|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-07-2315.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institut de Chimie des Substances Naturelles,
Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette,
France; Laboratory of Molecular Pharmacology, Department
of Pharmacy, University of Patras, Patras, Greece;
Division of Genetics, Cell and Developmental Biology, Department of
Biology, University of Patras, Patras, Greece; and
Institut de Myologie, INSERM U523, Paris, France.
The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), purified from
bone marrow and constitutively synthesized in vivo, belongs to the
family of negative regulators of hematopoiesis. It protects the stem
cell compartment from the toxicity of anticancer drugs and irradiation
and consequently contributes to a reduction in marrow failure. This
current work provides experimental evidence for another novel biologic
function of AcSDKP. We report that AcSDKP is a mediator of
angiogenesis, as measured by its ability to modulate endothelial cell
function in vitro and angiogenesis in vivo. AcSDKP at nanomolar
concentrations stimulates in vitro endothelial cell migration and
differentiation into capillary-like structures on Matrigel as well as
enhances the secretion of an active form of matrix metalloproteinase-1
(MMP-1). In vivo, AcSDKP promotes a significant angiogenic response in
the chicken embryo chorioallantoic membrane (CAM) and in the abdominal
muscle of the rat. Moreover, it induces the formation of blood vessels
in Matrigel plugs implanted subcutaneously in the rat. This is the first report demonstrating the ability of AcSDKP to interact directly with endothelial cells and to elicit an angiogenic response in vitro
and in vivo.
(Blood. 2003;101:3014-3020) The results of recent studies indicate that several
cytokines and interleukins initially thought to be specific for the
hematopoietic system are also capable of inducing responses in
endothelial cells.1-5 Alternatively, the ability of
angiogenic factors to affect several functions of hematopoietic cells
has also been reported.6-8 Indeed, the responsiveness of
both the hematopoietic and vascular system to the same factors probably
reflects the common origin of hematopoietic and endothelial
cells.9,10
The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), purified from bone
marrow and present in blood at nanomolar concentrations, is recognized
as a physiologic regulator of hematopoiesis.11 It inhibits
the proliferation of normal hematopoietic stem cells and committed
progenitors in vivo as well as in vitro11-14 and consequently reduces the damage to the stem cell compartment resulting from treatment with chemotherapeutic agents or ionizing
radiation.15-18 We have previously shown that AcSDKP is
degraded in the circulation by the angiotensin I-converting enzyme
(ACE)19,20 and that administration of specific inhibitors
of this protease (ACEIs) significantly reduces the proliferation of
primitive hematopoietic cells.21-23 The antiproliferative
ability of ACEIs, demonstrated both in vitro and in vivo, could
be mediated by endogenous AcSDKP, concentrations of which are
systematically increased following ACEI administration. Interestingly,
ACEIs also modulate angiogenesis,24-26 although the
mechanism of this activity remains unclear. It is therefore possible to
hypothesize that the increased levels of AcSDKP following
administration of ACEIs may contribute, at least in part, to the
angiogenic activity of these compounds.
Our hypothesis that AcSDKP has angiogenic activity follows also results
of recent studies on thymosin Angiogenesis, the formation of new capillaries by the sprouting of
pre-existing blood vessels, occurs both during embryonic development
and in postnatal life. It is a critical process not only during normal
growth, but also in pathologic situations, where inadequate vascular
growth contributes to health complications. Angiogenesis is a complex
multistep phenomenon. It involves the degradation of extracellular
matrix and the subsequent migration, proliferation, and differentiation
of endothelial cells. These cells form tubelike structures that
subsequently organize into a capillary network. Angiogenesis is
predominantly regulated by local factors that promote or inhibit
neovascularization.34
The findings presented in this paper contribute to the emerging
complex interactions of factors that regulate growth of the blood
vessels. We report here that the tetrapeptide AcSDKP, a negative
regulator of hematopoiesis, is a potent angiogenic factor both in vitro
and in vivo. In vitro, AcSDKP promotes an angiogenic response of
endothelial cells, namely, an increase in the secreted active form of
matrix metalloproteinase-1 (MMP-1), and the stimulation of cell
migration and differentiation into capillary-like structures on
Matrigel. In vivo, AcSDKP induces neovascularization when applied on
the surface of the chorioallantoic membrane (CAM) of the chick embryo
and when injected in the abdominal muscle of the rat. Moreover, it
stimulates vascular invasion in Matrigel plugs implanted subcutaneously in the rat. These data demonstrate the potential of AcSDKP as an
angiogenesis promoter that may prove to be an effective agent for
induction of therapeutic angiogenesis in the clinic.
