Connexin 26 Expression in Mammalian Cardiomyocytes.

Connexins are a family of membrane-spanning proteins named according to their molecular weight. They are known to form membrane channels mediating cell-cell communication, which play an essential role in the propagation of electrical activity in the heart. Cx26 has been described in a number of tissues but not in the heart, and its mutations are frequently associated with deafness and skin diseases. The aim of this study was to assess the possible Cx26 expression in heart tissues of different mammalian species and to demonstrate its localization at level of cardiomyocytes. Samples of pig, human and rat heart and H9c2 cells were used for our research. Immunohistochemical and molecular biology techniques were employed to test the expression of Cx26. Interestingly, this connexin was found in cardiomyocytes, at level of clusters scattered over the cell cytoplasm but not at level of the intercalated discs where the other cardiac connexins are usually located. Furthermore, the expression of Cx26 in H9c2 myoblast cells increased when they were differentiated into cardiac-like phenotype. To our knowledge, the expression of Cx26 in pig, human and rat has been demonstrated for the first time in the present paper.

Immunohistochemical localization of histidine-rich glycoprotein in human skeletal muscle: preferential distribution of the protein at the sarcomeric I-band.

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that is synthesized by parenchymal liver cells. Using Western blot analysis and immunoperoxidase techniques, we have previously shown the presence of HRG in human skeletal muscle. This paper reports the results of immunofluorescence experiments carried out on sections of human normal skeletal muscle biopsies to investigate the subcellular localization of HRG. The HRG localization was also compared with that of skeletal muscle AMP deaminase (AMPD1), since we have previously described an association of the enzyme with the protein. The obtained results give evidence for a preferential localization of HRG at the I-band level, where it shows the same distribution of actin and where AMPD1 is present in major concentration.

High concentration of C-type natriuretic peptide promotes VEGF-dependent vasculogenesis in the remodeled region of infarcted swine heart with preserved left ventricular ejection fraction.

BACKGROUND: Vasculogenesis is a hallmark of myocardial restoration. Post-ischemic late remodeling is associated with pathology and function worsening. At the same time, neo-vasculogenesis helps function improving and requires the release of vascular endothelial growth factor type A (VEGF-A). The vasculogenic role of C-type natriuretic peptide (CNP), a cardiac paracrine hormone, is unknown in infarcted hearts with preserved left ventricular (LV) ejection fraction (EF). We explored whether myocardial VEGF-dependent vasculogenesis is affected by CNP. METHODS AND RESULTS: To this end, infarcted swine hearts were investigated by magnetic resonance imaging (MRI), histological and molecular assays. At the fourth week, MRI showed that transmural myocardial infarction (MI) affected approximately 13% of the LV wall mass without impairing global function (LVEF>50%, n=9). Increased fibrosis, metalloproteases and capillary density were localized to the infarct border zone (BZ), and were associated with increased expression of CNP (p=0.03 vs. remote zone (RZ)), VEGF-A (p<0.001 vs. RZ), BNP, a marker of myocardial dysfunction (p<0.01 vs. RZ) and the endothelial marker, factor VIII-related antigen (p<0.01 vs. RZ). In vitro, CNP 1000 nM promoted VEGF-dependent vasculogenesis without affecting the cell growth and survival, although CNP 100 nM or a high concentration of VEGF-A halted vascular growth. CONCLUSIONS: CNP expression is locally increased in infarct remodeled myocardium in the presence of dense capillary network. The vasculogenic response requires the co-exposure to high concentration of CNP and VEGF-A. Our data will be helpful to develop combined myocardial delivery of CNP and VEGF-A genes in order to reverse the remodeling process.

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes.

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP=0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein.

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Embedding methods for poly(L-lactic acid) microfiber mesh/human mesenchymal stem cell constructs.

Fiber mesh scaffolds were recently investigated in tissue engineering as possible support for stem cell growth and differentiation, in order to repair lesion areas in clinical practice. In particular, the literature is focused on fiber mesh scaffolds constituted of biocompatible and resorbable polymeric structures, like poly(L-lactic acid) (PLLA). However, as regards the study of constructs constituted of PLLA microfibers and cells, only quantitative and SEM analyses were reported, lacking histological analysis. Histological evaluation of these constructs could give important information about cellular distribution in the scaffold, cell-scaffold interactions and extracellular matrix production. The purpose of our study was to find a valid method to analyze PLLA microfiber/cell constructs from both histological and histochemical angles. Biodegradable non-woven fiber meshes were prepared using hollow microfibers, based on PLLA. We first evaluated different embedding methods useable for histological analysis and the results showed that among the paraffin, Killik, and acrylic resin the only suitable medium was the latter. Then we employed the acrylic resin to embed the constructs made up of PLLA microfibers and bone marrow-derived human mesenchymal stromal cells, which we then analyzed with Toluidine Blue, PAS and Alcian Blue staining. These constructs, previously analyzed for cell viability by MTT and CCK-8 tests, showed viable/proliferating cells until 6 weeks of culture. The stainings performed on constructs confirmed viability data obtained with SEM and MTT/CCK-8 and supplied other information on the cell behaviors such as the distribution and organization onto the scaffold and the production of extracellular matrix molecules. In conclusion, this methodological study mainly suggests a suitable method to analyze PLLA microfiber/cell constructs, at the same time confirming and enriching the literature data on the compatibility between PLLA microfibers and hMSCs.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells.

