4-Hexylresorcinol induced angiogenesis potential in human endothelial cells

4-Hexylresorcinol (4HR) is able to increase angiogenesis. However, its molecular mechanism in the human endothelial cells has not been clarified. As endothelial cells are important in angiogenesis, we treated the human umbilical vein endothelial cells (HUVECs) with 4HR and investigated protein expressional changes by immunoprecipitation high-performance liquid chromatography (IP-HPLC) using 96 antisera. Here, we found that 4HR upregulated transforming growth factor-β (TGF-β)/SMAD/vascular endothelial growth factor (VEGF) signaling, RAF-B/ERK and p38 signaling, and M2 macrophage polarization pathways. 4HR also increased expression of caspases and subsequent cellular apoptosis. Mechanistically, 4HR increased TGF-β1 production and subsequent activation of SMADs/VEGFs, RAF-B/ERK and p38 signaling, and M2 macrophage polarization. Collectively, 4HR activates TGF-β/SMAD/VEGF signaling in endothelial cells and induced vascular regeneration and remodeling for wound healing.

Background 4-Hexylresorcinol (4HR) is a substituted phenol that is synthesized from resorcinol and caproic acid [1]. It is used as an antimicrobial in tooth pastes and skin lotions [2] and as a preservative for fresh fruits and vegetables [3]. It has bactericidal [4], anthelmintic [5], and potential antineoplastic activities [6], and thus, it is also used as an antiseptic in mouthwashes and skin wound cleansers [7]. 4HR may also inhibit oxidative DNA damage by enhancing the activities of antioxidant enzymes, including glutathione peroxidase and glutathione reductase, which facilitate the scavenging reactive oxygen species by glutathione [8], and thus, it is also used to prevent the enzymatic browning of shrimps and different fruits [9].
A recent study demonstrated that 4HR increases the expression level of vascular endothelial growth factor (VEGF) in RAW264.7 cells and angiogenesis in the animal model [10]. 4HR increases M2 markers, and broadspectrum matrix metalloproteinase (MMP) inhibitor (PD166793) can reduce 4HR-induced VEGF expression. However, MMPs are also highly expressed in the inflammatory phase, and the expression of MMPs is mostly regulated by hypoxic stress [11]. Interestingly, the action of PD166793 is mediated by chelating zinc ion [12]. Accordingly, zinc-dependent protein like transforming growth factor-β1 (TGF-β1) may be regulated by 4HR and induce VEGF and angiogenesis.
Immunoprecipitation high-performance liquid chromatography (IP-HPLC) had been used previously by several authors to detect organic compounds quantitatively, including peptides, but the technique used was complicated and of limited applicability [13,14]. Recently, a new IP-HPLC protocol was developed to determine protein expression levels in different biological fluids, such as blood serum, urine, saliva [15], inflammatory exudates [16][17][18], and different protein extracts from cells [19][20][21], liver [22], and cancer tissues [21]. Recent IP-HPLC results demonstrate that 4HR administration increases the expression of TGF-β1 in the osteoblast-like cells [23]. IP-HPLC is comparable to enzyme-linked immunosorbent assay (ELISA), but the former uses protein A/G agarose beads in buffer solution and ultraviolet spectroscopy to determine protein concentrations, whereas the latter uses fluorescence-conjugated antibodies fixed in plastic wells and fluoroscopy. Furthermore, multiple trials have shown that IP-HPLC can be used to rapidly determine multiple protein levels accurately (± 5% standard deviation) and reproducibly.
In this study, differentially expressed proteins by 4HR were screened by IP-HPLC in a human endothelial cell line (human umbilical vein endothelial cells [HUVECs]) using our antibody library. IP-HPLC results demonstrated that TGF-β1 played a key role in 4HR-induced activation of angiogenesis-associated signal pathway in HUVEC cells. To confirm this hypothesis, additional western blotting was done with TGF-β1 and its signal blocker.
About 70% confluent HUVECs grown on Petri dish surfaces were treated with 10 μg/mL 4HR (with a single dose given safely given in dog; 100-300 mg/kg, WHO food additives Series 35, 835) for 8, 16, or 24 h; control cells were treated with 1 mL of normal saline. Cultured cells were harvested with protein lysis buffer (PRO-PREP TM , iNtRON Biotechnology INC, Korea) and immediately preserved at − 70°C until required.
Briefly, protein samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, and 0.5% NP-40) and incubated in protein A/G agarose (Amicogen, Korea) columns on a rotating stirrer for 1 h at 4°C. After washing columns with PBS (phosphate-buffered saline solution), target proteins were eluted using 150 μL of IgG elution buffer (Pierce, USA). Immunoprecipitated proteins were analyzed using an HPLC unit (1100 series, Agilent, USA) equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, Korea). Elution was performed using 0.15M NaCl/20% acetonitrile solution at 0.4 mL/ min for 30 min, and proteins were detected using an ultraviolet spectrometer at 280 nm. Control and experimental samples were run sequentially to allow comparisons. For IP-HPLC, whole protein peak areas (mAU*s) were calculated after subtracting negative control antibody peak areas, and square roots of protein peak areas were calculated to normalize concentrations. Protein percentages in total proteins in experimental and control groups were plotted. Results were analyzed using the chi-squared test [19][20][21].

