Hesperidin Inhibits UVB-Induced VEGF Production and Angiogenesis via the Inhibition of PI3K/Akt Pathway in HR-1 Hairless Mice

Abstract

Hesperidin is a citrus flavanone glycoside with potent anti-inflammatory effects that interferes with UVB-stimulated angiogenesis in skin, but its molecular mechanisms of action remain unclear. Here, we investigated the effects of hesperidin on UVB-induced angiogenesis in HR-1 hairless mice. We found hesperidin treatment inhibited skin neovascularization skin induced by repetitive UVB light exposure. Exposure to UVB radiation induces the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-13 (MMP-13), and MMP-9, but we found all of these were inhibited by treatment with hesperidin. Using immunohistochemistry and Western blotting, we also found hesperidin inhibited the increase in hypoxia inducible factor-1 (HIF-1)α expression induced by UVB exposure. After discovering that UVB induces VEGF expression via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways, we found hesperidin reduces UVB-induced VEGF expression by inhibiting UVB-induced PI3K activity. This, in turn, reduces the UVB-induced Akt/p70S6K phosphorylation in human primary keratinocytes and fibroblast cells. Because it affects the mediators of angiogenesis, our data suggest hesperidin has an anti-angiogenic effect on the pathologic skin neovascularization induced by UVB light. Thus, hesperidin may prove useful in the treatment of skin injuries caused by UVB light exposure.

INTRODUCTION

Angiogenesis—the physiological process by which new capillaries are generated—is strongly associated with cancer but also with some pathological skin conditions.¹⁾ Normally tumors cannot escape the pre-vascular phase of cancer progression to grow beyond 1–2 mm in thickness without angiogenesis. With angiogenesis, however, cancers can develop far beyond this size and progress to invading other organs via metastasis.²⁾ This is why so many strategies for reducing angiogenesis are being evaluated in clinical trials. Some of these strategies attempt to interfere with tumor cell signaling pathways involved in releasing growth factors like vascular endothelial growth factor (VEGF) and proteolytic enzymes such as matrix metalloproteinases (MMPs), which are required for angiogenesis.³⁾ In transgenic mice, increased VEGF expression in the epidermis directly stimulates skin vascularization, increasing distorted and hyperpermeable blood vessels.⁴⁾ In addition, MMPs are associated with tissue invasion, cancer progression, and angiogenesis.⁵⁾ Recently, one study found exposure of skin to UVB light induces skin vascularization, while another study found that increased VEGF in hyperplastic epidermal cells increases dermal blood vessel formation.⁶⁾ Epidermal keratinocytes have also been implicated in the pathophysiology of cutaneous angiogenesis.⁷⁾ Although the precise molecular mechanisms by which UVB and skin damage induce angiogenesis remain unclear, we expect UVB-induced damage to keratinocytes induces angiogenesis via VEGF signaling. UVB-induced damage was already found to influence VEGF expression in human keratinocytes in several cutaneous diseases.⁸⁾ Moreover, UVB-induced changes in phosphoinositide 3-kinase (PI3K)/Akt signaling increase the expression of both VEGF and hypoxia inducible factor-1 (HIF-1α).^9,10) In addition to regulating its expression, activation of PI3K/Akt signaling also modulates the action of HIF-1α.¹¹⁾ These points are particularly important because HIF-1α itself regulates both VEGF and MMP expression, making it a central actor in both the response to hypoxia and an important regulator of angiogenesis.¹²⁾ Consequently, we expect pharmaceuticals developed to target PI3K/Akt signaling will suppress HIF-1α and angiogenesis and contribute significantly to the treatment of skin cancer.

Hesperidin is a flavanone glycoside from citrus fruits that has both antioxidant and anti-inflammatory characteristics.^13,14) Hesperidin suppresses the expression of protein kinase C, extracellular signaling-regulated kinases (ERKs), and c-Jun, thereby preventing the increase in MMP-2 activity induced by 12-O-tetradecanolyphorbol-13-acetate.¹⁵⁾ Yet, despite hesperidin’s anti-tumor effects, the detailed mechanisms by which it reduces angiogenesis remain unclear. In this study, we report on our discovery that hesperidin treatment inhibited UVB-induced skin vascularization in the HR-1 hairless mouse model. We found hesperidin suppressed UVB-induced PI3K/Akt, subsequently attenuating VEGF, MMP-9, and p70S6K expression in human primary skin cells.

