Devon Scott

Development and evaluation of microdevices for studying anisotropic biaxial cyclic stretch on cells

Mechanical effects on cells have received more and more attention in the studies of tissue engineering, cellular pathogenesis, and biomedical device design. Anisotropic biaxial cyclic stress, reminiscent of the in vivo cellular mechanical environment, may promise significant implications for biotechnology and human health. We have designed, fabricated and characterized a microdevice that imparts a variety of anisotropic biaxial cyclic strain gradients upon cells. The device is composed of an elastic membrane with microgroove patterns designed to associate cell orientation axes with biaxial strain vectors on the membrane and a Flexcell stretcher with timely controlled vacuum pressure. The stretcher generates strain profile of anisotropic biaxial microgradients on the membrane. Cell axes determined by the microgrooves are associated with the membrane strain profile to impose proper biaxial strains on cells. Using vascular smooth muscle cells as a cell model, we demonstrated that the strain anisotropy index of a cell was likely one of the determinant mechanical factors in cell structural and functional adaptations. The nuclear shape and cytoskeleton structure of smooth muscle cells were influenced by mechanical loading, but were not significantly affected by the strain anisotropy. However, cell proliferation has profound responses to strain anisotropy.

Mechanics and Function of the Pulmonary Vasculature: Implications for Pulmonary Vascular Disease and Right Ventricular Function

he relationship between cardiac function and the afterload against which the heart muscle must work to circulate blood throughout the pulmonary circulation is defined by a complex interaction between many coupled system parameters. These parameters range broadly and incorporate system effects originating primarily from three distinct locations: input power from the heart, hydraulic impedance from the large conduit pulmonary arteries, and hydraulic resistance from the more distal microcirculation. These organ systems are not independent, but rather, form a coupled system in which a change to any individual parameter affects all other system parameters. The result is a highly nonlinear system which requires not only detailed study of each specific component and the effect of disease on their specific function, but also requires study of the interconnected relationship between the microcirculation, the conduit arteries, and the heart in response to age and disease. Here, we investigate systems-level changes associated with pulmonary hypertensive disease progression in an effort to better understand this coupled relationship.

High pulsatility flow stimulates smooth muscle cell hypertrophy and contractile protein expression.

Proximal arterial stiffening is an important predictor of events in systemic and pulmonary hypertension, partly through its contribution to downstream vascular abnormalities. However, much remains undetermined regarding the mechanisms involved in the vascular changes induced by arterial stiffening. We therefore addressed the hypothesis that high pulsatility flow, caused by proximal arterial stiffening, induces downstream pulmonary artery endothelial cell (EC) dysfunction that in turn leads to phenotypic change of smooth muscle cells (SMCs). To test the hypothesis, we employed a model pulmonary circulation in which upstream compliance regulates the pulsatility of flow waves imposed onto a downstream vascular mimetic coculture composed of pulmonary ECs and SMCs. The effects of high pulsatility flow on SMCs were determined both in the presence and absence of ECs. In the presence of ECs, high pulsatility flow increased SMC size and expression of the contractile proteins, smooth muscle α-actin (SMA) and smooth muscle myosin heavy chain (SM-MHC), without affecting proliferation. In the absence of ECs, high pulsatility flow decreased SMC expression of SMA and SM-MHC, without affecting SMC size or proliferation. To identify the molecular signals involved in the EC-mediated SMC responses, mRNA and/or protein expression of vasoconstrictors [angiotensin-converting enzyme (ACE) and endothelin (ET)-1], vasodilator (eNOS), and growth factor (TGF-β1) in EC were examined. Results showed high pulsatility flow decreased eNOS and increased ACE, ET-1, and TGF-β1 expression. ACE inhibition with ramiprilat, ET-1 receptor inhibition with bosentan, and treatment with the vasodilator bradykinin prevented flow-induced, EC-dependent SMC changes. In conclusion, high pulsatility flow stimulated SMC hypertrophy and contractile protein expression by altering EC production of vasoactive mediators and cytokines, supporting the idea of a coupling between proximal vascular stiffening, flow pulsatility, and downstream vascular function.

