Protection From Ischemic Cell Death by the Induction of Cytoglobin

Protection From Ischemic Cell Death by the Induction of Cytoglobin J.I. Stagner, S.N. Parthasarathy, K. Wyler, and R.N. Parthasarathy ABSTRACT Methods to reduce ␤-cell loss after islet isolation and transplantation must be developed if islet transplantation is to become a preferred treatment for diabetes. Most recent research has focused on the reduction of toxicity from immunosuppressants and the enhancement of revascularization by growth factors such as vascular endothelial growth factor. Cytoglobin is an intracellular oxygen-binding protein found in islet ␤-cells, inducible by hypoxia. It is our hypothesis that cytoglobin induction and overexpression may improve survival and function of transplanted islets by preventing ischemic cell death. Lewis rat islets and MIN6 cells were transfected with the cytoglobin gene. Control and transfected cells and islets were held for 4 hours at 20% oxygen before glucose challenge. Another group of islets and cells was held for 4 hours at 20% and then 1% oxygen prior to glucose challenge. Untreated or transfected Lewis rat islets (n ⫽ 800) were transplanted beneath the renal capsule of streptozotocin diabetic Lewis rats. In another study, Sprague-Dawley islets were transfected and transplanted into streptozotocin diabetic Lewis rats. Fasting blood glucose was used as an indicator of islet function and survival. Cytoglobin transfected islets and cells retained the ability to secrete insulin at low oxygen concentrations in contrast to controls. Cytoglobin over expression reduced the development of central islet necrosis after 5 days in tissue culture. Cytoglobin inhibited the onset of immunorejection (14 ⫾ 2 days) as compared with controls islets (5 ⫾ 2 days). Cytoglobin induction may be a useful adjunct to islet transplantation.

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SOLATED ISLETS are avascular and therefore ischemic from the time of isolation through the period required for revascularization. Prolonged ischemia has profound deleterious effects on the islet, resulting in a significant loss of islet cells. It is Important to develop methods to increase islet survival and reduce the number of islets required to prevent diabetes in islet transplant recipients. Most recent work in this endeavor has explored methods to reduce the toxicity of the required immunosuppressant regimen1 as well as exploring the use of growth factors, such as vascular endothelial growth factor (VEGF), to enhance the onset and extent of islet revascularization.2– 4 These areas are important to increase islet survival, but are only half of the survival equation. The fact remains that islet cells remain hypoxic until the islet is fully revascularized and they remain at risk for ischemic cell death for up to 30 days after transplantation. Cytoglobin (CYTGB) is a newly discovered intracellular oxygen binding protein found in most mammalian tissues including islet ␤-cells.5–7 CYTGB is induced by hypoxia5–7 and is reported to function as an oxygen scavenging and distribution protein.5–7 It

both binds and releases oxygen to support aerobic metabolism as well as functioning as a terminal oxygenase to supply ATP for glycolysis.5–7 It is our hypothesis that the overexpression of CYTGB may enhance endogenous ␤-cell oxygen utilization and improve the survival and function of transplanted islet cells by the prevention of ischemic cell death. METHODS In Vivo Studies Lewis islets were isolated by standard collagenase/centrifugation methods. Streptozotocin diabetic Lewis rats received 800 untreated or CYTGB-transfected islets beneath the renal capsule to test the effects of CYTGB on the survival and function of a suboptimal islet number. In a parallel study 800 untreated or CYTGB-transfected From the Research Service, Department of Veterans Affairs Medical Center, Louisville, Kentucky, USA. Supported by grants from the Department of Veterans Affairs. Address reprint requests to J.I. Stagner, Research Service, Department of Veterans Affairs Medical Center, 800 Zorn Ave., Louisville, KY 40206, USA.

0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.10.001

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3452

Transplantation Proceedings, 37, 3452–3453 (2005)

INDUCTION OF CYTOGLOBIN

3453 Table 1. Effects of Cytoglobin on Cell Viability and Insulin Secretion

Control islets CYTGB transfected islets

Syngeneic Transplant, days to islet failure

Allotransplant, days to rejection

Islet cultures, insulin secretion (% of control at 1% oxygen)

30–60 90*

5⫾2 12 ⫾ 3*

7.5% 24–26%*

*P ⬍ .001 versus control, N ⫽ 10.

Sprague-Dawley islets were placed beneath the renal capsule of streptozotocin diabetic Lewis rats to test the effect of CYTGB on immune rejection. In each study, fasting blood glucose was used as an index of islet function and survival. Studies were carried out for over 90 days or until the development of hyperglycemia and frank diabetes (blood glucose ⬎11 mmol).

