In vivo fragmentation of by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis

Jin-Ping Li*†, Martha L. Escobar Galvis*‡, Feng Gong*‡§, Xiao Zhang¶, Eyal Zchariaʈ, Shula Metzger**, Israel Vlodavsky††, Robert Kisilevsky‡‡, and Ulf Lindahl*

*Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden; ¶Department of Public Health and Caring Science, Division of Molecular Geriatrics, Rudbeck Laboratory, Uppsala University, Box 609, SE-751 25 Uppsala, Sweden; Departments of ʈOncology and **Medicine, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel; ††Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel; and ‡‡Department of Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada K7L 3N6

Communicated by D. Carleton Gajdusek, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France, March 21, 2005 (received for review December 22, 2004) Amyloid diseases encompass >20 medical disorders that include The results demonstrate that overexpression of heparanase, amyloid protein A (AA) amyloidosis, Alzheimer’s disease, and type resulting in fragmentation of HS chains, affords protection 2 diabetes. A common feature of these conditions is the selective against amyloidosis. organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have gener- Materials and Methods ated transgenic mice that overexpress human heparanase and Animals and Amyloid Induction. The homozygous mouse strain have tested their susceptibility to amyloid induction. Drastic short- overexpressing human heparanase (hpa-tg) and the respective ening of heparan sulfate chains was observed in heparanase- control (ctr) mice (C57BL background) were generated as overproducing organs, such as liver and kidney. These sites selec- described (6) and maintained in the animal facility of the tively escaped amyloid deposition on experimental induction of Biomedical Center, Uppsala University. The animal experi- inflammation-associated AA amyloidosis, as verified by lack of ments were performed in compliance with Swedish legislation material staining with Congo Red, as well as lack of associated for animal welfare (approval number C176͞2). Amyloidosis was polysaccharide, whereas the same tissues from control animals induced as described (3). Briefly, amyloid enhancing factor were heavily infiltrated with amyloid. By contrast, the spleens of (AEF), prepared as AA amyloid fibrils (7), was administered i.p. transgenic mice that failed to significantly overexpress heparanase (200 ␮g per mouse, 200 ␮l, n ϭ 6 hpa-tg and n ϭ 6 ctr male mice, contained heparan sulfate chains similar in size to those of control 10 weeks old). Immediately after the administration of AEF, 0.5 spleen and remained susceptible to amyloid deposition. Our find- ml of AgNO3 (2% solution) was injected s.c. into the loose tissue ings provide direct in vivo evidence that heparan sulfate is essential of the back, between the shoulder blades. Mice were killed by for the development of amyloid disease. cervical dislocation 7 days after commencement of the induction protocol. Spleens, livers, and kidneys were dissected from each inflammation ͉ transgenic mice ͉ endo-glucuronidase ͉ Congo red staining animal, fixed overnight in a solution containing 96% ethanol, 1% glacial acetic acid, and 3% distilled water, and stored in 70% ethanol until processed for histological analysis. myloid diseases are characterized by deposition of disease- specific fibrillar proteins (amyloid) in various organs, lead- A Histochemical Analyses. Tissues in 70% ethanol were dehydrated ing to loss of function and to associated clinical symptoms. It has by using standard procedures and embedded in paraffin. Sec- been proposed that heparan sulfate (HS) may facilitate forma- ␮ ͞ tions of 8–10 m were stained with Congo red (8) to detect tion of the nidus and or protofilament around which amyloid amyloid deposition and with sulfated Alcian blue (SAB) (9) to fibrillogenesis takes place and impart stability to the amyloid detect sulfated glycosaminoglycans. The percent tissue area fibril in vivo (1, 2). This notion was supported by the marked ͞ occupied by birefringent Congo red-positive staining in polar- increase in fibrillogenesis in vitro exerted by HS on ized light was determined as described (3) by image analysis by ␤ various amyloidogenic polypeptides, including A and phos- using a program and apparatus from MCID M2 Imaging