Delivery of the high-mobility group box 1 box A peptide using heparin in the acute lung injury animal models

Song, J.H.,Kim, J.Y.,Piao, C.,Lee, S.,Kim, B.,Song, S.J.,Choi, J.S.,Lee, M. Elsevier Science Publishers 2016 Journal of controlled release Vol.234 No.-

<P>In this study, the efficacy of the high-mobility group box-1 box A (HMGB1A)/heparin complex was evaluated for the treatment of acute lung injury (ALI). HMGBIA is an antagonist against wild-type high-mobility group box-1 (wtHMGB1), a pro-inflammatory cytokine that is involved in ALls. HMGBIA has positive charges and can be captured in the mucus layer after intratracheal administration. To enhance the delivery and therapeutic efficiency of HMGBIA, the HMGBIA/heparin complex was produced using electrostatic interactions, with the expectation that the nano-sized complex with a negative surface charge could efficiently penetrate the mucus layer. Additionally, heparin itself had an anti-inflammatory effect. Complex formation with HMGBIA and heparin was confirmed by atomic force microscopy. The particle size and surface charge of the HMGB1A/heparin complex at a 1:1 weight ratio were 113 nm and 25 mV, respectively. Intratracheal administration of the complex was performed into an ALI animal model. The results showed that the HMGB1A,ffieparin complex reduced pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 beta, more effectively than HMGBIA or heparin alone. Hematoxylin and eosin staining confirmed the decreased inflammatory reaction in the lungs after delivery of the HMGB1A/heparin complex. In conclusion, the HMGB1Affieparin complex might be useful to treat ALI. (C) 2016 Elsevier B.V. All rights reserved.</P>

Cytoplasmic translocation of high-mobility group box-1 protein is induced by diabetes and high glucose in retinal pericytes

Kim, Junghyun,Kim, Chan-Sik,Sohn, Eunjin,Kim, Jin Sook SPANDIDOS PUBLICATIONS 2016 MOLECULAR MEDICINE REPORTS Vol. No.

<P>The aim of the present study was to assess the involvement of the high-mobility group box-1 (HMGB1) protein, receptor for advanced glycation end products (RAGE) and nuclear factor (NF)-κB signaling pathway in the development of diabetic retinopathy. Rat primary retinal pericytes were exposed to 25 mmol/l D-glucose for 48 h. Diabetic retinal vessels were prepared from streptozotocin-induced diabetic rats 12 weeks following the induction of diabetes. The expression of HMGB1 was detected using immunofluorescence staining. The expression of RAGE and the activity of NF-κB were analyzed using western blot and electrophoretic mobility shift assays, respectively. The results showed that HMGB1 was translocated to the cytoplasm of the high glucose-treated pericytes and diabetic retinal pericytes, whereas, in the control cells and the normal retinas, HMGB1 was expressed in the cell nuclei only. The expression of RAGE, a potential receptor for HMGB1, and the activity of NF-κB were also increased in the high glucose-treated pericytes, compared with the normal control cells. In addition, high glucose increased the binding of NF-κB to the RAGE promoter. These findings suggested that the cytoplasmic translocation of HMGB1 may be caused by diabetes and high glucose in retinal pericytes, and that the pathogenic role of HMGB1 may be dependent on the expression of RAGE and activation of NF-κB.</P>

HMGB1 Switches Alkylating DNA Damage-Induced Apoptosis to Necrosis

Su Yeon Lee(이수연),Eui Kyong Jeong(정의경),Hyun Min Jeon(전현민),Min Kyung Ju(주민경),Cho Hee Kim(김초희),Hye Gyeong Park(박혜경),Ho Sung Kang(강호성) 한국생명과학회 2011 생명과학회지 Vol.21 No.7

