Total TGF-beta Research Articles

Hormone-immunotherapy in endocrine dependent metastatic breast cancer patients

Insoluble and inactive protein aggregates known Hormone therapy is advised for ER+ metastatic breast cancer patients due to its efficacy concomitant with low toxicity; however, in most patients the occurrence of resistance is a not well yet understood hurdle to overcome. In these patients, during clinical benefit (CB) from conventional anti-estrogens, the addition of cycles of sequential immunotherapy could prolong the benefit and delay the arising of acquired hormone resistance. In order to validate this hypothesis, in 1992 we started an open exploratory clinical trial. Forty-two of these patients in CB during first line anti-estrogen salvage therapy also received beta-interferon (INF-beta) 3,000,000 IU i.m./day 3 days/week for 1-4 weeks and successively recombinant interleukin-2 (IL-2) 3,000,000 IU s.c./day 3 days/week for 5-8 weeks until progression. The immunotherapy cycle lasted 10 weeks and the patient continued anti-estrogen alone during 9-10 weeks, the 11th week being the first week of the successive cycle. At each control visit, routine laboratory examinations and serum measurement of a CEA, TPA, CA15.3 tumor marker (TM) panel were carried out, and an immunological assessment was made (total lymphocytes, CD4+, CD8+, NK cells, T-reg, IL-6, IL-10, IL-12, TNFa, TGFbeta1 and IFNgamma). The addition of INF-beta-IL-2 sequence significantly prolonged clinical benefit and overall survival from conventional antiestrogens. During CB as opposed to progression, a significant immune stimulation was observed. During CB also a significant CEA, TPA, CA15.3 decrease occurred 24–72 h after interleukin-2 administration. At the progression a significant increase for CEA and for all three markers (standardized values) was found 24–72 h after interleukin-2 administration. In patients who survived less than five years, the Treg cell increase occurred at a significantly shorter time interval than in those who survived longer than five years (20 vs. 45.5 months, respectively; P=0.001). To further confirm these promising results, a multicenter prospective phase II trial is going to be launched by the Cancer Center Institute of Tuscany in Italy. Note: This work was presented in Joint Event on 22nd Edition of International Conference on Immunology and Evolution of Infectious Diseases & 12th Edition of International Conference on Tissue Engineering and Regenerative Medicine during May 10-11, 2018 Frankfurt, Germany

Abstract LB-285: Pro-tumor N2 neutrophils contribute to the pre-metastatic niche.

Abstract Metastasis is the primary cause of cancer-related mortalities and the tissue microenvironment that supports tumor cell attachment and growth at metastatic sites has become an active area of investigation. Here we present evidence that pro-tumor N2 neutrophils are one of the bone marrow-derived cell types that establish this pre-metastatic niche. In the murine Lewis Lung adenocarcinoma (LLC) in vivo metastasis model, tumor cells metastasize to the lungs with increasing primary tumor growth over time. In these studies, immunocompetent mice injected subcutaneously with syngeneic LLC cells are sacrificed sequentially 10-32 days later, before and after established lung metastases. Neutrophil chemoattractants KC/CXCL1 and MIP-2/CXCL2 are significantly elevated in pre-metastatic tissue compared to normal lungs of healthy contemporary controls. MCP-1/CCL2, a marker of pro-tumor N2 neutrophils, increases steadily over time in pre-metastatic lung tissue of study mice and is significantly elevated in pre-metastatic tissue and in metastases compared to normal lung tissue. Isolated lung-infiltrating neutrophils also express N2 markers. In contrast, TNF-alpha, a marker for anti-tumor N1 cells, remains low or undetectable in lung tissue throughout metastatic tumor development. Neither active nor total TGF-beta, the putative control switch for a pro-tumor phenotype, is elevated in pre-metastatic tissue. Notably, levels of all cytokines are low or undetectable in normal lungs from naïve mice and in other organs of tumor-bearing mice (non-metastatic sites). Ly6G+ neutrophils were detected at a higher density in the lungs of pre-metastatic and metastatic mice compared to normal lungs, although the density of myeloperoxidase (MPO)-stained cells was similar in all groups. These data are consistent with recent reports that MPO activity is low or absent from tumor-associated neutrophils, although high in MDSC and naïve neutrophils. Neutrophil distribution in metastatic lung tissue is most concentrated around the tumor foci and tumor vasculature. Finally, studies of lung tissue from mice bearing LLC variants with different metastatic capacities are also consistent with our premise that neutrophils help establish the pre-metastatic niche. Understanding the factors that regulate the development and growth of malignant cells at metastatic sites is critical for improved clinical strategies in the prevention, diagnosis and treatment of metastases. Citation Format: Jered Cope Meyers, H. Keith Pittman, Hunter Story, Dare M. Imes, George A. Howard, Kathryn M. Verbanac. Pro-tumor N2 neutrophils contribute to the pre-metastatic niche. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-285. doi:10.1158/1538-7445.AM2013-LB-285

