Effect of a new PPAR-gamma agonist, lobeglitazone, on neointimal formation after balloon injury in rats and the development of atherosclerosis.

OBJECTIVE: The ligand-activated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is a key factor in adipogenesis, insulin sensitivity, and cell cycle regulation. Activated PPARγ might also have anti-inflammatory and antiatherogenic properties. We tested whether lobeglitazone, a new PPARγ agonist, might protect against atherosclerosis. METHODS: A rat model of balloon injury to the carotid artery, and high-fat, high-cholesterol diet-fed apolipoprotein E gene knockout (ApoE(-/-)) mice were studied. RESULTS: After the balloon injury, lobeglitazone treatment (0.3 and 0.9 mg/kg) caused a significant decrease in the intima-media ratio compared with control rats (2.2 ± 0.9, 1.8 ± 0.8, vs. 3.3 ± 1.2, P < 0.01). Consistent with this, in ApoE(-/-) mice fed a high-fat diet, lobeglitazone treatment (1, 3, and 10 mg/kg) for 8 weeks reduced atherosclerotic lesion sizes in the aorta compared with the control mice in a dose-dependent manner. Treatment of vascular smooth muscle cells with lobeglitazone inhibited proliferation and migration and blocked the cell cycle G0/G1 to S phase progression dose-dependently. In response to lobeglitazone, tumor necrosis factor alpha (TNFα)-induced monocyte-endothelial cell adhesion was decreased by downregulating the levels of adhesion molecules. TNFα-induced nuclear factor kappa-B (NF-κB) p65 translocation into the nucleus was also blocked in endothelial cells. Insulin resistance was decreased by lobeglitazone treatment. Circulating levels of high sensitivity C-reactive protein and monocyte chemoattractant protein-1 were decreased while adiponectin levels were increased by lobeglitazone in the high-fat diet-fed ApoE(-/-) mice. CONCLUSION: Lobeglitazone has antiatherosclerotic properties and has potential for treating patients with diabetes and cardiovascular risk.

Anti-diabetic efficacy of KICG1338, a novel glycogen synthase kinase-3ß inhibitor, and its molecular characterization in animal models of type 2 diabetes and insulin resistance.

Selective inhibition of glycogen synthase kinase-3 (GSK3) has been targeted as a novel therapeutic strategy for diabetes mellitus. We investigated the anti-diabetic efficacy and molecular mechanisms of KICG1338 (2-(4-fluoro-phenyl)-3H-imidazo[4,5-b]pyridine-7-carboxylic acid(4-methyl-pyridin-3-yl)-amide), a GSK3ß inhibitor, in three animal models: Otsuka Long-Evans Tokushima Fatty (OLETF) rats, leptin receptors-deficient db/db mice, and diet-induced obese (DIO) mice. Biochemical parameters including glucose tolerance tests and gene expressions associated with glucose metabolism were investigated. Glucose excursion decreased significantly by KICG1338-treated OLETF rats, accompanied by increase in insulin receptor substrate-1 and glucose transporter (GLUT)-4 expressions in muscle and decreased GLUT-2 expression in liver. Glucose-lowering effects were similarly observed in KICG1338-treated db/db and DIO mice. KICG1338 treatment increased adiponectin levels and decreased TNF-α levels. KICG1338 therapy also led to greater ß-cell preservation and less hepatic fat infiltration with decreased expressions of genes involved in inflammation and endoplasmic reticulum stress. These data demonstrate anti-diabetic efficacy of KICG1338, a novel GSK3ß inhibitor.

The PPARδ ligand L-165041 inhibits VEGF-induced angiogenesis, but the antiangiogenic effect is not related to PPARδ.

