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Papers by CRISTIAN RODRIGUEZ

Journal of Allergy and Clinical Immunology, 2020

BACKGROUND Fibrostenosis, the most serious eosinophilic esophagitis (EoE) complication, is mediat... more BACKGROUND Fibrostenosis, the most serious eosinophilic esophagitis (EoE) complication, is mediated by epithelial-mesenchymal transition (EMT). Transitioned cells contribute to pathogenesis by overproducing extracellular matrix. OBJECTIVE To determine whether RPC4046 (anti‒IL-13 monoclonal antibody) modulates EMT biomarkers in biopsies from adults with active EoE in a substudy of a double-blind, placebo-controlled phase 2 trial. METHODS Baseline and week-16 esophageal biopsies were taken from 69 patients randomized to weekly subcutaneous RPC4046 180 mg (n=19), 360 mg (n=26), or placebo (n=24). Duplex immunofluorescence slides stained for e-cadherin and vimentin were digitally analyzed by mapping each epithelial cell and recording fluorescence intensities. Endpoints included change from baseline to week 16 in percentage vimentin-positive epithelial cells (primary), total e-cadherin, and vimentin:e-cadherin ratio per cell (average of 47,000 cells/biopsy analyzed). RESULTS Mean percentage vimentin-positive cells decreased 0.94%, 2.75%, and 4.24% in the placebo, low-dose, and high-dose groups (P = 0.032, high-dose vs placebo). Mean e-cadherin expression per cell increased 5.6-fold in both dose groups versus placebo (high-dose P = 0.047). Increases in e-cadherin per cell from baseline to week 16 were correlated with improvements in histology, eosinophil counts, endoscopic findings, and symptoms. CONCLUSION RPC4046 significantly reduced EMT markers in adults with active EoE, with greater effects at 360 mg. Together with results for eosinophil density and clinical endpoints from the main trial, these data support the hypothesis that pharmacologic IL-13 inhibition ameliorates both inflammatory and remodeling pathways, and could potentially reduce the risk of fibrostenotic complications.

Clinical Gastroenterology and Hepatology, 2020

BACKGROUND & AIMS: The short-term efficacy of RPC4046, a monoclonal antibody against interleukin-... more BACKGROUND & AIMS: The short-term efficacy of RPC4046, a monoclonal antibody against interleukin-13, has been shown in patients with eosinophilic esophagitis (EoE). We investigated the long-term efficacy and safety of RPC4046 in an open-label, long-term extension (LTE) study in adults with EoE. METHODS: We analyzed data from 66 patients who completed the 16-week, double-blind, induction portion of a phase 2 study of RPC4046 (180 mg or 360 mg/wk) vs placebo and then completed a 52-week LTE, receiving open-label RPC4046 360 mg/wk. The study was conducted at 28 centers in 3 countries; patients were enrolled between September 2014 and January 2017. Outcomes were stratified by double-blind dose group and included esophageal eosinophil counts, EoE endoscopic reference score, EoE histologic scoring system score, symptom-based EoE activity index score, and safety. RESULTS: By week 12 of the LTE, esophageal eosinophil mean and peak counts, total EoE endoscopic reference scores, and EoE histologic scoring system grade and stage scores did not differ considerably between patients who originally received placebo vs RPC4046. Most patients maintained responses through week 52. Symptom remission (symptom-based EoE activity index score, £20) increased from 14% at LTE entry to 67% at LTE week 52 in placebo-RPC4046 patients and from 30% to 54% in RPC4046-RPC4046 (either dose) patients. Of the 28 patients who did not have a histologic response to RPC4046 during the double-blind induction phase, 10 patients (36%) achieved response during the LTE. The most common adverse events were upper respiratory tract infection (21%) and nasopharyngitis (14%). CONCLUSIONS: One year of treatment with RPC4046 is generally well tolerated and results in continued improvement and/or maintenance of endoscopic, histologic, and clinical measures of EoE disease activity relative to baseline. Trial registration: NCT02098473.

Nature communications, Jan 17, 2015

Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this pr... more Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this process are currently available. Here, an integrative computational analysis of the Cancer Genome Atlas OC data set and experimental validation identifies a zinc finger transcription factor ZNF304 associated with OC metastasis. High tumoral ZNF304 expression is associated with poor overall survival in OC patients. Through reverse phase protein array analysis, we demonstrate that ZNF304 promotes multiple proto-oncogenic pathways important for cell survival, migration and invasion. ZNF304 transcriptionally regulates β1 integrin, which subsequently regulates Src/focal adhesion kinase and paxillin and prevents anoikis. In vivo delivery of ZNF304 siRNA by a dual assembly nanoparticle leads to sustained gene silencing for 14 days, increased anoikis and reduced tumour growth in orthotopic mouse models of OC. Taken together, ZNF304 is a transcriptional regulator of β1 integrin, promotes cancer cel...

