The Presence of TGFß3 in Human Ovarian Intrafollicular Fluid and Its Involvement in Thromboxane Generation in Follicular Granulosa Cells through a Canonical TGFßRI, Smad2/3 Signaling Pathway and COX-2 Induction.

Ovarian follicular fluid (FF) has a direct impact on oocyte quality, playing key roles in fertilization, implantation, and early embryo development. In our recent study, we found FF thromboxane (TX) to be a novel factor inversely correlated with oocyte maturation and identified thrombin, transforming growth factor ß (TGFß), TNF-α, and follicular granulosa cells (GCs) as possible contributors to FF TX production. Therefore, this study sought to investigate the role of TGFß3 in regulating TX generation in human ovarian follicular GCs. TGFß3 was differentially and significantly present in the FF of large and small follicles obtained from IVF patients with average concentrations of 68.58 ± 12.38 and 112.55 ± 14.82 pg/mL, respectively, and its levels were correlated with oocyte maturity. In an in vitro study, TGFß3 induced TX generation/secretion and the converting enzyme-COX-2 protein/mRNA expression both in human HO23 and primary cultured ovarian follicular GCs. While TGFßRI and Smad2/3 signaling was mainly required for COX-2 induction, ERK1/2 appeared to regulate TX secretion. The participation of Smad2/3 and COX-2 in TGFß3-induced TX generation/secretion could be further supported by the observations that Smad2/3 phosphorylation and nuclear translocation and siRNA knockdown of COX-2 expression compromised TX secretion in GCs challenged with TGFß3. Taken together, the results presented here first demonstrated that FF TGFß3 levels differ significantly in IVF patients' large preovulatory and small mid-antral follicles and are positively associated with oocyte maturation. TGFß3 can provoke TX generation by induction of COX-2 mRNA/protein via a TGFßR-related canonical Smad2/3 signaling pathway, and TX secretion possibly by ERK1/2. These imply that TGFß3 is one of the inducers for yielding FF TX in vivo, which may play a role in folliculogenesis and oocyte maturation.

Novel involvement of PLD-PKCδ-CREB axis in regulating FGF-2-mediated pentraxin 3 production in human nasal fibroblast cells.

A higher expression level of mitogenic fibroblast growth factor-2 (FGF-2) has been reported in human nasal mucus of both chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). Meanwhile, we have shown that long pentraxin 3 (PTX3), an essential component of humoral innate immunity that is produced at sites of infection and inflammation, was overproduced in human nasal mucosae and secretions of CRSsNP. Therefore, this study was aimed to investigate how FGF-2 regulates PTX3 expression in human CRSsNP nasal mucosa-derived fibroblast cells (hNMDFs). The FGF-2 treatment caused ptx3 mRNA expression and PTX3 protein induction and secretion. In parallel, a differential expression of FGF receptor (FGFR)-1 to FGFR-4 was observed in hNMDFs and human nasal tissues. While conventionally known PI3K/Akt/mTOR and AP-1 pathways following FGFR activation were shown to be involved, the protein kinase Cδ (PKCδ) and cAMP response element-binding protein (CREB) were also found to be as critical signaling molecules in FGF-2-induced PTX3 induction. The PKCδ and CREB activation could be detected in total cells and in the cell nucleus. Accordingly, a novel CREB binding sequence was detected in the human ptx3 promoter region and could interact with activated CREB in cells challenged with FGF-2. Surprisingly, the phospholipase D (PLD), but not phosphoinositide- and phosphatidylcholine-phospholipase C, was necessarily required for PKCδ and CREB activation. Therefore, we demonstrated here for the first time that FGF-2 mediates PTX3 production not only through PI-3K/Akt/mTOR and AP-1 activation, but also through a novel FGFR-PLD-PKCδ-CREB cellular signaling pathway.

Expression of long pentraxin 3 in human nasal mucosa fibroblasts, tissues, and secretions of chronic rhinosinusitis without nasal polyps.

