Santhanam, Anantha V.

In the present study, we tested hypothesis that hyperglycemia impairs the neuro-protective function of cerebral microvessels. Type 1 diabetes (T1D) was induced by treatment of mice with streptozotocin (blood glucose >300 mg/dL for 2 months). Brain tissues and cerebral microvessels were isolated from diabetic and control mice. We observed significant decrease in the protein levels of soluble amyloid precursor protein-α (sAPPα, a product of α-cleavage of APP and a neurotrophic and neuroprotective molecule) in the total brain tissue of diabetic mice (n=5, P<0.05). Further examination of brain regions revealed that the reduction of protein levels of sAPPα was only detected in hippocampus and cortex but not in cerebellum (n=6, P<0.05). Our studies also revealed that there were no significant differences in the protein expression of APP and its non-amyloidogenic processing enzymes, α-secretases (a disintegrin and metalloprotease 10 [ADAM10], ADAM9, and ADAM17) in total brain tissue between T1D mice and control mice (n=5-6, P>0.05). The protein level of β-processing enzyme BACE1 was also not changed in T1D mouse brain. In contrast, the protein levels of APP and ADAM10 were significantly decreased in cerebral microvasculature of T1D mice, as compared to control mice (n=9-10, P<0.05). Immunofluorescent confocal microscopy also demonstrated that expressions of APP and ADAM10 in cerebral microvasculature (identified by co-staining with CD31) in hippocampus and cortex were reduced in T1D mice. Of note, cerebrovascular protein levels of ADAM9, ADAM17 and BACE1 were not affected by hyperglycemia (n=5-7, P>0.05). These results suggest that hyperglycemia impairs APP expression and α-processing in brain vasculature, thereby leading to the reduction of brain content of sAPPα. The selective loss of neuro-protective molecule sAPPα in hippocampus and cortex may be an important mechanism contributing to the development of cognitive dysfunction in diabetes.

We tested the hypothesis that endothelial PPARδ plays an important role in the protection of neurovascular unit. Cerebral microvessels were isolated from mice with endothelium-specific deficiency of PPARδ (ePPARδ-/-) and littermate wild type (WT) mice. After ex vivo incubation of cerebral microvessels for 24 h, supernatant was collected and levels of soluble amyloid precursor protein α [sAPPα, a product of non-amyloidogenic processing of amyloid precursor protein (APP)] were measured using an ELISA kit. The production of sAPPα in cerebral microvessels of ePPARδ-/- mice was reduced by 54%, as compared to WT mice (n=6-7, P<0.05). Western blot analysis demonstrated that microvascular expression of ADAM10 was reduced in ePPARδ-/- mice (80%) (n=18, P<0.05), while ADAM17 protein level was increased in by 132% (n=4, P<0.05), as compared to WT mice. Protein expressions of APP, ADAM9, and BACE1 were not significantly different between cerebral microvessels derived from ePPARδ-/- mice and WT mice. Real-time RT-PCR demonstrated that in brain microvessels of ePPARδ-/- mice mRNA levels of ADAM10 were also decreased by 56% (n=5, P<0.05). Furthermore, Western blot analysis of the brain regions showed that sAPPα content was selectively reduced in hippocampus of ePPARδ-/- mice by 80% (n=11, P<0.05). This was associated with significantly reduced expression of ADAM10 in the hippocampus (n=11, P<0.05). In contrast, protein levels of ADAM10, and sAPPα were not altered in cortex and cerebellum of ePPARδ-/- mice. In addition, we did not detect any changes in Aβ40 and Aβ42 levels (measured using ELISA assay) in all three examined brain regions. Since sAPPα is a potent neurotrophic and neuro-protective molecule, our results suggest that loss of endothelial PPARδ may selectively increase vulnerability of hippocampus. This mechanism may contribute to susceptibility of hippocampus to injury during initiation and progression of neuro-degenerative diseases.

