CAA deposits increased with age in Tg2576 mice
To assess progression of CAA deposits in the leptomeningial arteries, Aβ that was deposited in a β-pleated sheet structure was visualized utilizing a congo red derivative fluorescent dye, methoxy-X04 (). Consistent with previous reports (Hsiao et al., 1996
; Kawarabayashi et al., 2001
; Fryer et al., 2003
), we found no parenchymal or vascular amyloid deposits in 6 month old Tg2576 mice (). Substantial CAA deposits, however, were noted in 12 month-old Tg2576 mice (). These deposits were typically patchy in nature with spacing between affected vessel segments. By 15 months of age, CAA deposits in Tg2576 mice had substantially progressed to encompass almost the entire leptomeningeal arteriolar system without interruption ().
Progression of CAA in Tg2576 mice
Vessel dysfunction in young Tg2576 mice lacking CAA
Vasodilatory response to hypercapnia was decreased by 48% in young Tg2576 mice (pre-CAA) as compared to age-matched, littermate wild type (WT) mice (Tg2576 mice: 14.5 ± 2.5% vs. WT mice: 27.7 ± 5.1%; P= 0.0233) (). The attenuated vasodilatory response could not be attributed to differences between groups in extent of pCO2 change after induced hypercapnia (), other physiological parameters (), or baseline vascular diameters (). A similar reduction in vessel response to topical ACh and SNAP was also noted in young Tg2576 mice as compared to age-matched, littermate WT mice (ACh — Tg2576 mice: 11.4 ± 2.2% vs. WT mice: 20.5 ± 2.6%; P= 0.009; SNAP — Tg2576 mice: 20.0 ± 3.7% vs. WT mice: 33.9 ± 2.6%; P= 0.003) (). These data confirm that young Tg2576 mice have significant impairment in vascular reactivity and indicate that under our experimental conditions this is primarily the consequence of VSMC dysfunction.
Physiological parameters pre- and post-hypercapnia.
Further vessel dysfunction in older Tg2576 mice having extensive CAA
Hypercapnia-induced vasodilation was even further reduced by 85% in older Tg2576 mice having extensive CAA (Tg2576 mice: 4.1 ± 2.0% vs. WT mice: 25.3 ± 3.0%; P< 0.0001) (). To more specifically examine the relationship between CAA deposits and vessel function, we compared amyloid load across individual 25 μm vessel segments (reported as % CAA) to the vasodilatory function across the given vessel segment. In those segments having no or minimal CAA (i.e. 0-10% CAA), we noted significant reduction in hypercapnia-induced vasodilation as compared to vessel segments from age-matched, littermate WT mice (). Cerebrovascular function was further compromised in vessel segments having 11-20% CAA, and vascular response was completely abolished in vessel segments have >20% CAA (). These alterations in vasodilatory response could not be attributed to extent of pCO2 change after induced hypercapnia or to other physiological parameters between groups (). These data strongly implicate CAA as a causal factor in the cerebrovascular dysfunction of older Tg2576 mice.
Vascular function was severely reduced in CAA-affected vessels
Relationship between CAA and cerebral vessel structural integrity
To explore whether alterations in VSMC architecture and/or density accounted for the observed cerebrovascular dysfunction, we sought to rigorously characterize vessel wall changes in Tg2576 mice. In young mice, no CAA was found and VSMCs were arranged closely in parallel in all examined pial arterioles (). At 12 months of age, CAA deposits were found between VSMCs (). In vessels having small amounts of CAA (<20%), no or minimal disruption of VSMC topography was noted and circumferential amyloid deposition was distinctly rare (). On the other hand, when greater amounts of CAA were present (>20%), architectural VSMC changes became increasingly frequent and more severe, particularly when extent of CAA reached very high levels (>40%) (). At 15 months of age, VSMCs were extensively dismantled throughout the pial vessels, as CAA deposition had progressed to the point of near-continuous involvement of the arteriole system (). These architectural changes were not apparent in age-matched WT mice (data not shown).