Research ␣ phorylated tau (Alzheimer’s disease), -synuclein (Parkinson’s (St. Catherine’s, ON, Canada). All comparisons were made after disease), islet amyloid polypeptide (IAPP) (type 2 diabetes), and calibrating the apparatus against a set of standard spleen sections ␤ ␤ 2-microglobulin (chronic hemodialysis-related amyloid). A containing AA amyloid. fibrillogenesis is precluded by agents that block A␤:HS interac- tions (3). The in vivo evidence is circumstantial, primarily Immunohistochemical Detection of Heparanase. The analysis was observations of codistributed HS (HSPG), notably performed as described with minor modifications (6, 10, 11). perlecan, and amyloidogenic peptide in amyloid fibrils (2). Briefly, 8- to 10-␮m sections were deparaffinized and rehy- Heparanase is a mammalian endo-␤-D-glucuronidase that drated. The tissue was then denatured for 3 min in a microwave cleaves HS at a limited number of sites. Cloning of the human oven in citrate buffer (0.01 M, pH 6.0). Blocking steps included MEDICAL SCIENCES heparanase cDNA by several groups suggests that a single successive incubations in 3% H2O2 in methanol and 5% goat dominant HS-degrading endoglycosidase is expressed in mam- malian cells (4, 5). Our recent generation of transgenic mice overexpressing heparanase in various tissues revealed that the Abbreviations: AA, amyloid protein A; HS, heparan sulfate; ctr, control; hpa-tg, heparanase plays a role in diverse processes such as embryonic transgenic; SAB, sulfated Alcian blue. implantation, mammary gland morphogenesis, hair follicle †To whom correspondence should be addressed. E-mail: [email protected]. growth, and tissue repair (6). In the present study, the differ- ‡M.L.E.G. and F.G. contributed equally to this work. ential expression of heparanase has been exploited to assess the §Present address: Institution of Blood Transfusion, Box 130, Beijing 100850, China. role of HS in amyloid protein A (AA) amyloid generation in vivo. © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502287102 PNAS ͉ May 3, 2005 ͉ vol. 102 ͉ no. 18 ͉ 6473–6477 Downloaded by guest on September 28, 2021 serum. Tissue sections were incubated with anti-human hepara- nase antibodies (mAb 130) or with DMEM supplemented with 3.3% horse serum as control, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse an- tibodies (The Jackson Laboratory). Color was developed by using Zymed AEC substrate kit (Zymed) for 10 min, followed by counter staining with Mayer’s hematoxylin. The monoclonal mouse anti-human heparanase antibodies (mAb 130) are di- rected against the C terminus of the 50-kDa heparanase subunit and were produced as described (11). These antibodies do not recognize the mouse heparanase and were kindly provided by InSight Ltd (Rehovot, Israel).

In Vivo Radiolabeling and Purification of HS. Hpa-tg and ctr mice 35 (male, 10 weeks old) were injected i.p. with 0.5 mCi of Na SO4 (1 Ci ϭ 37 GBq) (Amersham Pharmacia Biosciences) and maintained for2hwithfreeaccesstowater and food. The animals were killed by cervical dislocation, and various organs (liver, kidney, spleen, lung, heart, and brain) were dissected. The heparan sulfate was isolated as described (12). Briefly, the organs were cut into small pieces and homogenized in Tris⅐HCl (50 mM, pH 7.4) extraction buffer containing 4 M urea and 1% Triton X-100 on ice. The homogenates were incubated at 4°C overnight with mild agitation and centrifuged at 2,800 ϫ g for 15 min. The supernatant was mixed with 4 M NaOH to a final concentration of 0.5 M and incubated at 4°C overnight. After neutralization with HCl, the samples were adjusted to 100 mM salt concentration by dilution with the Tris⅐HCl extraction buffer and applied on a DEAE-Sephacel column (2 ml) equilibrated in the same buffer. Columns were washed with acetate buffer (50 mM, pH 4.5) containing 4 M urea until there was no detectable radioactivity in the effluent. Elution was carried out by using the acetate buffer containing 1.5 M NaCl and 4 M urea, pH 4.5. The eluted radiolabeled material was pooled and desalted on a PD-10 column (Amersham Pharmacia Biosciences) in 10% ethanol and lyophilized. The samples were treated with chondroitinase ABC (1 unit͞ml) (Seikagaku, Tokyo) at 37°C overnight, and the free HS chains were recovered by purification on a 1-ml DEAE- Sephacel column connected to an HPLC system (12).

