Vascular involvement and cell damage in experimental AA and clinical ß2-microglobulin amyloidosis

Mar García-García1,2, Georges Mourad3, Mercè Durfort2, José García-Valero2 and Àngel Argilés1,

1 Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France, 2 Unit of Cell Biology, Department of Biochemistry and Physiology, Faculty of Biology, University of Barcelona, Barcelona, Spain and 3 Department of Nephrology, University Hospital Lapeyronie, Montpellier, France



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Amyloidosis is a highly prevalent disease characterized by the deposition of amyloid fibrils. Although several types of amyloidosis can be identified according to their protein constituents and suggest putative aetiological factors, the causes of amyloidosis remain unknown. Furthermore, the cellular participation and the ultrastructural particularities of amyloidosis have received little attention. The aim of our study was to evaluate the vascular participation in amyloidosis and the cellular consequences of this disease.

Methods. Two forms of amyloidosis were studied: experimental amyloid A (AA) and clinical ß2-microglobulin amyloidosis. We studied kidney, liver, and spleen in a mouse model, and examined surgically obtained carpal deposits from dialysis patients. We used light and electron microscopy with immunogold labelling for anti-ß2-microglobulin and anti-AA protein antibodies.

Results. AA amyloid fibril accumulation was associated with membrane lesions in basal, cytoplasmic organelle (endoplasmic reticulum, mitochondria), and nuclear membranes. Amyloid fibrils from ß2-microglobulin amyloidosis were also closely associated with elastic fibres and endothelial basement membrane. We observed proliferation of endothelial cells as well as basement membrane enlargement and disruption.

Conclusions. Vascular abnormalities, including endothelial enlargement, basement membrane modifications, and vascular proliferation were associated with amyloidosis. Amyloid fibrils have a high avidity for elastic fibres and are able to contact and damage the basement membrane, the cell and intracellular organelle membranes, as well as the nuclear envelope, suggesting a toxic effect of amyloid fibrils on cells.

Keywords: amyloid fibril toxicity; amyloidosis; endothelial cells; experimental amyloidosis; ß2-microglobulin



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Amyloidosis is a disease characterized by the tissue deposition of a fibrillar material positively stained with Congo red and which shows green birefringence under polarized light. It may involve many tissues and organs [1]. An experimental model of amyloidosis, established in mice, has been very helpful in determining the kinetics of appearance of amyloidosis and the participation of a variety of protein-derived compounds [2]. The form of amyloidosis most commonly observed in renal patients is called ß2-microglobulin or dialysis-related amyloidosis [3]. The mechanisms by which ß2-microglobulin precipitates into amyloid fibrils remain unclear.

Most studies examining the pathogenesis of dialysis-related amyloidosis have focused on ß2-microglobulin concentrations, biochemical modifications, or both [4]. However, serum ß2-microglobulin levels may not accurately predict the risk of appearance of dialysis-related amyloidosis [5], and none of the known modifications of ß2-microglobulin fully explains its ability to precipitate in amyloid fibrils [6]. Other investigations have focused on cell participation in the genesis of amyloid fibrils [79]. These studies found that the cells surrounding the amyloid deposits are mainly of monocyte-macrophage lineage [79]. In addition, recent evidence suggests that macrophages have mainly a degradative role in dialysis-related amyloidosis [reviewed in 10].

In the present study we analysed the participation of vessels and particularly vascular cells in this disease process. We analysed structural modifications of vessels and extracellular matrix with electron microscopy, and located amyloid proteins with coupling electron microscopy and immunogold labelling. We studied both experimental and human amyloidosis to identify common pathological features. Our findings demonstrate that there is a vascular involvement in both human and experimental amyloidosis, and that amyloid fibrils have an affinity for elastic fibres and cause membrane disruption in both forms of the disease.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Amyloid deposits
Experimental amyloidosis
Experimental amyloidosis (AA) was induced as previously described [2]. The primary amyloid-enhancing factor (AEF) was kindly provided by Dr Kisilevsky, Ontario, Canada. Swiss female mice (6–8 weeks old) received a single 200-µl (100-µg) intravenous injection of AEF into the lateral tail vein, and 200 µl (50 µg) of lipopolysacharide (LPS) intraperitoneally, every second day for 3 weeks. Two days after the last LPS injection, animals were injected subcutaneously with 0.5 ml of 2% AgNO3.

