Bergenin alleviates proliferative arterial diseases by modulating glucose metabolism in vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation and phenotypic switching are key mechanisms in the development of proliferative arterial diseases. Notably, reprogramming of the glucose metabolism pattern in VSMCs plays an important role in this process. PURPOSE: The aim of this study is to investigate the therapeutic potential and the mechanism underlying the effect of bergenin, an active compound found in Bergenia, in proliferative arterial diseases. METHODS: The effect of bergenin on proliferative arterial disease was evaluated using platelet-derived growth factor (PDGF)-stimulated VSMCs and a mouse model of carotid artery ligation. VSMC proliferation and phenotypic switching were evaluated in vitro using cell counting kit-8, 5-ethynyl-2-deoxyuridine incorporation, scratch, and transwell assays. Carotid artery neointimal hyperplasia was evaluated in vivo using hematoxylin and eosin staining and immunofluorescence. The expression of proliferation and VSMC contractile phenotype markers was evaluated using PCR and western blotting. RESULTS: Bergenin treatment inhibited PDGF-induced VSMC proliferation and phenotypic switching and reduced neointimal hyperplasia in the carotid artery ligation model. Additionally, bergenin partially reversed the PDGF-induced Warburg-like glucose metabolism pattern in VSMCs. RNA-sequencing data revealed that bergenin treatment significantly upregulated Ndufs2, an essential subunit of mitochondrial complex I. Ndufs2 knockdown attenuated the inhibitory effect of bergenin on PDGF-induced VSMC proliferation and phenotypic switching, and suppressed neointimal hyperplasia in vivo. Conversely, Ndufs2 overexpression enhanced the protective effect of bergenin. Moreover, Ndufs2 knockdown abrogated the effects of bergenin on the regulation of glucose metabolism in VSMCs. CONCLUSION: These findings suggest that bergenin is effective in alleviating proliferative arterial diseases. The reversal of the Warburg-like glucose metabolism pattern in VSMCs during proliferation and phenotypic switching may underlie this therapeutic mechanism.

Platelet-derived growth factor signaling in pericytes promotes hypothalamic inflammation and obesity.

BACKGROUND: Pericytes are a vital component of the blood-brain barrier, and their involvement in acute inflammation was recently suggested. However, it remains unclear whether pericytes contribute to hypothalamic chronic inflammation and energy metabolism in obesity. The present study investigated the impact of pericytes on the pathophysiology of obesity by focusing on platelet-derived growth factor (PDGF) signaling, which regulates pericyte functions. METHODS: Tamoxifen-inducible systemic conditional PDGF receptor ß knockout mice (Pdgfrb∆SYS-KO) and Calcium/calmodulin-dependent protein kinase type IIa (CaMKIIa)-positive neuron-specific PDGF receptor ß knockout mice (Pdgfrb∆CaMKII-KO) were fed a high-fat diet, and metabolic phenotypes before and 3 to 4 weeks after dietary loading were examined. Intracellular energy metabolism and relevant signal transduction in lipopolysaccharide- and/or platelet-derived growth factor-BB (PDGF-BB)-stimulated human brain pericytes (HBPCs) were assessed by the Seahorse XFe24 Analyzer and Western blotting. The pericyte secretome in conditioned medium from HBPCs was studied using cytokine array kit, and its impact on polarization was examined in bone marrow-derived macrophages (BMDMs), which are microglia-like cells. RESULTS: Energy consumption increased and body weight gain decreased after high-fat diet loading in Pdgfrb∆SYS-KO mice. Cellular oncogene fos (cFos) expression increased in proopiomelanocortin (POMC) neurons, whereas microglial numbers and inflammatory gene expression decreased in the hypothalamus of Pdgfrb∆SYS-KO mice. No significant changes were observed in Pdgfrb∆CaMKII-KO mice. In HBPCs, a co-stimulation with lipopolysaccharide and PDGF-BB shifted intracellular metabolism towards glycolysis, activated mitogen-activated protein kinase (MAPK), and modulated the secretome to the inflammatory phenotype. Consequently, the secretome showed an increase in various proinflammatory chemokines and growth factors including Epithelial-derived neutrophil-activating peptide 78 (C-X-C motif chemokine ligand (CXCL)5), Thymus and activation-regulated chemokine (C-C motif chemokine (CCL)17), Monocyte chemoattractant protein 1 (CCL2), and Growth-regulated oncogene α (CXCL1). Furthermore, conditioned medium from HBPCs stimulated the inflammatory priming of BMDMs, and this change was abolished by the C-X-C motif chemokine receptor (CXCR) inhibitor. Consistently, mRNA expression of CXCL5 was elevated by lipopolysaccharide and PDGF-BB treatment in HBPCs, and the expression was significantly lower in the hypothalamus of Pdgfrb∆SYS-KO mice than in control Pdgfrbflox/flox mice (FL) following 4 weeks of HFD feeding. CONCLUSIONS: PDGF receptor ß signaling in hypothalamic pericytes promotes polarization of macrophages by changing their secretome and contributes to the progression of obesity.

