IL‐17A is involved in the hyperplasia of blood vessels in local lesions of psoriasis by inhibiting autophagy

Increased angiogenesis is a pathological feature of psoriasis, but the pathomechanisms of angiogenesis in psoriasis are not clear. Interleukin‐17A (IL‐17A) is the major effect factor in the pathogenesis of psoriasis. Our results showed that IL‐17A can promote angiogenesis and cause endothelial cell inflammation. Autophagy plays an important role not only in regulating inflammation, but also in regulating angiogenesis. Whether angiogenesis in psoriasis is related to autophagy remains unclear. In this study, we treated human umbilical vein endothelial cells (HUVECs) with IL‐17A to simulate increased angiogenesis to study whether increased angiogenesis in psoriasis is related to autophagy.


| INTRODUC TI ON
2][3] Stephan Hailfinger et al. 4 showed that autophagy function was impaired in psoriasis.Impaired autophagy is presumably involved in inflammation and disturbed keratinocyte differentiation. 4Whether angiogenesis in psoriasis is related to autophagy remains unclear.
Since endothelial cells are between the walls of blood vessels and the blood, they are the primary target of inflammatory factors in inflammatory diseases. 5IL-17A belongs to the interleukin-17 family that are involved in the immune defense of contagious pathogens, the pathogenesis of cancers, some inflammatory syndromes, and autoimmune diseases. 6In psoriasis-like skin disease, dermal IL-17A overexpression can induce vascular inflammation and endothelial dysfunction. 7IL-17 can induce the expression of proinflammatory factors such as IL-8, CCL20, CCL2, IL-6, IL-1β, and TNFα, [8][9][10] among which CCL20, IL-8, and TNFα are known to play an significant role in the pathogenesis of psoriatic inflammation. 11,12[15] In tissue homeostasis, autophagy degrades the various cellular components (protein aggregates, damaged organelles) and the invading pathogens (such as viruses, bacteria, and protozoa).Nowadays, autophagy is emerging as an attractive therapeutic target. 16crotubule-associated protein light chain 3-I (LC3I) to LC3II conversion is commonly used as a marker for initiation, elongation, and autophagosome formation of autophagy.Accumulation of p62 indicates the inhibition of autophagosome degradation. 17Autophagy regulates various cellular functions.For example, induction of autophagy can improve endothelial dysfunction. 18Induction of autophagy is often involved in anti-inflammation. 19A recent study has shown that autophagy regulates tumor-related pathologic angiogenesis. 20As a major effector of the pathogenesis of psoriasis, IL-17A can promote angiogenesis of human umbilical vein endothelial cells (HUVECs). 21HUVEC is an important model cell for studying angiogenesis and endothelial cell function regulation. 22Therefore, we treated HUVECs with IL-17A to simulate increased angiogenesis in psoriasis to study whether increased angiogenesis in psoriasis is related to autophagy.

| Cell cultures
Human umbilical vein endothelial cells were isolated from human umbilical cord in our laboratory according to the methods described previously. 23Human dermal microvascular endothelial cells (HDMECs) were isolated from healthy subjects and lesional skin of patients with psoriasis according to the methods described previously. 24All experiments were conducted with the approval of the Medical Ethics Committee.HUVECs and HDMECs were cultured in endothelial cell growth medium-2 (EGM-2, Lonza) containing 2% fetal bovine serum in a 37°C incubator containing 5% CO 2 .Cells cultured less than six passages were used in experiments.For all experiments, the HUVECs were seeded in cell culture flask (Specification:

| Real-time quantitative PCR analysis
Total RNA was isolated from HUVECs using Trizol (Invitrogen).Reverse transcription of 0.8 μg RNA was performed with PrimeScript™ RT Master Mix (Takara).The target genes were amplified on an ABI StepOne Real-Time PCR System (Applied Biosystems) using TB Green® Premix Ex Taq™ II (Takara).Primer sequences for PCR are listed in Table 1.The mRNA expression levels of target genes were normalized to β-actin and expressed as the fold changes relative to the controls.

