Carthami flos extract against carbon tetrachloride-induced liver fibrosis via alleviating angiogenesis in mice.

BACKGROUND: Angiogenesis is a pathological phenomenon contribute to the development of chronic liver diseases, and anti-angiogenic therapy is an effective strategy to alleviate liver fibrosis. Carthami flos, a medicinal and edible herb, has the effects of improving blood circulation and regulating angiogenesis. However, the anti-angiogenic effect of Carthami flos in liver fibrosis remains unknown. METHODS: We investigated the protective effect and therapeutic mechanism of Carthami flos extract (CFE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. The liver injury and collagen deposition were observed and evaluated by conducting HE, Masson, and Sirius red staining, testing the serum biochemical indexes (ALT, AST, ALP, γ-GT), and measuring the contents of HYP and four indexes of liver fiber (Col-IV, LN, HA, PC-III). Simultaneously, the expressions of α-SMA and Collagen-I were detected to determine the activation of hepatic stellate cells (HSCs). Subsequently, we measured the expressions of angiogenesis-related proteins such as PDGFRB, ERK1/2, p-ERK1/2, MEK, p-MEK, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, and the mRNA levels of PDGFRB and VEGFA. Additionally, immunofluorescence staining and RT-qPCR assays were carried out to ascertain the expressions of continuous endothelial markers CD31, CD34 and vWF, and scanning electron microscope analysis was performed to observe the number of sinusoidal endothelial fenestrations. RESULTS: Herein, we found that CFE could significantly reduce liver injury and collagen deposition, like the same effect of colchicine. CFE significantly alleviated CCl4-induced liver injury and fibrosis, mainly manifested by reducing the levels of ALT, AST, ALP and γ-GT and decreasing the contents of HYP, Col-IV, LN, HA and PC-III. Additionally, CCl4 promoted the activation of HSCs by increasing the expressions of α-SMA and Collagen-I, while CFE could rectify the condition. Moreover, CFE treatment prevented the CCl4-induced the up-regulation of PDGFRB, p-MEK, p-ERK1/2, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, suggesting that CFE might provide the protection against abnormal angiogenesis. In the meantime, the gradual disappearance of sinusoidal capillarization after CFE treatment was supported by the decreased the contents of CD31, CD34 and vWF, as well as the increased number of sinusoidal endothelial fenestrae. CONCLUSION: In this study, the reduction of collagen deposition, the obstruction of HSCs activation, the inactivation of angiogenic signaling pathways and the weakening of hepatic sinusoidal capillarization jointly confirmed that CFE might be promising to resist angiogenesis in liver fibrosis via the PDGFRB/ERK/HIF-1α and VEGFA/AKT/eNOS signaling pathways. Nevertheless, as a potential therapeutic drug, the deeper mechanism of Carthami flos still needs to be further elucidated.

Label-Free Direct Mass Spectrometry Analysis of the Bystander Effects Induced in Chondrocytes by Chondrosarcoma Cells Irradiated with X-rays and Carbon Ions.

BACKGROUND: Radiation-induced bystander effects are induced changes in cells that were not themselves directly irradiated but were in the vicinity of a radiation path. Such effects, which occur in the microenvironment of an irradiated tumor, remain poorly understood and depend on the cell type and irradiation quality. This study aimed to evaluate bystander effects in non-irradiated chondrocytes that received conditioned medium from irradiated chondrosarcoma cells. METHODS: SW1353 chondrosarcoma cells were irradiated with X-rays and carbon ions, each at 0.1 Gy and 2 Gy, and the conditioned media of the irradiated cells were transferred to T/C-28A2 chondrocytes and Human Umbilical Venous Endothelial Cells (HUVECs). The whole proteome of bystander chondrocytes was analyzed by label-free mass spectrometry, and a comparative study was performed by dose and irradiation quality. HUVECs were evaluated for inflammatory cytokine secretion. RESULTS: The bystander response of chondrocytes to X-ray irradiation primarily affected the protein translation pathway (DHX36, EIF3B, EIF3D, EIF3M, EIF5, RPL6, RPLP0, RPS24, SYNCRIP), IL-12 (AIP, BOLA2, MIF, GAS6, MIF, PDGFRB) and the oxidative stress pathway (MGST3, PRDX2, PXDN, SOD2, TXN, TXNL1). Following carbon-ion irradiation, the G1/S pathway (PCBP4, PSMD12, PSME, XIAP) and mitotic G2 DNA damage checkpoint pathway (MRE11, TAOK1, UIMC1) were engaged. Changes in the regulation of chromosome separation (BCL7C, BUB3, CENPF, DYNC1LI1, SMARCA4, SMC4) were associated with only low-dose X-ray and carbon-ion irradiation. Modification of the protein translation pathway represented at least 30% of bystander effects and could play a role, possibly along with stress granules, in reduction in cellular metabolism to protect proteins. Stress granules were significantly enriched according to an interaction map. CONCLUSIONS: All these accessions corresponded to a window of the proteins modulated in response to the bystander effect. Our chondrosarcoma model clarified the nature of the bystander response of chondrocytes and may suggest several interesting new mechanisms that are specific to particular irradiation doses and qualities.

PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

PDGF-D Prodomain Differentially Inhibits the Biological Activities of PDGF-D and PDGF-B.

As a member of PDGF/VEGF (Platelet-derived growth factor/ Vascular endothelial growth factor) growth factors, PDGF-D regulates blood vessel development, wound healing, innate immunity, and organogenesis. Unlike PDGF-A and PDGF-B, PDGF-D has an additional CUB (Complement C1r/C1s, Uegf, Bmp1) domain at the N-terminus of its growth factor domain, and thus it is secreted in a latent, inactive complex, which needs to be proteolytically activated for its biological activities. However, how the CUB domain contributes to the latency and activation of the growth factor remains elusive. In this study, we modeled the dimeric structure of PDGF-D pro-complex and studied the inhibitory functions of PDGF-D prodomain on PDGF-B and PDGF-D signaling. In our model, the growth factor domain of PDGF-D forms a VEGF-D-like dimer through their ß1 and ß3 interactions. The hinge and CUB domains of PDGF-D bind at the opposite sides of the growth factor domain and exclude the PDGFR-ß (PDGF Receptor ß) D2 and D3 domains from recognizing the growth factor. In addition, we verified that PDGF-D prodomain could inhibit both PDGF-B and PDGF-D mediated PDGFR-ß transphosphorylation in a dose-dependent manner. However, PDGF-D prodomain could only inhibit the proliferation of NIH 3T3 cells stimulated by PDGF-D but not by PDGF-B, indicating its differential inhibitory activities toward PDGF-B and PDGF-D signaling.

Characterisation of PDGF-BB:PDGFRß signalling pathways in human brain pericytes: evidence of disruption in Alzheimer's disease.

Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-ß (PDGFRß) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer's disease (AD) is well documented. We found that PDGF-BB:PDGFRß signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB. We hypothesised that reduced PDGF-BB:PDGFRß signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRß signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD.

Endostatin inhibits fibrosis by modulating the PDGFR/ERK signal pathway: an in vitro study.

Accumulating evidence indicates that endostatin inhibits fibrosis. However, the mechanism is yet to be clarified. The aim of this study is to evaluate the effect of endostatin on platelet-derived growth factor-BB (PDGF-BB)- or transforming growth factor ß1 (TGF-ß1)-induced fibrosis in cultured human skin fibroblasts, and to further examine the molecular mechanisms involved. Human dermal fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) and serum-starved for 48 h before treatment. Cells were grouped as follows: "PDGF-BB", "PDGF-BB+ endostatin", "TGF-ß1", "TGF-ß1+endostatin", "endostatin", and "blank control". The fibroblasts were stimulated with either TGF-ß1 or PDGF-BB for 72 h in order to set up the fibrosis model in vitro. The cells were co-cultured with either TGF-ß1 or PDGF-BB and endostatin and were used to check the inhibiting effect of endostatin. A blank control group and an endostatin group were used as negative control groups. The biomarkers of fibrosis, including the expression of collagen I, hydroxyproline, and α-smooth muscle actin (α-SMA), were evaluated using an enzyme-linked immunosorbent assay (ELISA) and Western blot. The expression of phosphorylated PDGF receptor ß (p-PDGFRß), PDGFRß, phosphorylated extracellular signal-regulated kinase (p-ERK), and ERK was detected using Western blot and immunofluorescent staining was used to explore the mechanisms. Both PDGF-BB and TGF-ß1 significantly up-regulated the expression of collagen I, hydroxyproline, and α-SMA. Endostatin significantly attenuated both the PDGF-BB- and TGF-ß1-induced over-expression of collagen I, hydroxyproline, and α-SMA. PDGF-BB and TGF-ß1 both promoted the expression of PDGFR, ERK, and p-ERK. Endostatin inhibited the expression of PDGFR and p-ERK but did not affect the expression of total ERK. Endostatin inhibited hypertrophic scar by modulating the PDGFRß/ERK pathway. Endostatin could be a promising multi-target drug in future fibrosis therapy.

Inhibition of Glomerular Mesangial Cell Proliferation by siPDGF-B- and siPDGFR-ß-Containing Chitosan Nanoplexes.

