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Circulating factors resulting from immune dysfunction have been proposed as one of the causes for increased risk of graft loss associated with recurrence of focal segmental glomerulosclerosis (FSGS) after kidney transplant. However, the precise identity of the circulating factors and their sources remain incompletely characterized.In vivostudies in mouse models have implicated a role for immature bone marrow cells in the development of FSGS. Using single-cell RNA sequencing we have profiled >50,000 cells from bone marrow of FSGS patients with or without recurrence after kidney transplant and controls including healthy individuals and patients with end-stage renal disease due to non-FSGS causes. Bone marrow mononuclear cells from patients with recurrence of FSGS after transplant display an inflammatory phenotype with activation of cytokine and interferon signaling in neutrophils, T cells and B cells. We also observe a dramatically depleted B cell population in R-FSGS patients. Conditioned media from BMNCs of R-FSGS patients have higher levels of pro-inflammatory cytokine MIP-1α/CCL3, reduced anti-inflammatory chemokine CCL22 and cause injury in a human podocyte cell culture model. Our studies provide evidence for the role of bone marrow cells in FSGS associated inflammatory milieu and elucidate the transcriptional changes associated with the disease.

Significance Statement Posttranslational guanidinylation of ApoC3, a major constituent of triglyceride-rich lipoproteins, enhances ApoC3?s proinflammatory properties in uremia. Guanidinylated ApoC3 (gApoC3) is generated by guanidine and urea, and accumulates significantly in the plasma of patients with CKD, and in the kidneys of mouse models of CKD. In humanized mice, gApoC3 promotes kidney tissue fibrosis and suppresses regeneration after vascular injury. Importantly, in a large observational trial examining the clinical relevance of this posttranslational modification in patients with CKD, higher plasma gApoC3 intensity was associated with adverse renal and cardiovascular outcomes. Therefore, gApoC3 represents a novel mediator of kidney injury and CKD-associated vascular disease and offers a potential treatment target to halt progression and to prevent vascular disease in patients with CKD.Background Coexistent CKD and cardiovascular diseases are highly prevalent in Western populations and account for substantial mortality. We recently found that apolipoprotein C-3 (ApoC3), a major constituent of triglyceride-rich lipoproteins, induces sterile systemic inflammation by activating the NOD-like receptor protein-3 (NLRP3) inflammasome in human monocytes via an alternative pathway.Methods To identify posttranslational modifications of ApoC3 in patients with CKD, we used mass spectrometry to analyze ApoC3 from such patients and from healthy individuals. We determined the effects of posttranslationally modified ApoC3 on monocyte inflammatory response in vitro, as well as in humanized mice subjected to unilateral ureter ligation (a kidney fibrosis model) and in a humanized mouse model for vascular injury and regeneration. Finally, we conducted a prospective observational trial of 543 patients with CKD to explore the association of posttranslationally modified ApoC3 with renal and cardiovascular events in such patients.Results We identified significant posttranslational guanidinylation of ApoC3 (gApoC3) in patients with CKD. We also found that mechanistically, guanidine and urea induce guanidinylation of ApoC3. A 2D-proteomic analysis revealed that gApoC3 accumulated in kidneys and plasma in a CKD mouse model (mice fed an adenine-rich diet). In addition, gApoC3 augmented the proinflammatory effects of ApoC3 in monocytes in vitro. In humanized mice, gApoC3 promoted kidney tissue fibrosis and impeded vascular regeneration. In CKD patients, higher gApoC3 plasma levels (as determined by mass spectrometry) were associated with increased mortality as well as with renal and cardiovascular events.Conclusions Guanidinylation of ApoC3 represents a novel pathogenic mechanism in CKD and CKD-associated vascular injury, pointing to gApoC3 as a potential therapeutic target.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.

Urokinase plasminogen activator receptor (uPAR) is a multifaceted, GPI-anchored three-domain protein. Release of the receptor results in variable levels of soluble uPAR (suPAR) in the blood circulation. suPAR levels have been linked to many disease states. In this mini-review, we discuss suPAR as a key circulating molecule mediating kidney disease with a particular focus on differently spliced isoforms.