Peptides
Animals
Cell culture Immortalized EA.hy926 endothelial cells derived from fusion of human umbilical vein endothelial cells (HUVECs) with A549 lung carcinoma cells35 were generously provided by Dr E. C. J. Edgell (University of North Carolina, Chapel Hill, NC). Cells were grown in Dulbecco minimal essential medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal calf serum (FCS), 1% glutamine, and HAT (100 µM hypoxantine, 0.4 µM aminopterin, 16 µM thymidine from Invitrogen; Cergy-Pontoise, France). Bovine brain capillary (BBC) endothelial cells (kindly provided by J. Courty, University Paris XII) were maintained in DMEM containing 4.5 g/L glucose supplemented with 10% FCS, 1% glutamine, and 1 ng/mL bFGF. HUVECs purchased from Biowhittaker Europe (Verviers, Belgium) were grown in M199 medium supplemented with 20% FCS, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 15 mM sodium bicarbonate, and 1% glutamine. Lung human microvascular endothelial cells (HMVEC-Ls) purchased from Biowhittaker Europe were grown in EGM-2MV growth medium (Biowhittaker Europe) consisting of endothelial cell basal medium supplemented with human recombinant epidermal growth factor (10 ng/mL), hydrocortisone (1 µg/mL), gentamicin (50 µg/mL), amphotericin B (50 µg/mL), and fetal bovine serum (5% vol/vol). Cultures were maintained at 37°C and 5% CO2 in a humidified atmosphere. Cells were routinely harvested by trypsin treatment (0.5 g/L trypsin/0.2 g/L EDTA [ethylenediaminetetraacetic acid] from Invitrogen).Detection and measurement of AcSDKP levels AcSDKP concentration was measured using a highly specific competitive enzyme immunoassay (EIA) with acetylcholinesterase conjugate as a tracer (SPIbio, Massy, France). The quantitative limit of this assay was 0.5 pmol and the precision was characterized by a variation coefficient less than 10% in the 1- to 10-nM range. AcSDKP was quantified in the methanol extracts of EA.hy926 endothelial cells and in their serum-free conditioned media. Then 4 mL ice-cold methanol was added either to 1 mL sonicated cell suspension or to 1 mL conditioned medium both supplemented with lisinopril (Sigma) at 10 6 M final concentration to
prevent AcSDKP degradation. Following 30 minutes of incubation at 4°C
all samples were centrifuged for 15 minutes at 3500 rpm and the
supernatants were evaporated to dryness under vacuum (Speed Vac
Concentrator, Savant, Holbrook, NY). The extracts were then
diluted in 0.25 mL EIA buffer and used for the dosage. The results are
expressed in picomoles AcSDKP per milliliter supernatant or per
1 × 106 endothelial cells and are the mean of 4 experiments.
Matrix MMP enzyme-linked immunosorbent assay EA.hy926 cells at 90% confluence were rinsed twice with serum-free DMEM and incubated for 24 hours in serum-free DMEM in the absence or presence of AcSDKP at various concentrations. The conditioned media were then collected, centrifuged at 1200 rpm for 10 minutes, and stored at 20°C until analyzed. The amounts of MMP-1
(EC 3.4.24.7), MMP-2 (EC 3.4.24.24), and MMP-9 (EC 3.4.24.25), as
either free active or total (sum of active and proforms) enzyme, were determined in conditioned media using Biotrack cellular communication assays (Amersham Pharmacia Biotech, Orsay, France) according to the manufacturer's recommendations. Results are the mean of 6 independent experiments performed in triplicate.
Endothelial cell migration assay In vitro endothelial cell chemotactic evaluation was performed using 24-well Boyden chambers (8-µm pore polycarbonate polyvinylpyrrolidone-free filters; Costar, Avon, France). The bottom chamber was filled with 0.6 mL serum-free DMEM containing 0.1% Albumax in the absence or the presence of various concentrations of AcSDKP or 1 ng/mL bFGF. EA.hy926 endothelial cells resuspended in serum-free DMEM containing 0.1% Albumax were loaded into the upper chamber of a Transwell insert (105 cells in 0.1 mL) and incubated at 37°C for 6 hours. The filters were then removed and the nonmigrated cells on the top of the membranes were removed using cotton swabs. The migrated cells attached to the bottom of the membrane were fixed with methanol and stained with Diff-Quick (Dade Behring, Düdingen, Swizerland). Cell migration was quantified by counting cells in 10 random microscopic fields per filter at × 20 magnification using an Olympus IMT-2 inverted phase-contrast microscope. Assays were performed in triplicate and repeated 3 times. Results are expressed as the fold increase over control values (basal migration toward medium alone).Matrigel tube formation assay The spontaneous formation of capillary-like structures by endothelial cells on a basement membrane matrix preparation (Matrigel, Becton Dickinson, Bedford, MA) was used to assess the angiogenic potential of AcSDKP. The 4-well plates (Lab-Tech, Nunc, Roskilde, Denmark) were coated with 300 µL Matrigel (13 mg/mL) that was allowed to solidify for 1 hour at 37°C. EA.hy926 cells (2 × 105 cells/mL/well) were plated onto the surface of the Matrigel in serum-free DMEM containing AcSDKP at different concentrations or 1 ng/mL bFGF. Medium alone was used as a control. After 2, 4, 6, and 18 hours of incubation at 37°C in a 5% CO2 humidified atmosphere, cellular organization into tubular structures was investigated using an inverted phase-contrast photomicroscope and photographs