The Molecular Basis of VEGFR-1 Signal Transduction Pathways in Primary Human Monocytes
Objective— Arteriogenesis, the growth of preexisting arterioles into functional arteries, is dependent on the proper function of monocytes. Likewise, wound healing is monocyte-dependent. The activation of vascular endothelial growth factor receptor-1 (VEGFR-1) in monocytes induces a chemotactic response, triggers the expression of tissue factor, and gene expression of cytokines and chemokines. Little is known about intracellular signaling pathways mediating the biological functions triggered by VEGFR-1 in primary monocytes.
Methods and Results— Monocytes were isolated from peripheral venous blood of young healthy individuals using indirect magnetic labeling. Stimulation of monocytes with either vascular endothelial growth factor-A (VEGF-A) or placenta growth factor (PlGF-1) triggered VEGFR-1 autophosphorylation and phosphorylation of distinct downstream proteins: phosphatidylinositol-3 kinase (PI-3K), Akt, p38, and extracellular signal–regulated kinase-1/2 (ERK1/2). PI-3K appears to be a central regulator in VEGFR-1 signaling in monocytes as the activation of Akt, p38, and ERK1/2 are PI-3-K–dependent. In addition, Akt activation functions downstream of p38 kinase. VEGFR-1–mediated chemotaxis of monocytes is dependent on the activation of PI-3K, p38 kinase, Akt, and ERK1/2, when assessed in a modified Boyden chamber.
Conclusions— Both PlGF-1 and VEGF-A can activate VEGFR-1–dependent signaling pathways in primary human monocytes, leading to the activation of several intracellular signaling pathways. These pathways are critically involved in primary monocyte chemotaxis.
Angiogenesis and arteriogenesis are 2 distinct processes that are stimulated in the ischemic heart and related to ischemia in other tissues. Both processes can enhance regional blood flow and improve impaired muscle/organ function. Angiogenesis occurs by either sprouting or intussusceptive growth of capillaries, whereas arteriogenesis represents the growth of collateral arteries from preexisting ones.1 Monocytes2–4 and tissue resident precursor cells5 contribute to arteriogenesis by their recruitment to the growing vessel. Moreover, there is evidence that angiogenesis is monocyte-dependent, which was shown for angiogenesis in wound healing.6
The family of vascular endothelial growth factor (VEGF) and its receptors are crucially involved in the process of angiogenesis and arteriogenesis4 acting on 2 important cell types, ie, endothelial cells and monocytes.4,7 VEGF-A binds to 2 different receptor tyrosine kinases on vascular endothelial cells, namely VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1).8,9 VEGFR-2 is the major mediator of endothelial activation in response to VEGF and it mediates a large spectrum of endothelial functions.8,9 Monocytes can also be activated by members of the VEGF family. VEGFR-1 is the only VEGFR present on the surface of monocytes, and it mediates a chemotactic response to VEGF-A and tissue factor induction.10 Placenta growth factor (PlGF-1), another member of the VEGF family, was shown to promote cytokine and chemokine gene expression in monocytes and chemotaxis in the monocytic cell line THP-1.11 Critically, the presence of VEGFR-3 on atherosclerotic plaque macrophages has been recently argued in conflicting reports.12,13 However, macrophages, although most likely monocyte-derived, represent a cell population phenotypically different from monocytes and are activated under respective conditions.
Several pathological conditions were shown to negatively affect the process of arteriogenesis. Diabetes mellitus specifically impairs endothelial function14 and arteriogenesis, as the development and function of coronary collateral vessels is significantly reduced in patients with diabetes mellitus.15 The reduced collateral growth in diabetic individuals corresponds with a significantly altered ex vivo chemotactic response of monocytes to VEGF-A.16 At the same time, the kinase function of VEGFR-1 is intact in monocytes derived from diabetic individuals implying a signal transduction defect downstream from the receptor to be responsible for the impaired monocytic response.16 A similar impairment of VEGF-induced monocyte function has recently been shown for hypercholesterolemia17 and smoking.18 Circulating monocytes may therefore be used as biosensors to study the influence of different pathological conditions on cellular function.
