Striae reconstructed, a full thickness skin model that recapitulates the pathology behind stretch marks.

OBJECTIVE: Stretch marks are disfiguring skin lesions that often cause problems of self-esteem, but little effort has been put to studying this pathology. We therefore analysed cell cultures of dermal fibroblasts isolated from a striae albae, to thereafter reconstruct a full thickness skin model. METHODS: Human Dermal Fibroblasts (HDF) were isolated from a striae distensae (SD) lesion and from the adjacent non-lesioned skin. The dermis of two full thickness skin models was reconstructed with either striae- or normal-HDF, while the epidermis was in both reconstructed with Normal Human Epidermal Keratinocytes. RESULTS: Main observations and pertinent data: Gene expression analysis of cell cultures revealed a generalized decomposition of the Extra Cellular Matrix (ECM), since collagens type I and III, lysyl oxidase (LOX), biglycan, lumican and fibronectin were downregulated, while MMP3 was increased together with a decrease of its natural inhibitors (TIMP1, TIMP2 and PAI-1). These findings were statistically corroborated for key ECM elements at the protein level (COL1, MMP1 and TGFB1). Interestingly, striae albae fibroblasts retained a pro-inflammatory phenotype, as suggested by increased gene expression of CXCL8, HAS1 and TNFA. We next reconstructed a full thickness skin model (Striae Reconstructed) with dermal fibroblasts from striae albae. Gene expression analysis showed that the Striae Reconstructed elicited not only ECM decomposition, but also skin ageing, as indicated by the upregulation of P16, PTGS2 and SOD2. Discussion points: Although the epidermis was constructed with normal human epidermal keratinocytes, the Striae Reconstructed presented epidermal atrophy and a dramatic increase of ß1-integrin at the epidermal-dermal junction providing, for the first time to our knowledge, a rationale showing that the key cell player behind stretch marks are dermal fibroblasts rather than epidermal keratinocytes. CONCLUSION: New knowledge: Taken together, our findings shed new light into the aetiology of stretch marks and indicate that the Striae Reconstructed, a new model for in vitro testing and drug screening, may open new avenues for the treatment of stretch marks.

OBJECTIFS: Les vergetures sont des lésions cutanées défigurantes qui posent souvent des problèmes d'estime de soi, mais peu d'efforts ont été consacrés dans l'étude de cette pathologie. Nous avons donc analysé des cultures cellulaires de fibroblastes dermiques isolés d'un stria alba, afin de reconstruire ensuite un modèle de peau avec une pleine épaisseur. METHODES: Des fibroblastes dermiques humains (FDH) ont été isolés d'une lésion de Stria distensae (SD) et d'une peau adjacente sans lésion. Le derme de deux modèles de peau de pleine épaisseur a été reconstruit avec du HDF striae- ou normal, tandis que l'épiderme était reconstruit avec des kératinocytes humains normaux. RESULTATS: Principales observations et données pertinentes: L'analyse de l'expression génique de cultures cellulaires a révélé une décomposition généralisée de la matrice extra-cellulaire (MEC) car les collagènes de types I et III, la lysyl oxydase, le biglycane, le lumican et la fibronectine étaient régulés négativement, tandis que la MMP3 augmentait ses inhibiteurs naturels diminuaient (TIMP1, TIMP2 et PAI-1). Ces résultats ont été corroborés statistiquement pour les éléments clés de la MEC au niveau de la protéine (COL1, MMP1 et TGFB1). Il est intéressant de noter que les fibroblastes de Striae albae ont conservé un phénotype proinflammatoire, comme le suggère l'augmentation de l'expression des gènes de CXCL8, HAS1 et TNFA. Nous avons ensuite reconstruit un modèle de peau de pleine épaisseur (Stria Reconstructed) avec des fibroblastes dermiques de Striae albae. L'analyse de l'expression génique a montré que la reconstruction de Striae induisait non seulement la décomposition de la MEC, mais également le vieillissement de la peau, comme l'indique la régulation à la hausse de P16, PTGS2 et SOD2. Points de discussion : Bien que l'épiderme ait été construit avec des kératinocytes humains normaux, les stries reconstruites présentaient une atrophie épidermique et une augmentation spectaculaire du taux de ß1-intégrine au niveau de la jonction épidermo-dermique, fournissant pour la première fois une explication rationnelle qui démontre que les cellules principales impliquées dans la pathologie de la Striae sont les fibroblastes et non les kératinocytes. CONCLUSION: Nouvelles connaissances: Ensemble, nos résultats donnent une nouvelle lumière sur l'étiologie des vergetures et indiquent que le Striae Reconstructed, un nouveau modèle de test in vitro et de dépistage du médicament, pourrait être une avancée pour le traitement des vergetures.