The role of transforming growth factor-β1 and oxidative stress in podoconiosis pathogenesis

Podoconiosis (endemic nonfilarial elephantiasis) occurs in susceptible individuals who go barefoot in regions of irritant volcanic soil. Silicate particles absorbed via the skin are thought to induce an inflammatory process and a consequent endolymphangitis of the lower leg lymphatics. To establish which oxidative stress biomarkers play a part in the inflammatory process, and to test whether transforming growth factor (TGF)-beta1 also has a pathogenetic role. We enrolled 50 patients with early clinical stage disease, 43 patients with advanced stage disease and 35 local healthy controls. Oxidative stress biomarkers included serum total peroxides (TP), total antioxidant capacity (TAC), total nitrate plus nitrite (TN), malondialdehyde (MDA) and total superoxide dismutase (SOD) activity. The oxidative stress index (OSI) was also determined. Serum total TGF-beta1 was assayed using sandwich enzyme-linked immunosorbent assay. Compared with healthy controls, patients with early stage disease showed significantly higher mean levels of TP (P < 0.001), MDA (P < 0.05) and OSI (P < 0.01); and significantly lower mean concentrations of SOD (P < 0.001) and TGF-beta1 (P < 0.001). Mean levels of TGF-beta1 were even lower among patients with advanced stage disease (P < 0.001). Mean TAC levels were significantly lower among patients with advanced disease than either other group (P < 0.001). This is the first study, to our knowledge, to attempt to elucidate the molecular pathogenetic events in podoconiosis. We conclude that TGF-beta1 may have a pathogenetic role, with oxidative stress playing a minor role in the early stages of disease.

Loss of syndecan‐1 is associated with malignant conversion in skin carcinogenesis

Syndecan-1 (sdc-1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration, and response to growth factors. In keratinocytes, loss of sdc-1 delays wound healing, reduces migration, and increases Transforming growth factor beta (TGFbeta) 1 expression. In this study we show that sdc-1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc-1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. sdc-1 expression on wt mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc-1 null mice expressed suprabasal alpha3 and beta4 integrins; suprabasal beta4 integrin is a marker of a high risk for progression. While the proliferative response to phorbol-12-myristate-13-acetate (TPA) did not differ among the genotypes, sdc-1 null mice had an enhanced inflammatory response and retained higher levels of total TGFbeta1 within their skin after TPA treatment. sdc-1 null keratinocytes, transduced in vitro by oncogenic ras(Ha), expressed higher levels of beta4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wt ras(Ha)-transduced keratinocytes. When ras(Ha)-transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc-1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc-1 could be a useful marker for progression in neoplastic skin lesions.

TJN-331 Improves Anti-glomerular Basement Membrane Nephritis by Inhibiting the Production of Intraglomerular Transforming Growth Factor-.BETA.1

Transforming growth factor-beta1 (TGF-beta1) plays an important role in the development of glomerulonephritis. The study of experimental glomerulonephritis in rats was performed to examine the antinephritic effects of TJN-331, a new herbally-derived chemical compound. To clarify the action of TJN-331 ((E)-N-(3,4-dimethoxyphenethyl)-N-methyl-3-(3-pyridyl)-2-propenamide) on TGF-beta1 production, glomeruli were isolated from rats with antiglomerular basement membrane (GBM) nephritis and incubated for 48 h with test drugs in vitro. Next, we examined the effects of TJN-331 on rat anti-GBM nephritis induced by injection with anti-GBM serum. TJN-331 dose-dependently inhibited the increase in total and mature TGF-beta1 production from nephritic glomeruli, although it did not inhibit TGF-beta1 production from normal glomeruli. Administration of TJN-331, at a dose of 2 mg/kg/d, per os (p.o.), prevented proteinuria and increased crescent formation and adhesion of capillary walls to Bowman's capsule. The increases in mature TGF-beta1 protein production and TGF-beta1 staining score in nephritic rats were reversed by TJN-331 treatment. These results suggest that TJN-331 inhibits proteinuria and histopathological changes in glomeruli via suppression of TGF-beta1 production from inflamed glomeruli.

