Disturbed fluid flow reinforces endothelial tractions and intercellular stresses.

Disturbed fluid flow is well understood to have significant ramifications on endothelial function, but the impact disturbed flow has on endothelial biomechanics is not well understood. In this study, we measured tractions, intercellular stresses, and cell velocity of endothelial cells exposed to disturbed flow using a custom-fabricated flow chamber. Our flow chamber exposed cells to disturbed fluid flow within the following spatial zones: zone 1 (inlet; length 0.676-2.027 cm): 0.0037 ± 0.0001 Pa; zone 2 (middle; length 2.027-3.716 cm): 0.0059 ± 0.0005 Pa; and zone 3 (outlet; length 3.716-5.405 cm): 0.0051 ± 0.0025 Pa. Tractions and intercellular stresses were observed to be highest in the middle of the chamber (zone 2) and lowest at the chamber outlet (zone 3), while cell velocity was highest near the chamber inlet (zone 1), and lowest near the middle of the chamber (zone 2). Our findings suggest endothelial biomechanical response to disturbed fluid flow to be dependent on not only shear stress magnitude, but the spatial shear stress gradient as well. We believe our results will be useful to a host of fields including endothelial cell biology, the cardiovascular field, and cellular biomechanics in general.

A machine learning approach to predict cellular mechanical stresses in response to chemical perturbation.

Mechanical stresses generated at the cell-cell level and cell-substrate level have been suggested to be important in a host of physiological and pathological processes. However, the influence various chemical compounds have on the mechanical stresses mentioned above is poorly understood, hindering the discovery of novel therapeutics, and representing a barrier in the field. To overcome this barrier, we implemented two approaches: 1) monolayer boundary predictor and 2) discretized window predictor utilizing either stepwise linear regression or quadratic support vector machine machine learning model to predict the dose-dependent response of tractions and intercellular stresses to chemical perturbation. We used experimental traction and intercellular stress data gathered from samples subject to 0.2 or 2 µg/mL drug concentrations along with cell morphological properties extracted from the bright-field images as predictors to train our model. To demonstrate the predictive capability of our machine learning models, we predicted tractions and intercellular stresses in response to 0 and 1 µg/mL drug concentrations which were not utilized in the training sets. Results revealed the discretized window predictor trained just with four samples (292 images) to best predict both intercellular stresses and tractions using the quadratic support vector machine and stepwise linear regression models, respectively, for the unseen sample images.

Extracellular Matrix Composition Alters Endothelial Force Transmission.

ECM composition is important in a host of pathophysiological processes such as angiogenesis, atherosclerosis, and diabetes, for example and during each of these processes ECM composition has been reported to change over time. However, the impact ECM composition has on the endothelium’s ability to respond mechanically is currently unknown. Therefore, in this study we seeded human umbilical vein endothelial cells (HUVECs) onto soft hydrogels coated with an ECM concentration of 0.1 mg/mL at the following collagen I (Col-I) and fibronectin (FN) ratios: 100%Col-I, 75%Col-I-25%FN, 50%Col-I-50%FN, 25%Col-I-75%FN, and 100%FN. We subsequently measured tractions, intercellular stresses, strain energy, cell morphology, and cell velocity. Our results revealed huvecs seeded on gels coated with 50% Col-I - 50% FN to have the highest intercellular stresses, tractions, strain energies, but the lowest velocities and cell circularity. Huvecs seeded on 100% Col-I had the lowest tractions, cell area while havingthe highest velocities and cell circularity. In addition, cells cultured on 25% Col-I and 75% FN had the lowest intercellular stresses, but the highest cell area. Huvecs cultured on 100% FN yielded the lowest strain energies. We believe these results will be of great importance to the cardiovascular field, biomedical field, and cell mechanics. Summary: Study the influence of different Col-I - FN ECM compositions on endothelial cell mechanics and morphology.

Probing Endothelial Cell Mechanics Through Connexin 43 Disruption.

The endothelium has been established to generate intercellular stresses and suggested to transmit these intercellular stresses through cell-cell junctions, such as VE-Cadherin and ZO-1, for example. Although the previously mentioned molecules reflect the appreciable contributions both adherens junctions and tight junctions are believed to have in endothelial cell intercellular stresses, in doing so they also reveal the obscure relationship that exists between gap junctions and intercellular stresses. Therefore, to bring clarity to this relationship we disrupted expression of the endothelial gap junction connexin 43 (Cx43) by exposing confluent human umbilical vein endothelial cells (HUVECs) to a low (0.2 µg/mL) and high (2 µg/mL) concentration of 2,5-dihydroxychalcone (chalcone), a known Cx43 inhibitor. To evaluate the impact Cx43 disruption had on endothelial cell mechanics we utilized traction force microscopy and monolayer stress microscopy to measure cell-substrate tractions and cell-cell intercellular stresses, respectively. HUVEC monolayers exposed to a low concentration of chalcone produced average normal intercellular stresses that were on average 17% higher relative to control, while exposure to a high concentration of chalcone yielded average normal intercellular stresses that were on average 55% lower when compared to control HUVEC monolayers. HUVEC maximum shear intercellular stresses were observed to decrease by 16% (low chalcone concentration) and 66% (high chalcone concentration), while tractions exhibited an almost 2-fold decrease under high chalcone concentration. In addition, monolayer cell velocities were observed to decrease by 19% and 35% at low chalcone and high chalcone concentrations, respectively. Strain energies were also observed to decrease by 32% and 85% at low and high concentration of chalcone treatment, respectively, when compared to control. The findings we present here reveal for the first time the contribution Cx43 has to endothelial biomechanics.

Advice for starting a mechanobiology lab.

Starting a new lab in mechanobiology and a new lab in general can be an exciting, yet daunting experience. Depending on your previous position, you have now found yourself with significantly more responsibility in more ways than one. For those considering taking such a career path, I present in this commentary advice that will hopefully be useful and provide food for thought.

Protection from phenytoin-induced congenital malformations by coadministration of the antiepileptic drug stiripentol in a mouse model.

To test the hypothesis that the cytochrome P-450-inhibiting antiepileptic drug (AED) stiripentol (STP) can reduce the incidence of phenytoin (PHT) induced congenital malformations, we chronically administered the AEDs to three inbred mouse strains. The frequency of congenital defects in PHT-treated animals was dosage dependent, ranging from 7 to 55%. When STP was coadministered orally with PHT, there was a significant reduction in fetal defects in SWV and C57BL/6J mouse strains. A replication of the experiment was conducted with addition of a seventh group of mice that received the high dosage of PHT together with STP. In the replicate, all three strains demonstrated a significant reduction in fetal defects in fetuses exposed to PHT (60 mg/kg) and STP (200 mg/kg) as compared with PHT (60 mg/kg) monotherapy. These results strongly suggest that oxidative metabolites activated by cytochrome P-450 are the primary teratogenic molecules involved in PHT-induced teratogenesis and that clinical management of pregnant epileptic patients may be improved through heightened awareness of these drug interactions.