tom: anti-chondroitin sulfate (CS). Identical positions (tubules) are ..... Ramadori R, Sipe JD, Colten HR (1985) Expression and regulation of the murine serum ...
Virchows Archiv B Cell Pathol (1991) 60:321-328
HrchowsArchivB CellPathology IncludingMolecular
9 Springer-Verlag 1991
Characterization of proteoglycans and glycosaminoglycans in bovine renal AA-type amyloidosis Th.A. Niewold, J.M. Flores Landeira I, L.P.W.J. van den Heuvel 2, A. Ultee, P.C.J. Tooten, and J.H. Veerkamp 2 Department of Pathology, Veterinary Faculty, University of Utrecht, The Netherlands i Department of Histology and Pathological Anatomy, Veterinary Faculty, Universidad Complutense, Madrid, Spain 2 Department of Biochemistry, University of Nijmegen, The Netherlands Received October 16, 1990 / Accepted April 9, 1991
Summary. Highly sulfated glycosaminoglycans (GAG) or proteoglycans (PG), especially heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG), are considered to be intimately associated with amyloid deposits in different types of amyloidosis. Based on this relationship an important role for HS has been suggested in amyloidogenesis. The present immunohistological and ultrastructural study shows that in bovine renal AAamyloidosis, sulfated GAG/PG was not restricted to amyloid deposits proper and that areas without GAP/ PG were also present within the amyloid. Both glomerular and papillary amyloid contained HS (PG), and the latter also contained chondroitin sulfate (CS) and dermatan sulfate (DS), suggesting a correlation between the location of the amyloid and the type of GAG/PG deposited. Amyloid P component (AP) had a distribution similar to that of HSPG, confirming their affinity-based relationship. The GAG types found ultrastructurally in amyloid fibril preparations of glomerular and papillary amyloid isolated from the same kidney, reflected the immunohistological findings. HS was shown to be the predominant GAG in all papillary amyloid fibril extracts. Taking into account the chemico-physical properties of HS, it cannot be excluded that this predominance is introduced by the purification procedure. These results suggest that the association of GAG/PG and amyloid is not necessarily mutually obligatory and that the proposed importance of GAG in amyloidogenesis is disputable. Key words: Bovine AA-amyloidosis - Heparan sulfate - Chondroitin sulfate - Dermatan sulfate proteoglycan - Gel filtration - Electron microscopy
Introduction Apart from qualitative analysis by histochemical methods, the presence and increased amount of GAG in amyOffprint requests to: Th. A. Niewold, Yalelaan l, Postbox 80158, NL-3508 TD, Utrecht, The Netherlands
loidotic organs was established by isolation and subsequent characterization. Recent studies (Snow and Kisilevsky 1985; Snow et al. 1987a, 1987b) report heparin/heparan sulfate (H/HS) to be responsible for the increased GAG content of amyloid-containing liver, spleen and kidney, in both human patients and experimentally induced murine amyloidosis. Linker and Carney (1987) produced similar results in human organs, whereas in induced murine amyloidosis they found chondroitin sulfate (CS) to be the major GAG. Furthermore, the presence of highly sulfated GAG or PG has been demonstrated in amyloid deposits and on the amyloid fibril proper (Snow et al. 1987c, 1989). Based on the codeposition of GAG with the AA-protein, the coaccumulation of H/HS and the close ultrastructural relationship between PG or GAG and amyloid fibrils, an important role was suggested for GAG, especially H/HS, in amyloid fibril formation (Kisilevsky et al. 1986; Snow et al. 1987c). In addition, purified murine SAA2 (the amyloidogenic SAA isotype) was shown to have an increased fl-sheet structure upon binding to HS but not to CS (McCubbin et al. 1988). Although the presence of SAA mRNA in extrahepatic sites might implicate local production of AA-amyloid (Ramadori et al. 1985), circulating SAA appeared to be the major source of tissue AA deposition (Tape et al. 1988). In contrast, the origin of GAG in amyloidosis is not clear. Since in hepatic cirrhosis only a small increase in GAG content is found, the GAG response in amyloidosis is not likely to be a general reaction to an alterative stimulus (Linker and Carney 1987). In relation to this, one might ask if the GAG response in amyloidosis is of a localized or systemic nature. If systemic, GAG could originate from the circulation (Snow et al. 1987a). The presence of GAG in amyloid could then be explained by either entrapment of GAG based on chemico-physical affinity, as with amyloid P-component (AP) (Pepys 1986). The binding of AP to amyloid deposits is probably based on the presence of HS therein (Hamazaki 1987). AP was reported to occur abundantly outside amyloid deposits (Shirahama et al. 1985), possibly reflecting the distribution of HS (Hamazaki 1987). In
