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J. Biol. Chem., Vol. 278, Issue 33, 31128-31135, August 15, 2003
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Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Stimulates an Inflammatory Response*
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From the
Wallace H. Coulter Department of
Biomedical Engineering at Georgia Tech and Emory University and the
Division of Cardiology and
¶Department of Surgery, Emory University,
Atlanta, Georgia 30322
Received for publication, January 21, 2003 , and in revised form, May 22, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NF
B-dependent mechanism. We suggest that BMP4 is a mechanosensitive,
inflammatory factor playing a critical role in early steps of atherogenesis in
the lesion-prone areas. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endothelial cells are constantly exposed to shear stress (a dragging force generated by blood flow), which controls cellular structure and function such as regulation of vascular tone and diameter, vessel wall remodeling, hemostasis, and inflammatory responses (1). The importance of various types of shear stress is highlighted by the focal development of atherosclerosis (2). Atherosclerosis preferentially occurs in the arterial regions exposed to unstable shear stress conditions in branched or curved arteries, whereas straight arteries exposed to unidirectional laminar shear (LS)1 are relatively lesion-free (1–3). Atherosclerosis is now known as an inflammatory disease caused by endothelial dysfunction (3, 4). One of the first visible markers of endothelial dysfunction in the lesion-prone areas is up-regulation of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 (3–6). These endothelial adhesion molecules play essential roles in adhesion and recruitment of monocytes to the subendothelial layer (3, 4).
How do unstable shear conditions such as low and oscillating shear stress (OS) cause inflammation in those lesion-prone areas, whereas LS exerts athero-protective effects? The opposite effects of LS and OS may be determined by differential expression of genes and proteins, ultimately inducing anti- and pro-inflammatory and atherogenic responses. Recently, several studies (7–10) have begun to address the initial question to determine the expression profiles of mechanosensitive genes. However, the functional importance of those genes has not been clearly established.
Here, we report identification of a mechanosensitive gene, BMP4, by DNA microarray analyses and subsequent verification by a variety of additional approaches in both cultured endothelial cells and human coronary arteries. More importantly, we discovered a novel role of BMP4 as an inflammatory cytokine, providing a potential mechanistic link from shear forces to inflammatory responses and atherogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endothelial Cells—Mouse aortic endothelial cells (MAEC) were cultured and used at passages 4–8 as described by us (11). Human aortic endothelial cells (HAEC) purchased from Clonetics were cultured using the EGM-2 bullet kit (Clonetics) and used at passages 4–8.
Shear Stress Studies—Confluent endothelial monolayers grown in 100-mm tissue culture dishes were exposed to an arterial level of unidirectional LS (15 dyn/cm2) in the growth medium by rotating a Teflon cone (0.5° cone angle) as described previously by us (12). To mimic unstable shear conditions in vivo, endothelial cells were exposed to OS with directional changes of flow at 1 Hz cycle (±5 dyn/cm2) by rotating the cone back and forth using a stepping motor (Servo Motor) and a computer program (DC Motor Company, Atlanta, GA). In some studies, 5 dyn/cm2 unidirectional LS was used for comparison to OS (±5 dyn/cm2).
Preparation of Cell Lysates and Immunoblotting—Following experimental treatments, endothelial cell lysates were prepared and analyzed by Western blot analysis as described by us (13, 14). Briefly, cells were washed in ice-cold phosphate-buffered saline and lysed in 0.1 ml of boiling lysis buffer A (10 mM Tris-HCl, pH 7.6, 1 mM sodium vanadate, and 1% SDS). The lysate was further homogenized by repeated aspiration through a 25-gauge needle. Protein content of each sample was measured by using a Bio-Rad DC assay (15). To detect secreted BMP4 in conditioned media, endothelial monolayers were first washed in serum-free Dulbecco's modified Eagle's medium supplemented with minimum non-essential amino acids and pyruvic acid and exposed to OS, LS, or static conditions for 1 day. The conditioned media were then centrifuged at 1,000 x g for 10 min. Aliquots (2 ml) of the supernatant were collected and placed on ice with 10 ml of ice-cold acetone to precipitate protein for 30 min. Samples were pelleted by centrifugation (15,000 x g for 10 min) and resuspended in 100 µl of sample buffer for SDS-PAGE (13, 14). Aliquots of cell lysates (20 µg of protein each) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with a primary antibody overnight at 4 °C and then with a secondary antibody conjugated with alkaline phosphatase (1 h at room temperature), which were detected by a chemiluminescence method (15). The intensities of immunoreactive bands in Western blots were analyzed by using the NIH Image program. The following primary antibodies were used: a monoclonal BMP4 antibody, rabbit ICAM1 antibody, goat VCAM1 antibody, and goat actin antibody (Santa Cruz Biotechnology).
