Increased expression of transglutaminase-1 and PPARgamma after vitamin E treatment in human keratinocytes

In skin, vitamin E acts as the predominant lipophilic antioxidant with a protective function against irradiation and oxidative stress. In addition to that, vitamin E can also modulate signal transduction and gene expression. To study whether the four natural tocopherol analogues (alpha-, beta-, gamma-, delta-tocopherol) can influence transcriptional activity by modulating the activity of nuclear receptors, a human keratinocytes cell line (NCTC 2544) was transfected with plasmids containing the luciferase reporter gene under control by direct repeat elements (DR1-DR4), representing binding sites for four different classes of nuclear receptors. In this model, the tocopherols positively modulated only the reporter construct containing a consensus element for peroxisome proliferator-activated receptors (PPARs). The induction was strongest with gamma-tocopherol and was most likely the direct consequence of stimulation of PPARgamma protein expression in keratinocytes. Vitamin E treatment also led to increased expression of a known PPARgamma target gene involved in terminal keratinocytes differentiation, the transglutaminase-1.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

Increased expression of IL-12p70 and IL-23 by multiple dendritic cell and macrophage subsets in plaque psoriasis

BACKGROUND: Psoriasis is a chronic immune-mediated skin disease, in which interleukins 12 and 23 have been postulated to play a critical role. However, the cellular source of these cytokines in psoriatic lesions are still poorly defined and their relative contribution in inducing skin inflammation has been discussed controversially. OBJECTIVES: To investigate immunoreactivity of the bioactive forms of IL-12 and IL-23 in plaque psoriasis and to characterize the dendritic cell (DC) and macrophage subsets responsible for the production of these cytokines. METHODS: Immunohistochemistry was performed on normal skin (n=11) as well as non-lesional (n=11) and lesional (n=11) skin of patients with plaque psoriasis using monoclonal antibodies targeting the bioactive forms of IL-12 (IL-12p70) and IL-23 (IL-23p19/p40) on serial cryostat sections using the alkaline phosphatase-antialkaline phosphatase. Co-localization of IL-12 and IL-23 with different dendritic cells and macrophage cell markers (CD1a, CD11c, CD14, CD32, CD68, CD163, CD208/DC-LAMP) was performed using double immunofluorescence staining. RESULTS: Immunoreactivity for IL-12 and IL-23 was significantly enhanced in lesional psoriatic skin as compared to non-lesional and normal skin. No difference was observed between IL-12 and IL-23 immunoreactivity in any skin types. Both IL-12 and IL-23 immunoreactivity was readily detected mainly in CD11c+, CD14+, CD32+, CD68+ and some CD163+, DC-LAMP+ cells. IL-12 and occasionally IL-23 were also found in some CD1a+ dendritic cells. In addition, an enhanced expression mainly of IL-23 was observed in keratinocytes. CONCLUSIONS: Bioactive forms of IL-12 and IL-23 are highly expressed in various DC and macrophage subsets and their marked in situ production suggest that both cytokines have crucial pathogenic role in psoriasis.

Increased expression of putative cancer stem cell markers in the bone marrow of prostate cancer patients is associated with bone metastasis progression.

BACKGROUND The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. METHODS Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. RESULTS For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. CONCLUSIONS Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer.

Increased expression of heat shock protein 90 in keratinocytes and mast cells in patients with psoriasis.

BACKGROUND Psoriasis is a chronic inflammatory skin disease and various stress factors mediate inflammation. Heat shock protein (HSP) 90 plays an important role in cell survival; cytokine signaling, such as interleukin-17 receptor signaling; and immune responses. OBJECTIVE We sought to elucidate protein expression and distribution of HSP90 in psoriasis. METHODS HSP90 expression and its cellular source were analyzed on normal-appearing, nonlesional, lesional, and ustekinumab-treated psoriatic skin using immunohistochemistry and double immunofluorescence. RESULTS HSP90α, the inducible isoform of HSP90, was significantly up-regulated in epidermal keratinocytes and mast cells of lesional skin and down-regulated after ustekinumab therapy. LIMITATIONS There was a limited sample size. CONCLUSIONS HSP90 from keratinocytes and mast cells is a key regulator of psoriatic inflammation and HSP90 inhibitors may represent a novel therapeutic approach to the disease.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression.

