TGF-beta1 mRNA Expression Research Articles

Abstract 6415: CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel

Abstract Background: Regulatory T cells (Tregs) have a strong immunosuppressive function, inhibit anti-tumor immunity, and promote the occurrence and development of tumors. In addition, a high infiltration of Treg cells is associated with poor survival in various types of cancer. Since CD25 is highly expressed on Treg cells, it is a potential target protein for Treg depletion. Methods: The binding affinity(Kd) of CD25 aptamer to rhCD25 protein was calculated through bio-layer interferometry(BLI). The cell binding of Cy5-labelled CD25 aptamer was detected by flow cytometry, and the internalization was visualized by confocal microscopy. The cell internalization rate was determined using the MFI value of flow cytometry. Competitive ELISA, western blot, and qPCR were performed to investigate the effect of CD25 aptamer on CD25/IL-2 binding. In vitro cytotoxicity of prototype-ApDC was determined by CCK-8 assay. Moreover, CD25-dependent cytotoxicity of prototype-ApDC was evaluated by Karpas299(Treg-like):HuT78(Teff-like) 1:1 co-culture model using FACS analysis. Results: CD25 aptamer binds human CD25 protein with 4.64nM affinity. CD25 aptamer showed selective binding and internalization to CD25-positive Karpas299 and had no interaction with CD25-negative Daudi. The internalization half-time of CD25 aptamer in karpas299 was determined to 9.9 min (95% CI 8.2~12.6min). Interestingly, CD25 aptamer was identified as IL-2R antagonist by evaluating competitive ELISA and the level of phosphor-STAT5 and downstream mRNA expression of TGF-beta. The IC50 value of prototype-ApDC was 24nM to Karpas299 while >1uM to Daudi. These results show that prototype-ApDC has selective cytotoxicity against CD25-expressing cells. Furthermore, in a study of an in vitro co-culture model mimicking the Treg-rich intratumoral T cell environment, prototype-ApDC treatment induced CD25-specific depletion resulting in escalating the ratio of HuT89/Karpas299 with increasing exposure time. Conclusions: Our CD25-targeted aptamer and its drug-conjugated ApDC have demonstrated the selective blocking of IL-2R-STAT5 signaling and induction of CD25-positive cell-specific depletion, respectively. Moreover, in the co-culture system of the Treg-like and Teff-like cells, CD25-ApDC also proved its cytotoxic selectivity. Therefore, one potential strategy in cancer patients is to use CD25-ApDC with excellent tumor permeability as an anticancer agent with a mechanism of Treg cell depletion through specific targeting of CD25 overexpressed in tumor-infiltrating Treg cells. Citation Format: Daekyun Lee, Ju-hyung Kang, Inu Song. CD25-targeted aptamer-drug conjugate (CD25-ApDC) depletes and blocks regulatory T cells selectivel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6415.

