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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From The Division of Nephrology, Department of
Medicine, Children's Hospital; the Department of Pediatrics, Harvard
Medical School; and the Department of Pathology, Beth Israel Deaconess
Medical Center, Boston, MA.
This study addresses a mechanism by which lymphocytes may promote
vascular endothelial growth factor (VEGF) expression and angiogenesis
in immune inflammation. Resting human umbilical endothelial cells (HUVECs) were found to express low levels of VEGF messenger RNA
(mRNA) by reverse transcription polymerase chain reaction and
ribonuclease protection assay with little or no change in expression
following activation by cytokines, including tumor necrosis factor- It is well established that leukocyte
recruitment into tissues is associated with angiogenesis in a variety
of inflammatory conditions, including allograft
rejection.1-5 Indeed, the same factors that mediate
recruitment (eg, cytokines, chemokines, and adhesion molecules)
may themselves promote angiogenesis.6-8 Vascular endothelial growth factor (VEGF) is a potent angiogenesis factor and
has been demonstrated to be functional in acute and chronic inflammation.2,9,10 VEGF is produced by endothelial cells, macrophages, activated T cells, and a variety of other cell
types.6,11,12 It exists as 5 different isoforms composed
of 206, 189, 165, 145, and 121 amino acids by alternative splicing of a
single gene.12-14 VEGF binds to high-affinity tyrosine
kinase receptors KDR/Flk-1 and Flt-1, resulting in endothelial
proliferation in vitro and in angiogenesis and increased vascular
permeability in vivo.14 In addition, VEGF binds
to mononuclear cells, resulting in activation responses and chemotactic
activity.15 Despite an abundance of angiogenesis factors,
VEGF appears to be most critical in vivo since VEGF
gene-knockout mice (including heterozygotes) fail to develop
blood vessels, with resultant embryonic lethality.16 While
hypoxia is the most potent stimulus for VEGF
expression,17-19 other factors known to induce VEGF
include some cytokines, oncogenes, prostaglandins, modulators of
protein kinase C, nitric oxide, and stimulators of adenylate
cyclase.6
Interactions between CD40 ligand (CD40L, also called CD154) and CD40
have been found to have pluripotent functions in inflammation, predominantly in the effector phase of the immune
response.20,21 CD40, a 50 kd type I
transmembrane-glycoprotein member of the tumor necrosis factor receptor
(TNF-R) gene family is expressed by numerous cell types including most
professional antigen-presenting cells (APCs), monocytes, and
endothelial cells.21-24 CD40L, a 33-kd type II
membrane-protein member of the TNF family, is predominantly expressed
by activated CD4+ T cells and platelets25,26
although it is also expressed on many other cell types.20
Signaling via CD40 mediates immunoglobulin isotype switching in B
cells; the expression of costimulatory molecules, notably B7-family
molecules on APCs; the activation of monocytes; and adhesion molecule
expression by endothelial cells, as well as the expression of cytokines
and chemokines characteristic of the effector phase of the immune
response.23,24,27
In this study, we have examined pro-inflammatory signals that mediate
VEGF expression. Our data provide insight into the role of the T cell
in leukocyte-induced angiogenesis and show that CD40L-CD40 interactions
are potent for VEGF expression and VEGF-induced angiogenesis in vitro
and in vivo.
