Angiotensin II type 2 receptor regulates the development of pancreatic endocrine cells in mouse embryos.

BACKGROUND: We previously identified a local renin-angiotensin system (RAS) regulating the differentiation of an isolated population of human pancreatic progenitor cells. Major RAS components that regulate organogenesis have been also described in embryos; however, it is not known whether a local RAS is present in the fetal pancreas. We now hypothesize that angiotensin II type 1 (AT1 ) and type 2 (AT2 ) receptors are expressed in mouse embryonic pancreas and involved in regulating endocrine cell development. RESULTS: Differential expression of AT1 and AT2 receptors was observed in the mouse pancreata in late embryogenesis. Systemic AT2 , but not AT1 , receptor blockade during the second transition in pancreatic development (from embryonic day 12.0 onward) reduced the ß-cell to α-cell ratio of the neonate islets, impaired their insulin secretory function and the glucose tolerance of the pups. Studies with pancreas explants ex vivo revealed regulation by AT2 receptors of the differentiation of pancreatic progenitors into insulin-producing cells and of the proliferation of the differentiated cell, actions that did not result from reduced angiogenesis as a secondary effect of AT2 receptor antagonism. CONCLUSIONS: These data revealed an AT2 receptor-mediated mechanism regulating pancreatic endocrine cell development in vivo.

Angiotensin II type 2 receptor is critical for the development of human fetal pancreatic progenitor cells into islet-like cell clusters and their potential for transplantation.

Local renin-angiotensin systems (RASs) regulate the differentiation of tissue progenitors. However, it is not known whether such systems can regulate the development of pancreatic progenitor cells (PPCs). To address this issue, we characterized the expression profile of major RAS components in human fetal PPC preparations and examined their effects on the differentiation of PPCs into functional islet-like cell clusters (ICCs). We found that expression of RAS components was highly regulated throughout PPC differentiation and that locally generated angiotensin II (Ang II) maintained PPC growth and differentiation via Ang II type 1 and type 2 (AT(1) and AT(2)) receptors. In addition, we observed colocalization of AT(2) receptors with critical ß-cell phenotype markers in PPCs/ICCs, as well as AT(2) receptor upregulation during differentiation, suggesting that these receptors may regulate ß-cell development. In fact, we found that AT(2) , but not AT(1) , receptor was a key mediator of Ang II-induced upregulation of transcription factors important in ß-cell development. Furthermore, lentivirus-mediated knockdown of AT(2) receptor suppressed the expression of these transcription factors in ICCs. Transplantation of AT(2) receptor-depleted ICCs into immune-privileged diabetic mice failed to ameliorate hyperglycemia, implying that AT(2) receptors are indispensable during ICC maturation in vivo. These data strongly indicate that a local RAS is involved in governing the functional maturation of pancreatic progenitors toward the endocrine lineage.

Altered neutrophil-to-lymphocyte ratio in patients with non-affective first episode psychosis and its relationship with symptom severity and cognitive impairment.

Signatures of immune dysregulation as clinical biomarker for psychosis have remained unclear. We aimed to compare the Neutrophil-to-lymphocyte ratio (NLR) of patients with acute non-affective first-episode psychosis (FEP) with healthy controls after accounting for emotional states. We also explored the associations of NLR with symptom severity, onset profile and cognitive functions. The NLR was enumerated from complete blood count taken within a week of assessment. All FEP patients were rated on the Positive and Negative Syndrome Scale (PANSS) and the Clinician Global Impression-Severity (CGI-S) with verbal memory and executive functions assessed with the Cambridge Neuropsychological Test Automated Battery. Prevailing emotional state was measured with Beck Depression Inventory-II and Beck Anxiety Inventory. Out of seventy-nine consecutive FEP patients presenting to the study site, twenty-seven subjects were eligible and recruited. Twenty-seven age-/sex-matched controls were recruited. FEP patients had an NLR of 1.886 over the controls after accounting for scores on emotional states. The NLR of FEP patients was positively associated with CGI-S scores, PANSS positive symptom, disorganization and excitation scores. There was no significant correlation between NLR with the duration of untreated psychosis and cognitive performances. These findings support using NLR as a clinical biomarker in FEP, purporting further prospective study to measure NLR changes in the course of treatment.

Effects of hyperglycemia on angiotensin II receptor type 1 expression and insulin secretion in an INS-1E pancreatic beta-cell line.