Statistical analysis
Proportional data (%) of the experimental and control groups were plotted into line graphs and star plots, and analyses were repeated two to six times until standard deviations were ≤ ± 5%. Line graphs revealed the similarities of the expression pattern between the relevant proteins, and star plots revealed the differences in the expression levels of the whole objective proteins. Results were analyzed using the chi-squared test. The expressions of control housekeeping proteins, that is, β-actin, α-tubulin, and GAPDH, were nonresponsive (≤ 5%) to 12, 24, or 48 h of 4HR treatment.

Effects of 4HR on the expressions of RAS signaling proteins in HUVECs
The expressions of RAS signaling proteins were variable in HUVECs treated with 4HR for 24 h. K-RAS expression gradually decreased by 16.2% at 24 h, H-RAS expression decreased by 9% at 8 h but increased by 3.7% at 24 h versus nontreated control, while N-RAS increased by 2% at 16 h and by 1.6% at 24 h. Downstream signal proteins SOS1/2 and STAT3 tended to be decreased by 11.3% and 5% at 16 h, respectively.
These results indicate that 4HR significantly suppressed NFkB signaling in HUVECs through upregulation of

Effects of 4HR on the expressions of apoptosis-related proteins in HUVECs
4HR affected the expressions of p53-mediated apoptosisrelated proteins, particularly p53 protein, which was decreased by 16.9% after treatment for 16 h as compared with nontreated controls, and decreased the expressions of proapoptotic proteins, BCL2-associated death promoter (BAD; 11.5% at 16 h), BCL2 homologous antagonist/killer (BAK; 10% at 24 h), apoptosis regulator BAX (11.5% at 24 h), and apoptotic protease activating factor 1 (APAF-1; 23.4% at 24 h) but increased the expression of B cell lymphoma 2 (BCL2; 11.4% at 8 h) and apoptosis inducing factor (AIF; 13.5% at 8 h). On the other hand, the expressions of apoptosis executor proteins such as caspase 9 and c-caspase 9 were increased by 13.9% at 24 h and by 19% at 16 h, respectively (Fig. 2B1, B2). These results indicate that 4HR activated caspase 9 and c-caspase 9 via AIF signaling in the lack of upregulation of pro-apoptotic factors, including BAD, BAK, BAX, and APAF-1. HUVECs treated with 4HR showed decreases in the expressions of FAS-mediated apoptosis signaling proteins as compared with nontreated controls, although they showed an increase in the expression of FAS ligand (FASL; 28.7% at 24 h). After treatment with 4HR for 24 h, the expression of death receptors on cell surfaces, that is, FAS, was decreased by 10.9% at 16 h and that of FAS-associated protein with death domain (FADD) was also decreased by 11.9% at 16 h, but FLICE-like inhibitory protein (FLIP) expression was increased by 29.7% at 24 h, whereas HUVECs treated with 4HR showed increases in the expressions of apoptosis executor proteins, c-caspase 8 (by 8.6% at 8 h), c-caspase 10 (18.9%   Fig. 2)B1, B2). These results indicate 4HR rarely produced single-strand DNA breaks, which require repair by PARP-1.