Reagents
Cell isolation and culture
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords as previously described30 and were cultured in M199 medium (BioWhittaker, Walkersville, MD) containing 20% fetal bovine serum (FBS) (Gibco-BRL Products, Gaithersburg, MD), endothelial cell growth supplement, 1% penicillin/streptomycin, L-glutamine, and heparin. Single-donor HUVECs were purchased from Clonetics (Walkersville, MD) and were cultured in complete endothelial medium (EGM BulletKit, Clonetics) as supplied and according to the recommended instructions. HUVECs were subcultured and used at passage 3 to 5. The human renal cell carcinoma cell line 786-O was cultured in Dulbecco modified Eagle medium containing 10% FBS, 1% penicillin/streptomycin, and L-glutamine. This cell line has been shown to express high constitutive levels of VEGF and was used in our studies as a positive control.28 RNA extraction and reverse transcription polymerase chain reaction Transfer RNA was isolated from cultured cells by means of the Ultraspec RNA Isolation System (Biotecx Laboratories, Houston, TX) according to the manufacturer's instructions and was quantified by spectrophotometry. Complementary DNA (cDNA) was prepared by reverse transcription of RNA with the use of random hexamer primers (100 ng/µL) and Moloney murine leukemia virus reverse transcriptase (50 U/µL) (Gibco-BRL) in a 50-µL reaction. We used 10 µL cDNA for each polymerase chain reaction (PCR) amplification reaction. PCR was performed with Taq DNA polymerase with the use of the manufacturer's buffer (Boehringer Mannheim, Indianapolis, IN). Sequence-specific primers were used for amplification of the human VEGF gene (sense primer 5'-TCACCGCCTCGGCTTGTCACA-3', antisense primer 5'-ATGAACTTTCTGCTGTCTTGG-3').We used Ribonuclease protection assay Equal amounts of mRNA were analyzed by ribonuclease protection assay (RPA) by means of the RiboQuant multiprobe template (PharMingen) according to the manufacturer's instructions. Briefly, RNA was hybridized overnight with the 32P-labeled RNA probe, which had previously been synthesized from the template set. Single-stranded RNA and free probe were digested by ribonuclease A and T1. Subsequently, protected RNA was analyzed on a 5% denaturing polyacrylamide gel. VEGF and cytokine transcripts were analyzed either by means of a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) or by means of autoradiography for 4 hours to 5 days and subsequent quantification by densitometry (Alpha Innotech). For quantification, the VEGF signals for each sample of the blot were normalized by expressing the density of its signal to the sum of the corresponding signals of the housekeeping genes GAPDH and L32.Transfection and luciferase assay Single-donor HUVECs (Clonetics) were seeded at 2.5 × 105 cells per well in 3.0 mL EBM basal medium (Clonetics) containing 0.5% FBS, but no other supplements, in 6-well tissue culture plates for 24 hours. Transfection was then performed by lipofection by means of the GenePORTER Transfection Reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions. Briefly, 1 µg DNA and 3 µL GenePORTER were separately diluted in 500 µL EBM each and then rapidly mixed and incubated for 30 to 45 minutes at room temperature. Culture medium was aspirated, and 1 mL DNA-GenePORTER mix was carefully added to each well and incubated at 37°C. Following 3 to 4 hours, 1 mL EBM containing 1% FBS was added to each well, and the incubation was continued for 6 to 24 hours in the absence or presence of soluble CD40L (sCD40L) at 37°C in 5% CO2, with the use of duplicate wells for each condition. Subsequently, cells were lysed in buffer, and protein concentration was determined by Bradford assay.31 Using equal amounts of total cellular protein, we analyzed luciferase activity at room temperature by means of a MicroLumat LB96P luminometer (EG & G Berthold, Bad Wildbad, Germany) for 10 seconds according to the manufacturer's instructions (Promega).In vivo models of VEGF expression and angiogenesis We evaluated the effect of human sCD40L in vivo in human skin that was transplanted onto CB.17 severe combined immune deficient (SCID) mice (obtained from Massachusetts General Hospital, Boston, MA) as described.2,32 Briefly, the mice were anesthetized by means of ketamine and xylazine, according to the guidelines of the Animal Care and Use Committee, Children's Hospital, Boston, MA, and received approximately 1 cm2 full-thickness human neonatal foreskin grafts. After 4 to 6 weeks, when the human skin had engrafted, a total volume of 60 µL, containing 40 µL Matrigel (Becton Dickinson, Bedford, MA), together with 5 µg human sCD40L (Ancell) was injected into the human skin. After 7 days, the skin was harvested, snap-frozen in isopentane/liquid nitrogen, and analyzed by immunohistochemistry for the expression of VEGF. An adjacent portion of the human skin was fixed in formalin, embedded in paraffin, and processed for von Willebrand factor (vWF) staining.We also used a well-established in vivo model of angiogenesis in athymic NCr nude mice as described33,34 to determine the effect of CD40L-CD40 interaction on angiogenesis. Briefly, mice received subcutaneous injection of 35 µL Matrigel together with 35 µL murine sCD40L in one flank, and 35 µL Matrigel together with 35 µL heat-inactivated murine sCD40L in the other flank as control. The site of each injection was marked with indelible ink. After 7 days, the undersurface of the skin was exposed and photographed for evaluation of angiogenesis. In addition, all skin samples were formalin-fixed, paraffin-embedded, and processed for routine histology. Immunohistochemistry Immunohistochemistry was performed on 4-µm cryostat sections or on 10-µm-thick paraffin-embedded sections as described.2,32,35 Briefly, specimens were fixed in acetone (frozen) or were deparaffined, and primary antibody diluted in phosphate-buffered saline (PBS) was applied to the specimens for 1 hour at room temperature or overnight at 4°C in a humidified container. Optimal concentration of the antibody was determined by simultaneous staining of positive control tissues (human tonsil or inflamed skin). Subsequently, specimens were incubated with a species-specific peroxidase-conjugated secondary antibody (Jackson Immunoresearch, Westgrove, PA) and were developed with 0.25 mg/mL 3-amino-ethylcarbazole in 2% N, N-dimethylformamide and 0.1 mol/L sodium acetate buffer (pH 5.2) with 0.03% hydrogen peroxide to produce a rose-colored reaction product. Negative controls were skin sections incubated with an isotype-matched primary antibody or in PBS alone. Finally, specimens were counterstained in hematoxylin and were mounted in glycerol gelatin.
