Identification of plants' functional counterpart of the metazoan mediator of DNA Damage checkpoint 1.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

STAT3 activation in microglia increases pericyte apoptosis in diabetic retinas through TNF-ɑ/AKT/p70S6 kinase signaling.

Diabetic retinopathy (DR) is one of the vascular complications associated with diabetes mellitus. Pericyte loss is an early characteristic phenomenon in DR. However, the mechanism by which pericyte apoptosis occurs in DR is not fully understood. We have focused on the increased STAT3 activation in diabetic retinas because STAT3 activation is associated with inflammation, and persistent chronic inflammation is closely related to retinal lesions. In this study, we demonstrated that STAT3 was activated by IFN-γ and IL-6 that highly expressed in diabetic retinas. We identified TNF-α as a potent inducer of pericyte apoptosis in diabetic retinas from the gene expression analysis and found that STAT3 activation in microglia increased TNF-α expression in the diabetic retinas. We also demonstrated that increased TNF-α expression in microglia caused pericyte apoptosis through downregulating AKT/p70S6 kinase signaling. Moreover, we took advantage of mice lacking STAT3 in microglia and demonstrated that STAT3 ablation in microglia reduced the pericyte apoptosis and TNF-α expression in the diabetic retinas. These results suggest that STAT3 activation in microglia plays an important role in pericyte apoptosis in the diabetic retinas through increased TNF-α expression and provide STAT3 activation in microglia as a potential therapeutic target for preventing pericyte loss in DR.

Hyperglycemia-induced oxidative stress promotes tumor metastasis by upregulating vWF expression in endothelial cells through the transcription factor GATA1.

Diabetes mellitus (DM) characterized by hyperglycemia is a chronic metabolic disorder that leads to many symptoms and vascular complications. Despite the close association between DM and cancer progression, the response and role of endothelial cells (ECs) under diabetic conditions in tumor metastasis remain to be elucidated. In this study, we sought to determine whether and how ECs under diabetic conditions contribute to tumor metastasis. We have taken advantage of syngeneic mouse tumor models of Lewis lung carcinoma (LLC) and melanoma (B16F10) cells and a streptozotocin (STZ)-induced hyperglycemia model. We demonstrated that hyperglycemia increased the metastasis of LLC and B16F10 cells in an experimental metastasis model with an intravenous injection of the tumor cells. We also found that hyperglycemia promoted lung metastasis of tumor cells by increasing the adhesiveness of ECs to facilitate the adhesion of tumor cells to ECs rather than affecting the metastatic behavior of tumor cells themselves. From the analysis of gene expression in primary lung ECs from STZ-treated mice, we identified that vWF promoted the adhesion of tumor cells to ECs and the transendothelial migration of tumor cells. Mechanistically, hyperglycemia-induced oxidative stress in ECs, and increased oxidative stress enhanced vWF expression in ECs through an increase in the transcription factor GATA1. These results provide evidence for the role of vWF in ECs in promoting hyperglycemia-induced tumor metastasis and potential therapeutic targets for the regulation of vWF expression in ECs and hyperglycemia-induced tumor metastasis.

Enhanced eosinophil-mediated inflammation associated with antibody and complement-dependent pneumonic insults in critical COVID-19.

Despite the worldwide effect of the coronavirus disease 2019 (COVID-19) pandemic, the underlying mechanisms of fatal viral pneumonia remain elusive. Here, we show that critical COVID-19 is associated with enhanced eosinophil-mediated inflammation when compared to non-critical cases. In addition, we confirm increased T helper (Th)2-biased adaptive immune responses, accompanying overt complement activation, in the critical group. Moreover, enhanced antibody responses and complement activation are associated with disease pathogenesis as evidenced by formation of immune complexes and membrane attack complexes in airways and vasculature of lung biopsies from six fatal cases, as well as by enhanced hallmark gene set signatures of Fcγ receptor (FcγR) signaling and complement activation in myeloid cells of respiratory specimens from critical COVID-19 patients. These results suggest that SARS-CoV-2 infection may drive specific innate immune responses, including eosinophil-mediated inflammation, and subsequent pulmonary pathogenesis via enhanced Th2-biased immune responses, which might be crucial drivers of critical disease in COVID-19 patients.

