Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation
✍ Scribed by P. Odetti; I. Aragno; R. Rolandi; S. Garibaldi; S. Valentini; L. Cosso; N. Traverso; D. Cottalasso; M. A. Pronzato; U. M. Marinari
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 280 KB
- Volume
- 16
- Category
- Article
- ISSN
- 1520-7552
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✦ Synopsis
Background The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modi®cations in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation.
Methods Tendon ®brils were obtained from the tails of 8-month-old rats (BB/WOR/MOL\BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL\WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon ®brils and in ®brils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon ®brils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC.
Results Incubated ®brils revealed modi®cations in radius (228t5 nm) and gap depth (3.65t0.10 nm) that closely reproduce diabetes-induced damage (236t3 and 3.20t0.04 nm respectively) and were signi®cantly different from the pattern seen in non-diabetic ®brils (151t1 and 2.06t0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82t1.43 nmol/mg and 2.23t0.24 pmol/mg collagen respectively) and in glucose-incubated ®brils (9.27t0.55 nmol/mg and 5.15t0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29t0.08 nmol/mg and 0.88t0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modi®ed by non-enzymatic glycation.
Conclusion This is the ®rst description obtained with SFM of diabetesinduced ultrastructural changes in collagen ®brils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modi®cations in collagen ®brils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modi®cation.