Disruption of vascular smooth muscle cells was noted in CAA-affected vessels
We also examined VSMC density. In WT mice of all examined ages, we found ~20 VSMCs per 100-μm vessel segment (). In young Tg2576 mice, VSMC density did not differ from age-matched WT mice. In older Tg2576 mice, however, decreased overall VSMC density was noted (Tg2576 mice: 15.5 ± 0.5 cells/100 μm vs. WT mice: 20.6 ± 1.3 cells/100 μm; P < 0.05). A correlative analysis between CAA load and VSMC density was performed, and a strong correlation between CAA severity (measured as % CAA) and VSMC density was noted (R2= 0.506; P < 0.001) (). Specifically, unaffected or mildly affected vessels (i.e. 0-20% CAA) had no VSMC loss, moderately affected vessels (i.e. 21-40% CAA) had a non-significant trend for VSMC loss, and severely affected vessels (i.e. >40% CAA) had substantial and significant VSMC loss (). Finally, a positive correlation between vessel caliber and severity of CAA deposits was noted (R2= 0.3588, P < 0.001) (). These data suggest that mild amounts of CAA have no or minimal effect on vessel wall integrity, but as increasing amounts of CAA develop significant disruptions of vessel wall integrity including frank loss of VSMCs occur.
Correlation between CAA coverage and VSMC loss
γ-secretase inhibition restores vascular function in Tg2576 mice
Our results and those of others (Zhang et al., 1997
; Iadecola et al., 1999
; Niwa et al., 2000b
; Niwa et al., 2002b
; Niwa et al., 2002a
; Paris et al., 2004
; Park et al., 2004
; Park et al., 2005
; Park et al., 2008
) suggest that soluble Aβ can cause significant vessel dysfunction. However, decreasing endogenous Aβ levels or blocking its function followed by assessment of vasoreactivity has not been performed. We hypothesized that acute depletion of soluble Aβ via γ-secretase inhibition would improve vessel function. We assessed vessel reactivity in young Tg2576 mice in the presence and absence of significant extracellular brain and plasma human Aβ utilizing the blood-brain barrier permeable γ–secretase inhibitor, LY411575 (Cirrito et al., 2003
; Best et al., 2005
). Similar to previous results (Cirrito et al., 2005b
), subcutaneous administration of LY411575 (3 mg/kg) in young Tg2576 mice resulted in almost complete depletion of soluble human Aβ1-x
pool in the interstitial fluid (ISF) up to 36 h after drug administration (). It also markedly reduced the soluble human Aβ1-x
pool in plasma (vehicle-treated group: 17.2 ± 2.1 ng/ml vs. LY511475-treated group: 3.3 ± 0.9 ng/ml, P
< 0.05) (data not shown). While LY411575 did not affect baseline vessel diameters in leptomeningeal arteries (), it did substantially restore cerebral vasodilatory responses in young Tg2576 mice (but not WT mice) (). Vessel function was not improved after administration of the inactive form of the γ–secretase inhibitor, LY424196 (). These data implicate soluble human Aβ as a causative agent in cerebrovascular impairment of young Tg2576 mice. The effect of murine Aβ on cerebrovascular function, however, appeared negligible, as LY411575 treatment in WT mice decreased murine Aβ in ISF (data not shown) but did not alter cerebrovascular function ().
Blockade of Aβ production restored vascular function in Tg2576 mice
We next examined whether γ-secretase inhibition was capable of restoring vasomotor function in 12-month-old Tg2576 mice having extensive CAA. We found that LY411575 treatment produced a small but significant improvement in cerebral arteriole function as compared to vehicle-treated animals (). Importantly, this partial restoration of vasodilatory response to hypercapnia resulted entirely from functional improvement in vessels having small amounts of CAA (i.e. ≤ 20% CAA), while no effect was noted in more severely affected vessels (i.e. >20% CAA) (). Of note, acute LY411575 treatment had no appreciable effect on CAA severity within vessel walls (), strongly suggesting that reduction in soluble human Aβ was responsible for the improved vessel function.