The effect of oral treatment of royal jelly on the expression of the PDGF-ß gene in the skin wound of male mice.

AIMS OF THE STUDY: Royal jelly (RJ) is one of the most widely used drugs in traditional medicine. One of its important applications is the repair of skin damage, although the path of its mechanism is still unknown. Platelet-derived growth factor-beta (PDGF-beta) is one of the important factors in wound healing and it has been observed that PDGF-ß expression decreases with increasing age. In this study, for the first time, the effect of RJ on skin wounds has been investigated through the expression of PDGF-ß and tissue studies. MATERIALS AND METHODS: 25 small laboratory male BALB/c mice were selected randomly and after creating a 5 mm wound on the back of their neck, they were treated with doses of 2.5, 10, and 40 mg/kg body weight, After sampling from the healed wound in 9th day, histopathological studies and the expression of PDGF-ß gene were performed by Real-time PCR method. RESULTS: The findings of the present study showed that royal jelly caused a significant increase in PDGF-ß (10.99 times) compared to the healthy group. Also, royal jelly increased the formation of covering tissue or epithelium, the synthesis of collagen, the presence of inflammatory cells, and the formation of new blood vessels. CONCLUSION: The oral treatment of royal jelly is probably effective in skin wound healing by changing the expression of PDGF-ß.

Determination of the effectiveness of Dorema ammoniacum gum on wound healing: an experimental study.

OBJECTIVE: For a long time, natural compounds have been used to accelerate wound healing. In this study, the topical effects of ammoniacum gum extract on wound healing were investigated in white male rats. METHOD: Following skin wound induction in aseptic conditions, 48 Wistar rats were divided into six equal groups; phenytoin cream 1% (standard), untreated (control), Eucerin (control), and 5%, 10% and 20% ointments of Dorema ammoniacum gum extract (treatment groups). All experimental groups received topical drugs daily for 14 days. The percentage of wound healing, hydroxyproline content, histological parameters, and growth factors (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-α) were measured in experimental groups. RESULTS: The areas of the wounds in the treatment groups were significantly decreased compared with the wound areas of control groups at 5, 7 and 10 days after wounding. On the 12th day, the wounds in the treatment groups were completely healed. Hydroxyproline contents were significantly increased in the treatment groups compared with the control groups (p<0.001). In histological evaluation, the re-epithelialisation, increasing thickness of the epithelial layer, granulation tissue and neovascularisation parameters in the treatment groups showed significant increases compared with the control groups. Also, serum levels of TGF-ß, PDGF, EGF and VEGF in the treatment groups were significantly increased compared to the control groups. CONCLUSION: The topical application of ammoniacum gum extract significantly increases the percentage of wound healing in rats and reduces the time of wound closure.

Icariside II Attenuates Vascular Remodeling Via Wnt7b/CCND1 Axis.

ABSTRACT: Angioplasty often fails due to the abnormal proliferation of vascular smooth muscle cells (VSMCs). Success rates of angioplasty may increase following the administration of an agent that effectively ameliorates aberrant vascular remodeling. Icariside II (ICS-II) is a natural flavonol glycoside extract from the Chinese herbal medicine Epimedii that possesses several medicinal qualities that are beneficial in humans. Nevertheless, the role of ICS-II in addressing aberrant vascular remodeling have yet to be clarified. The current investigation studies the molecular effects of ICS-Ⅱ on balloon-inflicted neointimal hyperplasia in rats in vivo and on platelet-derived growth factor-induced vascular proliferation in primary rat aortic smooth muscle cells (VSMCs) in vitro. ICS-II was found to be as effective as rapamycin, the positive control used in this study. ICS-II inhibited neointimal formation in injured rat carotid arteries and notably reduced the expression of Wnt7b. ICS-Ⅱ significantly counteracted platelet-derived growth factor-induced VSMCs proliferation. Cell cycle analysis showed that ICS-II triggered cell cycle arrest during the G1/S transition. Western blot analysis further indicated that this cell cycle arrest was likely through Wnt7b suppression that led to CCND1 inhibition. In conclusion, our findings demonstrate that ICS-II possesses significant antiproliferative qualities that counteracts aberrant vascular neointimal hyperplasia. This phenomenon most likely occurs due to the suppression of the Wnt7b/CCND1 axis.

