Expression Of TGF-beta Research Articles

The Sphingosine-1-Phosphate Receptor Modulator, FTY720, Reverses Diastolic Dysfunction and Hypertrophy in Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) patients present with progressively worsening hypertrophy, diastolic dysfunction and fibrosis, ending in heart failure with preserved ejection fraction. At the molecular level, a common pathology found is an increase in myofilament-calcium (Ca) sensitivity. Previously, we have shown that oxidative modification of myofilament proteins, as a result of increased oxidative stress in HCM, may be a direct mediator of this increase. FTY720 is an immunomodulating drug, clinically used in the treatment of multiple sclerosis, which we have previously identified to have anti-fibrotic and anti-hypertrophic effects in a model of pressure-overload-induced hypertrophy. We sought to understand whether agonism of the sphingosine-1-phosphate receptor by FTY720 would be of therapeutic benefit in HCM, and its effects on oxidative modification. Methods: We treated an HCM mouse model linked to a mutation in tropomyosin (Tm-E180G) and non-transgenic (NTG) littermates with FTY720 or vehicle for six weeks. We assessed cardiac function and morphology using echocardiography and pressure-volume (PV) relations and the myofilament-Ca-response using detergent-treated (skinned) fibers. We also investigated if signaling pathways were altered. Results: We found that FTY720-treated Tm-E180G mice had a significant reduction in left atrial size, E/A and E/Em ratios compared to significantly increased baseline measurements as assessed by echocardiography. PV relations revealed significant improvements in the end- diastolic pressure volume relationship, preload recruitable stroke work, and relaxation constant (Tau). FTY720-treated NTG mice displayed a significant decrease in ejection fraction and rate of maximum pressure development in early systole compared to vehicle-treated NTG mice assessed by PV relations. Skinned fiber bundlesrevealed a significant decrease in myofilament- Ca-responsiveness in FTY720-treated Tm-E180G mice. We attributed these functional improvements to a down-regulation of S-glutathionylation of cardiac myosin binding protein-C in FTY720-treated Tm-E180G mice. The increases in oxidative modification in vehicle treated Tm- E180G mice were due to an up-regulation of NADPH oxidase 2 enzyme expression (NOX2). We attributed the and fibrosis and hypertrophy to increased periostin, TGF-beta expression, and GATA4 phosphorylation respectively. These maladaptive changes were reversed by FTY720 treatment. Conclusions: In Tm-E180G mice, FTY720 improves diastolic function and morphology by reversing oxidative modification of myofilament proteins, resulting in a decrease in myofilament- Ca-responsiveness, via down-regulation of NOX2 and other maladaptive remodeling signals.

PP.07.14] RENAL DOPAMINERGIC SYSTEM DYSFUNCTION ASSOCIATED TO HIGH BLOOD PRESSURE AND INFLAMMATION IN FRUCTOSE OVERLOAD INDUCED METABOLIC SYNDROME

Objective: The renal dopaminergic system (RDS) promotes sodium excretion and anti-inflammatory actions. Fructose overload (FO) produces in the rat hemodynamic and metabolic changes similar to the human metabolic syndrome. These changes are associated to an impairment of the RDS, leading to renal inflammation, sodium retention and blood pressure elevation. The aim of this study was to evaluate RDS state and its relation to hypertension and overexpression of renal inflammatory markers in the FO experimental model. Design and method: Six groups of male Sprague Dawley rats (six weeks at the beginning of the treatment) were studied: Control (C4, C8 and C12, tap water to drink) or FO (F4, F8 and F12, 10% w/v of fructose solution to drink), during 4, 8 and 12 weeks (n = 8/group/period). Urinary L-dopa and dopamine (DA) (HPLC), diuresis and albuminuria were determined. Systolic blood pressure (SBP) (tail cuff) and metabolic parameters were measured. Western blot analysis of renal expression of dopamine 1 receptor (D1R), NFkappa-beta, IL-6, TNF-alpha, TGF-beta1 and nephrin were performed. Results: Fructose overload increased SBP (mmHg, C4: 121 ± 8 vs. F4: 145 ± 1*; C8: 130 ± 4 vs. F8: 161 ± 10#; C12: 133 ± 5 vs. F12: 163 ± 4#), which positively correlated (R2 = 0.78; p < 0.002) to urinary L-dopa/DA index (C4: 0.49 ± 0.05 vs. F4: 1.9 ± 0.09#; C8: 0.53 ± 0.06 vs. F8: 2.35 ± 0.1#; C12: 0.54 ± 0.07 vs. F12: 2.57 ± 0.2#). A significant decrease in the D1R expression was related to a significant increase in nFkappa-beta, IL-6, TNF-alpha, TGF-beta1 expression since week 4. Microalbuminuria (C12:13.11 ± 1.4 vs F12:57.6 ± 2.5#) and a decrease in nephrin expression (C12: 1.00 ± 0.10 vs. F12: 0.73 ± 0.05#) were observed only at week 12. (*p < 0.05, #p < 0.01 vs. C). Conclusions: Fructose overload was associated to an L-dopa/DA index increase and D1R expression decrease since week 4 of treatment. The renal dopaminergic system dysfunction was accompanied by an increase in blood pressure levels and renal expression of inflammatory markers in all experimental periods studied. Alteration of L-dopa/DA index could be an earlier marker of renal dysfunction than the structural damage evidenced by microalbuminuria and decreased nephrin expression in week 12 of fructose treatment.

