Effects of leptin-modified human placenta-derived mesenchymal stem cells on angiogenic potential and peripheral inflammation of human umbilical vein endothelial cells (HUVECs) after X-ray radiation

Effects of leptin-modified human placenta-derived mesenchymal stem cells on angiogenic potential and peripheral inflammation of human umbilical vein endothelial cells (HUVECs) after X-ray radiation 转染瘦素人胎盘源间充质干细胞对经X射线辐照后人脐静脉内皮细胞的成血管潜能和周围炎症的影响 2 first-author 1 3 1 1 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:liu_zh@jlu.edu.cn †E-mail:liu_zh@jlu.edu.cn liu_zh@jlu.edu.cn Department of Prosthodontics, Hospital of Stomatology, Jilin University, Changchun 130021, China Department of Prosthodontics, Hospital of Stomatology, Jilin University, Changchun 130021, China Department of Thoracic Surgery, the Second Hospital of Jilin University, Changchun 130041, China Department of Thoracic Surgery, the Second Hospital of Jilin University, Changchun 130041, China Department of Radiology, the Second Hospital of Jilin University, Changchun 130041, China Department of Radiology, the Second Hospital of Jilin University, Changchun 130041, China Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor ( VEGF ) and basic fibroblast growth factor ( bFGF )) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF ). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation. 目的 2 放创复合伤是一种以血管损伤和促炎细胞因子缺乏为特征的难愈性创伤.瘦素(leptin)的直接应用在血管生成和炎症中起着重要作用.本研究构建了一种可持续稳定的leptin表达系统--leptin修饰的人胎盘来源间充质干细胞(HPMSCs/leptin),并探究其对经X射线辐照后的人脐静脉内皮细胞(HUVECs)的成血管潜能及周围炎症的影响和潜在机制. 创新点 2 可持续稳定的leptin表达系统(HPMSCs/leptin)促进受X射线辐照后HUVECs的成血管潜能及外周炎症反应,有助于解决放创复合伤伤口愈合过程中血管损伤和促炎因子缺乏的问题. 方法 2 利用慢病毒载体将leptin基因转染HPMSCs获得HPMSCs/leptin.采用X射线单次照射HUVECs,剂量为20 Gy.细胞迁移侵袭实验技术(Transwell)检测照射后HUVECs的迁移情况.在Transwell体系的基础上,建立HPMSCs与受辐照HUVECs共培养体系.CCK-8比色法测定细胞增殖.酶联免疫吸附法(ELISA)检测促炎细胞因子(粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-1α(IL-1α)、IL-6和IL-8)的分泌.实时荧光定量聚合酶链式反应(RT-qPCR)检测促血管生成因子(VEGF和bFGF)mRNA的表达.蛋白免疫印迹法(western blot)检测核因子κB(NF-κB)和JAK/STAT信号通路的相关分子表达. 结论 2 可持续稳定的leptin表达系统(HPMSCs/leptin)具有更好的细胞增殖、迁移和成血管潜能.HPMSCs/leptin单独培养和HPMSCs/leptin与受辐照HUVECs共培养体系中,促炎细胞因子的分泌增加与NF-κB和JAK/STAT信号通路的相互作用有关.HPMSCs/leptin可能促进X射线照射后HUVECs的成血管潜能和外周炎症反应. Leptin Angiogenesis Pro-inflammatory cytokines X-ray radiation Human placenta-derived mesenchymal stem cells (HPMSCs) Human umbilical vein endothelial cells (HUVECs) 瘦素表达系统 血管生成 促炎细胞因子 X线辐射 人脐静脉内皮细胞 Introduction 1 Combined radiation-wound injury (CRWI) is a refractory wound, which occurs in most malignant tumor patients with postoperative or preoperative radiation therapy. Compared with normal wound healing, the CRWI is refractory to healing mainly due to but not limited: (1) blood vessel damage brings about tissue anoxia and ischemic injury (van den Brenk et al., 1974a 1974a , 1974b 1974b ; Hao et al., 2009 2009 ); (2) inhibition of the hematopoietic function leads to a decrease in the total number of inflammatory cells and the cells recruited to the wound site (Hao et al., 2009 2009 ). Thus, angiogenesis and pro-inflammation are the critical points of CRWI healing. Leptin, a 16-kDa adipose tissue-specific cytokine, is mainly secreted from white adipose tissue (Adya et al., 2015 2015 ; Park and Ahima, 2015 2015 ). Leptin possesses multiple established physiological roles in the stable control of body weight, lipid metabolism, hematopoiesis, and body heat production (Frühbeck, 2006 2006 ). Importantly, the widespread expression of leptin receptors in peripheral tissues contributes to the high functional polymorphism of this hormone, including metabolism, bone remodeling, wound healing, and reproduction (Lancha et al., 2012 2012 ). Apart from the metabolic role, leptin is also considered as an angiogenic molecule (Sierra-Honigmann et al., 1998 1998 ; Cao et al., 2001 2001 ; Park et al., 2001 2001 ; Kurtovic et al., 2015 2015 ; Manjunathan and Ragunathan, 2015 2015 ), which has been widely studied in wound healing (Liapaki et al., 2008 2008 ; Umeki et al., 2014 2014 ; Tadokoro et al., 2015 2015 ). Recently, it was reported that leptin plays a pro-inflammatory role in regulating immune responses and facilitating numerous autoimmune diseases (Abella et al., 2017 2017 ). In addition, leptin promotes the stimulation and proliferation of various immune cells to generate and secrete inflammatory cytokines (Pérez-Pérez et al., 2017 2017 ). However, the effective concentration of local direct application of leptin often varies with individual differences and experimental conditions. Thus, it is worth considering the establishment