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From the Department of Pathology, University of Oklahoma HealthSciences Center, Oklahoma City, Oklahoma
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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.8-10
To add even further to its
extensive range of known functions, CAP37 has been demonstrated to
stimulate the reversible contraction of fibroblasts and endothelial
cells7
and to activate endothelial cell protein kinase
C.11
Recently, CAP37 released from stimulated PMNs was
reported to be taken up and sequestered in nearby endothelial
mitochondria and has been suggested to protect against
apoptosis.12
We have shown the presence of CAP37 in the endothelium of Alzheimer’s
brain microvessels13
and have shown it to be induced in
rat brain endothelial cells in response to stimulation with
inflammatory molecules TNF-, IL-1, and LPS.13
Here,
we report the presence of CAP37 in endothelium associated with
atherosclerotic plaques, and in and around foam cells, and cholesterol
clefts in complex plaques. There is strong evidence that both
Alzheimer’s disease and atherosclerosis are inflammatory-modulated
diseases14,15
in which inflammation and associated
mediators can exacerbate or augment the disease. We believe that the
association of CAP37 in both these diseases lends credence to our
hypothesis that it is an important mediator of inflammation leading to
the exacerbation or augmentation of the chronic inflammatory responses
observed in Alzheimer’s disease and atherosclerosis.
The aims of this study were to demonstrate the presence of CAP37 in atherosclerotic lesions, show its induction in endothelial cell culture, and confirm that it is CAP37 by nucleotide sequence analysis. We demonstrate that LPS induces CAP37 protein and mRNA expression in vitro in a time-dependent manner. Furthermore, isolated mRNA from activated human umbilical vein endothelial cells (HUVECs) shows sequence identity with an extensive region of PMN-CAP37. This is the first demonstration of endogenous endothelial-CAP37 (E-CAP37) as confirmed by sequence analysis and suggests that, because of its induction and location in the endothelium and its known monocyte- and endothelial-activating capabilities, CAP37 has the potential to modulate monocyte/endothelial dynamics at the vessel wall.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Sections of atherosclerotic vessels (carotid, iliac, coronary and femoral arteries, and aorta) were obtained from the Department of Pathology, University of Oklahoma archival tissue bank.
Cell Culture
Rat aorta endothelial cells (RAECs) were isolated and maintained in Dulbecco’s modified Eagle’s medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), as previously described.16 Cells were used within the first 15 passages.
Human endothelial cells were isolated from umbilical cords (HUVECs) (University Hospital, Oklahoma City, OK), according to methods modified from Jaffe and colleagues.17 Umbilical veins were washed, flushed with phosphate-buffered saline, and the lumen filled with 0.125% trypsin/Dulbecco’s modified Eagle’s medium for 15 minutes. The cell suspension was centrifuged for 5 minutes at 250 x g, and the pellet resuspended in endothelial growth media (EGM; Clonetics, San Diego, CA) supplemented with bovine brain extract (Clonetics). The cells were passaged at a 1 to 4 split ratio and were used in the first six passages.
Human lung microvessel endothelial cells (HMVEC-Ls) were purchased from Clonetics and maintained in Endothelial Growth Media-2 (Clonetics). Cells were used within passages 4 to 11.
Immunohistochemistry
Immunohistochemistry on human atherosclerotic tissue sections was performed using our published methodology.13 The antiserum used was previously characterized by enzyme-linked immunosorbent assay and shown to be specific for CAP37.13 Briefly, 5-µm sections were incubated at 37°C with the primary anti-CAP37 antiserum (1:500 to 1:2000), followed by biotinylated goat anti-rabbit antiserum (Vectastain Elite, Vector Laboratories, Burlingame, CA), and then incubated in ABC reagent (Vectastain Elite). Color was developed with diaminobenzidine (Research Genetics, Huntsville, AL) for 2 to 6 minutes. Sections were counterstained with hematoxylin. Antibody controls included normal rabbit serum and immunoadsorbed rabbit anti-CAP37 antiserum, as we have described.13
For immunocytochemical analysis of rat aorta cells the media was removed and replaced with serum-free Dulbecco’s modified Eagle’s medium overnight before start of the experiment. RAECs were incubated with 10 µg/ml of Salmonella minnesota LPS (Sigma, St. Louis, MO) for 0.5, 2, 4, 6, and 24 hours. Untreated cells at each time point were included as a control. The adherent cells on the LAB TEK slides were immunostained for CAP37 as described above13 except the RAEC slides were fixed in ice-cold 100% methanol and the primary rabbit anti-human CAP37 antiserum was used at a 1:10 dilution.