Functional complement activity is decisive for the development of chronic synovitis, osteophyte formation and processes of cell senescence in zymosan-induced arthritis

Synovial inflammation plays a critical role in the symptoms and structural progression of arthritis which leads to irreversible damage of the adjacent cartilage and bone. Activation of complement system is strongly implicated as a factor in the pathogenesis of chronic synovitis in human rheumatoid arthritis (RA). In this study, we show that the depletion of functional complement activity at the time of the initiation of zymosan-induced arthritis, significantly reduced the expression of TGF-beta1/3, BMP2 and pSmad2 and decreased the number of Sudan Black B positive cells in the synovium. Also, the excessive synthesis of proteoglycans and glycosaminoglycans was diminished. The appearance of apoptotic and senescent cells among the adherent bone marrow cells cultivated in vitro was not observed in complement depleted mice. Therefore, the lack of functional complement prevented the development of chronic synovitis, osteophyte formation and the generation of pathologic senescent arthritic cells.

Theurapeutic effectiveness of rat bone marrow stem cells in Poly Cystic Ovary Syndrome Mice Model on folliculogenesis, TGF-β, GDF-9 expression, and estrogen, TNF- and androgen Levels

Objectives: to identify therapeutic effectiveness of Rat Bone Marrow stem cell in PCOS rats model on folliculogenesis, TGF-beta and GDF-9 expression and on estrogen, TNF-a and androgen levels.Material and Methods: this study is a laboratory experimental research with using animal testing. PCOS was induced by the administration of testosterone propionate hormone into 30 mice. The subjects of this study are divided into 2 groups: stem cell group and control group. The mice were injected with testosterone then vaginal swab was performed to determine the mice cycle. After determining mice in anestrous cycle, stem cell was injected. TNF-a was measured with immunohistochemistry and androgen was examined using ELISA. The data was measured by student t-test.Result: The average number of TNF-a expression in control group was lower than stem cell group (5.35 vs 2.34; p= 0.0026). The average androgen level for stem cell group was lower than mean for control group (2.31 vs 0.40; p= 0.0026).Conclusion: In this study of polycystic model mice, stem cell decreased the expression of TNF-a and androgen level

Discrete modeling of macrophage differentiation

Abstract The process through which macrophages differentiate into either an M1 or M2 state is thought to be well elucidated. Literature suggests that only a handful of receptors and their ligands play a role in deciding macrophage fate. These receptors share many signaling pathways and work together to create either an M1 or M2 phenotype. While these phenotype generalizations are broad, the M1 and M2 states have been used as placeholders to describe drastically different metabolic phenotypes. To determine if this complex process could be controlled by a handful of ligands and receptors, we used discrete modeling—a modeling method that uses Boolean logic and a simple notation that qualitatively describe a system—to create a model of the differentiation pathway as it is currently understood. From there, we began testing the model, seeing if we could elicit either M1 or M2 states by altering the levels of cytokines and chemokines in the model to simulate a macrophage being exposed to different stimuli. According to literature, M1 activation is generally elicited by: LPS, RAGE, and Interferon gamma; and M2 activation by: CSF-1, IL-13, and IL-4. In order to determine if a phenotype was successfully observed, we defined M1 activation as high expression of TNF-alpha, IL-12 and CD-48; and M2 activation as high expression of IL-10, TGF-beta and CD-36. With this model, we were able to simulate the process of M1 and M2 differentiation using changes in ligand concentrations to elicit the metabolic phenotypes of each activation state. This means that the current understanding of the differentiation pathway is sufficient to build functional differentiation models. This opens up opportunities to explore the broad metabolic variation within activation states.

