The myocardial infarct-exacerbating effect of cell-free DNA is mediated by the high-mobility group box 1–receptor for advanced glycation end products–Toll-like receptor 9 pathway

IntroductionDamage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE).MethodsC57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60 minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer.ResultsFree HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40′/60′ IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0% ± 5.2% and 24.3% ± 3.5% (P < .05 vs control 54.3% ± 3.4%). However, combined treatment with deoxyribonuclease I + anti-HMGB1 monoclonal antibody did not further attenuate IS (29.3% ± 4.9%). In C57BL/6 mice undergoing 20′/60′ IRI, injection of 40′/5′ plasma upon reperfusion increased IS by more than 3-fold (to 19.9 ± 4.3; P < .05). This IS exacerbation was abolished by pretreating the plasma with deoxyribonuclease I or by depleting the HMGB1 by immunoprecipitation, or by splenectomy. The infarct-exacerbating effect also disappeared in RAGE KO mice and Toll-like receptor 9 KO mice. Injection of 40′/0′ coronary perfusate upon reperfusion similarly increased IS. The levels of HMGB1 and cfDNA were significantly elevated in the 40′/0′ coronary perfusate and 40′/reperfusion (min) plasma but not in those with 10′ ischemia. In C57BL/6 mice without IRI, 40′/5′ plasma significantly increased the interleukin-1β protein and messenger RNA expression in the spleen by 30 minutes after injection. Intravenous bolus injection of recombinant HMGB1 (0.1 μg/g) or mitochondrial DNA (0.5 μg/g) 5 minutes before reperfusion did not exacerbate IS (P = not significant vs control). However, combined administration of recombinant HMGB1 + mitochondrial DNA significantly increased IS (P < .05 vs individual treated groups) and this infarct-exacerbating effect disappeared in RAGE KO mice and splenectomized C57BL/6 mice. The accumulation of cfDNA in the spleen after combined recombinant HMGB1 + mitochondrial DNA treatment was significantly more elevated in C57BL/6 mice than in RAGE KO mice.Conclusions:Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and both contribute importantly and interdependently to post-IRI by a common RAGE-Toll-like receptor 9–dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.     

Contribution of human adipose tissue–derived stem cells and the secretome to the skin allograft survival in mice

Background

Despite considerable evidence showing the immunosuppressive properties of mesenchymal stem cells (MSCs) in vitro, such properties have not been fully demonstrated in vivo. The aim of this study was to evaluate the effect of MSCs and/or MSC secretome in inducing tolerance in a mouse skin transplantation model.

Methods

After receiving full-thickness skin allotransplantation on the back of the mouse, the recipient mice were infused with phosphate-buffered saline, adipose tissue–derived stem cells (ASCs), conditioned media (CM), and control media. Specifically, ASCs (1.0 × 10⁶/0.1 mL) were transplanted to ASC-infused mice and 25-fold concentrated CM, which had been obtained from ASC culture were infused to CM-infused mice. Graft survival rates and the parameters reflecting immunologic consequences were assessed.

Results

The serum level of proinflammatory cytokine interleukin 6 decreased in mice treated with ASCs or CM compared with the control groups after infusion (P < 0.05). Interferon gamma, interleukin 10, and tumor necrosis factor alpha messenger RNA levels in the skin graft seemed to be decreased in the ASC-infused mice and CM-infused mice. Hyporesponsiveness was identified in mixed lymphocyte reaction assay at 30-d posttransplantation in ASC- or CM-infused mice. And, administering ASCs and CM markedly increased skin allograft survival compared with control animals (P < 0.001).

Conclusions

These findings suggest that ASCs and their secretome have the potential to induce immunologic tolerance. Moreover, our results demonstrate that the immunosuppressive properties of ASCs are mediated by the ASC secretome. Our approach could provide insights into a promising strategy to avoid toxicities of chemical immunosuppressive regimen in solid organ transplantation.     