Considering the importance of VEGFR-1–stimulated monocytes in vascular biology,3,4 we have characterized intracellular signal transduction pathways mediating VEGF-A– and PlGF-1–induced monocyte chemotaxis after activation of VEGFR-1. In the present study we show that both PlGF-1 and VEGF-A can activate VEGFR-1 in primary human monocytes. Stimulation of monocytes with PlGF-1 or VEGF-A leads to the activation of several intracellular signaling molecules including phosphatidylinositol-3 kinase (PI-3K), Akt, extracellular signal-regulated kinase-1/2 (ERK1/2), and p38 mitogen-activated protein kinases (MAPK). In addition, we show that PlGF-1– and VEGF-A–induced activation of PI-3K, Akt, ERK1/2, and p38 is crucially involved in mediating the signal for monocyte chemotaxis.
For experimental procedures, please see the supplemental materials, available online at http://atvb.ahajournals.org.
Isolation of Monocytes from Peripheral Venous Blood
Monocytes from healthy volunteers, mean age 30±3.4 years, were used for this study. Blood samples were collected with informed consent and permission of local ethical committee. Donors with concurrent inflammatory or malignant disease, and those with a history of smoking were excluded from these investigations. For detailed monocyte isolation procedure, please see Experimental Procedures in the supplemental materials.
Immunoprecipitation and In Vitro Kinase Assay
Assays were performed as previously published9 with minor modifications (please see Experimental Procedures in the supplemental materials).
Lipid-Kinase Assay—PI-3 Kinase Assay
Lysates of ligand-stimulated monocytes were used for immunoprecipitation at 4°C overnight with a polyclonal antiserum raised against p85 subunit of PI-3 kinase (see Reagents). Lipid-kinase assay was performed as previously described9; for details please see the supplemental materials.
Western Blot Analysis of Akt, p38, and ERK1/2
Monocytes (5×106 cells/assay) were pretreated, where indicated, with pharmacological inhibitors and subsequently incubated with 100 μmol/L Na3VO4 for additional 5 minutes. Cells were stimulated with either VEGF-A or PlGF-1 before solubilization in ice-cold lysis buffer. Protein concentration was measured in the supernatant of lysates. Sample aliquots containing equal amounts of protein were boiled in β-mercaptoethanol–containing sample buffer and separated on 7.5% to 12% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a transfer onto nitrocellulose membranes (Amersham). Subsequently, membranes were blocked with 5% semi-dry milk solution and incubated with specific primary antibodies. Antibodies recognizing phosphorylated forms of Akt, p38, and ERK1/2 were used at a dilution of 1:1000. Horseradish peroxidase-conjugated secondary antibodies were added and the specific proteins were detected using Supersignal chemiluminescence substrate (Pierce). The intensity of bands was quantified by scanning using an LAS documentation system. To monitor protein loading, blots were stripped and reprobed using 1:1000 or 1:2500 dilutions of antibodies recognizing AKT or p38, correspondingly.
Monocyte chemotaxis was performed in the modified 48-well Boyden chamber (Nucleopore); please see Experimental Procedures in the supplemental materials.
In the chemotaxis experiments data are given as mean±SEM. The probability of difference between the samples without stimulus (chemokinesis) or with a stimulus (chemotaxis) was evaluated using Kruskal–Wallis test. Subsequently, Mann–Whitney test was performed to estimate the level of significance. Level of significance P<0.01 was considered significant.
PlGF-1 and VEGF-A Activate VEGFR-1 and Induce Phosphorylation of Various Intracellular Proteins in Monocytes
After stimulation of VEGFR-1 with either PlGF-1 or VEGF-A, tyrosine phosphorylation of a number of proteins could be observed (Figure 1A). Four distinct proteins (190, 120, 66, 50 kDa) showed the strongest level of phosphorylation in a ligand-concentration–dependent fashion. The maximal phosphorylation was observed at 10 ng/mL for both VEGF-A and PlGF-1 with a 2- to 4-fold increase (Figure 1A).