MO432: The ESRD Progression Delayed by Transplanting 3D Printed Omental Patch

Abstract BACKGROUND AND AIMS Renal fibrosis is one of the pathological hallmarks of chronic kidney disease (CKD) that can further progress to end-stage renal diseases (ESRD). Haemodialysis and kidney transplantation are the only feasible treatments of ESRD. In this study, a new treatment with autologous omental tissue was assessed to delay the progression of renal fibrosis. The omental tissue, a fatty structure, has been widely used in surgical procedures due to its immunologic, angiogenic characteristics and anti-fibrotic factors that enhance tissue repairs, cell proliferation and regeneration in a damaged tissue. Finally, we hypothesized that 3D printed omental patch can prevent renal fibrosis and help regenerate the damaged kidney in the animal model. The 3D printing can print cells and extracellular matrix uniformly to promote cell survival and differentiation during transplantation. The printing also can adjust the patch stiffness, providing a suitable environment for kidney recovery. METHOD Animal models of CKD were randomly divided into four groups: (A) sham-operated, (B) sham-operated + omental patch, (C) unilateral ureteral obstruction (UUO)-operated (Control) and (D) UUO-operated + omental patch. Micronized omental tissue as bio-ink was 3D printed in the form of a regular patch deformation. Then, the 3D printed omental patch was transplanted subcapsular layer of the kidney. All of the patches used in those groups were transplanted after 3 days of modelling. Kidneys were assessed histologically [Masson's Trichrome (MT), Sirius Red], by immunohistochemistry (α-SMA), molecular work (Bulk RNA sequencing, western blot) after 2 weeks of transplantation. RESULTS Omental patch treated group has been shown histologically less tubulointerstitial fibrosis and tubule atrophy by MT, Sirius Red staining. Particularly, the TGF beta mRNA expression level of the 3D printed omental treated group was decreased in the kidney, whereas the HGF level was significantly increased. Also, in terms of western blotting, pro-inflammatory and renal fibrosis markers (TGF-b, α-SMA) were all decreased. Moreover, in bulk RNA sequencing, we confirmed that STAT 3 kidney repair pathway and TF-beta signalling. From all those assessments we suggest that the induction of myofibroblasts and collagen deposition are all inhibited in the omental patch treated kidney model. The autologous 3D omental patch can prevent tubular atrophy and decreased tubulointerstitial fibrosis and this further delays the progression of ESRD. CONCLUSION The study demonstrated that the omental patch delays fibrosis and repairs tubule structure in the damaged kidney by providing protective effects to kidney disease. These studies indicate the feasibility of the use of the autologous omental patch in chronic kidney disease therapeutic effects and new treatment of renal replacement therapy (RRT).

Effects of Combined Inulin and VSL#3 on Susceptibility to Dextran Sodium Sulfate‐Induced Colitis

Inflammatory bowel disease (IBD) is a chronic intestinal disease that includes Crohn's disease and ulcerative colitis. Previous studies have found alterations in the microbiome in IBD and in animal models of IBD, underscoring the role of the intestinal microbiota in IBD pathogenesis. Prebiotics are molecules in foods that support the growth or activity of beneficial microbes. Inulin, a soluble fiber molecule, is a commonly available prebiotic, and previous studies suggest that it can reduce symptoms in dextran sodium sulfate (DSS)-induced colitis in mice. VSL#3 is a commercially available mixture of 8 probiotic species that has shown beneficial effects in models of intestinal inflammation. This study aimed to determine whether pretreatment with 1% inulin for 1 week, followed by 7 days of 3% DSS administration with inulin plus VSL#3 treatment, altered the severity of disease in C57Bl/6 mice. Clinical signs of colitis, including weight loss, fecal occult blood, and fecal consistency, were monitored daily during the DSS treatment period. At the end of the experiment, samples were collected for histological scoring, and for RNA isolation and analysis of colonic pro-inflammatory cytokine expression by RT-PCR. At the end of DSS treatment, the disease activity index was not different between control colitic mice or those treated with inulin and VSL#3. In addition, colon length and colon wet weight were similar in the two groups. Treatment with inulin+VSL#3 did not significantly alter expression of mRNA for COX-2, TGF-beta, or KC in the colon of mice with DSS colitis. In conclusion, a combination of inulin + VSL#3 treatment did not significantly alter susceptibility to DSS-induced colitis.

Induction of CD4+CD25+ Regulatory T Cells from In Vitro Grown Human Mononuclear Cells by Sparteine Sulfate and Harpagoside.