To assess the kinetics and organ involvement of experimental amyloidosis properly, mice were killed at 4 h, 1 day, 4 days and 1, 2, 3 and 4 weeks after the initial induction. The spleen, liver, and kidney were removed and examined for amyloid deposition.

Patients
Amyloid deposits were obtained from the carpal tunnel of three dialysis patients, aged 65±4 years with dialysis-related amyloidosis. Their renal diseases included two chronic glomerulonephritis cases and one autosomal dominant polycystic kidney disease. None of the patients had systemic diseases known to be associated with amyloidosis.

Sample preparation and immunohistochemistry
Congo red staining
Congo red, morphology studies, and immunogold methods were performed as previously described [11]. Unfixed amyloid deposits were immersed in OCT compound (Miles Inc., Diagnostics Division, Elkhart, USA), frozen and stored at -70°C. Cryostat sections of 10 µm were transferred onto gelatin-coated (0.3%) glass slides and stained with Congo red (Searle Scientific Services, High Wycombe Bucks, UK). Briefly, sections were dehydrated in 70% ethanol for 5 min and then treated with an alkaline-saturated salt solution. The stained sections were mounted in a medium compatible with organic solvents and observed under polarized light with a Zeiss microscope (Zeiss, Oberkochen, West Germany).

Morphology studies
Small blocks of amyloid deposits were fixed in 4% (w/v) paraformaldehyde (PFA) and 0.1% (v/v) glutaraldehyde (GA) in 0.1 mol/l phosphate-buffered saline (PBS), pH 7.4, for 2 h at 4°C, dehydrated in graded ethanols, and embedded in Lowicryl K4M (Chemische Werke Lowi, Wald-Kraiburg, Germany). Sections were transferred onto glass slides and stained with 0.5% methylene blue and 0.5% borax for 30 s at 90°C. The stained sections were then mounted in DPX medium and observed under a Polyvar 2 optical microscope (Reichert-Jung, Wien, Austria) under immersion oil.

For ultrastructural studies, after fixation with 4% PFA and 0.15 GA, small pieces of amyloid deposits were post-fixed in 2.5% GA (v/v) in PBS. The samples were then progressively treated in graded acetone-resin solutions, post-fixed with 0.1% osmium tetroxide, and embedded in Spurr (Agar Scientific Ltd, Stanstead, UK). Ultrathin sections were obtained using an Ultracut E system (Reichert-Jung) and counterstained with uranyl acetate and lead citrate before examination using a transmission electron microscope.

Immunogold methods
For the ultrastructural studies, blocks of approximately 1 mm3 of amyloid deposits were fixed and embedded in Lowicryl K4M. Ultrathin sections (60–90 nm) were obtained using an Ultracut E system and mounted on formvar-coated and etched gold grids.

Before labelling, sections were rinsed twice with 0.1 mol/l glycine–PBS for 5 min and incubated with 2% ovalbumin in PBS for 30 min to block unspecific antibody–antigen complexes. The grids with human ß2-microglobulin amyloidosis were then incubated with polyclonal anti-ß2-microglobulin (dilution 1:500; Nordic, Tilburg, The Netherlands) and diluted in 1% ovalbumin, and the grids containing murine AA amyloidosis were incubated with a polyclonal Ab against SAA (Dako, Glostrup, Denmark) and diluted in 1% ovalbumin for 2 h. After three 15-min rinses in glycine–PBS, bound antibodies were visualized with 10 nm protein A gold (pAg) (kindly provided by Dr J. W. Slot, University of Utrecht, The Netherlands). The sections were finally rinsed with PBS and double distilled water prior to counterstaining with aqueous uranyl acetate and lead citrate. Controls were performed by omitting the primary antibody. Observations were carried out with Hitachi H-600 AB (Japan) and Philips EM-300 (The Netherlands) transmission electron microscopes.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Experimental amyloidosis
Amyloid deposits were consistently observed in spleen, liver, and kidney in mice with induced AA amyloidosis. After the first week following induction, amyloid deposits were observed perifollicularly in the spleen. After the second week, liver amyloid deposits (mainly around the central vein) were frequently observed. The last organ showing involvement was the kidney (where amyloid deposits had a glomerular distribution).

