Decreased Macrophage Infiltration Research Articles

Induction of Nitric-Oxide Metabolism in Enterocytes Alleviates Colitis and Inflammation-Associated Colon Cancer

SummaryNitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

NSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.

Reduced atherosclerosis lesion size, inflammatory response in miR-150 knockout mice via macrophage effects

Atherosclerosis is considered to be a chronic inflammatory disease that can lead to severe clinically important cardiovascular events. miR-150 is a small noncoding RNA that significantly enhances inflammatory responses by upregulating endothelial cell proliferation and migration, as well as intravascular environmental homeostasis. However, the exact role of miR-150 in atherosclerosis remains unknown. Here, we investigated the effect of miR-150 deficiency on atherosclerosis development. Using double-knockout (miR-150-/- and ApoE-/-) mice, we measured atherosclerotic lesion size and stability. Meanwhile, we conducted in vivo bone marrow transplantation to identify cellular-level components of the inflammatory response. Compared with mice deficient only in ApoE, the double-knockout mice had significantly smaller atherosclerotic lesions and displayed an attenuated inflammatory response. Moreover, miR-150 ablation promoted plaque stabilization via increases in smooth muscle cell and collagen content and decreased macrophage infiltration and lipid accumulation. The in vitro experiments indicated that an inflammatory response with miR-150 deficiency in atherosclerosis results directly from upregulated expression of the cytoskeletal protein, PDZ and LIM domain 1 (PDLIM1), in macrophages. More importantly, the decreases in phosphorylated p65 expression and inflammatory cytokine secretion induced by miR-150 ablation were reversed by PDLIM1 knockdown. These findings suggest that miR-150 is a promising target for the management of atherosclerosis.

EPO Derivative ARA290 Attenuates Early Renal Allograft Injury in Rats by Targeting NF-κB Pathway

BackgroundThe goal of this study was to analyze the protective role of ARA290 in early renal allograft injury by using a rat model of renal allograft. MethodsLewis rats were divided into 3 groups: sham, University of Wisconsin solution (UW), and ARA290. A rat model of renal allograft was established by anastomosis using a titanium ring pin stapler. The kidneys were removed 24 hours after transplantation to accomplish the following: (1) examine the protective effect of ARA290 on renal morphology and function; (2) investigate the underlying mechanism by determining the binding affinity of nuclear factor-κB (NF-κB) to DNA by using an electrophoretic mobility shift assay; (3) observe the effect of ARA290 on macrophage infiltration using immunohistochemistry; and (4) detect messenger RNA (mRNA) expression of inflammatory mediators by using reverse transcriptase polymerase chain reaction. ResultsSerum creatinine and blood urea nitrogen levels in the ARA290 group were significantly lower than those in the UW group. Kidney tissue samples from the UW group exhibited morphologic abnormalities and marked macrophage infiltration compared with those in the sham group. In the ARA290 group, renal morphology was greatly improved with decreased macrophage infiltration. The binding affinity of NF-κB to DNA in the ARA290 group was markedly lower than that in the UW and sham groups. The mRNA expression of NF-κB downstream effectors (monocyte chemotactic protein-1; regulated on activation, normal T cell expressed and secreted; intercellular adhesion molecule-1; and vascular cell adhesion molecular-1) was significantly downregulated in the ARA290 group compared with that in the UW group. ConclusionsARA290 protects against early renal allograft injury in rats by reducing macrophage infiltration, improving renal morphology, inhibiting mRNA expression of inflammatory mediators, and weakening the binding affinity of NF-κB to DNA.

Alteration of the Tumor Stroma Using a Consensus DNA Vaccine Targeting Fibroblast Activation Protein (FAP) Synergizes with Antitumor Vaccine Therapy in Mice.

Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines.Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data.Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8+ and CD4+ immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8+ T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8+ T-cell infiltration.Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190-201. ©2018 AACR.

Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis

Acute kidney injury (AKI), characterized by aggressive inflammatory responses and destruction of renal resident cells, can cause abrupt kidney dysfunction. To date, effective therapy for AKI is lacking. In this study, we evaluated the renoprotective effect of wogonin, an herbal active compound, using a cisplatin-induced AKI mouse model. In vivo results show that wogonin substantially suppressed the increased levels of serum creatinine and blood urea nitrogen (BUN) almost to the normal level. Wogonin also attenuated tubular damage, shown by PAS staining, electron microscopy and molecular analysis of KIM-1. In addition, wogonin suppressed kidney inflammation as indicated by a >60% decrease in macrophage infiltration, a >50% reduction in inflammatory cytokine production and inhibited NF-κB activation in the injured kidney. Mechanistically, molecular docking results show that wogonin effectively inhibited RIPK1 by occupying the ATP-binding pocket of the enzyme, which is a key regulator of necroptosis. Moreover, inhibition of RIPK1, or RIPK3, reversed the protective effects of wogonin in cisplatin-treated HK2 cells, indicating wogonin works in a RIPK1/RIPK3-dependent manner. Surprisingly, wogonin enhanced the anti-proliferative effect of cisplatin on human hepatoma HepG2 cells. Thus, our findings suggest wogonin may be a renoprotective adjuvant for cisplatin-based anticancer therapy.

Rapeseed Protein-Derived Antioxidant Peptide Rap Ameliorates Nonalcoholic Steatohepatitis and Related Metabolic Disorders in Mice

Rapeseed protein hydrolysates have recently shown in vitro antioxidant and anti-inflammatory activities. However, scant data exist about their in vivo activities. Here we report that the peptide DHNNPQIR (hereinafter referred to as RAP-8), a natural peptide derived from rapeseed protein, has excellent in vivo efficacy in mouse models of nonalcoholic steatohepatitis (NASH) and liver fibrosis. We demonstrated that RAP-8 significantly reduced hepatic steatosis and improved insulin resistance and lipid metabolism. Furthermore, RAP-8 showed markedly reduced hepatic inflammation, fibrosis, liver injury, and metabolic deterioration. In particular, RAP-8 directly suppressed expression of fibrosis-related genes α-smooth muscle actin (α-Sma) and collagen type I (Col-1α) in the liver of mice in vivo. In addtion, RAP-8 significantly decreased macrophage infiltration and reduced secretion of proinflammatory cytokines. Finally, we found that RAP-8 administration significantly decreased oxidative stress-induced apoptosis in liver injury induced by CCl4. Therefore, our results suggest that RAP-8 could be an attractive therapeutic candidate for treatment of NASH and NASH-related metabolic deterioration. Funding Statement: The Program for Guangdong Introducing Innovative and Enterpreneurial Teams (2016ZT06Y337). X.J. supported by the Thousand Young Talents Program. Declaration of Interests: All authors participated in this study declare no potential, perceived, or real conflict of interest and have agreed to the content of this manuscript. Ethics Approval Statement: The experimental procedure used in this study met the guidelines of the Animal Care and Use Committee of the Sun Yat-sen University.

Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated via mTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.

SIRT2 decreases atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice by modulating macrophage polarization

Compelling evidence has demonstrated that the M1 macrophage phenotype is central to atherosclerotic lesion development. SIRT2, an NAD+-dependent sirtuin deacetylase, is involved in modulating macrophage polarization. However, the role of SIRT2 in atherosclerotic progression remains unknown. Female LDL receptor knockout (LDLr-/-) mice were randomly divided into four groups for treatment with saline, empty lentivirus, lentivirus-SIRT2, or shRNA-SIRT2 for 4 weeks. Thereafter, the mice were fed an atherogenic high-fat diet (HFD) for another 8 weeks. Atherosclerotic plaques were assessed in the aortic sinus by morphometry, immunohistochemistry and immunofluorescence analyses. Aortic levels of macrophage polarization markers were analysed by Western blot and immunofluorescence analyses. We found that lentivirus-SIRT2-treated LDLr-/- mice had decreased plaque areas in the aortic sinus and developed a more stable plaque phenotype, as shown by decreased macrophage infiltration and apoptosis. In addition, treatment with lentivirus-SIRT2 significantly reduced the expression of iNOS (inducible nitric oxide synthase) and increased the levels of ARG-1 (arginase-1) in atheromas. These findings suggest that SIRT2 inhibited atherosclerotic plaque progression and enhanced plaque stability in LDLr-/- mice by inhibiting macrophage polarization towards the M1 phenotype.