CONTEXT: A local pancreatic islet renin-angiotensin system has been identified and found to be upregulated in type 2 diabetes mellitus. Inhibition of this system improves beta-cell function and structure. The effects of hyperglycemia, a condition observed in diabetes, on angiotensin II type 1 receptor (AT1R) expression and beta-cell secretory function have yet to be explored. OBJECTIVE: This study investigated the effects of chronic hyperglycemia (glucotoxicity) on the expression of AT1Rs, and possibly thereby on oxidative stress-induced insulin release, in an INS-1E beta-cell line. SETTINGS: INS-1E beta-cells cultured and incubated in different glucose concentrations with a varying time course. MAIN OUTCOME MEASURES: Immunocytochemistry was employed for the precise localization of AT1Rs in INS-1E cells. The effects of hyperglycemia-induced AT1R expression changes in gene and protein levels were examined by real-time RT-PCR and Western blot analysis, respectively. AT1R activation-mediated oxidative stress was assessed by changes in NADPH oxidase expression, and the level of superoxide production was determined by nitroblue tetrazolium (NBT) assay. Glucotoxicity-induced AT1R activation-mediated secretory dysfunction was also assessed by insulin release from INS-1E cells. RESULTS: AT1R immunoreactivity was found to be localized specifically on the cell membrane. Chronic hyperglycemia resulted in dose-dependent upregulation of AT1R gene and protein expression accompanied by concomitantly-enhanced oxidative stress. Glucose-stimulated insulin secretion via AT1R activation was impaired by hyperglycemia. CONCLUSION: These data indicate that hyperglycemia-induced AT1R activation impairs insulin secretion; this impairment may be mediated via AT1R-dependent oxidative stress.

NADPH Oxidase-Dependent Reactive Oxygen Species Stimulate ß-Cell Regeneration Through Differentiation of Endocrine Progenitors in Murine Pancreas.

AIMS: Reactive oxygen species (ROS) act as second messengers for redox modification of transcription factors essential for differentiation. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of ROS, has been shown to regulate differentiation of various progenitor cells, while its role in pancreatic endocrine cell differentiation is unclear. This study was aimed at this knowledge gap. RESULTS: Our results showed that ROS levels were dynamically changed during pancreas development concomitant with endocrine cell differentiation induced by modest exogenous ROS in rudiment cultures. NOX4, but not NOX2, the member of NADPH oxidase, was expressed persistently in endocrine lineage and showed high activity in critical pancreas development phase. Inhibition of NADPH oxidase activity impeded the differentiation of endocrine progenitors in vitro, and exogenous ROS reversed this effect. Studies performed in streptozotocin (STZ)-injected neonatal rats showed that diphenyleneiodonium (DPI) obstructed ß-cell regeneration through the suppression of neurogenin 3 (NGN3) expression, but not Ki67-labeling ß-cells, indicating that ROS stimulation promoted differentiation beyond proliferation of ß-cells. Inhibition of NADPH oxidase also reduced expression of SRY (sex-determining region Y)-box 9 (SOX9), a transcriptional regulator of Ngn3, in endocrine precursor cells, both in vivo and in vitro. Overexpression of SOX9 attenuated the reduction of NGN3 induced by suppression of NADPH oxidase. INNOVATION AND CONCLUSION: This is the first study to demonstrate NADPH oxidase, especially NOX4-dependent ROS that promotes pancreatic progenitor cell differentiation into endocrine cells both in vitro and in vivo, probably through the regulation of SOX9. We provide evidence that NADPH oxidase-dependent ROS-mediated signaling is necessary for endocrine cell differentiation, which provides a potential strategy for efficient generation of insulin-producing cells in clinical application.

Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas.

The growth and development of pancreatic islet cells are regulated by various morphogens. Vitamin A modulates in vitro differentiation of islet cells and vitamin D affects beta-cell insulin secretion, while both vitamin ligands act through heterodimerization with the retinoid X receptor (RXR). However, their effects in modulating pancreatic development have not been determined. In this study, cultured human pancreatic progenitor cells (PPCs) isolated from human fetal pancreas were stimulated to differentiate into islet-like cell clusters (ICCs). RT-PCR, Western blotting and immunocytochemistry were used to examine the expression and localization of vitamin D receptor (VDR), retinoic acid receptor (RAR), and RXR in PPCs. The effects of added all-trans retinoic acid (atRA, a form of vitamin A), calcitriol (activated vitamin D) and of these ligands together on PPC cell viability, proliferation and apoptosis were assessed by MTT, BrdU and ELISA assays, respectively. Post-treatment neurogenin-3 (NGN3) expression, necessary for islet-cell lineage development, was examined by real-time RT-PCR. Results showed that RAR, RXR and VDR were expressed in PPCs. RAR and RXR were localized in nuclei, and the VDR in nuclei, cytoplasm and plasma membrane. atRA and calcitriol each increased PPC viability and proliferation; atRA additionally decreased PPC apoptosis. Co-addition of atRA and calcitriol had no additive effects on cell viability but did increase ngn3 responses. In conclusion, RAR, RXR and VDR are expressed in human fetal PPCs and PPC proliferation can be promoted by calcitriol, atRA or both together, data valuable for elucidating mechanisms underlying islet development and for developing clinical islet transplantation.