Analysis of HS. Purified metabolically 35S-labeled free HS chains Fig. 1. Heparanase mRNA and protein in different organs of hpa-tg vs. ctr were desalted on a PD-10 column (10% ethanol) and lyophilized. mice. (A) Total RNA was hybridized with a 560-bp fragment of the mouse heparanase cDNA (Upper) and reprobed with a MTN ␤-actin probe (Lower). For identification of the products, samples of 5,000 cpm were Overexpression of human heparanase is apparent in all organs analyzed, incubated with bacterial heparinase (1 milliunit) and hepariti- although at markedly different levels. Only a slight overexpression was noted nase (1 milliunit) (Seikagaku) in 100 ␮l of 50 mM Tris⅐HCl in the spleen. Hpa, heparanase. (B) Immunohistochemical staining with anti- buffer (pH 7.2) in the presence of 1.6 mM CaCl2 and 0.005% human heparanase antibody (mAb 130). Abundant heparanase protein ex- BSAfor4hat37°C, and the digests were analyzed by gel pression (reddish color) is seen in the liver and kidney, but not in the spleen, chromatography on a Superose-12 column. For size analysis, of the hpa-tg mice (magnification ϫ40). samples (Ϸ 5,000 cpm in a volume of 100 ␮l) were applied to a similar column (Amersham Pharmacia Biosciences) equilibrated in Tris⅐HCl buffer (50 mM, pH 7.4) containing 1 M NaCl and Biosciences). Hybridization was carried out at 68°C in Ex- 0.1% Triton X-100. Effluent fractions of 0.5 ml were collected pressHyb solution (Clontech) as described in the user’s manual, and analyzed after addition of OptiPhase HiSafe 3 in a Beckman with extensive prehybridization (4–16 h). To check the loaded ␤ scintillation counter (12). amount of RNA, the blots were reprobed with a MTN -actin probe from Clontech. The blots were exposed onto Phosphor- Northern Blot Analysis. One hpa-tg and one ctr mouse (10 weeks Imager screens and scanned in a Fuji scanner. Image analysis was old) were killed by cervical dislocation. The organs were dis- carried out by using IMAGE GAUGE. sected and frozen immediately in liquid nitrogen. Total RNA was prepared from the different organs by using either TRIzol Results or the SV Total RNA Isolation System (Promega) according to Expression of Heparanase mRNA and Protein. Despite a marked the manufacturer’s instructions. Total RNA (25 ␮g, estimated by reduction in overall HS chain length, the heparanase transgenic OD260) was separated by electrophoresis in a 1.2% denaturing (hpa-tg) mice seemed healthy, were fertile, and had a normal life agarose gel containing 0.66 M formaldehyde. After blotting onto span (6). Heparanase overexpression showed consistent organ a Hybond NX membrane (Amersham Pharmacia Biosciences) selectivity, and this trait was exploited in the present study to according to standard procedures, the RNA was hybridized with investigate the pathophysiological role of HS in AA amyloido- a purified PCR fragment, corresponding to nucleotides 175–734 genesis in vivo. The expression pattern of heparanase transcripts, of the mouse heparanase cDNA, which was labeled with determined by Northern blot analysis (Fig. 1A), was found to [32P]dCTP by using the Ready-To-Go kit (Amersham Pharmacia correlate well with immunohistochemical staining of the hepara-

6474 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502287102 Li et al. Downloaded by guest on September 28, 2021 nase protein (Fig. 1B) and with enzyme activity measurements in various organs (6). Although liver and kidney showed the highest expression levels, spleen of the hpa-tg mice differed only marginally from that of the ctr mice and showed little or no expression of human heparanase (Fig. 1 A and B). The amounts of mRNA obtained from heart were substantially smaller than those from other organs, judging from the low levels of ␤-actin signal, yet clearly revealed overexpression in the hpa-tg mouse.

