Pharmacological inhibition of CSF1R by GW2580 reduces microglial proliferation and is protective against neuroinflammation and dopaminergic neurodegeneration.

Increased pro-inflammatory cytokine levels and proliferation of activated microglia have been found in Parkinson's disease (PD) patients and animal models of PD, suggesting that targeting of the microglial inflammatory response may result in neuroprotection in PD. Microglial proliferation is regulated by many factors, but colony stimulating factor-1 receptor (CSF1R) has emerged as a primary factor. Using data mining techniques on existing microarray data, we found that mRNA expression of the CSF1R ligand, CSF-1, is increased in the brain of PD patients compared to controls. In two different neurotoxic mouse models of PD, acute MPTP and sub-chronic LPS treatment, mRNA and protein levels of CSF1R and CSF-1 were significantly increased. Treatment with the CSF1R inhibitor GW2580 significantly attenuated MPTP-induced CSF1R activation and Iba1-positive cell proliferation, without a reduction of the basal Iba1-positive population in the substantia nigra. GW2580 treatment also significantly decreased mRNA levels of pro-inflammatory factors, without alteration of anti-inflammatory mediators, and significantly attenuated the MPTP-induced loss of dopamine neurons and motor behavioral deficits. Importantly, these effects were observed in the absence of overt microglial depletion, suggesting that targeting CSF1R signaling may be a viable neuroprotective strategy in PD that disrupts pro-inflammatory signaling, but maintains the beneficial effects of microglia.

Hepatic Mitochondrial Defects in a Nonalcoholic Fatty Liver Disease Mouse Model Are Associated with Increased Degradation of Oxidative Phosphorylation Subunits.

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic mitochondrial dysfunction characterized by reduced ATP synthesis. We applied the 2H2O-metabolic labeling approach to test the hypothesis that the reduced stability of oxidative phosphorylation proteins contributes to mitochondrial dysfunction in a diet-induced mouse model of NAFLD. A high fat diet containing cholesterol (a so-called Western diet (WD)) led to hepatic oxidative stress, steatosis, inflammation and mild fibrosis, all markers of NAFLD, in low density cholesterol (LDL) receptor deficient (LDLR-/-) mice. In addition, compared with controls (LDLR-/- mice on normal diet), livers from NAFLD mice had reduced citrate synthase activity and ATP content, suggesting mitochondrial impairment. Proteome dynamics study revealed that mitochondrial defects are associated with reduced average half-lives of mitochondrial proteins in NAFLD mice (5.41 ± 0.46 versus 5.15 ± 0.49 day, p < 0.05). In particular, the WD reduced stability of oxidative phosphorylation subunits, including cytochrome b-c1 complex subunit 1 (5.9 ± 0.1 versus 3.4 ± 0.8 day), ATP synthase subunit α (6.3 ± 0.4 versus 5.5 ± 0.4 day) and ATP synthase F(0) complex subunit B1 of complex V (8.5 ± 0.6 versus 6.5 ± 0.2 day) (p < 0.05). These changes were associated with impaired complex III and F0F1-ATP synthase activities. Markers of mitophagy were increased, but proteasomal degradation activity were reduced in NAFLD mice liver, suggesting that ATP deficiency because of reduced stability of oxidative phosphorylation complex subunits contributed to inhibition of ubiquitin-proteasome and activation of mitophagy. In conclusion, the 2H2O-metabolic labeling approach shows that increased degradation of hepatic oxidative phosphorylation subunits contributed to mitochondrial impairment in NAFLD mice.

HDL flux is higher in patients with nonalcoholic fatty liver disease.

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS (n = 7), NASH (n = 8), and healthy controls (n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher (P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio (P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL-1·min-1) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins' turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.

MitoNEET Deficiency Alleviates Experimental Alcoholic Steatohepatitis in Mice by Stimulating Endocrine Adiponectin-Fgf15 Axis.

