The objective of this study was to develop an efficient dual-ligand based PEGylated liposomal delivery system that had target specificity as well as properties that would enhance cellular uptake. ...PEGylated liposomes (PEG-LP) were prepared by the lipid film hydration method by adding distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG2000) to a lipid mixture. The cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand with affinity for Integrin α
vβ
3 was coupled to the distal end of the PEG on the PEG-LP (RGD-PEG-LP). Stearylated octaarginine (STR-R8) was incorporated on the surface of the RGD-PEG-LP as dual-ligand (R8/RGD-PEG-LP) that functions as a cell penetrating peptide (CPP). RGD-PEG-LP and R8/RGD-PEG-LP were preferentially taken up by caveolae-mediated and clathrin-mediated endocytosis pathways, respectively. Compared to PEG-LP, R8/RGD-PEG-LP showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin α
vβ
3 expressing cells. However, the amount of cellular uptake or gene expression by the single ligand versions was negligible, even in Integrin α
vβ
3 expressing cells. No remarkable difference in cellular uptake or gene expression was observed for cells in which the expression of targeted receptors was absent. It can be concluded that dual-ligand modified PEG-LP possesses a strong capability for the efficient internalization of PEG-LP and consequently would be an effective tool for the targeted delivery of macromolecules or chemotherapeutics through accelerated cellular uptake.
Display omitted
Heterogeneity of tumor endothelial cells Hida, Kyoko; Ohga, Noritaka; Akiyama, Kosuke ...
Cancer science,
November 2013, Letnik:
104, Številka:
11
Journal Article
Recenzirano
Odprti dostop
Tumor blood vessels play important roles in tumor progression and metastasis. Thus, targeting tumor blood vessels is an important strategy for cancer therapy. Tumor endothelial cells (TECs) are the ...main targets of anti‐angiogenic therapy. Although tumor blood vessels generally sprout from pre‐existing vessels and have been thought to be genetically normal, they display a markedly abnormal phenotype, including morphological changes. The degree of angiogenesis is determined by the balance between the positive and negative regulating molecules that are released by tumor and host cells in the microenvironment. Reportedly, tumor blood vessels are heterogeneous with TECs differing from normal endothelial cells (in contrast to the conventional view). We recently compared characteristics of different TECs isolated from highly and low metastatic tumors. We found TECs from highly metastatic tumors had more proangiogenic phenotypes than those from low metastatic tumors. Elucidating the variety of TEC phenotypes and identifying TEC molecular signatures should lead to more complete understanding of the mechanisms of tumor progression, discovery of new therapeutic targets, and development of biomarkers. This review considers current studies on TEC heterogeneity and discusses the therapeutic implications of these findings.
Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that ...biglycan, a small leucine‐rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC‐derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope‐type nanodevice (MEND), which contains a unique pH‐sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg–Gly–Asp–D–Phe–Lys) (cRGD) into MEND because αVβ3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD‐MEND‐encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS‐RC‐2 tumor‐derived TECs and induced in vitro RNAi‐mediated gene silencing. MEND was then injected intravenously into OS‐RC‐2 tumor‐bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT‐PCR indicated that biglycan was knocked down by biglycan siRNA‐containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA‐containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.
Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. In this study, we targeted biglycan in TECs using an in vivo siRNA delivery system known as a multifunctional envelope‐type nanodevice (MEND). We report, for the first time, that TEC‐specific marker inhibition using an in vivo siRNA delivery system can cause therapeutic effects in tumors.
Development of quantitative analysis software has enabled application of several standardised uptake values (SUV) for bone analysis in single photon emission computed tomography (SPECT). The present ...retrospective study aimed to develop a reliable method of monitoring bone inflammatory activity in antiresorptive agent-related osteonecrosis of the jaw (ARONJ) using SPECT quantitative analysis software. Fifteen ARONJ patients underwent SPECT before and after anti-inflammatory therapy. We calculated the mean maximum SUV (SUVmax) of the bilateral cranial bones using quantitative analysis software and used this as the control C. We attempted to adjust the SUVmax of the lesion L as follows: adjusted SUVmax (aSUVmax) = L - C. The optimum threshold to calculate the metabolic bone volume (MBV) (cm
) was C + 3. The threshold values obtained for each case were input to calculate MBV at each osteomyelitis site. Retrospectively, we compared aSUVmax and MBV of each patient's ARONJ before and after anti-inflammatory therapy. The patients' high aSUVmax or large MBV of the ARONJ reduced rapidly, reflecting individual clinical findings after treatment. Application of SPECT quantitative analysis software to monitor bone inflammatory activity in ARONJ could improve the prognosis-deciding abilities of clinicians and enable them to treat ARONJ effectively.
Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in ...pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to αVβ3 integrin, which is selectively expressed at high levels in TECs. We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5′ rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy.
