Natural γδT17 cell development and functional acquisition is governed by the mTORC2-c-Maf-controlled mitochondrial fission pathway.

Natural IL-17-producing γδ T cells (γδT17 cells) are unconventional innate-like T cells that undergo functional programming in the fetal thymus. However, the intrinsic metabolic mechanisms of γδT17 cell development remain undefined. Here, we demonstrate that mTORC2, not mTORC1, selectively controls the functional fate commitment of γδT17 cells through regulating transcription factor c-Maf expression. scRNA-seq data suggest that fetal and adult γδT17 cells predominately utilize mitochondrial metabolism. mTORC2 deficiency results in impaired Drp1-mediated mitochondrial fission and mitochondrial dysfunction characterized by mitochondrial membrane potential (ΔΨm) loss, reduced oxidative phosphorylation (OXPHOS), and subsequent ATP depletion. Treatment with the Drp1 inhibitor Mdivi-1 alleviates imiquimod-induced skin inflammation. Reconstitution of intracellular ATP levels by ATP-encapsulated liposome completely rescues γδT17 defect caused by mTORC2 deficiency, revealing the fundamental role of metabolite ATP in γδT17 development. These results provide an in-depth insight into the intrinsic link between the mitochondrial OXPHOS pathway and γδT17 thymic programming and functional acquisition.

The role of cluster of differentiation 163-positive macrophages in wound healing: a preliminary study and a systematic review.

This is a literature assessment of essential information and current knowledge that pertains to the potential role for cluster of differentiation (CD) 163+ macrophages in different wound healing models, including extremely rapid tissue regeneration for regenerative medicine purposes. We intend to focus on the beneficial strategies that activate macrophage performance in order to advance the CD163+ macrophage-based therapy approaches to accelerate wound healing. We conducted an extensive literature search of peer reviewed articles obtained from the PubMed, Google Scholar, Scopus, Web of Science, and Cochrane databases by using the keywords "wound healing, CD163+ macrophages, diabetes mellitus, and burn." There were no limitations in terms of publication date. Our search resulted in 300 papers from which 17 articles were screened according to the inclusion criteria. We divided the selected articles into four distinct groups: healthy humans (n = 5); healthy animals (n = 7); humans with diabetes (n = 2); and animals with diabetes (n = 3). CD163 is a biomarker of the M2c macrophage subtype in mammals. Functions of M2c macrophages include angiogenesis, matrix maturation, and phagocytosis, and they activate prior to wounding. M2c produces many cytokines and growth factors, and also contains receptors for numerous cytokines and growth factors. Induction of M2c macrophages from tissue-resident macrophages in the wound bed by a suitable agent, such as delivery of intracellular ATP, appears to induce rapid granulation tissue formation without hypertrophic scarring and significantly reduces the lag time of the wound healing process.

Enhanced Skin Incisional Wound Healing With Intracellular ATP Delivery via Macrophage Proliferation and Direct Collagen Production.

This study sought to use a newly developed intracellular ATP delivery to enhance incisional wound healing to reduce surgical wound dehiscence and to explore possible mechanism for this effect. Thirty-five adult New Zealand white rabbits were used. Skin incisions were made on the back and closed. ATP-vesicles were mixed with a neutral cream for one side of the wounds while the neutral cream alone was used on the other side of the wounds. Laser speckle contrast imaging (LSCI), biomechanical, histological, and immunohistochemical analyses were performed 7 and 14 days after surgery, and macrophage culture was used to test the enhanced collagen production ability. Among them, 10 were used for wound perfusion study and 25 were used for wound biomechanical and histological/immunohistochemical studies. Wound tissue perfusion was reduced after surgery especially in early days. Wound tissue tensile strength, breaking stress, and elasticity were all much higher in the ATP-vesicle treated group than in the cream treated group at days 7 and 14. The healing was complemented by earlier macrophage accumulation, in situ proliferation, followed by direct collagen production. The results were further confirmed by human macrophage culture. It was concluded that intracellular ATP delivery enhanced healing strength of incisional wounds via multiple mechanisms.

Long non-coding RNA ZFAS1 is a major regulator of epithelial-mesenchymal transition through miR-200/ZEB1/E-cadherin, vimentin signaling in colon adenocarcinoma.

