Mi Jeong Sung

Metabolomic Analysis of Livers and Serum from High-Fat Diet Induced Obese Mice

Liver and serum metabolites of obese and lean mice fed on high fat or normal diets were analyzed using ultraperformance liquid chromatography-quadrupole-time-of-flight mass spectrometry, gas chromatography-mass spectrometry, and partial least-squares-discriminant analysis (PLS-DA). Obese and lean groups were clearly discriminated from each other on PLS-DA score plot and major metabolites contributing to the discrimination were assigned as lipid metabolites (fatty acids, phosphatidylcholines (PCs), and lysophosphatidylcholines (lysoPCs)), lipid metabolism intermediates (betaine, carnitine, and acylcarnitines), amino acids, acidic compounds, monosaccharides, and serotonin. A high-fat diet increased lipid metabolites but decreased lipid metabolism intermediates and the NAD/NADH ratio, indicating that abnormal lipid and energy metabolism induced by a high-fat diet resulted in fat accumulation via decreased β-oxidation. In addition, this study revealed that the levels of many metabolites, including serotonin, betaine, pipecolic acid, and uric acid, were positively or negatively related to obesity-associated diseases. On the basis of these metabolites, we proposed a metabolic pathway related to high-fat diet-induced obesity. These metabolites can be used to better understand obesity and related diseases induced by a hyperlipidic diet. Furthermore, the level changes of these metabolites can be used to assess the risk of obesity and the therapeutic effect of obesity management.

Angiogenic role of adrenomedullin through activation of Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells

Adrenomedullin (AM) is a multifunctional peptide in human pheochromocytoma. To evaluate whether AM could be an angiogenic factor, we examined its effect on kinases and angiogenic processes. AM induced tyrosine phosphorylation of Akt and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase1/2 (ERK1/2) by using distinct signaling pathways in human umbilical vein endothelial cells (HUVECs). AM also phosphorylated focal adhesion kinase, and phosphatidylinositol 3′‐kinase inhibitor inhibited AM‐induced focal adhesion kinase phosphorylation. Pretreatment with high concentrations of AM22‐52, a putative AM receptor antagonist, partially suppressed AM‐induced phosphorylation of Akt, ERK1/2, and focal adhesion kinase. AM and vascular endothelial growth factor produced increases in DNA synthesis and migration in HUVECs. AM induced tube formation in HUVECs, and its effect was inhibited by pretreatment with phosphatidylinositol 3′‐kinase inhibitor or ERK1/2 inhibitor. AM induced sprouting in porcine pulmonary arterial endothelial cells and promoted neovessel formation in a mouse Matrigel plug assay. Inhibitors of phosphatidylinositol 3′‐kinase and ERK1/2 inhibited AM‐induced endothelial sprouting in vitro and angiogenesis in vivo. AM exerts angiogenic activity through activation of Akt, MAPK, and focal adhesion kinase in endothelial cells.

Genistein protects the kidney from cisplatin-induced injury.

Oxidative stress and inflammation contribute to the pathogenesis of cisplatin-induced nephrotoxicity. We found that genistein, a tyrosine kinase inhibitor with broad specificities, and which also has estrogen-like activity, had protective effects on cisplatin-induced renal injury in mice. Genistein significantly decreased reactive oxygen species production, the expression of intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 proteins, as well as the translocation of the p65 subunit of nuclear factor-kappaB into the nucleus and the infiltration of macrophages, all of which were increased in the kidney by cisplatin treatment. Genistein also decreased cisplatin-induced apoptosis by regulating p53 induction in kidney. Genistein significantly reduced reactive oxygen species production in cisplatin-treated normal human kidney HK-2 cells. These studies show that genistein or similar compounds might be useful in prevention of cisplatin-induced renal injury.

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.