of a sustained and stable leptin expression system. Human placenta-derived mesenchymal stem cells (HPMSCs) belong to extra-fetal tissue-derived stem cells (Duscher et al., 2016 2016 ) and can be isolated from discarded placental tissue (Alviano et al., 2007 2007 ). The HPMSCs have plenty of inherent advantages: no invasive procedures, easier to access, fewer ethical restrictions, considerable multipotency, low immunogenicity, inability to form invasive colonies, and immunomodulatory effects (Liu et al., 2009 2009 ; Lee et al., 2012 2012 ; Sabapathy et al., 2012 2012 ; Duscher et al., 2016 2016 ). These advantages suggest that HPMSCs may be ideal leptin carriers and could be employed to establish a sustained and stable gene expression system. To this end, in this study, we transfected leptin gene into HPMSCs by lentiviral vectors and investigated the effects of leptin-modified HPMSCs (HPMSCs/leptin) on angiogenesis and pro-inflammation of irradiated human umbilical vein endothelial cells (HUVECs). We speculated that the leptin expression system may be a positive regulator of angiogenesis and pro-inflammation during the healing of CRWI. Materials and methods 1 Cells, agents, and antibodies 2 The human placenta and written informed consent for this study were provided by the Second Hospital of Jilin University (Changchun, China). The inclusion criteria of human placenta included full-term eutocous placenta without fetal malformation and being obtained from healthy lying-in women without infectious disease. HPMSCs were extracted, cultured, and identified in the Laboratory of the School of Stomatology, Jilin University. Construction of leptin was via a gene synthesis method, which was supported by Synbio Technologies (Suzhou, China). Recombinant lentiviral vectors pEB-copGFP (T2A) PURO-leptin (L.v.-pEB-copGFP (T2A) PURO-leptin) and pEB-copGFP (T2A) PURO (L.v.-pEB-copGFP (T2A) PURO) were constructed by Guangzhou Fit-gene Biotech Co., Ltd. (China). Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG), DMEM, penicillin-streptomycin, trypsin, and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA), and glutamine was obtained from Thermo Fisher Scientific (NJ, USA). HiFiScript gDNA Removal cDNA Synthesis Kit and UltraSYBR Mixture (High ROX) were obtained from Cowin Biosciences (Beijing, China). Transwell systems with 8.0 and 0.4 μm polyethylene terephthalate (PET) membranes were purchased from Corning (NY, USA). Anti-bodies against leptin, nuclear factor-κB (NF-κB) p65, inhibitor of NF-κB (IκBα), signal transducer and activator of transcription 3 (STAT3), β-Actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and enhanced chemiluminescence (ECL) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H+L) and cell counting kit-8 (CCK-8) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8 were purchased from Neobioscience Technology Co., Ltd. (Shenzhen, China). Experimental groups 2 HPMSCs were randomly divided into three groups, including HPMSCs (non-transfected), HPMSCs/NC (negative control, transfected with L.v.-pEB-copGFP (T2A) PURO), and HPMSCs/leptin (transfected with L.v.-pEB-copGFP (T2A) PURO-leptin). These groups were cultured under the same condition. Transfection of HPMSCs with lentiviral vectors 2 We cultured HPMSCs in DMEM-LG supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO 2 -containing humidified atmosphere. Cells were passaged and plated in a six-well plate, at a density of 5×10 6 cells/well. When it reached approximately 40%–50% confluence, the medium was changed to antibiotic-free DMEM-LG containing 10% FBS and cells were cultured at 37 °C in 5% CO 2 for 1 h. Respectively, 1 mL L.v.-pEB-copGFP (T2A) PURO-leptin supernatant, L.v.-pEB-copGFP (T2A) PURO supernatant, and phosphate-buffered saline (PBS) were added to the planned three groups and each group was incubated at the same condition for 6 h. The infected cells were transferred to DMEM-LG supplemented with 10% FBS and 1% penicillin-streptomycin, and cultured at 37 °C in 5% CO 2 and 95% relative humidity. After 48 h, the HPMSCs that successfully expressed L.v.