Colon adenocarcinoma is a common cause of cancer-related deaths worldwide. Epithelial-mesenchymal transition is a major regulator of cancer metastasis, and increased understanding of this process is essential to improve patient outcomes. Long non-coding RNA (lncRNA) are important regulators of carcinogenesis. To identify lncRNAs associated with colon carcinogenesis, we performed an exploratory differential gene expression analysis comparing paired colon adenocarcinoma and normal colon epithelium using an RNA-sequencing data set. This analysis identified lncRNA ZFAS1 as significantly increased in colon cancer compared to normal colon epithelium. This finding was validated in an institutional cohort using laser capture microdissection. ZFAS1 was also found to be principally located in the cellular cytoplasm. ZFAS1 knockdown was associated with decreased cellular proliferation, migration, and invasion in two colon cancer cell lines (HT29 and SW480). MicroRNA-200b and microRNA-200c (miR-200b and miR-200c) are experimentally validated targets of ZFAS1, and this interaction was confirmed using reciprocal gene knockdown. ZFAS1 knockdown regulated ZEB1 gene expression and downstream targets E-cadherin and vimentin. Knockdown of miR-200b or miR-200c reversed the effect of ZFAS1 knockdown in the ZEB1/E-cadherin, vimentin signaling cascade, and the effects of cellular migration and invasion, but not cellular proliferation. ZFAS1 knockdown was also associated with decreased tumor growth in an in vivo mouse model. These results demonstrate the critical importance of ZFAS1 as a regulator of the miR-200/ZEB1/E-cadherin, vimentin signaling cascade.

The role and function of IκKα/ß in monocyte impairment.

Following major trauma, sepsis or surgery, some patients exhibit an impaired monocyte inflammatory response that is characterized by a decreased response to a subsequent bacterial challenge. To investigate this poorly understood phenomenon, we adopted an in-vitro model of endotoxin tolerance utilising primary human CD14 + monocytes to focus on the effect of impairment on IκKα/ß, a critical part of the NFκB pathway. Impaired monocytes had decreased IκKα mRNA and protein expression and decreased phosphorylation of the IκKα/ß complex. The impaired monocyte secretome demonstrated a distinct cytokine/chemokine footprint from the naïve monocyte, and that TNF-α was the most sensitive cytokine or chemokine in this setting of impairment. Inhibition of IκKα/ß with a novel selective inhibitor reproduced the impaired monocyte phenotype with decreased production of TNF-α, IL-6, IL-12p70, IL-10, GM-CSF, VEGF, MIP-1ß, TNF-ß, IFN-α2 and IL-7 in response to an LPS challenge. Surgical patients with infection also exhibited an impaired monocyte phenotype and had decreased SITPEC, TAK1 and MEKK gene expression, which are important for IκKα/ß activation. Our results emphasize that impaired monocyte function is, at least in part, related to dysregulated IκKα/ß activation, and that IκKα/ß is likely involved in mounting a sufficient monocyte inflammatory response. Future studies may wish to focus on adjuvant therapies that augment IκKα/ß function to restore monocyte function in this clinically important problem.

The microRNA-200 family acts as an oncogene in colorectal cancer by inhibiting the tumor suppressor RASSF2.

This study aimed to determine whether manipulation of the microRNA-200 (miR-200) family could influence colon adenocarcinoma cell behavior. The miR-200 family has a significant role in tumor suppression and functions as an oncogene. In vitro studies on gain and loss of function with small interfering RNA demonstrated that the miR-200 family could regulate RASSF2 expression. Knockdown of the miR-200 family in the HT-29 colon cancer cell line increased KRAS expression but decreased signaling in the MAPK/ERK signaling pathway through reduced ERK phosphorylation. Increased expression of the miR-200 family in the CCD-841 colon epithelium cell line increased KRAS expression and led to increased signaling in the MAPK/ERK signaling pathway but increased ERK phosphorylation. Functionally, knockdown of the miR-200 family led to decreased cell proliferation in the HT-29 cells; therefore, increased miR-200 family expression could increase cell proliferation in the CCD-841 cell line. The present study included a large paired miR array dataset (n=632), in which the miR-200 family was significantly found to be increased in colon cancer when compared with normal adjacent colon epithelium. In a miR-seq dataset (n=199), the study found that miR-200 family expression was increased in localized colon cancer compared with metastatic disease. Decreased expression was associated with poorer overall survival. The miR-200 family directly targeted RASSF2 and was inversely correlated with RASSF2 expression (n=199, all P<0.001). Despite the well-defined role of the miR-200 family in tumor suppression, the present findings demonstrated a novel function of the miR-200 family in tumor proliferation.