MATERIALS AND METHODS

Chemicals and Reagents

Hesperidin was obtained from the Sigma-Aldrich Corporation. (St. Louis, MO, U.S.A.). LY294.002 was obtained from the Cayman Chemical Company (Ann Arbor, MI, U.S.A.). A Mineralight UV Display Lamp was acquired from UVP, LLC (Phoenix, AZ, U.S.A.). Antibodies specific for p-Akt (Ser 473) (#9271), Akt (#9272), p-PI3K (Tyr458) (#4228), PI3K (#4225), p-p70S6K (Thr389) (#9205), p70S6K (#9202), VEGF (#2463), β-actin (#4970) and MMP-13 (#94808) were acquired from Cell Signaling Technology (Beverly, MA, U.S.A.). Antibodies specific for p-mitogen-activated protein extracellular kinase (MEK) (Ser 218/222) (sc-271914), MEK (sc-365800), HIF-1 α (sc-13515), and MMP-9 (sc-13520) were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.). The CD31 (MA5-13188) antibody was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, U.S.A.).

Experimental Animals

Male HR-1 mice were obtained from Japan SLC, Inc. (Sizuoka, Japan) and housed at 24 °C and 50% humidity under a 12 h/12 h light/dark cycle in a climate-controlled room. Prior to the study, the mice were allowed to acclimate for 1 week with ad libitum access to food and water. All the experimental protocols were approved by the Korea Institute of Oriental Medicine Animal Care and Use Committee (No. 11-061: Daejeon, Korea). The mice were randomly placed into three groups (n = 5 mice/group): untreated control mice, UVB-treated vehicle control mice, and UVB-treated/hesperidin-treated mice. UVB-treated mice from the hesperidin group received oral administration 0.1 mL of water containing hesperidin at a concentration of 100 mg/kg body weight per day. The mice in the UVB-treated vehicle control group were provided only with drinking water, while those in the untreated control group were neither irradiated nor did they receive any oral treatments.

UVB Irradiation in Mice

As described earlier,¹⁶⁾ mice were exposed to 302-nm UVB light via a UVM-225D Mineralight UV Display Lamp. UV intensity was measured with a HD2102-2 UV meter (Delta OhM, Padova, Italy). The experiment investigated the effect of orally administered hesperidin in the dorsal skin of UVB-irradiated mice. The mice were exposed to UVB light 3 times at 48 h intervals every week for 12 weeks, with the light intensity increasing from 60 mJ/cm² per exposure in the first week to 90 mJ/cm² per exposure in week seven and every week thereafter.

Immunohistochemistry and Morphometric Analysis of Cutaneous Blood Vessels

For immunohistochemistry, we followed a protocol that was previously described.¹⁷⁾ Briefly, paraffin-embedded tissues were cut into 4 µm thick sections using a microtome. After a room temperature 1-h blocking incubation in 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS), the sections were incubated with anti-CD31 (1 : 200) and anti-p-Akt (1 : 200) primary antibodies. Then, the sections were washed and further incubated with biotinylated secondary antibodies followed by HRP-conjugated streptavidin. Images were captured with an Olympus BX41 microscope (Olympus Corporation, Tokyo, Japan) and quantified using ImageJ software (NIH, Bethesda, MD, U.S.A.). Three different fields per section located within 10⁴ µm² of the epidermal–dermal junction were examined at 40× magnification so that the number of the vessels per mm², average vessel size, and relative blood vessel-occupied dermal area could be quantified. In order to compare the statistical comparison, 5 sections per a mouse (n = 5 mice, each group) was analyzed.

