Katušić, Zvonimir S.

The arteriovenous fistula (AVF) is the preferred hemodialysis vascular access, but it is complicated by high failure rates and attendant morbidity. This study provides the first description of a murine AVF model that recapitulates two salient features of hemodialysis AVFs, namely, anastomosis of end-vein to side-artery to create the AVF and the presence of chronic kidney disease (CKD). CKD reduced AVF blood flow, observed as early as 3 days after AVF creation, and increased neointimal hyperplasia, venous wall thickness, thrombus formation, and vasculopathic gene expression in the AVF. These adverse effects of CKD could not be ascribed to preexisting alterations in blood pressure or vascular reactivity in this CKD model. In addition to vasculopathic genes, CKD induced potentially vasoprotective genes in the AVF such as heme oxygenase-1 (HO-1) and HO-2. To determine whether prior HO-1 upregulation may protect in this model, we upregulated HO-1 by adeno-associated viral gene delivery, achieving marked venous induction of the HO-1 protein and HO activity. Such HO-1 upregulation improved AVF blood flow and decreased venous wall thickness in the AVF. Finally, we demonstrate that the administration of carbon monoxide, a product of HO, acutely increased AVF blood flow. This study thus demonstrates: 1) the feasibility of a clinically relevant murine AVF model created in the presence of CKD and involving an end-vein to side-artery anastomosis; 2) the exacerbatory effect of CKD on clinically relevant features of this model; and 3) the beneficial effects in this model conferred by HO-1 upregulation by adeno-associated viral gene delivery.

We tested hypothesis that activation of the prostacyclin (PGI 2 ) receptor (IP receptor) signaling pathway in cerebral microvessels plays an important role in the metabolism of amyloid precursor protein (APP). In human brain microvascular endothelial cells activation of IP receptor with the stable analogue of PGI 2 , iloprost, stimulated expression of amyloid precursor protein and a disintegrin and metalloprotease 10 (ADAM10), resulting in an increased production of the neuroprotective and anticoagulant molecule, soluble APPα (sAPPα). Selective agonist of IP receptor, cicaprost, and adenylyl cyclase activator, forskolin, also enhanced expression of amyloid precursor protein and ADAM10. Notably, in cerebral microvessels of IP receptor knockout mice, protein levels of APP and ADAM10 were reduced. In addition, iloprost increased protein levels of peroxisome proliferator-activated receptor δ (PPARδ) in human brain microvascular endothelial cells. PPARδ-siRNA abolished iloprost-augmented protein expression of ADAM10. In contrast, GW501516 (a selective agonist of PPARδ) upregulated ADAM10 and increased production of sAPPα. Genetic deletion of endothelial PPARδ (ePPARδ −/− ) in mice significantly reduced cerebral microvascular expression of ADAM10 and production of sAPPα. In vivo treatment with GW501516 increased sAPPα content in hippocampus of wild type mice but not in hippocampus of ePPARδ −/− mice. Our findings identified previously unrecognized role of IP-PPARδ signal transduction pathway in the production of sAPPα in cerebral microvasculature.

AbstractIn this study, we tested the hypothesis that reduced bioavailability of tetrahydrobiopterin (BH4) is a major mechanism responsible for pathogenesis of endothelial dysfunction in cerebral microvessels of transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (APP) (Tg2576 mice). Endothelial nitric oxide synthase (eNOS) protein expression was significantly increased in cerebral vasculature of Tg2576 mice. In contrast, bioavailability of BH4 was significantly reduced (p < 0.05). Moreover, superoxide anion production was increased in cerebral microvessels of Tg2576 mice (p < 0.05). Incubation with NOS inhibitor, Nω‐nitro‐L‐arginine methyl ester, decreased superoxide anion indicating that uncoupled eNOS is most likely the source of superoxide anion. Increasing BH4 bioavailability either exogenously by BH4 supplementation or endogenously by treatment with the selective peroxisome proliferator‐activated receptor – delta activator GW501516 (2 mg/kg/day, 14 days) attenuated eNOS uncoupling and decreased superoxide anion production in cerebral microvessels of Tg2576 mice (p < 0.05). Treatment with GW501516 restored the biological activity of endothelial nitric oxide in cerebral microvessels of Tg2576 mice, as indicated by the increased nitrite/nitrate content and 3,5‐cyclic guanosine monophosphate levels (p < 0.05). Our studies indicate that sub‐optimal BH4 bioavailability in cerebral vasculature is an important contributor to oxidant stress and endothelial dysfunction in Tg2576 mouse model of Alzheimer's disease. imageExisting evidence suggests that Aβ peptides‐induced up‐regulation of expression and activity of NADPH oxidase causes increased production of superoxide anion (.O2−). .O2− can also be converted to hydrogen peroxide (H2O2) by enzymatic activity of superoxide dismutase (SOD) or spontaneous dismutation. Elevation of .O2− and H2O2 might cause oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) and subsequent uncoupling of endothelial nitric oxide synthase (eNOS) (a) thus reducing levels of nitric oxide (NO) and 3′,5′‐cyclic guanosine monophosphate (cGMP). Supplementation of BH4 or activation of PPARδ prevents detrimental effects of eNOS uncoupling by restoring bioavailability of BH4 and scavenging of .O2−, respectively (b). Activation of PPARδ also increases expression of catalase thereby inactivating H2O2. Generation of H2O2 by uncoupled eNOS in cerebral microvessels of Tg2576 mice is hypothetical.