MitoNEET (mNT) (CDGSH iron-sulfur domain-containing protein 1 or CISD1) is an outer mitochondrial membrane protein that donates 2Fe-2S clusters to apo-acceptor proteins. In the present study, using a global mNT knock-out (mNTKO) mouse model, we investigated the in vivo functional role of mNT in the development of alcoholic steatohepatitis. Experimental alcoholic steatohepatitis was achieved by pair feeding wild-type (WT) and mNTKO mice with Lieber-DeCarli ethanol-containing diets for 4 weeks. Strikingly, chronically ethanol-fed mNTKO mice were completely resistant to ethanol-induced steatohepatitis as revealed by dramatically reduced hepatic triglycerides, decreased hepatic cholesterol level, diminished liver inflammatory response, and normalized serum ALT levels. Mechanistic studies demonstrated that ethanol administration to mNTKO mice induced two pivotal endocrine hormones, namely, adipose-derived adiponectin and gut-derived fibroblast growth factor 15 (Fgf15). The elevation in circulating levels of adiponectin and Fgf15 led to normalized hepatic and serum levels of bile acids, limited hepatic accumulation of toxic bile, attenuated inflammation, and amelioration of liver injury in the ethanol-fed mNTKO mice. Other potential mechanisms such as reduced oxidative stress, activated Sirt1 signaling, and diminished NF-κB activity also contribute to hepatic improvement in the ethanol-fed mNTKO mice. In conclusion, the present study identified adiponectin and Fgf15 as pivotal adipose-gut-liver metabolic coordinators in mediating the protective action of mNT deficiency against development of alcoholic steatohepatitis in mice. Our findings may help to establish mNT as a novel therapeutic target and pharmacological inhibition of mNT may be beneficial for the prevention and treatment of human alcoholic steatohepatitis.

The Detrimental Role Played by Lipocalin-2 in Alcoholic Fatty Liver in Mice.

We have previously shown that the ethanol-mediated elevation of lipocaline-2 (LCN2) is closely associated with the development of alcoholic fatty liver disease (AFLD) in mice. Herein, we aimed to understand the functional significance of LCN2 induction by ethanol and to explore its underlying mechanisms. We evaluated the effects of LCN2 in an in vitro cellular alcoholic steatosis model and in an animal study using wild-type and LCN2 knockout mice fed for 4 weeks with an ethanol-supplemented Lieber-DeCarli diet. In the cellular model of alcoholic steatosis, recombinant LCN2 or overexpression of LCN2 exacerbated ethanol-induced fat accumulation, whereas knocking down LCN2 prevented steatosis in hepatocytes exposed to ethanol. Consistently, removal of LCN2 partially but significantly alleviated alcoholic fatty liver injury in mice. Mechanistically, LCN2 mediates detrimental effects of ethanol in the liver via disrupted multiple signaling pathways, including aberrant nicotinamide phosphoribosyltransferase-sirtuin 1 axis, perturbed endocrine metabolic regulatory fibroblast growth factor 15/19 signaling, and impaired chaperone-mediated autophagy. Finally, compared with healthy human livers, liver samples from patients with AFLD had lower gene expression of several LCN2-regualted molecules. Our study demonstrated a pivotal and causal role of LCN2 in the development of AFLD and suggested that targeting the LCN2 could be of great value for the treatment of human AFLD.

All-trans-retinoic acid ameliorates hepatic steatosis in mice by a novel transcriptional cascade.

UNLABELLED: Mice deficient in small heterodimer partner (SHP) are protected from diet-induced hepatic steatosis resulting from increased fatty acid oxidation and decreased lipogenesis. The decreased lipogenesis appears to be a direct consequence of very low expression of peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2), a potent lipogenic transcription factor, in the SHP(-/-) liver. The current study focused on the identification of a SHP-dependent regulatory cascade that controls PPAR-γ2 gene expression, thereby regulating hepatic fat accumulation. Illumina BeadChip array (Illumina, Inc., San Diego, CA) and real-time polymerase chain reaction were used to identify genes responsible for the linkage between SHP and PPAR-γ2 using hepatic RNAs isolated from SHP(-/-) and SHP-overexpressing mice. The initial efforts identify that hairy and enhancer of split 6 (Hes6), a novel transcriptional repressor, is an important mediator of the regulation of PPAR-γ2 transcription by SHP. The Hes6 promoter is specifically activated by the retinoic acid receptor (RAR) in response to its natural agonist ligand, all-trans retinoic acid (atRA), and is repressed by SHP. Hes6 subsequently represses hepatocyte nuclear factor 4 alpha (HNF-4α)-activated PPAR-γ2 gene expression by direct inhibition of HNF-4α transcriptional activity. Furthermore, we provide evidences that atRA treatment or adenovirus-mediated RAR-α overexpression significantly reduced hepatic fat accumulation in obese mouse models, as observed in earlier studies, and the beneficial effect is achieved by the proposed transcriptional cascade. CONCLUSIONS: Our study describes a novel transcriptional regulatory cascade controlling hepatic lipid metabolism that identifies retinoic acid signaling as a new therapeutic approach to nonalcoholic fatty liver diseases.