DNA Microarray Analysis—DNA microarray analyses were performed with an Affymetrix murine gene chip containing 12,000 genes (U74Av2; Affymetrix) and a Motorola murine genome chip containing 10,000 genes, according to the protocols provided by each manufacturer (16). Affymetrix chips were scanned and analyzed at the DNA core facility at Emory University School of Medicine; studies with Motorola chips, the entire process from reverse transcription to hybridization, scanning to initial data analysis, were performed by using the manufacturer's protocol and laboratory (Chicago, IL).
Quantitative Real-time PCR—Real-time PCR for BMP4 was carried out as previously described (17). Briefly, 4 µg of total RNA was reverse-transcribed by using random primers and a Superscript-II kit (Invitrogen) to synthesize first-strand cDNA. The cDNA was purified using a microbiospin 30 column (Bio-Rad) in Tris buffer and stored at –20 °C until used. The cDNA was amplified using a LightCycler (Roche Applied Science) RT-PCR machine. The mRNA copy numbers were determined based on standard curves generated with murine BMP4 and 18 S templates. The 18 S primers (50 nM at 61 °C annealing temperature; Ambion) were used as an internal control for real-time PCR using a LightCycler and capillaries (Roche Applied Science), recombinant Taq polymerase (Invitrogen), and Taq start antibody (Clontech). A quantitative RT-PCR using BMP4 primer pair (forward, 5'-CTGCGGGACTTCGAGGCGACACTTCT-3', reverse, 5'-TCTTCCTCCTCCTCCTCCCCAGACTG-3') using endothelial RNA sample yielded a 130-base pair fragment on agarose gel electrophoresis. This pair of primers was verified by a nested PCR using other BMP4 primer pairs (forward, 5'-ATGGACTGTTATTATGCCTTGTTTTCTGTCAACACCATGATTC-3', reverse, 5'-CCACGTATAGTGAATGGCGACGGCAGTTCTT-3', and forward, 5'-GTCAACACCATGATTCCTGGTAACCGAATGCTGA-3', reverse, 5'-TTATACGGTGGAAGCCCTGTTCCCAGTCAG-3') and by running DNA gels. Real-time PCR for BMP4 was carried out using the annealing temperature 65 °C and extension time for 7 s in the PCR buffer (20 mM Tris-Cl, pH 8.4, at 25 °C, 4mM MgCl2 to which was added 250 µg/ml bovine serum albumin, 200 µM deoxynucleotides) containing SYBR green (1:84,000 dilution), 0.05 unit/µl Taq DNA polymerase, and Taq Start antibody (1:100 dilution).
Fluorescence-activated Cytometry Sorting (FACS) Analysis—Treated cells were dissociated into single-cell suspensions using 0.25% trypsin-EDTA and resuspended in a FACS buffer (Hank's buffered solution containing 5% fetal bovine serum). Aliquots of cell suspensions were incubated with ICAM1 antibody (R&D Systems) for 20 min on ice, washed twice with FACS buffer, and incubated with secondary antibody (fluorescein-5-isothiocyanate- or phycoerytherin-conjugated; Chemicon) for 20 min on ice in the dark. Then samples were washed again, fixed in 1% paraformaldehyde, and analyzed by FACS (Calibur; Becton-Dickinson) using CellQuest software. The fluorescence intensity of ICAM1 and forward cell scattering of 30,000 cells were measured, and the geometric means calculated from histograms were shown. In some studies, HAEC were transfected with either BMP4 (Dr. Elizabeth J. Robertson, Harvard University) cloned in a bicistronic pAdTrack CMV vector (Dr. Bert Vogelstein, The Johns Hopkins University) or an empty vector, both expressing green fluorescent protein (GFP), using LipofectAMINE 2000. In these experiments, ICAM1 expression was measured in the red phycoerytherin channel, while the green channel was used to monitor GFP expression. GFP expression was determined by FACS analysis and fluorescence microscopy (20–30% transfection efficiency). Because expression of GFP was similar among different treatment groups within the same experiment, we did not need to normalize ICAM1 expression data to the GFP level. Human recombinant noggin was either a kind gift from Dr. Arturo Alvarez-Buylla (University of California at San Francisco) or purchased from R&D Systems and used in all experiments at 50 ng/ml (18–20).
