Kristie Aamodt

Islet Microenvironment, Modulated by Vascular Endothelial Growth Factor-A Signaling, Promotes β Cell Regeneration

Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature, islet microenvironment, and β cell mass, we transiently increased VEGF-A production by β cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to β cell loss. After withdrawal of the VEGF-A stimulus, β cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing β cells. Bone marrow-derived macrophages (MΦs) recruited to the site of β cell injury were crucial for the β cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated β cells will improve strategies aimed at β cell regeneration and expansion.

Prosaposin inhibits tumor metastasis via paracrine and endocrine stimulation of stromal p53 and Tsp-1

Metastatic tumors can prepare a distant site for colonization via the secretion of factors that act in a systemic manner. We hypothesized that non- or weakly metastatic human tumor cells may act in an opposite fashion by creating a microenvironment in distant tissues that is refractory to colonization. By comparing cell lines with different metastatic potential, we have identified a tumor-secreted inhibitor of metastasis, prosaposin (Psap), which functions in a paracrine and endocrine fashion by stimulating the expression of thrombospondin-1 (Tsp-1) in fibroblasts present in both primary tumors and distant organs, doing so in a p53-dependent manner. Introduction of Psap in highly metastatic cells significantly reduced the occurrence of metastases, whereas inhibition of Psap production by tumor cells was associated with increased metastatic frequency. In human prostate cancer, decreased Psap expression was significantly associated with metastatic tumors. Our findings suggest that prosaposin, or other agents that stimulate p53 activity in the tumor stroma, may be an effective therapy by inhibition of the metastatic process.

Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding

Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers.

Functional Endothelial Progenitor Cells from Cryopreserved Umbilical Cord Blood

Umbilical cord blood (UCB) is recognized as an enriched source of endothelial progenitor cells (EPCs) with potential therapeutic value. Because cryopreservation is the only reliable method for long-term storage of UCB cells, the clinical application of EPCs depends on our ability to acquire them from cryopreserved samples; however, the feasibility of doing so remains unclear. In this study we demonstrate that EPCs can be isolated from cryopreserved UCB-derived mononuclear cells (MNCs). The number of outgrowth EPC colonies that emerged in culture from cryopreserved samples was similar to that obtained from fresh UCB. Furthermore, EPCs obtained from cryopreserved MNCs were phenotypically and functionally indistinguishable from freshly isolated ones, including the ability to form blood vessels in vivo. Our results eliminate the necessity of performing cell isolation procedures ahead of future clinical needs and suggest that EPCs derived from cryopreserved UCB may be suitable for EPC-related therapies.

Induction of erythropoiesis using human vascular networks genetically engineered for controlled erythropoietin release

For decades, autologous ex vivo gene therapy has been postulated as a potential alternative to parenteral administration of recombinant proteins. However, achieving effective cellular engraftment of previously retrieved patient cells is challenging. Recently, our ability to engineer vasculature in vivo has allowed for the introduction of instructions into tissues by genetically modifying the vascular cells that build these blood vessels. In the present study, we genetically engineered human blood-derived endothelial colony-forming cells (ECFCs) to express erythropoietin (EPO) under the control of a tetracycline-regulated system, and generated subcutaneous vascular networks capable of systemic EPO release in immunodeficient mice. These ECFC-lined vascular networks formed functional anastomoses with the mouse vasculature, allowing direct delivery of recombinant human EPO into the bloodstream. After activation of EPO expression, erythropoiesis was induced in both normal and anemic mice, a process that was completely reversible. This approach could relieve patients from frequent EPO injections, reducing the medical costs associated with the management of anemia. We propose this ECFC-based gene-delivery strategy as a viable alternative technology when routine administration of recombinant proteins is needed.

Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity

Aims/hypothesisTissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells.MethodsTo investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca2+ indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells.ResultsIslet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity.Conclusions/interpretationOur results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.

Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration

Numerous compounds stimulate rodent β-cell proliferation; however, translating these findings to human β-cells remains a challenge. To examine human β-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled β-cells with high specificity using both nuclear and cytoplasmic markers. β-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ β-cells, whereas treatment with other compounds had limited to no effect on human β-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human β-cell proliferation, thus allowing for increased testing of candidate human β-cell mitogens.

Signals in the pancreatic islet microenvironment influence β-cell proliferation

The progressive loss of pancreatic β‐cell mass that occurs in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing or restoring β‐cell mass. While factors that seem to influence β‐cell proliferation in specific contexts have been described, reliable stimulation of human β‐cell proliferation has remained a challenge. Importantly, β‐cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between β‐cells and these other components influences β‐cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on β‐cell proliferation may lead to the development of successful approaches to increase or restore β‐cell mass in diabetes.