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1.
bioRxiv ; 2024 Mar 08.
Article in English | MEDLINE | ID: mdl-38496681

ABSTRACT

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines a novel membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validated the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supported the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrated the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yielded ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

2.
J Stomatol Oral Maxillofac Surg ; 123(4): e169-e172, 2022 09.
Article in English | MEDLINE | ID: mdl-35472483

ABSTRACT

Aneurysmal bone cysts (ABCs) are benign, non-neoplastic bone lesions, which contain blood and demonstrate a destructive pattern. They rarely occur in the head and neck region, with the mandible being the most common site of craniofacial origin. They develop in the second decade of life and their etiology is obscure. ABCs can sometimes demonstrate distinct radiological features and imaging is also necessary for preoperative planning. Surgery is the mainstay of treatment. The ABC should be excised in toto and usually osteotomies and primary reconstruction are necessary. We present a case of sizeable ABC of the mandibular ramus that was excised intraorally, without the need of combined approaches or reconstruction.


Subject(s)
Bone Cysts, Aneurysmal , Adolescent , Bone Cysts, Aneurysmal/diagnosis , Bone Cysts, Aneurysmal/pathology , Bone Cysts, Aneurysmal/surgery , Head , Humans , Mandible/pathology , Mandible/surgery
3.
Curr Opin Neurobiol ; 50: 56-63, 2018 06.
Article in English | MEDLINE | ID: mdl-29316506

ABSTRACT

Many neuroscience questions center around understanding how the molecules and wiring in neural circuits mechanistically yield behavioral functions, or go awry in disease states. However, mapping the molecules and wiring of neurons across the large scales of neural circuits has posed a great challenge. We recently developed expansion microscopy (ExM), a process in which we physically magnify biological specimens such as brain circuits. We synthesize throughout preserved brain specimens a dense, even mesh of a swellable polymer such as sodium polyacrylate, anchoring key biomolecules such as proteins and nucleic acids to the polymer. After mechanical homogenization of the specimen-polymer composite, we add water, and the polymer swells, pulling biomolecules apart. Due to the larger separation between molecules, ordinary microscopes can then perform nanoscale resolution imaging. We here review the ExM technology as well as applications to the mapping of synapses, cells, and circuits, including deployment in species such as Drosophila, mouse, non-human primate, and human.


Subject(s)
Microscopy , Neurons/cytology , Neurosciences/instrumentation , Neurosciences/methods , Synapses/physiology , Animals , Humans
4.
J Control Release ; 221: 48-61, 2016 Jan 10.
Article in English | MEDLINE | ID: mdl-26646543

ABSTRACT

A myriad of pathologies affect the gastrointestinal tract, citing this affected area as a significant target for therapeutic intervention. One group of therapeutic agents, antisense and oligonucleotides and small interfering RNAs, offer a promising platform for treating a wide variety of diseases ranging from cancer to auto-immune diseases. Current delivery methods are carried out either systemically or locally into diseased areas, both of which involve needles. The challenge in orally administering this type of treatment lies in the complications that arise due to the vast environmental extremes found within the gastrointestinal tract, owing to the fact that, as the drug travels down the gastrointestinal tract, it is subjected to pH changes and interactions with bacteria and a variety of digestive and protective enzymes including proteases, DNAses, and RNAses. Overcoming these challenges to allow the practical application of these drugs is a priority that has invoked a multitude of research in the chemical, biological, and material sciences. In this review, we will address common gastrointestinal pathologies, the barriers to oral-based therapies and antisense-interfering technologies, the approaches that have already been applied for their delivery, and the current status of antisense drug therapy clinical trials for gastrointestinal-related disorders.


Subject(s)
Drug Delivery Systems , Gastrointestinal Diseases/therapy , Gene Transfer Techniques , Oligonucleotides, Antisense/administration & dosage , RNA, Small Interfering/administration & dosage , Animals , Gastrointestinal Diseases/pathology , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/pathology , Humans , Needles , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/therapeutic use , RNA, Small Interfering/chemistry , RNA, Small Interfering/therapeutic use
5.
ACS Nano ; 7(10): 8616-26, 2013 Oct 22.
Article in English | MEDLINE | ID: mdl-24047542

ABSTRACT

Cell penetrating peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different peptides was evaluated both in vitro and in vivo and correlated with the peptide physical and chemical properties. In vitro, these particles were internalized primarily through macropinocytosis. When the peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. In vivo, peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuropeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the in vivo delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII.


