrolyze a numberof regulatory peptides, including angiotensin (Ang) I andendothelin-1 (ET-1).8–11 The potent vasoconstrictive peptideET-1 is produced by proteolytic cleavage of inactive propep-tide by the metalloendopeptidase endothelin-converting en-zyme.12 The highest level of ET-1 is found in the endotheli-um, which also contains the highest level of CathA.

Received August 8, 2007; accepted February 8, 2008.From the Department of Medical Genetics (V.S., M.F., S.E., A.V.P.), CHU Sainte Justine Research Center, and CHUM Research Center (J.P., J.L.,

J.T.), University of Montreal, Montreal, Quebec, Canada; Cardiovascular Research Program (A.H.), The Hospital for Sick Children and Department ofLaboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Department of Medicinal Biotechnology (Y.K., K.I.), GraduateSchool of Pharmaceutical Sciences, University of Tokushima, Shomachi, Japan; and Department of Anatomy and Cell Biology (M.C., C.R.M., A.V.P.),Faculty of Medicine, McGill University, Montreal, Quebec, Canada.

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.

Correspondence to Alexey V. Pshezhetsky, PhD, Department of Medical Genetics, CHU Sainte Justine, 3175 Côte Ste-Catherine, Montreal, QuebecH3T 1C5, Canada. E-mail alexei.pchejetski@umontreal.ca

© 2008 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.107.733212

1973

Molecular Cardiology

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Coincidentally, the endothelial cells of CathA knockout miceand galactosialidosis patients exhibit extensive cytoplasmicvacuolization.7,13

Studies of cultured fibroblasts from galactosialidosis pa-tients demonstrated that these cells lack endothelin-degradingcapacity, whereas brain autopsies showed high ET-1–specificimmunoreactivity.14,15 Because in tissues and in the circula-tion, ET-1 is also cleaved by an abundant neutral endopepti-dase, NEP,16 in vivo experiments are required to understandwhether CathA is a nonredundant ET-1–degrading enzyme.The previously described CathA-knockout mice are notsuitable for physiological and behavioral studies because theydevelop progressive and diffuse edema, tremor, and ataxiadue to secondary Neu1 deficiency.13

To explore the physiological role of CathA, we generateda transgenic mouse model in which the normal CathA wasreplaced with a catalytically inactive enzyme that forms LMCand activates Neu1. Our studies indicate that CathA plays anonredundant role in the regulation of blood pressure throughinactivation of ET-1 and that as a component of the elastin-binding protein complex, it participates in the biogenesis ofelastic fibers.

MethodsAnimalsA 5.4-kb fragment of the mouse CathA (Ctsa) gene containing exons2 to 15 was amplified by an Expand long-template polymerase chainreaction (PCR) system (Roche, Basel, Switzerland) with genomicDNA of 129/Sv mice and the primers 5�-CCC TAA AGT TCC TAGGAG GG CATG-3� (I2F) and 5�-TTA GCG GAG GAC TCC TCTCT GCTGA-3� (I15R) and cloned with a TOPO XL PCR kit(Invitrogen, Carlsbad, Calif). The fragment was inserted in pBlue-script vector (Stratagene, La Jolla, Calif) in front of the thymidinekinase gene. The c.571AGC3GCA (p.S190A) mutation was gen-erated by PCR mutagenesis with the primers 5�-TGG AGT CGCAGA ACG ACC CAA AGA ACA GC-3� (E3F), 5�-GCA TAT GCCTCT CCT GTC AGG AAA AGT TTG TTG-3� (E6R), 5�-CAGGAG AGG CAT ATG CTG GCA TCT ACA TCC (E6F), and5�-CAC ACT CTG GGT CTT TGT TGTC-3� (E8R); the mutagenicnucleotide sequence is underlined. The fragments were linked byoverlap amplification with primers E3F and E8R and subcloned withPstI sites. A neomycin (Neo)-resistant cassette flanked by loxP siteswas inserted into the NheI site in intron 7. The targeting construct(Figure 1A) linearized with NotI digestion was electroporated intoR1 embryonic stem (ES) cells. G418- and ganciclovir-resistant EScells were screened for homologous recombination by PCR (Figure1A) with the allele-specific primers 5�-TCC CGG AGA TGT GCGCCA TC-3� (I1F) and 5�-CGG GGC TGC TAA AGC GCA T-3�(NeoR) and Southern blot (Figure 1B). Targeted ES clones weremicroinjected into C57BL/6J blastocysts and implanted into pseu-dopregnant female mice. Male chimeras were mated with C57BL/6Jfemales, and offspring were genotyped by Southern blot as describedabove. The heterozygous mice were bred to produce homozygousCathAS190A-Neo mice or mated with FVB/N-TgN-EIIa-Cre strain (TheJackson Laboratory, Bar Harbor, Me) to remove the Neo cassette.Absence of the Neo gene was confirmed by PCR amplification ofgenomic DNA with the E3F and E8R primers followed by NdeIdigestion (Figure 1C). All mice were bred and maintained in theCanadian Council on Animal Care–accredited animal facilities of theCHU Ste Justine Research Center. Approval for the experiments wasgranted by the animal care and use committees of the CHU SteJustine Research Center and CHUM. CathA mRNA levels in tissueswere studied by Northern blotting with a full-length [�-32P] dCTP-labeled mouse CathA cDNA as a probe. The expression of CathA

