Santhanam, Anantha Vijay R.

Cerebrovascular effects of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) inactivation have not been systematically studied. In the present study we employed cultured human brain microvascular endothelial cells (BMECs), BACE1-knockout (BACE1 −/− ) mice and conditional (tamoxifen-induced) endothelium-specific BACE1-knockout (eBACE1 −/− ) mice to determine effect of BACE1 inhibition on expression and function of endothelial nitric oxide synthase (eNOS). Deletion of BACE1 caused upregulation of eNOS and glypican-1 (GPC1) in human BMECs treated with BACE1-siRNA, and cerebral microvessels of male BACE1 −/− mice and male eBACE1 −/− mice. In addition, BACE1siRNA treatment increased NO production in human BMECs. These effects appeared to be independent of amyloid β-peptide production. Furthermore, adenoviral-mediated overexpression of BACE1 in human BMECs down-regulated GPC1 and eNOS. Treatment of human BMECs with GPC1siRNA suppressed mRNA and protein levels of eNOS. In basilar arteries of male eBACE1 −/− mice, endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to NO donor, DEA-NONOate, were not affected, consistent with unchanged expression of eNOS and phosphorylation of eNOS at Ser1177 in large cerebral arteries. In aggregate, our findings suggest that under physiological conditions, inactivation of endothelial BACE1 increases expression of eNOS in cerebral microvessels but not in large brain arteries. This effect appears to be mediated by increased GPC1 expression.

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.

Background: Optimal availability of tetrahydrobiopterin (BH4) is a critical determinant of nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) in the vascular endothelium. Biosynthesis of BH4 is regulated by the enzymatic activation of GTP cyclohydrolase I (GTPCH-I). While the physiological role of GTPCH-I and BH4 have been extensively characterized in peripheral vasculature, their role in regulation of cerebral vascular function has not been investigated. Methods: The role of GTPCH-I in regulation of cerebral vascular function was studied in cerebral microvessels isolated from wild-type (WT) mice and from mice with endothelium-targeted overexpression of GTPCH-I (eGTPCH-I Tg) mice. Vascular protein expression, intracellular levels of biopterin and cGMP (second messenger of NO) as well as production of NO and superoxide anions were determined. Results: Endothelium targeted over-expression of GTPCH-I resulted in significant increase in levels of BH4, as well as its oxidized product, 7,8-dihydrobiopterin (7,8-BH2). Importantly, ratio of BH4 to 7,8-BH2, indicative of BH4 available for eNOS activation, was significantly increased in cerebral microvessels of eGTPCH-I Tg mice. However, protein expression of eNOS, levels of nitrate/nitrite - indicative of NO production remained unchanged between cerebral microvessels of WT mice and eGTPCH-I Tg mice. Furthermore, increased BH4 biosynthesis did not affect production of superoxide anions or expression of antioxidant enzymes. Moreover, intracellular levels of cGMP, reflective of NO signaling and activation of soluble guanylate cyclase, were not affected in eGTPCH-I Tg mice. Conclusion: Our results suggest that, despite a significant increase in BH4 bioavailability, generation of endothelial NO in cerebral microvessels remained unchanged in eGTPCH-I Tg mice. We conclude that under physiological conditions the levels of BH4 are optimal for activation of eNOS and NO/cGMP signaling in wild-type mice.

Background: While reported neuroprotective effects of erythropoietin (EPO) make it an appealing candidate for its evaluation in protection of neurovascular unit during cerebrovascular or neurodegenerative disorders, effects of EPO on cerebral microvessels, the vascular component of neurovascular unit, have not been studied to date. The present study was designed to determine the effects of EPO in cerebral microvessels derived from wild-type mice and also from hph-1 mice, a genetic mouse model of BH4 deficiency. Methods: Hph-1 mice and wild-type littermates (C57BL/6 background) were administered recombinant human EPO (1000 U/kg/day) intraperitoneally for 3 days. Following treatment, mice were killed by injection of an overdose of pentobarbital, brains were removed and cerebral microvessels were isolated. Results: Treatment of wild-type mice with EPO did not affect BH4 bioavailability, superoxide anion production or basal cGMP levels. We have reported that cerebral microvessels of hph-1 mice demonstrated reduced bioavailability of BH4, increased production of superoxide anions and impaired endothelial NO/cGMP signaling. Treatment of hph-1 mice with EPO attenuated the levels of 7,8-dihydrobiopterin (7,8-BH2; P<0.05, n=5), oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8-BH2 (P<0.05, n=5), indicative of increased bioavailability of BH4 for eNOS activation. Increased superoxide anion production in cerebral microvessels of hph-1 mice were attenuated by EPO treatment (P<0.05, n=5). While eNOS expression remained unchanged, levels of cGMP were significantly increased on EPO treatment in hph-1 mice (P< 0.05, n=6). Furthermore, EPO treatment selectively increased expression of manganese superoxide dismutase. Conclusion: The ability of EPO to attenuate oxidative stress and restore bioavailability of endothelial NO in cerebral microvessels may help to explain mechanisms responsible for cerebrovascular protective effects of EPO.

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.