Music exposure induced prolongation of cardiac allograft survival and generated regulatory CD4⁺ cells in mice

In clinical practice, music has been used to decrease stress, heart rate, and blood pressure and to provide a distraction from disease symptoms. We investigated sound effects on alloimmune responses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA, H2^K) underwent transplantation of a C57BL/6 (B6, H2^b) heart and were exposed to 1 of 3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6 different single sound frequencies for 7 days. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Cell-proliferation, cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graft exposed to opera and classical music had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single sound frequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreated and eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively). Adoptive transfer of whole splenocytes, CD4⁺ cells, and CD4⁺CD25⁺ cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and >50 days, respectively). Cell-proliferation, interleukin (IL)-2 and interferon-γ were suppressed in opera-exposed mice, whereas IL-4 and IL-10 from opera-exposed recipients were up-regulated. Flow cytometry studies showed an increased CD4⁺CD25⁺Foxp3⁺ cell population in splenocytes from opera-exposed mice. In conclusion, exposure to some types of music may induce prolonged survival of fully allogeneic cardiac allografts and generate CD4⁺CD25⁺Foxp3⁺ regulatory cells.     

Heat-induced changes in the expression and localisation of PGC-1α in the mice testis

The functions of mammalian testis are temperature-sensitive. There are various testicular factors, which express in response to heat as a mechanism of defence. PGC-1α and HSP70 have poetical role in the protection from oxidative stress in various tissues, including testis. The expression of PGC-1α and HSP70 has been shown in the testis, and it has also been documented that heat modulates the expression of PGC-1α and HSP70. However, heat-dependent changes in the localisation and expression of PGC-1α have not been investigated so far. Thus, we studied the expression and localisation pattern of PGC-1α in the testis of heat-treated mice along with marker of proliferation (PCNA, GCNA), serum testosterone levels, MDA levels and HSP70. The results showed a significant increase in PGC-1α and HSP70 and MDA levels in the testis of heat-treated mice along with a decrease in PCNA, GCNA and serum testosterone levels. The immunolocalisation study showed intense immunostaining of PGC-1α in the Leydig cell and germ cells of the heat-treated testis, with pronounced damaged in the histoarchitecture. The results showed that increase expression of PGC-1α in germ cells and Leydig cells of testis could be a counter mechanism to cope up with oxidative stress in coordination with HSP70.     

Therapeutic potential of transgenic mesenchymal stem cells engineered to mediate anti–high mobility group box 1 activity: targeting of colon cancer

Background

Mesenchymal stem cells (MSCs) are being developed as a new clinically relevant stem cell type to be recruited into and to repair injured tissue. A number of studies have focused on the therapeutic potential of MSCs by virtue of their immunomodulatory properties. Systemically administered MSCs can also migrate to sites of malignancies. Because of this latter phenomenon, we transfected human MSCs to secrete anti–high mobility group box (HMGB) 1 proteins. They were then injected into mice bearing human colon cancer to evaluate their efficacy as an antineoplastic agent.

Materials and methods

The ABOX gene was used in this model, which encodes part of the HMGB1 protein and acts as an HMGB1 antagonist. It was cotransduced by electroporation with a FLAG-tag to visualize the secreted ABOX protein, levels of which in supernatants from cultured transfected MSCs were quantified by immunofluorescence imaging using an anti-FLAG antibody. Antiangiogenic effects were evaluated in vitro using a novel optical assay device for the quantitative measurement of cellular chemotaxis assessing the velocity and direction of endothelial cell movement stimulated by supernatant from tumor cells. We found that ABOX proteins released from transfected MSCs suppressed migration in this assay. Finally, MSCs were injected subcutaneously into Nonobese diabetic/severe combined immunodeficiency mice bearing human colon cancer from a cell line, which secreted large amounts of HMGB1. Ten days after MSC injection, mice were sacrificed and tumors evaluated by immunohistochemistry.

Results

From 12 ho through 7 d after gene transfection, ABOX proteins secreted from MSCs could be detected by immunofluorescence and enzyme-linked immunosorbent assay. Quantitative measurement of cellular chemotaxis demonstrated that ABOX proteins secreted from transfected MSCs decreased the velocity and interfered with the direction of movement of vascular endothelial cells. Moreover, in an in vivo human colon cancer xenograft model, injection of anti-HMGB1–transfected MSCs resulted in a decreased tumor volume due to the antiangiogenic properties of the secreted ABOX proteins.

