The rs39335 polymorphism of the RELN gene is not associated with otosclerosis in a southern Italian population.

Otosclerosis, the single most common cause of hearing impairment in white adults, is characterised by bone dystrophy localized to the otic capsule and isolated endochondral bone sclerosis with alternating phases of bone resorption and formation. Conductive hearing loss develops when otosclerotic foci invade the stapedio-vestibular joint (oval window) and interfere with free motion of the stapes, but affected subjects frequently develop profound sensorineural hearing loss. The aetiology of otosclerosis is unknown. In the last years, several association studies have been performed and have suggested that single nucleotide polymorphisms in some genes may be implicated in development of otosclerosis. The strongest association has been demonstrated for the reelin gene, located on chromosome 7q22.1, which encodes an extracellular matrix protein. The involvement of reelin in the pathogenesis of otosclerosis is controversial; it was identified in European and North African populations, but was excluded in an Indian population. To analyze the role of reelin in otosclerosis, it has been studied in a case-control analysis for the polymorphism rs39335 in a southern Italy population. In this population, the pathogenic link between the rs39335 variant and otosclerosis was excluded.

Neuroprotective strategies in hippocampal neurodegeneration induced by the neurotoxicant trimethyltin.

The selective vulnerability of specific neuronal subpopulations to trimethyltin (TMT), an organotin compound with neurotoxicant effects selectively involving the limbic system and especially marked in the hippocampus, makes it useful to obtain in vivo models of neurodegeneration associated with behavioural alterations, such as hyperactivity and aggression, cognitive impairment as well as temporal lobe epilepsy. TMT has been widely used to study neuronal and glial factors involved in selective neuronal death, as well as the molecular mechanisms leading to hippocampal neurodegeneration (including neuroinflammation, excitotoxicity, intracellular calcium overload, mitochondrial dysfunction and oxidative stress). It also offers a valuable instrument to study the cell-cell interactions and signalling pathways that modulate injury-induced neurogenesis, including the involvement of newly generated neurons in the possible repair processes. Since TMT appears to be a useful tool to damage the brain and study the various responses to damage, this review summarises current data from in vivo and in vitro studies on neuroprotective strategies to counteract TMT-induced neuronal death, that may be useful to elucidate the role of putative candidates for translational medical research on neurodegenerative diseases.

Histological and histomorphometric evaluation of implant with nanometer scale and oxidized surface. in vitro and in vivo study.

BACKGROUND: The biological fixation of an implant to bone is influenced by numerous factors, including surface chemistry and surface topography. Various methods have been developed to create rough implant surfaces in order to improve the clinical performance of implants and to guarantee a stable mechanical bone-implant interface. Anodic oxidation is a dental implant surface modification technique that results in oxide layer growth up to a thickness of 1–10 micron. The purpose of this study was to evaluate the performance of the surface through the osteoblasts cells growth and the influence of oxidixed surface on BIC percent, in the human posterior maxilla after 2 months of unloaded healing. MATERIAL AND METHODS: In vitro commercially available primary human osteoblasts (NHOst) from both femur and tibia of different donor systems (Lonza Walkersville Inc, Walkersville, MD, USA) were grown in Osteoblast Growth Media (OBM) (Lonza). Osteogenic differentiation was induced for a period of 4 weeks by the OGM medium (OBM basal medium supplemented with 200nM of hydrocortisone-21-hemisuccinate and 7.5 mM of glycerophosphate). The viability of NHOst cells seeded test A and B was measured by the quantitative colorimetric MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2Htetrazoliumbromide test) (Promega, Milan, Italy). One custom-made 2 x 10-mm site evaluation implant (SEI) with nanometer scale and oxidized surface (test) ( Evo Plan 1 Health s.r.l. - Amaro, UD, Italy), and one SEI with hydroxyapatite sandblasted surface (control) (Osseogrip Plan 1 Health s.r.l. – Amaro, UD, Italy), were placed in the posterior maxilla of 15 patients. Patients received one of each type of SEI placed on controlateral side. RESULTS: The proliferation rate studied by the MTT assay showed that during the incubation time, starting at 24 h, an increased proliferation rate was evident in Test B respect to Test A. After 2 months of unloaded healing BIC percent was significantly higher in oxidized implants. BIC percent mean values for the Osseogrip surface was 36,133 +/-4,888 ER and 53,533 +/- 5,180 ER for the Evo surface(P = 0,028). CONCLUSION: These results seem to confirm that implant surface topography entails mechanical restrictions to the spread and locomotion of the cells involved in bone healing.