Tumor necrosis factor alpha-stimulated gene-6 (TSG-6) inhibits the inflammatory response by inhibiting the activation of P38 and JNK signaling pathway and decreases the restenosis of vein grafts in rats.

This study aims to explore the effects of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) on vascular inflammatory response and vascular injury in grafted vein wall of rats and its possible mechanism. Vascular grafting model was established by modified cuff. The effect of TSG-6 on the inflammatory response and vascular injury of vein graft was investigated. The activation of mast cells and macrophages after LPS stimulation was observed by lentivirus-mediated upregulation or downregulation of TSG-6 expression. The results showed that rhTSG-6 treatment could significantly inhibit the proliferation of venous bridge, decrease macrophage infiltration and smooth muscle cell proliferation. The expression levels of TNF-α and IL-1 in treated group were significantly lower than that of untreated group (P<0.05), while the expression of IL-10 in treated group were significantly higher than that of untreated group (P<0.05). The expression levels of P38, p-P38, JNK and p-JNK in venous bridge of rats were significantly lower than those of untreated rats (P<0.05), while there was no significant difference in the expression level of ERK and p-ERK (P>0.05). TSG-6 could inhibit the proliferation of mast cells and macrophages and the release of inflammatory cytokines by down regulating the expression levels of P38, p-P38, JNK and p-JNK. TSG-6 can inhibit the inflammatory response of transplanted vein grafts in rats and reduce vascular injury by downregulation of P38 and JNK signaling pathway.

DBZ (Danshensu Bingpian Zhi), a Novel Natural Compound Derivative, Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice.

BackgroundDBZ (Danshensu Bingpian Zhi), a synthetic derivative of a natural compound found in traditional Chinese medicine, has been reported to suppress lipopolysaccharide‐induced macrophage activation and lipid accumulation in vitro. The aim of this study was to assess whether DBZ could attenuate atherosclerosis at early and advanced stages.Methods and ResultsThe effects of DBZ on the development of atherosclerosis were studied using apolipoprotein E–deficient (apoE−/−) mice. For early treatment, 5‐week‐old apoE−/− mice were fed a Western diet and treated daily by oral gavage with or without DBZ or atorvastatin for 10 weeks. For advanced treatment, 5‐week‐old apoE−/− mice were fed a Western diet for 10 weeks to induce atherosclerosis, and then they were randomly divided into 4 groups and subjected to the treatment of vehicle, 20 mg/kg per day DBZ, 40 mg/kg per day DBZ, or 10 mg/kg per day atorvastatin for the subsequent 10 weeks. We showed that early treatment of apoE−/− mice with DBZ markedly reduced atherosclerotic lesion formation by inhibiting inflammation and decreasing macrophage infiltration into the vessel wall. Treatment with DBZ also attenuated the progression of preestablished diet‐induced atherosclerotic plaques in apoE−/− mice. In addition, we showed that DBZ may affect LXR (liver X receptor) function and that treatment of macrophages with DBZ suppressed lipopolysaccharide‐stimulated cell migration and oxidized low‐density lipoprotein–induced foam cell formation.Conclusions DBZ potentially has antiatherosclerotic effects that involve the inhibition of inflammation, macrophage migration, leukocyte adhesion, and foam cell formation. These results suggest that DBZ may be used as a therapeutic agent for the prevention and treatment of atherosclerosis.

Grape-seed Polyphenols Play a Protective Role in Elastase-induced Abdominal Aortic Aneurysm in Mice

Abdominal aortic aneurysm (AAA) is a kind of disease characterized by aortic dilation, whose pathogenesis is linked to inflammation. This study aimed to determine whether grape-seed polyphenols (GSP) has anti-AAA effects and what mechanism is involved, thus to find a way to prevent occurrence and inhibit expansion of small AAA. In our study, AAA was induced by incubating the abdominal aorta of the mice with elastase, and GSP was administrated to the mice by gavage at different doses beginning on the day of the AAA inducement. In in vivo experiments, 800 mg/kg GSP could significantly reduce the incidence of AAA, the dilatation of aorta and elastin degradation in media, and dramatically decrease macrophage infiltration and activation and expression of matrix metalloproteinase (MMP) −2 and MMP-9 in the aorta, compared to the AAA model group. Meanwhile, 400 mg/kg GSP could also but not completely inhibit the occurrence and development of AAA. In in vitro experiments, GSP dose-dependently inhibited mRNA expression of interleukin (IL)-1β, IL-6 and monocyte chemoattractant protein-1 (MCP-1), and significantly inhibited expression and activity of MMP-2 and MMP-9, thus prevented elastin from degradation. In conclusion, GSP showed great anti-AAA effects and its mechanisms were related to inhibition of inflammation.

