BMP‐2 induces human mononuclear cell chemotaxis and adhesion and modulates monocyte‐to‐macrophage differentiation

Abstract Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor which leads to atherosclerosis, an inflammatory disease characterized by the infiltration of mononuclear cells in the vessel. Bone morphogenetic protein (BMP)‐2 is a cytokine which has been recently shown to be elevated in atherosclerosis and T2DM and to contribute to vascular inflammation. However, the role of BMP‐2 in the regulation of mononuclear cell function remains to be established. Herein, we demonstrate that BMP‐2 induced human monocyte chemotaxis via phosphoinositide 3 kinase and mitogen‐activated protein kinases. Inhibition of endogenous BMP‐2 signalling, by Noggin or a BMP receptor inhibitor, interfered with monocyte migration. Although BMP‐2 expression was increased in monocytes from T2DM patients, it could still stimulate their migration. Furthermore, BMP‐2 interfered with their differentiation into M2 macrophages. Finally, BMP‐2 both induced the adhesion of monocytes to fibronectin and endothelial cells (ECs), and promoted the adhesive properties of ECs, by increasing expression of adhesion and pro‐inflammatory molecules. Our data demonstrate that BMP‐2 could exert its pro‐inflammatory effects by inducing monocyte migration and adhesiveness to ECs and by interfering with the monocyte differentiation into M2 macrophages. Our findings provide novel insights into the mechanisms by which BMP‐2 may contribute to the development of atherosclerosis.

risk factors such as type 2 diabetes mellitus (T2DM) accelerate monocyte recruitment, adhesion on the endothelium and their infiltration into the vessel wall. There monocytes start to proliferate, differentiate to macrophages and finally into foam cells leading to increased local inflammation and plaque development. 1,3 Elevated numbers and increased recruitment of monocytes have been linked to atherosclerotic plaque formation, while inhibition of monocytes leads to decreased size of atherosclerotic plaques. 1,3 Additionally, monocytes play an important role in the formation of new collaterals as they are recruited to sites of arteriogenesis by cytokines such as vascular endothelial growth factor A (VEGFA). Our previous studies demonstrated that T2DM results in impaired VEGFA-induced monocyte migratory responses and it has been suggested that this may contribute to the decreased formation of collateral vessels in patients with T2DM. 3,4 Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily and play a crucial role in development, cell differentiation, bone formation and vascular function. 5,6 BMPs signal through transmembrane heteromeric complexes of type I and II serine-threonine kinase receptors. 5,6 BMP binding induces constitutively active type II receptors to transphosphorylate and activate type I receptors, which in turn phosphorylate the intracellular receptor-associated (R)-Smads Smad1/5/8. R-Smads form then complexes with the common Smad4 and translocate into the nucleus where they regulate transcription of target genes. 5,6 Besides the Smad-mediated signalling pathway BMPs activate also additional pathways including mitogen-activated protein kinases (MAPKs) p38, phosphoinositide 3-kinase (PI3K) and others. BMP signalling can be modulated by extracellular ligand antagonists, such as noggin, chordin or cerberous. 5 Several studies have suggested that BMPs are involved in vascular inflammation, vessel calcification and atherosclerosis. 7-10 BMP-2 is expressed in calcified atherosclerotic plaques 11,12 and in calcified arteries in mouse models. 13,14 Moreover, increased BMP-2 plasma levels were associated with atherosclerosis and coronary calcification in T2DM patients. 15 In addition, BMP-2 can induce EC activation 10 and promote calcification of ECs, myofibroblasts and SMCs. 16,17 Furthermore, interference with the BMP-2 pathway hinders atherosclerosis, while enhanced BMP-2 activity results in increased atherosclerotic lesion formation in mice. 9,10,18 It was suggested that the effects of BMP-2 on atherosclerosis development are due to the effects of BMP-2 signalling on EC function. Whether the effects of BMP-2 on atherosclerosis development are also due to its effects on monocyte function is not known.
This study focuses on the characterization of the effects of BMP-2 on mononuclear cell function, as well as their interaction with ECs and addresses the mechanisms involved therein. To this extent, we used human peripheral blood monocytes and demonstrated that BMP-2 expression is increased in monocytes from T2DM patients. BMP-2 is a potent monocyte chemoattractant and interferes with monocyte differentiation into M2 macrophages.
Moreover, we demonstrate that induces ECs-mononuclear cell interactions. Altogether our results provide novel insights into the mechanisms by which BMP-2 exerts its pro-inflammatory effects and in this way may contribute to the development of atherosclerosis.

| Human primary monocyte isolation
Monocytes were isolated as described previously 19,20 Monocytes were isolated by means of density gradient centrifugation, followed by a magnetic-bead-based immunological isolation assay. In brief, density centrifugation was performed with Histopaque (1.077 g/mL) separation (Sigma-Aldrich Inc.) to isolate peripheral blood mononuclear cells. Subsequently, monocytes were extracted from the mononuclear fraction using the MACS monocyte isolation kit II (Miltenyi Biotec GmbH, Bergische Gladbach, Germany), according to the manufacturer's protocol.

