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receptor and Src
1Biomedical Engineering Department, Northwestern University, Evanston, Illinois 60208-3107; and 2Mathematical Sciences Department, Worcester Polytechnic Institute, Worcester, Massachusetts 01609
Submitted 7 May 2003 ; accepted in final form 7 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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receptor and Src in the regulation of such processes. In
experimental models with vascular polymer implants, SMCs migrated from the
vessel media into the neointima of the implant under defined fluid shear
stress. In a nonuniform shear model, blood shear stress suppressed the
expression of PDGF- receptor and the phosphorylation of Src in a shear
level-dependent manner, resulting in the formation of mitogen gradients, which
were consistent with the gradient of cell density as well as the alignment of
SMCs. In contrast, uniform shear stress in a control model elicited an even
influence on the activity of mitogenic molecules without modulating the
uniformity of cell density and did not significantly influence the direction
of SMC alignment. The suppression of the PDGF- receptor tyrosine kinase
and Src with pharmacological substances diminished the gradients of mitogens
and cell density and reduced the influence of nonuniform shear stress on SMC
alignment. These observations suggest that PDGF- receptor and Src
possibly serve as mediating factors in nonuniform shear-induced formation of
cell density gradients and alignment of SMCs in the neointima of vascular
polymer implants.
signal transduction; mitogen gradients; cell density gradients; cell migration; cell alignment
Previous studies have demonstrated that blood shear stress exerts an inhibitory effect on the proliferation of vascular cells (1, 33, 35, 37, 45). The presence of nonuniform shear stress influences the distribution of cell density and induces the formation of cell density gradients, which are associated with smooth muscle cell (SMC) migration and alignment in the direction of cell density gradients (39). In contrast, uniform fluid shear stress in a control model does not influence either the distribution of cell density or the alignment of SMCs (39). These observations suggest that nonuniform fluid shear stress may influence the direction of SMC migration and alignment via the mediation of cell density gradients. In the present study, we investigated a possible mechanism for such regulatory processes.
The transduction of fluid shear stress signals has been a topic of focus in
vascular research. While the mechanisms remain poorly understood, growth
factor-related signaling pathways have been shown to mediate shear
stress-dependent cellular activities
(1,
7–9,
11,
14,
21,
36,
44,
48,
59,
63). Among various growth
factors, platelet-derived growth factor (PDGF)-BB has been demonstrated to
regulate the proliferation of vascular SMCs
(13,
20,
27,
31,
45,
47,
49,
56). This growth factor binds
to the PDGF- receptor and induces the autophosphorylation of the
intracellular tyrosine kinase domain of the receptor, leading to the
activation of downstream signaling molecules, including Src, a nonreceptor
tyrosine kinase that regulates cell migration and proliferation
(50,
57,
58). A previous investigation
(45) has demonstrated that
fluid shear stress inhibits PDGF-BB-related signaling activities in vascular
cells. Thus it is possible that the presence of nonuniform fluid shear stress
may induce graded suppression of PDGF-BB-related signaling molecules and
induces the formation of mitogen gradients, which in turn regulate the
distribution of cell density. The present study was designed to verify this
potential mechanism using experimental models with vascular polymer implants
subject to defined flow fields with uniform and nonuniform shear stress.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Male 3-mo-old Sprague-Dawley rats (Harlan; Indianapolis, IL) were used in
this study. Two experimental models, including a uniform and nonuniform shear
stress model, were established using methods described in a related paper
(39). Briefly, to create a
nonuniform shear model, a polypropylene cylinder of 0.3 mm diameter coated
with polyethylene glycol was implanted in the center of the inferior vena cava
(
3 mm in diameter) in a direction perpendicular to blood flow
(38,
41). To create a uniform shear
model, a polytetrafluoroethylene (PTFE) membrane patch, 3, 0.5, and 0.01
mm in length, width, and thickness, respectively, was inserted into the vena
cava and attached to the endothelium in the circumferential direction of the
vessel with 10-O suture stitches. Because the membrane patch is thin, fluid
shear stress on the patch is similar to that on the endothelium. Observations
were carried out at days 1, 3, 5, 7, and 10 after surgery
with a focus on day 5 because of the presence of peak mitogen
activities and SMC migration. The analysis of shear stress and measurement of
SMC alignment and cell density are described in a related paper
(39). Experimental procedures
were approved by the Animal Care and Use Committee of Northwestern
University.