| Flow cytometry analysis
Apoptosis was detected by flow cytometry (Miltenyi Biotec) using Annexin V/Propidium Iodide (PI) Apoptosis Assays Kit (KeyGenBio TECH).In each treatment group, 1 ~ 5 × 10 5 HUVECs were harvested and washed twice with PBS, followed by resuspension in 500 μL binding buffer.Subsequently, Annexin V-FITC (5 μL) and PI (5 μL)   were added to the cell culture and incubated at room temperature in dark for 5-15 min.Annexin V+/PI− and Annexin V+/PI+ apoptotic cells were determined by flow cytometry.Apoptotic cells were calculated as the percentage of Annexin V-positive cells.

| Caspase-3 activity
The activity of caspase-3, which plays a central role in apoptosis, 26 in HUVECs was determined by Caspase 3 Activity Assay Kit (Beyotime).Total protein concentration of cultured cells was determined by Bradford Protein Assay Kit (Beyotime).According to the manufacturer's instructions, reaction mixture, containing 40 μL of detection buffer, 50 μL of sample (protein concentration reach 1-3 mg/ mL), and 10 μL of AC-DEVD-PNA (substrate of Caspase-3) was incubated at 37°C for 60-120 min.Caspase-3 activity was measured with a microplate analyzer (PerlonQ Instruments) at absorbance wavelength of 405 nm.

| Tube formation assay
This assay was carried out according to the method described by Zhou et al. 23 On the day before the experiment, the Matrigel (Corning) was thawed at 4°C overnight, and pipette tips and 96-well plate used in the experiment were also precooled in the refrigerator.A total of 50 μL Matrigel was added to each well of precooled 96well plate, then incubated at 37°C for 1 h HUVECs were seeded into Matrigel-coated wells at a density of 5 × 10 5 cells/mL, and cultured at 37°C incubator containing 5% CO 2 for 6 h.The formation of capillary-like structures was observed and captured under an inverted microscope (Olympus).The numbers of junctions and meshes were counted with ImageJ software (National Institutes of Health).

| Immunofluorescence
A coverslip was placed in the 6-well plate, and the HUVECs at logarithmic growth stage were seeded into the 6-well plate (2 × 10 4 cells/mL).When the cells grew to 80% confluence, autophagy and inflammation were induced by rapamycin and IL-17A, respectively.
Afterward, the cells were immobilized with 4% paraformaldehyde for 15 min, then infiltrated with 0.1% Triton X-100 for 15 min, and blocked with 5% BSA for 30 min at room temperature.The cells were then incubated overnight at 4°C with primary antibody LC3B (1 μg/ mL), followed by 1-h incubation with a FITC-conjugated goat secondary antibody to Rabbit IgG (1: 500, Boster) at room temperature.
Nuclear DNA was labeled with DAPI (Boster).The LC3 fluorescent staining were observed under inverted two-photon confocal microscope (Olympus) and the fluorescence intensity was analyzed.

| ELISA assay
The content of inflammatory cytokines (IL-8, CCL20) and human vascular endothelial growth factor (VEGF) in supernatants were detected by enzyme-linked immunosorbent assay (ELISA) kit (WesTang) in light of manufacturer's protocols.Concentrations of IL-8, CCL20, and VEGF in the supernatant were measured with a microplate analyzer (PerlonQ Instruments) at absorbance wavelength of 405 nm.

| NO/NOS assay
The intracellular levels of NO and total NOS activity were measured using commercial kits (Jiancheng).The total NOS activity (U/mg) was detected by measuring the absorbance of wavelength 530 nm with UV spectrophotometer (UV-2102PC, UNICO), and the levels of NO (μmol/L) was detected by measuring the absorbance of 550 nm with a microplate analyzer (PerlonQ Instruments) according to the manufacturer's protocols.