-pEB-copGFP (T2A) PURO (HPMSCs/NC and HPMSCs/leptin) were screened from selection medium I (DMEM-LG containing 10% FBS, 1% penicillin-streptomycin, and 1 μg/mL puromycin), and then the screened cells were expanded in selection medium II (DMEM-LG containing 10% FBS, 1% penicillin-streptomycin, and 0.5 μg/mL puromycin). Finally, these cells were cryopreserved for the subsequent assays. The expression of copGFP was detected by an inverted fluorescence microscope (MF51, MSHOT, China). The expression of leptin mRNA was determined by real-time quantitative polymerase chain reaction (RT-qPCR) assay. GAPDH was selected as an internal control. Primers for leptin and GAPDH are shown in Table 1 1 . The expression of leptin protein was determined by western blot assay. β-Actin was selected as an internal control. Primary antibody against leptin (1:200 dilution) and secondary antibody (HRP-labeled goat anti-rabbit IgG (H+L), 1:8000 dilution) were used. RT-qPCR: real-time quantitative polymerase chain reaction; GAPDH : glyceraldehyde-3-phosphate dehydrogenase; VEGF : vascular endothelial growth factor; bFGF : basic fibroblast growth factor Gene Forward primer sequence (5'→3') Reverse primer sequence (5'→3')   GAPDH TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG   Leptin CATTTCACACACGCAGTCAGT CTGGAAGGCATACTGGTGAGG   VEGF TCACCAAGGCCAGCACATAG TTTCTCCGCTCTGAGCAAGG   bFGF CAAAAACGGGGGCTTCTTCC GTTGTAGCTTGATGTGAGGGTC Cell proliferation of HPMSCs, HPMSCs/NC, and HPMSCs/leptin 2 Anabiotic cells of the three groups were cultured in DMEM-LG supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in 5% CO 2 . When approximately 80% confluence was reached, cells were trypsinized, centrifuged, and resuspended. After the cell density was adjusted to 5×10 4 cells/mL, the cells were seeded in a 96-well plate, with approximately 5×10 3 cells in 100 μL cell suspension per well. After cells adhered, 10 μL CCK-8 solution (at a 1:10 ratio) was added per well and incubated at 37 °C in 5% for 4 h. The optical density at 450 nm (OD 450 ) was measured by a microplate reader (318CT, Peiou, China). RT-qPCR assay for VEGF and bFGF in HPMSCs, HPMSCs/NC, and HPMSCs/leptin 2 We isolated the total RNA from the three groups by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. The concentration of RNA was measured by an SMA4000 ultramicrospectrophotometer (Metrinton, China). The integrity of RNA was analyzed by 1.0% (0.01 g/mL) agarose gel electrophoresis. DNA was removed, and cDNA was synthesized by reverse transcription using HiFiScript gDNA Removal cDNA Synthesis Kit. Primers for vascular endothelial growth factor ( VEGF ), basic fibroblast growth factor ( bFGF ), and GAPDH are shown in Table 1 1 . GAPDH was selected as an internal control. The relative expression of these target genes was calculated by fold change=2 −ΔΔ C T , Δ C T = C T ( leptin ) – C T ( GAPDH ) . ELISA for pro-inflammatory cytokines of HPMSCs, HPMSCs/NC, and HPMSCs/leptin 2 Human GM-CSF, IL-1α, IL-6, and IL-8 secretion levels in the medium of HPMSCs, HPMSCs/NC, HPMSCs/leptin were detected using the appropriate ELISA kits, and the OD 450 value was measured by a microplate reader (ELX800, BioTek, USA). The NC consisted of DMEM-LG containing 10% FBS and 1% penicillin-streptomycin only. All ELISAs were carried out according to the manufacturer’s instructions. Detection of NF-κB p65, IκBα, and STAT3 proteins in HPMSCs/NC and HPMSCs/leptin by western blot assay 2 Anabiotic cells of each group were harvested and lysed. Total protein was extracted and quantified according to the bicinchoninic acid (BCA) method. Upon denaturation by heating at 100 °C for 5 min, the proteins were separated using 12% sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) and electrically transferred onto a nitrocellulose membrane with 300 mA constant current for 90 min. The membrane was blocked in 5% skimmed milk Tris-buffered saline and Tween 20 (TBST) solution at room temperature (RT) for 1 h. The blot was washed with TBST three times for 5 min each and incubated with a primary antibody against NF-κB p65 (1:500 dilution), IκBα (1:500 dilution), and STAT3 (1:500 dilution) at RT overnight. The blot was washed with TBST and incubated with a secondary antibody (HRP-labeled goat anti-rabbit IgG (H+L), 1:8000 dilution) at RT for 2 h. After being washed, the protein bands were visualized using a chemiluminescence reagent. Cell culture and treatment 2 HUVECs were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin at 37 °C in 5% CO 2 atmosphere. When approximately 90% confluence was reached, HUVECs were irradiated by an X-ray irradiator (MultiRad 225, Faxitron, USA) at a single dose of 20 Gy. Transwell migration assay 2 Transwell migration assay was conducted using a Transwell system with an 8.0 μm PET membrane. A suspension of approximately 1.0×10 5 irradiated HUVECs in 100 μL serum-free DMEM was added to the upper chamber, while a suspension of approximately 1.0×10 4 HPMSCs/leptin or HPMSCs/NC in DMEM-LG was added to the lower