Katušić, Zvonimir S.

Experiments were designed to characterize the mechanism of vasopressin action in small arteries of brain stem and cerebrum and to determine the role of L-arginine pathway in reactivity of these vessels to vasopressin. Secondary branches of canine basilar arteries (425 +/- 63 microns ID, n = 6) and middle cerebral arteries (466 +/- 30 microns ID, n = 6) were dissected and mounted on glass microcannulas in organ chambers. Changes in intraluminal diameter of the pressurized arteries were measured using a video dimension analyzer. Vasopressin caused endothelium-dependent relaxation in the brain stem arteries [-log half-maximal effective concentration (EC50) = 9.2 +/- 0.4, n = 5] but not in the branches of middle cerebral arteries. In contrast, bradykinin caused identical endothelium-dependent relaxations in arteries of both regions (-log EC50 = 8.0 +/- 0.2, n = 5, and 7.7 +/- 0.1, n = 4 for brain stem and cerebrum, respectively). Relaxations to vasopressin (but not to bradykinin) were reduced in the presence of V1-vasopressinergic antagonist [1-(beta-mercapto-beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine]arginine vasopressin [d(CH2)5-Tyr(Me)AVP;10(-7) M], pertussin toxin (100 ng/ml), and NG-monomethyl-L-arginine (L-NMMA; 10(-4) M). The inhibitory effect of L-NMMA was prevented by L-arginine (3 x 10(-4) M) but not D-arginine (3 x 10(-4) M). These studies suggest that vasopressin causes endothelium-dependent relaxation in canine brain stem arteries. The effect of the neuropeptide appears to be mediated by activation of endothelial V1-vasopressinergic receptors coupled to nitric oxide synthase. This signal transduction pathway is not functional in endothelial cells of branches of middle cerebral arteries.

Experiments were designed to compare the reactivity of canine basilar arteries to endothelium-derived relaxing factor (EDRF) and nitric oxide. Preparations with endothelium activated by bradykinin were used to study relaxations induced with EDRF, whereas the inhibitory effect of nitric oxide was studied in preparations without endothelium. All experiments were performed in the presence of indomethacin. EDRF- and nitric oxide-induced relaxations were significantly augmented in the presence of superoxide dismutase plus catalase but were reduced in the presence of methylene blue, LY 83583, and hemoglobin. M & B 22984 did not affect relaxations to either EDRF or nitric oxide. These results indicate that in the canine basilar artery EDRF is not an oxygen-derived free radical. The similar responsiveness of the basilar artery to EDRF and nitric oxide is consistent with the proposal that in the canine basilar artery nitric oxide is the factor.

The calcium ionophore A23187 causes endothelium-dependent contractions in canine basilar arteries. Removal of the endothelium, or treatment with indomethacin or superoxide dismutase (SOD), prevented the endothelium-dependent excitatory effect of the calcium ionophore. Catalase and deferoxamine were without effect. Superoxide anion generated by xanthine plus xanthine oxidase in the presence of catalase caused contractions of the vascular smooth muscle, which were abolished by SOD or heat inactivation of xanthine oxidase. The A23187-induced production of prostaglandins F2 alpha and E2 and thromboxane B2 was abolished by the removal of endothelium and by treatment with indomethacin but was not affected by the presence of SOD plus catalase. These observations are consistent with the hypothesis that superoxide anion, rather than prostaglandins generated by hydroperoxidase activity of cyclooxygenase, is an endothelium-derived contracting factor in canine cerebral arteries.

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.

Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the Toll-like receptor-4 (TLR4) receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-κB and its downstream gene monocyte chemoattractant protein-1(MCP-1); in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor Nrf2 and its downstream gene heme oxygenase-1 (HO-1). TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histological injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4+/+ and TLR4−/− mice. We conclude that 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; and 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in virtually all other AKI models studied to date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what occurs in vivo.

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 .

Amyloid precursor protein (APP) is evolutionary conserved protein expressed in endothelial cells of cerebral and peripheral arteries. In this review, we discuss mechanisms responsible for expression and proteolytic cleavage of APP in endothelial cells. We focus on physiological and pathological implications of APP expression in vascular endothelium.

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.