Myeloid-specific deletion of NOX2 prevents the metabolic and neurologic consequences of high fat diet.

High fat diet-induced obesity is associated with inflammatory and oxidative signaling in macrophages that likely participates in metabolic and physiologic impairment. One key factor that could drive pathologic changes in macrophages is the pro-inflammatory, pro-oxidant enzyme NADPH oxidase. However, NADPH oxidase is a pleiotropic enzyme with both pathologic and physiologic functions, ruling out indiscriminant NADPH oxidase inhibition as a viable therapy. To determine if targeted inhibition of monocyte/macrophage NADPH oxidase could mitigate obesity pathology, we generated mice that lack the NADPH oxidase catalytic subunit NOX2 in myeloid lineage cells. C57Bl/6 control (NOX2-FL) and myeloid-deficient NOX2 (mNOX2-KO) mice were given high fat diet for 16 weeks, and subject to comprehensive metabolic, behavioral, and biochemical analyses. Data show that mNOX2-KO mice had lower body weight, delayed adiposity, attenuated visceral inflammation, and decreased macrophage infiltration and cell injury in visceral adipose relative to control NOX2-FL mice. Moreover, the effects of high fat diet on glucose regulation and circulating lipids were attenuated in mNOX2-KO mice. Finally, memory was impaired and markers of brain injury increased in NOX2-FL, but not mNOX2-KO mice. Collectively, these data indicate that NOX2 signaling in macrophages participates in the pathogenesis of obesity, and reinforce a key role for macrophage inflammation in diet-induced metabolic and neurologic decline. Development of macrophage/immune-specific NOX-based therapies could thus potentially be used to preserve metabolic and neurologic function in the context of obesity.

BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions.

Endothelial Nitric Oxide Synthase-Independent Pleiotropic Effects of Pitavastatin Against Atherogenesis and Limb Ischemia in Mice.

Aim: Statins have a protective impact against cardiovascular diseases through not only lipid-lowering effects but also pleiotropic effects, including activation of the endothelial nitric oxide synthase (eNOS) system. We aimed to clarify the protective effects of a statin against atherogenesis and ischemia in eNOS−/− mice.Methods: Study 1. eNOS−/− Apolipoprotein E (ApoE)−/− mice were treated with a vehicle or pitavastatin (0.3 mg/kg/day) for 4 weeks. Study 2. eNOS−/− mice were also treated with a vehicle or the same dose of pitavastatin for 2 weeks prior to hind-limb ischemia.Results: In Study 1, pitavastatin attenuated plaque formation and medial fibrosis of the aortic root with decreased macrophage infiltration in eNOS−/− ApoE−/− mice. PCR array analysis showed reductions in aortic gene expression of proatherogenic factors, including Ccl2 and Ccr2 in pitavastatin-treated double mutant mice. In addition, pitavastatin activated not only atherogenic p38MAPK and JNK but also anti-atherogenic ERK1/2 and ERK5 in the aorta of the double mutant mice. In Study 2, pitavastatin prolonged hind-limb survival after the surgery with increased BCL2-to-BAX protein ratio and inactivated JNK. Enhanced expression of anti-apoptotic genes, including Vegf, Api5, Atf5, Prdx2, and Dad1, was observed in the ischemic limb of pitavastatin-treated eNOS−/− mice. Furthermore, pitavastatin activated both aortic and skeletal muscle AMPK in the eNOS-deficient vascular injury models.Conclusion: Pitavastatin exerts eNOS-independent protective effects against atherogenesis and hindlimb ischemia in mice, which may occur via modifications on key molecules such as AMPK and diverse molecules.

