Vascular Endothelial Growth Factor siRNA Research Articles

Co-delivery of Doxorubicin and VEGF siRNA using carcinoembryonic antigen-targeted chitosan nanoparticles for colorectal cancer

Developing a targeted nanocarrier-based therapeutic approach is highly effective in delivering therapeutics to specific cancer cells. Combining two or more therapeutic approaches with different mechanisms of action can effectively maintain their unique benefits while considerably improving cancer treatment. Doxorubicin (DOX) binds to DNA by intercalation and triggers a cascade of biochemical processes in tumor cells leading to death. Anti-VEGF medicines may sensitize cancer cells to chemotherapeutics by limiting blood supply and increasing penetration by disrupting tumor vasculature. In this study, DOX and vascular endothelial growth factor siRNA (VEGF siRNA) loaded chitosan nanoparticles (ChNPs) were prepared by using the ionic gelation method. The DOX-siRNA-ChNPs surface was then functionalized with carcinoembryonic antigen (CEA) targeted monoclonal antibody (DOX-siRNA-ChNPs-CMAb), thereby delivering therapeutic moieties into the cancer milieu. The cytotoxicity and cellular uptake of the developed nanocarrier were tested in-vitro on HT-29, which revealed that DOX-siRNA-ChNPs-CMAb possessed the highest cytotoxicity when compared with other prepared formulations and free DOX. The cellular uptake using fluorescence microscopy displayed a significantly higher uptake of DOX-siRNA-ChNPs-CMAb (targeted) than free DOX and DOX-siRNA-ChNPs (non-targeted). Apoptosis analysis in flow cytometry using annexin-V/PI staining demonstrated a substantially higher apoptosis rate of HT-29 cells using DOX-siRNA-ChNPs-CMAb than DOX-siRNA-ChNPs. Moreover, DOX-siRNA-ChNPs-CMAb were studied in CRC induced animal models, showing that DOX-siRNA-ChNPs-CMAb specifically targets CRC cells without showing organ toxicity. Thus, the novel approach of simultaneous delivery of DOX and siRNA using CMAb conjugated ChNPs demonstrated significant targeting in both in-vitro and in-vivo with minimal organ toxicity.

Assessment of the synergic effect of Avastin and VEGF siRNA on inhibition of the cell proliferation, invasion and angiogenic factor expression of human breast cancer cells

Vascular endothelial growth factor (VEGF) is a key factor in angiogenesis, tumor growth and metastasis. Various small molecules and several humanized monoclonal antibodies against VEGF have been developed for inhibition of angiogenesis in different cancers. The aim of this work was to investigate the effect of combination of VEGF siRNA and Avastin on breast cancer MCF-7 cell line behavior.

Regulation of inflammation by VEGF/BDNF signaling in mouse retinal Müller glial cells exposed to high glucose.

The inflammatory changes seem to play an important role in the development of diabetic retinopathy (DR). Anti-VEGF therapy has been testified to inhibit inflammation in animal models of diabetes, but the detailed mechanisms during this process are not yet clear. Müller glial cells (MGCs) in the mammalian retina are deeply involved in DR, while the BDNF overexpression reduces inflammation in diabetic mice. In this research, we aimed to explore the relationship between VEGF and BDNF in mouse retinal MGCs during inflammation of diabetes. We examined the expression of glutamine-synthetase (GS), glial fibrillary acidic protein (GFAP), vascular-endothelial growth factor (VEGF), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) at different time points after mouse retinal MGCs exposed to high glucose (25mM). We also explored changes in the expression of brain-derived neurotrophic factor (BDNF), nuclear factor kappa B (NF-κB), IL-1β, and TNF-α in MGCs after treatments with anti-VEGF, VEGF siRNA, BDNF siRNA, BDNF recombination protein, and NF-κB inhibitor. In mouse retinal MGCs exposed to high glucose, BDNF was increased after treatments with anti-VEGF or VEGF siRNA. BDNF was decreased in MGCs from VEGF overexpressed mice. Moreover, the expressions of NF-κB, IL-1β, and TNF-α changed with BDNF: NF-κB, IL-1β, and TNF-α were increased after treatments with BDNF siRNA; NF-κB, IL-1β, and TNF-α were decreased after treatments with BDNF recombination protein. VEGF may regulate cytokines (IL-1β and TNF-α) by BDNF/NF-κB signaling pathway. The regulation of the VEGF/BDNF/NF-κB signaling pathway may be a significant therapeutic strategy for DR.