Effects of 4HR on the expressions of inflammatory proteins in HUVECs
4HR influenced the expressions of inflammatory proteins positively or negatively in HUVECs depending on the types of M1/M2 macrophage polarization. The proteins upregulated by 4HR usually belong to M2 macrophage polarization proteins, which were interleukin-10 (IL-10; 15 Fig. 3A1, A2).
These results indicate that HUVECs treated with 4HR for 16 h may have strong angiogenetic potential with concurrent elevation of TGF-β/SMAD signaling, RAF-B/ERK and p38 signaling, and M2 macrophage polarization and that 4HR-induced activation of caspases and subsequent cellular apoptosis in the reduction of NFkB signaling compensate by stimulating the expressions of TGF-βs in HUVECs.
4HR-treated HUVECs showed alternative cellular apoptosis caused by activation of different caspases (cleaved caspase-3, caspase-8, caspase-9, and caspase-10) despite the reduction of p53-and FAS-mediated pro-apoptotic signaling. Although the present study did not elucidate whether 4HR could damage the mitochondrial membrane, it was thought that the relatively innocuous 4HR, which did not elicit any oxidative stress in cells [19], produced abortive mitochondrial biogenesis by upregulation of PGC-1α and AIF but downregulation of AMPK (energy consumption) simultaneously in HUVECs resulting in alternative apoptosis by activation of caspases released from 4HR-involved mitochondria.
This 4HR-induced cellular apoptosis would be slowly progressed with no activation of NFkB signaling and compensate by stimulating TGF-β production in HUVECs. Actually, in the present study, 4HR-treated HUVECs showed dominant expressions of TGF-β1, TGF-β2, and TGF-β3 despite consistent downregulation of FGF-1, FGF-2, FGF-7, growth hormone, growth hormone releasing hormone, PDGF-A, and c-erbB-2 (HER2; some data not shown). The dominant expressions of TGF-β1, TGF-β2, and TGF-β3 were very characteristic in 4HR-treated HUVECs. However, when TGF-β ligands bind to TGF-β receptors (heteromeric complex of type I and type II TGF-β receptors), it is expected that the SMAD2/3/4 pathway is activated and undergoes target gene transcription such as VEGFs and BMPs and that RAF-B/ERK and p38 signaling are activated and crosstalk with TGF-β/SMAD signaling. These TGF-β signaling cascades were found as marked upregulation and activation of RAF-B, SMADs, ERK-1, p38, and VEGFs in the present 4HR-treated HUVECs. In addition, the administration of A83-01, a Smad pathway blocker, showed marked reduction of VEGF expression, which was expected to be increased by 4HR treatment.
In the previous study, we found that 4HR induced potent de novo angiogenesis in both in vitro and in vivo experiments [10]. 4HR treatment increased VEGF expression in RAW264.7 cells, and it is HIF independent. The present study explored the molecular mechanism of 4HR-induced angiogenesis in HUVECs and observed that 4HR-treated HUVECs showed dominant expressions of TGF-βs concurrently with upregulation of SMADs and VEGFs. In addition, it has been confirmed that TGF-β1 stimulates SMAD pathway and increases VEGF-A expression in in vitro culture of HUVECs. Therefore, it is suggested that 4HR-induced angiogenesis in HUVECs is characteristic with serial activation of cellular angiogenetic factors in the TGF-β/SMAD/VEGF pathways independent from the ordinary angiogenesis transcription factor (HIF-1α) and matrix angiogenetic factors (FGF-2, PDGF-A, MMP-2, and MMP-10).