Reduced immunogenicity of pancreatic progenitor cells derived from first-trimester human fetal pancreas.

The relatively low immunogenic and tumorigenic nature of fetal stem cells makes them attractive candidates for transplantation. Pancreatic progenitor cells (PPCs) derived from human fetal pancreas that are amenable to growth and differentiation into transplantable insulin-producing islet-like cell clusters (ICCs) have been reported recently; however, the immunological nature of these cells has yet to be characterized. We thus investigated and compared the immunogenicity of pancreatic progenitor cells and islet-like cell clusters from first- and second-trimester human fetal pancreas. Polymerase chain reaction demonstrated that pancreatic progenitor cells and islet-like cell clusters express immune-related genes of major histocompatibility complex, MHC-I and MHC-II, complement component 3 (C3), chemokine ligand (CCL19), and tumor necrosis factor super family (TNFSF10), but no expression of the co-stimulatory genes, CD80 and CD86. Interestingly, pancreatic progenitor cells showed a differential expression of MHC-I and MHC-II with advancing gestational age with a greater expression in pancreatic progenitor cells from the second trimester. Pre-incubation of the second-trimester cells with interferon-γ (IFN-γ) increased MHC molecule expression. Functional alloreactivity of pancreatic progenitor cells was investigated via mixed lymphocyte reactions (MLRs). Relative to first-trimester pancreatic progenitor cells, second-trimester pancreatic progenitor cells induced a greater extent of proliferation of peripheral blood mononuclear cells (PBMCs) and resulted in more IFN-γ production in phytohaemagllutinin-stimulated peripheral blood mononuclear cells following co-culture. Results of the study indicated that first-trimester pancreatic progenitor cells and islet-like cell clusters have a distinctively lower immunogenicity relative to second-trimester pancreatic progenitor cells, even after a pro-inflammatory cytokine challenge.

PDZ-domain containing-2 (PDZD2) drives the maturity of human fetal pancreatic progenitor-derived islet-like cell clusters with functional responsiveness against membrane depolarization.

We recently reported the isolation and characterization of a population of pancreatic progenitor cells (PPCs) from early trimester human fetal pancreata. The PPCs, being the forerunners of adult pancreatic cell lineages, were amenable to growth and differentiation into insulin-secreting islet-like cell clusters (ICCs) upon stimulation by adequate morphogens. Of note, a novel morphogenic factor, PDZ-domain containing-2 (PDZD2) and its secreted form (sPDZD2) were ubiquitously expressed in the PPCs. Our goals for this study were to evaluate the potential role of sPDZD2 in stimulating PPC differentiation and to establish the optimal concentration for such stimulation. We found that 10(-9)M sPDZD2 promoted PPC differentiation, as evidenced by the upregulation of the pancreatic endocrine markers (PDX-1, NGN3, NEURO-D, ISL-1, NKX 2.2, NKX 6.1) and INSULIN mRNA. Inhibited endogenous production of sPDZD2 suppressed expression of these factors. Secreted PDZD2 treatment significantly elevated the C-peptide content of the ICCs and increased the basal rate of insulin secretion. However, they remained unresponsive to glucose stimulation, reflected by a minimal increase in GLUT-2 and GLUCOKINASE mRNA expression. Interestingly, sPDZD2 treatment induced increased expression of the L-type voltage-gated calcium channel (Ca(v)1.2) in the ICCs, triggering calcium ion influx under KCl stimulation and conferring an ability to secrete insulin in response to KCl. Pancreatic progenitor cells from 10- and 13-week fetal pancreata showed peak expression of endogenous sPDZD2, implying that sPDZD2 has a specific role in islet development during the first trimester. In conclusion, our data suggest that sPDZD2 promotes functional maturation of human fetal PPC-derived ICCs, thus enhancing its transplanting potentials.