Citations

Summary Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.

Abstract The induced pluripotent stem cell (iPSC) technology enables derivation of patient-specific pluripotent stem cells from adult somatic cells without using an embryonic cell source. Redifferentiation of iPSCs from diabetic patients into pancreatic islets will allow patient-specific disease modeling and autologous cell replacement therapy for failing islets. To date, diabetes-specific iPSCs have been generated from patients with type 1 diabetes using integrating retroviral vectors. However, vector integration into the host genome could compromise the biosafety and differentiation propensities of derived iPSCs. Although various integration-free reprogramming systems have been described, their utility to reprogram somatic cells from patients remains largely undetermined. Here, we used nonintegrating Sendai viral vectors to reprogram cells from patients with type 1 and type 2 diabetes (T2D). Sendai vector infection led to reproducible generation of genomic modification-free iPSCs (SV-iPSCs) from patients with diabetes, including an 85-year-old individual with T2D. SV-iPSCs lost the Sendai viral genome and antigens within 8–12 passages while maintaining pluripotency. Genome-wide transcriptome analysis of SV-iPSCs revealed induction of endogenous pluripotency genes and downregulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16INK4a, p15INK4b, and p21CIP1. SV-iPSCs and iPSCs made with integrating lentiviral vectors demonstrated remarkable similarities in global gene expression profiles. Thus, the Sendai vector system facilitates reliable reprogramming of patient cells into transgene-free iPSCs, providing a pluripotent platform for personalized diagnostic and therapeutic approaches for diabetes and diabetes-associated complications.

More than two decades have passed since genetically modified HIV was used for gene delivery. Through continuous improvements these early marker gene-carrying HIVs have evolved into safer and more effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production. Accordingly, lentivector technologies now have widespread use in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, in vivo imaging, generating transgenic animals, induction of pluripotent cells, stem cell modification and lineage tracking, or site-directed gene editing. Moreover, in the present high-throughput ‘-omics’ era, the commercial availability of premade lentiviral vectors, which are engineered to express or silence genome-wide genes, accelerates the rapid expansion of this vector technology. In the present review, we assess the advances in lentiviral vector technology, including basic lentivirology, vector designs for improved efficiency and biosafety, protocols for vector production and infection, targeted gene delivery, advanced lentiviral applications and issues associated with the vector system.

BACKGROUND B-type natriuretic peptide (BNP), a key cardiac hormone in cardiorenal homeostasis, is produced as a 108 amino acid prohormone, proBNP1-108, which is converted to a biologically active peptide BNP1-32 and an inactive N-terminal (NT)-proBNP1-76. The widely accepted model is that the normal heart releases a proteolytically processed BNP1-32 and NT-proBNP, whereas the diseased heart secretes high amounts of unprocessed/glycosylated proBNP1-108 or inappropriately processed BNPs. In contrast, circulating proBNP1-108 has recently been identified in healthy individuals, indicating that the normal heart also secretes unprocessed proBNP1-108. However, the mechanism of proBNP1-108 secretion from the normal heart remains elusive. Our goal was to determine the molecular mechanisms underlying proBNP1-108 intracellular trafficking and secretion from the normal heart. METHODS We expressed preproBNP in cardiomyocytes, and determined the subcellular localization and dominant intracellular and extracellular forms of BNP. RESULTS Intracellular immunoreactive BNPs were first accumulated in the Golgi apparatus, and then distributed throughout the cytoplasm as secretory vesicles. The predominant intracellular form of BNP was nonglycosylated proBNP1-108, rather than BNP1-32. Glycosylated proBNP1-108, but not nonglycosylated proBNP1-108, was detected as the major extracellular form in the culture supernatants of preproBNP-expressing cell lines and primary human cardiomyocytes. Ablation of O-glycosylation of proBNP1-108 at T71 residue, near the convertase recognition site, reduced the extracellular proBNP1-108 and increased extracellular BNP1-32. CONCLUSIONS Intracellular proBNP trafficking occurs through a conventional Golgi–endoplasmic reticulum pathway. Glycosylation of proBNP1-108 controls the stability and processing of extracellular proBNP1-108. Our data establish a new BNP secretion model in which the normal cardiac cells secrete glycosylated proBNP1-108.

Background— Diastolic dysfunction associated with high blood pressure (BP) leads to cardiac remodeling and fibrosis and progression to congestive heart failure. B-type natriuretic peptide (BNP) has BP-lowering, antifibrotic, and antihypertrophic properties, which makes BNP an attractive agent for attenuating the adverse cardiac remodeling associated with hypertension. In the current study, we tested the effects of sustained cardiac proBNP gene delivery on BP, cardiac function, and remodeling in spontaneously hypertensive rats (SHR). Methods and Results— We used the myocardium-tropic adeno-associated virus serotype 9 (AAV9) vector to achieve continuously enhanced cardiac rat proBNP expression. In SHR, a single systemic administration of AAV9 vector allowed long-term cardiac BNP overexpression, resulting in reductions in systolic and diastolic BP for 9 months after injection. Left ventricular (LV) thickness, LV end-systolic dimensions, and LV mass were reduced, whereas ejection fraction was significantly increased, in BNP-treated compared with untreated SHR. Circumferential systolic strain and strain rate of the early phase of diastole were improved in BNP-treated compared with untreated SHR. Noncardiac overexpression of BNP via AAV2 vector was not associated with changes in BP and plasma BNP in SHR. Furthermore, normal Wistar rats injected with AAV9 proBNP vector showed significantly reduced heart weights 4 weeks after injection without BP reduction. Conclusions— AAV9 vector facilitates sustained cardiac proBNP overexpression and improves LV function in hypertensive heart disease. Long-term proBNP delivery improved both systolic and diastolic function. The effects on cardiac structure and function occurred independently of BP-lowering effects in normal Wistar rats.

