Dense, Continuous Membrane Labeling and Expansion Microscopy Visualization of Ultrastructure in Tissues.

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.

A systematic methodology for proteome-wide identification of peptides inhibiting the proliferation and migration of endothelial cells.

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.

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model.

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.

Novel anti-angiogenic peptides derived from ELR-containing CXC chemokines.

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.

Expansion microscopy: development and neuroscience applications.

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.

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells.

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.

What's Next for Gastrointestinal Disorders: No Needles?

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.

Rational design of a biomimetic cell penetrating peptide library.

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.

Rationally designed tumor-penetrating nanocomplexes.

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.

Therapeutic effect of orally administered microencapsulated oxaliplatin for colorectal cancer.

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.

Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery.

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).

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.

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.

The influence of scaffold elasticity on germ layer specification of human embryonic stem cells.

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.

A peptide derived from type 1 thrombospondin repeat-containing protein WISP-1 inhibits corneal and choroidal neovascularization.

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.

Multiscale models of angiogenesis.

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.

Peptides derived from type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins inhibit neovascularization and suppress tumor growth in MDA-MB-231 breast cancer xenografts.

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.

Anti-angiogenic peptides identified in thrombospondin type I domains.

Thrombospondin 1, the prototypical protein of the thrombospondin protein family, is a potent endogenous inhibitor of angiogenesis. Although the effects of the thrombospondin 1 on neovascularization have been well studied, little is known about the anti-angiogenic potency of other proteins or peptide fragments derived from the proteins in this family. Here we identify a set of 18 novel, anti-angiogenic 17- to 20-amino acid peptides that are derived from proteins containing type I thrombospondin motifs. We have named these peptides adamtsostatin-4, adamtsostatin-16, adamtsostatin-18, cartilostatin-1, cartilostatin-2, fibulostatin-6.2, fibulostatin-6.3, papilostatin-1, papilostatin-2, properdistatin, scospondistatin, semastatin-5A.1, semastatin-5A.2, semastatin-5B, thrombostatin containing-1, thrombostatin contaning-3, thrombostatin contaning-6, and wispostatin-1 to reflect their origin. We further demonstrate that these peptides inhibit the proliferation and migration of human umbilical vein endothelial cells in vitro. The anti-proliferative and anti-migratory properties of the identified peptides may be important in maintaining angiogenic homeostasis in vivo and make these peptides suitable candidates for use as anti-angiogenic pharmaceutical agents in numerous therapeutic applications.

Identification of novel short peptides derived from the alpha 4, alpha 5, and alpha 6 fibrils of type IV collagen with anti-angiogenic properties.

Angiogenesis, or neovascularization, is tightly controlled by positive and negative regulators, many of which reside in the extracellular matrix. We have now identified eight novel 19- to 20-residue peptides derived from the alpha4, alpha5, and alpha6 fibrils of type IV collagen, which we have designated tetrastatins, pentastatins, and hexastatins, respectively. We have shown that these endogenous peptides suppress the proliferation and migration of HUVECs in vitro. By performing clustering analyses of the sequences using sequence similarity criteria and of the experimental results using a hierarchical algorithm, we report that the clusters identified by the experimental results coincide with the sequence-based clusters, indicating a tight relationship between peptide sequence and anti-angiogenic potency. These peptides may have potential as anti-angiogenic therapeutic agents.

A biochemical model of matrix metalloproteinase 9 activation and inhibition.

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.

Distinct modes of collagen type I proteolysis by matrix metalloproteinase (MMP) 2 and membrane type I MMP during the migration of a tip endothelial cell: insights from a computational model.

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the proteolytic processing of extracellular matrix (ECM) structural proteins under physiological and pathological conditions. During sprouting angiogenesis, the MMPs expressed by a single "tip" endothelial cell exhibit proteolytic activity that allows the cells of the sprouting vessel bud to migrate into the ECM. Membrane type I matrix metalloproteinase (MT1-MMP) and the diffusible matrix metalloproteinase MMP2, in the presence of the tissue inhibitor of metalloproteinases TIMP2, constitute a system of proteins that play an important role during the proteolysis of collagen type I matrices. Here, we have formulated a computational model to investigate the proteolytic potential of such a tip endothelial cell. The cell expresses MMP2 in its proenzyme form, pro-MMP2, as well as MT1-MMP and TIMP2. The interactions of the proteins are described by a biochemically detailed reaction network. Assuming that the rate-limiting step of the migration is the ability of the tip cell to carry out proteolysis, we have estimated cell velocities for matrices of different collagen content. The estimated velocities of a few microns per hour are in agreement with experimental data. At high collagen content, proteolysis was carried out primarily by MT1-MMP and localized to the cell leading edge, whereas at lower concentrations, MT1-MMP and MMP2 were found to act in parallel, causing proteolysis in the vicinity of the leading edge. TIMP2 is a regulator of the proteolysis localization because it can shift the activity of MT1-MMP from its enzymatic toward its activatory mode, suggesting a tight mechanosensitive regulation of the enzymes and inhibitor expression. The model described here provides a foundation for quantitative studies of angiogenesis in extracellular matrices of different compositions, both in vitro and in vivo. It also identifies critical parameters whose values are not presently available and which should be determined in future experiments.