Distribution and time-course of 4-hydroxynonenal, heat shock protein 110/105 family members and cyclooxygenase-2 expression in the hippocampus of rat during trimethyltin-induced neurodegeneration.

Trimethyltin (TMT), an organotin compound considered a useful tool to obtain an experimental model of neurodegeneration, exhibits neurotoxicant effects selectively localised in the limbic system and especially in the hippocampus, which are different in the rat and in mice. In the rat hippocampus, we investigated the expression of aldehyde 4-hydroxynonenal, a major bioactive marker of membrane lipid peroxidation, heat shock protein (HSP) 110/105 family members, markers of oxidative stress, and the neuroinflammatory marker cyclooxygenase-2 after TMT-intoxication at various time points after treatment. Our data show that TMT-induced neurodegeneration in the rat hippocampus is associated specifically with oxidative stress and lipid peroxidation, but not with HSP expression, indicating species-specific differences in the neurotoxicity of TMT between rats and mice.

Transcritpional effects of S100B on neuroblastoma cells: perturbation of cholesterol homeostasis and interference on the cell cycle.

S100B is a Ca2+ binding protein mainly secreted by astrocytes in the vertebrate brain that is considered a multifunctional cytokine and/or a damage-associated molecular pattern (DAMP) protein and a marker of brain injury and neurodegeneration when measured in different body fluids. It has been widely shown that this protein can exert diverse effects in neural cultures depending on its concentration, having detrimental effects at micromolar concentrations. The molecular mechanisms underlying this effect are still largely unknown. This study attempts to delineate the genome-wide gene expression analysis of the events associated with exposure to micromolar concentration of S100B in a human neuroblastoma cell line. In this experimental condition cells undergo a severe perturbation of lipid homeostasis along with cell cycle arrest. These mechanisms might reasonably mediate some aspects of the S100B-related detrimental effects of S100B, although obvious differences between mature neurons and neuroblastoma cells have to be considered.

Nicotine reorganizes cytoskeleton of vascular endothelial cell through platelet-derived growth factor BB.

BACKGROUND: Cigarette smoking has been directly linked to atherosclerosis formation and vascular graft failures but the role of nicotine in these processes is not yet completely understood. We investigated the release of platelet-derived growth factor BB (PDGF BB) by the bovine aortic endothelial cell (EC) after nicotine administration at concentrations similar to those found in plasma of active and passive smokers and the role of PDGF BB, autocrinally released, in EC cytoskeletal modification. METHODS: EC were stimulated in a serum-free medium for 72 h with (-)-nicotine (from 6 x 10(-4) to 6 x 10(-8) M). The release of PDGF BB was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on EC was also determined. The EC cytoskeleton was studied with specific antibodies anti-alpha-actin fibers and anti-vimentin and the modification induced by PDGF BB was assessed by blocking PDGF BB activity with specific antibodies. RESULTS: The greatest PDGF BB release was noted at a (-)-nicotine concentration of 6 x 10(-6) M (P < 0.001). The addition of antibody anti-PDGF BB to EC exposed to (-)-nicotine decreased tritiated thymidine uptake by 20% (P < 0.001). EC exposed to (-)-nicotine concentrations of 6 x 10(-6) and 6 x 10(-8) M had a significant alteration in the expression of alpha-actin fibers and vimentin as compared with control. Administration of the antibody anti-PDGF BB in the culture medium reversed cytoskeletal alteration. CONCLUSIONS: Nicotine enhanced the release of PDGF BB by EC which in turn caused an alteration in cytoskeletal organization.

Nicotine-induced smooth muscle cell proliferation is mediated through bFGF and TGF-beta 1.