Cancer Research, 2014

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Anti-angiogenesis targeted ... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Anti-angiogenesis targeted therapy using RNA interference is a powerful tool to inhibit tumor growth and metastasis Here, we developed chitosan/thioaptamer nanoparticles that are capable of cell-type specific binding and delivery of siRNA into tumor-associated endothelial cells Thioaptamer (TA) was created by partial substitution of oxygen with sulfur on the aptamer phosphate backbone at the 5′dA position to enhance binding affinity and stability A combinatorial DNA aptamer library was established for cell-based selection method known as SELEX (systematic evolution of ligands by exponential enrichment) Endothelial cells isolated from both human ovarian tumors and normal human ovarian tissues were used to screen for selective thioaptamers After 10 cycles of cell-SELEX, two candidates, Endo28 and Endo31 showed highly specific binding to both human microvascular endothelial cells (HMVEC) and vasculature of human ovarian cancer tissue Endo28 decorated chitosan nanoparticles (CH/TA-siRNA) were ∼200 nm, positively charged (22mV), and spherical in shape The incorporation of thioaptamer Endo28 and siRNA to chitosan was confirmed using 1HNMR and PCR CH/TA-siRNA nanoparticles were intact and stable in the presence of serum at 37° C Intravenous administration of CH/TA nanoparticles into mice with HeyA8 ovarian tumors resulted in successful binding of aptamer to endothelial cells as well as delivery of siRNA into tumors TEM7 is a tumor biomarker found on the surface of endothelial cells TEM7 silencing using siRNA reduced endothelial tube formation and migration In orthotopic mouse models of ovarian cancer, murine TEM7 siRNA loaded nanoparticles showed substantial reduction in tumor growth as well as reduced angiogenesis These data have implications for new anti-angiogenesis approaches Citation Format: Dahai Jiang, Lingegowda S. Mangala, Hongyu Wang, Sherry Wu, Lokesh G. Rao, Cristian Rodriguez-Aguayo, Sunila Pradeep, David E. Volk, Gabriel Lopez-Berestein, Anil K. Sood. Tumor vasculature targeting using cell-specific thioaptamer decorated chitosan nanoparticle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2014-4468

Cancer Research, 2014

Background. Posttranscriptional regulation is a critical control point for the expression of gene... more Background. Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. The mRNA-binding protein hnRNP A1 is required for the processing of miR-18a, an inhibitor of k-ras expression. In this study we hypothesized that downmodulation of hnRNP A1 by miR-25 and miR-15a controls the expression of miR-18a and k-ras by modulating nuclear and cytoplasmic miRNA processing at the posttranscriptional level. Methods. In this study, we determined the expression of hnRNP A1 at mRNA and protein levels in three parental ovarian cancer cells lines (Skov3ip1, HeyA8, and A2780) and their chemotherapy-resistant derivatives (Skov3-TR, HeyA8 MDR, and A2780 cp20). We also predicted in silico which miRNAS can target hnRNP A1 and confirmed the miRNA expression by real-time PCR. Moreover, we inhibited miR-25 and mir-15a to determine their tumorigenic potential in vitro. We also correlated the expression of miR-25 and miR-15a and/or miR-18a and k-ras expression in the TCGA data. Results. Downregulation of hnRNP A1 at the RNA and protein levels was observed in the taxane-resistant cell lines but not in the cisplatin-resistant cell line. Furthermore, we predicted in silico that miR-25and miR-15a can target hnRNP A1 and confirmed the overexpression of these microRNAs in SKOV3-TR (5- and 3-fold, respectively) and HeyA8 MDR (3-fold) by real-time PCR. We also analyzed overall survival, trying to find a correlation between k-ras and miR-15a (high 23.77, low 33.97 months), k-ras and miR-25 (high 16.95, low 35.21 months), and the combination of k-ras, miR-25, and miR18a (k-ras high, miR25 high, mir18a low, 20.73 months; k-ras low, miR25 low, mir18a high, 31.49 months). Comparison of these data with a validation set yielded similar results. Conclusion. These results underscore the importance of a previously uncharacterized circuit of posttranscriptional regulation between miR-25 or -15a and RNA-binding protein hnRNP A1 that regulates miR-18a and k-ras expression. This circuit represents a unique opportunity for novel therapeutics approaches. Citation Format: Cristian Rodriguez-Aguayo, Paloma Monroig, Maria I. Almeida, Cristina Ivan, Vianey Gonzalez-Villasana, Maitri Y. Shah, Anil K. Sood, George Calin, Gabriel Lopez-Berestein. Downregulation of hnRNP A1 by miRNAs as a new mechanism of survival in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 539. doi:10.1158/1538-7445.AM2014-539

Cancer Research, 2013

Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensiti... more Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensitivity to paclitaxel. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is the cardiac isoform of the most important glycolytic regulator, phosphofructokinase-2, From the TCGA database of >300 ovarian cancers, the PFKFB2 gene ranked in the top 33% of overexpressed genes and in the top 19% of genes with copy number gain. We found that PFKFB2 mRNA was upregulated in 10 of 12 ovarian cancer cell lines when expression of the gene was compared to that in normal ovarian epithelial cells in culture. Knockdown of PFKFB2 with siRNA markedly inhibited cell proliferation and increased paclitaxel sensitivity in p53 wild type ovarian cancer cell lines as demonstrated in short-term cytotoxicity and long-term clonogenic assays. Liposome encapsulated PFKFB2 siRNA significantly inhibited growth (P Citation Format: Shu Zhang, Weiqun Mao, Anil K. Sood, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Zong-Fang Li, Robert C. Bast, Xiao-Feng Le. Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2013-5433

Molecular Cancer Therapeutics, 2014

Nature Communications, 2014

Author contributions P.Z. and L.M. conceived and designed the project. P.Z. performed and analyze... more Author contributions P.Z. and L.M. conceived and designed the project. P.Z. performed and analyzed most of the experiments. L.W. and K.K.A. performed tumor radiosensitivity studies. C.R.-A., G.L.-B., A.K.S. and Y.H. assisted with the nanoliposomal miRNA delivery study. Y.Y. and H.L. performed computational data analysis. B.G.D. and W.A.W. established the radioresistant subline. D.C. made some constructs. Y.S. maintained shRNA and ORF clones. M.J.Y. performed histopathological analysis. Y.L. and D.C.D. provided Zeb1-deficient MEFs. X.Y. synthesized miRNA mimics. J.C. provided DR-GFP-expressing U2OS cells. P.Z. and L.M. wrote the manuscript with input from all other authors. Competing financial interests X.Y. is an employee of AM Biotechnologies LLC. The remaining authors declare no competing financial interests. Accession codes The miRNA qPCR array data have been deposited in the Gene Expression Omnibus database under accession code GSE62511.