Amyloid and Glycosaminoglycan Deposition in Different Organs. A widely used experimental model for amyloid induction (3) was applied to hpa-tg and ctr mice of the same genetic background. Mice (six hpa-tg and six ctr) received a single i.p. injection of amyloid-enhancing factor (AEF), likely the nidus for AA amy- loid (13) along with a s.c. injection of AgNO3 as an inflammatory stimulus (see Materials and Methods). Seven days later, the organs were collected and examined for amyloid deposition by staining with Congo red. Representative sections of liver, kidney, and spleen (i.e., the preferred predilection targets in AA amy- loidosis) are shown in Fig. 2A.Inctr mice, substantial amyloid deposits were found around the central veins of the liver, in the renal papillae, and in the perifollicular areas of the spleen. In contrast, the hpa-tg mice showed virtually no amyloid in the kidney or the liver, whereas the perifollicular splenic amyloid seemed similar in hpa-tg and ctr mice. These observations were substantiated by image analysis, which showed the mean spleen area occupied by amyloid in both wild-type and transgenic groups of mice to be Ϸ7.5% (Fig. 2B). In contrast, the mean liver areas occupied by AA amyloid in the wild-type (Ϸ2.0% of area) and transgenic groups (Ϸ0.4%) differed considerably (P ϭ 0.0013). A similar 4- to 5-fold difference was noted in the kidneys. The renal amyloid was localized exclusively to the papillae (the only site of kidney amyloidogenesis at this early stage of induction), where the number of lesions per high- powered field were 23.1 Ϯ 14.0 and 4.9 Ϯ 2.3 in the wild-type and transgenic animals, respectively (Fig. 2B). Comparison of Congo red staining for amyloid (Fig. 2A) with immunohistochemical staining for heparanase (Fig. 1B), in adjacent serial tissue sections, revealed an inverse correlation between heparanase overexpression and amyloid formation. The amyloid-resistant organs (liver and kidney) of the hpa-tg mice displayed abundant heparanase, as opposed to the very low expression levels in the amyloid-susceptible spleen of the same animals. Similar results were seen in all six animals of each group. Codeposition of AA amyloid and HS may also be demon- strated by staining tissue sections with SAB, a dye used to visualize sulfated glycosaminoglycans (9). Although liver from amyloid-induced ctr mice showed abundant SAB staining, pri- marily around central veins and extending into the liver sinusoids (Fig. 2C), sites coinciding with Congo red staining (Fig. 2A), the transgenic hpa-tg liver was almost devoid of SAB-positive ma- Fig. 2. Visualization of amyloid and glycosaminoglycan deposition in dif- terial (Fig. 2C). Spleen sections, on the other hand, stained ferent organs of hpa-tg vs. ctr mice. (A) Sections stained for amyloid with strongly with SAB in perifollicular patterns (Fig. 2C) matching Congo red, viewed under crossed-polars, show representative areas of liver the deposition of AA amyloid (Fig. 2A), and with no discernible (Top), kidney (Middle), and spleen (Bottom). Seven days after amyloid induc- tion, ctr mice had developed amyloidosis (green-stained deposits) in all three difference between ctr and hpa-tg tissue. The amyloid-associated tissues (Left), whereas the liver of hpa-tg mice showed barely detectable SAB-staining material of mouse spleen was previously identified amyloid, and the kidney of hpa-tg mice showed no detectable amyloid as HS by its sensitivity to nitrous acid treatment (14, 15) and to deposition (Top Right and Middle Right). In contrast, the hpa-tg spleen HS lyase digestion, but not to chondroitinase ABC digestion (Bottom Right) showed as abundant amyloid as the ctr tissue. The light-blue (16). We conclude that the fragmented HS resulting from objects in the liver sections are blood vessels. (Middle Right) The arrow heparanase overexpression failed to support amyloidogenesis, indicates the margin of the renal papilla (magnification ϫ115). (B) Image MEDICAL SCIENCES hence secondary associated accumulation of polysaccharide. analysis of amyloid deposition, after staining with Congo red. Ctr, control; Tg, heparanase transgenic. (C) Sections stained with SAB reveal codeposition of glycosaminoglycans in amyloid deposits in the liver (Upper) and spleen Molecular Size of HS Extracted from Different Organs. The predicted (Lower)ofhpa-tg vs. ctr mice. Both the liver (area around the central vein and effect of heparanase overexpression on HS molecular size was adjacent liver parenchyma) and spleen (perifollicular area) of ctr mice (Left) 35 verified by in vivo labeling of the polysaccharide with [ S]sulfate, are strongly stained. Essentially no staining is detected in the hpa-tg liver followed by isolation of HS from the various organs and gel (Upper Right; arrow indicates small focus of AA amyloid) as opposed to an chromatography (see Materials and Methods). Notably, the total intense staining of the hpa-tg spleen (Lower Right), similar to that of the ctr amounts of 35S-labeled HS, relative to the wet weight of each spleen (magnification ϫ115).