세포괴사는 세포막의 파열, HMGB1을 포함한 세포 내용물의 세포외부로의 방출 등을 수반하는 세포죽음이다. HMGB1은 핵 단백질로 전사조절자로 작용하지만 세포괴사에 의해 세포 밖으로 방출되면 염증을 유발하고 암을 촉진하는 cytokine으로 작용한다. HMGB1의 과발현은 암 발생 및 항암제 저항과 밀접한 연관성을 가지고 있지만, 그 기작에 대한 연구는 미흡한 실정이다. 본 연구에서는, HMGB1이 항암제에 의한 세포 죽음에 미치는 영향을 조사하였다. 그 결과, HMGB1은 MCF-7, MDA-MB231, MDA-MB361 세포에서 cisplatin에 의한 세포사멸을 억제하고 세포운명을 세포괴사로 바꾼다는 사실을 확인하였다. HMGB1의 세포사멸-세포괴사 전환 작용을 4-HC를 처리한 세포에서도 관찰되었다. 그러나, HMGB1은 docetaxel (DOC)에 의한 세포사멸에는 영향을 주지 않음을 확인하였다. MTS를 이용하여 항암제에 의한 세포 죽음에 미치는 영향을 조사한 결과, necrotic core가 형성된 8일째 MCF-7 MTS에서 cisplatin에 의한 세포사멸이 세포괴사로 바뀌는 반면, DOC에 의한 세포사멸은 세포괴사로 전환되지 않는 것을 확인하였다. 또한 spheroid에서 HMGB1 receptor인 RAGE의 발현이 증가함을 확인하였다. 이러한 결과를 통해, HMGB1이 alkylating agent에 의한 세포사멸을 세포괴사로 전환시킴을 알 수 있었다. 따라서, alkylating agent에 의한 항암제 효능을 나타내기 위해선, 이들 항암제의 부작용 즉 세포괴사를 억제하는 전략이 필요한 것으로 생각된다. Necrosis is characterized by the cell membrane rupture and release of the cellular contents, including high-mobility group box 1 protein (HMGB1), into the extracellular microenvironment. HMGB1 acts as a transcriptional regulator in nuclei, but exerts a pro-inflammatory and tumor-promoting cytokine activity when released into the extracellular space. Its overexpression is associated with tumor progression and chemoresistance. Thus, HMGB1 acts as a clinically important molecule in tumor biology. In this study, we examined whether HMGB1 affects cell death induced by anti-cancer drugs. Here we show that HMGB1 prevented cisplatin (alkylating agent)-induced apoptosis and switched the cell fate to necrosis in MCF-7, MDA-MB231, and MDA-MB361 cells. Similar apoptosis-to-necrosis switch effects of HMGB1 were observed in cells treated with 4-HC, another alkylating agent. In contrast, HMGB1 did not exert any significant effects on docetaxel (DOC)-induced apoptosis in MCF-7 cells. We also show that cisplatin-induced apoptosis was switched to necrosis in MCF-7 multicellular tumor spheroids (MTS) that were cultured for 8 days and had necrotic cores, but DOC-induced apoptosis was prevented without the apoptosis-to-necrosis switch. Finally, the levels of RAGE, a receptor of HMGB1, were increased with extended culture of MTS. These findings demonstrate that HMGB1 switches alkylating agent-induced apoptosis to necrosis, suggesting that the strategy to prevent necrosis occurring as an undesirable action of alkylating agent-based chemotherapy should be delineated to improve the efficacy of chemotherapy for cancer.

Lung epithelial binding peptide-linked high mobility group box-1 A box for lung epithelial cell-specific delivery of DNA

Kim, Hyun Ah,Park, Ji Hwan,Cho, Su Hee,Lee, Minhyung Informa Healthcare 2011 JOURNAL OF DRUG TARGETING Vol.19 No.7