PLoS ONE, 2011

Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and ... more Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and therapeutic options are limited. Although the polycomb group gene, Bmi-1 that regulates the self-renewal of normal stem and progenitor cells has been implicated in the pathogenesis of many human malignancies, yet a role for Bmi-1 in influencing chemotherapy response has not been addressed before. Here we demonstrate that silencing Bmi-1 reduces intracellular GSH levels and thereby sensitizes chemoresistant ovarian cancer cells to chemotherapeutics such as cisplatin. By exacerbating ROS production in response to cisplatin, Bmi-1 silencing activates the DNA damage response pathway, caspases and cleaves PARP resulting in the induction apoptosis in ovarian cancer cells. In an in vivo orthotopic mouse model of chemoresistant ovarian cancer, knockdown of Bmi-1 by nanoliposomal delivery significantly inhibits tumor growth. While cisplatin monotherapy was inactive, combination of Bmi-1 silencing along with cisplatin almost completely abrogated ovarian tumor growth. Collectively these findings establish Bmi-1 as an important new target for therapy in chemoresistant ovarian cancer.

Advances in Delivery Science and Technology, 2014

Exosomes are nano-sized vesicles that are capable of transferring intracellular proteins or nucle... more Exosomes are nano-sized vesicles that are capable of transferring intracellular proteins or nucleic acids contents to the microenvironment and throughout the body. Our current knowledge about the communication mechanisms between exosomes and their target cells still remains limited. The interaction between exosomes and their target cells is likely to require exquisite specificity. The pairing between membrane fusion and trafficking proteins such as SNAREs or Rab proteins may be one of the mechanisms associated with specificity. Exosomes may stimulate antitumor response by presenting tumor antigens to dendritic cells; on the other hand, their contribution to angiogenesis, invasion, and metastasis of cancer may counteract these effects. Exosomes are known to transport miRNA; however, our knowledge of the interactions of these large non-coding RNA family and their effects and specificity on close or far away target cells is mostly unknown. As we improve our understanding of the biological fate of exosomal-miRNA, we will most likely obtain insights into the mechanisms involved in these events. Exosome mediated drug delivery is a promising strategy to efficiently deliver antitumor miRNAs or siRNAs to target cancer cells and to deliver chemotherapeutics for the treatment of malignant tumors. Here, we review some of the current knowledge on these communication mechanisms and address the potential roles of exosomes in cancer development, diagnosis, and development of therapeutics.

Cancer Research, 2013

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer (PC... more Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer (PC) is characterized by a prominent stromal reaction. Recently, the contribution of the stromal elements to the initiation and progression of the disease has come to be appreciated. In this context, targeting this tumor microenvironment represents a potential strategy to improve the delivery and efficacy of chemotherapeutic agents leading a better prognosis for the patients. Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic cancer. In the current study, we observed that PSC possess α-naphthyl acetate esterase (ANAE) activity, which is a specific marker enzyme for monocytes and macrophages, these cells can be differentiated/activated in a similar fashion as a monocytes/macrophages, suggesting that PSC behave in a similar fashion as macrophages, and hence can be treated as such. Based on these data we targeted PSC in vitro and in vivo by using bisphosphonates (BPs) that are macrophages inhibitors. BPs inhibited proliferation and inactivated PSC in vitro through inhibition of protein prenylation, and increased cell apoptosis and cell cycle arrest in G1. Furthermore, BPs inactivated PSC, inhibited tumor growth (p<0.0005), tumor weight (p<0.001) and number of nodules (p<0.001) in an orthotopic animal model of pancreas. These antitumor effects were enhanced when BPs were combined with a taxane. These data suggest that BPs could disrupt the pancreatic cancer, which could offer a better prognosis to PC patients. Citation Format: Vianey Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Thiru Arumugam, Zobeida Cruz-Monserrate, Defeng Deng, Enrique Fuentes-Mattei, Rosa F. Hwang, Huamin Wang, Guillermo N. Armaiz Pena, Burcu Aslan, Yolanda Gutierrez-Puente, Anil K. Sood, Craig Logsdon, Gabriel Lopez-Berestein. Targeting pancreatic stellate cells (PSC) to treat pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2013-4959

Cancer cell, Jan 8, 2014

Small noncoding miRNAs represent underexplored targets of genomic aberrations and emerging therap... more Small noncoding miRNAs represent underexplored targets of genomic aberrations and emerging therapeutic targets. The 3q26.2 amplicon is among the most frequent genomic aberrations in multiple cancer lineages including ovarian and breast cancers. We demonstrate that hsa-miR-569 (hereafter designated as miR569), which is overexpressed in a subset of ovarian and breast cancers, at least in part due to the 3q26.2 amplicon, alters cell survival and proliferation. Downregulation of TP53INP1 expression by miR569 is required for the effects of miR569 on survival and proliferation. Targeting miR569 sensitizes ovarian and breast cancer cells overexpressing miR569 to cisplatin by increasing cell death both in vitro and in vivo. Thus targeting miR569 could potentially benefit patients with the 3q26.2 amplicon and subsequent miR569 elevation.

Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 16, 2015

Purpose:Zoledronic acid (ZA) is being increasingly recognized for its anti-tumor properties, but ... more Purpose:Zoledronic acid (ZA) is being increasingly recognized for its anti-tumor properties, but the underlying functions are not well understood. In this study, we hypothesized that ZA inhibits ovarian cancer (OC) angiogenesis preventing Rac1 activation. Experimental Design: The biological effects of ZA were examined using a series of in vitro (cell invasion, cytokine production, Rac1 activation, reverse-phase protein array and in vivo (orthotopic mouse models) experiments. Results: There was significant inhibition of OC (HeyA8-MDR and OVCAR-5) cell invasion as well as reduced production of pro-angiogenic cytokines in response to ZA treatment. Furthermore, ZA inactivated Rac1 and decreased the levels of Pak1/p-38/matrix metalloproteinase-2 in OC cells. In vivo, ZA reduced tumor growth, angiogenesis and cell proliferation and inactivated Rac1 in both HeyA8-MDR and OVCAR-5 models. These in vivo antitumor effects were enhanced in both models when ZA was combined with nab-paclitaxel. C...

PLoS ONE, 2011

NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphori... more NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphorin and vascular endothelial growth factor families. NRP-2 has been detected on the surface of several types of human cancer cells, but its expression and function in gastrointestinal (GI) cancer cells remains to be determined. We sought to determine the function of NRP-2 in mediating downstream signals regulating the growth and survival of human gastrointestinal cancer cells. In human gastric cancer specimens, NRP-2 expression was detected in tumor tissues but not in adjacent normal mucosa. In CNDT 2.5 cells, shRNA mediated knockdown NRP-2 expression led to decreased migration and invasion in vitro (p,0.01). Focused gene-array analysis demonstrated that loss of NRP-2 reduced the expression of a critical metastasis mediator gene, S100A4. Steady-state levels and function of b-catenin, a known regulator of S100A4, were also decreased in the shNRP-2 clones. Furthermore, knockdown of NRP-2 sensitized CNDT 2.5 cells in vitro to 5FU toxicity. This effect was associated with activation of caspases 3 and 7, cleavage of PARP, and downregulation of Bcl-2. In vivo growth of CNDT 2.5 cells in the livers of nude mice was significantly decreased in the shNRP-2 group (p,0.05). Intraperitoneal administration of NRP-2 siRNA-DOPC decreased the tumor burden in mice (p = 0.01). Collectively, our results demonstrate that tumor cell-derived NRP-2 mediates critical survival signaling in gastrointestinal cancer cells.

Nature Communications, 2013

Users may view, print, copy, download and text and data-mine the content in such documents, for t... more Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Journal of the American College of Cardiology, 2014

background: Fatty acid binding protein 4 (FABP4), which is almost exclusively expressed in adipoc... more background: Fatty acid binding protein 4 (FABP4), which is almost exclusively expressed in adipocytes and macrophages, is regulated by peroxisome-proliferator-activated receptor-γ (PPARγ) and contributes to the development of insulin resistance and atherosclerosis in mice. Importantly, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in ApoE-/-mice and FABP4 may be a potential drug target in diseases like diabetes and atherosclerosis. However, the FABP4-related pathway in atherosclerosis has not been fully characterized. methods: Liposomal siRNAs were prepared by using the thin-film hydration method. To determine how FABP4 silencing affects pathways associated with atherosclerotic plaque development, we performed reverse phase protein array (RPPA) analysis in mouse macrophage RAW264 cells treated with liposomal FABP4 siRNA. results: Western blot analysis showed that liposomal FABP4 siRNA treatment induced a reduction of 80% compared with control siRNA treatment in mouse macrophage RWA264 cells (Figure: A, B). RPPA analysis revealed that the downregulation of FABP4 significantly increased the expression of the anti-apoptotic-related proteins BCL2 and BcL-XL and of the autophagy inhibitor protein Beclin-1. Furthermore, the downregulation of FABP4 significantly inhibited the expression of Bax, a key regulator of apoptosis (Figure: A, C). conclusion: FABP4 may be a key molecular regulator in atherosclerotic plaque apoptosis and autophagy.

Journal of the American College of Cardiology, 2014

background: Fatty acid binding protein 4 (FABP4) deficiency protects apolipoprotein E-deficient (... more background: Fatty acid binding protein 4 (FABP4) deficiency protects apolipoprotein E-deficient (ApoE-/-) mice against developing atherosclerosis, suggesting that FABP4 may be a potential drug target for treating atherosclerosis. methods: We developed a liposomal siRNA delivery system for silencing FABP4 as a potential treatment for atherosclerosis. We analyzed the shortterm distribution of liposomal FABP4 siRNA and the expression of FABP4 in aortic atherosclerotic tissues of ApoE-/-mice. Liposomal siRNAs were prepared by using the thin-film hydration method and were labeled with quantum dots (QD). ApoE-/-mice were intravenously injected twice in 1 week with either liposomal control siRNA-QD or liposomal FABP4 siRNA-QD. Mouse aortas were harvested, and we used immunofluorescence staining to analyze the distribution of liposomal siRNA. The expression of FABP4 was examined by using immunohistochemical staining. results: Immunofluorescence staining of ApoE-/-mouse aortas showed the successful uptake of fluorescently labeled liposomal siRNA into atherosclerotic plaque and the colocalization of these liposomes with FABP4. Additionally, liposomal FABP4 siRNA significantly reduced FABP4 expression in atherosclerotic plaques as compared with liposomal control siRNA (Figure). conclusion: The intravenous injection of liposomal FABP4 siRNA silences FABP4 expression in the aortas of ApoE-/-mice and may protect against the development of atherosclerosis.

Journal of Liposome Research, 2014

Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular dis... more Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE(-/-)) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE(-)(/)(-) mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.