chamber. The non-migrating cells on the upper surface of the PET membrane were wiped off by sterile cotton swabs after 48-h incubation. Cells which had migrated to the lower surface of the PET membrane were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet for 10 min. Lastly, images were randomly captured in the objective field by an inverted fluorescence microscope (MF51, Mshot, China). Construction of co-culture systems of irradiated HUVECs and HPMSCs/NC or HPMSCs/leptin 2 The mesenchymal stem cells (HPMSCs/NC, HPMSCs/leptin) and irradiated HUVECs were co-cultured using a Transwell system with 0.4 μm PET membrane. A 400-μL suspension containing approximately 1.0×10 5 irradiated HUVECs was added to the lower chamber. Then, a 200-μL suspension containing approximately 1.0×10 4 HPMSCs/NC or HPMSCs/leptin was added to the upper chamber. The co-culture systems of irradiated HUVECs and HPMSCs/NC or HPMSCs/leptin were incubated at 37 °C in 5% CO 2 . Cell proliferation of the irradiated HUVECs co-cultured with HPMSCs/NC or HPMSCs/leptin 2 Co-cultured with HPMSCs/NC or HPMSCs/leptin for 1, 2, 3, and 4 d, the irradiated HUVECs were collected and seeded in 96-well plates at a density of 2×10 3 cells/well and incubated at 37 °C in 5% CO 2 . After 24 h, these cells were treated with 10 μL CCK-8 solution for another 2 h. The plates were subsequently subjected to a microplate reader (ELX800, BioTek, USA) for measuring absorbance at 450 nm. ELISA for the irradiated HUVEC-derived pro-inflammatory cytokines in co-culture systems 2 Human GM-CSF, IL-1α, IL-6, and IL-8 secretion levels in the medium of co-culture systems were detected using the appropriate ELISA kits, and the OD 450 value was measured by a microplate reader (ELX800, BioTek, USA). The NC for ELISA consisted of DMEM containing 10% FBS and 1% penicillin-streptomycin only. All ELISAs were carried out according to the manufacturer’s instructions. Western blot assay for NF-κB p65, IκBα, and STAT3 proteins of the irradiated HUVECs 2 After being co-cultured with HPMSCs/NC or HPMSCs/leptin for 72 h, the irradiated HUVECs were collected, and the expression levels of NF-κB p65, IκBα, and STAT3 proteins were detected by western blotting as described in Section 2.7. Statistical analysis 2 All statistical data were analyzed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean±standard deviation (SD). Comparisons among three groups were made using a one-way analysis of variance (ANOVA) complemented with the Bonferroni correction. Comparisons between the two groups were measured using a two-tailed unpaired Student’s t -test. At an α level of 0.05, P < 0.05 was considered to indicate a statistically significant difference. Results 1 Transfection efficiency of HPMSCs with lentiviral vectors 2 The structure of L.v.-pEB-copGFP (T2A) PURO-leptin is shown in Fig. 1a 1a . After transfection of the leptin gene with lentiviral vectors, the copGFP fluorescence of HPMSCs/NC and HPMSCs/leptin was observed under an inverted fluorescence microscope (Fig. 1b 1b ). The expression of leptin mRNA (Fig. 1c 1c ) and leptin protein (Fig. 1d 1d ) in HPMSCs/leptin was higher than that in the other two groups. (a) Structure of L.v.-pEB-copGFP (T2A) PURO-leptin. (b) Fluorescence microscope analysis of transfection (scale bar=200 μm). RT-qPCR assay (c) and western blot assay (d) of leptin expression. Lane 1: HPMSCs/leptin; Lane 2: HPMSCs/NC; Lane 3: HPMSCs; LTR: long terminal repeat; L.v.: lentiviral vector; RT-qPCR: real-time quantitative polymerase chain reaction; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control. Data are expressed as mean±standard deviation (SD), n =3. *** P < 0.001 Better cell proliferation in HPMSCs/leptin 2 The CCK-8 assay (Fig. 2 2 ) showed that HPMSCs/leptin had a better cell proliferation than HPMSCs/NC or HPMSCs, which suggested that leptin could promote cell proliferation of HPMSCs. HPMSCs/leptin exhibited better cell proliferation, compared with HPMSCs and HPMSCs/NC. Data are expressed as mean±standard deviation (SD), n =4. *** P < 0.001 compared with other groups. HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control; CCK-8: cell counting kit-8; OD 450 : optical density at 450 nm Increased expression of VEGF and bFGF in HPMSCs/leptin 2 The RT-qPCR showed that the relative expression of VEGF and bFGF in HPMSCs/leptin was higher than that in HPMSCs/NC and HPMSCs (Fig. 3 3 ). This confirmed that leptin could increase the expression of VEGF and bFGF , and further, promote cell proliferation and migration. Leptin increased the expression