Numerous studies have shown that microbiomes play an important role in the pathogenesis of chronic rhinosinusitis (CRS). In addition to a known short pentraxin, C-reactive protein, long pentraxin 3 (PTX3) belongs to pentraxin family which detects conserved microbial pentraxin motifs and mobilizes early defense against foreign invaders, but its participation in CRS remains unclear. In the present study, through an intensive screening, peptidoglycan (PGN) was selected as a main material to investigate the action mechanism of a cell wall component on CRS without nasal polyps (CRSsNP) nasal mucosa-derived fibroblasts and the PTX3 expression in human nasal mucosa tissue and discharge. The PGN not only enhanced PTX3 mRNA and protein production in cells but also caused marked PTX3 secretion into extracellular space. The pharmacological interventions indicated that the PTX3 induction was mediated mainly through toll-like receptor 2 (TLR2), phosphoinositide-phospholipase C (PI-PLC), protein kinase C (PKC), NF-κB, and cAMP response element binding protein (CREB), which was further confirmed by the observations that a direct PKC activator (phorbol ester) had a similar inductory effect on PTX3 expression/production and the siRNA interference knockdown of PKCµ/δ, NF-κB, and CREB compromised PTX3 production. Meanwhile, PTX3 was found to be overexpressed/produced in nasal mucosa and discharge/secretion of the CRSsNP patients. Collectively, we first demonstrated here that PGN enhances PTX3 expression and release in nasal fibroblasts through TLR2, PI-PLC, PKCµ/δ, NF-κB, and CREB signaling pathways. The PTX3 is overexpressed in nasal mucosa and discharge/secretion of CRSsNP patients, revealing its possible importance in CRSsNP development and progression. KEY MESSAGES: Long pentraxin 3 (PTX3) is highly expressed in nasal mucosa and discharge/secretion of patients of chronic rhinosinusitis without nasal polyps (CRSsNP). The bacteria cell wall component-peptidoglycan (PGN) causes PTX3 expression in CRSsNP nasal mucosa-derived fibroblasts, contributing to the PTX3 increase in tissues. PGN induces PTX3 expression through a previously known IκB/NF-κB and a novel PKCµ/δ and CREB signaling pathway. The PTX3 may be used as a biomarker for CRS.

Thrombin induces cyclooxygenase-2 expression and prostaglandin E2 release via PAR1 activation and ERK1/2- and p38 MAPK-dependent pathway in murine macrophages.

Thrombin levels increase at sites of vascular injury and during acute coronary syndromes. It is also increased several fold by sepsis with a reciprocal decrease in the anti-thrombin III levels. In this study we investigate the effects of thrombin on the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in macrophages. Thrombin-induced COX-2 protein and mRNA expression in RAW264.7 and primary cultured peritoneal macrophages. A serine proteinase, trypsin, also exerted a similar effect. The inducing effect by thrombin in macrophages was not affected by a lipopolysaccharide (LPS)-binding antibiotic, polymyxin B, excluding the possibility of LPS contamination. The increase of COX-2 expression by thrombin was functionally linked to release of PGE(2) and PGI(2) but not thromboxane A(2) into macrophage culture medium. Thrombin-induced COX-2 expression and PGE(2) production were significantly attenuated by PD98059 and SB202190 but not by SP600125, suggesting that ERK1/2 and p38 MAPK activation were involved in this process. This was supported by the observation that thrombin could directly activate ERK1/2 and p38 MAPK in macrophages. A further analysis indicated that the proteinase-activated receptor 1 (PAR1)-activating agonist induced effects similar to those induced by thrombin in macrophages and the PAR1 antagonist-SCH79797 could attenuate thrombin-induced COX-2 expression and PGE(2) release. Taken together, we provided evidence demonstrating that thrombin can induce COX-2 mRNA and protein expression and PGE(2) production in macrophages through PAR1 activation and ERK1/2 and p38 MAPK-dependent pathway. The results presented here may explain, at least in part, the possible contribution of thrombin and macrophages in these pathological conditions.

Litebamine, a phenanthrene alkaloid from the wood of Litsea cubeba, inhibits rat smooth muscle cell adhesion and migration on collagen.