Study on the establishment of HMLD model and the expression of KL-6/TGF-beta in rat

Objective: To establish an animal model of hard metal lung disease (HMLD) in rats, and to screen the indications for diagnosis of HMLD. Methods: The rats were randomly divided into 5 groups, each group included 8 rats: saline group, pure cobalt group, pure tungsten carbide group, silica group and hard metal (HM) group. 10 mg subjects were administered in each group by using the pulmonary endotracheal tube. After 8 week, the lung CT scan and lung tissue pathology were observed, the serum and bronchoalveolar lavage fluid (BALF) were collected for KL-6, TGF-beta1 and TGF-beta2. Results: The lung tissue structure of HM group was destroyed, a large number of nuclear giant cells and epithelial like cells appeared in the stroma, and uncommon CT scan images appeared in the lung. KL-6, TGF-beta1, TGF-beta2 expression in each group was not the same, the difference was statistically significant (P<0.05) . The expression of KL-6 and TGF-beta1 in serum was not identical in all the groups, the difference was statistically significant (P<0.05) . The expression of TGF-beta2 had no significant difference between the groups (P>0.05) . Conclusion: Rats can be successfully established HMLD model, rats in vivo lung CT scan images appear abnormal, which are provided with assistant diagnostic value for HMLD. The expression of KL-6 and TGF-beta2 in serum and BALF on HMLD rats are not highly specific, and TGF-beta1 has reference value in the rat HMLD auxiliary diagnosis.

Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes

ABSTRACTTransforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFβ-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

The Sphingosine‐1‐Phosphate Analog, FTY720, Reverses Diastolic Dysfunction and Hypertrophy in Familial Hypertrophic Cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) patients present with progressively worsening hypertrophy, diastolic dysfunction and fibrosis, ending in heart failure with preserved ejection fraction. At the molecular level, a common pathology found is an increase in myofilament‐calcium (Ca) sensitivity. Previously, we have shown that oxidative modification of myofilament proteins, as a result of increased oxidative stress in HCM, may be a direct mediator of this increase. FTY720 is an immunomodulating drug, clinically used in the treatment of multiple sclerosis, which we have previously identified to have anti‐fibrotic and anti‐hypertrophic effects in a model of pressure‐overload‐induced hypertrophy. We sought to understand whether agonism of the sphingosine‐1‐phosphate receptor by FTY720 would be of therapeutic benefit in HCM, and its effects on oxidative modification.MethodsWe treated an HCM mouse model linked to a mutation in tropomyosin (Tm‐E180G) and non‐transgenic (NTG) littermates with FTY720 or vehicle for six weeks. We assessed cardiac function and morphology using echocardiography and pressure‐volume (PV) relations and the myofilament‐Ca‐response using detergent‐treated (skinned) fibers. We also investigated if signaling pathways were altered.ResultsWe found that FTY720‐treated Tm‐E180G mice had a significant reduction in left atrial size, E/A and E/Em ratios compared to significantly increased baseline measurements as assessed by echocardiography. PV relations revealed significant improvements in the end‐diastolic pressure volume relationship, preload recruitable stroke work, and relaxation constant (Tau). FTY720‐treated NTG mice displayed a significant decrease in ejection fraction and rate of maximum pressure development in early systole compared to vehicle‐treated NTG mice assessed by PV relations. Skinned fiber bundles revealed a significant decrease in myofilament‐Ca‐responsiveness. We attributed these functional improvements to a down‐regulation of S‐glutathionylation of cardiac myosin binding protein‐C in FTY720‐treated Tm‐E180G mice. The increases in oxidative modification in vehicle treated Tm‐E180G mice were due to an up‐regulation of NADPH oxidase 2 enzyme expression (NOX2), and fibrosis and hypertrophy to increased periostin, TGF‐beta expression, and GATA4 phosphorylation. These maladaptive changes were reversed by FTY720 treatment.ConclusionsIn Tm‐E180G mice, FTY720 improves diastolic function and morphology by reversing oxidative modification of myofilament proteins, resulting in a decrease in myofilament‐Ca‐responsiveness, via down‐regulation of NOX2 and other maladaptive remodeling signals.Support or Funding InformationR01 HL128468 (BMW, RJS) and F31 HL127996 (DMR)

The effect of the colostral cells on gene expression of cytokines in cord blood cells