Regulatory T cells (Tregs) are key players in the regulation of inflammatory responses. In this study, two natural molecules, namely, sparteine sulfate (SS) and harpagoside (Harp), were investigated for their ability to induce Tregs in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy volunteers and grown in the presence or absence of ConA, with TGF-beta, SS or Harp. Expression of the mRNA of FoxP3, TGF-beta, IL-10 and GAPDH was assessed via q-PCR. The expression of Treg markers including CD4, CD25, CD127 and FoxP3 was measured via flow cytometry. The secretion of IL-10 and TGF-beta by cultured cells was assessed by ELISA. Furthermore, the suppressive role of SS and Harp on PBMCs in vitro was tested via allogeneic mixed lymphocyte reaction (MLR). Data obtained show that both compounds increased the expression of FoxP3, TGF-beta and IL-10 mRNA in resting PBMCs but to a lesser extent in activated cells. Moreover, they significantly increased the percent of CD4+CD25+FoxP3+CD127− Tregs in activated and naïve PBMCs. Functionally, both compounds caused a significant reduction in the stimulation index in allogeneic MLR. Together, our data demonstrate for the first time that SS and Harp can induce human Tregs in vitro and therefore have great potential as anti-inflammatory agents.

Epigenetic regulation of TLR2‐mediated periapical inflammation

To determinethe methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n=17) and symptomatic apical periodontitis (SAP) (n=17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P<0.05). TLR2 depicted a hypomethylated DNA profile at the CpG island in SAPwhen compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P<0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P<0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P<0.05). TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

Deep Infiltrating Endometriosis and Activation and Memory Surface Markers and Cytokine Expression in Isolated Treg Cells.

It is not yet clear whether regulatory T (Treg) cells are active, and whether they play a favorable or adverse effect on endometrial foci implantation. Our aim was to evaluate activation and memory surface markers in Treg isolated from peritoneal fluid (PF) and peripheral blood (PB) of women with deep endometriosis and to assess its cytokine mRNA expression. This case-control study included 49 women with deep infiltrating endometriosis and 20 healthy controls. It was analyzed PF and PB of both groups. Cell surface markers GITR, TNFRII, HLA-DR, ICOS, CTLA-4, CD45RA, and CD45RO were evaluated in Treg (CD3+CD4+CD25+CD127lowFoxp3+) cells by flow cytometry. Additionally, Foxp3, TGF-beta, IL-10, IL-6, and TNF-alpha mRNA expression was assessed by real-time PCR in Treg cells (CD4+CD25+CD127dim/-) isolated using magnetic microbeads. Women with endometriosis had higher percentages of TNFRII+ Treg and CTLA-4+ Treg in their PB, and lower percentages of ICOS+ Treg and CD45RO+ Treg in their PF. The groups displayed no differences in mRNA expression. Regardless of the group, in PF, the percentage of Treg cells overall and of CD45RA+ Treg cells were significantly lower, whereas the percentage of TNFRII+ Treg and CD45RO+ Treg were significantly higher than in PB. Foxp3 and TGF-beta mRNA expression were also higher in PF than in PB. Our results indicated that Treg cells in women with endometriosis have a distinct profile of activation and memory markers, but similar cytokine expression. Moreover, we could observe clearly that Treg cells have distinct profile regarding their origin site.

The comparison of the mrna expressions of tgfbeta1, bfgf, igf-1, ngf and matrix metalloprotease iii genes in cervical and lumbar disc tissues

Aim: To evaluate and compare the mRNA expression of antioxidant genes between the cervical and lumbar degenerated disc tissues. Material and Methods: Obtained degenerated Nucleus Pulposus (DNP) materials were divided into two groups, which were cervical DNP (Group I) (n=20) and lumbar DNP (Group II) (n=20). There are 6 men and 14 women in group I with a mean age of 43.8 years and 13 men and 7 women in group II with the mean age of 40.3 years (28 to 54). All cervical DNP materials were obtained between C5 and C6 and all lumbar DNP materials were obtained between L4 and L5 levels. The TGFβ1, bFGF, IGF-1, NGF and Matrix metalloprotease III (MMP III) gene expressions of DNP materials were determined by polymerase chain reaction, Real-time polymerase chain reaction and western blotting techniques.Results: When the results of the two groups were compared by polymerase chain reaction, the expressions of TGFβ1, bFGF, IGF-1, NGF and MMP III was found lower in lumbar DNP group. Also the TGFβ1, bFGF, IGF-1, NGF, and Matrix metalloprotease III genes showed a decrease in gene expression levels when they were analyzed by Real-time polymerase chain reaction. Conclusion: These data showed that; decrease of the TGFβ1, bFGF, IGF-1, NGF and MMP III genes in the degenerated lumbar disc tissues, may related with the possibility of molecular background of the disease pathogenesis.