Smooth muscle cells (SMCs) play an important role in the development of atherosclerosis and restenosis after angioplasty and coronary bypass grafting. The pathogenesis of these vascular diseases includes the abnormal production of extracellular matrix (ECM) proteins by SMCs and their interactions with this newly synthesized and preexisting ECM. Litebamine, a natural phenanthrene alkaloid from the wood of Litsea cubeba, has been shown to inhibit platelet aggregation and thromboxane B(2) formation, suggesting its antithrombotic activity. In the present study we examined litebamine effects on vascular SMC adhesion and migration. Our results indicated that litebamine inhibited rat aortic SMCs (RASMCs) and A10 thoracic SMCs adhesion to collagen but not to other matrix proteins, suggesting its specificity on collagen. This inhibition was possibly resulted from that litebamine attenuated immobilized collagen-induced focal adhesion kinase (FAK) phosphorylation and actin cytoskeleton reorganization in RASMCs, as determined by Western blotting and immunofluorescence microscopy. In a functional study, litebamine also inhibited platelet-derived growth factor (PDGF)-induced RASMC migration but did not affect PDGF-induced matrix metalloproteinases (MMPs) secretion. Strikingly, among the tested kinases involved in PDGF-induced migration, only PDGF-induced phosphatidylinositol-3 kinase (PI-3K) activation was inhibited by litebamine. Taken together, we demonstrated here that litebamine can functionally inhibit vascular SMC adhesion and migration and elucidated its possible mechanisms of action. As SMC adhesion and migration are critical events in disease-related vascular remodeling, this compound may have beneficial effects in preventing cardiovascular diseases.

Lycopene inhibits TNF-alpha-induced endothelial ICAM-1 expression and monocyte-endothelial adhesion.

Inflammatory mediators such as TNF-alpha and interleukin (IL)-1beta, and IL-8, which can enhance binding of low-density lipoprotein (LDL) to endothelium and upregulate expression of leukocyte adhesion molecules on endothelium during atherogenesis. Lycopene, a natural carotenoid from tomato and other sources, has been shown to prevent cardiovascular diseases in epidemiological studies. However, its anti-inflammatory action mechanism remains unclear. In the present study, we studied the effect of lycopene on TNF-alpha-induced signaling in human umbilical endothelial cells (HUVECs). We found that TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression in HUVECs was inhibited by lycopene, whereas cyclooxygenase-2 (COX-2) and platelet-endothelial cell adhesion molecule (PECAM-1) expression were not affected. A further analysis indicated that lycopene attenuated TNF-alpha-induced IkappaB phosphorylation, NF-kappaB expression, and NF-kappaB p65 translocation from cytosol to nucleus. In line with this, TNF-alpha-induced NF-kappaB-DNA but not AP1-DNA complexes formation was inhibited by lycopene, as determined by the electrophoretic mobility shift assay (EMSA). On the other hand, lycopene did not affect TNF-alpha-induced p38 and extracellular matrix-regulated kinase1/2 (ERK1/2) phosphorylation and interferon-gamma (IFN-gamma)-induced signaling, suggesting that lycopene primarily affects TNF-alpha-induced NF-kappaB signaling pathway. In a functional study, lycopene dose-dependently attenuated monocyte adhesion to endothelial monolayer but not that adhesion to extracellular matrix. Taken together, we provided here the first evidence showing that lycopene is able to inhibit TNF-alpha-induced NF-kappaB activation, ICAM-1 expression, and monocyte-endothelial interaction, suggesting an anti-inflammatory role of lycopene and possibly explaining in part why lycopene can prevent cardiovascular diseases.

Involvement of NADPH oxidase and NF-κB activation in CXCL1 induction by vascular endothelial growth factor in human endometrial epithelial cells of patients with adenomyosis.

Chemokines were known to participate in inflammation and angiogenesis but have been recently recognized to be involved in embryonic implantation and endometrium-related pathologies. Among these chemokines, the CXC chemokines, such as CXCL1, have potential roles to work as biomarkers to identify patients with uterine adenomyosis. In this study, human endometrial epithelial cells (HEECs) were derived from patients' endometrium with adenomyosis. The inductive effects of CXCL1 production by various mediators/growth factors were investigated in the HEECs. Of the tested mediators, VEGF was found to be the most effective. The immunohistochemistry and RT-PCR analysis revealed a positive staining for VEGF and CXCL1 at the epithelium and the presence of CXCL1 in the human endometrium specimens, respectively. The CXCL1 induction by VEGF could be reduced by the antagonist for VEGF receptor (VEGFR), and by the inhibitors for NADPH oxidase and NF-κB signaling pathway. However, it was not affected by sex hormones and the inhibitors for MAPKs, PI-3K, protein kinase A and C. In parallel, VEGF induced p47 phox NADPH oxidase activation, IκBα phosphorylation, NF-κB translocation and NF-κB-DNA complex formation in the HEECs. Moreover, the CXCL1 released by the HEECs with VEGF stimulation attracted vascular endothelial cell migration. Taken together, we show that VEGF and CXCL1 are expressed in epithelium of the endometrium with adenomyosis and demonstrate here for the first time that VEGF is capable of inducing CXCL1 expression in HEECs through VEGFR, p47 phox NADPH oxidase and NF-κB signaling pathway, which is functionally required for attracting vascular endothelial cell migration.

