Katušić, Zvonimir S.

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.

Under physiological conditions, β-site amyloid precursor protein (APP)-cleaving enzyme 2 (BACE2) cleaves APP within Aβ sequence thereby functioning like a α-secretase. However, BACE2 could also function as a conditional β-secretase during aging, contributing to Alzheimer’s disease pathogenesis. To date the physiological functions of BACE2 in endothelium are largely unknown. The present study is therefore designed to investigate the role of BACE2 in APP metabolism in human BMECs. Cultured human BMECs (passage 5-6 or passage 22) were treated with BACE2siRNA (30 nM, for 3 days), levels of soluble APPα (sAPPα, a neurotrophic product of non-amyloidogenic processing of APP) and Aβ40 in the supernatant were measured. In human BMECs (passage 5-6), genetic inactivation of BACE2 significantly decreased production of sAPPα (n=12, P<0.05), but had no effect on production of Aβ40 (n=9, P>0.05). BACE2siRNA treatment significantly suppressed APP protein expression (n=7, P<0.05), but augmented protein levels of BACE1 (n=7, P<0.05). Genetic inactivation of BACE2 did not change protein levels of mature ADAM10 (n=7, P>0.05). Thus, reduced sAPPα secretion by BACE2siRNA treatment is likely caused not only by decreased α-secretase-like function of BACE2, but also by reduced APP expression. We further examined the effects of BACE2siRNA in senescent human BMECs. In cultured human BMECs (passage 22), protein expressions of senescent markers (p 21Cip1 and p 16INK4a ) were significantly increased (n=4, P<0.05). Genetic inactivation of BACE2 in senescent human BMECs also significantly suppressed secretion of sAPPα (n=8, P<0.05), but did not affect Aβ40 production (n=8, P>0.05). BACE2-siRNA treatment significantly inhibited protein expressions of APP and mature ADAM10 (n=7, P<0.05), but did not change BACE1 protein expression (n=7, P>0.05). Thus in senescent human BMECs, reduced APP expression and impaired α-processing may play important roles in the decreased sAPPα production. Since our previous studies have demonstrated that endothelial production of sAPPα significantly contributes to the sAPPα content in the hippocampus, our current findings suggests that inhibition of BACE2 could impair protective function of sAPPα in the hippocampus.

GTP Cyclohydrolase‐I (GTPCHI) is the rate‐limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), which is an essential cofactor for the enzymatic activity of endothelial nitric oxide synthase (eNOS). To better understand the role of GTPCHI in maintaining eNOS function, we analyzed GTPCHI subcellular localization and activity in human umbilical cord vein endothelial cells (HUVEC). Western blot analysis showed similar levels of GTPCHI protein expression in both Triton X‐100 ‐soluble and ‐insoluble fractions, suggesting membrane targeting of GTPCHI. Further analysis of membrane‐associated proteins using sucrose gradient ultracentrifugation revealed that GTPCHI protein expression was localized to caveolar microdomains along with caveolin‐1(Cav‐1) and eNOS. HPLC analysis for GTPCHI activity confirmed the localization of GTPCHI to caveolar microdomains as well as golgi‐associated membrane fractions. Using an in vitro GST pull‐down assay with recombinantGST‐caveolin‐1 fusion protein incubated with the lysate of HUVEC, we demonstrated specific binding of GTPCHI to caveolin‐1. To determine if caveolin‐1 may regulate GTPCHI activity in vivo, we analyzed aortas from Cav‐1 −/ − mice and were able to detect a two‐fold increase in BH4 production and GTPCHI activity in the aortas of Cav‐1 −/ − mice when compared to eNOS −/ − mice or wild type mice. These studies reveal that BH4 synthesis can occur in specialized membrane compartments in close proximity to eNOS and may be subjected to posttranslational regulation by caveolin‐1 which has an inhibitory effect on GTPCHI enzyme activity. Funded by NIH grant HL‐53524.

