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1.
NPJ Vaccines ; 9(1): 5, 2024 Jan 04.
Article in English | MEDLINE | ID: mdl-38177171

ABSTRACT

Schistosomiasis, a challenging neglected tropical disease, affects millions of people worldwide. Developing a prophylactic vaccine against Schistosoma mansoni has been hindered by the parasite's biological complexity. In this study, we utilized the innovative phage-display immunoprecipitation followed by a sequencing approach (PhIP-Seq) to screen the immune response of 10 infected rhesus macaques during self-cure and challenge-resistant phases, identifying vaccine candidates. Our high-throughput S. mansoni synthetic DNA phage-display library encoded 99.6% of 119,747 58-mer peptides, providing comprehensive coverage of the parasite's proteome. Library screening with rhesus macaques' antibodies, from the early phase of establishment of parasite infection, identified significantly enriched epitopes of parasite extracellular proteins known to be expressed in the digestive tract, shifting towards intracellular proteins during the late phase of parasite clearance. Immunization of mice with a selected pool of PhIP-Seq-enriched phage-displayed peptides from MEG proteins, cathepsins B, and asparaginyl endopeptidase significantly reduced worm burden in a vaccination assay. These findings enhance our understanding of parasite-host immune responses and provide promising prospects for developing an effective schistosomiasis vaccine.

2.
Proc Natl Acad Sci U S A ; 120(34): e2220269120, 2023 08 22.
Article in English | MEDLINE | ID: mdl-37579172

ABSTRACT

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.


Subject(s)
COVID-19 , Humans , Ligands , COVID-19/metabolism , Ceramides/metabolism , Lung/metabolism , Endothelium, Vascular/metabolism , Receptors, Cell Surface/metabolism , Carrier Proteins/metabolism , Sphingomyelin Phosphodiesterase/metabolism
4.
Cancer Gene Ther ; 27(5): 301-310, 2020 05.
Article in English | MEDLINE | ID: mdl-31130731

ABSTRACT

Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are reemerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium, but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor (TNF), a purely cytotoxic gene for antitumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP-HSVtk) utilizing ganciclovir (GCV) for a suicide gene therapy. Both AAVP constructs demonstrated antitumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.


Subject(s)
Antineoplastic Agents/administration & dosage , Brain Neoplasms/therapy , Drug Delivery Systems/methods , Genetic Vectors/administration & dosage , Glioblastoma/therapy , Animals , Bacteriophages/genetics , Brain Neoplasms/blood supply , Brain Neoplasms/diagnosis , Brain Neoplasms/genetics , Cell Line, Tumor , Dependovirus/genetics , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Female , Ganciclovir/administration & dosage , Gene Transfer Techniques , Genes, Transgenic, Suicide/genetics , Genetic Vectors/genetics , Glioblastoma/blood supply , Glioblastoma/diagnosis , Glioblastoma/genetics , Humans , Mice , Molecular Imaging/methods , Molecular Targeted Therapy/methods , Simplexvirus/genetics , Thymidine Kinase/genetics , Tumor Necrosis Factor-alpha/genetics , Viral Proteins/genetics , Xenograft Model Antitumor Assays
5.
PLoS Genet ; 15(12): e1008482, 2019 12.
Article in English | MEDLINE | ID: mdl-31846472

ABSTRACT

The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10-21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Profiling/methods , Neovascularization, Pathologic/genetics , Oxygen/adverse effects , Retina/chemistry , Aged , Animals , Breast Neoplasms/mortality , Disease Models, Animal , Female , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , High-Throughput Nucleotide Sequencing , Humans , Mice , Middle Aged , Neovascularization, Pathologic/chemically induced , Neovascularization, Pathologic/mortality , Retina/drug effects , Sequence Analysis, RNA
6.
Lung Cancer ; 130: 169-178, 2019 04.
Article in English | MEDLINE | ID: mdl-30885340

