Beneficial islet inflammation in health depends on pericytic TLR/MyD88 signaling.

While inflammation is beneficial for insulin secretion during homeostasis, its transformation adversely affects ß cells and contributes to diabetes. However, the regulation of islet inflammation for maintaining glucose homeostasis remains largely unknown. Here, we identified pericytes as pivotal regulators of islet immune and ß cell function in health. Islets and pancreatic pericytes express various cytokines in healthy humans and mice. To interfere with the pericytic inflammatory response, we selectively inhibited the TLR/MyD88 pathway in these cells in transgenic mice. The loss of MyD88 impaired pericytic cytokine production. Furthermore, MyD88-deficient mice exhibited skewed islet inflammation with fewer cells, an impaired macrophage phenotype, and reduced IL-1ß production. This aberrant pericyte-orchestrated islet inflammation was associated with ß cell dedifferentiation and impaired glucose response. Additionally, we found that Cxcl1, a pericytic MyD88-dependent cytokine, promoted immune IL-1ß production. Treatment with either Cxcl1 or IL-1ß restored the mature ß cell phenotype and glucose response in transgenic mice, suggesting a potential mechanism through which pericytes and immune cells regulate glucose homeostasis. Our study revealed pericyte-orchestrated islet inflammation as a crucial element in glucose regulation, implicating this process as a potential therapeutic target for diabetes.

Pericytes modulate islet immune cells and insulin secretion through Interleukin-33 production in mice.

Introduction: Immune cells were recently shown to support ß-cells and insulin secretion. However, little is known about how islet immune cells are regulated to maintain glucose homeostasis. Administration of various cytokines, including Interleukin-33 (IL-33), was shown to influence ß-cell function. However, the role of endogenous, locally produced IL-33 in pancreatic function remains unknown. Here, we show that IL-33, produced by pancreatic pericytes, is required for glucose homeostasis. Methods: To characterize pancreatic IL-33 production, we employed gene expression, flow cytometry, and immunofluorescence analyses. To define the role of this cytokine, we employed transgenic mouse systems to delete the Il33 gene selectively in pancreatic pericytes, in combination with the administration of recombinant IL-33. Glucose response was measured in vivo and in vitro, and morphometric and molecular analyses were used to measure ß-cell mass and gene expression. Immune cells were analyzed by flow cytometry. Resuts: Our results show that pericytes are the primary source of IL-33 in the pancreas. Mice lacking pericytic IL-33 were glucose intolerant due to impaired insulin secretion. Selective loss of pericytic IL-33 was further associated with reduced T and dendritic cell numbers in the islets and lower retinoic acid production by islet macrophages. Discussion: Our study demonstrates the importance of local, pericytic IL-33 production for glucose regulation. Additionally, it proposes that pericytes regulate islet immune cells to support ß-cell function in an IL-33-dependent manner. Our study reveals an intricate cellular network within the islet niche.

Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions.

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).

The postnatal pancreatic microenvironment guides ß cell maturation through BMP4 production.

Glucose homeostasis depends on regulated insulin secretion from pancreatic ß cells, which acquire their mature phenotype postnatally. The functional maturation of ß cells is regulated by a combination of cell-autonomous and exogenous factors; the identity of the latter is mostly unknown. Here, we identify BMP4 as a critical component through which the pancreatic microenvironment regulates ß cell function. By combining transgenic mouse models and human iPSCs, we show that BMP4 promotes the expression of core ß cell genes and is required for proper insulin production and secretion. We identified pericytes as the primary pancreatic source of BMP4, which start producing this ligand midway through the postnatal period, at the age ß cells mature. Overall, our findings show that the islet niche directly promotes ß cell functional maturation through the timely production of BMP4. Our study highlights the need to recapitulate the physiological postnatal islet niche for generating fully functional stem-cell-derived ß cells for cell replacement therapy for diabetes.

The Role of Vascular Cells in Pancreatic Beta-Cell Function.

Insulin-producing ß-cells constitute the majority of the cells in the pancreatic islets. Dysfunction of these cells is a key factor in the loss of glucose regulation that characterizes type 2 diabetes. The regulation of many of the functions of ß-cells relies on their close interaction with the intra-islet microvasculature, comprised of endothelial cells and pericytes. In addition to providing islet blood supply, cells of the islet vasculature directly regulate ß-cell activity through the secretion of growth factors and other molecules. These factors come from capillary mural pericytes and endothelial cells, and have been shown to promote insulin gene expression, insulin secretion, and ß-cell proliferation. This review focuses on the intimate crosstalk of the vascular cells and ß-cells and its role in glucose homeostasis and diabetes.

From virus to diabetes therapy: Characterization of a specific insulin-degrading enzyme inhibitor for diabetes treatment.