BACKGROUND: Cigarette smoking influences and enhances the development of atherosclerosis. We investigated if nicotine, an important constituent of cigarette smoking, has a stimulatory effect on bovine smooth muscle cell proliferation in vitro through the mediation of bFGF and TGF-beta 1. METHODS: Bovine aortic smooth muscle cells (SMC) were stimulated with (-)-nicotine at various concentrations ranging from 6 x 10(-4) mol/L to 6 x 10(-8) mol/L. SMC viability and count were assessed. The presence of bFGF and TGF-beta 1 in serum-free conditioned media was determined by the inhibition antibody-binding assay, and the mitogenic activity of (-)-nicotine on SMC was analyzed by the 3H-thymidine uptake. Polymerase chain reaction was used to study the expression of bFGF and TGF-beta 1. RESULTS: The bFGF release after (-)-nicotine stimulation was greater than in the controls, whereas TGF-beta 1 release was lower. The greatest mitogenic activity was found at a (-)-nicotine concentration of 6 x 10(-6) mol/L. The addition of monoclonal antibody anti-bFGF decreased the 3H-thymidine uptake of SMC exposed to (-)-nicotine, whereas the addition of monoclonal antibody anti-TGF-beta 1 increased the 3H-thymidine uptake of stimulated SMC. bFGF mRNA expression was significantly higher in SMC exposed to (-)-nicotine than in the controls, but TGF-beta 1 mRNA expression was significantly lower in SMC exposed to 6 x 10(-6) mol/L (-)-nicotine than in SMC treated with the other concentrations of (-)-nicotine and in controls. CONCLUSIONS: Nicotine is a potent regulator of bFGF and TGF-beta 1 production and release by aortic SMC, and it seems to play an important role in the development and progression of atherosclerosis and neointimal fibrous hyperplasia.

Nicotine induces platelet-derived growth factor release and cytoskeletal alteration in aortic smooth muscle cells.

BACKGROUND: Cigarette smoking is implicated in atherosclerotic plaque formation, but the role of nicotine in this process is not completely understood. The release of platelet-derived growth factor (PDGF) by the bovine aortic smooth muscle cell (SMC) after nicotine administration at a concentration similar to that ingested by active and passive smokers and the role of PDGF in SMC cytoskeletal modification were studied. METHODS: SMC, harvested with enzymatic digestion from calf aorta, were stimulated in a serum-free medium for 72 hours with (-)-nicotine (from 6 x 10(-4) mol/L to 6 x 10(-8) mol/L). The release of PDGF was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on SMCs was also determined. The SMC cytoskeleton was studied with specific antibodies anti-alpha-actin fibers, anti-vimentin, and anti-beta-tubulin, and the modification induced by PDGF was assessed by blocking PDGF activity with specific antibodies. RESULTS: The greatest PDGF release (1.24 +/- 0.14 ng/10(4) cells vs control 0.43 +/- 0.07 ng/10(4) cells) was noted at a (-)-nicotine concentration of 6 x 10(-7) mol/L (P < .001). The addition of monoclonal antibody anti-PDGF decreased the tritiated thymidine uptake of SMCs exposed to (-)-nicotine compared with the control (29% vs 5%-P < .001). SMCs exposed to (-)-nicotine concentration of 6 x 10(-7) mol/L and 6 x 10(-8) mol/L had a significant alteration in the expression of alpha-actin fibers, vimentin, and beta-tubulin compared with control. The administration of antibody anti-PDGF in the culture medium reversed cytoskeletal alteration. CONCLUSIONS: Nicotine enhanced the release of platelet-derived growth, which in turn caused an alteration in cytoskeletal organization.

Basic fibroblast growth factor and myointimal hyperplasia after experimental polytetrafluoroethylene arterial grafting.