Amyloid deposits in the liver were observed as fibril bundles located around the vessels, occupying the subendothelial space (Figure 1AGo). There was increased cellularity and thickening of the subendothelial space and the basal lamina, as well as the abnormal presence of collagen (Figure 1DGo). Animals sacrificed after 4 weeks of amyloid induction (late stages of amyloidosis) presented amyloid deposits that occupied most of the analysed tissue, and showed a decrease in the cellular infiltrate. Liver tissue from a control animal is shown in Figure 1BGo for comparison.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 1.  Murine experimental model of amyloidosis. (A) Early deposition of amyloid fibrils (arrows) in the subendothelial space of a central vein (CV) in the liver at 3 weeks after the first injection. The endothelium is thickened and the parenchyma is infiltrated by leukocytes associated with vascular diverticles (*) (x660). (B) Liver from a control animal (x900). (C) Immunolocalization of AA amyloid fibrils in mouse liver. Polymerization of amyloid fibrils (am) caused disorganization of the basal lamina. The amyloid fibrils were in contact with the plasma membrane (arrows) of endothelial cells (en) and occasional loss of continuity of the basal lamina can be observed (arrowheads) (x30 000). (D) Amyloid fibrils (am) in mouse liver were also associated with banded collagen (arrows) and disorganized endothelial cells (en) (x33 000). (E) Spleen from a mouse at 2 weeks after the first injection. AA amyloid fibrils (am) enter the cell after disorganization of the plasma membrane, causing cell damage by regression and swelling of endoplasmic reticulum (er) and mitochondria (m) endomembranes (x13 000). (F) Intracellular localization of AA amyloid fibrils (am) in an endothelial cell from mouse spleen after amyloidosis induction. A clear lesion of the nuclear envelope with loss of continuity (arrowheads) can be observed (x33 000).

 
Immunogold labelling with AA antibodies showed the presence of AA amyloid fibrils closely related to the basal lamina of the endothelial cells (Figure 1CGo). Basal lamina and plasma membrane were disorganized (Figure 1CGo, DGo). Plasma membranes had loss of continuity, particularly in their basal domain, and were interrupted by amyloid fibrils that invaded the cytoplasm, provoking a general disorganization of the endomembranes. Mitochondria (Figure 1EGo) and the nuclear envelope (Figure 1FGo) were the most altered endocellular compartments. As a consequence of the cell damage, the endothelium had a focal loss of its barrier function.

ß2-Microglobulin amyloid deposits
Vascular involvement and membrane modifications were also studied in human ß2-microglobulin amyloidosis to determine whether these pathological findings observed in experimental amyloidosis also participate in dialysis-related amyloidosis. We observed findings that were very consistent among our patients. Amyloid deposits consisted of an intercellular amorphous material, including macrophage-like cells and blood vessels that supplied this abnormal tissue (Figure 2Go). They were clearly separated from the surrounding tissue by a loose connective matrix. The cells and amyloid fibrils were grouped in regions usually associated with a single blood vessel. These areas were separated from each other by a layer of connective tissue that included thin bundles of collagen fibres and some elastic fibres. These elastic fibres were characteristically associated with the highest ordered amyloid fibrils (Figure 3Go).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.  Semi-thin sections of dialysis-related amyloidosis with methylene blue staining. (A) Amyloid deposit (am) surrounded by connective tissue (ct) with an obvious cellular infiltrate (x370). (B) A second zone from the same patient with amyloid clumps (am) in close association with the long processes of the cells (arrows) (x370). (C) A third region of the amyloid deposit from the same patient showing a clear predominance of amyloid fibrils among the cellular elements.

 


View larger version (178K):
[in this window]
[in a new window]
 
Fig. 3.  Ultrastructural location of amyloid fibrils. Electron micrograph showing the ultrastructural localization of ß2-microglobulin amyloid fibrils (am) forming around the elastic fibres (ef) in close association with macrophage processes (arrows) (x21 000).

 
Amyloid regions displayed considerable structural diversity within a single amyloid deposit. The pattern of cellular infiltrate was equivalent to what we had previously identified as macrophages [9]. The cell density varied greatly from region to region (Figure 2AGo, BGo, CGo). In turn, macrophages also displayed morphological dissimilarities. Those surrounding vessels had an irregular profile and abundant cytoplasm, mainly occupied by lysosomes. In contrast, macrophages distant from the vessels had reduced cytoplasm with fewer lysosomes as well as condensed chromatin and long cytoplasmic processes. The former cell type was associated mainly with the regions having scarce amyloid material and high cellularity, whereas the latter cell type was found in zones with a predominance of amorphous amyloid material and hypocellularity.