Li et al. PNAS ͉ May 3, 2005 ͉ vol. 102 ͉ no. 18 ͉ 6475 Downloaded by guest on September 28, 2021 Fig. 4. Proposed models of HS function in normal and heparanase- overexpressing tissues. (A) Polyvalent interaction. Efficient polymerization Fig. 3. Molecular size of HS purified from hpa-tg and ctr organs. HS samples, and deposition of amyloid peptides is promoted by intact HS (Left), but not by labeled in vivo with 35S were isolated from the indicated organs as described fragments of HS generated upon cleavage of HS by heparanase (Right). (B) in Materials and Methods. The labeled polysaccharides were susceptible to Monomeric͞oligomeric interactions with proteins. HS side chains of a cell- digestion with heparin lyases (data not shown). Analysis by gel chromatog- surface HS proteoglycan bind a ligand (e.g., growth factor, morphogen) and raphy on a Superose-12 column showed a pronounced reduction in the size of its receptor. Complex formation (here shown to involve a monomeric receptor HS extracted from hpa-tg (open circles) vs. ctr (filled circles) liver and kidney. only) is promoted by the intact HS chain (Left) as well as by HS fragments A significant decrease in the size of HS was noted in the heart and lung, as released by heparanase (Right). compared with a marginal decrease in the spleen and brain of the hpa-tg vs. ctr mice. Arrows mark the peak elution position of a standard 12-kDa heparin. observations point toward a therapeutic potential for oligosac- organ, did not differ significantly between hpa-tg and ctr mice charides with the appropriate structure. Indeed, low-molecular (data not shown). Gel chromatography, however, revealed a weight heparin, with a molecular size (Ϸ5 kDa) similar to the HS pronounced reduction in the average chain length of [35S]HS fragments extracted from heparanase-overexpressing organs, isolated from organs (i.e., liver and kidney) in which heparanase was found to prevent AA and A␤-peptide fibril formation in was markedly overexpressed (Fig. 3). Thus, liver HS was reduced vitro, and to arrest the progression of AA amyloidosis in mice in size from an average Ϸ35 kDa (ctr)toϷ3 kDa (hpa-tg), and (19). Further, agents (such as small-molecule sulfonates and renal HS was degraded to products largely Ͻ10 kDa. In contrast, sulfates) that inhibit the binding of HS to amyloid precursor HS in spleen and brain, both organs with minor expression of protein͞peptide are effective anti-amyloid compounds both in transgenic heparanase (Fig. 1), was only marginally affected, as vivo and in vitro (3, 20). We also demonstrated that degradation indicated by the essentially overlapping elution patterns of HS of HS chains, through exposure to bacterial heparinase III, derived from hpa-tg and ctr mice (Fig. 3). markedly inhibits conversion of cellular prion protein (PrPc) into an abnormal conformer (PrPSc) in cultured neuroblastoma cells Discussion (21). A similar effect was obtained in response to inhibition of Application of hpa-tg mice in the present study provided HS chain elongation and͞or sulfation. Moreover, partial resto- straightforward evidence for an essential role of HS in AA ration of PrPSc deposition was induced by exogenously added HS amyloidosis. The variable overexpression of heparanase in dif- (21), further emphasizing the contributory involvement of HS in ferent organs provided a reliable internal control. Unlike most amyloid disorders. Precise characterization of HS͞protein inter- organs, the spleen essentially escaped excessive heparanase actions at the molecular level may provide clues to the genera- action, retained almost full-size HS chains, and remained sus- tion of drugs against amyloid diseases. ceptible to amyloid development. The lack of a significant Our findings have bearing on the structure-function