this respect, it is i m p o r t a n t to establish whether the G A G , and in particular HS deposition, is really part o f the p r i m a r y process o f amyloidogenesis, or whether it is coincidal or a secondary reaction to the presence o f a m y l o i d deposits. Initially G A G s were not f o u n d in isolated amyloid fibrils (Pras et al. 1971, 1972), but later studies showed them to be present (Gorevic et al. 1980; Linker and C a r n e y 1987; Snow and Kisilevsky 1985; Snow et al. 1987a, 1987b). A possible explanation for this discrepancy could be c o n t a m i n a t i o n o f amyloid fibril preparations by n o n - a m y l o i d tissue c o m p o n e n t s ( U n s w o r t h et al. 1982). Furthermore, the biochemical characterization is h a m p e r e d by differences in extractability o f G A G , depending on the tissue affected, the type o f G A G , the age o f the process (Linker and C a r n e y 1987) and the extraction procedure (Van den Heuvel et al. 1988). To examine the relationship o f G A G to amyloid deposition and to the amyloid fibril, we p e r f o r m e d a series o f investigations in the kidney, in view o f the different locations o f a m y l o i d deposits. Histological study was c o m b i n e d with biochemical analysis. Tissue sections o f seven bovine kidneys were examined histologically by staining with Alcian Blue/MgClz for sulfated G A G ; by this technique a consistently weak staining o f the a m y loid, as o p p o s e d to other tissue c o m p o n e n t s , was obtained. Therefore, i m m u n o - h i s t o c h e m i c a l d e m o n s t r a t i o n o f H S P G and CS was performed because o f its greater sensitivity. Using the same technique, the distributions o f amyloid protein A (AA) and amyloid P c o m p o n e n t (AP) were investigated. Samples were stained with the Alcian Blue a n a l o g Cuprolinic Blue (CB) for ultrastructural d e m o n s t r a t i o n o f G A G / P G . F r o m the same kidney, glomerular and papillary a m y l o i d were separately isolated and stained with CB. Furthermore, amyloid fibrils were isolated, assayed for sulfated G A G and the protein c o m p o s i t i o n analyzed by gel filtration.
fate) and connective tissue (chondroitinsulfate) were used. After the incubations the sections were stained with the Alcian blueMgCI2 technique according to Scott and Dorling (1965). The following techniques were applied to kidneys from Cows 1 and 7 only.
bnmunohistochemistry. Immuno-staining was performed on 5 p.m paraffin sections with the immunogold silver staining (IGSS) according to the instructions of the manufacturer (Janssen Life Sciences Products, Olen, Belgium). Primary antibodies used were: rabbit anti-bovine protein AA antibodies, affinity-purified as described (Niewold et al. 1987), rabbit anti-bovine amyloid P-component (AP) IgG (a generous gift of Dr. M.B. Pepys, London) and rabbit anti-HSPG antibodies directed against core protein of human renal basement membrane (Van den Heuvel et al. 1989). Murine monoclonal IgM anti-chondroitinsulfate antibodies specific for GAG-part of chondroitinsulfate A and C (CS-56 BioYeda, Rehovot, Israel) were used for the demonstration of CS, in this case by the indirect peroxidase method.
Electron microscopy. Samples of cortex and medulla (Cows 1 and 7) were fixed overnight at room temperature in 0.025 M sodium acetate buffer (pH 5.6) containing 2.5% glutaraldehyde, 0.2% Cuprolinic Blue (BDH Ltd, Poole, UK) and 0.1-0.4 M MgCI2. The samples were then washed three times in the same solution without Cuprolinic Blue and subsequently washed three times in 1% NazWO4. Samples were dehydrated in ascending concentrations of ethanol, the 30% and 50% ethanol containing 1% Na2WQ (Van Kuppevelt et al. 1984a). Nitrous acid oxidation was performed according to Van Kuppevelt et al. (1984b). Samples were routinely embedded in Durcupan and ultrathin sections collected on copper or nickel grids.
