Transforming growth factor‐β1 signalling triggers vascular endothelial growth factor resistance and monocyte dysfunction in type 2 diabetes mellitus

Abstract Type 2 diabetes mellitus (T2DM) leads to monocyte dysfunction associated with atherogenesis and defective arteriogenesis. Transforming growth factor (TGF)‐β1, placenta growth factor (PlGF)‐1 and vascular endothelial growth factor (VEGF)A play important roles in atherogenesis and arteriogenesis. VEGF‐receptor (VEGFR)‐mediated monocyte migration is inhibited in T2DM (VEGFA resistance), while TGF‐β1‐induced monocyte migration is fully functional. Therefore, we hypothesize that TGF‐β antagonises the VEGFA responses in human monocytes. We demonstrate that monocytes from T2DM patients have an increased migratory response towards low concentrations of TGF‐β1, while PlGF‐1/VEGFA responses are mitigated. Mechanistically, this is due to increased expression of type II TGF‐β receptor in monocytes under high‐glucose conditions and increased expression of soluble (s)VEGFR1, which is known to interfere with VEGFA signalling. VEGFA resistance in monocytes from T2DM patients can be rescued by either experimental down‐regulation of TGF‐β receptor expression in vitro or by functional blocking of TGF‐β signalling using either a TGF‐β receptor kinase inhibitor or a TGF‐β neutralizing antibody. Our data demonstrate that both T2DM and high‐glucose potentiate the TGF‐β pathway. TGF‐β signalling impairs VEGFR‐mediated responses in T2DM monocytes and in this way contributes to mononuclear cell dysfunction, provide novel insights into T2DM vascular dysfunction.

has been shown that monocytes from T2DM patients display vascular endothelial growth factor (VEGF)A impaired function, 6 since they show a reduced migratory response towards VEGFA and placental growth factor (PlGF)-1, earlier described as VEGFA resistance. 6,7 Considering the important role of monocytes in arteriogenesis it has been suggested that the decreased formation of collateral vessels in patients with T2DM may be due to an impaired migratory response of monocytes to VEGFA. 7 Reduced VEGFA responses were shown to be due to increased baseline activity of signalling cascades such as phosphoinositide 3-kinase (PI3K), extracellular-signal regulated Kinases (ERK) p44/42 mitogen-activated protein kinases (MAPK) and p38 MAPK. 6 Although some of the underlying molecular mechanisms of VEGFA resistance have been characterised, 6 the exact basis of this remains largely unclear.
Transforming growth factor (TGF)-β1 is the prototype member of the TGFβ family of cytokines, which play a crucial role in embryonic development and adult tissue homeostasis. 8,9 TGFβ signals via heterotetrameric receptor complexes consisting of type I and type II receptors. Upon ligand binding, the type II receptor phosphorylates and activates the type I receptor which propagates the signal into the cells by activating the SMAD proteins which will regulate gene transcription. In addition to the SMAD pathway, TGFβ binding to its receptors leads to the activation of the non-SMAD pathways in a cell-dependent manner. 8 TGFβ signalling plays an important role in angiogenesis and arteriogenesis and perturbation of the TGFβ pathway leads to (cardio)vascular abnormalities. 9 Several studies have provided evidence suggesting that the cross-talk of VEGFA and TGFβ signalling pathways play an important role in angiogenesis. 8,10 However, there are no studies on the interplay between the TGFβ and VEGFA signalling pathways in monocyte function. Interestingly, it has been shown, that the level of TGF-β1 is elevated in the serum of patients with T2DM 11 and in the tissue of diabetic mice models. [12][13][14][15] We have shown previously that despite the fact that monocytes from T2DM patients do not respond to VEGFA and PlGF-1-induced chemotaxis, their chemotaxis towards TGF-β1 is fully functional. 16 We hypothesized that the TGFβ pathway interferes with the VEGF pathway in human monocytes contributing to VEGFA resistance.
Here, we show that TGF-β1 and VEGFA/PlGF-1 signalling do antagonize each other in inducing monocyte migration. High-glucose levels enhance TGFβ responses and by this contribute to mononuclear cell dysfunction and reduced VEGFA-responsiveness.

| Cells and reagents
The experiments were performed with peripheral blood human CD14 ++ CD16monocytes isolated from healthy volunteers and patients. The cells were cultured in RPMI 1640 GlutaMAX (Gibco) or RPMI without glucose (Gibco) supplemented with 5 mmol/L glucose (Sigma-Aldrich) and 15 mmol/L mannitol (Sigma-Aldrich) (normal glucose, NG) or 20 mmol/L glucose (high glucose, HG). The medium was supplemented with 10% foetal bovine serum (FBS) (Sigma-Aldrich) and 1% Penicillin Streptomycin (Gibco). Human VEGFA and PlGF-1 were obtained from Reliatech and TGF-β1 from Peprotech. TGF-β1 kinase receptor inhibitor LY-364947 was obtained from Tocris Bioscience, DMSO from CalBiochem. The IgG2B Isotype antibody was obtained from R&D system, whereas the TGFβ neutralizing antibody (a kind gift of Dr E. de Heer, Leiden University Medical Center) was isolated from the 2G7 hybridoma as described before. 17

| Characterization of healthy volunteers and patients
The present study conforms to the principles of the Declaration of  Table 1. Monocytes were also isolated from human blood leukocyte reduction chambers from healthy subjects recruited by the blood bank of the University Hospital Münster.