Cell Culture

Human epidermal keratinocytes (HEKs) and Human dermal fibroblasts (HDFs) were cultured as previously described.¹⁸⁾ HDFs were cultured in fibroblast medium supplemented with 5% fetal bovine serum (FBS), 1% fibroblast growth supplement, and 1% penicillin and streptomycin (P/S). HEKs were obtained from Lonza (Walkersville, MD, U.S.A.) and cultured in KGM-Gold SingleQuots™ medium supplemented with standard growth factors. Both cell types were maintained in a humidified 5% CO₂ incubator at 37 °C.

UVB Light

After a 24-h preincubation with hesperidin at the indicated concentrations, HEK and HDFs were washed with PBS and exposed to 302-nm UVB light (20 mJ/cm²) for 1 min using a UVP cross linker (Ultra-Violet Products Ltd., Cambridge, U.K.). Then, after washing in PBS, the growth medium was replenished and the cells were incubated for a further 6 h.

Immunofluorescence Analysis

Using a microtome, paraffin-embedded tissues were sliced into 4 µm-thick sections. After a 1-h room temperature blocking step in 5% BSA in PBS, the sections were placed in a humidified chamber overnight at 4 °C to bind the anti-VEGF (1 : 200) and anti-HIF-1α (1 : 200) primary antibodies. After being fully washed, the cells were then incubated with Alexa 488-conjugated secondary antibody (1 : 2000; Thermo Fisher Scientific, Waltham, MA, U.S.A.) for 1 h before being counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for 15 min. Finally, the sections were washed, mounted in VectaShield (Vector Laboratories, Burlingame, CA, U.S.A.), and imaged using a DM 2500 fluorescence microscope (Leica, Wetzlar, Germany). The immunofluorescence expressions of VEGF and HIF-1α was quantified using ImageJ software (NIH). In order to compare the statistical comparison, 5 sections per a mouse (n = 5 mice, each group) was analyzed.

RNA Interference

Small interfering RNAs (siRNA) against PI3K (p110α) or control siRNAs (Cell Signaling Technology) were transiently transfected as previously described.¹⁸⁾ The transfected cells were then incubated for 24 h, lysed, and assessed by Western blotting.

Western Blotting

We used a standard Western blot protocol that was previously described¹⁸⁾ with the following: anti-MMP-9 (1 : 1000), anti-MMP-13 (1 : 1000), anti-VEGF (1 : 1000), anti-HIF-1α (1 : 1000), anti-p70S6K (1 : 1000), and anti-β-actin (1 : 1000). Western blot analysis was performed three independent experiments (n = 3, each group) and the protein expression levels were determined by measuring signal intensities as captured with a Las-3000 image analyzer (FUJIFILM, Tokyo, Japan). The protein expression levels were quantified using ImageJ software (NIH).

Statistical Analyses

The data presented here are means ± standard deviation (S.D.) unless otherwise noted. Statistical significance was determined via one-way ANOVA using GraphPad Prism 7 (San Diego, CA, U.S.A.). Student’s t-tests were also used to compare relative differences. p < 0.05 was considered statistically significant.

RESULTS

Effect of Hesperidin Treatment on Expression of VEGF, HIF-1α, and MMPs in HR-1 Mice Exposed to UVB Light

Angiogenesis levels represent an integration of the various pro-angiogenic stimulators like VEGF. The receptors for VEGF are expressed primarily on endothelial cells but can also be found on other cell types.¹⁹⁾ When we evaluated hesperidin’s effects on angiogenesis in the skin of HR-1 mice exposed to UVB light three times a week for 12 weeks, we found a suppression of the expression of VEGF normally induced by UVB light (Fig. 1A). HIF-1α may transactivate angiogenic factors like MMP-2, VEGF, and MMP-9.²⁰⁾ When we treated HR-1 mice exposed to UVB light with hesperidin, we found a downregulation of the HIF-1α expression normally induced by UVB light in the skin (Fig. 1B). We then confirmed by Western blot that hesperidin prevented the increase in VEGF, HIF-1α, MMP-9, and MMP-13 expression normally induced by UVB light (Fig. 1C).