Cyclic peptides nanospheres: A ‘2-in-1′ self-assembled delivery system for targeting nucleus and cytoplasm

Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.

Endogenous microRNA triggered enzyme-free DNA logic self-assembly for amplified bioimaging and enhanced gene therapy via in situ generation of siRNAs

BackgroundSmall interfering RNA (siRNA) has emerged as a kind of promising therapeutic agents for cancer therapy. However, the off-target effect and degradation are the main challenges for siRNAs delivery. Herein, an enzyme-free DNA amplification strategy initiated by a specific endogenous microRNA has been developed for in situ generation of siRNAs with enhanced gene therapy effect on cervical carcinoma.MethodsThis strategy contains three DNA hairpins (H1, H2/PS and H3) which can be triggered by microRNA-21 (miR-21) for self-assembly of DNA nanowheels (DNWs). Notably, this system is consistent with the operation of a DNA logic circuitry containing cascaded “AND” gates with feedback mechanism. Accordingly, a versatile biosensing and bioimaging platform is fabricated for sensitive and specific analysis of miR-21 in HeLa cells via fluorescence resonance energy transfer (FRET). Meanwhile, since the vascular endothelial growth factor (VEGF) antisense and sense sequences are encoded in hairpin reactants, the performance of this DNA circuit leads to in situ assembly of VEGF siRNAs in DNWs, which can be specifically recognized and cleaved by Dicer for gene therapy of cervical carcinoma.ResultsThe proposed isothermal amplification approach exhibits high sensitivity for miR-21 with a detection limit of 0.25 pM and indicates excellent specificity to discriminate target miR-21 from the single-base mismatched sequence. Furthermore, this strategy achieves accurate and sensitive imaging analysis of the expression and distribution of miR-21 in different living cells. To note, compared to naked siRNAs alone, in situ siRNA generation shows a significantly enhanced gene silencing and anti-tumor effect due to the high reaction efficiency of DNA circuit and improved delivery stability of siRNAs.ConclusionsThe endogenous miRNA-activated DNA circuit provides an exciting opportunity to construct a general nanoplatform for precise cancer diagnosis and efficient gene therapy, which has an important significance in clinical translation.Graphic abstract

Dual-targeted lung cancer therapy via inhalation delivery of UCNP-siRNA-AS1411 nanocages.

Objective:Although great progress has been made in the field of siRNA gene therapy, safe, efficient, and targeted delivery of siRNA are still major challenges in siRNA therapeutics.Methods:We developed an up-conversion nanoparticle-based nanocage system. This system protected the siRNA from being degraded by nucleases in organisms and selectively delivered the siRNAs to the tumor sites, due to modifications of targeted molecules on the surfaces of nanocages and local inhalation.Results:The siRNAs delivered by the up-conversion nanoparticle nanocages were protected from degradation in transit to the tumor sites, where they accumulated. Compared with the passive target and control groups, the up-conversion nanoparticles based on the nanocage system showed a tumor suppressive effect after approximately 3 weeks of treatment.Conclusions:The up-conversion nanoparticle nanocages efficiently delivered vascular endothelial growth factor siRNAs to tumor sites. Mice with lung tumors treated with tumors targeting up-conversion nanoparticle nanocages showed steady body weight changes, high tumor inhibition ratios, and longer survival times.