Previous studies demonstrated that treatment with erythropoietin (EPO) prevents endothelial dysfunction and medial thickening after wire‐induced injury of mouse carotid artery. In the present study, we tested the hypothesis that stimulatory effect of EPO on expression and activity of SOD1 is an important mechanism underlying the vascular protective effect of EPO. This hypothesis was based on observation that treatment with recombinant human EPO (1000 U/kg, s.c., biweekly for 2 weeks) significantly enhanced SOD1 protein expression by 31% in arteries of wild‐type mice (P<0.05; n=3). Analysis of the vascular effects of EPO was performed in SOD1‐deficient (SOD1 −/− ) mice. Two weeks after wire‐induced injury vascular reactivity of carotid arteries was studied in‐vitro under pressurized condition, and the arteries were histologically assessed. Endothelium‐dependent relaxations to acetylcholine were impaired in carotid arteries of SOD1 −/− mice (P<0.05 vs. control SOD1 −/− mice; n=7‐8) while endothelium‐independent relaxations to nitric oxide donor, DEA‐NONOate, were unaffected. This was associated with increased medial cross sectional area (33±2 x10 −3 mm 2 ; P<0.05 vs. controls: 28±1 x10 −3 mm 2 ; n=9‐10). Treatment with EPO did not affect endothelial dysfunction or medial thickening of injured carotid arteries in SOD1 −/− mice (n=7‐10). Furthermore, EPO did not prevent increased superoxide anion production in aortas of SOD1 −/− mice. These findings suggest that the vascular protective effects of EPO are dependent on activation of SOD1.

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.

The role of oxidative stress and antioxidant enzymes in the aging related dysfunction of EPCs has not been studied. We hypothesized that impaired antioxidant capacity contributes to dysfunction of EPCs. Early EPCs isolated from healthy young (24±3 year‐old, n=9) and aged (71±5 year‐old, n=9) males were examined. The levels of protein expression and enzymatic activity of glutathione peroxidase 1 (GPX1) were decreased significantly in the EPCs from aged individuals in compared to EPCs from young individuals (n=9, P<0.05). After 24 h of serum starvation EPC numbers in young group and aged group were 0.6±0.2 and 0.4±0.1, respectively (expressed as ratio to the cell number under normal culture conditions, n=6, P<0.05). H 2 O 2 (1mM) treatment for 24 h significantly decreased EPC number to 37±13% in aged, compared with young individuals (67±13%, n=3, P<0.05). Furthermore, under normal culture conditions the numbers of senescent β‐galactosidase (β‐gal) positive EPCs were not significantly different between young and aged subjects (46.1±8.4%, and 58.3±7.6, respectively, n=3). However, H 2 O 2 (50 μM) treatment significantly increased the β‐gal positive cells in aged EPCs (73±9%), compared with young EPCs (43±11%, n=3, P<0.05). These results suggest that EPCs of older individuals are more susceptible to oxidative stress induced by H 2 O 2 and serum starvation, resulting in decreased survival and increased senescence. Reduced expression of GPX1 may contribute to EPC dysfunction with aging.

Background and Purpose: Molecular mechanisms underlying increased risk of Alzheimer’s disease in hypertensive patients are poorly defined. Conflicting results have been reported regarding the effects of angiotensin II (Ang II) on expression of amyloid precursor protein (APP) and enzymes responsible for proteolytic cleavage of APP in the brain. However, no previous studies have directly assessed the effects of Ang II on metabolisms of APP in human endothelial cells. Current study was designed to determine the effects of Ang II on the expression and processing of APP in cultured human brain microvascular endothelial cells (hBMECs). Methods: Human BMECs were treated with increasing concentrations of Ang II (0.1nM to 100nM; incubation time = 24h). The protein expression of APP, α-secretase [a disintegrin and metalloprotease 10, (ADAM10), ADAM9, and ADAM17], β-secretase (β-site APP-cleaving enzyme, BACE1), NADPH oxidase subunits and endothelial nitric oxide synthase (eNOS) were measured by western blot. Production and release of amyloid β-peptides (Aβ) from endothelium were detected by ELISA. Results: In hBMECs, Ang II had no effect on the expression of APP (n=7-9, P>0.05) and α-secretase (ADAM10, n=7-9, P>0.05; ADAM9, n=4-7, P>0.05; ADAM17, n=4-7, P>0.05). Treatment with Ang II at 1nM concentration induced significant increase in BACE1 expression (n=13, P<0.05), however, we did not observe significant changes in production of Aβ40 (n=11, P>0.05) or Aβ42 (n=11, undetectable). The higher concentration of Ang II (100nM) increased expression of NOX4 (n=6, P<0.05). In addition, Ang II (1nM and 10nM) significantly up-regulated eNOS protein levels (n=8-10, P<0.05). Conclusion: Our findings suggest that in hBMECs: a) Ang II does not affect expression of APP and α-secretase-mediated cleavage of APP, and b) Ang II increases expression of BACE1 but does not elevate production and release of Aβ peptides. We also confirm previously reported ability of Ang II to increase expression of NOX4 and eNOS. We speculate that under pathological conditions, increased expression of BACE1 induced by Ang II may predispose cerebrovascular endothelium to elevated amyloidogenic processing of APP.

