Amyloid Beta Peptides and Th1 Cytokines Modulate Human Brain Vascular Smooth Muscle Tonic Contractile Capacity In Vitro: Relevance to Alzheimer's Disease?

Abstract

Alzheimer's Disease (AD) is a neurodegenerative condition characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular "plaques". The cleavage of amyloid precursor protein (APP) yields several Aβ peptides. Although Aβ toxicity to neurons has been described extensively, its effects on other components of the neurovasculature such as vascular smooth muscle cells have been less well characterized. AD is now also recognized as a neurovascular disease characterized by cerebral microbleeds and disturbances in autoregulation. AD is also a neuroinflammatory condition in which several proinflammatory cytokines are elevated and may contribute to the intensification of AD severity. Cerebral autoregulation (the mechanism by which brain blood flow is maintained despite changes in perfusion pressure) is extremely tightly controlled in the brain and shows disturbances in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow might could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen gel contractility assay to evaluate the influence of Aβ25-35, Aβ1-40 and Aβ1-42 peptides and inflammatory cytokines on the tonic contractility of human brain vascular smooth muscle cells (HBVSMC) as an in vitro model of cerebral autoregulation. We found that 5 and 10 μM Aβ1-42 significantly depressed HBVSM contractility, while Aβ1-40 5-20 μM had no effect on contractility. Conversely, Aβ25-35 (1-50 μM) increased contractility. Interestingly, the inflammatory cytokines TNF-α (20 ng/mL), IL-1β (20 ng/mL) and IFN-γ (1000 U/mL) also depressed HBVSM tonic contractility alone and in combination. These data suggest that both the inflammatory milieu in AD as well as the abundance of Aβ peptides may promote autoregulatory failure and increase brain susceptibility to dysregulated perfusion and microbleeds which are an important and devastating characteristic of AD.

Keywords: Alzheimer’s disease; inflammatory cytokine; tonic contraction; vascular smooth muscle.

Figure 1

Aβ1-42 peptide decreased HBVSMC contractility. (a) Human brain vascular smooth muscle cells (HBVSMC) treated with 5 or 10 μM of Aβ1-42 exhibit significantly larger area after 3 days of treatment (days 3–5). (b) Aβ1-42 decreased HBVSMC contractility at day 3 significantly compared with control n = 4, * p < 0.05, ** p < 0.01, **** p < 0.001 for 5 uM treatment vs. control on each day, # p < 0.05, ## p < 0.01 for 10 uM treatment vs. control each day; one-way ANOVA with Dunnett’s post-testing; data are mean ± SEM. (c) Representative figure of HBVSMC/collagen gels incubated with or without 5 μM of Aβ1-42 peptides over the period of 3 days. Red dashed lines indicate the outline of the HBVSMC/collagen gels which were used to calculate the area.

Figure 1

Aβ1-42 peptide decreased HBVSMC contractility. (a) Human brain vascular smooth muscle cells (HBVSMC) treated with 5 or 10 μM of Aβ1-42 exhibit significantly larger area after 3 days of treatment (days 3–5). (b) Aβ1-42 decreased HBVSMC contractility at day 3 significantly compared with control n = 4, * p < 0.05, ** p < 0.01, **** p < 0.001 for 5 uM treatment vs. control on each day, # p < 0.05, ## p < 0.01 for 10 uM treatment vs. control each day; one-way ANOVA with Dunnett’s post-testing; data are mean ± SEM. (c) Representative figure of HBVSMC/collagen gels incubated with or without 5 μM of Aβ1-42 peptides over the period of 3 days. Red dashed lines indicate the outline of the HBVSMC/collagen gels which were used to calculate the area.

Figure 2

Aβ1-40 peptides did not alter HBVSMC contractility. At 5, 10 or 20 μM. Aβ1-40 did not significantly affect HBVSMC contractility at 5, 10 or 20 μM over 5 days. n = 4, data are mean ± SEM.

