Effect of vaginally administered fumagillin on immunohistochemical distribution of leukemia inhibitory factor,interleukin 6, transforming growth factor beta and vascular endothelial growth factor in implantation stage endometrium of the rhesus monkey

Intravaginal administration of an anti-angiogenic agent, fumagillin, during blastocyst implantation inhibits pregnancy establishment in a dose-related manner in the rhesus monkey. In the present study, mated female rhesus monkeys were vaginally inserted with tampons containing vehicle (group 1; n = 5) and test agent (fumagillin, 4 mg/animal; group 2; n = 6) on cycle day 20, and endometrial tissue samples were collected on cycle day 24 from all monkeys and processed for histological examination and immunohistochemical localization for LIF, IL-6, TGF-beta and VEGF. Concentrations of estradiol-17 beta, progesterone and chorionic gonadotrophin in peripheral circulation were determined. From the serum profiles of the hormones, 2 monkeys in group 1, and 1 monkey in group 2 appeared pregnant. However, endometrial morphology revealed histological evidence of pregnancy in 3 out of 6 fumagillin-treated animals. Histometric analysis of immunohistochemical staining in epithelial, stromal and vascular compartments revealed that per cent areas occupied by immunoprecipitate for the cytokines studies did not change in epithelial and stromal compartments, except that for TGF-beta which was higher (P < 0.05) in epithelial compartment in group 2. No change was observed in immunoprecipitation areas for IL-6 in epithelial, stromal and vascular compartments. On the other hand, changes (P < 0.05) for LIF, TGF-beta and VEGF were evident in the vascular compartment. It is possible that disparate responses observed in glandular, stromal and vascular compartments in implantation stage endometrium following fumagillin treatment actually caused from associated decline in progesterone concentration in peripheral circulation. It is also possible that fumagillin, an angiostatic agent, affects the synthesis and secretion of cytokines primarily in the vascular compartment of implantation stage endometrium, and thereby manifests differential responses in epithelial, stromal and vascular compartments.     

Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5)

Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay.     

Effect of transforming growth factor beta 1 (TGF-beta 1) on nitric oxide production and lipid peroxidation in oral mucosal wound healing

Wound healing is a highly orchestrated process including complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important modulators. Exogenous TGF-beta treatment has been shown to accelerate wound healing in normal and impaired animal models. Nitric oxide (NO) also plays a key role in wound healing. The objective of this study is to examine the effects of exogenous TGF-beta 1 treatment on NO and lipid peroxidation levels in the process of oral wound healing on different days. In this study, we used 5-month-old New Zealand albino male rabbits. After a standard surgical incision in the diestema region, the rabbits were divided into controls and TGF-beta 1 implanted groups. NO levels and malondialdeyhde (MDA) levels which are indicators of lipid peroxidation were determined by spectrophotometry. In the TGF-beta 1 implanted groups, both NO and MDA levels significantly increased only on the third day after wounding when compared to control groups. We found decreased MDA levels parallel to NO levels on the fifth day after wounding. These findings suggest that TGF-beta 1 affects mucosal wound healing by altering NO production on different days of wounding. TGF-beta 1 may regulate NO production by its dual effect in as both an activator and inhibitor an in oral mucosal healing.     

白花丹醌对瘦素刺激人肝星状细胞转化生长因子-β_1表达的影响

目的研究白花丹醌对瘦素刺激体外培养人HSC-LX2TGF-β1 mRNA和蛋白表达的影响。方法体外培养HSC-LX2,瘦素(leptin)刺激24h,各药物与细胞共孵育24h后,采用荧光PCR检测各组TGF-β1 mRNA的表达和免疫组织化学方法检测TGF-β1蛋白表达情况。结果与leptin组比较,白花丹醌各剂量组作用24h后,HSC-LX2细胞中TGF-β1 mRNA的水平明显降低,尤以中、高剂量组明显(P<0.01);免疫组化结果显示白花丹醌各剂量组对HSC-LX2细胞TGF-β1蛋白表达有一定的抑制作用,且作用呈剂量依赖性。结论白花丹醌抗肝纤维化作用机制之一是从mRNA和蛋白水平抑制TGF-β1表达,从而抑制HSCECM的合成,发挥抗肝纤维化作用。     