NAD(P)H:quinone Oxidoreductase 1-Compromised Human Bone Marrow Endothelial Cells Exhibit Decreased Adhesion Molecule Expression and CD34+ Hematopoietic Cell Adhesion

NAD(P)H:quinone oxidoreductase 1 (NQO1) deficiency resulting from a homozygous NQO1*2 polymorphism has been associated with an increased risk of benzene-induced myeloid toxicity and a variety of de novo and therapy-induced leukemias. Endothelial cells in human bone marrow form one of the two known hematopoietic stem cell microenvironments and are one of the major cell types that express NQO1 in bone marrow. We have used a transformed human bone marrow endothelial cell (TrHBMEC) line to study the potential impact of a lack of NQO1 activity on adhesion molecule [endothelial leukocyte adhesion molecule 1 (E-selectin), vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1] expression and functional adhesion to bone marrow progenitor cells. We used both 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1, and anti-NQO1 small interfering RNA to abrogate NQO1 activity. Real-time reverse transcription-polymerase chain reaction data demonstrated a significant inhibition of tumor necrosis factor (TNF)α-induced E-selectin mRNA levels after ES936 pretreatment. Immunoblot assays demonstrated a significant reduction in TNFα-stimulated E-selectin, VCAM-1, and ICAM-1 proteins after inhibition or knockdown of NQO1. The mechanisms underlying this effect remain undefined, but modulation of nuclear factor-κB (p65), c-Jun, and activating transcription factor 2, transcriptional regulators of adhesion molecules, were observed after inhibition or knockdown of NQO1. Decreased level of E-selectin, VCAM-1, and ICAM-1 also resulted in a functional deficit in adhesion. A parallel plate flow chamber study demonstrated a marked reduction in CD34+ cell (KG1a) adhesion to NQO1-deficient TrHBMECs relative to controls. The reduced adhesive ability of TrHBMECs may affect the function of the vascular stem cell niche and also may contribute to the increased susceptibility of polymorphic individuals lacking NQO1 to leukemias and hematotoxicants such as benzene.

Effect of Vessel Stiffening and High Pulsatility Flow on Contractile Function and Proliferation of Small Arterial Cells

Recent studies have identified arterial stiffening as a predictor of some vascular diseases such as pulmonary hypertension, which is characterized by dysfunction of small arteries. Stiffening is shown to cause changes in blood flow, extending high pulsatile flow into small arteries that normally experience steady flow conditions (Chui 2004). However, few studies have investigated the mechanisms underlying the effects of arterial stiffening on vascular remodeling. We hypothesized that arterial stiffness effects dysfunction of downstream vascular endothelium and smooth muscle through changes in flow pulsatility. Previously we developed a flow system to study the influence of pulse flow waves, by modulating upstream stiffness, on downstream mimetic vascular cell co-culture. With this system, the present study examines contractile and proliferating protein expressions of smooth muscle cell (SMC) co-cultured with endothelial cell (EC). The endothelium, directly interfaces with the blood flow, and transduces mechanical signals to underlying SMC (Stegemann 2005). We recently showed that high pulsatile flow induced EC dysfunction. Therefore, we further asked whether high pulsatility flow would cause characteristic changes of small arterial SMC in the hypertension condition such as smooth muscle hyperplasia (increased cell proliferation) and hypertrophy (increased contractile proteins) (Voelkel 1997), and whether these changes would be mediated by EC dysfunction.© 2010 ASME

Devlopment of a Cell Coculture Microfluidic Shear Device for Mechano-Transmission Study

We have developed a microfluidic shear device that allows for the study of cell communication in a dynamically controlled biochemical and biomechanical environments simulating cells’ living environments in vivo. Such study may help to improve our understanding in the effects of hypertension-relevant and vascular development-relevant flow shear stress on cell behaviors. Endothelial cells may be a key factor for transmitting the blood flow conditions from the endothelial lining to interstitial layers and smooth muscle cells. The interstitial flow stress and the shear stress induced signaling factors may greatly alter vascular biology of these deep layers. Endothelial cells act as a mechano-transducer by converting shear stress into biochemical signaling factors. The biochemical factors diffuse to smooth muscle cells and further alter the biological structure of vascular tissues. Also, the flow shear stress will be transmitted to the interstitial tissue layer through the pores resulted from the pores in the fenestrated endothelial lining. Studies in both the mechano-transduction process and the mechano-transmission process will benefit from a biomimetic flow shear device with co-cultured cells. Our device will allow the co-culture of endothelial cells and smooth muscle cells to study these biomechanical processes. The pulmonary arterial cells are used as a model in the study. The microfluidic device developed here will be used to enhance the understanding of pulmonary vascular disease pathogenesis due to the variations in the flow shear stress.Copyright

Anisotropic Strain Effects on Vascular Smooth Muscle Cell Physiology

Biological development is a complex and highly-regulated process, a significant part of which is controlled by mechanostimulus, or the strain imparted on a cell by its environment. Mechanostimulus is important for stem cell differentiation, from cytoskeletal assembly to cell-cell and cell-matrix adhesion [1]. The mechanics of cells and tissues play a critical role in organisms, under both physiological and pathological conditions; abnormal mechanotransduction — the mechanism by which cells sense and respond to strain — has been implicated in a wide range of clinical pathologies [2,3].Copyright