Endothelial Denudation
To investigate the role of endothelial cells in the regulation of shear stress-induced SMC activities, we removed the endothelium of the host vena cava for polymer implantation in selected cases, resulting in the formation of neointima on the implant without the coverage of endothelial cells. Briefly, a balloon-tipped catheter was inserted into the inferior vena cava through a side branch, and the balloon was inflated with PBS to a diameter slightly larger than that of the vena cava. The catheter was moved back and forth five times for the removal of endothelial cells. A polyethylene cylinder was implanted into the vena cava, as described in Experimental Models, Measurements, and Analyses. Results were compared between specimens with and without endothelial denudation at day 5. The absence of endothelial cells was verified by immunohistochemistry as described in Immunohistochemistry.
Immunohistochemistry
For the detection of endothelial cells, whole neointimal tissues were collected from polymer implants at selected observation times, fixed in 4% formaldehyde in PBS at 4°C for 20 min, incubated with an anti-factor VIII antibody (1:10, Biomeda) at 37°C for 1 h, subsequently incubated with a rhodamine-conjugated secondary antibody under the same conditions, and observed en face using an Olympus fluorescence microscope.
The relative expression of PDGF- receptor and the relative
phosphorylation activity of Src were detected by immunohistochemistry.
Specimens from the uniform and nonuniform shear models were collected at
selected observation times and cut into serial transverse cryosections of 10
µm thickness. Sections were selected at an axial location with a Reynolds
number of 10 from the nonuniform shear model and from the uniform shear
model with comparable shear stress. Three sections from each location of a
selected specimen were incubated with an anti-PDGF-BB and anti-PDGF-
receptor antibody (R&D Systems) and an anti-Src pY418 phosphospecific
antibody (Biosources), respectively, at 37°C for 1 h and subsequently
incubated with a corresponding rhodamine-conjugated secondary antibody under
the same conditions. Each section was then incubated with an anti-SMC
-actin antibody (Chemicon) mixed with 20 nM Hoechst 33258 (for cell
nucleus labeling) and subsequently incubated with a fluorescein-conjugated
secondary antibody at 37°C for 1 h. Specimens were observed using a
fluorescence microscope.
For the nonuniform shear model, the fluorescent intensity of labeled molecules from each section was measured in areas selected at circumferential locations of 0, 8, 20, 28, 36, 47, and 55° (Fig. 1), covering a range from the leading stagnation edge to the maximal shear stress location, under identical optical conditions using a cooled charge-coupled device camera and a MetaMorph imaging system. The intensity of background fluorescence was measured in areas outside the tissue section and used for the calculation of a relative expression or phosphorylation index (intensity of labeled molecules/intensity of background). For the uniform shear stress model, fluorescent intensity for each type of molecules was measured and analyzed from the leading, middle, and trailing sites of each selected transverse section using the methods described above.
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The expression of PDGF- receptor and the phosphorylation activity of
PDGF- receptor tyrosine kinase and Src were verified by immunoblotting
as described previously (32).
This method was also used for the verification of the influence of protein
kinase inhibitors on the activity of the PDGF- receptor tyrosine kinase
and Src. The encapsulating neointima of the nonuniform shear model was
collected and homogenized (5 specimens/assay) in RIPA buffer containing 100 mM
Tris, 0.15 M NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 10 µg/ml
aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 mM
Na3VO4, 2 µg/ml pepstatin, 50 mM NaF, and 5 mM EDTA.
The homogenate was centrifuged at 15,000 g for 6 min. The supernatant
was collected for further analyses. Protein concentrations were determined
using the Bradford method.
The expression of PDGF-BB and PDGF- receptor was detected by
immunoblotting. Total proteins (50 µg/lane) were separated by SDS-PAGE and
then transferred to a polyvinylidene difluoride membrane. The membrane was
incubated with an anti-PDGF-BB or anti-PDGF- receptor antibody (1:1,000,
R&D Systems) at 37°C for 1 h and subsequently incubated with a
horseradish peroxidase-conjugated secondary antibody under the same
conditions. Protein signals were detected using a chemiluminescent method. A
relative expression index was defined as the ratio of the density of a protein
in the encapsulating thrombus to that in the vena cava wall.