<P>High mobility group box-1 A box (HMGB1A) is an anti-inflammatory peptide originating from HMGB1. A previous report demonstrated that recombinant HMGB1A could deliver DNA into cells. Lung epithelial-specific gene delivery is required for the gene therapy of various lung diseases such as acute lung injury. In this study, a lung epithelial-specific DNA carrier was produced by linking the lung epithelial binding peptide (LEBP) to HMGB1A. An LEBP-linked HMGB1A (LEBP-HMGB1A) expression vector, pET21a-LEBP-HMGB1A, was constructed. LEBP-HMGB1A was expressed in BL21 strain and purified by consecutive applications of nickel affinity chromatography and cationic exchange chromatography. In a gel retardation assay, LEBP-HMGB1A completely retarded DNA at a 5:1 weight ratio (peptide:DNA). LEBP-HMGB1A/DNA complexes were prepared at various weight ratios, to which a fixed amount of polyethylenimine (2 ??kDa, PEI2k) was added to increase the proton buffering effect of the complex. LEBP-HMGB1A had the highest transfection efficiency to L2 lung epithelial cells at a 20:1 weight ratio (peptide:DNA). At this ratio, LEBP-HMGB1A had a higher transfection efficiency than poly-L-lysine (PLL) as well as HMGB1A without LEBP. A cytotoxicity assay showed that LEBP-HMGB1A was not toxic to L2 cells. Therefore, LEBP-HMGB1A may be useful in developing gene therapies for lung diseases.</P>

The box a domain of high mobility group box‐1 protein as an efficient siRNA carrier with anti‐inflammatory effects

Lee, Sanghyun,Song, Hojung,Kim, Hyun Ah,Oh, Binna,Lee, Dong Yun,Lee, Minhyung Wiley Subscription Services, Inc., A Wiley Company 2012 Journal of cellular biochemistry Vol.113 No.1

<P><B>Abstract</B></P><P>High mobility group box‐1 (HMGB‐1) is a DNA binding nuclear protein and pro‐inflammatory cytokine. The box A domain of HMGB‐1 (rHMGB‐1A) exerts an anti‐inflammatory effect, inhibiting wild‐type HMGB‐1 (wtHMGB‐1). In this study, HMGB‐1A was evaluated as an siRNA carrier with anti‐inflammatory effects. HMGB‐1A was expressed and purified by consecutive nickel chelate chromatography, cationic exchange chromatography, and polymixin B chromatography. Purified rHMGB‐1A demonstrated an anti‐inflammatory effect, reducing tumor necrosis factor‐α (TNF‐α) in wtHMGB‐1 or lipopolysaccharide (LPS) activated macrophages. In gel retardation assay, rHMGB‐1A formed a stable complex with siRNA at or above a 1:2 weight ratio (siRNA:rHMGB‐1A). A heparin competition assay showed that an siRNA/rHMGB‐1A complex released siRNA more easily than an siRNA/polyethylenimine (PEI, 25 kDa) complex. Luciferase siRNA/rHMGB‐1A reduced firefly luciferase expression at a similar level as luciferase siRNA/PEI complex. Furthermore, TNF‐α siRNA/rHMGB‐1A synergistically reduced TNF‐α expression in LPS activated macrophages. Therefore, rHMGB‐1A may be useful as an siRNA carrier with anti‐inflammatory effects in siRNA therapy for various inflammatory diseases. J. Cell. Biochem. 113: 122–131, 2012. © 2011 Wiley Periodicals, Inc.</P>

VEGF receptor binding peptide-linked high mobility box group-1 box A as a targeting gene carrier for hypoxic endothelial cells

Han, Jee Seung,Kim, Hyun Ah,Lee, Sanghyun,Lee, Minhyung Wiley Subscription Services, Inc., A Wiley Company 2010 Journal of cellular biochemistry Vol.110 No.5