protein was studied by Western blotting with polyclonal rabbitanti-CathA antibodies.17

Purification of LMCTotal glycoproteins purified from 40 g of combined mouse liver andkidney tissues with affinity chromatography on concanavalinA-Sepharose3 were dialysed against 20 mmol/L sodium acetatebuffer, pH 4.75, which contained 0.15 mol/L NaCl, concentrated to�65 mg of protein per milliliter, applied on an FPLC Superose 6column (GE Healthcare, Baie d’Urfe, Canada), and eluted with thesame buffer at a flow rate of 0.4 mL/min. Thirty 0.5-mL fractionswere collected and analyzed for sialidase, GAL, and carboxypepti-dase activities. The molecular masses of the eluted proteins weredetermined with the calibration curve obtained with protein molec-ular weight standards (GE Healthcare).

Enzyme AssaysSialidase, �-galactosidase, and �-hexosaminidase activities wereassayed with the corresponding fluorogenic 4-methylumbelliferylglycoside substrates as described previously.3 Carboxypeptidaseactivity 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 TissuesAt 1, 4, and 8 months of age, mice were anesthetized with sodiumpentobarbital and euthanized by exsanguination. Lungs, spleen,brain, testis, and liver were fixed by immersion in 2.5% glutaralde-hyde in 0.1 mol/L cacodylate buffer for 48 hours at 4°C. The tissueswere 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 AortaHistological sections of skin, lungs, and aortas derived from 17-week–old mice were stained with Movat’s pentachrome,18 whichshows elastin as black. The distribution of black-stained materialentirely overlapped with the immunostaining by the specific anti-elastin antibodies performed on the parallel sections. At least 5 micewere studied for each phenotype.

Isolation and Culture of Primary Neural CellsFrom Cerebella and CerebraPrimary neural cell cultures were prepared from wild-type andCathAS190A mice aged 1 to 2 days. Pooled cerebra from 6 to 7 brainswere passed through a nylon mesh with a 40-�m pore size (BDFalcon, BD Biosciences, Mississauga, Canada) in Hank’s balancedsalt solution (Invitrogen). The dissociated cells were cultured inDMEM, supplemented with 10% FCS, 5 �g/mL insulin, andantibiotics.

Assessment of Elastin and Other Extracellular MatrixComponents Produced by Cultured Skin FibroblastsSkin fibroblasts isolated by collagenase digestion were cultured inDMEM, supplemented with 10% FCS and antibiotics as describedpreviously.6 Seven-day-old confluent cultures were fixed in 100%cold methanol. The deposition of ECM components was thendetected with antibodies to tropoelastin, collagen I, and fibronectinas described previously.19

Measurement of Insoluble Elastin Produced byCultured CellsSkin fibroblasts were grown to confluence in 10-cm cell culturedishes in quadruplicates. A total of 20 �Ci of [3H]-valine was addedto each dish along with fresh media at day 4. After 72 hours, levelsof insoluble elastin were measured as described previously.6,19

Blood Pressure Measurements by RadiotelemetryMale CathAS190A-Neo mice and appropriate littermate controls wereimplanted with TA11PA-C10 radiotelemetry sensors (Data Sciences

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International, St Paul, Minn) in the left carotid artery for directmeasurement of arterial pressure and heart rate as described previ-ously.20,21 Immobilization stress was performed by placing the micein a transparent restraining plastic holder routinely used for tail-cuffmeasurement of blood pressure.22 At least 6 mice were studied foreach genotype.