Intracellular ATP Delivery Causes Rapid Tissue Regeneration via Upregulation of Cytokines, Chemokines, and Stem Cells.

We have reported accelerated wound healing induced by intracellular ATP delivery in rabbits, through early massive accumulation, in situ proliferation, and M2 polarization of macrophages. Granulation tissue started to grow within first 24 h of treatment and continued the growth till the wound cavity is completely covered. However, the mechanisms underlying this macrophage response are totally unclear because no one has ever reported this before. In this study, we performed a preliminary exploration of the possible mechanisms by focusing on the roles of cytokines, growth factors, and stem cells in this process. Among the 33 adult rabbits, 18 were used for cytokine measurements and the remaining were used for histological and immunohistochemical studies. Four wounds were created on the ventral side of each ear. Two wounds on one side were treated with ATP-vesicles (10 mM ATP), and the other two were treated with controls (normal saline or Regranex). Dressing changes were made daily and the rabbits were sacrificed at 5 h, 12 h, and 1, 2, 3, 4, 6, 9, 15, and 26 days after wounding. Tissue samples were analyzed for cytokines and growth factors using real-time PCR and immunohistochemical staining. The control wounds showed an immediate increase in proinflammatory cytokines after wound creation but no further increase after this initial spike. The growth factor levels in the control wounds remained unchanged throughout the study. Conversely, the wounds treated with ATP-vesicles showed significantly higher expression of MCP-1 and stem cell markers (CD44, CD106, CD146, and CD34) at day 1, significantly higher IL-1ß and TNF-α expression from day 1-4, and significantly higher VEGF-A, VEGF-D, and VEGFR-2 expression from day 4-6 when compared to the controls. The significant upregulation of these factors corresponded to the very early and rapid macrophage accumulation, in situ proliferation, and M2 polarization, resulting in unprecedented rapid granulation tissue generation due to direct macrophage collagen production and neovascularization.

Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study.

We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery-extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.

TRPA1 mediates the effects of hypothermia on the monocyte inflammatory response.

INTRODUCTION: Hypothermia is a well-known risk factor for postoperative complications because it prolongs the monocyte inflammatory response. The purpose of this study was to investigate whether temperature-activated ion channels (transient receptor protein channels [TRP] A1 and V1) mediate the effects of temperature on monocytes. METHODS: Primary human monocytes were isolated and stimulated with lipopolysaccharide at 32°C or 39°C. RNA was isolated for analysis of microRNA (miR)-155 expression, and cytokines in the supernatant were measured with an enzyme-linked immunosorbent assay. Specific inhibitors of TRPA1 (HC- 030031) and a specific activator of TRPV1 (capsaicin) were used to block or activate TRPA1 and TRPV1, respectively. Statistical analysis was performed using the Wilcoxon signed-rank test. RESULTS: TRPM8 mRNA was not expressed in primary human monocytes, whereas TRPA1 and TRPV1 were expressed. TRPV1 mRNA expression was suppressed at 32°C but not at 39°C. TRPA1 was induced strongly at 32°C and 39°C. Immunofluorescence microscopy confirmed that monocytes express TRPA1 and TRPV1 on their cell surface. Interleukin-10 secretion was increased by blocking TRPA1 (77.8 ± 3 2.8 pg/mL) and activating TRPA1 (79.4 ± 16.1 pg/mL) after 24 hours at 32°C (control 37.4 ± 17.1 pg/mL, P < .05). At 36 hours, tumor necrosis factor secretion was decreased after TRPA1 blockade (2,321 ± 439 pg/mL) and TRPV1 activation (2,137 ± 411 pg/mL) compared with control (2,567 ± 495 pg/mL, P < .05). Furthermore, miR-155 expression also was suppressed at 24 hours by TRPA1 blockade and TRPV1 activation (both P < .05). Silencing of TRPA1 normalized monocyte IL-10 secretion at 32°C. CONCLUSION: These results demonstrate that hypothermia mediates its effects on monocytes through TRPA1. Blockade of TRPA1 or activation of TRPV1 may be used to modify the effects of hypothermia on the monocyte inflammatory response.