STAT3 activation in microglia exacerbates hippocampal neuronal apoptosis in diabetic brains.

Diabetes mellitus (DM) characterized by hyperglycemia leads to a variety of complications, including cognitive impairment or memory loss. The hippocampus is a key brain area for learning and memory and is one of the regions that is most sensitive to diabetes. However, the pathogenesis of diabetic neuronal lesion is not yet completely understood. We focused on the association of microglia activation and brain lesions in diabetes. In this study, we investigated whether and how signal transducer and activator of transcription 3 (STAT3) activation in microglia affects neuronal lesions in diabetic brains. Using a streptozotocin-induced type 1 DM model, we showed enhanced hippocampal neuronal apoptosis that was associated with increased STAT3 activation. We found that hyperglycemia increased the expression of inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6, in the diabetic hippocampus. In particular, IFN-γ induced autocrine activation of microglia, and STAT3 activation is important for this process. We also demonstrated that STAT3 activation in microglia increased tumor necrosis factor-α (TNF-α) expression; subsequently, TNF-α increased neuronal apoptosis by increasing reactive oxygen species (ROS) levels in the neuronal cells. We also took advantage of mice lacking STAT3 in microglia and demonstrated that depletion of microglial STAT3 reduced neuronal apoptosis in the diabetic hippocampus. Taken together, these results suggest that STAT3 activation in microglia plays an important role in hyperglycemia-induced neuronal apoptosis in the diabetic hippocampus and provide a potential therapeutic benefit of STAT3 inhibition in microglia for preventing diabetic neuronal lesions.

A muscular hypotonia-associated STIM1 mutant at R429 induces abnormalities in intracellular Ca2+ movement and extracellular Ca2+ entry in skeletal muscle.

Stromal interaction molecule 1 (STIM1) mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism, which is involved in the physiological functions of various tissues, including skeletal muscle. STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.

Angiopoietin 1 attenuates interleukin-6-induced endothelial cell permeability through SHP-1.

The regulation of endothelial cell (EC) permeability is critical for the physiological homeostasis of blood vessels and tissues. The elevation of pro-inflammatory cytokines is highly associated with lesions, such as the increased vascular permeability of diabetic retinas. We have previously reported that interleukin-6 (IL-6) increases EC permeability through the downregulation of tight junction protein expression. Angiopoietin 1 (Ang1) has an anti-permeability function, but the effect of Ang1 on vascular permeability induced by inflammatory cytokines is unclear. In the present study, we investigated the effect of Ang1 on IL-6-induced EC permeability and its underlying molecular mechanisms. We demonstrated that Ang1 inhibited the IL-6-induced increase in EC permeability by inhibiting the reductions in the levels of tight junction protein ZO-1 and occludin, which was related to the decrease in vascular endothelial growth factor (VEGF) secretion through the inhibition of STAT3 activation by Ang1. Mechanistically, Ang1 induced the dissociation of the tyrosine phosphatase SHP-1 from the Tie2 receptor and increased the binding of SHP-1 to JAK1, JAK2, and STAT3, which are IL-6 downstream signaling proteins. We conclude that SHP-1 plays an important role in the Ang1-induced inhibition of JAK/STAT3 signaling. These results provide evidence for a potential beneficial role of Ang1 in suppressing the vascular permeability induced by the pro-inflammatory cytokine IL-6 in diabetic retinopathy.

Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT.

Although many methods to assess sensitization have been investigated to replace animal testing, it is still imperative to develop an in vitro method to minimize the use of animals and to classify sensitizers. Recently, an assay using the human keratinocyte cell line (HaCaT) was developed as an alternative method. Our aim was to optimize this method and validate its ability to assess sensitization. The highest dose that resulted in 75% cell viability was determined for each test substance. Then, serial dilutions of the dose were applied to measure the levels of secreted proinflammatory cytokines. To optimize the assay, statistical analyses were performed to determine whether all of the doses tested were necessary to maintain the predictive values. Exclusion of the 0.5× dose did not change the predictive values drastically. To validate the optimized method, 22 substances were evaluated without the 0.5× dose, resulting in overall predictive values of 83.3% for sensitivity, 80.0% for specificity, and 81.8% for accuracy, which are comparable to results from other validated assays. These results suggest that statistical analysis can assist in development of alternative in vitro methods and that the optimized HaCaT cell assay is reproducible.