relation- amyloid deposition in the liver and kidney, both expressing high ships of HS in development and homeostasis. Targeted disrup- levels of heparanase, can therefore be confidently ascribed to the tion of genes encoding various involved in HS biosyn- marked decrease in the molecular size of HS observed in these thesis has demonstrated that HS is essential for normal tissues. Interestingly, the spleen of control mice is the first organ embryonic development (12, 22–24). The multiple regulatory affected by AA amyloid, followed by the liver and then the kidney. Also, a higher amount of AA amyloid is deposited in functions of HS are generally ascribed to interactions with a spleen vs. the liver (17). Formation of amyloid fibrils͞plaques variety of bioactive proteins, including growth factors and their seems to proceed through intermediate stages of polymerization receptors, morphogens, cytokines, enzymes, and extracellular- that are thought to depend on interactions with HS structures matrix proteins (25, 26). Molecular studies suggest that the (1). We propose that this process critically depends on the majority of such interactions can be accommodated within a macromolecular state of the HS constituent, and that a minimal 10-mer saccharide sequence (25, 26). A putative 10-mer ‘‘func- Ϸ chain length is a prerequisite for efficient fibril polymerization tional unit’’ of an HS chain would have a Mr of 3,000, thus and deposition in vivo (Fig. 4A). Free HS oligosaccharides approaching the size of HS fragments extracted from several generated by heparanase can presumably bind amyloid mono- organs of the hpa-tg mice (Fig. 3). The relatively modest mers, which are then unable to polymerize and assemble into phenotypic alterations observed in these mice (6) could there- larger aggregates. Conversely, infusion in rat brain of perlecan- fore conceivably reflect the functional competence of such carrying full-size HS chains was shown to promote deposition of fragments (Fig. 4B), in accord with crystallography data (27, 28). A␤ amyloid, the hallmark of Alzheimer’s disease (18). These The same fragments, however, are too short to promote assembly

6476 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502287102 Li et al. Downloaded by guest on September 28, 2021 of amyloid aggregates that are critical to the pathogenesis of amyloidosis. Current studies on mice that overexpress a secreted amyloid diseases. form of heparanase in the brain may provide information to Finally, it is recalled that HS has been implicated in other verify this prediction. major diseases involving amyloid formation, such as Alzheimer’s disease, type 2 diabetes, and Parkinson’s disease. We predict that We thank Mr. Lee Boudreau for able technical assistance. This work was these conditions may be alleviated by means of appropriately supported by Swedish Research Council Grant 32X-15023, Swedish depolymerized heparin͞HS-related compounds. Indeed, treat- Cancer Society Grant 4708-B02-01XAA, European Commission Grant ment of APP-transgenic mice (a model of Alzheimer’s disease) QLK3-CT-2002-02049, Swedish Foundation for Strategic Research with low-molecular-weight heparin (Mr Ϸ 5,000) attenuated Grant A303:156e, Polysackaridforskning AB (Uppsala), the Center for plaque formation and improved neuropathology of the animals the Study of Emerging Diseases (CSED), Israel Science Foundation (19, 29). It is important to establish whether A␤ aggregation in Grant 532͞02, Canadian Institutes of Health Research Grant MOP- Alzheimer’s disease, as well as amyloid generation in other 3153, and a Detweiler Traveling Fellowship from the Royal College of conditions, depends on HS chain length as shown here for AA Physicians and Surgeons of Canada.

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