Peroxisome proliferator-activated receptor (PPAR)δ is known to be expressed ubiquitously and involved in lipid and glucose metabolism. Recent studies have demonstrated that PPARδ is expressed in endothelial cells (ECs) and plays a potential role in endothelial survival and proliferation. Although PPARα and PPARγ are well recognized to play anti-inflammatory, antiproliferative, and antiangiogenic roles in ECs, the general effect of PPARδ on angiogenesis in ECs remains unclear. Thus, we investigated the effect of the PPARδ ligand L-165041 on vascular EC proliferation and angiogenesis in vitro as well as in vivo. Our data show that L-165041 inhibited VEGF-induced cell proliferation and migration in human umbilical vein ECs (HUVECs). L-165041 also inhibited angiogenesis in the Matrigel plug assay and aortic ring assay. Flow cytometric analysis indicated that L-165041 reduced the number of ECs in the S phase and the expression levels of cell cycle regulatory proteins such as cyclin A, cyclin E, CDK2, and CDK4; phosphorylation of the retinoblastoma protein was suppressed by pretreatment with L-165041. We confirmed whether these antiangiogenic effects of L-165041 were PPARδ-dependent using GW501516 and PPARδ siRNA. GW501516 treatment did not inhibit VEGF-induced angiogenesis, and transfection of PPARδ siRNA did not reverse this antiangiogenic effect of L-165041, suggesting that the antiangiogenic effect of L-165041 on ECs is PPARδ-independent. Together, these data indicate that the PPARδ ligand L-165041 inhibits VEGF-stimulated angiogenesis by suppressing the cell cycle progression independently of PPARδ. This study highlights the therapeutic potential of L-165041 in the treatment of many disorders related to pathological angiogenesis.

HB-EGF induces cardiomyocyte hypertrophy via an ERK5-MEF2A-COX2 signaling pathway.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates the EGF receptor. Transactivated by angiotensin II, ET-1, and various growth factors in cardiomyocytes, HB-EGF is known to induce cardiac hypertrophy via the PI3K-Akt, MAP kinase, and JAK-STAT pathways. However, little is known about the potential involvement of the ERK5 pathway in HB-EGF-induced cardiac hypertrophy. In the present report, we identify and characterize a novel MEK5-ERK5 pathway that is involved in HB-EGF-induced cardiomyocyte hypertrophy. HB-EGF (10ng/ml) significantly increased [(3)H]-leucine incorporation and atrial natriuretic factor (ANF) mRNA expression in H9c2 cells. In addition, HB-EGF activated a MEK5-ERK5 pathway. Pretreatment with the EGFR inhibitor AG1478 attenuated the activation of ERK5. Blockade of MEK5-ERK5 signaling using MEK5 siRNA reduced the ability of HB-EGF to increase cell size and the expression of ANF mRNA, suggesting the involvement of an EGFR-ERK5 pathway in HB-EGF-induced cardiomyocyte hypertrophy. We further analyzed cyclooxygenase-2 (COX-2). HB-EGF enhanced the expression of COX-2, a response mediated by MEK5-ERK5 signaling, while the COX-2 inhibitor rofecoxib attenuated HB-EGF-induced ANF mRNA expression, suggesting that COX-2 is also associated with HB-EGF-induced cardiomyocyte hypertrophy. It has been known that ERK5 activates the myocyte enhancer factor (MEF) 2 family of transcription factor, we next tested whether activation of MEF2A contributes to HB-EGF-induced COX-2 expression. Inhibition of MEF2A using siRNA attenuated HB-EGF-induced COX-2, ANF expression and cell size. In conclusion, HB-EGF induces cardiomyocyte hypertrophy through an EGFR-ERK5-MEF2A-COX-2 pathway. Our findings will help us to better understand the molecular mechanisms behind HB-EGF-induced cardiomyocyte hypertrophy.

PPARdelta ligand L-165041 ameliorates Western diet-induced hepatic lipid accumulation and inflammation in LDLR-/- mice.