Fig. 1. Effects of Hesperidin Treatment on VEGF, HIF-1α, and MMPs Expression in HR-1 Mice Exposed to UVB Light

Immunohistochemical analysis for (A) VEGF and (B) HIF-1α expression was carried out on 4 µm-thick paraffin sections (n = 5 mice, each group). Magnification, 400×. Scare bar, 100 µm. (C) The expression level of skin proteins was analyzed by Western blotting and quantified using ImageJ software (n = 3, each group). All values are presented as mean ± standard deviation (S.D.) ^# p < 0.01 for the UVB-treated vehicle control group vs. the UVB-treated/hesperidin-treated group. (Color figure can be accessed in the online version.)

Effect of Hesperidin on the Angiogenesis and CD31 Experssion Induced in HR-1 Mice by UVB Light

Acute UVB light exposure is known to induce significant angiogenesis in both humans and mice.²¹⁾ We therefore asked whether hesperidin affects the increase in cutaneous angiogenesis induced by UVB light. After confirming that UVB light irradiation increased the number, area, and size of cutaneous blood vessels, we found that hesperidin treatment significantly reduced all these variables (Fig. 2A). This indicates hesperidin reduces UVB-induced angiogenesis. We then found hesperidin treatment inhibited the expression of CD-31 (also known as PECAM-1), which is an endothelial cell-specific adhesion molecule, and reduced the enlarged blood vessels characteristic of UVB-irradiated skin (Fig. 2B). Using H&E staining, we found hesperidin treatment reduced the epidermal hypertrophy and thickness of UVB-irradiated skin (Fig. 2C). Hesperidin treatment may, therefore, protect skin from the effects of chronic UVB exposure by inhibiting UVB-induced angiogenesis and CD31 expression.

Fig. 2. Hesperidin Inhibits UVB-Induced Dermal Angiogenesis in HR-1 Mice

(A) Vessel number, area, and size were measured in fields-of-view within 10⁴ µm² of the epidermal-dermal junction. (B) Immunohistochemical analysis of CD31 expression was performed on 4 µm-thick paraffin sections. (C) Representative photomicrographs of H&E staining from each treatment group are shown. Magnification, 400×. Scare bar, 100 µm. All values are presented as mean ± S.D. (n = 5 mice, each group). ** p < 0.01 for the untreated control group vs. UVB-treated control group. ^# p < 0.05 for the UVB-treated vehicle control group vs. the UVB-treated/hesperidin-treated group. (Color figure can be accessed in the online version.)

Hesperidin Inhibits the Akt Phosphorylation in HR-1 Mice Induced by UVB Light

PI3K/Akt signaling has been implicated in UVB-induced skin malignancies. PI3K/Akt signaling regulates cellular apoptosis, autophagy, and senescence.²²⁾ To clarify the molecular mechanisms underlying the anti-angiogenic effects of hesperidin, we investigated Akt phosphorylation in mouse skin. Figure 3 shows the skin of HR-1 mice exposed to UVB, revealing significantly elevated levels of Akt phosphorylation. We found mice treated with hesperidin showed significantly less Akt phosphorylation than UVB-exposed vehicle control mice.

Fig. 3. Hesperidin Inhibits the UVB-Induced Akt Activation in the Skin of HR-1 Mice

(A) Immunohistochemical analysis of p-Akt expression in 4 µm-thick paraffin sections. (B) The relative area comparison of p-AKT positive area per epidermis area in each group. The p-AKT positive area was quantified using ImageJ software. Magnification, 400×. Scare bar, 100 µm. All values are presented as mean ± S.D. (n = 5 mice, each group). * p < 0.05 for the untreated control group vs. UVB-treated control group. ^# p < 0.05 for the UVB-treated vehicle control group vs. the UVB-treated/hesperidin-treated group. (Color figure can be accessed in the online version.)