OBJECTIVE: To assess the role of polyclonal antibodies to basic fibroblast growth factor (bFGF) in inhibiting myointimal hyperplasia after insertion of polytetrafluoroethylene (PTFE) grafts in rats. DESIGN: Experimental study. SETTING: University laboratory, Italy. ANIMALS: 24 inbred Lewis rats. INTERVENTIONS: A segment of PTFE I cm long was interposed in the abdominal aorta. The animals were randomised in two groups, n = 12 in each. The first were given polyclonal antibodies to bFGF at the time of operation, and for the first two postoperative days; and the second were given non-specific IgG at the same time periods. MAIN OUTCOME AND MEASURES: Two animals died during the immediate postoperative period of anaesthetic complications. 12 animals (6 in each group) were killed 7 days postoperatively (24 hours after injection of 5-bromo-deoxyuridine BrdU) to assess smooth muscle cell proliferation. The remaining 10 animals (5 in each group) were killed after 1 month to assess the degree of anastomotic myointimal hyperplasia. RESULTS: Antibodies to bFGF resulted in less smooth muscle cell proliferation at the anastomoses as well as anastomotic myointimal hyperplasia. Smooth muscle cell proliferation was reduced to about half in animals treated with anti-bFGF antibodies. Neointimal thickness was reduced in treated animals. CONCLUSIONS: We conclude that after PTFE arterial grafting there is increased production of bFGF at the anastomotic regions that leads to smooth muscle cell proliferation and formation of myointimal hyperplasia. Agents that reduce the production of bFGF may also reduce the development of myointimal hyperplasia after PTFE arterial grafting.

Nicotine regulates basic fibroblastic growth factor and transforming growth factor beta1 production in endothelial cells.

Nicotine, a constituent of cigarette smoking, may induce atherosclerosis through the production of growth factors. The pattern of bFGF and TGF beta1 production and release by bovine aortic endothelial cells (EC) stimulated with nicotine (from 6 x 10(-4) to 6 x 10(-8) M) was studied. EC viability and count were assessed. The presence of bFGF and TGF beta1 in serum-free conditioned media was determined by the inhibition antibody-binding assay and Western blot analysis. Mitogenic activity of nicotine on EC was also determined. Polymerase chain reaction (PCR) was used to study the expression of bFGF and TGF beta1. The bFGF release after nicotine stimulation was greater than controls, whereas TGF beta1 release was lower. At a nicotine concentration of 6 x 10(-6) M we noted the greatest mitogenic activity. The addition of monoclonal antibody anti-bFGF decreased the tritiated thymidine uptake of EC exposed to nicotine but the addition of monoclonal antibody anti-TGF beta1 had no significant effect. bFGF mRNA expression was significantly higher in EC exposed to nicotine than in controls, whereas TGF beta1 mRNA expression was not modified. From these data we concluded that nicotine regulates bFGF production and release and TGF beta1 release and may have a key role in the development and progression of atherosclerosis.

Thrombin induces production of growth factors from aortic smooth muscle cells.

BACKGROUND: Myointimal hyperplasia is a common complication of arterial recontructive surgery. The serine protease thrombin has a major role in vessel wall healing and eventual myointimal hyperplasia formation. The aim of this study was to determine the effect of thrombin on the production of PDGF AA and bFGF by arterial smooth muscle cells. MATERIALS AND METHODS: Bovine smooth muscle cells were stimulated with thrombin in a serum-free culture. The release of PDGF AA and bFGF was assessed by ELISA. The effect of thrombin on the proliferation of confluent monolayers of bovine smooth muscle cells was determined by tritiated thymidine uptake. RESULTS: Smooth muscle cells stimulated with thrombin released more PDGF AA (P < 0.001) and bFGF (P < 0.001) than the control. Addition of anti-PDGF AA and anti-bFGF antibodies to the medium of smooth muscle cell cultures neutralized the mitogenic effect of thrombin (P < 0.001). CONCLUSIONS: The findings of our study suggest that thrombin may lead to myointimal hyperplasia formation through induction of PDGF and bFGF production by smooth muscle cells.

Suppression of smooth muscle cell proliferation after experimental PTFE arterial grafting: a role for polyclonal anti-basic fibroblast growth factor (bFGF) antibody.