Stimulation of monocytes for 5 minutes with either PlGF-1 or VEGF-A (10 ng/mL) resulted in a considerable phosphorylation of VEGFR-1 (Figure 1B). Further increase in the ligand concentrations to 50 ng/mL did not result in higher VEGFR-1 activity. Similar results were obtained when VEGFR-1 was specifically immunoprecipitated and subsequently immunoblotted with an antiphosphotyrosine antibody (supplemental Figure I). Critically, the latter assay, in contrast to in vitro kinase assay, required 4 times higher number of monocytes, namely 20×106/sample, which can be obtained from 60 to 80 mL of blood.
The concentration 10 ng/mL was chosen for further experiments with both ligands. Critically, the concentrations of 1 ng/mL of both PlGF-1 and VEGF-A, used in chemotaxis assay (see below and Figure 5) were able to stimulate tyrosine phosphorylation overall (Figure 1A) as well as activation of a set of important signaling molecules (supplemental Figure V).
PlGF-1 and VEGF-A Activate the PI-3K/Akt Signaling Pathway in Monocytes
PlGF-1 was shown to stimulate Akt phosphorylation in the monocytic cell line THP-1.11 This stimulation was dependent on the activation of PI-3 kinase. Stimulation of primary human monocytes with either PlGF-1 or VEGF-A for 5 minutes resulted in an increased activity of PI-3K as measured by the ability of PI-3K to phosphorylate the substrate PI at 3′ position in a lipid-kinase assay (Figure 2). Both VEGF-A and PlGF-1 (10 ng/mL) caused a 2.7- and 1.7-fold increase, respectively, in the formation of phosphatidylinositol phosphate (PIP) as compared with the nonstimulated control.
Both VEGF-A and PlGF-1 induced Akt (Ser473) phosphorylation by about 2.6- and 5.2-fold, correspondingly (Figure 3A). Pretreatment of monocytes with 10 μmol/L of LY294002 (Figure 3A) completely abolished Akt (Ser473) phosphorylation in response to either VEGF-A or PlGF-1. Similar results were observed after monocyte pretreatment with Wortmannin, another PI-3K inhibitor (supplemental Figure III).
P38 Kinase Regulates Akt (Ser473) Phosphorylation
In neutrophils, Akt was found in the signaling complexes together with members of the p38 MAPK signaling pathway.19 Akt phosphorylation on Ser473 residue was found to be dependent on p38 kinase activation in vascular smooth muscle cells.20 Stimulation of monocytes with either VEGF-A or PlGF-1 leads to a 1.95- or 2.4-fold induction of Akt (Ser473) phosphorylation (Figure 3B). Preincubation of monocytes with p38 kinase inhibitor SB203580 completely abrogated VEGF-A– and PlGF-1–stimulated Akt phosphorylation, indicating that either p38 itself or a downstream member of p38 signaling pathway inflict Akt phosphorylation.
PI-3K Mediates VEGF-A–/PlGF-1–Induced Activation of Two MAPKs, p38 and ERK1
Recent studies have shown that migratory responses of monocytes to various chemoattractants were dependent on MAPK signaling pathways.21–23 Stimulation of monocytes with either VEGF-A or PlGF-1 (10 ng/mL) results in the phosphorylation of ERK1 (5.8-fold and 10-fold increase, respectively) and in a somewhat weaker phosphorylation of ERK2 (Figure 4). Preincubation of monocytes with the PI-3K inhibitor LY294002 for 30 minutes before ligand stimulation completely abrogated ERK1 phosphorylation. The fairly detectable ligand-induced phosphorylation of ERK2 may be attributed to its tremendously higher baseline activation (Figure 4).
Stimulation of monocytes with VEGF-A or PlGF-1 leads to phosphorylation of p38 kinase with a 4- and 10-fold increase in p38 kinase activity, respectively (Figure 4). Pretreatment of monocytes with LY294002 suppressed p38 kinase phosphorylation to undetectable levels.
Biological Effects of VEGFR-1–Dependent Akt Activation in Primary Human Monocytes Are Independent of ERK1/2 Pathway Activation
VEGFR-1–mediated phosphorylation of ERK1/2 in THP-1 cells was shown to depend on Akt. We used a recently developed inhibitor of Akt (Ser473) phosphorylation, AktVIII inhibitor,24 to test this hypothesis in primary monocytes. AktVIII inhibitor effectively abolishes more than 90% of both baseline and VEGFR1-ligand–induced Akt (Ser473) phosphorylation in primary human monocytes at 1 μmol/L (supplemental Figure IVA). Pretreatment of monocytes with 1μmol/L of AktVIII inhibitor did not affect phosphorylation of neither ERK1/2 nor p38 (supplemental Figure IVB). This indicates that VEGFR-1–induced biological effects in primary monocytes are mediated by Akt independently of ERK1/2 pathway.