of VEGF and bFGF in HPMSCs/leptin. Data are expressed as mean±standard deviation (SD), n =3. *** P < 0.001. VEGF : vascular endothelial growth factor; bFGF : basic fibroblast growth factor; RT-qPCR: real-time quantitative polymerase chain reaction; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control Upregulated production of pro-inflammatory cytokines in HPMSCs/leptin 2 ELISA was used to quantify the concentration of secreted pro-inflammatory cytokines (human GM-CSF, IL-1α, IL-6, and IL-8) in HPMSCs/leptin, HPMSCs/NC, and HPMSCs (Fig. 4 4 ). The levels of IL-6, GM-CSF, and IL-1α secreted by HPMSCs/leptin in culture supernatant were higher than the average levels of the other two groups. However, the concentration of IL-8 did not reveal a significant difference among groups. Taken together, our current work showed that leptin promoted HPMSCs/leptin to secrete the human GM-CSF, IL-1α, and IL-6. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the concentrations of secreted pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) in HPMSCs/leptin, HPMSCs/NC, and HPMSCs. Leptin increased the human GM-CSF, IL-1α, and IL-6 secreted by HPMSCs. Data are expressed as mean±standard deviation (SD), n =3. One way analysis of variance (ANOVA) with Bonferroni correction was performed among three groups to compare the quantity of the concentrations of secreted pro-inflammatory cytokines. ** P < 0.01, *** P < 0.001. HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control Promoted expression of NF-κB p65 and STAT3 proteins in HPMSCs/leptin 2 Cells of each group were collected and lysed. The total proteins were collected and then separated by 12% (0.12 g/mL) SDS-PAGE. Results revealed that the expression of NF-κB p65 and STAT3 proteins was observed in the HPMSCs/leptin, but not in the HPMSCs/NC (Fig. 5 5 ). IκBα was not observed in any group. These results suggested that leptin could facilitate the expression of NF-κB p65 and STAT3 proteins in HPMSCs/leptin. NF-κB: nuclear factor-κB; IκBα: inhibitor of NF-κB; STAT3: signal transducer and activator of transcription 3; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control Promoted migration of the irradiated HUVECs in HPMSCs/leptin 2 Transwell migration assay was employed to assess the migration of irradiated HUVECs (Fig. 6a 6a ). Results showed that the cell migration of irradiated HUVECs was significantly stimulative in the Transwell system of irradiated HUVECs and HPMSCs/leptin (Figs. 6b and 6c 6b and 6c ), which confirmed that HPMSCs/leptin could promote the migration of irradiated HUVECs. (a) Transwell migration system was employed to investigate the migration of irradiated HUVECs induced by HPMSCs/NC or HPMSCs/leptin. (b, c) Compared with HPMSCs/NC, HPMSCs/leptin promoted the migration of irradiated HUVECs (scale bar=100 μm). Data are expressed as mean±standard deviation (SD), n =3. ** P < 0.01. HUVECs: human umbilical vein endothelial cells; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control; PET: polyethylene terephthalate Promoted cell proliferation of the irradiated HUVECs in HPMSCs/leptin 2 Based on the Transwell system, co-culture systems of HPMSCs/NC or HPMSCs/leptin and irradiated HUVECs were constructed (Fig. 7a 7a ). CCK-8 assay showed that, compared with those co-cultured with HPMSCs/NC, the irradiated HUVECs possessed better cell proliferation co-cultured with HPMSCs/leptin (Fig. 7b 7b ), which suggested that HPMSCs/leptin could promote cell proliferation of irradiated HUVECs. (a) Co-culture systems of irradiated HUVECs and HPMSCs/NC or HPMSCs/leptin. (b) HPMSCs/leptin promoted cell proliferation of irradiated HUVECs in the co-culture system of HPMSCs/leptin and irradiated HUVECs. Data are expressed as mean±standard deviation (SD), n =4. *** P < 0.001 vs. HPMSCs/NC. CCK-8: cell counting kit-8; HUVECs: human umbilical vein endothelial cells; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control; PET: polyethylene terephthalate; OD 450 : optical density at 450 nm Upregulated secretion of pro-inflammatory cytokines in co-culture systems of HPMSCs/leptin and irradiated HUVECs 2 ELISA was used to quantify the concentrations of secreted pro-inflammatory cytokines (human GM-CSF, IL-1α, IL-6, and IL-8) in co-culture systems (Fig. 8 8 ). The levels of IL-6, GM-CSF, and IL-1α secreted by the co-culture system of HPMSCs/leptin and irradiated HUVECs in culture supernatant were higher than the average levels of the co-culture system of HPMSCs/NC and irradiated HUVECs. However, the concentration of IL-8 did not