The phosphorylation of PDGF- receptor tyrosine kianse and Src was
detected by immunoprecipitation and immunoblotting. The homogenate supernatant
(containing 100 µg total protein) was incubated with Sepharose 2B beads and
cleared with centrifugation. The precleared supernatant was incubated with
either an anti-PDGF- receptor or anti-Src antibody (1:100, Biosource) at
4°C for 4 h, followed by incubation with 30 µg protein A-Sepharose 2B
beads (Sigma) at 4°C for 12 h. After centrifugation at 200 g for
3 min, the beads were collected, washed, resuspended in Laemmli sample buffer,
boiled for 5 min, and centrifuged at 200 g for 3 min. The supernatant
was collected and used for SDS-PAGE analyses and protein transfer as described
above. Membranes containing the PDGF- receptor were incubated with an
anti-PDGF- receptor or anti-phosphotyrosine antibody (PY20), and
membranes containing Src were incubated with an anti-Src or anti-Src pY418
phosphospecific antibody. The expression and phosphorylation of PDGF-
receptor and Src were detected as described above. The relative
phosphorylation activity of these molecules was defined as the ratio of the
density of a phosphorylated protein to that of the same protein expressed in
the same specimen.
Administration of Pharmacological Inhibitors
Selective PDGF- receptor tyrosine kinase and Src inhibitors, AG-1296
(Biosciences) (28) and pp1
(Bio-Mol) (19), respectively,
were delivered one at a time to the nonuniform shear model via an osmotic pump
(Alza) (46). The osmotic pump
was filled with either 10 µM AG-1296 or 10 µM pp1 and implanted in the
rat abdominal cavity immediately after the implantation of the cylinder. The
outlet of the osmotic pump was connected to a branch of the vena cava upstream
to the implanted cylinder. The contained substance was delivered into blood at
a constant rate of 8 µl/h for up to 10 days.
Statistics
Means ± SD were calculated for measured parameters. Student's t-test (two tailed) was used to determine the significance of difference between two selected groups. ANOVA was used to determine the significance of difference between more than two groups. A difference was considered statistically significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Nonuniform shear model. PDGF-BB and PDGF- receptor are
expressed in vascular cells, and their activities are regulated by fluid shear
stress, suggesting a possible involvement of these mitogenic factors in
shear-related events. To test the possibility that these mitogenic factors may
participate in the regulation of shear-induced formation of cell density
gradients and alterations in the direction of SMC migration and alignment, we
examined the relative expression of these factors and the relative
phosphorylation of PDGF- receptor tyrosine kinase and a downstream
nonreceptor tyrosine kinase, Src, with a focus on the relationship with fluid
shear stress and cell density distribution.
In the nonuniform shear model, the expression of PDGF-BB was upregulated in
mainly the surface cells (endothelial cells and/or SMCs) of the encapsulating
neointima in regions near the zero-shear stagnation and flow separation edges
(perpendicular to blood shear stress), whereas that in the shear stress region
between the stagnation and flow separation edges was significantly lower (see
Ref. 39 for the definition of
these locations). A gradient of PDGF-BB expression was observed from the
stagnation edge to the maximal shear location in the direction of blood shear
stress (Fig. 2). A similar
pattern was observed for the expression of PDGF- receptor and the
phosphorylation of Src on Y418, although these molecules appeared in not only
the surface but also in the subsurface cells
(Fig. 2). An inverse
relationship was found between the level of fluid shear stress and the
relative activity of mitogenic molecules
(Fig. 3).
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The expression of PDGF-BB and PDGF- receptor was verified by
immunoblotting. As shown in Fig.
4, the relative expression of PDGF-BB from the entire neointima of
the implant increased after the implantation surgery, reached a peak at
day 5, and decreased afterward. In contrast, the relative expression
of PDGF- receptor increased first and reached a relatively stable level
after day 3. Changes with time were statistically significant for
both PDGF-BB and PDGF- receptor (ANOVA). Immunoblotting was also used to
verify the phosphorylation of PDGF- receptor tyrosine kinase and Src in
the nonuniform shear model at day 5 (maximal SMC migration) and
day 10 (significantly reduced SMC migration). As shown in Figs.
5 and
6, these kinases were
phosphorylated at day 5, and the relative phosphorylation for both
kinases was significantly reduced at day 10.
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Uniform shear model. To verify the role of nonuniform shear stress
in the graded activation of mitogenic molecules, we analyzed and compared the
pattern of PDGF-BB and PDGF- receptor expression and Src Y418
phosphorylation between the nonuniform and uniform shear models. In the
uniform shear model, PDGF-BB was mainly expressed in the surface cells of the
implanted PTFE patch, whereas the PDGF- receptor was expressed in
surface as well as subsurface cells, as observed in the nonuniform shear
model. However, unlike that observed in the nonuniform shear model, no
apparent gradients of PDGF-BB, PDGF- receptor, and Src were observed in
the direction of blood shear stress (Fig.
