Katušić, Zvonimir S.

Tetrahydrobiopterin (BH 4 ) is an essential cofactor required for enzymatic activity of endothelial NO synthase. Recently, it has been shown that vascular protective effects of erythropoietin (EPO) are dependent on activation of endothelial NO synthase. Therefore, our objective was to characterize the effect of EPO on the biosynthesis of BH 4 in the vascular wall. Incubation of isolated C57BL/6J mouse aortas for 18 hours with recombinant human EPO (1 to 50 U/mL) caused a concentration-dependent increase in intracellular BH 4 levels and activity of GTP-cyclohydrolase I. Maximal biosynthesis of BH 4 was detected at therapeutic concentrations of 5 U/mL. Removal of the endothelium abolished EPO-induced biosynthesis of BH 4 demonstrating that the vascular endothelium is a major source of BH 4 . Treatment with a selective phosphatidylinositol 3-kinase inhibitor wortmannin significantly reduced BH 4 biosynthesis stimulated by EPO. The stimulatory effect of EPO on vascular GTP-cyclohydrolase I activity, BH 4 production, and phosphorylation of endothelial NO synthase was also detected in vivo in mice treated with recombinant human EPO. These effects of EPO were abolished in protein kinase Bα/Akt1-deficient mice. In addition, EPO significantly increased systolic blood pressure and the number of circulating platelets in Akt1-deficient mice. Our results demonstrate that EPO stimulates biosynthesis of BH 4 in vascular endothelium and that the increase in BH 4 levels is caused by de novo biosynthesis of BH 4 via the phosphatidylinositol 3-kinase/Akt1 pathway. This effect is most likely designed to provide optimal intracellular concentration of the cofactor necessary for EPO-induced elevation of endothelial NO synthase activity.

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI 2 ) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI 2 biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-α. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI 2 analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI 2 synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator–activated receptor (PPAR)δ agonist GW501516 but not by the selective PGI 2 receptor agonist cicaprost. Downregulation of PPARδ by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPARδ siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI 2 synthase siRNA, COX-1 siRNA, or PPARδ siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI 2 /PPARδ pathway is an important mechanism underlying proangiogenic function of EPCs.

In the present study we hypothesized that endothelial progenitor cells (EPCs) enhance production of vasoprotective substances in cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM-2) for 7 days to yield EPCs. Rabbit basilar arteries were exposed to autologous EPCs (≈5×10 5 cells) in vitro or in vivo. Twenty-four hours after intracisternal delivery of autologous EPCs, basilar arteries were isolated and expression of vasoregulatory proteins, production of prostacyclin (PGI 2 ), and cAMP were determined. Arteries transplanted with EPCs demonstrated increased protein expression of cyclooxygenase-2 and PGI 2 in adventitia, media, and endothelium. Furthermore, production of PGI 2 and arterial content of cAMP, second messenger for PGI 2 , were significantly augmented after transplantation of EPCs. In contrast, production of thromboxane A 2 was significantly reduced, whereas production of prostaglandin E 2 remained unchanged. The increased production of PGI 2 and arterial content of cAMP were inhibited only by a selective cyclooxygenase-2 inhibitor, NS-398. In vitro or in vivo treatment of basilar artery with conditioned media from EPCs also caused increase in cyclooxygenase-2 and PGI 2 synthase protein expression associated with elevation of cAMP. Our results suggest that in cerebral arteries, paracrine effect of EPCs promotes vasoprotection by increasing PGI 2 production and intracellular concentration of cAMP. This effect appears to be mediated by activation of arachidonic acid metabolism via stimulation of cyclooxygenase-2/PGI 2 synthase pathway.

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.

We studied the arachidonic acid (AA)-mediated modulation of large-conductance Ca2+-activated K+ (BK) channels in coronary arterial smooth myocytes from lean control and Zucker Diabetic Fatty (ZDF) rats. A total of 1 μmol/l AA enhanced BK current by 274% in lean and by 98% in ZDF rats. After incubation with 10 μmol/l indomethacin, 1 μmol/l AA increased BK currents by 80% in lean and by 70% in ZDF rats. Vasoreactivity studies showed that the dilation of small coronary arteries produced by 1 μmol/l AA was reduced by 44% in ZDF rats. [3H]6-keto-prostagladin F1α ([3H]6-keto-PGF1α,), the stable metabolite of prostacyclin (PGI2), was the major [3H]AA metabolite produced by coronary arteries of lean vessels, but ZDF vessels produced only 15% as much [3H]6-keto-PGF1α. BK channel activation and vasorelaxation by iloprost were similar in lean and ZDF rats. Immunoblots showed a 73% reduction in PGI2 synthase (PGIS) expression in ZDF vessels compared with lean vessels, and there was no change in cyclooxygenase (COX) and BK channel expressions. Real-time PCR studies showed that mRNA levels of PGIS, COX-1, and COX-2 were similar between lean and ZDF vessels. We conclude that PGI2 is the major AA metabolite in lean coronaries, and AA-mediated BK channel activation is impaired in ZDF coronaries due to reduced PGIS activity.