Although peroxisome proliferator-activated receptor delta (PPARdelta) has been implicated in energy metabolism and lipid oxidation process, detailed roles of PPARdelta in lipid homeostasis under pathologic conditions still remain controversial. Thus, we investigated the effect of PPARdelta ligand L-165041 on Western diet-induced fatty liver using low-density lipoprotein receptor-deficient (LDLR(-/-)) mice. LDLR(-/-) mice received either L-165041 (5mg/kg/day) or vehicle (0.1N NaOH) with Western diet for 16 weeks. According to our data, L-165041 drastically reduced lipid accumulation in the liver, decreasing total hepatic cholesterol and triglyceride content compared to the vehicle group. Gene expression analysis demonstrated that L-165041 lowered hepatic expression of PPARgamma, apolipoprotein B, interleukin 1 beta (IL-1beta), and interleukin-6. In contrast, L-165041 increased hepatic expressions of PPARdelta, lipoprotein lipase (LPL), and ATP-binding cassette transporter G1 (ABCG1). Our data suggest that L-165041 might be effective in preventing Western diet-induced hepatic steatosis by regulating genes involved in lipid metabolism and the inflammatory response.

PPARdelta activation inhibits angiotensin II induced cardiomyocyte hypertrophy by suppressing intracellular Ca2+ signaling pathway.

Peroxisome proliferator-activated receptors delta (PPARdelta) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARdelta in cardiac cells except that PPARdelta ligand treatment protects cardiac hypertrophy by inhibiting NF-kappaB activation. Thus, in the present study, we examined the effect of selective PPARdelta ligand L-165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L-165041 (10 microM) inhibited AngII-induced [(3)H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L-165041 pretreatment significantly inhibited AngII-induced intracellular Ca(2+) increase and subsequent phosphorylation of FAK. Further experiment using Ca(2+) ionophore A23187 confirmed that Ca(2+) induced FAK phosphorylation, and this was also blocked by L-165041 pretreatment. In addition, overexpression of PPARdelta using adenovirus significantly inhibited AngII-induced intracellular Ca(2+) increase and FAK expression, while PPARdelta siRNA treatment abolished the effect of L-165041. These data indicate that PPARdelta ligand L-165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca(2+)/FAK/ERK signaling pathway in a PPARdelta dependent mechanism.

PPAR delta agonist L-165041 inhibits rat vascular smooth muscle cell proliferation and migration via inhibition of cell cycle.

The peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors consists of three subtypes (alpha, beta/delta, and gamma). PPAR delta is ubiquitously expressed and involved in lipid and glucose metabolism. However, the effect of PPAR delta on vascular smooth muscle cell (VSMC) proliferation and migration has not been fully elucidated yet. Here, we investigated the effect of L-165041, a selective ligand for PPAR delta, on PDGF-induced rat VSMC proliferation. Our data show that L-165041 inhibited rat VSMC proliferation in a dose dependent manner by blocking G(1) to S phase progression and repressing the phosphorylation of retinoblastoma protein (Rb). Furthermore, L-165041 inhibited PDGF-induced expression of cyclin D1 and CDK4. These effects less likely involve PPAR gamma pathway because PPAR gamma antagonist GW9662 pretreatment failed to reverse the inhibitory effect of L-165041 on rVSMC proliferation and migration. For in vivo studies, L-165041 was administered to Sprague-Dawley rats using osmotic pumps before and after the carotid balloon injury, and L-165041 decreased neointima formation after the carotid injury. In conclusion, our results suggest that PPAR delta ligand L-165041 can be a therapeutic agent to control pathologic cardiovascular conditions such as restenosis and atherosclerosis.

Anti-proliferative effect of licochalcone A on vascular smooth muscle cells.

Licochalcone A, a flavonoid found in licorice root (Glycyrrhiza glabra), is known for its anti-microbial activity and its reported ability to inhibit cancer cell proliferation. In the present study, we investigated whether licochalcone A inhibits rat vascular smooth muscle cell (rVSMC) proliferation. Our data indicate that 5 microM licochalcone A inhibited platelet-derived growth factor (PDGF)-induced rVSMC proliferation, possibly through its ability to block the progression of the cell cycle from G1 to S phase. In addition, 5 microM licochalcone A significantly inhibited the PDGF-induced expression of cyclin A, cyclin D1, CDK2, and CDK4, and the phosphorylation of Rb. Licochalcone A also reversed the decrease in p27(kip1) expression reduced by PDGF. Finally, licochalcone A inhibited the PDGF-induced activation of extracellular signal-regulated kinase (ERK)1/2. Together, these data provide the first evidence that licochalcone A can regulate rVSMC proliferation and suggest that licochalcone A inhibits the proliferation of rVSMCs by suppressing the PDGF-induced activation of the ERK1/2 pathway and Rb phosphorylation, resulting in cell cycle arrest.

