A perfusable vascularized full-thickness skin model for potential topical and systemic applications.

Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance ofin vitroapplications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing (a) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, (b) three perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, (c) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

Corrective effects of hepatotoxicity by hepatic Dyrk1a gene delivery in mice with intermediate hyperhomocysteinemia.

Hyperhomocysteinemia results from hepatic metabolism dysfunction and is characterized by a high plasma homocysteine level, which is also an independent risk factor for cardiovascular disease. Elevated levels of homocysteine in plasma lead to hepatic lesions and abnormal lipid metabolism. Therefore, lowering homocysteine levels might offer therapeutic benefits. Recently, we were able to lower plasma homocysteine levels in mice with moderate hyperhomocysteinemia using an adenoviral construct designed to restrict the expression of DYRK1A, a serine/threonine kinase involved in methionine metabolism (and therefore homocysteine production), to hepatocytes. Here, we aimed to extend our previous findings by analyzing the effect of hepatocyte-specific Dyrk1a gene transfer on intermediate hyperhomocysteinemia and its associated hepatic toxicity and liver dysfunction. Commensurate with decreased plasma homocysteine and alanine aminotransferase levels, targeted hepatic expression of DYRK1A in mice with intermediate hyperhomocysteinemia resulted in elevated plasma paraoxonase-1 and lecithin:cholesterol acyltransferase activities and apolipoprotein A-I levels. It also rescued hepatic apolipoprotein E, J, and D levels. Further, Akt/GSK3/cyclin D1 signaling pathways in the liver of treated mice were altered, which may help prevent homocysteine-induced cell cycle dysfunction. DYRK1A gene therapy could be useful in the treatment of hyperhomocysteinemia in populations, such as end-stage renal disease patients, who are unresponsive to B-complex vitamin therapy.

One-carbon cycle alterations induced by Dyrk1a dosage.

Hyperhomocysteinemia due to cystathionine beta synthase deficiency confers diverse clinical manifestations. It is characterized by elevated plasma homocysteine levels, a common amino acid metabolized by remethylation to methionine or transsulfuration to cysteine. We recently found a relationship between hepatic Dyrk1A protein expression, a serine/threonine kinase involved in signal transduction in biological processes, hepatic S-adenosylhomocysteine activity, and plasma homocysteine levels. We aimed to study whether there is also a relationship between Dyrk1a and cystathionine beta synthase activity. We used different murine models carrying altered gene coy numbers for Dyrk1a, and found a decreased cystathionine beta synthase activity in the liver of mice under-expressing Dyrk1a, and an increased in liver of mice over-expressing Dyrk1a. For each model, a positive correlation was found between cystathionine beta synthase activity and Dyrk1a protein expression in the liver of mice, which was confirmed in a non-modified genetic context. The positive correlation found between liver Dyrk1a protein expression and CBS activity in modified and non-modified genetic context strengthens the role of this kinase in one carbon metabolism.