Conclusions

MSC modified to secrete HMGB1 antagonist proteins have therapeutic antineoplastic potential. These findings may contribute to future novel targeting strategies using autologous bone marrow–derived cells as gene delivery vectors.     

Effect of all-trans retinoic acid on the expression of Sp1 and c-met in glomerular mesangial cells induced by TGF-β

肝细胞生长因子(HGF)是一种多效性的细胞因子,c-met是HGF现已知的唯一的受体,HGF与c-met结合后,发挥相应的生物学效应[1].Sp1蛋白是众多基因的基本转录因子,能与c-met基因启动子区域的多重GC盒结合,是调节肾脏细胞表达c-met的重要转录因子[2].全反式维甲酸(ATRA)能抑制肾脏纤维化,保护肾功能.本研究探讨ATRA对肾小球系膜细胞Sp1、c-met表达的影响,以进一步了解ATRA对肾脏的保护机制.     

Effect of all-trans retinoic acid on the expression of Sp1 and c-met in glomerular mesangial cells induced by TGF-β

肝细胞生长因子(HGF)是一种多效性的细胞因子,c-met是HGF现已知的唯一的受体,HGF与c-met结合后,发挥相应的生物学效应[1].Sp1蛋白是众多基因的基本转录因子,能与c-met基因启动子区域的多重GC盒结合,是调节肾脏细胞表达c-met的重要转录因子[2].全反式维甲酸(ATRA)能抑制肾脏纤维化,保护肾功能.本研究探讨ATRA对肾小球系膜细胞Sp1、c-met表达的影响,以进一步了解ATRA对肾脏的保护机制.     

High-power helium–neon laser irradiation inhibits the growth of traumatic scars in vitro and in vivo

This study explored the inhibitory effect of the high-power helium–neon (He–Ne) laser on the growth of scars post trauma. For the in vitro study, human wound fibroblasts were exposed to the high-power He–Ne laser for 30 min, once per day with different power densities (10, 50, 100, and 150 mW/cm²). After 3 days of repeated irradiation with the He–Ne laser, fibroblast proliferation and collagen synthesis were evaluated. For in vivo evaluation, a wounded animal model of hypertrophic scar formation was established. At postoperative day 21, the high-power He–Ne laser irradiation (output power 120 mW, 6 mm in diameter, 30 min each session, every other day) was performed on 20 scars. At postoperative day 35, the hydroxyproline content, apoptosis rate, PCNA protein expression and FADD mRNA level were assessed. The in vitro study showed that the irradiation group that received the power densities of 100 and 150 mW/cm² showed decreases in the cell proliferation index, increases in the percentage of cells in the G0/G1 phase, and decreases in collagen synthesis and type I procollagen gene expression. In the in vivo animal studies, regions exposed to He–Ne irradiation showed a significant decrease in scar thickness as well as decreases in hydroxyproline levels and PCNA protein expression. Results from the in vitro and in vivo studies suggest that repeated irradiation with a He–Ne laser at certain power densities inhibits fibroblast proliferation and collagen synthesis, thereby inhibits the growth of hypertrophic scars.     

Reduced trabecular bone mass and strength in mice overexpressing Gα11 protein in cells of the osteoblast lineage

G protein-coupled receptors (GPCRs) require G proteins for intracellular signaling to regulate a variety of growth and maintenance processes, including osteogenesis and bone turnover. Bone maintenance events may be altered by changes in the activity or level of G proteins, which then modify signaling in bone cells such as osteoblasts. We have previously reported increased levels of Gα₁₁ protein and signaling to phospholipase C/protein kinase C pathways in response to dexamethasone in osteoblastic UMR 106-01 cells. Here we generated pOBCol3.6-GNA11 transgenic mice that overexpress Gα₁₁ protein in cells of the osteoblast lineage (G₁₁-Tg mice). G₁₁-Tg mice exhibit an osteopenic phenotype characterized by significant reductions in trabecular bone mineral density, thickness, number and strength. The numbers of osteoblasts and osteocytes were unchanged in G₁₁-Tg bone, but early markers of osteoblast differentiation, Alp and Bsp, were increased while the late stage differentiation marker Ocn was not changed suggesting reduced osteoblast maturation in G₁₁-Tg trabecular bone which was accompanied by a decreased bone formation rate. Furthermore, in vitro cultures of G₁₁-Tg primary osteoblasts show delayed osteoblast differentiation and mineralization. Histological analyses also revealed increased osteoclast parameters, accompanied by elevated mRNA expression of Trap and Ctsk. mRNA levels of Rankl and M-csf were elevated in vitro in bone marrow stromal cells undergoing osteogenesis and in trabecular bone in vivo. Together, these findings demonstrate that increasing Gα₁₁ protein expression in osteoblasts can alter gene expression and result in a dual mechanism of trabecular bone loss.     