Smart Polymeric Nanoparticles with pH-Responsive and PEG-Detachable Properties (II): Co-Delivery of Paclitaxel and VEGF siRNA for Synergistic Breast Cancer Therapy in Mice

BackgroundThe dual-loaded nano-delivery system can realize chemotherapeutic drug and small interfering RNA (siRNA) co-loading as well as enhance the therapeutic effect of drugs on tumors through a synergistic effect, while reducing their toxic and side effects on normal tissues.MethodsPreviously, we developed layered smart nanoparticles (NPs) to co-deliver survivin siRNA as well as small molecule drugs for lung cancer. In this study, we used such smart NPs to co-deliver paclitaxel (PTX) and siRNA against vascular endothelial growth factor (VEGF) gene for breast cancer therapy in mice models. For the prepared NPs, characterizations such as particle size, zeta potential, gel electrophoresis imaging and in vitro stability were investigated. Then, 4T1 cells were used to evaluate the in vitro VEGF silencing capacity, tumor cell inhibitory and anti-apoptotic abilities. Finally, an orthotopic model of mouse breast cancer was established to evaluate the in vivo antitumor effects and safety properties of PTX-siRNAVEGF-NPs.ResultsWe prepared PTX-siRNAVEGF-NPs with particle size of 85.25 nm, PDI of 0.261, and zeta potential of 5.25 mV. The NPs with VEGF siRNA effectively knocked down the expression of VEGF mRNA. Cell counting kit-8 (CCK-8) and apoptosis assays revealed that the PTX-siRNAVEGF-NPs exhibited antiproliferation effect of PTX on 4T1 cells. The in vivo anti-tumor study indicated that PTX-siRNAVEGF-NPs could exert an antitumor effect by inhibiting the formation and development of new blood vessels in tumor tissues, thereby cutting off nutrient and blood supplies required for tumor tissue growth. Both the anti-tumor efficacy and in vivo safety of the PTX-siRNAVEGF-NPs group were better than that of the PTX-NPs and siRNAVEGF-NPs groups.ConclusionThe combination of PTX and VEGF siRNA exerts good antitumor effect on 4T1 tumor cells. This study provides a theoretical and practical basis for breast cancer therapy.

Lactate Consumption via Cascaded Enzymes Combined VEGF siRNA for Synergistic Anti‐Proliferation and Anti‐Angiogenesis Therapy of Tumors

Lactate, as the most abundant component with concentrations of 4-40mm in tumors, contributes to the regulation of metabolic pathways, angiogenesis, and immunosuppression, exhibiting remarkable potential in cancer treatment. Therefore, a codelivery strategy that combined the cascaded enzymes Lactate oxidase/Catalase (LOx/CAT) and vascular endothelial growth factor (VEGF) siRNA (siVEGF) to suppress tumor proliferation and angiogenesis synergistically is creatively proposed. In brief, the cationic liposomes (LIP) encapsulated with LOx/CAT and siVEGF via hydrophilic interaction and electrostatic adsorption followed by coating with PEGylated phenylboronic acid (PP) is established (PPL@[LOX+CAT]). Moreover, a simple 3-aminophenylboronic acid (PBA)-shielded strategy via fructose (Fru) is applied to further enhance the targeting efficiency in the tumor site. The obtained co-encapsulated nanoparticles (NPs) can simultaneous intracellular release of LOx/CAT and siVEGF, and the collaborative use of LOx and CAT can promote lactate consumption even under a hypoxic tumor microenvironment (TME) without producing systemic toxicity. The combined application of lactate depletion and VEGF silencing demonstrated the efficient migration suppression of 4T1 cells in vitro and superior antitumor and antimetastatic properties in vivo. This work offers a promising tumor treatment strategy via integrating cascaded enzymes and gene therapy, and explores a promising therapy regimen for 4T1 triple-negative breast cancer.

Magnetic Resonance Imaging and Rna Interference of Tumor with Reducible Poly(Urethane-Amine) Nanocomposite System

Gadolinium-based small-molecule contrast agents are commonly utilized in clinics. However, these agents have a short retention time in vivo and possess a certain level of cytotoxicity. The reducible polymer poly(urethane-amine) can increase the relaxation rate and blood circulation time of small-molecule gadolinium chelates. Moreover, the polymer can be used as a gene carrier to bind small interfering RNA (siRNA). We synthesized an active targeting integrated diagnosis and treatment nanocomposite system with reducible poly(urethane-amine) as the carrier. This system incorporated functional units such as MRI contrast agent Gd-diethylenetriamine pentacetate (Gd-DTPA) and siRNA-vascular endothelial growth factor (siRNA-VEGF), and folic acid (FA) modified onto the surface of the polymer directed it to actively target tumor cells, thus improving the imaging effect of MRI. Meanwhlie, siRNA-VEGF carried by the system could reduce the expression of VEGF in tumor cells and inhibited tumor growth. The system showed good biocompatibility and was an integrated system of tumor diagnosis and treatment.

