HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction Implications for Statin Treatment in HIV Patients.

RATIONALE: Even in antiretroviral therapy-treated patients, HIV continues to play a pathogenic role in cardiovascular diseases. A possible cofactor may be persistence of the early HIV response gene Nef, which we have demonstrated recently to persist in the lungs of HIV+ patients on antiretroviral therapy. Previously, we have reported that HIV strains with Nef, but not Nef-deleted HIV strains, cause endothelial proinflammatory activation and apoptosis. OBJECTIVE: To characterize mechanisms through which HIV-Nef leads to the development of cardiovascular diseases using ex vivo tissue culture approaches as well as interventional experiments in transgenic murine models. METHODS AND RESULTS: Extracellular vesicles derived from both peripheral blood mononuclear cells and plasma from HIV+ patient blood samples induced human coronary artery endothelial cells dysfunction. Plasma-derived extracellular vesicles from antiretroviral therapy+ patients who were HIV-Nef+ induced significantly greater endothelial apoptosis compared with HIV-Nef-plasma extracellular vesicles. Both HIV-Nef expressing T cells and HIV-Nef-induced extracellular vesicles increased transfer of cytosol and Nef protein to endothelial monolayers in a Rac1-dependent manner, consequently leading to endothelial adhesion protein upregulation and apoptosis. HIV-Nef induced Rac1 activation also led to dsDNA breaks in endothelial colony forming cells, thereby resulting in endothelial colony forming cell premature senescence and endothelial nitric oxide synthase downregulation. These Rac1-dependent activities were characterized by NOX2-mediated reactive oxygen species production. Statin treatment equally inhibited Rac1 inhibition in preventing or reversing all HIV-Nef-induction abnormalities assessed. This was likely because of the ability of statins to block Rac1 prenylation as geranylgeranyl transferase inhibitors were effective in inhibiting HIV-Nef-induced reactive oxygen species formation. Finally, transgenic expression of HIV-Nef in endothelial cells in a murine model impaired endothelium-mediated aortic ring dilation, which was then reversed by 3-week treatment with 5 mg/kg atorvastatin. CONCLUSIONS: These studies establish a mechanism by which HIV-Nef persistence despite antiretroviral therapy could contribute to ongoing HIV-related vascular dysfunction, which may then be ameliorated by statin treatment.

Endotoxin Preconditioning Reprograms S1 Tubules and Macrophages to Protect the Kidney.

Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis.

Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes.

AIMS/HYPOTHESIS: Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development. METHODS: MIN6 and EndoC-ßH1 beta cell lines and human islets were treated with IL-1ß, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed. RESULTS: Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants. CONCLUSIONS/INTERPRETATION: We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.

Mig6 haploinsufficiency protects mice against streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: EGF and gastrin co-administration reverses type 1 diabetes in rodent models. However, the failure of this to translate into a clinical treatment suggests that EGF-mediated tissue repair is a complicated process and warrants further investigation. Thus, we aimed to determine whether EGF receptor (EGFR) feedback inhibition by mitogen-inducible gene 6 protein (MIG6) limits the effectiveness of EGF therapy and promotes type 1 diabetes development. METHODS: We treated Mig6 (also known as Errfi1) haploinsufficient mice (Mig6 (+/-)) and their wild-type littermates (Mig6 (+/+)) with multiple low doses of streptozotocin (STZ), and monitored diabetes development via glucose homeostasis tests and histological analyses. We also investigated MIG6-mediated cytokine-induced desensitisation of EGFR signalling and the DNA damage repair response in 832/13 INS-1 beta cells. RESULTS: Whereas STZ-treated Mig6 (+/+) mice became diabetic, STZ-treated Mig6 (+/-) mice remained glucose tolerant. In addition, STZ-treated Mig6 (+/-) mice exhibited preserved circulating insulin levels following a glucose challenge. As insulin sensitivity was similar between Mig6 (+/-) and Mig6 (+/+) mice, the preserved glucose tolerance in STZ-treated Mig6 (+/-) mice probably results from preserved beta cell function. This is supported by elevated Pdx1 and Irs2 mRNA levels in islets isolated from STZ-treated Mig6 (+/-) mice. Conversely, MIG6 overexpression in isolated islets compromises glucose-stimulated insulin secretion. Studies in 832/13 cells suggested that cytokine-induced MIG6 hinders EGFR activation and inhibits DNA damage repair. STZ-treated Mig6 (+/-) mice also have increased beta cell mass recovery. CONCLUSIONS/INTERPRETATION: Reducing Mig6 expression promotes beta cell repair and abates the development of experimental diabetes, suggesting that MIG6 may be a novel therapeutic target for preserving beta cells.