ABSTRACT

OBJECTIVES: The ability of tumor cells to drive angiogenesis is an important cancer hallmark that positively correlates with metastatic potential and poor prognosis. Therefore, targeting angiogenesis is a rational therapeutic approach and dissecting proangiogenic pathways is important, particularly for malignancies driven by oncogenic KRAS, which are widespread and lack effective targeted therapies. Based on published studies showing that oncogenic RAS promotes angiogenesis by upregulating the proangiogenic NF-κB target genes IL-8 and VEGF, that NF-κB activation by KRAS requires the IKKß kinase, and that targeting IKKß reduces KRAS-induced lung tumor growth in vivo, but has limited effects on cell growth in vitro, we hypothesized that IKKß targeting would reduce lung tumor growth by inhibiting KRAS-induced angiogenesis. MATERIALS AND METHODS: To test this hypothesis, we targeted IKKß in KRAS-mutant lung cancer cell lines either by siRNA-mediated transfection or by treatment with Compound A (CmpdA), a highly specific IKKß inhibitor, and used in vitro and in vivo assays to evaluate angiogenesis. RESULTS AND CONCLUSIONS: Both pharmacological and siRNA-mediated IKKß targeting in lung cells reduced expression and secretion of NF-κB-regulated proangiogenic factors IL-8 and VEGF. Moreover, conditioned media from IKKß-targeted lung cells reduced human umbilical vein endothelial cell (HUVEC) migration, invasion and tube formation in vitro. Furthermore, siRNA-mediated IKKß inhibition reduced xenograft tumor growth and vascularity in vivo. Finally, IKKß inhibition also affects endothelial cell function in a cancer-independent manner, as IKKß inhibition reduced pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Taken together, these results provide a novel mechanistic understanding of how the IKKß pathway affects human lung tumorigenesis, indicating that IKKß promotes KRAS-induced angiogenesis both by cancer cell-intrinsic and cancer cell-independent mechanisms, which strongly suggests IKKß inhibition as a promising antiangiogenic approach to be explored for KRAS-induced lung cancer therapy.


Subject(s)
Endothelial Cells/physiology , I-kappa B Kinase/metabolism , Lung Neoplasms/blood supply , Oxazines/pharmacology , Piperidines/pharmacology , Pyridines/pharmacology , Animals , Cell Line, Tumor , Cell Movement , Humans , I-kappa B Kinase/antagonists & inhibitors , I-kappa B Kinase/genetics , Interleukin-8/genetics , Interleukin-8/metabolism , Lung Neoplasms/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mutation/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Neovascularization, Pathologic , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , RNA, Small Interfering/genetics , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays
7.
Proc Natl Acad Sci U S A ; 116(6): 2300-2305, 2019 02 05.
Article in English | MEDLINE | ID: mdl-30670660

ABSTRACT

Endothelial heterogeneity has important implications in health and disease. Molecular markers selectively expressed in the vasculature of different organs and tissues are currently being explored in targeted therapies with promising results in preclinical and clinical studies. Noteworthy is the role that combinatorial approaches such as phage display have had in identifying such markers by using phage as nanoparticles and surrogates for billions of different peptides, screening noninvasively the vascular lumen for binding sites. Here, we show that a new peptide motif that emerged from such combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.


Subject(s)
Amino Acid Motifs , Brain/metabolism , Cerebrovascular Circulation/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Ligands , Retinal Vessels/drug effects , Retinal Vessels/metabolism , Amino Acid Sequence , Animals , Brain/blood supply , Cell Surface Display Techniques , Endothelium, Vascular/ultrastructure , Female , Fluorescent Antibody Technique , Mice , Peptides/chemistry , Peptides/metabolism , Protein Binding
8.
Front Microbiol ; 9: 1177, 2018.
Article in English | MEDLINE | ID: mdl-29922261

ABSTRACT

Leishmaniasis is caused by trypanosomatid protozoa of the genus Leishmania, which infect preferentially macrophages. The disease affects 12 million people worldwide, who may present cutaneous, mucocutaneous or visceral forms. Several factors influence the form and severity of the disease, and the main ones are the Leishmania species and the host immune response. CD100 is a membrane bound protein that can also be shed. It was first identified in T lymphocytes and latter shown to be induced in macrophages by inflammatory stimuli. The soluble CD100 (sCD100) reduces migration and expression of inflammatory cytokines in human monocytes and dendritic cells, as well as the intake of oxidized low-density lipoprotein (oxLDL) by human macrophages. Considering the importance of macrophages in Leishmania infection and the potential role of sCD100 in the modulation of macrophage phagocytosis and activation, we analyzed the expression and distribution of CD100 in murine macrophages and the effects of sCD100 on macrophage infection by Leishmania (Leishmania) amazonensis. Here we show that CD100 expression in murine macrophages increases after infection with Leishmania. sCD100 augments infection and phagocytosis of Leishmania (L.) amazonensis promastigotes by macrophages, an effect dependent on macrophage CD72 receptor. Besides, sCD100 enhances phagocytosis of zymosan particles and infection by Trypanosoma cruzi.