Beneficial effect of maternal milk is acknowledged, but there is still question whether maternal milk from allergic mother is as good as from healthy one. In our study, we have assayed the effect of cells from colostrum of healthy and allergic mothers on gene expression of cytokines in cord blood cells of newborns of healthy and allergic mothers. Cytokines typical for Th1 (IL-2, IFN-gamma), Th2 (IL-4, IL-13), Tregs (IL-10, TGF-beta), and IL-8 were followed. We were not able to detect significant influence of colostral cells on gene expression of cytokines in cord blood after 2-day coculture using Transwell system. There was no difference in gene expression of cytokines in nonstimulated cord blood cells of newborns of healthy and allergic mothers, but generally increased gene expression of cytokines except IL-10 and TGF-beta after polyclonal stimulation was detected in cord blood cells of children of allergic mothers. There was no difference in IL-10 expression in stimulated cord blood cells of children of healthy and allergic mothers. Gene expression of TGF-beta was even decreased in stimulated cord blood cells of children of allergic mothers in comparison to healthy ones. We have not observed difference in the capacity of colostral cells of healthy and allergic mothers to influence gene expression of cytokines in cord blood cells, but we have described difference in the reactivity of cord blood cells between children of allergic and healthy mothers.

Adenovirus-Mediated CCR7 and BTLA Overexpression Enhances Immune Tolerance and Migration in Immature Dendritic Cells.

Our previous report revealed that immature dendritic cells (imDCs) with adenovirus-mediated CCR7 overexpression acquired an enhanced migratory ability but also exhibited the lower immune tolerance observed in more mature cells. In the present study, we aimed to investigate whether BTLA overexpression was sufficient to preserve immune tolerance in imDCs with exogenous CCR7 overexpression. Scanning electron microscopy and surface antigens analysis revealed that BTLA overexpression suppressed DC maturation, an effect further potentiated in CCR7 and BTLA cooverexpressing cells. Correspondingly, in vitro chemotaxis assays and mixed lymphocyte reactions demonstrated increased migratory potential and immune tolerance in CCR7 and BTLA coexpressing cells. Furthermore, CCR7 and BTLA cooverexpressed imDCs suppressed IFN-γ and IL-17 expression and promoted IL-4 and TGF-beta expression of lymphocyte, indicating an increase of T helper 2 (Th2) regulatory T cell (Treg). Thus, these data indicate that CCR7 and BTLA cooverexpression imparts an intermediate immune phenotype in imDCs when compared to that in CCR7- or BTLA-expressing counterparts that show a more immunocompetent or immunotolerant phenotype, respectively. All these results indicated that adenovirus-mediated CCR7 and BTLA overexpression could enhance immune tolerance and migration of imDCs. Our study provides a basis for further studies on imDCs in immune tolerance, with the goal of developing effective cellular immunotherapies for transplant recipients.

Immunopathological characterization of cryptoglandular anal fistula: a pilot study investigating its pathogenesis.

The pathogenesis of cryptoglandular anal fistula (AF) is still under debate. Tissue inflammation could play a primary role. The pathological process of epithelial mesenchymal transition (EMT) might be involved but has never been investigated. In a prospective pilot study, 12 patients with an AF had a fistulectomy. The excised track was divided into proximal (intrasphincteric) and distal (extrasphincteric) parts which were subjected to standard histopathological examination. The cytokines IL-8 and IL-1beta were analysed as markers of inflammation, while EMT was evaluated by expression of TGF-beta, Vimentin, Zeb-1, Snail and E-cadherin. The mRNA and protein expression of these molecules was investigated by real-time PCR (RT-PCR), Western blot analysis and immunohistochemistry and was compared with that of the normal adjacent tissue. Chronic inflammation and granulation tissue and a stratified epithelium were evident on standard histopathological examination. The cytokine IL-8 was more expressed in the proximal than the distal part of the track (fold increase 4.34 vs 3.60), while the reverse was found for IL-1beta (fold increase 1.33 vs 2.01); both were more intensely expressed compared with the normal anal mucosa. EMT was demonstrated, in both proximal and distal parts of the track, with an increase of TGF-beta, Vimentin, Zeb-1 and Snail and a mean decrease of E-cadherin. Western blot analysis and immunohistochemistry confirmed the protein expression. The study suggests that chronic inflammation is present in cryptoglandular fistulas. The inflammatory pattern might be different in the proximal than in the distal part of the fistula track. The cytokines IL-1beta and IL-8 could play a possible role in fistula formation. The study demonstrates for the first time the potential importance of EMT in the pathogenesis of cryptoglandular AF.

Phenotype of regenerative epithelium in idiopathic interstitial pneumonias.

