Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.     

Inhibition of corneal neovascularization by rapamycin

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.     

Selenadiazole Derivatives Inhibit Angiogenesis‐Mediated Human Breast Tumor Growth by Suppressing the VEGFR2‐Mediated ERK and AKT Signaling Pathways

Selenadiazole derivatives (SeDs) have been found to show promise in chemo‐/radiotherapy applications by activating various downstream signaling pathways. However, the functional role of SeDs on angiogenesis, which is pivotal for tumor progression and metastasis, has not yet been elucidated. In the present study, we have examined the antiangiogenic activities of SeDs and elucidated their underlying mechanisms. The results showed that the as‐synthesized SeDs not only enhanced their anticancer activities against several human cancer cells but also showed more potent inhibition on human umbilical vein endothelial cells (HUVECs). The in vitro results suggested that SeDs, especially 1 a , dose‐dependently inhibited the vascular endothelial growth factor (VEGF)‐induced cell migration, invasion, and capillary‐like structure formation of HUVECs. Compound 1 a also significantly suppressed VEGF‐induced angiogenesis in a Matrigel plug assay as part of a C57/BL6 mice assay by means of down regulation of VEGF. Furthermore, we found that 1 a significantly inhibited MCF‐7 human breast tumor growth in nude mice without severe systematic cytotoxicity. Compound 1 a was more effective in inhibiting cell proliferation and induced a much more pronounced apoptosis effect in endothelial cells than MCF‐7 cells, which implies that endothelial cells might be the primary target of 1 a . Further mechanistic studies on tumor growth inhibition effects and neovessel formation suppression demonstrated that 1 a inhibited cell viability of MCF‐7 and HUVECs by induction of cell apoptosis, accompanied by poly(adenosine diphosphate ribose)polymerase (PARP) cleavage and caspase activation. Additionally, the 1 a ‐induced antiangiogenesis effect was achieved by abolishing the VEGF‐VEGFR2‐ERK/AKT (ERK=extracellular signal–regulated kinases; AKT=protein kinease B) signal axis and enhanced the apoptosis effect by triggering reactive oxygen species (ROS)‐mediated DNA damage. Taken together, these results clearly demonstrate the antiangiogenic potency of SeDs and the underlying molecular mechanisms.     

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound- healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.     

Buffalo Colostrum β-lactoglobulin Inhibits VEGF-Induced Angiogenesis by Interacting with G protein-Coupled Receptor Kinase

β-lactoglobulin (β-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as β-lg. The sequences IIVTQ f[1–5] and LSFNPTQLEEQCHV f(149–162) of m/z 933⁺ and 851²⁺ were found to match N- and C-extreme of β-lg while IDALNENK f(84–91) and TPEVDDEALEKFDK f(125–138) sequences deduced for m/z 916⁺ and 818²⁺ were in compliance to buffalo milk β-lg. Considering the sequence similarity of β-lg to glycodelin, a proven angiogenic protein, similar role for β-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled β-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64 kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.     

人工细胞外基质对血管内皮细胞生存的影响

通过物理吸附方法, 利用胶原、 聚赖氨酸和融合蛋白VEGF-Fc对聚苯乙烯培养板表面进行改性, 以研究细胞外基质材料对血管内皮细胞的影响. 结果表明, 3种蛋白显著提高了聚苯乙烯表面的亲水性. 内皮细胞的黏附、 增殖、 细胞骨架蛋白染色和血管性血友病因子(vWF)免疫染色实验结果表明, 胶原、 聚赖氨酸和VEGF-Fc基质均能有效提高血管内皮细胞的黏附, 其中胶原可与VEGF协同作用促进内皮细胞分化表型的表达; VEGF-Fc基质兼具了VEGF的生物学活性, 可促进内皮细胞的黏附和增殖以及vWF功能性蛋白的表达. 本研究为诱导材料表面内皮化和血管新生的生物活性材料的设计开发提供了新思路.     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G₀/G₁ cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.     

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.     

Surface Modification of Multiple Bioactive Peptides to Improve Endothelialization of Vascular Grafts

Endothelialization is an effective approach to prevent thrombus formation and enhance vascular graft survival. Surface modification of biomolecules has been proved to be effective in regulating endothelial cell behaviors. In this study, several peptides including YIGSR, RGD, and REDV sequences are covalently immobilized on the surface of electrospun silk fibroin scaffolds and the effects of combined application of these peptides on cell behaviors are studied. The results show that, compared with the scaffolds modified with single peptides, the scaffolds modified with dual peptides (YIGSR+RGD) could significantly enhance the proliferation of human umbilical vein endothelial cells (HUVECs). However, the combination of REDV+RGD or YIGSR+REDV does not promote the adhesion or proliferation of HUVECs. Notably, YIGSR‐modified scaffolds improved HUVEC migration significantly in comparison to REDV‐ or RGD‐modified groups. Moreover, its combination with either of these two peptides also presents excellent effect on cell migration. Thus, all the data suggest that the combined application of peptides might be a promising method to enhance the endothelialization of small‐diameter vascular grafts.     

