Ying-Yue Li

Induction of early inflammatory gene expression in a murine model of nonresuscitated, fixed-volume hemorrhage.

The etiology of many end-organ problems associated with hemorrhage has been attributed to the inflammatory response to hemorrhage. In a murine model of nonresuscitated, fixed-volume hemorrhage, we sought to elucidate the role that hemorrhagic insult alone plays in the generation of the early inflammatory cascade. Differences could be appreciated as early as 1 h post-hemorrhage, with consistent differences detected by 3 h in all of the major cytokine genes studied. Significant upregulation of IL-1&bgr;, IL-6, TNF-&agr;, and IL-10 mRNA expression was observed in both the liver and lung samples of mice subjected to fixed-volume hemorrhage when compared with sham-hemorrhaged mice. The cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) genes also were upregulated in the livers and lungs of hemorrhaged mice. Finally, expression of the genes that encode the Toll-like receptors (TLR)-2 and -4 was increased by hemorrhage. Taken collectively, these data demonstrate that the initial inflammatory cascade associated with hemorrhage occurs within hours after the initial hemorrhagic event, and can be associated with significant modulation of expression of key pro- and anti-inflammatory cytokine, enzyme, and TLR genes, suggesting that these may be possible new therapeutic targets.

Antiapoptotic action of hypoxia-inducible factor-1α in human endothelial cells

Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor involved in the adaptive response to hypoxia and consists of HIF-1α and HIF-1β subunits. Indirect evidence suggests that HIF-1α may exert both proapoptotic and antiapoptotic actions in response to hypoxia. In this study, we evaluated the effects of RNA interference (RNAi) targeting HIF-1α messenger RNA (mRNA) on apoptosis in primary cultured human umbilical vascular endothelial cells (HUVECs) exposed to anoxia and reoxygenation (A/R). HUVECs were transfected with specific 21-nt small interfering RNA (siRNA) duplexes targeting HIF-1α mRNA sequences or scrambled RNA duplexes and subjected either to normoxia for 251/2 h or to anoxia for 11/2 h, and subsequently normoxia for 24 h (A/R). Control samples were subjected to A/R but not transfected. HUVECs apoptosis was evaluated by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and by activated caspase-3 immunostaining and immunoblotting. The efficacy of RNAi was assessed by knockdown of HIF-1α mRNA and protein expression via in situ hybridization, real-time quantitative PCR, immunohistochemistry, and Western blotting. When compared with normoxic cultures, A/R significantly upregulated HIF-1α mRNA and protein expression in HUVECs, but did not appreciably alter the percentage of apoptotic cells. In contrast, a significantly greater proportion of HUVECs transfected with specific siRNA duplexes and exposed to A/R demonstrated evidence of apoptosis when compared with nontransfected cells. Transfection with specific siRNA duplexes knocked down HIF-1α mRNA and protein expression in A/R-treated cells by approximately 60%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF-1α expression. These findings strongly suggest that HIF-1α exerts an antiapoptotic role in HUVECs stressed by anoxia.

Akt phosphorylation and kinase activity are down-regulated during hibernation in the 13-lined ground squirrel

Hibernation in mammals is a reversible state of suspended animation associated with tolerance to an otherwise lethal reduction of core body temperature and metabolism. An integral aspect of hibernation is tolerance to a profound decrease of cerebral perfusion. Identification of regulatory mechanisms that control hibernation in ground squirrels can guide efforts to develop improved treatment for stroke and brain trauma. In this study, we show in multiple tissues that S473 phosphorylation of Akt (Protein kinase B), a phosphatidylinositol-3 kinase-regulated serine/threonine kinase, was significantly reduced (P<0.001) as was its kinase activity (P=0.023) in the 13-lined ground squirrel, Spermophilus tridecemlineatus, during hibernation. T308 phosphorylation of Akt was relatively preserved. Brain immunohistochemical staining confirmed these results. In hibernating animals, reduction of immunoreactive phospho (S473)-Akt was noted throughout the brain. Akt is a key molecule in the insulin/insulin-like growth factor signal transduction pathway, which plays a critical role in the balance between survival and apoptosis. The data presented here raise the possibility that down-regulation of Akt phosphorylation plays a regulatory role in hibernation. This would resemble dauer larva formation in Caenorhabditis elegans where Akt inhibition is associated with energy conservation, fat storage, expression of antioxidant enzymes and growth arrest.

HIF‐1α has an anti‐apoptotic effect in human airway epithelium that is mediated via Mcl‐1 gene expression

Hypoxia‐inducible factor‐1α (HIF‐1α) and myeloid cell leukemia‐1 (Mcl‐1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF‐1α and Mcl‐1 to evaluate their possible anti‐apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF‐1α or Mcl‐1 mRNA and then immediately exposed to A/R. As controls, non‐transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF‐1α and Mcl‐1 mRNA and protein expression using real‐time quantitative RT‐PCR (Q‐PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF‐1α or Mcl‐1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non‐transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF‐1α or Mcl‐1. Unexpectedly, knockdown of HIF‐1α induced parallel knockdown of Mcl‐1 mRNA and protein expression, whereas Mcl‐1 knockdown had no noticeable effect on HIF‐1α expression. Thus, although both of these proteins were shown to be anti‐apoptotic, the action of HIF‐1α appeared to be mediated in part via Mcl‐1. J. Cell. Biochem. 97: 755–765, 2006.

RNA interference targeting Akt promotes apoptosis in hypoxia-exposed human neuroblastoma cells

Overactivation of the PI3 kinase/Akt pathway plays an essential role in the development and progression of various tumors. Akt is a key component of this pathway and hyperactivated in different tumors including neuroblastoma and glioma. In the present study, we tested the therapeutic efficacy of siRNA targeting Akt in inducing apoptotic cell death in NBFL cells (a human neuroblastoma cell line) subjected to anoxia/reoxygenation (A/R), a process that has been shown to modulate growth and progression of malignant tumors. We observed that siRNA targeting Akt effectively induced apoptotic cell death in NBFL cells (as determined by TUNEL assay and activated caspase-3 immunoreactivity) under normoxic conditions, an effect that was greatly enhanced under conditions of A/R. These findings underscore the importance of Akt signaling in promoting survival of neuroblastoma cells and may have potential therapeutic applications.