Journal of Allergy and Clinical Immunology, 2020

BACKGROUND Fibrostenosis, the most serious eosinophilic esophagitis (EoE) complication, is mediat... more BACKGROUND Fibrostenosis, the most serious eosinophilic esophagitis (EoE) complication, is mediated by epithelial-mesenchymal transition (EMT). Transitioned cells contribute to pathogenesis by overproducing extracellular matrix. OBJECTIVE To determine whether RPC4046 (anti‒IL-13 monoclonal antibody) modulates EMT biomarkers in biopsies from adults with active EoE in a substudy of a double-blind, placebo-controlled phase 2 trial. METHODS Baseline and week-16 esophageal biopsies were taken from 69 patients randomized to weekly subcutaneous RPC4046 180 mg (n=19), 360 mg (n=26), or placebo (n=24). Duplex immunofluorescence slides stained for e-cadherin and vimentin were digitally analyzed by mapping each epithelial cell and recording fluorescence intensities. Endpoints included change from baseline to week 16 in percentage vimentin-positive epithelial cells (primary), total e-cadherin, and vimentin:e-cadherin ratio per cell (average of 47,000 cells/biopsy analyzed). RESULTS Mean percentage vimentin-positive cells decreased 0.94%, 2.75%, and 4.24% in the placebo, low-dose, and high-dose groups (P = 0.032, high-dose vs placebo). Mean e-cadherin expression per cell increased 5.6-fold in both dose groups versus placebo (high-dose P = 0.047). Increases in e-cadherin per cell from baseline to week 16 were correlated with improvements in histology, eosinophil counts, endoscopic findings, and symptoms. CONCLUSION RPC4046 significantly reduced EMT markers in adults with active EoE, with greater effects at 360 mg. Together with results for eosinophil density and clinical endpoints from the main trial, these data support the hypothesis that pharmacologic IL-13 inhibition ameliorates both inflammatory and remodeling pathways, and could potentially reduce the risk of fibrostenotic complications.

Clinical Gastroenterology and Hepatology, 2020

BACKGROUND & AIMS: The short-term efficacy of RPC4046, a monoclonal antibody against interleukin-... more BACKGROUND & AIMS: The short-term efficacy of RPC4046, a monoclonal antibody against interleukin-13, has been shown in patients with eosinophilic esophagitis (EoE). We investigated the long-term efficacy and safety of RPC4046 in an open-label, long-term extension (LTE) study in adults with EoE. METHODS: We analyzed data from 66 patients who completed the 16-week, double-blind, induction portion of a phase 2 study of RPC4046 (180 mg or 360 mg/wk) vs placebo and then completed a 52-week LTE, receiving open-label RPC4046 360 mg/wk. The study was conducted at 28 centers in 3 countries; patients were enrolled between September 2014 and January 2017. Outcomes were stratified by double-blind dose group and included esophageal eosinophil counts, EoE endoscopic reference score, EoE histologic scoring system score, symptom-based EoE activity index score, and safety. RESULTS: By week 12 of the LTE, esophageal eosinophil mean and peak counts, total EoE endoscopic reference scores, and EoE histologic scoring system grade and stage scores did not differ considerably between patients who originally received placebo vs RPC4046. Most patients maintained responses through week 52. Symptom remission (symptom-based EoE activity index score, £20) increased from 14% at LTE entry to 67% at LTE week 52 in placebo-RPC4046 patients and from 30% to 54% in RPC4046-RPC4046 (either dose) patients. Of the 28 patients who did not have a histologic response to RPC4046 during the double-blind induction phase, 10 patients (36%) achieved response during the LTE. The most common adverse events were upper respiratory tract infection (21%) and nasopharyngitis (14%). CONCLUSIONS: One year of treatment with RPC4046 is generally well tolerated and results in continued improvement and/or maintenance of endoscopic, histologic, and clinical measures of EoE disease activity relative to baseline. Trial registration: NCT02098473.

Nature communications, Jan 17, 2015

Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this pr... more Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this process are currently available. Here, an integrative computational analysis of the Cancer Genome Atlas OC data set and experimental validation identifies a zinc finger transcription factor ZNF304 associated with OC metastasis. High tumoral ZNF304 expression is associated with poor overall survival in OC patients. Through reverse phase protein array analysis, we demonstrate that ZNF304 promotes multiple proto-oncogenic pathways important for cell survival, migration and invasion. ZNF304 transcriptionally regulates β1 integrin, which subsequently regulates Src/focal adhesion kinase and paxillin and prevents anoikis. In vivo delivery of ZNF304 siRNA by a dual assembly nanoparticle leads to sustained gene silencing for 14 days, increased anoikis and reduced tumour growth in orthotopic mouse models of OC. Taken together, ZNF304 is a transcriptional regulator of β1 integrin, promotes cancer cel...