The regulatory effect of ERK1／2 signal pathway on production of TNFα induced by LPS in mice Kupffer cells

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Sleeve gastrectomy–induced endocrine changes in the remnant stomachs of premenopausal and postmenopausal rats: role of the estrogen receptors

BackgroundAlthough alterations in the plasma levels of leptin, glucagon-like peptide-1, and gastrin were linked with bariatric surgery outcomes, gastric production of these peptides was not elucidated before.ObjectiveThe aim was to evaluate the impact of estrogen depletion and estrogen receptors (ERs) on sleeve gastrectomy (SG)-induced alterations in gastric hormone production, gastric mucosal integrity, and bone mass.SettingPhysiology Research Lab at the University.MethodsFemale Sprague-Dawley rats underwent ovariectomy or sham operation (control), and 2 months later SG or sham SG was performed. Rats received either nonselective agonist 17 β, ER-α agonist, ER-β agonist, or vehicle for 3 weeks. Trunk blood and gastric tissues were collected for biochemical measurements, while histopathologic examination was performed in gastric and femur samples.ResultsIn the presence of intact ovaries, SG-induced weight loss was accompanied by reductions in the gastric synthesis of leptin and gastrin, while gastric glucagon-like peptide-1 was additionally decreased when SG was performed at the postmenopausal state. SG elevated the depleted serum estradiol levels of menopause, implicating a beneficial effect, but the occurrence of severe gastric mucosal injury was triggered. On the other hand, using ER agonists upregulated gastrin-expressing cells, ameliorated gastric injury, and improved bone loss.ConclusionsSG, either at premenopausal or postmenopausal state, resulted in considerable loss in bone mass, along with reductions in the gastric levels of gastrin and leptin. Functional status of the ovaries needs to be taken into consideration when monitoring the outcomes of SG, and ER agonists could be of value in controlling SG-induced complications.     

Impact of PCI-neo-HGF on the expression of TGF-β1 and proliferation of glomerular mesangial cells in rats

肾小球系膜细胞(GMC)的过度增生是导致肾小球硬化及肾间质纤维化的重要机制之一[1].肝细胞生长因子(HGF)是一种多效性的细胞因子,其可通过加速细胞外基质降解,阻断小管上皮细胞转分化等实现对肾脏的保护[2-4].目前HGF对正常及增生的GMC是否有抑制作用尚不明确.本研究采用可在体内持续平稳表达的PCI-neo-HGF质粒进行研究[5],主要探讨HGF是否能抑制正常及脂多糖(LPS)刺激后的大鼠GMC的增生,以及这种作用是否与抑制转化生长因子β1(TGF-β1)的表达相关.     

Impact of PCI-neo-HGF on the expression of TGF-β1 and proliferation of glomerular mesangial cells in rats

肾小球系膜细胞(GMC)的过度增生是导致肾小球硬化及肾间质纤维化的重要机制之一[1].肝细胞生长因子(HGF)是一种多效性的细胞因子,其可通过加速细胞外基质降解,阻断小管上皮细胞转分化等实现对肾脏的保护[2-4].目前HGF对正常及增生的GMC是否有抑制作用尚不明确.本研究采用可在体内持续平稳表达的PCI-neo-HGF质粒进行研究[5],主要探讨HGF是否能抑制正常及脂多糖(LPS)刺激后的大鼠GMC的增生,以及这种作用是否与抑制转化生长因子β1(TGF-β1)的表达相关.