Erythropoietin (EPO) has been recognized as a tissue protective cytokine. Recently, it has been shown that vascular protective effects of EPO are dependent on activation of endothelial nitric oxide synthase (eNOS). Tetrahydrobiopterin (BH 4 ) is an essential cofactor required for enzymatic activity of eNOS. Therefore, our objective was to characterize the effect of EPO on biosynthesis of BH 4 in vascular wall. Incubation of isolated wild-type (C57BL/6J) mouse aortas for 18 hours at 37°C in minimal essential medium supplemented with recombinant human EPO (1–50 U/ml) caused concentration-dependent increase in intracellular levels of BH 4 as determined by HPLC analysis. Maximum biosynthesis of BH 4 was detected at therapeutic concentrations of 5 U/mL (15.8±1.3 pmol/mg protein; P<0.05 vs control: 8.2±0.4 pmol/mg protein; n=6 – 8). Oxidative products of BH 4 , 7,8-dihydrobiopterin, were unaffected by EPO indicating that EPO does not affect oxidation of BH 4 . Removal of the endothelium abolished EPO-induced biosynthesis of BH 4 (P<0.05; n=5) demonstrating that the vascular endothelium is a major source of BH 4 . Treatment of intact isolated wild-type mouse aortas with a selective phosphatidylinositol (PI)3-kinase inhibitor wortmannin (1 μM) significantly reduced BH 4 biosynthesis by EPO (8.4±0.6 pmol/mg protein; P<0.05; n=6). Stimulatory effect of EPO on production of BH 4 in aorta was also detected in wild-type mice treated with recombinant human EPO (1000 U/kg, s.c. biweekly) for 14 days (P<0.05; n=6). This vascular effect was abolished in protein kinase B (Akt) 1-deficient mice treated with EPO (P<0.05; n=5). Furthermore, aortic GTP cyclohydrolase I (GTPCH I) enzymatic activity was augmented in EPO treated wild-type mice (0.48±0.12 pmol neopterin/mg protein; P<0.05 vs control: 0.23±0.05 pmol neopterin/mg; n=4) but not in EPO treated Akt 1-deficient mice (0.21±0.03 pmol neopterin/mg; n=4), indicating that the selective increase in BH 4 levels was caused by de-novo biosynthesis of BH 4 via Akt/GTPCH I pathway. Our results demonstrate that EPO stimulates biosynthesis of BH 4 in vascular endothelium. This effect is most likely designed to provide optimal intracellular concentration of cofactor necessary for EPO-induced elevation of eNOS activity.

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO is recognized as a tissue protective cytokine. In this study, we tested the hypothesis that EPO promotes recovery of injured vascular endothelium. A murine model of wire‐induced injury of common carotid artery was used to examine the therapeutic effect of EPO. Recombinant human EPO (1000 U/kg, s.c., biweekly) was administered for 2 weeks after which vascular reactivity of isolated carotid arteries was studied in vitro under pressurized condition using a video dimension analyzer. Changes in vascular structure were histologically assessed. Injured arteries exhibited impairment in endothelium‐dependent relaxations to acetylcholine and increased thickness of the medial cross sectional area (P<0.05; n=5–7). EPO normalized the vasorelaxant response of injured arteries to acetylcholine and prevented the increase in medial cross sectional area (30±1 x 10 ‐3 mm 2 ; P<0.05 vs. without EPO: 37±3 x 10 ‐3 mm 2 ; n=6). The number of circulating endothelial progenitor cells (EPCs), defined as CD34 + /Flk‐1 + cells in the peripheral blood, was significantly reduced 2 weeks after vascular injury (0.016±0.005%; P<0.05 vs. non‐injured control: 0.09±0.01%), but this was entirely restored by EPO (0.212±0.08%; P<0.05; n=5). Our results indicate that EPO accelerates recovery of the injured carotid artery endothelium, and such salutary effects of EPO may reflect augmentation in circulating EPCs induced by EPO.

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.

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A 2 (TXA 2 ). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA 2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.