taken at × 4 and × 10 magnifications. Each assay was performed in triplicate and the results are the mean of 5 independent experiments. Results are expressed as the percentage increase in tube formation compared with control values.In vivo Matrigel plug angiogenesis assay An aliquot (0.5 mL) of Matrigel containing either saline or AcSDKP at different concentrations was injected subcutaneously into the mid-abdominal region of Sprague-Dawley rats. After 7 days, the animals were killed and the Matrigel plugs removed and fixed in 4% formaldehyde solution. Preparations were subsequently embedded in paraffin, sectioned, stained with hematoxylin-eosin-saffron, and examined for the in-growth of blood vessel. Ten random fields (31.4 mm2 total) localized on the border of plugs were analyzed for capillary growth. Photographs were taken at × 10 magnification. The experiment was repeated 3 times with 4 rats per condition in each experiment. Results are expressed as the percentage increase observed in the vessel counts (Matrigel containing AcSDKP) compared to control values (Matrigel alone).Chicken embryo CAM assay The in vivo chicken embryo CAM angiogenesis model was used to investigate the angiogenic potency of AcSDKP. Leghorn fertilized eggs (Pindos, Epirus, Greece) were incubated for 4 days at 37°C; thereafter, a window was opened on the eggshell, exposing the CAM. The window was covered with sterile tape and the eggs were returned to the incubator. At day 9 of embryo development, 20 µL distilled water containing different amounts of AcSDKP (0.002-200 pmol) was applied on an area of 1 cm2 of the CAM restricted by a plastic ring. Following 48 hours of subsequent incubation at 37°C, CAMs were fixed in situ, excised from the eggs, placed on slides, and left to air-dry. Photographs were taken using a stereoscope equipped with a digital camera. The total length of the vessels was measured as previously described.44 Water alone was used as a control. HARP, recognized as a potent angiogenic inducer in the chicken embryo CAM,36 was used as a positive control and AcSDDKP, a stereoisomer of AcSDKP, as a negative control. Each sample was tested 3 times using 10 to 20 eggs for each data point. Results are expressed as the percentage increase in the vessel length compared to control values.In vivo effects of AcSDKP on vascularization of the abdominal muscle To assess the effects of AcSDKP on normal vasculature, we performed local intramuscular injections into the abdominal wall of adult Wistar rats. All animals were approximately 300 g and injections were performed percutaneously using a 30-gauge needle, injecting 300 µL volume into the ventrolateral muscles of the abdomen. A dose of 5 µg AcSDKP/kg/injection was delivered twice a day for 5 consecutive days. Sterile saline was injected as an excipient. Rats were housed individually and allowed food and water ad libitum. Animals were analyzed at day 7 and day 28 to investigate vascularization of the ventrolateral muscles of the abdomen. Ketamine-xylazine (Centravet, Plancoet, France) anesthesia was administered intraperitoneally and a median abdominal incision was performed. Careful dissection of the skin allowed visualization of muscular layer of the abdominal wall. A small catheter was inserted into the descending aorta below the renal arteries and above the bifurcation of the iliac arteries. India ink was used to visualize the vasculature and to assay vascular permeability. Analysis focused on the epigastric arteries and the ascending branch of the lateral femoral arteries. All animals were killed by an excess dose of anesthetic agents. Three independent experiments with 4 animals per control and treated group (2 from day 7 and 2 from day 28) were included in this study.Statistical analysis All results were expressed as the mean ± SEM. Significance of differences was determined with the Student t test with significance at P < .05.
Endothelial cells contain and secrete AcSDKP AcSDKP concentrations were measured in growing EA.hy926 endothelial cells and in the conditioned media of nonconfluent and confluent EA.hy926 cells. As shown in Figure 1, the intracellular concentration of AcSDKP is related to the growth phase of the cells and varies from 0.78 ± 0.07 to 2.53 ± 0.44 pmol/106 cells. The concentrations of AcSDKP present in the serum-free supernatants of either nonconfluent or confluent endothelial cells were found to be between 1.03 ± 0.07 and 1.13 ± 0.06 pmol, respectively, at the second and seventh day of culture.
AcSKP induces directional migration of endothelial cells Regarding the ability of angiogenic factors to induce migration of endothelial cells, we tested different concentrations of AcSDKP, ranging from 1013 M to 105 M using an in
vitro chemotactic assay. As shown in Figure
2, AcSDKP induced chemotaxis of EA.hy926
cells in a dose-dependent manner, with a typical bell-shaped curve. The
maximal stimulation of EA.hy926 cell migration (1.57-fold increase) was
observed with 1011 M AcSDKP. This was about 20% lower
than the chemotactic activity of bFGF (1.98-fold increase) included as
a positive control and used at 5 × 1011 M solution (1 ng/mL). Similar results were obtained with BBC cells with
109 M AcSDKP. A 1.63 ± 0.23-fold increase
(P < .01) after 4 hours in migration was induced compared
with untreated control cells.
