Exposure of Induced Pluripotent Stem Cell-Derived Vascular Endothelial and Smooth Muscle Cells in Coculture to Hemodynamics Induces Primary Vascular Cell-Like Phenotypes.

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation.

Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis.

A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies.

Hemodynamics associated with atrial fibrillation directly alters thrombotic potential of endothelial cells.

An experimental in vitro model of the hemodynamics that occur in atrial fibrillation (AFib) in the left atrial appendage (LAA) was developed to study changes in human endothelial cell thrombotic potential. We applied human-derived sinus rhythm and AFib hemodynamic shear stress patterns to primary human endothelial cells (ECs) in culture. We found that ECs exposed to AFib hemodynamics have increased thrombotic potential as measured by increased expression of pro-thrombotic gene markers and fibrin deposition on the endothelium.

Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging.

Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction.

Objective: Collateral arteriogenesis, the growth of existing arterial vessels to a larger diameter, is a fundamental adaptive response that is often critical for the perfusion and survival of tissues downstream of chronic arterial occlusion(s). Shear stress regulates arteriogenesis; however, the arteriogenic significance of reversed flow direction, occurring in numerous collateral artery segments after femoral artery ligation, is unknown. Our objective was to determine if reversed flow direction in collateral artery segments differentially regulates endothelial cell signaling and arteriogenesis.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation.

Objectives: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions.

Approach And Results: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins.

Human haemodynamic frequency harmonics regulate the inflammatory phenotype of vascular endothelial cells.

Haemodynamic variations are inherent to blood vessel geometries (such as bifurcations) and correlate with regional development of inflammation and atherosclerosis. However, the complex frequency spectrum characteristics from these haemodynamics have never been exploited to test whether frequency variations are critical determinants of endothelial inflammatory phenotype. Here we utilize an experimental Fourier transform analysis to systematically manipulate individual frequency harmonics from human carotid shear stress waveforms applied in vitro to human endothelial cells.

Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect.

Organotypic systems in drug metabolism and toxicity: challenges and opportunities.

Introduction: The frequent failure of high-throughput screening cell-based tools to accurately predict in vivo responses, coupled with limitations of animal models in predicting human safety or drug efficacy, impairs the de-risking process for biotechnology/pharmaceutical companies as they make important decisions to enter human clinical trials. Organotypic systems strive to fill the gap between these screening and in vivo studies and provide a solution.

Areas Covered: The authors examine the various approaches to recreate physiological response on the bench and trace the evolution of organotypic systems, while discussing intrinsic challenges and opportunities that lie ahead.

Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

Background: Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.

Methodology/principal Findings: This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates.

Three-dimensional phase contrast angiography of the mouse aortic arch using spiral MRI.

Atherosclerosis is a complex disease whose spatial distribution is hypothesized to be influenced by the local hemodynamic environment. The use of transgenic mice provides a mechanism to study the relationship between hemodynamic forces, most notably wall shear stress (WSS), and the molecular factors that influence the disease process. Phase contrast MRI using rectilinear trajectories has been used to measure boundary conditions for use in computational fluid dynamic models.

Hemodynamic activation of beta-catenin and T-cell-specific transcription factor signaling in vascular endothelium regulates fibronectin expression.

Objective: The goal of this study was to assess the activity of β-catenin/T-cell-specific transcription factor (TCF) signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium.

Methods And Results: Histological staining identified preferential nuclear localization of β-catenin in the endothelium of atheroprone aorta before and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with β-catenin nuclear localization and fibronectin deposition.

Matrix rigidity regulates cancer cell growth and cellular phenotype.

Background: The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines.

Methodology/principal Findings: In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate.

Atheroprone hemodynamics regulate fibronectin deposition to create positive feedback that sustains endothelial inflammation.

Rationale: The extracellular matrix protein fibronectin (FN) is focally deposited in regions of atherosclerosis, where it contributes to inflammatory signaling.

Objective: To elucidate the mechanism by which FN deposition is regulated by local shear stress patterns, its dependence on platelet-endothelial cell adhesion molecule (PECAM)-1 mechanotransduction and the role this pathway plays in sustaining an atheroprone/proinflammatory phenotype.

Methods And Results: Human endothelial cells were exposed in vitro to atheroprone or atheroprotective shear stress patterns derived from human carotid arteries.

Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis.

Vascular smooth muscle cell (SMC) phenotypic modulation plays a key role in atherosclerosis and is classically defined as a switch from a 'contractile' phenotype to a 'synthetic' phenotype, whereby genes that define the contractile SMC phenotype are suppressed and proliferation and/or migratory mechanisms are induced. There is also evidence that SMCs may take on a 'proinflammatory' phenotype, whereby SMCs secrete cytokines and express cell adhesion molecules, e.g.