Inhibition of insulin-degrading enzyme (IDE) is a possible target for treating diabetes. However, it has not yet evolved into a medical intervention, mainly because most developed inhibitors target the zinc in IDE's catalytic site, potentially causing toxicity to other essential metalloproteases. Since IDE is a cellular receptor for the varicella-zoster virus (VZV), we constructed a VZV-based inhibitor. We computationally characterized its interaction site with IDE showing that the peptide specifically binds inside IDE's central cavity, however, not in close proximity to the zinc ion. We confirmed the peptide's effective inhibition on IDE activity in vitro and showed its efficacy in ameliorating insulin-related defects in types 1 and 2 diabetes mouse models. In addition, we suggest that inhibition of IDE may ameliorate the pro-inflammatory profile of CD4+ T-cells toward insulin. Together, we propose a potential role of a designed VZV-derived peptide to serve as a selectively-targeted and as an efficient diabetes therapy.

Pericytes contribute to the islet basement membranes to promote beta-cell gene expression.

ß-Cells depend on the islet basement membrane (BM). While some islet BM components are produced by endothelial cells (ECs), the source of others remains unknown. Pancreatic pericytes directly support ß-cells through mostly unidentified secreted factors. Thus, we hypothesized that pericytes regulate ß-cells through the production of BM components. Here, we show that pericytes produce multiple components of the mouse pancreatic and islet interstitial and BM matrices. Several of the pericyte-produced ECM components were previously implicated in ß-cell physiology, including collagen IV, laminins, proteoglycans, fibronectin, nidogen, and hyaluronan. Compared to ECs, pancreatic pericytes produce significantly higher levels of α2 and α4 laminin chains, which constitute the peri-islet and vascular BM. We further found that the pericytic laminin isoforms differentially regulate mouse ß-cells. Whereas α2 laminins promoted islet cell clustering, they did not affect gene expression. In contrast, culturing on Laminin-421 induced the expression of ß-cell genes, including Ins1, MafA, and Glut2, and significantly improved glucose-stimulated insulin secretion. Thus, alongside ECs, pericytes are a significant source of the islet BM, which is essential for proper ß-cell function.

NF-κB activity during pancreas development regulates adult ß-cell mass by modulating neonatal ß-cell proliferation and apoptosis.

NF-κB is a well-characterized transcription factor, widely known for its roles in inflammation and immune responses, as well as in control of cell division and apoptosis. However, its function in ß-cells is still being debated, as it appears to depend on the timing and kinetics of its activation. To elucidate the temporal role of NF-κB in vivo, we have generated two transgenic mouse models, the ToIß and NOD/ToIß mice, in which NF-κB activation is specifically and conditionally inhibited in ß-cells. In this study, we present a novel function of the canonical NF-κB pathway during murine islet ß-cell development. Interestingly, inhibiting the NF-κB pathway in ß-cells during embryogenesis, but not after birth, in both ToIß and NOD/ToIß mice, increased ß-cell turnover, ultimately resulting in a reduced ß-cell mass. On the NOD background, this was associated with a marked increase in insulitis and diabetes incidence. While a robust nuclear immunoreactivity of the NF-κB p65-subunit was found in neonatal ß-cells, significant activation was not detected in ß-cells of either adult NOD/ToIß mice or in the pancreata of recently diagnosed adult T1D patients. Moreover, in NOD/ToIß mice, inhibiting NF-κB post-weaning had no effect on the development of diabetes or ß-cell dysfunction. In conclusion, our data point to NF-κB as an important component of the physiological regulatory circuit that controls the balance of ß-cell proliferation and apoptosis in the early developmental stages of insulin-producing cells, thus modulating ß-cell mass and the development of diabetes in the mouse model of T1D.

Beta cell dysfunction in diabetes: the islet microenvironment as an unusual suspect.

Cells in different tissues, including endocrine cells in the pancreas, live in complex microenvironments that are rich in cellular and acellular components. Intricate interactions with their microenvironment dictate most cellular properties, such as their function, structure and size, and maintain tissue homeostasis. Pancreatic islets are populated by endocrine, vascular and immune cells that are immersed in the extracellular matrix. While the intrinsic properties of beta cells have been vastly investigated, our understanding of their interactions with their surroundings has only recently begun to unveil. Here, we review current research on the interplay between the islet cellular and acellular components, and the role these components play in beta cell physiology and pathophysiology. Although beta cell failure is a key pathomechanism in diabetes, its causes are far from being fully elucidated. We, thus, propose deleterious alterations of the islet niche as potential underlying mechanisms contributing to beta cell failure. In sum, this review emphasises that the function of the pancreatic islet depends on all of its components. Graphical abstract.

