Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2.

BACKGROUND/AIMS: Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. METHODS: A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-ß1 and TGFß type I receptor (TGFßRI), and protein expression was assessed by immunoblotting and immunofluorescence. RESULTS: Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-ß1 expression, and silencing of TGF-ß1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFßRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. CONCLUSION: Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

HMGB1 increases RAGE expression in vascular smooth muscle cells via ERK and p-38 MAPK-dependent pathways.

The increased expression of receptors for advanced glycation end-product (RAGE) is known as a key player in the progression of vascular remodeling. However, the precise signal pathways regulating RAGE expression in vascular smooth muscle cells (VSMCs) in the injured vasculatures are unclear. Given the importance of mitogen-activated protein kinase (MAPK) signaling in cell proliferation, we investigated the importance of MAPK signaling in high-mobility group box 1 (HMGB1)-induced RAGE expression in VSMCs. In HMGB1 (100 ng/ml)-stimulated human VSMCs, the expression of RAGE mRNA and protein was increased in association with an increase in AGE-induced VSMC proliferation. The HMGB1-induced RAGE expression was attenuated in cells pretreated with inhibitors for ERK (PD98059, 10 µM) and p38 MAPK (SB203580, 10 µM) as well as in cells deficient in ERK and p38 MAPK using siRNAs, but not in cells deficient of JNK signaling. In cells stimulated with HMGB1, the phosphorylation of ERK, JNK, and p38 MAPK was increased. This increase in ERK and p38 MAPK phosphorylation was inhibited by p38 MAPK and ERK inhibitors, respectively, but not by JNK inhibitor. Moreover, AGE-induced VSMC proliferation in HMGB1-stimulated cells was attenuated in cells treated with ERK and p38 MAPK inhibitors. Taken together, our results indicate that ERK and p38 MAPK signaling are involved in RAGE expression in HMGB1-stimulated VSMCs. Thus, the ERK/p38 MAPK-RAGE signaling axis in VSMCs was suggested as a potential therapeutic target for vascular remodeling in the injured vasculatures.

Combination therapy with cilostazol, aripiprazole, and donepezil protects neuronal cells from ß-amyloid neurotoxicity through synergistically enhanced SIRT1 expression.

Alzheimer's disease (AD) is a multi-faceted neurodegenerative disease. Thus, current therapeutic strategies require multitarget-drug combinations to treat or prevent the disease. At the present time, single drugs have proven to be inadequate in terms of addressing the multifactorial pathology of AD, and multitarget-directed drug design has not been successful. Based on these points of views, it is judged that combinatorial drug therapies that target several pathogenic factors may offer more attractive therapeutic options. Thus, we explored that the combination therapy with lower doses of cilostazol and aripiprazole with add-on donepezil (CAD) might have potential in the pathogenesis of AD. In the present study, we found the superior efficacies of donepezil add-on with combinatorial mixture of cilostazol plus aripiprazole in modulation of expression of AD-relevant genes: Aß accumulation, GSK-3ß, P300, acetylated tau, phosphorylated-tau levels, and activation of α-secretase/ADAM 10 through SIRT1 activation in the N2a Swe cells expressing human APP Swedish mutation (N2a Swe cells). We also assessed that CAD synergistically raised acetylcholine release and choline acetyltransferase (CHAT) expression that were declined by increased ß-amyloid level in the activated N2a Swe cells. Consequently, CAD treatment synergistically increased neurite elongation and improved cell viability through activations of PI3K, BDNF, ß-catenin and a7-nicotinic cholinergic receptors in neuronal cells in the presence of Aß1-42. This work endorses the possibility for efficient treatment of AD by supporting the synergistic therapeutic potential of donepezil add-on therapy in combination with lower doses of cilostazol and aripiprazole.

Cilostazol add-on therapy for celecoxib synergistically inhibits proinflammatory cytokines by activating IL-10 and SOCS3 in the synovial fibroblasts of patients with rheumatoid arthritis.

Cilostazol (an inhibitor of phosphodiesterase type III) has potent anti-inflammatory effects, and celecoxib (a COX-2 specific inhibitor) has been reported to improve the unsatisfactory profile of NSAIDs. This study investigated the synergistic anti-arthritic potential of a multitarget-based cotreatment, in which cilostazol was used as an add-on therapy for celecoxib, using the synovial fibroblasts of RA patients (RASFs). Increased COX-2 protein expression and PGE2 synthesis by LPS (1 µg/ml) were significantly and synergistically attenuated by cotreatment with 3 µM cilostazol and 30 µM celecoxib, whereas monotherapy with either cilostazol or celecoxib showed little effects. IL-10 mRNA levels in LPS-treated RASFs were moderately increased by pretreating cilostazol (1-10 µM) or celecoxib (10-50 µM) monotherapy, but 3 µM of cilostazol add-on for 30 µM celecoxib treatment synergistically increased IL-10 mRNA levels and IL-10 release to culture media. Cilostazol and celecoxib cotreatment similarly showed synergistic increase in SOCS3 mRNA levels. Accordingly, LPS-induced increases in IL-1ß and IL-6 mRNA and TNF-α release were significantly and synergistically diminished by cilostazol and celecoxib cotreatment. Moreover, synovial cell proliferation was significantly suppressed by cotreatment. Summarizing, cotreatment with cilostazol and celecoxib exhibited a synergistic increase in IL-10 production and SOCS3 expressions, thereby resulted in synergistic decreases in IL-1ß mRNA, IL-6 mRNA expression and TNF-α synthesis in association with synergistic decreases in COX-2 and PGE2 protein expression in the RA synovial fibroblasts. In conclusion, these observations suggest low concentrations of cilostazol and celecoxib cotreatment may ensure a synergistic anti-arthritic potential.

7-Oxygenated cholesterol molecules differentially affect the expression of zonula occludens-1 in vascular smooth muscle cells and monocyte/macrophage cells.

To investigate the effects of 7-oxygenated cholesterol molecules on the expression of tight junction proteins, we examined the outcomes effects of 7-ketocholesterol (7K), 7α-hydroxycholesterol (7αOHChol) and 7ß-hydroxycholesterol (7ßOHChol) on the expression of the tight-junction protein zonula occludens-1 (ZO-1) using vascular cells. Vascular smooth muscle cells (VSMCs) constitutively express ZO-1, and this expression remained unaffected in the presence of cholesterol. However, the level of ZO-1 protein decreased after exposure to 7K and, to a lesser extent, 7αOHChol and 7ßOHChol. ZO-1 was translocated to the nucleus following treatment with 7K; this translocation was inhibited by z-VAD-fmk, a pan-caspase inhibitor. ZO-1 protein was found to disintegrate in the aorta of ApoE knockout mice fed a high cholesterol diet, whereas it remained intact in the wild-type control. THP-1 monocyte/macrophage cells, which show no expression of ZO-1, were not influenced by treatment with cholesterol, 7K, and 7ßOHChol. However, the treatment of THP-1 cells with 7αOHChol resulted in ZO-1 expression, which largely remained localized on the cytoplasmic membrane. These results indicate the varying effects of 7-oxygenated cholesterol molecules on the expression and localization of ZO-1 depending on cell types, and suggest the contribution of 7-oxygeneted cholesterol molecules to the structural alteration of tight junctions.

SURF4 has oncogenic potential in NIH3T3 cells.

SURF4, which is located in the Surfeit gene cluster, encodes for a conserved integral membrane protein containing multiple putative transmembrane regions. However, the physiological role of SURF4 has not been determined. We found that SURF4 demonstrated aberrant amplification and increased expression in the tumor tissues of several human cancer patients. Overexpression of SURF4 led to increased cell proliferation, migration, and maintenance of anchorage-independent growth. In addition, NIH3T3 cells overexpressing SURF4 induced tumor growth in the mice. Collectively, our findings demonstrate that SURF4 has the potential for inducing cellular transformation and cell migration in vitro and has oncogenic transformation ability in vivo.

Caffeic acid phenethyl ester protects against photothrombotic cortical ischemic injury in mice.