Human IL-8 regulates smooth muscle cell VCAM-1 expression in response to endothelial cells exposed to atheroprone flow.

Objective: Interleukin-8 (IL-8) is a soluble human-specific chemokine implicated in the development of the chronic inflammatory disease atherosclerosis. Recently, we showed that atheroprone hemodynamics induced IL-8 secretion from endothelial cells (ECs) concurrent with increased EC/smooth muscle cell (SMC) VCAM-1 expression in a human hemodynamic coculture model. Despite an IL-8 association with inflammation, we show here that blocking IL-8 activity during atheroprone flow resulted in increased levels of EC/SMC VCAM-1 expression.

Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury.

Lipoma preferred partner (LPP) localizes to focal adhesions/dense bodies, is selectively expressed in smooth muscle cells (SMC) and enhances cell migration. SMCs cultured on denatured collagen or on a rigid substrate, up regulated expression of LPP, its partner palladin, tenascin C (TN-C), phosphorylated focal adhesion kinase (pFAK) and exhibited robust stress fibers. In an endothelial (EC)/SMC hemodynamic flow system, shear stress waveforms mimicking atheroprone flow, applied to the EC layer, significantly decreased expression of SMC LPP and palladin.

PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns.

Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF alphaalpha-, alphabeta-, and betabeta-membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM alpha-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels.

Capillary sprout endothelial cells exhibit a CD36 low phenotype: regulation by shear stress and vascular endothelial growth factor-induced mechanism for attenuating anti-proliferative thrombospondin-1 signaling.

Endothelial cells acquire distinctive molecular signatures in their transformation to an angiogenic phenotype that are indicative of changes in cell behavior and function. Using a rat mesentery model of inflammation-induced angiogenesis and a panel of known endothelial markers (CD31, VE-cadherin, BS-I lectin), we identified a capillary sprout-specific endothelial phenotype that is characterized by the marked down-regulation of CD36, a receptor for the anti-angiogenic molecule thrombospondin-1 (TSP-1). TSP-1/CD36 interactions were shown to regulate angiogenesis in this model as application of TSP-1 inhibited angiogenesis and blockade of both TSP-1 and CD36 accelerated angiogenesis.

Endothelial cell PECAM-1 promotes atherosclerotic lesions in areas of disturbed flow in ApoE-deficient mice.

Objective: Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis.

Methods And Results: We crossed C57BL/6 Pecam1(-/-) mice with apolipoprotein E-deficient (Apoe(-/-)) mice.

p21-activated kinase signaling regulates oxidant-dependent NF-kappa B activation by flow.

Disturbed blood flow induces inflammatory gene expression in endothelial cells, which promotes atherosclerosis. Flow stimulates the proinflammatory transcription factor nuclear factor (NF)-kappaB through integrin- and Rac-dependent production of reactive oxygen species (ROS). Previous work demonstrated that NF-kappaB activation by flow is matrix-specific, occurring in cells on fibronectin but not collagen.

GRP78 upregulation by atheroprone shear stress via p38-, alpha2beta1-dependent mechanism in endothelial cells.

Objective: The initiation of atherosclerosis is in part dependent on the hemodynamic shear stress environment promoting a proinflammatory phenotype of the endothelium. Previous studies demonstrated increased expression of ER stress protein and unfolded protein response (UPR) regulator, GRP78, within all vascular cells in atherosclerotic lesions and its regulation in the endothelium by several atherosclerotic stressors; however, regulation of GRP78 by shear stress directly has not been established.

Method And Results: Using an in vitro model to simulate human arterial shear stress waveforms, atheroprone or atheroprotective flow was applied to human endothelial cells.

Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming.

Atherosclerosis is an inflammatory disease that preferentially forms at hemodynamically compromised regions of altered shear stress patterns. Endothelial cells (EC) and smooth muscle cells (SMC) undergo phenotypic modulation during atherosclerosis. An in vitro coculture model was developed to determine the role of hemodynamic regulation of EC and SMC phenotypes in coculture.

Arterial shear stress regulates endothelial cell-directed migration, polarity, and morphology in confluent monolayers.

Hemodynamic regulation of directional endothelial cell (EC) migration implies an essential role of shear stress in governing EC polarity. Shear stress induces reorientation of the microtubule organizing center toward the leading edge of migrating cells in a Cdc42-dependent manner. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors.

Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis.

Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability.