Ronald J. Korthuis

Activation of AMPK stimulates heme oxygenase-1 gene expression and human endothelial cell survival

The present study determined whether AMP-activated protein kinase (AMPK) regulates heme oxygenase (HO)-1 gene expression in endothelial cells (ECs) and if HO-1 contributes to the biological actions of this kinase. Treatment of human ECs with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) stimulated a concentration- and time-dependent increase in HO-1 protein and mRNA expression that was associated with a prominent increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) protein. Induction of HO-1 was also observed in rat carotid arteries after the in vivo application of AICAR. Induction of HO-1 by AICAR was blocked by the AMPK inhibitor compound C, the adenosine kinase inhibitor 5-iodotubercidin, and by silencing AMPK-α(1/2) and was mimicked by the AMPK activator A-769662 and by infecting ECs with an adenovirus expressing constitutively active AMPK-α(1). AICAR also induced a significant rise in HO-1 promoter activity that was abolished by mutating the antioxidant responsive elements of the HO-1 promoter or by the overexpression of dominant negative Nrf2. Finally, activation of AMPK inhibited cytokine-mediated EC death, and this was prevented by the HO inhibitor tin protoporphyrin-IX or by silencing HO-1 expression. In conclusion, AMPK stimulates HO-1 gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.

Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of BK channels

The objectives of this study were to determine the role of calcium-activated, small (SK), intermediate (IK), and large (BK) conductance potassium channels in initiating the development of an anti-inflammatory phenotype elicited by preconditioning with an exogenous hydrogen sulfide (H(2)S) donor, sodium hydrosulfide (NaHS). Intravital microscopy was used to visualize rolling and firmly adherent leukocytes in vessels of the small intestine of mice preconditioned with NaHS (in the absence and presence of SK, IK, and BK channel inhibitors, apamin, TRAM-34, and paxilline, respectively) or SK/IK (NS-309) or BK channel activators (NS-1619) 24 h before ischemia-reperfusion (I/R). I/R induced marked increases in leukocyte rolling and adhesion, effects that were largely abolished by preconditioning with NaHS, NS-309, or NS-1619. The postischemic anti-inflammatory effects of NaHS-induced preconditioning were mitigated by BKB channel inhibitor treatment coincident with NaHS, but not by apamin or TRAM-34, 24 h before I/R. Confocal imaging and immunohistochemistry were used to demonstrate the presence of BKα subunit staining in both endothelial and vascular smooth muscle cells of isolated, pressurized mesenteric venules. Using patch-clamp techniques, we found that BK channels in cultured endothelial cells were activated after exposure to NaHS. Bath application of the same concentration of NaHS used in preconditioning protocols led to a rapid increase in a whole cell K(+) current; specifically, the component of K(+) current blocked by the selective BK channel antagonist iberiotoxin. The activation of BK current by NaHS could also be demonstrated in single channel recording mode where it was independent of a change in intracellular Ca(+) concentration. Our data are consistent with the concept that H(2)S induces the development of an anti-adhesive state in I/R in part mediated by a BK channel-dependent mechanism.

Heme oxygenase-1 deficiency leads to alteration of soluble guanylate cyclase redox regulation

Heme oxygenase-1 knockout, Hmox1(−/−), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in Hmox1(−/−) mice. The contractile responses [superior mesenteric arteries (SMA), from female Hmox1(−/−) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in Hmox1(−/−) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in Hmox1(−/−) than Hmox1(+/+) mice (p < 0.001). Branches from Hmox1(−/−) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4′-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. Hmox1(−/−) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were Hmox1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of Hmox1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of Hmox1(−/−) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both α1and β1 subunit levels were reduced to <50% of Hmox1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of Hmox1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.

Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion-Induced Leukocyte-Endothelial Adhesive Interactions and Delayed Neuronal Death: Role of Large Conductance, Ca2+-activated K+ Channels

Please cite this paper as: Wang, Kalogeris, Wang, Jones and Korthuis (2010). Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion‐Induced Leukocyte‐Endothelial Adhesive Interactions and Delayed Neuronal Death: Role of Large Conductance, Ca2+‐activated K+ Channels. Microcirculation17(6), 427–438.

Isoform-selective 5'-AMP-activated protein kinase-dependent preconditioning mechanisms to prevent postischemic leukocyte-endothelial cell adhesive interactions

We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α(1)- and α(2)-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α(2)-isoform-dependent mechanism.

Vascular receptors as new substrates for matrix metalloproteinases in hypertension and other inflammatory states

the matrix metalloproteinase (MMP) family consists of a large group of zinc- and calcium-requiring enzymes that include interstitial collagenases, gelatinases, elastases, stromelysins, and secreted as well as membrane-type MMPs ([2][1], [13][2]). These hydrolytic enzymes act synergistically to

Filling GAPs in the understanding of cardioprotection induced by GPCR activation: RGS proteins modulate ischaemic injury

This editorial refers to ‘Gαi2-mediated protection from ischaemic injury is modulated by endogenous RGS proteins in the mouse heart’ by R.E. Waterson et al. , pp. 45–52, this issue. nnBrief periods of vascular occlusion followed by reperfusion (I/R) have been shown to confer protection against myocardial injury and necrosis induced by subsequent exposure to prolonged I/R, a phenomenon referred to as ischaemic preconditioning. Initially discovered by Murry et al. 1 in canine hearts 25 years ago, subsequent work has shown that preconditioning induces two periods of protection: an initial, early phase that arises with minutes of exposure to the bouts of preconditioning I/R, persists for 1–4 h, and then disappears, followed by the re-emergence of a protected phenotype 24 h later (delayed preconditioning).2,3 More recently, the phenomenon of ischaemic post-conditioning has been described, wherein the short bouts of I/R are initiated at the onset of reperfusion to induce cardioprotection.4 These preconditioning protocols (IPC) activate endogenous cell survival programmes that appear to exist in every species, organ, and tissue tested and may also operate in humans.2–4 Moreover, IPC represents the most powerful cardioprotective intervention yet discovered. As a consequence, an intense research effort has ensued in an attempt to elucidate the signalling mechanisms that mediate IPC so that practical therapies can be developed for patients who are predisposed to ischaemic disease. This work has resulted in identification of a number of pharmacological agents that mimic IPC, including adenosine, acetylcholine, opioids, calcitonin gene-related peptide, bradykinin, angiotensin II, and endothelin.2–4 Because each of these endogenously produced agonists serves as a ligand for Gαi protein-coupled receptors (GPCRs), there is growing interest in developing therapies that potentiate and/or sustain their activity in cardiovascular disease. Since it is difficult to predict when myocardial ischaemia will occur, …