The epithelial alteration in interstitial pneumonias is one of the repair processes at the sites of disease activity. Regenerative epithelial cells may participate in remodeling of the lung. To determine the phenotype of regenerative epithelial cells in usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP), the expression of Clara cell 10KD protein (CC10), cytokeratin (CK) 14 and 17, surfactant apoprotein (SP)-A, KL-6/MUC1, transforming growth factor (TGF) beta2 were examined in 25 patients with UIP, 9 patients with NSIP and normal lung tissues from 10 patients with lung cancer. In honeycomb lesions of UIP, non-ciliated columnar cells mainly expressed CC10, cuboidal cells expressed CC10, CK17, CK14 and SP-A in descending order. Fibroblastic foci are covered by CK17, CK14, CC10, and a few SP-A positive flattened or cuboidal cells. Regenerative epithelium in NSIP mainly comprised cuboidal cells expressing SP-A, CC10 and CK17. KL-6 was more remarkably expressed in cuboidal and non-ciliated columnar cells both in UIP and NSIP. Expression of TGFbeta2 was observed in cuboidal and flattened epithelium. In severe fibrotic areas, CC10 expressing cells were more prominent, while SP-A positive cells were more prominent in less fibrotic areas. Regenerative epithelial cells in remodeling area in UIP may be derived from bronchiolar basal cells and Clara cells, while most of those in NSIP may be derived from type II pneumocytes. The different origin of regenerative epithelium may reflect the severity and extent of the injury and the degree of consequent fibrosis in UIP and NSIP.

The impact of Fli1 deficiency in B cells on the development of systemic sclerosis

Fli1 is a potential predisposing factor of systemic sclerosis (SSc), whose deficiency is involved in its fibrotic and vascular processes. Given that B cells from Klf5+/−; Fli1+/− mice, spontaneously developing SSc-like tissue fibrosis and vasculopathy, represent SSc-like features such as higher CD 19 expression and autoantibody production, Fli1 deficiency may also contribute to its immunological aspect, such as B cell activation. Here, we confirmed decreased Fli1 expression in SSc-derived B cells and further explored the role of Fli1-deficient B cells in SSc utilizing B cell-specific Fli1 knockout (BcKO) mice. Fli1-deficient B cells revealed lower expression of CD22, an inhibitory receptor, and higher expression of BAFF receptor than control B cells. Consistently, Fli1-deficient B cells were constitutively activated with higher IL-6 secretion and autoantibody production. Furthermore, the proportion of regulatory B cells was decreased and Th17 skewed immune polarization was noted in Fli1 BcKO mice. As for the fibrotic and vascular aspects, of note, tissue fibrosis and SSc-like vascular changes were evident in the skin and lung of Fli1 BcKO mice. Relevant to tissue fibrosis, TGF-beta and IL-6 expression was elevated in the fibrotic tissue and cultivated Fli1-deficient B cells. More importantly, the direct interaction with Fli1-deficient B cells activated pro-angiogenic gene program and altered the expression of cell adhesion molecules promoting Th2/Th17 cell infiltration in endothelial cells. Collectively, Fli1-deficient B cells induces endothelial cell activation and Th17 skewed immune polarization, eventually leading to SSc-like tissue fibrosis and vasculopathy, indicating a pivotal contribution of Fli1 deficiency in B cells to the pathogenesis of SSc.

Abstract 273: T cell Sphingosine 1-Phosphate Receptor 1 Signaling Possesses Intrinsic Anti-fibrotic Effects in Murine Heart

T lymphocyte infiltration into myocardium has been observed in patients with myocardial infarction and diabetic cardiomyopathy. T cell trafficking from primary lymphoid organs into blood depends on S1P receptor 1 (S1P 1 ) activation and S1P concentration gradients. However, effects of T cell S1P 1 signaling activation on cardiac fibrosis and function remain unknown. Mice with conditional knockout of T cell S1P 1 were generated by cross-bred of S1P 1 loxp-loxp mice with Lck-Cre transgenic mice. T cell S1P 1 mutant mice (TS1P 1 KO) and wildtype littermate (WT) were utilized. To explore the effect of T cell S1P 1 on cardiac fibrosis and myocardial function, additional groups with reconstitution of T cells containing S1P 1 were conducted by injection of freshly prepared T cells from WT into TS1P 1 KO. Body weight, glucose levels, and behavior change were recorded twice a week. After 11 weeks, Langendorff heart perfusion was prepared. Cardiac contractile force was recorded. Trichrome staining was used to evaluate the extent of cardiac fibrosis. CD4, CD8, and Foxp3 regulatory T cells in the blood and spleen were quantified with flow cytometry. Expression of TGF-beta and CD34 in mouse myocardium was determined by using immunohistochemistry. Comparing with WT control, TS1P 1 KO mice exerted increased cardiac fibrosis and low cardiac contractile force (P&lt;0.05). TGF-beta expression and CD34 stained cells were significantly enhanced in TS1P 1 KO mice. Both CD4 and CD8 T cells in the blood and spleen were reduced significantly in TS1P 1 KO. Reconstitution of T cells with S1P 1 into TS1P1KO attenuated cardiac fibrosis and myocardial dysfunction. In conclusion, T cell S1P 1 signaling plays a critical role in anti-fibrosis and cardioprotection.

