A Dialogue between the Hypoxia-Inducible Factor and the Tumor Microenvironment

The hypoxia-inducible factor is the key protein responsible for the cellular adaptation to low oxygen tension. This transcription factor becomes activated as a result of a drop in the partial pressure of oxygen, to hypoxic levels below 5% oxygen, and targets a panel of genes involved in maintenance of oxygen homeostasis. Hypoxia is a common characteristic of the microenvironment of solid tumors and, through activation of the hypoxia-inducible factor, is at the center of the growth dynamics of tumor cells. Not only does the microenvironment impact on the hypoxia-inducible factor but this factor impacts on microenvironmental features, such as pH, nutrient availability, metabolism and the extracellular matrix. In this review we discuss the influence the tumor environment has on the hypoxia-inducible factor and outline the role of this factor as a modulator of the microenvironment and as a powerful actor in tumor remodeling. From a fundamental research point of view the hypoxia-inducible factor is at the center of a signaling pathway that must be deciphered to fully understand the dynamics of the tumor microenvironment. From a translational and pharmacological research point of view the hypoxia-inducible factor and its induced downstream gene products may provide information on patient prognosis and offer promising targets that open perspectives for novel “anti-microenvironment” directed therapies

[Skin substitutes reconstructed in the laboratory: application in burn treatment]

International audienceThe development of skin substitutes started 25 years ago with the cultivation of keratinocytes to replace the epidermis of extensively burned patients. It is now possible to reproduce in vitro the two layers of skin, epidermis and dermis. Cultured epidermises are now usually used in burn centers dealing with the more severe patients. They are provided by hospital or private laboratories. Dermal substrates are some collagen matrices, which act in vivo as a guide for the reconstruction of a neodermis. Living dermis include living fibroblasts. Different models are now available for clinical use. Living skin equivalent is obtained by coculturing fibroblast and keratinocytes on a collagen support. Clinical essays are going on for chronic wounds. We present the different skin equivalent models and their clinical applications.The development of skin substitutes started 25 years ago with the cultivation of keratinocytes to replace the epidermis of extensively burned patients. It is now possible to reproduce in vitro the two layers of skin, epidermis and dermis. Cultured epidermises are now usually used in burn centers dealing with the more severe patients. They are provided by hospital or private laboratories. Dermal substrates are some collagen matrices, which act in vivo as a guide for the reconstruction of a neodermis. Living dermis include living fibroblasts. Different models are now available for clinical use. Living skin equivalent is obtained by coculturing fibroblast and keratinocytes on a collagen support. Clinical essays are going on for chronic wounds. We present the different skin equivalent models and their clinical applications

Epidermal and dermal characteristics in skin equivalent after systemic and topical application of skin care ingredients.

International audienceEffects of active ingredients from topical and systemic skincare products on structure and organization of epidermis, dermal-epidermal junction (DEJ), and dermis were examined using an in vitro reconstructed skin equivalent (SE). Imedeen Time Perfection (ITP) ingredients (a mixture of BioMarine Complex, grape seed extract, tomato extract, vitamin C) were supplemented systemically into culture medium. Kinetin, an active ingredient from Imedeen Expression Line Control Serum, was applied topically. Both treatments were tested separately or combined. In epidermis, all treatments stimulated keratinocyte proliferation, showing a significant increase of Ki67-positive keratinocytes (P < 0.05). Kinetin showed a twofold increase of Ki67-positive cells, ITP resulted in a fivefold, and ITP+kinetin showed a nine-fold increase. Differentiation of keratinocytes was influenced only by kinetin since filaggrin was found only in kinetin and kinetin+ITP samples. At the DEJ, laminin 5 was slightly increased by all treatments. In dermis, only ITP increased the amount of collagen type I. Both kinetin and ITP stimulated formation of fibrillin-1 and elastin deposition. The effect of kinetin was seen in upper dermis. It stimulated not only the amount of deposited fibrillin-1 and elastin fibers but also their organization perpendicularly to the DEJ. ITP stimulated formation of fibrillin-1 in deeper dermis. In summary, the combination of topical treatment with kinetin and systemic treatment with ITP had complementary beneficial effects in the formation and development of epidermis and dermis.Effects of active ingredients from topical and systemic skincare products on structure and organization of epidermis, dermal-epidermal junction (DEJ), and dermis were examined using an in vitro reconstructed skin equivalent (SE). Imedeen Time Perfection (ITP) ingredients (a mixture of BioMarine Complex, grape seed extract, tomato extract, vitamin C) were supplemented systemically into culture medium. Kinetin, an active ingredient from Imedeen Expression Line Control Serum, was applied topically. Both treatments were tested separately or combined. In epidermis, all treatments stimulated keratinocyte proliferation, showing a significant increase of Ki67-positive keratinocytes (P < 0.05). Kinetin showed a twofold increase of Ki67-positive cells, ITP resulted in a fivefold, and ITP+kinetin showed a nine-fold increase. Differentiation of keratinocytes was influenced only by kinetin since filaggrin was found only in kinetin and kinetin+ITP samples. At the DEJ, laminin 5 was slightly increased by all treatments. In dermis, only ITP increased the amount of collagen type I. Both kinetin and ITP stimulated formation of fibrillin-1 and elastin deposition. The effect of kinetin was seen in upper dermis. It stimulated not only the amount of deposited fibrillin-1 and elastin fibers but also their organization perpendicularly to the DEJ. ITP stimulated formation of fibrillin-1 in deeper dermis. In summary, the combination of topical treatment with kinetin and systemic treatment with ITP had complementary beneficial effects in the formation and development of epidermis and dermis

Optimization and characterization of an engineered human skin equivalent.