Isolation of amyloidfibrils. Renal cortical slices (Cow 1) were sieved
Materials and methods
to yield glomeruli as described by Hol et al. (1984). Papillae from the same kidney were collected. From both preparations amyloid fibrils were isolated according to Pras et al. (1968). Briefly, tissue was homogenized in 0.15 M NaCI on ice for 5 min. The homogenate was centrifuged at 50000 g for 30 min. The procedure was repeated with the pellet until the supernatant showed an OD28 o < 0.1. Subsequently, the pellet was repeatedly homogenized and centrifuged using distilled water. Amyloid fibrils were present from the third papillary and the second glomerular distilled water extract on. Fibrils were stained with 1% phosphotungstic acid for negativecontrast electron microscopy. Staining with Cuprolinic Blue (containing 0.2 M MgCI2) of isolated fibrils and subsequent embedding in Durcupan was performed in the same manner as the tissue samples (see above).
Source of tissue. Bovine kidneys were obtained from a local slaugh-
Superose 12 gel filtration. Lyophilized samples were dissolved in
terhouse; six amyloidotic kidneys containing both glomerular and papillary amyloid (Cows 1-6) and one normal kidney (Cow 7) were selected. Samples were fixed in buffered formalin and embedded in paraffin wax. Part of the material was snap frozen in liquid nitrogen. The kidney of Cow 1 was used for isolation of amyloid fibrils.
6 M guanidine, 0.5 M Tris-HCl (pH 8.5), containing 1 mM dithiothreitol and 0.1 mM EDTA (GuHC1-TED buffer). After 24 h the solution was centrifuged for 15 min at 50000 g and applied to a Superose 12 column eluted with the same buffer at a rate of 0.3 ml/min. Reference proteins (MW) were: bovine serum albumin (67000), ovalbumin (43 000), chymotrypsinogen A (25000) and ribonuclease A (13700) (Pharmacia, Uppsala, Sweden) to which were added aprotinin (6500) (Sigma Chemical Company, St. Louis MO, USA) and bovine insulin (3000) (BDH Biochemicals Ltd, Poole, England). As an additional standard purified bovine SAA was used, isolated from acute phase serum by cholesteryl hemisuccinate affinity chromatography and subsequent gel filtration, as described by Niewold and Tooten (1990). The composition of the samples was estimated from the peak areas at 280 nm.
Histochem&try. Paraffin-embedded and cryostat sections of 5 ~tm thickness were used. They were routinely stained with 1% Congo red according to Romhanyi (1971). Incubation with chondroitinase ABC from Proteus vulgaris (Sigma Chemical Company, St. Louis, USA) was performed using 0.5 units of enzyme in 0.1 M Trisacetate buffer (pH 8.0) per section, incubated at 37~ for 2 h. A control section was incubated with buffer only. Nitrous acid oxidation was performed using 20% of nitrous acid: 33% acetic acid (1:1, v/v) applied directly to the sections and incubated at room temperature for 6 h. A control section was incubated with distilled water. Sections of tissues used as positive controls were as follows: pig tracheal cartilage for chondroitinase ABC and normal bovine kidney for nitrous acid treatment. As internal controls mast cells (heparin), glomerular basement membrane (heparan-sul-
Determination ofsulphated GAG. GAG-content was assayed using a spectrophotometric procedure with dimethylmethylene blue (Reubsaet et al. 1985). Proteoglycans were identified by degradation with heparitinase, chondroitinase ABC, chondroitinase AC and by cellulose acetate electrophoresis (Van den Heuvel et al. 1988).
Immunoelectron microscopy. Grids with ultrathin sections were washed three times in PBG (PBS pH 7.4 containing 0.2% gelatin and 0.5% bovine serum albumin) and subsequently incubated for 1 h with affinity-purified rabbit anti-bovine protein AA antibodies, dilution 1:1000 in PBG. The grids were then washed six times in PBG, followed by incubation for 1 h with gold-labeled goat anti-rabbit antibodies (Auroprobe EM GAR G15, Janssen Life Sciences Products, Olen, Belgium) diluted 1 : 50 in PBG. Thereafter, the grids were washed six times in PBG, seven times in PBS and incubated for 10 min in distilled water. Sections were viewed either uncontrasted, contrasted with uranyl acetate only, or contrasted with uranyl acetate and lead citrate. All incubations were performed at room temperature (modified after Boonstra et al. 1985).