Effect of Korean Magnolia obovata Extract on Platelet-Derived Growth Factor-Induced Vascular Smooth Muscle Cells.

OBJECTIVE: To investigate the effects of Korean Magnolia obovata crude extract (KME) on plateletderived growth factor (PDGF)-BB-induced proliferation and migration of vascular smooth muscle cells (VSMCs). METHODS: KME composition was analyzed by high-performance liquid chromatography (HPLC). VSMCs were isolated from the aorta of a Sprague-Dawley rat, incubated in serum free-Dulbecco's modified Eagle's medium in the presence or absence of KME (10, 30, 100, and 300 µg/mL), then further treated with PDGF-BB (10 ng/mL). VSMC proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and VSMC migration was determined using the Boyden chamber and scratch wound healing assays. Western blot analysis was used to detect phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (p-ERK1/2), protein kinase B (p-Akt), and stress-activated protein kinase/c-Jun NH2-terminal kinase (p-SAPK/JNK). The antimigration and proliferation effects of KME were tested using aortic sprout outgrowth. RESULTS: The HPLC analysis identified honokiol (0.45 mg/g) and magnolol (0.34 mg/g) as the major components of KME. KME (30, 100, and 300 µg/mL) significantly decreased the proliferation and migration of PDGF-BB-stimulated (10 ng/mL) VSMCs and the PDGF-BB-induced phosphorylation of EKR1/2, Akt, and SAPK/JNK (P<0.05). Furthermore, PDGF-BBinduced VSMCs treated with 300 µg/mL of KME showed reduction in aortic sprout outgrowth. CONCLUSION: KME could inhibit abnormal proliferation and migration of VSMCs by down-regulating the phosphorylation of EKR1/2 and Akt. Thus, KME might be a functional food for preventing vascular disorders.

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway.

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.

Comparative study of different anticoagulants and coagulants in the evaluation of clinical application of platelet-rich plasma (PRP) standardization.

To investigate the impact of different anticoagulants and coagulants with autologous platelet-rich plasma (PRP) in order to evaluate the clinical application of PRP standardization. Bone marrow stem cells (BMSCs) were seeded into autologous PRP gel scaffolds with different anticoagulants (EDTA, heparin sodium HS, and sodium citrate SC) as well as control group (the whole blood group). Quality of PRP was evaluated and flow cytometric assay was used to detect the activity of the platelet (CD62p, PAC-1). BMSCs were also seeded into PRP with different coagulants (Thrombin, Collagen-I, ADP) as well as PRP un-activated (negative group) and L-DMEM complete culture without PRP (control group). The effects of different coagulants with PRP on proliferation, osteogenic differentiation of BMSCs were analyzed by methyl thiazolyl tetrazolium assay (MTT), ALP staining, Von Kossa staining, Confocal microscopic observation, RT-PCR and Western Blot at the morphological, cellular and molecular levels. Different anticoagulants (EDTA, HS, and SC) could affect the quality of PRP. EDTA group revealed the best quality and activity (CD62p, PAC-1). With different coagulants (Thrombin, Collagen-I and ADP) in the proliferation of BMSCs, the MTT assay showed that the proliferation of BMSCs was increased in all groups with time. On the sixth day of culture, the cell number of each PRP group was significantly higher than that in the control group (P < 0.05), while the most rapidly increasing was found in Collagen-I group. The cumulative release of growth factor (TGF-ß1, PDGF) at each time point in the PRP gel of the four groups was higher than that in the control group (P < 0.05). Collagen-I was considered as the best PRP coagulant. When thrombin was used as a platelet coagulant, the release of growth factor in PRP was rapid and direct, while the release of growth factor in Collagen-I-activated PRP was sustained and slow, and the total release of ADP-activated PRP growth factors was the lowest. The study demonstrated the similar outcome in osteogenic differentiation. In terms of gene expression and western bolt, the PCR results showed that the expression levels of OCN gene and RUNX2 protein in each PRP group were higher than that in the control group (P < 0.05). Different anticoagulants caused different degrees of lysis and spontaneous activation of platelets, which lead to different quality of PRP. Compared with HS and SC, EDTA could maintain the structural integrity of platelets, reduce their spontaneous activation, and increase the release of PRP growth factors for a longer period of time, thus ensuring the biomass of PRP. In addition, different coagulants also showed different results in the proliferation as well as osteogenic differentiation of BMSCs. Compared with Thrombin and ADP, Collagen-I may be a better choice.