10.
Sci Adv ; 2(10): e1600611, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27819042

ABSTRACT

Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.


Subject(s)
Angiogenesis Inhibitors , Endothelial Cells/metabolism , Peptide Library , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/metabolism , Vascular Endothelial Growth Factor A , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/genetics , Angiogenesis Inhibitors/pharmacology , Animals , Endothelial Cells/cytology , Humans , Mice , Protein Domains , Retinal Neovascularization/drug therapy , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/pharmacology
11.
Int J Mol Sci ; 17(9)2016 Aug 24.
Article in English | MEDLINE | ID: mdl-27563889

ABSTRACT

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with (111)InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.


Subject(s)
Atherosclerosis/diagnosis , Molecular Imaging/methods , Peptides/metabolism , Animals , Antigens, CD/chemistry , Antigens, Differentiation, Myelomonocytic/chemistry , Atherosclerosis/metabolism , Disease Models, Animal , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptides/chemistry , Receptors, Cell Surface/chemistry , Receptors, LDL/deficiency , Receptors, LDL/genetics
12.
Front Pharmacol ; 7: 509, 2016.
Article in English | MEDLINE | ID: mdl-28066254

ABSTRACT

Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy.

13.
J Ophthalmol ; 2015: 892043, 2015.
Article in English | MEDLINE | ID: mdl-26491556
14.
Sci Transl Med ; 7(309): 309ra165, 2015 Oct 14.
Article in English | MEDLINE | ID: mdl-26468327

ABSTRACT

Blood vessel growth from preexisting vessels (angiogenesis) underlies many severe diseases including major blinding retinal diseases such as retinopathy of prematurity (ROP) and aged macular degeneration (AMD). This observation has driven development of antibody inhibitors that block a central factor in AMD, vascular endothelial growth factor (VEGF), from binding to its receptors VEGFR-1 and mainly VEGFR-2. However, some patients are insensitive to current anti-VEGF drugs or develop resistance, and the required repeated intravitreal injection of these large molecules is costly and clinically problematic. We have evaluated a small cyclic retro-inverted peptidomimetic, D(Cys-Leu-Pro-Arg-Cys) [D(CLPRC)], and hereafter named Vasotide, that inhibits retinal angiogenesis by binding selectively to the VEGF receptors VEGFR-1 and neuropilin-1 (NRP-1). Delivery of Vasotide via either eye drops or intraperitoneal injection in a laser-induced monkey model of human wet AMD, a mouse genetic knockout model of the AMD subtype called retinal angiomatous proliferation (RAP), and a mouse oxygen-induced model of ROP decreased retinal angiogenesis in all three animal models. This prototype drug candidate is a promising new dual receptor inhibitor of the VEGF ligand with potential for translation into safer, less-invasive applications to combat pathological angiogenesis in retinal disorders.


Subject(s)
Macular Degeneration/drug therapy , Neovascularization, Pathologic/drug therapy , Peptides, Cyclic/therapeutic use , Peptidomimetics/therapeutic use , Retinopathy of Prematurity/drug therapy , Animals , Drug Resistance , Haplorhini , Humans , Immunophilins/antagonists & inhibitors , Macular Degeneration/immunology , Macular Degeneration/physiopathology , Mice , Mice, Knockout , Models, Animal , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/physiopathology , Peptides, Cyclic/chemistry , Peptidomimetics/chemistry , Retinopathy of Prematurity/immunology , Retinopathy of Prematurity/physiopathology , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor Receptor-1/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors
15.
Cancer ; 121(14): 2411-21, 2015 Jul 15.
Article in English | MEDLINE | ID: mdl-25832466

ABSTRACT

BACKGROUND: Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors ("vascular zip codes") within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS: The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα-based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS: BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m(2) ). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS: These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients.