Reversal of liver fibrosis through AG490 inhibitor-mediated inhibition of the TGFbeta1-STAT3 pathway

To investigate the role of TGF-beta1 and STAT3 signaling in liver fibrosis using a rat model system and to determine the therapeutic mechanism of AG490 in relation to this signaling pathway. Rats were randomly divided into a control group and DENA-induced liver fibrosis model group, and then subdivided into AG490 treatment groups. During fibrosis development, liver tissue samples were collected at different time points (0, 4 and 8 weeks) and evaluated according to the Scheuer scoring system. Expression of STAT3, TGFbeta1, alpha-SMA, E-cadherin, MMP2 and TIMP1 was measured by PCR (mRNA) and immunohistochemistry and western blotting (protein). Increasing degrees of inflammation and fibrosis were observed in liver tissues of DENA-treated rats throughout model establishment. The mRNA expression of TGFbeta1 and STAT3 was significantly increased in DENA-induced rats with advanced fibrosis (AF) compared to those with early fibrosis (EF) (P = 0.034 and P = 0.012 respectively). The protein expression of TGF-beta1, phospho-Smad2, alpha-SMA, E-cadherin, STAT3 and phospho-STAT3 was significantly increased in DENA-induced rats with AF compared to the unmodeled control group (P = 0.048, P = 0.003, P = 0.002, P = 0.028, P = 0.009 and P = 0.039). The protein expression of E-cadherin was lower in the DENA-induced rats with AF than in those with EF (P = 0.026). STAT3 and TGF-beta1 co-expression was detected in AF tissues. DENA-induced AG490-treated rats with AF showed substantially lower protein expression of STAT3, TGF-beta1, MMP2 and TIMP1 compared to DENA-induced untreated rats with AF (P = 0.006, P = 0.018, P = 0.010 and P = 0.005); in addition, the degrees of fibrosis and inflammation were also greatly reduced in the DENA-induced AG490-treated rats with AF compared to DENA-induced untreated rats with AF (P = 0.042 and P = 0.021). Conclusions STAT3 signal transduction may regulate the TGF-beta1 pathway and affect liver fibrosis, especially in the advanced phase. AG490 can inhibit TGFbeta1-STAT3 signaling, resulting in reversal of liver fibrosis.

Abstract 321: Macrophage Elavl1 Regulates Fibrogenesis Through Post-transcriptional Mechanism Under Diabetic Conditions