For immunocytochemical analysis of surface-expressed and
cell-associated CAP37 in human endothelial cells, the media was
replaced with serum-free endothelial cell basal medium (EBM)
(Clonetics) 6 hours before the start of the experiment. HUVECs were
incubated in the absence or presence of 10 ng/ml of TNF- for 10 and
18 hours. Samples were either fixed only or fixed and permeabilized
essentially as described by Gräbner and
colleagues.18
Cells were stained as above using 5% normal
donkey serum (Jackson, West Grove, PA) to block nonspecific binding,
rabbit anti-human CAP37 (1:750) at room temperature, biotin-sp-donkey
F(ab')2 anti-rabbit IgG, (1:500, Jackson) and
peroxidase-conjugated streptavidin (2 µg/ml, Jackson) for
amplification of signal. Staining using normal rabbit serum was
included as a control.
Northern Blot Analysis
Total cellular RNA was isolated from RAECs.19 Thirty µg of total RNA per well were run on a 1% agarose/formaldehyde gel at 80 mA for 1.5 hours. The RNA was transferred to nylon membrane overnight in standard saline citrate (3 mol/L sodium chloride, 0.3 mol/L sodium citrate) transfer buffer and crosslinked to the membrane with a UV crosslinker. CAP37 mRNA was detected by hybridizing a 32P-labeled CAP37 cDNA probe (6.5 µg of probe at 50 µCi/blot), prepared with the Prime-it II Random Primer kit (Stratagene, La Jolla, CA), by incubating with the membrane at 60°C overnight. After a low [2x standard saline citrate buffer, 0.1% sodium dodecyl sulfate (SDS), room temperature] and high (0.1x standard saline citrate buffer, 0.1% SDS, 60°C) stringency wash the membrane was exposed to autoradiograph film at -80°C. To demonstrate the integrity and relative amounts of sample RNA, total cellular RNA (5 µg) was run on a 1% agarose/formaldehyde gel and visualized by ethidium bromide staining.
Western Blot Analysis
Human umbilical vein endothelial cells were grown to confluency,
serum-starved for 6 hours before start of the experiment, and treated
for 12 hours with 10 ng/ml of TNF- (Boehringer-Mannheim,
Indianapolis, IN). Cells were lysed in 1% SDS/TE [1% SDS (Fisher,
Fair Lawn, NJ); TE, 1 mol/L Tris, 0.5 mol/L ethylenediaminetetraacetic
acid, pH 8 (Fisher)] and 50 µg of lysate were loaded per lane onto a
12.5% SDS-polyacrylamide gel electrophoresis gel and transferred to
nitrocellulose membrane (Schleicher and Schuell, Keene, NH) for Western
analysis.6
Briefly, blots were probed for CAP37 using a
monospecific polyclonal rabbit antisera against human CAP37 (1:1000)
and alkaline-phosphatase-conjugated donkey anti-rabbit IgG at 1:1000
(Jackson), and color developed with Nitro BT/5-bromo-4-chloro-3-indolyl
phosphate 
-toluidine salt (Fisher). An identical blot was probed
with normal rabbit serum to show specificity of the reaction. Twenty
µg of PMN extract was included as a positive control for CAP37.
