Differences in extracellular matrix production and basic fibroblast growth factor response in skin fibroblasts from sporadic and familial Alzheimer's disease

Abstract

Extracellular matrix (ECM) molecules and growth factors, such as fibroblast growth factor (FGF), play a crucial role in Alzheimer's disease (AD). The purpose of this investigation was to determine whether phenotypic alterations in ECM production are present in non-neuronal AD cells associated with different FGF expression and response. Synthesis of glycosaminoglycans (GAG) and collagen were measured in skin fibroblasts from patients with familial, sporadic AD (FAD and SAD respectively), and from age-matched controls by radiolabeled precursors. Proteoglycans (PG), metalloprotease (MMP)-1, and FGF gene expressions were measured by reverse transcription-polymerase chain reaction. The results showed different ECM neosynthesis and mRNA levels in the two AD fibroblast populations. FAD accumulated more collagen and secreted less GAG than SAD. Biglycan PG was upregulated in FAD while betaglycan, syndecan, and decorin were markedly downregulated in SAD fibroblasts. We found a significant decrease of MMP1, more marked in FAD than in SAD fibroblasts. Constitutive FGF expression was greatly reduced in both pathological conditions (SAD>FAD). Moreover, an inverse high affinity/low affinity FGF receptor ratio between SAD and FAD fibroblasts was observed. FGF treatment differently modulated ECM molecule production and gene expression in the two cell populations. These observations in association with the changes in FGF gene expression and in the FGF receptor number, suggest that cellular mechanisms downstream from FGF receptor binding are involved in the two different forms of AD.

Figure 1

Cell number and FGF2 effects (+) on control (C), sporadic (SAD) and familial (FAD) Alzheimer’s disease fibroblasts. Fibroblasts were mantained in serum–free MEM for 24h with and without FGF2 (20 ng/ml). The values were the mean ± SD of three independent experiments each performed in quintuplicate for n=10 subjects for group. Data were analyzed by ANOVA. Differences vs. respective control: *F–test significant at 99%. Differences vs. respective untreated group: §F–test significant at 99%; ns = not significant.

Figure 2

Cellular, secreted and total GAG production from control (C), sporadic (SAD) and familial (FAD) Alzheimer’s disease fibroblasts and FGF2 effects (+). The values were the mean ± SD of three independent experiments each performed in quintuplicate for n=10 subjects for group. Data were analyzed by ANOVA. Differences vs. respective control: *F–test significant at 99%. Differences vs. respective untreated group: §F–test significant at 99%; §§F–test significant at 95%; ns = not significant.

Figure 3

Cellular, secreted and total collagen production from control (C), sporadic (SAD) and familial (FAD) Alzheimer’s disease fibroblasts and FGF2 effects (+). The values were the mean ± SD of three independent experiments each performed in quintuplicate for n=10 subjects for group. Data were analyzed by ANOVA. Differences vs. respective control: *F–test significant at 99%; NS = not significant. Differences vs. respective untreated group: §F–test significant at 99%; §§F–test significant at 95%; ns = not significant.

Figure 4

Expression data (mRNA levels) for betaglycan, decorin, syndecan, biglycan, MMP1 and FGF2 obtained from control (C), sporadic (SAD) and familial (FAD) Alzheimer’s disease fibroblasts treated or not with FGF2 (+). The mRNA levels were quantified by real–time quantitative PCR. Values were the mean ± SD of three independent experiments each performed in triplicate for n=10 subjects for group. Data were analyzed by ANOVA. The results were expressed as fold change in β–actin normalized mRNA values. Differences vs. mRNA levels in each respective control: *F–test significant at 99%; **F–test significant at 95%; NS = not significant. Differences vs. mRNA levels in each untreated group: §F–test significant at 99%; ns = not significant

Figure 5

Scatchard analysis of ¹²⁵I–FGF2 binding to low affinity receptors in control, sporadic (SAD) and familial (FAD) fibroblasts, and FGF2 effect. Specifically bound (B) anf free (F) ligands were measured as described in Materials and Methods. Values were the mean ± SD of three independent experiments each performed in triplicate for n=10 subjects for group. Difference vs. number of receptors in each respective control: *F–test significant at 99%. Differences vs. number of receptors in respective untreated group: §F–test significant at 99%; §§F–test significant at 95%.

Figure 6

Scatchard analysis of ¹²⁵I–FGF2 binding to high affinity receptors in control, sporadic (SAD) and familial (FAD) fibroblasts, and FGF2 effect. Specifically bound (B) and free (F) ligands were measured as described in Materials and Methods. Values were the mean ± SD of three independent experiments each performed in triplicate for n=10 subjects for group. Difference vs. number of receptors in each respective control: *F–test significant at 99%. Differences vs. number of receptors in respective untreated group: §F–test significant at 99%; §§F–test significant at 95%.