| IL-17A is involved in angiogenesis by inhibiting autophagy through AMPK signaling pathway
Increased angiogenesis is a pathological feature of psoriasis, but the pathomechanisms of angiogenesis in psoriasis are not clear.IL-17A is a crucial effector cytokine in the pathogenesis of psoriasis.Previous studies 21 were consistent with our findings, indicating that IL-17A can induce angiogenesis in HUVECs (Figure 2A,B).IL-17A also inhibited autophagy in HUVECs.Whether IL-17A is involved in angiogenesis by inhibiting autophagy is unknown.Rapamycin has been reported to induce autophagy. 27To assess whether autophagy affects angiogenesis, we successfully induced autophagy in HUVECs using rapamycin (Figure 1D,E Our results indicated that IL-17A is involved in the hyperplasia of blood vessels in psoriasis by inhibiting autophagy (Figure 4F).We also examined whether the autophagy of HUVECs can affect inflammation, proliferation, and differentiation of epidermal cells.
We induced enhancement and inhibition of autophagy in HUVECs, respectively, and then indirectly cocultured them with HaCaT cells.
PCR was used to detect mRNA levels of genes related to inflammation (IL-6, IL-8), proliferation (proliferating cell nuclear antigen, PCNA), and differentiation (keratin 5, Involucrin) in HaCaT cells.As shown in Figure 1F, the enhancement of autophagy in HUVECs inhibits the inflammation, proliferation, and differentiation of HaCaT cells, while the inhibition of autophagy has the opposite effect.As a highly conserved cellular energy sensor, AMPK is also involved in the regulation of angiogenesis. 29Literature studies have shown that a simple AMPK inhibitor Dor (CC) can significantly exacerbate psoriasis lesions. 30 AMPK promotes autophagy by phosphorylating ULK1 at Ser555, while mTOR inhibits autophagy by phosphorylating ULK1 at Ser757. 33 shown in Figure 3D,E, treatment of HUVECs with IL-17A increased mTOR activity, evidenced by increased ratio of phosphorylated mTOR(Ser2448)/mTOR, an inhibitory site of autophagy. 34In contrast, Rap decreased the ratio of p-mTOR(Ser2448)/mTOR in IL-17A-treated HUVECs (Figure 3D,E).Inhibition of either AMPK or autophagy overrode Rap-induced activation of ULK1 activity and reduction of mTOR activity in IL-17A-treated HUVECs (Figure 3D-G).These results suggested that regulation of IL-17A-induced HUVECs inflammation by autophagy is mediated by AMPK/mTOR/ULK1 signaling pathway.

| Enhanced autophagy suppresses IL-17A-induced apoptosis in HUVECs
To assess the influence of IL-17A on HUVEC apoptosis, we used both flow cytometric analysis and caspase-3 activity assay.Results

| Enhanced autophagy decreases IL-17A-induced VEGF production in HUVECs
Because VEGF can promote angiogenesis, we assessed next whether regulation of angiogenesis by IL-17A and/or autophagy is mediated by VEGF.ELISA assay revealed that IL-17A increased VEGF content in the supernatant of HUVECs (Figure 3C).Induction of autophagy reversed the effect of IL-17A on VEGF production (Figure 3C).However, inhibition of AMPK with Dor did not block the effect of Rap on VEGF production in IL-17A-treated HUVECs (Figure 3C), suggesting that autophagy regulates VEGF production through signaling pathways other than AMPK.

| Regulation of IL-17A-induced NO reduction by autophagy is mediated by the AMPK signaling
Psoriasis is featured by decreased NO-dependent vasodilation. 35 determine whether IL-17A decreases NO production in HUVECs,