On the other hand, 4HR-treated HUVECs expressed a higher level of M2 macrophage polarization proteins (cytokines) than nontreated controls and a lower level of M1 macrophage polarization proteins. The upregulation of M2 macrophage polarization cytokines might autonomously stimulate HUVECs to undergo cytological changes appropriate for angiogenesis, subsequently followed by HUVEC differentiation via TGF-β/SMAD/ VEGF signaling in vitro. Our previous study reported that 4HR induced a strong wound-healing effect with de novo angiogenesis associated with M2 macrophage infiltration in in vivo animal experiments [25,26]. In this study, however, the HUVEC culture contained no macrophages, so there was no cellular interaction between HUVECs and macrophages, resulting in a diminished angiogenetic effect of M2 macrophage polarization cytokines on HUVECs. Among 4HR-induced angiogenic effects, M2 macrophage polarization proteins will be more greatly amplified in in vivo animal experiments, where macrophages can be infiltrated, than in in vitro cell culture. In addition, 4HR can increase the expression level of M2 markers in RAW264.7 cells directly [10]. The global protein expression changes are illustrated in Fig. 4 using 51 representative proteins selected from 6 major molecular signaling pathways. It was found that 4HR-treated HUVECs showed concurrent upregulation of TGF-β/SMAD/VEGF signaling, RAF-B/ERK and p38 signaling, and M2 macrophage polarization and that 4HR-induced activation of caspases and subsequent cellular apoptosis were closely relevant to the overexpression of TGF-βs in HUVECs. If the protein expression patterns obtained from precision IP-HPLC analysis were similar to each other at their level (%) and time after 4HR treatment, the concurrent protein expression changes in the same functional groups may have implications for the signal transduction or cross-talk to achieve the final goals of objective proteins. Therefore, two major angiogenetic pathways induced by 4HR were identified from the above global protein expression data analyzed by IP-HPLC: the caspase activation/TGF-β/ SMAD/VEGF pathway and reduced NFkB signaling/upregulation of M2 macrophage polarization proteins/ endothelial cell differentiation in HUVECs (Fig. 5).
Angiogenesis is a vital step for uneventful wound healing. The materials inducing M2 macrophage polarization are required for angiogenesis and wound remodeling [27]. 4HR is an agent for M2 macrophage polarization [10]. In this study, 4HR increased the expression level of Fig. 5 Schematic drawings for the proposed mechanism. Apoptotic stress on the mitochondria is induced by application of 4HR. This stress induces TGF-β1 expression, and secreted TGF-β1 protein will bind to ALK5. Then, the downstream signal is generated by the RAS/Smads pathway. This signal will increase the expression of VEGFs proteins that are responsible for endothelial cell differentiation (Fig. 5). In future perspective, 4HR incorporating materials may be developed for the maxillofacial regeneration. Actually, 4HR incorporated xenograft has been shown reduced foreign body reaction [28] and accelerated degradation [29]. Bone grafts with 4HR suppress NFkB signaling and increase bone regeneration [30].

Conclusion
Collectively, 4HR-induced angiogenic factors (VEGFs) were controlled by TGF-β1 overexpression and subsequent activation of SMADs/VEGFs, RAF-B/ERK and p38 signaling, and M2 macrophage polarization. Therefore, it is assumed that 4HR activates TGF-β/SMAD/ VEGF signaling and induced vascular regeneration and remodeling for wound healing. In particular, the overexpression of TGF-βs in 4HR-treated HUVECs might be ascribed to the increase of apoptosis via FAS-mediated signaling, and the dominant TGF-β1 expression might induce the protein expressions of M2 macrophage polarization proteins, which subsequently stimulate wound-healing procedures.