Gary L. Wright

Erythropoietin receptor expression in adult rat cardiomyocytes is associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury

Erythropoietin (EPO), the principal hematopoietic cytokine that regulates mammalian erythropoiesis, exhibits diverse cellular effects in non‐hematopoietic tissues. The physiologic functions of EPO are mediated by its specific cell‐surface receptor EPOR. In this study, we demonstrate EPOR expression in adult rat cardiac myocytes and examine the direct effects of EPO on the heart to investigate whether recombinant EPO may exert an acute cardioprotective effect during ischemia‐reperfusion injury. To determine whether EPO is cardioprotective, isolated rat hearts were perfused for 10 min in the Langendorff‐mode with Krebs‐Henseleit buffer in the absence or presence of brief recombinant EPO treatment while left‐ventricular‐developed pressure (LVDP) was measured continuously to assess contractile function. The hearts were then subjected to 20 min of normothermic global ischemia followed by 25 min of reperfusion. The post‐ischemic recovery of LVDP in the untreated control hearts was 26 ± 5% of their baseline LVDP, whereas hearts pretreated with EPO exhibited significantly improved post‐ischemic recovery to 57 ± 7%. We used 31P nuclear magnetic resonance (NMR) spectroscopy to determine whether modulation of intracellular pH and/or high‐energy phosphate levels during ischemia contributed to EPO‐mediated cardioprotection. These experiments revealed that the rapid cardioprotective effect of EPO during ischemia‐reperfusion injury was associated with preservation of ATP levels in the ischemic myocardium.

Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling

Langendorff‐perfused rat hearts treated with EPO exhibited significantly improved postischemic recovery of left ventricular developed pressure (LVDP) and reduced infarct size compared with control hearts. Perfusion with the mitogen/extracellular signal‐regulated kinase (MEK) inhibitor U0126 just before and concomitant with EPO treatment abolished EPO‐induced phosphorylation of the MEK substrate extracellular signal‐regulated kinase (ERK) but had no effect of EPO‐mediated cardioprotection. EPO treatment of the perfused hearts induced translocation of protein kinase C (PKC) ε isoform to the membrane fraction of the hearts and the protective effect of EPO was significantly inhibited by the PKC catalytic inhibitor chelerythrine added before and concomitant with EPO. These data demonstrate that EPO‐mediated activation of the PKC signaling pathway before or during ischemia is required for the cardioprotective effect of EPO during ischemia‐reperfusion injury. Perfusion with the phosphatidylinositol 3‐kinase (PI3K) inhibitors LY294002 or wortmannin just before and concomitant with EPO treatment attenuated EPO‐induced phosphorylation of the PI3K substrate Akt but had no effect on EPO‐mediated cardioprotection. However, when wortmannin was added during EPO treatment and continued during reperfusion, EPO‐mediated cardioprotection was significantly inhibited. We also show that postischemia EPO treatment at the onset of reperfusion significantly improved recovery of LVDP and reduced infarct size. Postischemia cardioprotection by EPO required the PI3K pathway but was not affected by inhibition of PKC at the time of EPO treatment.

VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase

The growth factor, vascular endothelial growth factor (VEGF), induces angiogenesis and promotes endothelial cell (EC) proliferation. Affymetrix gene array analyses show that VEGF stimulates the expression of a cluster of nuclear‐encoded mitochondrial genes, suggesting a role for VEGF in the regulation of mitochondrial biogenesis. We show that the serine threonine kinase Akt3 specifically links VEGF to mitochondrial biogenesis. A direct comparison of Akt1 vs. Akt3 gene silencing was performed in ECs and has uncovered a discrete role for Akt3 in the control of mitochondrial biogenesis. Silencing of Akt3, but not Akt1, results in a decrease in mitochondrial gene expression and mtDNA content. Nuclear‐encoded mitochondrial gene transcripts are also found to decrease when Akt3 expression is silenced. Concurrent with these changes in mitochondrial gene expression, lower O2 consumption was observed. VEGF stimulation of the major mitochondrial import protein TOM70 is also blocked by Akt3 inhibition. In support of a role for Akt3 in the regulation of mitochondrial biogenesis, Akt3 silencing results in the cytoplasmic accumulation of the master regulator of mitochondrial biogenesis, PGC‐1α, and a reduction in known PGC‐1α target genes. Finally, a subtle but significant, abnormal mitochondrial phenotype is observed in the brain tissue of AKT3 knockout mice. These results suggest that Akt3 is important in coordinating mitochondrial biogenesis with growth factor‐induced increases in cellular energy demands.—Wright, G. L., Maroulakou, I. G., Eldridge, J., Liby, T. L., Sridharan, V., Tsichlis, P. N., Muise‐Helmericks, R. C. VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase. FASEB J. 22, 3264–3275 (2008)

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.

