Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro

INTRODUCTION

Diabetic retinopathy is the primary cause of adult blindness in the world. Obesity, which is due to weight gain caused by increased adipose tissue, is usually related to lifestyle-related cardiovascular and metabolic diseases, for instance, diabetes, hypertension, and hyperlipidemia which lead to a higher risk of developing vascular disease or atherosclerosis.Adipose tissue is a vital endocrine organ that secretes many bioactive substances, such as leptin, chemerin and apelin

,collectively known as adipocytokines

. Evidence suggests that adipocytokines play a pathophysiological role in complications related to obesity and diabetes

. Leptin is an adipocytokine that acts directly on the hypothalamus, regulating energy intake and consumption

. Leptin receptors are expressed not only in the hypothalamus but also in various peripheral tissues

. Leptin concentrations in the peripheral blood of obese people is proportional to the degree of obesity,suggesting that leptin may play a pathophysiological role in obesity-related complications

. In addition, leptin is closely related to energy metabolism and insulin resistance

. Serum leptin concentrations were found to be significantly higher in hyperplastic diabetic retinopathy patients than non-proliferative retinopathy patients

. In proliferative diabetic retinopathy patients, intravitreous leptin concentrations were higher

,suggesting that leptin may play a role in proliferative diabetic retinopathy. The most crucial pathological change of proliferative diabetic retinopathy is the formation of retinal neovascularization

. However, it remains unclear whether leptin is involved in the process. Thus, this study aimed to clarify the role and mechanism of action of leptin on retinal neovascularization

.

MATERIALS AND METHODS

The Matrigel tube formation experiment was used to detect the formation of tubular structures in control, 100 ng/mL leptin- and AG-490- (100 ng/mL leptin+10 μmol/L AG-490) treated cells. We found that inhibition of the JAK/STAT pathway by AG-490 inhibited the number of tube formations in RF/6A cells induced by 100 ng/mL leptin, significantly (

=80.50,

=0.001; Figure 7).

RF/6A cells in the logarithmic growth phase were plated at a density of 5×10

/mL in 96-well plates (100 μL cell suspension per well). Cells were treated as described above for 24h, then cultured at 37

C for a further 24h. Next, cells were cultured for 4h at 37

C with 10 μL CCK8 in each well. Plates were read at OD 450 using an Enzyme standard instrument (ThermoFisher Scientific, MULTISKAN MK3).

In the final stages of varieties of ocular diseases, including diabetic retinopathy and retinal vein occlusion, retinal neovascularization often leads to catastrophic vision loss. In this study, it was demonstrated that leptin can stimulate RF/6A cells' proliferation, migration, and tube formation

.As an adipocytokine, leptin was found to be expressed nearly exclusively in adipose tissue in mammals

. It acts directly on the hypothalamus to regulates energy intake and expenditure.It has been shown that leptin expressed in retina, choroid,sclera, vitreous and tears

. Serum levels of leptin were closely related to obesity and obesity-associated microvascular complications

. Our findings are similar to previous studies

. Cell migration, proliferation, and tube formation are vital factors in the formation of new blood vessels

.Our data suggest that leptin could promote new blood vessels formation. Furthermore, our findings confirm that leptin could activate the JAK2/STAT3 signaling pathway in RF/6A cells,and that inhibition of the JAK2/STAT3 signaling pathway through AG-490 blocks leptin-induced endothelial cells'proliferation, migration, and tube formation.

2.1.7 稳定性试验 分别精密吸取“2.1.3”项制备的同一姜黄供试品溶液（1号）10 μL，分别在0、2、4、6、8、12、18、24 h按“2.1.1”项下色谱条件测定，姜黄样品中姜黄素、去甲氧基姜黄、双去甲氧基姜黄素的峰面积RSD分别为0.8%、1.4%、2.0%，结果表明供试品溶液在24 h内稳定。