Figure 3

Aβ25-35 peptide enhanced HBVSMC contractility. (a) HBVSMC in collagen gel treated with various concentrations of Aβ25-35 exhibit a smaller area at 3 days of treatment. This effect was not observed after 4 days of treatment. (b) At 3 days in culture, Aβ25-35 treatment significantly enhanced HBVSMC contractility at all concentrations tested, from 1–50 μM compared with control n = 4, * p < 0.05, **** p<0.0001 versus control; one-way ANOVA with Dunnett’s post-testing; data shown are mean ± SEM.

Figure 4

Inflammatory cytokines depress HBVSMC contractility. (a) 1000 U/mL of IFNγ treatment on HBVSMC resulted in greater area (measured in cm²) after 3 days. (b) HBVSMC treated with 1000 U/mL of IFNγ for 4 days showed a significantly lower contractility (expressed in % of contraction vs. control) compared with control treated HBVSMC. (c) 20 ng/mL of TNFα treatment on HBVSMC resulted in greater areas after 1 day up to 5 days. (d) HBVSMC treated with TNFα for 4 days showed a significantly lower contractility compared to control HBVSMC. (e) 20 ng/mL of IL-1b treatment on HBVSMC in collagen gel resulted in a larger area after 1 day up to 5 days. (f) HBVSMC treated with 20 ng/mL of IL-1b for 4 days showed a significantly lower contractility compared to control HBVSMC. (g) A combined treatment consisting of 1000 U/mL of IFNγ and 20 ng/mL of TNFα on HBVSMC in collagen gel resulted in even greater areas (measured in cm²) after 1 day. This effect persisted throughout the study (5 days). (h) HBVSMC treated with a combination of 1000 U/mL of IFNγ and 20 ng/mL of TNFα for 4 days showed a significantly lower contractility. (i) A combined treatment consisting of 20 ng/mL of IL-1b and 20 ng/mL of TNFα on HBVSMC in collagen gel resulted in a larger area (measured in cm²) after 1 day. This effect persisted throughout the study (5 days). (j) HBVSMC treated with a combination of 20 ng/mL of IL-1b and 20 ng/mL of TNFα for 4 days showed a significantly lower contractility. n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control; Student’s t-test; data are mean ± SEM.

Figure 4

Inflammatory cytokines depress HBVSMC contractility. (a) 1000 U/mL of IFNγ treatment on HBVSMC resulted in greater area (measured in cm²) after 3 days. (b) HBVSMC treated with 1000 U/mL of IFNγ for 4 days showed a significantly lower contractility (expressed in % of contraction vs. control) compared with control treated HBVSMC. (c) 20 ng/mL of TNFα treatment on HBVSMC resulted in greater areas after 1 day up to 5 days. (d) HBVSMC treated with TNFα for 4 days showed a significantly lower contractility compared to control HBVSMC. (e) 20 ng/mL of IL-1b treatment on HBVSMC in collagen gel resulted in a larger area after 1 day up to 5 days. (f) HBVSMC treated with 20 ng/mL of IL-1b for 4 days showed a significantly lower contractility compared to control HBVSMC. (g) A combined treatment consisting of 1000 U/mL of IFNγ and 20 ng/mL of TNFα on HBVSMC in collagen gel resulted in even greater areas (measured in cm²) after 1 day. This effect persisted throughout the study (5 days). (h) HBVSMC treated with a combination of 1000 U/mL of IFNγ and 20 ng/mL of TNFα for 4 days showed a significantly lower contractility. (i) A combined treatment consisting of 20 ng/mL of IL-1b and 20 ng/mL of TNFα on HBVSMC in collagen gel resulted in a larger area (measured in cm²) after 1 day. This effect persisted throughout the study (5 days). (j) HBVSMC treated with a combination of 20 ng/mL of IL-1b and 20 ng/mL of TNFα for 4 days showed a significantly lower contractility. n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control; Student’s t-test; data are mean ± SEM.

Figure 5

Disturbances in autoregulation produced by Aβ1-42 and inflammatory cytokines (TNF-α, IL-1β and IFN-γ) could lead to impaired (−) cerebral perfusion and contribute to microbleeds and dysregulated Aβ clearance. Aβ25-35 may enhance constriction (+). We were unable to observe effects of Aβ1-40 on human brain smooth muscle contractility.