Measurement of ET-1 Degradation Rate in MouseBlood and TissuesCathAS190A mice and their wild-type siblings (5 mice per group) witha 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-1peptide (Bachem Bioscience, King of Prussia, Pa) in saline at a doseof 10 nmol/kg body weight. After 15 minutes, blood was collectedthrough cardiac puncture and immediately centrifuged to separateplasma. Lungs and liver were dissected and frozen in liquid nitrogen.

For peptide extraction, tissues (200 mg) were homogenized in 1mol/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 at20 000g for 10 minutes, and supernatant was applied to a Sep-PacC18 column (Waters, Milford, Mass). Columns were washed with 3volumes of 0.1% TFA in water, and peptides were eluted with 60%acetonitrile/0.1% TFA and lyophilized. Samples were reconstitutedin 0.1% TFA in DMSO for ELISA analysis or 10% acetonitrile/0.1%TFA for tandem mass spectrometry analysis. Quantitative assay ofET-1 by ELISA was performed with a kit from IBL (Toronto,Canada).

Measurement of ET-1 With Multiple-ReactionMonitoring Tandem Mass SpectrometrySamples were analyzed in duplicate with an 1100 Series nanoflowliquid chromatography system (Agilent Technologies, Santa Clara,Calif) and a 4000 QTrap mass spectrometer (Applied Biosystems,Foster City, Calif). The peptides were enriched on a Zorbax300SB-C18 trap column (Agilent Technologies) and separated by

Figure 1. Generation of CathAS190A-Neo and CathAS190A mice. A, The structure of the mouse Ctsa gene (top), the targeting construct (mid-dle), and the mutant locus after the homologous recombination (bottom). Exons are shown as solid boxes; PGK-neomycin (Neo) cas-sette flanked with loxP sites (arrowheads) and PGK-thymidine kinase (TK) genes are shown as white boxes. The c.571AGC�GCAmutation created a unique NdeI restriction site. The Neo cassette introduced a novel HindIII restriction site used for genotyping bySouthern blotting with the external 5� probe (thick line). Restriction sites are shown as H (HindIII), S (SstII), and N (NotI). B, TargetedCtsa locus after in vivo Cre-mediated excision of the Neo gene. C, Targeted allele-specific PCR amplification of 2040-bp DNA fragmentof CathAS190A-Neo mouse with I1F and NeoR primers. NdeI digestion of PCR product generated 2 fragments of 1320 and 720 bp,respectively. D, Southern hybridization of HindIII- and SstII-digested DNA from wild-type and CathAS190A heterozygous mice in F1 prog-eny indicating the germline (5.6 kb) and targeted (4.1 kb) alleles. E, PCR amplification of the CathA gene in F2 progeny with primersE3F and E8R generated a 2170-bp fragment in wild-type mice, 2170- and 2220-bp fragments in heterozygous CathAS190A mice, and a2220-bp fragment in homozygous CathAS190A mice.

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reverse-phase chromatography on a PicoFrit column (New Objec-tive, Woburn, Mass) packed with Biobasic C18. Acquired spectrawere analyzed by Analyst software (Applied Biosystems).

Statistical AnalysisStatistical analysis has been performed with 2-tailed t test, Welch’smodification of 2-tailed unpaired t test (Table), and ANOVA orrepeated-measures ANOVA tests.

The authors had full access to and take full responsibility for theintegrity of the data. All authors have read and agree to themanuscript as written.

