VEGFR endocytosis regulates the angiogenesis in a mouse model of hindlimb ischemia.

BACKGROUND: The regulation of angiogenesis in the treatment of cardiovascular diseases has been widely studied and the vascular endothelial growth factor (VEGF) families and VEGF receptor (VEGFR) have been proven to be one of the key regulators. The VEGFR endocytosis has been recently proved to be involved in the regulation of angiogenesis. Our previous study showed that the upregulation of VEGFR endocytosis enhanced angiogenesis in vitro. In this research, we utilized mice with induced hindlimb ischemia, as a model to investigate the role of VEGFR endocytosis in the regulation of angiogenesis in vivo. Our goal was to observe the effect of revascularization with different degrees of VEGFR endocytosis after injecting atypical protein kinase C inhibitor (αPKCi) and dynasore, which could respectively promote and inhibit the VEGFR endocytosis. METHODS: We induced the hindlimb ischemia in adult male mice by ligating the hindlimb artery. By directly injecting the ischemic muscles with endothelial progenitor cells (EPCs) alone or EPCs + αPKCi/EPCs + dynasore or control medium (sham group), we divided the mice into four groups and detected lower limb blood flow using a laser Doppler blood perfusion imager. We also measured the immunohistochemistry (IHC) of markers for angiogenesis, such as CD31 and alpha smooth muscle actin (α-SMA) in the ischemic hindlimb tissues. RESULTS: We demonstrated VEGFR endocytosis played an important role in the angiogenesis of the ischemic hindlimb model in vivo. By using atypical PKC inhibitor that increase the VEGFR endocytosis, the angiogenesis in the mice model was promoted. Treatment with EPCs + αPKCi showed greater effects on blood perfusion recovery and increased the α-SMA-positive vessels. CONCLUSIONS: The regulation of VEGFR endocytosis represents a valuable method of improving angiogenesis and thus revascularization in ischemic disease model.

Cell-permeable NF-κB inhibitor-conjugated liposomes for treatment of glioma.

Application of liposomes-based drug delivery in treatment of glioma has been hampered by the poor permeability of blood-brain barrier and the low uptake efficiency by glioma tissues. Moreover, many chemotherapy drugs promote the activation of the NF-κB, which plays a role in the development and progression of cancer and chemoresistance. In this report, CB5005 peptide, designed for its dual function in cell membrane penetration and NF-κB inhibition, was conjugated to PEGylated liposomes loaded with doxorubicin (CB5005-LS/DOX) or a fluorescent dye (CB5005-LS/dye). These CB5005-modified liposomes were utilized for targeting and penetrating glioma. Both qualitative and quantitative evaluations of CB5005-LS/dye showed that modification by CB5005 significantly increased cellular uptake of the liposomes by glioma cells, and substantially improved permeability of the liposomes into tumor spheroids. Intracellular localization studies demonstrated that CB5005-modified liposomes could not only penetrate into glioma cells but also deliver DOX into the nucleus. Cytotoxicity assay indicated that compared with the unmodified DOX liposomes (LS/DOX), CB5005-LS/DOX increased the efficiency of killing glioma cells by more than fivefold. In vivo imaging illustrated that CB5005-modified liposomes, via intravenous injection, distributed fluorescence into the brain and accumulated at tumor xenograft and intracranial glioblastoma in different animal models. More importantly, CB5005-LS/DOX treatment significantly prolonged the survival time of nude mice bearing intracranial glioblastoma. In summary, CB5005-modified liposomes represent a promising drug delivery system for cancer treatment attributing to its unique ability not only to transfer drugs to the tumor sites but also to function as a synergist for chemotherapy of glioma and other human tumors.

The role of the Wnt/ß-catenin signaling pathway in the proliferation of gold nanoparticle-treated human periodontal ligament stem cells.

BACKGROUND: Several studies have confirmed that gold nanoparticles (AuNPs) of specific concentration and size exert a boosting effect on cell proliferation; however, the mechanism through which this effect occurs remains unknown. This study explores the canonical Wnt signaling pathway in AuNP promotion of human periodontal ligament stem cell (hPDLSC) proliferation. METHODS: MTS was employed to evaluate hPDLSC proliferation. The interference of LRP5 and ß-catenin was steered by shRNA plasmids and siRNA, respectively, at which point the expression of MYC, CCND1, AXIN2, and POU5F1 had been estimated via real-time PCR. The expressions of LRP5 and ß-catenin were detected via western blot assay. RESULTS: The proliferation of hPDLSCs treated with 60 nm AuNPs at 56 µM was clearly elevated. In contrast, ß-catenin siRNA significantly decreased cell viability. The LRP5 shRNA plasmid did not consistently impact cells. The expressions of these four genes downstream of the Wnt/ß-catenin signaling pathway were not significantly overexpressed in response to the interference of shRNA plasmid/siRNA with the treatment of AuNPs. CONCLUSIONS: These results suggest that the Wnt/ß-catenin signaling pathway plays a significant role in the process of AuNP promotion of hPDLSC proliferation.