Effects of inflammatory cytokines on VEGF expression To investigate mechanisms by which CD4+ T cells may stimulate VEGF-induced angiogenesis in immune reactions, we initially assessed the expression of VEGF mRNA in resting HUVECs, or HUVECs cultured with optimal concentrations of the cytokines IL-1 (0.1 to
10 ng/mL), TNF- (0.1 to 100 U/mL), IL-4 (0.1 to 500 U/mL), or
IFN- (0.1 to 1000 U/mL). By reverse transcription PCR (RT-PCR) and RPA, we found that resting HUVECs express low and variable levels of
VEGF mRNA and that activation of HUVECs with cytokines induced, at
best, a weak enhancement in VEGF expression, even in concentrations that markedly induced the expression of E-selectin,
intracellular adhesion molecule 1, and vascular cell adhesion molecule
1 in control cultures (Figure 1).
Ligation of CD40 induces VEGF expression in endothelial cells and monocytes We next wished to define whether a cell-surface molecule on activated CD4+ T cells mediates VEGF expression. In initial studies, using semiquantitative RT-PCR, we found that treatment of HUVECs with cell membranes derived from 6-hour PHA-activated CD4+ T cells resulted in a 2- to 2.5-fold induction in VEGF mRNA expression (not shown). We next assessed the temporal expression of VEGF following stimulation of HUVECs with sCD40L. Our HUVECs,35 as well as others,36,37 express CD40 constitutively as well as following stimulation in a homogenous distribution. HUVECs were treated with increasing concentrations of sCD40L, and VEGF mRNA was examined by RPA. As illustrated in Figure 2A, we found a marked increase in VEGF expression as early as 1 hour following activation with sCD40L, with peak expression occurring between 2 and 5 hours and expression remaining elevated for 48 hours. Furthermore, CD40-dependent VEGF expression was dose-dependent (Figure 2B) and was inhibited by coculture in the presence of neutralizing monoclonal antibodies to CD40 (Figure 2C) or CD40L (not shown). Parallel experiments, using purified recombinant human sCD40L (Ancell), instead of culture supernatant (Bristol Myers Squibb), yielded equivalent results.
Since leukocyte subsets, especially monocytes and CD4+ T
cells, are known to be a potent source of VEGF in vitro and in
vivo,11,38,39 we also wished to assess the effect of CD40
ligation on VEGF expression by these cells. After purification,
monocytes were cultured in the absence or presence of sCD40L, and mRNA
expression was again assessed by RPA. Our findings were that resting
monocytes (immediate harvest) express low but variable levels of VEGF
that was induced moderately by plastic adherence and in a more
pronounced way by treatment with LPS (1 µg/mL) (Figure
3A). Treatment with sCD40L resulted in a
marked induction of VEGF mRNA expression as described above for
endothelial cells. In contrast, while activation of CD4+ T
cells with PHA induced VEGF mRNA expression, treatment with sCD40L
alone or in combination with PHA had no effect on CD4+
T-cell VEGF expression (Figure 3B). This is consistent with the low
level of expression of CD40 on CD4+ T cells and the lack of
dependence of CD4+ T cells on CD40 signaling for activation
response.21
CD40-signals induce VEGF promoter activity Having established that ligation of CD40 induces VEGF mRNA expression in endothelial cells and monocytes, we next wished to assess whether CD40 signals regulate transcriptional activity of VEGF. For these experiments, HUVECs were transiently transfected with a full-length (2.6 kb) luciferase reporter construct under the control of the human VEGF promoter by means of a standard lipofection procedure as described.40,41 Luciferase activity was assessed in transfected cell lysates following a 6- to 24-hour incubation in the presence or absence of human sCD40L. Consistently, we found enhanced luciferase activity following treatment with sCD40L (Figure 4A). Furthermore, the relative fold induction in luciferase activity was similar to the relative fold induction in mRNA expression (Figure 4B), suggesting a predominant transcriptional control mechanism for CD40-dependent regulation of VEGF.