Soluble urokinase receptor (suPAR) is a circulatory molecule that activates αvβ3 integrin on podocytes, causes foot process effacement, and contributes to proteinuric kidney disease. While active integrin can be targeted by antibodies and small molecules, endogenous inhibitors haven't been discovered yet. Here we report what we believe is a novel renoprotective role for the inducible costimulator ligand (ICOSL) in early kidney disease through its selective binding to podocyte αvβ3 integrin. Contrary to ICOSL's immune-regulatory role, ICOSL in nonhematopoietic cells limited the activation of αvβ3 integrin. Specifically, ICOSL contains the arginine-glycine-aspartate (RGD) motif, which allowed for a high-affinity and selective binding to αvβ3 and modulation of podocyte adhesion. This binding was largely inhibited either by a synthetic RGD peptide or by a disrupted RGD sequence in ICOSL. ICOSL binding favored the active αvβ3 rather than the inactive form and showed little affinity for other integrins. Consistent with the rapid induction of podocyte ICOSL by inflammatory stimuli, glomerular ICOSL expression was increased in biopsies of early-stage human proteinuric kidney diseases. Icosl deficiency in mice resulted in an increased susceptibility to proteinuria that was rescued by recombinant ICOSL. Our work identified a potentially novel role for ICOSL, which serves as an endogenous αvβ3-selective antagonist to maintain glomerular filtration.

Background and Aims Sepsis is the main contributor to the development of acute kidney injury (AKI) in critically ill patients. Plasma soluble urokinase plasminogen activator receptor (suPAR) is a circulating risk factor for AKI and a new prognostic marker for renal outcome prediction. We analyzed the pathophysiological role and kinetic properties of suPAR in septic AKI in critically ill patients and in a murine model of septic AKI. Method 200 critically ill patients were enrolled prospectively after meeting Sepsis-3 criteria. Serum suPAR levels were measured at 0, 12, 24, 48, 72, 96, 120 and 168-hour after enrollment (n=1440) and the need for RRT within 7 days (predefined criteria) was assessed as the primary outcome measure. Polybacterial sepsis was induced by cecal slurry injection in three mouse strains, respectively wild type (WT, N=16), uPAR-knockout (KO, N=15), and suPAR transgenic overexpression (OE, N=14). Results No or mild AKI occurred in 62 patients (31.0%), moderate or severe AKI without the need for RRT in 102 patients (51.0%), criteria for RRT were met in 36 patients (18.0%) and 7 patients (3.5%) died within the 7-day period. Compared to all other maximum AKI stages and AKI disease courses within 7 days, patients requiring RRT showed significantly higher suPAR levels at all time-points. Patients with suPAR levels ≥ 12.7 ng/mL (highest quartile) had an unadjusted odds ratio (OR) of 9.73 (95% confidence interval [CI], 4.31-21.98) and an adjusted odds ratio of 5.22 (95% CI, 2.16-12.65) for the need for RRT; and 7.47 (95% CI, 3.54-15.74) and 4.44 (95% CI, 1.98-9.97) for RRT or death within 7 days compared to patients with levels < 12.7 ng/mL. Compared to KO mice, WT and OE mice showed a significantly greater impairment of renal function, structure and tubular apoptosis 24 hours after induction of sepsis. The inflammation levels with respect to Interleukin 6 were comparable between different strains. Kaplan-Meier analysis revealed a survival benefit of KO mice over OE mice within 24h (86.7% vs. 50.0%, p=0.033). Conclusion SuPAR distinguishes between divergent AKI stages/courses and the need for RRT at any time within 7 days after sepsis diagnosis. Our experimental data suggest that suPAR is a pathophysiological driver of septic AKI and may serve as a target for future interventional strategies.

The kidney is an organ involved in cross talk with many human organs. The link between the immune system and the kidney has been studied in some detail, although data precisely elucidating their interaction are sparse, in particular with regard to the function of the kidney filter apparatus. Current research suggests that an understanding of the impairment of this cross talk between the bone marrow, as a fundament of the immune system and the kidney will provide meaningful insights into the pathophysiological mechanisms of impaired kidney filter function. Circulating factors have long been implicated in the pathogenesis of idiopathic nephrotic syndrome, particularly focal segmental glomerulosclerosis (FSGS) and its recurrence. Soluble urokinase receptor (suPAR) has emerged as a circulating factor responsible for FSGS and also as an early predictive marker for the development of various renal diseases. The bone marrow has recently been revealed as a predominant source of suPAR with deleterious effects on the kidney filter. These new findings have led to bone marrow or hematopoietic stem cell transplants being considered as potential therapeutic options for preventing the post-transplantation recurrence of FSGS or even as a treatment for the original disease associated with high suPAR levels. Whereas bone marrow transplantation for patients with pre-existing chronic kidney disease is challenging, recent clinical trials have demonstrated the promising outcome of combined bone marrow and kidney transplantation in patients with kidney failure. In this review, with its brief update on suPAR, we describe the critical new role of the bone marrow in the pathogenesis of the kidney disease process and the functional connection between these two organs through the soluble mediator, suPAR. We also comment on the feasibility of bone marrow transplants for the treatment of patients with chronic renal failure arising from recurrent FSGS.

Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and αvβ3 integrin, whereby APOL1 protein variants G1 and G2 exhibited higher affinity for suPAR-activated avb3 integrin than APOL1 G0. APOL1 G1 or G2 augments αvβ3 integrin activation and causes proteinuria in mice in a suPAR-dependent manner. The synergy of circulating factor suPAR and APOL1 G1 or G2 on αvβ3 integrin activation is a mechanism for CKD.

Soluble uPAR is known to contribute to certain types of chronic kidney disease, and myeloid cells from the bone marrow have now been shown to be a key source of this factor. Excess levels of protein in urine (proteinuria) is a hallmark of kidney disease that typically occurs in conjunction with diabetes, hypertension, gene mutations, toxins or infections but may also be of unknown cause (idiopathic)1. Systemic soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor implicated in the onset and progression of chronic kidney disease (CKD)2, such as focal segmental glomerulosclerosis (FSGS)3,4. The cellular source(s) of elevated suPAR associated with future and progressing kidney disease is unclear, but is likely extra-renal, as the pathological uPAR is circulating and FSGS can recur even after a damaged kidney is replaced with a healthy donor organ. Here we report that bone marrow (BM) Gr-1lo immature myeloid cells are responsible for the elevated, pathological levels of suPAR, as evidenced by BM chimera and BM ablation and cell transfer studies. A marked increase of Gr-1lo myeloid cells was commonly found in the BM of proteinuric animals having high suPAR, and these cells efficiently transmit proteinuria when transferred to healthy mice. In accordance with the results seen in suPAR-associated proteinuric animal models, in which kidney damage is caused not by local podocyte-selective injury but more likely by systemic insults, a humanized xenograft model of FSGS resulted in an expansion of Gr-1lo cells in the BM, leading to high plasma suPAR and proteinuric kidney disease. Together, these results identify suPAR as a functional connection between the BM and the kidney, and they implicate BM immature myeloid cells as a key contributor to glomerular dysfunction.

Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and α

Despite documentation of successful therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with lung cancer, the response rate of patients treated with this therapy remains low. The present study investigated whether L-ascorbic acid serves an adjuvant role in vitro when combined with the EGFR tyrosine kinase inhibitor gefitinib (Iressa®) in lung cancer cell lines. A total of three human lung cancer cell lines were used. The antiproliferative effects and changes in the cell cycle and expression of intracellular signaling molecules, including extracellular signal-regulated kinases (Erk), signal transducer and activator of transcription 3 (Stat3) and protein kinase B (Akt), were measured in cells treated with gefitinib and/or L-ascorbic acid at various concentrations. When combined with gefitinib, L-ascorbic acid exhibited an additive effect on cell proliferation in all gefitinib-sensitive and gefitinib-resistant cell lines. A decrement of ~40% was observed with a low dose 0.5 mM L-ascorbic acid and gefitinib in the relatively gefitinib-resistant A549 cell line (85.6±5.4% with gefitinib alone vs. 52.7±7.3% with combination therapy; P=0.046). The downregulation of intracellular signaling cascades, including EGFR, Akt, Erk and Stat3, was also observed. L-Ascorbic acid serves an adjuvant role when administered in combination with gefitinib; however, the degree of inhibition of cell proliferation differs between lung cancer cell lines.

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

Corticotropin releasing hormone (CRH) is a major regulator of the stress response. This study examined whether CRH regulates interleukin-18 expression on microglia, BV2. Our data show that CRH enhanced IL-18 expression and significantly induced the secretion of functional IL-18 protein. Furthermore, CRH induced IL-18 production could be blocked by N-acetyl-L-cystein (NAC), which suggests that reactive oxygen intermediates (ROI) may be involved in regulating IL-18. Indeed, it was also found that CRH increased the generation of ROI. Taken together, these results indicate that CRH is an important mediator that regulates IL-18 expression in the brain during stress.