Adenoviral Gene Transfer of Active Human Transforming Growth Factor-β2 Elevates Intraocular Pressure and Reduces Outflow Facility in Rodent Eyes

Purpose. Glaucoma is a leading cause worldwide of blindness and visual impairment. Transforming growth factor-beta2 (TGFbeta2) has been implicated in the pathogenesis of primary open-angle glaucoma (POAG) based on elevated levels in glaucomatous aqueous humor and its ability to induce extracellular matrix (ECM) remodeling in the trabecular meshwork (TM). The goal of this study was to generate a rodent model of POAG using viral gene transfer of human TGFbeta2. Methods. Latent (hTGFbeta2(WT)) or active (C226S, C228S; hTGFbeta2(226/228)) TGFbeta2-encoding cDNA was cloned into the pac.Ad5.CMV.K-N.pA shuttle vector for generation of replication-deficient adenovirus. Empty adenovirus (Ad5.CMV.K-N.pA) was used as a control. Adenoviral expression of active and total TGFbeta2 was assayed in vitro by the transduction of Chinese hamster ovary and trabecular meshwork cells. BALB/cJ mice or Wistar rats were injected either intracamerally or intravitreally with the adenovectors and assessed for changes in intraocular pressure (IOP) using the rebound tonometer. At peak IOP, aqueous outflow facility and total TGFbeta2 levels in aqueous humor were measured. Mouse eye morphology was assessed by hematoxylin and eosin staining. Results. Adenoviral gene transfer of hTGFbeta2(226/228), but not hTGFbeta2(WT), to the rodent eye elevated IOP in rat (43%, P < 0.001) and mouse (110%, P < 0.001) and reduced aqueous humor outflow facility in the mouse. The TGFbeta2-induced ocular hypertension correlated with anterior segment TGFbeta2 expression levels (P < 0.0001). Conclusions. The adenoviral TGFbeta2 rodent model displays the glaucoma risk factors of elevated IOP and decreased aqueous outflow facility and may potentially serve as a model for studying glaucoma.

Anti-fibrotic effect of thalidomide through inhibiting TGF-β-induced ERK1/2 pathways in bleomycin-induced lung fibrosis in mice

This study is designed to confirm the anti-fibrotic effect of thalidomide on bleomycin-induced lung fibrosis in a mouse model and to identify whether this anti-fibrotic effect is associated with inhibition of the transforming growth factor-beta (TGF-beta)-induced extracellular signal-regulated kinase1/2 (ERK1/2). C57BL/6 female mice were administered blomycin sulfate. In cultured human lung fibroblasts, expressions of type I collagen, fibronectin, and either TGF-beta or IL-6 were measured after thalidomide treatment by reverse transcription-polymerase chain reaction (RT-PCR). Expressions of ERK1/2, type I collagen, fibronectin, and TGF-beta1 from lung tissues of blomycin-induced mice and from mouse lung fibroblasts were evaluated using RT-PCR and western blotting. Thalidomide administration significantly inhibits TGF-beta1 mRNA expression in a dose-dependant manner following administration of IL-6 and IL-6R. In the analysis of BAL fluids, total BAL inflammatory cell counts, TGF-beta1, and IL-6 levels in thalidomide-treated mice were significantly reduced when compared with bleomycin-treated mice (p < 0.01, p < 0.01, and p < 0.001, respectively). Thalidomide inhibited total ERK1/2 and phospho-ERK1/2 expression after TGF-beta1 stimulation in the RT-PCR and western blotting. The results of our study suggest that the anti-fibrotic effect of thalidomide on lung fibrosis may be related to suppression of the TGF-beta1-induced ERK1/2 signaling pathway.