trans-3,4'-Dimethyl-3-hydroxyflavanone, a hair growth enhancing active component, decreases active transforming growth factor beta2 (TGF-beta2) through control of urokinase-type plasminogen activator (uPA) on the surface of keratinocytes

trans-3,4'-Dimethyl-3-hydroxyflavanone (t-flavanone) is a synthetic compound with hair growth enhancing activity that is effective against male pattern alopecia. t-Flavanone was designed as a derivative of astilbin, the active hair growth enhancing component of Hypericum perforatum extracts. This study was designed to elucidate the mechanism of hair growth enhancement by t-flavanone. We investigated the effects of t-flavanone on transforming growth factor beta (TGF-beta), a known catagen-inducing factor induced in hair papilla cells by male hormone. When t-flavanone was added to cocultures of human hair papilla cells and human keratinocytes, there was no change in the total level of TGF-beta2. However, levels of active TGF-beta2 were reduced, suggesting the involvement of t-flavanone in the activation pathway of TGF-beta2. In order to investigate the effects of t-flavanone on TGF-beta2 activation by human keratinocytes, we evaluated the level of active TGF-beta2 converted from the inactive form in t-flavanone-treated human keratinocytes. The amount of active TGF-beta2 was reduced compared with controls suggesting that t-flavanone suppresses the TGF-beta2 activation cascade in human keratinocytes. We then examined the activity of urokinase-type plasminogen activator (uPA), the rate-limiting enzyme in the TGF-beta2 activation cascade, in t-flavanone-treated human keratinocytes. We found that t-flavanone reduces uPA activity on the keratinocyte surface. t-Flavanone is a hair growth enhancing component that has a novel mechanism of action which suppresses TGF-beta2 activation, and thereby is expected to have therapeutic effects on other types of alopecia in addition to male pattern alopecia.     

Loreclezole inhibition of recombinant alpha1beta1gamma2L GABA(A) receptor single channel currents

Loreclezole had two different effects on GABA(A) receptor (GABAR) currents. When applied to GABARs that contained a beta2 or beta3 subunit subtype, but not a beta1 subtype, loreclezole potentiated the peak current evoked by sub-maximal concentrations of GABA. Loreclezole also increased the rate and degree of apparent desensitization of GABAR whole-cell currents, an effect that was independent of the beta subunit subtype, suggesting that potentiation and inhibition of GABAR current by loreclezole occurred through separate sites. We used patch-clamp recording from outside-out and inside-out patches from L929 fibroblasts transiently transfected with rat GABAR subunits to examine the properties of inhibition of alpha1beta1gamma2L single channel currents by loreclezole. Loreclezole decreased the mean open time of the channel by decreasing the average durations of the open states. Loreclezole also increased the occurrence of a closed component with an average duration near 20 ms. Inhibition by loreclezole was not voltage-dependent. Loreclezole was equally effective when applied to the intracellular side of the receptor, suggesting that its binding site was readily accessible from both sides of the membrane. Pre-application of loreclezole effectively inhibited the GABAR current in macropatches, indicating that binding did not require an open channel. These findings were consistent with a mechanism of allosteric modulation at a site formed by the membrane spanning regions of the receptor.     

Development of high-throughput radioligand binding assays for interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF-alpha) in isolated membrane preparations.

Cytokine binding has been studied in a variety of intact cells, and in isolated receptor preparations. Each approach is associated with limitations with regard to screening large numbers of samples on a repetitive basis. In order to provide a more reproducible system of screening for compounds which modify IL-1 alpha and TNF-alpha binding, we have developed isolated membrane preparations for studying agents which can alter the association of these ligands with their receptors. These results demonstrate IL-1 alpha binding to BALB/c 3T3 cell membranes and TNF-alpha binding to HeLa S3 cell membranes, and indicate that this is a viable approach to high-throughput screening.     

Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).     

鼻息肉复发与血管内皮生长因子和转化生长因子-β1的表达

目的　探讨血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)和转化生长因子 β1(transforminggrowthfactor β1,TGF β1)的表达与鼻息肉复发关系。方法　 6 0例内窥镜鼻窦术后鼻息肉复发病人 ,用SP免疫组化染色法比较VEGF和TGF β1的表达。结果　VEGF和TGF β1在鼻息肉组织的血管内皮细胞和腺体细胞的表达明显高于中鼻甲组织 (P <0 0 1) ;鼻息肉临床分型与VEGF和TGF β1的表达无关。结论　VEGF和TGF β1的表达可能与鼻息肉复发有关 ,术后定期合理的鼻窦内窥镜随访是预防和减少复发的重要手段。     

Low doses of 2,3,7,8-tetrachlorodibenzo- p-dioxin increase transforming growth factor beta and cause myocardial fibrosis in marmosets ( Callithrix jacchus)

Epidemiological studies have suggested an association between exposure to dioxins and cardiovascular morbidity and mortality. However, cardiotoxic effects of low doses of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in animals have not been reported so far. We studied the hearts of male marmosets ( Callithrix jacchus)after treatment with single subcutaneous doses of 1, 10 or 100 ng TCDD/kg body weight or vehicle (toluene/DMSO 1+2 v/v, 100 microl/kg body weight). The animals were killed 2 or 4 weeks after treatment. Tissue samples of left ventricular myocardium were stained with picrosirius red and examined histologically along with quantitative image analysis. Extracellular matrix proteins were additionally analysed by western blotting. Monkeys showed no overt signs of toxicity nor did their relative heart weights differ significantly depending on treatment. Histology revealed an increase of picrosirius red-positive area above control values in 2 of 4 (1 ng TCDD/kg body weight), 6 of 12 (10 ng/kg) and 6 of 10 (100 ng/kg) marmosets. Western blotting confirmed these histological findings showing an increase of collagen, fibronectin and laminin in the hearts of TCDD-treated animals. Western blotting additionally showed an increased concentration of transforming growth factor beta1 (TGF-beta1) as well as TGF-beta receptor type I which could be a functional link to the effects on extracellular matrix. Our findings might explain the association of TCDD exposure with increased cardiovascular mortality observed in epidemiological studies and should stimulate further research on the role of changes in the extracellular matrix in the toxic effects of dioxins and related substances on other organs.     

Specific inhibition of hypoxia-inducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids

Screening using a reporter under the control of the hypoxia-response element (HRE) identified several flavonoids and homoisoflavonoids that inhibit the activation of HRE under hypoxic conditions. Among various compounds, isorhamnetin, luteolin, quercetin, and methyl ophiopogonanone B (MOB) were effective at 3 to 9 microg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by MOB in HepG2 cells, but the effective doses were 10 to 20 microg/ml. MOB caused destabilization of hypoxia-inducible factor (HIF)-1alpha, as revealed by Western blotting, that was dependent on proteasome activity and the tumor suppressor, p53. The tubular formation and migration of human umbilical vein endothelial cells was also inhibited by MOB. MOB is expected to act as an inhibitor of angiogenesis.     

地塞米松抑制转化生长因子β1对人α1(Ⅰ)前胶原基因的转录激活

目的探讨地塞米松与转化生长因子β1(TGFβ1)之间的相互作用及对人α1(Ⅰ)前胶原基因启动转录的影响.方法人皮肤及瘢痕成纤维细胞原代、传代培养.采用FuGENE转染试剂,分别瞬间转染含人α1(Ⅰ)胶原基因5′侧翼序列-2.5kb与报告基因氯霉素乙酰基转移酶(CAT)的重组体phCOL2.5至人皮肤及瘢痕成纤维细胞.ELISA法测定地塞米松及TGFβ1作用24h后,转染了phCOL2.5的2种成纤维细胞的报告基因CAT表达量.结果地塞米松能抑制转染了phCOL2.5重组体的人皮肤及瘢痕成纤维细胞CAT表达量,且能拮抗TGFβ1对转染了phCOL2.5重组体的2种成纤维细胞CAT表达的上调作用(P＜0.05).结论在正常皮肤及瘢痕成纤维细胞中,地塞米松均能抑制人α1(Ⅰ)前胶原基因的启动转录,且能拮抗TGFβ1对人α1(Ⅰ)前胶原基因的转录激活.     