The vessels observed around amyloid deposits were mainly from precapillary sphincter regions, capillaries, and pericytic venulae. Arterioles and venulae irrigating the surrounding connective tissue were observed only occasionally (Figure 4Go). The capillaries and pericytic venulae showed an increased number of endothelial cells, the presence of buttons of cell proliferation (Figure 4BGo), and enlargement of subendothelial spaces due to deposition of amyloid material (Figure 4AGo). Macrophages and pericytes were associated with these vessel areas of amyloid deposits (Figure 4CGo).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.  Analysis of vessels from dialysis-related amyloidosis. (A) Vessel walls are thickened by the deposition of amyloid material in the subendothelial spaces (arrows). Numerous long-shaped pericytes and macrophages (mo) are observed around the vessels. The connective tissue (ct) and a venula (v) are also observed in this section (x530). (B) Angiogeneic activity: the vessel displays an increased number of endothelial cells grouped in proliferation buttons (arrows) (x325). (C) Pericytic venulae (pv) and capillaries (c) are always associated with development of amyloid deposition areas (am) (x530).

 
Electron microscopy showed irregular endothelial cells with dense cytoplasm filled with multiple transcytotic vesicles. The plasma membrane showed numerous evaginations at both the apical and basal domains, and occasional overlapping with neighbouring cells (Figure 5AGo). The basal lamina was irregular and presented as multi-layered in some zones (Figure 5AGo, BGo) or lacking the lamina densa in other areas (Figure 5CGo). Two distinguishable cell types were observed penetrating the endothelial basal lamina: round-shaped cells with macrophage-like features including lysosomes and vesicles (Figure 5AGo) and myofibroblast-like cells with thick bundles of cytoskeleton filaments and associated dense bodies (Figure 5BGo).



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 5.  Ultrastructural analysis of the vessel walls in ß2-microglobulin amyloidosis. (A) Endothelial cells (en) with a multi-layered basal lamina (mbl). Desmosome like junctions were present between neighbouring cells (arrow). The basal lamina contains pericytes displaying macrophage-like features (p). The vessel lumen is labelled as (L) (x11 000). (B) Pericytes with myofibroblast characteristics (bundles of microfilaments packaged by dense bodies (arrows)) were also observed. The basal lamina was also multi-layered (mbl) and was between myofibroblast-like cells, endothelial cells (en), and the vessel lumen (L) (x11 000). (C) Immunolocalization of ß2-microglobulin fibrils (am). Endothelial cells (en) of the vessels, associated with ß2-microglobulin amyloid deposits, were characterized by numerous pinocytic vesicles and extended cytoplasmic processes at the apical and basal membrane domains. Labelled ß2-microglobulin was again in close association with the basal lamina (bl), which had embedded pericytes (p) (x19 000).

 
Immunogold labelling showed the presence of ß2-microglobulin both around the vessels and in close contact with the basal lamina (Figure 5CGo). In agreement with the light microscopy findings, it was also associated with macrophage-like cells.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Our study clearly shows vascular abnormalities in amyloid deposits. The vascular involvement in amyloidosis was consistent and present in both the experimental model and in human ß2-microglobulin amyloidosis. Three different observations are of importance: (i) the vascular compartment was a preferential site of amyloid deposition; (ii) it was strongly associated with the cellular infiltrate; (iii) it reacted actively as was shown by vascular proliferation or angiogenesis.

The location of amyloid fibrils in the subendothelial space has been classically observed in several types of amyloidoses, including hereditary cerebral haemorrhage amyloidosis [12] and Alzheimer's disease [13]. In ß2-microglobulin amyloidosis, there is no agreement about the vascular distribution of amyloid fibrils. Gal et al. [14], studying necropsy specimens from dialysis patients, found that amyloid material was preferentially observed in the wall of blood vessels of the osteoarticular system, gastrointestinal tract, heart, and lungs. In contrast, Noël et al. [15] failed to find a significant multiorgan involvement that included a vascular distribution of deposits in 23 patients with ß2-microglobulin amyloidosis. Our study showed the presence of amyloid deposits in the subendothelial space of blood vessels. However, we also found that amyloid deposits are not homogeneous in terms of amyloid fibril density, the presence of cells, and the proportion of blood vessels. These disparities may contribute to the differences reported by these authors.

