Cadmium exposure alters steroid receptors and proinflammatory cytokine levels in endothelial cells in vitro: a potential mechanism of endocrine disruptor atherogenic effect.

BACKGROUND: Cadmium (Cd) is a widespread environmental pollutant that causes alterations in human health acting as endocrine disruptor. Recent data suggest that cardiovascular system might be a contamination target tissue, since Cd is found in atheromatic plaques. Thus, the purpose of this study was to evaluate the consequence of Cd exposure of endothelial cells in vitro to evaluate detrimental effect in vascular system by a potential sex-steroid hormone receptor-dependent mechanism(s). METHODS: To this aim, Human Umbilical Vein Endothelial Cells (HUVECs) were cultured and exposed to several concentrations of cadmium chloride (CdCl2) for different interval times. RESULTS: CdCl2 exposure of HUVECs induced a significant increase of ERß and Cyp19a1 at both mRNA and protein levels, while a drastic dose-dependent decrease of AR expression level was observed after 24 h of exposure. On the contrary, an increase of PhARser308 as well as a reduction of PhGSK-3ßser9 and PhAKTser473 was detected after 1 h treatment. This effect was consistently reduced by GSK inhibition. Furthermore, CdCl2 abolished DHT-induced cell proliferation in HUVECs suggesting an antagonist-like effect of Cd on AR-mediated signaling. Remarkable, after 6 h CdCl2-treatment, a relevant increase in TNF-α, IL-6 and IL-8 mRNA was observed and this effect was blocked by the presence of an ERß-selective antagonist. Moreover, Cd-induced TxR1 overexpression, likely, correlated with the activation of p38 MAPK/NF-κB pathway. CONCLUSION: In conclusion, our study demonstrates for the first time that Cd alters sex-steroid hormone receptors level and activity likely affecting intracellular signaling linked to a proinflammatory state in endothelial cells. This alteration might possibly lead to endothelial cell injury and vascular dysfunction and could be a mechanism of gender-specific atherogenic damages induced by endocrine disruptors and, thus, induce atherogenic events with increased risk of cardiovascular diseases in individuals exposed to this endocrine disruptor.

Correction to: Cadmium exposure alters steroid receptors and proinflammatory cytokine levels in endothelial cells in vitro: a potential mechanism of endocrine disruptor atherogenic effect.

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Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PURPOSE: Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). METHODS: Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. RESULTS: Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. CONCLUSIONS: Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

The endocrine disruptor cadmium alters human osteoblast-like Saos-2 cells homeostasis in vitro by alteration of Wnt/ß-catenin pathway and activation of caspases.

PURPOSE: The pollutant Cadmium (Cd) is widespread in the environment and causes alterations of human health by acting as an endocrine disruptor. Bone tissue seems to be a crucial target of Cd contamination. Indeed, we have previously demonstrated that this endocrine disruptor induces osteoblast apoptosis and necrosis. Thus, aim of this study was to further evaluate the effect of Cd on osteoblasts homeostasis, investigating potential modification of the Wnt/ß-catenin intracellular pathway, the intracellular process involved in programmed cellular death and the cytoskeletal alterations. MATERIAL AND METHODS: To this purpose, human osteoblastic Saos-2 cells, a human osteosarcoma osteoblast-like cell line, were cultured and treated with Cd. RESULTS: Osteoblastic cells were treated for 6 h with 10µM Cd, which induced nuclear translocation of ß-catenin and increased expression of Wnt/ß-catenin target genes. Longer exposure to the same Cd concentration induced osteoblastic cell apoptosis. To better characterize the intracellular events involved in these Cd-induced alterations, we evaluated the effect of Cd exposure on actin filaments and proteins associated to cytoskeletal actin, characterized by the presence of LIM domains. Long (15, 24 h) exposure of osteoblasts to Cd reduced LIM proteins expression and induced actin filaments destruction and a significant caspase-3 activation after 24 h. In addition, to prove that Cd induces osteoblastic cells apoptosis after long exposure, we performed TUNEL assay which demonstrated increase of cell apoptosis after 24 h. CONCLUSION: In conclusion, our study shows that osteoblasts exposed to Cd for short intervals of time demonstrated an increase in cell proliferation through a Wnt/ß-catenin dependent mechanism, likely as a compensatory mechanism in response to cell injury. Longer exposure to the same Cd concentration induced cells apoptosis through cytoskeleton disruption-mediated mechanisms and caspase activation.

Biochemical and immunomorphological evaluation of hepatocyte growth factor and c-Met pathway in patients with critical limb ischemia.

OBJECTIVES: Hepatocyte growth factor (HGF), the c-Met receptor, and hypoxia-inducible factor (HIF) are crucial for regenerative processes including ischemic wound healing. The aims of the present study are (a) to analyze the tissue c-Met and HIF-1α expression in skin from patients with critical limb ischemia (CLI); (b) to compare the serum HGF levels of CLI and control subjects. METHODS: This is a prospective, controlled, single-center study. Thirty-seven patients were enrolled. A skin sample adjacent to the ischemic lesion was taken from 20 patients with CLI; skin samples were taken from the surgical wounds of 17 patients surgically treated for abdominal aortic aneurysm as healthy controls. Serum samples were taken in all cases. Samples were formalin fixed, paraffin embedded, and routinely processed. Tissue inflammation was histologically assessed. Immunohistochemistry was performed with antibodies against total c-Met receptor, activated Met (p-Met), and HIF-1α. RT-polymerase chain reaction was used to quantify HIF-1α mRNA. The enzyme-linked immunosorbent assay was performed to evaluate serum HGF levels. RESULTS: With immunohistochemistry, while total c-Met was unchanged, different patterns of p-Met positivity were observed between CLI and control cases (p < .001). In particular, CLI skin showed a total negativity or membrane positivity for p-Met (19/20 cases), while control skin mainly showed cytoplasmic positivity in the epidermal basal layer (16/17 cases). HIF-1α was diffusely lost in CLI, but HIF-1α mRNA was threefold higher than in controls. Finally, mean serum HGF levels were 590.5 pg/mL and 2380.0 pg/mL in CLI and control groups respectively (p < .001). CONCLUSIONS: In CLI patients a significant decrease in serum HGF levels, concomitant with a loss of skin HIF-1α stabilization and a lack of c-Met phosphorylation were seen, probably driving a decrease in wound-healing functions. The next hypothesis is that HGF application might reactivate the c-Met receptor, stabilizing the normal wound healing process.

