**5. Conclusion**

226 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

Various candidate transgenes are being examined for their potential in protecting β-cells under various stresses including cytokine-exposure and oxidative stress. The rational choice of therapeutic genes is helped by understanding the mechanism of -cell destruction which has been the subject of this chapter. Potential targets will be reviewed in this section. Target genes studied to date encode regulators of the cytokine signal transduction pathways, molecules that inhibit -cell apoptosis, antioxidant enzymes, immunoregulatory proteins

Apoptosis plays a major role in β-cell death in T1DM (see section 2.). The transfer of antiapoptotic genes as a strategy to counteract islet destruction has been explored. Candidate genes include those expressing cytoprotective and anti-apoptotic heat shock proteins (Hsps) and anti-apoptotic Bcl-2 family proteins. Hsp70 is one of the major heat shock proteins in mammals and is thought to be responsible for the relative resistance of human β-cells to cytokine-induced stress and death (Burkart et al., 2000). Hsp70 can protect cells under conditions of stress by directly inhibiting the transduction of the apoptotic signal, by decreasing the amount of oxidative stress and also by reducing ER stress via its chaperone activity. It has been shown that pre-conditioning by heat shock could protect pancreatic islet cells from insults by NO, ROS and the cytotoxic drug streptozocin and this increased resistance correlated with induced expression of Hsp70 (Bellmann et al., 1995). Another Hsp that is potentially capable of protecting β-cells is heme oxygenase (HO-1), also known as Hsp32. HO-1 exerts its cytoprotective effects mainly by reduction of oxidative stress (McCabe et al., 2006) and overexpression of HO-1 could protect cytokine-exposed islet cells from apoptosis (Pileggi et al., 2001, Ye & Laychock, 1998). Bcl-2 family proteins, such as the anti-apoptotic Bcl-2, are major regulators of the apoptotic signalling cascade. It has been suggested that an impaired induction of anti-apoptotic Bcl-2 plays a role in cytokineinduced dysfunction and cell death of human islet cells relative to porcine islets (Piro et al., 2001). Moreover, overexpression of Bcl-2 was shown to protect β-cells from cytokineinduced apoptosis (Y. Liu et al., 1996) and increase the longevity of islet grafts after transplantation (Contreras et al., 2001). Several mechanisms by which Bcl-2 might exert βcell protection have been suggested (McCabe et al., 2006). These include inhibition of cytochrome c release from mitochondria, inhibition of ER stress-induced apoptosis and blocking of Ca2+ release from the ER. It was shown that Bcl-2 overexpression can reduce ER stress-induced apoptosis in islet cells (Contreras et al., 2003). Both of these mechanisms have been associated with cytokine-induced β-cell death. Another candidate transgene may be the gene encoding the cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (cFlip) as its overexpression has been shown to inhibit the activation of caspase-8 in

Inhibition of NF-κB, a main effector of cytokine-signalling, was shown to reduce cytokineinduced apoptosis in rodent β-cells *in vitro* (Baker et al., 2001, Heimberg et al., 2001) and *in vivo* (Eldor et al., 2006) and Fas-induced apoptosis in human islet cells (Giannoukakis et al., 2000). It should be noted that active NF-κB has been shown to be an essential factor in mediating glucose-stimulated insulin secretion (Norlin et al., 2005) and while NF-κB inhibition may protect β-cells from apoptosis it may also interfere with insulin secretion.

and pro-survival cytokines (McCabe et al., 2006).

β-cells exposed to TNFα (Cottet et al., 2002).

**4.2 Anti-cytokine gene transfer** 

**4.1 Anti-apoptotic gene transfer** 

In recent years basic biomedical research has delivered a wealth of knowledge about the pathways by which inflammatory cytokines sensitise β-cells to cell death during the course of T1DM pathogenesis. Although the picture is still incomplete, we have learned about the major stresses to which β-cells are exposed. Some of the molecular players mediating these stresses have been identified. In particular, pro-inflammatory cytokines IL-1β, TNFα and IFNγ have been implicated as main mediators of β-cell stress and death during T1DM. It emerges that these cytokines synergistically activate transcriptional programs that lead to NO signalling, oxidative stress, ER stress, as well as modulation of Bcl-2 family protein expression. How these pathways precisely intersect has not yet been fully clarified. Studies elucidating these mechanisms may provide the knowledge to improve therapy. Islet transplantation, a therapeutic approach that would overcome the need of continuous insulin administration, is still in its infancy. Modern gene transfer techniques offer a huge potential for improvement to islet transplantation as it can help overcoming the cellular and autoimmune-mediated stress transplanted islets are exposed to. The experiments mentioned at the end of this chapter are encouraging that the accumulating knowledge of the molecules and pathways mediating β-cell stress will help to develop gene therapeutic approaches alleviating these stresses, thus improving survival of transplanted islets.

#### **6. Acknowledgement**

We are grateful to Dr. Sandra Healy for critical reading of this manuscript and Anna McCormick for providing the microscopy images of mouse pancreatic tissue sections. LV is

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funded by an Irish Cancer Society Scholarship (CRS10VIN). Our work is in part supported by a grant from Science Foundation Ireland (09/RFP/BIC2371).