| DISCUSS ION
In the traditional view, autophagy is the degradation of cellular components and pathogens.However, previous studies also showed that either inhibition or impairment of autophagy flux aggravates the endothelial inflammation, 36 while restoration of autophagic flux can protect vascular endothelium from inflammatory damages. 37rrespondingly, we demonstrated inhibitory effect of autophagy on inflammation in HUVECs, consistent with prior findings. 38However, other study showed that activation of autophagy can promote angiotensin II-induced inflammatory response through enhanced HDAC4 expression in RAECs. 39These discrepant results could be attributable to the inflammation induced by different inducers, that is, IL-17A and Angiotensin II.Further researches are needed to validate this postulation.
Previous studies revealed that IL-17A promotes angiogenesis of HUVECs, 21 while cytokines, such as IL-6, IL-8, and VEGF are wellknown angiogenic factors. 40Hence, IL-17A-induced upregulation of cytokines and VEGF production can contribute to angiogenesis. 40,41 showed here that induction of autophagy inhibited IL-17Ainduced cytokine production and angiogenesis in HUVECs, strongly suggesting a pivotal role of autophagy in IL-17A-induced endothelial cells angiogenesis and inflammation.Interestingly, our results showed that IL-17A promoted angiogenesis by inhibiting autophagy through the inactivation of AMPK.In contrast, other studies showed that activations of AMPKα and autophagy promote angiogenesis after ischemia. 42These results suggest that AMPK plays positive and negative influence on angiogenesis depending on the conditions.
This assumption is supported by the evidence that under an energy crisis, such as hypoxia, ischemia, and exercise, AMPK is activated and controls cellular energy homeostasis by promoting ATP synthesis to maintain body energy balance, but under pathological circumstance, such as cancer, AMPK is inactivated and angiogenesis is abnormally increased, and the activation of AMPK can inhibit angiogenesis. 29 the contrary, a recent study showed that VEGF promotes angiogenesis by activating AMPK to trigger transient autophagy prior to activation of mTOR, suggesting that autophagy may exert different functions in regulating angiogenesis. 43VEGF can also trigger the Akt/mTOR pathway, 44 resulting in an inhibition autophagy, 33 which could be a mechanism by which IL-17A impairs endothelial autophagic flux.There are other pathways involved in angiogenesis, for example, Wu et al. 45  different sites of ULK1. 33,46Previous studies demonstrated that activation of AMPK can inactivate mTOR, 46,47 resulting in induction of autophagy, 31,38 consequently leading to an inhibition of inflammation. 48Both the present and the previous studies showed that induction of autophagy is via activation of AMPK/mTOR/ULK1 pathway. 32Evidence suggested that AMPK is involved in the initiation of autophagy by directly phosphorylating multiple sites in ULK1 (S317, S467, S555, T575, S637, and S777).Activation of AMPK enhances ULK1 function, resulting in the upregulation of autophagosome formation. 46,49,50However, our results suggest that AMPK is also involved in the late stage of autophagy because inhibition of AMPK not only decreased LC3-II but also increased p62 protein expression.Tang et al. 51 showed that CQ not only increased the level of p62 but also decreased the LC3 ratio in the presence of autophagy activator, which is consistent with our results.Ahmed Nadeem et al. 52 found that BET bromodomain inhibitors inhibited imiquimodinduced psoriasis-like inflammation through RORC/IL-17A pathway.
This indicates that IL-17A exert an important role in the regulation of inflammation, which is the same as our study.It is possible that autophagy and BET bromodomain inhibitors have a little correlation, which can be further studied.
The relationship between apoptosis and autophagy has been demonstrated scrupulously. 28But the results are controversial.
Evidence indicates that autophagy exhibits antiapoptotic effect in some cases, 28,53 while under certain condition, activation of autophagy triggers apoptosis. 28,54It is further hypothesized that removing or inhibiting key proteins in apoptosis could relieve the inhibition of autophagy or switch the cellular stress response from apoptosis to the state of increased autophagy. 28Caspase-3 is exploited intracellularly as a key protease controlling apoptosis. 26,55Both this study and previous studies showed that induction of autophagy inhibits caspase-3 activity, 56 and attenuates IL-17A-induced increase in caspase-3 activity.Therefore, we speculated that induction of autophagy attenuates the inhibition of IL-17A on autophagy by inhibiting caspase-3 activity, resulting in an enhanced autophagy and decreased apoptosis.NO/NOS system is involved in regulating endothelial functions. 57Reduced NO levels can be paralleled by impaired endothelial function, and an increase in the expression levels of MCP-1(CCL2), a biomarker of endothelial dysfunction, 15 which recruits mononuclear phagocytes. 58,59Moreover, NO serves not only as a vasodilator, but also as an anti-inflammatory signaling molecule, which plays an important role in apoptosis. 60,61Inflammation decreases NO bioavailability, 58  Our study showed that IL-17A significantly decreased the intracellular levels of total NOS activity in HUVECs.So we analyzed that most of the total NOS reduced by IL-17A in HUVECs were eNOS.And endothelial dysfunction is closely related to NO primarily produced by eNOS.But some studies showed that IL-17A has been shown to induce iNOS. 62,63iNOS acts as an inflammatory mediator and is associated with inflammation. 63NO is produced by iNOS in the immune system. 64Our study did not conflict with theirs, IL-17A induces a different type of NOS, which have different functional properties.
In summary, IL-17A is involved in angiogenesis by inhibiting autophagy.IL-17A-induced inflammation results in a reduction in NO bioavailability and an increase in the expression of CCL2, leading to endothelial dysfunction.Induction of autophagy attenuates IL-17Ainduced functional changes in endothelial cells accompanied by the activation of AMPK pathway, suggesting that autophagy may be a therapeutic target for psoriasis.

