In humans, the localization of atherosclerotic lesions coincides
with the presence of altered hemodynamics ("disturbed blood flows")
where endothelial cells exhibit an altered phenotype. The cell mechanical
environment at these atherogenesis-prone regions is a complex one
in which the shear stress force on the endothelial surface changes
its magnitude and direction in space and time at the macroscopic
(monolayer level) and microscopic (individual cell) level.
Experimental models, in which endothelial cell morphology, function,
and gene regulation are studied in a well-controlled fluid mechanical
environment, are essential to the investigation of the cellular
and molecular mechanisms by which complex hemodymanics may alter
endothelial cell function leading to lesion development. However,
the effect of fluid forces in vascular endothelial biology has been
traditionally investigated in vitro using flow models in
which endothelial monolayers are exposed to unidirectional flows
of uniform shear stress magnitudes characteristic of normal segments
of the vasculature. I developed the first in vitro model
to study the effect of complex flow characteristics associated with
arterial disease in endothelial cell function. These in vitro studies
recreated the spatial and temporal variations of fluid shear stresses
experienced in vivo by the endothelium in areas prone to atherosclerosis
(i.e. carotid sinus). The cell culture environment was engineered
so that the principal hemodynamic characteristics of the circulation
at arterial bifurcations (secondary flows, flow separation, reattachment
and recirculation) were reproduced in a well-controlled fashion.
Therefore, advancing the state of the art in engineering the cell
environment for cell culture studies.
Using this in vitro model we demonstrated for the first
time that shear stress gradients (rather than shear stress
magnitude) is the key element of disturbed flow fields responsible
for local manifestations of "endothelial dysfunction" in vitro
that are consistent with cellular alterations seen in vivo
in areas of altered hemodynamics. Specifically, localized increased
cell DNA synthesis and evidence of flow-induced cell migration and
cell loss were reported for the first time (DePaola, et al Arteriosclerosis
and Thrombosis 12, 1992).
We have further investigated the role of steady and unsteady disturbed
flows in lesion development by evaluating various spatial and temporal
alterations in endothelial cell function associated with early atherosclerosis.
Results have demonstrated that cell proliferation, cell migration,
cell density, monolayer permeability to macromolecules, endothelial-monocyte
adhesion, monolayer remodeling, and gap junctional intercellular
communication are dynamically regulated by macroscopic shear stress
gradients in vitro. The current data strongly support the existence
of a mechanistic link between gap junction gene expression and cell
proliferation and show evidence of cell signaling processes initiated
by flow. These studies (currently ongoing) are providing fundamental
information expected to contribute to the identification of physical
mechanisms involved in cell pathobiology and to the understanding
of the role of hemodynamics in early arterial disease.
My research work on endothelial intercellular communication has
been conducted in close collaboration with Dr. Polacek and Dr. Davies
from the Institute for Medicine and Engineering at Penn. We have
reported the first in vitro study on gap junctional regulation by
fluid forces and demonstrated that spatial gradients in fluid
shear stress (associated with disturbed flow regions) induce
local changes in endothelial gap junction-mediated intercellular
communication. The results demonstrate that gene expression, protein
organization, and function of the gap junctional protein connexin
43 is regionally mapped to hemodynamic forces.
Differences between hemodynamically defined regions of the endothelial
layer demonstrated compartmentalization of cell-cell communication
in vitro. This finding is highly relevant since it could
aid the understanding of the focal nature of atherosclerotic lesions.
The altered communication rate observed in areas of disturbed flow
in vitro may constitute localized areas of altered cell function.
Motivated by the above findings we are continuing our collaborative
work with the IME under the hypothesis that gradients in fluid
shear stress induce regional changes in endothelial gap junctional
intercellular communication (GJIC) which in turn, alter cell phenotype,
contributing to "functional compartmentalization" (adjacent regions
with altered cell function) of the endothelial monolayer in vivo
and in vitro. This project, funded by NIH, will be one of the
main research activities in my laboratory for the next 5 years.
The long-term objective of this research is to investigate the role
of hemodynamic forces in endothelial cell communication and to identify
the cellular and molecular mechanisms by which flow regulates endothelial
GJIC, contributing to localized vessel wall remodeling and the development
of atherosclerotic lesions.
More recently in collaboration with the IME at Penn (Dr. D. Polacek
and Dr. P.F. Davies) we are investigating the hypothesis that within
any defined region of the vessel wall there is significant heterogeneity
of endothelial signaling and gene expression regulated by differential
shear stress from cell to cell. This study involves the analysis
of gene expression in single cells, and group of cells, removed
from precise hemodynamic locations in vitro and in vivo.
The expression of known shear stress-responsive genes is being evaluated
and new hemodynamic genes are expected to be identified. Our in
vitro flow studies consider the complex spatial and temporal
flow characteristics found in regions prone to atherosclerosis (flow
disturbance). Hemodynamic gene expression is being studied based
on spatial and temporal variations in shear stress defined at the
local cell surface. Well-characterized and precise experimental
models of spatially defined flow are combined with regional and
single-cell gene-expression profiling to investigate the relationships
linking hemodynamics to vessel-wall pathobiology.