Cancer Research, 2014

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Anti-angiogenesis targeted ... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Anti-angiogenesis targeted therapy using RNA interference is a powerful tool to inhibit tumor growth and metastasis Here, we developed chitosan/thioaptamer nanoparticles that are capable of cell-type specific binding and delivery of siRNA into tumor-associated endothelial cells Thioaptamer (TA) was created by partial substitution of oxygen with sulfur on the aptamer phosphate backbone at the 5′dA position to enhance binding affinity and stability A combinatorial DNA aptamer library was established for cell-based selection method known as SELEX (systematic evolution of ligands by exponential enrichment) Endothelial cells isolated from both human ovarian tumors and normal human ovarian tissues were used to screen for selective thioaptamers After 10 cycles of cell-SELEX, two candidates, Endo28 and Endo31 showed highly specific binding to both human microvascular endothelial cells (HMVEC) and vasculature of human ovarian cancer tissue Endo28 decorated chitosan nanoparticles (CH/TA-siRNA) were ∼200 nm, positively charged (22mV), and spherical in shape The incorporation of thioaptamer Endo28 and siRNA to chitosan was confirmed using 1HNMR and PCR CH/TA-siRNA nanoparticles were intact and stable in the presence of serum at 37° C Intravenous administration of CH/TA nanoparticles into mice with HeyA8 ovarian tumors resulted in successful binding of aptamer to endothelial cells as well as delivery of siRNA into tumors TEM7 is a tumor biomarker found on the surface of endothelial cells TEM7 silencing using siRNA reduced endothelial tube formation and migration In orthotopic mouse models of ovarian cancer, murine TEM7 siRNA loaded nanoparticles showed substantial reduction in tumor growth as well as reduced angiogenesis These data have implications for new anti-angiogenesis approaches Citation Format: Dahai Jiang, Lingegowda S. Mangala, Hongyu Wang, Sherry Wu, Lokesh G. Rao, Cristian Rodriguez-Aguayo, Sunila Pradeep, David E. Volk, Gabriel Lopez-Berestein, Anil K. Sood. Tumor vasculature targeting using cell-specific thioaptamer decorated chitosan nanoparticle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2014-4468

Cancer Research, 2014

Background. Posttranscriptional regulation is a critical control point for the expression of gene... more Background. Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. The mRNA-binding protein hnRNP A1 is required for the processing of miR-18a, an inhibitor of k-ras expression. In this study we hypothesized that downmodulation of hnRNP A1 by miR-25 and miR-15a controls the expression of miR-18a and k-ras by modulating nuclear and cytoplasmic miRNA processing at the posttranscriptional level. Methods. In this study, we determined the expression of hnRNP A1 at mRNA and protein levels in three parental ovarian cancer cells lines (Skov3ip1, HeyA8, and A2780) and their chemotherapy-resistant derivatives (Skov3-TR, HeyA8 MDR, and A2780 cp20). We also predicted in silico which miRNAS can target hnRNP A1 and confirmed the miRNA expression by real-time PCR. Moreover, we inhibited miR-25 and mir-15a to determine their tumorigenic potential in vitro. We also correlated the expression of miR-25 and miR-15a and/or miR-18a and k-ras expression in the TCGA data. Results. Downregulation of hnRNP A1 at the RNA and protein levels was observed in the taxane-resistant cell lines but not in the cisplatin-resistant cell line. Furthermore, we predicted in silico that miR-25and miR-15a can target hnRNP A1 and confirmed the overexpression of these microRNAs in SKOV3-TR (5- and 3-fold, respectively) and HeyA8 MDR (3-fold) by real-time PCR. We also analyzed overall survival, trying to find a correlation between k-ras and miR-15a (high 23.77, low 33.97 months), k-ras and miR-25 (high 16.95, low 35.21 months), and the combination of k-ras, miR-25, and miR18a (k-ras high, miR25 high, mir18a low, 20.73 months; k-ras low, miR25 low, mir18a high, 31.49 months). Comparison of these data with a validation set yielded similar results. Conclusion. These results underscore the importance of a previously uncharacterized circuit of posttranscriptional regulation between miR-25 or -15a and RNA-binding protein hnRNP A1 that regulates miR-18a and k-ras expression. This circuit represents a unique opportunity for novel therapeutics approaches. Citation Format: Cristian Rodriguez-Aguayo, Paloma Monroig, Maria I. Almeida, Cristina Ivan, Vianey Gonzalez-Villasana, Maitri Y. Shah, Anil K. Sood, George Calin, Gabriel Lopez-Berestein. Downregulation of hnRNP A1 by miRNAs as a new mechanism of survival in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 539. doi:10.1158/1538-7445.AM2014-539

Cancer Research, 2013

Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensiti... more Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensitivity to paclitaxel. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is the cardiac isoform of the most important glycolytic regulator, phosphofructokinase-2, From the TCGA database of >300 ovarian cancers, the PFKFB2 gene ranked in the top 33% of overexpressed genes and in the top 19% of genes with copy number gain. We found that PFKFB2 mRNA was upregulated in 10 of 12 ovarian cancer cell lines when expression of the gene was compared to that in normal ovarian epithelial cells in culture. Knockdown of PFKFB2 with siRNA markedly inhibited cell proliferation and increased paclitaxel sensitivity in p53 wild type ovarian cancer cell lines as demonstrated in short-term cytotoxicity and long-term clonogenic assays. Liposome encapsulated PFKFB2 siRNA significantly inhibited growth (P Citation Format: Shu Zhang, Weiqun Mao, Anil K. Sood, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Zong-Fang Li, Robert C. Bast, Xiao-Feng Le. Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2013-5433

Molecular Cancer Therapeutics, 2014

Nature Communications, 2014

Author contributions P.Z. and L.M. conceived and designed the project. P.Z. performed and analyze... more Author contributions P.Z. and L.M. conceived and designed the project. P.Z. performed and analyzed most of the experiments. L.W. and K.K.A. performed tumor radiosensitivity studies. C.R.-A., G.L.-B., A.K.S. and Y.H. assisted with the nanoliposomal miRNA delivery study. Y.Y. and H.L. performed computational data analysis. B.G.D. and W.A.W. established the radioresistant subline. D.C. made some constructs. Y.S. maintained shRNA and ORF clones. M.J.Y. performed histopathological analysis. Y.L. and D.C.D. provided Zeb1-deficient MEFs. X.Y. synthesized miRNA mimics. J.C. provided DR-GFP-expressing U2OS cells. P.Z. and L.M. wrote the manuscript with input from all other authors. Competing financial interests X.Y. is an employee of AM Biotechnologies LLC. The remaining authors declare no competing financial interests. Accession codes The miRNA qPCR array data have been deposited in the Gene Expression Omnibus database under accession code GSE62511.