Remodeling of the actin cytoskeleton in the contracting A7r5 smooth muscle cell.

It has been proposed that the reorganization of components of the actin cytomatrix could contribute to force development and the low energy cost of sustained contraction in contractile cells which lack a structured sarcomere (A.S. Battistella-Patterson, S. Wang and G.L. Wright (1997) Can J Physiol Pharmacol75: 1287–1299). However, there has been no direct evidence of an apropos actin reorganization specifically linked to the contractile response in cells of this type. Remodeling of the α- and β-actin domains was studied in A7r5 smooth muscle cells during phorbol 12,13 dibutyrate (PDB)-induced contraction using immunohistologic staining and β-actin-green fluorescent protein (β-actin-GFP) fusion protein expression. Cell stained with phalloidin as well as cells expressing β-actin-GFP showed densely packed actin stress cables, arranged in parallel and extending across the cell body. PDB caused approximately 85% of cells to contract with evidence of forcible detachment from peripheral adhesion sites seen in many cells. The contraction of the cell body was not uniform but occurred along a principal axis parallel to the system of densely packed β-actin cables. During the interval of contraction, the β-actin cables shortened without evidence of disassembly or new cable formation. The use of cytochalasin to inhibit actin polymerization resulted in the dissolution of the actin cables at the central region of the cell and caused the elongation of precontracted cells. In unstimulated cells, α-actin formed cables similar in arrangement to the cell spanning β-actin cables. Within a short interval after PDB addition; however, the majority of α-actin cables disassembled and reformed into intensely fluorescing column-like structures extending vertically from the cell base at the center of clusters of α-actin filaments. The α-actin columns of contracting cells showed strong colocalization of α-actinin suggesting they could be structurally analogous to the dense bodies of highly differentiated smooth muscle cells. The results indicate that the α- and β-actin domains of A7r5 cells undergo a highly structured reorganization during PDB-induced contraction. The extent and nature of this restructuring suggest that remodeling could play a role in contractile function.

Prolyl hydroxylase inhibitor treatment confers whole-animal hypoxia tolerance

Aim:  Recently a family of O2‐dependent prolyl hydroxylase domain‐containing enzymes (PHD) has been identified as a cellular oxygen‐sensing mechanism. Reduced prolyl hydroxylase activity initiates a signalling cascade that includes the accumulation, as well as the activation, of hypoxia‐inducible factor (HIF‐1α). In turn the transcription factor HIF‐1α, and other targets of the PHD, elicit a myriad of incompletely understood cellular responses. In these studies we have tested: (1) whether a small‐molecule prolyl hydroxylase inhibitor (PHI) can effectively activate the oxygen‐sensing pathway when administered systemically to mice, and (2) whether the activation of the PHD signalling pathway at the cellular level results in whole‐animal hypoxic tolerance.

A hypertensive substance found in the blood of spontaneously hypertensive rats

A substance has been obtained from the blood of spontaneously hypertensive rats which produces a hypertensive elevation of the blood pressure in normotensive rats. The substance is dialyzable and is associated with the erythrocyte membrane. It appears to be relatively long-lived in its effect on arterial pressure. The erythrocyte fractions that exhibit pressor activity also stimulate the in vitro uptake of calcium by aortas obtained from normotensive animals. This suggests that the hypertensive factor or related substances may influence the calcium metabolism of vascular tissue.

Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme‐selective increases in the protein levels of PKC isozymes of osteoblastic UMR‐108 cells upon growth to confluence which may be regulated at the transcriptional or the post‐transcriptional level. The results from UMR‐108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994 . J Bone Miner Res 9(7):999–1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton.

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts.

Background. Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. Methods. IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with &agr;-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. Results. After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2′-deoxyuridine labeling and increases of graft weight were only ∼3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. Conclusion. Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.

Ca2+-dependent actin remodeling in the contracting A7r5 cell

Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the α-actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775–781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca2+]i by A23187 and thapsigargin on α- and β-actin remodeling. The effects of A23187 and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the α- and β-actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, β-actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca2+]i resulted in partial or complete dissolution of α-actin cables without further remodeling. In addition, PDBu-mediated α-actin remodeling was blocked in the presence of A23187. Increased [Ca2+]i also caused dispersal of α-actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for α-actin cytoskeletal structure. The incubation of cells in calcium-free media prevented α-actin dissolution by A23187/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of α-actin cables by increased [Ca2+]i. The results suggest that the sustained elevation of [Ca2+]i beyond a threshold level initiates depolymerization of α-actin but not β-actin. It further appears that PDBu-induced α-actin remodeling requires Ca2+ but increases of [Ca2+]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits α-actin dissolution in the presence of A23187/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing α-actin structure in the activated cell.