Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Vascular endothelial growth factor A (VEGFA) modulates bovine placenta steroidogenesis in vitro

Our objectives were to investigate the possible role of VEGFA in bovine placenta steroid synthesis and to determine whether cloned derived placental cells present similar responses as non-cloned ones. Placental cells from cloned (term) and non-cloned (days 90, 150, 210 and term) pregnancies were isolated and treated with VEGFA (50 ng/ml) for 24, 48 or 96 h. Progesterone (P-4) and estrone sulfate (E1S) were assessed by RIA, while aromatase P450-positive cells were quantified using the point counting test. The percentages of steroidogenic and non-steroidogenic populations were determined by flow cytometry. VEGFA augmented or decreased P-4 and E1S concentrations as well as aromatase P450-positive cell density, depending on gestational age and time in culture. The percentage of steroidogenic cells was lower than that of non-steroidogenic ones for each culture time (P < 0.05). VEGFA treatment did not change the proportion of steroidogenic and non-steroidogenic cells. Placental cells derived from cloned pregnancies presented higher concentrations of E1S and P4 than the non-cloned group. However, aromatase P450-positive cells were similar between groups (P > 0.05). VEGFA treatment altered P-4 and E1S levels in placental cells depending on type of gestation. These results suggest that VEGFA acts locally in the bovine placenta to modulate steroidogenesis during gestation, but in a different pattern between cloned and non-cloned derived placental cells at term. Therefore, this factor can be considered an important regulator of placental development and function. (C) 2012 Elsevier Ltd. All rights reserved.

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract Introduction Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway. Methods During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay. Results Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α. Conclusion We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.

Forced Expression of Nanog in Human Bone Marrow-Derived Endothelial Cells Activates Other Six Pluripotent Genes

Human endothelial cells (ECs) have the ability to make up the lining of blood vessels. These cells are also capable of neovascularization and revascularization and have been applied in various clinical situations. With the aim of understanding the effect of NANOG superexpression on ECs, we transduced the Nanog gene into the ECs. Nanog is highly expressed in embryonic stem cells (ESCs) and is essential for pluripotency and self-renewal. However, Nanog can also be expressed in somatic stem cells, and this gene is related to great expansion capacity in vitro. We found that ECs expressing Nanog showed expression of other stemness genes, such as Sox2, FoxD3, Oct4, Klf4, c-myc, and beta-catenin, that are not normally expressed or are expressed at very low levels in ECs. Nanog is one of the stemness genes that can activate other stemness genes, and the upregulation of the Nanog gene seems to be critical for reprogramming cells. In this study, the introduction of Nanog was sufficient to alter the expression of key genes of the pluripotent pathway. The functional importance of Nanog for altering the cell expression profile and morphology was clearly demonstrated by our results.

The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas

Abstract: Background Pancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors. Methods Paraffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant. Results In the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival. Conclusions Pancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive. Virtual slides. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

Abstract Background Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The objective of the present study was to evaluate the effects of enamel matrix derivative (EMD), TGF-β1, and the combination of both factors (EMD+TGF-β1) on human osteoblastic cell cultures. Methods Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of EMD, TGF-β1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP), osteopontin (OPN) and alkaline phosphatase (ALP) immunodetection, total protein synthesis, ALP activity and bone-like nodule formation. Results All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days. At day 7, EMD group showed higher cell proliferation compared to TGF-β1, EMD + TGF-β1 and the control group. OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group. Conclusions The exposure of human osteoblastic cells to EMD, TGF-β1 and the combination of factors in vitro supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell number, and reduced ALP activity levels and matrix mineralization.