Sodium ascorbate (vitamin C) has a reputation for inconsistent effects upon malignant tumor cells, which vary from growth stimulation to apoptosis induction. Melanoma cells were found to be more susceptible to vitamin C toxicity than any other tumor cells. The present study has shown that sodium ascorbate decreases cellular iron uptake by melanoma cells in a dose- and time-dependent fashion, indicating that intracellular iron levels may be a critical factor in sodium ascorbate-induced apoptosis. Indeed, sodium ascorbate-induced apoptosis is enhanced by the iron chelator, desferrioxamine (DFO) while it is inhibited by the iron donor, ferric ammonium citrate (FAC). Moreover, the inhibitory effects of sodium ascorbate on intracellular iron levels are blocked by addition of transferrin, suggesting that transferrin receptor (TfR) dependent pathway of iron uptake may be regulated by sodium ascorbate. Cells exposed to sodium ascorbate demonstrated down-regulation of TfR expression and this precedes sodium ascorbate-induced apoptosis. Taken together, sodium ascorbate-mediated apoptosis appears to be initiated by a reduction of TfR expression, resulting in a down-regulation of iron uptake followed by an induction of apoptosis. This study demonstrates the specific mechanism of sodium ascorbate-induced apoptosis and these findings support future clinical trial of sodium ascorbate in the prevention of human melanoma relapse.

We recently reported that interleukin-18 (IL-18) is highly expressed in malignant skin tumours such as melanomas, and may play a key role in the malignancy of such tumours. This study was designed to investigate the mechanisms of IL-18 regulation by vitamin C in B16F10 murine melanoma cells. Cells were treated with vitamin C, and the expression of IL-18 was measured by reverse transcription-polymerase chain reaction and intracellular flow cytometry analysis. Decreased IL-18 production and a significant reduction in IL-18 mRNA transcript were detected in cells treated with vitamin C. The effect of vitamin C treatment was blocked by the antioxidant N-acetyl-L-cysteine, suggesting that vitamin C affects IL-18 expression by up-regulating intracellular reactive oxygen intermediate (ROI) levels. To investigate whether the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the downregulation of IL-18 production, cells were pretreated with SB203580, an inhibitor of p38 MAPK, prior to the addition of vitamin C. This pretreatment blocked the decrease in IL-18 production. However, vitamin C treatment enhanced the expression of phosphorylated p38 MAPK. Taken together, we conclude that vitamin C increases intracellular ROI levels, and regulates IL-18 production through the MAPK signalling pathway.

L-ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate-induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl- L-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate-treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate-induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate-induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8-independent manner.

Based on our recent observation that enhanced IL-18 expression positively correlates with malignant skin tumors, such as SCC and melanoma, we examined the possible role of UVB, known to be associated with skin cancer development, in the enhancement of IL-18 production using primary human epidermal keratinocytes and human keratinocyte cell line HaCaT. After cells were exposed to UVB irradiation in vitro, IL-18 production was examined by Northern blot analysis and ELISA, and it was found that IL-18 production is enhanced by UVB irradiation in a dose- and time-dependent manner. In addition, we confirmed that it is functionally active form of IL-18 using the inhibitor of caspase-1. The effect of UVB irradiation was blocked by antioxidant, N -acetyl- l -cysteine (NAC), which suggested the involvement of reactive oxygen intermediates (ROI) in the signal transduction of UVB irradiation-enhanced IL-18 synthesis. We also found that UVB irradiation increased AP-1 binding activity by using EMSA with AP-1-specific oligonucleotide. Furthermore, inhibitors of UVB-induced AP-1 activity, such as PD98059, blocked enhanced IL-18 production, indicating that AP-1 activation is required for UVB-induced IL-18 production. Taken together, our results suggest that UVB irradiation-enhanced IL-18 production is selectively mediated through the generation of ROI and the activation of AP-1.

Summary Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell–associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α–induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

β2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-α–induced vascular inflammation in vivo. Rescue experiments show that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. Studies with blocking anti-PDI antibodies and αLβ2 or αMβ2 null mice suggest that extracellular PDI regulates αMβ2 integrin-mediated adhesive function of neutrophils during vascular inflammation. Consistently, we show that neutrophil surface PDI is important for αMβ2 integrin-mediated adhesion of human neutrophils under shear and static conditions and for binding of soluble fibrinogen to activated αMβ2 integrin. Confocal microscopy and biochemical studies reveal that neutrophil surface PDI interacts with αMβ2 integrin in lipid rafts of stimulated neutrophils and regulates αMβ2 integrin clustering, presumably by changing the redox state of the integrin. Thus, our results provide the first evidence that extracellular PDI could be a novel therapeutic target for preventing and treating inappropriate neutrophil sequestration.