VEGF Production Is Regulated by the AKT/ERK1/2 Signaling Pathway and Controls the Proliferation of Toxoplasma gondii in ARPE-19 Cells.

The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.

Experimental abdominal aortic aneurysm growth is inhibited by blocking the JAK2/STAT3 pathway

BackgroundThe JAK/STAT pathway is a vital transcription signaling pathway that regulates gene expression and cellular activity. Our recently published study highlighted the role of IL-17A in abdominal aortic aneurysm (AAA) formation and rupture. IL-17A has been proven to upregulate vascular endothelial growth factor (VEGF) expression in some diseases. However, no study has demonstrated the relationships among JAK2/STAT3, IL-17A and VEGF. Therefore, we hypothesized that IL-17A may up-regulate VEGF expression via the JAK2/STAT3 signaling pathway to amplify the inflammatory response, exacerbate neovascularization, and accelerate AAA progression. MethodsTo fully verify our hypothesis, two separate studies were performed: i) a study investigating the influence of JAK2/STAT3 on AAA formation and progression. ii) a study evaluating the relationship among IL-17A, JAK2/STAT3 and VEGF. Human tissues were collected from 7 AAA patients who underwent open surgery and 7 liver transplantation donors. All human aortic tissues were examined by histological and immunohistochemical staining, and Western blotting. Furthermore, mouse aortic tissues were also examined by histological and immunohistochemical staining and Western blotting, and the mouse aortic diameters were assessed by high-resolution Vevo 2100 microimaging system. ResultsAmong human aortic tissues, JAK2/STAT3, IL-17A and VEGF expression levels were higher in AAA tissues than in control tissues. Group treated with WP1066 (a selective JAK2/STAT3 pathway inhibitor), IL-17A, and VEGF groups had AAA incidences of 25%, 40%, and 65%, respectively, while the control group had an incidence of 75%. Histopathological analysis revealed that the IL-17A- and VEGF-related inflammatory responses were attenuated by WP1066. Thus, blocking the JAK2/STAT3 pathway with WP1066 attenuated experimental AAA progression. In addition, in study ii, we found that IL-17A siRNA seemed to attenuate the expression of IL-17A and VEGF in vivo study; treatment with VEGF siRNA decreased the expression of VEGF, while IL-17A expression remained high. In an in vitro study, rhIL-17A treatment increased JAK2/STAT3 and VEGF expression in macrophages in a dose-dependent manner. ConclusionBlocking the JAK2/STAT3 pathway with WP1066 (a JAK2/STAT3 specific inhibitor) attenuates experimental AAA progression. During AAA progression, IL-17A may influence the expression of VEGF via the JAK2/STAT3 signaling pathway. This potential mechanism may suggest a novel strategy for nonsurgical AAA treatment.

Hypoxia Mediates Runt-Related Transcription Factor 2 Expression via Induction of Vascular Endothelial Growth Factor in Periodontal Ligament Stem Cells.

Periodontitis is characterized by the loss of periodontal tissues, especially alveolar bone. Common therapies cannot satisfactorily recover lost alveolar bone. Periodontal ligament stem cells (PDLSCs) possess the capacity of self-renewal and multilineage differentiation and are likely to recover lost alveolar bone. In addition, periodontitis is accompanied by hypoxia, and hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor in the response to hypoxia. Thus, we aimed to ascertain how hypoxia affects runt-related transcription factor 2 (RUNX2), a key osteogenic marker, in the osteogenesis of PDLSCs. In this study, we found that hypoxia enhanced the protein expression of HIF-1α, vascular endothelial growth factor (VEGF), and RUNX2 ex vivo and in situ. VEGF is a target gene of HIF-1α, and the increased expression of VEGF and RUNX2 proteins was enhanced by cobalt chloride (CoCl2, 100 μmol/L), an agonist of HIF-1α, and suppressed by 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1, 10 μmol/L), an antagonist of HIF-1α. In addition, VEGF could regulate the expression of RUNX2, as RUNX2 expression was enhanced by human VEGF (hVEGF165) and suppressed by VEGF siRNA. In addition, knocking down VEGF could decrease the expression of osteogenesis-related genes, i.e., RUNX2, alkaline phosphatase (ALP), and type I collagen (COL1), and hypoxia could enhance the expression of ALP, COL1, and osteocalcin (OCN) in the early stage of osteogenesis of PDLSCs. Taken together, our results showed that hypoxia could mediate the expression of RUNX2 in PDLSCs via HIF-1α-induced VEGF and play a positive role in the early stage of osteogenesis of PDLSCs.