Subject(s)
Biomimetics , Peptide Library , Peptides/pharmacokinetics , Amino Acid Sequence , Drug Design , Endocytosis , Humans , Molecular Sequence Data , Peptides/chemistry , Pinocytosis , Tissue Distribution
6.
Nat Biotechnol ; 31(7): 653-8, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23792629

ABSTRACT

Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.


Subject(s)
Endocytosis/genetics , Lipids/administration & dosage , Metal Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , Carrier Proteins , Gene Silencing , Gene Transfer Techniques , Humans , Intracellular Signaling Peptides and Proteins , Lipids/chemistry , Lipids/genetics , Membrane Glycoproteins , Metal Nanoparticles/chemistry , Microscopy, Confocal , Niemann-Pick C1 Protein , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Signal Transduction/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
7.
ACS Nano ; 6(10): 8484-7, 2012 Oct 23.
Article in English | MEDLINE | ID: mdl-23088785

ABSTRACT

Small interfering RNA (siRNA) therapeutics have broad potential uses in medicine but require safe and effective delivery vehicles to function. An ideal delivery system should encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell surface, and release its cargo in the desired intracellular compartment. One approach to the design of delivery vehicles that meets all of these requirements utilizes the systematic assembly of multiple components that can address each barrier. This rational approach was adopted by Ren et al., who designed novel myristoylated tandem peptides that consist of a tumor-targeting module and a cell-penetrating module, as described in this issue of ACS Nano. These tandem peptides were formulated with siRNAs into nanocomplexes for cell-specific delivery to a variety of tumor cell lines. The correlation of the structural properties of the nanocomplex to cell-type-specific activity via a computational approach identified the valence of the tumor-targeting ligand and overall nanocomplex charge as important parameters for the activity of the formulations. The in vivo gene silencing potency of these peptide-based nanocomplex formulations was demonstrated by Ren et al. in an ovarian cancer model. Tumor-penetrating nanocomplexes carrying a siRNA sequence against a novel oncogene (ID4) led to a significant reduction in tumor burden and an 80% increase in mouse survival. As such, the combination of a systematic approach with computational modeling can be advantageous for improving the delivery and potency of siRNA therapeutics.


Subject(s)
Nanocapsules/therapeutic use , Neoplasms, Experimental/physiopathology , Peptides/pharmacokinetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Receptors, Cell Surface/metabolism , Transfection/methods , Humans
8.
Nat Nanotechnol ; 7(6): 389-93, 2012 Jun 03.
Article in English | MEDLINE | ID: mdl-22659608

ABSTRACT

Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t(1/2) ≈ 24.2 min) than the parent siRNA (t(1/2) ≈ 6 min).


Subject(s)
DNA , Drug Delivery Systems/methods , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing/drug effects , Nanoparticles , Neoplasms, Experimental/drug therapy , RNA, Small Interfering , Animals , DNA/chemistry , DNA/genetics , DNA/pharmacology , Female , Folic Acid/chemistry , Folic Acid/pharmacology , Gene Expression Regulation, Neoplastic/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology
9.
Biomaterials ; 33(18): 4752-61, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22472433

ABSTRACT

Colorectal cancer is a significant source of morbidity and mortality in the United States and other Western countries. Oral delivery of therapeutics remains the most patient accepted form of medication. The development of an oral delivery formulation for local delivery of chemotherapeutics in the gastrointestinal tract can potentially alleviate the adverse side effects including systemic cytotoxicity, as well as focus therapy to the lesions. Here we develop an oral formulation of the chemotherapeutic drug oxaliplatin for the treatment of colorectal cancer. Oxaliplatin was encapsulated in pH sensitive, mucoadhesive chitosan-coated alginate microspheres. The microparticles were formulated to release the chemotherapeutics after passing through the acidic gastric environment thus targeting the intestinal tract. In vivo, these particles substantially reduced the tumor burden in an orthotopic mouse model of colorectal cancer, and reduced mortality.