The second aspect of vascular participation in the pathogenesis of amyloidosis is the carrier function. It is clear that for amyloid deposits to appear, the substrate and cofactors (normally proteins) as well as the cells need to reach the precise location where amyloidogenesis occurs. We previously reported the presence of albumin, transferrin, haemopexin, and other serum proteins in ß2-microglobulin amyloidosis [16] and Graeber et al. [17] identified pre-albumin, albumin, immunoglobulins, and amyloid P component, as well as complement factors in amyloid deposits from Alzheimer's disease patients. The presence of serum proteins in amyloid deposits suggests an increased permeability of blood vessels in these regions. Therefore, blood vessels not only provide a path to reach amyloid deposits, but the barrier function of the endothelium may also be altered, allowing serum proteins to reach the subendothelial and extravascular spaces and precipitate into amyloid fibrils.

Although cellular infiltrates have been less frequently studied than proteins in amyloidogenesis, certain details are well established. The density of these cells is relatively small (<200 cells/0.2 mm2) and >90% of them express the CD14 antigen, indicating that they are of macrophage origin [9]. Although macrophages have been thought to participate in the formation of amyloid fibrils [18,19], they may also contribute to a secondary process aimed at clearing the affected tissues of amyloid deposits by phagocytosis [11]. Regardless of whether macrophages are at the origin of amyloid fibril formation or whether they are a reactive phenomenon, the higher density of cells around blood vessels is in keeping with a vascular provenance of the cellular infiltrate.

In addition to providing a path for amyloid components to reach amyloid deposits and the altered barrier function of endothelium, blood vessels display characteristics suggesting an active participation in the disease. They have an increased number of cells, proliferation buttons, numerous transcytotic vesicles, and thickened or multi-layered basal lamina. These features, controlled by pericytes, are observed in angiogenesis and are associated with an increased vascular permeability [20].

Finally, our study showed that cellular membranes in close contact with amyloid fibrils were damaged. This was true for the nuclear envelope as well as for the cytosolic endomembranes, indicating a toxic effect of the amyloid fibrils that may result in cellular damage and death. We have recently shown that there is an impaired processing of ß2-microglobulin from amyloid fibrils occurring in the lysosomes of macrophages [11]. The unprocessed ß2-microglobulin is retained in the cell and can result in cell lysis. Our findings in experimental amyloidosis suggest a series of sequential events in amyloidogenesis. The animals analysed after 1 week of amyloid induction had spleen deposits in vascular areas showing a large proportion of cells. The animals having longer periods of amyloidosis presented with large clumps of amyloid fibrils and sparsely distributed blood vessels and cells. These findings are in agreement with a toxic effect of undigested amyloid proteins that would produce cellular death. As we have observed in ß2-microglobulin amyloidosis [11], the cells would then be replaced by amyloid fibrils which would become predominant in areas of amyloid deposits along with cellular debris that would form between amyloid fibrils.

In summary, the present findings suggest that blood vessels participate in amyloidogenesis through a variety of mechanisms: blood vessels are sites of amyloid fibril deposition, they facilitate the access of the serum proteins and blood cells into amyloid deposits, and they react with cell proliferation and angiogenesis factors. Our sequential analysis of experimental murine amyloidosis supports a toxic effect of amyloid fibrils that produce cell death followed by immersion of cellular debris in accumulated amyloid fibrils.



   Acknowledgments
 
We thank Almudena García and the staff of Serveis Científico-Tècnics (Universitat de Barcelona) for technical assistance. We are indebted to Baxter Healthcare Co., McGaw Park, Illinois, USA for contributing to this work through its Extramural Grant Program. MG was a recipient of a grant from the Comissió Interdepartamental de Recerca i Tecnologia (CIRIT)—Direcció General de Recerca, Generalitat de Catalunya.