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

Constitutive expression of cyclooxygenase-2 in the neuromuscular compartment of normal human colon.

Prostaglandins regulate various functions throughout the gastrointestinal system. Their biosynthesis depends on cyclooxygenase isoforms, named COX-1 and COX-2. The initial hypothesis that COX-2 is an inducible enzyme has been challenged and its constitutive expression in the stomach has been established. In this study, an immunohistochemical analysis was performed to evaluate the distribution and cellular localization of COX-2 in normal human colon. Colonic surgical specimens were processed for COX-2, protein HuC/HuD, neurofilament, S-100 protein and CD117/c-kit immunodetection. COX-2 protein was found to be constitutively expressed in the colonic wall: detectable amounts were localized in mucosal, submucosal and muscular layers, mainly in the neuromuscular compartment. In particular, COX-2 was expressed in muscularis mucosae, submucosal ganglia, longitudinal muscle layer and myenteric ganglia, the neurons of which displayed different degrees of immunostaining. Intramuscular interstitial cells of Cajal, regarded as important sites for the regulation of enteric neuromuscular activity, were also partly COX-2 immunoreactive. This study provides a detailed mapping of COX-2 expression in human colon, and allows better understanding of the roles played by this isoenzyme in gut physiology.

Morphological features of ovine embryonic lung fibroblasts cultured on different bioactive scaffolds.

Tissue regeneration with autologous cell transplantation is one of the most important goals in clinical research. In this field, the development of bioactive materials that provide microenvironments for cell-matrix interactions mimicking biological conditions is required. In recent years, many synthetic materials have been developed as scaffolds and many procedures for the surface modification of these materials have been applied using biological molecules. In this study, we analyzed the morphology and the molecule production by ovine embryonic lung fibroblasts cultured on three different sponge-like matrices based on poly(L-lactic acid) (PLLA): agarose/PLLA, crosslinked and uncrosslinked gelatin/PLLA. The matrices were produced by using an emulsion freeze-drying method leading to the formation of sponge-like materials with high porosity and with interconnection between the pores. In vitro MTT test demonstrated that transplanted cells were viable and metabolically active. Morphological analysis revealed that fibroblasts adhered to and penetrated the polymeric structures. Moreover, all the different matrices supported fibroblast production of proteoglycans, glycoproteins, and matrix molecules such as elastin, collagen I, and fibronectin. These data suggest that the tested bioactive scaffolds may support the growth and extracellular matrix molecule production of fibroblasts allowing in vitro connective tissue regeneration.

Immunohistochemical demonstration of the small GTPase RhoA on epoxy-resin embedded sections.

The purpose of the present study was to establish a method for light microscopical immunohistochemical localization of the small G protein RhoA on specimens treated and embedded for routine transmission electron microscopy. There are advantages in antigen immunolocalization on resin semi-thin sections compared to cryostat or paraffin sections: the preservation of morphological details, the well-defined immunoprecipitate localization and the possibility to correlate the immunohistochemical results with those obtained by electron microscope on neighbouring sections. These advantages are particularly useful for the subcellular localization of low molecular weight proteins such as RhoA, a small G protein able to cycle from the inactive cytoplasmic form to the plasma membrane-bound active form.

Renal cell cultures for the study of growth factor interactions underlying kidney organogenesis.