The Effect and Mechanism of Vascular Endothelial Growth Fac-tor (VEGF) on Tumor Angiogenesis in Gallbladder Carcinoma

To investigate the effect of vascular endothelial growth factor (VEGF) on tumor angiogenesis in gallbladder carcinoma. Fifty one patients with gallbladder carcinoma were enrolled as observation group. Thirty healthy people were included as control group. Chemically synthesized siRNA sequences targeting VEGF was transfected with VEGF-siRNA. A blank group (group B), a negative control group (transfected with independent sequence, group C), and an inhibition group (transfected with VEGF siRNA, group D) were established. Physiological saline was set as group A. The expression of VEGF was detected by qRT-PCR. The expression of VEGF protein was detected by Western blot. MVD was used to measure microvessel density. CCK-8, Transwell and flow cytometry were used to detect cell proliferation, invasion and apoptosis. The tumor volume of nude mice and VEGF mRNA expression in group D was significantly smaller than that in group B and C (P<0.05). The MVD density in group B and C was significantly higher than that in group D (P<0.01). The proliferation of cells was detected from the 3rd day, and the proliferation of cells in the blank and negative control groups was faster than that of the inhibition group (P<0.05). The apoptosis rate of the blank group and the negative control group was lower than that of the inhibition group (P<0.001). VEGF is highly expressed in serum of patients with cholangiocarcinoma, it promotes angiogenesis, proliferation and invasion of gallbladder cancer cells, and inhibits apoptosis of tumor cells.

Development of a Biocompatible Copolymer Nanocomplex to Deliver VEGF siRNA for Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat. TNBC patients have significantly higher expression of vascular endothelial growth factor (VEGF) in tumors compared to non-TNBC patients. VEGF not only exerts its pro-angiogenic effects on endothelial cells but also acts as a survival and autocrine growth factor for VEGF receptor (VEGFR) expressing cancer cells. Silencing the expression of VEGF is therefore a potential therapy for TNBC.Methods: A novel biocompatible linear copolymer poly[bis(ε-Lys-PEI)Glut-PEG] (PLEGP) was developed to deliver VEGF siRNA for TNBC therapy. The copolymer is composed of lysine and glutaric acid, a natural metabolite of amino acids in the body. Low-molecular weight polyethyleneimine (PEI) was grafted to the copolymer to efficiently condense siRNA into nanocomplex without inducing cytotoxicity. Various in vitro studies were performed to evaluate the stability, cellular uptake, tumor penetration, and biological activities of the VEGF siRNA nanocomplex. The anti-tumor activities of the nanocomplex was also evaluated in an orthotopic TNBC mouse model.Results: PEIs with different molecular weights were evaluated, and the copolymer PLEGP1800 was able to easily form a stable nanocomplex with siRNAs and protect them from serum degradation. The siRNA/PLEGP1800 nanocomplex exhibited negligible cytotoxicity but showed high cellular uptake, high transfection efficiency, and high tumor penetration. In vitro activity studies showed that the siRNA nanocomplex significantly inhibited migration and invasion of TNBC cells. Moreover, the VEGF siRNA nanocomplex efficiently inhibited tumor growth in an orthotopic TNBC mouse model and down-regulated VEGF expression in the tumor.Conclusion: PLEGP1800 is a safe and efficient copolymer to deliver siRNAs for TNBC therapy. It could potentially be applied to other cancers by changing the cargo and incorporating tumor-specific ligands.

