Austin, Susan A.

Background: Amyloid precursor protein (APP) is expressed in neuronal and non-neuronal tissues in the brain, including cerebrovascular endothelium. However, the physiological role of APP in cerebral vasculature is not completely understood. The present study was designed to determine the effects of inactivation of APP in cerebral microvasculature. Methods: Effect of genetic inactivation of APP was studied both in vitro and in vivo . Cultured human brain microvascular endothelial cells (hBMECs) were incubated with APP-siRNA in vitro , while control-siRNA treated hBMECs served as controls. To study the effect of genetic inactivation of APP in vivo , cerebral microvessels were obtained from APP-deficient (APPKO) mice. Cerebral microvessels from wild-type (C57BL/6) littermates served as controls. Results: Silencing APP expression in hBMECs resulted in selective reduction in endothelial nitric oxide synthase (eNOS) expression (P<0.05, n=6), while expressions of inducible NOS and prostacyclin (PGI2) synthase remained unchanged. Furthermore, loss of APP in hBMECs resulted in significantly increased production (P<0.05, n=5) of superoxide anions, as determined by quantitation of 2-hydroxyethidium from dihydroethidium using HPLC. In line with the results obtained from in vitro studies, cerebral microvessels of APPKO mice also demonstrated increased production of superoxide anions. Furthermore, levels of cGMP, second messenger of endothelial NO, were significantly attenuated in cerebral microvessels of APPKO mice, while levels of cAMP remained unchanged. Conclusions: Our results suggest that genetic inactivation of APP results in oxidative stress and impairment of endothelial NO signaling. We speculate that APP exerts vascular protective effects in the cerebral circulation under physiological conditions.

Several cardiovascular risk factors are associated with an increased incidence of Alzheimer’s disease (AD); however, the mechanistic link is unknown. A common feature of cardiovascular risk factors is endothelial dysfunction, specifically, loss of bioavailability of endothelial nitric oxide (NO). Recently, we demonstrated that loss of endothelial NO led to increased protein levels of amyloid precursor protein (APP), β-site APP cleaving enzyme 1 (BACE1) and beta amyloid (Aβ) peptide. APP, the parent protein, when sequentially cleaved by BACE1 and γ-secretase generates amyloidogenic, Aβ, the primary component of AD plaques. Our data was the first to demonstrate a possible mechanism linking endothelial dysfunction, via loss of endothelial NO, with the upregulation of important AD related proteins in the cerebral microvasculature. Therefore, we sought to determine if supplementation of cultured human brain microvascular endothelial cells with NO, by utilizing the NO donor, DETA NONOate, would attenuate APP and BACE1 protein. DETA NONOate, 10 -8 to 10 -6 M, reduced expression of APP and BACE1 as compared to untreated control cells (n=3). To test the effects of NO supplementation in vivo, we treated endothelial nitric oxide synthase deficient (eNOS -/- ) mice with 30 mg/kg nitroglycerine b.i.d. or vehicle via subcutaneous injections for 3 days. No difference was observed in blood pressure between animals treated with vehicle (n=7) or nitroglycerine (n=6) (P>0.05) or before and after nitroglycerine treatment (n=6, P>0.05). As expected, cerebral microvessles of eNOS -/- animals treated with nitroglycerine displayed increased levels of cGMP as compared to vehicle (n=7-8; P<0.05). Moreover, APP and BACE1 protein levels were statistically lower in cerebral microvessels from eNOS -/- mice treated with nitroglycerine as compared to vehicle treated (n=6-7, APP, P<0.05; BACE1, P<0.05). These data indicate that supplementation with NO is able to suppress APP and BACE1 levels in the cerebrovascular endothelium. Our findings suggest that preservation of NO/cGMP signaling may be an important therapeutic target in preventing and treating mild cognitive impairment as well as AD.

Aging and other cardiovascular risk factors are associated with increased incidence of Alzheimer's disease (AD). A common feature of cardiovascular risk factors is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelial NO on amyloid precursor protein (APP) related phenotype in aged (14–15 month) endothelial nitric oxide synthase deficient (eNOS −/− ) mice. APP, β‐site APP cleaving enzyme 1, and amyloid beta (Aβ) levels were significantly higher in the brains of aged eNOS −/− mice as compared to aged wild type mice. APP and Aβ 1–40 were increased in hippocampal tissue of eNOS −/− mice as compared to wild type mice. Aged eNOS −/− mice displayed an increased inflammatory phenotype as compared to aged wild type mice. Importantly, aged eNOS −/− mice performed worse in a radial arm maze test of spatial learning and memory as compared to aged wild type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline. Research was supported by NIH grants HL‐111062 and HL‐91867, the Mayo Alzheimer's Disease Research Center (Z.S.K.), AHA scientist development grant (0835436N, A.V.S.), AHA postdoctoral fellowship 12POST8550003 (S.A.A.), Clinical Pharmacology Training Grant (T32 GM08685) (Trainee S.A.A.), the Samuel Johnson Foundation for Genomics of Addiction Program at Mayo Clinic (D.‐S.C.) and the Mayo Foundation.

