Antcin K inhibits VEGF-dependent angiogenesis in human rheumatoid arthritis synovial fibroblasts.

Antrodia cinnamomea is a well-known medicinal mushroom in Taiwan that exhibits anti-inflammatory biological activities. In rheumatoid arthritis (RA), chronic inflammation and angiogenesis driven by proinflammatory cytokines reflect the severity of the disease. Although biological treatments have improved the outlook for RA, no healing exists. Moreover, the available pharmacotherapies do not work for all patients and drug safety is a major consideration. Investigations into plant-based medicines hope to reveal better, more tolerable agents. We examined whether Antcin K, a phytosterol isolated from A. cinnamomea, has anti-angiogenic activity in RA. The GSE12021 gene dataset from the Gene Expression Omnibus (GEO) database was examined for levels of vascular endothelial growth factor (VEGF) expression in 10 RA and 10 osteoarthritis (OA) synovial tissue samples. In clinical samples, VEGF expression was analyzed by immunohistochemical staining and ELISA in normal and RA synovial tissue, as well as OA and RA synovial fluid. Collagen-induced arthritis (CIA) and control tissue was stained with hematoxylin and eosin (H&E) for histological changes; Safranin O/Fast Green staining examined histopathological changes and evidence of bone erosion. Human RA synovial fibroblasts (RASFs) were incubated with Antcin K and cell viability was examined by the MTT assay. VEGF mRNA expression was detected in RASFs using qPCR. Antcin K significantly inhibited VEGF expression and ameliorates endothelial progenitor cell (EPC) migration and tube formation in RASFs by downregulating the phospholipase C-γ/protein kinase C-α pathway. Antcin K also induced anti-angiogenic effects in human RASFs without cytotoxicity. PRACTICAL APPLICATIONS: Analysis of GEO dataset samples and human synovial fluids or synovial tissues revealed higher VEGF levels in rheumatoid arthritis (RA) samples compared with osteoarthritis (OA) and healthy control samples. VEGF levels were also higher in mice with collagen-induced arthritis (CIA) than in healthy controls. Antcin K markedly suppressed VEGF expression in human RA synovial fibroblasts and inhibited the migration and tube formation of epithelial progenitor cells (EPCs) by downregulating the phospholipase C-γ/protein kinase C-α pathway. Further investigations are warranted to examine the effects of Antcin K in other angiogenesis-associated disorders.

Apelin Promotes Endothelial Progenitor Cell Angiogenesis in Rheumatoid Arthritis Disease via the miR-525-5p/Angiopoietin-1 Pathway.

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. The adipokine apelin (APLN) plays critical roles in several cellular functions, including angiogenesis. We report that APLN treatment of RA synovial fibroblasts (RASFs) increased angiopoietin-1 (Ang1) expression. Ang1 antibody abolished endothelial progenitor cell (EPC) tube formation and migration in conditioned medium from APLN-treated RASFs. We also found significantly higher levels of APLN and Ang1 expression in synovial fluid from RA patients compared with those with osteoarthritis. APLN facilitated Ang1-dependent EPC angiogenesis by inhibiting miR-525-5p synthesis via phospholipase C gamma (PLCγ) and protein kinase C alpha (PKCα) signaling. Importantly, infection with APLN shRNA mitigated EPC angiogenesis, articular swelling, and cartilage erosion in ankle joints of mice with collagen-induced arthritis. APLN is therefore a novel therapeutic target for RA.

Hypoxia induced mitogenic factor (HIMF) triggers angiogenesis by increasing interleukin-18 production in myoblasts.

Inflammatory myopathy is a rare autoimmune muscle disorder. Treatment typically focuses on skeletal muscle weakness or inflammation within muscle, as well as complications of respiratory failure secondary to respiratory muscle weakness. Impaired respiratory muscle function contributes to increased dyspnea and reduced exercise capacity in pulmonary hypertension (PH), a debilitating condition that has few treatment options. The initiation and progression of PH is associated with inflammation and inflammatory cell recruitment and it is established that hypoxia-induced mitogenic factor (HIMF, also known as resistin-like molecule α), activates macrophages in PH. However, the relationship between HIMF and inflammatory myoblasts remains unclear. This study investigated the signaling pathway involved in interleukin-18 (IL-18) expression and its relationship with HIMF in cultured myoblasts. We found that HIMF increased IL-18 production in myoblasts and that secreted IL-18 promoted tube formation of the endothelial progenitor cells. We used the mouse xenograft model and the chick chorioallantoic membrane assay to further explore the role of HIMF in inflammatory myoblasts and angiogenesis in vivo. Thus, our study focused on the mechanism by which HIMF mediates IL-18 expression in myoblasts through angiogenesis in vitro and in vivo. Our findings provide an insight into HIMF functioning in inflammatory myoblasts.

Kuei-Lu-Er-Xian-Jiao extract enhances BMP-2 production in osteoblasts.

Osteoporosis is a common skeletal disorder, resulting from an imbalance in bone resorption relative to formation. Bone morphogenetic protein (BMP) is a key regulator in bone formation and osteoblastic differentiation. Hence, compounds that promote BMP expression may be suitable candidates for osteoporosis treatment. This study examined the effects of the traditional Chinese medicinal agent, Kuei-Lu-Er-Xian-Jiao (KLEXJ), on BMP-2 production in osteoblasts. We found that KLEXJ extract promoted osteoblastic differentiation marker ALP activity and increased BMP-2 production; pretreatment with PI3K and Akt inhibitors, or small interfering RNA (siRNA), reduced these effects. KLEXJ also enhanced PI3K and Akt phosphorylation. Treatment of osteoblastic cells with NF-κB inhibitors (TPCK or PDTC) markedly inhibited KLEXJ-enhancement of ALP activity and BMP-2 production. KLEXJ also significantly promoted p65 phosphorylation, while treatment with PI3K and Akt inhibitors antagonized KLEXJ-enhanced p65 phosphorylation. Thus, KLEXJ enhances ALP activity and BMP-2 production of osteoblasts through the PI3K/Akt/ NF-κB signaling pathway and hence may be suitable in the treatment of osteoporosis.