reveal a significant difference between groups. Taken together, HPMSCs/leptin increased the production of the pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) in the co-culture system of HPMSCs/leptin and irradiated HUVECs. The concentrations of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) in the co-culture system of HPMSCs/leptin or HPMSCs/NC and irradiated HUVECs were calculated. HPMSCs/leptin upregulated the production of human GM-CSF, IL-1α, and IL-6 in the co-culture system of HPMSCs/leptin and irradiated HUVECs. Data are expressed as mean±standard deviation (SD), n =3. Two-tailed unpaired Student’s t -test was performed between two groups. * P < 0.05, *** P < 0.001. ELISA: enzyme-linked immunosorbent assay; HUVECs: human umbilical vein endothelial cells; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control Promoted expression of STAT3 protein in the irradiated HUVECs co-cultured with HPMSCs/NC 2 The irradiated HUVECs of each co-culture system were collected and lysed. The total proteins of irradiated HUVECs were collected and then separated by 12% (0.12 g/mL) SDS-PAGE. As shown in Fig. 9 9 , this revealed that the expression of STAT3 protein was observed in the irradiated HUVECs that were co-cultured with the HPMSCs/leptin, but not in the irradiated HUVECs that were co-cultured with the HPMSCs/NC. The irradiated HUVECs of each co-culture system expressed NF-κB p65 protein, and there was no significant difference between the two co-culture systems. IκBα was not observed in any group. These results suggested that leptin could promote the expression of STAT3 protein in irradiated HUVECs. NF-κB: nuclear factor-κB; IκBα: inhibitor of NF-κB; STAT3: signal transducer and activator of transcription 3; HPMSCs: human placenta-derived mesenchymal stem cells; NC: negative control; HUVECs: human umbilical vein endothelial cells Discussion 1 CRWI is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis (Umeki et al., 2014 2014 ; Kurtovic et al., 2015 2015 ; Manjunathan and Ragunathan, 2015 2015 ; Tadokoro et al., 2015 2015 ; Nwadozi et al., 2019 2019 ) and inflammation (Sun et al., 2018 2018 ; Yu et al., 2019 2019 ). However, the effective concentration of local direct application of leptin often varies with individual differences and experimental conditions. Therefore, it is worth considering establishing a sustained and stable leptin expression system to explore the mechanism. In our examination, we successfully transfected leptin into HPMSCs using the lentiviral vectors method, and obtained HPMSCs/leptin. Compared with our previous work, we improved the transfection efficiency of HPMSCs/leptin (Jin et al., 2014 2014 ). HPMSCs/leptin exhibited better cell proliferation, migration, and angiogenic potential. In addition, increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6), both in the single HPMSCs/leptin and in the co-culture system of HPMSCs/leptin and irradiated HUVECs, was associated with the interaction of the NF-κB signaling pathway and the Janus kinase (JAK)/STAT signaling pathway. Our study indicated that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs following X-ray radiation. Different from the methods of most related works (Umeki et al., 2014 2014 ; Kurtovic et al., 2015 2015 ; Manjunathan and Ragunathan, 2015 2015 ; Tadokoro et al., 2015 2015 ; Sun et al., 2018 2018 ; Nwadozi et al., 2019 2019 ; Yu et al., 2019 2019 ), in which leptin protein was directly added to medium, our study established a gene expression system that allowed HPMSC expression of leptin in a sustained and stable manner. By observing the copGFP fluorescence and detecting the expression of leptin mRNA and leptin protein in the HPMSCs/leptin, our work confirmed the success of constructing a leptin expression system. HPMSCs/leptin exhibited better cell proliferation than non-leptin-modified HPMSCs. This is consistent with our previous work (Jin et al., 2014 2014 ). Many previous studies have proved that leptin stimulates VEGF and bFGF expression in angiogenesis (Park et al., 2001 2001 ; Manjunathan and Ragunathan, 2015 2015 ). In our study, the expression of VEGF and bFGF in HPMSCs/leptin was significantly higher than that in non-leptin-modified HPMSCs. It is confirmed that leptin could promote HPMSCs to express more VEGF and bFGF , and further promote angiogenic potential. Our findings confirm that leptin increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) in the single HPMSCs/leptin. It is generally known that leptin is a pro-inflammatory factor (Abella et al., 2017 2017 ; Pérez-Pérez et al., 2017 2017 ; Yu et al., 2019 2019 ), which is able to promote the secretion levels of numerous pro-inflammatory cytokines in a variety of tissues (Nwadozi et al., 2019 2019 ; Yu et al., 2019 2019 ). Our work found that leptin could upregulate the production of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) in HPMSCs/leptin. This suggests that HPMSCs/leptin could increase the secretion levels of pro-inflammatory cytokines of itself; nevertheless, up to now, there has been no study examining this phenomenon. Our findings suggest that increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) in the single HPMSCs/leptin is associated with the interaction of the NF-κB signaling pathway and the JAK/STAT signaling pathway. It has been reported that leptin induces IL-6 expression in ligamentum flavum cells through NF-κB (Sun et al., 2018 2018 ). In addition, IL-6/gp130 is related to the activation of the JAK/STAT signaling pathway in the inflammatory processes of vascular diseases (Grote et al., 2005 2005 ). Thus, leptin upregulates pro-inflammatory cytokines and is implicated in the NF-κB signaling pathway, even the JAK/STAT signaling pathway. NF-κB p65 is an essential member of NF-κB heterodimeric protein, which induces the transcription of pro-inflammatory cytokines (Sun et al., 2018 2018 ) and is mainly involved in inflammatory responses (Baldwin, 1996 1996 ). In the HPMSCs/leptin, we observed that leptin promotes the expression of NF-κB p65 and upregulates the production of the pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6). IκBα is an inhibitory protein that maintains balance by inhibiting the inflammatory response caused by signal transduction through non-covalent interactions with NF-κB dimers in unstimulated cells. The degradation of IκBα allows the activation of NF-κB complex to translocate into the nucleus and induce gene expression (Baeuerle, 1998 1998 ). In our present study, we observed no IκBα expression in HPMSCs/leptin or HPMSCs/NC. The former may activate NF-κB complex via leptin stimulation, while the latter may be determined by the property of HPMSCs. In addition, leptin binds to the leptin receptor long isoform and transmits extracellular signals through the JAK2/STAT3 signaling pathway. STAT3 is a kind of STATs, which can be induced by leptin (Sierra-Honigmann et al., 1998 1998 ) to form active STAT3 dimers. These translocate into the nucleus to regulate gene expression and alter target cell proliferation and differentiation (Grote et al., 2005 2005 ; Abella et al., 2017 2017 ). Moreover, the function of activated STAT3 can be altered by association with NF-κB (Walker and Smith, 2005 2005 ). In HPMSCs/leptin, to some extent, the result that leptin facilitates the expression of NF-κB p65 and STAT3 could be attributed to the interaction between the NF-κB signaling pathway and the JAK/STAT signaling pathway. In HPMSCs/leptin, to some extent, our findings provide evidence for the angiogenic potential of leptin in irradiated HUVECs. Surgery and radiation are necessary for most malignant tumor patients; however, to no small extent, postoperative or preoperative radiation therapy usually results in refractory wounds (Powers et al., 1967 1967 ). This wound is characterized by tissue anoxia and ischemic injury caused by blood vessel damage (van den Brenk et al., 1974a 1974a , 1974b 1974b ; Hao et al., 2009 2009 ). This is called CRWI. It has been proved that a low dose (0.3 Gy) of ionizing radiation promotes the angiogenic potential of adipocyte conditioned medium from mature adipocytes, which differentiated from irradiated pre-adipocytes (Marques et al., 2019 2019 ). Moreover, under low (0.2 or 1.0 Gy) doses of radiation, the irradiated HUVECs induce the expression of pro-angiogenic microRNA and promote the formation of capillary-like tubes (Vincenti et al., 2011 2011 ). High (20.0 Gy) doses of radiation can induce the production of angiostatic chemokine and apoptosis in human endothelial cells (Chang et al., 2009 2009 ). Given this fact, we placed HUVECs under an X-ray irradiator at a single dose of 20.0 Gy (van den Brenk et al., 1974a 1974a ; Mustoe et al., 1989 1989 ). We designed Transwell migration systems and co-culture systems to test and verify whether HPMSCs/leptin is capable of promoting cell migration and proliferation when cells are irradiated. In the Transwell migration systems, the irradiated HUVECs were placed in the upper chamber; meanwhile, the HPMSCs/leptin or the HPMSCs/NC were placed in the lower chamber. We found that