In Vitro Studies Lewis rat islets and MIN6 cells (a gift from Dr J. Miyazaki, Osaka, Japan) were transfected with the CYTGB gene by incubation with Fugene6 for 4 hours. Islets and cells were held overnight in CMRL 1066 media, 5.5 mmol glucose, prior to testing or transplantation. Untreated or CYTGB-transfected cells were placed in a controlled atmosphere of 20% oxygen for 4 hours. The media was changed from 5.5 to 11.0 mmol glucose to test for glucose responsiveness and insulin secretion. Another group of islets and cells was held for 4 hours at 20% oxygen and then 1% oxygen for 4 hours prior to glucose challenge. Control and CYTGB-transfected islets were retained in tissue culture at 5.5 mmol glucose and 20% oxygen for 5 days. Islets were stained with propidium iodide to mark dead cells and the diameters of the necrotic centers versus the outer islet diameter were compared as an index of central islet necrosis and ischemic cell death.2,3 Statistical significance was calculated by Student’s t-test.

RESULTS

Rats receiving syngeneic control islets initially maintained normoglycemia (6.6 ⫾ 0.6 mmol) for up to 30 days, but failed within 30 to 60 days after transplantation. CYTGBtransfected islets maintained glucose levels of 4.6 ⫾ 0.3 mmol for the 90 days of study. As shown in Table 1, CYTGB reduced central islet necrosis in islets cultured at 20% oxygen. There was no difference in insulin secretion between control and CYTGB-transfected islets at 20% oxygen; however, CYTGB increased insulin secretion in contrast to control islets held at 1% oxygen. DISCUSSION

The results obtained suggest that CYTGB induction and overexpression supports the survival and function of isolated and transplanted islets and ␤-cells by a variety of mechanisms. The reduction of central islet necrosis by CYTGB suggests that ␤-cells may bind and be able to utilize available oxygen and survive during prolonged ischemic conditions both in vitro and in vivo. The observation that CYTGB expression maintained insulin secretion, albeit at a lower rate, during hypoxic conditions, suggests that

CYTGB preserves the metabolic pathways responsible for insulin secretion. The prolongation of islet function after transplantation despite a suboptimal islet transplant mass suggests that islet cell survival was improved in the presence of CYTGB, thereby suggesting that the number of islets in a transplant could be further reduced from what is now accepted as a minimal number. Immunostaining (figures not shown) for CYTGB and VEGF showed that CYTGB transfection induced the presence of CYTGB and VEGF, whereas VEGF transfection induced the presence of VEGF but not CYTGB in transplanted islets. These results suggest that CYTGB may induce the induction and synthesis of VEGF, or that they may have a separate but parallel induction mechanism. The inhibition of immune rejection from CYTGB was significant because no immunosuppressants were used. We ascribe the inhibition of rejection to both the presence of healthier islets, less ischemic necrosis, and therefore a reduced soluble or particulate antigen burden. The last effect may be direct from improved islet survival and oxygen utilization or may be an indirect effect of VEGF as previously reported.2,3 Overexpression of CYTGB in isolated and transplanted islets has a beneficial effect on islet cell survival and function. It may well be that the enhanced expression of CYTGB in isolated islets may be a useful adjunct to islet transplantation. REFERENCES 1. Ryan EA, Lakey RT, Rajotte RV, et al: Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50:710, 2001 2. Stagner JI, Mokshagundam S, Wyler K, et al: Beta cell sparing in transplanted islets by vascular endothelial growth factor. Transplant Proc 36:1178, 2004 3. Stagner JI, Parthasarathy R, Wyler K, et al: Overexpression of vascular endothelial growth factor enhances islet survival. Transplantation 74:337, 2002 4. Stagner JI, Samols E: The induction of a capillary bed by endothelial cell growth factor prior to islet transplantation may prevent islet ischemia. Transplant Proc 22:824, 1990 5. Burmester T, Ebner B, Weich B, et al: Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues. Molec Biol Evolution 19:416, 2002 6. Hamdane D, Kiger L, Dewilde S, et al: The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin. J Biol Chem 278:51713, 2003 7. Ascenzi P, Bocedi A, de Sanctis D, et al: Neuroglobin and cytoglobin. Two new entries in the hemoglobin superfamily. Biochem Mol Biol Educ 32:305, 2004