Functional effect of CD40 ligation for VEGF-induced angiogenesis To assess the functionality of CD40-dependent VEGF expression in vitro, we developed a modified endothelial cell growth assay. HUVECs were seeded in gelatin-coated tissue culture plates in medium containing 5% FBS but no endothelial cell growth supplement overnight. Subsequently, HUVECs were cultured with sCD40L or recombinant human VEGF (1 to 10 ng/mL) in the absence or presence of a neutralizing anti-human VEGF antibody (0.1 to 10 µg/mL). As illustrated in Figure 5, we found marked growth of HUVECs and increased HUVEC density following treatment with sCD40L for 48 to 72 hours. Furthermore, we found that neutralizing anti-VEGF antibody completely inhibited the effect of sCD40L on HUVEC growth. Positive control cultures treated with VEGF resulted in a similar increase in cell density, and the effect of optimal concentrations of sCD40L was similar to that found for VEGF at a concentration of 5 ng/mL (Figure 5A-B). Negative control cultures treated with heat-inactivated sCD40L or murine sCD40L (generated identically as human sCD40L) were similar to untreated controls. Thus, CD40-dependent VEGF expression is of functional consequence for HUVEC proliferation in vitro.
In vivo induction of VEGF expression and angiogenesis by sCD40L We next wished to assess the effect of CD40L-CD40 interactions on VEGF expression and angiogenesis in vivo. First, we evaluated the effect of human sCD40L (used in the in vitro studies described above) for VEGF expression in vivo. For these studies, we used a model in SCID mice bearing human skin transplants as described.2,32 We injected human sCD40L into the human skin grafts (n = 4) and evaluated the expression of VEGF after 7 days. As illustrated in Figure 6, we found a marked induction in VEGF expression in all sCD40L-treated skins compared with controls (nontransplanted skin, and transplanted but untreated skin) (n = 5). In addition, the number of vWF-expressing vessels appeared to be more numerous (2- to 3-fold by semiquantitative grid counting) in the sCD40L-treated skin specimens compared with untreated controls (not shown).
We next used an established model in the athymic nude
mouse33,34 to determine the effect of sCD40L on
angiogenesis. Mice (n = 7) received subcutaneous injections of
Matrigel containing murine sCD40L on one flank and Matrigel containing
heat-unactivated murine sCD40L on the contralateral flank. Additional
control animals received Matrigel implants with saline only instead of
sCD40L. On day 7, animals were anesthetized, and the undersurface of
the injection site was exposed (Figure
7). Consistently, there was more bleeding
around the incision site of the sCD40L-treated skin (Figure 7B)
compared with control skin (Figure 7A). As illustrated, in the
sCD40L-treated skins, there was an increased number of grossly visible
small tortuous blood vessels with multiple bifurcations and
trifurcations as compared with negative control implants with heat-inactivated murine sCD40L or saline (not shown). Findings were
highly consistent within each of the groups. These observations confirm
that ligation of CD40 in vivo results in VEGF expression and
angiogenesis.