25 cm 2
) (1 × 10 5 cells/mL) and the following experiments were carried out when the adherent culture reached 70%-80% fusion degree.For induction of inflammation, HUVECs were stimulated with recombinant human IL-17A at a dose of 50 ng/mL (Peprotech) for 24 h.HUVECs were treated with 200 nM rapamycin (Rap, Solarbio) and 50 μM chloroquine (CQ, MCE), respectively, for 1 h to induce and inhibit autophagy.Dorsomorphin dihydrochloride (Dor, MCE) was used as an inhibitor of AMPK.In some experiments, HUVECs were pretreated with 20 μM of Dor for 2 h prior to IL-17A stimulation.The untreated HUVECs served as the controls.Five independent replicates were performed for each experiment.
) to detect the effect of autophagy on IL-17A-induced angiogenesis.As shown in Figure1D,E, we found that treatment of HUVECs with Rap notably increased the ratio of LC3II/I and decreased the p62 proteins as compared to untreated cells by western blot.Immunofluorescence showed that LC3 fluorescent puncta were significantly increased in HUVECs treated with Rap as compared to untreated cells (Figure 2G,H).It was noteworthy that induction of autophagy significantly inhibited IL-17A-induced increase in the numbers of junctions and meshes (Figure 2B,C,F).To further confirm the role of autophagy in IL-17Ainduced angiogenesis, chloroquine (CQ), an inhibitor of lysosomal acidification, 28 was employed to inhibit the late stage of autophagy.CQ not only increased p62 proteins, but also decreased the ratio of LC3II/I in the HUVECs-treated with both IL-17A and Rap (Figure 1I,J,M,N).CQ reversed the effects of Rap on IL-17A-induced increases in the numbers of junctions and meshes (Figure 2C,D,F).