Differential regulation of epidermal langerhans cell migration by interleukins (IL)-1alpha and IL-1beta during irritant- and allergen-induced cutaneous immune responses

Tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-18 are all known to contribute to the regulation of epidermal Langerhans cells (LC) migration and the subsequent accumulation of dendritic cells (DC) in draining lymph nodes following skin sensitization. However, the cytokine signals that control these responses following skin irritation have yet to be defined. We demonstrate that IL-1alpha, a cytokine associated with skin injury and inflammation, is able to stimulate the activation and migration from the epidermis of LC and their subsequent accumulation in skin-draining lymph nodes. Stimulation of these responses by IL-1alpha required the local availability of TNF-alpha. Using specific neutralizing antibodies, LC migration induced following skin sensitization with oxazolone (Ox) was found to be dependent upon IL-1beta and independent of a requirement for IL-1alpha. However, the converse was true following stimulation of responses with the nonsensitizing skin irritant sodium lauryl sulfate (SLS). Here, the loss of LC from the epidermis and the accumulation of DC in draining lymph nodes required IL-1alpha and not IL-1beta. Despite utilizing different IL-1 isoforms for LC mobilization, the phenotypic characteristics of DC arriving in draining lymph nodes in response to Ox and SLS were similar with respect to the membrane determinants MHC class II, B7-1, B7-2, and intercellular adhesion molecule-1. These data suggest that contact sensitization and skin irritation employ subtly different cytokine networks in the regulation of LC migration, both involving TNF-alpha but demonstrating differential requirements for IL-1 cytokines. The proposal is that different forms of cutaneous trauma may achieve LC migration through distinct molecular mechanisms.     

Activation of genes for spasmolytic peptide, transforming growth factor alpha and for cyclooxygenase (COX)-1 and COX-2 during gastric adaptation to aspirin damage in rats

Background:

NSAIDs, such as aspirin (ASA), cause widespread mucosal damage, but repeated ASA insults appear to induce mucosal tolerance (adaptation) to this?injury. The mechanism of the gastric adaptation to the damage induced by ASA has not been fully explained.

Aim:

To determine the role of the mucosal gene expression for spasmolitic peptide (SP) (a member of trefoil peptides) and transforming growth factor alpha (TGFα) as well as for cyclooxygenase (COX)-1 and COX-2 during gastric adaptation to ASA in rats.

Methods:

Gastric lesions were produced by ASA (100 mg/kg in 1.5 mL of 0.2 M HCl) applied intragastrically (i.g.) as a single dose, every day for 5 days. Control rats were given 1.5 mL of vehicle (0.2 M HCl i.g.) as a single dose, during 5 consecutive days. Gastric blood flow (GBF) was measured by H₂-gas clearance technique and gastric mucosal specimens were taken for the assessment of cell?proliferation rate in gastric mucosa by bromodeoxyuridine (BrdU) uptake, mucosal generation of prostaglandin E₂ measured by radioimmunoassay, and for expression of SP, TGFα COX-1 and COX-2 mRNA as determined by RT-PCR. To quantify the relative amounts of mRNA for SP and TGFα, southern blotting analysis of the PCR products was performed and the intensity of PCR products was compared with that of β-actin used as a standard.

