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Effect of Graft Preservation and Acute Rejection on Hypoxia-Inducible Factor-1 in Rat Cardiac Allografts
Effect of Graft Preservation and Acute Rejection on Hypoxia-Inducible Factor-1 in Rat Cardiac Allografts M.A.I. Keränen, A.I. Nykänen, R. Krebs, R. Tuuminen, H. Sandelin, P.K. Koskinen, and K.B. Lemström ABSTRACT Hypoxia plays an integral part in cardiac transplantation as prolonged graft preservation is an individual risk factor for the development of cardiac allograft vasculopathy (CAV). In this study we characterized the role of hypoxia-inducible factor-1 (HIF-1) during prolonged graft preservation, ischemia-reperfusion (I/R), acute rejection, and chronic rejection. Heart transplantations were performed from Dark Agouti (DA) to Wister-Furth (allo) or DA to DA (syn) rats, without immunosuppression (acute rejection model, harvested at day 5) or with cyclosporine (chronic rejection model, harvested at day 60). To study the effect of preservation on HIF-1 regulation, normal DA hearts were subjected to different cold ischemia times with or without 45 minutes of additional warm ischemia. The role of I/R was studied by harvesting syngrafts at different time points after reperfusion. Real-time reverse-transcriptase polymerase chain reaction quantified total HIF-1 mRNA, while enzyme-linked immunosorbent assay and immunohistochemistry quantified and localized HIF-1 protein. Our results show that HIF-1 nuclear immunoreactivity is increased during graft preservation and I/R leads to loss of nuclear HIF-1 immunoreactivity. Acute rejection induced HIF-1 in mRNA level. Our findings thus indicated that HIF-1 is activated during transplantation and suggested that manipulation of the HIF-1 pathway might reveal new therapeutic options to manage CAV.
P
ROLONGED GRAFT ISCHEMIA is a risk factor for cardiac allograft vasculopathy (CAV),1 but the mechanisms are unknown. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor adapting cells to hypoxia. HIF-1 is heterodimer composed of ␣ and  subunits.2 Both of the subunits are constitutively expressed, but HIF-1 ␣ is modified by oxygen-dependent prolyl-hydroxylation, allowing the binding of the von Hippel-Lindau protein (pVHL), which targets HIF-1 ␣ for ubiquitinylation and proteasomal degradation.3 As the role of HIF-1 in the transplantation setting is unknown, we sought to characterize the activation of HIF-1 during graft preservation, ischemia-reperfusion, and rejection in transplanted hearts.
METHODS The effect of prolonged allograft preservation, ischemia-reperfusion, and acute rejection on activation of HIF-1 and its downstream genes was investigated using rat heterotopic heart transplantation models. The effect of graft preservation time was assessed by subjecting the nontransplanted (Dark Agouti [DA]) hearts to various cold ischemia times (0, 2, or 4 hours) with or without additional warm ischemia (45 minutes). The effect of ischemiareperfusion was assessed by harvesting the syngrafts (DA ¡ DA) at
various times after reperfusion of the transplanted heart: (15 minutes, 30 minutes, 1 hour, 4 hours, 24 hours, and 120 hours). The effect on the alloimmune response was assessed by comparing allografts (DA ¡ Wister-Furth) to syngenic grafts during acute rejection. Real-time reverse transcriptase polymerase chain reaction was used to quantify mRNA and immunohistochemistry and enzyme-linked immunosorbent assay to measure protein levels of HIF-1. As HIF-1 is located in the nuclei, we performed quantitative analysis of HIF-1-positive cells. Total score was based on HIF-1␣ immunohistochemistry, as 0 ⫽ no, 1 ⫽ mild, 2 ⫽ moderate, and 3 ⫽ strong immunoreactivity.
RESULTS
In nontransplanted hearts, prolonged cold ischemia activated the HIF-1 pathway in a preservation time-dependent fashion, which was aggravated by warm ischemia (Fig 1A). The effect of ischemia on the activation of HIF-1 pathway From the Transplantation Laboratory, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland. Address reprint requests to Mikko Antti Ilmari Keränen, MD, Transplantation Laboratory, University of Helsinki, Haartmaninkatu 3, PO Box 21, Helsink 00014, Finland.
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.057
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3372
Transplantation Proceedings, 38, 3372–3373 (2006)
HYPOXIA-INDUCIBLE FACTOR-1 IN RATS
P<0.0005
2 1 0
C 3
Cold Cold+warm
HIF-1+ Score
HIF-1+ Score
3
B
2 1 0
0-hour 2-hour 4-hour
3 HIF-1+ Score
A
3373
0
¼½ 1 4 24 120 Hours after reperfusion
P<0.05
P<0.05
2 1 0
Syn Allo Syn Allo 5 days 56 days
Fig 1. Activation of HIF-1 pathway (score 0 –3 according to HIF-1␣ immunoreactivity) during graft preservation (A), reperfusion (B), and chronic rejection (C). Data are given as means ⫾ standard error of the means by Kruskall-Wallis with Dunn correction (A) and Mann-Whitney U test.
was most prominent at 2 hours. No changes in mRNA levels were observed during graft preservation. Ischemia-reperfusion showed no effect on the HIF-1 mRNA levels, but declined total HIF-1 protein toward baseline values (Fig 1B). In cell-specific analysis, HIF-1 nuclear immunoreactivity decreased shortly after reperfusion in postcapillary venules (PCV) and arterial endothelial cells remaining elevated in vascular smooth muscle cells (vSMC) and cardiomyocytes, although eventually decreasing. In cardiac allografts, acute and chronic rejection significantly induced HIF-1 mRNA and protein production (Fig 1C) and cell-specific analysis located HIF-1 protein in PCV and vSMC. DISCUSSION
The results suggested that activation of the HIF-1 pathway played an important role during ischemia-reperfusion and acute and chronic rejection in cardiac allografts. We hy-
pothesized that during graft preservation, the HIF-1 pathway elicits a protective function, but during rejection it may also enhance the development of CAV. Uncovering the biologic effects of HIF-1 may open new therapeutic strategies to prevent CAV.
