L-arginine and arginine ethyl ester enhance proliferation of endothelial cells and preadipocytes – How an arginine ethyl ester-releasing biomaterial could support endothelial cell growth in tissue engineering

Abstract

Adipose tissue engineering is a promising solution for the reconstruction of soft tissue defects. An insufficient neovascularisation within the scaffolds that leads to necrosis and tissue loss is still a major shortcoming of current tissue engineering attempts. Biomaterials, which release angiogenic factors such as L-arginine, could overcome this challenge by supporting the neovascularisation of the constructs. L-arginine is insoluble in organic solvents and thus cannot be incorporated into commonly used polymers in contrast to its ethyl ester. Here, we compared the effects of arginine and its ethyl ester on endothelial cells and preadipocytes, and generated an arginine ethyl ester-releasing, angiogenic polymer. We cultivated adipose tissue-derived endothelial cells and preadipocytes in arginine-free medium supplemented with L-arginine or L-arginine ethyl ester and assayed the proliferation rate and the degree of adipogenic differentiation, respectively. Additionally, we prepared arginine ethyl ester-releasing poly(D,L-lactide) foils, and investigated their impact on endothelial cell proliferation. We could demonstrate that arginine ethyl ester like arginine significantly increased the proliferation of endothelial cells and preadipocytes without inhibiting an induced adipogenic conversion of the preadipocytes. Further, we could show that the arginine ethyl ester-releasing polymer significantly increased endothelial cell growth. The present data are helpful guidance for generating angiogenic biomaterials that promote endothelial cell growth, and thereby could support neovascularisation within tissue engineering approaches.

Keywords

Arginine arginine ethyl ester endothelial cells preadipocytes angiogenesis

1. Introduction

Current applications for the reconstruction of soft tissue defects that occur after deep burns or tumour resections remain disappointing and confirm the need to identify new approaches [1]. The in vitro generation of fat tissue appears promising to overcome the present limitations of reconstructive surgery. Adipose-derived stem cells, also termed preadipocytes, emerged as an ideal cell type for tissue engineering. These cells exhibit a high proliferation and differentiation capacity in vitro and have the advantage of being more tolerant to ischemia compared to mature adipocytes [2,3]. However, a major shortcoming of current tissue engineering attempts is the absence of neovascularisation within the cell-seeded scaffolds leading to cell necrosis and tissue loss. Coculturing of preadipocytes with endothelial cells (ECs) could promote the formation of new vessels within the growing cell conglomerate. Springhorn et al. established a method for the isolation of microvascular ECs (MVECs) from adipose tissue by applying a CD31 antibody conjugated to microbeads [4]. We suggest that these adipose tissue-derived MVECs are the appropriate cell type for supporting neovascularisation in adipose tissue engineering. Promoting a sufficient proliferation of these cells by disposing angiogenic factors into the scaffold represents a promising approach. It has been shown that nitric oxide (NO) plays a key role in promoting angiogenesis, the process of new vessel generation leading to neovascularisation [5–7]. Vascular endothelial growth factor (VEGF), an EC-specific mitogen [8], and NO have been demonstrated to reciprocally enhance their syntheses [9–11]. Jozkowicz and colleagues found that an increase in NO production was involved in VEGF-induced EC proliferation, whereas decreased NO levels resulted in an impairment of several endothelial functions [12]. L-arginine (Arg), the physiological precursor of NO in vascular ECs [13,14], appears as a promising angiogenic candidate for tissue engineering. It has been shown that poly(D,L-lactide) represents an appropriate biomaterial for the cultivation of preadipocytes as well as for endothelial cells [15,16]. However, Arg is insoluble in organic solvents and thus cannot be incorporated into commonly used biomaterial polymers like PDLLA. Here, we established a method to incorporate the ethyl ester of Arg (ArgOET) into PDLLA and investigated whether ArgOET has the same angiogenic properties as Arg. For this, we compared the impact of ArgOET with Arg on the proliferation of human adipose tissue-derived MVECs. Additionally, we tested the influence of these substances on preadipocyte proliferation and differentiation. Further, we prepared ArgOET-releasing PDLLA (ArgOET-PDLLA) and tested its potential as an angiogenic biomaterial.

