Pasquale Somma

Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet

Several experimental and clinical studies have shown that oxidized low-density lipoprotein and oxidation-sensitive mechanisms are central in the pathogenesis of vascular dysfunction and atherogenesis. Here, we have used p66Shc−/− and WT mice to investigate the effects of high-fat diet on both systemic and tissue oxidative stress and the development of early vascular lesions. To date, the p66Shc−/− mouse is the unique genetic model of increased resistance to oxidative stress and prolonged life span in mammals. Computer-assisted image analysis revealed that chronic 21% high-fat treatment increased the aortic cumulative early lesion area by ≈21% in WT mice and only by 3% in p66Shc−/− mice. Early lesions from p66Shc−/− mice had less content of macrophage-derived foam cells and apoptotic vascular cells, in comparison to the WT. Furthermore, in p66Shc−/− mice, but not WT mice, we found a significant reduction of systemic and tissue oxidative stress (assessed by isoprostanes, plasma low-density lipoprotein oxidizability, and the formation of arterial oxidation-specific epitopes). These results support the concept that p66Shc−/− may play a pivotal role in controlling systemic oxidative stress and vascular diseases. Therefore, p66Shc might represent a molecular target for therapies against vascular diseases.

Chronic treatment with sulfhydryl angiotensin-converting enzyme inhibitors reduce susceptibility of plasma LDL to in vitro oxidation, formation of oxidation-specific epitopes in the arterial wall, and atherogenesis in apolipoprotein E knockout mice

The effects of chronic treatment with the new sulfhydryl angiotensin-converting enzyme (ACE)-inhibitor, zofenopril, in comparison with the classical sulfhydryl ACE-inhibitor captopril or enalapril or placebo on the development of atherosclerosis were determined in apolipoprotein-E knockout (apoE(-/-)) mice. Groups of 2-month-old male mice received either placebo (N=10), 0.05 mg/kg/day of zofenopril (N=10), 1 mg/kg/day of zofenopril (N=10), 5 mg/kg/day of captopril (N=10) or 0.5 mg/kg/day of enalapril (N=8). After 29 weeks of treatment, computer-assisted imaging analysis revealed that zofenopril reduced the aortic cumulative lesion area by 78% at 0.05 mg/kg/day and by 89% at 1 mg/ml/day of zofenopril compared to that of the placebo (P<0.0001). Captopril reduced by 52% aortic lesions compared to placebo (P<0.01 vs. placebo; P<0.05 vs. zofenopril at both doses). Enalapril did not reduce aortic lesions. Furthermore, 0.05 mg/kg/day of zofenopril reduced susceptibility of plasma LDL to in vitro oxidation compared to captopril, enalapril or placebo, as shown by significant reduction of malondialdehyde content (P<0.001 vs. placebo or enalapril; P<0.05 vs. captopril), as well as by the prolongation of lag-time (P<0.01 vs. placebo or enalapril P<0.05 vs. captopril). More importantly, mice treated with 1 mg/ml/day of zofenopril had a significant decrease in the intimal immunohistochemical presence of oxidation-specific epitopes on oxLDL (NA59 monoclonal antibody, P<0.01), macrophages derived foam cells (F4/80 monoclonal antibody, P<0.05) and native LDL (NP monoclonal antibody, P<0.01) compared to placebo, captopril or enalapril. Thus, chronic treatment with the new sulfhydryl ACE-inhibitor zofenopril has antiatherosclerotic and antioxidant effects in the arterial wall of hypercholesterolemic apoE(-/-) mice. This protection was significantly higher than that reached with captopril and at lower doses of the drug. Treatment with 0.5 mg/kg/day of enalapril did not provide any protective effect.

Clinical response, vascular change, and angiogenesis in gonadotropin-releasing hormone analogue-treated women with uterine myomas

Objective: Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) are involved in the pathogenesis of leiomyomas and influence angiogenesis, which is necessary for growth of leiomyomas. Gonadotropin-releasing hormone analogue (GnRH-a) treatment might modify the growth factor expression and the blood supply in myomas. We investigated the effects of GnRH-a treatment on some clinical parameters, on the immunohistochemical expression of bFGF, VEGF, and PDGF, and on the vasculature of leiomyomas. Methods: Thirty-one women were treated with leuprolide acetate for 3 months; 55 untreated patients formed the control group. Hematologic parameters were assessed at the admission, after GnRH-a treatment, and after surgery. Uterine volume was evaluated by ultrasonography. The immunoexpression of bFGF, VEGF, and PDGF and of the endothelial markers CD34 and CD105, as well as the vascular pattern, were studied in leiomyomas, comparing treated and untreated patients. Results: Hematologic parameters improved and uterine volumes decreased after GnRJH-a treatment. The immunoexpression of bFGF, VEGF, and PDGF decreased in treated myomas, together with the total number of vessels and the angiogenetic vessels. Conclusion: This study confirms the clinical response of uterine shrinkage after GnRH-a treatment. A pathogenetic role of bFGF, VEGF, and PDGF in myoma growth and vascularization is suggested. Finally, this study indirectly confirms the importance of the vasculature in leiomyoma growth.

