Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Summary

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Abstract

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 µg/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Introduction

The field that refers to the study of the effects of physical forces and of mechanical properties on cellular and tissue physiology and pathology is known as mechanobiology¹. A few useful techniques that have been utilized in mechanobiology are monolayer stress microscopy and traction force microscopy. Traction force microscopy allows for the computation of tractions generated at the cell-substrate interface, while monolayer stress microscopy allows for the computation of intercellular stresses generated between adjacent cells within a monolayer^2,3,4

Protocol

1. Making polyacrylamide (PA) gels

1. Preparation of Petri dish
   1. Prepare bind silane solution by mixing 200 mL ultrapure water with 80 µL acetic acid and 50 µL of 3-(trimethoxysilyl)propyl methacrylate. Bind silane is a solution used to functionalize the glass bottom Petri dish surface for hydrogel attachment.
   2. Stir the bind silane solution on a stir plate for at least 1 h.
   3. Treat the center well of the Petri dish with bind silane solution for 45 min.
   4. Remove bind silane solution and rinse the Petri dish with ultra-pure water 2x-3x.
   5. Dry the Petri dish surface and store at room temp....

Results

Phase contrast images of control, 0.2 µg/mL, and 2 µg/mL chalcone treated monolayers were taken 30 minutes before chalcone treatment (Figure 1A-C) and 2 hours after chalcone treatment (Figure 1D-F). Cell-induced bead displacements (µm) were observed to decrease in both low dose chalcone and high dose chalcone conditions (Figure 2E,F) w.......

Discussion

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 cells¹⁵. While it .......

Materials

Name	Company	Catalog Number	Comments
18 mm coverslip	ThermoFisher	18CIR-1	Essential to flatten polyacrylamide gels
2% bis-acrylamide	BIO-RAD	1610143	Component of polyacrylamide gel
2′,5′-Dihydroxychalcone	SIGMA	IDF00046	To disrupt Cx43 structure
3-(Trimethoxysilyl)propyl methacrylate	SIGMA	2530-85-0	Stock solution to make bind silane mixture with acetic acid and ultra-pure water
40% Acrylamide	BIO-RAD	1610140	Component of polyacrylamide gel
Acetic acid	Fisher-Sceintific	64-19-7	Essential to make bind saline solution
Alexa Fluro 488 goat anti-mouse IgG;	ThermoFisher	Catalog # A-11001	Secondary antibody
Ammonium persulfate	BIO-RAD	1610700	Polyacrylamide gel polymerizing agent
Bovine Serum Albumin (BSA)	SIGMA	9048-46-8	To make blocking solution
Bovine Type I Atelo-Collagen Solution, 3 mg/mL, 100 mL	Advance Biomatrix	5005-100ML	Use as a extracellular matrix
Corning Cell Culture Phosphate Buffered Saline (1x)	Fisher-Sceintific	21040CV	Buffer Saline needed for cell culture
Dimethyl Sulfoxide, Fisher BioReagents	Fisher-Sceintific	67-68-5	To dissolve chalcone and make stock solution
Fluoromount-G with DAPI	ThermoFisher	00-4959-52	Mounting medium for immunostaing used to stain for DAPI
Fluroscent microsphere Carboxylate-modified beads	ThermoFisher	F8812	0.5 micron carboxylate-modified beads (red), 2% solids
HEPES buffer solution 1 M	SIGMA	7365-45-9	Essential to
LVES	ThermoFisher	A1460801	Essential HUEVC media 200 supplement
Medium 200	ThermoFisher	M200500	Essential media for HUVEC cell culture
Mouse monoclonal Cx43 antibody (CX - 1B1)	ThermoFisher	Catalog #13-8300	Primary antibody for Cx43
Petri dish (35 mm dia)	CellVis	D35-20-1.5H	35 mm petri dish with a 20 mm center well
Sulfo-SANPAH Crosslinker 100 mg	Proteochem	102568-43-4	Essential to functionalize polyacrylamide gel surface
SYLGARD 184 Silicone Elastomer Kit	DOW corning	2646340	Silicon elastomer with curing agent to make PDMS
TEMED	BIO-RAD	1610801	Polyacrylamide gel polymerizing agent
Triton-X 100	SIGMA	9002-93-1	To permeabilize cells
Trypsin -EDTA	ThermoFisher	25300054	Used to detach cells