Possible mechanism of the stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells: involvement of basic fibroblast growth factor

To investigate the possible mechanism of the stimulatory effect of a hot water extract from Artemisia leaf (Artemisia princeps PANPANINI) (AFE) on the proliferation of endothelial cells, cells from bovine aorta were cultured for 72 h in RPMI1640 medium supplemented with 10% fetal calf serum in the presence of 5 micrograms/ml AFE. The AFE treatment significantly increased the cell number after culture, while in the presence of 10 micrograms/ml unfractionated heparin, AFE conversely decreased it. This implied that AFE enhanced the cell growth promotion by basic fibroblast growth factor (bFGF). The accumulation of bFGF was significantly increased in the culture medium, in the low-affinity (glycosaminoglycans-binding) fraction, and in the cell extract fraction, but was unchanged in the high-affinity (receptor-binding) fraction. The contents of [35S]sulfate-labeled glycosaminoglycans in both cell layer and the medium were not increased by AFE treatment. The proliferation of A10 cells, an established cell line of smooth muscle cells from murine aorta, was not stimulated by AFE. A10 cells did not produce a significant amount of bFGF in the presence or absence of AFE. Thus, the production of bFGF was considered to be involved in AFE stimulation of cell proliferation. In conclusion, it was suggested that AFE stimulated endothelial cell proliferation by increasing the production of bFGF rather than by an increase in the number of bFGF receptors and the content of glycosaminoglycans in the cell layer. The enhanced reserve of bFGF in the low-affinity fraction of cell layer and in the medium would cause the AFE-stimulated proliferation of endothelial cells.     

Release of prostaglandin E(1) from N-(2-hydroxypropyl)methacrylamide copolymer conjugates by bone cells

Bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-PGE(1) conjugates, containing cathepsin K sensitive spacers, were incubated with induced osteoclasts and osteoblasts, their precursors, and control non-skeletal cells. The release of PGE(1) was monitored by an HPLC assay. In both murine and human cell lines, osteoclasts appeared to be the most active cells in the cleavage (PGE(1) release). Incubation with osteoblasts also resulted in fast PGE(1) release, whereas precursor and control cells released PGE(1) with a substantially slower rate than bone cells (apparently through ester bond cleavage). Experiments in the presence of inhibitors revealed that other enzymes, in addition to cathepsin K, were participating in the cleavage of the conjugate. Confocal fluorescence studies exposed internalization of the conjugate by endocytosis with ultimate localization in the lysosomal/endosomal compartment.     

The role of adiponectin in the production of IL-6, IL-8, VEGF and MMPs in human endothelial cells and osteoblasts: implications for arthritic joints

This study was performed to evaluate the contribution of adiponectin to the production of interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-13 in human endothelial cells and osteoblasts in arthritic joints. Cultured human umbilical vascular endothelial cells (HUVECs) and osteoblasts were stimulated with adiponectin (1 or 10 μg ml⁻¹) or IL-1β (0.1 ng ml⁻¹) in the presence or absence of hypoxia for 24 h. The protein expression patterns were examined by analyzing culture supernatants using the enzyme-linked immunosorbent assay (ELISA). Adiponectin significantly stimulated the production of VEGF, MMP-1 and MMP-13 in osteoblasts but not in endothelial cells, whereas it significantly stimulated the production of IL-6 and IL-8 in both endothelial cells and osteoblasts. The increase in VEGF production induced by adiponectin was significantly greater than that induced by IL-1β. The production of IL-6 and IL-8 in adiponectin-stimulated endothelial cells was approximately 10-fold higher than that in IL-1β-stimulated endothelial cells; in osteoblasts, adiponectin-induced IL-6 and IL-8 secretion was approximately twofold higher than that induced by IL-1β. In addition, IL-8 production in endothelial cells was approximately sevenfold higher than in osteoblasts. However, IL-6 levels were similar between the two cell types, suggesting that adiponectin may be involved in the production of IL-8 in endothelial cells, which may have an important role in neutrophil recruitment to arthritic joints. Furthermore, the increases in protein expression induced by adiponectin were differentially regulated by hypoxia. In conclusion, adiponectin has a more important role than does IL-1β in the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts at physiological concentrations.     

Honokiol ameliorates endothelial dysfunction through suppression of PTX3 expression,a key mediator of IKK/IκB/NF-κB,in atherosclerotic cell model

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IκB kinase (IKK)/IκB/nuclear factor-κB (NF-κB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IκB phosphorylation and the expression of two NF-κB subunits (p50 and p65) in the IKK/IκB/NF-κB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.     

Effects of Various Cell Surface Engineering Reactions on the Biological Behavior of Mammalian Cells

Cell surface engineering technologies can regulate cell function and behavior by modifying the cell surface. Previous studies have mainly focused on investigating the effects of cell surface engineering reactions and materials on cell activity. However, they do not comprehensively analyze other cellular processes. This study exploits covalent bonding, hydrophobic interactions, and electrostatic interactions to modify the macromolecules succinimide ester-methoxy polyethylene glycol (NHS-mPEG), distearoyl phosphoethanolamine-methoxy polyethylene glycol (DSPE-mPEG), and poly-L -lysine (PLL), respectively, on the cell surface. This work systematically investigates the effects of the three surface engineering reactions on the behavior of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts, including viability, growth, proliferation, cell cycle, adhesion, and migration. The results reveals that the PLL modification method notably affects cell viability and G2/M arrest and has a short modification duration. However, the DSPE-mPEG and NHS-mPEG modification methods have little effect on cell viability and proliferation but have a prolonged modification duration. Moreover, the DSPE-mPEG modification method highly affects cell adherence. Further, the NHS-mPEG modification method can significantly improve the migration ability of HUVECs by reducing the area of focal adhesions. The findings of this study will contribute to the application of cell surface engineering technology in the biomedical field.