Flow Cytometry
Human umbilical vein endothelial cells that were serum-starved for
6 hours were incubated in the absence or presence of 10 ng/ml of
TNF- for 10 hours and 18 hours. Permeabilized and nonpermeabilized
cells were fixed and stained essentially as described by Gräbner
and colleagues.18
The cells were first blocked with 4%
normal donkey serum (Jackson), then incubated at 4°C with rabbit
anti-human CAP37 antisera (1:300), and followed by biotin-sp-donkey
F(ab')2 anti-rabbit IgG, (1:200, Jackson) at
4°C. For detection the cells were incubated with fluorescein dichloro
triazinyl amino fluorescein (DTAF)-conjugated streptavidin
(Jackson) at 2 µg/ml at 4°C. Cells were analyzed by flow cytometry
(FACSCalibur; Becton Dickinson, San Jose, CA). Unstained cells and
cells stained with normal rabbit serum were included as controls.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Endothelial cells from umbilical cords from four donors were
incubated for 1 to 24 hours at 37°C with 10 ng/ml of TNF-
(Boehringer-Mannheim). The supernatant was aspirated and the cells
homogenized with Trizol (Life Technologies, Inc., Gaithersburg, MD)
according to the manufacturer’s instructions for total RNA isolation.
cDNA was prepared using Superscript II reverse transcriptase and oligo (dt)12–18 (Life Technologies, Inc.) essentially according to the manufacturer’s protocol with an additional 30-minute incubation at 50°C before termination of the reaction. cDNA was amplified in the polymerase chain reaction with primers designed for a 468-bp internal fragment (5'-GTGCTGGGTGCCTATGACCTGAGG-3', 5'-AAGAGCGCCACTCGGGTGAAGAA-3') flanking exons and introns of HL60-CAP37. The PCR reaction mix (1.5 mmol/L MgCl2, 0.3 mmol/L dNTPs, 0.3 U Taq polymerase (Life Technologies, Inc.), 0.4 µmol/L primer mix, and cDNA in a 25-µl total volume) was amplified for 30 cycles on a Biometra T Gradient thermocycler followed by visualization on a 1% agarose gel with 0.5 µg/ml ethidium bromide. RNA samples with no reverse transcriptase were included in the PCR reaction to demonstrate lack of genomic DNA contamination.
Cloning and Sequencing of E-CAP37
RT-PCR was performed essentially as above using primers designed
for an internal (5'-CAGAATCAAGGCAGGCACTTCTGC-3',
5'-GAGAACACCATCGATCCAGTCTCG-3') 597-bp fragment of CAP37. The
products were excised and eluted from the agarose gel with Gene Clean
II (Bio101, Vista, CA) and ligated into a pCR2.1 vector (The Original
TA Cloning Kit; Invitrogen, Carlsbad, CA), and cloned in INVF’
E. coli (One Shot Chemically Competent E. coli,
Invitrogen) according to the manufacturer’s protocol. Plasmids from
transformants were sequenced by the Oklahoma Medical Research
Foundation Sequencing Facility in both forward and reverse directions
using the T7 and M13 reverse primers for four clones from three
separate induction experiments. The resulting sequences were aligned
using Pôle Bio-Informatique Lyonnais, Network Protein Sequence
@nalysis20
for DNA and the consensus sequence
blasted against the HL60-CAP37 cDNA sequence.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tissue sections from human atherosclerotic lesions showed strong
staining for CAP37 in the endothelium associated with the plaque area
(Figure 1A)
. CAP37 was also detected in
and around foam cells and cholesterol clefts in plaques with advanced
disease (Figure 1B)
. Normal endothelium away from the injured
endothelium associated with the plaque did not stain for CAP37 (Figure 1C)
. Antibody controls using immunoadsorbed antisera to
CAP3713
showed no staining for CAP37 (Figure 1D)
indicating the specificity of the reaction obtained in Figure 1
; A, B,
and C.
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Because CAP37 was detected in the endothelium of atherosclerotic
plaques but not in normal endothelium, we hypothesized that CAP37 was
induced in response to injurious and/or inflammatory mediators. To
explore this possibility we obtained endothelial cells from various
vascular beds including rat aorta (RAECs), umbilical vein (HUVECs), and
human lung microvessel (HMVEC-Ls) and treated these cell cultures with
LPS and cytokines including TNF- and IL-1. We used
immunocytochemical, Northern blot analysis, and RT-PCR to detect CAP37.
