Glucocorticoids inhibit tetrahydrobiopterin-dependent endothelial function.

Tetrahydrobiopterin (BH4) acts as an important co-factor for endothelial nitric oxide synthase (eNOS). Glucocorticoids have been shown to inhibit expression of the rate-limiting enzyme for tetrahydrobiopterin synthesis, GTP cyclohydrolase, in other cell types. We hypothesized that endothelium-dependent vasodilator responses would be blunted in rats made hypertensive with dexamethasone. Further, we hypothesized that treatment of rat vascular segments with dexamethasone would result in attenuation of endothelial function accompanied by decreased GTP cyclohydrolase expression. We report that endothelium-dependent relaxation responses to the calcium ionophore A23187 are reduced in aortic rings from dexamethasone-hypertensive rats compared with sham values. Dexamethasone incubation abolishes contraction to Nomega-nitro-L-arginine (L-NNA, 10(-5) M) in endothelium-intact aortic rings, and inhibits expression of GTP cyclohydrolase. We conclude that inhibition of BH4 synthesis by glucocorticoid regulation of GTP cyclohydrolase expression may contribute to reduced endothelium-dependent vasodilation characteristic of glucocorticoid-induced hypertension.

Hibernation triggers and myocardial protection.

BACKGROUND: Hypothermic cardioplegia provides myocellular protection, yet postischemic dysfunction remains a significant problem. Interestingly, the subcellular changes in hibernation parallel the altered biology of induced cardiac ischemia but are well tolerated by hibernated mammalian myocardium. An uncharacterized factor derived from hibernating animals, hibernation induction trigger (HIT), has been shown to induce hibernation in active animals and afford myocardial protection after ischemia-reperfusion injury. Therefore, it was of interest to further characterize the cardioprotective effects of HIT in the setting of ischemia-reperfusion injury. METHODS AND RESULTS: To determine whether HIT could improve myocardial recovery after global ischemia, isolated rabbit hearts received either standard cardioplegia or HIT in the cardioplegia or underwent preperfusion with HIT before cardioplegia. Alternatively, to determine whether HIT requires metabolic alteration, additional rabbits had in vivo pretreatment with HIT from 15 minutes to 5 days before ischemia. All hearts underwent 2 hours of global ischemia at 34 degrees C. Recovery of postischemic isovolumic developed pressure, coronary flows, and MVO2 were compared. Compared with vehicle pretreatment, HIT pretreatment (1 hour) significantly enhanced indexes of functional recovery, including developed pressure (38 +/- 3 versus 69 +/- 7 mm Hg) and coronary flow (46 +/- 2 versus 82 +/- 11 mL/min). In addition, ultrastructural morphology was preserved but only with in vivo pretreatment. Liver protein content was not increased in rabbits treated from 12 hours to 5 days with HIT versus controls, belying a protein neosynthesis mechanism. However, the temporal sequences suggested conversion of an inactive HIT profactor to an active form. CONCLUSIONS: Administration of serum derived from hibernating black bears to rabbits affords protection against ischemia-reperfusion injury compared with vehicle (saline)-treated animals in a rabbit isolated heart preparation. It is apparent that HIT deserves further identification and mechanistic study in the setting of ischemia-reperfusion injury.

Use of "natural" hibernation induction triggers for myocardial protection.

BACKGROUND: Hypothermic cardioplegia provides adequate myocellular protection, yet stunning and dysfunction remain significant problems. Interestingly, the subcellular changes of hibernation parallel the altered biology of induced cardiac ischemia, but are well tolerated by hibernating mammalian myocardium. Hibernation induction trigger (HIT) from winter-hibernating animal serum induces hibernation in active animals. Hibernation induction trigger is opiate in nature and is similar to the delta 2 opioids. METHODS: To determine whether HIT could improve myocardial recovery following global ischemia, we gave 37 isolated rabbit hearts either standard cardioplegia or cardioplegia containing summer-active woodchuck, hibernating woodchuck, or black bear HIT serum or a delta 2 opioid, D-Ala2-Leu5-enkephalin, before 2 hours of global ischemia. RESULTS: Hibernation induction trigger appeared not to have an active mechanism during ischemia, as all hearts had equal recovery. In contrast, when examining for a preischemia mechanism, 23 additional rabbits received 3 days pretreatment with summer-active woodchuck or HIT hibernating woodchuck or black bear serum, or were preperfused with D-Ala2-Leu5-enkephalin or D-pen2,5-enkephalin, a-delta 1 opioid, again before 2 hours of global ischemia. Postischemic ventricular function, coronary flows, myocardial oxygen consumption, and ultrastructural preservation were all significantly improved with HIT and D-Ala2-Leu5-enkephalin pretreatment. CONCLUSION: "Natural" HIT protection is superior to standard cardioplegia alone and may have clinical application.

Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.

Activation of the complement cascade and subsequent assembly of the membrane attack complex (MAC) occur in a number of pathophysiological settings. When formed on the surface of endothelial cells in sublytic concentrations, the MAC can induce a number of proinflammatory activities, including the secretion of soluble mediators (eg, interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) and the up-regulation of cell surface adhesion molecules. Available data indicate that MAC-induced cell activation may occur through several complex signal transduction pathways, but little is known about the intranuclear mechanisms by which complement-derived products promote the up-regulation of inflammatory mediators. Using purified distal complement proteins (C5-9) to assemble functional MAC on early-passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction. Formation of sublytic concentrations of MAC promoted an increase in nuclear factor (NF)-kappa B DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-kappa B was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-kappa B subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Preincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-induced increases in IL-8 and MCP-1 mRNA concentrations and protein secretion. A direct cause and effect linkage between MAC assembly and NF-kappa B activation was established through examination of the pharmacological effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 is a recently engineered 26-amino-acid peptide that contains a lipophilic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of the NF-kappa B p50 subunit. This study provides direct in vitro evidence that the distal complement system (MAC) can promote proinflammatory endothelial cell activation, specifically, increases in IL-8 and MCP-1 mRNA concentrations and protein secretion, and that cytosolic to nuclear translocation of NF-kappa B is necessary for this response.