Introduction: Protein disulfide isomerase (PDI), a cell-surface localized oxidoreductase, plays a crucial role in regulating integrin-mediated platelet functions. Although PDI is detected on the neutrophil surface, its role in neutrophil recruitment during vascular inflammation remains unknown. Objectives: To determine the physiological role of neutrophil surface PDI in integrin-mediated neutrophil recruitment during vascular inflammation. Methods and Results: Using real-time fluorescence intravital microscopy in myeloid-specific PDI knockout mice, we have demonstrated that neutrophil PDI is required for neutrophil recruitment into the site of TNF-alpha-induced cremaster venular inflammation. Infusion of wild-type PDI but not functionally-inactive PDI rapidly rescued the defect of neutrophil recruitment in the PDI knockout mice. Cell-impermeable PDI inhibitors also perturbed neutrophil recruitment to the site of TNF-alpha-inflamed venular inflammation in wild-type mice. Neutrophil recruitment was further inhibited when blocking anti-PDI antibodies were infused into alphaLbeta2 but not alphaMbeta2 knockout mice, suggesting that extracellular PDI regulates alphaMbeta2-mediated neutrophil recruitment. To further investigate the role of neutrophil surface PDI in integrin-mediated neutrophil adhesion during vascular inflammation, adhesion assays were performed using human neutrophils treated with cell-impermeable PDI inhibitors. PDI inhibition impaired human neutrophil adhesion to TNF-alpha-activated endothelial cells under venous shear and to ICAM-1-coated surfaces under static conditions. Neutrophil adhesion was further inhibited when anti-PDI was combined with anti-alphaL but not anti-alphaM antibodies. PDI inhibitors also perturbed fibrinogen binding to activated alphaMbeta2 integrin. Using PDI shRNA in neutrophil-like HL60 cells, we found that knockdown of PDI gene diminished alphaMbeta2-mediated cell adhesion to activated endothelial cells under shear. Immunoprecipitation assays and confocal microscopy revealed that surface PDI-alphaMbeta2 interaction is enhanced upon neutrophil activation and regulated by the isomerase activity. Conclusion: Our results indicate that neutrophil surface PDI regulates the alphaMbeta2-ligand interaction, thereby playing an important role in neutrophil recruitment to the activated endothelium during vascular inflammation.

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over-expression of cyclooxygenase (COX)-2 and type I insulin-like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX-2 expression and IGF-I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX-2 expression and IGF-I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK-MEL-2 without induction of apoptosis. At that moment, IGF-II production was decreased, followed by the inhibition of COX-2 activity. IGF-IR expression was also down-regulated by vitamin C treatment. It coincided with the result from the inhibition of COX-2 by NS-398 and COX-2 siRNA. In addition, the decreased IGF-IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C-induced IGF-II and IGF-IR down-regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX-2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK-MEL2 via the down-regulation of IGF-II production and IGF-IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX-2 expression.

Although ascorbate (Vitamin C) has been shown to inhibit cell growth and induce cell death in variety of cancer cells, results reported in other studies are inconsistent with this conclusion. It was previously reported that ascorbate induces apoptosis in human breast cancer cells. However, the molecular mechanism for this is not clear. In this study, we demonstrate that ascorbate induces cell death through the apoptosis-inducing factor (AIF) in the human breast cancer cell lines, SK-BR3 and Hs578T, but not in a normal breast cell line, Hs578. Ascorbate treatment caused the nuclear translocation of AIF, which is retained in the mitochondria in healthy cells, but caspase cleavage is not induced. Moreover, MG132, an inhibitor of AIF release from mitochondria, blocked the induction of cell death. Furthermore, cells that had been treated with human AIF-specific siRNA resisted cell death induced by ascorbate, implying that the translocation of AIF from mitochondria to the nucleus is responsible for ascorbate-mediated cell death. Therefore, these results suggest that ascorbate activates a caspase-independent and AIF-mediated cell death pathway in human breast cancer cells, SK-BR3, and Hs578T.

CD54 is a cell surface adhesive glycoprotein, which is expressed in most cells. Interaction between CD54 and its ligands is involved in several cellular events including activation, proliferation, and cell death and also cell-to-cell adhesion. In the present study, we found that cross-linking of CD54 on Burkitt lymphoma cell lines, Raji and Ramos, induced apoptosis. We investigated that cross-linking of CD54 on Raji and Ramos using immobilized anti-CD54 mAb (clone 6.5B5) leads to apoptosis. CD54-induced apoptosis took place in association with an increase of intracellular reactive oxygen species (ROS) and a loss of the mitochondrial membrane potential and also the activation of caspases 3 and 9, resulting in the degradation of the proteolytic poly (ADP-ribose) polymerase. Pretreatment of each N-acetyl cystein and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (a broad caspase inhibitor) blocked apoptosis. Cross-linking of CD54 immediately induced expression of fasL, which was inhibited by pretreatment of N-acetyl cystein. NOK-1 (antagonistic anti-fasL), ZB4 (antagonistic anti-fas), and N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketon (caspase 8 inhibitor) effectively rescued cells from apoptosis via adjacent fas-fasL interaction but did not block ROS generation. Taken together, it is concluded that engagement of CD54 on B lymphoma cell lines by anti-CD54 mAb may trigger fasL expression through ROS generation and may subsequently induce apoptosis in adjacent fas-fasL interaction.