In this study, we aimed to investigate the neuroprotective effects of caffeic acid phenethyl ester (CAPE), an active component of propolis purified from honeybee hives, on photothrombotic cortical ischemic injury in mice. Permanent focal ischemia was achieved in the medial frontal and somatosensory cortices of anesthetized male C57BL/6 mice by irradiation of the skull with cold light laser in combination with systemic administration of rose bengal. The animals were treated with CAPE (0.5-5 mg/kg, i.p.) twice 1 and 6 h after ischemic insult. CAPE significantly reduced the infarct size as well as the expression of tumor necrosis factor-α, hypoxiainducible factor-1α, monocyte chemoattractant protein-1, interleukin-1α, and indoleamine 2,3-dioxygenase in the cerebral cortex ipsilateral to the photothrombosis. Moreover, it induced an increase in heme oxygenase-1 immunoreactivity and interleukin-10 expression. These results suggest that CAPE exerts a remarkable neuroprotective effect on ischemic brain injury via its anti-inflammatory properties, thereby providing a benefit to the therapy of cerebral infarction.

α-Isocubebene modulates vascular tone by inhibiting myosin light chain phosphorylation in murine thoracic aorta.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms, but the mechanism responsible for this activity has not been determined. To determine the role played by ICB on the regulation of vascular tone, we investigated the inhibitory effects of ICB on vascular contractile responses by adrenergic α-receptor agonists. In addition, we investigated the role on myosin light chain (MLC) phosphorylation and cytosolic calcium concentration in vascular smooth muscle cells (VSMC). In aortic rings isolated from C57BL/6J mice, ICB significantly attenuated the contraction induced by phenylephrine (PE) and norepinephrine (NE), whereas ICB had no effects on KCl (60 mM)-induced contraction. In vasculatures precontracted with PE, ICB caused marked relaxation of aortic rings with or without endothelium, suggesting a direct effect on VSMC. In cultured rat VSMC, PE or NE increased MLC phosphorylation and increased cytosolic calcium levels. Both of these effects were significantly suppressed by ICB. In conclusion, our results showed that ICB regulated vascular tone by inhibiting MLC phosphorylation and calcium flux into VSMC, and suggest that ICB has anti-hypertensive properties and therapeutic potential for cardiovascular disorders related to vascular hypertension.

5-Lipoxygenase in monocytes emerges as a therapeutic target for intimal hyperplasia in a murine wire-injured femoral artery.

Given the importance of leukotrienes in vascular inflammation induced by local tissue injury, this study investigated the role for 5-lipoxygenase (5-LO) in monocytes in the development of intimal hyperplasia. As a mechanistic study, the importance of monocyte 5-LO in monocyte-macrophage differentiation with subsequent infiltration in neointima was evaluated. In a mouse model of wire-injured femoral artery, intimal hyperplasia started as early as 2wks after injury, and luminal area and blood flow were reduced due to increased neointima formation. Time-dependent increases in macrophage infiltration were observed in neointima and showed a positive relationship with neointima volume. In 5-LO-deficient (KO) mice or wild-type (WT) mice treated with an inhibitor of 5-LO activating protein (MK886, 1 and 10mg/kg), intimal hyperplasia and macrophage infiltration into neointima were reduced, but monocyte adhesion to injured luminal surface was not inhibited, which suggested 5-LO participates in monocyte-macrophage differentiation. In an in vitro study, monocyte-macrophage differentiation was found to be increased by high mobility group box 1 protein (HMGB1), but this effect was attenuated in cells isolated from 5-LO-KO mice. Furthermore, macrophage infiltration and intimal hyperplasia were more prominent in 5-LO-KO mice transplanted with monocytes from WT mice than in 5-LO-KO mice transplanted with monocytes from 5-LO-KO mice. Taken together, it was suggested that 5-LO in monocytes played a pivotal role in monocyte-macrophage differentiation and subsequent infiltration of macrophage in neointima, leading to vascular remodeling after vascular injury.

27-Hydroxycholesterol up-regulates CD14 and predisposes monocytic cells to superproduction of CCL2 in response to lipopolysaccharide.