Gold Nanoparticles Compromise TNF-α-Induced Endothelial Cell Adhesion Molecule Expression Through NF-κB and Protein Degradation Pathways and Reduce Neointima Formation in A Rat Carotid Balloon Injury Model.

The aim of this study was to investigate the anti-inflammatory effects and mechanism of action of the gold nanoparticles (AuNPs) on vascular injury. In vitro vascular endothelial cell (EC) inflammation and in vivo rat carotid balloon injury models were used. The expression of TNF-α-induced cell adhesion molecules (CAMs) was suppressed by the AuNPs in human umbilical vein ECs and aortic ECs. The AuNPs reduced TNF-α-induced intracellular ROS production and NF-κB signaling pathways and enhanced CAM protein degradation by increasing their ubiquitination. However, they did not interfere with the mTOR pathway for protein synthesis and TNF-αbinding to ECs. These effects led to a reduction of monocyte adhesion to EC monolayers in vitro and endothelial CAM expression and monocyte/macrophage level in the vascular injured areas, contributing to a substantial decrease of arterial neointima formation in the rat carotid balloon injury model. The serum gold concentration was 99.5±18 ng/ml after three-day oral administration. Moreover, incubation of the AuNPs with serum and albumin led to an increase of particle sizes of the AuNPs. Collectively, we provide the first evidence that demonstrates that AuNPs possess anti-inflammatory bioactivity on vascular ECsin vitro and can reduce arterial neointima hyperplasia during vascular injury in vivo.

Effects of all-trans retinoic acid, retinol, and ß-carotene on murine macrophage activity.

Previous studies have demonstrated that vitamin A and carotenoids regulate immune function in lymphocytes and splenocytes, and that the carotenoid lutein regulates matrix metalloproteinase-9 (MMP-9) production in macrophages. In this study, we investigated the effects of all-trans retinoic acid (atRA, a bioactive vitamin A metabolite), retinol (vitamin A), and ß-carotene (vitamin A precursor) on the activity of murine RAW264.7 and peritoneal macrophages. Our results indicated that atRA and retinol could induce GM-CSF and IL-16 expression, whereas all these tested substances enhanced MMP-9 production. Interestingly, the expression of GM-CSF, IL-16, and MMP-9 was distinctly regulated by these three substances. AtRA and retinol affected GM-CSF and IL-16 expression mainly through RA receptor ß (RARß). However, atRA induced MMP-9 production was via RARα activation and retinol and ß-carotene caused MMP-9 production via RARα and ß activation. These were supported by the observations that the RARα and ß agonists/antagonists differentially affected MMP-9 production and that atRA and ß-carotene enhanced RARE-mediated and MMP-9 promoter luciferase activity. In parallel, while the MMP-9 induction by atRA was not affected by the MAPKs inhibitors, its induction by retinol and ß-carotene was repressed by the inhibitor targeting ERK1/2. Finally, we show that all the tested substances could functionally enhance macrophage phagocytosis. Taken together, we provide evidence here for the first time that atRA, retinol, and ß-carotene differentially regulate GM-CSF, IL-16, and MMP-9 production in macrophages, explaining at least in part why these vitamin A-related substances are beneficial for immunity.

The carotenoid lutein enhances matrix metalloproteinase-9 production and phagocytosis through intracellular ROS generation and ERK1/2, p38 MAPK, and RARß activation in murine macrophages.

