Won Sun Park

Blockade of Indoleamine 2,3-Dioxygenase Protects Mice against Lipopolysaccharide-Induced Endotoxin Shock

Suppression of an excessive systemic inflammatory response is a promising and potent strategy for treating endotoxic sepsis. Indoleamine 2,3-dioxygenase (IDO), which is the rate-limiting enzyme for tryptophan catabolism, may play a critical role in various inflammatory disorders. In this study, we report a critical role for IDO in the dysregulated immune response associated with endotoxin shock. We found that IDO knockout (IDO−/−) mice and 1-methyl-d-tryptophan-treated, endotoxin-shocked mice had decreased levels of the cytokines, TNF-α, IL-6, and IL-12, and enhanced levels of IL-10. Blockade of IDO is thought to promote host survival in LPS-induced endotoxin shock, yet little is known about the molecular mechanisms that regulate IDO expression during endotoxin shock. In vitro and in vivo, IDO expression was increased by exogenous IL-12, but decreased by exogenous IL-10 in dendritic cells and splenic dendritic cells. Interestingly, whereas LPS-induced IL-12 levels in serum were higher than those of IL-10, the balance between serum IL-12 and IL-10 following challenge became reversed in IDO−/−- or 1-methyl-d-tryptophan-treated mice. Our findings demonstrate that the detrimental immune response to endotoxin shock may occur via IDO modulation. Restoring the IL-12 and IL-10 balance by blocking IDO represents a potential strategy for sepsis treatment.

Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases.

Background and purpose:  Cilostazol is a specific inhibitor of 3′‐5′‐cyclic adenosine monophosphate (cAMP) phosphodiesterase, which is widely used to treat ischemic symptoms of peripheral vascular disease. Although cilostazol has been shown to exhibit vasodilator properties as well as antiplatelet and anti‐inflammatory effects, its cellular mechanism in microglia is unknown. In the present study, we assessed the anti‐inflammatory effect of cilostazol on the production of pro‐inflammatory mediators in lipopolysaccharide (LPS)‐stimulated murine BV2 microglia.

Differential regulation of indoleamine 2,3-dioxygenase by lipopolysaccharide and interferon gamma in murine bone marrow derived dendritic cells

Indoleamine 2,3‐dioxygenase (IDO) is a rate‐limiting enzyme in the l‐tryptophan‐kynurenine pathway, which converts an essential amino acid, l‐tryptophan, to N‐formylkynurenine. The expression of IDO increases when inflammation is induced by wounding, infection or tumor growth. Although recent studies have suggested that IDO expression is up‐regulated by IFN‐γ in various cell types and that the induction of IDO can also be mediated through an IFN‐γ‐independent mechanism, these mechanisms still remain unknown. In this study, we investigated whether lipopolysaccharide (LPS) induces the expression of IDO through an IFN‐γ‐mediated signaling pathway or not. IFN‐γ‐induced expression of IDO expression was inhibited only by JAK inhibitor I. However, LPS‐induced expression of IDO was inhibited by LY294002 and SP600125 but not by JAK inhibitor I, SB203580, or U0126. These findings clearly indicate that LPS can induce the IDO expression via an IFN‐γ‐independent mechanism and PI3 kinase and JNK in the LPS‐induced pathway leading to IDO expression.

A Rho Kinase/Myocardin-Related Transcription Factor-A–Dependent Mechanism Underlies the Sphingosylphosphorylcholine-Induced Differentiation of Mesenchymal Stem Cells Into Contractile Smooth Muscle Cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue–derived mesenchymal stem cells (hADSCs) to smooth muscle cells (SMCs). In the present study, we characterized contractile and ion channel properties of SMCs differentiated from hADSCs (hADSC-SMCs) as a result of SPC treatment, and we investigated the molecular mechanisms involved in the SPC-induced differentiation. Using in vitro collagen gel lattice contraction and whole cell patch clamp, we showed that the hADSC-SMCs expressed functional L-type voltage-gated Ca2+ channels and contractile activities in response to KCl, carbachol, and the L-type Ca2+ channel opener Bay K8644, whereas the L-type Ca2+ channel blocker nifedipine abrogated the contractility of hADSC-SMCs. Furthermore, hADSC-SMCs expressed functional big conductance Ca2+-activated K+ (BKCa) channels, and the BKCa channel blocker iberiotoxin potentiated the Bay K8644-stimulated contractility of the hADSC-SMCs, indicating that these cells exhibited SMC-like contractile characteristics. SPC activated RhoA in hADSCs and pretreatment with the Rho kinase inhibitor Y27632 or by overexpression of dominant-negative mutants of RhoA or Rho kinase completely abrogated the SPC-induced differentiation of hADSCs into SMCs. SPC also increased the expression levels of myocardin-related transcription factor (MRTF)-A, a transcription factor involved in smooth muscle differentiation, in hADSCs. Small interference RNA–mediated depletion of endogenous MRTF-A abolished the SPC-induced differentiation of hADSCs into SMCs. Furthermore, SPC promoted nuclear translocation of MRTF-A, and pharmacological inhibition of Rho kinase blocked this effect. These results suggest that SPC induced differentiation of hADSCs into contractile SMCs through a mechanism involving RhoA/Rho kinase–dependent nuclear translocation of MRTF-A.

Pathophysiology of voltage-gated K+ channels in vascular smooth muscle cells: modulation by protein kinases.