Pancreatic Pericytes in Glucose Homeostasis and Diabetes.

Glucose homeostasis relies on tightly regulated insulin secretion from pancreatic beta-cells, and its loss in diabetes is associated with the dysfunction of these cells. Beta-cells reside in the islets of Langerhans, which are highly vascularized by a dense capillary network comprised of endothelial cells and pericytes. While the requirement of the endothelium for the proper pancreatic function is well established, the role of pancreatic pericytes has only recently begun to unveil. Recent studies described multiple roles for pancreatic pericytes in glucose homeostasis, highlighting their function as both regulators of islet blood flow and as a source of critical signals that support proper beta-cell function and mass. Furthermore, recent findings point to the contribution of pericytic abnormalities to beta-cell dysfunction in type 2 diabetes, implicating the involvement of pancreatic pericytes in both the initiation and the progression of this disease. This newly gained data implicate pancreatic pericytes as critical components of the cellular network required for glucose regulation.

Pancreatic pericytes originate from the embryonic pancreatic mesenchyme.

The embryonic origin of pericytes is heterogeneous, both between and within organs. While pericytes of coelomic organs were proposed to differentiate from the mesothelium, a single-layer squamous epithelium, the embryonic origin of pancreatic pericytes has yet to be reported. Here, we show that adult pancreatic pericytes originate from the embryonic pancreatic mesenchyme. Our analysis indicates that pericytes of the adult mouse pancreas originate from cells expressing the transcription factor Nkx3.2. In the embryonic pancreas, Nkx3.2-expressing cells constitute the multilayered mesenchyme, which surrounds the pancreatic epithelium and supports multiple events in its development. Thus, we traced the fate of the pancreatic mesenchyme. Our analysis reveals that pancreatic mesenchymal cells acquire various pericyte characteristics, including gene expression, typical morphology, and periendothelial location, during embryogenesis. Importantly, we show that the vast majority of pancreatic mesenchymal cells differentiate into pericytes already at embryonic day 13.5 and progressively acquires a more mature pericyte phenotype during later stages of pancreas organogenesis. Thus, our study indicates the embryonic pancreatic mesenchyme as the primary origin to adult pancreatic pericytes. As pericytes of other coelomic organs were suggested to differentiate from the mesothelium, our findings point to a distinct origin of these cells in the pancreas. Thus, our study proposes a complex ontogeny of pericytes of coelomic organs.

Pancreas organogenesis: Approaches to elucidate the role of epithelial-mesenchymal interactions.

Pancreas organogenesis depends on proper interactions of endoderm-derived epithelial cells, which will form the exocrine and endocrine cells of the adult organ, with their surrounding mesenchymal layer. Research on the role of pancreatic mesenchyme, pioneered by Golosow and Grobstein in the 1960's, revealed these cells regulate multiple events during pancreas development. Still, much is unknown regards the molecular basis of epithelial-mesenchymal interactions in this process. Here, we review in vivo and ex vivo approaches to study mesenchymal requirements for mammal pancreas organogenesis, and how gained knowledge is being translated toward the development of cell replacement therapy for diabetes.

Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer.

The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) suggests that successful treatment might rely on simultaneous targeting of multiple genes, which can be achieved by RNA interference-based therapeutic strategies. Here we show a potent combination of microRNA and siRNA delivered by an efficient nanocarrier to PDAC tumors. Using proteomic-microRNA profiles and survival data of PDAC patients from TCGA, we found a novel signature for prolonged survival. Accordingly, we used a microRNA-mimic to increase miR-34a together with siRNA to silence PLK1 oncogene. For in vivo dual-targeting of this combination, we developed a biodegradable amphiphilic polyglutamate amine polymeric nanocarrier (APA). APA-miRNA-siRNA polyplexes systemically administered to orthotopically inoculated PDAC-bearing mice showed no toxicity and accumulated at the tumor, resulting in an enhanced antitumor effect due to inhibition of MYC oncogene, a common target of both miR-34a and PLK1. Taken together, our findings warrant this unique combined polyplex's potential as a novel nanotherapeutic for PDAC.

Pancreatic Pericytes Support ß-Cell Function in a Tcf7l2-Dependent Manner.