更多的时候因为“传统”发酵，引来了许多盗墓贼或梁上君子，我也不例外，剥离了传统与现代与生活，模糊了审美的视线，使美的发现愈来愈难。于是，困惑中我在画外追问：前人的生活与现代人的生活？我的灵魂因此而漂泊，在漂泊中发现，生活带来最初的冲动是源于心动，是无法抵御和抑制的激情奔腾。我的画是从这里开始，以验证着一个为个性化的漂泊灵魂寻找归宿和充满着执着与真正的人生的追恋。

The Matrigel tube formation assay revealed that the number of tubular structures that developed in control, 50, 100, and 200 ng/mL leptin groups was 38.6±2, 50.1±3.5, 67.7±5.2, and 84±5.6,respectively (

=68.7,

=0.001). These findings suggest that leptin can promote RF/6A cell tube formation (Figure 3).

Data are analyzed using SPSS 20.0 software and given as the mean±standard deviation. Comparison of the means between groups was analyzed by single-factor ANOVA,while the LSD method was used to compare two groups.

＜0.05 was considered to be statistically significant.

RESULTS

AG-490 is a tyrosine kinase inhibitor that inhibits STAT-3 activation by selectively blocking JAK2. Thus, JAK/STAT-3 activation is inhibited using AG-490 selectively. To verify the inhibitory effect of AG-490 on the JAK/STAT signaling pathway, cells were separated into control, 100 ng/mL leptin,and AG-490 (100 ng/mL leptin+10 μmol/L AG-490) groups.We found that AG-490 could inhibit leptin-induced JAK2 and STAT3 phosphorylation (P-JAK2:

=63.29,

=0.001;P-STAT3:

=70.68,

=0.001; Figure 4).

Migration of endothelial cells is essential during early angiogenesis.To determine the effect of leptin on angiogenesis, we used the Transwell assay to measure the cell migration ability of RF/6A cells. We found that leptin led to an obvious increase in the migration of endothelial cells after 24h significantly.The number of migrating cells in the control, 50, 100, and 200 ng/mL leptin groups were 43±4.5, 65±6.5, 83.5±7.2, and 112.1±7.6, respectively (

=145.8,

=0.001; Figure 2).

Cells were lysed and then total protein was extracted. Samples were separated by SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. Overnight at 4

C, membranes were incubated with the following primary antibodies: GAPDH (1:1000), JAK2 (1:1000), p-JAK2 (1:1000),STAT3 (1:1000), and p-STAT3 (1:2000). The JAK2 and p-STAT3 antibodies were purchased from Cell Signaling Technologies (USA), the p-JAK2 antibody was obtained from Abcam (UK), the STAT3 antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd. (China) and the GAPDH antibody was purchased from Hangzhou Xianzhi Biological Co., Ltd. (China). After washing, membranes were incubated with the corresponding secondary HRP-conjugated antibodies at 37

C for 2h. Protein bands were analyzed by BandScan.

To determine the effect of leptin on the JAK/STAT signaling pathway, we used Western blot analyses to detect the protein expression levels of JAK2, p-JAK2, STAT3, and p-STAT3.It was found that incubation with leptin increased the phosphorylation levels of JAK2 and STAT3 were significantly increased in RF/6A cells significantly (P-JAK2:

=47.33,

=0.001; P-STAT3:

=111.86,

=0.001).

The effects of different concentrations of leptin on cell proliferation were examined using the CCK8 assay. As shown in Figure 1, varying concentrations of leptin (20, 50, 100, and 200 ng/mL) led to a significant dose-dependent increase in RF/6A proliferation after 24h (

＜0.05).

The role of the JAK/STAT signaling pathway on leptin-induced cell proliferation was examined using AG-490, the selective JAK/STAT signaling pathway inhibitor. The effects of the following treatment groups: control,100 ng/mL leptin, and AG-490 (100 ng/mL leptin+10 μmol/L AG-490) on cell proliferation were assessed using the CCK8 assay. We found that AG-490 could significantly inhibit the proliferation of RF/6A cells induced by 100 ng/mL leptin(

=131.99;

=0.001; Figure 5).