We investigated the possibility that a cholesterol-rich milieu can accelerate response to pathogen-associated molecular patterns in order to elucidate mechanisms underlying aggravation of atherosclerosis after bacterial infection. The consumption of a high-cholesterol diet resulted in enhanced the expression of CD14 in arteries of ApoE(-/-) mice. 27-Hydroxycholesterol (27OHChol), the most abundant cholesterol oxide in atherosclerotic lesions, induced the significant expression of CD14 by THP-1 monocytic cells, but not by vascular smooth muscle cells or Jurkat T cells. Additions of lipopolysaccharide (LPS) to 27OHChol-treated THP-1 monocytic cells resulted in superinduction in terms of the gene transcription of CCL2 and the secretion of its gene product. In contrast, cholesterol did not cause increased the expression of CD14 in the aforementioned cells, and the addition of LPS to cholesterol-treated monocytic cells did not result in enhanced the expression of CCL2. The conditioned medium isolated from THP-1 cells exposed to 27OHChol plus LPS further induced the migration of monocytic cells in comparison with conditioned media obtained from THP-1 cells treated with 27OHChol or LPS alone. Treatment with 27OHChol also resulted in the enhanced secretion of MMP-9 and soluble CD14 (sCD14), and the secretion of sCD14 was blocked by a selective MMP-9 inhibitor. The inhibition of the ERK pathway resulted in significantly attenuated the secretion of sCD14 via mechanisms that were distinct from those by PI3K inhibition. We propose that 27OHChol can prime monocytes/macrophages by up-regulation of CD14 such that LPS-mediated inflammatory reaction is accelerated, thereby contributing to aggravated development of atherosclerotic lesions by enhancing recruitment of monocytic cells after infection with Gram-negative bacteria.

Suppression of RANKL-induced osteoclast differentiation by cilostazol via SIRT1-induced RANK inhibition.

Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage hematopoietic lineages and degrade bone matrix by secretion of lytic enzymes. The regulation of osteoclast differentiation provides a potential strategy for treatment of bone-lytic damage. In this study, cilostazol, an inhibitor of type III phosphodiesterase, inhibited RANKL [receptor activator of nuclear factor kappa B (RANK) ligand]-induced RANK expression in bone marrow-derived monocyte/macrophage precursors (BMMs) and Raw 264.7 cells by inhibiting PU.1 via SIRT1 activation. RANKL-induced RANK expression was attenuated by cilostazol and rSIRT1 in Raw 264.7 cells, and these were blocked by sirtinol. In line with these, cilostazol elevated SIRT1 mRNA and protein levels in 12-24h and increased SIRT1 activity, and these effects were inhibited by sirtinol. Furthermore, the RANKL-induced nuclear expression of PU.1, a transcription factor required for macrophage differentiation, was suppressed by cilostazol. Additionally, marked RANKL-induced RANK immunofluorescence staining in Raw 264.7 cells was attenuated by cilostazol and rSIRT1, and both attenuations were prevented by sirtinol. Extensive RANK staining of knee synovial tissues in a mouse model of collagen-induced arthritis (CIA) was markedly reduced by cilostazol (30mg/kg/day). In line with these results, both RANKL- and M-CSF-induced differentiation of BMMs to multinucleated TRAP(+) giant cells and resorption pit formation were inhibited by cilostazol associated with a decrease in TRAP (a marker enzyme of osteoclasts) activity. In conclusion, cilostazol activates SIRT1, which suppresses the nuclear translocation of PU.1, and thus, inhibits RANKL-stimulated RANK expression and causes anti-osteoclast formation in BMMs in vitro and in their murine model of CIA.

HMGB1 induces angiogenesis in rheumatoid arthritis via HIF-1α activation.