Subject(s)
Antineoplastic Agents/therapeutic use , Bone Neoplasms/prevention & control , Interleukin-11 Receptor alpha Subunit/metabolism , Peptides/therapeutic use , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Bone Neoplasms/secondary , Drug Administration Schedule , Humans , Interleukin-11 Receptor alpha Subunit/drug effects , Kidney/drug effects , Male , Maximum Tolerated Dose , Middle Aged , Peptides/pharmacology , Proteinuria/chemically induced , Treatment Outcome
16.
Proc Natl Acad Sci U S A ; 112(12): 3770-5, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25775553

ABSTRACT

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.


Subject(s)
Down-Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , MicroRNAs/metabolism , Retinal Neovascularization/genetics , Retinal Vessels/metabolism , 3' Untranslated Regions , Animals , Base Sequence , Cell Line, Tumor , Disease Models, Animal , Female , Gene Expression Regulation , Genes, Reporter , Humans , Male , Mice , Molecular Sequence Data , Neovascularization, Pathologic/genetics , Retinopathy of Prematurity/pathology , Sequence Homology, Nucleic Acid , Vascular Endothelial Growth Factor A/metabolism
17.
Proc Natl Acad Sci U S A ; 109(5): 1637-42, 2012 Jan 31.
Article in English | MEDLINE | ID: mdl-22307623

ABSTRACT

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.


Subject(s)
CD13 Antigens/metabolism , Lung Neoplasms/enzymology , Animals , CD13 Antigens/genetics , Cell Line, Tumor , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Mice , Mice, Inbred C57BL , Mice, Knockout
18.
PLoS One ; 6(8): e21702, 2011.
Article in English | MEDLINE | ID: mdl-21829599

ABSTRACT

Thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4+CD25+ T regulatory cells we targeted thymic CD4+CD25+ cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4+CD25+ T cells. After four rounds of selection on CD4+CD25+ enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4+CD25+ cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.


Subject(s)
Bacteriophages/genetics , CD4 Antigens/immunology , Interleukin-2 Receptor alpha Subunit/immunology , T-Lymphocytes/immunology , Thymus Gland/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Forkhead Transcription Factors/immunology , Humans , Receptors, Calcitriol/immunology
19.
PLoS Negl Trop Dis ; 4(11): e864, 2010 Nov 02.
Article in English | MEDLINE | ID: mdl-21072227

ABSTRACT

BACKGROUND: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. METHODS: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. FINDINGS AND CONCLUSIONS: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.


Subject(s)
Chagas Disease/parasitology , Endothelium, Vascular/parasitology , Glycoproteins/chemistry , Glycoproteins/metabolism , Neuraminidase/chemistry , Neuraminidase/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Tropism , Trypanosoma cruzi/enzymology , Amino Acid Motifs , Animals , Cells, Cultured , Chagas Disease/metabolism , Endothelial Cells/metabolism , Endothelial Cells/parasitology , Endothelium, Vascular/metabolism , Female , Glycoproteins/genetics , Humans , Intermediate Filament Proteins/metabolism , Mice , Mice, Inbred C57BL , Neuraminidase/genetics , Organ Specificity , Protein Binding , Protozoan Proteins/genetics , Trypanosoma cruzi/chemistry , Trypanosoma cruzi/genetics , Trypanosoma cruzi/physiology
20.
Proc Natl Acad Sci U S A ; 107(11): 5112-7, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20190181

ABSTRACT

Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated (D)(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, (D)(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.


Subject(s)
Drug Design , Peptide Library , Peptides/pharmacology , Receptors, Vascular Endothelial Growth Factor/metabolism , Signal Transduction/drug effects , Amino Acid Sequence , Animals , Disease Models, Animal , Drug Resistance/drug effects , Endothelial Cells/drug effects , Endothelial Cells/pathology , Humans , Ligands , Mice , Molecular Sequence Data , Neuropilin-1/metabolism , Peptides/chemistry , Peptides/therapeutic use , Retina/drug effects , Retina/pathology , Retinal Neovascularization/drug therapy , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Structure-Activity Relationship
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