Alpha-lipoic acid attenuates cisplatin-induced acute kidney injury in mice by suppressing renal inflammation

BACKGROUND Cisplatin is a chemotherapeutic agent used in treatment of malignant tumours. However, cisplatin produces various side effects, such as nephrotoxicity, neurotoxicity, emetogenesis and ototoxicity. Inflammation is an important mechanism of cisplatin nephrotoxicity. Alpha-lipoic acid (alpha-LA) has anti-inflammatory effects that inhibit both adhesion molecule expression in human endothelial cells and monocyte adhesion by suppressing the nuclear factor-kappaB (NF-kappaB) signalling pathway. The goals of this study were to investigate the anti-inflammatory effects of alpha-LA during cisplatin-induced renal injury and to examine the mechanisms of protection. METHODS C57BL/6 mice were given cisplatin (20 mg/kg) with or without alpha-LA treatment (100 mg/kg for 3 days). Renal function, histological changes, adhesion molecule expression and inflammatory cell infiltration were examined. The effect of alpha-LA on NF-kappaB activity was evaluated by examining nuclear translocation and phosphorylation of NF-kappaB p65 subunits in kidney tissue. RESULTS Cisplatin-induced decreases in renal function, measured by blood urea nitrogen, serum creatinine level and renal tubular injury scores, were attenuated by alpha-LA treatment. alpha-LA decreased the tissue levels of tumour necrosis factor-alpha, the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), and suppressed the infiltration of CD11b-positive macrophages. alpha-LA also attenuated the cisplatin-induced increases in the phosphorylation and nuclear translocation of NF- kappaB p65 subunits in kidney tissue. CONCLUSIONS These results suggest that alpha-LA treatment ameliorates cisplatin-induced acute kidney injury by reducing inflammatory adhesion molecule expression and NF-kappaB activity.

COMP–Angiopoietin-1 Ameliorates Renal Fibrosis in a Unilateral Ureteral Obstruction Model

Injury to the renal microvasculature may be a major factor in the progression of renal disease; therefore, protection of endothelial cells (EC) in renal vasculature may have a therapeutic role in renal fibrosis. Recently, a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, was developed. The contribution of COMP-Ang1 in renal interstitial fibrosis, however, remains to be clarified. This study investigated the effects of COMP-Ang1 on peritubular capillary EC in the renal cortex and the renal fibrogenic process that is triggered by unilateral ureteral obstruction. COMP-Ang1 preserved renal platelet-EC adhesion molecule-1-and Tie2-positive EC. Morphologic examination indicated less tubular injury and tubulointerstitial fibrosis in mice that received COMP-Ang1 than vehicle-treated mice. Interstitial type I collagen and myofibroblast accumulation were significantly suppressed by COMP-Ang1 treatment. COMP-Ang1 increased Tie2 and Akt phosphorylation in ureteral obstructed kidneys. Renal surface microvasculature and renal blood flow were higher after treatment with COMP-Ang1 than with vehicle. COMP-Ang1 treatment decreased monocyte/macrophage infiltration, tissue levels of TGF-beta1, and Smad 2/3 phosphorylation and increased Smad 7 in the obstructed kidney. These results demonstrate that COMP-Ang1 treatment can decrease the progression of renal fibrosis in unilateral ureteral obstruction. COMP-Ang1 may be an endothelium-specific therapeutic modality in fibrotic renal disease.

Protective Effect of α-Lipoic Acid in Lipopolysaccharide-Induced Endothelial Fractalkine Expression

Fractalkine is a unique chemokine that functions as a chemoattractant as well as an adhesion molecule on endothelial cells activated by proinflammatory cytokines. Alpha-lipoic acid (LA), a naturally occurring dithiol compound, is an essential cofactor for mitochondrial bioenergetic enzymes. LA improves glycemic control, reduces diabetic polyneuropathies, and mitigates toxicity associated with heavy metal poisoning. The effects of LA on processes associated with sepsis, however, are unknown. We evaluated the antiinflammatory effect of LA on fractalkine expression in a lipopolysaccharide-induced endotoxemia model. Tumor necrosis factor-&agr; (TNF-&agr;) and interleukin-1&bgr; (IL-1&bgr;) significantly induced fractalkine mRNA and protein expression in endothelial cells. LA strongly suppressed TNF-&agr;– or IL-1&bgr;–induced fractalkine expression in endothelial cells by suppressing the activities of nuclear factor-&kgr;B and specificity protein-1. LA also decreased TNF-&agr;– or IL-1&bgr;–stimulated monocyte adhesion to human umbilical vein endothelial cells. As shown by immunohistochemistry, fractalkine protein expression was markedly increased by treatment with lipopolysaccharide in arterial endothelial cells, endocardium, and endothelium of intestinal villi. LA suppressed lipopolysaccharide-induced fractalkine protein expression and infiltration of endothelin 1-positive cells into the heart and intestine in vivo. LA protected against lipopolysaccharide-induced myocardial dysfunction and improved survival in lipopolysaccharide-induced endotoxemia. These results suggest that LA could be an effective agent to reduce fractalkine-mediated inflammatory processes in endotoxemia.