AcSDKP induces endothelial cell differentiation on Matrigel An important step in angiogenesis is the ability of endothelial cells to differentiate in response to specific stimuli and to migrate out of the existing blood vessels to organize new capillaries. To investigate the morphogenic potential of AcSDKP we used an in vitro assay that allowed the visualization of multicellular tubelike structures that resemble microvascular networks on Matrigel. We first evaluated the optimal time course for EA.hy926 endothelial cells to respond to AcSDKP used at 1011 M and 109 M
according to its previously determined maximal chemotactic activity. As
shown in Figure 3A, the formation of
tubelike structures (visible rings and cords) by EA.hy926 cells plated
on Matrigel was significantly stimulated in the presence of
1011 M and 109 M AcSDKP after 2 hours of
incubation. The maximal stimulatory effect of the tetrapeptide
(corresponding to 54% increase in tube formation) was measured after 6 hours of incubation. The data presented in Figure 3B indicate that
AcSDKP stimulated tube formation by EA.hy926 cells in a dose-dependent
manner. Stimulation was detectable at 1015 M and reached
its maximum when AcSDKP was present at 1011 M. Higher
concentrations of AcSDKP were less effective at inducing EA.hy926 cell
morphogenesis, resulting in a bell-shaped dose-response curve similar
to that observed for many angiogenic factors. The stimulatory effect of
AcSDKP at 1011 M was greater than the activity of bFGF
assessed at 1 ng/mL (5 × 1011 M). The accelerated
formation of tubelike structures in the presence of AcSDKP was also
observed for other endothelial cells. Thus, after 6 hours of
treatment with 109 M AcSDKP, the increase in tube
formation was 112% ± 20% for BBC cells, 33% ± 3% for
HMVEC-Ls, and 31% ± 10% for HUVECs.
AcSDKP activates MMP-1 Angiogenic promoters induce several responses in endothelial cell cultures including the secretion of matrix MMPs, which are thought to play a critical role in endothelial cell migration during angiogenesis. To determine whether AcSDKP stimulated the secretion of MMPs, EA.hy926 cells were cultured for 24 hours in the presence of AcSDKP. Levels of secreted MMP-1, MMP-2, and MMP-9 were measured in the culture medium using a specific enzyme-linked immunosorbent assay (ELISA) that allows quantification of both total and active forms of these proteins. Taking into account the maximal chemotactic activity of AcSDKP observed at 109 M and 1011 M, we used the tetrapeptide
at these 2 concentrations. As shown in Table
1, AcSDKP induces a significant increase
in the active form of MMP-1, without modifying the secretion of the
total amount of this protein with a maximal effect observed at
109 M (84% over the control). Secretion of MMP-2 was not
increased in the presence of AcSDKP. MMP-9 was not detected under any
of the experimental conditions.
Angiogenic effect of AcSDKP in CAM assay The chicken embryo CAM was used as a suitable model to investigate the ability of AcSDKP to modulate angiogenesis in vivo under physiologic conditions. A range of doses between 0.002 and 200 pmol/cm2 AcSDKP was assayed on the CAM at day 9 of embryo development. As shown in Figure 4, AcSDKP induced angiogenesis in a dose-dependent manner, reaching the maximal stimulatory effect at 200 pmol/cm2. The stereoisomer of the tetrapeptide, AcSDDKP, assayed at 2 pmol/cm2, had no effect on CAM vascular density. HARP, a potent inductor of angiogenesis in the CAM and assayed at 100 pmol/cm2, exhibited similar stimulatory potency to AcSDKP at 200 pmol/cm2.
Angiogenic effect of AcSDKP in the in vivo Matrigel plug assay To further assess the angiogenic properties of AcSDKP in vivo, we evaluated blood vessel invasion into Matrigel plugs implanted subcutaneously in rats. As shown in Figure 5, AcSDKP significantly stimulates the vascular invasion of Matrigel plugs removed 7 days after implantation. Histologic analysis of Matrigel sections (Figure 5A) identifies new blood vessel development in the AcSDKP-containing plug (upper panel) as compared to the control plug (lower panel). The quantitative evaluation of vessels present in the border region of plugs indicates that AcSDKP induces a significant angiogenic response when used at concentrations of 105 M, 107 M, and 109 M
(Figure 5B).
AcSDKP increases vascular density in abdominal muscle of rat Skeletal muscle is one of the most plastic tissues in the body. To assess angiogenic effect of AcSDKP on the perfusion of abdominal muscles, the tetrapeptide was administrated at 5 µg/kg/injection delivered twice a day for 5 consecutive days and the vascular pattern visualized by India ink angiography. After 7 or 28 days, comparative analysis of the vascular density in the left (treated with AcSDKP) versus the right (control) side of abdominal muscle showed a greater diffusion of the dye in the AcSDKP-injected muscles than that in the contralateral muscles (Figure 6). Effects of the injections of AcSDKP were limited to the region of administration and there was no angioma-like structure or inflammatory infiltration observed in either side. Furthermore, the effects on normal vasculature persisted for up to day 28.