Targeting Negative Surface Charges of Cancer Cells by Multifunctional Nanoprobes.

A set of electrostatically charged, fluorescent, and superparamagnetic nanoprobes was developed for targeting cancer cells without using any molecular biomarkers. The surface electrostatic properties of the established cancer cell lines and primary normal cells were characterized by using these nanoprobes with various electrostatic signs and amplitudes. All twenty two randomly selected cancer cell lines of different organs, but not normal control cells, bound specifically to the positively charged nanoprobes. The relative surface charges of cancer cells could be quantified by the percentage of cells captured magnetically. The activities of glucose metabolism had a profound impact on the surface charge level of cancer cells. The data indicate that an elevated glycolysis in the cancer cells led to a higher level secretion of lactate. The secreted lactate anions are known to remove the positive ions, leaving behind the negative changes on the cell surfaces. This unique metabolic behavior is responsible for generating negative cancer surface charges in a perpetuating fashion. The metabolically active cancer cells are shown to a unique surface electrostatic pattern that can be used for recovering cancer cells from the circulating blood and other solutions.

Functionalized cell nucleus-penetrating peptide combined with doxorubicin for synergistic treatment of glioma.

UNLABELLED: Clinical application of cell-penetrating peptides (CPPs) in cancer therapy is greatly restricted due to lack of tissue selectivity and tumor-targeting ability. CB5005, a rationally designed CPP that targets and inhibits intracellular NF-κB activation, is constituted by a unique membrane-permeable sequence (CB5005M) cascading to a NF-κB nuclear localization sequence (CB5005N). In vitro cellular evaluation confirmed that CB5005 was effectively taken up by brain capillary endothelial cell bEnd.3 and glioma cells U87. The intracellular localization analysis further demonstrated that CB5005 could not only penetrate into the cells but also enter into their nuclei. More interestingly, CB5005 permeated deeply into the tumor spheroids of U87 cell. In vivo imaging illustrated that the fluorescence-labeled CB5005 distributed itself into the brain and accumulated at the tumor site after intravenous injection. Given the important role of over expressed NF-κB in tumor growth and development, we further investigated CB5005 for its potential in treatment of glioma. When combined administration in vitro with doxorubicin (DOX), CB5005 exhibited a synergistic effect in killing U87 cells. In a nude mice xenograft model, CB5005 inhibited the growth of tumor when applied alone, and displayed a synergistic anti-tumor effect with DOX. In conclusion, CB5005 functioned simultaneously as a cell penetrating peptide and a tumor growth inhibitor, therefore can work as a potential synergist for chemotherapy of human tumor. STATEMENT OF SIGNIFICANCE: Clinical application of cell-penetrating peptides in cancer therapy is restricted due to lack of tissue selectivity and tumor-targeting ability. In this manuscript, we reported a rationally designed peptide, named CB5005, which had an attractive capability of translocation into the cell nucleus and blocking nuclear translocation of endogenous NF-κB protein. CB5005 had unique affinity with brain and glioma, and could rapidly accumulate in these tissues after intravenous injection. Furthermore, CB5005 showed a synergistic effect on inhibiting gliomas when administrated with doxorubicin. This is the first literature report on this multi-functionalized peptide, which can work as a potential synergist for chemotherapy of tumor. This work should be of general interest to scientists in the fields of biomaterials, biology, pharmacy, and oncology.

S100A4 upregulation suppresses tissue ossification and enhances matrix degradation in experimental periodontitis models.

AIM: S100A4, also known as fibroblast-specific protein 1 or metastasin 1, is not only highly expressed in growth-stimulated cultured cells and metastatic tumor cells, but also in the periodontal ligament. The aim of this study was to investigate the roles of S100A4 in the pathogenesis of periodontitis and its regulatory mechanisms in inflammatory milieu. METHODS: Experimental periodontitis was induced in rats by submarginal silk ligatures. TRAP activity and S100A4 expression in periodontal ligaments were examined using immunohistochemistry and immunofluorescence methods. IL-1ß-treated human periodontal ligament cells (hPDLCs) were used as in vitro model of experimental periodontitis. S100A4 mRNA and protein were assessed using qRT-PCR and Western blot, respectively. hPDLCs were transfected with either S100A4 overexpression plasmids or shRNAs plasmids. The mineralization in hPDLCs was evaluated with a 12-d osteogenic induction assay, and the expression of ALP, OCN, MMP-2 and MMP-13 was analyzed by qRT-PCR. RESULTS: In the periodontal ligaments of rats with experimental periodontitis, TRAP activity and S100A4 protein staining were considerably more intense compared with those in the control rats. Treatment of hPDLCs with IL-1ß (10, 50 and 100 ng/mL) dose-dependently increased the mRNA and protein levels of S100A4. Transfection with shRNAs markedly increased mineralized nodule formation and the osteogenic-related markers ALP and OCN levels in hPDLCs, whereas the overexpression of S100A4 significantly reduced mineralized nodule formation, and increased the matrix degradation enzymes MMP-2 and MMP-13 levels in hPDLCs. CONCLUSION: S100A4 is upregulated in the experimental rat periodontitis and in IL-1ß-treated hPDLCs, where S100A4 suppresses osteogenic differentiation and enhances matrix degradation. Thus, S100A4 is a potential target for the treatment of periodontitis.