Overexpression of the Cytokine Osteopontin Identifies Aggressive Laryngeal Squamous Cell Carcinomas and Enhances Carcinoma Cell Proliferation and Invasiveness

Purpose: Osteopontin is a secreted cytokine that binds to the cell surface CD44v6 receptor. We studied osteopontin and CD44v6 expression in laryngeal squamous cell carcinomas and correlated osteopontin expression levels with clinicopathologic tumor features. Experimental Design: We used immunohistochemistry, immunoblotting, and reverse transcription-PCR to study osteopontin expression in 58 laryngeal squamous cell carcinomas. Cultured squamous carcinoma cells were treated with exogenous osteopontin or with RNA interference to knockdown osteopontin expression. Results: Osteopontin expression was higher in all the invasive carcinomas than in patient-matched normal mucosa. Its expression levels were significantly correlated with tumor stage and grade and with the presence of lymph node and distant metastases. Osteopontin positivity was negatively correlated with overall survival (P = 0.03). Osteopontin expression was paralleled by intense cell surface reactivity for CD44v6. Treatment of squamous carcinoma cells with recombinant osteopontin sharply increased proliferation and Matrigel invasion in comparison with the untreated cells parallel to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase signaling cascade. Osteopontin knockdown by RNA interference, anti-CD44 antibodies, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition prevented these effects. Conclusions: These results identify osteopontin as a marker and a potential therapeutic target in cases of aggressive laryngeal squamous cell carcinomas.

Histopathologically Proven Prevention of Post-Prostatectomy Cavernosal Fibrosis with Sildenafil

Aim: To evaluate whether a dose of 50 mg preserved the architecture of the corpora cavernosa biopsy in man. Methods: 21 patients (54–70 years old) who underwent radical prostatectomy for prostate cancer were treated with sildenafil citrate (50 mg, 3 times a week for 2 months) soon after surgery. They underwent cavernous biopsy before surgery and after 2 months of sildenafil treatment. Biopsy tissues were fixed in formalin, stained with Masson’s trichrome method, and evaluated with the Eureka Interface system with a per-area analysis, and elastic fibers were counted on 10–12 fields (×400) of five serial sections. Results: Two months after surgery the percent of connective tissue in cavernosa samples in all patients did not differ from that before surgery, being between 30 and 40% in the per-area analysis. Similarly, the elastic fiber count did not differ significantly before and after surgery. Conclusions: Sildenafil prevented the progression of fibrosis in prostatectomized patients. Its efficacy seems to result from an antiproliferative effect exerted on fibroblasts.

Beneficial effects of ACE-inhibition with zofenopril on plaque formation and low-density lipoprotein oxidation in watanabe heritable hyperlipidemic rabbits.

The effects of angiotensin-converting enzyme (ACE)-inhibition with zofenopril on the development of atherosclerosis and low-density lipoprotein (LDL) oxidation were determined in Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Rabbits received either placebo (n = 6) or 0.5 mg/kg/day of zofenopril (n = 6). After 6 weeks of treatment, the computer-assisted analysis revealed that zofenopril reduced the aortic and common carotid corrected cumulative lesion area by 34% and 39%, respectively (p < 0.05 vs placebo-treated group). The intimal/medial ratio of the largest fatty streaks was 0.426+/-0.158 in the zofenopril-treated group and 0.875+/-0.238 in the placebo-treated group (p < 0.05). Furthermore, we found in the zofenopril-treated group smaller lesions with an intimal/medial ratio of zofenopril also reduced plasmatic LDL oxidation, as shown by significant reduction of malondialdehyde content (p < 0.01) and relative agarose gel mobility (p < 0.05), as well as by the prolongation of the lag-time (p < 0.05). Compared to zofenopril-treated rabbits, arterial sections of the placebo-group had significant increase in the intimal presence of macrophages-derived foam cells (p < 0.05), ox-LDL (p < 0.01), and native LDL (p < 0.01) detected by immunocytochemistry with RAM-11, MDA2 and NP1533975 monoclonal antibodies, respectively. To investigate the amount of platelet accumulation in the atherosclerotic plaque we also measured platelet-associated radioactivity. Autologous platelets were labeled with 111Indiumoxine and injected intravenously. After 2 hours, WHHL were sacrificed and arterial sections were counted for platelet-associated radioactivity. In the placebo-treated group, platelet radioactivity was 0.52+/-0.12 equivalent of radioactivity per mg of tissue in the common carotid and 0.25+/-0.18 in the abdominal aorta; in contrast, rabbits treated by zofenopril had 0.20+/-0.12 in the common carotid and 0.06+/-0.01 in the abdominal aorta. These data indicate that ACE-inhibition with zofenopril has antiatherosclerotic and antioxidant effects in WHHL-rabbits. Our results also shows that these effects could be linked to a reduced wall-associated platelet deposition at the site of atherosclerotic lesions.