ABSTRACTA novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. StandardMus musculuslaboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whetherMus paharicould serve as a model for XMRV, primaryMus paharifibroblasts and mice were infected with cell-free XMRV. Infection of cellsin vitroresulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV intoMus paharimice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP+CD4+T cells and CD19+B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3in vivo. These data demonstrate infection ofMus pahariby XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infectionin vivo. These data support the use ofMus paharias a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.

Rationale : Induced pluripotent stem cells (iPS) allow derivation of pluripotent progenitors from somatic sources. Originally, iPS were induced by a stemness-related gene set that included the c-MYC oncogene. Objective : Here, we determined from embryo to adult the cardiogenic proficiency of iPS programmed without c-MYC, a cardiogenicity-associated transcription factor. Methods and Results : Transgenic expression of 3 human stemness factors SOX2, OCT4, and KLF4 here reset murine fibroblasts to the pluripotent ground state. Transduction without c-MYC reversed cellular ultrastructure into a primitive archetype and induced stem cell markers generating 3-germ layers, all qualifiers of acquired pluripotency. Three-factor induced iPS (3F-iPS) clones reproducibly demonstrated cardiac differentiation properties characterized by vigorous beating activity of embryoid bodies and robust expression of cardiac Mef2c, α-actinin, connexin43, MLC2a, and troponin I. In vitro isolated iPS-derived cardiomyocytes demonstrated functional excitation-contraction coupling. Chimerism with 3F-iPS derived by morula-stage diploid aggregation was sustained during prenatal heart organogenesis and contributed in vivo to normal cardiac structure and overall performance in adult tumor-free offspring. Conclusions : Thus, 3F-iPS bioengineered without c-MYC achieve highest stringency criteria for bona fide cardiogenesis enabling reprogrammed fibroblasts to yield de novo heart tissue compatible with native counterpart throughout embryological development and into adulthood.

Background— Nuclear reprogramming provides an emerging strategy to produce embryo-independent pluripotent stem cells from somatic tissue. Induced pluripotent stem cells (iPS) demonstrate aptitude for de novo cardiac differentiation, yet their potential for heart disease therapy has not been tested. Methods and Results— In this study, fibroblasts transduced with human stemness factors OCT3/4, SOX2, KLF4, and c-MYC converted into an embryonic stem cell–like phenotype and demonstrated the ability to spontaneously assimilate into preimplantation host morula via diploid aggregation, unique to bona fide pluripotent cells. In utero, iPS-derived chimera executed differentiation programs to construct normal heart parenchyma patterning. Within infarcted hearts in the adult, intramyocardial delivery of iPS yielded progeny that properly engrafted without disrupting cytoarchitecture in immunocompetent recipients. In contrast to parental nonreparative fibroblasts, iPS treatment restored postischemic contractile performance, ventricular wall thickness, and electric stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Conclusions— Fibroblasts reprogrammed by human stemness factors thus acquire the potential to repair acute myocardial infarction, establishing iPS in the treatment of heart disease.

Abstract Purpose: Various studies have shown the importance of the GAIP interacting protein, COOH-terminus (GIPC, also known as Synectin) as a central adaptor molecule in different signaling pathways and as an important mediator of receptor stability. GIPC/Synectin is associated with different growth-promoting receptors such as insulin-like growth factor receptor I (IGF-IR) and integrins. These interactions were mediated through its PDZ domain. GIPC/Synectin has been shown to be overexpressed in pancreatic and breast cancer. The goal of this study was to show the importance of GIPC/Synectin in pancreatic cancer growth and to evaluate a possible therapeutic strategy by using a GIPC-PDZ domain inhibitor. Furthermore, the effect of targeting GIPC on the IGF-I receptor as one of its associated receptors was tested. Experimental Design: The in vivo effects of GIPC/Synectin knockdown were studied after lentiviral transduction of luciferase-expressing pancreatic cancer cells with short hairpin RNA against GIPC/Synectin. Additionally, a GIPC-PDZ–targeting peptide was designed. This peptide was tested for its influence on pancreatic cancer growth in vitro and in vivo. Results: Knockdown of GIPC/Synectin led to a significant inhibition of pancreatic adenocarcinoma growth in an orthotopic mouse model. Additionally, a cell-permeable GIPC-PDZ inhibitor was able to block tumor growth significantly without showing toxicity in a mouse model. Targeting GIPC was accompanied by a significant reduction in IGF-IR expression in pancreatic cancer cells. Conclusions: Our findings show that targeting GIPC/Synectin and its PDZ domain inhibits pancreatic carcinoma growth and is a potential strategy for therapeutic intervention of pancreatic cancer.