Role of Inflammation in Classification of Diabetic Macular Edema by Optical Coherence Tomography.

Diabetic macular edema (DME) is the abnormal accumulation of fluid in the subretinal or intraretinal spaces in the macula in patients with diabetic retinopathy and leads to severely impaired central vision. Technical developments in retinal imaging systems have led to many advances in the study of DME. In particular, optical coherence tomography (OCT) can provide longitudinal and microstructural analysis of the macula. A comprehensive review was provided regarding the role of inflammation using OCT-based classification of DME and current and ongoing therapeutic approaches. In this review, we first describe the pathogenesis of DME, then discuss the classification of DME based on OCT findings and the association of different types of DME with inflammation, and finally describe current and ongoing therapeutic approaches using OCT-based classification of DME. Inflammation has an important role in the pathogenesis of DME, but its role appears to differ among the DME phenotypes, as determined by OCT. It is important to determine how the different DME subtypes respond to intravitreal injections of steroids, antivascular endothelial growth factor agents, and other drugs to improve prognosis and responsiveness to treatment.

Retinal pigment epithelium-derived transforming growth factor-ß2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor-2 expression.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-ß secretion, particularly TGF-ß2. However, it is largely unclear whether and how TGF-ß2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-ß2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-ß2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-ß2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-ß2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-ß2 expression in RPE cells under pathologic conditions.

Interaction between microglia and retinal pigment epithelial cells determines the integrity of outer blood-retinal barrier in diabetic retinopathy.

Inner and outer blood-retinal barriers (BRBs), mainly composed of retinal endothelial cells and retinal pigment epithelial (RPE) cells, respectively, maintain the integrity of the retinal tissues. In this study, we aimed to investigate the mechanisms of the outer BRB disruption regarding the interaction between RPE and microglia. In mice with high-fat diet-induced obesity and streptozotocin-induced hyperglycemia, microglia accumulated on the RPE layer, as in those after intravitreal injection of interleukin (IL)-6, which is elevated in ocular fluids of patients with diabetic retinopathy. Although IL-6 did not directly affect the levels of zonula occludens (ZO)-1 and occludin in RPE cells, IL-6 increased VEGFA mRNA in RPE cells to recruit microglial cells. In microglial cells, IL-6 upregulated the mRNA levels of MCP1, MIP1A, and MIP1B, to amplify the recruitment of microglial cells. In this manner, IL-6 modulated RPE and microglial cells to attract microglial cells on RPE cells. Furthermore, IL-6-treated microglial cells produced and secreted tumor necrosis factor (TNF)-α, which activated NF-κB and decreased the levels of ZO-1 in RPE cells. As STAT3 inhibition reversed the effects of IL-6-treated microglial cells on the RPE monolayer in vitro, it reduced the recruitment of microglial cells and the production of TNF-α in RPE tissues in streptozotocin-treated mice. Taken together, IL-6-treated RPE and microglial cells amplified the recruitment of microglial cells and IL-6-treated microglial cells produced TNF-α to disrupt the outer BRB in diabetic retinopathy.

Microgravity induces autophagy via mitochondrial dysfunction in human Hodgkin's lymphoma cells.

Gravitational forces can impose physical stresses on the human body as it functions to maintain homeostasis. It has been reported that astronauts exposed to microgravity experience altered biological functions and many subsequent studies on the effects of microgravity have therefore been conducted. However, the anticancer mechanisms of simulated microgravity remain unclear. We previously showed that the proliferation of human Hodgkin's lymphoma (HL) cells was inhibited when these cells were cultured in time-averaged simulated microgravity (taSMG). In the present study, we investigated whether taSMG produced an anticancer effect. Exposure of human HL cells to taSMG for 2 days increased their reactive oxygen species (ROS) production and NADPH oxidase family gene expression, while mitochondrial mass, ATPase, ATP synthase, and intracellular ATP levels were decreased. Furthermore, human HL cells exposed to taSMG underwent autophagy via AMPK/Akt/mTOR and MAPK pathway modulation; such autophagy was inhibited by the ROS scavenger N-acetylcysteine (NAC). These results suggest an innovative therapeutic approach to HL that is markedly different from conventional chemotherapy and radiotherapy.

