Jérôme Aubert

Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation

The multilineage differentiation capacity of mouse embryonic stem (ES) cells offers a potential testing platform for gene products that mediate mammalian lineage determination and cellular specialization. Identification of such differentiation regulators is crucial to harnessing ES cells for pharmaceutical discovery and cell therapy. Here we describe the use of episomal expression technology for functional evaluation of cDNA clones during ES-cell differentiation in vitro. Several candidate cDNAs identified by subtractive cloning and expression profiling were introduced into ES cells in episomal expression constructs. Subsequent differentiation revealed that the Wnt antagonist Sfrp2 stimulates production of neural progenitors. The significance of this observation was substantiated by forced expression of Wnt-1 and treatment with lithium chloride, both of which inhibit neural differentiation. These findings reveal the importance of Wnt signaling in regulating ES-cell lineage diversification. More generally, this study establishes a path for rapid and direct validation of candidate genes in ES cells.

Leukemia Inhibitory Factor and Its Receptor Promote Adipocyte Differentiation via the Mitogen-activated Protein Kinase Cascade

Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived fromlifr −/− ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor γ2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPβ and C/EBPδ is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPβ and C/EBPδ genes via the mitogen-activated protein kinase cascade.

Neurovascular and Neuroimmune Aspects in the Pathophysiology of Rosacea

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea.

Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

Transcriptional Profiling Shows Altered Expression of Wnt Pathway– and Lipid Metabolism–Related Genes as Well as Melanogenesis-Related Genes in Melasma

Melasma is a commonly acquired hyperpigmentary disorder of the face, but its pathogenesis is poorly understood and its treatment remains challenging. We conducted a comparative histological study on lesional and perilesional normal skin to clarify the histological nature of melasma. Significantly, higher amounts of melanin and of melanogenesis-associated proteins were observed in the epidermis of lesional skin, and the mRNA level of tyrosinase-related protein 1 was higher in lesional skin, indicating regulation at the mRNA level. However, melanocyte numbers were comparable between lesional and perilesional skin. A transcriptomic study was undertaken to identify genes involved in the pathology of melasma. A total of 279 genes were found to be differentially expressed in lesional and perilesional skin. As was expected, the mRNA levels of a number of known melanogenesis-associated genes, such as tyrosinase, were found to be elevated in lesional skin. Bioinformatics analysis revealed that the most lipid metabolism-associated genes were downregulated in lesional skin, and this finding was supported by an impaired barrier function in melasma. Interestingly, a subset of Wnt signaling modulators, including Wnt inhibitory factor 1, secreted frizzled-related protein 2, and Wnt5a, were also found to be upregulated in lesional skin. Immunohistochemistry confirmed the higher expression of these factors in melasma lesions.

Molecular and Morphological Characterization of Inflammatory Infiltrate in Rosacea Reveals Activation of Th1/Th17 Pathways

Rosacea is a common chronic inflammatory skin disease of unknown etiology. Our knowledge about an involvement of the adaptive immune system is very limited. We performed detailed transcriptome analysis, quantitative real-time reverse-transcriptase-PCR, and quantitative immunohistochemistry on facial biopsies of rosacea patients, classified according to their clinical subtype. As controls, we used samples from patients with facial lupus erythematosus and healthy controls. Our study shows significant activation of the immune system in all subtypes of rosacea, characterizing erythematotelangiectatic rosacea (ETR) already as a disease with significant influx of proinflammatory cells. The T-cell response is dominated by Th1/Th17-polarized immune cells, as demonstrated by significant upregulation of IFN-γ or IL-17, for example. Chemokine expression patterns support a Th1/Th17 polarization profile of the T-cell response. Macrophages and mast cells are increased in all three subtypes of rosacea, whereas neutrophils reach a maximum in papulopustular rosacea. Our studies also provide evidence for the activation of plasma cells with significant antibody production already in ETR, followed by a crescendo pattern toward phymatous rosacea. In sum, Th1/Th17 polarized inflammation and macrophage infiltration are an underestimated hallmark in all subtypes of rosacea. Therapies directly targeting the Th1/Th17 pathway are promising candidates in the future treatment of this skin disease.