Mesangioproliferative glomerulonephritis is a disease that has a high incidence in humans. In this disease, the proliferation of glomerular mesangial cells and the production of extracellular matrix are important. In recent years, the RNAi technology has been widely used in the treatment of various diseases due to its capability to inhibit the gene expression with high specificity and targeting. The objective of this study was to decrease mesangial cell proliferation by knocking down PDGF-B and its receptor, PDGFR-ß. To be able to use small interfering RNAs (siRNAs) in the treatment of this disease successfully, it is necessary to develop appropriate delivery systems. Chitosan, which is a biopolymer, is used as a siRNA delivery system in kidney drug targeting. In order to deliver siRNA molecules targeted at PDGF-B and PDGFR-ß, chitosan/siRNA nanoplexes were prepared. The in vitro characterization, transfection studies, and knockdown efficiencies were studied in immortalized and primary rat mesangial cells. In addition, the effects of chitosan nanoplexes on mesangial cell proliferation and migration were investigated. After in vitro transfection, the PDGF-B and PDGFR-ß gene silencing efficiencies of PDGF-B and PDGFR-ß targeting siRNA-containing chitosan nanoplexes were 74 and 71% in immortalized rat mesangial cells and 66 and 62% in primary rat mesangial cells, respectively. siPDGF-B- and siPDGFR-ß-containing nanoplexes indicated a significant decrease in mesangial cell migration and proliferation. These results suggested that mesangial cell proliferation may be inhibited by silencing of the PDGF-B signaling pathway. Gene silencing approaches with chitosan-based gene delivery systems have promise for the efficient treatment of renal disease.

p19(Arf) limits primary vitreous cell proliferation driven by PDGF-B.

Arf encodes an important tumor suppressor, p19(Arf), which also plays a critical role to control hyperplasia in the primary vitreous during mouse eye development. In the absence of Arf, mice are born blind and display a phenotype closely mimicking severe forms of the human eye disease, persistent hyperplastic primary vitreous (PHPV). In this report, we characterize p19(Arf) expression in perivascular cells that normally populate the primary vitreous and express the Arf promoter. Using a new ex vivo model, we show that these cells respond to exogenous Tgfß, despite being isolated at a time when Tgfß has already turned on the Arf promoter. Treatment of the cells with PDGF-B ligand doubles the population of cells in S-phase and ectopic expression of Arf blunts that effect. We show this effect is mediated through Pdgfrß as expression of Arf represses expression of Pdgfrß mRNA and protein to approximately 60%. p53 is not required for Arf-dependent blockade of PDGF-B driven proliferation and repression of Pdgfrß protein as ectopic expression of Arf is still able to inhibit the 2-fold increase in the S-phase fraction of cells upon treatment with PDGF-B. Finally, induction of mature miR-34a, a microRNA previously identified to be regulated by p19(Arf) does not depend on p53 while the expression of the primary transcript does require p53. These data corroborate that, as in vivo, p19(Arf) functions to inhibit PDGF-B driven proliferation ex vivo.

Anti-proliferative actions of 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone in vascular smooth muscle cells.

Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rß or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G(0)/G(1) phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G(0)/G(1) phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G(0)/G(1) phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.

Inhibitory effect of soluble platelet-derived growth factor receptor ß on intraosseous growth of breast cancer cells in nude mice.

Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor ß (PDGFRß) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRß signaling using soluble PDGFRß (sPDGFRß) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRß. A stable transfectant (TNBCT/Bo-sPDGFRß) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRß cells. Intratibial injection of TNBCT/Bo-sPDGFRß cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRß is useful for the treatment of breast cancer bone metastasis.

Regulation of ERK1/2 activity upon contact inhibition in fibroblasts.

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Despite its generally accepted importance for maintaining tissue homeostasis knowledge about the underlying molecular mechanisms of contact inhibition is still scarce. Since the MAPK ERK1/2 plays a pivotal role in the control of proliferation, we investigated regulation of ERK1/2 phosphorylation which is downregulated in confluent NIH3T3 cultures. We found a decrease in upstream signaling including phosphorylation of the growth factor receptor adaptor protein ShcA and the MAPK kinase MEK1/2 in confluent compared to exponentially growing cultures whereas involvement of ERK1/2 phosphatases in ERK1/2 inactivation is unlikely. Treatment of confluent, serum-deprived cultures with PDGF-B resulted in similar phosphorylation of ERK1/2 and induction of DNA-synthesis as detected in sparse, serum-deprived cultures. In contrast, ERK1/2 phosphorylation and DNA-synthesis could not be stimulated in confluent, serum-deprived cultures exposed to EGF. Our data indicate that PDGFR- and EGFR signaling are differentially inhibited in confluent cultures of NIH3T3 cells.