2. Materials and methods

2.1. Materials

Collagenase solution type I, PBS, fetal calf serum (FCS) and DMEM mod. (w/o arginine, w/o ferric nitrate) were purchased from Biochrom (Berlin, Germany). Trypsin and DMEM/Ham’s F12 were from PAA Laboratories (Colbe, Germany). Endothelial Cell Growth Medium was received from PromoCell GmbH (Heidelberg, Germany). L-arginine monohydrochloride was purchased from Merck KGaA (Darmstadt, Germany), L-arginine ethyl ester dihydrochloride, triiodo-L-thyronine, transferrin, dexamethasone, basal fibroblast growth factor (bFGF), isobutylmethylxanthine (IBMX) and Oil Red O were from Sigma (Deisenhofen, Germany). All other materials were of best quality and purchased from diverse conventional suppliers.

2.2. Preparation of ArgOET-releasing PDLLA (ArgOET-PDLLA)

PDLLA foils containing 5% (m/m) L-arginine ethyl ester dihydrochloride were prepared from a solution of 4.75% (m/m) poly(D,L-lactide) (Resomer R 208, Boehringer Ingelheim, Germany) and 0.25% (m/m) L-arginine ethyl ester dihydrochloride in a $3 : 1$ (v/v) mixture of chloroform and methanol. 30 g each of this solution were cast into PTFE dishes ( $d = 75 mm$ ). After drying at room temperature and removal of residual solvent in vacuum, the foils were cut into pieces of ( $8.25 \pm 0.16$ ) mg corresponding to a loading of ( $1.50 \pm 0.16$ ) µmol L-arginine ethyl ester dihydrochloride per piece.

2.3. Measurement of ArgOET release from ArgOET-PDLLA

For each measurement, one ArgOET-PDLLA piece of ( $8.25 \pm 0.16$ ) mg was incubated in 500 µl DMEM mod. (referred to as arginine-free medium (AFM)) at 37°C and 5% CO₂. After 1, 2, 3, 4, 5, 6, 24, 48, 72 h, the ArgOET concentration was determined in triplicates using the L-Arginin ELISA Kit (Immundiagnostik AG, Germany). Applicability of the kit for the detection of L-arginine ethyl ester was tested (data not shown).

2.4. Isolation of stromal vascular fraction (SVF) cells

The SVF was isolated from freshly excised human subcutaneous adipose tissue of male and female patients (mean age: ( $44.4 \pm 14.8$ ) years, BMI: ( $27.5 \pm 4.2$ ) kg/m²) who underwent elective operations (e.g. abdominoplasty) at the Department of Plastic Surgery and Hand Surgery – Burn Center of the University Hospital Aachen. The local Ethical Committee gave ethical approval for this study, and all patients provided informed consent. Fibrous tissue and visible blood vessels were removed from adipose tissue, before it was minced and digested by collagenase solution type I (0.2%; ratio tissue to enzyme was $1 : 1$ ) at 37°C for 45 min with constant shaking. After filtration through a 250 µm nylon sieve (Verseidag Techfab, Geldern, Germany), the fat layer was removed and the cell suspension centrifuged at 300 g for 10 min. After resuspension of the pelleted SVF in DMEM/Ham’s F12 supplemented with 10% FCS and 10 ng/ml bFGF (referred to as preadipocyte proliferation medium, PPM), cells were seeded and cultured at 37°C and 5% CO₂. Subconfluent SVF cells were trypsinised and selected using the CD31 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. The CD31⁻ cell fraction was designated preadipocytes, the CD31⁺ fraction microvascular endothelial cells (MVEC).