To determine the role of the JAK/STAT signaling pathway on leptin-induced cell migration, the Transwell assay was used to assess the levels of migration in control,100 ng/mL leptin, and AG-490 (100 ng/mL leptin+10 μmol/L AG-490)-treated cells. We found that inhibition of the JAK/STAT pathway by AG-490 significantly inhibited the migration of RF/6A cells induced by 100 ng/mL leptin (

=150.31;

=0.001;Figure 6).

RF/6A cells were purchased from the Cell Bank of Typical Cell Culture Preservation Committee, Chinese Academy of Sciences. Cell culture consumables were purchased from Biyuntian Biotechnology Company (China). RF/6A cells growing in good condition were digested by 0.25% trypsin, collected and centrifuged at 1000 rpm for 5min, and then moistened with phosphate buffered saline (PBS) twice to remove the residual serum.According to different treatment, RF/6A cells were randomly divided into four groups: control, 50, 100, and 200 ng/mL leptin groups. In leptin treatment group, different concentrations of leptin (0, 50, 100, and 200 ng/mL) were added to the culture medium for culture. Recombinant human leptin was purchased from Peprotech (USA). In leptin+AG-490 treatment group,leptin (100 ng/mL) and AG-490 (10 μmol/L) were added to the cell culture medium. AG-490 inhibitor was purchased from AbMole (USA). RF/6A cells were treated for 24h, then assayed as outlined below.

DISCUSSION

Treated RF/6A cells were digested with 0.25% trypsin, collected and diluted(1×10

cells/mL). Medium containing 10% FBS (800 μL) was placed into a 24-well plate containing a Transwell chamber.One hour later, 200 μL of cell suspension was added to the upper section of the Transwell chamber and incubated at 37

C in an atmosphere of 5% CO

for 24h. Cells were washed with PBS and fixed in 10% methanol for 30min after the Transwell chamber was removed. The membrane was stained with 5%crystal violet at room temperature for 20min. After washing with PBS, samples were observed and photographed under the inverted microscope (Nikon, ECLIPSE Ts2).

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The JAK/STAT pathway was initially regarded as a transmitter of interferon signaling on cells

. The JAK family is comprised of non-receptor tyrosine protein kinases and includes JAK1,JAK2, JAK3, and Tyk2. The JAK family has a C-terminal tyrosine kinase domain, while the N-terminus plays a regulatory role when JAK couples with the receptor protein. The JAK/STAT signaling pathway relays signals from the membrane to the nucleus,

tyrosine phosphorylation, leading to the activation and nuclear translocation of STATs

. Activation of the upstream JAK2 kinase by cytokines or growth factors results in tyrosine phosphorylation and activation of STAT3,which in turn regulates the transcription of target genes and mediates diverse biological effects including cell proliferation,differentiation, and apoptosis

. The activation of STATs is transient in normal cells, lasting only a few minutes or hours.In tumor cells, abnormal and persistent activation of STAT3 is closely associated with the biological behavior of the tumor and its pathogenesis

.

随着我国经济的不断发展，人们的生活水平逐渐提高，如今水果市场的销量有了非常大的提升。在这样的情况下，提升葡萄产量、提早葡萄上市时间对于提升果农的经济收益有着非常大的帮助。通过应用大棚进行促成栽培，合理的对早中晚品种进行搭配，可以使葡萄更早的上市，延长葡萄的销售期。因此对于葡萄大棚促成栽培技术的研究对于我国葡萄种植有着非常重要的意义。本文通过对葡萄大棚促成栽培技术的技术要点进行了分析，希望可以为果农和研究人员提供一些参考。

In addition to cell proliferation, differentiation and immune regulation

, the JAK2/STAT3 signaling pathway also plays a role in the process of neovascularization

and the regulation of VEGF expression. It has been shown that JAK2/STAT3 can activate VEGF expression in some studies

,while inhibition of the JAK2/STAT3 pathway inhibits VEGF expression

. Abnormal activation of STAT3 regulates the STAT3/VEGF signaling pathway and promotes VEGF expression

. VEGF expression can be down-regulated by inhibiting the JAK2/STAT3 pathway

. The well-established JAK/STAT pathway inhibitor AG-490, also known as Tyrphostin AG-490, was used in our study to specifically block the phosphorylation and activation of JAK2 and STAT3

.Consistent with previous studies, leptin can activate the JAK/STAT signaling pathway

. Thus, leptin may increase the expression of VEGF in retinal endothelial cells by activating the JAK2/STAT3 pathway, and then promotes neovascularization.