OBJECTIVES: To determine the role of polyclonal anti-basic Fibroblast Growth Factor (bFGF) antibody in inhibiting the proliferation of smooth muscle cells after experimental polytetrafluorethilene (PTFE) arterial grafting. MATERIALS: In 14 male inbred Lewis rats (weight 250 mg) a 1 cm long segment of PTFE was interposed at the level of abdominal aorta. Animals were randomised to receive polyclonal anti-bFGF antibody (group A: n = seven animals) or aspecific immunoglobulin (group B: n = seven animals). Anti-bFGF antibody or aspecific immunoglublin were given intraperitoneally at the end of operation, and for the first 2 postoperative days. Animals were sacrificed 7 days after surgery, 24 h after intraperitoneal injection of BromodeoxyUridin (BrdU) to label proliferating smooth muscle cells. RESULTS: One animal in each group died in the immediate postoperative period due to anaesthetic problems. All grafts were patent at the time of sacrifice. BrdU labelling index was statistically higher in the control group B animals at the level of the anastomotic regions (proximal anastomosis: group B 7.9% vs. group A 4.1%. Distal anastomosis: group B 5.1% vs. group A 2.6% p = 0.009) and at the level of PTFE graft (group B 3.8% vs. group A 2.6% p = 0.002), while there was no statistical difference between the control thoracic aorta of the two groups. MAIN CONCLUSIONS: bFGF plays a major role in the proliferation of smooth muscle cells at the level of the anastomoses after arterial PTFE grafting. Agents able to block the action of bFGF may be useful in inhibiting the formation of myointimal hyperplasia.

[High-porosity and normal-porosity prostheses. Differences in growth factor release]. / Protesi ad alta porosità od a porosità normale. Differenze nel rilascio di fattori di crescita.

BACKGROUND: The majority of mid- and long-term synthetic vascular graft failures are due to anastomotic neointimal fibrous hyperplasia. The major cause of this phenomenon is the absence of an endothelial lining within prosthetic grafts. We investigated the PDGF-BB and bFGF release by umbilical vein endothelial cell cultured on precoated standard porosity or high porosity expanded polytetrafluoroethylene (ePTFE) vascular grafts. METHODS: Endothelial cell harvested from umbilical veins were cultured into standard (30 microns internodal distance) or high porosity (60 microns internodal distance) cPTFE disks. ePTFE disks uncoated or precoated with collagen type I, fibronectin and Matrigel were used and endothelial cell cultured into plastic wells coated as ePTFE disks or uncoated plastic wells served as controls. Scanning electron microscopy study assessed endothelial cell coverage. The presence of PDGF-BB and bFGF in serum-free conditioned media from endothelial cells cultured into ePTFE grafts and endothelial cell cultured into wells was determined by the inhibition antibody-binding assay 48 hours after insertion. RESULTS: Endothelial cell coverage was similar in uncoated and coated ePTFE grafts and no differences were observed between standard or high porosity grafts. The release of PDGF-BB and bFGF was significantly higher either for standard porosity or high porosity ePTFE grafts as compared with endothelial cell cultured into plastic wells (p < 0.05 and p < 0.05, respectively). The release of PDGF-BB and bFGF was independent from the various substrates either for endothelial cell cultured into standard or high porosity ePTFE grafts or plastic wells. CONCLUSIONS: Our findings pointed out that in ePTFE grafts smooth muscle cellsc proliferated only under endothelial cell at the anastomoses, along seeded synthetic grafts or in areas of transmural ingrowth where smooth muscle cells followed endothelial cell migrating into the graft. This is probably due to an alteration of the interactions between the endothelial cell, the matrices and the synthetic prosthesis.

Oxidised LDL (OxLDL) induces production of platelet derived growth factor AA (PDGF AA) from aortic smooth muscle cells.

