Contribution of reactive oxygen species to cerebral amyloid angiopathy,vasomotor dysfunction,and microhemorrhage in aged Tg2576 mice

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer’s Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE—a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within walls of leptomeningeal and cortical arterioles. Among the several types of amyloid proteins causing CAA, fibrillar amyloid β (Aβ) is by far the most common (1). This pathological form of Aβ is also the major constituent of neuritic plaques in patients with Alzheimer’s disease (AD) (2). Aβ is a 39- to 43-amino acid peptide that is produced from the amyloid precursor protein (APP) via sequential proteolytic cleavage processed by β- and γ-secretases (3, 4). Aβ₄₀ is the predominant Aβ species present in CAA whereas Aβ₄₂ is the major Aβ species present in neuritic plaques. CAA is a very common disorder, pathologically affecting about one-third of all elderly patients (>60 y of age) and about 90% of patients with AD (5, 6). CAA is a well-recognized cause of intracerebral hemorrhage (7, 8). It is also a major contributor to ischemic stroke and dementia (2, 9–12)—two conditions in which CAA-induced impairment in cerebral arteriole function is likely to play a fundamental role (13).Multiple lines of evidence indicate that soluble Aβ monomers and insoluble Aβ fibrils in the form of CAA cause significant cerebrovascular (CV) impairment. Ex vivo studies with isolated cerebral arterioles show that synthetic Aβ₄₀ (and to a lesser degree Aβ₄₂) induces direct vessel constriction, enhanced response to vasoconstrictors, and reduced response to vasodilators (14–22). Similar results have been demonstrated with synthetic Aβ₄₀ topically applied to the cerebral cortex (23, 24), results that are generally supported by in vivo studies (20, 23, 25). For example, Iadecola and coworkers have shown that young APP transgenic mice (Tg2576) exposed to elevated levels of Aβ₄₀ and Aβ₄₂ (but no CAA) have reduced baseline cerebral blood flow (CBF) and decreased CBF responses to topical vasodilators (23, 24, 26). We have shown similar CV deficits in young Tg2576 mice (13). Moreover, we provided the most direct evidence to date that endogenous soluble Aβ plays a causal role in these CV deficits when we found that depletion of soluble Aβ via γ-secretase inhibition restores CV function in young Tg2576 mice (13).Fibrillar Aβ in the form of CAA produces even greater degrees of CV impairment. Evidence for this notion comes from several experimental studies from different laboratories that show reduced pial arteriole responses (27) and diminished CBF responses (27, 28) to a variety of vasodilatory stimuli in aged APP mice with CAA vs. young APP mice without CAA. Our past work examining pial arteriole function in young vs. aged Tg2576 mice shows similar age-dependent CV deficits (13). Moreover, multiple additional observations from our study show that CAA (and not prolonged exposure to soluble Aβ and/or mutant APP) is the principle cause of the severe CV dysfunction noted in aged Tg2576 mice: (i) The severity of the vasomotor deficits noted in these mice is dependent on the presence and extent of CAA; (ii) even small amounts of CAA are associated with profound vasomotor impairment; and (iii) the CV dysfunction noted in CAA-ladened arteries is poorly responsive to depletion of soluble Aβ via γ-secretase inhibition (13).Regarding the mechanism of soluble Aβ-induced CV deficits, increased reactive oxygen species (ROS) are strongly implicated. Cerebral arterioles exposed to exogenous Aβ₄₀ develop significant oxidative stress (29), and various anti-ROS strategies have been shown to improve Aβ₄₀-induced vessel dysfunction (16, 23). Similarly, cerebral arterioles of young APP mice producing elevated levels of endogenous Aβ₄₀ and Aβ₄₂ (but no CAA) display oxidative stress (19), and the CV deficits found in these mice can be attenuated by both genetic and pharmacologic anti-ROS interventions (15, 19, 20, 30). In particular, ROS derived from NADPH oxidase—one of two major sources of ROS in the cerebrovasculature (31–33)—have been implicated (28–30, 34, 35).Regarding the mechanism of CAA-induced CV deficits, far less is known; however, three recent findings suggest that ROS may play a role. First, CAA-affected vessels were shown to have significantly greater oxidative stress than CAA-free vessels of aged Tg2576 mice (36). Second, genetic knockdown of mitochondrial superoxide dismutase 2 (SOD2)—which increases mitochondria-derived ROS—was shown to exacerbate CAA pathology in aged APP mice (37). Third, genetic depletion of the catalytic subunit Nox2 of NADPH oxidase was shown to reduce oxidative stress and improve CV function in aged Tg2576 mice (28, 35). Importantly, the latter studies did not examine for the presence of CAA, nor did they assess for the effect of CAA on cerebral arteriole function (28, 35). To address this critical knowledge gap, we examined the effect of the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, on CAA-induced CV dysfunction in aged Tg2576 mice. The effect of these agents on CAA formation and CAA-related microhemorrhage was also examined.