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway. J. Cell. Biochem. 102: 1002–1010, 2007. © 2007 Wiley-Liss, Inc.

Long-term treatment with glucagon-like peptide (GLP)-1 or its analog can improve insulin sensitivity. However, continuous administration is required due to its short half-life. We hypothesized that continuous production of therapeutic levels of GLP-1 in vivo by a gene therapy strategy may remit hyperglycemia and maintain prolonged normoglycemia. We produced a recombinant adenovirus expressing GLP-1 (rAd-GLP-1) under the cytomegalovirus promoter, intravenously injected it into diabetic ob/ob mice, and investigated the effect of this treatment on remission of diabetes, as well as the mechanisms involved. rAd-GLP-1–treated diabetic ob/ob mice became normoglycemic 4 days after treatment, remained normoglycemic over 60 days, and had reduced body weight gain. Glucose tolerance tests found that exogenous glucose was cleared normally. rAd-GLP-1–treated diabetic ob/ob mice showed improved β-cell function, evidenced by glucose-responsive insulin release, and increased insulin sensitivity, evidenced by improved insulin tolerance and increased insulin-stimulated glucose uptake in adipocytes. rAd-GLP-1 treatment increased basal levels of insulin receptor substrate (IRS)-1 in the liver and activation of IRS-1 and protein kinase C by insulin in liver and muscle; increased Akt activation was only observed in muscle. rAd-GLP-1 treatment reduced hepatic glucose production and hepatic expression of phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fatty acid synthase in ob/ob mice. Taken together, these results show that a single administration of rAd-GLP-1 results in the long-term remission of diabetes in ob/ob mice by improving insulin sensitivity through restoration of insulin signaling and reducing hepatic gluconeogenesis.

It is known that B lymphoma cells are sensitive to cytotoxic chemotherapy, but primary or secondary chemoresistance frequently occurs and is the major cause of death in these patients. However, the mechanisms by which lymphoma cells acquire resistance to cytotoxic drugs are not fully understood. Recently, it was reported that B cells secrete immunoglobulin and produce cytokines after interacting with NK cells, thus indicating the importance of NK/B interactions. In this study, we investigated the mechanism of resistance to cytotoxic chemotherapy induced in cocultures of NK cells and Raji cells. Normally, Raji cells are doxorubicin-sensitive, but Raji cells cocultured with NK cells become doxorubicin-resistant. In addition, we detected the upregulation of CD69 and CD70 on Raji cells cocultured with NK cells, suggesting that Raji cells are activated by NK cells. We also found that the resistance of Raji cells to doxorubicin increased when they had been treated with NK cell coculture supernatant. Furthermore, boiled culture supernatant did not inhibit doxorubicin-mediated cell death, indicating that soluble factors are involved. Finally, we confirmed that NK cells produce TNF alpha, and that doxorubicin-sensitive Raji cells become doxorubicin-resistant after TNF alpha treatment. Taken together, these results suggest that B lymphoma cell resistance to doxorubicin-mediated cell death is induced by coculture with NK cells, because of TNF alpha secretion.

Burkitt lymphoma (BL) is a tumor with the characteristics of germinal center B cells. We previously reported that the CM1 (centrocyte/-blast marker 1) molecule is expressed only in germinal center B cells, specifically, in a subpopulation of centroblasts and centrocytes. In the present study, we investigated the apoptosis induced by anti-CM1 in the Ramos and Raji human BL cell lines. The Ramos is protected from apoptosis by the crosslinking of sIgM and the calcium ionophore by the ligation of CD40 with anti-CD40 monoclonal antibodies (mAb) or soluble CD40 ligand (sCD40L). In this investigation on the effect of CM1 on apoptosis in BL cell lines, we found that cellular signaling by CM1 induces apoptosis and decreases cell viability, in BL cell lines cultured for 24 hours with protein-G agarose beads conjugated anti-CM1 mAb. Stimulation by CD40 ligated with sCD40L protected Raji cells from CM1-induced apoptosis, but did not protect Ramos cells. Furthermore, after anti-CM1 mAb stimulation, CD95 expression was upregulated and CD40 expression was unaltered or slightly decreased in Ramos cells, whereas CD95 was downregulated and CD40 was slightly upregulated in Raji cells. The engagement of CD40 by sCD40L enhanced CD95 expression, but the level of CM1 expression was unchanged in Ramos. However, sCD40L downregulated both CD95 and CM1 expression in Raji. In addition, the caspase-8 specific inhibitor blocked CM1-induced apoptosis in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization was observed only in Raji cells. Moreover, the effector caspase inhibitor, z-DEVD, blocked CM1-mediated apoptosis in both cell lines. We found that CM1-induced apoptosis is achieved via different initiation pathways, which are cell-type dependent.