ResultsGeneration of Mice With Deficient CathA ActivityTo define the physiological role of CathA activity, wegenerated a mouse strain with the targeted point mutationc.571AGC�GCA in the Ctsa gene, which replaced thenucleophile of the CathA active site, Ser190, with Ala (Figure1). R1 ES cells were electroporated with the targeting vectorthat contained the above mutation in exon 6 and a PGK-Neocassette inserted in intron 7. Targeted ES cells were injectedinto C57BL/6 embryos and transfected to pseudopregnantfemales. The chimeras thus obtained were bred with C57BL/6mice to obtain germline transmission (CathAS190A-Neo strain).Mice heterozygous for CathAS190A-Neo (Figure 1C, D) werecrossed with mice carrying a Cre-expressing transgene23 toexcise the PGK-Neo cassette. Littermates were genotyped forthe presence of the PGK-Neo gene and CathAS190A allele(Figure 1E) and mated with each other to obtain animalshomozygous for the CathAS190A allele.

Both the CathAS190A-Neo and CathAS190A mice were vital andfertile, grew normally, and had a normal lifespan, but theyhad 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 muta-tion.24 CathAS190A mice, however, had normal sialidase activ-ity, whereas in tissues of CathAS190A-Neo mice, the sialidaseactivity was reduced to �10% (Table). CathA mRNA levelswere reduced dramatically in tissues of CathAS190A-Neo mice,

consistent with the previously reported hypomorphic effect ofthe Neo gene,25,26 whereas CathAS190A mice expressed normalCathA mRNA levels (Figure 2A). Both Western blotting andimmunohistochemistry showed normal levels of CathA pro-tein in the tissues of CathAS190A mice (Figure 2B and 2C).

To confirm that the inactive S190A CathA mutant iscapable of forming a complex with Neu1 and GAL, weperformed a gel-filtration analysis of concentrated glycopro-tein extracts from the combined kidney and liver tissues ofthe wild-type, CathAS190A, and CathAS190A-Neo animals. In thetissue extracts of the wild-type mice, both an �1200-kDaLMC and a 120-kDa CathA homodimer6,17 were detected bythe presence of enzymatic activity peaks that eluted from thecolumn with correspondingly appropriate retention times(Figure 2D, peaks I and II). Similar profiles for GAL andNeu1 activities were also observed in the fractions obtainedby gel filtration of the tissue extracts of CathAS190A mice, butno carboxypeptidase activity was detected (Figure 2D), al-though CathA protein was present in the expected amountaccording to Western blot analysis (Figure 2D, inset). BothCathA activity and protein were absent from the fractionscollected after gel filtration of the tissue extracts ofCathAS190A-Neo mice (not shown).

CathAS190A Mice Have Pathological Changes inElastin-Rich TissuesPathological examination of CathAS190A mice performed at theage of 1 and 8 months did not reveal any gross changes in thevisceral organs. Similarly, microscopic investigation of tissuesections (Figure 3A) showed normal morphology in mosttissues, including those with high expression of CathAactivity, such as kidney, liver, testis, and brain. However,light microscopic histochemical examination demonstratedpathological changes in elastin-rich tissues such as skin,arteries, and lungs (Figure 3B). The dermis of CathAS190A miceshowed a significant decrease in elastic fibers as detected bythe pentachrome Movat method (Figure 3B) and by immu-nohistochemistry with specific anti-elastin antibodies (Figure

Table. CathA, GAL, and Sialidase Activities in Tissue Extracts of CathAS190A-Neo Mice, CathAS190A Mice, andTheir Wild-Type Littermates

Fraction CathAS190A-Neo WT CathAS190A WT

Specific CathA activity, nmol � h�1 � mg�1

Liver 2.2�0.9* 20.8�2.1 3.0�1.8* 26.4�2.1

Kidney 0.9�0.17* 18.4�1.0 2.2�1.1* 16.9�3.8

Lungs 0.04�0.02* 0.41�0.19 0.06�0.03* 0.47�0.07

Specific GAL activity, nmol � h�1 � mg�1

Liver 57�11 74�24 87�4.5 74�19

Kidney 223�17* 313�26 324�18 322�30

Lungs 73�18 73�25 82�19 92�23

Specific sialidase activity, nmol � h�1 � mg�1

Liver 0.2�0.02* 3.5�0.5 3.6�0.4 4.2�0.4

Kidney 1.8�0.7* 14.2�1.2 25.9�8 17.8�2.7

Lungs 0.4�0.04* 1.6�0.09 1.62�0.6 1.4�0.9

Data show mean�SD of measurements in 15 mice. Activity of the control lysosomal enzyme N-acetyl-�-D-glucosaminidase wasnot statistically different between the strains. WT indicates wild type.