Hyaluronan oligosaccharides promote diabetic wound healing by increasing angiogenesis.

Hyaluronan (also known as hyaluronic acid) oligosaccharides (O-HA) can promote angiogenesis and wound healing; however, there are few reports on whether O-HA also plays a role in healing wounds of diabetic patients. In this study, we prepared a special ointment containing a mixture of hyaluronan fragments from 2 to 10 disaccharide units and investigated its effects on healing the wounds of diabetic rats. We found that O-HA significantly increases proliferation, migration, and tube formation of endothelial cells under high glucose conditions, and topical administration of O-HA ointment promotes wound healing by increasing angiogenesis in the wounded area of the skin. The underlying mechanisms are that O-HA increases the phosphorylation of Src and ERK, and expression of TGF beta1, thereby increasing angiogenesis. This suggests that topical application of O-HA could be a useful method by which to treat diabetic wounds in clinical practice.

Abstract SY03-01: Macrophage PI3Kgamma drives cancer immune suppression

Abstract Innate immune cells can switch from a proinflammatory state that defends against pathogens and to an anti-inflammatory state that repairs damaged tissue. Proinflammatory myeloid cells activate cytotoxic T cells to eliminate pathogens, but anti-inflammatory myeloid cells inhibit T-cell-mediated immunity, stimulate angiogenesis, and stimulate fibrosis, all of which promote tumor progression. The predominant isoform of PI(3)Kinase in myeloid cells, PI3Kgamma, controls this switch between immune stimulation and immune suppression during inflammation and cancer. Myeloid cell PI3Kgamma and its effectors Akt1, mTor, and S6Kinase stimulate C/EBPbeta-dependent expression of immunosuppressive factors, including Arginase and TGFbeta, while also suppressing NFkappaB-dependent expression of proinflammatory cytokines such as IL12 and IFNgamma, thereby inducing tumor immune suppression and consequent tumor growth and metastasis. In contrast, inhibition of PI3Kgamma activity by genetic or pharmacological means suppresses C/EPBbeta-mediated transcription of anti-inflammatory factors and stimulates NFkappaB-mediated transcription of the proinflammatory cytokines IL12 and IFNgamma, thereby restoring CD8+ T-cell-dependent cytotoxicity that inhibits tumor growth and metastasis. Therefore, inhibitory targeting of PI3Kgamma indirectly restores cytotoxic T-cell immune responses that elicit tumor suppression without unwanted side effects. Our studies highlight the strong therapeutic potential of targeting this kinase to control inflammation and cancer. This work was supported by grants to JAV from the NIH (5R01CA126820), the Landon Foundation-AACR (12-60-27-VARN), the Lustgarten Foundation for Pancreatic Research and the Whitworth Foundation for Cancer Research. Citation Format: Judith A. Varner. Macrophage PI3Kgamma drives cancer immune suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY03-01.

Abstract 718: Macrophage PI3Kgamma signaling promotes cancer immune suppression

Abstract Innate immune cells can switch from a pro-inflammatory state that defends against pathogens and to an anti-inflammatory state that repairs damaged tissue. Pro-inflammatory myeloid cells activate cytotoxic T cells to eliminate pathogens, but anti-inflammatory myeloid cells inhibit T cell mediated immunity, stimulate angiogenesis, and stimulate fibrosis, all of which promote tumor progression. The predominant isoform of PI(3)Kinase in myeloid cells, PI3Kgamma, controls the switch between immune stimulation and immune suppression during inflammation and cancer. Myeloid cell PI3Kgamma and its effectors Akt1, mTor, and S6Kinase stimulate C/EBPbeta-dependent expression of immunosuppressive factors, including Arginase and TGFbeta, but suppress NFkappaB-dependent expression of pro-inflammatory cytokines such as IL12 and IFNgamma, thereby inducing tumor immune suppression and consequent tumor growth and metastasis. In contrast, inhibition of PI3Kgamma activity by genetic or pharmacological means suppresses C/EPBbeta-mediated transcription of anti-inflammatory factors and stimulates NFkappaB-mediated transcription of the pro-inflammatory cytokines IL12 and IFNgamma, thereby restoring CD8+ T cell-dependent cytotoxicity that inhibits tumor growth and metastasis. Therefore, inhibitory targeting of PI3Kgamma indirectly restores cytotoxic T cell immune responses that elicit tumor suppression without unwanted side effects. Our studies highlight the strong therapeutic potential of targeting this kinase to control inflammation and cancer. This work was supported by grants to JAV from the NIH (5R01CA126820), the Landon Foundation-AACR (12-60-27-VARN), the Lustgarten Foundation for Pancreatic Research and the Whitworth Foundation for Cancer Research. Citation Format: Megan M. Kaneda, Judith A. Varner. Macrophage PI3Kgamma signaling promotes cancer immune suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 718.