Early studies have demonstrated the ability of dietary carotenoids to enhance immune response, but the mechanism underlying their influence on macrophage activity remains unclear. Here, we investigated the effects of carotenoids on macrophage activity. Carotenoids, including lutein and lycopene, enhanced MMP-9 activity in RAW264.7 macrophages. Lutein was chosen as a representative and analyzed further in this study. It increased the synthesis, activity, and release of MMP-9 in murine RAW264.7 and primary-cultured peritoneal macrophages. MMP-9 induction by lutein was through the transcriptional regulation of mmp-9. It was blunted by the MAPK inhibitors targeting ERK1/2 and p38 MAPK, the reagents that inhibit free radical signaling, and the inhibitors and siRNA targeting RARß. Moreover, lutein induced Nox activation and intracellular ROS production at an early stage of treatment. This carotenoid also caused ERK1/2 and p38 MAPK activation, RARß expression, and RAR interaction with its responsive element in the promoter region. These findings suggest the involvement of ROS, MAPKs, and RARß activation in lutein-driven MMP-9 expression and release. Interestingly, lutein enhanced the phagocytic activity of macrophages, and the secreted MMP-9 appeared to be involved in this process. In summary, we provide evidence here for the first time that the carotenoid lutein induces intracellular ROS generation and MAPK and RARß activation in macrophages, leading to an increase in MMP-9 release and macrophage phagocytosis. Our results demonstrate that lutein exerts an immunomodulatory effect on macrophages.

Chrysin restores PDGF-induced inhibition on protein tyrosine phosphatase and reduces PDGF signaling in cultured VSMCs.

Previous studies have shown that an increased intake of dietary flavonoids is associated with a decreased risk of cardiovascular diseases (CVDs). PDGF is a major mitogen for vascular smooth muscle cell (VSMC) and participates in the pathogenesis of many CVDs. The study investigated whether the flavone chrysin affected PDGF functions in VSMCs and neointma formation in rat artery. We found that chrysin concentration-dependently inhibited PDGF-induced proliferation and chemotaxis and reduced PDGF signaling in VSMCs. Chrysin attenuated H(2)O(2) signaling and PDGF-induced reactive oxygen species production and NADPH oxidase activation but did not interfere with PDGF binding to VSMCs. The further analyses revealed that chrysin relieved PDGF-induced inhibition on activity of protein tyrosine phosphatase (PTP) and reduced PDGF-induced oxidation of PTP cysteinyl active site. Moreover, it inhibited PDGF receptor autophosphorylation induced by low-dose vanadate (an inhibitor for PTP). The effect of chrysin, but not of the flavonoid (-)-epigallocatechin-3-gallate and antioxidant N-acetylcysteine, on PDGF signaling and PTP activity was reversed by depletion of intracellular glutathione (GSH), suggesting an involvement of chrysin on GSH/glutaredoxin system for PTP reactivation. Finally, to demonstrate the effectiveness of chrysin in vivo, we showed that oral administration of chrysin before and after angioplasty could reduce neointima formation in balloon-injured carotid artery in rats. In conclusion, we provide here evidence that chrysin can regulate intracellular PTP activity during PDGF signaling, inhibits PDGF-induced VSMC proliferation and chemotaxis, and reduces arterial intima hyperplasia in vivo.

Lycopene binding compromised PDGF-AA/-AB signaling and migration in smooth muscle cells and fibroblasts: prediction of the possible lycopene binding site within PDGF.

Platelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of smooth muscle cells (SMCs) and fibroblasts. Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that natural carotenoid lycopene can directly bind to PDGF-BB and affect its related functions in vascular SMCs. In this study we examined lycopene effect on PDGF-AA/-AB-induced signaling and migration in SMCs and fibroblasts. We found that lycopene inhibited PDGF-AA-induced SMC and fibroblast migration in a concentration-dependent manner. Lycopene reduced PDGF-AA signaling, including phosphorylation in PDGFR-alpha and its downstream protein kinases/enzymes. It also inhibited PDGF-AB-induced signaling and cell migration. However, lycopene did not affect PDGF-induced reactive oxygen species production and H2O2-induced PDGFR phosphorylation. The binding analysis revealed that lycopene but not beta-carotene could directly bind to PDGF-AA in vitro and in plasma and this binding competitively inhibited lycopene interaction with PDGF-BB, suggesting that lycopene bound to PDGF-AA/-BB at a homologous/similar region within PDGF. Moreover, the docking and binding analyses predicted that the lycopene-binding region within PDGF was located at loop 2 region. Taken together, we provide here evidence that lycopene interacts with PDGF-AA/-AB and compromises their intracellular signaling, leading to a marked inhibition on PDGF-AA/-AB-induced migration in both SMCs and fibroblasts. We also predicted its binding region within PDGF and proved its anti-vascular injury effect in vivo. The results, together with our previous findings, suggest lycopene special affinity/effect for PDGF family and its possible application in prevention in vascular diseases and malignancy.