Immunohistochemical Study—Frozen sections of human coronary arteries obtained from patients undergoing heart transplants were prepared (17) and stained with antibodies specific for BMP4 (1:1,000 dilution, goat antibody, Santa Cruz Biotechnology), ICAM-1 (1:50 dilution, mouse antibody; R&D), or von Willebrand factor (1:100 dilution, mouse antibody; Dako) for 2 h at room temperature, washed, and followed by incubation with secondary antibodies (anti-goat IgG or anti-mouse IgG) conjugated to alkaline phosphatase. Then the slides were washed and developed with a DAKO kit (DAKO). Photographs of the slides were taken using a Zeiss microscope. Fifteen different human coronary arteries, containing various stages of atherosclerosis from minimally diseased to fatty streak to advanced atheroma stage, from six different patients were examined.
Monocyte Adhesion—Monocyte binding was determined under no-flow conditions using THP-1 monocytes (ATCC) by the method described by Chappel et al. (21). Briefly, THP-1 cells (5 x 105 cells/ml) were labeled with a fluorescent dye 2',7'-bis(carboxyethyl)-5 (6)-carboxyfluorescein-AM (BCECF; Molecular Probes) (1 mg/ml) in serum-free RPMI medium for 45 min at 37 °C. Following exposure to shear stress or BMP treatments in the presence or absence of noggin or vehicle, the endothelial cells were washed in RPMI medium before adding BCECF-loaded THP-1 cells (1:1 ratio). After a 30-min incubation at 37 °C under no-flow conditions, unbound monocytes were removed by washing the endothelial dishes five times with Hank's phosphate-buffered saline. Bound monocytes were quantified by either counting the cells under a fluorescent microscope or by measuring the fluorescent intensity of cell lysates by fluorescence spectrophotometry using a plate reader. Both assays showed similar results. Some studies were performed with MAEC pretreated with 5 µg/ml mouse-ICAM1 antibody (YN1; Southern Biotechnology) (22).
NFB Assay—NFB activity was determined
by using a NFB reporter construct, NFB-SEAP vector (1 µg;
Clontech) expressing a secreted form of placental alkaline phosphatase driven
by 4-B sequences in tandem. This construct was co-transfected with 0.5
µg of either pAdTrack BMP4 or empty vector control using LipofectAMINE
2000. Six hours post-transfection, conditioned media were centrifuged and heat
treated at 65 °C (to inactivate endogenous alkaline phosphatase) for 30
min, followed by chemiluminescence alkaline phosphatase assay according to the
manufacturer's instructions.
Statistical Analysis—Statistical significance was assessed by Student's t test using the Microcal Origin statistical package.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential Regulation of the BMP4 Gene by LS and OS in Endothelial Cells—To identify the genes that may be responsible for the athero-protective and pro-atherogenic effects of LS and OS, respectively, we performed DNA microarray studies using cultured MAEC. Exposing MAEC to LS, but not OS, for 1 day using the modified "cone-and-plate" device (12) induced a cell shape alignment to the direction of the flow from a typical polygonal "cobblestone shape" found in static cultured cells (Fig. 1).
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The total RNAs prepared from these cells were used to determine mRNA
expression profiles by using Affymetrix and/or Motorola DNA chips according to
the manufacturers' protocols. The analyses of these studies showed that LS
exposure significantly and consistently inhibited BMP4 mRNA level in MAEC by
more than 60–80% of static control levels
(Fig. 2A). Unlike LS,
however, exposure of endothelial cells to OS did not inhibit BMP4
mRNA expression (Fig.