High-mobility group box protein 1 (HMGB1), a nonhistone nuclear protein and a cytokine mediator, is implicated in the pathogenesis of rheumatoid arthritis (RA). Extracellular HMGB1 binds to its receptors and triggers downstream signal cascade leading to the perpetuation of synovitis and local tissue invasion. Here, we investigated a novel role of HMGB1 in regulating hypoxia-inducible factor (HIF)-1α to mediate angiogenesis in RA synovium. HIF-1α mRNA levels and activities in synovial fibroblasts from RA patients were enhanced by HMGB1. Pharmacological inhibition of TLR4 and NF-kappaB activation blocked the HMGB1-dependent upregulation of HIF-1α mRNA expression and its activity, suggesting the involvement of transcriptional regulation. HMGB1 stimulated expression of vascular endothelial growth factor (VEGF), and inhibition of HIF-1α attenuated HMGB1-induced VEGF. Conditioned media derived from HMGB1-stimulated synovial fibroblasts enhanced tube formation in human microvascular endothelial cells by upregulating HIF-1α. In the joint tissues of mice with collagen-induced arthritis, treatment with anti-HMGB1 neutralizing antibody prevented blood vessel formation in association with decreased expression of HIF-1α. These observations support the idea that increased HMGB1 induces an extension of inflamed synovium by accelerating angiogenesis in RA through enhancement of HIF-1α activation. Therefore, inhibition of HMGB1 could prove beneficial for the treatment of angiogenesis in RA.

Osteopontin plays a key role in vascular smooth muscle cell proliferation via EGFR-mediated activation of AP-1 and C/EBPß pathways.

Osteopontin (OPN) is known as an active player in the progression of vascular remodeling diseases, however, the precise role in the proliferation of vascular smooth muscle cells (VSMC) is unclear. Thus, this study investigated the role of OPN in VSMC proliferation induced by 4-hydroxynonenal (HNE), and identified the intracellular signaling pathways involved in 4-HNE-induced OPN production. In VSMC primary cultured from rat thoracic aorta as well as in VSMC in the media of aorta, HNE enhanced OPN expression in concentration-dependent manners. Both the proliferation of cultured VSMC and PCNA positive cells in aortic tissues were also increased by HNE, which were attenuated in OPN-deficient cells and aortic tissues isolated from OPN-deficient mice, indicating a pivotal role of OPN in HNE-induced VSMC proliferation. In the promoter assay, HNE increased OPN promoter activity, which was attenuated when the regions harboring AP-1 and C/EBPß binding sites were mutated. The increased bindings of AP-1 and C/EBPß to the OPN promoter were also demonstrated by ChIP analysis. In addition, the increases in both OPN expression and the activities of AP-1 and C/EBPß by HNE were attenuated by AG1478, an EGFR antagonist. Based on these results, it was suggested that HNE induced OPN expression in VSMC via signaling pathways involving AP-1 and C/EBPß, leading to increases in VSMC proliferation and subsequent vascular remodeling.

SIRT1 prevents pulmonary thrombus formation induced by arachidonic acid via downregulation of PAF receptor expression in platelets.

SIRT1, a class III histone deacetylase, is critically involved in cellular response to stress and modulates cardiovascular risk factors. However, its role in thrombus formation is largely unknown. Thus, this study investigated the effect of SIRT1 on pulmonary thrombus formation, and then identified its role in the modulation of platelet aggregation. In isolated human platelets, cell aggregation was increased by various platelet activators, such as platelet activating factor (PAF), arachidonic acid (AA), ADP, and thrombin. AA- and PAF-mediated platelet aggregations were suppressed by WEB2086, a PAF receptor (PAFR) antagonist. Pulmonary thrombus formation induced by PAF or AA was also attenuated by WEB2086, suggesting that PAFR plays a key role in AA-induced platelet aggregation. In platelets isolated from SIRT1-TG mice as well as in platelets treated with resveratrol or reSIRT1, PAFR expression was decreased, whereas this expressional downregulation by SIRT1 activators was inhibited in platelets treated with MG132 (a proteasome inhibitor) or NH4Cl (a lysosome inhibitor). Furthermore, platelet aggregation induced by AA was markedly attenuated by resveratrol and reSIRT1. Likewise, the increased pulmonary thrombus formation in mice treated with AA was also attenuated by SIRT1 activators. In line with these results, pulmonary thrombus formation was markedly attenuated in SIRT1-TG mice. Taken together, this study showed that SIRT1 downregulates PAFR expression on platelets via proteasomal and lysosomal pathways, and that this downregulation inhibits platelet aggregation in vitro and pulmonary thrombus formation in vivo.