| Monocyte chemotaxis
Chemotaxis assays were performed with Boyden chamber methodology as previously described 19

| Macrophage differentiation
Freshly isolated monocytes were cultured in RPMI medium (containing 10% FBS and 1.25% Penicillin/Streptomycin). Monocyte differentiation into macrophages was induced by addition of 50 ng/mL M-CSF. Differentiation into M1 or M2 macrophages was induced by addition of 10 ng/mL IL-1β or 10 ng/ml IL-4, respectively, after 7 days of differentiation.

| Protein analysis
Monocytes or endothelial cells were stimulated with different concentrations of the indicated ligands and for the indicated lengths of time. Thereafter, cells were processed to lysates and subjected to Western blot analysis as described previously. 19

| Adhesion assay of mononuclear cells to Fibronectin
Forty-eight-well plates were coated with 10 μg/mL fibronectin (Sigma-Aldrich) overnight at 37°C. Unspecific binding sites were blocked with 1% BSA in PBS at least 1 hour at 37°C. Besides, 1% BSA coated wells served as negative control. Primary monocytes were stimulated with and without 50 ng/mL BMP-2 for 60 minutes at 37°C. Afterwards, cells were plated (1 x 10 5 cells/well) and allowed to adhere for 15 minutes at 37°C. Subsequently, nonadhered cells were washed away by swinging gently 2-5 times with prewarmed PBS. Pictures were taken of the well-middle, and the cells were counted.

| Adhesion assay of mononuclear cells to endothelial cells
Human Mouse brain endothelial cells bEnd5 were grown in DMEM (high glucose, 4.5 g/L), supplemented with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin, on plates coated with 1% gelatin in a humidified 37°C incubator with 5% CO 2 . For adhesion assays using mouse bEnd5 endothelial cell line, the cells were seeded on 48-well plates and allowed to grow for 2 days until they reached confluence.
They were stimulated overnight with the ligands, prior to the addition of labelled mononuclear cells. In some experiments, endothelial cells were preincubated for 30 minutes in the presence or absence of inhibitors dissolved in DMSO and then the ligands or medium only (control) were added. Then, the adhesion assay was performed as described above.

| PCR expression analysis
Total RNA was extracted using the NucleoSpin RNAkit (Macherery-Nagel) and first strand cDNA synthesis and real-time PCR was performed as previously described. 21 Gene expression levels were  Tables S1 and S2.

| Statistical analysis
The data are presented as mean ± SEM using GraphPad Prism. Data were analysed using the nonparametric Kruskal-Wallis test with Dunn for the comparison of more than two groups. For the comparison of two groups with parametric distribution of the data the t test and for nonparametric distributed data the unpaired t test or Wilcoxon signed-rank test was used. The generalized linear mixed model (GLMM) was used for the analysis of the results from the migration assays with monocytes from patients. A probability (P-) value of <0.05 was considered statistically significant. P values: *P < 0.05, **P < 0.01, ***P < 0.001.

| BMP-2 induces mononuclear cell chemotaxis
BMP-2 can induce migration of various cells. 22 To analyse the effects of BMP-2 on mononuclear cell movement, we performed chemotaxis assays. Our results demonstrated that BMP-2 induces monocyte chemotaxis in a dose-dependent manner ( Figure 1A). The effects of BMP-2 were neutralized by addition of noggin, a natural antagonist of BMP-2/4/7 5 , which binds to BMPs and inhibits the binding to their receptors. In conclusion, our results suggest that BMP-2 acts as a potent monocyte chemoattractant.
To characterize the role of endogenous BMP-2/4 signalling on mononuclear cell migration, monocytes were treated with noggin or the BMP type I receptors (ALK2 and ALK3) LDN193189 prior to the migration assay ( Figure 1B).  Figure 1D). Treatment of monocytes with a p38 kinase inhibitor prior to the chemotaxis assay inhibited both basal and BMP-2induced monocyte chemotaxis ( Figure 1D). Finally, although inhibition of the ERK1/2 pathway did not affect basal monocyte motility, it interfered with BMP-2-induced monocyte migration ( Figure 1E).

| T2DM induces BMP-2 expression but it does not affect BMP-2-induced monocyte migration
Increased BMP-2 levels are associated with atherosclerosis in T2DM patients. 15 T2DM is a cardiovascular risk factor, which results in monocyte dysfunction 3,4 ). We have analysed BMP-2 mRNA expression in monocytes from non-T2DM (nT2DM) and T2DM patients.
Our results show that BMP-2 gene expression is increased in mono-

M2 macrophages
After entering a tissue monocytes differentiate into macrophages. 23 M1 macrophages promote inflammatory responses, while M2 macrophages are anti-inflammatory and play an important role in resolution of inflammation, tissue repair and wound healing. 24 To characterize the effects of BMP-2 on monocyte differentiation into macrophages, monocytes were differentiated into macrophages and then in M1 or M2 macrophages in the presence or absence of BMP-2 ( Figure 3A).