Oncostatin M receptor β deficiency attenuates atherogenesis by inhibiting JAK2/STAT3 signaling in macrophages

Oncostatin M (OSM) is a secreted cytokine mainly involved in chronic inflammatory and cardiovascular diseases through binding to OSM receptor β (OSMR-β). Recent studies demonstrated that the presence of OSM contributed to the destabilization of atherosclerotic plaque. To investigate whether OSMR-β deficiency affects atherosclerosis, male OSMR-β−/−ApoE−/− mice were generated and utilized. Here we observed that OSMR-β expression was remarkably upregulated in both human and mouse atherosclerotic lesions, which were mainly located in macrophages. We found that OSMR-β deficiency significantly ameliorated atherosclerotic burden in aorta and aortic root relative to ApoE-deficient littermates and enhanced the stability of atherosclerotic plaques by increasing collagen and smooth muscle cell content, while decreasing macrophage infiltration and lipid accumulation. Moreover, bone marrow transplantation of OSMR-β−/− hematopoietic cells to atherosclerosis-prone mice displayed a consistent phenotype. Additionally, we observed a relatively reduced level of JAK2 and signal transducer and activator of transcription (STAT)3 in vivo and under Ox-LDL stimulation in vitro. Our findings suggest that OSMR-β deficiency in macrophages improved high-fat diet-induced atherogenesis and plaque vulnerability. Mech­anistically, the protective effect of OSMR-β deficiency on atherosclerosis may be partially attributed to the inhibition of the JAK2/STAT3 activation in macrophages, whereas OSM stimulation can activate the signaling pathway.

P01.32 NDRG1 induced downregulation of CCL2 influences the migration of macrophages and impacts the tumor microenvironement

AbstractBACKGROUND: Malignant gliomas belong to the most aggressive neoplasms in humans. The interactions and intercellular communication between malignant and non-malignant cells in the tumor microenvironment are tumor-promoting and critically to improve the understanding of the disease. N-myc downstream regulated gene 1 (NDRG1) is a stress inducible gene and key determinant of resistance towards alkylating chemotherapy in glioblastoma. NDRG1 promotes invasive growth and acts antiapoptotic in hepatocellular carcinoma. In human pancreatic cancer cells NDRG1 suppresses chemokine expression, decreases macrophage infiltration and microvascular density. Further understanding the influence of NDRG1 on the tumor microenvironment may lead to new therapeutic approaches in glioma. METHODS: To analyze the NDRG1 effects on the glioma microenvironment, we used human and murine NDRG1 knockdown (KD) cells. In orthotopic xenograft experiments control and KD microenvironments were compared for infiltrating immune cells dependent on NDRG1. Interaction and signaling mechanisms were evaluated in vitro using western blots, cytokine and migration assays and treatment with the NF-kB inhibitor JSH-23 as well as SGK1 inhibitor EMD638683 to assess the influence of NDRG1. Results: Orthotopic NDRG1 KD tumors were significantly larger compared to control tumors. Ex vivo flow cytometry analyses of the tumor microenvironment revealed a significant increase in peripheral macrophages, dendritic cells and monocytic myeloid derived suppressor cells in NDRG1 KD tumors. In vitro cytokine array analyses showed an increase of the chemokine CCL2 in NDRG1 KD cells compared to control cells. This was confirmed by ELISA. Macrophages showed an increased migration rate in the NDRG1 knockdown environment. To analyze molecular mechanisms CCL2 secretion was blocked with a NF-κB inhibitor (JSH-23) and stimulated with a SGK1 inhibitor (EMD638683). We found that NDRG1 induced NF-κB signaling attenuation is mediated through decreases in IKK α expression and IκBα phosphorylation. CONCLUSION: We identified CCL2 as a NDRG1-induced NF-κB target gene that triggers macrophage recruitment. Our findings support an immunomodulatory effect of NDRG1 on the tumor microenvironment and provide novel insights into the biological relevance of the tumor stroma and the function of NDRG1 with important implications for the development of cancer therapeutics.