Platelet rich concentrate enhances mesenchymal stem cells capacity to repair focal cartilage injury in rabbits.

BACKGROUND: It has been previously suggested that the use of regenerative promoters, which include bone marrow-derived mesenchymal stem cells (MSCs) or natural growth factors supplement such as platelet-rich concentrate (PRC) could promote cartilage regeneration. However, the notion that the concurrent use of both promoters may provide a synergistic effect that improves the repair outcome of focal cartilage injury has not been previously demonstrated. This study was thus conducted to determine whether the concomitant use of PRC could further enhance the reparative potential of MSCs encapsulated in alginate transplanted into focal cartilage injury in rabbits. METHODS: Artifically created full thickness cartilage defects were made on the weight-bearing region of medial femoral condyles in bilateral knees of New Zealand White rabbits (N = 30). After one month, the right knee was treated with either i) PRC (n = 10), ii) MSCs (n = 10), or, iii) a combination of PRC and MSCs (PRC + MSC) (n = 10), all encapsulated in alginate. The left knee remained untreated (control). Rabbits were sacrificed at 3 and 6 months after treatment. Cartilage tissue regeneration was accessed using ICRS morphologic scoring, histologic grading by O'Driscoll scoring, immunohistochemical staining and quantitative analysis of glycosaminoglycans (GAG) per total protein content. RESULTS: At 3 months, transplantation using PRC alone was equally effective as MSCs in inducing the repair of cartilage defects. However, PRC + MSC resulted in significantly higher ICRS and O'Driscoll scores (p < 0.05) as compared to other groups. The regenerated tissues from the PRC + MSC group also had stronger staining for Safranin-O and collagen type II. By 6 months, in addition to superior ICRS and O'Driscoll scores as well as stronger staining, glycosaminoglycan per total protein content was also significantly higher (p < 0.05) in the PRC + MSC group (3.4 ±â€¯0.3 µg/mg) as compared to the MSC (2.6 ±â€¯0.2 µg/mg) or PRC (2.1 ±â€¯0.2 µg/mg) groups. CONCLUSION: PRC enhances the reparative effects of MSC in treating focal articular cartilage injuries.

Wedelolactone, a plant coumarin, prevents vascular smooth muscle cell proliferation and injury-induced neointimal hyperplasia through Akt and AMPK signaling.

Wedelolactone (WDL) is a natural compound derived from Chinese herbal medicine Eclipta prostrate L, and has been reported to exhibit various effects potentially beneficial for human health. However, the possible preventive effects of WDL toward vascular remodeling and mechanisms involved have not been investigated to date. In this study, we investigated the effects of WDL on proliferation induced by platelet-derived growth factor (PDGF) in primary rat aortic smooth muscle cells (VSMCs) and on neointimal hyperplasia resulted from balloon injury in rats. WDL exhibited strong inhibitory effects against PDGF-induced VSMC proliferation. Cell cycle analysis revealed that WDL induced G0/G1 arrest and prevented cell cycle from entering S phase. Immunoblot analysis suggested that the cell cycle arrest induced by WDL was through Akt suppression and adenosine 5'-monophosphate-activated protein kinase (AMPK) activation, with a subsequent cyclin-dependent kinase inhibitor p21 induction and cyclin D1 inhibition. We also observed that WDL notably reduced neointima-to-media area ratio of balloon-injured rat common carotid arteries (CCAs) in comparison with those untreated balloon-injured CCAs. The regulation of WDL on protein expressions of Akt, AMPK and cyclin D1 in vivo were also consistent with that in vitro. Taken together, our results suggest WDL exhibits potential preventive effects toward vascular remodeling and neointimal hyperplasia through the reduction of VSMC proliferation via inhibition of Akt and activation of AMPK.

Inhibition of Proliferation of Vascular Smooth Muscle Cells by Cucurbitanes from Momordica charantia.

