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Anoxia and reoxygenation of human endothelial cells decrease ceramide glucosyltransferase expression and activates caspases¹

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, and
^* Department of Emergency Medicine, Beth Israel Deaconess Hospital, Harvard Medical School, Boston, Massachusetts, USA

²Correspondence: Center for Experimental Therapeutics and Reperfusion Injury, Thorn 705, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA. E-mail: gstahl{at}zeus.bwh.harvard.edu

SPECIFIC AIMS

This study investigated the effect of brief periods of oxidative stress (3 h of anoxia and 0–5 h of reoxygenation) on glucosylceramide synthase (GCS) expression and caspase activity in human umbilical vein endothelial cells (HUVECs). We pharmacologically inhibited GCS and/or ceramide synthase after normoxic and/or oxidative stress to investigate the role of these enzymes in HUVEC caspase activity. We investigated the effect of myocardial ischemia and reperfusion on GCS mRNA expression in the rat myocardium.

PRINCIPAL FINDINGS

1. 1. Caspase activity increased in a time-dependent manner after reoxygenation of anoxic human endothelial cells. Reoxygenation of the anoxic HUVECs was necessary to induce an increase in caspase activity. (Fig. 1 ).
   
2. 2. ROCK-1 cleavage increases in HUVECs after brief periods of oxidative stress compared with normoxic cells (Fig. 1 , insert).
   
3. 3. GCS mRNA (real time PCR) and protein (Western blot) expression decreased in a time-dependent manner after reoxygenation of anoxic HUVECs.
   
4. 4. Inhibition of GCS in normoxic cells increases HUVEC caspase activity.
   
5. 5. Inhibition of N-acyl-sphinganine dehydrogenase during HUVEC anoxia and reoxygenation decreases caspase activity. Fumonisin B1 significantly attenuated oxidative stress induced increase in caspase activity (Fig. 2 ).
   
6. 6. Myocardial GCS mRNA expression decreases in rat heart after myocardial ischemia and reperfusion.
   

View larger version (29K): [in this window] [in a new window]   Figure 1. Increased caspase activity after oxidative stress in HUVECs. HUVECs subjected to anoxia (3 h) and 3 or 5 h of reoxygenation demonstrated a significant increase in caspase activity (i.e., free rhodamine-110). Bars represent means; brackets represent SE; numbers in bars represent number of experiments. Insert: Western analysis of ROCK-1 expression in normoxic (N) and after 3 h anoxia and 5 h reoxygenation (A/R). Increased cleavage of ROCK-1 was observed in HUVECs subjected to A/R. *P < 0.05 compared with normoxic cells.

View larger version (22K): [in this window] [in a new window]   Figure 2. Fumonisin B1 attenuates caspase activity after oxidative stress. HUVECS treated with Fumonisin B1 did not demonstrate an increase in caspase activity compared with normoxic cells. Vehicle-treated cells exposed to anoxia (3 h) and reoxygenation (5 h) demonstrated a significant increase in caspase activity compared with all groups. Bars represent means; brackets represent SE for 3 individual experiments. *P < 0.05 compared with all other groups.

CONCLUSIONS AND SIGNIFICANCE

The molecular mechanisms involved in induction of apoptosis in HUVECs after oxidative stress are not fully defined. We demonstrate in this study that GCS is down-regulated at the mRNA and protein level after a brief 3 h exposure to anoxic conditions and reoxygenation. Inhibition of glucosylceramide synthase (transferase) in normoxic HUVECs increased caspase activity to anoxic/reoxygenation levels. Further, inhibition of N-acyl-sphinganine dehydrogenase (ceramide synthase) inhibited the increase in caspase activity observed after anoxia and reoxygenation. We also observed a significant decrease in CGS mRNA expression in vivo in the rat myocardium after ischemia and reperfusion. Collectively, these data suggest that after brief periods of oxidative stress, caspase activity is a result of increased ceramide levels from a down-regulation of GCS expression (Fig. 3 ). This is the first time that changes in GCS expression have been demonstrated after oxidative stress, and suggests that alterations in GCS activity may affect apoptosis in endothelial cells. It is not known whether the decrease in GCS expression in vivo is a result of decreased endothelial cell or myocyte expression. Further, will inhibition of ceramide synthase in vivo decrease apoptosis in the heart and lead to decreased tissue injury after MI/R?

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0806fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0806fje

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This Article Full Text (PDF) All Versions of this Article: 17/6/723 02-0806fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZHAO, H. Articles by STAHL, G. L. Search for Related Content PubMed PubMed Citation Articles by ZHAO, H. Articles by STAHL, G. L.