2A). We also found that LS exposure up-regulated a well
known mechanosensitive gene, endothelial nitric oxide synthase (eNOS), mRNA
level by more than 5-fold (5.6 ± 1.2, n = 3) above static
controls. In addition, eNOS protein level was also increased by 
2-fold
above controls as determined by Western blot analysis using a monoclonal
antibody (data not shown), providing further confidence in our results.
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BMPs play an important role in bone formation, embryonic development, and differentiation (23, 24). Although BMP4 protein has been found previously in calcified atherosclerotic plaques (25), its expression and functional importance in endothelial cells have not been determined. Therefore, we decided to verify the microarray results by independent methods at the levels of mRNA and protein as well as the functional roles of BMP4 in endothelial biology and pathobiology.
First, we verified the BMP4 mRNA data by using a quantitative real-time PCR method. Exposure of endothelial cells to LS almost eliminated the BMP4 mRNA level (n = 6, p < 0.001) (Fig. 2B). In contrast, OS marginally, statistically not significant, increased BMP4 mRNA level compared with that of static control (n = 3). These results confirmed the DNA microarray results.
Next, BMP4 protein expression was determined by immunoblot studies. BMP4
protein is synthesized as an inactive precursor (48–55 kDa) that is
proteolytically cleaved by proprotein convertases, and the active 23-kDa
protein is secreted (23,
24). In endothelial cell
lysates, the BMP4 precursor was detected as a 54-kDa protein, and the mature
form (p23) was detected in the conditioned media collected from static or
shear-exposed cells (Fig.
2C). Exposure of cells to LS significantly down-regulated
expression of BMP4 precursor in a time-dependent manner
(Fig. 2C). After
16–24 h of LS exposure, BMP4 precursor expression was virtually
undetectable (Fig. 2C,
left panel, p < 0.05). In contrast, exposure of MAEC to OS
significantly increased BMP4 precursor protein level by 2-fold above control
(Fig. 2C, middle
panel, p < 0.05). Consistent with the cell lysate result, the
conditioned media of MAEC exposed to LS (15 dyn/cm2) showed a
barely detectable amount of secreted form of BMP4 (p23)
(Fig 2C, right
panel). In contrast, OS exposure did not significantly change the p23
BMP4 level in the conditioned medium (Fig.
2C, right panel, p < 0.05). Because the cells
were exposed to LS (15 dyn/cm2) and OS (±5
dyn/cm2), we next determined whether it was the shear magnitude
difference that accounted for our results observed so far. To address this
question, we compared the effects of LS and OS using the same magnitudes (5
dyn/cm2 LS versus ±5 dyn/cm2 OS). As
shown as Fig. 2C,
right panel, at the same shear magnitude, OS-exposed cells had more
than 3-fold BMP4 protein than that of LS. However, the higher LS magnitude (15
dyn/cm2) showed a much lower amount of BMP4 than that of lower LS (5
dyn/cm2). These results show that LS exposure inhibits BMP4
expression in a force-dependent manner, whereas OS maintains high BMP4
expression.
BMP4 Expression in the Selective Patches of Endothelial Cells over Foam
Cell Lesions in Human Coronary Arteries— Next, using the human
coronary arteries we determined whether BMP4 protein is expressed in
endothelial cells of human atherosclerotic lesions. The coronary arteries
exhibiting a spectrum of atherosclerotic lesion complexity were obtained from
patients undergoing heart transplants and examined by immunohistochemical
staining (17). BMP4 protein
expression was not apparent in the intimal endothelial cells in relatively
normal, "minimally diseased" human coronary arteries
(Fig. 3A) or in
advanced lesions (data not shown). As shown in
Fig. 3D, one exception
was found in the endothelial cells (arrows) overlying foam cell
lesions that were stained strongly against the BMP4 antibody. As shown in
Fig. 3C,
isotype-matched nonspecific mouse IgG used as a negative control further
supported the specificity of BMP4 staining. In contrast, the medial smooth
muscle cells and macrophages (Fig. 3,
A and D) were most intensely stained against a
monoclonal BMP4 antibody (smooth muscle cells and macrophages identified by
-actin and CD-68 staining, respectively; data not shown). To verify the
identity of endothelial cells, the serial sections were stained with a von
Willebrand factor antibody (endothelial marker,
Fig. 3, B and
E), demonstrating the location of BMP4 staining in select
areas of endothelium. Furthermore, immunostaining with an ICAM1 staining
showed that the expression of this pro-inflammatory adhesion molecule was
selectively increased in the similar endothelial areas expressing BMP4
(Fig. 3, D and
F, arrowheads). On the other hand, we failed to
detect VCAM-1 in the adjacent serial sections (data not shown). This result is
consistent with the finding reported by Endress et al.