Effects of Hesperidin on the Expression of MMP-9, MMP-13, and VEGF in HDFs and HEKs Exposed to UVB Light

MMPs and VEGF are pro-angiogenic effectors that reportedly promote cancer invasiveness. After finding that UVB light induced MMP-9, MMP-13, and VEGF expression in skin, we asked whether hesperidin affects this increase. We found that treatment of skin cells exposed to UVB light with 5–20 µM hesperidin for 24 h reduced their expression of MMP-9, MMP-13, and VEGF (Figs. 4A, B). This suggests hesperidin represses the increase in MMP-9, MMP-13, and VEGF expression induced in skin cells upon UVB light exposure.

Fig. 4. Hesperidin Inhibits the UVB-Induced Expression of MMP-9, MMP-13, and VEGF in HDFs and HEKs

(A) HDFs and (B) HEKs cells were exposed to the indicated concentrations of hesperidin for 24 h before being a 1-min exposure to UVB light (20 mJ/cm²). Then, after washing and a 4-h recovery incubation, cell lysates were analyzed by Western blot and quantified using ImageJ software (n = 3). All values are presented as mean ± S.D. * p < 0.05 ** p < 0.01 for the untreated control group vs. UVB-treated control group. ^# p < 0.01 for the UVB-treated vehicle control group vs. the UVB-treated/hesperidin-treated group.

Hesperidin Reduces the Phosphorylation of PI3K/Akt Signaling Pathway Components Induced in Primary Skin Cells by UVB Light

UVB light and arsenite both increase the expression of HIF-1α and VEGF via PI3K signaling.²³⁾ We examined the effects of hesperidin on PI3K/Akt and p70S6K expression. We found that hesperidin inhibited the phosphorylation of Akt and p70S6K proteins induced by exposure to UVB light (Fig. 5A). Angiogenesis is reportedly regulated by the MEK/ERK signaling pathway.²⁴⁾ Thus, we investigated the effects of hesperidin on ERK signaling via Western blotting. We found hesperidin decreased UVB-induced MEK phosphorylation (Fig. 5A). We then used the PI3K inhibitor LY294002 and Western blotting to confirm hesperidin’s inhibition of PI3K/Akt signaling. We found pretreatment with hesperidin or LY294002 inhibited both Akt expression and p70S6K phosphorylation (Fig. 5B). We also examined the changes in PI3K/Akt expression induced by transfection with PI3K 110α-specific siRNAs. This allowed us to confirm that hesperidin treatment can significantly repress PI3K/Akt expression in HEKs (Fig. 5C). Thus, hesperidin effectively reduces UVB-induced angiogenesis in human primary skin cells.

Fig. 5. Hesperidin Suppresses the UVB-Induced p70S6K Expression and Akt Phosphorylation in HEKs

(A) HEKs treated with hesperidin for 24 h were washed and given a 1-min UVB light (20 mJ/cm²) exposure. (B) HEKs treated with hesperidin for 24 h with or without a 1 h LY294002 for 60 min were washed with PBS and given a 1-min UVB light (20 mJ/cm²) exposure. (C) HEKs transfected with control siRNAs or PI3K (p110α) siRNAs were treated with 20 µM hesperidin for 24 h before being washed and given a 1-min UVB light (20 mJ/cm²) exposure. p-PI3K and p-Akt levels were quantified using ImageJ software in the resulting HEK lysates via Western blot (n = 3). All values are presented as mean ± S.D. ^# p < 0.01 for the UVB-treated vehicle control group vs. the UVB-treated/hesperidin-treated group.

DISCUSSION

For the last 20 years, angiogenesis has remained one of the major focal points of cancer research, but increased angiogenesis also occurs in noncancerous skin diseases like rosacea and psoriasis.²⁵⁾ Angiogenesis in skin also seems to be important in the appearance of light-induced characteristics associated with age, since the inhibition of angiogenesis reduces the formation of wrinkles after exposure to UVB light.²⁶⁾ Increased angiogenesis has also been associated with skin thickening, and IR light irradiation aggravates UVB-induced skin wrinkling in hairless mice.^27,28) The angiogenic factor VEGF is an important regulator of skin angiogenesis. Recently, some groups have attempted to suppress VEGF expression as an anti-angiogenic and anti-tumor intervention. Further studies on the regulation of VEGF in skin may help improve our understanding and treatment of angiogenesis-related skin disorders.