Inducible pluripotent stem cells: not quite ready for prime time?

PURPOSE OF REVIEW: Inducible pluripotent stem (iPS) cells derived from somatic cells represent a novel renewable source of tissue precursors. The potential of iPS cells is considered to be at least equivalent to that of human embryonic stem cells, facilitating the treatment or cure of diseases such as diabetes mellitus, spinal cord injuries, cardiovascular disease, and neurodegenerative diseases, but with the potential added benefit of evading the adaptive immune response that otherwise limits allogeneic cell-based therapies. This review discusses recent advances in pluripotency induction and the use of iPS cells to produce differentiated cells, while highlighting roadblocks to the widespread use of this technology in the clinical arena. RECENT FINDINGS: Whereas ethical and safety issues surrounding the use of human embryonic stem cells for the treatment of disease continue to be debated, use of iPS cells may be viewed as a more widely acceptable compromise. Since the first descriptions of inducible pluripotency from somatic cells, multiple laboratories have collectively made tremendous strides both in developing alternative, more clinically acceptable, induction strategies and in demonstrating the proof-of-principle that iPS cells can be differentiated into a variety of cell types to reverse mouse models of human disease. SUMMARY: Although the prospect of using patient-specific iPS cells has much appeal from an ethical and immunologic perspective, the limitations of the technology from the standpoint of reprogramming efficiency and therapeutic safety necessitate much more in-depth research before the initiation of human clinical trials.

IRS1 deficiency protects ß-cells against ER stress-induced apoptosis by modulating sXBP-1 stability and protein translation.

Endoplasmic reticulum (ER) stress is among several pathological features that underlie ß-cell failure in the development of type 1 and type 2 diabetes. Adaptor proteins in the insulin/insulin-like-growth factor-1 signaling pathways, such as insulin receptor substrate-1 (IRS1) and IRS2, differentially impact ß-cell survival but the underlying mechanisms remain unclear. Here we report that ß-cells deficient in IRS1 (IRS1KO) are resistant, while IRS2 deficiency (IRS2KO) makes them susceptible to ER stress-mediated apoptosis. IRS1KOs exhibited low nuclear accumulation of spliced XBP-1 due to its poor stability, in contrast to elevated accumulation in IRS2KO. The reduced nuclear accumulation in IRS1KO was due to protein instability of Xbp1 secondary to proteasomal degradation. IRS1KO also demonstrated an attenuation in their general translation status in response to ER stress revealed by polyribosomal profiling. Phosphorylation of eEF2 was dramatically increased in IRS1KO enabling the ß-cells to adapt to ER stress by blocking translation. Furthermore, significantly high ER calcium (Ca(2+)) was detected in IRS1KO ß-cells even upon induction of ER stress. These observations suggest that IRS1 could be a therapeutic target for ß-cell protection against ER stress-mediated cell death by modulating XBP-1 stability, protein synthesis, and Ca(2+) storage in the ER.

Mitogen-inducible gene 6 triggers apoptosis and exacerbates ER stress-induced ß-cell death.

The increased insulin secretory burden placed on pancreatic ß-cells during obesity and insulin resistance can ultimately lead to ß-cell dysfunction and death and the development of type 2 diabetes. Mitogen-inducible gene 6 (Mig6) is a cellular stress-responsive protein that can negatively regulate the duration and intensity of epidermal growth factor receptor signaling and has been classically viewed as a molecular brake for proliferation. In this study, we used Mig6 heterozygous knockout mice (Mig6(+/-)) to study the role of Mig6 in regulating ß-cell proliferation and survival. Surprisingly, the proliferation rate of Mig6(+/-) pancreatic islets was lower than wild-type islets despite having comparable ß-cell mass and glucose tolerance. We thus speculated that Mig6 regulates cellular death. Using adenoviral vectors to overexpress or knockdown Mig6, we found that caspase 3 activation during apoptosis was dependent on the level of Mig6. Interestingly, Mig6 expression was induced during endoplasmic reticulum (ER) stress, and its protein levels were maintained throughout ER stress. Using polyribosomal profiling, we identified that Mig6 protein translation was maintained, whereas the global protein translation was inhibited during ER stress. In addition, Mig6 overexpression exacerbated ER stress-induced caspase 3 activation in vitro. In conclusion, Mig6 is transcriptionally up-regulated and resistant to global translational inhibition during stressed conditions in ß-cells and mediates apoptosis in the form of caspase 3 activation. The sustained production of Mig6 protein exacerbates ER stress-induced ß-cell death. Thus, preventing the induction, translation, and/or function of Mig6 is warranted for increasing ß-cell survival.