The present study was performed in four renal cell lines to evaluate their capability to: (1) produce and express transforming growth factor alpha (TGFalpha), its respective receptor, the epidermal growth factor receptor (EGFr) and the small G protein, RhoA, and (2) exhibit morphogenetic properties when grown on Matri-cell substrates. The cell lines were derived from normal (Madin-Darby canine kidney cells), embryonic (SK-NEP-1 and 293 cells), and cancerous (human renal adenocarcinoma cells) kidneys. TGFalpha messenger ribonucleic acid, evaluated by a nonradioactive in situ hybridization technique, was found to be expressed in all the cell lines. Large amounts of TGFalpha peptide were observed in all four cell lines, while EGFr was highly expressed only in cancerous ACHN and embryonic-tumor SK-NEP-1 cells. RhoA peptide was found in appreciable amounts in SK-NEP-1 and 293 cells (compared to the other two cell lines). The morphogenetic properties of the four cell lines were assessed, by culturing them on Matri-cell dishes: SK-NEP-1 cells alone were found to grow in three-dimensional structures forming clusters and worm-like cellular aggregates. This feature was displayed by SK-NEP-1 cells but not by the other three cell lines, and may be connected with the contemporary presence of RhoA, EGFr, and TGFalpha found in significant amounts only in the SK-NEP-1 cell line.

TGF-alpha mRNA expression in renal organogenesis: a study in rat and human embryos.

The peptides belonging to the epidermal growth factor (EGF) family play a significant role in kidney development by binding the EGF receptor. Transforming growth factor-alpha (TGF alpha), a member of this family, is thought to be the fetal ligand of the EGF receptor. The present study aims to localize the TGF alpha transcripts in rat and human embryonic kidneys using a nonradioactive in situ hybridization method on paraffin-embedded embryonic samples. The results obtained in this study, beside demonstrating the usefulness of the nonradioactive technique for the detection of TGF alpha mRNA in paraffin sections, allowed TGF alpha-producing cells to be seen in developing kidneys. TGF alpha mRNA and its respective peptide were found in the primitive mesonephric structures and within metanephric blastema and ureteric bud cells. The presence of the TGF alpha gene transcript in the developing rat and human kidney suggests that the TGF alpha peptide is of embryonic origin and that it may contribute to renal organogenetic processes together with other growth factors.

A morphological study of the expression of the small G protein RhoA in resting and activated MDCK cells.

The small G protein Rho subfamily controls several cellular events such as growth, movement, proliferation and differentiation by rearranging actin and cytoskeleton proteins. Most of these effects are mediated by the activation of growth factor and extracellular matrix molecule receptors, suggesting a role for Rho molecules in the transduction pathway of these receptors. Despite the importance of Rho peptides in fundamental cellular events, data on their subcellular immunolocalisation are sparse: here we investigated the expression and subcellular localisation of RhoA in resting (cultured on plastic) and activated (Matri-cell or hepatocyte growth factor) MDCK cells by immunoperoxidase and immunogold techniques. Resting MDCK cells contain detectable amounts of RhoA mainly localised in the cytoplasm; RhoA expression is significantly enhanced by Matri-cell substrates that promote translocation of RhoA at the membrane level. This enhancing effect is reduced after exposure to hepatocyte growth factor.

Use of the comet test in the evaluation of multidrug resistance of human cell lines.

The comet test is a reported method for measuring DNA damage in individual mammalian cells. In the present report, the ability of this test to detect multidrug resistance (MDR) was evaluated. For this purpose, two human leukemia, well-characterized parental cell lines, HL60 and CEM, and their derived multidrug-resistant cells, HL60/DNR and CEM/VBL, were cultured with or without different anti-cancer agents. To evaluate the comet test, two DNA-damaging agents were used: daunorubicin (DNR), which is involved in MDR, and ambamustine (AMBA), which is independent from MDR. Moreover, in order to evaluate the specificity of the comet test, the activity of vinblastine (VBL), an MDR-related, DNA-independent anti-cancer drug, was also tested. Finally, the specificity of the comet test in detecting MDR was confirmed by culturing parental or resistant cells with DNR with or without the revertant agent verapamil (VER). Results confirm that the comet test is able to predict cellular chemoresistance when DNA damaging agents are tested. Finally, experiments on the role of the comet test in evaluating certain aspects of DNA repair are discussed.

The effect of cytokines, including IL4, IL7, stem cell factor, insulin-like growth factor on childhood acute lymphoblastic leukemia.