*Significantly different from wild-type by Welch’s modification of 2-tailed unpaired t test (P�0.01).

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3C and online-only Data Supplement Figure I). The elasticarteries of CathAS190A mice exhibited a peculiar reduction ofelastic fibers in the tunica adventitia. The deficiency in elasticfibers was also noted within the alveolar septae of the lung inCathAS190A mice and coincided with an apparent enlargementof the alveolar diameter, which resembled an initial stage ofthe emphysema. Finally, cultured skin fibroblasts ofCathAS190A mice also showed impaired deposition of insolubleelastin, as measured by [3H]-valine incorporation, and adiscriminatory reduction in the number of elastic fibers, asassessed by quantitative immunohistochemistry. Of note,cultured skin fibroblasts of CathAS190A mice produced normallevels of fibronectin and collagen (Figure 3D).

Changes in Cardiovascular Function inCathA-Deficient MiceTo test the hypothesis that CathA, as the ET-1–degradingenzyme, is involved in hemodynamic regulation, we mea-sured heart rate, blood pressure (BP), natriuresis, and diuresisin CathAS190A and control mice by radiotelemetry and meta-bolic cages. BP and heart rate were measured at rest (over a10-day period), after the stress caused by 30 minutes ofimmobilization or after intravenous injection of ET-1 (0.1nmol per kg of body weight). The same measurements wereperformed after the mice were challenged with a high-saltdiet (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 inBP during immobilization stress, although the differencecompared with the wild-type group did not reach statisticalsignificance (Figure 4B). A significant (P�0.038) differencein systolic and diastolic BP between wild-type and CathAS190A

mice was also detected for the mice challenged by a high-saltdiet (Figure 4D) and between these groups in the immobili-zation stress experiment (Figure 4E). CathAS190A mice kept ona high-salt diet also consumed more water and produced moreurine than their wild-type siblings (not shown) and showed adifferent response to the intravenous injections of ET-1.Although in wild-type mice, intravenous administration ofET-1 resulted in an �40% increase in BP that lasted for atleast 50 minutes, BP in the CathAS190A mice remainedmostly unchanged (Figure 4F).

Figure 2. Expression of LMC components in CathA-defic-ient mice. A, Northern blot analysis of total liver RNA fromCathAS190A-Neo and CathAS190A mice (�/�) and their wild-type lit-termates (�/�) hybridized with CathA cDNA and mouse �-actincDNA. B, Western blotting of CathA protein in tissues of wild-type (�/�) and CathAS190A (�/�) mice. MW indicates molecularweight. C, Immunohistochemical detection of CathA protein inlung tissues of a wild-type and CathAS190A mouse. Slides werestained with antibodies against the 32-kDa protein chain ofCathA (CathA) or secondary antibodies only (Control). Magnifi-cation 100. D, FPLC gel filtration and Western blot analysis(insets) of concentrated glycoprotein extracts from liver and kid-ney tissues of mice homozygous for the CathAS190A allele (upperpanel) and their wild-type littermates (lower panel). Glycoprotein

Figure 2 (Continued). extracts were analyzed by gel filtration ona Superose 6 HR column and collected fractions were assayedfor GAL activity (�, left axis), CathA activity (‘, left axis), andNeu1 activity (□, right axis). The positions of the elution peaksof the molecular weight standards and void volume (V0) areshown by arrows. The 25 �L of indicated gel-filtration fractions,corresponding to the LMC (peak I), were analyzed by Westernblot with antibodies against the 32-kDa protein chain of CathA.