Background and Purpose— Aging is a major risk for stroke and a highly complex biological process believed to involve multiple mechanisms. Mutant mice that express low levels of the spindle assembly checkpoint protein BubR1 are known to develop several aging-associated phenotypes at a very young age, including cataracts, lordokyphosis, loss of subcutaneous fat, and impaired wound healing. However, whether BubR1 acts to prevent vascular aging has not yet been established. The present study was designed to investigate the vascular phenotype of mutant mice with low levels of BubR1. Methods— Morphological, functional, and biochemical analyses were performed on aortas and carotid arteries of 3- to 5-month-old BubR1 mutant mice and wild-type littermates. Results— Arterial wall thickness and inner diameter were significantly reduced in BubR1 mutant mice. Arterial walls of BubR1 mutant mice had low numbers of medial smooth muscle cells. Masson trichrome staining showed profound fibrosis in arterial walls of BubR1 mutant. In agreement with these morphological changes, functional analysis of pressurized isolated carotid arteries of BubR1 mutant mice demonstrated reduced elastic properties. Endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to the nitric oxide donor DEA-NONOate were significantly reduced in carotid arteries of BubR1 mutant mice. Furthermore, enzymatic activity of nitric oxide synthase and levels of cyclic GMP were significantly reduced in aortas of mutant mice, but production of superoxide anions was significantly increased. Conclusions— These findings demonstrate that BubR1 insufficiency in mice results in phenotypic changes reminiscent of vascular aging in humans and suggest a role for BubR1 in suppressing the vascular aging process.

Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K ATP channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress‐responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine‐ or metabolic‐mediated vasodilation necessary to support myocardial perfusion in the wild‐type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K ATP channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1containing K ATP channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock.—Kane, G. C., Lam, C‐F., O'Cochlain, F., Hodgson, D. M., Reyes, S., Liu, X‐K., Miki, T., Seino, S., Katusic, Z. S., Terzic, A. Gene knockout of the KCNJ8encoded Kir6.1 KATP channel imparts fatal susceptibility to endotoxemia. FASEB J. 20, 2271–2280 (2006)

Diabetic vascular dysfunction is associated with an increase in reactive oxygen species (ROS). In this study, we hypothesized that hyperglycemia-induced ROS generation would impair the function of large conductance Ca 2+ -activated K + (BK) channels, which are major determinants in vasorelaxation. We found that when cultured in high glucose (HG) (22 mmol/L), HEK293 cells showed a reduction in expressed hSlo current densities, as well as slowed activation and deactivation kinetics. When human coronary smooth muscle cells were cultured in HG, similar findings were observed for the BK currents. HG enhanced superoxide dismutase and suppressed catalase (CAT) expression in HEK293 cells, leading to a significant increase in intracellular ROS. The effects of HG were mimicked by hydrogen peroxide (H 2 O 2 ), and hSlo functions were restored by CAT gene transfer. Peroxynitrite inhibited hSlo current density but did not change channel kinetics. The hSloC911A mutant was insensitive to the effects of HG and H 2 O 2 . Hence, imbalance of antioxidant enzymes plays a critical role in ROS generation in HG, impairing hSlo functions through H 2 O 2 -dependent oxidation at cysteine 911. This may represent an important fundamental mechanism that contributes to the impairment of vasodilation in diabetes.

The National Heart, Lung, and Blood Institute (NHLBI) convened a conference call working group, consisting of experts in stroke and cerebrovascular biology on January 28, 2005. The purpose of this working group was to develop a prioritized set of recommendations for NHLBI to establish a focused and comprehensive set of research activities in cerebrovascular biology and disease. Three thematic areas of research emerged: (1) molecular and cellular neurobiology of cerebral blood vessels, focusing on genomics and proteomics, neurovascular signaling and cerebrovascular embryogenesis, development and plasticity; (2) resource development, involving the development of new methodological approaches for normal and altered function of the neurovascular unit, collaborative research, and training in cerebrovascular pathobiology; and (3) cerebrovascular diseases and translational approaches, addressing vascular mechanisms of disease, the role of risk factors, importance of biomarkers with the ultimate goal of developing new treatments.