- Top of page
- Authors' Contributions
Type-1 diabetes mellitus (T1DM) is characterized by autoimmune destruction of pancreatic β cells with serious complications, such as retinopathy, nephropathy or ischemic heart disease . According to the database of International Diabetes Federation (IDF), 285 million people, that is 6.4% of the global population, currently suffer from diabetes and 438 million people are predicted to develop diabetes by 2030 [2, 3]. Although a majority of these individuals will develop type-2 diabetes, approximately 4 million patients with T1DM are estimated to die of diabetic complications annually. Therefore, despite various treatments, including longstanding availability of insulin, cell therapy for T1DM is of interest, especially for brittle or otherwise difficult-to-control disease, and for achieving better physiological control of blood sugar levels. To date, cell therapy with intraportal transplantation of pancreatic islets has been most useful for treating refractory cases of T1DM [4-6]. However, intraportally transplanted islets have shown limited benefits in the long term because their engraftment and persistence have been suboptimal with early as well as later progressive attrition. The introduction of glucocorticoid-free immunosuppressive regimen by the Edmonton group improved success of islet transplantation but restrictions remained [7, 8], for example, needs for transplanting islets isolated from multiple donors, since islets isolated from a single pancreas are insufficient, or repeated infusion of islets . According to the Collaborative Islet Transplant Registry, second infusion of islets may provide greater islet function in the long term, but allosensitization to multiple donors could pose further potential problems for subsequent transplants of islets or other organs, for example, kidneys .
The loss of transplanted islets is driven by multiple mechanisms. Necessary procedures and processes for islet isolation, for example, enzymatic dissociation of pancreatic tissue and overnight culture, may decrease islet viability by anoikis  or stress-activated intracellular signaling . The microenvironment in portal vein may exacerbate these issues, due to vascular, immunologic [12, 13] and other factors, such as lack of extracellular matrix components [14, 15]. These types of differences likely contribute to immediate or early loss of 50–70% of transplanted islets [16-18]. Moreover, blockade of blood flow in the portal vein by transplanted islets serving as emboli likely induces hepatic ischemia and inflammatory responses with potential for further damage to transplanted islets . These problems require development of alternative implantation sites for pancreatic islets to improve therapeutic outcomes .
Previously, we reported that vascularized segments of small intestine, which is depleted of intestinal mucosa, can support transplanted pancreatic islets . Here, we considered that if vascularized segments of the small intestine provided superior microenvironment along with revascularization of transplanted islets, then hyperglycemia should be better corrected despite transplantation of fewer islets. In this study, we analyzed the mechanisms by which pancreatic islets engrafted, vascularized and functioned in vascularized small intestinal segments over the long term. This permitted evaluation of whether transplantation of fewer pancreatic islets was sufficient for glycemic control in rats with streptozotocin (STZ)-induced hyperglycemia. To reproduce the effects of impure preparations of islets that are often transplanted in humans, we examined the outcomes after transplanting nonhomogeneously purified preparations of syngeneic islets.
- Top of page
- Authors' Contributions
Although intraportal islet transplantation represents an effective therapy for patients with T1DM, there are numerous challenges currently facing the clinical application of islet transplantation that need to be overcome in order to expand its indication. The first is extensive loss of islets immediately after intraportal transplantation, which requires transplantation of islets from more than one donor pancreas or repeated islet transplantation [22, 23]. Mechanisms underlying losses of transplanted islets include exposure of islets to portal blood with an instant blood-mediated inflammatory reaction (IBMIR) and acute destruction of transplanted islets . In addition, transplanted islets may produce localized ischemia within the liver, with activation of monocytes/macrophages (Kupffer cells). These Kupffer cells play central roles in hepatic inflammatory responses and secrete an array of substances, including arachidonic acid metabolites, cytokines and peptides, that can directly affect survival of intraportal islets [24, 25]. Furthermore, Kupffer cells play fundamental roles in initiation/amplification of immune responses via antigen processing and presentation. Indeed, depletion of macrophages reduced inflammation after embolization of islets and inert beads into portal vein . Thus, alternative implantation sites with appropriate microenvironments supporting islet engraftment and function have been of considerable interest, for example, the omental pouch , intramuscular [28, 29], intra-bone marrow , intrapancreatic [31, 32], intra-ocular , an isolated venous sac  as well as prevascularized implantable devices [35-37].