In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

Thrombospondin 1 (TSP-1), which is contained in platelet α-granules and released with activation, has been shown to activate latent TGF-β1 in vitro, but its in vivo role is unclear as TSP-1-null (Thbs1−/−) mice have a much less severe phenotype than TGF-β1-null (Tgfb1−/−) mice. We recently demonstrated that stirring and/or shear could activate latent TGF-β1 released from platelets and have now studied these methods of TGF-β1 activation in samples from Thbs1−/− mice, which have higher platelet counts and higher levels of total TGF-β1 in their serum than wild type mice. After either two hours of stirring or shear, Thbs1−/− samples demonstrated less TGF-β1 activation (31% and 54% lower levels of active TGF-β1 in serum and platelet releasates, respectively). TGF-β1 activation in Thbs1−/− mice samples was normalized by adding recombinant human TSP-1 (rhTSP-1). Exposure of platelet releasates to shear for one hour led to near depletion of TSP-1, but this could be prevented by preincubating samples with thiol-reactive agents. Moreover, replenishing rhTSP-1 to human platelet releasates after one hour of stirring enhanced TGF-β1 activation. In vivo TGF-β1 activation in carotid artery thrombi was also partially impaired in Thbs1−/− mice. These data indicate that TSP-1 contributes to shear-dependent TGF-β1 activation, thus providing a potential explanation for the inconsistent in vitro data previously reported as well as for the differences in phenotypes of Thbs1−/− and Tgfb1−/− mice.

Deficient activation and different expression of transforming growth factor-beta isoforms in active proliferative diabetic retinopathy and neovascular eye disease.

An increased expression and secretion of angiogenic growth factors was proposed to occur in proliferative diabetic retinopathy and other neovascularizing retinal diseases. However, a loss of anti-angiogenic factors also might promote retinal neovascularization. Therefore we investigated the active and latent vitreous levels of the subtypes of the endothelial anti-mitogen transforming growth factor-beta in vitreous of 58 patients. Four groups of patients were compared: Controls without retinal hypoxia, patients with quiescent and active proliferative diabetic retinopathy (PDR), and patients with severe retinal hypoxia resulting in rubeosis iridis. Whereas the amount of total TGF-beta in the four groups did not differ significantly, latent TGF-beta isoform expression showed complex alterations in ocular vitreous. Levels of active TGF-beta of patients with active PDR (79.5 +/- 28 pg/ml; n = 8) were decreased to 20% of the control levels (378 +/- 55 pg/ml; n = 12; p = 0.0005) and 25% of the mean concentration in quiescent PDR (346 +/- 64 pg/ml; n = 9; p = 0.0021). Levels in rubeosis (52 +/- 10 pg/ml; n = 10) did not differ significantly from those found in active PDR but were decreased to 15% of those in patients with quiescent PDR (p = 0.0004). Furthermore a highly significant inverse correlation between active TGF-beta and alpha2-antiplasmin, a liver produced inhibitor of the activation of TGF-beta by plasmin was noted (r = -0.59; n = 28; p = 0.001). We conclude that deficient activation of TGF-beta occurs in active proliferative diabetic retinopathy and in hypoxic angiogenesis most likely as a consequence of a blood retina barrier breakdown and influx of alpha2-antiplasmin from serum. The disinhibition of endothelial cell proliferation may be a central component in the process of neovascularization.

Heme Arginate Suppresses Cardiac Lesions and Hypertrophy in Deoxycorticosterone Acetate-Salt Hypertension