Display omitted
Members of the bone morphogenetic protein (BMP) family have been implicated in the development and maintenance of vascular systems. Whereas members of the BMP-2/4 and osteogenic protein-1 groups ...signal via activin receptor-like kinase (ALK)-2, ALK-3 and ALK-6, BMP-9 and BMP-10 have been reported to bind to ALK-1 in endothelial cells. However, the roles of BMP-9-ALK-1 signaling in the regulation of endothelial cells have not yet been fully elucidated. Here, using various systems, we examined the effects of BMP-9 on the proliferation of endothelial cells. Vascular-tube formation from ex vivo allantoic explants of mouse embryos was promoted by BMP-9. BMP-9, as well as BMP-4 and BMP-6, also induced the proliferation of in-vitro-cultured mouse embryonic-stem-cell-derived endothelial cells (MESECs) by inducing the expression of vascular endothelial growth factor receptor 2 and Tie2, a receptor for angiopoietin-1. A decrease in ALK-1 expression or expression of constitutively active ALK-1 in MESECs abrogated and mimicked the effects of BMP-9 on the proliferation of MESECs, respectively, suggesting that BMP-9 promotes the proliferation of these cells via ALK-1. Furthermore, in vivo angiogenesis was promoted by BMP-9 in a Matrigel plug assay and in a BxPC3 xenograft model of human pancreatic cancer. Consistent with these in vivo findings, BMP-9 enhanced the proliferation of in-vitro-cultured normal endothelial cells from dermal tissues of adult mice and of tumor-associated endothelial cells isolated from tumor xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium tested in the present study via ALK-1.
Objectives
The aim of the present study was to evaluate the effectiveness of platelet-rich fibrin (PRF) as a wound-healing accelerator in patients undergoing oral bisphosphonate therapy and requiring ...tooth extractions.
Materials and methods
A total of 102 patients were divided into a PRF group and control group. The patients received oral bisphosphonate therapy for osteoporosis for an average of 32 months. Blood was collected and PRF was introduced into the socket of the PRF group only. Monitoring of mucosal healing was conducted for 3 months in both groups, and radiographic evaluation in the sockets was performed in the PRF group. Delayed recovery was defined as exposed bone and vulnerable granulation tissue without epithelization after 4 weeks and resolving by 8 weeks.
Results
There were no intraoperative complications, and none of the patients exhibited onset of medication-related osteonecrosis of the jaw (MRONJ). Delayed recovery was observed in 9 out of 73 control patients (12%), whereas 29 PRF patients exhibited complete epithelialization of the socket within 1 month. The prevalence of delayed recovery was significantly higher in the control group than the PRF group (
P
< 0.05). Multivariate logistic regression analysis revealed that risk factors and use of PRF were independent significant factors to relate to delayed recovery (
P
= 0.02).
Conclusions
Early epithelization was confirmed in all PRF patients. Thus, PRF may reduce the risk of delayed recovery in patients undergoing oral bisphosphonate therapy.
Clinical relevance
PRF may be useful in preventing MRONJ in patients receiving oral bisphosphonate (BP).
Tumor blood vessels are thought to contain genetically normal and stable endothelial cells (ECs), unlike tumor cells, which typically display genetic instability. Yet, chromosomal aberration in human ...tumor-associated ECs (hTECs) in carcinoma has not yet been investigated. Here we isolated TECs from 20 human renal cell carcinomas and analyzed their cytogenetic abnormalities. The degree of aneuploidy was analyzed by fluorescence in situ hybridization using chromosome 7 and chromosome 8 DNA probes in isolated hTECs. In human renal cell carcinomas, 22–58% (median, 33%) of uncultured hTECs were aneuploid, whereas normal ECs were diploid. The mechanisms governing TEC aneuploidy were then studied using mouse TECs (mTECs) isolated from xenografts of human epithelial tumors. To investigate the contribution of progenitor cells to aneuploidy in mTECs, CD133+ and CD133− mTECs were compared for aneuploidy. CD133+ mTECs showed aneuploidy more frequently than CD133− mTECs. This is the first report showing cytogenetic abnormality of hTECs in carcinoma, contrary to traditional belief. Cytogenetic alterations in tumor vessels of carcinoma therefore can occur and may play a significant role in modifying tumor- stromal interactions.
There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those ...resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.
Tumor angiogenesis is necessary for progression and metastasis of solid tumor. Tumor blood vessels are morphologically different from their normal counterparts. In this study, we isolated tumor ...endothelial cells (TECs) and revealed their abnormalities. We have compared the gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and found that several genes were upregulated in TECs. Expression of the chemokine receptor CXCR7 mRNA was higher in TECs than in NECs. However, information regarding the expression of CXCR7 in the tumor vessels of renal cell carcinoma is limited. CXCR7 and its ligand CXCL12 have been implicated in tumor cell survival. In this study, the expression of CXCR7 in the tumor vessels of renal cell carcinoma (RCC) was investigated. Real‐time PCR revealed higher expression level of CXCR7 in cultured TECs than in cultured NECs. Furthermore, similar to mouse TECs, immunostaining revealed strong expression of CXCR7 in vivo in human tumor vessels. These findings suggest that CXCR7 is a novel TEC marker and a target for antiangiogenic therapy for RCC.
PDF