REFERENCES 1. Taylor DO, Edwards LB, Boucek MM, et al: Registry of the International Society for Heart and Lung Transplantation: twentysecond official adult heart transplant report—2005. J Heart Lung Transplant 24:945, 2005 2. Wang GL, Jiang BH, Rue EA, et al: Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 92:5510, 1995 3. Hirota K, Semenza GL: Regulation of hypoxia-inducible factor 1 by prolyl and asparaginyl hydroxylases. Biochem Biophys Res Commun 338:610, 2005
P
ROLONGED GRAFT ISCHEMIA is a risk factor for cardiac allograft vasculopathy (CAV),1 but the mechanisms are unknown. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor adapting cells to hypoxia. HIF-1 is heterodimer composed of ␣ and  subunits.2 Both of the subunits are constitutively expressed, but HIF-1 ␣ is modified by oxygen-dependent prolyl-hydroxylation, allowing the binding of the von Hippel-Lindau protein (pVHL), which targets HIF-1 ␣ for ubiquitinylation and proteasomal degradation.3 As the role of HIF-1 in the transplantation setting is unknown, we sought to characterize the activation of HIF-1 during graft preservation, ischemia-reperfusion, and rejection in transplanted hearts.
METHODS The effect of prolonged allograft preservation, ischemia-reperfusion, and acute rejection on activation of HIF-1 and its downstream genes was investigated using rat heterotopic heart transplantation models. The effect of graft preservation time was assessed by subjecting the nontransplanted (Dark Agouti [DA]) hearts to various cold ischemia times (0, 2, or 4 hours) with or without additional warm ischemia (45 minutes). The effect of ischemiareperfusion was assessed by harvesting the syngrafts (DA ¡ DA) at
various times after reperfusion of the transplanted heart: (15 minutes, 30 minutes, 1 hour, 4 hours, 24 hours, and 120 hours). The effect on the alloimmune response was assessed by comparing allografts (DA ¡ Wister-Furth) to syngenic grafts during acute rejection. Real-time reverse transcriptase polymerase chain reaction was used to quantify mRNA and immunohistochemistry and enzyme-linked immunosorbent assay to measure protein levels of HIF-1. As HIF-1 is located in the nuclei, we performed quantitative analysis of HIF-1-positive cells. Total score was based on HIF-1␣ immunohistochemistry, as 0 ⫽ no, 1 ⫽ mild, 2 ⫽ moderate, and 3 ⫽ strong immunoreactivity.
RESULTS
In nontransplanted hearts, prolonged cold ischemia activated the HIF-1 pathway in a preservation time-dependent fashion, which was aggravated by warm ischemia (Fig 1A). The effect of ischemia on the activation of HIF-1 pathway From the Transplantation Laboratory, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland. Address reprint requests to Mikko Antti Ilmari Keränen, MD, Transplantation Laboratory, University of Helsinki, Haartmaninkatu 3, PO Box 21, Helsink 00014, Finland.
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.057
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3372
Transplantation Proceedings, 38, 3372–3373 (2006)
HYPOXIA-INDUCIBLE FACTOR-1 IN RATS
P<0.0005
2 1 0
C 3
Cold Cold+warm
HIF-1+ Score
HIF-1+ Score
3
B
2 1 0
0-hour 2-hour 4-hour
3 HIF-1+ Score
A
3373
0
¼½ 1 4 24 120 Hours after reperfusion
P<0.05
P<0.05
2 1 0
Syn Allo Syn Allo 5 days 56 days
Fig 1. Activation of HIF-1 pathway (score 0 –3 according to HIF-1␣ immunoreactivity) during graft preservation (A), reperfusion (B), and chronic rejection (C). Data are given as means ⫾ standard error of the means by Kruskall-Wallis with Dunn correction (A) and Mann-Whitney U test.
was most prominent at 2 hours. No changes in mRNA levels were observed during graft preservation. Ischemia-reperfusion showed no effect on the HIF-1 mRNA levels, but declined total HIF-1 protein toward baseline values (Fig 1B). In cell-specific analysis, HIF-1 nuclear immunoreactivity decreased shortly after reperfusion in postcapillary venules (PCV) and arterial endothelial cells remaining elevated in vascular smooth muscle cells (vSMC) and cardiomyocytes, although eventually decreasing. In cardiac allografts, acute and chronic rejection significantly induced HIF-1 mRNA and protein production (Fig 1C) and cell-specific analysis located HIF-1 protein in PCV and vSMC. DISCUSSION
The results suggested that activation of the HIF-1 pathway played an important role during ischemia-reperfusion and acute and chronic rejection in cardiac allografts. We hy-
pothesized that during graft preservation, the HIF-1 pathway elicits a protective function, but during rejection it may also enhance the development of CAV. Uncovering the biologic effects of HIF-1 may open new therapeutic strategies to prevent CAV.
REFERENCES 1. Taylor DO, Edwards LB, Boucek MM, et al: Registry of the International Society for Heart and Lung Transplantation: twentysecond official adult heart transplant report—2005. J Heart Lung Transplant 24:945, 2005 2. Wang GL, Jiang BH, Rue EA, et al: Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 92:5510, 1995 3. Hirota K, Semenza GL: Regulation of hypoxia-inducible factor 1 by prolyl and asparaginyl hydroxylases. Biochem Biophys Res Commun 338:610, 2005