HB-EGF induces delayed STAT3 activation via NF-kappaB mediated IL-6 secretion in vascular smooth muscle cell.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates EGF receptor, and is expressed in a variety of tissues, predominantly in the lung, heart, brain and skeletal muscle. HB-EGF is known to induce vascular smooth muscle cell (VSMC) proliferation by activating PI3K-Akt and MAPK pathway. However, our preliminary data showed that Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway was also involved in HB-EGF induced VSMC proliferation. More interestingly, HB-EGF (10 ng/ml) induced a biphasic activation of STAT3 (early at 5 min and late at 60-120 min). Therefore, we tried to elucidate the underlying mechanism of this delayed STAT3 activation by HB-EGF in VSMCs. First, we examined the effect of HB-EGF on interleukin-6 (IL-6) mRNA expressions, since IL-6 have been implicated in the regulation of STAT3 activation. According to our data, HB-EGF increased transcription of IL-6, cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). The secretion of IL-6 was also increased by HB-EGF. Furthermore, these HB-EGF-mediated up-regulation of IL-6 mRNA expression and secretion were inhibited by NF-kappaB inhibitor Bay117082 (2.5 microM) treatment suggesting involvement of NF-kappaB pathway. Again, the late activation of STAT3 by HB-EGF was abolished by both Bay117082 and IL-6 neutralizing antibody (1 microg/ml) indicating IL-6 is a key molecule in the delayed activation of STAT3 by HB-EGF. In addition, IL-6 neutralizing antibody inhibited both HB-EGF conditioned media induced STAT3 activation and HB-EGF induced VSMC proliferation. In conclusion, IL-6 plays an important role in the delayed activation of STAT3 and VSMC proliferation induced by HB-EGF.

Berberine-induced LDLR up-regulation involves JNK pathway.

Berberine, an herbal alkaloid, has been reported to have a lipid lowering effect by stabilizing hepatic LDLR mRNA in an ERK-dependent manner rather than promoting transcriptional activity. However, considering the complexity of interconnected signal pathways in biological processes, it is highly possible that there exist signal pathway(s) other than ERK pathway which contribute to the berberine-induced up-regulation of LDLR. In the present study, we examined possible involvement of other signal pathways in berberine-induced hepatic LDLR up-regulation. As evidenced by RT-PCR, berberine-induced LDLR mRNA expression was inhibited by JNK inhibitor SP600125 pretreatment. Furthermore, we demonstrate that putative c-jun binding site of LDLR promoter is important in berberine-induced LDLR transcription using luciferase assay. The result of EMSA also shows that berberine induces c-jun binding to LDLR promoter and this is decreased by SP600125 pretreatment. The present study demonstrates that berberine increases transcriptional activity of LDLR promoter and this involves JNK pathway.

15d-PGJ2 stimulates HO-1 expression through p38 MAP kinase and Nrf-2 pathway in rat vascular smooth muscle cells.

15d-PGJ(2), a potent endogenous ligand for peroxisome proliferators activated receptor-gamma, is a cyclopentenone-type prostaglandin produced by many different types of cells. Pertinent to its effect on vascular smooth muscle cell (VSMC), antiproliferative effects have been most frequently reported. In the present study, we investigated the effect of 15d-PGJ(2) on HO-1 expression that has been reported to inhibit VSMC proliferation. According to our data, 15d-PGJ(2) significantly induced ROS/NO production and HO-1 expression in rVSMCs. We also observed 15d-PGJ(2)-induced translocation of Nrf-2. In addition, ROS scavenger pretreatment suppressed 15d-PGJ(2)-induced HO-1 expression while PPARgamma antagonist did not, suggesting nuclear translocation of Nrf-2 and subsequent HO-1 expression was ROS dependent rather than PPARgamma dependent. Furthermore, an inhibitor of p38 MAPK abolished 15d-PGJ(2)-induced HO-1 expression. These data suggest that 15d-PGJ(2)-induced up-regulation of HO-1 is independent of PPARgamma but dependent of ROS and p38 MAPK pathway. The present study reports for the first time that 15d-PGJ(2) induces HO-1 expression possibly using Nrf-2 pathway as a response to ROS in VSMCs.