Effects of IL-33/ST2 pathway in acute inflammation on tissue damage, antioxidative parameters, magnesium concentration and cytokines profile

AimThe aim of this study was to examine the role of IL-33/ST2 pathway in a pathogenesis of acute inflammation and its effects on tissue damage, antioxidative capacity, magnesium concentration and cytokine profile in acutely inflamed tissue. Material and methodsMale mice were randomly divided in four groups: wild-type control group (WT-C), ST2 knockout control group (KO-C), wild-type inflammatory group (WT-I), and ST2 knockout inflammatory group (KO-I). Acute inflammation was induced in WT-I and KO-I by intramuscular injection of turpentine oil, while mice in WT-C and KO-C were treated with saline. After 12h, animals were euthanized, and blood was collected for determination of creatine kinase (CK) and aspartate transaminase (AST) activity. The treated tissue was used for histopathological analysis, determination of volume density of inflammatory infiltrate (Vdii) and necrotic fiber (Vdnf), gene expression of interleukin (IL)-33, ST2, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-12p35, and transforming growth factor beta (TGF-beta), concentration of magnesium (Mg), copper (Cu), selenium (Se), manganese (Mn) and reduced glutathione (GSH), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity. ResultsPresence of inflammatory infiltration and necrosis in the treated tissue was histopathologically confirmed in WT-I and KO-I. Vdii was significantly higher in WT-I when compared to KO-I, whereas Vdnf did not significantly differ between WT-I and KO-I. CK and AST significantly increased in both inflammatory groups when compared to corresponding control groups. However, the values of CK and AST were significantly higher in WT-I than in KO-I. Mg in the treated tissue was significantly lower in WT-I in comparison to WT-C and KO-I, while there was no significant difference between KO-C and KO-I. There was no significant difference in Cu, Se, and Mn in the treated tissue between WT-C, KO-C, WT-I and KO-I. Gene expression of IL-33 in the treated tissue increased in both inflammatory groups when compared to the corresponding control groups, but it was significantly higher in KO-I than in WT-I. Gene expression of ST2 in the treated tissue was significantly higher in WT-I than in WT-C. Gene expression of TNF-alpha, IL-6, and IL-12p35 in the treated tissue was significantly higher in WT-I and KO-I than in the corresponding control groups, and IL-6 was significantly higher in KO-C than in WT-C. TGF-beta gene expression in the treated tissue was significantly higher in KO-I when compared to WT-I, while there was no difference between WT-C and KO-C. SOD activity decreased at the site of acute inflammation in both inflammatory groups, while the GPx activity increased. GSH in the treated tissue was significantly higher in KO-I than in KO-C or WT-I. ConclusionThe results of our study have indicated, to our knowledge for the first time, that IL-33/ST2 pathway plays a role in enhancing inflammation and tissue damage at the site of acute inflammation by affecting the concentration of magnesium and GSH, important for antioxidative capacity, as well as gene expression of anti-inflammatory cytokine TGF-beta.

