Probing Endothelial Cell Mechanics through Connexin 43 Disruption

Abstract

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 2,5dihydroxychalcone (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.

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

“…Our group, as well as others, has been successfully using TFM and MSM to probe the influence of cell-cell junctions in various pathological and physiological cellular processes in vitro 7,15,18,27 . For example, Hardin et al presented a very insightful study that suggests intercellular stress transmission guides paracellular gap formation in endothelial cells 15 .…”

“…These intercellular stresses have been suggested to be important for tissue growth and repair, wound healing, and cancer metastasis 12,[15][16][17] . In addition, intercellular stresses have been suggested to also be important in endothelial cell migration and endothelial barrier function 17,18 . While cell-cell junctions such as tight junctions and adherens junctions have both been suggested to play a critical role in endothelial intercellular stress generation and transmission, the role of gap junctions remains elusive.…”

“…Evidence of Cx43's importance may be found in the fact that genetic deletion of Cx43 in mice results in hypotension 24 and has adverse effects on angiogenesis 25 . In addition, Cx43 has been documented to be important for cell migration and proliferation and in the progression of atherosclerosis 18,[22][23][24][25] In this protocol, we used TFM and MSM to investigate whether traction and intercellular stress generation within the confluent, endothelial monolayer would be impacted by the disruption of the endothelial gap junction Cx43. We disrupted Cx43 with 2,5dihydroxychalcone (chalcone), a molecule documented to inhibit Cx43 expression 26 .…”

Variable fluid flow regimes alter human brain microvascular endothelial cell–cell junctions and cytoskeletal structure

“…In addition to the brain, the endothelium is found throughout the body and is connected by cell-cell junction proteins. Cell-cell junction proteins consist of the following types: tight junctions (TJs), gap junctions (GJs), and adherens junctions (AJs) (Colgan et al, 2007;Colgan et al, 2008;Islam & Steward Jr, 2019;Noria, Cowan, Gotlieb, & Langille, 1999;Taddei et al, 2008). AJ's role in the endothelium is closely linked to intercellular adhesion, which is seen in AJ-specific proteins such as cadherins.…”

Advice for starting a mechanobiology lab

“…1) for 4 years. My research interest includes investigating the mechanics of the cardiovascular system (Islam and Steward Jr 2018;Islam et al 2017;Warren et al 2016;Wu et al 2017), cerebrovascular system, neurovascular unit, and gut epithelium. My lab is currently funded by the NIH.…”