2.5. Determination of proliferation

2000 preadipocytes or MVECs were plated in 24-well plates and cultured in PPM and Endothelial Cell Growth Medium (referred to as ECM), respectively. After 24 h medium was changed to AFM (consisting of DMEM mod. supplemented with 2.5% FCS) and 25, 100 and 500 µg/ml L-arginine monohydrochloride (Arg) and L-arginine ethyl ester dihydrochloride (ArgOET), respectively, was added. Proliferation was assayed at day 1, 3, 5, 7 and 9 using the alamarBlue reagent from Invitrogen (Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, cells were incubated with 10% (v/v) alamarBlue for 2 h, and then fluorescence was measured at 560 nm with a FLUOstar Optima microplate reader (BMG LABTECH GmbH, Offenburg, Germany). The fluorescence is proportional to number of living cells. Values on day 1 with AFM were set to 100%. Additionally, 2000 MVECs were cultured in AFM, incubated with the prepared ArgOET-PDLLA (one foil of ( $8.25 \pm 0.16$ ) mg per 500 µl AFM) and proliferation was assayed for three days as described above.

2.6. Determination of adipogenic conversion

Preadipocytes were plated in 6-well plates at confluence and cultured in PPM for 24 h. Adipogenic conversion was promoted by changing the medium to DMEM/Ham’s F12 supplemented with 66 nM insulin, 1 µM dexamethasone, 0.5 mM IBMX, 0.1 µg/ml pioglitazone, 1 nM triiodo-L-thyronine and 10 µg/ml transferrin. 500 µg/ml Arg or ArgOET were added at day 1, 3 and 5 of differentiation. On day 6 the medium was used as before but without IBMX and pioglitazone. After 14 days degree of differentiation was assayed using Oil Red O staining and subsequent spectrophotometric quantification. Briefly, cells were fixed with 4% paraformaldehyde and incubated in Oil Red O staining solution for 15 min. Oil Red O was eluted with 100% isopropanol and OD was measured at 500 nm with a FLUOstar Optima microplate reader (BMG LABTECH GmbH, Offenburg, Germany). Value of standard differentiation was set to 100%.

2.7. Statistical analysis

One-way ANOVA with Bonferroni’s post test was performed using GraphPad Prism version 5.0c (GraphPad Software, La Jolla, USA). Data are given as arithmetical means ± SEM. Given p values are compared to control. When p values were less than 0.05, differences were considered to be statistically significant.

3. Results

3.1. Effect of Arg and ArgOET on MVEC proliferation

To investigate the impact of Arg and ArgOET on proliferation we incubated primary human adipose tissue-derived MVECs in arginine-free medium (AFM) supplemented with different concentrations of Arg or ArgOET and assayed proliferation every other day. Values on day 1 were set to 100% for each individual experiment and proliferation rates were then calculated relative to day 1 (Fig. 1(A)). As expected, the untreated control demonstrated a low proliferation rate of $332.7 \pm 17.1 %$ at day 9. Application of Arg and ArgOET, respectively, increased MVEC proliferation at all tested concentrations approx. 2-fold compared to untreated control. Differences between control and treated cells were significant at day 7 ( $p < 0.05$ ) and day 9 ( $p < 0.01$ ). Despite the fact that proliferation levels were higher with ArgOET than with Arg, the differences were not statistically significant, as well as the differences between the concentrations tested.

Fig. 1.