331 WANG et al. 3 . 3 |
We found that HUVECs treated with Dor enhanced the ratio of LC3II/I and the p62 proteins compared with untreated cells, indicating that Dor treatment inhibited autophagy in HU-VECs (Figure 1G,H,K,L).Meanwhile, Dor treatment increased the angiogenesis of HUVECs as indicated in Figure 1O,P,Q.So we determined whether AMPK is involved in both autophagy and IL-17Ainduced angiogenesis in HUVECs.Our results showed that IL-17A decreased the ratio of phosphorylated AMPKα(Thr172)/AMPKα (Figure 2J,k), while induction of autophagy with Rap increased the ratio of p-AMPKα(Thr172)/AMPKα (Figure 2J,K), indicating that IL-17A and autophagy inversely regulate AMPK pathway in IL-17Atreated HUVECs.The efficiency of Dor for AMPK inactivation was confirmed as shown that Dor significantly decreased the ratio of p-AMPKα(Thr172)/AMPKα (Figure 2J,K).Moreover, inhibition of AMPK with Dor reversed the effects of Rap on IL-17A-induced increases in the numbers of junctions and meshes (Figure 2C,E).Dor also largely reversed Rap-induced increase in autophagy activity as demonstrated by Dor-induced decreases in the ratio of LC3-II/I and increases in p62 proteins in the HUVECs-treated with both IL-17A and Rap (Figure 1I,J,M,N).These results indicated that IL-17A is involved in angiogenesis by inhibiting autophagy through AMPK signaling pathway.|Regulation of IL-17A-induced HUVECs inflammation by autophagy is mediated by the AMPK/mTOR/ULK1 signaling pathwayTo assess the link between inflammation and autophagy, induction of autophagy with Rap attenuated the IL-17A-induced accumulation of p62 proteins (Figure1J,N) and expression levels of mRNA for proinflammatory cytokines (Figure1C), while increasing the accumulation of LC3 fluorescent puncta in IL-17A-treated cells (Figure2I,L).And the content of CCL20 and IL-8 in the culture supernatant was decreased following induction of autophagy with Rap in IL-17Atreated HUVECs (Figure3A,B).As shown in Figure1I,J,M,N, CQ reversed the effects of Rap on expression levels of the LC3-II and p62 proteins in IL-17A-treated HUVECs.Likewise, CQ attenuated the effects of Rap on CCL20 and IL-8 levels in the supernatant of IL-17A-treated HUVECs (Figure 3A,B).These results suggested that induction of autophagy can attenuate IL-17A-induced inflammatory response in HUVECs.Since previous study showed that increased AMPK activity is associated with autophagy and inhibition of inflammation, 31 next we determined whether AMPK is also involved in autophagy and IL-17A-induced inflammation in HUVECs.As shown in the previous results, IL-17A and autophagy inversely regulate AMPK pathway in IL-17A-treated HUVECs.Since either increase in autophagy activity or activation of AMPK can inhibit inflammation, we next used Dor to demonstrate whether induction of autophagy suppresses inflammation through AMPK activation.Dor largely reversed Rap-induced increase in autophagy activity as demonstrated by Dor-induced decreases in the ratio of LC3-II/I and increases in p62 proteins in the HUVECs-treated with both IL-17A and Rap (Figure 1I,J,M,N).Moreover, Dor prevented Rap-induced inhibition of proinflammatory cytokine expression (CCL20 and IL-8) in the supernatant of IL-17A-treated HUVECs (Figure 3A,B).In addition, inhibition of autophagy with CQ exhibited similar effects to Dor on Rap-induced changes in the ratio of p-AMPKα(Thr172)/AMPKα, autophagy activity as well as cytokine levels in IL-17A-treated HUVECs (Figure 2J,K).These results suggested that regulation of IL-17Ainduced HUVECs inflammation by autophagy is mediated by AMPK signaling pathway.Since mTOR-ULK1 (Unc-51 like kinase 1) signal, a downstream signal pathway of AMPK, can activate autophagy and inhibit inflammation, 32 we next assessed the involvement of ULK1 in the IL-17A-induced inflammation in HUVECs.In comparison to untreated HUVECs, IL-17A treatment significantly increased the ratio of phosphorylated ULK1(Ser757)/ULK1 (Figure 3D,G) and decreased the ratio of phosphorylated ULK1(Ser555)/ULK1 (Figure 3D,F), indicating inactivation of ULK1.In contrast, Rap dramatically decreased the ratio of p-ULK1(Ser757)/ULK1 (Figure 3D,G), while increasing the ratio of p-ULK1(Ser555)/ULK1(Figure 3D,F) in the IL-17A-treated HUVECs.