The immunocytochemical data presented here (Figure 2)
were obtained from RAECs. CAP37
protein was detected in LPS-treated endothelial cells as early as 2
hours. Maximum staining was obtained between 4 hours (Figure 2A)
and 6
hours. Staining was reduced, but still evident at 24 hours. Untreated
cells (Figure 2B)
did not stain at any of the time points with
anti-CAP37 antiserum. Antibody controls using normal rabbit serum
showed virtually no background staining. Similar studies using HUVECs
and HMVEC-Ls indicated expression of CAP37 in response to LPS (data not
shown).
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. We did not detect CAP37 mRNA in
unstimulated cultures at any time point. An HL-60 cell line (abundant
in CAP37 mRNA)21
was used as a positive control.
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. We performed these studies by incubating HUVECs in
the absence or presence of 10 ng/ml of TNF-. CAP37 mRNA induction
was assessed by RT-PCR. Kinetic studies performed throughout a 24-hour
time period indicated that CAP37 mRNA significantly increased after
TNF- stimulation. Initial up-regulated expression was observed as
early as 1 hour and in general persisted for a 6-hour time period
(Figure 4)
. In certain experiments,
up-regulated expression of CAP37 was observed as late as 12 hours. In
contrast to the Northern blot data using RAECs, we observed
constitutive CAP37 mRNA expression in HUVECs using RT-PCR. RT-PCR was
performed with primers designed to flank exons and introns of CAP37 so
that any genomic DNA contamination would be readily apparent (appearing
as a PCR product much larger than that obtained from cDNA). In
addition, the control PCR reactions using RNA samples containing no
reverse transcriptase did not detect genomic DNA contamination.
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Final confirmation that we were in fact dealing with CAP37 was obtained from sequence data. We cloned an extensive region of E-CAP37 and compared the cDNA sequence to the known HL60-CAP3721 sequence. This comparison demonstrated complete identity with the known amino acid sequence of PMN-CAP37 between residues 19 through 217.
Endothelial CAP37 Is Mainly Cell-Associated
To determine whether the induced form of CAP37 was cell associated
or released we undertook a series of experiments that included
immunocytochemistry, flow cytometry, enzyme-linked immunosorbent assay,
and Western blot analysis. In the immunocytochemical studies, we
treated HUVECs with TNF- and compared the staining pattern for CAP37
in fixed cells with permeabilized cells. Figure 5A
indicates that there is virtually no
CAP37 detected when cells are fixed but not permeabilized indicating
that there is very minimal, if any, cell surface-expressed CAP37. This
is true regardless of whether cells are treated with TNF- (Figure 5A)
or remain untreated (Figure 5B)
. On the other hand, when
TNF--treated cells are permeabilized we observe dramatic staining
for CAP37 indicating that the major component of endothelial CAP37 is
cell associated (Figure 5C)
. The staining is punctate throughout the
cytoplasm with visible perinuclear localization. Untreated cells
indicate a minimal amount of intracellular staining (Figure 5D)
in
comparison to the treated cells. Antibody controls using normal rabbit
serum show absence of staining in TNF--treated cells (Figure 5E)
.
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TNF--treated cells that were permeabilized indicated up to a
fivefold increase of CAP37 expression over untreated cells (Figure 6)
. Once again, the data indicated that
there is a low level of constitutive expression of CAP37 protein in
HUVECs. No detectable surface expression of CAP37 was observed in
nonpermeabilized cells using flow cytometry irrespective of whether
cells were treated or untreated (data not shown). To determine whether
CAP37 is released from treated endothelial cells, we used enzyme-linked
immunosorbent assay to analyze supernatants from TNF--treated HUVEC
cultures. Levels of released CAP37 from treated HUVECs were twofold
higher than untreated cells (data not shown). It was clear that the
amount and proportion of CAP37 released from HUVECs was in general much
less than the amount and proportion of CAP37 released from
PMNs.6
Almost 90% of total CAP37 is released from PMNs
after phagocytosis.6
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-treated cells was performed to provide information regarding
the molecular mass and processing of the various CAP37 species. Figure 7
indicates an extremely interesting
finding. The released form of CAP37 from HUVECs seems to have a
molecular mass closely correlating with the major form of PMN-derived
protein, and is clearly a single species. However, there seem to be two
forms of the cell-associated form of endothelial CAP37, one that
migrates with a molecular mass of 
26 kd and another stronger band at
33 kd. Because of the differential glycosylation of PMN-derived
CAP37, the protein migrates as a smear on SDS-polyacrylamide gel
electrophoresis with a range of molecular mass between 24 to 37 kd.