In hypertension, elevated levels of oxidative/inflammatory mediators including nuclear factor kappaB (NF-kappaB), activating protein (AP-1), c-Jun-NH2-terminal kinase (JNK), and cell-regulatory proteins such as transforming growth factor beta (TGF-beta), trigger the mobilization of extracellular matrix (ECM) leading to fibrosis, hypertrophy and impairment of cardiac function. Although the heme oxygenase (HO) system is cytoprotective, its effects on cardiac fibrosis and hypertrophy in deoxycorticosterone acetate (DOCA-salt) hypertension are not completely elucidated. Here, we report cardioprotection by the HO inducer, heme arginate against histopathological lesions in DOCA-hypertension. Treatment with heme arginate restored physiological blood pressure, and abated cardiac hypertrophy (3.75 +/- 0.12 vs. 3.19 +/- 0.09 g/kg body wt; n =16, P < 0.01), left-to-right ventricular ratio (6.67 +/- 0.62 vs. 4.39 +/- 0.63; n = 16, P < 0.01), left ventricular mass (2.48 +/- 0.14 vs. 2.01 +/- 0.09 g/kg body wt; n = 16, P < 0.01) and left-ventricular wall thickness (2.82 +/- 0.16 vs. 1.98 +/- 0.14 mm; n = 16, P < 0.01), whereas the HO inhibitor, chromium mesoporphyrin, exacerbated hypertrophy and cardiac lesions. The suppression of cardiac hypertrophy was accompanied by a robust increase in HO-1, HO activity, cyclic guanosine monophosphate (cGMP), ferritin and the total antioxidant capacity, whereas 8-isoprostane, NF-kappaB, JNK, AP-1, TGF-beta, fibronectin and collagen-I were significantly abated. Correspondingly, histopathological parameters that depict progressive cardiac damage, including fibrosis, interstitial/perivascular collagen deposition, scarring, muscle-fiber thickness, muscular hypertrophy and coronary-arteriolar thickening were abated. Our study suggests that upregulating the HO system lowers blood pressure, potentiates the antioxidant status in tissues, suppresses oxidative stress/mediators such as NF-kappaB, AP-1 and cJNK, and suppresses the mobilization of ECM proteins like TGF-beta, collagen and fibronectin, with corresponding reduction of cardiac histopathological lesion and hypertrophy.

Inhibition of latent transforming growth factor-β1 activation by lentivirus-mediated short hairpin RNA targeting the CD36 gene in NR8383 cells

CD36, a cell surface receptor for thrombospondin-1 (TSP-1), is believed to interact with latent transforming growth factor-beta1 (L-TGF-beta1) thereby activating its fibrogenic bioactivity. In this study, a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the rat CD36 gene (Lv-shCD36) is developed and tested. To observe the inhibitory effect of Lv-shCD36 on the activation of L-TGF-beta1, a rat alveolar macrophage cell line (NR8383), infected with either Lv-shCD36 or Lv-shCD36-NC (non-silenced control lentivirus), was treated with 0.1 microg/ml bleomycin, which is known to stimulate alveolar macrophages to release increasing amounts of TGF-beta1. The results show that Lv-shCD36 can suppress expression of CD36 mRNA and protein in bleomycin-treated NR8383 cells. By quantifying active and total TGF-beta1 in the supernatant, it was discovered that the quantity of total TGF-beta1 is not significantly different between the three groups, while the quantity and percent of active TGF-beta1 in the Lv-shCD36 group was significantly lower than in either the bleomycin-treated group or the Lv-shCD36-NC group, respectively (P < 0.05). These results suggest that Lv-shCD36 can inhibit activation of L-TGF-beta1 secreted in bleomycin-treated NR8383 cells by decreasing the expression of CD36 on the cell membrane, thereby reducing binding of CD36 to TSP-1.

Active TGF‐β1 correlates with myofibroblasts and malignancy in the colorectal adenoma‐carcinoma sequence

Transforming growth factor-beta1 (TGF-beta1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF-beta1 levels in homogenates of colorectal neoplasia. In contrast to total TGF-b levels, showing a stepwise increase in the mucosa-adenoma-carcinoma sequence, active TGF-beta1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF-beta1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF-beta1 levels correlated significantly with plasminogen activator inhibitor (PAI)-1, alpha-smooth muscle actin (SMA) and several matrix-remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF-beta1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF-beta1 and SMA expression in tumors indicates that tumor-promoting myofibroblasts might arise as a result of increased TGF-beta1 activation. These data underline the significance of the interaction between malignant cells and (myo)-fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer.

Biologically active TGF-beta 1 is increased in cerebrospinal fluid while it is reduced in serum in multiple sclerosis patients.