Polymorphism in TCF7L2, a component of the canonical Wnt signaling pathway, has a strong association with ß-cell dysfunction and type 2 diabetes through a mechanism that has yet to be defined. ß-Cells rely on cells in their microenvironment, including pericytes, for their proper function. Here, we show that Tcf7l2 activity in pancreatic pericytes is required for ß-cell function. Transgenic mice in which Tcf7l2 was selectively inactivated in their pancreatic pericytes exhibited impaired glucose tolerance due to compromised ß-cell function and glucose-stimulated insulin secretion. Inactivation of pericytic Tcf7l2 was associated with impaired expression of genes required for ß-cell function and maturity in isolated islets. In addition, we identified Tcf7l2-dependent pericytic expression of secreted factors shown to promote ß-cell function, including bone morphogenetic protein 4 (BMP4). Finally, we show that exogenous BMP4 is sufficient to rescue the impaired glucose-stimulated insulin secretion of transgenic mice, pointing to a potential mechanism through which pericytic Tcf7l2 activity affects ß-cells. To conclude, we suggest that pancreatic pericytes produce secreted factors, including BMP4, in a Tcf7l2-dependent manner to support ß-cell function. Our findings thus propose a potential cellular mechanism through which abnormal TCF7L2 activity predisposes individuals to diabetes and implicates abnormalities in the islet microenvironment in this disease.

Neonatal pancreatic pericytes support ß-cell proliferation.

OBJECTIVE: The maintenance and expansion of ß-cell mass rely on their proliferation, which reaches its peak in the neonatal stage. ß-cell proliferation was found to rely on cells of the islet microenvironment. We hypothesized that pericytes, which are components of the islet vasculature, support neonatal ß-cell proliferation. METHODS: To test our hypothesis, we combined in vivo and in vitro approaches. Briefly, we used a Diphtheria toxin-based transgenic mouse system to specifically deplete neonatal pancreatic pericytes in vivo. We further cultured neonatal pericytes isolated from the neonatal pancreas and combined the use of a ß-cell line and primary cultured mouse ß-cells. RESULTS: Our findings indicate that neonatal pancreatic pericytes are required and sufficient for ß-cell proliferation. We observed impaired proliferation of neonatal ß-cells upon in vivo depletion of pancreatic pericytes. Furthermore, exposure to pericyte-conditioned medium stimulated proliferation in cultured ß-cells. CONCLUSIONS: This study introduces pancreatic pericytes as regulators of neonatal ß-cell proliferation. In addition to advancing current understanding of the physiological ß-cell replication process, these findings could facilitate the development of protocols aimed at expending these cells as a potential cure for diabetes.

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Pancreatic Mesenchyme Regulates Islet Cellular Composition in a Patched/Hedgehog-Dependent Manner.

Pancreas development requires restrained Hedgehog (Hh) signaling activation. While deregulated Hh signaling in the pancreatic mesenchyme has been long suggested to be detrimental for proper organogenesis, this association was not directly shown. Here, we analyzed the contribution of mesenchymal Hh signaling to pancreas development. To increase Hh signaling in the pancreatic mesenchyme of mouse embryos, we deleted Patched1 (Ptch1) in these cells. Our findings indicate that deregulated Hh signaling in mesenchymal cells was sufficient to impair pancreas development, affecting both endocrine and exocrine cells. Notably, transgenic embryos displayed disrupted islet cellular composition and morphology, with a reduced ß-cell portion. Our results indicate that the cell-specific growth rates of α- and ß-cell populations, found during normal development, require regulated mesenchymal Hh signaling. In addition, we detected hyperplasia of mesenchymal cells upon elevated Hh signaling, accompanied by them acquiring smooth-muscle like phenotype. By specifically manipulating mesenchymal cells, our findings provide direct evidence for the non-autonomous roles of the Hh pathway in pancreatic epithelium development. To conclude, we directly show that regulated mesenchymal Hh signaling is required for pancreas organogenesis and establishment of its proper cellular composition.

Islet Pericytes Are Required for ß-Cell Maturity.

ß-Cells rely on the islet microenvironment for their functionality and mass. Pericytes, along with endothelial cells, make up the dense islet capillary network. However, although the role of endothelial cells in supporting ß-cell homeostasis has been vastly investigated, the role of pericytes remains largely unknown. Here, we focus on contribution of pericytes to ß-cell function. To this end, we used a transgenic mouse system that allows diphtheria toxin-based depletion of pericytes. Our results indicate that islets depleted of their pericytes have reduced insulin content and expression. Additionally, isolated islets displayed impaired glucose-stimulated insulin secretion, accompanied by a reduced expression of genes associated with ß-cell function. Importantly, reduced levels of the transcription factors MafA and Pdx1 point to ß-cell dedifferentiation in the absence of pericytes. Ex vivo depletion of pericytes in isolated islets resulted in a similar impairment of gene expression, implicating their direct, blood flow-independent role in maintaining ß-cell maturity. To conclude, our findings suggest that pericytes are pivotal components of the islet niche, which are required for ß-cell maturity and functionality. Abnormalities of islet pericytes, as implicated in type 2 diabetes, may therefore contribute to ß-cell dysfunction and disease progression.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.