Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

Summary

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Abstract

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.⁹ is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Introduction

Vascular endothelial cells form the inner cellular lining of blood vessels in the closed cardiovascular system of higher species. They form the interface between the blood and tissues and are characterized by luminal and abluminal surfaces. The endothelium is a diverse, active, and adaptive system that regulates blood flow, nutrient trafficking, immunity, and the growth of new blood vessels¹. In the body, endothelial cells normally exist in an environment where they are exposed to the frictional force of circulation, shear stress². Shear stress is an important regulator of endothelial cell gene expression

Protocol

1. Preparation of Exogenous Reference RNA

NOTE: Choose an exogenous reference RNA that does not exist in the species or model of interest. For mammalian systems, firefly luciferase RNA may be used.

1. Linearization of exogenous reference RNA plasmid
   1. Prepare exogenous reference RNA at least 48 h prior to the anticipated RNA extraction. Obtain or manufacture a cDNA clone of the chosen exogenous reference RNA, such as a firefly luciferase cDNA clone in a plas.......

Discussion

Shear stress is a physiologic condition that modulates endothelial function, in part, by affecting steady-state gene expression^2,5. Models of gene regulation in various shear stress conditions will contribute to a greater understanding of endothelial function. This pragmatic workflow includes a flow circuit using a parallel-plate flow chamber adapted from Lane et al.⁹ and represents laminar, non-pulsatile flow. The overall set-up .......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	gibco	25300-062
10 mL Syringe	BD	302995
10 mm2 Culture Dish	Sarstedt	83.3902
30 mL Syringe	BD	302832
4-Way Stopcocks	Discofix	D500
Aluminum foil
BEACH	Darwin Chambers Company	MN: HO85, SN: 4947549
Cell Scrapers
CO2 Meter	BioSphenix, Ltd.	MN: P120, SN: 0342
CO2 Sensor	BioSphenix, Ltd.	MN: C700, SN: 52852
Distilled water	gibco	15230-170
Dulbecco's phosphate-buffered saline (DPBS) -/-	gibco	14190-144
Endothelial Cell Growth Medium 2	Promo Cell	C-22011
Endothelial Cell Growth Medium 2 Supplement Mix	Promo Cell	C-39216
Fibronectin (pure)	Sigma-Aldrich	11051407001
Filter (0.20 um)	Sarstedt	83.1826.001
Flow Dampener and Cap	U of T glass blowing shop
Flow Meter: 400 Series Console	Transonic Scisense Inc.	T402
Flow Meter: 400 Series Tubing	Transonic Scisense Inc.	TS410
Flow Reservoir and Cap	U of T glass blowing shop
Flow Sensor	Transonic Scisense Inc.	ME4PXL
Isotemp 737F Oven	Fisher Scientific	FI-737F
J cloth	J cloth
Microscope Slide (25 x 75 x 1 mm)	Fisherfinest	12-544-4
Paper sterilization pouch	Cardinal Health	92713
Pump (Masterflex L/S Economy Drive)	Cole-Parmer	7554-90
Pump Head (Masterflex L/S Easy Load)	Cole-Parmer	7518-00
Rectangular 4 Well Dish	Thermo Scientific	267061
Tweezers
Name	Company	Catalog Number	Comments
Tubing
Masterflex C-Flex L/S 25 Soft Tubing	Cole-Parmer	06424-25
Masterflex C-Flex L/S 14 Soft Tubing	Cole-Parmer	06424-14
Masterflex C-Flex L/S 16 Soft Tubing	Cole-Parmer	06424-16
Masterflex PharMed BPT L/S 13 Hard Tubing	Cole-Parmer	06508-13
Masterflex PharMed BPT L/S 14 Hard Tubing	Cole-Parmer	06508-14
Name	Company	Catalog Number	Comments
Luer
3/16" Male Luer	Cole-Parmer	45518-08	For #25 tubing
1/8" Male Luer	Cole-Parmer	30800-24	For #16 tubing
1/8" Female Luer	Cole-Parmer	30800-08	For #16 tubing
1/16" Male Luer	Cole-Parmer	45518-00	For #14 tubing
1/16" Female Luer	Cole-Parmer	45508-00	For #14 tubing
Name	Company	Catalog Number	Comments
Knockdown reagents
Oligofectamine Reagent	Invitrogen	12252-011
Opti-MEM I Reduced Serum Medium	gibco	31985-070
Name	Company	Catalog Number	Comments
In vitro transcription
Generuler 1kb+ DNA ladder	Thermo Scientific	SM1331
MEGAclear Kit	Ambion	AM1908
mMESSSEGE mMACHINE SP6 Transcription Kit	Ambion	AM1340
pSP-luc+	Promega	E4471
Supercoiled DNA Ladder	New England BioLabs Inc.	N0472S
UltraPure Agarose	Invitrogen	16500-500
UltraPure Ethidium Bromide	Invitrogen	15585011
XhoI Restriction Enzyme	New England BioLabs Inc.	R0146S
Name	Company	Catalog Number	Comments
RNA extraction
Beta-mercaptoethanol	Sigma	M3148-100mL
RNeasy Mini Kit	Qiagen	74104