The polypeptide transforming growth factor-beta 1 (TGF-beta 1) is a product of activated monocytes, among other inflammatory cells, and it affects immune responsiveness, cellular growth and differentiation. TGF-beta 1 has potent T-cell inhibiting activities. It may play an important role in limiting autoimmune inflammation. We were interested about levels of biologically active and total TGF-beta 1 in serum and CSF in patients suffering from multiple sclerosis. We measured biologically active and total TGF-beta 1 in serum and CSF using ELISA-technique in 64 MS patients with 57 during acute exacerbation of MS and 7 in remission (primary-relapsing: n = 59; primary-progressive: n = 5), 20 healthy subjects, and 21 patients with other non-inflammatory neurological diseases (OND). Biologically active TGF-beta 1 in serum was reduced in MS patients compared to controls, on the other hand total TGF-beta 1 was elevated in CSF compared to patients with OND. Biologically active TGF-beta 1 in CSF correlated positively with the duration of the acute relapse in patients with primary-relapsing MS. The more relapses the patients had the higher was biologically active TGF-beta 1 in CSF. Total TGF-beta 1 in CSF correlated with macrophages in CSF and albumin quotient. We found that an elevated level of biologically active TGF-beta 1 in CSF might be useful as an indicator of disease limitation while active TGF-beta 1 in serum is reduced in multiple sclerosis. Measuring TGF-beta 1 in body fluids by ELISA techniques produces valid results and might be used for further studies focusing on the role of this cytokine in MS.

TJN-259 Improves Mesangial Lesions in Experimental Immunoglobulin A Nephropathy in ddY Mice

TJN-259 is a chemical substance based on the structural features of the botanically derived ingredient acteoside. This study was performed in order to elucidate the antinephritic effects of TJN-259 in experimental immunoglobulin A (IgA) nephropathy. In this study, 28-week-old ddY mice were used as a spontaneous model of IgA nephropathy. With regard to spontaneous IgA nephropathy, we investigated the effects of TJN-259 administered from 28 to 40 weeks. In addition, an accelerated model of IgA nephropathy was experimentally induced in ddY mice by oral administration of bovine serum albumin, followed by reticuloendothelial blocking by colloidal carbon injection and heminephrectomy. At 10 weeks after the 3rd carbon injection, we also examined the effects of TJN-259 on accelerated IgA nephropathy. To investigate the effects of TJN-259 on transforming growth factor (TGF)-beta1 production in accelerated IgA nephropathy, kidneys were isolated and measured TGF-beta1 by the enzyme-linked immunosorbent assay (ELISA) method. The administration of TJN-259 to mice with spontaneous IgA nephropathy decreased the incidence of mesangial expansion as well as the number of nuclei per glomerular cross-section in comparison with that of non-treated mice. In addition, TJN-259 treatment prevented the increase in the incidence of mesangial expansion, crescent formation, and segmental sclerosis in glomeruli in accelerated IgA nephropathy. TJN-259 also inhibited the increased immunostaining score of collagen type IV and TGF-beta1 in glomeruli of accelerated IgA nephropathy. Treatment with TJN-259 inhibited the increases in renal total and mature TGF-beta1 protein levels in accelerated type IgA nephropathy. TJN-259 failed to inhibit the increase in serum IgA levels in both models. These results suggest that TJN-259 was an effective treatment against IgA nephropathy in ddY mice, acting via the suppression of TGF-beta1 production in glomeruli.

Expression of TGFβ1 and Its Signaling Components by Peripheral Lymphocytes in Systemic Lupus Erythematosus

Transforming growth factor beta1 (TGFbeta1) is an important immunosuppressive cytokine. Defects in its production by lymphocytes and the failure of TGFbeta1 to regulate immunological functions have been described in SLE. Expression of TGFbeta1 and the related signaling pathway was studied in the peripheral lymphocytes of SLE patients. The total plasma TGFbeta1 level in active and inactive SLE patients compared to healthy controls was also measured. TGFbeta1 and all downstream signaling elements were expressed in normal cells. However, in more than 50% of SLE patients the isolated T cell population showed no TGFbeta1 mRNA expression and at least one member of the TGFbeta1 pathway was also missing (TGFbeta-RI, Smad2 and Smad3) in more than half of the patients. Total plasma TGFbeta1 level was increased in both active and inactive SLE groups compared to normal controls (p< 0.05). These data raise questions about the availability of TGFbeta1 signaling in lymphocytes in SLE patients, however, the elevated total plasma TGFbeta1 level suggests that the failure of TGFbeta1 effects is not the consequence of low level of this cytokine in SLE.

Thrombospondin 1 (TSP-1) Partially Contributes to Shear or Stirring- Dependent TGF-beta1 Activation In Vitro and During Platelet-Rich Thrombi Formation In Vivo.