Results:

ASA applied once produced numerous gastric erosions, but with repeated ASA doses the adaptation to this NSAID developed, the area of gastric lesions being reduced by 86% after six consecutive ASA insults. This adaptation to ASA was accompanied by approximately a 90% reduction in prostaglandin E₂ biosynthesis, by a significant rise in BrdU uptake by glandular cells predominantly in the neck region of gastric glands and by expression of SP (SP/β-actin ratio; 0.96 ± 0.08 in ASA-adapted mucosa vs. 0.38 ± 0.05 in the control mucosa) and TGFα (TGFα/β-actin ratio: 0.97 ± 0.07 in ASA-adapted mucosa vs. 0.77 ± 0.06 in the control mucosa). COX-1 expression was detected in vehicle-control gastric mucosa and after single exposure to ASA or after six consecutive ASA insults, while COX-2 mRNA was not detected in vehicle-control gastric mucosa, but appeared after single ASA insult and was sustained after subsequent ASA doses.

Conclusions:

(i) Gastric adaptation to aspirin injury involves enhanced cell proliferation which appears to be mediated by increased expression of SP and TGFα, and (ii) rapid upregulation of COX-2 expression following single and repeated ASA insults may represent a compensatory response to suppression of prostaglandin generation by this NSAID.

     

Effect of an angiotensin II receptor blocker and two angiotensin converting enzyme inhibitors on transforming growth factor-beta (TGF-beta) and alpha-actomyosin (alpha SMA), important mediators of radiation-induced pneumopathy and lung fibrosis

Progressive, irreversible fibrosis is one of the most clinically significant consequences of ionizing radiation on normal tissue. When applied to lungs, it leads to a complication described as idiopathic pneumonia syndrome (IPS) and eventually to organ fibrosis. For its high mortality, the condition precludes treatment with high doses of radiation. There is widespread interest to understand the pathogenetic mechanisms of IPS and to find drugs effective in the prevention of its development. This report summarizes our experience with the protective effects of L 158,809, an angiotensin II (ANG II) receptor blocker, and two angiotensin converting enzyme (ACE) inhibitors in the development of IPS and the role of transforming growth factor beta (TGF-beta) and of alpha-actomyosin (alpha SMA) in pathogenesis of radiation induced pulmonary fibrosis in an experimental model of bone marrow transplant (BMT). Male WAG/Riji/MCV rats received total body irradiation and a regimen of cyclophosphamide (CTX) in preparation for bone marrow transplant. While one group of animals remained untreated, the remainders were subdivided into three groups, each of them receiving either the ANG II receptor blocker or one of the two ACE inhibitors (Captopril or Enalapril). Each of the three drugs was administered orally from 11 days before the transplant up to 56 days post transplant. At sacrifice time the irradiated rats receiving only CTX showed a chronic pneumonitis with septal fibrosis and vasculitis affecting, in particular, small caliber pulmonary arteries and arterioles. Their lung content of hydroxyproline was also markedly elevated in association with the lung concentrations of thromboxane (TXA2) and prostaglandin (PGI(2)), (two markers of pulmonary endothelial damage). A significant increase of alpha actomyosin staining was observed in vessels, septa and macrophages of the same animals which also overexpressed TGF-beta. When L 158,809, Captopril and Enalapril were added to the radiation and cytoxan treatment, a significant amelioration of the histological damage as well as the overexpression of alpha SMA was observed. Lung concentrations of hydroxyproline, PGI(2), TXA2 and TGF-beta were also observed in these animals so that the values of these compounds were closer to those measured in untreated control rats than to their irradiated and cytoxan treated counterparts. Angiotensin II plays an important role in the regulation of TGF-beta and alpha SMA, two proteins involved in the pathogenesis of pulmonary fibrosis. The finding that ACE inhibitors or ANG II receptor blockers protect the lungs from radiation induced pneumonitis and fibrosis reaffirms the role that ANG II plays in this inflammatory process and suggests an additional indication of treatment of this condition, thus opening a new potential pharmacologic use of these drugs.     

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 micrograms or 200 micrograms MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 micrograms MDP enhanced LAF activity whereas 200 micrograms caused inhibition. Increased dilution of 200 micrograms exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulated in vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential therapeutic action.