Transforming growth factor beta and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection

The course of leprosy depends of the host immune response which ranges from the lepromatous pole (LL) to the tuberculoid pole (TT). A comparative study was conducted in 60 patients with the LL and TT The results showed a mean expression of TGF-beta of 339 +/- 99.4 cells/field for TT and of 519.2 +/- 68.2 cells/field for LL. Frequency of apoptosis was 6.3 +/- 1.8 in TT and 14.0 +/- 6.1 in LL. A correlation (p = 0.0251) between TGF-beta and caspase-3 in the LL was found. This finding indicates a role of TGF-beta and apoptosis in the immune response in leprosy. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Fluctuation of Anti-Endothelial Cell Antibody Titers in Mixed Connective Tissue Disease

Background: Antibodies directed against endothelial cell surface antigens have been described in many disorders and have been associated with disease activity. Since the most prominent histopathologic feature in mixed connective tissue disease (MCTD) is the widespread and unique proliferative vascular lesion, our aim was to evaluate the frequency of anti-endothelial cell antibodies (AECA) in this condition. Objectives: To evaluate the frequency of AECA in this disease and assess its clinical and laboratory associations. Methods: Seventy-three sera from 35 patients with MCTD (Kasukawa's criteria), collected during a 7 year period, were tested for immunoglobulins G and M (IgG and IgM) AECA by cellular ELISA, using HUVEC (human umbilical vein endothelial cells). Sera from 37 patients with systemic lupus erythematosus (SLE), 22 with systemic sclerosis (SSc) and 36 sera from normal healthy individuals were used as controls. A cellular ELISA using HeLa cells was also performed as a laboratory control method. Results: IgG-AECA was detected in 77% of MCTD patients, 54% of SLE patients, 36% of SSc patients and 6% of normal controls. In MCTD, IgG-AECA was associated with vasculitic manifestations, disease activity and lymphopenia, and was also a predictor of constant disease activity. Immunosuppressive drugs were shown to reduce IgG-AECA titers. Since antibodies directed to HeLa cell surface were negative, AECA was apparently unrelated to common epitopes present on epithelial cell lines. Conclusions: AECA are present in a large proportion of patients with MCTD and these antibodies decrease after immunosuppressive treatment. IMAJ 2012; 14:84-87

Lyophilized bovine colostrum as a source of immunoglobulins and insulin-like growth factor for newborn goat kids

The objective of this study was to evaluate lyophilized bovine colostrum as an alternative source of passive immunity and insulin like growth factor I (IGF-I) for goat kids, considering newborns consuming non-maternal colostrum. Twenty-nine male newborns received 5% of body weight of lyophilized bovine (LBC) or goat colostrum (GC), both with 55 mg/mL of IgG, at 0, 7 and 14 h of life. Blood samples were collected at 0, 7, 14, 18, 24, 36, 48, 72 and 96 h of life to determine serum IgG, total protein (TP), IGF-I and apparent efficiency of IgG absorption at 7, 14, 18 and 24 h (AEA(7hr), AEA(14hr), AEA(18hr), AEA(total), respectively). In LBC, the values of serum IgG at 14, 18,24 and 48 h (13.1, 13.4, 14.1 and 14.6 mg/mL, respectively) were higher than the values at 0 and 7 h (0.04 and 6.9 mg/mL, respectively). In GC, the serum IgG at 18 h (9.3 mg/mL) was higher than the value at 7 h (5.5 mg/mL). AEA(7hr) and AEA(14hr) in LBC were the same (19.2 and 18.5%, respectively, P>0.05) and the values of AEA(18hr) and AEA(total), 9.3 and 9.5%, respectively, were equal and smaller than AEA(7hr), and AEA(14hr). In GC, AEA(7hr), 20.8%, was higher than AEA(14hr), 16.1% (P<0.05) and AEA(18hr), and AEA(total), 9.2 and 8.0%, respectively, were equal and smaller than AEA(7hr) and AEA(14hr). The serum TP and IGF-I were not affected by colostrum feeding. Considering the variables study in the present work, lyophilized bovine colostrum constitutes a promising alternative substitute to goat colostrum in newborn goat kids, since the supply of immunoglobulins and IGF-I was suitable for the kids. (c) 2012 Elsevier B.V. All rights reserved.