Effect of L-arginine (Arg) and L-arginine ethyl ester (ArgOET) on the (A) proliferation of MVECs, and the (B) proliferation and (C) differentiation of preadipocytes. (A) MVECs were cultured in arginine-free medium (AFM) for 9 days in the absence (control) or presence of Arg or ArgOET at concentrations indicated. Proliferation was evaluated every other day by alamarBlue assay. Data represent means ± SEM of 7 independent experiments, and are demonstrated relative to control at day 1, which is set to 100%. (B) Preadipocytes were cultured in arginine-free medium (AFM) for 9 days in the absence (control) or presence of Arg or ArgOET at concentrations indicated. Proliferation was evaluated every other day by alamarBlue assay. Data represent means ± SEM of 4 independent experiments, and are demonstrated relative to control at day 1, which is set to 100%. (C) Preadipocytes were cultured in standard differentiation medium for 14 days in the absence (control) or presence of 500 µg/ml Arg or ArgOET. Differentiation was evaluated with Oil Red O staining and elution assay. Data represent means ± SEM of 3 independent experiments, and are demonstrated relative to control, which is set to 100%.

3.2. Effect of Arg and ArgOET on preadipocyte proliferation

To test whether Arg and ArgOET, respectively, have the same mitogenic effect on primary human preadipocytes, cells were cultivated as described above. As shown in Fig. 1(B), preadipocytes demonstrated a low proliferation rate in AFM similar to that of MVECs ( $354.5 \pm 38.7 %$ at day 9). Accordingly, all tested concentrations of Arg and ArgOET increased preadipocyte proliferation approx. 2-fold compared to untreated control ( $p < 0.05$ at day 7 and $p < 0.01$ at day 9). Differences in proliferation rates between concentrations tested and between Arg and ArgOET treatment were not statistically significant.

3.3. Effect of Arg and ArgOET on adipogenic conversion

An adequate adipogenic differentiation of the preadipocytes is crucial for successful adipose tissue engineering. To test the influence of Arg and ArgOET on preadipocyte differentiation the cells were cultured in standard differentiation medium and 500 µg/ml Arg and ArgOET, respectively, were added. As demonstrated in Fig. 1(C), there was no difference in the degree of differentiation between control in standard differentiation medium and application of Arg. ArgOET slightly increased preadipocyte differentiation to $120 \pm 15 %$ compared to untreated control but differences were not statistically significant.

3.4. Effect of ArgOET-releasing PDLLA on MVEC proliferation

Based on the promising results with ArgOET as a potential mitogenic factor for MVECs and preadipocytes, we generated ArgOET-releasing PDLLA foils. The maximum concentration that could be achieved in each sample of 500 µl was $1.50 \pm 0.16 µmol$ , i.e. approx. 600 µg/ml ArgOET. As shown in Fig. 2(A), the PDLLA foils released ArgOET continuously over time. After 72 h, the ArgOET concentration in the samples was $484.0 \pm 32.8 µg/ml$ corresponding to approx. 81% of total ArgOET loading. To test the potential of ArgOET-PDLLA as an EC growth promoting biomaterial, MVECs were cultivated in 500 µl AFM and one piece of the ArgOET-PDLLA foil per well was added to the medium. As demonstrated in Fig. 2(B), incubation of MVECs with ArgOET-PDLLA for 3 days increased MVEC growth more than 2-fold compared to control in AFM ( $275 \pm 32 %$ and $123 \pm 9 %$ , respectively; $p < 0.001$ ).

Fig. 2.

ArgOET-PDLLA. (A) ArgOET release over time and (B) effect on MVEC proliferation. (A) For each measurement, one ArgOET-PDLLA foil of $8.25 \pm 0.16 mg$ was incubated in 500 µl arginine-free medium (AFM) and ArgOET concentration was measured using the L-arginine ELISA Kit. Data represent means ± SEM of 3 independent experiments. (B) MVECs were cultured in 500 µl AFM for 3 days in the absence (control) or presence of $8.25 \pm 0.16 mg$ ArgOET-PDLLA. Proliferation was evaluated every day by alamarBlue assay. Data represent means ± SEM of 4 independent experiments, and are demonstrated relative to control at 24 h, which is set to 100%.