We have investigated the effects of some interleukins, such as interleukin (IL) 4, IL7, stem cell factor (SCF) and insulin-like growth factor (IGF-1), known to be involved in human lymphopoiesis, on proliferation, clonal growth and differentiation of cells from two acute lymphoblastic leukemia (ALL) derived pre-B cell lines, that is, Nalm 1, Nalm 6 and purified blasts from 37 childhood ALL. IL4 did not display any promoting activity, an inhibitory effect being observed in two patients. IL7 showed an heterogeneous responsiveness, not related to immunophenotype or cytogenetic features, proliferation and clonal growth being observed in a minority of ALL. In other patients no or even inhibitory effects on proliferation were observed. In one case this inhibition of DNA synthesis was accompanied by maturation of the cells, as demonstrated by the induced expression of surface immunoglobulins (slg); other IL7 treated samples failed to express slg, but showed a decreased expression of terminal deoxynucleotidyl transferase and cALL antigen, suggesting that the cells have a potential of limited maturation by IL7. SCF, known to synergize with IL7 in the most primitive stages of normal B cell development, did not enhance the IL7 response in B cell precursor ALL. Finally IGF-1 failed to induce a proliferative response and clonal growth in BCP ALL either alone or in combination with IL7.

c-Jun and GST-pi expression in human plasma cells.

Bone marrow samples from 33 patients affected by MM and MGUS, and 8 patients not affected by lymphoproliferative diseases were studied for expression of c-Jun (a component of the transcription factor AP-1) and glutathione-S-transferase pi (GST-pi) using immunocytochemical methods. A high and frequent expression of these two proteins was found both in MM and MGUS patients (31/33 patients positive for c-Jun and 29/33 patients positive for GST-pi) and in controls not affected by monoclonal gammopathy (7/8 patients positive for both c-Jun and GST-pi). No statistically significant correlation was found between c-Jun- and GST-pi-positive plasma cells. The expression of these two proteins was not related to clinical or laboratory data. Our results seem to confirm a possible role of the transcriptional complex AP-1 in activating GST-pi promoter in human plasma cells.

GST-pi and P-170 co-expression in multiple myeloma.

Bone marrow samples from 40 patients affected by multiple myeloma either treated or untreated were examined for expression of glutathione-S-transferase pi (GST-pi), P-glycoprotein and the protein product of ras oncogenes family, p-21, on plasma cells, by immunocytochemical detection. 72% of evaluated samples were positive for P-170 and 82% for GST-pi without any correlation with clinical or prognostic parameters. A significant relationship between GST-pi expression and P-170 positivity was found and co-expression was observed in 91% of evaluated samples. Expression of P-170 and GST-pi was found both in treated and untreated patients. However, patients evaluated before and after therapy showed an increase in the percentage of plasma cells positive for GST-pi or P-170 or both. Expression of p-21 was not associated with these mechanisms of drug resistance. These data suggest that different resistance mechanisms are present in multiple myeloma.

Evaluation of resistance index of several anticancer agents on parental and resistant P-388 cell lines.

Multidrug resistance is frequently detected in haematological malignancies and in acute leukaemias with a poor prognosis. In the last few years, several reports seem to suggest that the new anthracycline derivative idarubicin and the anthraquinone mitoxantrone have some advantages in the management of untreated or relapsed acute leukaemias compared with older anthracyclines. This could be due to a different interaction of these drugs with multidrug resistance. To evaluate this possibility, we compared the activity of doxorubicin (DOXO), epirubicin (EPI), idarubicin (IDA) and mitoxantrone (MITO) on a murine, multidrug resistant, leukaemic cell line (P-388/Dx) cultured in vitro. ID50 of IDA and MITO was in the ng range whereas that of DOXO and EPI was in the microgram(s) range. Moreover, IDA has a resistance index of 50 whereas DOXO has one of 250. Verapamil is able to almost completely abolish the resistance to IDA. Efflux experiments confirm that verapamil increases IDA intracellular concentration. IDA and MITO appear to be less involved in multidrug resistance than older anthracyclines.

Induction of multidrug resistance (MDR) by transfection of MCF-10A cell line with c-Ha-ras and c-erbB-2 oncogenes.

To investigate the relationship between oncogene activation and appearance of multidrug resistance (MDR) we transfected the human breast epithelial cell line MCF-10A, negative for the expression of the P-glycoprotein, with c-Ha-ras and/or c-erbB-2 oncogenes. The appearance of the MDR phenotype was then studied by evaluating mdr-1 mRNA expression, the presence of P-glycoprotein on the cell membrane and the onset of doxorubicin resistance, together with the effect of the reversing agent verapamil. We found that only MCF-10A transfected with both c-Ha-ras and c-erbB-2 oncogenes acquired the MDR phenotype.

Idarubicin is active on MDR cells: evaluation of DNA synthesis inhibition on P388 cell lines.

Multidrug resistance is frequently found in patients affected by hematological malignancies and has been related to a poor prognosis of acute leukemia. In the present paper we report results concerning the activity of idarubicin, an anthracycline derivative, on the leukemic P388 and P388 doxorubicin-resistant cell lines. The results clearly show that idarubicin inhibits DNA synthesis in the resistant cell line more actively than doxorubicin.