In Vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P<0.05); B16F10 exhibited an increase in cell number within 7 days (P<0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P<0.05) compared to inside was observed for all cell types. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MU) values indicated higher cell viability in samples cultured with HGF compared to CGF (P=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non-CM was observed at 48 and 72 hours (P<0.05). Conclusions: ADM is not suitable as a three-dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. J Periodontol 2011;82:293-301.

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation

P>Aim To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. Methodology Samples of LPS solution (50 mu gmL-1), Ca(OH)(2) suspension (25 mg mL-1) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 mu L of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey`s test at 5% significance level. Results The mean and SE (in mu mol L-1) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00 +/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). Conclusions Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells

Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

NAD(P)H Oxidase Participates in the Palmitate-Induced Superoxide Production and Insulin Secretion by Rat Pancreatic Islets

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose-stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free-albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI-diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase-I (CPT-I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47(PHOX) translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up-regulated the protein content of p47(PHOX) and the mRNA levels of p22(PHOX), gp91(PHOX), p47(PHOX), proinsulin and the G protein-coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate. J. Cell. Physiol. 226: 1110-1117, 2011. (C) 2010 Wiley-Liss, Inc.

Glucose-Induced Regulation of NHEs Activity and SGLTs Expression Involves the PKA Signaling Pathway

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 +/- A 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (similar to 40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (similar to 50%, P < 0.05) and the plasma membrane (similar to 100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

Relationship between structure and photoactivity of porphyrins derived from protoporphyrin IX

Protoporphyrin (Pp IX) derivatives were prepared to study the relationship between photosensitizer structure and photoactivity, with an emphasis on understanding the role of membrane interactions in the efficiency of photosensitizers used in photodynamic therapy (PDT). The synthetic strategies described here aimed at changing protoporphyrin periferic groups, varying overall charge and oil/water partition, while maintaining their photochemical properties. Three synthetic routes were used: (1) modification of Pp IX at positions 3(1) and 8(1) by addition of alkyl amine groups of different lengths (compounds 2-5), (2) change of Pp IX at positions 13(3) and 17(3), generating alkyl amines (compounds 6 and 7), a phosphate amine (compound 8), and quarternary ammonium compounds (compounds 9 and 10), and (3) amine-alkylation of Hematoporphyrin IX (Hp IX) at positions 3(1), 8(1), 13(3) and 17(3) (compound 12). Strategy 1 leads to hydrophobic compounds with low photocytotoxicity. Strategy 2 leads to compounds 6-10 that have high levels of binding/incorporation in vesicles, mitochondria and cells, which are indicative of high bioavailability. Addition of the phosphate group (compound 8), generates an anionic compound that has low liposome and cell incorporation, plus low photocytotoxicity. Compound 12 has intermediate incorporation and photocytotoxic properties. Compound modification is also associated with changes in their sub-cellular localization: 30% of 8 (anionic) is found in mitochondria as compared to 95% of compound 10 (cationic). Photocytotoxicity was shown to be highly correlated with membrane affinity, which depends on the asymmetrical and amphiphilic characters of sens, as well as with sub-cellular localization.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(]V) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 mu M, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells. (C) 2008 Elsevier Ltd. All rights reserved.