   Notes
 
Correspondence and offprint requests to: Àngel Argilés, Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France. Email: angel.argiles{at}igh.cnrs.fr Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Kisilevsky R, Fraser PE. A beta amyloidogenesis: unique, or variation on a systemic theme? Crit Rev Biochem Mol Biol1997; 32: 361–404[Abstract]
  2. Snow AD, Kisilevsky, R. Temporal relationship between glycosaminoglycan accumulation and amyloid deposition during experimental amyloidosis. A histochemical study. Lab Invest1985; 53: 37–44[ISI][Medline]
  3. Gejyo F, Yamada T, Odani S et al. A new form of amyloid protein associated with chronic hemodialysis was identified as ß2m. Biochem Biophys Res Commun1985; 129: 701–706[ISI][Medline]
  4. Argilés A. ß2-microglobulin amyloidosis. Nephrology1996; 2: 373–386[ISI]
  5. Gejyo F, Homma N, Suzuki Y, Arakawa M. Serum levels of ß2 microglobulin, as a new form of amyloid protein in patients undergoing long-term hemodialysis. N Engl J Med1986; 314: 585–586[ISI][Medline]
  6. Argilés A, García-García M, Derancourt J et al. ß2-microglobulin isoforms in healthy individuals and in amyloid deposits. Kidney Int1995; 48: 1397–1405[ISI][Medline]
  7. Depierreux M, Goldman M, Fayt I et al. Osteoarticular amyloidosis associated with haemodialysis: an immunoultrastructural study. J Clin Pathol1988; 41: 158–162[Abstract]
  8. Nishi S, Ogino S, Marayama Y et al. Electron-microscopic and immunohistochemical study of ß2m-related amyloidosis. Nephron1990; 56: 357–363[ISI][Medline]
  9. Argilés A, Mourad G, Kerr P et al. Cells surrounding haemodialysis-associated amyloid deposits are mainly macrophages. Nephrol Dial Transplant1994; 9: 662–667[Abstract]
  10. Argilés A, Mourad G, Gouin-Charnet A, Schmitt-Bernard CF. Antiproteases and cells in the pathogenesis of ß2-microglobulin amyloidosis: The role of {alpha}2-macroglobulin and macrophages. Nephron2000; 86: 1–11[ISI][Medline]
  11. García-García M, Argilés A, Gouin-Charnet A et al. Impaired lysosomal processing of beta 2 microglobulin by infiltrating macrophages in dialysis amyloidosis. Kidney Int1999; 55: 899–906[ISI][Medline]
  12. Cohen DH, Feiner H, Jensson O, Frangione B. Amyloid fibril in hereditary cerebral hemorrhage amyloidosis is related to the gastroenteropancreatic neuroendocrine protein gamma trace. J Exp Med1983; 158: 623[Abstract]
  13. Perlmutter LS. Microvascular pathology and vascular basal lamina components in Alzheimer's disease. Mol Neurobiol1994; 9: 33–40[ISI][Medline]
  14. Gal R, Kortzets A, Schwartz A et al. Systemic distribution of ß2-microglobulin amyloidosis in patients who undergo long-term hemodialysis. Arch Pathol Lab Med1994; 118: 718–721[ISI][Medline]
  15. Noël LH, Zingraff J, Bardin T et al. Tissue distribution of dialysis amyloidosis. Clin Nephrol1987; 27: 175–178[ISI][Medline]
  16. Argilés A, Mourad G, Axelrud-Cavadore C et al. Analyse immunochimique des depots amyloides secondaire a l'hemodialyse de suppleance au long-cours. Néphrologie1987; 8: 51–54
  17. Graeber MB, Streit WJ, Kreutzberg GW. Identity of ED2-perivascular cells in rat brai. J Neurosci Res1989; 22: 103–106[ISI][Medline]
  18. Kisilevsky R, Lyon AW, Young I. A critical analysis of postulated pathogenetic mechanisms in amyloidogenesis. Crit Rev Clin Lab Sci1992; 29: 59–82[ISI][Medline]
  19. Ohashi K, Hara M, Kawai R et al. Cervical discs are most susceptible to ß2-microglobulin amyloid deposition in the vertebral column. Kidney Int1992; 41: 1646–1652[ISI][Medline]
  20. Diaz-Flores L, Gutierrez R, Varela H. Angiogenesis: an update. Histol Histopathol1994; 9: 807–843[ISI][Medline]
Received for publication: 7.12.01
Accepted in revised form: 8. 3.02





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by García-García, M.
Articles by Argilés, A.
PubMed
PubMed Citation
Articles by García-García, M.
Articles by Argilés, A.