The exact physiological function of amyloid-β precursor protein (APP) in endothelial cells is unknown. Endothelium-specific APP-deficient (eAPP −/− ) mice were created to gain new insights into the role of APP in the control of vascular endothelial function. Endothelium-dependent relaxations to acetylcholine were significantly impaired in basilar arteries of global APP knockout (APP −/− ) and eAPP −/− mice ( P < 0.05). In contrast, endothelium-independent relaxations to nitric oxide (NO)-donor diethylamine-NONOate were unchanged. Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated in large cerebral arteries of APP −/− mice and eAPP −/− mice as compared to respective wild-type littermates ( P < 0.05). Furthermore, basal levels of cyclic guanosine monophosphate (cGMP) were also significantly reduced in large cerebral arteries of APP-deficient mice ( P < 0.05). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate (cAMP) was not affected by genetic inactivation of APP in endothelial cells. By using siRNA to knockdown APP in cultured human brain microvascular endothelial cells we also found a significant downregulation of eNOS mRNA and protein expressions in APP-deficient endothelium ( P < 0.05). These findings indicate that under physiological conditions, expression of APP in cerebral vascular endothelium plays an important protective function by maintaining constitutive expression of eNOS .

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.

Rationale: The exact etiology of sporadic Alzheimer disease (AD) is unclear, but it is interesting that several cardiovascular risk factors are associated with higher incidence of AD. The link between these risk factors and AD has yet to be identified; however, a common feature is endothelial dysfunction, specifically, decreased bioavailability of nitric oxide (NO). Objective: To determine the relationship between endothelial derived NO and the expression and processing of amyloid precursor protein (APP). Methods and Results: We used human brain microvascular endothelial cells to examine the role of NO in modulating APP expression and processing in vitro. Inhibition of endothelial nitric oxide synthase (eNOS) with the specific NOS inhibitor L-NAME ( N G -nitro- l -arginine methyl ester) led to increased APP and BACE1 (β-site APP-cleaving enzyme1) protein levels, as well as increased secretion of the amyloidogenic peptide amyloid β (Aβ) (control 10.93±0.70 pg/mL; L-NAME 168.21±27.38 pg/mL; P <0.001). To examine the role of NO in modulation of APP expression and processing in vivo, we used brain and cerebral microvessels from eNOS-deficient (eNOS −/− ) mice. Brain tissue from eNOS −/− mice had statistically higher APP and BACE1 protein levels, as well as increased BACE1 enzyme activity and Aβ (Aβ 1 - 42 wild-type control, 0.737pg/mg; eNOS −/− , 1.475 pg/mg; P <0.05), compared with wild-type controls (n=6 to 8 animals per background). Brain microvessels from eNOS −/− mice also showed statistically higher BACE1 protein levels as compared with wild-type control. Conclusions: Our data suggest that endothelial NO plays an important role in modulating APP expression and processing within the brain and cerebrovasculature. The NO/cGMP pathway may be an important therapeutic target in preventing and treating mild cognitive impairment, as well as AD.

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO has been recognized as a tissue-protective cytokine. In this study, we tested the hypothesis that endothelial NO synthase (eNOS) plays a key role in the vascular protective effect of EPO. A murine model of wire-induced injury of carotid artery was used to examine the effect of EPO on endothelial repair and arterial wall architecture. Recombinant human EPO (1000 U/kg, SC, biweekly) was administered for 2 weeks in wild-type and eNOS-deficient mice after which reactivity of isolated carotid arteries was studied in vitro, and the vasculature was histologically assessed. Injured arteries exhibited impairment of endothelium-dependent relaxations to acetylcholine ( P <0.05). This was associated with increased medial cross-sectional area ( P <0.05). EPO upregulated expression of phosphorylated Ser1177-eNOS and normalized the vasodilator response to acetylcholine ( P <0.05). Furthermore, EPO prevented the injury-induced increase in medial cross-sectional area ( P <0.05). The vascular protective effects of EPO were abolished in eNOS-deficient mice. Most notably, EPO significantly increased systolic blood pressure and enhanced medial thickening of injured carotid arteries in eNOS-deficient mice ( P <0.05). Our results demonstrate that EPO prevents aberrant remodeling of the injured carotid artery. The protective effects of EPO are critically dependent on activation of eNOS.