In 2001, Sageshima et al  investigated the potential of intestinal subserosa as a place for islet transplantation. The islets were transplanted in clusters and resulted in normoglycemia in rats. Recently, the utility of islet transplantation under the gastric submucosa was also examined in large animal models [39-41].
In 2003, we reported that isolated small intestinal segment denuded from mucosa is a suitable site for hepatic fragment transplantation [42, 43]. Recently, we have shown that also islet cells can successfully engraft into Small Intestinal Submucosa (SIS) used as a vascularized reservoir . However, previously we did not elucidate mechanisms of islet engraftment and reorganization and efficacy of impure islets, especially when transplanted in low numbers.
Our findings established that use of semi-pure islet preparation did not impair islet engraftment, vascularization and function. It may be that presence of additional cells could have benefited islet engraftment and long-term maintenance of graft function and survival. For instance, presence of endothelial cells or mesenchymal cells in such preparations could have aided angiogenesis and revascularization of transplanted islets. Our histological studies revealed that transplanted islets were morphologically intact and expressed insulin as well as glucagon over the 1-year study period. We considered that the evidence of revascularization of islets in small intestinal segment was noteworthy. Expression of angiogenic genes, as confirmed by RT-PCR analysis, permitted revascularization of transplanted islets. This revascularization process should be of crucial importance for survival of transplanted islets. For instance, pretreating islets as well as the implantation site with proangiogenic factors, such as VEGF and/or FGF, was shown to accelerate islet revascularization . Recent studies showed that VEGF-A was responsible for high Endothelial Cell (EC) numbers in islets [45, 46], and 10-fold fewer endothelial fenestrae were observed in VEGF-A-deficient islets . These findings suggest that suitable capillary network is required for secretory function of islets, which would have benefited from islet vascularization in the intestinal segment. Similarly, direct vascularization of transplanted islets should have contributed to their engraftment, survival and function in the SIS. Such vascularization through sprouting of new vessels cannot obviously be expected to occur in the portal vein. Other regulators of islet graft survival and reorganization should include extracellular matrix components, which are abundantly present in small intestinal submucosa, and collagens types I, III and VI, glycosaminoglycans, proteoglycans and fibronectin among others.
Moreover, we identified the expression in the SIS of transcription factors required for maintenance of β cells, including Pdx1 and Pax6. Therefore, it should be appropriate to consider that survival, engraftment and excellent long-term function of transplanted islets resulted from superior revascularization and angiogenic signaling. This possibility is in agreement with previous descriptions of trophic and cytoprotective properties in SIS for islet cells ex vivo . Isolation and demucosation of intestinal segment give the possibility to disperse islets throughout submucosal layer, which is essential for adequate gas exchange, islet engraftment and prompt vascularization. Importantly, the relatively large surface area obtained would adequately accommodate relatively large volumes of impure islet preparations that are currently used for autologous and allogeneic transplants that are otherwise concerning for intrahepatic implantation.
Although creation of intestinal segments requires surgical methods, we did not observe the evidence of intestinal perforation or other complications in rats. Reanastomosed small intestine appeared healthy without any evidence of gastrointestinal obstruction.
Therefore, these studies of islet transplantation in small intestine segment suggest that this approach should be worthy of further development. Such studies should strengthen the rationale for cell therapy in people with islet transplantation in small intestinal segment. The safety issues have to be carefully investigated in large animal models. Nevertheless, studies in small animals indicate that this site provides excellent conditions for survival and engraftment of implanted islets, without inflammatory and fibrotic components.
Our findings offer new horizons for developing effective cell therapy applications to restore β cell function in patients with diabetes.