The cucurbitaceous plant Momordica charantia L., named "bitter melon", inhabits Asia, Africa, and South America and has been used as a traditional medicine. The atypical proliferation of vascular smooth muscle cells (VSMCs) plays an important role in triggering the pathogenesis of cardiovascular diseases. Platelet-derived growth factor (PDGF) is regarded as the most powerful growth factor in promoting the intimal accumulation of VSMCs. The current study features the identification of six new cucurbitane-type triterpenoids (1-6) from the fruits of M.  charantia, utilizing diverse chromatographic and spectroscopic techniques. In particular, the 2D structure of 1 was confirmed utilizing the long-range HSQMBC NMR pulse, capable of measuring heteronuclear long-range correlations (4-6JCH). The cucurbitanes were also assessed for their inhibitory activity against PDGF-induced VSMC proliferation. This current study may constitute a basis for developing those chemotypes into sensible pharmacophores alleviating cardiovascular disorders.

α-Iso-Cubebene Inhibits PDGF-Induced Vascular Smooth Muscle Cell Proliferation by Suppressing Osteopontin Expression.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms. Thus, we examined the effect of ICB on vascular smooth muscle cell (VSMC) proliferation, a key feature of diverse vascular diseases. When VSMCs primary cultured from rat thoracic aorta were stimulated with PDGF (1-10 ng/ml), cell proliferation and osteopontin (OPN) expression were concomitantly up-regulated, but these effects were attenuated when cells were treated with MPIIIB10, a neutralizing monoclonal antibody for OPN. In aortic tissues exposed to PDGF, sprouting VSMC numbers increased, which was attenuated in tissues from OPN-deficient mice. Furthermore, VSMC proliferation and OPN expression induced by PDGF were attenuated dose-dependently by ICB (10 or 30 µg/ml). Reporter assays conducted using OPN promoter-luciferase constructs showed that the promoter region 538-234 bp of the transcription start site was responsible for transcriptional activity enhancement by PDGF, which was significantly inhibited by ICB. Putative binding sites for AP-1 and C/EBPß in the indicated promoter region were suggested by TF Search, and increased binding of AP-1 and C/EBPß in PDGF-treated VSMCs was demonstrated using a ChIP assay. The increased bindings of AP-1 and C/EBPß into OPN promoter were attenuated by ICB. Moreover, the PDGF-induced expression of OPN was markedly attenuated in VSMCs transfected with siRNA for AP-1 and C/EBPß. These results indicate that ICB inhibit VSMC proliferation by inhibiting the AP-1 and C/EBPß signaling pathways and thus downregulating OPN expression.

Arborinane Triterpenoids from Rubia philippinensis Inhibit Proliferation and Migration of Vascular Smooth Muscle Cells Induced by the Platelet-Derived Growth Factor.

The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are associated with cardiovascular diseases and related complications. Such deleterious proliferation and migration events are triggered by cytokines and growth factors, and among them, platelet-derived growth factor (PDGF) is recognized as the most potent inducer. Despite the genus Rubia being researched to identify valuable commercial and medicinal virtues, Rubia philippinensis has rarely been investigated. Nine arborinane-type triterpenoids (1-9) were identified from this underutilized plant species. In particular, 4 was identified as the first arborinane derivative carrying a ketocarbonyl motif at C-19. The presence of the cyclopentanone moiety and the associated configurational assignment were determined by utilizing NOE and coupling constant analysis. These compounds were assessed for their inhibitory potential on PDGF-induced proliferation and the migration of VSMCs. Treatment with 5 µM compound 5 (62.6 ± 10.7%) and compound 9 (41.1 ± 4.7%) impeded PDGF-stimulated proliferation without exerting cytotoxicity. Compound 7 exhibited antimigration activity in a dose-dependent manner (38.5 ± 3.0% at 10 µM, 57.6 ± 3.2% at 30 µM). These results suggest that the arborinane-type triterpenoids may be a pertinent starting point for the development of cardiovascular drugs capable of preventing the intimal accumulation of VSMCs.

Development of a reliable automated screening system to identify small molecules and biologics that promote human ß-cell regeneration.

Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.

The inhibition of calpains ameliorates vascular restenosis through MMP2/TGF-ß1 pathway.