Thrombospondin 1 (TSP-1) has been shown to activate latent TGF-beta1 in vitro, but its in vivo role is unclear because TSP-1-null mice have a much less severe phenotype than TGF-beta1-null mice. TSP-1 is stored in platelet alpha-granules and released, along with latent TGF-beta1, with activation. Thus, it is strategically located to contribute to TGF-beta1 activation at sites of platelet deposition. We recently demonstrated that stirring and/ or shear could activate latent TGF-beta1 released from platelets, probably in part through thiol-disulfide exchange and we have identified TSP-1 in human platelet releasates by immunoblotting and observed that it labeled with a thiol-reactive biotin-maleimide probe (MPB) by LC/MS/MS analysis, indicating the presence of free thiols. Remarkably, we found that the amount of TSP-1 detected by immunoblotting in human platelet releasates decreased with stirring or shear and that pretreatment of platelet releasates with the thiol-reactive compounds MPB, NEM or BMCC largely prevented the loss of TSP-1 with shear or stirring. To further assess the role of TSP-1 in TGF-beta1 activation, we have now studied the effect of stirring and shear on TGF-beta1 activation in samples from WT and TSP-1-null mice. Unstirred sera or platelet releasates from WT and TSP-1-null mice had similar levels of total TGF-beta1 [serum, 91 ± 15 ng/mL in WT (n=23) and 108 ± 15 ng/ mL in TSP-1-null mice (n=23; p=0.13); platelet releasates, 58 ± 14 ng/mL in WT (n=14) and 53 ± 16 ng/mL in TSP-1-null mice (n=14; p=0.45)]. With either stirring or shear, the TSP-1-null samples demonstrated less activatibility, but the defect was only partial [stirring increased active TGF-beta1 in serum from 0.6 ± 0.2 ng/mL to 2.2 ± 0.8 ng/mL in WT mice and from 0.5 ± 0.2 ng/mL to 1.6 ± 0.5 ng/mL in TSP-1-null mice (n=23) (p=0.05 using ANOVA analysis for the linear model comparing two-way interactions of stirred samples between WT and TSP-1-null mice); stirring increased active TGF-beta1 in platelet releasates from 0.3 ± 0.2 ng/mL to 2.4 ± 1.5 ng/mL in WT mice (n=14) and from 0.2 ± 0.2 ng/mL to 1.1 ± 0.6 ng/mL in TSP-1-null mice (n=14) (p=0.005)]. To assess in vivo TGF-beta1 activation we induced carotid artery thrombi with FeCl3 in WT and TSP-1-null mice. When thrombi were removed 5 min after total occlusion, the thrombus extracts from WT and TSP-1-null mice contained similar amounts of total and active TGF-beta1 [total TGF-beta1, 5.6 ± 2.4 ng/mL in WT and 5.5 ± 1.6 ng/mL in TSP-1-null mice (n=9) (p=0.9) and active TGF-beta1 (expressed as a percentage of total), 1.7 ± 0.7% in WT and 2.5 ± 1.4% in TSP-1-null mice (n=9) (p=0.15)]. When thrombi were removed 120 min after total occlusion, however, the percentage of active TGF-beta1 in the thrombus extracts was higher in WT than TSP-1-null mice [4.5 ± 2.0% in WT mice (n=9) and 3.3 ± 1.5% in TSP- 1-null mice (n=9) (p=0.02 using ANOVA analysis comparing interactions between time and type of mice)]. We conclude that:TSP-1 released from human platelets undergoes a shear-induced, thiol-dependent conformational change,TSP-1 contributes partially to shear or stirring-dependent TGF-beta1 activation in vitro in mice, andTime-dependent activation of TGF-beta1 released from thrombi formed in vivo in mice is also partially dependent on TSP-1.These data suggest a significant, but minor role of TSP-1 in TGF-beta1 activation in vivo and thus provide an explanation for the less severe phenotype of TSP-1-null mice than TGF-beta1-null mice.

