RESUMO
Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Matriz Extracelular/química , Mecanotransdução Celular/fisiologia , Animais , Materiais Biomiméticos/química , Células Cultivadas , Ratos , Ratos Endogâmicos F344 , Estresse Mecânico , Propriedades de Superfície , Alicerces Teciduais , Proteínas de Sinalização YAPRESUMO
While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands µm(-2)) and global (0 to 71 000 ligand µm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVß3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD µm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD µm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell-matrix interface may serve as design principles when developing future generations of implantable biomaterials.
Assuntos
Algoritmos , Materiais Biocompatíveis/química , Integrinas/química , Oligopeptídeos/química , Engenharia de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Galvanoplastia , Ligantes , Teste de Materiais , Rotação , TêxteisAssuntos
Materiais Biocompatíveis/química , Materiais Biomiméticos/química , Adesão Celular/fisiologia , Elastina/química , Eletroquímica/métodos , Matriz Extracelular/fisiologia , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Reagentes de Ligações Cruzadas/química , Módulo de Elasticidade , Humanos , Teste de Materiais , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , RotaçãoRESUMO
The dipeptidyl peptidase-IV inhibitor saxagliptin (Onglyza) can undergo a thermodynamically favored cyclization to form the corresponding cyclic amidine. The kinetics and mechanism of this conversion were examined to develop a commercial synthesis that afforded saxagliptin with only trace levels of this key byproduct. Important findings of this work are the identification of a profound solvent effect and the determination of an autocatalytic pathway. Both of these phenomena result from transition structures involving proton transfer.
Assuntos
Adamantano/análogos & derivados , Amidinas/química , Dicetopiperazinas/química , Dipeptídeos/química , Inibidores da Dipeptidil Peptidase IV/química , Hipoglicemiantes/química , Prótons , Adamantano/química , Ciclização , Dipeptidil Peptidase 4/química , Estabilidade de Medicamentos , Humanos , Ligação de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Solventes/química , TermodinâmicaRESUMO
Sleep loss, as well as concomitant fatigue and risk, is ubiquitous in today's fast-paced society. A biomathematical model that succeeds in describing performance during extended wakefulness would have practical utility in operational environments and could help elucidate the physiological basis of sleep loss effects. Eighteen subjects (14 males, 4 females; age 25.8 +/- 4.3 years) with low levels of habitual caffeine consumption (<300 mg/day) participated. On night 1, subjects slept for 8 h (2300-0700 h), followed by 77 h of continuous wakefulness. They were assigned randomly to receive placebo or caffeine (200 mg, i.e., two sticks of Stay Alert gum) at 0100, 0300, 0500, and 0700 during nights 2, 3, and 4. The psychomotor vigilance test (PVT) was administered periodically over the 77-h period of continuous wakefulness. Statistical analysis reveals lognormality in each PVT, allowing for closed-form median calculation. An iterative parameter estimation algorithm, which takes advantage of MatLab's (R2007a) least-squares nonlinear regression, is used to estimate model parameters from subjects' PVT medians over time awake. In the model, daily periodicity is accounted for with a four-component Fourier series, and a simplified binding function describes asymptotic fatigue. The model highlights patterns in data that suggest (1) the presence of a performance inhibitor that increases and saturates over the period of continuous wakefulness, (2) competitive inhibition of this inhibitor by caffeine, (3) the persistence of an internally driven circadian rhythm of alertness, and (4) a multiplicative relationship between circadian rhythm and performance inhibition. The present inhibitor-based minimal model describes performance data in a manner consistent with known biochemical processes.