Obeticholic acid's effect on HDL function in MASH varies by diabetic status.

Inflammation and oxidative stress are the key factors in the pathogenesis of both metabolic dysfunction-associated steatohepatitis (MASH) and atherosclerosis. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, improves hepatic inflammation and fibrosis in patients with MASH. However, it also reduces HDL cholesterol, suggesting that OCA may increase cardiovascular disease (CVD) risk in patients with MASH. We assessed HDL cholesterol efflux function, antioxidant (paraoxonase and ceruloplasmin activity), pro-inflammatory index, and particle sizes in a small group of patients with and without diabetes (n = 10/group) at baseline and after 18 months of OCA treatment. Patients on lipid-lowering medications (statins, fibrates) were excluded. At baseline, ferritin levels were higher in patients with MASH without diabetes (336.5 [157.0, 451.0] vs. 83 [36.0, 151.0] ng/mL, p < 0.005). Markers of HDL functions were similar in both groups. OCA therapy significantly improved liver histology and liver enzymes but increased alkaline phosphatase levels in nondiabetic patients with MASH (p < 0.05). However, it did not have any significant effect on cholesterol efflux and the antioxidant paraoxonase functions. In nondiabetics, ceruloplasmin (CP) antioxidant activity decreased (p < 0.005) and the pro-inflammatory index of HDL increased (p < 0.005) due to OCA therapy. In contrast, in diabetics, OCA increased levels of pre-ß-HDL-the HDL particles enhanced protective capacity (p = 0.005) with no alteration in HDL functionality. In all patients, serum glucose levels were negatively correlated with OCA-induced change in pro-inflammatory function in HDL (p < 0.001), which was primarily due to diabetes (p = 0.05). These preliminary results suggest a distinct effect of OCA therapy on diabetic and nondiabetic patients with MASH and warrant a future large-scale study.

Voltage-Gated Proton Channel Hv1 Regulates Neuroinflammation and Dopaminergic Neurodegeneration in Parkinson's Disease Models.

Although the precise mechanisms for neurodegeneration in Parkinson's disease (PD) are unknown, evidence suggests that neuroinflammation is a critical factor in the pathogenic process. Here, we sought to determine whether the voltage-gated proton channel, Hv1 (HVCN1), which is expressed in microglia and regulates NADPH oxidase, is associated with dopaminergic neurodegeneration. We utilized data mining to evaluate the mRNA expression of HVCN1 in the brains of PD patients and controls and uncovered increased expression of the gene encoding Hv1, HVCN1, in the brains of PD patients compared to controls, specifically in male PD patients. In an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 4 × 16 mg/kg) mouse model of PD, Hvcn1 gene expression was increased 2-fold in the striatum. MPTP administration to wild-type (WT) mice resulted in a ~65% loss of tyrosine hydroxylase positive neurons (TH+) in the substantia nigra (SN), while a ~39% loss was observed in Hv1 knockout (KO) mice. Comparable neuroprotective effects of Hv1 deficiency were found in a repeated-dose LPS model. Neuroprotection was associated with decreased pro-inflammatory cytokine levels and pro-oxidant factors in both neurotoxicant animal models. These in vivo results were confirmed in primary microglial cultures, with LPS treatment increasing Hvcn1 mRNA levels and Hv1 KO microglia failing to exhibit the LPS-mediated inflammatory response. Conditioned media from Hv1 KO microglia treated with LPS resulted in an attenuated loss of cultured dopamine neuron cell viability compared to WT microglia. Taken together, these data suggest that Hv1 is upregulated and mediates microglial pro-inflammatory cytokine production in parkinsonian models and therefore represents a novel target for neuroprotection.

Ginseng Berry Extract Prevents Atherogenesis via Anti-Inflammatory Action by Upregulating Phase II Gene Expression.

Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE) and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1) and glutamine-cysteine ligase (GCL). Furthermore, KGBE administration suppressed NF-κB-mediated expression of atherogenic inflammatory genes (TNF-α, IL-1ß, iNOS, COX-2, ICAM-1, and VCAM-1), without altering serum cholesterol levels, in ApoE(-/-) mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-κB activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-α-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-κB-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis.