F I G U R E 1
Autophagic flux is impaired in the IL-17A-induced inflammatory response of human umbilical vein endothelial cells (HUVECs).(A, B) Expression levels of the autophagy marker LC3 (LC3-I and LC3-II) proteins and p62 protein by western blot in psoriatic (n = 5) and normal HDMECs (n = 5).Bar graph showed that the ratio of LC3II/I and the p62 proteins in psoriatic HDMECs were significantly increased compared with normal HDMECs.(C) Changes in expression levels of mRNA for cytokines were assessed by qRT-PCR.Experiments included the following groups: Control (untreated cells), IL-17A (treated with 50 ng/mL IL-17A alone for 24 h), and Rap+IL-17A (treated with 200 nM Rap for 1 h, followed by IL-17A stimulation).(D) Expression levels of the autophagy marker LC3 (LC3-I and LC3-II) proteins by western blot.Bar graph showed that the ratio of LC3II/I was significantly increased in cells treated with 200 nM Rap for 1 h compared with untreated cells (Control).(E) Expression levels of p62 protein by western blot.Expression levels were normalized with β-Actin.Bar graph showed that the ratio of p62/β-actin was significantly reduced in Rap-treated cells compared with untreated cells (Control).(F) Changes in mRNA levels of genes related to inflammation (IL-6, IL-8), proliferation (PCNA), and differentiation (keratin 5, Involucrin) in HaCaT culture alone, HaCaT + Rap-HUVECs, and HaCaT + CQ-HUVECs were assessed by qRT-PCR.First, Rap induced the enhancement of autophagy in HUVECs, and CQ induced the inhibition of autophagy in HUVECs, and then cocultured with HaCaT cells for 3 days, respectively.(G, H) Expression levels of the autophagy marker LC3 (LC3-I and LC3-II) proteins by western blot in Control, Dor.(K) and (L) Bar graph showed that HUVECs treated with Dor enhanced the ratio of LC3II/I and the p62 proteins compared with untreated cells.(I) and (M) Expression levels of LC3 (LC3-I and LC3-II) proteins by western blot.Experiments included the following groups: Control (untreated cells), IL-17A (treated with 50 ng/mL IL-17A alone for 24 h), Rap+IL-17A (pretreated with 200 nM Rap for 1 h before IL-17A stimulation), CQ + Rap+IL-17A (treated with 50 μM CQ for 1 h, followed by Rap and IL-17A treatment), and Dor + Rap+IL-17A (treated with 20 μM Dor for 2 h, followed by Rap and IL-17A treatment).(J) and (N) Expression levels of p62 protein by western blot.(O) and (P) Quantification of the numbers of junctions and meshes in Control (untreated cells), Dor.Representative photomicrographs of capillary-like structures in (Q).The data are expressed as the mean ± Standard deviation (SD).'A, B, D, E, G, H, O, P, and Q' were analyzed by the two-tailed Student's t-test and 'C, F, I, and J' were analyzed by one-way analysis of variance (ANOVA) followed by the least significant difference method (LSD), n = 5/group.*p < 0.05, **p < 0.01.| 333 WANG et al. of flow cytometric analysis demonstrated an increase in the apoptosis in IL-17A treatment versus untreated controls (Figure 4A,B,D).Similarly, IL-17A treatment significantly increased caspase-3 activity in comparison to untreated controls (Figure 4E).Interestingly, induction of autophagy with Rap decreased apoptotic cells (Figure 4B-D) and caspase-3 activity (Figure 4E) in IL-17A-treated HUVECs.These results suggested that autophagy can inhibit IL-17-induced apoptosis in HUVECs.
NO content and NOS activity were measured in HUVECs treated with or without IL-17A.The results revealed that IL-17A significantly decreased the intracellular levels of NO and NOS activity in HUVECs (Figure3H,I).However, Rap dramatically increased NO levels and NOS activity in IL-17A-treated HUVECs.Moreover, inhibition of either autophagy or AMPK markedly blocked the effect of Rap on NO content and NOS activity in IL-17A-treated HUVECs (Figures3H,I