PLoS ONE, 2011

Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and ... more Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and therapeutic options are limited. Although the polycomb group gene, Bmi-1 that regulates the self-renewal of normal stem and progenitor cells has been implicated in the pathogenesis of many human malignancies, yet a role for Bmi-1 in influencing chemotherapy response has not been addressed before. Here we demonstrate that silencing Bmi-1 reduces intracellular GSH levels and thereby sensitizes chemoresistant ovarian cancer cells to chemotherapeutics such as cisplatin. By exacerbating ROS production in response to cisplatin, Bmi-1 silencing activates the DNA damage response pathway, caspases and cleaves PARP resulting in the induction apoptosis in ovarian cancer cells. In an in vivo orthotopic mouse model of chemoresistant ovarian cancer, knockdown of Bmi-1 by nanoliposomal delivery significantly inhibits tumor growth. While cisplatin monotherapy was inactive, combination of Bmi-1 silencing along with cisplatin almost completely abrogated ovarian tumor growth. Collectively these findings establish Bmi-1 as an important new target for therapy in chemoresistant ovarian cancer.

Advances in Delivery Science and Technology, 2014

Exosomes are nano-sized vesicles that are capable of transferring intracellular proteins or nucle... more Exosomes are nano-sized vesicles that are capable of transferring intracellular proteins or nucleic acids contents to the microenvironment and throughout the body. Our current knowledge about the communication mechanisms between exosomes and their target cells still remains limited. The interaction between exosomes and their target cells is likely to require exquisite specificity. The pairing between membrane fusion and trafficking proteins such as SNAREs or Rab proteins may be one of the mechanisms associated with specificity. Exosomes may stimulate antitumor response by presenting tumor antigens to dendritic cells; on the other hand, their contribution to angiogenesis, invasion, and metastasis of cancer may counteract these effects. Exosomes are known to transport miRNA; however, our knowledge of the interactions of these large non-coding RNA family and their effects and specificity on close or far away target cells is mostly unknown. As we improve our understanding of the biological fate of exosomal-miRNA, we will most likely obtain insights into the mechanisms involved in these events. Exosome mediated drug delivery is a promising strategy to efficiently deliver antitumor miRNAs or siRNAs to target cancer cells and to deliver chemotherapeutics for the treatment of malignant tumors. Here, we review some of the current knowledge on these communication mechanisms and address the potential roles of exosomes in cancer development, diagnosis, and development of therapeutics.

Cancer Research, 2013

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer (PC... more Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer (PC) is characterized by a prominent stromal reaction. Recently, the contribution of the stromal elements to the initiation and progression of the disease has come to be appreciated. In this context, targeting this tumor microenvironment represents a potential strategy to improve the delivery and efficacy of chemotherapeutic agents leading a better prognosis for the patients. Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic cancer. In the current study, we observed that PSC possess α-naphthyl acetate esterase (ANAE) activity, which is a specific marker enzyme for monocytes and macrophages, these cells can be differentiated/activated in a similar fashion as a monocytes/macrophages, suggesting that PSC behave in a similar fashion as macrophages, and hence can be treated as such. Based on these data we targeted PSC in vitro and in vivo by using bisphosphonates (BPs) that are macrophages inhibitors. BPs inhibited proliferation and inactivated PSC in vitro through inhibition of protein prenylation, and increased cell apoptosis and cell cycle arrest in G1. Furthermore, BPs inactivated PSC, inhibited tumor growth (p<0.0005), tumor weight (p<0.001) and number of nodules (p<0.001) in an orthotopic animal model of pancreas. These antitumor effects were enhanced when BPs were combined with a taxane. These data suggest that BPs could disrupt the pancreatic cancer, which could offer a better prognosis to PC patients. Citation Format: Vianey Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Thiru Arumugam, Zobeida Cruz-Monserrate, Defeng Deng, Enrique Fuentes-Mattei, Rosa F. Hwang, Huamin Wang, Guillermo N. Armaiz Pena, Burcu Aslan, Yolanda Gutierrez-Puente, Anil K. Sood, Craig Logsdon, Gabriel Lopez-Berestein. Targeting pancreatic stellate cells (PSC) to treat pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2013-4959

Cancer cell, Jan 8, 2014

Small noncoding miRNAs represent underexplored targets of genomic aberrations and emerging therap... more Small noncoding miRNAs represent underexplored targets of genomic aberrations and emerging therapeutic targets. The 3q26.2 amplicon is among the most frequent genomic aberrations in multiple cancer lineages including ovarian and breast cancers. We demonstrate that hsa-miR-569 (hereafter designated as miR569), which is overexpressed in a subset of ovarian and breast cancers, at least in part due to the 3q26.2 amplicon, alters cell survival and proliferation. Downregulation of TP53INP1 expression by miR569 is required for the effects of miR569 on survival and proliferation. Targeting miR569 sensitizes ovarian and breast cancer cells overexpressing miR569 to cisplatin by increasing cell death both in vitro and in vivo. Thus targeting miR569 could potentially benefit patients with the 3q26.2 amplicon and subsequent miR569 elevation.

Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 16, 2015

Purpose:Zoledronic acid (ZA) is being increasingly recognized for its anti-tumor properties, but ... more Purpose:Zoledronic acid (ZA) is being increasingly recognized for its anti-tumor properties, but the underlying functions are not well understood. In this study, we hypothesized that ZA inhibits ovarian cancer (OC) angiogenesis preventing Rac1 activation. Experimental Design: The biological effects of ZA were examined using a series of in vitro (cell invasion, cytokine production, Rac1 activation, reverse-phase protein array and in vivo (orthotopic mouse models) experiments. Results: There was significant inhibition of OC (HeyA8-MDR and OVCAR-5) cell invasion as well as reduced production of pro-angiogenic cytokines in response to ZA treatment. Furthermore, ZA inactivated Rac1 and decreased the levels of Pak1/p-38/matrix metalloproteinase-2 in OC cells. In vivo, ZA reduced tumor growth, angiogenesis and cell proliferation and inactivated Rac1 in both HeyA8-MDR and OVCAR-5 models. These in vivo antitumor effects were enhanced in both models when ZA was combined with nab-paclitaxel. C...

PLoS ONE, 2011

NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphori... more NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphorin and vascular endothelial growth factor families. NRP-2 has been detected on the surface of several types of human cancer cells, but its expression and function in gastrointestinal (GI) cancer cells remains to be determined. We sought to determine the function of NRP-2 in mediating downstream signals regulating the growth and survival of human gastrointestinal cancer cells. In human gastric cancer specimens, NRP-2 expression was detected in tumor tissues but not in adjacent normal mucosa. In CNDT 2.5 cells, shRNA mediated knockdown NRP-2 expression led to decreased migration and invasion in vitro (p,0.01). Focused gene-array analysis demonstrated that loss of NRP-2 reduced the expression of a critical metastasis mediator gene, S100A4. Steady-state levels and function of b-catenin, a known regulator of S100A4, were also decreased in the shNRP-2 clones. Furthermore, knockdown of NRP-2 sensitized CNDT 2.5 cells in vitro to 5FU toxicity. This effect was associated with activation of caspases 3 and 7, cleavage of PARP, and downregulation of Bcl-2. In vivo growth of CNDT 2.5 cells in the livers of nude mice was significantly decreased in the shNRP-2 group (p,0.05). Intraperitoneal administration of NRP-2 siRNA-DOPC decreased the tumor burden in mice (p = 0.01). Collectively, our results demonstrate that tumor cell-derived NRP-2 mediates critical survival signaling in gastrointestinal cancer cells.

Nature Communications, 2013

Users may view, print, copy, download and text and data-mine the content in such documents, for t... more Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Journal of the American College of Cardiology, 2014

background: Fatty acid binding protein 4 (FABP4), which is almost exclusively expressed in adipoc... more background: Fatty acid binding protein 4 (FABP4), which is almost exclusively expressed in adipocytes and macrophages, is regulated by peroxisome-proliferator-activated receptor-γ (PPARγ) and contributes to the development of insulin resistance and atherosclerosis in mice. Importantly, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in ApoE-/-mice and FABP4 may be a potential drug target in diseases like diabetes and atherosclerosis. However, the FABP4-related pathway in atherosclerosis has not been fully characterized. methods: Liposomal siRNAs were prepared by using the thin-film hydration method. To determine how FABP4 silencing affects pathways associated with atherosclerotic plaque development, we performed reverse phase protein array (RPPA) analysis in mouse macrophage RAW264 cells treated with liposomal FABP4 siRNA. results: Western blot analysis showed that liposomal FABP4 siRNA treatment induced a reduction of 80% compared with control siRNA treatment in mouse macrophage RWA264 cells (Figure: A, B). RPPA analysis revealed that the downregulation of FABP4 significantly increased the expression of the anti-apoptotic-related proteins BCL2 and BcL-XL and of the autophagy inhibitor protein Beclin-1. Furthermore, the downregulation of FABP4 significantly inhibited the expression of Bax, a key regulator of apoptosis (Figure: A, C). conclusion: FABP4 may be a key molecular regulator in atherosclerotic plaque apoptosis and autophagy.

Journal of the American College of Cardiology, 2014

background: Fatty acid binding protein 4 (FABP4) deficiency protects apolipoprotein E-deficient (... more background: Fatty acid binding protein 4 (FABP4) deficiency protects apolipoprotein E-deficient (ApoE-/-) mice against developing atherosclerosis, suggesting that FABP4 may be a potential drug target for treating atherosclerosis. methods: We developed a liposomal siRNA delivery system for silencing FABP4 as a potential treatment for atherosclerosis. We analyzed the shortterm distribution of liposomal FABP4 siRNA and the expression of FABP4 in aortic atherosclerotic tissues of ApoE-/-mice. Liposomal siRNAs were prepared by using the thin-film hydration method and were labeled with quantum dots (QD). ApoE-/-mice were intravenously injected twice in 1 week with either liposomal control siRNA-QD or liposomal FABP4 siRNA-QD. Mouse aortas were harvested, and we used immunofluorescence staining to analyze the distribution of liposomal siRNA. The expression of FABP4 was examined by using immunohistochemical staining. results: Immunofluorescence staining of ApoE-/-mouse aortas showed the successful uptake of fluorescently labeled liposomal siRNA into atherosclerotic plaque and the colocalization of these liposomes with FABP4. Additionally, liposomal FABP4 siRNA significantly reduced FABP4 expression in atherosclerotic plaques as compared with liposomal control siRNA (Figure). conclusion: The intravenous injection of liposomal FABP4 siRNA silences FABP4 expression in the aortas of ApoE-/-mice and may protect against the development of atherosclerosis.

Journal of Liposome Research, 2014

Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular dis... more Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE(-/-)) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE(-)(/)(-) mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.