Due to the shortage of human primary retinal vascular endothelial cells, RF/6A cells have been widely used in experiments of angiogenesis, cell differentiation and various drug and environmental treatments in the choroid and retina

. In this experiment, the RF/6A cells were selected because they were easy to obtain, stable and already widely used. Although RF/6A cells have been used for many years, they may also be different from real human retinal vascular endothelial cells.Further research is required to determine whether leptin has the same effect on human retinal microvascular endothelial cells and retinal neovascularization

.

In conclusion, our study shows that leptin can activate the JAK2/STAT3 signaling pathway, resulting in increased RF/6A cell proliferation, migration, and tube formation, which may participate in neovascularization. If it can be proved by more studies that leptin promotes the angiogenesis of retinal endothelial cells

activation of the JAK2/STAT3 signaling pathway, the inhibitor or antagonist of this pathway could prevent the neovascularization of diabetic retinopathy. Further research is required to demonstrate whether leptin has the same effect on retinal neovascularization

.

答案来自第二次世界大战。二战期间，美国海军开发了声呐和回声探测之类的新技术，以寻找德国潜艇。到了和平时期，美国海军发现留下来了一些令人印象深刻的海底地形图。海洋研究继续得到了资助，并形成了第一张详尽的海底地图，由美国地质学家玛丽·萨普（Marie Tharp）和布鲁斯·希森（Bruce Heezen）于20世纪50年代末公开出版。

在工程伦理教育中，要注重对个性，即人的个性特征的概括，包括人的遗传特征，人的独立性，人的发展的丰富性。工程师根据自己的喜好开展活动，选择自己的方向，使他们的共性追求与个性特征相统一。马克思说过：“有个性的个人与偶然的个人之间的差别，不仅是逻辑的差别，而且是历史的事实。”所以，在工程伦理教育中，要突出工程师的个性形成，彰显社会实践、环境、遗传和伦理教育的作用。基于此，对工程师个性的“培塑”应包括三个方面，即培育和发展自主性、能动性和创造性，发挥工程伦理教育在培育工程师个性发展过程中的导向作用，造就合格的工程师。

Supported by the Matching Funds of the National Natural Science Foundation of China (No.XYFYPT-2020-01); the Natural Science Foundation of Shaanxi Province (No.2020JM-685; No.2021JM-547); the Fundamental Research Funds for the Central Universities(No.1191329116); the Foundation of Xi'an Health Committee(No.2020MS07).

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1 Li R, Du JH, Yao Y, Yao GM, Wang XD. Adiponectin inhibits high glucose-induced angiogenesis

inhibiting autophagy in RF/6A cells.

2019;234(11):20566-20576.

2 Du JH, Li X, Li R, Xu L, Ma RR, Liu SF, Zhang Z, Sun HZ. Elevation of serum apelin-13 associated with proliferative diabetic retinopathy in type 2 diabetic patients.

2014;7(6):968-973.

3 Du JH, Li R, Xu L,

. Increased serum chemerin levels in diabetic retinopathy of type 2 diabetic patients.

2016;41(1):114-120.

4 Funcke JB, Scherer PE. Beyond adiponectin and leptin: adipose tissue-derived mediators of inter-organ communication.

2019;60(10):1648-1684.

5 Cao HM. Adipocytokines in obesity and metabolic disease.

2014;220(2):T47-T59.

6 Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals.

1998;395(6704):763-770.

7 Guzmán A, Hernández-Coronado CG, Rosales-Torres AM, Hernández-Medrano JH. Leptin regulates neuropeptides associated with food intake and GnRH secretion.

(

) 2019;80(1):38-46.

8 Jiang L, Su HR, Wu XY,

. Leptin receptor-expressing neuron Sh2b1 supports sympathetic nervous system and protects against obesity and metabolic disease.