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CathA-Deficient Mice Have Decreased DegradationRate of ET-1Cultured embryonic brain cells of CathAS190A mice secreted asignificantly higher level of ET-1 (Figure 5A); however, thecomparisons of ET-1 levels in tissues of CathAS190A mice andtheir wild-type siblings measured with both ELISA (Figure5B) and immunohistochemistry (not shown) were not con-clusive. To determine whether the ET-1 degradation rate wasdifferent for the wild-type and CathA-deficient mice, wemeasured the concentration of the exogenous ET-1 in lungsand plasma 15 minutes after the intravenous injection (0.1nmol per kg of body weight). Because antibody-based assayscould not differentiate between the active full-length ET-1peptide and the inactive peptide generated from ET-1 byCathA and lacking the C-terminal Trp residue, the ET-1

levels were assayed by multiple-reaction monitoring tandemmass spectrometry. The ET-1 concentration was determinedby spiking the samples with known concentrations of ET-1standard and comparing the mass spectrometry intensities tothose of nonspiked samples. The data showed that 15 minutesafter the intravenous ET-1 injection, its concentration in lungs(Figure 5C) or plasma (not shown) was on average 3-foldhigher in CathAS190A mice than in their wild-type siblings,which suggests that in CathAS190A mice, the degradation rate ofET-1 is considerably reduced.

DiscussionDespite the fact that ubiquitously expressed lysosomal car-boxypeptidase, CathA, was described almost 7 decades ago,27

its biological function related to carboxypeptidase activity

Figure 3. Morphological findings in mouse tissues and fibroblast cultures. A, Representative micrographs of tissues obtained from wild-type (�/�) and CathAS190A (�/�) mice. No evident structural defects can be observed. B, Representative micrographs of elastin-richtissues from wild-type (�/�) and CathAS190A (�/�) mice showing pathological changes. Dermis shows decrease of elastic fibers(arrows); arteries show lack of elastic fibers in the tunica adventitia; and lungs show enlargement of alveolar diameter consistent withthe beginning of emphysema-like process. C, Representative immunohistochemistry micrographs showing the apparent lack of elasticfibers (arrows). D, Impaired deposition of insoluble elastin as measured by [3H]-valine incorporation and a discriminatory reduction innumber of elastic fibers as assessed by quantitative immunohistochemistry (inset) in cultured skin fibroblasts of CathAS190A mice. Dataare mean�SD of 4 independent experiments. *P�0.001. ECM indicates extracellular matrix.

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remained mainly unknown. Mice with targeted disruption ofthe Ctsa gene and human sialidosis patients with CathAmutations develop splenomegaly, skeletal abnormalities, andneuronal death.7,13 The present results show that these defectsare caused by the secondary deficiency of Neu1 and not bythe lack of CathA activity.

However, mutant mice in the present study that expressednormal levels of a catalytically inactive CathA proteinshowed a significant loss of elastic fibers in elastin-richtissues, such as the skin and the tunica adventitia of elasticarteries. Also, the lungs, which are normally rich in elasticfibers, showed an unusual enlargement of the alveolar sacsand thinning of the alveolar septae. Previously, we haveidentified that CathA is a component of the elastin-bindingprotein complex, a nonintegrin cell-surface receptor ex-pressed in all elastin-producing cells.6 The complex alsocontains Neu1 and the elastin-binding protein (EBP), analternatively spliced product of the GAL gene.28 We alsoreported that cultured fibroblasts from galactosialidosis pa-tients produce sufficient amounts of microfibrillar compo-nents and tropoelastin, which fail to assemble into elasticfibers.6 The present study constitutes the first in vivo dem-onstration that CathA activity in the elastin-binding protein

complex is a prerequisite for the efficient assembly of elasticfibers.

The development of arterial hypertension, previously re-ported in galactosialidosis but not in sialidosis patients,29,30 isanother clinical feature that could not be explained just on thebasis of impaired lysosomal catabolism of sialylated glyco-conjugates. We show that CathAS190A mice fed a normal and ahigh-salt diet had elevated BP compared with their wild-typesiblings. The heart rate values were similar for both strains,which suggests that the observed difference in BP relates to avascular effect. Because previous data showed that CathA caninactivate ET-1 in vitro,11 the elevated BP in CathA-deficientmice could be attributed to higher circulating levels of thispeptide. Indeed, mass spectroscopy studies showed that thesemice had a reduced degradation rate of ET-1 in blood andtissues.

