**3. Discussion**

Culture media used to generate tissue-engineered skin substitutes are supplemented with a cAMP stimulator, which ensures epithelial cell proliferation [15,17]. In the present study, it was shown that even if the psoriatic skin models reconstructed with either CT or ISO displayed pathological features of psoriasis, CT remains more e ffective for stimulating the cells towards a psoriatic phenotype with hyperproliferative epidermal cells. Psoriatic substitutes produced with CT also displayed higher cAMP levels, as well as higher AC9 expression, than other conditions. These results show that higher cAMP levels would be associated with a stronger psoriatic phenotype, such as epidermal hyperproliferation and altered epidermal di fferentiation, therefore supporting studies in which cAMP levels are found to be increased in psoriatic skin.

Our data sugges<sup>t</sup> that, in the presence of healthy keratinocytes, both cAMP enhancers (CT and ISO) stimulate the production of cAMP in the same manner, although the concentration of CT used in the experiment was lower than the concentration of ISO. Therefore, these results are in accordance with previous studies, which have reported that even if CT and ISO are both cAMP stimulators of epidermal cells, CT is better than ISO for the improvement of keratinocyte growth since it may increase the overall growth rate of epidermal cells by reducing the doubling time of cells [15]. In vitro studies reported by Green et al. have shown that using 10−<sup>6</sup> M of isoproterenol produced an increase in keratinocyte colony size but had less e ffect on cAMP levels than 10−<sup>6</sup> M of CT. Indeed, CT was the strongest agen<sup>t</sup> in a ffecting cAMP concentration among the four di fferent cAMP stimulators tested [15]. Moreover, these results support the use of both cAMP inducers, at appropriate concentrations, when producing healthy reconstructed skin models, and are in accord with results reported by Cortez Ghio et al. [38]. Isoproterenol (ISO) is therefore a good candidate for replacing CT since it presents several advantages regarding safety and regulations [38]. Indeed, CT is known to be potentially toxic and therefore requires more precautions in its manipulation [39,40].

In psoriatic substitutes, CT induced a stronger psoriatic phenotype, as well as higher levels of cAMP, than ISO when using the same concentration of each inducer as for the healthy models. This suggests that CT would be a better cAMP stimulator for lesional cells and that unlike for healthy substitutes, ISO is not recommended to stimulate cAMP in lesional cells. This incapacity of ISO to stimulate cAMP production in psoriatic substitutes could be attributed to a beta-adrenergic defect in psoriatic skin. Although Archer et al. once reported that psoriasis is not associated with impaired beta-adrenergic reactivity [41], most studies have since reported a decrease in responsiveness to beta-adrenergic stimuli, such as isoproterenol, in the stimulation of cAMP production in psoriatic tissues [16,30,42–44]. This decreased responsiveness would be linked to a lower expression of the beta-adrenergic receptor in psoriatic skin, which was confirmed in the present study with immunofluorescence staining. Di fferent beta-2-adrenergic receptor polymorphisms may also contribute to the pathogenesis [30,45]. This defect of the beta-2-adrenergic receptor in psoriatic skin does not a ffect cAMP stimulation by CT, since the CT mechanism of action is based on direct activation of AC9. Finally, in 1978, Das et al. reported that the topical application of 0.1% isoproterenol sulphate mixed with white Vaseline and applied to the psoriatic plaques of twelve patients induced a significant decrease in the scaliness and cell turnover of the treated psoriatic skin [46]. Even if no other literature was found on a possible commercial development of this formulation, it appears that isoproterenol could be a potential topical or systemic treatment for psoriatic patients.

In the present study, a stronger psoriatic phenotype and higher levels of cAMP were found in psoriatic substitutes as compared with healthy substitutes, especially with CT, for which the di fference

was significant. These results therefore imply that higher levels of cAMP would be found in psoriatic skin, which is in contrast to the levels of cAMP monitored in psoriatic skin in the late seventies [33]. However, these results are in accordance with studies on the e ffects of CT on psoriasis in vivo, in which it was revealed that the psoriatic epidermis accumulates much more cAMP than uninvolved body regions or normal human epidermis [18,21]. According to the present study, the higher levels of cAMP found in psoriatic epidermis could be attributed to enhanced AC9 expression. AC9 is mostly expressed in the brain, spinal cord, liver, heart, lung, kidney, muscle, and adrenal gland, but little is known about its localization in the skin, particularly in the epidermis [24,47,48]. Finally, enhanced levels of cAMP in psoriatic skin are more consistent with the levels of downstream mediators identified in the literature during the past decade [49–51]. Indeed, it is relatively well established that psoriatic skin displays increased levels of phosphorylated CREB, which controls cellular functions, such as the regulation of gene expression [52].

In summary, our study showed that, although both cAMP inducers stimulate the production of psoriatic skin substitutes displaying hyperproliferating epidermis and disturbed epidermal di fferentiation, CT induced a stronger psoriatic phenotype than ISO. Moreover, enhanced cAMP levels were found in the epidermis of our psoriatic substitutes, which could be attributed to enhanced expression of AC9 in psoriatic skin. Therefore, our study answers a highly controversial question, suggesting that cAMP levels are increased in psoriatic skin. Finally, cAMP levels were higher in the CT psoriatic substitutes than in the ISO psoriatic substitutes, confirming a defect of the beta2-adrenergic receptors in psoriatic skin. This study is of particular interest since to our knowledge it is one of the first to determine the concentrations of cAMP in a psoriatic reconstructed skin model produced by tissue engineering.

## **4. Materials and Methods**