Patients with diabetes are predisposed to increased risk of cardiovascular diseases. Persistent interaction of infiltrating macrophages and resident fibroblasts play a critical role in cardiac fibrosis. However, the signaling mechanism is not clear. We hypothesized that macrophage ELAV1 (mRNA stabilizing protein) modulates profibrotic mediators and extracellular matrix turnover by binding to 3′UTR and regulating the mRNA stability of TGF-beta and MMP-9 in hyperglycemic conditions. Mice receiving intramyocardial injection of HuR-specific shRNA showed significant reduction in infarct size and fibrosis area. Reduced fibrosis was associated with decrease in TGF-beta and MMP-9 expression in the myocardium. Conditioned media (CM) from high glucose (HG) treated macrophages significantly increased profibrogenic response (increased mRNA expression of Col1a1, Col3a1 and fibronectin) in fibroblast cell line as compared to fibroblasts incubated with CM from low glucose (LG)-treated macrophages. Knockdown of ELAV1 in HG-treated macrophages abrogated the profibrotic effects in fibroblasts. Indirect immunofluroscence of bone marrow-derived macrophages (BMM) demonstrated that HG increases nuclear ELAV1 export to the cytoplasm. Pharmacological inhibition of Protein kinase C-delta (PKCd) blocked HG-induced ELAV1 nuclear to cytoplasmic translocation. In vitro, stable knockdown of ELAV1 in mouse macrophage cell line RAW 264.7 reduced mRNA expression of TGF-beta and MMP-9 following LPS challenge, accompanied by a marked reduction in the mRNA stability of these genes. Our study here establishes an ELAV1/TGF-beta/MMP-9/PKC-delta signaling axis in the macrophages controlling the profibrogenic responses in fibroblasts, the major contributor in the pathogenesis of fibrosis. Therefore, targeting this signaling pathway might be of therapy value for cardiac fibrosis in diabetic patients.

Effect of regular circus physical exercises on lymphocytes in overweight children.

Obesity associated with a sedentary lifestyle can lead to changes in the immune system balance resulting in the development of inflammatory diseases. The aim of this study was to compare lymphocyte activation mechanisms between overweight children practicing regular circus physical exercises with non-exercised children. The study comprised 60 pubescent children randomly divided into 4 groups: Overweight Children (OWC) (10.67 ± 0.22 years old), Overweight Exercised Children (OWE) (10.00 ± 0.41 years old), Eutrophic Children (EC) (11.00 ± 0.29 years old) and Eutrophic Exercised Children (EE) (10.60 ± 0.29 years old). OWE and EE groups practiced circus activities twice a week, for 4.3 ± 0.5 and 4.4 ± 0.5 months, respectively. Percentage of T regulatory cells (Treg) and the expression of CD95 and CD25 in CD4+ lymphocytes were evaluated by flow cytometry. Lymphocyte proliferation capacity was measured by [14C]-thymidine incorporation and mRNA expression of IL-35, TGF-beta, IL-2 and IL-10 by real-time PCR. Lymphocyte proliferation was higher in OWC and OWE groups compared with the EC (3509 ± 887; 2694 ± 560, and 1768 ± 208 cpm, respectively) and EE (2313 ± 111 cpm) groups. CD95 expression on lymphocytes was augmented in the EC (953.9 ± 101.2) and EE groups (736.7 ± 194.6) compared with the OWC (522.1 ± 125.2) and OWE groups (551.6 ± 144.5). CTLA-4 expression was also lower in the OWC and OWE groups compared with the EC and EE groups. Percentage of Treg, IL-35, and IL-10 mRNA expression were lower in the OWC and OWE groups compared with the EC and EE groups. In conclusion, overweight children present altered immune system balance characterized by elevated lymphocyte proliferation due to a decrease in T regulatory cell percentage. These effects were partially reverted by moderate physical exercise, as demonstrated by decreased lymphocyte proliferation.

Transforming growth factor beta1 and aldosterone

It is well established that blocking the renin-angiotensin-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of the RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here, we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. TGFbeta1 suppresses the adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps, from 10 to 300% of normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10% hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. These results identify TGFbeta signalling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism, as well as assist in the development of novel therapeutic strategies for hypertension.

Abstract 017: Hyperglycemia-induced Posttranscriptional Modification Of Proinflammatory Cytokine Mrna Stability Is Mediated Via Hur/PKC-delta Signaling Pathway

