Specimens were observed under a fluorescence microscope. was then injected intravenously into OS\RC\2 tumor\bearing FJX1 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. siRNA delivery system known as a multifunctional envelope\type nanodevice (MEND). We report, for the first time, that TEC\specific marker inhibition using an siRNA delivery system can cause therapeutic effects in tumors. AbbreviationsDTDiphtheria toxinECEndothelial cellsEGFEpidermal growth factorEGFREpidermal growth factor receptorMENDMultifunctional envelope\type nano deviceNECNormal endothelial cellsTECTumor endothelial cellsVEGFVascular endothelial growth factor 1.?INTRODUCTION Angiogenesis, the formation of new blood vessels, plays a vital role in tumor progression. Angiogenic factors such as VEGF are released from tumor cells and other stromal cells PX20606 trans-isomer to stimulate the formation of new capillaries for tumorigenesis. Without angiogenesis, tumors remain dormant at diameters of 1C2?mm. 1 Dr. Folkman proposed antiangiogenic therapy as an anticancer therapy. 2 The important targets of antiangiogenic therapy are TECs, which line the inner surface of tumor blood vessels. Anti\VEGF drugs such as bevacizumab, which is a humanized anti\VEGF monoclonal antibody, 3 have become successful therapeutics and have improved clinical outcomes. However, as VEGF signaling is also required in normal physiologic angiogenesis, anti\VEGF agents have several adverse effects. 4 Therefore, understanding TEC biology is essential for developing novel antiangiogenic drugs that specifically target TECs with less harmful effects on NECs. In the past two decades, it has been reported that TECs exhibited some abnormal phenotypes compared with NECs. 5 For instance, NEC and TEC gene expression patterns are different. 6 , 7 , 8 , 9 , 10 , 11 TECs proliferate and migrate faster than NECs. 12 , 13 Several upregulated genes in TECs are associated with angiogenesis. 11 , 14 , 15 , 16 , 17 , 18 Targeting these molecules may serve as a novel antiangiogenic therapy. Biglycan, a member of the small leucine\rich proteoglycan family, exists in the ECM as a matrix component and an essential signaling molecule. 19 It is also known as a damage\associated molecular pattern. Secreted biglycan binds with Toll\like receptor 2 (TLR2) and TLR4 on immune cells such as macrophages, causing an inflammatory response through the nuclear factor\kappa B (NF\B) pathway. 20 We previously observed that biglycan expression was upregulated in TECs of several types of tumors such as melanoma 14 , 21 and breast cancer 22 in the murine model and renal, lung, colon, and liver cancers in human clinical cases. 14 , 21 TECs utilize biglycan in an autocrine manner for migration through TLRs. 14 Biglycan secreted from TECs actively promoted tumor cell intravasation in a paracrine manner and caused distant metastasis. 21 Therefore, biglycan in TECs is a potential target PX20606 trans-isomer for cancer therapy. However, no biglycan inhibitors, including compounds and neutralizing antibodies, are commercially available. RNAi is a useful tool for target\specific inhibition because it regulates target genes in a sequence\dependent PX20606 trans-isomer manner. delivery of siRNA to the target organ is potentially a powerful tool for therapeutic applications. PX20606 trans-isomer 23 However, siRNA delivery requires several carriers such as nanodrug delivery systems (DDSs) because siRNA is rapidly degraded in the body. 24 Nano DDSs can target the blood vessels of tumor tissues as they can target an organ using functional devices. 25 We previously developed a liposomal siRNA system known as the multifunctional envelope\type nanodevice (MEND). 26 , 27 , 28 , 29 We incorporated cyclo(ArgCGlyCAspCDCPheCLys) (cRGD) into MEND to specifically target TECs because V3 integrin, a cRGD receptor, is selectively expressed in TECs at high levels. 30 This RGD\MEND was used to specifically deliver siRNA into tumor blood vessels. 31 VEGFR2?silencing using the TEC\targeting RGD\MEND induced antiangiogenic effect and antitumor effect. 31 , 32 In this study, this TEC\targeting RGD\MEND was used to investigate the therapeutic effect of biglycan inhibition in TECs. 2.?MATERIALS AND METHODS 2.1. Isolation of TECs and NECs and cell culture The human renal clear cell carcinoma cell line.