From cellular to molecular mechanobiology.

Mechanobiology at the cellular level is concerned with what phenotypes that cells exhibit to maintain homeostasis in their normal physiological mechanical environment, as well as what phenotypical changes that cells have to make when their environment is altered. Mechanobiology at the molecular level aims to understand the molecular underpinning of how cells sense, respond to, and adapt to mechanical cues in their environment. In this Perspective, we use our work inspired by and in collaboration with Professor Shu Chien as an example with which we connect the mechanobiology between the cellular and molecular levels.

Mechanochemical coupling of formin-induced actin interaction at the level of single molecular complex.

Formins promote actin assembly and are involved in force-dependent cytoskeletal remodeling. However, how force alters the formin functions still needs to be investigated. Here, using atomic force microscopy and biomembrane force probe, we investigated how mechanical force affects formin-mediated actin interactions at the level of single molecular complexes.

Force-history dependence and cyclic mechanical reinforcement of actin filaments at the single molecular level.

The actin cytoskeleton is subjected to dynamic mechanical forces over time and the history of force loading may serve as mechanical preconditioning. While the actin cytoskeleton is known to be mechanosensitive, the mechanisms underlying force regulation of actin dynamics still need to be elucidated. Here, we investigated actin depolymerization under a range of dynamic tensile forces using atomic force microscopy.

Regulation of actin catch-slip bonds with a RhoA-formin module.

The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated.

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds.

As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold.

Effect of zinc and nitric oxide on monocyte adhesion to endothelial cells under shear stress.

This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm(2) or 1 dyne/cm(2)) or reversing shear stress (time average 1 dyne/cm(2)) for 24 h. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure.

Endothelial metallothionein expression and intracellular free zinc levels are regulated by shear stress.

We examined the effects of fluid shear stress on metallothionein (MT) gene and protein expression and intracellular free zinc in mouse aorta and in human umbilical vein endothelial cells (HUVECs). Immunostaining of the endothelial surface of mouse aorta revealed increased expression of MT protein in the lesser curvature of the aorta relative to the descending thoracic aorta. HUVECs were exposed to high steady shear stress (15 dyn/cm(2)), low steady shear stress (1 dyn/cm(2)), or reversing shear stress (mean of 1 dyn/cm(2), 1 Hz) for 24 h.

Endothelial cell responses to atheroprone flow are driven by two separate flow components: low time-average shear stress and fluid flow reversal.

To simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress to mimic the hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average: 1 dyn/cm(2), maximum: +11 dyn/cm(2), minimum: -11 dyn/cm(2), 1 Hz), arterial steady shear stress (15 dyn/cm(2)), and low steady shear stress (1 dyn/cm(2)) on gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed that most differentially expressed genes were similarly regulated by all three shear stress regimens compared with static culture.

Modulating the functional contributions of c-Myc to the human endothelial cell cyclic strain response.

This study addresses whether pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction in human umbilical vein endothelial cells (HUVEC) or human aortic endothelial cells (HAEC). mRNA and protein expression of c-Myc under physiological (6-10%) and pathological cyclic strain conditions (20%) were studied. Both c-Myc mRNA and protein expression increased 2-3-fold in HUVEC cyclically strained at 20%.

Transmigration across activated endothelium induces transcriptional changes, inhibits apoptosis, and decreases antimicrobial protein expression in human monocytes.

We investigated the hypothesis that transmigration drives monocyte transcriptional changes. Using Agilent whole human genome microarrays, we identified over 692 differentially expressed genes (2x, P<0.05) in freshly isolated human monocytes following 1.

The impact of biomechanics in tissue engineering and regenerative medicine.

Biomechanical factors profoundly influence the processes of tissue growth, development, maintenance, degeneration, and repair. Regenerative strategies to restore damaged or diseased tissues in vivo and create living tissue replacements in vitro have recently begun to harness advances in understanding of how cells and tissues sense and adapt to their mechanical environment. It is clear that biomechanical considerations will be fundamental to the successful development of clinical therapies based on principles of tissue engineering and regenerative medicine for a broad range of musculoskeletal, cardiovascular, craniofacial, skin, urinary, and neural tissues.