Epigenetic consequences of an adverse intrauterine environment

The aim of this study was to examine the role of IL-33/ST2 pathway in a pathogenesis of acute inflammation and its effects on tissue damage, antioxidative capacity, magnesium concentration and cytokine profile in acutely inflamed tissue.Male mice were randomly divided in four groups: wild-type control group (WT-C), ST2 knockout control group (KO-C), wild-type inflammatory group (WT-I), and ST2 knockout inflammatory group (KO-I). Acute inflammation was induced in WT-I and KO-I by intramuscular injection of turpentine oil, while mice in WT-C and KO-C were treated with saline. After 12 h, animals were euthanized, and blood was collected for determination of creatine kinase (CK) and aspartate transaminase (AST) activity. The treated tissue was used for histopathological analysis, determination of volume density of inflammatory infiltrate (Vdii) and necrotic fiber (Vdnf), gene expression of interleukin (IL)-33, ST2, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-12p35, and transforming growth factor beta (TGF-beta), concentration of magnesium (Mg), copper (Cu), selenium (Se), manganese (Mn) and reduced glutathione (GSH), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity.Presence of inflammatory infiltration and necrosis in the treated tissue was histopathologically confirmed in WT-I and KO-I. Vdii was significantly higher in WT-I when compared to KO-I, whereas Vdnf did not significantly differ between WT-I and KO-I. CK and AST significantly increased in both inflammatory groups when compared to corresponding control groups. However, the values of CK and AST were significantly higher in WT-I than in KO-I. Mg in the treated tissue was significantly lower in WT-I in comparison to WT-C and KO-I, while there was no significant difference between KO-C and KO-I. There was no significant difference in Cu, Se, and Mn in the treated tissue between WT-C, KO-C, WT-I and KO-I. Gene expression of IL-33 in the treated tissue increased in both inflammatory groups when compared to the corresponding control groups, but it was significantly higher in KO-I than in WT-I. Gene expression of ST2 in the treated tissue was significantly higher in WT-I than in WT-C. Gene expression of TNF-alpha, IL-6, and IL-12p35 in the treated tissue was significantly higher in WT-I and KO-I than in the corresponding control groups, and IL-6 was significantly higher in KO-C than in WT-C. TGF-beta gene expression in the treated tissue was significantly higher in KO-I when compared to WT-I, while there was no difference between WT-C and KO-C. SOD activity decreased at the site of acute inflammation in both inflammatory groups, while the GPx activity increased. GSH in the treated tissue was significantly higher in KO-I than in KO-C or WT-I.The results of our study have indicated, to our knowledge for the first time, that IL-33/ST2 pathway plays a role in enhancing inflammation and tissue damage at the site of acute inflammation by affecting the concentration of magnesium and GSH, important for antioxidative capacity, as well as gene expression of anti-inflammatory cytokine TGF-beta.

Effect of DaChaiHu Decoction on pancreatic fibrosis induced by DBTC combined with alcohol and the mechanism of TGF-beta/Smad signaling pathway

To investigate the effects of transforming growth factor-β (TGF-β) and Smad in chronic pancreatitis (CP) induced by dibutyltin dichloride (DBTC) combined with ethanol, and the effect for prevention and treatment of pancreatic fibrosis by DaChaiHu Decoction (tradeiional Chinese medicine). Ninety-six healthy male Kunming mice were randomly divided into control group (Con group), chronic pancreatitis (CP group) group, Dachai Hu decoction treatment group (DCHD group) (n=32). Then the mice were treated with DBTC 8 mg/kg by tail vein injection and fed with 10% ethanol replacing the normal drinking water to replicate mouse CP model. After three days of modeling the mice were randomly divided into CP group and DCHD group. The mice in DCHD group were administered intragastrically with DaChaiHu Decoction (1 g/ml, 6 g/kg·d) at the following 8 weeks. After modeling for 1 week, 2 weeks, 4 weeks and 8 weeks, the mice were anesthetized and sacrificed(n=8). The morphology and the degree of fibrosischanges of pancreatic tissue were detected by HE staining. The protein expressions of type I collagen and TGF beta R I, p-Smad 2/3 and Smad 7 in pancreatic tissue were detected by Western blot assay. The expressions of MMP-1/TIMP-1 mRNA inpancreatic tissue were detected by RT-PCR. Compared with control group, DBTC combined with ethanol induced CP with increased activity of serum amylase and hyaluronic acid level. While in DCHD group, the activity of amylase and hyaluronic acid level were decreased significantly. Compared with control group, the protein expressions of COLA1, TGF beta R I, p-Smad 2/3 in CP Group were elevated,but the expression of Smad 7 was significantly reduced; In DCHD treatment group,the expression of TGF beta R I, p-Smad 2/3 and COLA1 were reduced, and the protein expression of Smad 7 was increased. In 2w and 4w, the level of MMP-1mRNA continued to decrease, while TIMP-1mRNA expression was increased significantly (P<0.01). At each time point of DCHD group, the expression of MMP-1 were markedly increased and TIMP-1 were reduced, compared with those of the CP group (P<0.05). DaChaiHu Decoction play a role in the prevention and treatment of chronic pancreatitis and the development of fibrosis, the mechanism may related to inhibit the activation of TGF beta/Smad signaling pathway, and regulate the balance between synthesis and degradation of extracellular matrix.