The present study examined the heme oxygenase (HO) system in an in vivo murine model of pathological shear stress induced by partial carotid artery ligation. In this model, along with upregulation of vasculopathic genes, HO-1 is induced in the endothelium and adventitia, whereas HO-2 is mainly upregulated in the endothelium. Within minutes of ligation, NF-κB, a transcription factor that upregulates vasculopathic genes and HO-1, is activated. Failure to express either HO-1 or HO-2 exaggerates the reduction in carotid blood flow and exacerbates vascular injury. After artery ligation, comparable induction of HO-2 occurred in HO-1+/+and HO-1−/−mice, whereas HO-1 induction was exaggerated in HO-2−/−mice compared with HO-2+/+mice. Upregulation of HO-1 by an adeno-associated viral vector increased vascular HO-1 expression and HO activity and augmented blood flow in both ligated and contralateral carotid arteries. Acute inhibition of HO activity decreased flow in the ligated carotid artery, whereas a product of HO, carbon monoxide (CO), delivered by CO-releasing molecule-3, increased carotid blood flow. In conclusion, in the partial carotid artery ligation model of pathological shear stress, this study provides the first demonstration of 1) upregulation and vasoprotective effects of HO-1 and HO-2 and the vasorelaxant effects of CO as well as 2) vascular upregulation of HO-1 in vivo by an adeno-associated viral vector that is attended by a salutary vascular response. Induction of HO-1 may reside in NF-κB activation, and, along with induced HO-2, such upregulation of HO-1 provides a countervailing vasoprotective response in pathological shear stress in vivo.

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37°C) to an adenoviral vector [107, 108, or 109plaque-forming units (pfu)/ml] encoding either the iNOS gene or the β-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (109pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10−9–10−3M) and diethylaminodiazen-1-ium-1,2-dioate (10−10–10−5M) were significantly shifted to the right in iNOS gene (109pfu/ml)-transduced rings compared with control and β-galactosidase-transduced rings ( P < 0.05, n = 5–6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings ( P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O[Formula: see text]·) production were elevated in iNOS-transduced rings ( P < 0.05, n = 7 for cGMP; P < 0.01, n = 6–9 for O[Formula: see text]· production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.

Although hydrogen peroxide (H2O2) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding β-galactosidase (Adβgal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by β-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adβgal-transduced cells (536.8 ± 31 vs. 1,875.1 ± 132.9 vs. 1,347.5 ± 93.7 dpm/well, respectively; P < 0.05 vs. control and Adβgal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 ± 1.8% of cell counts in control, P < 0.05; and 45 ± 2% of cell count in Adβgal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H2O2(50 μM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H2O2is an important modulator of endothelial cell proliferation.

AbstractThis study was designed to determine whether treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Wild‐type and GTP cyclohydrolase I (GTPCH‐I)‐deficient hph1 mice were administered EPO (1000 U/kg/day, s.c., 3 days). Cerebral microvessels of hph1 mice demonstrated reduced tetrahydrobiopterin (BH4) bioavailability, increased production of superoxide anions and impaired endothelial NO signaling. Treatment of hph1 mice with EPO attenuated the levels of 7,8‐dihydrobiopterin, the oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8‐dihydrobiopterin. Moreover, EPO decreased the levels of superoxide anions and increased NO bioavailability in cerebral microvessels of hph1 mice. Attenuated oxidation of BH4 and inhibition of endothelial NO synthase uncoupling were explained by the increased expression of antioxidant proteins, manganese superoxide dismutase, and catalase. The protective effects of EPO observed in cerebral microvessels of hph1 mice were also observed in GTPCH‐I siRNA‐treated human brain microvascular endothelial cells exposed to EPO (1 U/mL or 10 U/mL; 3 days). Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of BH4 and endothelial NO in the cerebral microvascular endothelium. image We demonstrate that treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of tetrahydrobiopterin (BH4) and endothelial nitric oxide.

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear hormone receptor that is mainly involved in lipid metabolism. Recent studies have suggested that PPAR-δ agonists exert vascular protective effects. The present study was designed to characterize vascular function in mice with genetic inactivation of PPAR-δ in the endothelium. Mice with vascular endothelial cell-specific deletion of the PPAR-δ gene (ePPARδ−/− mice) were generated using loxP/Cre technology. ePPARδ−/− mice were normotensive and did not display any sign of metabolic syndrome. Endothelium-dependent relaxations to ACh and endothelium-independent relaxations to the nitric oxide (NO) donor diethylammonium ( Z)-1-( N, N-diethylamino)diazen-1-ium-1,2-diolate were both significantly impaired in the aorta and carotid arteries of ePPARδ−/− mice ( P < 0.05). In ePPARδ−/− mouse aortas, phosphorylation of endothelial NO synthase at Ser1177 was significantly decreased ( P < 0.05). However, basal levels of cGMP were unexpectedly increased ( P < 0.05). Enzymatic activity of GTP-cyclohydrolase I and tetrahydrobiopterin levels were also enhanced in ePPARδ−/− mice ( P < 0.05). Most notably, endothelium-specific deletion of the PPAR-δ gene significantly decreased protein expressions of catalase and glutathione peroxidase 1 and resulted in increased levels of H2O2 in the aorta ( P < 0.05). In contrast, superoxide anion production was unaltered. Moreover, treatment with catalase prevented the endothelial dysfunction and elevation of cGMP detected in aortas of ePPARδ−/− mice. The findings suggest that increased levels of cGMP caused by H2O2 impair vasodilator reactivity to endogenous and exogenous NO. We speculate that chronic elevation of H2O2 predisposes PPAR-δ-deficient arteries to oxidative stress and vascular dysfunction.