Abstract 2607: TGF-beta 1 as a Diagnostic Biomarker for Acute Aortic Dissection Type A in a Mouse Model of Marfan Syndrome

Objectives: Acute aortic dissection type A is the main cause of mortality in patients with Marfan Syndrome (MFS). The aim of this study was to develop a procedure to surgically induce an acute aortic dissection type A in a mouse model of MFS, and to discover a diagnostic biomarker of the aortic lesion. Methods: Ten fibrillin-1 deficient (Fbn1C1039G/+) and ten wild-type (WT) mice at 8 months of age were intubated, ventilated and the aorta exposed via hemisternotomy. We hypothesized that an aortic dissection type A could be induced by performing an aortic clamp injury. Serum samples were collected before and after induction (30 min) of the aortic lesion from the right femoral vein. Total (acid activated) TGF-beta 1 concentrations were measured by ELISA. Echo measurements of the aortic root were obtained from a parasternal long axis view in all mice. Results: Transthoracic echocardiography of the aortic root performed prior to surgery showed significantly larger diameters in Fbn1C1039G/+ mice compared to WT (p&lt;0.0001). Aortic clamp injury led in Fbn1C1039G/+ and WT mice to a reproducible aortic wall hematoma, and intimal tears. Hematoxylin-eosin stained histological sections revealed aortic dissection of the medial layer in Fbn1C1039G/+ mice but not in WT (p&lt;0.001). Elastin-stained sections revealed a significantly higher elastic fiber fragmentation and disarray in Fbn1C1039G/+ aortas compared to WT (p&lt;0.001). Aortic dissection in Fbn1C1039G/+ mice was limited to the ascending aorta. Mean TGF-beta 1 serum concentrations significantly increased after inducing the aortic lesion in Fbn1C1039G/+ mice (p=0.01; 130 ng/ml vs 412.9 ng/ml) but not in WT mice (p=0.2). Sham operated Fbn1C1039G/+ and WT mice showed no difference in TGF-beta 1 serum concentrations before and after surgery (p=0.7; n=4). Conclusions: We present a reproducible and in the short-term non-lethal mouse model of a surgically induced acute aortic dissection type A in MFS. TGF-beta 1 serum concentrations after inducing the aortic lesion significantly increased in Fbn1C1039G/+ mice but not in WT and Sham operated mice. TGF-beta 1 is a promising diagnostic biomarker for an acute aortic dissection type A in MFS.

Role of the Chemokine Receptor CXCR2 in Bleomycin-Induced Pulmonary Inflammation and Fibrosis

Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.

Transforming growth factor-beta 1 (TGF-beta 1)- and beta 2-like activities in malignant pleural effusions caused by malignant mesothelioma or primary lung cancer.

We investigated the levels of TGF-beta in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF-beta levels in MPE caused by MESO were 283.9 +/- 219.2 pm (mean +/- s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF-beta 1- and beta 2-like activities in MPE using specific polyclonal antibodies. Although TGF-beta 1-like activity could be detected in all cases, TGF-beta 2-like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF-beta and TGF-beta 2-like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF-beta may help further characterize the clinical features of MESO.

Studies of TGF-β 1-3 in Serosal Fluid During Abdominal Surgery and Their Effect on In Vitro Human Mesothelial Cell Proliferation

Increased transforming growth factor-beta (TGF-beta) levels are associated with fibrosis, affected cell proliferation, and postsurgical adhesion development, but the knowledge regarding TGF-beta response to the surgical trauma is limited. This study investigated TGF-beta(1-3) isoforms and fibrinolytical factors in peritoneal serosal fluid during abdominal surgery, together with the in vitro effect of TGF-beta(1-3) on human mesothelial cell proliferation. Total as well as biologically active TGF-beta(1-3) and fibrinolytic factors: t-PA, uPA, and PAI-1 were measured in serosal fluid and plasma from 23 patients undergoing colorectal cancer surgery. In vitro proliferation of human primary mesothelial cell cultures upon TGF-beta(1-3) stimulation was also investigated. Total TGF-beta1 and TGF-beta2 levels were similar in serosal fluid and plasma while active fractions were increased in serosal fluid. In contrast, total fraction of TGF-beta3 was higher in serosal fluid compared with plasma, while levels of active fractions did not differ. Plasminogen activators (t-PA, uPA) were elevated while the inhibitor (PAI-1) was decreased in serosal fluid compared with plasma. The in vitro mesothelial cell proliferation studies revealed that high TGF-beta(1-3) concentrations decreased cell proliferation, while lower concentrations of TGF-beta1 increased mesothelial cell proliferation. This human study shows increased active TGF-beta levels in peritoneal serosal fluid, compared with plasma, during abdominal surgery and that TGF-beta1 at physiological concentrations increased human mesothelial cell proliferation in vitro. TGF-beta cytokines may be involved in postsurgical adhesion formation.