Cerebrovascular transforming growth factor-beta contributes to inflammation in the Alzheimer's disease brain

Abstract

Inflammatory mechanisms are thought to contribute to lesion pathogenesis and neuronal cell death in Alzheimer's disease. Transforming growth factor-beta (TGF-beta) plays a central role in the response of the brain to injury, and is increased in the brain in Alzheimer's disease. In this study we determine whether expression of TGF-beta is abnormal in the microvasculature in Alzheimer's disease and whether TGF-beta affects vascular production of pro-inflammatory cytokines, interleukin (IL)-1 beta, and tumor necrosis factor (TNF)-alpha. Microvessels isolated from the cortices of Alzheimer's disease patients and age-matched controls are analyzed for microvessel-associated and released TGF-beta. Results from Western blot analysis and enzyme-linked immunosorbent assay indicate a higher level of TGF-beta in Alzheimer's disease vessels compared to controls. To determine whether TGF-beta affects vascular release of inflammatory factors, cultured brain endothelial cells are treated with TGF-beta and levels of IL-1 beta and TNF-alpha determined. Both enzyme-linked immunosorbent assay and Western blot analyses show that untreated endothelial cells express little IL-1 beta or TNF-alpha, but incubation with TGF-beta results in robust expression of these factors by brain endothelial cells. Our results suggest that vessel-derived TGF-beta contributes to inflammatory processes in the Alzheimer brain.

Figure 1.

Microvessels were isolated from the brains of patients with AD (n = 10) or nondemented elderly controls (n = 5). Brain microvessels were washed and incubated for 6 hours with serum-free DMEM containing 1% lactalbumin hydrogenase, centrifuged, and the supernatant assayed for TGF-β using an ELISA-based assay. *, P < 0.05, significantly different from control-derived microvessels.

Figure 2.

Microvessels were isolated from the brains of patients with AD or nondemented elderly controls. Microvessel lysates (50 μg/lane) were loaded for SDS-polyacrylamide gel electrophoresis, and Western blot analyses and were performed using antibodies to TGF-β. Lanes 1–3, non-AD control-derived microvessels; lanes 4–6, AD-derived vessels.

Figure 3.

Confluent brain endothelial cells were incubated in serum-free DMEM with 0.1% bovine serum albumin and then exposed to different concentrations of TGF-β for 24 hours. Control (unstimulated) cells were incubated in media alone. The supernatants were assayed for IL-1β (right) and TNF-α (left) using an ELISA-based assay. *, P < 0.05; **, P < 0.01;***, P < 0.005, significantly different from unstimulated endothelial cells.

Figure 4.

Confluent endothelial cells were incubated in serum-free DMEM with 0.1% bovine serum albumin and then exposed to different concentrations of TGF-β for 24 hours (lanes 2–4 and 6–8). Control (unstimulated) cells were incubated in media alone (lanes 1 and 5). Endothelial cells were washed, scraped, and samples prepared for SDS-polyacrylamide gel electrophoresis. Western blot analysis was performed using antibodies to IL-1β (left) and TNF-α (right).