Results
Histochemistry In the six amyloidotic kidneys, Alcian blue (AB) staining was increased at the lowest salt concentration compared with the normal kidney. Part of this increase was located in the areas of interstitial fibrosis, which did not stain with either Congo red or anti-AA antibodies. Amyloid deposits stained blue, but less intensely than other tissue components. The glomerular amyloid staining was evidently weaker than the papillary deposits. At a concentration o f 0.55 M MgC12 the blue staining of the amyloid was greatly diminished, whereas it was not in the fibrotic areas. At 0.75 M the latter showed no AB staining as did part of the amyloid deposits. Mast cells, containing heparin and serving as an internal control, were stained with Alcian blue at all salt concentrations. Treatment with nitrous acid resulted in a slight loss of AB staining intensity in parts o f the papillary amyloid, whereas the staining of mast cells was completely abolished. After chondroitinase ABC digestion most of the AB staining of vascular walls and the fibrotic areas was lost, whereas staining of the glomerular amyloid and parts of the papillary amyloid was not affected. No essential difference concerning sulfated G A G was observed between the cryostat and paraffin sections (results not shown).
Immunohistochemistry All areas showing Congo red birefringence stained with anti-bovine protein AA antibodies (Fig. 1). The papillary amyloid deposits stained very inhomogeneously with the anti-HSPG antibodies, whereas the glomerular amyloid showed a more homogeneous but weaker staining. Arteriolar amyloid was not stained with anti-HSPG. In the papillae some parts of the tubular BM, some fibroblasts and parts of the amyloid showed intense staining with anti-HSPG (Fig. 1). In the lumen of the blood vessels a weak reaction was visible. Tubular epithelial cells showed strong staining, especially at the basal side. In the amyloidotic glomeruli the G B M stained weakly with anti-HSPG compared with the normal GBM. Pre-treatment with nitrous acid resulted in an enhanced staining of the tissue sections with anti-HSPG (not shown). This phenomenon can be explained by the
Table 1. Immunohistochemical analysis of amyloidotic kidney Anti-AA Anti-AP Anti-HSPG b Anti-CS Glomerular Amyloid masses Papillary Amyloid masses Arteriolar amyloid Glomerular/tubular Basement membrane GBM/TBM Mesangial cells Endothelium Epithelium Tubular lumen Fibroblasts Fibrotic areas Blood plasma
+
+
+
-
+ +
+a trace
_~ a
_}_ a
-
-
_ -
+c + + + + + trace +
+~ + + + + + trace +
_ + + + +
a Inhomogeneous staining pattern b Enhancement of staining on treatment with nitrous acid; no effect on other staining including Congo red c GBM adjacent to amyloid deposits showed weak or no staining
removal of HS chains from the core protein, resulting in unmasking of the antigen. Using the anti-AP antibodies a similar pattern was obtained to that of anti-HSPG, i.e. a diffuse weak staining of the glomerular amyloid and an inhomogeneous staining of papillary amyloid, as well as staining of tubular epithelium and some fibroblasts. Since AP has affinity for HS (Hamazaki 1987), co-occurrence of both compounds was to be expected. Furthermore, the contents of some of the cortical and papillary tubules and blood vessels stained heavily with anti-AP, as did the BM of glomerulus and tubular epithelium. The evident staining with anti-AP of non-amyloid areas in amyloidotic kidneys described here is similar to that reported by Shirahama et al. (1985). The anti-CS antibodies stained the fibrotic areas and part of the vascular walls, but not the glomerular amyloid deposits, indicating the absence of CS in the latter as in arteriolar amyloid. In the papillae the staining of the amyloid showed a heterogeneous pattern (Fig. 1). Comparison with adjacent Congo red-stained sections, showed that the anti-CS antibodies stained some parts of amyloid deposits markedly and other parts not at all. Blood plasma showed a very strong reaction. The immunohistochemical results are summarized in Table 1.