This work provides evidence for the angiogenic properties of the
tetrapeptide AcSDKP, which belongs to the family of negative physiologic regulators of hematopoiesis. We have demonstrated the
ability of this peptide to induce functional angiogenesis in vitro and
in vivo. As previously reported, AcSDKP is present in plasma
at nanomolar concentrations and has a wide tissue distribution but is
most abundant in hematopoietic organs such as the thymus, spleen, and
bone marrow.37,38 Measurable tetrapeptide concentrations have also been detected in the intestine, heart, stomach, and lung,
which are highly vascular organs.38 Using a highly
specific and sensitive EIA, we have shown that the EA.hy926 human
endothelial cell line produces AcSDKP and releases it at nanomolar
concentrations. The intracellular concentration of AcSDKP increases at
the beginning of culture and decreases progressively as the cells
approach confluence. These data are consistent with the recently
reported presence of AcSDKP in extracts of bone marrow endothelial
cells39,40 and suggest that AcSDKP produced constitutively
by the endothelium may act in an autocrine manner to induce
angiogenesis. Here we report that AcSDKP indeed acts in vitro as a
positive regulator of endothelial cell motility and differentiation, 2 essential components for new vessel formation. Maximal chemotactic
activity was obtained at 10 In view of the findings obtained in vitro with cultured endothelial cells, it was important to extend the study with AcSDKP into in vivo models. The angiogenic potential of AcSDKP was clearly evident in vivo in the CAM assay. The intensity of the vasoproliferative response defined as vascular index was similar to that observed for HARP, a potent inductor of neovascularization.36 Subsequent work has demonstrated the ability of AcSDKP to induce the recruitment of new vessels into a Matrigel plug implanted in animals at nanomolar concentrations. One important event in endothelial cell invasion is the interaction of endothelial cells with the protein components of the extracellular matrix such as fibronectin and vitronectin.48 Preliminary data have shown the increased adherence of EA.hy926 cells on fibronectin in the presence of AcSDKP (data not shown). These findings are consistent with results obtained using the Matrigel plug assay. Taking into account the in vivo angiogenic potency of AcSDKP demonstrated in 2 widely used assays for studying angiogenesis as well as the ability of AcSDKP to stimulate neovascularization in normal mammalian muscle, it is possible to hypothesize that the administration of this peptide may improve the insufficient vascularization in the animal model. Indeed, pharmacologic stimulation of angiogenesis is currently under investigation as an important adjunct to conventional therapeutic strategies for the treatment of ischemia, wound healing, and tissue transplantation.49 In vivo, AcSDKP certainly exhibits its angiogenic activity on bone marrow endothelium providing a possible mechanism for the enhancement of stem cell engraftment observed following AcSDKP administration at the time of transplantation. It has been shown that AcSDKP significantly increases the survival of lethally irradiated mice that receive transplants of the unfractionated bone marrow cells. This effect was mediated by an increased homing of hematopoietic precursors in the bone marrow as early as 1 day after transplantation.50 However, another report suggests that AcSDKP may improve the engraftment of ex vivo expanded hematopoietic stem cells through a mechanism unrelated to its activity as a stem cell cycle inhibitor.51 Given the results presented in this report, we hypothesize that AcSDKP mediates homing of hematopoietic stem cells from the peripheral blood to the bone marrow via its activity on endothelial cells. Bone marrow endothelial cells are intimately involved not only in the homing of hematopoietic progenitor cells to the bone marrow but also in the regulation of their proliferation and differentiation.52,53 The mechanism of inhibitory action of AcSDKP toward hematopoietic stem cell proliferation, however, is far from clear. Nevertheless, evidence suggests that AcSDKP could exert its effect on hematopoietic cells indirectly through its action on accessory cells. The present findings may allow speculation that the interaction of AcSDKP with endothelial cells may regulate putative secondary signals leading directly to the inhibition of primitive hematopoietic cells progression into S phase. Putative regulation may have an impact on endothelial adhesion molecules or the production of specific cytokines. Angiogenesis is an important process in a variety of pathologic
disorders, including tumor growth and metastasis.54 It has long been thought that leukemia and other hematologic malignancies differ from solid tumors in that they do not stimulate or require angiogenesis. However, it is now clear that a large number of hematologic disorders including leukemia, multiple myeloma,
polycythemia vera, and myelofibrosis are angiogenesis dependent and
reveal significantly increased neovascularity.55,56 It has
been found that the microvessel density in bone marrow is tightly
correlated with the progression of all these diseases and that the
level of intracellular VEGF and FGF can be considered as a predictor of
outcome in patients with myeloproliferative diseases.57,58 Interestingly, it was observed that the serum concentrations of AcSDKP
are frequently higher in adult patients with hematologic malignancies
than in healthy subjects.59 These results are consistent with the previous findings that the up-expression of T Taken together, these reports provide the first evidence that AcSDKP has angiogenic potential in addition to its role as an inhibitor of primitive hematopoietic cell proliferation. Moreover, the data suggest that AcSDKP may have therapeutic potential for the clinical treatment of deficient angiogenesis-related diseases. However, a detailed analysis of the role of AcSDKP in maintaining normal vascularization needs to be undertaken. Furthermore, the reported in vivo efficacy of AcSDKP to promote the growth of new vessels suggests that it may be a potential target for therapeutic antiangiogenic strategies targeting mainly tumor dormancy or stabilization. The molecular mechanisms responsible for the angiogenic activity of AcSDKP are currently under investigation. Whatever the modes of action of AcSDKP, it is clear that it exhibits a number of interesting biologic activities that are possibly a consequence of molecular cross-talk due to the redundancy within the numerous angiogenic and hematopoietic pathways supporting these processes. In fact, the angiogenic property of AcSDKP may be pivotal in controlling both hematopoiesis and bone marrow engraftment.