Tanshinone IIA inhibits angiogenesis in human endothelial progenitor cells in vitro and in vivo.

Accumulating evidence reports that bone marrow-derived endothelial progenitor cells (EPCs) regulate angiogenesis, postnatal neovascularization and tumor metastasis. It has been suggested that understanding the molecular targets and pharmacological functions of natural products is important for novel drug discovery. Tanshinone IIA is a major diterpene quinone compound isolated from Danshen (Salvia miltiorrhiza) and is widely used in traditional Chinese medicine (TCM). Evidence indicates that tanshinone IIA modulates angiogenic functions in human umbilical vein endothelial cells. However, the anti-angiogenic activity of tanshinone IIA in human EPCs has not been addressed. Here, we report that tanshinone IIA dramatically suppresses vascular endothelial growth factor (VEGF)-promoted migration and tube formation of human EPCs, without cytotoxic effects. We also show that tanshinone IIA markedly inhibits VEGF-induced angiogenesis in the chick embryo chorioallantoic membrane (CAM) model. Importantly, tanshinone IIA significantly attenuated microvessel formation and the expression of EPC-specific markers in the in vivo Matrigel plug assay in mice. Further, we found that tanshinone IIA inhibits EPC angiogenesis through the PLC, Akt and JNK signaling pathways. Our report is the first to reveal that tanshinone IIA reduces EPC angiogenesis both in vitro and in vivo. Tanshinone IIA is a promising natural product worthy of further development for the treatment of cancer and other angiogenesis-related pathologies.

Adiponectin promotes VEGF-A-dependent angiogenesis in human chondrosarcoma through PI3K, Akt, mTOR, and HIF-α pathway.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. On the other hand, angiogenesis is a critical step in tumor growth and metastasis. However, the relationship of adiponectin with vascular endothelial growth factor-A (VEGF-A) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study we first demonstrated that the expression of adiponectin was correlated with tumor stage of human chondrosarcoma tissues. In addition, we also found that adiponectin increased VEGF-A expression in human chondrosarcoma cells and subsequently induced migration and tube formation in human endothelial progenitor cells (EPCs). Adiponectin promoted VEGF-A expression through adiponectin receptor (AdipoR), phosphoinositide 3 kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF)-1α signaling cascades. Knockdown of adiponectin decreased VEGF-A expression and also abolished chondrosarcoma conditional medium-mediated tube formation in EPCs in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. Therefore, adiponectin is crucial for tumor angiogenesis and growth, which may represent a novel target for anti-angiogenic therapy in human chondrosarcoma.

Berberine attenuates CCN2-induced IL-1ß expression and prevents cartilage degradation in a rat model of osteoarthritis.

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator that is abundantly expressed in osteoarthritis (OA). Interleukin-1ß (IL-1ß) plays a pivotal role in OA pathogenesis. Berberine exhibits an anti-inflammatory effect, but the mechanisms by which it modulates CCN2-induced IL-1ß expression in OA synovial fibroblasts (OASFs) remain unknown. We showed that CCN2-induced IL-1ß expression is mediated by the activation of αvß3/αvß5 integrin-dependent reactive oxygen species (ROS) generation, and subsequent activation of apoptosis signal-regulating kinase 1 (ASK1), p38/JNK, and nuclear factor-κB (NF-κB) signaling pathways. This IL-1ß expression in OASFs is attenuated by N-acetylcysteine (NAC), inhibitors of ASK1, p38, or JNK, or treatment with berberine. Furthermore, berberine also reverses cartilage damage in an experimental model of collagenase-induced OA (CIOA). We observed that CCN2 increased IL-1ß expression via αvß3/αvß5 integrins, ROS, and ASK1, p38/JNK, and NF-κB signaling pathways. Berberine was found to inhibit these signaling components in OASFs in vitro and prevent cartilage degradation in vivo. We suggest a novel therapeutic strategy of using berberine for managing OA.

Curcumin induces cell apoptosis in human chondrosarcoma through extrinsic death receptor pathway.

Chondrosarcoma is a soft tissue sarcoma with a poor prognosis that is unresponsive to conventional chemotherapy. Surgical treatment leads to severe disability with high rates of local recurrence and life threat. Curcumin, an active compound in turmeric and curry, has been proven to induce tumor apoptosis and inhibit tumor proliferation, invasion, angiogenesis, and metastasis of cancer cells. In this study, we investigated the anticancer effects of curcumin in human chondrosarcoma cells. Curcumin induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. Curcumin induced upregulation of Fas, FasL, and DR5 expression in chondrosarcoma cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced curcumin-induced cell death. In addition, p53 involved in curcumin-mediated Fas, FasL, and DR5 expression and cell apoptosis in chondrosarcoma cells. Most importantly, animal studies revealed a dramatic 60% reduction in tumor volume after 21 days of treatment. Thus, curcumin may be a novel anticancer agent for the treatment of chondrosarcoma.