Pivotal role of ATP in macrophages fast tracking wound repair and regeneration.

Chronic wounds occurring during aging or diabetes pose a significant burden to patients. The classical four-phase wound healing process has a 3-6 day lag before granulation starts to appear and it requires an intermediate step of activation of resident fibroblasts during the remodeling phase for production of collagen. This brief communication discusses published articles that demonstrate how the entire wound healing process can be fast tracked by intracellular ATP delivery, which triggers a novel pathway where alternatively activated macrophages play absolutely critical and central roles. This novel pathway involves an increase in proinflammatory cytokines (TNF, IL-1ß, IL-6) and a chemokine (MCP-1) release. This is followed by activation of purinergic receptor (a family of plasma membrane receptors found in almost all mammalian cells), production of platelets and platelet microparticles, and activation of ATP-dependent chromatin remodeling enzymes. The end result is a massive influx and in situ proliferation of macrophages, increases in vascular endothelial growth factors that promote neovascularization, and most prominently, the direct production of collagen.

MicroRNA-155 potentiates the inflammatory response in hypothermia by suppressing IL-10 production.

Therapeutic hypothermia is commonly used to improve neurological outcomes in patients after cardiac arrest. However, therapeutic hypothermia increases sepsis risk and unintentional hypothermia in surgical patients increases infectious complications. Nonetheless, the molecular mechanisms by which hypothermia dysregulates innate immunity are incompletely understood. We found that exposure of human monocytes to cold (32°C) potentiated LPS-induced production of TNF and IL-6, while blunting IL-10 production. This dysregulation was associated with increased expression of microRNA-155 (miR-155), which potentiates Toll-like receptor (TLR) signaling by negatively regulating Ship1 and Socs1. Indeed, Ship1 and Socs1 were suppressed at 32°C and miR-155 antagomirs increased Ship1 and Socs1 and reversed the alterations in cytokine production in cold-exposed monocytes. In contrast, miR-155 mimics phenocopied the effects of cold exposure, reducing Ship1 and Socs1 and altering TNF and IL-10 production. In a murine model of LPS-induced peritonitis, cold exposure potentiated hypothermia and decreased survival (10 vs. 50%; P < 0.05), effects that were associated with increased miR-155, suppression of Ship1 and Socs1, and alterations in TNF and IL-10. Importantly, miR-155-deficiency reduced hypothermia and improved survival (78 vs. 32%, P < 0.05), which was associated with increased Ship1, Socs1, and IL-10. These results establish a causal role of miR-155 in the dysregulation of the inflammatory response to hypothermia.

Rapid granulation tissue regeneration by intracellular ATP delivery--a comparison with Regranex.

This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles) while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA) and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.

Post-transcriptional regulation of IGF1R by key microRNAs in long-lived mutant mice.

Long-lived mutant mice, both Ames dwarf and growth hormone receptor gene-disrupted or knockout strains, exhibit heightened cognitive robustness and altered IGF1 signaling in the brain. Here, we report, in both these long-lived mice, that three up-regulated lead microRNAs, miR-470, miR-669b, and miR-681, are involved in posttranscriptional regulation of genes pertinent to growth hormone/IGF1 signaling. All three are most prominently localized in the hippocampus and correspond to reduced expression of key IGF1 signaling genes: IGF1, IGF1R, and PI3 kinase. The decline in these genes' expression translates into decreased phosphorylation of downstream molecules AKT and FoxO3a. Cultures transfected with either miR-470, miR-669b, or miR-681 show repressed endogenous expression of all three genes of the IGF1 signaling axis, most significantly IGF1R, while other similarly up-regulated microRNAs, including let-7g and miR-509, do not induce the same levels of repression. Transduction study in IGF1-responsive cell cultures shows significantly reduced IGF1R expression, and AKT to some extent, most notably by miR-681. This is accompanied by decreased levels of downstream phosphorylated forms of AKT and FoxO3a upon IGF1 stimulation. Suppression of IGF1R by the three microRNAs is further validated by IGF1R 3'UTR reporter assays. Taken together, our results suggest that miR-470, miR-669b, and miR-681 are all functionally able to suppress IGF1R and AKT, two upstream genes controlling FoxO3a phosphorylation status. Their up-regulation in growth hormone signaling-deficient mutant mouse brain suggests reduced IGF1 signaling at the posttranscriptional level, for numerous gains of neuronal function in these long-lived mice.

