Effects of substrate stiffness on phosphorylation of endothelial nitric oxide synthase and nitric oxide bioavailability / Forough Ataollahi
Blood vessels are exposed to mechanical loading (pulse) approximately 75 times per minute. Mechanical loading triggers nitric oxide (NO) production, which is an essential mediator in blood vessel tone regulation. However, disorders, such as aging and atherosclerosis, affect NO produced in each pu...
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| Format: | Thesis |
| Published: |
2015
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| Online Access: | http://studentsrepo.um.edu.my/8764/4/Effects_of_Substrate_Stiffness_on_Phosphorylation_of_Endothelial_Nitric_Oxide_Synthase_and_Nitric_Oxide_Bioavailability.pdf http://studentsrepo.um.edu.my/8764/ |
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| Summary: | Blood vessels are exposed to mechanical loading (pulse) approximately 75 times per
minute. Mechanical loading triggers nitric oxide (NO) production, which is an
essential mediator in blood vessel tone regulation. However, disorders, such as aging
and atherosclerosis, affect NO produced in each pulse because of blood vessel
stiffness. The mechanisms involved in reducing NO production in stiff blood vessels
are not fully understood, because the mechanical environment in blood vessels is a
complex system consisting of shear stress, tensile loading, and pressure that each of
those involves specific mechanisms. This study aims to determine the effect of
substrate stiffness on endothelial NO synthase (eNOS) phosphorylation and NO
production under tensile loading.
Bovine aortic endothelial cells were isolated through incorporation of a new method
in conventional enzymatic digestion, and were characterized by CD.31, Von
Willbrand Factor, 1,1’-dioctadecyl-1,3,3,3’,3’-tetramethylindocarbocyanine
perchlorate acetylated LDL, and angiogenesis behavior. Then, cells were seeded on
the substrates with different stiffness. Substrates were prepared through mixing
polydimethylsiloxane (PDMS) gel with 5 wt% and 10 wt% alumina (Al2O3), and
were characterized by mechanical, structural, and morphological analysis.
Approximately 10% of the strains with 1 Hz frequency were applied on the cells
seeded on the substrates for 3 h. NO production was then measured in the culture
medium, and the intensity of eNOS phosphorylated at Serin1177 was detected in cell
lysate through enzyme-linked immunosorbent assay (ELISA) kits.
The incorporation of filter paper in conventional enzymatic digestion enhanced the
purity of isolated endothelial cells to approximately 90%. Membrane characterization
showed that Al2O3 particles were distributed properly in the PDMS base and did not affect the surface roughness. Moreover, adding Al2O3 to the PDMS base increased
membrane stiffness. The Young’s modulus of the membranes was 0.331, 0.592, and
1.076 MPa for pure PDMS, PDMS/5% Al2O3, and PDMS/10% Al2O3, respectively.
The stretch loading results showed that 10% stretch of cells seeded on compliant
substrates (pure PDMS) with 1 Hz frequency and 3 h duration enhanced the
fleuroscnce intensity of p-eNOS at Serin1177 to 1.6 and increased NO bioavailability
to 600 μg/ml. However, p-eNOS intensity was 1.07 and 1.06 in PDMS/5% Al2O3 and
PDMS/10% Al2O3, respectively, which were significantly less than the pure PDMS.
Moreover, NO concentration in response to tensile loading decreased three times in
cells seeded on PDMS/5% Al2O3 compared with those in pure PDMS, and was
undetectable in PDMS/10% Al2O3.
This study presented the findings related to the phosphorylation of eNOS and NO
production in response to tensile loading. In addition, this study showed that tensile
loading elevated eNOS phosphorylation and NO production dependent on substrate
stiffness.
Keywords: bovine aortic endothelial cells, polydimethylsiloxan, alumina, stretch,
endothelial nitric oxide synthase, nitric oxide. |
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