In this study, we define the ability of CD40L-CD40 interactions for the expression of VEGF and angiogenesis in vitro and in vivo. Our findings establish a mechanistic association among the inflammatory response and VEGF-induced angiogenesis and are consistent with the ability of early activated T cells and monocytes to initiate angiogenesis.2,5,8,11,12,38,39,42 There are several pathophysiologic implications of our findings for immune inflammation, including allograft rejection. One possibility is that CD40L expressed by early activated T cells (and perhaps by activated platelets) stimulates local endothelial cell and tissue monocyte/macrophage production of VEGF in the course of recruitment into local sites of inflammation. This possibility is consistent with our recent observations that angiogenesis occurs very early in the course of inflammation and is temporally and spatially associated with T-cell infiltrates.2 Furthermore, our data are consistent with the original studies by Auerbach,3-5 in which it was concluded that leukocyte-induced angiogenesis is mediated by CD4+ T cells, or products of CD4+ T cells that have biologic effects on local cells in the course of acute inflammation. The expression of CD40 and CD40L has been previously reported to be prominent in pathologic processes known to be associated with high levels of VEGF, such as tumors, at sites of chronic inflammation and in allografts undergoing rejection.10,12,20,21,35,38,43 Indeed, the prominent expression of CD40 at sites of inflammation is consistent with a potent function in the effector angiogenesis response,35,44 and we have also found a spatial correlation between the expression of CD40 and the expression of VEGF in skin allografts undergoing rejection (M.M. and D.M.B., unpublished observations, December 1999 to March 2000). Activated leukocytes that are recruited into inflammatory sites may already produce VEGF as well as other angiogenesis factors as a function of their state of activation.6,11,38 It is well established that monocytes known to be critical mediators of leukocyte-induced angiogenesis do so via the induced expression of many angiogenesis factors, and monocyte activation may occur in the absence of CD40 ligation.1,8,39 Thus, the effect of CD40 signals on angiogenesis in vivo observed in this study may be redundant. However, a large body of literature has concluded that CD40 signals are most potent for monocyte activation responses and that CD40 signals may promote the expression of additional angiogenesis factors.21,45 Furthermore, blockade of CD40L-CD40 interactions has been found to inhibit acute inflammation, chronic inflammation, and chronic diseases such as atherosclerosis and rheumatoid arthritis known to be dependent on monocyte activation responses and characterized by profound angiogenesis.21,46 Thus, it is likely that the biological effects of CD40L-CD40 interactions for the angiogenesis response in chronic inflammation is of great pathologic significance. It is possible that the effects of sCD40L for angiogenesis in vivo
observed in our studies may be in part a result of non-VEGF mechanisms
and positive-feedback loops. Ligation of CD40 results in the expression
of multiple cytokines, chemokines, and adhesion molecules that
themselves may have effects on VEGF expression and the angiogenesis
reaction.21,45 For instance, the production of TNF- In our studies, the overall increase in VEGF mRNA expression
(approximately 4-fold induction) was similar to that previously reported for hypoxic-induced transcriptional regulation of
VEGF.19 In addition, the relative fold induction of VEGF
mRNA expression in HUVECs and in monocytes was similar to that found
following CD40-dependent activation of our VEGF promoter construct,
suggesting a predominant transcriptional control mechanism. To date,
hypoxia is the best-characterized stimulus for VEGF expression and for the VEGF-induced angiogenesis response in
vivo.17-19 Hypoxia stimulates VEGF expression by
both transcriptional and post-transcriptional mechanisms.19 Transcriptional regulation is mediated by
well-described hypoxia-inducible factor-1 (HIF-1) and Sp-1 binding
elements. In addition, several transactivating factor intermediaries
including mutant src and ras oncogenes have been
described to regulate VEGF expression.17,48 Ligation of
CD40 is known to regulate multiple genes via transcriptional
mechanisms.21,49 CD40 signals result in the activation of
the TNF-receptor-associated factor (TRAF) family molecules, the
nuclear factor (NF)- In summary, these studies define that CD40L-CD40 interactions promote VEGF expression and angiogenesis in vitro and in vivo. Our findings have important implications for the understanding of acute and chronic inflammation, including atherosclerosis and allograft rejection, in which CD40L-CD40 interactions are thought to play a pathophysiologic role.
Dr Harald F. Dvorak, Dr Donald R. Senger, and Dr Karen Moulton for helpful discussions. M.M. received the TEAM Management Young Investigator Award of the American Society of Transplantation at the 18th Annual Scientific Meeting on May 16, 1999, for this work.
Submitted March 16, 2000; accepted August 4, 2000.
Supported by National Institute of Health grants DK53606 and AI46756 and the National Kidney Foundation (Massachusetts affiliate).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David M. Briscoe, Division of Nephrology, Children's Hospital, 300 Longwood Ave, Boston, MA 02115; e-mail: briscoe{at}a1.tch.harvard.edu.
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Top
Abstract
Introduction
2361 to +298
relative to the transcription start site) VEGF promoter fragment was
used in transfection assays as described.
-actin (Stratagene, La Jolla, CA) as an internal
control. PCR reactions were performed under the following
conditions: 1 cycle at 94°C for 5 minutes followed by 40 cycles at
94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. The
last cycle was extended to 7 minutes at 72°C. Messenger RNA (mRNA)
extracted from PBMCs served as positive control. The amplified products were resolved by electrophoresis in an ethidium bromide-stained 1.5%
agarose gel and were analyzed by densitometry by means of an
AlphaImager 2000 system (Alpha Innotech, San Leandro, CA).
B and c-Jun amino-terminal kinase/activator
protein (AP)-1 transactivating factor(s) and resultant signaling. But
the VEGF promoter does not contain putative NF-