Inhibition of glioma proliferation and migration by magnetic nanoparticle mediated JAM-2 silencing.

Brain invasion is a biological hallmark of glioma that leads to its aggressiveness and prognosis. Junctional adhesion molecule-2 (JAM-2) was found to be overexpressed in human glioma. In this study, the effects of JAM-2 silencing mediated by cell-penetrating magnetic nanoparticles were investigated on glioma cell proliferation and migration in vitro and in vivo. The results showed that the deregulation of JAM-2 in glioma cell lines could cause a dramatic decrease in cell proliferation and migration in vitro. The expression level of cytoskeleton remodeling and migration associated protein genes appeared to be a downstream factor of JAM-2. Furthermore, silencing of JAM-2 expression in implanted glioma cells was found to impair in vivo tumor growth significantly. These data provide new evidence for the role of JAM-2 in the progression of glioma and show its great potential in human glioma gene therapeutics.

Bioreducible polyethylenimine-delivered siRNA targeting human telomerase reverse transcriptase inhibits HepG2 cell growth in vitro and in vivo.

One new siRNA sequence was found efficient for human telomerase reverse transcriptase (hTERT) gene silencing in vitro in five types of human cancer cells. Then, a biodegradable polyethylenimine containing multiple disulfide bonds (SS-PEI) was successfully applied as a potent non-viral carrier for intracellular delivery of the hTERT siRNA in vitro and in vivo. The SS-PEI could strongly bind siRNA to form nano-sized and positively-charged complexes, but which were readily destabilized to sufficiently release siRNA in a reducing environment. Transfection experiments showed that the complexes of SS-PEI/hTERT siRNA were able to transfect HepG2 cells in vitro, inducing reduced levels of hTERT mRNA and hTERT protein, decreased telomerase activity, cell growth inhibition and significant cell apoptosis. Besides, treatment with the complexes of SS-PEI/hTERT siRNA could inhibit HepG2 tumor growth in a xenograft mouse model. Importantly, the SS-PEI revealed relatively low cytotoxicity in vitro and at an appropriate dose had no adverse effect on liver and kidney functions in vivo. The results of this study indicate that SS-PEI/siRNA-induced hTERT gene silencing provides a promising method for human cancer gene therapy.

Highly efficient drug delivery nanosystem via L-phenylalanine triggering based on supramolecular polymer micelles.

An intelligent drug delivery nanosystem has been developed based on biodegradable supramolecular polymer micelles (SMPMs). The drug release can be triggered from SMPMs responsively by a bioactive agent, L-phenylalanine in a controlled fashion. The SMPMs are constructed from ethylcellulose-graft-poly(ε-caprolactone) (EC-g-PCL) and α-cyclodextrin (α-CD) derivate via host-guest and hydrophobic interactions. It has been found that these SMPMs have disassembled rapidly in response to an additional L-phenylalanine, due to great affinity discrepancy to α-CD between L-phenylalanine and PCL. Experiments have been carried out on trigger-controlled in vitro drug release of the SMPMs loaded with a model porphyrin based photosensitizer THPP. The result shows that the SMPMs released over 85% THPP in 6 h, which is two orders magnitudes faster than that of control. Also investigated is the photodynamic therapy (PDT) of THPP-loaded SMPMs with and without L-phenylalanine on MCF-7 carcinoma cell line. An effective trigger-concentration dependent lethal effect has been found showing promise in clinical photodynamic therapy.

Preparation of highly fluorescent magnetic nanoparticles for analytes-enrichment and subsequent biodetection.

Bifunctional nanoparticles with highly fluorescence and decent magnetic properties have been widely used in biomedical application. In this study, highly fluorescent magnetic nanoparticles (FMNPs) with uniform size of ca. 40 nm are prepared by encapsulation of both magnetic nanoparticles (MNPs) and shell/core quantum dots (QDs) with well-designed shell structure/compositions into silica matrix via a one-pot reverse microemulsion approach. The spectral analysis shows that the FMNPs hold high fluorescent quantum yield (QY). The QYs and saturation magnetization of the FMNPs can be regulated by varying the ratio of the encapsulated QDs to MNPs. Moreover, the surface of the FMNPs can be modified to offer chemical groups for antibody conjugation for following use in target-enrichment and subsequent fluorescent detection. The in vitro immunofluorescence assay and flow cytometric analysis indicate that the bifunctional FMNPs-antibody bioconjugates are capable of target-enrichment, magnetic separation and can also be used as alternative fluorescent probes on flow cytometry for biodetection.