Peter Mack

Cell migration into scaffolds under co-culture conditions in a microfluidic platform

Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.

Biomechanical forces promote embryonic haematopoiesis

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3–5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41+c-Kit+ haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta–gonads–mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

Hemodynamic Environments from Opposing Sides of Human Aortic Valve Leaflets Evoke Distinct Endothelial Phenotypes In Vitro

The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the “atheroprotective” transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.

Biomechanical regulation of endothelium-dependent events critical for adaptive remodeling

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling.

A high-throughput system to probe and direct biological functions driven by complex hemodynamic environments

Abstract One of the most critical challenges in modeling physiological and pathological disease states is the requirement for incorporating dynamic effects into the cellular microenvironment. In particular, vascular biology research has been limited by the inability to culture endothelial and other vascular cells in the presence of a hemodynamic waveform, which is critical to recapitulation of the in vivo microenvironment and to the realization of physiologically relevant phenotypic and genotypic expression. Here we describe technologies capable of applying precise hemodynamic waveforms to cultured endothelial and other cell types in multiwell plates using micromechanical motor-driven cones spinning in each of 96 microwells. Physiological responses are demonstrated, including atherogenic and atheroprotective morphologic and gene expression profiles. This technology has the capability to enable rapid and efficient biological screening for a wide range of drug development applications.

Stress-Induced Mechanotransduction: Some Preliminaries

Mechanical stimuli affect nearly every aspect of cellular function, yet the underlying mechanisms of transduction of force into biochemical signals are not clearly understood. One hypothesis is that forces transmitted via individual proteins, either at the site of cell adhesion to its surroundings or within the stress-bearing members of the cytoskeleton, cause conformational changes that change their binding affinity to other intracellular molecules. This altered equilibrium state can subsequently initiate biochemical signaling cascades of produce immediate structural changes. This paper addresses the distribution of forces within the cell resulting from specific mechanical stimuli, computed using a 3-D multi compartment, continuum, viscoelastic finite element model, and uses these to estimate the forces transmitted by individual proteins and protein complexes. These levels of force are compared to those known to produce conformational changes in cytoskeletal proteins, as speculated from magnetocytometry observations and computed by molecular dynamics.Copyright