Carbon monoxide promotes VEGF expression by increasing HIF-1alpha protein level via two distinct mechanisms, translational activation and stabilization of HIF-1alpha protein.

Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates HIF-1 (hypoxia-inducible transcription factor-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with CORM-2 (CO-releasing molecule) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced HO-1 (hemeoxygenase-1) expression and increased nuclear HIF-1α protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1α siRNA and a hemeoxygenase inhibitor, indicating that CO stimulates VEGF expression via up-regulation of HIF-1α protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70(S6k) and eIF-4E as well as phosphorylating their upstream signal mediators Akt and ERK. These translational signal events and HIF-1α protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1α. In addition, CORM-2 also increased stability of the HIF-1α protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1α degradation pathway. CORM-2 increased HIF-1α/HSP90α interaction, which is responsible for HIF-1α stabilization, and HSP90-specific inhibitors decreased this interaction, HIF-1α protein level, and VEGF expression. Furthermore, HSP90α knockdown suppressed CORM-2-induced increases in HIF-1α and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1α protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1α.

Effect of recombinant IL-10 on cultured fetal rat alveolar type II cells exposed to 65%-hyperoxia.

BACKGROUND: Hyperoxia plays an important role in the genesis of lung injury in preterm infants. Although alveolar type II cells are the main target of hyperoxic lung injury, the exact mechanisms whereby hyperoxia on fetal alveolar type II cells contributes to the genesis of lung injury are not fully defined, and there have been no specific measures for protection of fetal alveolar type II cells. OBJECTIVE: The aim of this study was to investigate (a) cell death response and inflammatory response in fetal alveolar type II cells in the transitional period from canalicular to saccular stages during 65%-hyperoxia and (b) whether the injurious stimulus is promoted by creating an imbalance between pro- and anti-inflammatory cytokines and (c) whether treatment with an anti-inflammatory cytokine may be effective for protection of fetal alveolar type II cells from injury secondary to 65%-hyperoxia. METHODS: Fetal alveolar type II cells were isolated on embryonic day 19 and exposed to 65%-oxygen for 24 h and 36 h. Cells in room air were used as controls. Cellular necrosis was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry, and cell proliferation was studied by BrdU incorporation. Release of cytokines including VEGF was analyzed by ELISA, and their gene expressions were investigated by qRT-PCR. RESULTS: 65%-hyperoxia increased cellular necrosis, whereas it decreased cell proliferation in a time-dependent manner compared to controls. 65%-hyperoxia stimulated IL-8-release in a time-dependent fashion, whereas the anti-inflammatory cytokine, IL-10, showed an opposite response. 65%-hyperoxia induced a significant decrease of VEGF-release compared to controls, and similar findings were observed on IL-8/IL-10/VEGF genes expression. Preincubation of recombinant IL-10 prior to 65%-hyperoxia decreased cellular necrosis and IL-8-release, and increased VEGF-release and cell proliferation significantly compared to hyperoxic cells without IL-10. CONCLUSIONS: The present study provides an experimental evidence that IL-10 may play a potential role in protection of fetal alveolar type II cells from injury induced by 65%-hyperoxia.

The farnesyltransferase inhibitor LB42708 suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting ras-dependent mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signal pathways.

Farnesyltransferase (FTase) inhibitors induce growth arrest and apoptosis in various human cancer cells by inhibiting the post-translational activation of Ras. FTase inhibitors also function to suppress the release of vascular endothelial growth factor (VEGF) from tumor cells by inhibiting Ras activation; however, the effects of FTase inhibitors on VEGF-induced angiogenesis in endothelial cells have not been studied. We have investigated the antiangiogenic effect and molecular mechanism of 4-((1-((1-((4-bromophenyl)methyl)-1H-imidazol-5-yl)methyl)-4-(1-napthalenyl)-1H-pyrrol-3-yl)carbonyl)-(9C1)-morpholine (LB42708), a selective nonpeptidic FTase inhibitor, using in vitro and in vivo assay systems. LB42708 inhibited VEGF-induced Ras activation and subsequently suppressed angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase/p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric-oxide synthase pathways in endothelial cells without altering FAK/Src activation. In addition, this inhibitor suppressed VEGF-induced endothelial cell cycle progression at the G(1) phase by suppressing cyclin D1 expression and retinoblastoma phosphorylation as well as up-regulating the cyclin-dependent kinase inhibitors p21 and p27. Knockdown of Ras by short interfering RNA revealed similar inhibitory effects on VEGF-induced angiogenic signal events compared with LB42708. Moreover, the inhibitory effects of LB42708 were significantly higher than those of 4-(2-(4-(8-chloro-3,10-dibromo-6,11-dihydro-5H-benzo-(5,6)-cyclohepta(1,2-b)-pyridin-11(R)-yl)-1-piperidinyl)-2-oxo-ethyl)-1-piperidinecarboxamide (SCH66336), a well known FTase inhibitor. LB42708 suppressed tumor growth and tumor angiogenesis in both xenograft tumor models of Ras-mutated HCT116 cells and its wild-type Caco-2 cells, indicating its potential application in the treatment of both Ras-mutated and wild type tumors. These data indicate that the antitumor effect of LB42708 can be associated with direct inhibition of VEGF-induced tumor angiogenesis by blocking Ras-dependent MAPK and PI3K/Akt signal pathways in tumor-associated endothelial cells.