P38, ERK1/2 MAPK, and Akt/PKB Signaling Pathways Are Involved in VEGF-A–/PlGF-1–Induced Chemotaxis of Human Monocytes
VEGF-A is an established chemoattractant for primary human monocytes.10 We therefore investigated which intracellular signaling pathways are involved in the motogenic response of monocytes. Thus, monocyte chemotaxis was studied in the presence of various kinase inhibitors. Monocytes responded with a significant (P<0.01) 45±3.4% and 35±4.2% increase in chemotaxis when stimulated with either 1 ng/mL of VEGF-A or PlGF-1 (Figure 5). Ligand concentration of 1 ng/mL was established to be optimal for chemotaxis.16 The lower concentration of PlGF-1 or VEGF-A required in chemotaxis assay, as compared with 10 ng/mL used in biochemical analysis of enzyme phosphorylation, may be attributed to the longer period of stimulation in chemotaxis assay sufficient to induce the biological response. A stronger chemotactic response was observed after stimulation with fMLP (130% increase over the unstimulated baseline value, P<0.001). Further, we used established pharmacological inhibitors at effective concentrations shown to specifically inhibit their targets.
Monocyte migration toward VEGF-A or PlGF-1 was significantly (P<0.01) suppressed in the presence of the AktVIII inhibitor by 29±7.0% and 34±6.4%, correspondingly (Figure 5). In the presence of the ERK1/2 pathway inhibitor PD98059, VEGF-A– and PlGF-1–induced chemotaxis was significantly (P<0.01) decreased by 29±4.5% and 38±6.5%, respectively. Pretreatment with the p38 kinase inhibitor SB203580 resulted in a strong suppression (P<0.01) of VEGF-A– and PlGF-1–induced monocyte chemotaxis by 52±7.3% and 48±1.9%. Comparable inhibition of monocyte chemotaxis by 55±6.3% and 54±7.6% was observed using the PI-3K inhibitor LY294002 (P<0.01; Figure 5).
When assessing fMLP-induced monocyte-migration (10 nmol/L), ie, growth factor–independent stimulation of monocyte migration, various kinase inhibitors abrogated ligand-stimulated locomotion with a comparable pattern (Figure 5). Notably, monocytes could still respond to fMLP stimulation with residual chemotaxis in the presence of the different inhibitors. This residual chemotactic response varied from 33% in the presence of p38 kinase inhibitor to 72% in the presence of Akt inhibitor. This may imply stimulus-dependent activation of different signaling pathways in the chemotactic response of primary monocytes.
In this study, we were able to significantly extend our knowledge on the signal transduction properties of VEGFR-1 in primary human monocytes. Critically, this is the first article describing the intracellular signaling pathways involved in the process of VEGFR-1–mediated monocyte migration. Several previous studies reported distinct biological responses of primary human monocytes or monocytic cells in response to VEGFR-1 ligands without showing VEGFR-1 activation.10,11,25 Because of the involvement of monocyte migration in arteriogenesis and because of the previously described signal transduction defects in diabetes,16 hypercholesterolemia,17 and smoking,18 these data are of crucial importance for our understanding of monocyte (dys)function as well as physiological and defective arteriogenesis.
Monocytes express significant level of VEGFR-1 mRNA, but no VEGFR-2 mRNA (supplemental Figure II).10 Activation of VEGFR-1 induces migration of human monocytes.10 Macrophages carrying a mutant VEGFR-1 lacking the tyrosine kinase domain (VEGFR-1 TK−/−) showed markedly decreased migration toward VEGF.26 Mice carrying VEGFR-1 TK−/− are characterized by slower growth of some tumors27 and reduced tumor metastasis.27 Impaired migration and activation of monocytes/macrophages in these mice is responsible for a delayed progression of rheumatoid arthritis.27 These observations indicate that the VEGFR-1 tyrosine kinase domain is required for physiological monocyte function, namely the induction of monocyte/macrophage migration.
In the present study, stimulation of human primary monocytes with either VEGF-A or PlGF-1 led to VEGFR-1 autophosphorylation in vitro. Ligand-induced autophosphorylation of tyrosine residues in the cytoplasmic region of VEGFR-1 is a key event coupling VEGFR-1 to intracellular signal transduction pathways. The intracellular signaling pathways activated after VEGFR-1 stimulation are poorly understood, mainly because of the comparably weak tyrosine kinase activity of VEGFR-1.27 VEGFR-1 was documented to undergo autophosphorylation at Tyr-1169, 1213, 1242, 1327, 1333.27
After VEGF stimulation, VEGFR-1 is autophosphorylated on Tyr-1213.27 PI-3K is a potential candidate mediating biological effects after Tyr-1213 autophosphorylation.27 PI-3K is a pivotal enzyme and a well-established mediator of locomotive function in monocytes and monocyte-like cells.11,28 We demonstrate that PI-3K plays a crucial role in VEGFR-1–mediated monocyte signaling as PI-3K activity increases on stimulation with either VEGF-A or PlGF-1. Several distinct signaling molecules such as Akt, p38, and ERK1/2 are activated in a PI-3K–dependent fashion. Our data are in line with recent data generated in the monocytic cell line THP-1, where PlGF stimulated Akt phosphorylation in a PI-3K–dependent fashion.11 It is important to note, however, that our novel data represent the first proof of VEGF signaling in primary human monocytes, where we can exclude any differentiation or dedifferentiation, which is always possible in stable or transformed cell lines such as THP-1.
In porcine aortic endothelial cells stably expressing human VEGFR-1 (PAEC/Flt-1),9 stimulation of VEGFR-1 did not result in ligand-induced activation of PI-3K.29 Likewise, activation of VEGFR-1 in PAEC/Flt-1 was unable to induce endothelial cell migration.9 Our novel data clearly demonstrate that this situation is different in primary human monocytes with a functional VEGFR-1–PI-3K axis.
The serine/threonine kinase Akt is one of the signal transduction mediators acting downstream of PI-3K.30 Akt activation plays a critical role in mediating cell proliferation, differentiation, and survival.31 In our study, VEGFR-1 propagated the phosphorylation of Ser473 of Akt in monocytes, which was strictly PI-3K–dependent. Two inhibitors of PI-3K, Wortmannin and LY294002, abolished VEGF-A– and PlGF-1–stimulated Akt activation to almost undetectable levels. Activation of Akt (Ser473 phosphorylation) was recently shown in monocytic THP-1 cells in response to PlGF.11 Similarly, the motogenic response after VEGFR-2 stimulation in endothelial cells requires subsequent activation of PI-3K and Akt.32
Previous reports suggested that phosphorylation of Ser473 was the result of autophosphorylation after PDK1-dependent Akt Thr308 phosphorylation or was attributable to sequential phosphorylation of Thr308 and then Ser473.33 It was recently described that p38 kinase is required for PIP3-stimulated activation of Akt Ser473 in human neutrophils.19 Our results provide initial evidence that p38 kinase32 participates in the signal transduction pathway leading to Akt-Ser473 activation in monocytes.
According to our new data, stimulation of VEGFR-1 in monocytes activates the p38 kinase pathway, which in turn is required for the VEGFR-1–mediated chemotactic response of monocytes. Activation of p38 revealed a rapid response (5 minutes) in contrast to a somewhat delayed activation profile in the monocytic cell line MonoMac6 after stimulation with MCP-1 or sFKN.34,35 Activation of p38 on stimulation of primary human monocytes with plasmin peaked at 15 minutes and was involved in plasmin-induced chemotaxis.21 All this implies that the kinetics of monocyte/monocytic cell activation are stimulus- and cell-dependent.