OBJECTIVES: Elevated concentrations of oxidised low density lipoproteins (OxLDL) are associated with accelerated atherogenesis. The aim of our study was to determine the effect of OxLDL on the proliferation rate and platelet derived growth factor (PDGF) AA production on aortic smooth muscle cells. High density lipoproteins (HDL), which are known to have a protective effect against atherosclerosis, were used as control. MATERIALS AND METHODS: Bovine aortic smooth muscle cells were grown in presence of increased concentrations of OxLDL and HDL and in presence of control medium culture (DMEM). Proliferation rate was assessed by 3H-thymidine uptake. PDGF AA production was determined by ELISA and Western Blot Analysis. RESULTS: OxLDL increased the proliferation rate of aortic smooth muscle cells as compared to DMEM and HDL (p < 0.001). The mitogenic activity of OxLDL on smooth muscle cells was reduced adding anti-PDGF AA antibodies (p < 0.001). PDGF AA production by aortic smooth muscle cells was increased after exposure to OxLDL as compared to DMEM (p < 0.001). HDL significantly reduced the production of PDGF AA by aortic smooth muscle cells (p < 0.001). CONCLUSIONS: Part of the atherogenic effect of OxLDL is mediated through the autocrine production of PDGF AA from aortic smooth muscle cells.

Release of PDGF-BB and bFGF by human endothelial cells seeded on expanded polytetrafluoroethylene vascular grafts.

BACKGROUND: The majority of endothelial cell (EC) seeded graft failures are due to anastomotic neointimal fibrous hyperplasia. We investigated the PDGF-BB and bFGF release in vitro by umbilical vein EC seeded on precoated expanded polytetrafluoroethylene (ePTFE) prostheses. MATERIALS: EC harvested from human umbilical veins were seeded into ePTFE (30 microns internodal distance, 1 cm2 in diameter) disks. ePTFE disks uncoated or precoated with collagen type I, fibronectin, and Matrigel were used, and EC seeded into plastic wells coated as ePTFE disks or uncoated plastic wells served as controls. Scanning electron microscopy study assessed EC coverage. The presence of bFGF and PDGF-BB in serum-free conditioned media from EC seeded into ePTFE grafts and EC seeded into wells was determined by the inhibition antibody-binding assay 72 h after seeding. RESULTS: EC coverage was similar in uncoated and coated ePTFE grafts. The release of PDGF-BB and bFGF by EC seeded into ePTFE grafts was significantly higher than that observed in EC seeded into plastic wells. The release of PDGF-BB and bFGF was independent from the various substrates used in the experiments in EC seeded into either ePTFE grafts or plastic wells. CONCLUSIONS: Our findings pointed out that in seeded ePTFE grafts, anastomotic smooth muscle cell proliferation and intimal thickening could take place underneath an intact endothelium because seeded EC may release several growth factors.

Formation of myointimal hyperplasia and cytokine production in experimental vein grafts.

BACKGROUND: The purpose of this study was to determine the correlation between progression and regression of myointimal hyperplasia (MH) and cytokine production in experimental vein grafts. Although the autologous vein is the best suitable bypass conduit for reconstruction of peripheral arteries, at the end of the first year thrombosis in the coronary and lower extremity circulation ranges from 20% to 50%. Many of these failures are caused by MH. METHODS: In 76 inbred Lewis rats, a 1 cm long segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngeneic Lewis rats. In 56 animals the arterial vein graft was explanted 3 days (n = 10), 7 days (n = 10), 4 weeks (n = 26), and 12 weeks (n = 10) after operation. In 20 animals the vein graft was explanted 4 weeks after being in the arterial system and reimplanted as iliac venovenous bypass in syngeneic Lewis rats. These grafts were explanted 2 weeks (n = 10) and 8 weeks (n = 10) later. Grafts were analyzed by light and electron microscopy, morphometric study, and histochemical analysis and were put in an organ culture to assess cytokine production. RESULTS: We observed MH formation in arterial vein grafts and MH regression in reimplanted vein grafts (p < 0.001). MH formation was correlated with production of platelet-derived growth factor, basic fibroblast growth factor, interleukin-1, and tumor necrosis factor-alpha. MH regression was correlated with transforming growth factor-beta 1 production. CONCLUSIONS: On the basis of the results of our study, we conclude that MH formation in experimental vein grafts depends on production of platelet-derived growth factor, basic fibroblast growth factor, interleukin-1, and tumor necrosis factor-alpha, and MH regression depends on transforming growth factor-beta 1 production. Cytokine therapy may represent a valuable new treatment to prevent vein bypass failures caused by MH.