Expression of adipokines and estrogen receptors in adipose tissue and placenta of patients with gestational diabetes mellitus

The purpose of this study was to assess the expression profile of genes with potential role in the development of insulin resistance (adipokines, cytokines/chemokines, estrogen receptors) in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placenta of pregnant women with gestational diabetes mellitus (GDM) and age-matched women with physiological pregnancy at the time of Caesarean section. qRT-PCR was used for expression analysis of the studied genes. Leptin gene expression in VAT of GDM group was significantly higher relative to control group. Gene expressions of interleukin-6 and interleukin-8 were significantly increased, whereas the expressions of genes for estrogen receptors alpha and beta were significantly reduced in SAT of GDM group relative to controls, respectively. We found no significant differences in the expression of any genes of interest (LEP, RETN, ADIPOR1, ADIPOR2, TNF-alpha, CD68, IL-6, IL-8, ER alpha, ER beta) in placentas of women with GDM relative to controls. We conclude that increased expression of leptin in visceral adipose depot together with increased expressions of proinflammatory cytokines and reduced expressions of estrogen receptors in subcutaneous fat may play a role in the etiopathogenesis of GDM.

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

The physiologic increase in expression of some type I IFN-inducible genes during pregnancy is not associated with improved disease activity in pregnant patients with rheumatoid arthritis

During pregnancy, most patients with rheumatoid arthritis (RA) experience a spontaneous improvement in their condition. Since type I interferons (IFN) have immunomodulatory properties, we investigated whether type I IFN-inducible genes are upregulated in pregnant patients with RA. Peripheral blood mononuclear cells were evaluated using quantitative real-time polymerase chain reaction for type I IFN-inducible genes (IFI 35, IFI44, IFI44L, IFIT3, OAS1, and Siglec1) in patients with RA and healthy women during and after pregnancy as well as in nonpregnant controls. IFN-alpha and IFN-beta levels in sera of patients and healthy donors were analyzed by enzyme linked immunosorbent assay. It was found that healthy women did not show a change of gene expression levels from the second trimester until postpartum, yet some type I IFN-inducible genes were significantly upregulated in pregnant and postpartum women compared with nonpregnant individuals. In patients with RA, a pronounced upregulation of IFI35 and IFI44 at the second trimester and a peak expression of Siglec1 at the third trimester were observed. Pregnancy levels of IFI35 and IFI44 in patients with RA were higher than those of nonpregnant patients with RA. No significant association of gene expression levels with disease activity was found. In the sera of patients and healthy women, IFN-beta was undetectable and IFN-alpha levels remained stable throughout pregnancy and postpartum. Thus, pregnancy can give rise to an increased expression of type I IFN-inducible genes, reflecting an upregulation of the innate immune system. However, an association of type I IFN-inducible genes with pregnancy induced disease amelioration seems unlikely.

Increased uptake of 111In-octreotide in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by an uncontrolled accumulation and activation of lung fibroblasts. A modulation of fibroblast activation has been observed in various systems with octreotide, a synthetic somatostatin analog with strong affinity for the somatostatin receptor subtype 2 (sst2). One aim of our study was to evaluate the expression of somatostatin receptors in the lungs of patients with IPF. A second aim was to evaluate the relationship between 111In-octreotide uptake and the effect of pulmonary fibrosis as assessed by lung function tests and parameters and by radiologic findings. METHODS: We investigated 11 patients with IPF, 6 patients with pulmonary fibrosis associated with systemic sclerosis (SSc), and 19 patients with disease not of the lung (control patients). The expression of somatostatin receptors was evaluated in vivo using 111In-octreotide scintigraphy. We evaluated the relationship between 111In-octreotide uptake and the activity of pulmonary fibrosis as assessed by lung function tests, bronchoalveolar lavage (BAL) cellularity, and high-resolution CT (HRCT) of the chest. Planar images and thoracic SPECT (24 h) were performed after injection of 222 MBq of 111In-octreotide. Lung uptake was quantified using the lung-to-background ratio (L/B). In addition, the expression of sst2 was evaluated in vitro, in frozen lung-tissue samples using autoradiography, and in human cultures of lung fibroblasts using a ligand-binding assay. RESULTS: Compared with lung uptake in control patients (median L/B, 1.25; range, 1.14-1.49), lung uptake was increased in all 11 IPF patients (median L/B, 2.63; range, 1.59-3.13; P < 0.001) and in 4 of 6 SSc patients (median L/B, 1.68; range, 1.42-2.16). The L/B was lower in SSc patients than in IPF patients (P = 0.011). Increased uptake correlated with the alteration of lung function (carbon monoxide diffusing capacity [rho = -0.655; P = 0.038], diffusing capacity for carbon monoxide and alveolar volume ratio [rho = -0.627; P = 0.047], vital capacity [rho = -0.609; P = 0.054], and total lung capacity [rho = -0.598; P = 0.058]) and with the intensity of alveolitis (total BAL cellularity [rho = 0.756; P = 0.045], neutrophil counts [rho = 0.738; P = 0.05]), and HRCT fibrosis score (rho = 0.673; P = 0.007). Autoradiography suggested that vascular structures were a prominent binding site. Lung fibroblasts expressed somatostatin receptors in vitro as measured by binding assay. CONCLUSION: Our preliminary results identified an increased expression of sst2 in (mainly idiopathic) pulmonary fibrosis. Lung uptake correlates with the alteration of lung function and with the intensity of alveolitis.