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

Cerebral amyloid angiopathy (CAA) is present in over half of the elderly population and in 80–90% of Alzheimer’s disease (AD) patients. CAA is defined by the deposition of beta amyloid (Aβ) in small cerebral arteries and capillaries. Cardiovascular risk factors are associated with an increased incidence of CAA. We utilized 18-month-old endothelial nitric oxide synthase (eNOS) heterozygous knockout ( +/− ) mice, a clinically relevant model of endothelial dysfunction, to examine the role of endothelial nitric oxide (NO) in vascular Aβ accumulation. eNOS +/− mice had significantly higher vascular levels of Aβ40 ( P < 0.05). Aβ42 was not detected. There was no difference in Aβ in brain tissue. Amyloid precursor protein and β-site APP cleavage enzyme 1 protein levels were unaltered, while levels of the α-secretase enzyme, a disintegrin and metalloproteinase 10, were significantly lower in eNOS + /− microvascular tissue ( P < 0.05). Insulin degrading enzyme and low-density lipoprotein receptor-related protein 1 were significantly increased in eNOS +/− microvascular tissue, most likely an adaptive response to locally higher Aβ concentrations. Lastly, catalase and CuZn superoxide dismutase were significantly elevated in eNOS +/− microvascular tissue ( P < 0.05). These data demonstrate decreased availability of endothelial NO leads to increased cerebrovascular concentration of Aβ along with compensatory mechanisms to protect the vasculature.

Rationale: Alzheimer’s disease has an unknown pathogenesis; however, cardiovascular risk factors are associated with a higher incidence of Alzheimer’s disease. A defining feature of endothelial dysfunction induced by cardiovascular risk factors is reduced bioavailable endothelial nitric oxide (NO). We previously demonstrated that endothelial NO acts as an important signaling molecule in neuronal tissue. Objective: We sought to determine the relationship between the loss of endothelial NO synthase (eNOS) and tau phosphorylation in neuronal tissue. Methods and Results: We used eNOS knockout ( −/− ) mice as well as an Alzheimer’s disease mouse model, amyloid precursor protein (APP)/PSEN1dE9 +/− (PS1) that lacked eNOS (APP/PS1/eNOS −/− ) to examine expression of tau kinases and tau phosphorylation. Brain tissue from eNOS −/− mice had statistically higher ratios of p25/p35, indicative of increased cyclin-dependent kinase 5 activity as compared with wild-type (n=8, P <0.05). However, tau phosphorylation was unchanged in eNOS −/− mice ( P >0.05). Next, we determined the role of NO in tau pathology in APP/PS1/eNOS −/− . These mice had significantly higher levels of p25, a higher p25/p35 ratio (n=12–14; P <0.05), and significantly higher cyclin-dependent kinase 5 activity (n=4; P <0.001). Importantly, APP/PS1/eNOS −/− mice also had significantly increased tau phosphorylation (n=4–6; P <0.05). No other changes in amyloid pathology, antioxidant pathways, or neuroinflammation were observed in APP/PS1/eNOS −/− mice as compared with APP/PS1 mice. Conclusions: Our data suggests that loss of endothelial NO plays an important role in the generation of p25 and resulting tau phosphorylation in neuronal tissue. These findings provide important new insights into the molecular mechanisms linking endothelial dysfunction with the pathogenesis of Alzheimer’s disease.

Abstract Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer's disease. A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14–15 month) endothelial nitric oxide synthase deficient ( eNOS −/− ) mice. APP, β‐site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS −/− mice as compared with LMA wild‐type controls. APP and Aβ 1‐40 were increased in hippocampal tissue of eNOS −/− mice as compared with wild‐type mice. LMA eNOS −/− mice displayed an increased inflammatory phenotype as compared with LMA wild‐type mice. Importantly, LMA eNOS −/− mice performed worse in a radial arm maze test of spatial learning and memory as compared with LMA wild‐type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline. image Cardiovascular risk factors are associated with increased incidence of Alzheimer's disease (AD). A common feature of these risk factors is decreased endothelial nitric oxide (NO). We observed, in mice deficient in endothelial nitric oxide synthase, increased amyloid precursor protein (APP), β‐site APP cleaving enzyme 1, amyloid beta levels, microglial activation, and impaired spatial memory. This suggests chronic loss of endothelial NO may be an important contributor to the pathogenesis of sporadic AD.