Combination antitumor immunotherapy with VEGF and PIGF siRNA via systemic delivery of multi-functionalized nanoparticles to tumor-associated macrophages and breast cancer cells

Given that vascular endothelial growth factor (VEGF) and placental growth factor (PIGF), over-expressed in breast cancer cells and M2-like tumor-associated macrophages (M2-TAMs) within tumor microenvironment (TME), work synergistically and independently in mediating tumor progression and immunosuppression, combinatorial immune-based approaches targeting them are expected to be a potent therapeutic modality for patients. Here, polyethylene glycol (PEG) and mannose doubly modified trimethyl chitosan (PEG = MT) along with citraconic anhydride grafted poly (allylamine hydrochloride) (PC)-based nanoparticles (NPs) (PEG = MT/PC NPs) with dual pH-responsiveness were developed to deliver VEGF siRNA (siVEGF)/PIGF siRNA (siPIGF) to both M2-TAMs and breast cancer cells for antitumor immunotherapy. With prolonged blood circulation and intelligent pH-sensitivity, PEG = MT/PC NPs were highly accumulated in tumor tissues and then internalized in M2-TAMs and breast cancer cells via mannose-mediated active targeting and passive targeting, respectively. With the charge-reversal of PC, PEG = MT/PC NPs presented effective endosomal/lysosomal escape and intracellular siRNA release, resulting in efficient gene silencing. Due to the synergism between siVEGF and siPIGF in anti-proliferation of tumor cells and reversal of the TME from pro-oncogenic to anti-tumoral, PEG = MT/PC/siVEGF/siPIGF NPs (PEG = MT/PC/siV-P NPs) exerted robust suppression of breast tumor growth and lung metastasis. This combination strategy may provide a promising alternative for breast cancer therapy.

Treatment of Arsenite Intoxication-Induced Peripheral Vasculopathy with Mesenchymal Stem Cells.

Arsenite (As), a notorious toxic metal, is ubiquitously distributed in the earth and poses a serious threat to human health. Histopathological lesions of As intoxication are known as thromboangiitis obliterans, which are resistant to current treatment and often lead to lower limb amputation. In this study, we attempt to find that treatment with mesenchymal stem cells (MSCs) may be effective for As-induced vasculopathy. We first conducted an in vitro study with a co-culture system containing human MSCs and human umbilical vein endothelial cells (HUVECs) and treated individual and co-cultured cells with various concentrations of arsenite. We also designed an in vivo study in which Sprague Dawley (SD) rats received periodic intraperitoneal (IP) injections of 16 ppm arsenite for 12 weeks. MSCs were harvested from BALB/c mice that were transplanted via tail vein injection. We found that there was significantly higher cellular viability in human mesenchymal stem cells (hMSCs) than in HUVECs under concentrations of arsenite between 15 and 25 μM. The Annexin V apoptosis assay further confirmed this finding. Cytokine array assay for As-conditioned media revealed an elevated vascular endothelial growth factor (VEGF) level secreted by MSCs, which is crucial for HUVEC survival and was evaluated by an siRNA VEGF knockdown test. In the in vivo study, we demonstrated early apoptotic changes in the anterior tibial vessels of As-injected SD rats with a Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, but these apoptotic changes were less frequently observed upon MSCs transplantation, indicating that the cytoprotective effect of MSCs successfully protected against As-induced peripheral vasculopathy. The feasibility of MSCs to treat and /or prevent the progression of As-induced vasculopathy is justified. Further clinical studies are required to demonstrate the therapeutic efficacy of MSCs in patients suffering from As intoxication with vasculopathy.

