Poster H10 - Investigating the Cooperation Between Endothelin B Receptor and Heparan Sulfate in Regulating Endothelin-1 Expression Using qPCR

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Christina I. Kyriacou (she/her/hers)

Student
University of Rochester
East Amherst, New York, United States

Co-Author(s)

Camden Holm, BS

PhD Student
Worcester Polytechnic Institute
SOUTHBOROUGH, Massachusetts, United States

Last Author(s)

Solomon Mensah

Assistant Professor
Worcester Polytechnic Institute
Sutton, Massachusetts, United States

Introduction: The endothelial glycocalyx (eGCX) is the luminal layer surrounding the
endothelial cell membrane made up of glycoproteins and glycolipids. Its fundamental
roles include modulating vascular permeability, mediating vascular tone, and
mechanotransduction. Mechanotransduction is the conversion of mechanical stimuli,
such as shear stress from blood flow, into biochemical signals by transmitting the force
to the cytoskeleton where it can influence biochemical processes. Endothelin-1 (ET-1) is
a signaling molecule that can cause vasoconstriction through its binding to the
endothelin B receptor (ETB) found on the endothelial cell membrane. ET-1 expression
is shear-dependent and may be regulated through cytoskeletal changes, implicating
potential cooperation with HS. Heparan sulfate (HS) is a glycosaminoglycan that makes
up 50% - 90% of the eGCX and is a known regulator of mechanotransduction in the cell.
This work aims to understand the relationship between ETB and HS. We hypothesize
that there is a cooperative relationship between ETB and HS that may affect ET-1
expression.

Materials and

Methods: To investigate this cooperation, human lung microvascular endothelial cells
(HLMVECs) were subjected to several shear stress magnitudes, 5, 15, and 25
dynes/cm2, and the expression changes of ET-1, ETB, and HS were assessed using
real time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cells
were cultured and seeded on glass coverslips. The fluid flow experiments were
conducted using a parallel-plate flow chamber in a sterile environment to impart shear
stress to the luminal surface of the cells on the coverslip. In addition to a static sample
that experienced no exposure to fluid flow, other HLMVECs were exposed to 5, 15, and
25 dynes/cm^2 of shear stress for 30 minutes. SYBR Green Fast Advanced Cells-to
CT™ Kit from ThermoFisher Scientific was used to perform RT-qPCR for ET-1, ETB,
and HS. Primers were designed using Primer-BLAST software from NCBI. Preliminary
qPCR results were analyzed using the 2^-ΔΔCt method.

Results, Conclusions, and Discussions: Primer design yielded few successful primers. Primers that functioned sufficiently
well were used to gather preliminary data. Fold change analysis using the 2^-ΔΔCt
method showed HS expression to be decreased for all the flow rates with the lowest at
15 dynes/cm^2. ET-1 expression increased significantly at the 5 dynes/cm^2 flow rate,
and insignificantly increased at 15 dynes/cm^2 and 25 dynes/cm^2. ETB expression
also increased significantly at 5 dynes/cm^2 and decreased at 15 dynes/cm^2. The ETB
expression at 25 dynes/cm^2 was insignificant. It is important to note that ET-1 and ETB
followed a similar pattern. These results are only from one round of experiments (n=1),
and further experimentation is ongoing to obtain more results before any claims can be
made.
Challenges in RT-qPCR protocol primer design created several delays in
collection gene expression data. The primers used for the preliminary results have at
this time not been fully validated. While ET-1 gene expression after exposure to shear
stress aligns with known expression profiles, HS gene expression contradicts what is
currently known about HS expression under shear stress. This is likely due to
insufficient RT-qPCR primer development and is the current focus of our research.
Continued refinement of primer choices will allow for a proper understanding of changes
in gene expression of ET-1, ETB, and HS, and will allow us to begin to understand the
potential cooperation between ETB and HS causing changes in ET-1 expression.
Defining this cooperation may lead to a greater understanding of cardiovascular
diseases such as hypertension.

Acknowledgements (Optional): This work was supported by NSF REU grant EEC2150076.