| Isolation and chemotaxis assays of CD14 ++ CD16monocytes
Monocytes were isolated as described before. 18 In brief, blood mononuclear cells were purified via density centrifugation.
CD14 ++ CD16monocytes were isolated from the mononuclear cell fraction with the MACS ® monocyte isolation kit II (Miltenyi).
The migratory response of the cells towards different ligands was analysed by using a modified Boyden chamber (Neuroprobe) chemotaxis assay as described previously. 16

| FACS analysis
Surface expression of the VEGFR1 on CD14 ++ CD16monocytes was determined by flow cytometric analysis using a FACSCalibur (BD). The relative fluorescence intensity (RFI) was calculated by dividing the mean of VEGFR1 stained cells with the mean of isotype-stained cells from all samples and used for further analysis.

| Protein analysis
Monocytes were cultured in different media with normal or high-glucose conditions as indicated. Thereafter, cells were processed to lysates and subjected to western blot analysis as described previously. 18 The intensity of the detected bands was quantified using ImageJ. The ratio of the intensity of the analysed protein to the intensity of detected actin for the corresponding sample was calculated and the values were normalized to control set as 1.

| Zymography
Matrix metalloproteinase (MMP)-2 and MMP-9 activity was determined by gelatin zymography. Condition medium was mixed with Tris-Glycine SDS Native sample buffer and loaded/run on a zymogram Tris-Glycine gelatin gel. 19 After the separation the gel was washed for 30 minutes with 2,5% Triton and then incubated overnight in Brij solution (16 hours) at 37°C. Gels were stained with Coomassie blue for 60 minutes and after distaining gels were imaged. The band intensity was calculated with ImageJ.

| ELISA assay
One millilitre of plasma was collected by centrifugation of 3 mL of patient whole blood sample containing 20 U/mL heparine (Ratiopharm). The total amount of TGF-β1 in the plasma was meas-

| Quantitative real-time PCR (qRT-PCR)
RNA isolation was performed with the NucleoSpin ® RNAII Kit  Table 1  used for the analysis of the results from the chemotaxis assays with monocytes from patients. Differences with values P < .05 were considered significant. *P < .05 **P < .01 ***P < .001.

| T2DM enhances monocyte migratory responses towards low concentrations of a TGF-β1 gradient
To characterize the molecular mechanisms of monocyte dysfunction and VEGFA resistance in T2DM we analysed the migratory response of CD14 ++ CD16monocytes isolated from subjects without T2DM and patients with T2DM towards PlGF-1 and TGF-β1. The patient characteristics are shown in Table 1. Although the migratory response of monocytes from patients with T2DM towards PlGF-1 was impaired ( Figure 1B), their migration towards TGF-β1 was not affected ( Figure 1A). Interestingly, monocyte migration towards low concentrations of TGF-β1 (1 ng/mL) was significantly increased only in T2DM patients compared to non-T2DM patients ( Figure 1A). Analysis of the total levels of TGF-β1 in the plasma of both patient groups using a TGFβ ELISA suggested that there are no differences in the concentration of total TGF-β1 ( Figure 3C).

| Interference with the TGFβ signalling rescues the impaired PlGF-1-induced migratory response of monocytic cells in highglucose conditions
To determine whether enhanced TGFβ signalling has a functional role in the impaired PlGF-1-induced monocyte migration in ( Figure 6A). Importantly, the TGFβ neutralizing antibody rescued the impaired PlGF-1-induced migration of monocytes from patients with T2DM ( Figure 6B). These results illustrate that inhibition of endogenous TGFβ and autocrine TGFβ signalling in monocytes from T2DM patients can fully restore their migratory response towards PlGF-1.

| D ISCUSS I ON
Monocytes from T2DM patients have an impaired migratory response towards VEGFA and PlGF-1 (ie VEGFA resistance). 6 To our knowledge, this is the first study to demonstrate that the TGFβ pathway contributes to VEGFA resistance and monocyte dysfunction in T2DM.
Our data strongly indicate that high glucose and T2DM lead to enhanced TGFβ signalling in human monocytes possibly by inducing the expression of the TβRII and TGF-β1, as suggested by our in vitro data. In line with our results, it was shown that the TGF-β1 levels are elevated in the serum of T2DM patients, 11 and in tissue of diabetic mice. 12,13 It was also shown that high glucose induces TGFβ in podocytes, 13 and in mesangial cells. 20 In contrast to the results from our TGF-β1 signalling by blocking the binding of TGF-β1 to TβRII. 28 Interestingly, metformin was shown to reduce sVEGFR1 secretion from primary human tissues. 29 In another study it was shown that inhibition of the TGFβ/Smad3 pathway in mice protected against diabetes mellitus during high-fat-induced obesity. 30

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding authors upon reasonable request.