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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 9, 2004 as doi:10.1096/fj.04-2022fje. |
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,1
* Molecular Neurobiology, Institute for Cellular and Molecular Biology, Porto, Portugal;
Neuropathology, Hospital Geral de Santo António, Porto; and
ICBAS, University of Porto, Portugal
1Correspondence: Rua Campo Alegre 823, Porto, Portugal 4150-180. E-mail: mjsaraiv{at}gaia.ibmc.up.pt
SPECIFIC AIM
Familial amyloid polyneuropathy (FAP) is characterized by the extracellular deposition of transthyretin (TTR) aggregates and amyloid fibrils in tissues, particularly in the peripheral nervous system (PNS), and is accompanied by changes in connective tissue. Changes in proteoglycan type and distribution could account at least in part for the altered physical properties observed during disease progression in tissues with TTR deposition, but information is lacking concerning the molecular events that underlie tissue remodeling in FAP. Identification of genes and proteins differentially expressed during pathological changes throughout the course of FAP is needed. FAP salivary glands (SG) were used to identify differentially expressed genes related to tissue remodeling during the course of FAP.
PRINCIPAL FINDINGS
1. Microarray analysis of FAP salivary glands identified increased expression of genes related to extracellular matrix remodeling
To test whether SG from FAP patients follow signaling pathways similar to those found in the nerve—namely, activation of NF-
B—we assessed nuclear translocation of this transcription factor. In all FAP SG biopsies, nuclear translocation of p65 occurred but was absent in all normal SG biopsies. Microarrays of SG from normal individuals and FAP patients were subsequently performed; selection criteria identified two genes differentially expressed coding the extracellular matrix (ECM) -related proteins biglycan and neutrophil gelatinase-associated lipocalin (NGAL), which were both up-regulated in FAP (2.7±0.9 and 6.7±3.4 times, respectively).
2. Up-regulation of the ECM-related proteins biglycan, NGAL, and MMP-9 occurs in tissues with TTR deposition
Microarray results were confirmed by RT-PCR and immunohistochemistry (IHC) for NGAL and biglycan on SG biopsies from FAP patients and normal individuals. Since NGAL exists as a complex with matrix metalloproteinase-9 (MMP-9) modulating its activity by protecting MMP-9 from degradation, we carried out IHC for MMP-9 in the same SG biopsies and observed up-regulation of NGAL expression in FAP tissues and colocalization with MMP-9; in normal SG, MMP-9 staining was absent. Increase of MMP-9 in SG from FAP patients was further demonstrated by gelatin zymography.
To establish whether the events observed in SG biopsies are relevant in other tissues that present TTR deposition—namely, the PNS—we performed immunohistochemical analysis for biglycan in FAP nerve biopsies at different stages of disease progression. In normal nerves, biglycan immunostaining was either absent or low. Prior to amyloid deposition (FAP 0), biglycan staining was markedly increased around blood vessels (Fig. 1
, left upper panel, arrow) whereas in the first stages of fibril deposition (FAP 1) it was also present in the endoneurium (Fig. 1
, left middle panel, E). At later stages (FAP 2 and FAP 3) biglycan immunostaining was observed in sites of amyloid deposition. In the case of NGAL, staining was absent in both normal and FAP 0 nerves (Fig. 1
, upper center panel); in FAP 1 and 2 nerves, staining was sometimes observed in axons (Fig. 1
, center middle panel, arrows). By TTR IHC on adjacent sections, NGAL staining was found in sites of TTR deposition (Fig. 1
, lower center panel, arrowheads). FAP nerves seemed to present increased NGAL staining observed only after the presence of fibrilar amyloid deposits in the nerve. MMP-9 immunostaining in FAP nerves accompanied the pattern observed for NGAL (Fig. 1)
.
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3. MMP-9 is able to degrade TTR aggregates and fibrils in vitro
It has been reported that MMP-9 is able to degrade ß amyloid. To test the hypothesis that MMP-9 might be able to degrade TTR aggregates and/or fibrils, we incubated the active form of the enzyme with soluble TTR, TTR aggregates, and TTR fibrils. In vitro, MMP-9 degraded TTR aggregates and fibrils whereas the soluble counterpart of the protein remained uncleaved, as shown by SDS-PAGE analysis (Fig. 2
). We next investigated the influence of components that are associated to fibrilar deposits such as the serum amyloid P component (SAP); in the presence of SAP, the ability of MMP-9 to degrade TTR fibrils was clearly diminished. To rule out a possible nonspecific effect of SAP, similar experiments were carried out replacing SAP by BSA; in the presence of BSA, MMP-9 retained the ability to degrade TTR fibrils (Fig. 2)
. Furthermore, SAP does not compete with TTR in the assay as it is not a substrate of MMP-9 (Fig. 2)
. TEM of the preparations confirmed that MMP-9 is able to degrade TTR fibrils.