EVIDENCE FOR A NOVEL REGULATORY PATHWAY ACTIVATED BY (CARBA)PROSTACYCLIN IN PREADIPOSE AND ADIPOSE CELLS

Prostacyclin, one of the major prostanoids generated in adipose tissue, has been previously described as an autocrine/ paracrine adipogenic effector, acting, in preadipose cells, by means of cAMP and free Ca2+ as cell surface receptor‐mediated messengers. The present study presents evidence for the first time that its stable analogue, carbaprostacyclin, is unique among prostanoids in regulating the expression of two differentiation‐dependent genes in preadipose and adipose cells in a way distinct from that elicited by its cell surface receptor. This regulation is likely mediated by some member(s) of the peroxisome proliferator‐activated receptor family and suggests that prostacyclin behaves as an intracrine effector of adipose cell differentiation.

The Global Error Assessment (GEA) model for the selection of differentially expressed genes in microarray data

MOTIVATION Microarray technology has become a powerful research tool in many fields of study; however, the cost of microarrays often results in the use of a low number of replicates (k). Under circumstances where k is low, it becomes difficult to perform standard statistical tests to extract the most biologically significant experimental results. Other more advanced statistical tests have been developed; however, their use and interpretation often remain difficult to implement in routine biological research. The present work outlines a method that achieves sufficient statistical power for selecting differentially expressed genes under conditions of low k, while remaining as an intuitive and computationally efficient procedure. RESULTS The present study describes a Global Error Assessment (GEA) methodology to select differentially expressed genes in microarray datasets, and was developed using an in vitro experiment that compared control and interferon-gamma treated skin cells. In this experiment, up to nine replicates were used to confidently estimate error, thereby enabling methods of different statistical power to be compared. Gene expression results of a similar absolute expression are binned, so as to enable a highly accurate local estimate of the mean squared error within conditions. The model then relates variability of gene expression in each bin to absolute expression levels and uses this in a test derived from the classical ANOVA. The GEA selection method is compared with both the classical and permutational ANOVA tests, and demonstrates an increased stability, robustness and confidence in gene selection. A subset of the selected genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). All these results suggest that GEA methodology is (i) suitable for selection of differentially expressed genes in microarray data, (ii) intuitive and computationally efficient and (iii) especially advantageous under conditions of low k. AVAILABILITY The GEA code for R software is freely available upon request to authors.

Vasoconstriction and anti-inflammatory properties of the selective α-adrenergic receptor agonist brimonidine

BACKGROUND The facial erythema of rosacea is recognized as the most prevalent and most difficult manifestation of rosacea to treat. A recent approach in patients with rosacea has been to reduce this erythema through vasoconstriction of cutaneous blood vessels by selectively targeting α2-adrenergic receptors with brimonidine. OBJECTIVE To further investigate the pharmacodynamic profile of brimonidine, its vasoconstrictive effects and its anti-inflammatory properties. METHODS The potency for the α1A, α1B, α2A, α2B and α2C receptors of brimonidine was measured, as well as performing a large target profiling study in order to determine the target selectivity profile of brimonidine. The vasoconstrictive effects of brimonidine were measured using ex vivo wire myography and human skin biopsy neuroinflammation models. The anti-inflammatory properties of brimonidine were measured using two in vivo mice ear inflammation models. RESULTS Brimonidine was found to be highly selective for the α2A adrenoreceptor (EC50 0.45nM) over the other α-adrenoreceptors. Additionally, the large target profiling study demonstrated the high selectivity of brimonidine with minimal off-target effects. The ex vivo wire myography model showed that brimonidine is a potent vasoconstrictor of human subcutaneous vessels with a diameter of less than 200μm (EC50 0.4nM). The ex vivo human skin biopsy neuroinflammation model demonstrated that brimonidine completely inhibited vasodilation induced by capsaicin. Both in vivo mouse ear inflammation models highlighted that brimonidine inhibited ear edema (up to 76%) when compared to vehicle. CONCLUSION The selectivity, vasoconstrictive and anti-inflammatory properties of brimonidine that have been described in these studies are in agreement with the benefits observed with this compound in the treatment of facial erythema in rosacea.

Characterization of Human Knee and Chin Adipose-Derived Stromal Cells

Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin) and limb (knee) fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs) from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies.