Gabriella Mincione

Increased Expression of Transforming Growth Factor-β1 as a Stabilizing Factor in Human Atherosclerotic Plaques

Background and Purpose— Transforming growth factor-β (TGF-β) is a growth factor/cytokine involved in vascular remodeling and atherogenesis. Recent studies in apolipoprotein E-deficient mice have demonstrated a pivotal role of TGF-β in the maintenance of the balance between inflammation and fibrosis in atherosclerotic plaques. Furthermore, inhibition of TGF-β signaling has been shown to accelerate plaque formation and its progression toward an unstable phenotype in mice. However, if this mechanism is operative also in humans is still unknown. The aim of this study was to characterize the expression of TGF-β1 in human carotid plaque and to correlate it with the extent of inflammatory infiltration and collagen content with the clinical signs of plaque instability. Methods— Plaques were obtained from patients undergoing carotid endoarterectomy and divided into symptomatic and asymptomatic according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for TGF-β1 expression by Immunocytochemistry, Western, and Northern blotting analysis. Immunocytochemistry was used to identify CD68+ macrophages, CD3 T lymphocytes, HLA-DR+ cells, and α-smooth muscle cells. Procollagen and interstitial collagen content were analyzed by immunohistochemistry and Sirius Red staining, respectively. Results— Plaque TGF-β1 mRNA was increased up to 3-fold in asymptomatic as compared with symptomatic plaques. Plaques from asymptomatic group had fewer (P<0.0001) macrophages and T lymphocytes compared with symptomatic plaques. TGF-β1 gene was transcriptionally active as demonstrated by increased (P<0.0001) TGF-β1 protein expression in asymptomatic plaques. Immunohistochemistry showed that TGF-β was mainly expressed in plaque shoulder and was associated with a comparable increase (P<0.0001) in plaque procollagen and collagen content. Conclusions— In conclusion, this study demonstrates the higher expression of TGF-β1 in human asymptomatic lesions and provides evidence that TGF-β1 may play an important role in the process of plaque stabilization.

Cyclin D1 and cyclin E expression in malignant thyroid cells and in human thyroid carcinomas

Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K‐ras‐transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin‐fixed, paraffin‐embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis. Int. J. Cancer 76:806–811, 1998.© 1998 Wiley‐Liss, Inc.

Increased Transforming Growth Factor-β Production and Gene Expression by Peripheral Blood Monocytes of Hypertensive Patients

Cultured human peripheral blood monocytes are known to secrete and express transforming growth factor-beta (TGF-beta), a multifunctional cytokine that can be involved in myocardial and vascular remodeling. In addition, monocytes/macrophages have been demonstrated to be colocalized with fibrosis of hypertrophied heart and in the vascular wall of hypertensive vessels. In this study, we tested TGF-beta production and mRNA expression in peripheral blood monocytes from hypertensive patients with myocardial hypertrophy and increased carotid myointimal thickness with respect to healthy normotensive control subjects. We found an increased TGF-beta activity in the conditioned medium of monocytes from hypertensive patients compared with control subjects as evaluated by inhibition of [3H]thymidine incorporation by mink lung epithelial cells (-83% and -18% in hypertensive and normotensive subjects; P<.001). Western blot analysis confirmed a significant difference in the amount of TGF-beta protein secreted in the conditioned medium of hypertensive patients compared with that of normotensive subjects. Finally, we also observed a 4.2- and 5.5-fold increase in the amount of TGF-beta1 and TGF-beta2 transcripts, respectively. Our results indicate an upregulation of the TGF-beta system in the peripheral blood monocytes of hypertensive patients with cardiovascular structural changes, suggesting a possible role of TGF-beta monocyte production in hypertensive disease.

Restored expression of transforming growth factor β type II receptor ink-ras-transformed thyroid cells, TGFβ-resistant, reverts their malignant phenotype

Transforming growth factor β1 (TGFβ1) inhibits the growth of normal rat epithelial thyroid cells (FRTL‐5 strain) by counteracting thyrotropin (TSH)‐stimulated DNA synthesis and by slowing the cells in the G1 phase of the cell cycle. Here, we have studied two clones of FRTL‐5 thyroid cell line transformed by the wild type (wt) v‐k‐ras oncogene (K.M.A1, K.M.A2) and one clone (A6) transformed by a temperature‐sensitive (ts) v‐k‐ras mutant. Anchorage‐dependent as well as anchorage‐independent growth of these k‐ras‐transformed cells was not inhibited by TGFβ1. TGFβ1 resistance appeared to be dependent by a functional p21 k‐ras, because A6 cell growth was partially inhibited at the nonpermissive temperature (39°C). To determine the basis for TGFβ1 resistance in k‐ras‐transformed thyroid cells, we looked for possible defects in the expression of type I (TβR‐I/ALK5) and type II TGFβ receptors (TβR‐II). Lower levels of type II receptors were present in all of the k‐ras‐transformed clones, as revealed by both Northern blot and cross‐linking experiments.

Enhanced expression of heregulin in c‐erb B‐2 and c‐Ha‐ras transformed mouse and human mammary epithelial cells

Heregulin β1 was found to stimulate the anchorage‐dependent, serum‐free growth of nontransformed human MCF‐10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor α, or amphiregulin, heregulin β1 was also able to induce the anchorage‐independent growth of MCF‐10A cells. In contrast, the anchorage‐dependent, serum‐free growth of c‐Ha‐ras or c‐erb B‐2 transformed MCF‐10A cells was unaffected by heregulin β1, whereas heregulin β1 was able to stimulate the anchorage‐independent growth of these cells. c‐Ha‐ras or c‐erb B‐2 (c‐neu) transformed MCF‐10A or mouse NOG‐8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell‐associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF‐10A cells and transformants also express c‐erb B‐3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha‐ras or erb B‐2 transformed mammary epithelial cells.

Selective Insulin Resistance Affecting Nitric Oxide Release But Not Plasminogen Activator Inhibitor-1 Synthesis in Fibroblasts From Insulin-Resistant Individuals

Objectives—Insulin activates several processes potentially dangerous for the arterial wall and hyperinsulinemia might be atherogenic. However, other insulin effects are protective for the vessel wall and thus anti-atherogenic. Aim of this study was to investigate whether insulin effects on potentially pro-atherogenic and anti-atherogenic processes were differently affected in cells from insulin-resistant individuals. Methods and Results—We determined insulin effect on nitric oxide (NO) production and plasminogen activator inhibitor (PAI)-1 synthesis in 12 fibroblast strains obtained from skin biopsy samples of 6 insulin-sensitive (IS) (clamp M >7 mg/kg body weight per minute) and 6 insulin-resistant (IR) (clamp M <5 mg/kg body weight per minute) healthy volunteers. Insulin effects on NO release and Akt phosphorylation were significantly impaired in fibroblasts from IR as compared with IS individuals. Conversely, there was not any difference between IR and IS strains in insulin ability to increase PAI-1 antigen levels and, after 24-hour insulin incubation, PAI-1 mRNA increase in IR strains was only slightly less than in IS strains. Insulin ability to induce MAPK activation was also comparable in IR and IS cells. Conclusions—We conclude that in cells from IR individuals, insulin action on anti-atherogenic processes, such as NO release, is impaired, whereas the hormone ability to stimulate atherogenic processes, such as PAI-1 release, is preserved.

Heregulin-dependent autocrine loop regulates growth of K-ras but not erbB-2 transformed rat thyroid epithelial cells.

The EGF‐like family of proteins, such as epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin (AR), betacellulin (BTC), cripto‐1 (CR‐1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL‐5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL‐5H2 cell line, overexpressing erbB‐2, Kimol cells, transformed by the wild‐type K‐ras and A6 clone, transformed by a temperature sensitive K‐ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF‐like growth factors and corresponding receptors (erbB‐2, erbB‐3, and erbB‐4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL‐5 cells, both K‐ras and erbB‐2‐transformed cells expressed elevated levels of erbB‐2 receptor. Moreover, both K‐ras transformed cells, Kimol and A6 cells, but no FRTL‐5H2 cells, were found able to express also high levels of erbB‐4 receptor and HRG/NDF ligand. Treatment of K‐ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB‐2 overexpressing FRTL‐5 cells, in K‐ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB‐2/erbB‐4 receptors in an autocrine fashion. J. Cell. Physiol. 176:383–391, 1998.

TGFβ inhibits rat thyroid cell proliferation without alterations in the expression of TSH-induced cell cycle-related genes

Transforming growth factor beta (TGF beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. TGF beta acts on thyroid cells by inhibiting cell proliferation and expression of differentiation markers, such as thyroglobulin production and iodide uptake. Exponentially growing thyroid cells cultures accumulate mostly in G0/G1 after exposure to TGF beta for 48 hours. TGF beta inhibits the TSH induced transition of quiescent thyroid cell from the G0 to the S phase. These effects on the thyroid cell growth, however, are not mediated by changes in the TSH-induced cell cycle-related genes expression; both immediate early and progression genes expression is unaffected by the TGF beta treatment.

Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA)

Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation.

Cysteinyl-leukotriene D4 induced IL-1β expression and release in rat vascular smooth muscle cells

Vascular cells, including smooth muscle cells (VSMC), may release interleukin 1 (IL-1) and transcribe its genes for both isoforms. Previous studies have shown that cysteinyl-leukotrienes can modulate cytokine production by monocytes and a cytokine-eicosanoid network has been suggested during atherosclerosis. In this study the effects of cysteinyl-leukotriene D4 (LTD4) on IL-1 beta production and IL-1 beta mRNA expression were tested on rat VSMC. LTD4 showed a significant dose-dependent (from basal production of 55 +/- 15 pg/ml to maximal production of 177 +/- 14 pg/ml) and time-dependent (peaking at 24 h 16 +/- 54 pg/ml) increase of IL-1 beta immunoreactivity in the supernatants of conditioned medium and cell lysates. Furthermore, LTD4 induced an increased mRNA expression which began at 1 h and peaked at 12 h incubation time. The production of IL-1 beta was inhibited by MK-571 (from 145 +/- 12 to 60 +/- 10 pg/ml), a specific receptor antagonist of LTD4 and partially reduced by IL-1 receptor antagonist (IL-1 ra) (from 160 +/- 12 to 85 +/- 5 pg/ml). These experiments suggest that cysteinyl-leukotrienes, potentially produced in the vascular wall by leukocytes or by transcellular metabolism, may be involved in local IL-1 production.