Propranolol increases vascular permeability through pericyte apoptosis and exacerbates oxygen-induced retinopathy.

Retinopathy of prematurity (ROP) is an eye disease that causes blindness due to delayed vascular growth, retinal ischemia, and resulting abnormal angiogenesis. Nonselective ß-antagonist propranolol is in clinical trials for the treatment of ROP due to its effect of reducing VEGF expression and inhibiting retinal angiogenesis in oxygen-induced ROP models (OIR), but the mechanism by which propranolol acts on ROP vessels is still unclear. In the present study, we have focused on the effect of propranolol on pericyte survival and vascular permeability. We demonstrated that propranolol increases pericyte apoptosis more sensitively than endothelial cells (ECs), thereby weakening EC tight junctions to increase endothelial permeability in co-cultures of pericytes and ECs. Mechanistically, pericyte apoptosis by propranolol was due to the inhibition of Akt signaling pathway. We also demonstrated that propranolol increases pericyte loss and vascular permeability of retinal vessels in a mouse model of OIR. These results suggest that propranolol may be negative for blood vessels in retinas of OIR, and that the efficacy of propranolol for the treatment of ROP needs to be more thoroughly verified.

Oleanolic Acids Inhibit Vascular Endothelial Growth Factor Receptor 2 Signaling in Endothelial Cells: Implication for Anti-Angiogenic Therapy.

Angiogenesis must be precisely controlled because uncontrolled angiogenesis is involved in aggravation of disease symptoms. Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) signaling is a key pathway leading to angiogenic responses in vascular endothelial cells (ECs). Therefore, targeting VEGF/VEGFR-2 signaling may be effective at modulating angiogenesis to alleviate various disease symptoms. Oleanolic acid was verified as a VEGFR-2 binding chemical from anticancer herbs with similar binding affinity as a reference drug in the Protein Data Bank (PDB) entry 3CJG of model A coordination. Oleanolic acid effectively inhibited VEGF-induced VEGFR-2 activation and angiogenesis in HU-VECs without cytotoxicity. We also verified that oleanolic acid inhibits in vivo angiogenesis during the development and the course of the retinopathy of prematurity (ROP) model in the mouse retina. Taken together, our results suggest a potential therapeutic benefit of oleanolic acid for inhibiting angiogenesis in proangiogenic diseases, including retinopathy.

Epithelial cell-derived cytokines CST3 and GDF15 as potential therapeutics for pulmonary fibrosis.

While wound healing is completed, the epithelium functions to normalize the interstitial context by eliminating fibroblasts excited during matrix reconstruction. If not, tissues undergo pathologic fibrosis. Pulmonary fibrosis is a fatal and hardly curable disorder. We here tried to identify epithelium-derived cytokines capable of ameliorating pulmonary fibrosis. Human lung fibroblasts were inactivated in epithelial cell-conditioned media. Cystatin C (CST3) and growth differentiation factor 15 (GDF15) were found to be enriched in the conditioned media and to inhibit the growth and activation of lung fibroblasts by inactivating the TGF-Smad pathway. In mouse and human lungs with interstitial fibrosis, CST3 and GDF15 expressions were markedly reduced, and the restoration of these cytokines alleviated the fibrotic changes in mouse lungs. These results suggest that CST3 and GDF15 are bona fide regulators to prevent excessive proliferation and activation of fibroblasts in injured lungs. These cytokines could be potential therapeutics for ameliorating interstitial lung fibrosis.

Plastid genome analysis of three Nemaliophycidae red algal species suggests environmental adaptation for iron limited habitats.