Restenosis limits the efficacy of vascular percutaneous intervention, in which vascular smooth muscle cell (VSMC) proliferation and activation of inflammation are two primary causal factors. Calpains influence VSMC proliferation and collagen synthesis. However, the roles of calpastatin and calpains in vascular restenosis remain unclear. Here, restenosis was induced by ligating the left carotid artery, and VSMCs were pretreated with platelet-derived growth factor (PDGF)-BB. Adenovirus vector carrying MMP2 sequence and specific small interfering RNA against calpain-1/2 were introduced. Finally, restenosis enhanced the expression of calpain-1/2, but reduced calpastatin content. In calpastatin transgenic mice, lumen narrowing was attenuated gradually and peaked on days 14-21. Cell proliferation and migration as well as collagen synthesis were inhibited in transgenic mice, and expression of calpain-1/2 and MMP2/transforming growth factor-ß1 (TGF-ß1). Consistently, in VSMCs pretreated with PDGF-BB, calpastatin induction and calpains inhibition suppressed the proliferation and migration of VSMCs and collagen synthesis, and reduced expression of calpain-1/2 and MMP2/TGF-ß1. Moreover, simvastatin improved restenosis indicators by suppressing the HIF-1α/calpains/MMP2/TGF-ß1 pathway. However, MMP2 supplementation eliminated the vascular protection of calpastatin induction and simvastatin. Collectively, calpains inhibition plays crucial roles in vascular restenosis by preventing neointimal hyperplasia at the early stage via suppression of the MMP2/TGF-ß1 pathway.

Inhibition of PDGF-induced migration and TNF-α-induced ICAM-1 expression by maltotetraose from bamboo stem extract (BSE) in mouse vascular smooth muscle cells.

SCOPE: Expression of intercellular adhesion molecule-1 (ICAM-1) on vascular smooth muscle cells (VSMCs) plays an important role in the progression of atherosclerosis. We investigated the effects of bamboo stem extract (BSE) on motility and ICAM-1 expression by using mouse MOVAS-1 cells. Active constituents of BSE exhibiting an inhibitory activity on TNF-α-induced ICAM1 expression were identified using HPLC. METHODS AND RESULTS: The effects of BSE on platelet-derived growth factor (PDGF)-BB-induced migration, tumor necrosis factor alpha (TNF-α)-induced expression of ICAM-1, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation were investigated. BSE inhibited migration of MOVAS-1 cells and sprout formation by mouse aorta explants. Reverse transcription PCR analysis and promoter reporter assays revealed that BSE suppressed ICAM-1 expression by inhibiting NF-κB activity. In addition, BSE reduced adhesion between VSMCs and monocytes. Several oligosaccharides were identified in BSE. Among the oligosaccharides contained in BSE, maltotetraose and stachyose were potent inhibitors of TNF-α-induced ICAM-1 expression. We confirmed that maltotetraose reduced PDGF-induced sprout formation by mouse aorta explants and inhibited TNF-α-induced NF-κB activation and ICAM-1 expression in MOVAS-1 cells. CONCLUSION: The BSE constituent maltotetraose may be beneficial in the suppression of early atherosclerosis development and could be developed as a dietary supplement for cardiovascular health.

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice.

OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.

Effect of hesperidin on TGF-beta1/Smad signaling pathway in HSC.

Liver fibrosis is a common pathological process for chronic liver injury caused by multiple etiological factors and an inevitable phase leading to liver cirrhosis. According to the previous studies, hesperidin (HDN) shows a very good protective effect on CCl4-induced chemical hepatic fibrosis in rats. In this experiment, based on the findings of the previous studies, a platelet-derived growth factor (PDGF)-induced HSC-T6 model was established to observe the inhibitory effect of HDN on HSC-T6 proliferation. The ELISA method was adopted to detect the content of collagen I in HSC-T6 supernatant. Transforming growth factor (TGF)-beta1, Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) mRNA expressions were measured by RT-PCR; TGF-beta1 and CT-GF protein expressions in HSC-T6 were determined by Western blot, in order to study HDN's effect on TGF-beta1 signaling pathway in HSC and its potential action mechanism. The results demonstrated that HDN could notably improve HSC-T6 proliferation, Collagen I growth and TGF-beta1, Smad2, Smad3 and CTGF mRNA.expressions. After being intervened with HDN, it could notably inhibit HSC-T6 proliferation and Collagen I growth, reduce TGF-beta1, Smad2, Smad3 and CTGF mRNA and TGF-beta1, CTGF protein expressions and increase Smad7 mRNA expression. HDN's antihepatic fibrosis effect may be related to the inhibition of HSC proliferation and activation by modulating TGF-beta/Smad signaling pathway.