and
4F).These results suggested that autophagy increases the levels of NO and NOS activity in IL-17A-treated HUVECs through activation of AMPK signaling.
found that IL-17A induced increased production of VEGF and angiogenesis via Stat3 pathway.Our study showed that the production of IL-17A-induced VEGF regulated by autophagy was not through AMPK, but possibly through other pathways.The underlying mechanisms by which autophagy regulates IL-17A-induced endothelial cells angiogenesis and inflammation is not clear.It is likely via AMPK-mTOR pathways.AMPK and mTOR have inverse effects on autophagy induction via phosphorylating F I G U R E 2 IL-17A is involved in angiogenesis by inhibiting autophagy through AMPK signaling pathway.Representative photomicrographs of capillary-like structures in (A) Control, (B) IL-17A, (C) Rap+IL-17A, (D) CQ+ Rap+IL-17A, and (E) Dor + Rap+IL-17A.Scale bar = 100 μm.(F) Quantification of the numbers of junctions and meshes in different groups; (H and I) Confocal microscopy images of LC3 immunofluorescence staining.The LC3 was stained with FITC (green) and the cell nucleus with DAPI (blue).Scale bar = 80 μm.(magnification = 10 × 20 = 200).(G and L) Quantification of intensity of immunofluorescence staining.(J) Representative western blot images of AMPKα, p-AMPKα(Thr172).(K) Expression levels of p-AMPKα(Thr172), presented as ratio of p-AMPKα to AMPKα.The data are expressed as the mean ± Standard deviation (SD).(G, H) were analyzed by the two-tailed Student's t-test and 'A-F, I-L' were analyzed by one-way analysis of variance (ANOVA) followed by the least significant difference method (LSD), n = 5/group.*p < 0.05, **p < 0.01.| 335 WANG et al.

F I G U R E 4 F I G U R E 3
Induction of autophagy decreases IL-17A-induced apoptosis in human umbilical vein endothelial cells.Experiments included the following groups: Control (untreated cells), IL-17A (treated with 50 ng/mL IL-17A alone for 24 h), Rap (treated with 200 nM alone for 1 h), and Rap+IL-17A (pretreated with 200 nM Rap for 1 h before IL-17A stimulation).(A-C) Representative images of apoptosis ratio in the controls (A), IL-17A (B) and Rap+IL-17A (C) detected using Annexin V/propidium iodide (PI) double staining by flow cytometry.Total apoptotic rate is calculated as percentage of the number of Annexin V-positive cells; (D) Apoptosis rate; (E) The caspase-3 activity was determined using a Caspase 3 Activity Assay Kit.(F) Schematic representation of the effects of autophagy on inflammation, angiogenesis, and endothelial function upon IL-17A signaling.Data are presented as mean ± SD and were analyzed by ANOVA followed by LSD, n = 5/group.*p < 0.05, **p < 0.01.Autophagy mediated by AMPK /mTOR/ ULK1 signaling pathway inhibits IL-17A-induced human umbilical vein endothelial cells inflammation.(A) CCL20 content in culture supernatants measured by ELISA.(B) IL-8 levels in culture supernatants measured by ELISA.(C) VEGF levels in supernatants measured by ELISA.(D) Representative western blot images of mTOR, p-mTOR(Ser2448), ULK1, p-ULK1(Ser555), and p-ULK1(Ser757) proteins.(E) Expression levels of p-mTOR(Ser2448), presented as ratio of p-mTOR to mTOR.(F, G) Expression levels of p-ULK1(Ser555) and p-ULK1(Ser757), respectively, normalized with ULK1.NOS activity (H) and NO content (I) were measured by respective commercial kit.Data are expressed as mean ± SD and were analyzed by ANOVA followed by LSD, n = 5/group.*p < 0.05, **p < 0.01.
leading to both endothelial dysfunction and alterations in inflammatory responses and apoptosis.In this study, we demonstrated that induction of autophagy can increase NO production, while inhibiting apoptosis and inflammation, leading to improvement of endothelial function.NO is synthesized by the NO synthase and there are three different types of NOS: Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).eNOS is mainly expressed in vascular endothelial cells, although it is also expressed in other special cell groups that have important circulatory effects.60