Vascular endothelial growth factor mediates ceramide 1-phosphate-stimulated macrophage proliferation

The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cell types including macrophages. However, the mechanisms involved in this action are not completely understood. In the present work we show that C1P stimulates the release of vascular endothelial growth factor (VEGF) in RAW264.7 macrophages, and that this growth factor is essential for stimulation of cell proliferation by C1P. The stimulation of VEGF release was dependent upon activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB-1 also known as Akt-1), and mitogen-activated protein kinase-kinase (MEK)/extracellularly regulated kinase-2 (ERK-2) pathways, as inhibition of these kinases with selective pharmacological inhibitors or with specific gene silencing siRNA, abrogated VEGF release. A key observation was that sequestration of VEGF with a neutralizing antibody, or treatment with VEGF siRNA abolished C1P-stimulated macrophage growth. Also, inhibition of the pathways involved in C1P-stimulated VEGF release inhibited the stimulation of macrophage growth by C1P. Moreover, blockade of VEGF receptor-2 (VEGFR-2), which is the primary receptor for VEGF, with the pharmacological inhibitor DMH4, or with specific VEGFR-2 siRNA, substantially inhibited C1P-stimulated cell growth. It can be concluded that stimulation of VEGF release is a key factor in the promotion of macrophage proliferation by C1P.

Glioma stem cells-derived exosomes promote the angiogenic ability of endothelial cells through miR-21/VEGF signal.

Glioma stem cells (GSCs) play an important role in glioblastoma prognosis. Exosomes (EXs) mediate cell communication by delivering microRNAs (miRs). Glioblastoma has a high level of miR-21 which could upregulate vascular endothelial growth factor (VEGF) expression. We hypothesized GSC-EXs can promote the angiogenic ability of endothelial cells (ECs) through miR-21/VEGF signal. GSCs were isolated from U-251 cells with stem cell marker CD133. GSCs transfected without or with scramble or miR-21 mimics were used to produce GSC-EXscon, GSC-EXssc and GSC-EXsmiR-21. Human brain ECs were co-cultured with vehicle, GSC-EXscon, GSC-EXssc or GSC-EXsmiR-21 plus VEGF siRNAs (siRNAVEGF). After 24 hours, the angiogenic abilities of ECs were evaluated. The levels of miR-21, VEGF and p-Flk1/VEGFR2 were determined. Results showed: 1) Over 90% of purified GSCs expressed CD133; 2) The levels of miR-21 and VEGF in GSCs and GSC-EXs were up-regulated by miR-21 mimic transfection; 3) Compared to GSC-EXscon or GSC-EXssc, GSC-EXsmiR-21 were more effective in elevating the levels of miR-21 and VEGF, and the ratio of p-Flk1/VEGFR2 in ECs; 4) GSC-EXsmiR-21 were more effective in promoting the angiogenic ability of ECs than GSC-EXscon or GSC-EXssc, which were remarkably reduced by siRNAVEGF pretreatment. In conclusion, GSC-EXs can promote the angiogenic ability of ECs by stimulating miR-21/VEGF/VEGFR2 signal pathway.

Dendrimer-Stabilized Gold Nanostars as a Multifunctional Theranostic Nanoplatform for CT Imaging, Photothermal Therapy, and Gene Silencing of Tumors.

Development of versatile nanomaterials combining diagnostic and therapeutic functionalities within one single nanoplatform is extremely important for tumor theranostics. In this work, the authors report the synthesis of a gold nanostar (Au NS)-based theranostic platform stabilized with cyclic arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified amine-terminated generation 3 poly(amidoamine) dendrimers. The formed RGD-modified dendrimer-stabilized Au NSs (RGD-Au DSNSs) are used as a gene delivery vector to complex small interfering RNA (siRNA) for computed tomography (CT) imaging, thermal imaging, photothermal therapy (PTT), and gene therapy of tumors. The results show that the RGD-Au DSNSs are able to compact vascular endothelial growth factor siRNA and specifically deliver siRNA to cancer cells overexpressing αv β3 integrin. Under near-infrared laser irradiation, the viability of cancer cells is only 20.2% after incubation with the RGD-Au DSNS/siRNA polyplexes, which is much lower than that of cells after single PTT or gene therapy treatment. Furthermore, in vivo results show that the RGD-Au DSNS/siRNA polyplexes enable tumor CT imaging, thermal imaging, PTT, and gene therapy after intratumoral injection. These results indicate that the developed multifunctional nanoconstruct is a promising platform for tumor imaging and combinational PTT and gene therapy.

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.