In this review, the pathological alteration and clinical relevance of voltage-gated K(+) (Kv) channels and their specific regulation by protein kinase-dependent signaling in vascular smooth muscle cells are described, particularly focusing on the pulmonary vasculature. The physiological relevance, channel characteristics, pharmacological modulation, and expression of Kv channels vary between different arterial beds and between subdivisions of arteries within those vascular beds. Although detailed signaling cascades regulating Kv channels are not clearly elucidated, it is known that the Kv channels in vascular smooth muscle cells can be tightly regulated by protein kinases C (PKC) and A (PKA). Alterations in Kv channel expression and function has been noted in pathological and pathophysiological conditions including hypertension (pulmonary and systemic), in diabetes and in individuals subjected to prolonged hypoxia (high altitude living). Vascular Kv channels are potential therapeutic targets in diseases such as pulmonary arterial hypertension and, therefore, it is important to understand the specific pharmacological modulation of Kv channel isoforms in different vascular beds.

Cilostazol Suppresses Superoxide Production and Expression of Adhesion Molecules in Human Endothelial Cells via Mediation of cAMP-Dependent Protein Kinase-Mediated Maxi-K Channel Activation

This study shows whether increased intracellular cAMP level by cilostazol is directly coupled to its maxi-K channel activation in human endothelial cells. Cilostazol (1 μM) increased the K+ currents in the human endothelial cells by activating maxi-K channels, which was abolished by iberiotoxin (100 nM), a maxi-K channel blocker. On incubation of human coronary artery endothelial cells with tumor necrosis factor-α (TNF-α) (50 ng/ml), monocyte adhesion significantly increased with increased superoxide generation and expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) accompanied by increased degradation of inhibitory κBα in cytoplasm and activation of nuclear factor-κB p65 in nucleus. All these variables were significantly suppressed by cilostazol (10 μM), which was antagonized by iberiotoxin (1 μM) and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) (300 nM, cAMP-dependent protein kinase inhibitor), but not by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindo-lo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823) (300 nM, cGMP-dependent protein kinase inhibitor). In the human endothelial cells transfected with siRNA-targeting maxi-K channels, cilostazol did not suppress the superoxide generation, VCAM-1 and MCP-1 expressions, and monocyte adhesion as contrasted with the wild-type cells. These findings were similarly evident with (3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one (BMS-204352), a maxi-K channel opener, and forskolin and dibutyryl cAMP. In conclusion, increased cAMP level by cilostazol is directly coupled to its maxi-K channel opening action via protein kinase activation in human endothelial cells, thereby suppressing TNF-α–stimulated superoxide production and expression of adhesion molecules.

Enhanced Efficacy of Therapeutic Cancer Vaccines Produced by Co-Treatment with Mycobacterium tuberculosis Heparin-Binding Hemagglutinin, a Novel TLR4 Agonist

Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism Mycobacterium tuberculosis includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs in vitro and in vivo. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated naïve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ, and induced T-cell-mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA(251-264)-pulsed ex vivo greatly strengthened immune priming in vivo, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy.

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.

Endothelin-1 acts via protein kinase C to block KATP channels in rabbit coronary and pulmonary arterial smooth muscle cells.

We investigated the effects of the vasoconstrictor endothelin-1 (ET-1) on the whole-cell ATP-sensitive K+ (KATP) currents of smooth muscle cells that were isolated enzymatically from rabbit coronary artery (CASMCs) and pulmonary artery (PASMCs). The size of the KATP current did not differ significantly between CASMCs and PASMCs. ET-1 reduced the KATP current in a concentration-dependent manner, and this inhibition was greater in PASMCs than in CASMCs (half-inhibition values of 12.20 nM and 1.98 nM in CASMCs and PASMCs, respectively). However, the level of inhibition induced by other vasoconstrictors (angiotensin II, norepinephrine, and serotonin) were not significantly different between CASMCs and PASMCs. Pretreatment with the protein kinase C (PKC) inhibitors staurosporine (100 nM) and GF 109203X (1 μM) prevented ET-1-induced inhibition of the KATP current in both arterial smooth muscle cell preparations. The PKC activators phorbol-12,13-dibutyrate (PDBu) and 1-olelyl-2-acetyl-sn-glycerol (OAG) reduced the KATP current in dose-dependent manner. Although the numbers of ET receptors were not significantly different between the 2 arterial smooth muscle cell preparations, the effects of PDBu and OAG were greater on PASMCs. ET-1-induced inhibition of the KATP current was unaffected by the PKA inhibitor Rp-cAMPs (100 μM) and PKA inhibitory peptide (5 μM).

Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts.

Glutathione peroxidase 1 (GPx1) plays an important role in preventing cardiac dysfunction following ischemia-reperfusion injury. However, its role in protecting cardiac mitochondria against reoxygenation-induced reactive oxygen species (ROS) generation in vivo is unclear. We examined the role of GPx1 in protecting cardiac mitochondria against hypoxia–reoxygenation (HR) damage by testing for alterations in cardiac mitochondrial function. We used a two-dimensional gel electrophoresis proteomics analysis to examine the effects of reoxygenation on cardiac protein in wild-type (GPx1+/+) and GPx1 knockout (GPx1−/−) mouse hearts. We identified 42 protein spots showing differential expression in the two groups. Sixteen of the proteins identified were located in mitochondria and were involved in a number of key metabolic pathways. To verify our proteomics findings functionally, we performed NADH autofluorescence measurements and ATP production assays. The reduced expression of oxidative phosphorylation proteins in GPx1−/− mice following HR treatment resulted in loss of the mitochondrial membrane potential and decreased mitochondrial respiration. Mitochondrial ROS production and oxidative mtDNA damage were increased markedly during reoxygenation in GPx1−/− hearts. We also found morphological abnormalities in cardiac mitochondria and myocytes in HR-treated GPx1−/−. This is the first report of the role of GPx1 in protecting cardiac mitochondria against reoxygenation damage in vivo. These findings will help clarify the mechanisms of HR injury and will aid in the development of antioxidant therapies to prevent cardiac mitochondrial dysfunction associated with reoxygenation.