Loss of oestrogen receptor β, high PCNA and p53 expression and aneuploidy as markers of worse prognosis in ovarian granulosa cell tumours

Aims:  Ovarian granulosa cell tumour (OGCT) is a sex‐cord stromal tumour with a general trend toward late relapse and/or metastasis. However, mortality rate corrected for long‐term follow‐up shows that about 50% of patients die within 20 years of diagnosis. Classical clinicopathological parameters are unable to predict the biological behaviour of OGCT. The involvement of a recently characterized subtype of oestrogen receptor, ERβ, in ovarian carcinogenesis has been hypothesized.

Effects of vitamin E and HMG-CoA reductase inhibition on cholesteryl ester transfer protein and lecithin-cholesterol acyltransferase in hypercholesterolemia.

BackgroundThe enzyme lecithin-cholesterol acyl transferase (LCAT) esterifies free cholesterol on highdensity lipoprotein (HDL) and the cholesteryl ester transfer protein (CETP) transfers cholesteryl esters to very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Using statins, contradictory findings have been made regarding CETP activity in normolipidemic individuals and in those with familial dysbetatlipoproteinemia. In contrast, LCAT activity appears to be unaffected by simvastatin. Antioxidants have also been proposed for use in anti-atherosclerotic treatment, because the oxidation of LDL may have a key role in the pathophysiology of atherogenesis. ObjectiveTo investigate, in hypercholesterolemic patients, whether a combination of pravastatin with the antioxidant, vitamin E, has greater effects on the activity of CETP and of LCAT than does pravastatin alone. MethodsThis placebo-diet-controlled multicenter trial included 220 hypercholesterolemic patients who were assigned randomly to groups to receive: diet and 20–40 mg pravastatin (n = 52), diet and pravastatin in combination with 100 mg/day vitamin E (100 IU) as DL-α-tocopherol (n = 56), diet and α-tocopherol (n = 60), or diet associated with placebo (n = 52). Plasma LCAT activity was determined using excess exogenous substrate, containing [3H]cholesterol. Plasma CETP activity was measured in the supernatant fraction after precipitation of endogenous apo B-containing lipoproteins with phosphotungstate-Mg2+. The exchange of cholesteryl esters between [14C]cholesteryl esterlabeled LDL and unlabeled HDL was measured during a 16-h incubation, while LCAT was inhibited. ResultsThe addition of pravastatin to the diet induced a significant decrease in plasma CETP activity (P< 0.05); this effect was less evident in the group cotreated with vitamin E. For the first time, it was shown that CETP concentrations increased significantly after vitamin E alone (P< 0.05). No significant differences in the plasma activity of LCAT were observed among the groups. ConclusionsPravastatin reduced CETP activity, but not that of LCAT. Addition of vitamin E prevented the decrease in CETP activity and had no effect on LCAT activity. The mechanism responsible for these effects is unknown, but could involve the prevention of radical-induced damage to CETP by vitamin E. Coronary Artery Dis 9:257–264

Squamous Cell Carcinoma of the Lower LIP: Fas/Fasl Expression, Lymphocyte Subtypes and Outcome

Squamous cell carcinoma (SCC) of the lip is a relatively common malignancy of the head and neck region. Tumour thickness, grading and perineural invasion are significant prognostic indicators. However, there is still the need of new reliable biological markers able to predict the prognosis of the single cases with an unfavourable biological behaviour unpredictable by the classic clinical-pathological parameters. 32 cases of (SCC) of the lower lip were analysed for their clinicopathologic features, and immunohistochemical expression of Fas/FasL in neoplastic cells and in inflammatory infiltrate. Moreover the density and phenotype of tumour-infiltrating lymphocytes (TIL) were analysed. The results were related with the follow-up of the patients ranging from 2 to 6 years. The cases with over-expression of Fas/FasL in neoplastic cells and Fas+ in T cells preferentially showed a more aggressive clinical behaviour (p<0.01). Moreover we found an alteration of the normal expression of CD4 and CD8 lymphocyte types in ten cases. This data suggest that the Fas/FasL pathway is involved in the close relation between neoplastic cells and T cells and so in the biological behaviour of these tumours.

Colonoscopic removal of a polypoid arteriovenous malformation.

A 53-year-old male presenting with a 3-month history of intermittent mild rectal bleeding was found, on double contrast barium enema, to have a large polyp on a long stalk in the sigmoid colon. Large bowel endoscopy confirmed the presence of a 2 cm pedunculated polyp which was removed using a diathermic snare, with slight bleeding following the procedure that did not require endoscopic haemostasis. Only after histologic examination was the polyp shown to be a colonic arteriovenous malformation. Endoscopically, arteriovenous malformations generally appear as flat or elevated bright red lesions. A pedunculated polypoid appearance is extremely uncommon. In this case, no gastrointestinal bleeding or polypoid recurrence was observed during the 12 months of clinical and endoscopic follow-up.