Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and β1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.

Several surfactant-like peptides undergo self-assembly to form nanotubes and nanovesicles having an average diameter of 30 -50 nm with a helical twist. The peptide monomer contains 7-8 residues and has a hydrophilic head composed of aspartic acid and a tail of hydrophobic amino acids such as alanine, valine, or leucine. The length of each peptide is Ϸ2 nm, similar to that of biological phospholipids. Dynamic light-scattering studies showed structures with very discrete sizes. The distribution becomes broader over time, indicating a very dynamic process of assembly and disassembly. Visualization with transmission electron microscopy of quickfreeze͞deep-etch sample preparation revealed a network of openended nanotubes and some vesicles, with the latter being able to ''fuse'' and ''bud'' out of the former. The structures showed some tail sequence preference. Many three-way junctions that may act as links between the nanotubes have been observed also. Studies of peptide surfactant molecules have significant implications in the design of nonlipid biological surfactants and the understanding of the complexity and dynamics of the self-assembly processes.amino acids ͉ charged and hydrophobic residues ͉ nonlipid surfactants ͉ simplicity to complexity ͉ prebiotic enclosures M olecular self-assembly recently has attracted considerable attention for its use in the design and fabrication of nanostructures leading to the development of advanced materials (1, 2). The self-assembly of biomolecular building blocks plays an increasingly important role in the discovery of new materials and scaffolds (3, 4), with a wide range of applications in nanotechnology and medical technologies such as regenerative medicine and drug delivery systems (5, 6). Recently, Hartgerink et al. (7) reported the design of a chimeric material consisting of a hydrophobic alkyl tail and a hydrophilic peptide containing phosphorylated serine with an RGD motif that facilitates directional alignment of mineralization of hydroxyapatite.We previously described a class of ionic self-complementary peptide that spontaneously self-assemble to form interwoven nanofibers in the presence of monovalent cations (8 -10). These nanofibers further form a hydrogel consisting of greater than 99.5% water. The constituent of the hydrogel scaffold is made of peptides with alternating hydrophilic and hydrophobic amino acids. Such a sequence has a tendency to form an unusually stable ␤-sheet structure in water (8 -10). When the peptides form a ␤-sheet, they exhibit two surfaces, a hydrophilic surface consisting of charged ionic side chains and a hydrophobic surface with hydrophobic side chains. As a result, the self-assembly of these peptides is facilitated by electrostatic interactions on one side and the hydrophobic interaction on the other, in addition to the conventional ␤-sheet hydrogen bond along the backbones. The self-assembling peptide scaffolds have been demonstrated to serve as substrate for tissuecell attachment, extensive neurite outgrowth, and formation of active n...

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

We examined whether the presence of the cell cortex might explain, in part, why previous studies using atomic force microscopy (AFM) to measure cell modulus (E) gave higher values with sharp tips than for larger spherical tips. We confirmed these AFM findings in human umbilical vein endothelial cells (HUVEC) and Schlemm's canal (SC) endothelial cells with AFM indentation ≤ 400 nm, two cell types with prominent cortices (312 ± 65 nm in HUVEC and 371 ± 91 nm in SC cells). With spherical tips, E (kPa) was 0.71 ± 0.16 in HUVEC and 0.94 ± 0.06 in SC cells. Much higher values of E were measured using sharp tips: 3.23 ± 0.54 in HUVEC and 6.67 ± 1.07 in SC cells. Previous explanations for this difference such as strain hardening or a substrate effect were shown to be inconsistent with our measurements. Finite element modeling studies showed that a stiff cell cortex could explain the results. In both cell types, Latrunculin-A greatly reduced E for sharp and rounded tips, and also reduced the ratio of the values measured with a sharp tip as compared to a rounded tip. Our results suggest that the cell cortex increases the apparent endothelial cell modulus considerably when measured using a sharp AFM tip.

Decreasing outflow facility during acute IOP elevation coincides with a reduction in available area for aqueous humor outflow and the confinement of outflow to the vicinity of CC ostia. These hydrodynamic changes are likely driven by morphologic changes associated with AP collapse and herniation of IW of AP into CC ostia.

Improving our ability to control capillary morphogenesis has implications for not only better understanding of basic biology, but also for applications in tissue engineering and in vitro testing. Numerous biomaterials have been investigated as cellular supports for these applications and the biophysical environment biomaterials provide to cells has been increasingly recognized as an important factor in directing cell function. Here, the ability of ionic self-assembling peptide gels to support capillary morphogenesis and the effect of their mechanical properties is investigated. When placed in a physiological salt solution, these oligopeptides spontaneously self-assemble into gels with an extracellular matrix (ECM)-like microarchitecture. To evaluate the ability of three-dimensional (3D) self-assembled peptide gels to support capillary-like network formation, human umbilical vein endothelial cells (HUVECs) were embedded within RAD16-I ((RADA)4) or RAD16-II ((RARADADA)2) peptide gels with various stiffness values. As peptide stiffness is decreased cells show increased elongation and are increasingly able to contract gels. The observation that capillary morphogenesis is favored in more malleable substrates is consistent with previous reports using natural biomaterials. The structural properties of peptide gels and their ability to support capillary morphogenesis in vitro make them promising biomaterials to investigate for numerous biomedical applications.

Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50 µM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 µm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusionfixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in ≥16 images/eye, and the average percent effective filtration length (PEFL=FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in ≥16 micrographs/eye, and the average percent separation length (PSL= SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54±0.34 (±SEM) to 2.36±0.54 µL/min/mmHg (58.2±18.9%) while control eyes changed from 1.67±0.41 to 1.71±0.39 µl/min/mmHg (6.0±9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p = 0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6±3.7%) compared to control eyes (18.2±4.5%; p<0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3±3.6%) than in controls (20.8±2.0%; p<0.001). A significant positive correlation was found between PEFL and PSL (p = 0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage Reprint Requests (Corresponding Author): Dr. Haiyan Gong, Department of Ophthalmology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, Email: hgong@bu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of th...

Appropriate choice of biomaterial supports is critical for the study of capillary morphogenesis in vitro as well as to support vascularization of engineered tissues in vivo. Self-assembling peptides are a class of synthetic, ionic, oligopeptides that spontaneously assemble into gels with an ECM-like microarchitecture when exposed to salt. In this paper, the ability of four different self-assembling peptide gels to promote endothelial cell adhesion and capillary morphogenesis is explored. Human umbilical vein endothelial cells (HUVECs) were cultured within ionic self-assembling peptide family members, RAD16-I ((RADA)(4)), RAD16-II ((RARADADA)(2)), KFE-8 ((FKFE)(2)), or KLD-12 ((KLDL)(3)). HUVECs suspended in RAD16-I or RAD16-II gels elongated and formed interconnected capillary-like networks resembling in vivo capillaries, while they remained round and formed clusters within KFE-8 or KLD-12 gels. As HUVECs attach to RAD16-I and RAD16-II significantly better than the other peptides, these differences appear to be explained by differences in cell adhesion. Although adhesion likely occurs via bound adhesion proteins, there appears to be no difference in protein binding to the peptides investigated. Results indicate that, although these oligopeptides have similar mechanisms of self- assembly, their primary sequence can greatly affect cell adhesion. Additionally, a subset of these biomimetic ECM-like materials support capillary morphogenesis and thus may be useful for supporting vascularization.