To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and ␤-cell mass, mice with ␤-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of ␤-cell secretagogues to islets, we showed that reduced insulin output is not a result of ␤-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation. Diabetes

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.

Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To survive and function, transplanted islets must revascularize because islet isolation severs arterial and venous connections; the current paradigm is that islet revascularization originates from the transplant recipient. Because isolated islets retain intraislet endothelial cells, we determined whether these endothelial cells contribute to the revascularization using a murine model with tagged endothelial cells (lacZ knock-in to Flk-1/VEGFR2 gene) and using transplanted human islets. At 3-5 weeks after transplantation beneath the renal capsule, we found that islets were revascularized and that the transplant recipient vasculature indeed contributed to the revascularization process. Using the lacZ-tagged endothelial cell model, we found that intraislet endothelial cells not only survived after transplantation but became a functional part of revascularized islet graft. A similar contribution of intraislet endothelial cells was also seen with human islets transplanted into an immunodeficient mouse model. In the murine model, individual blood vessels within the islet graft consisted of donor or recipient endothelial cells or were a chimera of donor and recipient endothelial cells, indicating that both sources of endothelial cells contribute to the new vasculature. These observations suggest that interventions to activate, amplify, or sustain intraislet endothelial cells before and after transplantation may facilitate islet revascularization, enhance islet survival, and improve islet transplantation. Diabetes 53:1318 -1325, 2004 P ancreatic islet transplantation holds great promise for the treatment of type 1 diabetes since recent advances in islet isolation and immunosuppression have led to greatly improved results (1-3). However, several major challenges currently prevent islet transplantation from being widely adapted as a treatment for type 1 diabetes. For example, less-toxic immunologic interventions are needed to prevent allograft rejection and the recurrence of the autoimmune process that originally caused type 1 diabetes. Another major challenge is that most patients must receive islets isolated from at least two pancreata to become insulin independent and often insulin independence is not permanent (4). Why islets from at least two pancreata are required to reverse diabetes is perplexing as the majority of the pancreas can be surgically removed without a normal individual becoming diabetic. One possible explanation for the requirement of islets from at least two pancreata is that many islets die in the first days after transplantation, before adequate vascular supply is reestablished. Davalli and colleagues (5-7) found that islet cell survival, islet insulin content, and ␤-cell mass declined 1-3 days after transplantation. This is the period when the islet graft is avascular, since islet isolation severs arterial and venous connections; until revascularized, transplanted islets are dependent on diffusion of nutrients and oxygen fr...

The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early but enriched in insulin-producing ␤ cells postnatally. Previous studies showed that the 5 conserved promoter regions areas I and II (Pdx1 PB ) direct endocrine cell expression, while an adjacent region (Pdx1 XB ) containing conserved area III directs transient ␤-cell expression. In this study, we used Cre-mediated lineage tracing to track cells that activated these regions. Pdx1 PB Cre mediated only endocrine cell recombination, while Pdx1 XB Cre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing areas I, II, and III was expressed throughout the embryonic day 10.5 (E10.5) pancreas and gradually became ␤ cell enriched, similar to endogenous Pdx1. These data suggested that sequences within area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated area III-containing reporter expression in a nonpancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development.The characterization of genetic regulatory elements and the transcription factors operating through these elements during pancreas development contributes to our understanding of insulin-producing ␤-cell formation. Perturbations in the transcription factors that bind important regulatory control elements can lead to defective pancreatic function and diabetes (2,19,41,48). Proper development of the endocrine and exocrine compartments of the pancreas requires several characterized transcription factors, including pancreas transcription factor 1a (Ptf1a) and the homeodomain transcription factor pancreas and duodenum homeobox 1 (Pdx1).The pancreas transcription factor 1 complex (PTF1) was first identified as an activator of exocrine-specific genes (35) and is comprised of an acinar-cell-enriched basic helix loop helix protein (bHLH), Ptf1a (p48); a ubiquitous bHLH protein (24), HEB; and the distinct mammalian Suppressor of Hairless (RBP-J) (28) or its paralogue (RBP-L) (2). Ptf1a is expressed as early as embryonic day 9.5 (E9.5) throughout the developing pancreas and is essential for pancreas formation and function in both mouse and human (23,25,28,42). Ptf1a null mice lack a ventral pancreatic bud and show an early arrest in dorsal bud outgrowth (23). Exocrine cells do not develop and there is limited endocrine development. The endocrine cells that do form are mislocalized to the spleen (25). Pdx1 is also expressed very early in pancreas development throughout both the dorsal and ventral pancreatic buds (13,18,27). After birth, Pdx1 is expressed at high levels in the i...