Amyloid-β precursor protein (APP) is an integral membrane protein expressed abundantly in the endothelium of cerebral arteries. However, the exact physiological function of APP in endothelial cells is unknown. Mice with vascular endothelium-specific deletion of APP gene (eAPP-KO) were generated using loxP/Cre technology to test the hypothesis that APP plays a key role in the control of vascular endothelial nitric oxide (NO) function. Vasoreactivity of isolated basilar arteries were studied in vitro under pressurized conditions and compared to control littermates. eAPP-KO mice were normotensive (118±2 mmHg vs. control mice: 113±2 mmHg; n=5) and plasma cholesterol and blood glucose levels were not affected by endothelium-specific genetic inactivation of APP. Endothelium-dependent relaxations to acetylcholine were significantly impaired in eAPP-KO mice (maximal relaxation: 30±4% vs. 45±5% for control littermates; P<0.05; n=7-8) while endothelium-independent relaxations to NO-donor diethylamine-NONOate were unchanged (n=5-6). Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated by 31% in cerebral arteries of eAPP-KO mice (P<0.05 vs. littermates; n=6). Furthermore, basal levels of cyclic guanosine monophosphate were also significantly reduced in cerebral arteries of eAPP-KO mice (0.64±0.09 pmol/mg; P<0.05 vs. littermates: 0.97±0.11 pmol/mg; n=11). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate were not affected by genetic inactivation of APP in endothelial cells (n=5-7). Moreover, superoxide anion levels as determined by HPLC analysis of 2-hydroxyethidium were unaltered in eAPP-KO mice cerebral arteries (1.4±0.2 nmol/mg vs. littermates: 1.2±0.1 nmol/mg; P=n.s.; n=7). Our results demonstrate that impaired endothelium-dependent relaxations to acetylcholine in eAPP-KO mice are caused by the reduced expression and function of eNOS. These findings indicate that under physiological conditions APP expression in cerebral vascular endothelium plays an important role in control of endothelial function.

Evidence continues to accumulate on the importance of endothelial progenitor cells (EPCs) in neovascularization of ischemic tissues and repair of injured endothelium. The present study was designed to determine the in vivo effect of EPCs on the vasomotor function of rabbit cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM‐2) for 7 days, yielding EPCs. Rabbits were injected intracisternally with either autologous EPCs (1x10 5 to 5x10 5 cells) or vehicle (EGM‐2) as control. After 24 hours, basilar arteries were isolated, vasomotor function was studied in organ chambers, and protein expression in the basilar arteries quantified by Western blot analysis. In rabbits treated with EPCs, contractions to either potassium chloride (80 mmol/L) or histamine (10 −9 to 10 −5 mol/L) were not affected. However, endothelium‐dependent relaxations to acetylcholine (10 −9 to 10 −5 mol/L) and endothelium‐independent relaxations to a NO donor, DEA‐NONOate (10 −9 to 10 −5 mol/L) were enhanced in basilar arteries from EPC‐treated rabbits. Western blot studies demonstrated increased expressions of cyclooxygenase‐2 and prostacyclin synthase (PGI 2 synthase) only in arteries exposed to EPCs. Results of the present study suggest that in vivo administration of EPCs could augment vasodilatation of the cerebral arteries by increasing vascular production of prostacyclin. Funded by NIH HL66958.

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.