Selective hydrolysis of 2,4-diaminopyrimidine systems: a theoretical and experimental insight into an old rule.

Hydrolysis of the amino groups in condensed 2,4-diaminopyrimidine systems (1) has been used as a common method for the synthesis of oxo-substituted pyrimidines. In particular, the treatment with 6 M HCl usually yields exclusively the 2-amino-4-oxopyrimidine isomer (2). During our work, we found that the hydrolysis of the amino groups present in some condensed 2,4-diaminopyrimidine systems unexpectedly afforded exclusively the 4-amino-2-oxopyrimidine isomer (3). In this paper, we present the experimental work and ab initio calculations carried out to understand this discrepancy. As a part of such study, eight compounds containing a 2,4-diaminopyrimidine moiety were calculated in gas phase and in aqueous solution, and some acid hydrolyses were reexamined. Results showed that the presence of an electron-donating nitrogen linked to C6 of the 2,4-diaminopyrimidine ring changes the preferred hydrolysis site to yield the 4-amino-2-oxopyrimidine isomer.

Synthesis and biological activity of 7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF) and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).

We recently described the syntheses of 12a-c, 4-amino-7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF), and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), in six steps from commercially available p-substituted methyl benzoates in 20-27% overall yields. Such analogues were tested in vitro against CCRF-CEM leukemia cells and showed that they are completely devoid of any activity, the IC(50) being higher than 20 microg/mL for all cases. To clarify if the presence of the carbonyl group in position C7, the distinctive feature of our synthetic methodology, is the reason for this lack of activity, we have now obtained the 7-oxo substituted analogues of 5-DATHF and DDATHF, 18a-c, in 10-30% overall yield. Testing of 18a-c in vitro against CCRF-CEM leukemia cells revealed that these compounds are totally inactive. A molecular modeling study of 18b inside the active site of the complex E. coliGARTFase-5-DATHF-GAR pointed to an electronic repulsion between the atoms of the 7-oxo group and the carbonyl group of Arg90 as a possible explanation for the inactivity of 18a-c.

Synthesis and biological activity of 4-amino-7-oxo-substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid and 5,10-dideaza-5, 6,7,8-tetrahydrofolic acid.

The 4-amino-7-oxo-substituted analogues of 5-deaza-5,6,7, 8-tetrahydrofolic acid (5-DATHF) and 5,10-dideaza-5,6,7, 8-tetrahydrofolic acid (DDATHF) were synthesized as potential antifolates. Treatment of the alpha,beta-unsaturated esters 11a-c, obtained in one synthetic step from commercially available para-substituted methyl benzoates (9a-c) and methyl 2-(bromomethyl)acrylate (10), with malononitrile in NaOMe/MeOH afforded the corresponding pyridones 12a-c. Formation of the pyrido[2,3-d]pyrimidines 13a-c was accomplished upon treatment of 12a-c with guanidine in methanol. After the hydrolysis of the ester group present in 13a-c, the resulting carboxylic acids 14a-c were treated with diethyl cyanophosphonate in Et3N/DMF and coupled with L-glutamic acid dimethyl ester to give 15a-c. Finally, the basic hydrolysis of 15a-c yielded the desired 4-amino-7-oxo-substituted analogues 16a-c in 20-27% overall yield. Compounds 16a-c were tested in vitro against CCRF-CEM leukemia cells. The results obtained indicated that our 4-amino-7-oxo analogues are completely devoid of any activity, the IC50 being higher than 20 microg/mL for all cases except 14c for which a value of 6.7 microg/mL was obtained. These results seem to indicate that 16a-c are inactive precisely due to the presence of the carbonyl group in position C7, the distinctive feature of our synthetic methodology.