Simultaneous quantification and antiatherosclerosis effect of the traditional Korean medicine, Hwangryunhaedok-tang.

BACKGROUND: Hwangryunhaedok-tang (HHT) is a traditional herbal medicine that is used for the treatment of fever, inflammation, gastritis, and hypertension. In this study, we performed simultaneous determination of the five components, geniposide (1), baicalin (2), coptisine (3), palmatine (4), and berberine (5) in HHT by using a high-performance liquid chromatography-photodiode array (HPLC-PDA) analysis. We also evaluated the antioxidative activity of HHT and compounds 1-5 by measuring their effects on low-density lipoprotein (LDL) oxidation and antiproliferative abilities in vascular smooth muscle cells (VSMCs). METHODS: Five compounds were separated within 40 min by using a Gemini C18 column (temp. 35°C; two-component gradient elution; flow rate 1.0 mL/min; detector 240 and 277 nm). The activities of HHT and compounds 1-5 were tested with the radical scavengers 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 2,2-diphenyl-1-picrylhydrazyl, in thiobarbituric acid reactive substance assays, and in relative electrophoretic mobility assays using CuSO4-induced LDL oxidation systems. The antiproliferative effects of samples on platelet-derived growth factor (PDGF)-induced VSMC proliferation were studied by using a cell proliferation assay. RESULTS: Regression analysis of the five major compounds showed good linearity (r (2) ≥ 0.9997) in different concentration ranges. The recoveries of the five compounds were in the range 86.31-110.78%, with relative standard deviations below 2.1%; those of intra- and interday precision were 0.04-3.78% and 0.04-1.69%, respectively. HHT reduced the oxidation properties of LDL induced by CuSO4 and inhibited cell proliferation in PDGF-treated VSMCs. Among the five components, compound 2 could effectively suppress LDL oxidation and PDGF-induced VSMC proliferation. CONCLUSIONS: The established HPLC-PDA method will help to improve quality control of HHT. The results demonstrate that HHT has antiatherosclerotic activity and that it functions by modulating LDL oxidation and VSMC proliferation. The effects of HHT may be attributed, at least I part, to compound 2.

Chrysanthemum boreale flower floral water inhibits platelet-derived growth factor-stimulated migration and proliferation in vascular smooth muscle cells.

CONTEXT: Chrysanthemum boreale Makino (Compositae) (CBM) is a traditional medicine that has been used for the prevention or treatment of various disorders; it has various properties including antioxidation, anti-inflammation, and antitumor. OBJECTIVE: The present study was designed to explore the in vitro effect of CBM flower floral water (CBMFF) on atherosclerosis-related responses in rat aortic smooth muscle cells (RASMCs). MATERIALS AND METHODS: CBMFF was extracted from CBM flower by steam distillation and analyzed using gas chromatography-mass spectrometry. The anti-atherosclerosis activity of CBMFF was tested by estimating platelet-derived growth factor (PDGF)-BB (10 ng/mL)-induced proliferation and migration levels and intracellular kinase pathways in RASMCs at CBMFF concentrations of 0.01-100 µM and analyzing ex vivo aortic ring assay. RESULTS: Gas chromatography-mass spectrometry showed that the CBMFF contained a total of seven components. The CBMFF inhibits PDGF-BB-stimulated RASMC migration and proliferation (IC50: 0.010 µg/mL). Treatment of RASMCs with PDGF-BB induced PDGFR-ß phosphorylation and increased the phosphorylations of MAPK p38 and ERK1/2. CBMFF addition prevented PDGF-BB-induced phosphorylation of these kinases (IC50: 008 and 0.018 µg/mL, for p38 MAPK and ERK1/2, respectively), as well as PDGFR-ß (IC50: 0.046 µg/mL). Treatment with inhibitors of PDGFR, P38 MAPK, and ERK1/2 decreased PDGF-BB-increased migration and proliferation in RASMCs. Moreover, the CBMFF suppressed PDGF-BB-increased sprout outgrowth of aortic rings (IC50: 0.047 µg/mL). DISCUSSION AND CONCLUSION: These results demonstrate that CBMFF may inhibit PDGF-BB-induced vascular migration and proliferation, most likely through inhibition of the PDGFR-ß-mediated MAPK pathway; therefore, the CBMFF may be promising candidate for the development of herbal remedies for vascular disorders.