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Purpose A recent study revealed that angiotensin receptor signaling mediates ocular inflammation and neovascularization. It was also found that prorenin undergoes nonproteolytic activation leading to upregulation of the renin-angiotensin system (RAS) when prorenin receptor interacts specifically with the handle region of prorenin. The purpose of the present study was to elucidate the role of the receptor-dependent nonproteolytic activation of prorenin in ocular inflammation in endotoxin-induced uveitis (EIU). Methods EIU was induced in Long-Evans rats by a single intraperitoneal injection of 100 microg lipopolysaccharide (LPS). Tissue localization of total prorenin, prorenin receptor, and activated prorenin in the EIU retina was examined by immunohistochemistry. To inhibit the prorenin receptor-mediated upregulation of the RAS, a decoy handle-region peptide (HRP) was intraperitoneally administered 24 hours before and immediately after the injection of LPS. Twenty-four hours after LPS injection, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. In addition, leukocyte infiltration into the vitreous cavity and protein concentration in the anterior chamber were also measured. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1, interleukin (IL)-6, and C-C chemokine ligand (CCL) 2/monocyte chemotactic protein (MCP)-1 were examined by RT-PCR and ELISA. Results Retinal vessels in rats with EIU were strongly positive for total prorenin, prorenin receptor, and activated prorenin. Systemic treatment with HRP resulted in dose- and time-dependent inhibition of the leukocyte adhesion and infiltration and the protein leakage, all of which were increased by the induction of EIU. Retinal mRNA expression and protein levels of ICAM-1, CCL2/MCP-1 and IL-6, induced in rats with EIU, were also significantly suppressed with application of HRP. Conclusions These findings demonstrate for the first time that nonproteolytically activated prorenin plays a significant role in the development of ocular inflammation in the EIU model. The present study suggests the potential use of HRP, a decoy peptide binding to the prorenin receptor, as a therapeutic agent to reduce ocular inflammation.

Purpose To examine whether the angiotensin II type 1 receptor (AT1-R) signaling plays a role in ocular inflammation in endotoxin-induced uveitis (EIU). Methods EIU was induced in C57BL/6 mice by a single intraperitoneal injection of 150 mug lipopolysaccharide (LPS). Tissue localization, mRNA expression, and protein levels of AT1-R in murine retinas were examined by immunohistochemistry, RT-PCR, and Western blot analyses, respectively. Telmisartan, an AT1-R antagonist widely used as an antihypertensive agent, was administered intraperitoneally at a dose of 10 mg/kg daily for 5 days until the injection of LPS. Twenty-four hours after administration, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1 were examined by RT-PCR and ELISA, respectively. Protein concentration and inflammatory cells in the aqueous humor were also measured. Results Retinal vessels were positive for AT1-R. In mice with EIU, retinal AT1-R mRNA and protein levels were significantly increased when compared to the normal control. EIU animals also showed significant increases in the number of inflammatory cells infiltrating the anterior chamber and adhering to the retinal vessels and in retinal ICAM-1 levels. Administration of telmisartan to EIU mice resulted in significant suppression of retinal ICAM-1 expression and leukocyte adhesion and infiltration compared with vehicle treatment. Protein concentration in the aqueous humor of telmisartan-treated EIU mice tended to be lower than that of vehicle-treated EIU mice, but the difference was not statistically significant. Conclusions AT1-R signaling blockade inhibited retinal ICAM-1 upregulation and leukocyte adhesion and infiltration in the EIU model. These results suggest the potential use of an AT1-R antagonist as a therapeutic agent to reduce ocular inflammation.