The authors thank Dr Cora J. Edgell for generously supplying EA.hy926 cells. We also thank Dr Josette Badet and Dr José Courty for their helpful advice during these studies and Dr Andrew Riches for his constant interest in our work. The authors wish to thank Dr André Balaton for his expertise with the Matrigel plug assay and Widad Farid for her expert technical assistance. We are extremely grateful to Dr Simon Robinson for critical reading of the manuscript.
Submitted July 31, 2002; accepted November 29, 2002.
Prepublished online as Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood-2002-07-2315.
Supported by a grant funded by Institut de Chimie des Substances Naturelles, CNRS (M.K.).
J.-M.L. and F.L. contributed equally to this work and share first authorship.
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: Joanna Wdzieczak-Bakala, ICSN, CNRS, 91198 Gif-sur-Yvette, France; e-mail: johanna.bakala{at}icsn.cnrs-gif.fr.
1. Ribatti D, Vacca A, De Falco G, Ria R, Roncali L, Dammacco F. Role of hematopoietic growth factors in angiogenesis. Acta Haematol. 2001;106:157-161[CrossRef][Medline] [Order article via Infotrieve].
2.
Dentelli P, Del Sorbo L, Rosso A, et al.
Human IL-3 stimulates endothelial cell motility and promotes in vivo new vessel formation.
J Immunol.
1999;163:2151-2159
3.
Ribatti D, Presta M, Vacca A, et al.
Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.
Blood.
1999;93:2627-2636
4.
Aoki Y, Jaffe ES, Chang Y, et al.
Angiogenesis and hematopoiesis induced by Kaposi's sarcoma-associated herpes virus-encoded interleukin-6.
Blood.
1999;93:4034-4043 5. Bussolino F, Colotta F, Bocchietto E, Guglielmetti A, Mantovani A. Recent developments in the cell biology of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor: activities on endothelial cells. Int J Clin Lab Res. 1993;23:8-12[Medline] [Order article via Infotrieve]. 6. Bikfalvi A, Han ZC. Angiogenic factors are hematopoietic growth factors and vice versa. Leukemia. 1994;8:523-529[Medline] [Order article via Infotrieve]. 7. Gerber HP, Malik AK, Solar GP, et al. VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature. 2002;417:954-958[CrossRef][Medline] [Order article via Infotrieve].
8.
Wilson EL, Rifkin DB, Kelly F, Hannocks MJ, Gabrilove JL.
Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures.
Blood.
1991;77:954-960 9. Ribatti D, Vacca A, Roncali L, Dammacco F. Hematopoiesis and angiogenesis: a link between two apparently independent processes. J Hematother Stem Cell Res. 2000;9:13-19[CrossRef][Medline] [Order article via Infotrieve]. 10. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998;125:725-732[Abstract].
11.
Lenfant M, Wdzieczak-Bakala J, Guittet E, Prome JC, Sotty D, Frindel E.
Inhibitor of hematopoietic pluripotent stem cell proliferation: purification and determination of its structure.
Proc Natl Acad Sci U S A.
1989;86:779-782 12. Guigon M, Bonnet D, Lemoine F, et al. Inhibition of human bone marrow progenitors by the synthetic tetrapeptide AcSDKP. Exp Hematol. 1990;18:1112-1115[Medline] [Order article via Infotrieve].
13.
Cashman JD, Eaves AC, Eaves CJ.
The tetrapeptide AcSDKP specifically blocks the cycling of primitive normal but not leukemic progenitors in long-term culture: evidence for an indirect mechanism.
Blood.
1994;84:1534-1542 14. Jackson JD, Yan Y, Ewel C, Talmage JE. Activity of acetyl-Ser-Asp-Lys-Pro (AcSDKP) on hematopoietic progenitors in short-term and long-term murine bone marrow cultures. Exp Hematol. 1996;24:475-481[Medline] [Order article via Infotrieve].
15.
Masse A, Ramirez LH, Bindoula G, et al.
The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Goralatide) protects mice from doxorubicin-induced toxicity: an improvement in both survival and hematological protection for early marrow stem cells and progenitors.
Blood.
1998;91:441-449 16. Bogden AE, Moreau JP, Gamba-Vitalo C, et al. Goralatide (AcSDKP), a negative growth regulator, protects the stem cell compartment during chemotherapy, enhancing the myelopoietic response to GM-CSF. Int J Cancer. 1998;76:38-46[CrossRef][Medline] [Order article via Infotrieve]. 17. Aidoudi S, Guigon M, Drouet V, Caen JP, Han ZC. The tetrapeptide AcSDKP reduces the sensitivity of murine CFU-MK and CFU-GM progenitors to aracytine in vitro and in vivo. Int J Hematol. 1996;68:145-155. 18. Watanabe T, Brown GS, Kelsey LS, et al. In vivo protective effects of tetrapeptide AcSDKP with or without granulocyte colony-stimulating factor on murine progenitor cell following sublethal irradiation. Exp Hematol. 1996;24:713-721[Medline] [Order article via Infotrieve].
19.
Rousseau A, Michaud A, Chauvet MT, Lenfant M, Corvol P.
The hemoregulatory peptide N-acetyl-Ser-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin I-converting enzyme.
J Biol Chem.