7).
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Influence of Mitogenic Molecules on Distribution of Cell Density and Pattern Formation of SMCs
To clarify the role of mitogenic molecules in the mediation of nonuniform
shear-induced formation of cell density gradients and the pattern formation of
SMCs, we applied the pharmacological inhibitors AG-1296 and pp1 for
PDGF- receptor tyrosine kinase and Src, respectively, to the nonuniform
shear model. Immunoblotting analyses demonstrated that AG-1296 significantly
suppressed the phosphorylation of PDGF- receptor tyrosine kinase as well
as Src (Figs. 5 and
6, respectively) and pp1
inhibited that of Src at day 5 when maximal SMC migration was
observed (Fig. 8). Immunohistochemical analyses showed that AG-1296 and pp1 both significantly
reduced the relative phosphorylation of Src on Y418 in mainly regions near the
zero-shear stagnation and flow separation edges without a significant
influence on the Src activity in the shear stress region between the
stagnation and flow separation edges (Fig.
2). Such an influence resulted in a shear stress-independent,
relatively uniform distribution of phosphorylated Src on Y418 in the shear
stress direction of the nonuniform shear model
(Fig. 3B).
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In correspondence to changes in the distribution of mitogenic molecules, a treatment with pharmacological inhibitors, including AG-1296 and pp1, significantly suppressed SMC migration in regions near the zero-shear stagnation and flow separation edges, whereas such a treatment did not significantly influence SMC migration in the shear stress region between the stagnation and flow separation edges. The influence of inhibitors resulted in a relatively uniform migration of SMCs in the axial direction of the implant (perpendicular to blood shear stress) ranging from the stagnation edge to the maximal shear stress location, thus reducing the circumferential gradient of cell density (parallel to blood shear stress) on the implant (Fig. 9). As a result, fewer SMCs were migrating in the circumferential direction of the implant, and the population of circumferentially aligned SMCs was significantly reduced compared with the control model without a protein kinase inhibitor (Fig. 10).
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Influence of Endothelial Denudation
Endothelial cells are directly exposed to fluid shear stress. It is
possible that these cells may mediate shear-induced events in SMCs. To verify
such a possibility, we examined PDGF-BB expression in the nonuniform shear
model without endothelial cells. The absence of endothelial cells in specimens
collected from endothelium-denuded blood vessels was confirmed by
immunohistochemical labeling of factor VIII, an endothelial cell-specific
marker (Fig. 11). In specimens
without endothelial cells, PDGF-BB was expressed in the surface cells, which
were SMC -actin positive, of the encapsulating neointima. As shown in
Fig. 2, the pattern of PDGF-BB
expression was similar to that with endothelial cells.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In a related report (39),
it was shown that fluid shear stress inhibited the proliferation of vascular
cells in the neointima of polymer structures implanted in the rat vena cava.
The presence of nonuniform shear stress influenced the distribution of cell
density, inducing the formation of cell density gradients. Because SMCs within
the neointima were not directly exposed to blood shear stress, the shear
influence may be transmitted by intermediating factors. While it is not
entirely understood what factors are involved, PDGF-BB and related signaling
molecules have been shown to promote the migration and proliferation of
vascular SMCs (13,
20,
27,
31,
45,
47,
49,
47,
56). Because the activity of
PDGF-BB and PDGF- receptor is regulated by fluid shear stress
(43,
45,
49,
53), it is possible that these
signaling molecules may serve as mediating factors for nonuniform
shear-induced formation of cell density gradients and alignment of SMCs. Here,
we focused on the role of these mitogenic factors.
As shown in this study, nonuniform shear stress was associated with graded
expression of PDGF-BB and PDGF- receptor and graded phosphorylation of
Src Y418 in the direction of blood shear stress. The level of shear stress was
inversely related to the relative activity of these mitogenic molecules. In
contrast, no apparent gradients were observed for these molecules in the
uniform shear model. These results support the hypothesis that fluid shear
stress may inhibit the activity of mitogenic molecules, and the presence of
nonuniform shear stress may induce the formation of mitogen gradients. Further
investigations demonstrated that the distribution of mitogenic molecules was
consistent with that of cell density, suggesting a role for mitogen gradients
in the formation of cell density gradients. Such a relationship was supported
by the observation that the distribution of bromodeoxyuridine (BrdU)-labeled
or proliferating cells, as found in a previous study
(41) by using a similar
experimental model, was consistent with that of the mitogenic molecules.