Influence of Drugs Carried in Lipid Nanoparticles in Coronary Disease of Rabbit Transplanted Heart

Coronary allograft vasculopathy is an inflammatory-proliferative process that compromises the long-term success of heart transplantation and currently has no effective prevention and treatment. Lipid nanoparticles, termed LDE can carry chemotherapeutic agents in the circulation and concentrates them in the heart. Twenty-eight rabbits fed a cholesterol-rich diet and submitted to heterotopic heart transplantation were treated with cyclosporine A (10 mg/kg daily) and allocated to four groups of 7 animals treated with intravenous LDE-methotrexate (MTX; 4 mg/kg weekly), with LDE-paclitaxel (PACLI; 4 mg/kg weekly), or with LDE-PACLI (4mg/kg weekly) and LDE-MTX (4 mg/kg weekly). A control group was treated with only weekly intravenous saline solution. Animals were euthanized 6 weeks later for morphometric, histologic, immunohistochemical, and gene expression analysis of the graft and native hearts. Compared with controls, grafts of rabbits treated with LDE-PACLI showed 50% reduction of coronary stenosis, and in the LDE-MTX and LDE-MTX/PACLI stenosis was approximately 18% less than in control, but this difference was not statistically significant. In the three treatment groups, macrophage infiltration was decreased. In the LDE-MTX group, gene expression of proinflammatory factors tumor necrosis factor-α, monocyte chemoattractant protein 1, interleukin 18, vascular cellular adhesion molecule 1, and matrix metalloproteinase 12 was strongly diminished, whereas expression of antiinflammatory interleukin 10 increased. In the LDE-PACLI and LDE-PACLI/MTX groups, proinflammatory and antiinflammatory gene expressions were not consistently changed by the treatments. LDE-PACLI promoted strong improvement of cardiac allograft vasculopathy, but the decrease in coronary stenosis by LDE-MTX and LDE-MTX/PACLI was not significant. All three treatments decreased macrophage infiltration in the graft. These results may encourage future clinical trials to test this new therapeutic approach to coronary allograft vasculopathy.

Induced Pluripotent Stem Cells Reduce Progression of Experimental Chronic Kidney Disease but Develop Wilms' Tumors.

The therapeutic effect of induced pluripotent stem cells (iPSs) on the progression of chronic kidney disease (CKD) has not yet been demonstrated. In this study, we sought to assess whether treatment with iPSs retards progression of CKD when compared with bone marrow mesenchymal stem cells (BMSCs). Untreated 5/6 nephrectomized rats were compared with CKD animals receiving BMSCs or iPSs. Renal function, histology, immunohistochemistry, and gene expression were studied. Implanted iPSs were tracked by the SRY gene expression analysis. Both treatments minimized elevation in serum creatinine, significantly improved clearance, and slowed down progression of disease. The proteinuria was reduced only in the iPS group. Both treatments reduced glomerulosclerosis, iPSs decreased macrophage infiltration, and TGF-β was reduced in kidneys from the BMSC group. Both types of treatments increased VEGF gene expression, TGF-β was upregulated only in the iPS group, and IL-10 had low expression in both groups. The SRY gene was found in 5/8 rats treated with iPSs. These 5 animals presented tumors with histology and cells highly staining positive for PCNA and Wilms' tumor protein antibody characteristics of Wilms' tumor. These results suggest that iPSs may be efficient to retard progression of CKD but carry the risk of Wilms' tumor development.

Endothelial Cell-Specific Deletion of P2Y2 Receptor Promotes Plaque Stability in Atherosclerosis-Susceptible ApoE-Null Mice.

Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in apolipoprotein E null (ApoE-/-) mice. Because P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R floxed mice. Endothelial P2Y2R deficiency reduced endothelial nitric oxide synthase activity and significantly altered ATP- and UTP (uridine 5'-triphosphate)-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE-/- background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathologic examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration, and increased smooth muscle cell and collagen content, leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE-/- mice. Expression and proteolytic activity of matrix metalloproteinase-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE-/- mice. Furthermore, EC-specific P2Y2R deficiency inhibited nitric oxide production, leading to significant increase in smooth muscle cell migration out of aortic explants. EC-specific P2Y2R deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE-/- mice through impaired macrophage infiltration acting together with reduced matrix metalloproteinase-2 activity and increased smooth muscle cell migration.