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and beta-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal beta-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal beta-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized beta-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized beta-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis. (Ant J Pathol 2012, 181:1017-1033; http://dx.doi.org/10.1016/j.ajpath.2012.05.026)

Endogenous testosterone increases leukocyte-endothelial cell interaction in spontaneously hypertensive rats

Aims: Inflammation may have an important role in the beginning and in the progress of cardiovascular diseases. Testosterone exerts important effects on vascular function, which is altered in arterial hypertension. Thus, the aim of this study was to evaluate the influence of endogenous testosterone on leukocyte behavior in post-capillary venules of the mesenteric bed of spontaneously hypertensive rats (SHR). Main methods: 18 week-old intact SHR, castrated SHR and normotensive rats (intact Wistar) were used. Blood pressure was measured by tail plethysmography and serum testosterone levels by ELISA. Leukocyte rolling, adhesion and migration were evaluated in vivo in situ by intravital microscopy. Key findings: Castration significantly reduced blood pressure and reversed the increased leukocyte rolling and adhesion observed in SHRs. Leukocyte counts and other hemodynamic parameters did not differ among groups. SHRs displayed increased protein expression of P-selectin and ICAM-1 in mesenteric venules when compared to intact Wistar. Castration of SHRs restored the protein expression of the cell adhesion molecules. Significance: The findings of the present study demonstrate the critical role of endogenous testosterone mediating the effects of hypertension increasing leukocyte-endothelial cell interaction. Increased expression of cell adhesion molecules contribute to the effects of endogenous testosterone promoting increased leukocyte rolling and adhesion in SHRs. (c) 2012 Elsevier Inc. All rights reserved.

Assessing the benefits of rosiglitazone in women with polycystic ovary syndrome through its effects on insulin-like growth factor 1, insulin-like growth factor-binding protein-3 and insulin resistance: a pilot study

Federal University of Sao Paulo

Transforming growth factor alpha immunoreactivity. A study in hepatocellular carcinoma and in non-neoplastic liver tissue

Background. Transforming growth factor alpha (TGF alpha) is an important mitogen that binds to epidermal growth factor receptor and is associated with the development of several tumors. Aims. Assessment of the immunoexpression of TGF alpha in hepatocellular carcinoma (HCC) and in non-neoplastic liver tissue and its relationship to morphological patterns of HCC. Material and methods. The immunohistochemical expression of TGF alpha was studied in 47 cases of HCC (27 multinodular, 20 nodular lesions). Five lesions measured up to 5 cm and 15 lesions above 5 cm. Thirty-two cases were graded as I or II and 15 as III or IV. The non-neoplastic tissue was examined in 40 cases, of which 22 had cirrhosis. HBsAg and anti-HCV were positive in 5/38 and 15/37 patients, respectively. The statistical analysis for possible association of immunostaining of TGF alpha and pathological features was performed through chi-square test. Results. TGF alpha was detected in 31.9% of the HCC and in 42.5% of the non-neoplastic. There was a statistically significant association between the expression of TGF alpha and cirrhosis (OR = 8.75, 95% CI = [1.93, 39.75]). The TGF alpha was detected more frequently in patients anti-HCV(+) than in those HBsAg(+). The immunoexpression of TGF alpha was not found related to tumor size or differentiation. In conclusion the TGF alpha is present in hepatocarcinogenesis in HBV negative patients. Further analysis is needed to examine the involvement of TGF alpha in the carcinogenesis associated with HCV and other possible agents. In addition, TGF alpha has an higher expression in hepatocyte regeneration and proliferation in cirrhotic livers than in HCC.

Adipose Tissue-Derived Stem Cell Treatment Prevents Renal Disease Progression

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 x 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 x 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4. IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-alpha, KC, RANTES, and IL-1 alpha. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.