PPARgamma activation induces CD36 expression and stimulates foam cell like changes in rVSMCs.

The purpose of the present study was to determine the role of peroxisome proliferator-activated receptor gamma (PPARgamma) activation in smooth muscle cell (SMC) derived form cell formation. Wild and mutant type PPARgamma were delivered by adenovirus then activated with troglitazone. The result of Oil Red O staining and FACS analysis showed that PPARgamma activation induced lipid accumulation in rVSMCs. Furthermore, PPARgamma activation reduced SMC marker genes such as alpha-actin while induced adipocyte differentiation marker genes and lipid metabolism-related genes as evidenced by RT-PCR and fluorescent immunocytochemistry. All these data demonstrate that PPARgamma activation can drive foam cell like change in rVSMCs. Our results strongly suggest that PPARgamma expression induces CD36 expression and adipocyte differentiation gene activation in the process of atherosclerosis and might be one of the crucial events in SMC derived foam cell formation.

Troglitazone inhibits endothelial cell proliferation through suppression of casein kinase 2 activity.

Troglitazone, an agonist of peroxisome proliferator activated receptor gamma (PPARgamma), has been reported to inhibit endothelial cell proliferation by suppressing Akt activation. Recently, it has been also proposed that phosphatase and tensin homolog deleted from chromosome 10 (PTEN) plays an important role in such effect of troglitazone. However, the mechanism of how troglitazone regulates PTEN remains to be elucidated. We therefore investigated the effects of troglitazone on casein kinase 2 (CK2), which is known to negatively regulate PTEN activity. Troglitazone significantly inhibited serum-induced proliferation of HUVEC in a concentration dependent manner. Serum-induced Akt and its downstream signaling pathway activation was attenuated by troglitazone (10 microM) pretreatment. The phosphorylation of PTEN, which was directly related to Akt activation, was decreased with troglitazone pretreatment and was inversely proportional to CK2 activity. DRB, a CK2 inhibitor, also showed effects similar to that of troglitazone on Akt and its downstream signaling molecules. In conclusion, our results suggest that troglitazone inhibits proliferation of HUVECs through suppression of CK2 activity rendering PTEN to remain activated, and this effect of troglitazone in HUVECs seems to be PPARgamma independent.

Berberine inhibits rat vascular smooth muscle cell proliferation and migration in vitro and improves neointima formation after balloon injury in vivo. Berberine improves neointima formation in a rat model.

Berberine, an alkaloid isolated from Chinese medicinal herbs, long been known for its anti-microbial activity and used to treat various infectious disorders in traditional Chinese medicine. In the present study, we have tested the hypothesis that berberine could inhibit vascular smooth muscle cell (VSMC) proliferation as it did in endothelial cells or cancer cells. Our results show that berberine significantly inhibits growth factor, mainly angiotensin II (AngII) and heparin binding epidermal growth factor (HB-EGF), induced VSMC proliferation and migration in vitro, and this effect is achieved by delaying or partially suppressing activation of Akt pathway rather than ERK pathway. Furthermore, we have examined its effect in vivo using a rat carotid artery injury model. A 28 days of chronic berberine treatment using an osmotic pump (100 microg kg(-1)d(-1), 2 weeks before and 2 weeks after the injury) improved neointima formation. The Neointima/Media ratio for control group and berberine treated group were 1.14+/-0.11 and 0.85+/-0.06 (p<0.05), respectively, and the reduction was approximately 25%. The result of the present study suggests a possibility of berberine being a potent agent to control restenosis after balloon angioplasty and warrants further study to gain a more complete understanding of its underlying mechanisms at a cellular level.