Electron microscopy In the normal glomerular basement membrane (GBM) (Cow 7) Cuprolinic Blue stained filamentous particles with maximum dimensions of 70 x 12 nm lining both aspects (Fig. 2A), thus resembling the basement membrane filaments (bmf I), sensitive to nitrous acid oxidation, considered to be H S P G (Van Kuppevelt et al. 1984b, 1985). In amyloidotic glomeruli (Cow 1), hardly any P G was seen in the G B M (Fig. 2B), as was also
Fig. 1. Immunohistochemical staining of adjacent tissue sections of bovine amyloidotic renal papilla (Bar = 40 I-tm). Top: antiprotein AA (AA). Center: anti-heparan sulfate proteoglycan (HSPG). Bottom: anti-chondroitin sulfate (CS). Identical positions (tubules) are marked A, B and C respectively. Note the difference in GAG/PG content between the two amyloid areas, one located between B and C containing both HSPG and CS, whereas the amyloid deposited between A and B contained CS only the case for amyloid deposits visualized with gold-labeled anti-AA antibodies (Fig. 2C). The apparent loss of HSPG from the amyloidotic GBM seen by light microscopy was affirmed by the paucity of CB-staining particles observed by electron microscopy, as also described in other types of glomerular nephrosis (Mijn-
derse et al. 1983; Vernier et al. 1983). Amyloidotic papillae were shown to have thickened tubular basement membranes (TBM) containing large amounts of HSPG, whether or not amyloid fibrils were present (Figs. 2D, E). In interstitial amyloid deposits some showed PG (Fig. 2 F, G), but others did not (Fig. 2 H), in good agree-
Fig. 2. Ultrastructural demonstration of proteoglycans in bovine kidney using Cuprolinic Blue (0.2 M MgC12). Demonstration of amyloid fibrils with anti-protein AA immunogold labeling (B-H, gold particles are 15 rim). Glomerulus. A. Normal glomerular basement membrane (GBM) showing the presence of HSPG filaments (max. dimensions: 70x 12 rim, arrows) lining both the podocyte (P) and the endothelial (E) side. B Glomerulus of amyloidotic cow, showing amyloid deposits at both sides of the GBM. Note the virtual absence of HSPG (arrow). C Glomerular amyloid deposit
showing the absence of PG (counterstain uranyl acetate). Renal papilla showed the presence of thickened tubular basement membrane (TBM), either with amyloid fibrils D or without E, in both cases showing abundant HSPG. Note the presence of CSPG (arrow) in the fibrotic interstitium. F, G and H Interstitial amyloid deposits containing either PG (arrow, F and G) or not H. G is also stained with uranyl acetate, showing immunodecorated amyloid fibrils and a collagen fibril (C)
Fig. 3A-E. Proteoglycan content of different amyloid fibril preparations isolated from the same bovine kidney, Cuprolinic Blue (0.2 M MgC12). In the third extract of papillary origin (P3) (A and B) two types of PG are seen: small ones (70 x 12 nm) representment with the imrnunohistochemical results. Cuprolinic Blue staining of the water extracts revealed the third papillary extract to contain two different filamentous types: small ones (maximum dimensions: 70 x 12 nm), resembling bmf I (HSPG) and heavily staining large ones (maximum dimensions: 100 • 30 nm), resembling the heavy-staining filaments (hsf), attributed to CSPG (Van Kuppevelt et al. 1985) (Fig. 3A and B, Table 2). The seventh extract contained PG only of the small filamentous type, as did the glomerular amyloid (Fig. D and E, Table 2). The PG-type found here correspond with those found immunohistologically in both locations.
Gel filtration
ing HSPG and larger ones (100 • 30 nm) representing CSPG. C control, showing P3 without CB. The seventh papillary extract (P7, D) and the second glomerular extract (G2, E) contain almost exclusively HSPG. (A: bar = 0.2 lam, B--E: bar= 0.2 lain) Table 2. Characterization of AA-amyloid fibrils from different water extracts of bovine renal papillae and of glomeruli Source/ extract no. Papilla 3
mg Ratio lag GAG/mg %HS EM PG c dry retarded dry weight /GAG b size wt /HMW a 300
0.60
95
73
Papilla 7 70 Papilla I l 30 Glomerulus2 8
1.38 1.72 0.41
37 24 nd
100 94 nd
70 x 12 nm 100 x 30 nm 70 x 12 nm nd 70• 12nm
The composition of the samples was estimated from the relative peak area at 280 nm on 6 M guanidine gel filtration b Heparitinase sensitive fraction c Ultrastructural characteristics established by Cuprolinic Blue staining nd: not determined
a
On gel filtration, the amyloid fibril preparations were resolved into three major fractions, as described before (Van Andel et al. 1986b). The relative contribution of high molecular weight (HMW) material was shown to decrease over the subsequent papillary extracts, a phenomenon similar to that described earlier (Van Andel et al. 1986b). The glomerular extract contained the largest amount of H M W material (Table 2).
chondroitinase ABC/chondroitinase AC and cellulose acetate electrophoresis. In the subsequent extracts a decrease in G A G - c o n t e n t was found that paralleled the decrease in the relative a m o u n t o f H M W material, established by gel filtration.