Patients with diabetes are predisposed to increased risk of cardiovascular diseases. Persistent interaction of infiltrating macrophages and resident fibroblasts play a critical role in cardiac fibrosis. However, the signaling mechanism is not clear. We hypothesized that macrophage ELAV1 (mRNA stabilizing protein) modulates profibrotic mediators and extracellular matrix turnover by binding to 3′UTR and regulating the mRNA stability of TGF-beta and MMP-9 in hyperglycemic conditions. Mice receiving intramyocardial injection of HuR-specific shRNA showed significant reduction in infarct size and fibrosis area. Reduced fibrosis was associated with decrease in TGF-beta and MMP-9 expression in the myocardium. Conditioned media (CM) from high glucose (HG) treated macrophages significantly increased profibrogenic response (increased mRNA expression of Col1a1, Col3a1 and fibronectin) in fibroblast cell line as compared to fibroblasts incubated with CM from low glucose (LG)-treated macrophages. Knockdown of ELAV1 in HG-treated macrophages abrogated the profibrotic effects in fibroblasts. Indirect immunofluroscence of bone marrow-derived macrophages (BMM) demonstrated that HG increases nuclear ELAV1 export to the cytoplasm. Pharmacological inhibition of Protein kinase C-delta (PKCd) blocked HG-induced ELAV1 nuclear to cytoplasmic translocation. In vitro, stable knockdown of ELAV1 in mouse macrophage cell line RAW 264.7 reduced mRNA expression of TGF-beta and MMP-9 following LPS challenge, accompanied by a marked reduction in the mRNA stability of these genes. Our study here establishes an ELAV1/TGF-beta/MMP-9/PKC-delta signaling axis in the macrophages controlling the profibrogenic responses in fibroblasts, the major contributor in the pathogenesis of fibrosis. Therefore, targeting this signaling pathway might be of therapy value for cardiac fibrosis in diabetic patients.

1132 EFFECT OF ADENOSINE 2B RECEPTOR ANTAGONIST MRS1754 ON FIBROSIS PROGRESSION AND REGRESSION IN MICE

during cancer and cirrhosis, but it also participates in the pathogenesis of metabolic syndrome. Recently, myostatin levels have been associated with fibrosis during NASH. However, no information is available on the possible direct role of myostatin in the biology of HSC. Aim of the study was to evaluate the effect of myostatin on biological actions and intracellular signaling in human HSC. Methods: We employed both an immortalized human HSC line (LX-2) and primary HSC isolated from human livers. Cell proliferation was evaluated by MTT. Cell migration was assessed with modified Boyden chambers. Gene expression and secretion of cytokines or ECM proteins were measured by qRT-PCR and ELISA, respectively. Intracellular signaling pathways were evaluated using phosphorylation-specific antibodies. Results: Transcripts for ActRIIB were expressed by HSC. Exposure to myostatin (50ng/ml) induced a significant increase in cell migration in both LX-2 and primary HSC, and time-dependently reduced cell proliferation. In addition, myostatin increased mRNA expression of TGF-beta and procollagen type1. We tested the ability of myostatin to activate intracellular signaling pathways in HSC. Myostatin rapidly and markedly induced phosphorylation of Smad3 and JNK. In contrast, p38MAPK and ERK1/2 were only modestly activated. Pretreatment of both LX-2 or primary HSC with the selective JNK inhibitor, SP600125 (20mM), caused a marked inhibition of myostatin-induced cell migration. Transcripts for myostatin and ActRIIB were measurable in normal liver. Induction of steatohepatitis by administration of a methionine and choline-deficient diet significantly increased the expression of both molecules. Conclusions: HSC express ActRIIB and respond to myostatin with increased chemotaxis, reduced proliferation, and increased expression of profibrogenic genes. At least some of these require JNK activation. Expression of myostatin and its receptor is present in the liver and up-regulated following chronic liver injury, indicating a possible role for this molecule in liver fibrosis. The first two Authors contributed equally to the present study.