Regulation of retinal angiogenesis by endothelial nitric oxide synthase signaling pathway.

Angiogenesis plays an essential role in embryo development, tissue repair, inflammatory diseases, and tumor growth. In the present study, we showed that endothelial nitric oxide synthase (eNOS) regulates retinal angiogenesis. Mice that lack eNOS showed growth retardation, and retinal vessel development was significantly delayed. In addition, the number of tip cells and filopodia length were significantly reduced in mice lacking eNOS. Retinal endothelial cell proliferation was significantly blocked in mice lacking eNOS, and EMG-2-induced endothelial cell sprouting was significantly reduced in aortic vessels isolated from eNOS-deficient mice. Finally, pericyte recruitment to endothelial cells and vascular smooth muscle cell coverage to blood vessels were attenuated in mice lacking eNOS. Taken together, we suggest that the endothelial cell function and blood vessel maturation are regulated by eNOS during retinal angiogenesis.

Mechanical stretch enhances the expression and activity of osteopontin and MMP-2 via the Akt1/AP-1 pathways in VSMC.

Hemodynamic forces causing mechanical stretch (MS) of vascular smooth muscle cells (VSMC) play an important role in vascular remodeling, but the underlying mechanism involved is not fully understood. Thus, this study investigated whether osteopontin (OPN) expression in VSMC was induced by MS, and identified the intracellular signaling pathways involved in OPN production. The plasma level of OPN and its expression in aortic tissue were increased in various animal models of hypertension including spontaneous hypertensive rats and hypertensive mice induced by angiotensin II or L-NAME. When aortic VSMC was stimulated with MS, OPN production was increased, which was markedly attenuated in VSMC treated with PI3K/Akt inhibitor as well as in Akt1-depleted cells, but not in Akt2-depleted cells, suggesting a pivotal role of Akt1 isoform in OPN expression in VSMC. In the promoter assay, MS increased OPN promoter activity, which was attenuated when the region harboring AP-1 binding sites was mutated. The MS-enhanced promoter activity and OPN expression were also decreased in cells treated with AP-1 siRNA or inhibitor. Moreover, MS-induced MMP-2 production was attenuated in cells treated with OPN siRNA or anti-OPN antibody as well as in OPN-deficient VSMC cultured from aorta of OPN deficient mice. In in vivo experiments, the expressions of OPN and MMP-2 were increased in the aortic tissues from hypertensive mice, but these increases were markedly attenuated in OPN-deficient mice with hypertension. In conclusion, these results suggested that OPN expression in the hypertensive vasculature was increased via signaling pathways that involve Akt1/AP-1, leading to vascular remodeling by increasing the production of MMP-2.

Insulin regulates monocyte trans-endothelial migration through surface expression of macrophage-1 antigen.

During the pathogenesis of atherosclerosis, adhesion of monocytes to vascular endothelium and subsequent migration across the endothelium has been recognized as a key process in the chronic inflammatory response in atherosclerosis. As type 2 diabetes is closely associated with the pathogenesis of atherosclerosis, we investigated whether monocyte adhesion and migration were affected by insulin. We found that insulin activated Akt and induced subsequent migration in THP-1. However, glucose and insulin-like growth factor-1, which is a growth factor that is structurally similar to insulin, were not effective. Insulin-dependent migration of THP-1 was blocked by inhibition of PI3K or Akt and by silencing of Akt1. Insulin-dependent migration of bone marrow-derived monocytic cells (BDMCs) was attenuated by inhibition of PI3K and Akt. In addition, BDMCs from Akt1(-/-) mice showed defects in insulin-dependent migration. Stimulation of THP-1 with insulin caused adhesion with human vein endothelial cells (HUVECs) that was blocked by silencing of Akt1. However, stimulation of HUVECs did not cause adhesion with THP-1. Moreover, BDMCs from Akt1(-/-) mice showed defects in insulin-dependent adhesion with HUVECs. Insulin induced surface expression of Mac-1, and neutralization of Mac-1 blocked insulin-induced adhesion of THP-1 as well as BDMCs. Surface expression of Mac-1 was blocked in THP-1 with silenced Akt1, and in BDMCs isolated from mice lacking Akt1. Finally, trans-endothelial migration of THP-1 and BDMCs was blocked by Mac-1-neutralizing antibody, in THP-1 with silenced Akt1 and in BDMCs from Akt1(-/-) mice. These results suggest that insulin stimulates monocyte trans-endothelial migration through Akt-dependent surface expression of Mac-1, which may be part of the atherogenesis in type 2 diabetes.