To confirm that nonuniform shear stress regulates the distribution of cell
density via a PDGF-BB and PDGF- receptor signaling mechanism, we applied
the selective PDGF- receptor tyrosine kinase and Src inhibitors AG-1296
(28) and pp1
(19), respectively, to the
nonuniform shear model. Immunoblotting analyses showed that a treatment with
AG-1296 significantly reduced the relative phosphorylation of PDGF-
receptor tyrosine kinase and Src, whereas pp1 suppressed the relative
phosphorylation of Src Y418. Both inhibitors diminished the responsiveness of
Src to fluid shear stress, as shown by immunohistochemistry, resulting in a
more uniform distribution of Src pY418 in the shear stress direction compared
with untreated specimens in the presence of the same field of nonuniform shear
stress. The irresponsiveness of Src to fluid shear stress was possibly due to
a significant overall inhibition of Src by AG-1296 or pp1, so that fluid shear
stress could not induce further suppression of the Src activity. As a result,
the gradient of cell density diminished significantly in the shear stress
direction even though nonuniform fluid shear stress was present. These
observations support the role of the PDGF- receptor and Src in mediating
the influence of nonuniform blood shear stress on the distribution of cell
density.
Potential Shear Stress Signaling Pathway for Regulating the Pattern Formation of Vascular SMCs
Fluid shear stress has long been hypothesized to influence the morphogenesis of blood vessels (30). Although there is little direct evidence for such a hypothesis, fluid shear stress has been demonstrated to influence the geometry and dimensions of vascular cells (3, 10, 15, 25, 40, 52) and cell proliferation via regulating the activity of mitogenic and signaling molecules (1, 2, 7–9, 11, 13, 14, 17, 22–24, 26, 36, 43, 44, 48, 54, 55, 59, 63–65), essential processes contributing to vascular morphogenesis and pathogenesis. The present study provides one more line of evidence that nonuniform fluid shear stress may play a role in regulating the pattern formation of vascular SMCs.
While information remains limited, results from previous investigations, as well as the present study, support a hypothetical signaling pathway for shear stress-induced SMC pattern formation. The presence of nonuniform shear stress may cause graded activation of mitogenic molecules. These molecules may induce graded cell proliferation, resulting in the formation of cell density gradients. The gradient of cell density may in turn influence the direction of SMC migration and alignment. However, it remains to be determined how fluid shear stress inhibits the expression of growth factors and how cell density gradients control the direction of cell migration.
In the present models, the surface cells of the encapsulating neointima of the implant were physically sheared by blood flow. However, the inhibitory influence of fluid shear stress on mitogen activities and BrdU incorporation was observed in not only surface cells but also in subsurface cells. These observations suggest that the surface cells may serve as sensors for the conversion of physical shearing friction to paracrine mediators, which can be transmitted to and influence subsurface cells. In particular, the expression of PDGF-BB, observed mainly in surface cells, was inversely related to the level of fluid shear stress. Such a growth factor may serve as a potential paracrine mediator for the influence of fluid shear stress.
The surface layer of the encapsulating neointima of the implant may contain several cell types, including endothelial cells, SMCs, and leukocytes (38). To understand the role of endothelial cells in the mediation of shear stress-dependent SMC pattern formation, we investigated the influence of fluid shear stress on SMC alignment in the nonuniform shear model without endothelial cells. A comparison with endothelialized specimens demonstrated little difference in the expression and distribution of PDGF-BB and in the pattern formation of SMCs. These observations suggest that either endothelial cells or SMCs may serve as shear stress sensors, capable of transmitting shear stress signals to subsurface cells. Leukocytes were attached to the implant during early thrombogenesis and were mainly localized to the interface between the implant and the encapsulating thrombus (38). These cells may not play a dominant role in the mediation of shear stress-dependent SMC pattern formation because of their rare presence at the surface.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-receptor autophosphorylation by the receptor blocking tyrphostin
AG1296. Biochemistry 36:
6260–6269, 1997.[Medline]
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) and without (
) endothelial cells and PDGF-
) and the relative phosphorylation of Src Y418 (
) in the
nonuniform shear stress model in the shear stress or circumferential direction
from 0 to 55° at day 5 (n = 6). These results are
compared with the distribution of shear stress (solid curved line without
symbols) in the same model. Changes with respect to circumferential locations
were significant for all molecules (P < 0.01 by ANOVA).
B: distribution of the relative phosphorylation of Src Y418 in the
presence of AG-1296 (