1995;270:3656-3661 20. Azizi M, Rousseau A, Ezan E, et al. Acute angiotensin converting enzyme inhibition increases the plasma level of the natural stem-cell regulator N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). J Clin Invest. 1996;97:839-844[Medline] [Order article via Infotrieve]. 21. Rousseau-Plasse A, Wdzieczak-Bakala J, Lenfant M, et al. Lisinopril, an angiotensin I-converting enzyme inhibitor, prevents in vivo the entry into cell cycle of murine hematopoietic stem cells following irradiation. Exp Hematol. 1998;26:1074-1079[Medline] [Order article via Infotrieve].
22.
Chisi JE, Wdzieczak-Bakala J, Thierry J, Briscoe CV, Riches AC.
Captopril inhibits the proliferation of hematopoietic stem and progenitor cells in murine long term bone marrow cultures.
Stem Cells.
1999;17:339-344 23. Chisi JE, Briscoe CV, Ezan E, Genet R, Riches A, Wdzieczak-Bakala J. Captopril inhibits in vitro and in vivo the proliferation of hematopoietic stem cells induced into cell cycle by cytotoxic drug administration or irradiation but has no effect on myeloid leukemia proliferation. Br J Haematol. 2000;109:563-570[CrossRef][Medline] [Order article via Infotrieve]. 24. Cameron NE, Cotter MA, Robertson S. Angiotensin converting enzyme inhibition prevents development of muscle and nerve dysfunction and stimulates angiogenesis in streptozotocin-diabetic rats. Diabetologia. 1992;35:12-18[CrossRef][Medline] [Order article via Infotrieve].
25.
Fabre J-E, Rivard A, Manger M, Silver M, Isner JM.
Tissue inhibition of angiotensin-converting enzyme activity stimulates angiogenesis in vivo.
Circulation.
1999;99:3043-3049
26.
Takeshita S, Tomiyama H, Yokoyama N, et al.
Angiotensin-converting enzyme inhibition improves defective angiogenesis in the ischemic limb of spontaneously hypertensive rats.
Cardiovasc Res.
2001;52:314-320 27. Wdzieczak-Bakala J, Fache MP, Lenfant M, Frindel E, Sainteny F. AcSDKP, an inhibitor of CFU-S proliferation, is synthesized in mice under steady-state conditions and secreted by bone marrow in long-term culture. Leukemia. 1990;4:235-237[Medline] [Order article via Infotrieve].
28.
Grillon C, Rieger K, Bakala J, et al.
Involvement of thymosin
29.
Safer D, Elzinga M, Nachmias VT.
Thymosin
30.
Bonnet D, Lemoine FM, Frobert Y, et al.
Thymosin
31.
Moscinski LC, Naylor PH, Olivier J, Goldstein AL.
Thymosin
32.
Malinda KM, Goldstein AL, Kleinman HK.
Thymosin 33. Koutrafouri V, Leondiadis L, Avgoustakis K, et al. Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model. Biochim Biophys Acta. 2001;1568:60-66[Medline] [Order article via Infotrieve]. 34. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol. 2001;61:253-270[CrossRef][Medline] [Order article via Infotrieve].
35.
Edgell CJS, McDonald CC, Graham JB.
Permanent cell line expressing human factor VIII-related antigen established by hybridization.
Proc Natl Acad Sci U S A.
1983;80:3734-3737 36. Papadimitriou E, Polykratis A, Courty J, Koolwijk P, Heroult M, Katsoris P. HARP induces angiogenesis in vivo and in vitro: implication of N or C terminal peptides. Biochem Biophys Res Commun. 2001;282:306-313[CrossRef][Medline] [Order article via Infotrieve]. 37. Pradelles P, Frobert Y, Creminon C, Liozon E, Masse A, Frindel E. Negative regulator of pluripotent hematopoietic stem cell proliferation in human white blood cells and plasma as analysed by enzyme immunoassay. Biochem Biophys Res Commun. 1990;170:986-993[CrossRef][Medline] [Order article via Infotrieve].
38.
Pradelles P, Frobert Y, Creminon C, Ivonine H, Frindel E.
Distribution of a negative regulator of haematopoietic stem cell proliferation (AcSDKP) and thymosin
39.
Huang WQ, Wang QR.
Bone marrow endothelial cells secrete thymosin 40. Li WM, Huang WQ, Huang YH, Jiang DZ, Wang QR. Positive and negative haematopoietic cytokines produced by bone marrow endothelial cells. Cytokine. 2000;12:1017-1023[CrossRef][Medline] [Order article via Infotrieve]. 41. Weisberg E, Sattler M, Ewaniuk DS, Salgia R. Role of focal adhesion proteins in signal transduction and oncogenesis. Crit Rev Oncog. 1997;8:343-358[Medline] [Order article via Infotrieve].
42.
Cheviron N, Grillon C, Carlier MF, Wdzieczak-Bakala J.
The antiproliferative activity of the tetrapeptide acetyl-N-SerAspLysPro, an inhibitor of haematopoietic stem cell proliferation, is not mediated by a thymosin 43. Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell. 1996;87:1153-1155[CrossRef][Medline] [Order article via Infotrieve]. 44. Risau W. Mechanisms of angiogenesis. Nature 1997;386:671-674[CrossRef][Medline] [Order article via Infotrieve].