Role of exercise-induced reactive oxygen species in the modulation of heat shock protein response.

The multiple roles that have been associated with heat shock proteins (HSPs), inside and outside cells are remarkable. HSPs have been found to play a fundamental role in multiple stress conditions and to offer protection from subsequent insults. Exercise, because of the physiological stresses associated with it, is one of the main stimuli associated with a robust increase of different HSPs in several tissues. Given the combination of physiological stresses induced by exercise, and the 'cross-talk' that occurs between signaling pathways in different tissues, it is likely that exercise induces the HSP expression through a combination of 'stressors', among which reactive oxygen species (ROS) could play a major role. Indeed, although an imbalance between ROS production and antioxidant levels results in oxidative stress, causing damage to lipids, proteins, and nucleic acids with a possible activation of the programed cell death pathway, at moderate concentrations ROS play an important role as regulatory mediators in signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and re-establish redox homeostasis. The aim of this review is to provide a critical update on the role of exercise-induced ROS in the modulation of the HSP's response, focusing on experimental results from animal and human studies where the link between redox homeostasis and HSPs' expression in different tissues has been addressed.

C-reactive protein and embolization during carotid artery stenting. A serological and morphological study.

INTRODUCTION: High-sensitivity C-Reactive Protein (hsCRP) levels are correlated with vulnerable carotid plaques, although their impact on the outcome of carotid revascularization is unknown. The aim of our study was to investigate the correlation between hsCRP and embolization during carotid artery stenting (CAS). METHODS: Patients with symptomatic carotid stenosis were submitted to CAS with distal protection filters. Serum hsCRP was analysed prior to CAS and patients were divided into two groups: Class I, patients presenting hsCRP < 5 mg/l and, Class II, patients presenting hsCRP≥5 mg/l. Plaques were categorised by ultrasound grey scale measurement as homogenous and dishomogenous. Afterwards CAS filters were analyzed microscopically and ultrastructurally to determine the type and the amount of debris present, based on percentage of surface involvement (SI) and pore occluded (PO) by embolic material. RESULTS: Fourteen patients underwent uneventful CAS, with a mean hsCRP of 11.5±18.4 mg/l. Eight patients were in Class I and six in Class II. All filters had microscopic debris. SI was 25.4% in Class I and 33.3% in Class II (p=ns), PO 22.9% and 33.3% respectively (p=0.049). Patients in Class II who also had a dishomogenous plaque showed greater SI and PO compared with patients in Class I with homogenous plaque (35.0% vs. 21.8% and 40.4% vs. 22.7% respectively, p<0.05). Microscopically embolic material was identified as atherosclerotic plaque fragments and platelet aggregates and was similar in both groups. DISCUSSION: High hsCRP levels are associated with significantly greater embolization during CAS in symptomatic patients, particularly in dishomogenous plaque. Although these results need further investigation due to the limited number of enrolled patients, this study suggests that CAS may not be indicated as a method of carotid revascularization in this setting.

Identification of carotid 'vulnerable plaque' by contrast-enhanced ultrasonography: correlation with plaque histology, symptoms and cerebral computed tomography.

INTRODUCTION: Indication to carotid revascularisation is commonly determined by percent of stenosis as well as neurological symptoms and clinical conditions. High plaque embolic potential is defined as 'vulnerability'; however, its characterisation is not universally used for carotid revascularisation. We investigated the role of contrast-enhanced ultrasonography (CEUS) to identify carotid vulnerable plaque. METHODS: Patients undergoing carotid endarterectomy were preoperatively evaluated by cerebral computed tomography (CT) scan and CEUS. Contrast microbubbles detected within the plaque indicated neovascularisation and were quantified by decibel enhancement (dB-E). Plaques were histologically evaluated for five features: (microvessel density, fibrous cap thickness, extension of calcification, inflammatory infiltrate and lipid core) and blindly scored 1-5 to assess plaque vulnerability. Analysis of variance (ANOVA), Fisher's and Student's t-test were used to correlate patients' characteristics, histological features and dB-E. RESULTS: In 22 patients, dB-E (range 2-7.8, mean 4.85 ± 1.9 SD) was significantly greater in symptomatic (7.40 ± 0.5) vs. asymptomatic (3.5 ± 1.4) patients (p = 0.002). A higher dB-E was significantly associated with thinner fibrous cap (<200 µm, 5.96 ± 1.5 vs. 3 ± 1, p = 0.01) and greater inflammatory infiltrate (3.2 ± 0.9 vs. 6.4 ± 1.2, p = 0.03). Plaques with vulnerability score of 5 had significantly higher dB-E compared with those with vulnerability score of 1 (7.6 ± 0.2 vs. 2.5 ± 0.6, respectively, p = 0.001). Preoperative ipsilateral embolic lesions at CT were correlated with higher dB-E (5.96 ± 1.5 vs. 3.0 ± 1.0, p = 0.01). CONCLUSION: CEUS with dB-E is indicative of the extent of plaque neovascularisation. It can be used therefore as a marker for vulnerable plaque.