It has been reported that vitamin C plays an effective role in the treatment and prevention of cancer, but its specific mechanism is still not fully understood. In particular, the effect of vitamin C on the treatment and prevention of colorectal cancer, which has been increasingly a common disease in South Korea due to westernized diet of many Koreans, is still largely unknown. Therefore, we have examined the effects of vitamin C on the induction of apoptosis on HCT-8, human colon cancer cell line. We have first confirmed that the optimal concentrations of vitamin C for the induction of apoptosis on human colon cancer cell line, HCT-8 is 2 mM of vitamin C. Here we demonstrated that vitamin C induced ER stress through redox imbalance as antioxidant and, increased cytosol calcium level. Then, we investigated the changes of ER-stress induced molecules. As a result, the phosphorylation of pro-apoptotic protein, Bad decreased and Bad, which was subsequently released from 14-3-3, was translocated to the mitochondria. In addition, we also found that the vitamin C increased expression of pro-apoptotic protein, Bax and tumor suppressor gene product, p53, which usually resulted in apoptosis, in a time dependent manner. Moreover, we have witnessed that vitamin C treatment disrupts mitochondrial membrane potential in HCT-8, human colon cancer cell line. Taken together, vitamin C induced redox imbalance, resulting in apoptosis in human colon cancer cells by both endoplasmic reticulum stress and Bad/p53/Bax-mediated activation of mitochondrial pathway.

Platelet-leukocyte interactions on activated endothelial cells play important roles in mediating pathological thrombosis and inflammation. Heterotypic platelet-leukocyte aggregation is mediated by the interaction of two crucial receptors and counter receptors; P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. In spite of extensive understanding of receptor-counter receptor interactions, it remains unclear how heterotypic cell-cell interactions are regulated under thrombo-inflammatory conditions. Using real-time fluorescence intravital microscopic analysis of Akt isoform-specific knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays an important role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, heterotypic platelet-neutrophil interactions on the activated endothelium were markedly reduced in Akt2 but not Akt1 or Akt3 KO mice. Studies with chimeric mice generated from bone marrow transplants on wild-type and Akt2 KO mice revealed that hematopoietic but not endothelial cell Akt2 regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. Using in vitro reconstituted systems in which platelets and neutrophils were treated with an Akt2 specific inhibitor or cells were isolated from WT and Akt KO mice, we observed that both platelet and neutrophil Akt2 play an important role in platelet-neutrophil aggregation under shear conditions. In particular, neutrophil Akt2 was critical for membrane translocation, activation, and adhesive function of alphaMbeta2 integrin. We found that the basal phosphorylation levels of Akt isoforms are significantly increased in neutrophils and platelets of patients with sickle cell disease (SCD), which is an inherited hematological disorder with vascular inflammation and occlusion. Also, SCD patients’ neutrophils show increased alphaMbeta2 integrin activation in the absence of an agonist, in comparison with healthy donors’ cells. Inhibition of Akt2 dose-dependently reduced heterotypic aggregation of patients’ neutrophils and platelets in vitro and inhibited neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow. Our results provide important genetic and pharmacologic evidence that neutrophil Akt2 regulates alphaMbeta2 integrin function and thus plays a critical role during neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD. Disclosures: No relevant conflicts of interest to declare.

Overexpression of cyclooxygenase-2 (COX-2) frequently occurs in several malignancies and the type I insulin-like growth factor receptor (IGF-IR) is important for growth, transformation, proliferation and protection from apoptosis. However the interaction between COX-2 expression and IGF-I axis in human melanoma remains obscure. Although inhibition of COX-2 could reduce tumor volumes in various murine models, selective COX-2 inhibitors could increase cardiovascular risk. Therefore, we studied the effects of vitamin C, which is known as the potent inhibitor of proliferation of human melanoma cell line, SK-MEL-2. We confirmed that the optimal concentration of vitamin C for the inhibition of proliferation without inducing apoptosis is 1.0 mM. ELISA for IGF-I and IGF-II conducted after treatment with NS-398 and vitamin C revealed that the production of IGF-II was decreased by COX-2 inhibition. RT-PCR for IGF-IR after COX-2 inhibition using NS-398 or COX-2 siRNA showed the inhibition of COX-2 expression led to dramatic down-regulation of IGF-IR expression which is restored by adding exogenous PGE2, which is produced by the enzymatic action of COX-2. Next we observed that both COX-2 and IGF-IR expression were significantly decreased after treatment of 1.0 mM vitamin C. Interestingly exogenously treated IGF-II induced IGF-IR expression and exogenous PGE2 treatment restored IGF-IR expression which is down-regulated by vitamin C. These findings suggest that vitamin C could modulate IGF-II production and then down-regulate the expression of IGF-IR by the inhibition of COX-2 expression, followed by suppression of the proliferation of human melanoma cell line, SK-MEL-2.

