L. Rodella

[PP.12.08] EFFECTS OF MELATONIN ON THE PRODUCTION OF ADIPONECTIN AND THE EXPRESSION OF ADIPONECTIN RECEPTOR IN THE VISCERAL ADIPOSE TISSUE OF AGING MICE.

Objective: It has been previously demonstrated that inflammation in adipose tissue may be implicated in vascular dysfunction (Circulation 2009; 119(12):1661–1670). A senescence-accelerated prone mouse (SAMP8) is a model of age-related cognitive decline and vascular dysfunction. Several studies demonstrated that SAMP8 suffers from increased oxidative stress and that accelerated senescence was associated with increased oxygen radicals synthesis. We have also recently demostrated that the anticontractile effect of perivascular fat is impaired in a senescence-accelerated prone mouse, compared with control senescence-resistant mice (SAMR1). A long-term treatment with melatonin seems to decrease contractile responses to norepinephrine in mesenteric small arteries of SAMP8, thus restoring an anticontractile effect, probably through antioxidant mechanisms. However, it is not know whether melatonin is able to modulate the production of adiponectin and/or the expression of adiponectin receptors. Therefore, the aim of the study was to investigate the production of adiponectin and/or the expression of adiponectin receptors in the visceral adipose tissue of SAMP8 before and after chronic treatment with melatonin. Design and method: We investigated 7 SAMP8 and 7 SAMR1 treated for 10 months with melatonin (MEL, 10 mg/kg/day), an endogenous indoleamine with antioxidant and vasculoprotective properties, as well as 7 SAMP8 and 7 SAMR1 untreated controls (CTR) kept untreated for 10 months. Visceral fat (perirenal fat) was obtained by dissection, and the adiponectin content, as well as the expression of adiponectin receptor were evaluated by immunohistochemistry. Results: Results are shown in the figure (* p < 0.05 vs SAMR1 CTR; # p < 0.05 vs SAMR1 + MEL; + p < 0.05 vs SAMP8 + MEL). The adiponectin content, as well as the expression of adiponectin receptor were reduced in untreated SAMP8 compared with untreated SAMR1. Treament with MEL was able to increase the production of both proteins in SAMP8. Figure. No caption available. Conclusions: Melatonin treatment is able to increase the production of adiponectin and the expression of adiponectin receptor in the visceral fat of aging mice. This observation might contribute to explain the improvement of the anticontractile action of perivascular fat observed in the mesenteric small resistance arteries of SAMP8 mice after chronic treatment with MEL.

Endothelial and Vascular Smooth Cell Dysfunctions: A Comprehensive Appraisal

Cardiovascular disease (CvDs) such as coronary artery disease, hypertension, congestive heart failure and stroke are the leading causes of death and disability in the Western World (Madamanchi et al., 2005; Thom, 1989). The majority of CvDs results from complication of atherosclerosis. Prevention of cardiovascular events is therefore urgently needed and is one of the major recent challenges of medicine. New molecular imaging approaches featuring the assessment of inflammatory processes in the vascular wall (on top of existing anatomic and functional vessel imaging procedures) could emerge as decisive tools for the understanding and prevention of cardiovascular events (Schafers et al., 2010).

FIBRONECTIN AND LAMININ CONTENT IN THE TUNICA MEDIA OF SUBCUTANEOUS SMALL RESISTANCE ARTERIES OF PATIENTS WITH ESSENTIAL HYPERTENSION: PP.11.428

Objective: We have previously demonstrated that a pronounced fibrosis, due to an increased content in type 1 collagen, may be observed both in the tunica media and in the adventitia of subcutaneous small resistance arteries of patients with essential hypertension and with primary aldosteronism (JCEM 2006, 91:2638–2642). However, it was suggested that, in the development of hypertensive microvascular remodeling, a relevant role may be played by laminin and fibronectin vascular content, at least in animal models of hypertension. Aim of this study was to evaluate the amount of fibronectin and laminin within the tunica media of subcutaneous small arteries of normotensive subjects and essential hypertensive patients. Design and Methods: We have investigated 6 normotensive control subjects and 10 essential hypertensive patients. All subjects were submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, the fibronectin and laminin content within the tunica media was evaluated by immunohistochemistry, with subsequent image analysis (% of area stained). Results: As expected, clinic blood pressure values were higher in essential hypertensive patients (153/89 ± 7/11 mm Hg) than in normotensive controls (116/81 ± 10/5 mm Hg, P < 0.01). The media to lumen ratio was significantly increased in essential hypertensive patients (0.100 ± 0.03) compared with normotensive controls (0.06 ± 0.02, p < 0.01). Fibronectin media content was significantly greater in essential hypertensive patients (7.41 ± 2.28 %), compared with normotensive controls (5.62 ± 0.40, P < 0.05). A significant correlation was observed between fibronectin media content and media to lumen ratio (r = 0.49, p < 0.05). No significant difference in laminin media content was observed between groups (3.7 ± 1.71 % in essential hypertensive patients, 5.63 ± 1.79 % in normotensive controls). Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, fibronectin, but not laminin media content is increased. Fibronectin might be therefore involved in the development of small resistance artery remodeling in humans.

Intraepidermal Injections of Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study

Regenerative medicine is a modern approach of dermatological treatment, using Epidermal Cells of the interfollicular epidermis (ESC) for their effect in skin regeneration in chronic ulcers and burns, melanoma, vitiligo, junctional epidermolysisbullosa. Intraepidermal injections of autologous epidermal cell suspension can be a new and very promising treatment for mano other cutaneous disorders as non scarring alopecia (alopecia areata, androgenic alopecia) or scarring alopecia (Lichern Plano Pilaris alopecia, Discoid Lupus Erithematosus alopecia), anti-aging therapies. The intraepidermal injection of an autologous epidermal cell suspension is a simple, fast and safe surgical procedure: a small, thin portion of the epidermis of the patient undergoes a treatment where a suspension with all the cells collected from the epidermis is injected into the skin. The epidermal grafts were then incubated in a trypsin solution (Trypsyn 0.5 g/ EDTA 0.2 solution, Sigma-Aldrich co) for 45 minutes at 37oC (Plasmatherm Barkley). After incubation, the trypsin solution was discarded and the tissues were washed with HBSS. The epidermis (thin yellow layer) was treated with a scalpel blade to separate the cells. The supernatant was suctioned through a sterile syringe and then cultured in Dulbeccos modified Eagles medium (DMEM; Sigma Aldrich, Saint Louis, USA) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (EuroClone, Devon, UK) and 1% penicillin/streptomycin solution (Sigma Aldrich) at 37 oC in a humidified atmosphere of 95% air and 5% CO2. The cells were seeding in a 25oC at day 0, the cells showed a heterogeneous appearance with regard to shape and size and their nucleus was almost detectable. At day 7, the cells maintained a heterogeneous appearance with regard to shape and size, even though more spheroidal elements were observed. Cells were generally in suspension, only some microspots of fibroblastoid-shaped cells were well attached to the flask surface m2 cell culture flask at a density of about 20000 cells/cm2. Our preliminary study show that an suspension contains a significant number of viable cells (59,40%, SD ± 6.07%) that survive at day 7 in culture (63.55% , SD ± 5.41%). The number of epidermal cells in each sample is significant as keratinocytes (in greater quantity) and melanocytes (1900 ±178 melanocytes/mm2 as reported by Khodadadi L) (6) are detected. Our research is currently continuing and it is focusing on the typing of the different cells in the suspension and evaluating the presence and the nature of stem cells.