Tie2 is a receptor type tyrosine kinase, and is expressed in the hematopoietic stem cells and endothelial cells. We have recently shown that Tie2 and its ligand Angiopoietin-1 (Ang1) signal play a crucial role for maintenance of long-term repopulating hematopoietic stem cells in adult bone marrow (Cell, Vol 118, 149, 2004). Although Tie2 deficient mice showed defect of endothelial cell development, it is not clear whether Tie2 is critical for the development of hematopoietic and lymphangiogenic endothelial cells. In order to clarify the function of Tie2 during the developmental stage, we have developed the cell culture system of ES cell differentiation. After removing LIF, ES cells were cultured on collagen type IV coated plates to promote the differentiation to mesodermal lineage for two days, and the cells were cultured on OP9 stromal cells. Using our system hematopoietic and endothelial progenitors were differentiated efficiently on OP9 cells from the different ES cell straines, E14, TT2, and R1. Expression study showed that ES cell derived cells expressed Tie2 and Flk1 at day 5 of culture on OP9 cells. When we compared the cell fraction sorted by Tie2 and Flk1 mAb regarding differentiation potential, Tie2+Flk1+ fraction was revealed to be an enriched fraction of progenitors for hematopoietic cells and PECAM-1+ endothelial cells. To detect the lymphangiogenic endothelial cells derived from ES cells, we prepared the monoclonal antibody against LYVE-1, which is the receptor for extracellular matrix, glycosaminoglycan. And we confirmed that LYVE-1 was expressed in the embryonic lymphatic endothelium. By using LYVE-1 mAb, we sorted out LYVE-1+ cells from differentiated ES cells and carried out RT-PCR assay. LYVE-1+ cells expressed lymphangiogenic endothelial cell-specific genes, VEGFR-3, Podoplanin, and Prox-1, moreover LYVE-1+ cells took up the DiI-Ac-LDL. These findings indicate that LYVE-1+ cells derived from ES cells have a character of lymphangiogenic endothelial cells. When we compared the cell fraction sorted by Tie2 and Flk1 mAb, LYVE-1+ cells were differentiated from Tie2+Flk1+ fraction dominantly, but not from the other fractions, Tie2-Flk1+, Tie2+Flk1−, and Tie2-Flk1− fraction. These findings suggest that Tie2 is crucial for development of lymphangiogenic endothelial cells as well as hematopoietic cells and endothelial cells. In order to analyze the function of Tie2 during the developmental stage, we differentiated Tie2−/− ES cells using our system. The LYVE-1+ and PECAM-1+ cells derived from Tie2−/− ES cells dramatically decreased as culturing days went by, and at day6 of culture the LYVE-1+ and PECAM-1+ cells derived from Tie2−/− ES cells were one sixth and one third of Tie2+/− cells respectively. When we added 100μM of caspase inhibitor in the culture media, the number of both LYVE-1+ cells and PECAM-1+ cells were recovered. These findings suggest that developmental defect of lymphangiogenic endothelial and endothelial cells are caused by apoptosis because of the blockage of Tie2 signaling. However we could not detect abnormal development of hematopoietic cells from Tie2−/− ES cells. In conclusion, Tie2+Flk1+ fraction derived from ES cells is an enriched fraction of progenitors for lymphangiogenic endothelial cells, and Tie2 signaling is dispensable for lymphangiogenic endothelial cell development as well as endothelial cell development as an anti-apoptotic signaling during ES cell differentiation, but Tie2 is not essential for hematopoietic development.

Transfer RNAs (tRNAs) contain a wide variety of posttranscriptional modifications that are important for accurate decoding. Mammalian mitochondrial tRNAs (mt-tRNAs) are modified by nuclear-encoded tRNA-modifying enzymes; however, the physiological roles of these modifications remain largely unknown. In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. The KO mice were highly susceptive to stress-induced mitochondrial remodeling and exhibited accelerated myopathy and cardiac dysfunction under stressed conditions. Furthermore, we demonstrate that the ms(2) modifications of mt-tRNAs were sensitive to oxidative stress and were reduced in patients with mitochondrial disease. These findings highlight the fundamental role of ms(2) modifications of mt-tRNAs in mitochondrial protein synthesis and their pathological consequences in mitochondrial disease.

SummaryThe premetastatic niche is a predetermined site of metastases, awaiting the influx of tumor cells. However, the regulation of the angiogenic switch at these sites has not been examined. Here, we demonstrate that the calcineurin and nuclear factor of activated T cells (NFAT) pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. Upregulation of the calcineurin pathway via deletion of its endogenous inhibitor Dscr1 leads to a significant increase in lung metastases due to increased expression of a newly identified NFAT target, Angiopoietin-2 (ANG2). Increased VEGF levels specifically in the lung, and not other organ microenvironments, trigger a threshold of calcineurin-NFAT signaling that transactivates Ang2 in lung endothelium. Further, we demonstrate that overexpression of DSCR1 or the ANG2 receptor, soluble TIE2, prevents the activation of lung endothelium, inhibiting lung metastases in our mouse models. Our studies provide insights into mechanisms underlying angiogenesis in the premetastatic niche and offer targets for lung metastases.

SummaryIn obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Brown adipose tissue plays a central role in the regulation of the energy balance by expending energy to produce heat. NAD(+)-dependent deacylase sirtuins have widely been recognized as positive regulators of brown adipose tissue thermogenesis. However, here we reveal that SIRT7, one of seven mammalian sirtuins, suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions. Whole-body and brown adipose tissue-specific Sirt7 knockout mice have higher body temperature and energy expenditure. SIRT7 deficiency increases the protein level of UCP1, a key regulator of brown adipose tissue thermogenesis. Mechanistically, we found that SIRT7 deacetylates insulin-like growth factor 2 mRNA-binding protein 2, an RNA-binding protein that inhibits the translation of Ucp1 mRNA, thereby enhancing its inhibitory action on Ucp1. Furthermore, SIRT7 attenuates the expression of batokine genes, such as fibroblast growth factor 21. In conclusion, we propose that SIRT7 serves as an energy-saving factor by suppressing brown adipose tissue functions. Sirtuins have been reported to positively regulate brown adipose tissue thermogenesis. Here the authors report that brown adipocytic SIRT7 suppresses whole-body energy expenditure and thermogenesis in mice, potentially by attenuating batokine gene expressions and Ucp1 mRNA translation.