Normal rabbit serum, used as a control antibody to probe an identical
blot, showed no reaction with HUVEC lysate, supernatant, or PMN extract
indicating the specificity of the antiserum used (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. This is the first demonstration of endogenous endothelial
CAP37. Thus, to confirm that the expressed protein we were dealing with
was unequivocally CAP37 or an isoform of CAP37 we undertook its
sequencing.
Sequence analysis demonstrated homology between E-CAP37 and PMN-CAP37,
with a complete match of 199 amino acids between residues 19 to 217.
This homologous region includes coding sequence for the domains of
PMN-CAP37 reported to have bactericidal22
and
endotoxin-neutralizing5
activity. The region reported to
activate protein kinase C11
in endothelial cells is also
included within this region. Mature PMN-CAP37 is a 222-amino acid
molecule3
with a calculated molecular mass of 24 kd.
Molecular masses ranging from 37 to 24 kd have been observed on
SDS-polyacrylamide gel electrophoresis because of its differential
glycosylation.3
Based on the calculated molecular mass of
endothelial CAP37 observed on our Western blots one would expect to
find differences/extensions at the amino- and/or carboxy-terminus end
of the molecule. We are currently performing 3'RACE and 5'RACE to
determine the complete sequence of inducible endothelial CAP37 to
define any upstream sequences that might have regulatory control.
Although the entire sequence is currently unavailable, the extent of
sequencing obtained so far strongly suggests that we are unequivocally
dealing with CAP37. It is not unusual for inducible and constitutively
expressed forms of the same molecule to have variations in size and
amino acid sequence. This has been well documented for
IL-12.23,24
In addition to the differences in molecular mass between inducible
E-CAP37 and PMN-derived CAP37, our data would suggest differences
relating to the processing of endothelial CAP37. Previous findings from
our laboratory indicate that PMN-derived CAP37 is easily released from
the granules of the PMNs on activation, with almost 90% of total CAP37
detected in supernatant fluids.6
On the other hand,
E-CAP37 seems to have a distinct cell-associated and released form. The
cell-associated protein migrated as a higher kd band whereas the
released protein migrated equivalently to the PMN-derived protein. The
two isoforms of IL-1 also demonstrate a differential pattern of
extracellular release, IL-1ß is easily released, whereas IL-1 is
not.25
We were unable to detect surface expression of
CAP37 on endothelial cells and therefore do not believe that there is a
membrane-anchoring component associated with the induced form. It is
possible that the higher molecular weight band may indicate a glycoform
of the protein, as glycosylation can be dependent on cell
type.26
To explore the identity of the mediators involved in the induction of
CAP37 in endothelium we undertook a series of in vitro
studies. In Figure 2
, we demonstrated immunohistochemically, that CAP37
is induced in endothelial cells in response to the injurious mediator,
LPS. Kinetic studies showed that CAP37 protein was induced in RAECs
in vitro by LPS as early as 30 minutes, peaked at 4 to 6
hours, and subsided by 24 hours. Corroborative studies using Northern
blot analysis demonstrated the expression of CAP37 mRNA to follow a
similar time course in which expression is no longer detected at 24
hours of LPS stimulation. The antiserum used for these experiments was
raised against human CAP37,13
and the probes used for the
Northern blot analysis were based on the human CAP37
sequence21
indicating that there is significant
conservation of CAP37 across species.6,11,27
Our studies
were performed using RAECs because we believed that endothelial cells
derived from the aorta would be the most appropriate site for studies
dealing with atherosclerosis. It is important to note that this
induction of CAP37 in endothelial cells does not seem to be limited to
the aorta. Other studies from our laboratory indicate that TNF- and
IL-1 can induce CAP37 in cultured endothelial cells from rat
cerebral microvessel endothelial cells13
and as described
in Figures 4 through 7
can also be induced in HUVECs and human lung
microvessel endothelial cells (not shown). What is interesting is that
PMN-CAP37 is entirely constitutive and cannot be induced. In fact,
mature PMNs lack mRNA for CAP37.21
In endothelial cells
the constitutive expression of basal mRNA and protein levels seemed to
vary. As seen in our figures with rat aorta we found no constitutive
levels even at the mRNA or protein level whereas our study with HUVECs
and human lung microvessels indicated some constitutive expression.