Subject(s)
Colorectal Neoplasms/drug therapy , Drug Compounding/methods , Organoplatinum Compounds/administration & dosage , Organoplatinum Compounds/therapeutic use , Administration, Oral , Animals , Body Weight/drug effects , C-Reactive Protein/metabolism , Colorectal Neoplasms/metabolism , Immunohistochemistry , Lactoferrin/metabolism , Male , Mice , Mice, Inbred C57BL , Organoplatinum Compounds/chemistry , Oxaliplatin
10.
Biomaterials ; 32(36): 9612-21, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21963156

ABSTRACT

Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate.


Subject(s)
Cell Lineage , Elasticity , Embryonic Stem Cells/cytology , Germ Layers/embryology , Tissue Scaffolds/chemistry , Cell Differentiation , Elastic Modulus , Humans , Microscopy, Electron, Scanning , Porosity
11.
Biomaterials ; 32(31): 7793-800, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21835461

ABSTRACT

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.


Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Embryonic Stem Cells/cytology , Gene Transfer Techniques , RNA, Small Interfering/metabolism , Animals , Cells, Cultured , Embryoid Bodies/cytology , Embryoid Bodies/metabolism , Embryonic Stem Cells/metabolism , Gene Knockdown Techniques , Green Fluorescent Proteins/metabolism , Humans , Mice , Protein Interaction Maps , Reverse Transcriptase Polymerase Chain Reaction , Viruses/metabolism
12.
BMC Cancer ; 10: 29, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20122172

ABSTRACT

BACKGROUND: Angiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays. METHODS: One family of peptides with high activity is derived from the alpha-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the alpha5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line. RESULTS: Pentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model. CONCLUSIONS: The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.


Subject(s)
Collagen Type IV/metabolism , Lung Neoplasms/metabolism , Peptide Fragments/pharmacology , Peptides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Movement , Cell Proliferation , Collagen Type IV/chemistry , Collagen Type IV/pharmacology , Disease Models, Animal , Disease Progression , Endothelial Cells/cytology , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice , Neoplasm Transplantation , Neovascularization, Pathologic , Peptide Fragments/chemistry , Peptides/chemistry , Umbilical Veins/pathology
13.
Neoplasia ; 11(12): 1285-91, 2009 Dec.
Article in English | MEDLINE | ID: mdl-20019836

ABSTRACT

Angiogenesis or neovascularization, the process of new blood vessel formation from preexisting microvasculature, involves interactions among several cell types including parenchymal, endothelial cells, and immune cells. The formation of new vessels is tightly regulated by a balance between endogenous proangiogenic and antiangiogenic factors to maintain homeostasis in tissue; tumor progression and metastasis in breast cancer have been shown to be angiogenesis-dependent. We previously introduced a systematic methodology to identify putative endogenous antiangiogenic peptides and validated these predictions in vitro in human umbilical vein endothelial cell proliferation and migration assays. These peptides are derived from several protein families including type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins. On the basis of the results from the in vitro screening, we have evaluated the ability of one peptide selected from each family named pentastatin-1, chemokinostatin-1, and properdistatin, respectively, to suppress angiogenesis in an MDA-MB-231 human breast cancer orthotopic xenograft model in severe combined immunodeficient mice. Peptides were administered intraperitoneally once per day. We have demonstrated significant suppression of tumor growth in vivo and subsequent reductions in microvascular density, indicating the potential of these peptides as therapeutic agents for breast cancer.


Subject(s)
Breast Neoplasms/prevention & control , Neovascularization, Pathologic/prevention & control , Oligopeptides/pharmacology , Xenograft Model Antitumor Assays , 3T3 Cells , Amino Acid Sequence , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Chemokines, CXC/chemistry , Collagen Type IV/chemistry , Female , Humans , Immunohistochemistry , Mammary Neoplasms, Experimental/blood supply , Mammary Neoplasms, Experimental/pathology , Mammary Neoplasms, Experimental/prevention & control , Mice , Mice, SCID , Molecular Sequence Data , Neovascularization, Pathologic/metabolism , Oligopeptides/chemical synthesis , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Thrombospondins/chemistry , Tumor Burden/drug effects
15.
IEEE Eng Med Biol Mag ; 28(2): 14-31, 2009.
Article in English | MEDLINE | ID: mdl-19349248