Akt1 isoform modulates phenotypic conversion of vascular smooth muscle cells.

In this study, we investigated the role of Akt1 isoform in phenotypic change of vascular smooth muscle cells (VSMCs) and neointima formation. Laminin-induced conversion of synthetic VSMCs into contractile VSMCs was measured by expression of marker proteins for contractile VSMCs and collagen gel contraction assay. Culture of synthetic VSMCs on laminin-coated plates induced expression of marker proteins for contractile VSMCs and showed contraction in response to angiotensin II (AngII) stimulation. Silencing integrin-linked kinase attenuated activation of Akt and blocked phenotypic conversion of VSMCs resulting in the loss of AngII-dependent contraction. Laminin-induced phenotypic conversion of VSMCs was abrogated by phosphatidylinositol 3-kinase inhibitor or in cells silencing Akt1 but not Akt2. Proliferation of contractile VSMCs on laminin-coated plate was enhanced in cells silencing Akt1 whereas silencing Akt2 did not affect. Promoter activity of myocardin and SM22α was enhanced in contractile phenotype and overexpression of myocardin stimulated promoter activity of SM22α in synthetic phenotype. Promoter activity of myocardin and SM22α was reduced in cells silencing Akt1 and promoter activity of SM22α was restored by overexpression of myocardin in cells silencing Akt1. However, silencing of Akt2 affected neither promoter activity of myocardin nor SM22α. Finally, neointima formation in carotid artery ligation and high fat-diet-induced atherosclerosis was facilitated in mice lacking Akt1. This study demonstrates that Akt1 isoform stimulates laminin-induced phenotypic conversion of synthetic VSMCs by regulating the expression of myocardin. VSMCs become susceptible to shifting from contractile to synthetic phenotype by the loss of Akt1 in pathological conditions.

WKYMVm-induced activation of formyl peptide receptor 2 stimulates ischemic neovasculogenesis by promoting homing of endothelial colony-forming cells.

Endothelial colony-forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G-protein-coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA-mediated silencing of FPR2, but not FPR3, abrogated WKYMVm-induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC-mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm-induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2-dependent mechanism.

Resistin derived from diabetic perivascular adipose tissue up-regulates vascular expression of osteopontin via the AP-1 signalling pathway.

Perivascular adipose tissue (PVAT) is implicated in the development of vascular diseases; however, the roles of PVAT on OPN expression in diabetic vasculature remain to be determined. This study investigated the role of adipokines derived from diabetic PVAT in regulating the vascular expression of OPN and explored the mechanisms involved. Aortic sections of ob/ob and high-fat diet (HFD)-induced obese (DIO) mice showed an increased expression of OPN, which was paralleled by increased amounts of PVAT characterized by enlargement of adipocytes. In the earlier phase of HFD feeding, macrophage infiltration was mainly localized to the area of PVAT at which adipocytes were enlarged, suggesting a potential link of activated adipocytes to macrophage infiltration. PVAT sections of ob/ob and DIO mice revealed a significantly greater number of macrophages with increased expression of adipokines, including resistin and visfatin. The distribution of resistin in PVAT mostly co-localized with macrophages, while visfatin was expressed in macrophages and other cells. In in vitro studies, OPN expression in vascular smooth muscle cells (VSMCs) co-cultured with PVAT of DIO mice was significantly increased, which was attenuated by a resistin-neutralizing antibody. Likewise, resistin up-regulated expression of OPN mRNA and protein in cultured VSMCs and the pivotal role of AP-1 in resistin-induced OPN transcription was demonstrated. Resistin produced by PVAT plays a pivotal role in the up-regulated expression of OPN in the diabetic vasculature via a signalling pathway that involves activation of AP-1.