Recently, activation of VEGFR-1 was implied in chemokine and cytokine gene expression and chemotaxis of the monocytic cell line THP-1.11 The chemotactic response to PlGF-1 was dependent on the activation of PI-3K, Akt, and ERK1/2 kinases. In the same study, ligand-induced activation of ERK1/2 was abolished to different extent in the presence of a PI-3K inhibitor as well as after transfection of the dominant-negative form of PI-3K into THP-1 cells. Our findings support and extend these data. We were able to show that the chemotactic response of primary human monocytes mediated by VEGFR-1 requires activation of PI-3K/Akt and ERK1/2. In primary human monocytes, we observed significant activation of these molecules at concentrations of 10 ng/mL for both VEGF-A and PlGF-1. In our study, the chemotactic response of primary monocytes was optimal at concentrations between 1 and 10 ng/mL for both PlGF-1 and VEGF-A, which corresponds with previously published data on VEGF-A–induced chemotaxis of primary human monocytes,10,16 mouse macrophages,26 and primary mouse monocytes (unpublished data 2006).
Our findings extend current knowledge on the signal transduction events mediated by VEGFR-1. Selvaraj et al suggested that a cross-talk between PI-3K/Akt and ERK1/2 pathways may converge on MEK1/2, which is the kinase upstream from ERK1/2.36 Our results indicate that activation of ERK1/2 in response to VEGFR-1 ligands is dependent on PI-3K but does not require Akt activation in primary human monocytes (supplemental Figure IVB).
The ligand-induced VEGFR-1–mediated monocyte chemotaxis was affected in the presence of distinct kinase inhibitors. Application of Akt inhibitor led to a 30% decrease in monocyte chemotaxis (Figure 5). This supports similar findings in THP-1 monocytic cells,11 where expression of dominant-negative Akt inhibited PlGF-induced chemotaxis by about 50%. Similar inhibition of VEGFR-1–mediated monocyte chemotaxis was observed when using an ERK1/2 pathway inhibitor.
A central role of PI3-K in VEGFR-1–mediated signaling in primary human monocytes was supported by its role in monocyte chemotaxis. Inhibition of PI3-K resulted in a 55% decrease of ligand-induced migration. Directed cell motility is driven by chemoattractants that bind to G protein–coupled receptors (eg, fMLP)28 or growth factors that signal through receptor tyrosine kinases (eg, VEGF). In both cases, studies have shown that PI-3K is an important mediator of these chemotactic responses. Moreover, neutrophils or peritoneal macrophages deficient in p110γ, a catalytic subunit of PI-3K, show a reduced chemotaxis toward fMLP.28,37
For the first time, we present data indicating that VEGFR-1 stimulation in primary monocytes induces a chemotactic response by activating distinct signaling pathways (Figure 6). The exclusive use of pharmacological inhibitors to dissect VEGFR-1–dependent signaling pathways in primary monocytes may be considered as a limitation of the current study. However, the primary nature of monocytes did not allow us to use either siRNA or vectors carrying dominant-negative or constitutively-active forms of enzymes to silence or activate specific signaling events. The latter would require at least 24 hours of monocyte culturing and would lead to their differentiation into macrophages.
In summary, VEGFR-1–mediated ligand-induced signal transduction in monocytes involves activation of PI-3K/Akt and 2 MAP kinase pathways, p38 and ERK1/2. Ligand-induced monocyte migration is dependent on the activation of PI-3K, p38 MAPK, and—to a somewhat lesser extent—on ERK1/2 and Akt. PI-3K plays a central role in monocyte signaling downstream of VEGFR-1, and activation of p38 and ERK1/2 pathways are strongly dependent on PI-3K activation. These data create a solid and valuable basis for the further elucidation of the recently described signal transduction defects responsible for impaired monocyte migration in diabetic,16 hypercholesterolemic,17 and smoking18 individuals. The identification of the underlying molecular defects may change our understanding of the interindividual differences in arteriogenesis (poor and good collateralizers) and may be of help to develop novel strategies for stimulating therapeutic angiogenesis/arteriogenesis.
Source of Funding
This work was supported in part by the European commission (VEGF-therapies QLK3-CT-2002-01955), the German Research Council DFG (Project Wa734/6-2 of Priority Research Project 1069 “Angiogenesis; Heisenberg.Scholarship Wa734/5-1 and DFG SFB452/B1), and the Cardiovascular Research Institute Maastricht (CARIM), all to J.W.
V.T. and G.F. contributed equally to this study.
Original received August 24, 2007; final version accepted November 29, 2007.
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