Background: The present study was designed to test the hypothesis that supplementation of tetrahydrobiopterin (BH4) to transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (Tg2576 mice) results in restoration of BH4 levels required for activation of endothelial nitric oxide synthase (eNOS), and in turn, prevents oxidative stress in cerebral microvasculature. Methods: Cerebral microvessels were obtained from 4-5 months old female wild-type and Tg2576 mice. Biopterin levels, enzymatic activity of GTP cyclohydrolase I (GTPCH-I) and superoxide production were measured by HPLC. The effects of supplementation of BH4 on oxidative stress were studied by injecting wild-type and Tg2576 mice subcutaneously with 100 mol/kg (b.w.) of BH 4 ([ 6R ]-5,6,7,8-tetrahydro-L-biopterin dihydrochloride; [ 6R ]-BH4). Results: Enzymatic activity of GTPCH-I, rate limiting enzyme in BH4 biosynthesis, was not different between cerebral microvessels of wild-type and Tg2576 mice. However, bioavailability of BH4, was significantly reduced in cerebral microvessels of Tg2576 mice (P<0.05, n=8). Production of superoxide anions was significantly elevated in cerebral microvessels of Tg2576 mice (P<0.01, n=6), indicative of oxidative stress. This increased superoxide anion production was abolished by L-NAME, a NOS inhibitor, suggestive of eNOS uncoupling (P<0.05, n=6). Supplementation of [ 6R ]-BH4 to wild-type and Tg2576 mice resulted in significant increase in BH4 bioavailability (P<0.05, n=6). Notably, supplementation of [ 6R ]-BH4 abrogated the increase in superoxide anion production in cerebral microvessels of Tg2576 mice (P<0.05, n=5), while superoxide anion production remained unchanged in cerebral microvessels of WT mice. Furthermore, the inhibitory effects of L-NAME on superoxide anion production in cerebral microvessels of Tg2576 mice were abolished following [ 6R ]-BH4 supplementation (P<0.05, n=4). Conclusion: Supplementation of [ 6R ]-BH4 restored bioavailability of BH4, thereby abrogating superoxide anion production derived from eNOS. Our results suggest that uncoupling of eNOS contributes to oxidative stress in cerebral microvessels of Tg2576 mice.

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.

Objectives- Oxidative stress and endothelial dysfunction precedes cerebral β-amyloid deposits in human Alzheimer’s disease as well as in transgenic mouse models expressing mutations in amyloid precursor protein (APP). In the present study, we hypothesized that uncoupling of endothelial nitric oxide synthase (eNOS) caused by loss of its essential co-factor, tetrahydrobiopterin (BH 4 ), in cerebral arteries and microvessels contributes, in part, to oxidative stress and cerebral vascular dysfunction in a APP transgenic mouse (Tg2576) that express the Swedish double mutation of human APP. In addition, we examined whether treatment with Peroxisome Proliferator Activated Receptor-δ (PPARδ) activator - GW501516 restores bioavailability of BH 4 and reverses oxidative stress in APP Tg2576 mice. Methods- APP Tg2576 mice (4-5 months old) were treated with GW501516 , a selective PPARδ activator (2 mg/kg/day, po, 14 days). Following treatment, cerebral arteries and microvessels were obtained. Biopterin levels, enzymatic activity of GTP cyclohydrolase I (GTPCH I) and superoxide production were measured by HPLC and protein expression was studied by Western blotting. Results- Cerebral arteries and microvessels demonstrated increased expression of eNOS, while the bioavailability of its essential co-factor BH 4 was significantly reduced, suggestive of eNOS uncoupling in APP Tg2576 mice. Furthermore, expressions of catalase and manganese superoxide dismutase (MnSOD) were decreased, while superoxide production was increased (P<0.01, n=6) in cerebral microvessels of APP Tg2576 mice. Treatment with GW501516 restored the BH 4 /BH 2 ratio in cerebral arteries and microvessels of APP Tg2576 mice (P<0.05, n-7-8), while the enzymatic activity of GTPCH-I remained unchanged (P>0.05, n=6). PPARδ activation also prevented the attenuation in expressions of MnSOD and catalase, and inhibited the increased superoxide production (P<0.05, n=6) in cerebral arteries and microvessels of APP Tg2576 mice. Interestingly, PPARδ activation significantly inhibited the over-expression of APP in cerebral microvessels obtained from APP Tg2576 mice (P<0.001, n=4). Conclusion- Our results suggest that endothelial dysfunction in APP Tg2576 mice may be caused, in part, by reduced bioavailability of BH 4 and uncoupling of eNOS. Treatment of APP Tg2576 mice with PPARδ agonist GW501516 exerted cerebral vascular protection by multiple mechanisms: (a) by inhibiting eNOS uncoupling via increased expressions of MnSOD and catalase, as well as, (b) by attenuating the increased expression of APP in the cerebral microvessels of APP Tg2576 mice.

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.

In animal models, transplantation of CD34 + cells has beneficial effect on cerebral ischemia and recovery from stroke, however, the mechanism underlying cerebrovascular effects of CD34 + cells is poorly understood. In the present study, CD34 + cells were obtained by magnetic cell sorting of rabbit peripheral blood mononuclear cells. Rabbit basilar arteries were exposed to autologous CD34 + cells (~5×10 5 cells) in vitro or in vivo . Arteries treated with endothelial growth medium‐2 (EGM‐2, vehicle) served as controls. Twenty four hours after intracisternal delivery of cells, basilar arteries were isolated and studied. In arteries treated with CD34 + cells either in vivo or in vitro , protein expression of phosphorylated Ser‐473‐Akt and phosphorylated Ser‐1177 endothelial nitric oxide synthase (eNOS) were significantly increased (n = 6, P<0.05), while expressions of eNOS, Akt, inducible NOS, cyclooxygenase‐1 (COX‐1) and COX‐2 were not affected (n = 6, P>0.05). In arteries exposed to CD34 + cells in vitro , cGMP content was significantly augmented (n = 4, P<0.01), whereas cAMP levels remained unaffected (n = 4, P>0.05). Our results suggest that in basilar artery, CD34 + cells stimulate phosphorylation of Akt and eNOS thereby increasing endothelial NO production. These findings may help to explain described therapeutic effect of transplanted CD34 + cells in cerebral circulation.