Gain of survival signaling by down-regulation of three key miRNAs in brain of calorie-restricted mice.

The decline in cognitive robustness with aging can be attributed to complex genetic pathways involving many cellular dysfunctions, cumulative over time, precipitating in frailty and loss of wellness in the elderly brain. The size and health of the neuronal cell population determines cognitive robustness in mammals. A transgenic mouse model over-expressing Bcl-2 has been shown to rescue neurons from naturally occurring cell death (NOCD). Here we show that in the brain of calorie-restricted (CR) mice, there is an age-dependent decreased expression of microRNAs mmu-miR-181a-1*, mmu-miR-30e and mmu-miR-34a, with a corresponding gain in Bcl-2 expression, and decreases in pro-apoptosis genes such as Bax and cleavage of Caspases. Functional characterization shows that these miRNAs repress Bcl-2 expression by the 3'UTR reporter assays, accompanied by loss of this gene's endogenous expression, and a gain in pro-apoptosome-specific proteins. Over-expression of these miRNAs increases the rate of apoptosis, accompanied by a decline in Bcl-2 expression in miRNA-transfected mouse and human cell lines. We report here that down-regulation of miR-34a, -30e, and -181a permits their shared target gene expression (Bcl-2) to remain at a high level without post-transcriptional repression, accompanied by concomitant low levels of Bax expression and Caspase cleaving; this chain event may be a part of the underlying mechanism contributing to the gain in neuronal survival in long-lived CR-fed mice.

Increased expression of miR-34a and miR-93 in rat liver during aging, and their impact on the expression of Mgst1 and Sirt1.

Age-dependent loss of oxidative defense is well recognized in rodent models, although the control mechanism is still obscure; a few studies have shown how microRNAs, a non-coding RNA species, regulate the expression of their target genes at the post-transcriptional level. In the current study, miR-34a and miR-93 are observed to increase in middle- and old-age rat liver, compared to young rats; the up-regulation of these two miRNAs is determined by qPCR through a grind-and-find approach, and histochemical in situ hybridization. Three commonly used miRNA target prediction programs suggest four candidate targets of miR-34a and miR-93: Sp1, Nrf2 (Nfe2l2), Sirt1 and Mgst1; their expression is found to be reduced inversely to the up-regulation of the two miRNAs by Western blotting of protein extracts, as well as immunofluorescence staining of intact liver tissues. Furthermore, the suppression of the four proteins by miR-34a/miR-93 is examined in HEK 293 cells by transfection and co-transfection; miR-34a represses all four proteins' expression, whereas miR-93 affects only Sp1, Sirt1 and Mgst1. Taken together, our study suggests a model of post-transcriptional repression, not only of genes involved in oxidative stress regulation and oxidative stress defense proteins, such as Sirt1 and Mgst1, but also of upstream transcription factors (TFs) regulating their activation, since Sp1 is the TF for both Sirt1 and Mgst1, and Nrf2 is the TF of Mgst1. Thus, up-regulation of both miR-34a and miR-93 constitutes an inescapable repression of two vital oxidative defense genes, by targeting not only the targets, but also transcription factors controlling their activation, a double dampening regulation at the post-transcriptional level.

Prosaposin in the secretome of marrow stroma-derived neural progenitor cells protects neural cells from apoptotic death.