This may reflect the species from which the cells are obtained, because
rat cerebral vessel showed no constitutive expression
either.13
It might quite simply be a matter of sensitivity
and that probes and antibodies based on the human protein are unable to
detect low levels of rat mRNA or protein for CAP37. A third possibility
is that the rat cells unlike the human cells are not primary cultures.
The major issues of what alters permeability and adhesiveness of the endothelial lining and which mediators are involved in monocyte recruitment in chronic inflammatory diseases have yet to be fully elucidated. In this example of atherosclerosis we speculate that CAP37 (either platelet, PMN, or endothelial derived) is responsible for endothelial cell contraction and permeability7,28 as well as monocyte migration into the intima. Our immunohistochemical data on atherosclerotic lesions demonstrate that the expression of CAP37 protein is not confined solely to the endothelium but is also detected throughout the cholesterol clefts, foam cells, and proliferating smooth muscle cells in the subintimal area of advanced lesions. The source of CAP37 in these advanced lesions remains somewhat equivocal. Our data would strongly suggest that the CAP37 expressed in the endothelium is endogenous E-CAP37. We also believe that the CAP37 in the smooth muscle cells is of endogenous origin, because ongoing studies in our laboratory indicate that CAP37 is expressed in proliferating smooth muscle cells (unpublished data). However, in addition to these endogenous sources of CAP37 there may be a component of the CAP37 associated with atherosclerotic plaques that is entirely exogenous. It is known that platelets contain CAP37,27 and platelets are intimately associated with atherosclerotic plaques.14 It is possible that some of this strong staining for CAP37 is of platelet origin or might be exogenous CAP37 taken up by endothelial cells. The levels of PMN-CAP37 contributing to the staining are probably low, because PMNs are seldom associated with atherosclerotic plaques.14 Our model would suggest that after injury to the endothelium, platelets and/or PMNs will adhere to it because of up-regulation of various adhesion molecules, and on activation will release CAP37. In addition, CAP37 is induced in endothelial cells in response to inflammatory cytokines. The presence of exogenous and endogenous CAP37 sets up a chemotactic gradient across the endothelium that ensures recruitment and migration of monocytes. CAP37 could also contribute to endothelial contraction7 further influencing the transmigration of leukocytes across the endothelium.
Additional support of our hypothesis that E-CAP37 is involved in inflammatory-mediated disease comes from our studies in Alzheimer’s disease.13 It is now well established that brain endothelial cells demonstrate an inflammatory phenotype in Alzheimer’s disease. We have demonstrated CAP37 in the blood vessels of Alzheimer’s disease brains and not in controls.13,29 We have shown its induction in rat cerebral resistance vessels after stimulation with cytokines or injurious/immunomodulatory agents such as LPS and ß-amyloid,13 a significant component of Alzheimer’s disease lesions. It would seem that CAP37 is a biologically significant molecule whose expression in the vasculature may modulate cell migration and activation with important consequences on the progression of disease.
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Acknowledgements |
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Footnotes |
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Supported by the Oklahoma Center for Advancement of Science and Technology, Public Health Service grant AI-28018-06 from the National Institute of Allergy and Infectious Disease, the American Heart Association, Established Investigator grant 9740193N, and an American Heart Association Heartland Affiliate Predoctoral award.
Current address of P. K.:US Oncology, Molecular/Cell Processing Center, 9000 Harry Hines Blvd., Suite 537, Dallas, TX, 75235.
Current address of S. C.-R.: University of Oklahoma College of Dentistry, 1001 Stanton L. Young Blvd., DCS 3044, Oklahoma City, OK, 73117.
Accepted for publication November 14, 2001.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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production in human monocytes. J Immunol 1999, 162:4240–4245
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, endothelium; *, foam cell. Original magnifications:
x400.