2020;11(1):1517.

9 Seoane-Collazo P, Martínez-Sánchez N, Milbank E, Contreras C.Incendiary leptin.

2020;12(2):E472.

10 Yadav A, Kataria MA, Saini V, Yadav A. Role of leptin and adiponectin in insulin resistance.

2013;417:80-84.

11 Ghadge AA, Khaire AA. Leptin as a predictive marker for metabolic syndrome.

2019;121:154735.

12 Afarid M, Attarzadeh A, Farvardin M, Ashraf H. The association of serum leptin level and anthropometric measures with the severity of diabetic retinopathy in type 2 diabetes mellitus.

2018;7(4):156-162.

13 Gariano RF, Nath AK, D'Amico DJ, Lee T, Sierra-Honigmann MR. Elevation of vitreous leptin in diabetic retinopathy and retinal detachment.

2000;41(11):3576-3581.

14 Hernández C, Lecube A, Castellanos JM, Segura RM, Garat M,García-Arumí J, Simó R. Intravitreous leptin concentrations in patients with proliferative diabetic retinopathy.

2004;24(1):30-35.

15 Roychowdhury S, Koozekanani D, Parhi K. Automated detection of neovascularization for proliferative diabetic retinopathy screening.

2016;2016:1300-1303.

16 Wang Y, Lin WW, Ju JH. microRNA-409-5p promotes retinal neovascularization in diabetic retinopathy.

2020;19(11):1314-1325.

17 Zhang YY, Chua S Jr. Leptin function and regulation.

2017;8(1):351-369.

18 Kükner A, Colakoğlu N, Serin D, Alagӧz G, Celebi S, Kükner AS.Effects of intraperitoneal vitamin E, melatonin and aprotinin on leptin expression in the Guinea pig eye during experimental uveitis.

2006;84(1):54-61.

19 Hao R, Liu Y, Li XM. Leptin's concentration in tears and dry eye: a clinical observational study.

2021;14(1):83-88.

20 Maberley D, Cui JZ, Matsubara JA. Vitreous leptin levels in retinal disease.

(

) 2006;20(7):801-804.

21 Sierra-Honigmann MR, Nath AK, Murakami C,

. Biological action of leptin as an angiogenic factor.

1998;281(5383):1683-1686.

22 Yu F, Fu RQ, Liu L, Wang XT, Wu TT, Shen W, Gui ZP, Mo XM, Fang B, Xia LG. Leptin-induced angiogenesis of EA.Hy926 endothelial cells via the Akt and Wnt signaling pathways

and

.

2019;10:1275.

23 Gentile MT, Pastorino O, Bifulco M, Colucci-D'Amato L. HUVEC tube-formation assay to evaluate the impact of natural products on angiogenesis.

2019;(148).

24 Sun LL, Li WD, Lei FR, Li XQ. The regulatory role of microRNAs in angiogenesis-related diseases.

2018;22(10):4568-4587.

25 Quintás-Cardama A, Verstovsek S. Molecular pathways: jak/STAT pathway: mutations, inhibitors, and resistance.

2013;19(8):1933-1940.

26 Boyle DL, Soma K, Hodge J, Kavanaugh A, Mandel D, Mease P,Shurmur R, Singhal AK, Wei N, Rosengren S, Kaplan I, Krishnaswami S, Luo Z, Bradley J, Firestein GS. The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis.

2015;74(6):1311-1316.

27 Xiong AL, Yang ZD, Shen YC, Zhou J, Shen Q. Transcription factor STAT3 as a novel molecular target for cancer prevention.

2014;6(2):926-957.

28 Xu YC, Zhang QY, Zhou J, Li ZL, Guo JY, Wang WN, Wang W.Down-regulation of SOX18 inhibits laryngeal carcinoma cell proliferation, migration, and invasion through JAK2/STAT3 signaling.

2019;39(7):BSR20182480.