(7).
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BMP4 Produced in Endothelial Cells by OS Stimulates Monocyte
Adhesion—The selective expression of BMP4 protein in endothelial
cells above foam cell lesions (an early form of atherosclerotic lesions)
prompted a speculation that BMP4 may be involved in the inflammatory responses
observed in lesion-prone areas
(3,
4). To begin to test the
hypothesis, MAEC were treated with increasing amounts of BMP4 for 24 h and
then monocyte adhesion to endothelium was determined. As a positive control,
some cells were treated with a well known inflammatory cytokine, TNF
(100 units/ml). BMP4 stimulated monocyte binding in a concentration-dependent
manner with a maximum activation of 4–7-fold over control
(Fig. 4A, p
< 0.05). As low as 0.1 ng/ml BMP4 induced a statistically significant
increase, whereas 50 ng/ml BMP4 induced a maximum effect. A similar effect of
BMP4 on monocyte adhesion was also observed by transfecting MAEC or HAEC with
a vector expressing mouse BMP4 (data not shown).
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OS has been shown to induce monocyte adhesion both in vivo and in cultured endothelial cells by increasing surface expression of adhesion molecules (21). Therefore, we used a BMP4 inhibitor, noggin (18, 20), to examine whether OS induces monocyte adhesion in endothelial cells in a BMP4-dependent manner. Exposure of endothelial cells to OS for 24 h significantly increased monocyte adhesion (Fig. 4B, p < 0.05). Treatment of MAEC with noggin (50 ng/ml) inhibited OS-induced monocyte adhesion (Fig. 4B).
In contrast, exposure of MAEC to LS for 24 h inhibited monocyte adhesion by
50% of static control level (Fig.
4C, p < 0.05) as expected
(26). Because LS exposure
significantly inhibited BMP4 expression in endothelial cells
(Fig. 2), we next examined
whether the inhibitory effect of LS on monocyte adhesion could be reversed by
BMP4 addition. For this study, we exposed MAEC in the presence of BMP4 during
shear or static control for 24 h, followed by monocyte adhesion assay. The
inhibitory effect of LS on monocyte adhesion was lost when MAEC were sheared
in medium supplemented with BMP4 (Fig.
4C, p < 0.05). Taken together, these results
suggest that BMP4 produced from endothelial cells by OS exposure leads to
monocyte adhesion.
BMP4 Stimulates Monocyte Adhesion by Inducing ICAM-1 Expression in an
NFB-dependent Manner—Next, we examined the
mechanism by which BMP4 increases monocyte adhesion to endothelial cells.
Adhesion of monocytes to endothelial cells is mediated by sequential
coordinated molecular interactions between the integrins expressed on monocyte
surface and several adhesion molecules expressed on the endothelial surface,
including ICAM-1, VCAM-1, and E-selectin
(3). Moreover, it has been
shown previously that expression of ICAM-1, VCAM-1, and E-selectin on
endothelial cell surface is increased in atherosclerosis-prone areas
(3). Therefore, we first
determined whether the endothelial expression of ICAM-1, VCAM-1, and
E-selectin was modified in response to OS by FACS analysis.
Exposure of HAEC to OS (1 day) increased ICAM-1 expression by 2.8-fold
above control (Fig.
5A, p < 0.05). For comparison, TNF
stimulated ICAM-1 expression 4–5-fold above control cells
(Fig. 5A). To
determine whether ICAM-1 expression induced by OS was mediated by a
BMP4-dependent mechanism, HAEC were either exposed to OS or static conditions
in the presence of a natural BMP antagonist noggin (50 ng/ml). Noggin
completely inhibited ICAM-1 expression in cells exposed to OS, demonstrating
that BMP mediates the OS effect (Fig.
5A). In contrast, noggin did not affect ICAM-1 expression
if it was induced by TNF, a non-BMP family member
(Fig. 5B, p
< 0.05), providing further support for the specificity of noggin toward
BMP.