Age-related changes in structure and extracellular matrix protein expression levels in rat tendons

The musculoskeletal system (muscle-tendon-bone) demonstrates numerous age-related changes, with modifications in tendons the least well studied, although increased predisposition to tendinopathy and rupture have been reported. In order to gain insights into the basis of age-associated increase in tendon injuries, we compared Achilles and tibialis anterior tendons and myotendinous junctions (MTJs) from 3- to 5- and 22- to 25-month-old rats for underlying structure and composition. Significant decreases were observed by qRT-PCR for collagen I, III, and V mRNA expression in tendons of old rats, but immunostaining detected no apparent differences in collagen I and V expression on the protein level. Tendons of old compared with young rats had decreased mRNA expression levels of proteoglycan 4 (PRG4) and elastin (Eln), but no differences in the mRNA expression of connective tissue growth factor, TGF-beta 1, or stromal cell-derived factor 1. For PRG4, immunostaining showed good correlation with qRT-PCR results. This is the first study to show reductions in PRG4 in tendons and MTJs of old rats. Decreased PRG4 expression in tendons could result in increased tendon stiffness and may be associated with decreased activity in the elderly. The diminished collagen mRNA expression in combination with decreased PRG4 and Eln mRNA expression may be associated with increased risk of tendon injury with aging.

Heart Rate Reduction by Ivabradine Improves Aortic Compliance in Apolipoprotein E-Deficient Mice

Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE^–/^–) mice. Methods: ApoE^–/^– mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (∼19%) in WT mice and by 133 ± 6 bpm (∼23%) in ApoE^–/^– mice. Compared to WT controls, ApoE^–/^– mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE^–/^– mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE^–/^– mice. Moreover, membrane translocation of p47^phox was inhibited. In ApoE^–/^– mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE^–/^– mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression.

Protective effect of TGF-β-Smads signal-based oxymatrine on myocardial fibrosis induced by acute myocardial infarction in rats

To study the protective effect of oxymatrine (OMT) on myocardial fibrosis induced by acute myocardial infarction in rats and its effect on TGF-beta-Smads signal pathway. Arteria coronaria ligation-induced acute myocardial infarction model was established in rats. The survived rats were randomly allotted into the model group, 50, 25, 12.5 mg x kg(-1) OMT groups, the 50 mg x kg(-1) captopril group, and the Sham-operated group which was treated as the model group without the arteria coranaria ligation. After 8 weeks of ligation, myocardial fibrosis was detected by HE and Masson staining, and the RT-PCR method were used to detect the expression of mRNA of TGF-beta-Smads signal system. The histopathological examination showed decrease in cardiocytes, deposition of extra-cellular matrix, and increase of collagen contents after 8 weeks of ligation. RT-PCR results showed that mRNA expressions of TGF-beta1, TbetaR1, Smad2, Smad3 and Smad4 significantly increased, but mRNA expression of Smad7 is remarkable lower than the sham-operated group. Treatment with OMT for 8 weeks could remarkably inhibit myocardial fibrosis, decrease mRNA expressions of TGF-beta1, TbetaR1, Smad2, Smad3, and Smad4, and increase mRNA expressions of Smad7. OMT has the inhibitory effect on the experimental myocardial fibrosis induced by AMI in rats. Its mechanism may be closely related to TGF-beta-Smads signal system.

Pulse Steroid Therapy Inhibits Murine Subglottic Granulation

Objective: Using a functional model of airway granulation tissue in subglottic stenosis, we investigated changes in inflammatory markers within granulation tissue in response to intraperitoneal dexamethasone injections. Changes in inflammatory markers will allow us to identify potential targets for immunological therapy. Method: Laryngotracheal complexes (LTCs) of donor mice underwent airway injury. Donor LTCs were transplanted into subcutaneous tissue of recipient mice in 2 groups: steroid-treated and untreated. The steroid-treated arm received intraperitoneal dexamethasone for 3 weeks. LTCs were then harvested. Granulation formation was measured; mRNA expression of TGF-beta and IL-1b was quantified. Results: At 3 weeks’ posttransplantation, there were statistically significant differences in observable granulation formation as well as mRNA expression of TGF-beta 1 and IL-1b in all groups within the steroid treated arm as compared to the untreated arm. Conclusion: Systemic steroids have been used to prevent formation of granulation tissue. However, the study of immunologic markers and the corresponding changes with steroid treatment has not been well studied in animal models. Using a previously described novel murine model, we begin to delineate inflammatory markers for potential therapeutic targets.