ABSTRACT

Vascular disease, cancer, stroke, neurodegeneration, diabetes, inflammation, asthma, obesity, arthritis--the list of conditions that involve angiogenesis reads like main chapters in a book on pathology. Angiogenesis, the growth of capillaries from preexisting vessels, also occurs in normal physiology, in response to exercise or in the process of wound healing.Why and when is angiogenesis prevalent? What controls the process? How can we intelligently control it? These are the key questions driving researchers in fields as diverse as cell biology, oncology, cardiology, neurology, biomathematics, systems biology, and biomedical engineering. As bioengineers, we approach angiogenesis as a complex, interconnected system of events occurring in sequence and in parallel, on multiple levels, triggered by a main stimulus, e.g., hypoxia.


Subject(s)
Models, Cardiovascular , Neovascularization, Physiologic , Computational Biology , Computer Simulation , Humans , Hypoxia-Inducible Factor 1/physiology , Matrix Metalloproteinases/physiology , Vascular Endothelial Growth Factor A/physiology
16.
Invest Ophthalmol Vis Sci ; 50(8): 3840-5, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19279315

ABSTRACT

PURPOSE: Ocular neovascularization is the primary cause of blindness in a wide range of prevalent ocular diseases including proliferative diabetic retinopathy, exudative age-related macular degeneration, and retinopathy of prematurity, among others. Antiangiogenic therapies are starting to give promising results in these diseases. In the present study the antiangiogenic potential of an 18-mer peptide derived from type 1 thrombospondin repeat-containing protein WISP-1 (wispostatin-1) was analyzed in vitro with human retinal endothelial cell proliferation and migration assays. The peptide was also tested in vivo in the corneal micropocket and the laser-induced choroidal neovascularization (CNV) mouse models. METHODS: Human retinal endothelial cells were treated with the WISP-1 peptide and in vitro migration and proliferation assays were performed. Also evaluated was the antiangiogenic effect of this peptide in vivo using the corneal micropocket assay and the laser-induced CNV model. RESULTS: Wispostatin-1 derived peptide demonstrated antimigratory and antiproliferative activity in vitro. Wispostatin-1 completely abolished bFGF-induced neovascularization in the corneal micropocket assay. The peptide also demonstrated significant inhibition of laser-induced CNV. CONCLUSIONS: An inhibitory effect of Wispostatin-1 on ocular neovascularization was found in vitro and in vivo. The identification of novel and potent endogenous peptide inhibitors provides insight into the pathogenesis of corneal and choroidal neovascularization. The results demonstrate potential for therapeutic application in prevalent ocular disease.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Choroidal Neovascularization/drug therapy , Corneal Neovascularization/drug therapy , Oncogene Proteins/therapeutic use , Peptides/therapeutic use , Amino Acid Sequence , Animals , CCN Intercellular Signaling Proteins , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Choroidal Neovascularization/pathology , Corneal Neovascularization/chemically induced , Corneal Neovascularization/pathology , Disease Models, Animal , Endothelium, Vascular/cytology , Humans , Injections , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oncogene Proteins/chemistry , Peptides/chemistry , Proto-Oncogene Proteins , Retinal Vessels/cytology , Vitreous Body
17.
Proc Natl Acad Sci U S A ; 105(37): 13775-80, 2008 Sep 16.
Article in English | MEDLINE | ID: mdl-18780781

ABSTRACT

We introduce a systematic computational methodology based on bioinformatics that has enabled us to identify and classify >120 endogenous peptide inhibitors of endothelial cell proliferation and migration. These peptides are derived from members of the type IV collagen, thrombospondin, and CXC chemokine protein families, as well as somatotropin hormones, serpins, and various kringle-containing proteins. Their activity in suppressing the proliferation and migration of endothelial cells in vitro provides proof of principle for the validity of this computational method. Interestingly, some of the peptides are derived from proteins known to be proangiogenic. By performing receptor neutralization studies, we have identified receptors to which these peptides bind. On the basis of this receptor-binding information, we evaluated several examples of peptide-based combinatorial screening strategies. In some cases, this combinatorial screening identified strong synergism between peptides. The current work provides a guideline for a computational-based peptidomics approach for the discovery of endogenous bioactive peptides.