Here, we explored how hesperidin affects the expression of angiogenesis-related factors like PI3K/Akt, VEGF, HIF-1α, and MMPs in the skin of HR-1 mice after exposure to UVB light. We found that hesperidin reduces VEGF expression after the induction of MMP-9 and MMP-13 by UVB light exposure (Fig. 1). Thus, indicates hesperidin can inhibit angiogenesis in vivo. MMPs provoke angiogenesis and vasculogenesis,²⁹⁾ with MMP-9 facilitating new vessel formation³⁰⁾ and permitting VEGF to interact with VEGF receptors.²⁴⁾ HIF-1α belongs to a family of transcription factors that regulate angiogenesis via downstream factors including VEGF³¹⁾ and the MMPs.^32,33) Our results have revealed that hesperidin treatment of mouse skin reduces the HIF-1α expression and therefore MMP-13 and MMP-9 expression induced by UVB light exposure (Fig. 1).

UVB light irradiation of skin promotes the formation of blood vessels in the dermis and induces hyperplasia of the epidermis.³⁴⁾ Hesperidin treatment reduces this increase in skin blood vessel number, area, and size, and significantly suppresses the expression of the endothelial cell adhesion molecule CD31. Although a direct mechanism explanation for the control mechanism of epidermal thickness in relation angiogenesis has not been established, hesperidin also reduces the thickening of the dorsal epidermis induced by UVB light (Fig. 2). UVB light activates PI3K signaling and thereby upregulates HIF-1α and VEGF expression.⁹⁾

Myricetin, another plant-derived flavonoid, reportedly suppresses the UVB-induced activity of Fyn kinase and as well as the activity of the Raf/MEK/ERK signaling pathways. Therefore, the inhibition of MEK/ERK signaling induced in mouse skin by myricetin may be secondary to inhibition of Fyn kinase.³⁵⁾

It has been reported that UV can induces VEGF upregulation in immortalized HaCaT cells developed from human adult skin keratinocytes.^36,37) According to report, the PI3K-Akt pathway play important regulatory roles in angiogenesis. Therefore, we investigated Akt pathway are involved in UVB-induced VEGF expression in skin. Our results hesperidin suppresses UVB-induced increases in Akt expression in mouse skin (Fig. 3). We also examined the effects of UVB light on MMP-9, MMP-13, and VEGF expression levels in primary human skin cells. We found hesperidin inhibited the increases in MMP-9, MMP-13, and VEGF expression induced in primary HDFs and HEKs upon UVB exposure (Fig. 4). In addition, we found hesperidin inhibited the induction of Akt/p70S6K signaling by UVB light (Fig. 5A). We therefore conclude that hesperidin administration may suppress PI3K expression because PI3K influences HIF-1α and/or VEGF expression in response to UVB light exposure. We also noted that both hesperidin and the PI3K inhibitor LY294002 effectively repress the increase in Akt/p70S6K expression stimulated by UVB light irradiation (Fig. 5B). Using siRNAs to downregulate the p110α regulatory subunit of PI3K, we confirmed disruption of PI3K can modulate antiangiogenic signaling.

Treatment with a PI3K-specific siRNA revealed that hesperidin inhibits UVB-induced PI3K/Akt activity (Fig. 5C). We therefore conclude that hesperidin is an effective inhibitor of UVB-induced angiogenesis via its downregulation of VEGF signaling. Future studies will further explain the mechanisms by which hesperidin prevents UVB-stimulated skin injury. In summary, we found hesperidin inhibits the neovascularization of the skin of HR-1 mice that is induced by UVB light exposure. It also inhibits PI3K signaling to repress VEGF, HIF-1α, and MMP expression in HR-1 mice and primary human skin cells.

Acknowledgments

This research was funded by Grant (KSN2013330) from the Korea Institute of Oriental Medicine (KIOM).

Conflict of Interest

The authors declare no conflict of interest.

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