HPMSCs/leptin significantly promoted the migration of irradiated HUVECs compared to the HPMSCs/NC. In the co-culture systems, the HPMSCs/leptin or the HPMSCs/NC were placed in the upper chamber, and the irradiated HUVECs were placed in the lower chamber. In this design, we co-cultured HPMSCs/leptin with irradiated HUVECs to investigate whether the HPMSCs/leptin have a positive effect on the cell proliferation of irradiated HUVECs. We found that HPMSCs/leptin promoted the cell proliferation of irradiated HUVECs. It has been reported that leptin alone and in synergism with VEGF or bFGF promotes the cell proliferation of bovine capillary endothelial (BCE) (Cao et al., 2001 2001 ). Also, leptin plays a pro-angiogenic role in skeletal myocytes by promoting the production of VEGF-A (Nwadozi et al., 2019 2019 ). VEGF and bFGF are well-known pro-angiogenic factors and have a synergistic pro-angiogenic effect on cell proliferation and migration of endothelial cells (Cao et al., 2001 2001 ; van Hove and Benoit, 2015 2015 ; Bai et al., 2018 2018 ; Shentu et al., 2018 2018 ). These facts suggest that leptin could promote cell migration and proliferation of irradiated HUVECs via improving the expression of VEGF and bFGF, and further to promote the angiogenesis potential of irradiated HUVECs. Our findings confirm that HPMSCs/leptin increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) in the co-culture system of HPMSCs/leptin and irradiated HUVECs. A previous study has shown that JAK/STAT signaling pathway is involved in regulating the inflammatory processes of many cells in the vessel wall (Grote et al., 2005 2005 ). In the co-culture systems, HPMSCs/leptin or HPMSCs/NC were placed in the upper chamber and irradiated HUVECs were placed in the lower chamber. In this experiment, we co-cultured HPMSCs/leptin with irradiated HUVECs to study the effects of HPMSCs/leptin on the secretion levels of pro-inflammatory cytokines in irradiated HUVECs. We found that in the co-culture system of HPMSCs/leptin and irradiated HUVECs significantly higher secretion levels of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) were detected, indicating that HPMSCs/leptin were capable of increasing the secretion levels of pro-inflammatory cytokines of irradiated HUVECs. This may help to maintain the normal progression of inflammatory response during CRWI healing. Nevertheless, so far, there has been no study examining this phenomenon. Our findings suggest that increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) in the co-culture system of HPMSCs/leptin and irradiated HUVECs was associated with the interaction of the NF-κB signaling pathway and the JAK/STAT signaling pathway. It has been shown that exposure of cells to ionizing radiation stimulates the signaling pathway to activate transcription factor NF-κB (Li and Karin, 1998 1998 ). In each co-culture system, the expression of NF-κB p65 and the degradation of IκBα were observed. However, only in the co-culture system of HPMSCs/leptin and irradiated HUVECs was the expression of STAT3 protein detected. Since the function of activated STAT3 can be altered by NF-κB (Walker and Smith, 2005 2005 ), and the JAK/STAT signaling pathway is related to the inflammatory process of many cells in the vascular wall (Grote et al., 2005 2005 ), the upregulation of pro-inflammatory cytokines secretion in the co-culture system of HPMSCs/leptin and irradiated HUVECs may be attributed to the interaction of the NF-κB signaling pathway and the JAK/STAT signaling pathway. Conclusions 1 In conclusion, we have succeeded in obtaining the HPMSCs/leptin, a sustained and stable leptin expression system, by transfecting lentiviral vectors (L.v.-pEB-copGFP (T2A) PURO-leptin) into HPMSCs. HPMSCs/leptin exhibited better cell proliferation, migration, and angiogenic potential. 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Leptin promotes monosodium urate crystal-induced inflammation in human and murine models of gout We thank Ji-zhou ZHANG (Department of Biochemistry, Basic Medical School, Jilin University, Changchun, China) for guiding the writing of this manuscript. Compliance with ethics guidelines Shu CHEN, Qian WANG, Bing HAN, Jia WU, Ding-kun LIU, Jun-dong ZOU, Mi WANG, and Zhi-hui LIU declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1900598 Project supported by the Special Fund for Cooperation of Local Government and College (Schools and Institutes) in Changchun, Jilin Province (No. 17DY024), China 2019-10-15 2020-01-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 21