COMP-angiopoietin-1 decreases lipopolysaccharide-induced acute kidney injury.

During sepsis endothelial dysfunction is an important pathogenetic mechanism in acute kidney injury (AKI). Lipopolysaccharide (LPS)-induced endotoxemia is associated with renal hemodynamic changes such as alterations of renal blood flow (RBF), vascular resistance, and glomerular filtration rate. We used adenoviral delivery of an engineered variant of native angiopoietin-1 (COMP-angiopoietin-1) containing anti-inflammatory and anti-permeability functions, to determine if regulation of renal endothelial cell dysfunction may have a beneficial role in preventing AKI during LPS-induced endotoxemia in mice. This treatment prevented the endotoxin-induced decrease of RBF and mean arterial pressure while improving glomerular filtration rate. Treatment also mitigated the effects of LPS on renal intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 protein expression, the number of ER-HR3-positive macrophages that infiltrated the kidney, serum nitrate/nitrite levels, renal inducible nitric oxide synthase protein expression, the induction of tubular epithelial reactive oxygen and nitrogen species, and renal microvascular permeability. Our findings show that COMP-angiopoietin-1, an endothelium-oriented therapeutic agent, protects against AKI caused by endotoxemia.

Vitisin A suppresses LPS-induced NO production by inhibiting ERK, p38, and NF-κB activation in RAW 264.7 cells.

Vitisin A, a resveratrol tetramer isolated from Vitis vinifera roots, exhibits antioxidative, anticancer, antiapoptotic, and anti-inflammatory effects. It also inhibits nitric oxide (NO) production. Here, we examined the mechanism by which vitisin A inhibits NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells. Vitisin A dose dependently inhibited LPS-induced NO production and inducible NO synthase (iNOS) expression. In contrast, the production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) was not altered by vitisin A. To investigate the signaling pathway for NO inhibition by vitisin A, we examined nuclear factor-kappaB (NF-kappaB) activation in the mitogen-activated protein kinase (MAPK) pathway, an inflammation-induced signal pathway in RAW 264.7 cells. Vitisin A inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation and suppressed LPS-induced NF-kappaB activation in RAW 264.7 cells. This suggests that vitisin A decreased NO production via downregulation of ERK1/2 and p38 and the NF-kappaB signal pathway in RAW 264.7 cells.

Resveratrol Suppresses Tumor Necrosis Factor-α-Induced Fractalkine Expression in Endothelial Cells

Up-regulation of fractalkine is involved in vascular and tissue damage in inflammatory conditions. Resveratrol has been shown to have anti-inflammatory, antioxidant, and antitumor activities. Its regulatory effects on expression of fractalkine in vascular endothelial cells and fractalkine receptor CX3CR1 in monocytes have not been studied. We evaluated the effects of resveratrol on fractalkine expression in human umbilical vein endothelial cells and CX3CR1 expression in THP-1 cells in response to treatment with tumor necrosis factor (TNF)-α. TNF-α significantly induced fractalkine mRNA and protein expression in endothelial cells. Resveratrol strongly suppressed TNF-α-induced fractalkine expression in endothelial cells through suppression of nuclear factor-κB and Sp1 activities. Resveratrol decreased the number of TNF-α-induced fractalkine-positive endothelial cells and CX3CR1-positive cells determined by flow cytometric analysis. Resveratrol suppressed TNF-α-stimulated monocytes adhesion to human umbilical vein endothelial cells. Immunohistochemical analysis revealed that resveratrol suppressed TNF-α-induced arterial endothelial fractalkine expression in heart, kidney, and intestine and decreased ED-1-positive cell infiltration in intestinal villi. Resveratrol may provide a new pharmacological approach for suppressing fractalkine/CX3CR1-mediated injury in inflammatory conditions.