Expression of CYP1A1 and CYP1B1 in human endothelial cells: regulation by fluid shear stress.

Aims: CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions.

Methods And Results: We investigated CYP1A1 and CYP1B1 expressions under different durations, levels, and patterns of shear stress.

Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF.

Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes.

Gene expression of endothelial cells due to interleukin-1 beta stimulation and neutrophil transmigration.

During the inflammatory response, endothelial cell (EC) functions and mechanics change dramatically. To understand these responses, the authors analyzed changes in EC gene expression in an in vitro model of inflammation using cDNA microarrays. After interleukin-1 beta (IL1beta) stimulation, over 2500 genes were differentially expressed, of which approximately 2000 had not been previously identified by microarray studies of IL1beta stimulation in human umbilical vein endothelial cells (HUVECs).

Cell adhesion under hydrodynamic flow conditions.

This unit describes a hydrodynamic assay to study the relative importance of various receptor/ligand interactions in cell-cell and cell-substrate adhesion and to quantitate the strength of their binding. The basic protocol describes how to assemble the single-chamber flow system with the substrate, add the cells in suspension, and record the experiment on videotape. Alternate protocols present assays to determine how monoclonal antibodies and stimulating and inhibiting agents affect the substrate and the perfusing cells in suspension.

Gene expression of endothelial cells under pulsatile non-reversing vs. steady shear stress; comparison of nitric oxide production.

Pulsations in arterial blood flow expose the endothelium to diverse mechanical forces that may differentially regulate endothelial cell (EC) phenotype. We postulated that pulsatile non-reversing shear stress (typical of the common carotid artery), would produce a more "athero-protective" gene expression pattern compared with steady shear stress of the same mean value. Transcriptional analysis of human umbilical vein endothelial cells (HUVEC) subjected to 24 h of pulsatile shear stress (average = 13 dyne/cm(2), range = 7-25 dyne/cm(2); 1 Hz) or steady shear stress (13 dyne/cm(2)) identified approximately 200 differentially expressed genes.

Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types.

The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC).

Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis.

Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination.

Enhanced platelet adhesion and aggregation by endothelial cell-derived unusually large multimers of von Willebrand factor.

Endothelial cells synthesize and secrete von Willebrand factor (VWF) multimers, including unusually large forms (ULVWF), which are usually cleaved into smaller multimers found in normal plasma (P-VWF). Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder characterized by systemic attachment of platelets to inadequately cleaved ULVWF multimers. We have compared ULVWF and P-VWF in their capacity to become immobilized onto surfaces in vitro and their ability to mediate platelet adhesion.

A validated system for simulating common carotid arterial flow in vitro: alteration of endothelial cell response.

Pulsations in blood flow alter gene and protein expressions in endothelial cells (EC). A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier-Stokes equation solution.

Oxidative stress produced with cell migration increases synthetic phenotype of vascular smooth muscle cells.

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC.

Therapeutic neovascularization: contributions from bioengineering.

A number of pathological entities and surgical interventions could benefit from therapeutic stimulation of new blood vessel formation. Although strategies designed for promoting neovascularization have shown promise in preclinical models, translation to human application has met with limited success when angiogenesis is used as the single therapeutic mechanism. While clinical protocols continue to be optimized, a number of exciting new approaches are being developed.

Integrin interactions with immobilized peptides in polyethylene glycol diacrylate hydrogels.

This study employs tissue-engineering technologies to evaluate neutrophil interactions with extracellular matrix (ECM)-mimetic peptides. We have used a polyethylene glycol (PEG) diacrylate derivative to form a hydrogel as a biologically inert surface. Covalent attachment of bioactive moieties to the hydrogel makes it bioactive.