Functionally, adult stem cells not only participate in replication and differentiation to various cell lineages, but also may be involved in rescuing cells from apoptosis. Identifying functional factors secreted by stem cells, as well as their target cells, may advance our understanding of stem cells' multifaceted physiologic functions. Here, we report that mouse bone marrow stromal cell-derived neuroprogenitor cells (mMSC-NPC) provide a protective function by secreting a key factor, prosaposin (PSAP), capable of rescuing mature neurons from apoptotic death. This factor is identified as the lead protein in the secretome of mMSC-NPC cultures by tandem mass spectroscopic profiling, and further validated by western blotting and immunocytochemistry. The secretome of MSC-NPC reduces toxin-induced cell death in cultures of rat pheochromocytoma neuronal cells, human ReNcell CX neurons, and rat cortical primary neurons; removal of PSAP by immunodepletion annuls this protective effect. This neuronal protection against toxin treatment was validated further by the recombinant PSAP peptide. Interestingly, the secretome of neuronal culture does not possess such a self-protective action. We suggest that upon injury, a subgroup of MSCs differentiates into neural/neuronal progenitor cells, and remains in this intermediate stem cell-like stage, defending injured neighboring mature neurons from apoptosis by secreting PSAP.

MicroRNA regulation in Ames dwarf mouse liver may contribute to delayed aging.

The Ames dwarf mouse is well known for its remarkable propensity to delay the onset of aging. Although significant advances have been made demonstrating that this aging phenotype results primarily from an endocrine imbalance, the post-transcriptional regulation of gene expression and its impact on longevity remains to be explored. Towards this end, we present the first comprehensive study by microRNA (miRNA) microarray screening to identify dwarf-specific lead miRNAs, and investigate their roles as pivotal molecular regulators directing the long-lived phenotype. Mapping the signature miRNAs to the inversely expressed putative target genes, followed by in situ immunohistochemical staining and in vitro correlation assays, reveals that dwarf mice post-transcriptionally regulate key proteins of intermediate metabolism, most importantly the biosynthetic pathway involving ornithine decarboxylase and spermidine synthase. Functional assays using 3'-untranslated region reporter constructs in co-transfection experiments confirm that miRNA-27a indeed suppresses the expression of both of these proteins, marking them as probable targets of this miRNA in vivo. Moreover, the putative repressed action of this miRNA on ornithine decarboxylase is identified in dwarf mouse liver as early as 2 months of age. Taken together, our results show that among the altered aspects of intermediate metabolism detected in the dwarf mouse liver--glutathione metabolism, the urea cycle and polyamine biosynthesis--miRNA-27a is a key post-transcriptional control. Furthermore, compared to its normal siblings, the dwarf mouse exhibits a head start in regulating these pathways to control their normality, which may ultimately contribute to its extended health-span and longevity.

Stepwise up-regulation of microRNA expression levels from replicating to reversible and irreversible growth arrest states in WI-38 human fibroblasts.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse genetic expression networks through their control of mRNA stability or translation. Their role in aging mechanisms has been proposed in various model systems. In this report, the expression profiling of 462 human miRNAs in the reversible growth arrest state of quiescence, and irreversible states of replicative senescence and hydrogen peroxide-induced premature senescence, are compared to young replicating lung fibroblasts. Greater numbers of up-regulated than down-regulated miRNAs are observed when cells stop proliferating, particularly in premature senescence, somewhat less in replicative senescence, and less still in quiescence. Several altered miRNA expressions are shared by the three growth arrest states, including the up-regulation of miR-34a, -624, -638 and miR-377, and the down-regulation of miR-365 and miR-512-5p. miRNAs up-regulated in both permanent growth arrest states but not in quiescence include let-7g, miR-26a, -136, -144, -195 and miR-200b. In each of the growth arrest states, miR-34a and let-7f have the most robust up-regulation in H(2)O(2)-induced premature senescence, followed by miR-638 and miR-663 in replicative senescence, and finally, miR-331-3p and miR-595 in quiescence. Our comprehensive evaluation of miRNA target correlations with known biomarkers for replicative senescence suggests that miRNAs may repress pathways controlling not only cell cycle traverse and proliferation, but also insulin-like signaling, DNA repair and apoptosis, all of which are cellular functions deficient in senescent human fibroblasts.

Secretome from mesenchymal stem cells induces angiogenesis via Cyr61.

It is well known that bone marrow-derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine-rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic-inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61-depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis-promoting activity, a key event needed for regeneration and repair of injured tissues. J. Cell. Physiol. 219: 563-571, 2009. (c) 2009 Wiley-Liss, Inc.

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.