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4. Cytokine balance occurs in tissues with TTR deposition
We have previously reported that FAP nerves show increased expression of proinflammatory cytokines in a stage prior to FAP 0 and that this increase is sustained in later stages of disease progression (FAP 1-3). Given the importance of cytokine balance in ECM remodeling, we selected the anti-inflammatory cytokine IL-10 to test the hypothesis that pro- and anti-inflammatory events may exist during the course of FAP. By semiquantitative IHC for IL-10 in FAP nerves, no statistically significant increase was seen in a stage prior to amyloid deposition in FAP 0 relative to normal nerves. However, in later FAP stages IL-10 staining was 
2-fold increased relative to FAP 0 and normal nerves. Therefore, in FAP there is an increased expression of the anti-inflammatory cytokine IL-10 that occurs only after the onset of amyloid deposition, following the overexpression of proinflammatory cytokines.
CONCLUSIONS AND SIGNIFICANCE
The present study represents the first assessment of molecular events related to ECM remodeling that occurs upon TTR deposition in FAP. We show that SG biopsies are a sensitive means to investigate molecular events related to TTR deposition during the pathogenesis of FAP; the genes found differentially expressed in FAP SG biopsies vs. normal SG biopsies were altered in the nerve. Similar to what has been reported in the nerve, SG biopsies with TTR deposition present activation and nuclear translocation of NF-B, that could account for the overexpression of biglycan, NGAL and MMP-9 as analysis of their promoters revealed the presence of NF-B binding sites.
In nerves with TTR fibrilar deposition, biglycan colocalized with TTR fibrils; furthermore, in FAP 0 nerves, biglycan immunostaining was already increased. This proteoglycan had already been reported in other amyloid deposits such as renal AA amyloidosis and light chain deposition disease. The observation that biglycan may also be associated with TTR fibrils further emphasizes the importance of this proteoglycan in amyloid-related disorders.
Similar to biglycan, NGAL and MMP-9 were increased in FAP relative to control samples. However, in the case of these two proteins, this was only evident in stages where fibrilar TTR was already present; furthermore, expression of NGAL and MMP-9 seemed to overlap, which correlates well with the fact that NGAL forms a complex with MMP-9, avoiding its degradation. The ability of MMP-9 to degrade ECM components and regulate the activity of soluble proteins suggests it has an important role in various physiological and pathological processes, particularly in PNS degeneration. In the present study we show that MMP-9 is able to degrade TTR aggregates and fibrils obtained in vitro. However, in FAP 0 nerves, in the absence of overt fibril deposition, MMP-9 levels were maintained unaltered relative to controls, therefore suggesting that in vivo TTR aggregates are not removed possibly due to the presence of other unknown tissue factors. In later stages of the disease, when TTR fibrils are formed, increased levels of this enzyme should lead to their degradation. Our data suggest, however, that as in vivo TTR fibrils are bound to other components such as SAP, proteolysis by MMP-9 is prevented. Up-regulation and/or activation of metalloproteinases in sites of early aggregate deposition may serve as a future therapeutic target.
Remodeling of the ECM is affected by cytokines, and interference with inflammation-related signaling cascades may be a promising strategy to prevent aberrant matrix turnover. On the other hand, ECM components can play a role in inflammation cascades; proteoglycans have been shown to be important mediators of cytokine binding, modulating their biological activity by enabling cytokines to be immobilized to different environments. In FAP nerves we observed an early increased expression of the anti-inflammatory cytokine IL-10 that occurs concomitantly with the overexpression of proinflammatory cytokines. It is conceivable that in this disorder, the levels of IL-10 may be insufficiently increased to promote down-regulation of inflammatory cytokines during the latter stages of disease evolution.
The observations reported here raise the possibility that abnormalities in basement membrane metabolism may be an early event and potentially play an integral part in the process FAP amyloidogenesis, as has been suggested in other amyloid-related disorders. Disclosing the molecular events that lead to neurodegeneration in FAP may contribute to future therapeutical approaches in this disorder and may apply to other amyloid-related disorders.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2022fje;
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