Temporal Dynamics of High-Density Lipoprotein Proteome in Diet-Controlled Subjects with Type 2 Diabetes.

We examined the effect of mild hyperglycemia on high-density lipoprotein (HDL) metabolism and kinetics in diet-controlled subjects with type 2 diabetes (T2D). 2H2O-labeling coupled with mass spectrometry was applied to quantify HDL cholesterol turnover and HDL proteome dynamics in subjects with T2D (n = 9) and age- and BMI-matched healthy controls (n = 8). The activities of lecithin-cholesterol acyltransferase (LCAT), cholesterol ester transfer protein (CETP), and the proinflammatory index of HDL were quantified. Plasma adiponectin levels were reduced in subjects with T2D, which was directly associated with suppressed ABCA1-dependent cholesterol efflux capacity of HDL. The fractional catabolic rates of HDL cholesterol, apolipoprotein A-II (ApoA-II), ApoJ, ApoA-IV, transthyretin, complement C3, and vitamin D-binding protein (all p < 0.05) were increased in subjects with T2D. Despite increased HDL flux of acute-phase HDL proteins, there was no change in the proinflammatory index of HDL. Although LCAT and CETP activities were not affected in subjects with T2D, LCAT was inversely associated with blood glucose and CETP was inversely associated with plasma adiponectin. The degradation rates of ApoA-II and ApoA-IV were correlated with hemoglobin A1c. In conclusion, there were in vivo impairments in HDL proteome dynamics and HDL metabolism in diet-controlled patients with T2D.

Co-localization of inducible nitric oxide synthase and phosphorylated Akt in the lesional skins of patients with melasma.

Activation of the inducible nitric oxide synthase (iNOS)/nitric oxide (NO) pathway in keratinocytes has been reported to be associated with the pathogenesis of melanogenesis. Akt activation plays an important role in the activation of the transcription factor nuclear factor (NF)-kappaB and subsequent elevation of iNOS expression. In the present study, we highly detected both iNOS protein and Akt phosphorylation in keratinocytes of the basal layer of the epidermis at the junction with the dermis of melasma skin biopsy specimens, but not in normal skin tissues, from nine patients using immunohistological analysis. iNOS protein and phosphorylated Akt were co-localized in the lesional skins, and their levels were highly correlated R2= 0.69). Furthermore, iNOS mRNA was also detected in an additional three skin biopsy specimens, but not in normal skin, by reverse transcription polymerase chain reaction. Our results describe that iNOS expression is elevated in human melasma lesions, probably via activation of the Akt/NF-kappaB pathway, indicating that NO production plays an important role in the mechanism of hyperpigmentation in human facial melasma.

The non-provitamin A carotenoid, lutein, inhibits NF-kappaB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways: role of H(2)O(2) in NF-kappaB activation.

Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells.

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.

The caspase-8/Bid/cytochrome c axis links signals from death receptors to mitochondrial reactive oxygen species production.