<P>High mobility group box-1 (HMGB-1) is a nuclear protein that can bind to and condense plasmid DNA. In this study, we developed a recombinant VEGF receptor binding peptide (VRBP) linked to HMGB-1 box A (VRBP-HMGB1A) as a targeting gene carrier to hypoxic endothelial cells. Hypoxic endothelial cells in ischemic tissues of solid tumors are important targets for gene therapy. A recombinant VRBP-HMGB1A expression vector, pET21a-VRBP-HMGB1A was constructed. VRBP-HMGB1A was over-expressed in BL21 strain and purified by nickel-chelate affinity chromatography. Complex formation between VRBP-HMGB1A and pCMV-Luc was confirmed by gel retardation assay. pCMV-Luc was retarded completely at a 2/1 weight ratio (peptide/plasmid). For transfection assays, calf pulmonary artery endothelial (CPAE) cells were incubated under hypoxia for 24 h, prior to transfection to induce the VEGF receptors on the cells. VRBP-HMGB1A/pCMV-Luc complexes were transfected to hypoxic CPAE cells. The highest transfection efficiency was at a 30/1 weight ratio (peptide/plasmid). In addition, VRBP-HMGB1A had higher efficiency than poly-L-lysine (PLL) specifically in hypoxic CPAE cells, However, VRBP-HMGB1A had lower efficiency than PLL in 293, H9C2, and normoxic CPAE cells. In MTT assay, VRBP-HMGB1A was less toxic than PLL to cells. In conclusion, VRBP-HMGB1A is a potential gene carrier for targeting hypoxic endothelial cells and thus, may be useful for cancer gene therapy. J. Cell. Biochem. 110: 1094–1100, 2010. Published 2010 Wiley-Liss, Inc.</P>

HMGB1 Promotes the Synthesis of Pro-IL-1β and Pro-IL-18 by Activation of p38 MAPK and NF-κB Through Receptors for Advanced Glycation End-products in Macrophages

He, Qiang,You, Hong,Li, Xin-Min,Liu, Tian-Hui,Wang, Ping,Wang, Bao-En Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.4

The high mobility group box-1 (HMGB1) protein and NALP3 inflammasome have been identified to play important roles in inflammation and cancer pathogenesis, but the relationships between the two and cancer remain unclear. The current study investigated the relationship between HMGB1 and the NALP3 inflammasome in THP-1 macrophages. HMGB1 was found unable to activate the NALP3 inflammasome and failed to induce the release of the IL-$1{\beta}$ and IL-18 in THP-1 macrophages. HMGB1 was also found significantly enhanced the activity of ATP to induce IL-$1{\beta}$ and IL-18 by the induction of increased expression of pro-IL-$1{\beta}$ and pro-IL-18. This process was dependent on activation of RAGE, MAPK p38 and NF-${\kappa}B$ signaling pathway. These results demonstrate that HMGB1 promotes the synthesis of pro-IL-$1{\beta}$ and pro-IL-18 in THP-1 macrophages by the activation of p38 MAPK and NF-${\kappa}B$ through RAGE. HMGB1 likely plays an important role in the first step of the release of the IL-$1{\beta}$ and IL-18, preparing for other cytokines to induce excessive release of IL-$1{\beta}$ and IL-18 which promote inflammation and cancer progression.

Basic, Research : The Pathogenic Role of the Interaction of the Receptor for Advanced Glycation End Productsligand and T Helper 17 Cells in Chronic Hepatitis B

( Hee Yeon Kim ),( Jong Young Choi ),( Chang Wook Kim ),( Chang Don Lee ),( Seung Kew Yoon ),( Si Hyun Bae ),( Nam Ik Han ),( Joo Yeon Jhun ),( Mi La Cho ),( Yang Mi Heo ) 대한간학회 2013 춘·추계 학술대회 (KASL) Vol.2013 No.1

Background: High-mobility group box 1 (HMGB1), a ligand for receptor for advanced glycation end products (RAGE), is released from necrotic hepatocytes, and contribute to the pathogenesis of chronic hepatitis. T helper 17 cells (Th17) also have been suggested to participate in the pathogenesis of chronic hepatitis B (CHB) infection. We investigated the role of HMGB1-RAGE-Th17 axis in the pathogenesis of CHB. Methods: The levels of HMGB1 and IL-17 expression were detected by Western blotting and real-time RT-PCR in patients with CHB. The effect of HMGB1-RAGE on the immune activity of Th17 cells and vice versa, was assessed by an induction assay. The levels of IL-17 expression was determined by RT-PCR and Western blotting after blocking RAGE. Results: The levels of HMGB1 and IL-17 expression were higher in CHB patients than in controls. The levels of IL-17 were significantly elevated when PBMCs were stimulated with HMGB1 in vitro, and vice versa. The levels of IL-17 expression was significantly reduced when PBMCs were treated with competitors for RAGE in vitro. Conclusions: HMGB1-RAGE induces the generation of IL- 17, which mediates the pathogenesis of CHB infection. It is suggested that HMGB1 might be a potential target for controlling hepatitis B infection by suppressing Th17 activity.