Calycosin protects HUVECs from advanced glycation end products-induced macrophage infiltration

Astragali radix is a traditional Chinese medicine that has long been used for treatment of diabetes and diabetes-associated disease, but its active component and mechanism on the disease is not well defined. Infiltration of leukocytes within the glomeruli and vasculature is one of the early and characteristic features of diabetic nephropathy. Advanced glycation end products (AGEs) play pivotal role in the progression of diabetic-associated diseases. The present study was designed to explore the therapeutic effect of calycosin, an active component from A. radix, on AGEs-induced macrophages infiltration in HUVECs. Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions; real-time PCR was carried out to determine mRNA expression of TGF-beta1, ICAM-1 and RAGE. Immunofluorescence was carried out to observe estrogen receptor-alpha, ICAM-1, RAGE expression and the phosphorylation status of ERK1/2 and NF-κB. Calycosin significantly reduced AGEs-induced macrophage migration and adhesion to HUVEC. Pre-treatment with calycosin strikingly down-regulated HUVEC TGF-beta1, ICAM-1 and RAGE expressions in both protein and mRNA levels. Furthermore, calycosin incubation significantly increased estrogen receptor expression and reversed AGEs-induced ERK1/2 and NF-κB phosphorylation and nuclear translocation in HUVEC, and this effect of calycosin could be inhibited by estrogen receptor inhibitor, ICI182780. These findings suggest that calycosin can reduce AGEs-induced macrophage migration and adhesion to endothelial cells and relieve the local inflammation; furthermore, this effect was via estrogen receptor-ERK1/2-NF-κB pathway.

Ethylenediaminetetraacetic acid induces antioxidant and anti-inflammatory activities in experimental liver fibrosis

BackgroundExperimental liver fibrosis induced by carbon tetrachloride (CCl4) is associated with oxidative stress, lipid peroxidation, and inflammation. This work was focused on elucidating the anti-inflammatory and antioxidant effects of ethylenediaminetetraacetic acid (EDTA) in this model of hepatotoxicityMethodsWistar male rats were treated with CCl4 and EDTA (60, 120, or 240 mg/kg). Morphometric analyses were carried out in Masson's stained liver sections to determine fibrosis index. Coagulation tests prothrombin time (PT) and partial thromboplastin time (PTT) were also determined. Gene expression for transforming growth factor beta (TGF-beta1), alpha1(I) procollagen gene (alpha1 Col I), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and superoxide dismutase (SOD) was monitored by real-time PCR. Antioxidant effect of EDTA was measured by its effects on lipid peroxidation; biological activity of ceruloplasmin (Cp), SOD, and catalase (Cat) were analyzed by zymography assays.ResultsAnimals with CCl4-hepatic injury that received EDTA showed a decrement in fibrosis (20%) and lipid peroxidation (22%). The mRNA expression for TNF-alpha (55%), TGF-beta1 (50%), IL-6 (52%), and alpha1 Col I (60%) was also decreased. This group of animals showed increased Cp (62%) and SOD (25%) biological activities. Coagulation blood tests, Cat activity, and gene expression for SOD were not modified by EDTA treatment.ConclusionThis study demonstrates that EDTA treatment induces the activity of antioxidant enzymes, decreases lipid peroxidation, hepatic inflammation, and fibrosis in experimental liver fibrosis induced by CCl4.

TWEAK Appears as a Modulator of Endometrial IL-18 Related Cytotoxic Activity of Uterine Natural Killers

BackgroundTWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity.Methodology/Principal FindingsEndometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions.Conclusions/SignificanceTWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human.