45.
Mignatti P, Tsuboi R, Robbins E, Rifkin DB.
In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases.
J Cell Biol.
1989;108:671-682 46. Unemori EN, Ferrara N, Bauer EA, Amento EP. Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells. J Cell Physiol. 1992;153:557-562[CrossRef][Medline] [Order article via Infotrieve]. 47. Fisher C, Gilbertson-Beadling S, Powers EA, Petzold G, Poorman R, Mitchell MA. Interstitial collagenase is required for angiogenesis in vitro. Dev Biol. 1994;162:499-510[CrossRef][Medline] [Order article via Infotrieve]. 48. Strömblad S, Cheresh DA. Cell adhesion and angiogenesis. Trends Cell Biol. 1996;6:462-468[CrossRef][Medline] [Order article via Infotrieve]. 49. Pepper MS. Positive and negative regulation of angiogenesis: from cell biology to the clinic. Vasc Med. 1997;1:253-266. 50. Suzuki A, Aizawa S, Araki S, et al. Enhanced engraftment of intravenously transplanted hematopoietic stem cells into bone marrow of irradiated mice treated with AcSDKP. Exp Hematol. 1998;26:79-83[Medline] [Order article via Infotrieve].
51.
Szilvassy SJ, Meyerrose TE, Grimes B.
Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells.
Blood.
2000;95:2829-2837 52. Rafii S, Mohle R, Shapiro F, Frey BM, Moore MA. Regulation of hematopoiesis by microvascular endothelium. Leuk Lymphoma. 1997;27:375-386[Medline] [Order article via Infotrieve]. 53. Suda T, Takakura N, Oike Y. Hematopoiesis and angiogenesis. Int J Hematol. 2000;71:99-107[Medline] [Order article via Infotrieve]. 54. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27-31[CrossRef][Medline] [Order article via Infotrieve]. 55. Yang R, Han ZC. Angiogenesis in hematologic malignancies and its clinical implications. Int J Hematol. 2002;75:246-256[Medline] [Order article via Infotrieve]. 56. Ribatti D, Vacca A, De Falco G, Roccaro A, Roncali L, Dammacco F. Angiogenesis, angiogenic factor expression and hematological malignancies. Anticancer Res. 2001;21:4333-4339[Medline] [Order article via Infotrieve].
57.
Aguayo A, Estey E, Kantarjian H, et al.
Cellular vascular endothelial growth factor is a predictor of outcome in patients with acute myeloid leukemia.
Blood.
1999;94:3717-3721 58. Molica S, Vitelli G, Levato D, Ricciotti A, Digiesi G. Clinicoprognostic implications of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in early B-cell chronic lymphocytic leukaemia. Br J Cancer. 2002;86:31-35[CrossRef][Medline] [Order article via Infotrieve]. 59. Liozon E, Pradelles P, Venot J, et al. Serum levels of a negative regulator of cell proliferation (AcSDKP) are increased in certain human haemopathies. Leukemia. 1993;7:808-812[Medline] [Order article via Infotrieve]. 60. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 2000;406:532-535[CrossRef][Medline] [Order article via Infotrieve]. 61. Hall AK. Differential expression of thymosin genes in human tumors and in the developing human kidney. Int J Cancer. 1991;48:672-677[Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  Y.-H. Liu, M. D'Ambrosio, T.-d. Liao, H. Peng, N.-E. Rhaleb, U. Sharma, S. Andre, H.-J. Gabius, and O. A. Carretero N-acetyl-seryl-aspartyl-lysyl-proline prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin Am J Physiol Heart Circ Physiol, February 1, 2009; 296(2): H404 - H412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Waeckel, J. Bignon, J.-M. Liu, D. Markovits, T. G. Ebrahimian, J. Vilar, B. Mees, O. Blanc-Brude, V. Barateau, S. Le ricousse-Roussanne, et al. Tetrapeptide AcSDKP Induces Postischemic Neovascularization Through Monocyte Chemoattractant Protein-1 Signaling Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 773 - 779. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wang, O. A. Carretero, X.-Y. Yang, N.-E. Rhaleb, Y.-H. Liu, T.-D. Liao, and X.-P. Yang N-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2099 - H2105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nakayama, N. Nara, Y. Kawakita, Y. Takeshima, M. Arakawa, M. Katoh, S. Morita, K. Iwatsuki, K. Tanaka, S. Okamoto, et al. Cloning of cDNA Encoding a Regeneration-Associated Muscle Protease Whose Expression Is Attenuated in Cell Lines Derived from Duchenne Muscular Dystrophy Patients Am. J. Pathol., May 1, 2004; 164(5): 1773 - 1782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fuchs, H. D. Xiao, J. M. Cole, J. W. Adams, K. Frenzel, A. Michaud, H. Zhao, G. Keshelava, M. R. Capecchi, P. Corvol, et al. Role of the N-terminal Catalytic Domain of Angiotensin-converting Enzyme Investigated by Targeted Inactivation in Mice J. Biol. Chem., April 16, 2004; 279(16): 15946 - 15953. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
4 (T