Biochemical determination of GAG
Discussion
The third papillary extract contained the highest proportion of G A G , which decreased in the next extracts tested. Only the third extract contained a considerable proportion of non-HS G A G (Table 2), that proved to consist of 40% chondroitin sulfate AC and 60% dermatan sulfate, as established by both digestion with heparitinase/
Based on their findings, Snow and colleagues (Snow and Kisilevsky 1985; Snow et al. 1987a-c, 1989) claimed heparin/heparan sulphate to be a constituent of different types of amyloid. If a specific type of G A G or PG plays an essential role in amyloidosis, one should expect the co-occurrence, co-accumulation and co-isolation of
GAG/PG and amyloid to be a constant feature. In the present study, by immunohistochemical techniques, no constant relationship between a specific GAG and bovine renal amyloid deposits could be demonstrated, whereas in all isolated amyloid fibril preparations HS was the major GAG-type. However, the predominance of HS in purified amyloid fibril preparations, also described by Snow et al. (1987c), could be caused by preferential extraction of other GAGs during the salt washes of the purification procedure, since human glomerular and tubular CSPG was demonstrated to be better extracted in water and salt solutions than HSPG (Van den Heuvel et al. 1988). As a consequence, the HS found in the water-extracted amyloid fibril preparations may represent the small water soluble fraction of the total HS concentrated in the amyloid enriched tissue residue. It also gives an explanation for the fact that CS/DS is absent from the later papillary amyloid extracts. This would implicate CS/DS to be present in purified amyloid preparations merely as a contaminant, a possibility that could also not be excluded for HS. Furthermore, the decrease in HS content in the subsequent amyloid fibril extracts points towards an extrinsic role for HS. In induced amyloidosis in mice, Snow et al. (1987a) found elevated serum levels of CS during amyloidogenic stimulation, although they found HS to be the predominant GAG in amyloidotic organs. In contrast, in murine induced amyloidosis Linker and Carney (1987) found CS to be the major GAG present. Although this finding may be suggestive of a systemic origin for G A G in amyloid deposits, they also found in vitro GAG synthesis in amyloidotic organ slices. Moreover, the major type of GAG formed appeared to be organ-dependent. Our finding that papillary amyloid deposits did contain CS/ DS, whereas the glomerular amyloid deposits did not, then suggests that at least the deposition of CS/DS in amyloid is dependent on the location of the amyloid. In primary familial amyloidosis of the heart, the major GAG appeared to be hyaluronic acid, not HS or CS (Dalferes et al. 1967). Interestingly, in the present study, arteriolar amyloid deposits seemed to be devoid of both CS and HS. Furthermore, in human hepatic AL-amyloid the HSPG found showed characteristics of hepatocellular heparan sulfate (to be published). Presuming GAGs do play an important role in amyloido- or fibrillogenesis, it seems that the presence of GAG, rather than a single type of GAG, is decisive. In other words, a common characteristic like the anionic charge may be relevant. However, in bovine material, low density lipoprotein showed binding to GAG based on the latter mechanism, whereas high density lipoprotein (the carrier of SAA) had no affinity for either HS or CS (Brantmeier et al. 1988; Kawahara et al. 1989). Thus, GAG-deposition may be an epiphenomenon or a post-amyloidogenic event. Although HS was demonstrated to increase the /~sheet structure of murine amyloidogenic SAA in vitro (McCubbin et al. 1988), the existence of amyloid fibrils virtually devoid of the high molecular weight fraction in which GAGs reside, was shown (Hol et al. 1984; Van Andel et al. 1986a). Furthermore, it appeared to be pos-
sible to reconstitute amyloid fibrils using the lower molecular weight fractions only (Pras etal. 1972; Prelli et al. 1987) or fragments of SAA (Baba et al. 1988), whereas the high molecular weight fraction could play a role in final morphogenesis of the amyloid fibril (Hol et al. 1984; Van Andel et al. 1986a). These findings are suggestive of a non-intrinsic role for HS (and AP), their presence in amyloid deposits being post-fibrillogenic. The fact that fibroblasts in culture stimulated by amyloid fibrils produce GAG (Palmoski and Brandt 1975), provides a possible mechanism for a specific localized secondary GAG response, in which process the type of cell involved may determine the type of GAG produced. The strong staining with anti-HSPG of tubular epithelial cells observed in the bovine amyloidotic kidney may fit into this hypothesis. However, in human renal AA-amyloidosis, endothelial cells were suggested as a possible source of HSPG in amyloid deposits (Norling et al. 1988). The question of the cellular origin of the amyloidassociated PG may be resolved by in situ hybridization using cDNA probes for PG core proteins. The latter were recently produced in our laboratory and are being characterized for further research in this matter.
Acknowledgement. The authors wish to thank Prof. Dr. E. Gruys for his useful suggestions and discussion.
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