Eosinophils Modulate CD4+ T Cell Responses via High Mobility Group Box-1 in the Pathogenesis of Asthma

심은진,이현승,방보람,조상헌,민경업,박형우,전은영 대한천식알레르기학회 2015 Allergy, Asthma & Immunology Research Vol.7 No.2

Eosinophils have been reported to modulate T cell responses. Previously, we reported that high-mobility group box 1 protein (HMGB1) played a keyrole in the pathogenesis of asthma. This study was conducted to test our hypothesis that eosinophils could modulate T cell responses via HMGB1 inthe pathogenesis of asthma characterized by eosinophilic airway inflammation. We performed in vitro experiments using eosinophils, dendritic cells(DCs), and CD4+ T cells obtained from a murine model of asthma. The supernatant of the eosinophil culture was found to significantly increase thelevels of interleukin (IL)-4 and IL-5 in the supernatant of CD4+ T cells co-cultured with DCs. HMGB1 levels increased in the supernatant of the eosinophilculture stimulated with IL-5. Anti-HMGB1 antibodies significantly attenuated increases of IL-4 and IL-5 levels in the supernatant of CD4+ T cellsco-cultured with DCs that were induced by the supernatant of the eosinophil culture. In addition, anti-HMGB1 antibodies significantly attenuatedthe expressions of activation markers (CD44 and CD69) on CD4+ T cells. Our data suggest that eosinophils modulate CD4+ T cell responses viaHMGB1 in the pathogenesis of asthma.

신장 허혈 재관류 손상에 미치는 Ethyl Pyruvate의 효과

정구용(Ku-Yong Chung),정규영(Gyu Young Jeong),안형준(Hyung Joon Ahn),주만기(Man Ki Ju),장혜경(Hye Kyung Chang),이우정(Woo Jung Lee),한평림(Pyung-Lim Han),김유선(Yu Seun Kim) 대한외과학회 2007 Annals of Surgical Treatment and Research(ASRT) Vol.72 No.5

Purpose: Reactive oxygen species (ROS) significantly contribute to ischemia-reperfusion injury, and are also associated with the gradual loss of renal function and renal failure following renal transplantation. Pyruvate is an endogenous antioxidant, but its use as a therapeutic agent for treating conditions mediated by oxidative stress is limited due to its poor stability in solution. However, ethyl pyruvate (EP), a soluble pyruvate derivative, has far greater stability than pyruvate; thus, may serve as a practical pyruvate precursor. Therefore, the ability of EP in the prevention of renal ischemia- reperfusion injury was assessed. Methods: Sprague-Dawley rats (n=54) were subjected to 40 minutes of renal warm ischemia. The animals were divided into three groups: the sham group without warm ischemia (n=18), the EP group (n=18, EP given before ischemia), and the ischemic control (n=18). The serum levels of creatinine and TNF-α were measured 1, 3 and 5 days after induction of ischemia. The expression of high mobility group box-1 (HMGB-1), a delayed inflammatory mediator, was also assessed by Western blot of renal specimens. Results: In the EP group, late improvements in the serum levels of creatinine and TNF-α were observed in comparison with the ischemic control. Based on this delayed effect, the expression of HMGB-1 was assessed in renal tissue. The HMGB-1 expression increased over time during the ischemia process, but EP suppressed this expression 3 and 5 days after renal ischemia-reperfusion injury. Conclusion: These results have demonstrated, for the first time, that EP ameliorates renal ischemia-reperfusion injury. EP attenuates the renal ischemia-reperfusion injury, at least in part, by suppressing the expression of HMGB-1, a late mediator of delayed inflammation.