CD40 has been shown to play a critical role in normal B-cell development and function. Interestingly, CD40 has also demonstrated to be expressed at high levels on some human cancers, including the breast cancer. However, the function of CD40 expressed on tumor cells is still not fully understood. In this study, therefore, the function of CD40 on tumor cells in the aspect of the production of Transforming Growth Factor-beta (TGF-β) was investigated after CD40 stimulation. First, high expression of CD40 on the human breast cancer cell line, MDA-MB231 was observed. The tumor cells were incubated for 24 hr in the presence of agonistic anti-CD40 antibodies or sCD40 ligands, the productions of TGF-β were dramatically increased in each cases. Human T cells purified from peripheral blood were stimulated with PHA and ionomycin. And then cells were cultured with MDA-MB231 for 24 hr. As a result, significantly increased production of TGF-β was observed. Taken together, TGF-β production by breast cancer cells increased via the ligation of CD40 on their surface with CD40L on the surface of T cells. Thus, considering that TGF-β serves dual roles not only in promoting tumor progression and metastasis, but also in immune suppression, these results give an important clue for understanding the mechanism of the immune suppression by tumor cells.

Malignancies of lung are the leading causes of cancer mortality in world wide and there are so many limitations in early detection of lung cancer, which eventually made the cancer detected at advanced stage. Conventional cytotoxic drugs have showed more increased response rate with grave toxicities. Recently, successful therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in lung cancer patients have been reported, although a mono-therapy still showed low response rate. Here, we investigated to elucidate whether the vitamin C has the adjuvant role as an antioxidant or other functions in EGFR tyrosine kinase inhibitor, gefitinib (Iressa ¢â), therapy in lung cancers. We used three kinds of human non-small cell lung cancer cell (NSCLC) lines depending on EGFR mutation, A549, Calu-3 and HCC827. A549 and Calu-3 are NSCLC cell lines which have wild type EGFR domain and showed relative resistance to gefitinib treatment. HCC827 is well known cell line of EGFR domain mutation. Vitamin C showed reduction of S phase in cell cycle and had function as an anti-oxidant. Also that could induce synergistic effects of EGFR tyrosine kinase inhibitor therapy in cellular proliferation and down-regulation of EGFR phosphorylation, AKT & ERK activation. Synergistic inhibitory effects of vitamin C with EGFR tyrosine kinase inhibitor therapy are showed in both wild and mutant EGFR cell lines. In this present study, we demonstrated an possible adjuvant role of vitamin C in EGFR tyrosine kinase inhibitor therapy in human NSCLC.

CM1 (Centrocyte/-blast Marker I) defined by a mAb developed against concanavalin-A activated PBMC, is expressed specifically on a subpopulation of centroblasts and centrocytes of human germinal center (GC) B cells. Burkitt lymphoma (BL) is a tumor consisting of tumor cells with the characteristics of GC B cell. Previously we reported that CM1 ligation with anti-CM1 mAb induced apoptosis in Ramos (IgM high ) and Raji IgM low cells. Methods & Results: In the present study, we observed that CM1 ligation with anti-CM1 mAb induced Fas ligand and Fas expression in Ramos cells, but not in Raji cells. Furthermore, anti-Fas blocking antibody, ZB4, blocked CM1-mediated apoptosis effectively in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization, which was measured by DiOC 6 , was observed only in Raji cells. In contrast to no significant change of Bax known as pro-apoptotic protein, anti-apoptotic protein Bcl-2 was significantly decreased in Raji cells. In addition, we observed that CM1 ligation increased release of mitochondrial cytochrome c and upregulated caspase-9 activity in Raji cells. Conclusion: These results suggest that apoptosis induced by CM1-ligation is mediated by Fas-Fas ligand interaction in Ramos cells, whereas apoptosis is mediated by down-regulation of Bcl-2 and subsequent decrease of mitochondrial membrane potential in Raji cells.