Wall Shear Stress Effects on Endothelial-Endothelial and Endothelial-Smooth Muscle Cell Interactions in Tissue Engineered Models of the Vascular Wall

Dalit Shav,Shmuel Einav,Ruth Gotlieb,David Elad,Uri Zaretsky,Rajesh Mohanraj

doi:10.1371/journal.pone.0088304

Abstract

Vascular functions are affected by wall shear stresses (WSS) applied on the endothelial cells (EC), as well as by the interactions of the EC with the adjacent smooth muscle cells (SMC). The present study was designed to investigate the effects of WSS on the endothelial interactions with its surroundings. For this purpose we developed and constructed two co-culture models of EC and SMC, and compared their response to that of a single monolayer of cultured EC. In one co-culture model the EC were cultured on the SMC, whereas in the other model the EC and SMC were cultured on the opposite sides of a membrane. We studied EC-matrix interactions through focal adhesion kinase morphology, EC-EC interactions through VE-Cadherin expression and morphology, and EC-SMC interactions through the expression of Cx43 and Cx37. In the absence of WSS the SMC presence reduced EC-EC connectivity but produced EC-SMC connections using both connexins. The exposure to WSS produced discontinuity in the EC-EC connections, with a weaker effect in the co-culture models. In the EC monolayer, WSS exposure (12 and 4 dyne/cm2 for 30 min) increased the EC-EC interaction using both connexins. WSS exposure of 12 dyne/cm2 did not affect the EC-SMC interactions, whereas WSS of 4 dyne/cm2 elevated the amount of Cx43 and reduced the amount of Cx37, with a different magnitude between the models. The reduced endothelium connectivity suggests that the presence of SMC reduces the sealing properties of the endothelium, showing a more inflammatory phenotype while the distance between the two cell types reduced their interactions. These results demonstrate that EC-SMC interactions affect EC phenotype and change the EC response to WSS. Furthermore, the interactions formed between the EC and SMC demonstrate that the 1-side model can simulate better the arterioles, while the 2-side model provides better simulation of larger arteries.

Highlights

The close proximity between the endothelial cells (EC) that compose the intima of blood vessels and the smooth muscle cells (SMC) composing the tunica media enables both these cells to interact with each other
We investigated the attachment of EC to the matrix as well as the interactions of EC-EC and EC-SMC in response to wall shear stresses (WSS) stimulation
Endothelial-matrix Interactions The focal adhesion kinase (FAK), which is an important protein in the focal adhesion complex that attaches cells to the matrix, was used to evaluate the EC-matrix interaction

Summary

Introduction

The close proximity between the endothelial cells (EC) that compose the intima of blood vessels and the smooth muscle cells (SMC) composing the tunica media enables both these cells to interact with each other. The impact of these SMC-EC interactions on the response of EC to WSS is still unclear and requires more research. Most research on the effects of WSS on EC biology has been done with EC alone, that only forms EC-EC interactions and without the interactions of EC with its neighboring SMC (ECSMC interactions) [4]. To investigate the interactions between EC and SMC, several co-culture techniques and models have been introduced. The main four co-culture models are : (1) direct culture of EC on SMC (1-side model) as illustrated in Figure 1B; (2) a culture of SMC and EC on opposite sides of different membranes (2-side models) as illustrated in Figure 1C; (3) a culture of EC on collagen gels or other polymers containing SMC (3 dimensional (3D) model) as illustrated in Figure 1E; and (4) the use of conditioned media as illustrated in Figure 1D, which is a useful method for assessing EC–SMC interactions through secretory mechanisms [5,6,7]

Objectives

Methods

Results

Conclusion