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Unlike ICAM-1, however, neither OS nor BMP4 had any effect on the surface
expressions of VCAM-1 and E-selectin in endothelial cells, whereas TNF
strongly increased both (Fig. 5, C
and D). Next, we examined the effect of LS on endothelial
expression of ICAM-1, VCAM-1, and E-selectin. Unlike OS, LS significantly
decreased VCAM-1 expression without any significant effect on ICAM-1
(Fig. 5E) or
E-selectin (data not shown). Unlike ICAM-1, the BMP4 effect on VCAM-1 was
marginal at best and not significant (p > 0.05, n = 3).
These results suggest that LS can inhibit monocyte adhesion directly by
down-regulating VCAM-1 expression.
To further investigate the role of BMP4 in ICAM-1 induction, HAEC were transfected with BMP4 vector or an empty control vector by a LipofectAMINE-mediated method. BMP4 stimulated ICAM-1 expression 3–4-fold above that of vector control cells (n = 3, p < 0.001) (Fig. 5F). As further controls, HAEC were mock-transfected with each transfection component, LipofectAMINE alone, empty vector alone, BMP4 vector alone, or not transfected at all. ICAM-1 expressions in all mock-transfected cells were similar to untransfected cells (Fig. 5F). These results demonstrate the specific effect of BMP4 on ICAM-1 induction.
Transfection with BMP4 in HAEC clearly increased expression of both BMP4 precursor (p54 found in cell lysate) and the secreted form (p23 found in conditioned medium) in comparison to controls vector as demonstrated by Western blot (Fig. 5G). This is the first report, as far as we are aware, showing that BMP4 acts as an inflammatory factor by stimulating a specific adhesion molecule, ICAM-1.
Next, we determined whether OS and BMP4 regulate ICAM-1 induction by an
NFB-dependent mechanism
(28–30).
HAEC were exposed to OS, BMP4, or static conditions in the presence or absence
of a NFB translocation inhibitor SN50
(31) or the inactive peptide
SN50M or the proteosome inhibitor MG132
(32). Either SN50 or MG132
completely prevented ICAM-1 expression induced by BMP4, whereas SN50M did not
inhibit ICAM-1 induction induced by BMP4
(Fig. 5F). These
results strongly suggest that NFB plays a critical role in ICAM-1
induction by BMP4.
To demonstrate further whether BMP4 directly stimulates NFB
activity, we examined the effect of BMP4 on NFB activation using an
NFB reporter construct. In this study, NFB-SEAP construct
expressing a secreted form of placental alkaline phosphatase driven by 4
B sequences in tandem was co-transfected with either BMP4 vector or
empty vector control by the LipofectAMINE method used above
(Fig. 5F). As shown in
Fig. 5H, expression of
BMP4 stimulated NFB activity by more than 3-fold above controls
(n = 3, *, p < 0.05).
Finally, by using a blocking antibody we examined whether OS-induced monocyte binding was ICAM-1-dependent. As shown in Fig. 6, monocyte adhesion induced by OS was prevented by treating MAEC with an ICAM1 blocking antibody. These results further demonstrate that chronic exposure of endothelial cells to OS induces monocyte adhesion in an ICAM1-dependent manner.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we identified BMP4 as a mechanosensitive and pro-inflammatory protein. Whereas the atheroprotective LS inhibited expression of BMP4 protein, the pro-atherogenic OS stimulated it. Through functional studies, we discovered a novel function of BMP4 as an inflammatory factor produced in endothelial cells by OS. We found that OS stimulates production of BMP4, which in turn induces ICAM-1 expression on endothelial surface in a NF
B-dependent manner, eventually leading to monocyte recruitment. In the current study, we presented most OS and LS results in comparison to static control conditions. However, it seems more appropriate to compare OS effects to LS conditions rather than to static conditions. Currently, most investigators in this field, including us, use static cultured cells as a physiologically "normal" control. This is necessary because of technical constraints of culturing cells under continuous shear conditions. The majority of arterial endothelial cells in vivo, however, are continuously exposed to LS and aligned to the direction of the flow. In contrast, endothelial cells in the hemodynamically defined lesion-prone areas are exposed to unstable and low wall shear conditions. Therefore, endothelial cells cultured under "static" conditions may represent low or no-shear conditions and may not represent true "control" conditions. Many in vivo studies examine the differences between the normal regions exposed to LS in straight arteries and the lesion-prone areas exposed to disturbed and low shear conditions in branched or curved arteries.