Ligation of the death receptors for TNF-α, FasL, and TRAIL triggers two common pathways, caspase-dependent intrinsic apoptosis and intracellular reactive oxygen species (ROS) generation. The apoptotic pathway is well characterized; however, a signaling linker between the death receptor and ROS production has not been clearly elucidated. Here, we found that death receptor-induced ROS generation was strongly inhibited by mitochondrial complex I and II inhibitors, but not by inhibitors of NADPH oxidase, lipoxygenase, cyclooxygenase or xanthine oxidase, indicating that ROS are mostly generated by the impairment of the mitochondrial respiratory chain. ROS generation was accompanied by caspase-8 activation, Bid cleavage, and cytochrome c release; it was blocked in FADD- and caspase-8-deficient cells, as well as by caspase-8 knockdown and inhibitor. Moreover, Bid knockdown abrogated TNF-α- or TRAIL-induced ROS generation, whereas overexpression of truncated Bid (tBid) or knockdown of cytochrome c spontaneously elevated ROS production. In addition, p53-overexpressing cells accumulated intracellular ROS via cytochrome c release mediated by the BH3-only protein Noxa induction. In a cell-free reconstitution system, caspase-8-mediated Bid cleavage and recombinant tBid induced mitochondrial cytochrome c release and ROS generation, which were blocked by Bcl-xL and antioxidant enzymes. These data suggest that anti-apoptotic Bcl-2 proteins play an important role in mitochondrial ROS generation by preventing cytochrome c release. These data provide evidence that the FADD/caspase-8/Bid/cytochrome c axis is a crucial linker between death receptors and mitochondria, where they play a role in ROS generation and apoptosis.

Myeloid Cell-Specific Lipin-1 Deficiency Stimulates Endocrine Adiponectin-FGF15 Axis and Ameliorates Ethanol-Induced Liver Injury in Mice.

Lipin-1 is a phosphatidate phosphohydrolase (PAP) required for the generation of diacylglycerol during glycerolipid synthesis, and exhibits dual functions in the regulation of lipid metabolism. Lipin-1 has been implicated in the pathogenesis of alcoholic liver disease (ALD). In the present study, we assessed lipin-1 function in myeloid cells in ALD using a myeloid cell-specific lipin-1 knockout (mLipin-1KO) mouse model. Utilizing the Gao-binge ethanol feeding protocol, matched mLipin-1KO mice and littermate loxP control (WT) mice were pair-fed with either an ethanol-containing diet or an ethanol-free diet (control). Surprisingly, deletion of lipin-1 in myeloid cells dramatically attenuated liver inflammatory responses and ameliorated liver injury that would normally occur following the ethanol feeding protocol, but slightly exacerbated the ethanol-induced steatosis in mice. Mechanistically, myeloid cell-specific lipin-1 deficiency concomitantly increased the fat-derived adiponectin and ileum-derived fibroblast growth factor (FGF) 15. In concordance with concerted elevation of circulating adiponectin and FGF15, myeloid cell-specific lipin-1 deficiency diminished hepatic nuclear factor kappa B (NF-κB) activity, limited liver inflammatory responses, normalized serum levels of bile acids, and protected mice from liver damage after ethanol challenge. Our novel data demonstrate that myeloid cell-specific deletion of lipin-1 ameliorated inflammation and alcoholic hepatitis in mice via activation of endocrine adiponectin-FGF15 signaling.

Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis.

The therapeutic effects of stem cell transplantation in ischemic disease are mediated by the production of paracrine bioactive factors. However, the bioactive factors secreted by human mesenchymal stem cells (hMSCs) and their angiogenic activity are not clearly identified or determined. We here found that hMSC-derived conditioned media (hMSC-CdM) stimulated in vitro angiogenic activity of endothelial cells and contained significant levels of various growth factors and cytokines, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-ß1). The angiogenic activity of hMSC-CdM was significantly inhibited by pretreatment with neutralizing antibodies against VEGF, MCP-1, and IL-6, but not against TGF-ß1 and HGF. A mixture of those inhibitory antibodies blocked CdM-mediated activation of angiogenic signals, as well as inhibited CdM-mediated in vivo angiogenesis. Moreover, local injection of CdM increased angiogenesis and promoted blood flow in mice with hindlimb ischemia, and these effects were inhibited by co-treatment with these inhibitory antibodies. These results indicate that hMSC-CdM represents a promising cell-free therapeutic strategy for neovascularization in ischemic diseases. These results suggest the combination of VEGF, MCP-1, and IL-6 as a commercial application for therapeutic angiogenesis.

Interleukin-10 down-regulates cathepsin B expression in fetal rat alveolar type II cells exposed to hyperoxia.

PURPOSE: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia. MATERIALS AND METHODS: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR. RESULTS: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia. CONCLUSION: Our study suggests that CB is enhanced in an oxygen- concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.