Elevated ET-1 values have been observed previously invascular and cardiovascular disorders such as acute myocar-dial infarction, congestive heart failure, ischemia, atheroscle-rosis, and hypercholesterolemia, as well as in patients withatrial and pulmonary hypertension.31 ET-1–deficient miceshow abnormal manifestations in fetal development andhemodynamics,32 whereas overexpression of human ET-1 in

Figure 4. Radiotelemetry measurements of systolic BP, diastolic BP, and mean arterial pressure (MAP). A, Diastolic BP in CathAS190A

mice (▫) and wild-type mice (�), systolic BP in CathAS190A mice (‚), and wild-type mice (ƒ) were recorded continuously under basal con-ditions in mice fed a normal salt diet. MAP was also recorded before, during, and after a 30-minute immobilization stress (B) or afterthe intravenous injection of ET-1 at a dose of 0.1 nmol per kg of body weight (C). The same measurements were performed after themice were challenged with a high-salt diet (D, E, and F). P values report differences between wild-type and CathAS190A mice. Data wereanalyzed with a general linear model (repeated-measures ANOVA). At least 6 mice were studied for each genotype.

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mice caused vascular remodeling and endothelial dysfunctionbut no increase in BP.33,34 The latter result is contrary to thepresent findings, which demonstrated elevated basal BPlevels in CathA–deficient mice. One possible explanation forthe difference in BP between CathA-deficient mice withreduced capacity of ET-1 degradation and the transgenic micestably secreting high levels of ET-1 could be the compensa-tory suppression of the renin-angiotensin system previouslyshown in men systemically infused with ET-1.35 In addition,CathA has a high activity against Ang I, converting it to Ang1-9, which competes with Ang I in the reaction catalyzed byangiotensin-converting enzyme and potentiates bradykininaction on its B2 receptor.9,36,37 It is tempting to speculate,therefore, that the CathA-deficient mice would fail to gener-ate this peptide and to release Ang II through the alternativestepwise pathway. Atrial natriuretic peptide is another regu-lator of BP that can be affected by the increased levels orreduced degradation rate of ET-1.38 Although in the present

study, we did not directly measure levels of atrial natriureticpeptide, the increased urinary volume and water consumptionin CathA-deficient mice challenged by a high-salt diet areconsistent with higher levels of this hormone.

Together, the present results demonstrate that CathA, inaddition to its known role in the lysosome, is an importantfactor that contributes to BP regulation and normal develop-ment of elastic fibers, a crucial component of the cardiovas-cular and respiratory systems.

AcknowledgmentsWe thank Marcos R. Di Falco for help in the tandem massspectrometry analysis, Lucie Sedova and Michal Abrahamowicz forhelp with the statistical analysis, Raffaela Ballarano for help inpreparation of the manuscript, and Professor Igor P. Ashmarin forhelpful advice.

Sources of FundingThis work was supported in part by operating grants from theCanadian Institutes of Health Research to Dr Pshezhetsky (FRN15079) and to Dr Tremblay (MOP 11463 and MOP 43859). DrSeyrantepe was supported by postdoctoral fellowships from theFonds de la recherche en santé du Québec and Fondation de l’HôpitalSainte-Justine.

DisclosuresNone.

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CLINICAL PERSPECTIVEOur studies in a pediatric genetic disorder, galactosialidosis (OMIM No. 256540), caused by an inherited defect oflysosomal carboxypeptidase (cathepsin) A/protective protein unexpectedly led to the hypothesis that cathepsin A may beinvolved in hemodynamic functions. Here, we demonstrate that gene-targeted mice in which the normal cathepsin A wasreplaced with an active site (Ser190Ala) mutant have a reduced degradation rate of the vasoconstrictor peptide endothelin-1and significantly increased arterial blood pressure. Cathepsin A–deficient mice also display scarcity of elastic fibers inaortic adventitia, lungs, and skin. Our results provide the first evidence that cathepsin A acts in vivo as anendothelin-1–inactivating enzyme and has a crucial role in elastogenesis, revealing a new facet of cardiovascular biologypertinent to hypertension, vascular resilience, and the cardiovascular complications of lysosomal diseases.

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