If we compare the OS effects on expression of BMP4 and ICAM-1 as well as monocyte adhesion to those of LS instead of static conditions, the effects of the pro-atherogenic force become more pronounced than what we have shown under "Results." For example, endothelial cells exposed to LS express BMP4 mRNA and protein at almost undetectable levels. Exposure to OS, however, dramatically up-regulated expression of BMP4 mRNA and protein (Fig. 2). This is consistent with our human coronary artery data showing that BMP4 expression is undetectable in normal (minimally diseased) arteries, whereas it is strongly expressed in endothelial patches overlying foam cell lesions. In typical studies, OS increases monocyte binding 8–10-fold above that of LS (data not shown).
BMPs are members of the transforming growth factor-
superfamily and
play important roles in bone formation, embryonic development, and
differentiation (23,
24). Although BMP4 protein has
been found previously in calcified atherosclerotic plaques
(25), its expression and
functional importance in endothelial cells have not been determined. There are
two types of signaling receptors specific for BMPs: BMPR-I and BMPR-II. It
appears they are both required for signaling
(20). Three BMP type I
receptors, BMPR-IA (also known as ALK3, Activin-Like
Kinase-3), BMPR-IB (ALK6), and ALK2 and one BMP type II receptor
have been identified (33).
Although somewhat variable depending upon species and vascular bed origins,
endothelial cells from mouse arteries as well as cultured murine and bovine
aortic endothelial cells have been shown to express both type I (ALK2, 3, and
6) and type II BMPRs (34).
Unlike their well known effects in bone formation and embryonic development,
the functional importance of BMPRs in vascular wall is not clear. One notable
exception is the link in vascular smooth muscle cells as demonstrated by the
loss-of-function mutations of the type II BMPR in familial primary pulmonary
hypertension and sporadic primary pulmonary hypertension
(35). In endothelial cells,
transfection with constitutively active mutants of ALK2, ALK3, and ALK6 has
been shown to stimulate expression of id gene and angiogenic
responses (34).
As far as we are aware, BMP4 has not been shown to induce inflammatory responses previously, especially in endothelial cells. Our finding that BMP4 stimulates monocyte adhesion by increasing surface expression of ICAM-1 in endothelial cells seems to be the most interesting and novel aspect of the current study.
Atherosclerosis is a focal and inflammatory disease preferentially occurring at the lesion-prone areas exposed to unstable and low shear stress (1–4). The branched, bifurcated, and curved arteries such as the lesser curvature of the ascending aorta, the outer wall across the apex of the carotid sinus, and the left descending coronary arteries are the preferential sites of atherosclerotic development (1–4). Endothelial cells in the lesion prone areas become dysfunctional and have been shown to express adhesion molecules including ICAM-1, VCAM-1, and E-selectin (3). Circulating blood monocytes and lymphocytes then bind to these adhesion molecules, migrate beneath endothelium, engulf lipids, and transform into macrophage foam cells, eventually becoming the site of advanced atherosclerotic plaques (3, 4).
Unexpectedly, the current study showed that OS and BMP4 selectively regulate expression of ICAM-1 without significantly affecting VCAM-1 and E-selectin. These seemingly conflicting results on ICAM-1 and VCAM-1 have been reported in mouse atherosclerosis models as well. Cybulsky et al. (5) showed that VCAM-1, but not ICAM-1, expression was up-regulated by a high fat diet in low density lipoprotein receptor–/– mice. In contrast, Nakashima et al. (36) reported that only ICAM-1, but not VCAM-1, expression was up-regulated in a disturbed flow-dependent manner in the lesion-prone areas such as the aortic sinus, whereas VCAM-1 expression was robustly increased by high fat diet feeding in ApoE–/– mouse. Evidence from other mouse atherosclerosis models lacking expression of ICAM-1 or VCAM-1 has shown the importance of both adhesion molecules (5, 27). In addition, this concept is consistent with data from Nagel et al. (37), who used cultured endothelial cells to show that ICAM-1 expression, but not VCAM-1 and E-selectin, was up-regulated by shear stress. Moreover, Endres et al. (6) reported that the early atherosclerotic lesions found in the outer wall of human carotid artery bifurcations showed increased expression of ICAM-1, but not VCAM-1 and E-selectin. However, expressions of VCAM-1 and E-Selectin did increase in advanced atherosclerotic plaques (7). These findings are consistent with our hypothesis that oscillatory shear stress selectively up-regulates ICAM-1 expression. Therefore, although both VCAM-1 and ICAM-1 are important in the pathogenesis of atherosclerosis, ICAM-1 expression in the lesion-prone areas seems to be regulated mainly by oscillatory shear stress, whereas VCAM-1 seems to be more responsive to high cholesterolemic conditions.