Retinal pigment epithelium damage enhances expression of chemoattractants and migration of bone marrow-derived stem cells

PURPOSE: To characterize chemoattractants expressed by the retinal pigment epithelium (RPE) after sodium iodate (NaIO3)-induced damage and to investigate whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE. METHODS: C57/BL6 mice were treated with a single intravenous injection of NaIO3 (50 mg/kg) to create RPE damage. At different time points real-time RT-PCR, ELISA, and immunohistochemistry were used to identify chemoattractants secreted in the subretinal space. Conditioned medium from NaIO3-treated mouse RPE was used in an in vitro assay to assess chemotaxis of stem cell antigen-1 positive (Sca-1+) BM mononuclear cells (MNCs). The expression of early ocular markers (MITF, Pax-6, Six-3, Otx) in migrated cells and in MNCs isolated from granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (FL)-mobilized and nonmobilized peripheral blood (PB) was analyzed by real-time RT-PCR. RESULTS: mRNA for stromal cell-derived factor-1 (SDF-1), C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) was significantly increased, and higher SDF-1 and C3 protein secretion from the RPE was found after NaIO3 treatment. A higher number of BMMNCs expressing early ocular markers migrated to conditioned medium from damaged retina. There was also increased expression of early ocular markers in PBMNCs after mobilization. CONCLUSIONS: Damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells (BMSCs). Retina-committed stem cells appear to reside in the BM and can be mobilized into the PB by G-CSF and FL. These stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage.

Modulation of bcl-xL in tumor cells regulates angiogenesis through CXCL8 expression

In this paper, we investigated whether bcl-xL can be involved in the modulation of the angiogenic phenotype of human tumor cells. Using the ADF human glioblastoma and the M14 melanoma lines, and their derivative bcl-xL-overexpressing clones, we showed that the conditioned medium of bcl-xL transfectants increased in vitro endothelial cell functions, such as proliferation and morphogenesis, and in vivo vessel formation in Matrigel plugs, compared with the conditioned medium of control cells. Moreover, the overexpression of bcl-xL induced an increased expression of the proangiogenic interleukin-8 (CXCL8), both at the protein and mRNA levels, and an enhanced CXCL8 promoter activity. The role of CXCL8 on bcl-xL-induced angiogenesis was validated using CXCL8-neutralizing antibodies, whereas down-regulation of bcl-xL through antisense oligonucleotide or RNA interference strategies confirmed the involvement of bcl-xL on CXCL8 expression. Transient overexpression of bcl-xL led to extend this observation to other tumor cell lines with different origin, such as colon and prostate carcinoma. In conclusion, our results showed that CXCL8 modulation by bcl-xL regulates tumor angiogenesis, and they point to elucidate an additional function of bcl-xL protein.

Extracts of Lindera obtusiloba induce antifibrotic effects in hepatic stellate cells via suppression of a TGF-beta-mediated profibrotic gene expression pattern

Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.