The red algal subclass Nemaliophycidae includes both marine and freshwater taxa that contribute to more than half of the freshwater species in Rhodophyta. Given that these taxa inhabit diverse habitats, the Nemaliophycidae is a suitable model for studying environmental adaptation. For this purpose, we characterized plastid genomes of two freshwater species, Kumanoa americana (Batrachospermales) and Thorea hispida (Thoreales), and one marine species Palmaria palmata (Palmariales). Comparative genome analysis identified seven genes (ycf34, ycf35, ycf37, ycf46, ycf91, grx, and pbsA) that were different among marine and freshwater species. Among currently available red algal plastid genomes (127), four genes (pbsA, ycf34, ycf35, ycf37) were retained in most of the marine species. Among these, the pbsA gene, known for encoding heme oxygenase, had two additional copies (HMOX1 and HMOX2) that were newly discovered and were reported from previously red algal nuclear genomes. Each type of heme oxygenase had a different evolutionary history and special modifications (e.g., plastid targeting signal peptide). Based on this observation, we suggest that the plastid-encoded pbsA contributes to the iron controlling system in iron-deprived conditions. Thus, we highlight that this functional requirement may have prevented gene loss during the long evolutionary history of red algal plastid genomes.

ß-Adrenergic receptor agonists attenuate pericyte loss in diabetic retinas through Akt activation.

Pericytes (PCs) are crucial in maintaining the quiescence of endothelial cells (ECs) and the integrity of EC tight junctions. Especially in diabetic retinopathy (DR), PC loss is one of the early pathologic changes in capillaries of diabetic retinas. Thus, preventing PC loss is beneficial for attenuating vision impairment in patients with DR. Although many studies have revealed the mechanism of PC loss in retinas, little is known about the mechanisms that increase PC survival. We focused on the effect of ß-adrenergic receptor agonists (ß-agonists) on PC loss in diabetic retinas. In this study, ß-agonists increased the cell viability of PCs by increasing PC survival and proliferation. Mechanistically, ß-agonist-induced protein kinase B activation in PCs reduced PC apoptosis in response to various stimuli. ß2-agonists more potently increased PC survival than ß1-agonists. ß2-Agonist reduced vascular leakage and PC loss in retinas of mice with streptozotocin-induced diabetes. In cocultures of PCs and ECs, ß2-agonists restored the altered permeability and ZO-1 expression in ECs induced by PC loss. We concluded that ß-agonists, especially ß2-agonists, increase PC survival, thereby preventing diabetes-induced PC loss in retinas. These results provide a potential therapeutic benefit of ß-agonists for preventing PC loss in DR.-Yun, J.-H., Jeong, H.-S., Kim, K.-J., Han, M. H., Lee, E. H., Lee, K., Cho, C.-H. ß-Adrenergic receptor agonists attenuate pericyte loss in diabetic retinas through Akt activation.

Accelerated tumor growth under intermittent hypoxia is associated with hypoxia-inducible factor-1-dependent adaptive responses to hypoxia.

Mounting evidence has revealed a causative role of intermittent hypoxia (IH) in cancer progression in mouse models of obstructive sleep apnea (OSA), but most studies have focused on the effects of IH following tumor implantation using an exposure to single IH frequency. Thus, we aimed to investigate 1) the potential effect of IH on the initial tumor growth in patients with OSA without an interaction with other mechanisms induced by IH in mice and 2) the influence of the IH frequency on tumor growth, which were tested using pre-conditioning with IH (Pre-IH) and 2 different IH frequencies, respectively. Pre-IH was achieved by alternatively maintaining melanoma cells between normoxia (10 min, 21% O2) and hypoxia (50 min, 1% O2) for 7 days (12 cycles per day) before administering them to mice. The conditions for IH-1 and IH-2 were 90 s of 12% FiO2 followed by 270s of 21% FiO2 (10 cycles/h), and 90 s of 12% FiO2 and 90 s of 21% FiO2 (20 cycles/h), respectively, for 8 h per day. Tumor growth was significantly higher in the Pre-IH group than in the normoxia group. In addition, the IH-2 group showed more accelerated tumor growth compared to the normoxia and IH-1 groups. Immunohistochemistry and gene-expression results consistently showed the up-regulation of molecules associated with HIF-1α-dependent hypoxic adaptation in tumors of the Pre-IH and IH-2 groups. Our findings reveal that IH increased tumor progression in a frequency-dependent manner, regardless of whether it was introduced before or after in vivo tumor cell implantation.

Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy.

Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro-inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL-6-induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO-1 and occludin. In a co-culture model with microglia and endothelial cells under a high glucose condition, the microglia-derived IL-6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL-6-induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO-1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123-1134, 2017. © 2016 Wiley Periodicals, Inc.