Our findings on the selective effect of OS on ICAM-1 expression are consistent with previous reports in cultured endothelial cells using a similar cone-and-plate shear system (37). However, using a parallel plate shear device and human umbilical endothelial cells, OS has been shown to stimulate VCAM-1 and E-selectin in addition to ICAM-1 (21). These discrepancies may be due in part to subtle differences in flow profiles generated in the two different shear devices. Most importantly, however, BMP4 increased expression of ICAM-1, but not VCAM-1 and E-selectin, demonstrating its selective effect on ICAM-1.
VCAM-1 expression and monocyte adhesion are kept very low in most healthy arteries by unknown mechanisms (7, 36). In our current study, we found that chronic exposure of endothelial cells to LS (a physiological condition expected for healthy straight arteries in vivo) virtually eliminated VCAM-1 expression. This may be an important mechanism by which LS acts as a potent anti-inflammatory and anti-atherogenic force. Our results suggest that LS inhibits monocyte adhesion by multiple mechanisms, including the direct inhibition of VCAM-1 expression as well as down-regulation of BMP4 protein, disinhibiting its effect on ICAM-1 expression. The inhibitory effect of LS on VCAM-1 has been shown to be regulated by the NO-dependent mechanisms (26).
Based on the current data and literature, we propose that BMP4 plays a
critical role as a mechanosensitive and pro-inflammatory cytokine mediating
the opposite effects of OS and LS. Our working hypothesis is shown in
Fig. 7: The endothelial cells
in lesion-prone areas (Fig. 7,
dark line) experience low and disturbed shear stress, including OS,
which induces endothelial BMP4 expression. BMP4 then initiates inflammatory
cascades in an NFB-dependent manner by stimulating ICAM-1 surface
expression in those activated endothelial cells. ICAM-1 expression allows
monocyte adhesion and foam cell lesion formation, eventually leading to
atherosclerotic plaque development. In contrast, LS acts as a potent
anti-inflammatory and anti-atherogenic force by not only inhibiting BMP4
expression but also directly down-regulating VCAM-1 expression. Interestingly,
it has been shown (38) that LS
induces expression of two signaling molecules, SMAD 6 and 7, which are known
to inhibit BMP4 action, providing an additional mechanism by which LS prevents
BMP4-dependent responses. The identification of BMP4 as an inflammatory
cytokine may provide not only opportunities for better understanding of
vascular biology and atherogenesis but also novel diagnostic and therapeutic
approaches in atherosclerosis.
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FOOTNOTES |
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* This work was supported by Grants HL71014, HL67413, and HL70531 from the National Institute of Health (to H. J. and W. R. T.), NASA (NAG2-1431), and the Whitaker Foundation (H. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed: Wallace H. Coulter Dept. of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, GA 30322. Tel.: 404-712-9654; Fax: 404-727-3330; E-mail: hanjoong.jo{at}bme.gatech.edu.
1 The abbreviations used are: LS, laminar shear; BMP4, bone morphogenic
protein-4; OS, oscillatory shear; ICAM-1, intercellular adhesion molecule-1;
VCAM1, vascular cell adhesion molecule-1; MAEC, mouse aortic endothelial
cells; HAEC, human aortic endothelial cells; FACS, fluorescence-activated
cytometry sorting; ALK, activin-like kinase.
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ACKNOWLEDGMENTS |
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We thank Dr. Elizabeth Robertson for providing the BMP4 construct, Drs. Daniel Lim and Arturo Alvarez-Buylla for noggin, Dr. Rod Riedel for recombinant BMP4, and Dr. Robert A. Swerlick for help with FACS studies.
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