Subject(s)
Cell Movement , Computational Biology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Peptides/analysis , Peptides/metabolism , Proteome/metabolism , Algorithms , Amino Acid Sequence , Cell Proliferation , Cells, Cultured , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/classification , Phylogeny , Proteome/chemistry , Proteome/genetics , Sequence Alignment , Sequence Homology, Amino Acid
18.
J Cell Biochem ; 104(4): 1356-63, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18307172

ABSTRACT

Angiogenesis is tightly regulated by numerous endogenous pro- and anti-angiogenic proteins and peptides. Among these are the CXC chemokines, a set of multifunctional peptides. CXC chemokines containing the ELR motif act as pro-angiogenic agents by regulating both endothelial cell proliferation and migration. Here we show that a set of six 22-24-amino acid peptides derived from the pro-angiogenic ELR-containing CXC chemokines exhibit notable anti-proliferative and anti-migratory activity in vitro; we call these peptides chemokinostatins. The ability of the identified peptides to inhibit the basic components of angiogenesis even though they are derived from pro-angiogenic proteins contributes towards the understanding of the diverse role of the CXC chemokine family in angiogenesis.


Subject(s)
Angiogenesis Inhibitors/chemistry , Chemokines, CXC , Peptide Fragments/pharmacology , Peptides/chemical synthesis , Amino Acid Motifs , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Peptide Fragments/chemical synthesis , Peptides/pharmacology , Umbilical Veins
19.
J Biol Chem ; 282(52): 37585-96, 2007 Dec 28.
Article in English | MEDLINE | ID: mdl-17848556

ABSTRACT

Matrix metalloproteinases (MMPs) are a class of extracellular and membrane-bound proteases involved in an array of physiological processes, including angiogenesis. We present a detailed computational model of MMP9 activation and inhibition. Our model is validated to existing biochemical experimental data. We determine kinetic rate constants for the processes of MMP9 activation by MMP3, MMP10, MMP13, and trypsin; inhibition by the tissue inhibitors of metalloproteinases (TIMPs) 1 and 2; and MMP9 deactivation. This computational approach allows us to investigate discrepancies in our understanding of the interaction of MMP9 with TIMP1. Specifically, we find that inhibition due to a single binding event cannot describe MMP9 inhibition by TIMP1. Temporally accurate biphasic inhibition requires either an additional isomerization step or a second lower affinity isoform of MMP9. We also theoretically characterize the MMP3/TIMP2/pro-MMP9 and MMP3/TIMP1/pro-MMP9 systems. We speculate that these systems differ significantly in their time scales of activation and inhibition such that MMP9 is able to temporarily overshoot its final equilibrium value in the latter. Our numerical simulations suggest that the ability of pro-MMP9 to complex TIMP1 increases this overshoot. In all, our analysis serves as a summary of existing kinetic data for MMP9 and a foundation for future models utilizing MMP9 or other MMPs under physiologically well defined microenvironments.


Subject(s)
Gene Expression Regulation, Enzymologic , Matrix Metalloproteinase 9/metabolism , Biochemistry/methods , Cell Membrane/metabolism , Computer Simulation , Kinetics , Models, Biological , Models, Chemical , Models, Theoretical , Protein Isoforms , Temperature , Time Factors , Tissue Inhibitor of Metalloproteinase-1/chemistry
20.
Int J Biochem Cell Biol ; 39(12): 2314-23, 2007.
Article in English | MEDLINE | ID: mdl-17707681

ABSTRACT

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18-20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Peptide Fragments/pharmacology , Thrombospondin 1/chemistry , ADAMTS Proteins , Amino Acid Sequence , Angiogenesis Inhibitors/chemical synthesis , CCN Intercellular Signaling Proteins , Cells, Cultured , Cluster Analysis , Complement C6/chemistry , Connective Tissue Growth Factor , Cysteine-Rich Protein 61 , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Humans , Immediate-Early Proteins/chemistry , Immunoglobulins/chemistry , Insulin-Like Growth Factor Binding Proteins/chemistry , Intercellular Signaling Peptides and Proteins/chemistry , Molecular Sequence Data , Netrin Receptors , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Receptors, Cell Surface/chemistry , Repressor Proteins , Sequence Homology, Amino Acid , Thrombospondins/chemistry , Transcription Factors/chemistry
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