29 Cheng X, Yeung PKK, Zhong K, Zilundu PLM, Zhou LH, Chung SK.Astrocytic endothelin-1 overexpression promotes neural progenitor cells proliferation and differentiation into astrocytes via the Jak2/Stat3 pathway after stroke.

2019;16(1):227.

30 Zhang JW, Sikka S, Siveen KS, Lee JH, Um JY, Kumar AP, Chinnathambi A, Alharbi SA, Basappa, Rangappa KS, Sethi G, Ahn KS. Cardamonin represses proliferation, invasion, and causes apoptosis through the modulation of signal transducer and activator of transcription 3 pathway in prostate cancer.

2017;22(1):158-168.

31 Leone Roberti Maggiore U, Ferrero S. Methodological concerns regarding levels of vascular endothelial growth factor (VEGF) in serum of women with endometriosis.

2015;8(4):485-486.

32 Dhapare S, Sakagami M. Salvianolic acid B as an anti-emphysema agent I:

stimulation of lung cell proliferation and migration,and protection against lung cell death, and

lung STAT3 activation and VEGF elevation.

2018;53:107-115.

33 Chen M, Zou WY, Chen MM, Cao L, Ding JH, Xiao W, Hu G.Ginkgolide K promotes angiogenesis in a middle cerebral artery occlusion mouse model

activating JAK2/STAT3 pathway.

2018;833:221-229.

34 Ataie-Kachoie P, Morris DL, Pourgholami MH. Minocycline suppresses interleukine-6, its receptor system and signaling pathways and impairs migration, invasion and adhesion capacity of ovarian cancer cells:

and

studies.

2013;8(4):e60817.

35 Yun S, Yun CW, Lee JH, Kim S, Lee SH. Cripto enhances proliferation and survival of mesenchymal stem cells by up-regulating JAK2/STAT3 pathway in a GRP78-dependent manner.

(

)2018;26(5):464-473.

36 Lei Z, Duan HJ, Zhao TF, Zhang YX, Li GQ, Meng JH, Zhang SZ,Yan WQ. PARK2 inhibits osteosarcoma cell growth through the JAK2/STAT3/VEGF signaling pathway.

2018;9(3):375.

37 Qian CJ, Wang JJ, Yao J, Wang L, Xue M, Liu WL, Si JM.Involvement of nuclear JAK2 signaling in AG490-induced apoptosis of gastric cancer cells.

(

) 2013;296(12):1865-1873.

38 Joung YH, Na YM, Yoo YB, Darvin P, Sp N, Kang DY, Kim SY,Kim HS, Choi YH, Lee HK, Park KD, Cho BW, Kim HS, Park JH, Yang YM. Combination of AG490, a Jak2 inhibitor, and methylsulfonylmethane synergistically suppresses bladder tumor growth

the Jak2/STAT3 pathway.

2014;44(3):883-895.

39 Erkasap S, Erkasap N, Bradford B, Mamedova L, Uysal O, Ozkurt M,Ozyurt R, Kutlay O, Bayram B. The effect of leptin and resveratrol on

/

pathways and Sirt-1 gene expression in the renal tissue of ischemia/reperfusion induced rats.

2017;118(8):443-448.

40 Mullen M, Gonzalez-Perez RR. Leptin-induced JAK/STAT signaling and cancer growth.

(

) 2016;4(3):E26.

41 Pijet M, Pijet B, Litwiniuk A, Pajak B, Gajkowska B, Orzechowski A. Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways.

2013;61(2):445-454.

42 Wu K, Tan XY, Xu YH, Chen QL, Pan YX. JAK and STAT members of yellow catfish Pelteobagrus fulvidraco and their roles in leptin affecting lipid metabolism.

2016;226:14-26.

43 Warden C, Barnett JM, Brantley MA Jr. Taurocholic acid inhibits features of age-related macular degeneration

.

2020;193:107974.

44 Jiang HY, Wu MJ, Liu YM, Song LP, Li SF, Wang XW, Zhang YF,Fang JX, Wu SZ. Serine racemase deficiency attenuates choroidal neovascularization and reduces nitric oxide and VEGF levels by retinal pigment epithelial cells.

2017;143(3):375-388.