Arterial Uptake of Macromolecules in
Response to Changing Shear Levels
The accumulation of macromolecules, such as low-density lipoprotein,
in the arterial intima is an important component of the atherosclerotic
disease process. We hypothesize that the transport of macromolecules
into the arterial wall, and hence their cumulative uptake over time, is
modulated by changes in the local shear stress that are caused by
corresponding changes in the magnitude or direction of the flow of blood
past the arterial endothelial surface. To test this hypothesis and
investigate the mechanism of this modulation, we are carrying out both
in vivo animal studies and in vitro flow chamber studies.
In vivo studies
A series of experiments has been carried out to measure the effects of
changing wall shear stress on macromolecular uptake in living swine, and
we are now carrying out a second series to examine the corresponding
effect at the molecular level. These acute experiments utilize a femoral
arteriovenous shunt to modulate in vivo the wall shear stress in porcine
iliac arteries. In the uptake experiments, the entry of serum albumin
into these arteries during the course of the experiment was quantified
by tagging the protein with Evans blue dye (EBD) and measuring the
intimal accumulation of the tag using en face photographic densitometry
(see figure).
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This color image illustrates the EBD
staining of
porcine arteries at the conclusion
of an experiment.
A digital photograph of
the vessels is obtained for image processing and EBD
quantitation.
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The uptake experiments showed that, when the time-average wall shear
stress in one external iliac artery is varied by periodically opening
and closing the ipsilateral femoral shunt, intimal albumin uptake in
this vessel increases relative to the unshunted contralateral control
vessel, provided that the period of the shear stress alternation exceeds
about one minute. Furthermore, the size of this increase, during an
experiment of fixed duration, becomes larger when the frequency with
which these changes are imposed is reduced. This frequency dependence,
and the time course of uptake in response to single step changes in
shear, can be explained by a model in which adaptation of the
endothelial lining to changes in shear is accompanied by transient
increases in endothelial permeability.
To understand the molecular mechanisms underlying this behavior, we are
currently characterizing the differences in gene and protein expression
between the shunted and unshunted arteries. A microarray technique is
used to identify differentially expressed genes within the pig genome,
with the hope of finding the important pathways that respond to
hemodynamic changes. Real time PCR and western blots are also used in
this study, for gene and protein quantitation.
Flow chamber studies
Although the most realistic data are obtained from in vivo studies, in
vitro flow chamber experiments can provide better characterized flow
environments, and better control of flow changes. Therefore, it is also
being used in our laboratory to study the adaptive responses of
endothelial cells to hemodynamic changes.
We have developed a computer-controlled time-lapse microscopy system
with auto-focusing, which enables monitoring of cell morphological
changes over long period of time. Microarray, RT-PCR and western blot
protocols have been established in our lab to quantitate gene and
protein expression. We are currently developing a method to measure the
permeability of cultured endothelial cells in our flow chamber. These
techniques can be used to correlate the temporal molecular responses,
morphological remodeling and permeability changes of the endothelial cells.