4. Discussion

The achievement of an adequate neovascularisation within a cell-seeded scaffold poses a huge challenge and represents a general shortcoming of current tissue engineering attempts. Thus, innovative methods are required to promote an appropriate neovascularisation within the constructs to avoid necrosis and tissue loss. A promising approach is the coculture of the target tissue-derived precursor cells with ECs, which could provide the basis for the formation of new blood vessels. A prerequisite for this is to promote an appropriate proliferation of the ECs within the scaffold without inhibiting the proliferation and differentiation of the target precursor cells. Since NO has been proven to promote migration, proliferation and survival of ECs [17] we here investigated the impact of Arg, the physiological precursor of NO, on primary human adipose tissue-derived MVECs and preadipocytes.

We could demonstrate that Arg significantly enhanced the proliferation of primary human MVECs compared to control, without further supplement of EC-typical growth factors like EGF and VEGF. An important and new finding is that Arg had the same mitogenic effect on primary human preadipocytes. Additionally, we could show that Arg did not impair an induced adipogenic conversion of the precursor cells, a prerequisite for successful adipose tissue engineering. In contrast to free Arg, the ethyl ester of the amino acid can be incorporated into commonly used biocompatible polymers, a precondition for the generation of drug-releasing scaffolds. Therefore, we compared the effects of ArgOET with that of Arg. We could show that the ethyl ester of Arg had the same positive impact on the proliferation of human AT-derived MVECs and preadipocytes as Arg itself. Further, ArgOET, like Arg, did not impair the differentiation of preadipocytes. Based on these results, we generated ArgOET-releasing PDLLA as an angiogenic biomaterial. We could show that these PDLLA foils continuously released ArgOET over time and significantly enhanced MVEC proliferation.

Current studies revealed that adipogenesis and angiogenesis mutually enhance each other. Fukumura et al. demonstrated that angiogenesis is required for sufficient adipogenesis; vice versa an efficient neovascularisation is triggered by adipogenic conversion [18]. In accordance, the application of several angiogenesis inhibitors significantly decreased body weight and adipose tissue mass in mice [19]. Nakagami and colleagues found that autologous transplantation of adipose tissue-derived cells resulted in an increase in the capillary density in the murine ischemic hindlimb model [20]. Accordingly, the introduction of fat into the rabbit cornea induced neovascularisation [21]. In an in vitro model of adipose tissue development it has been shown that during differentiation the increase of adipogenic marker gene expression was accompanied by an increase in the expression of angiogenic marker genes [22]. The background for this reciprocal regulation of angiogenesis and adipogenesis is the secretion of angiogenic factors from pre/adipocytes as well as the release of adipogenic mediators from ECs. In this context, Kern and colleagues showed that cultured adipose tissue-derived MVECs secreted IGF and IGF-binding protein to the medium that bind to adipocytes resulting in an increase of lipoprotein lipase activity [23]. Varzaneh et al. demonstrated that MVECs isolated from adipose tissue secreted extracellular matrix components that stimulate preadipocyte differentiation [24], whereas Hutley et al. found that MVEC-conditioned media induced the proliferation of preadipocytes [25]. On the other hand, leptin and insulin, mediators particularly secreted by adipocytes, influence angiogenesis. Holmang et al. found that a moderate hyperinsulinaemia resulted in a strong enhancement of capillary formation in muscles of rats [26]. In accordance, Sierra-Honigmann and colleagues showed that leptin had a mitogenic effect on several human EC types, and that leptin application resulted in an intensive angiogenic response in rats [27]. Further, it has been demonstrated that preadipocytes secrete several angiogenic GFs, and reduce apoptosis in ECs [28].

In conclusion, the present data are helpful guidance for generating angiogenic biomaterials that could promote ECs growth, and thereby support neovascularisation within tissue engineering constructs. The next step towards successful adipose tissue engineering applications will be to load ArgOET-releasing biomaterials with factors that further promote adipogenesis. By designing such a combined construct with stronger differentiation and vascularisation potential, new generation tissue engineering models will have more freedom to expand in size.

Footnotes

Acknowledgement

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich Transregio 37.

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