International audienceSkin equivalents (SEs) have been designed to meet both basic and applied research needs. The successful application of tissue-engineered SEs requires that the reconstituted tissues be endowed with the correct organization and function. A large body of experimental evidence now supports the notion that the inducing effects of mesenchymal tissue on epithelial cell morphogenesis are mediated, at least in part, by extracellular matrix components in addition to cell-cell interactions. A coculture model including both fibroblasts and keratinocytes was used to study the effects of progressive serum reduction on epidermal differentiation, quality of dermal and dermal-epidermal junctions, and expression of extracellular matrix proteins. The cells were successively added to a dermal substrate composed of collagen, glycosaminoglycans, and chitosan. The main aim of this study was to optimize this model for pharmacotoxicological trials. Control skin equivalents were cultured with medium containing 10% serum throughout the production process. Serum content was reduced to 1 and 0% at the air-liquid interface and compared with control skin equivalents. First, we demonstrated that serum deprivation at the air-liquid interface improves keratinocyte terminal differentiation. Second, we showed that, in the absence of serum, the specific characteristics of the SE are maintained, including epidermal and dermal ultrastructure, the expression of major dermal extracellular matrix components (human collagen types I, III, and V, fibronectin, elastin, and fibrillin 1), and the dermal-epidermal junction (laminin, human type IV collagen, alpha6 integrin). Furthermore, our results indicate that coculture models using keratinocytes and fibroblasts have both morphological and functional properties required for biologically useful tissues.Skin equivalents (SEs) have been designed to meet both basic and applied research needs. The successful application of tissue-engineered SEs requires that the reconstituted tissues be endowed with the correct organization and function. A large body of experimental evidence now supports the notion that the inducing effects of mesenchymal tissue on epithelial cell morphogenesis are mediated, at least in part, by extracellular matrix components in addition to cell-cell interactions. A coculture model including both fibroblasts and keratinocytes was used to study the effects of progressive serum reduction on epidermal differentiation, quality of dermal and dermal-epidermal junctions, and expression of extracellular matrix proteins. The cells were successively added to a dermal substrate composed of collagen, glycosaminoglycans, and chitosan. The main aim of this study was to optimize this model for pharmacotoxicological trials. Control skin equivalents were cultured with medium containing 10% serum throughout the production process. Serum content was reduced to 1 and 0% at the air-liquid interface and compared with control skin equivalents. First, we demonstrated that serum deprivation at the air-liquid interface improves keratinocyte terminal differentiation. Second, we showed that, in the absence of serum, the specific characteristics of the SE are maintained, including epidermal and dermal ultrastructure, the expression of major dermal extracellular matrix components (human collagen types I, III, and V, fibronectin, elastin, and fibrillin 1), and the dermal-epidermal junction (laminin, human type IV collagen, alpha6 integrin). Furthermore, our results indicate that coculture models using keratinocytes and fibroblasts have both morphological and functional properties required for biologically useful tissues

Supplementation with a complex of active nutrients improved dermal and epidermal characteristics in skin equivalents generated from fibroblasts from young or aged donors.

International audienceCultured skin equivalent (SE, Mimeskin) was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan dermal substrate. In order to examine donor age effect, fibroblasts from 19- (young) or 49- (aged) year-old females were used. Culture medium was supplemented with nutrients complex containing soy extract, tomato extract, grape seed extract, white tea extract, sodium ascorbate, tocopherol acetate, zinc gluconate and BioMarine complex. Epidermal and dermal structure and composition were examined after 42 and 60 days of culture. In untreated samples, SE generated from young fibroblasts was superior to SE from aged fibroblasts in all characteristics. Those include number and regularity of keratinocyte layers, number of keratinocytes expressing proliferation marker Ki67, content of collagen type I, fibrillin-1, elastin, and SE lifespan. Effects of nutritional supplementation were observed in SE from both young and aged fibroblasts, however, those effects were more pronounced in SE from aged fibroblasts. In epidermis, the treatment increased number of keratinocyte layers and delayed epidermal senescence. The number of cells expressing Ki67 was nine folds higher than those of controls, and was similar to that of young cell SE. In dermis, the treatment increased mRNA synthesis of collagen I, fibrillin-1 and elastin. In conclusion, skin cell donor age had major important effect on formation of reconstructed SE. Imperfections in epidermal and dermal structure and composition as well as life span in SE from aged cells can be improved by supplementation with active nutrients.Cultured skin equivalent (SE, Mimeskin) was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan dermal substrate. In order to examine donor age effect, fibroblasts from 19- (young) or 49- (aged) year-old females were used. Culture medium was supplemented with nutrients complex containing soy extract, tomato extract, grape seed extract, white tea extract, sodium ascorbate, tocopherol acetate, zinc gluconate and BioMarine complex. Epidermal and dermal structure and composition were examined after 42 and 60 days of culture. In untreated samples, SE generated from young fibroblasts was superior to SE from aged fibroblasts in all characteristics. Those include number and regularity of keratinocyte layers, number of keratinocytes expressing proliferation marker Ki67, content of collagen type I, fibrillin-1, elastin, and SE lifespan. Effects of nutritional supplementation were observed in SE from both young and aged fibroblasts, however, those effects were more pronounced in SE from aged fibroblasts. In epidermis, the treatment increased number of keratinocyte layers and delayed epidermal senescence. The number of cells expressing Ki67 was nine folds higher than those of controls, and was similar to that of young cell SE. In dermis, the treatment increased mRNA synthesis of collagen I, fibrillin-1 and elastin. In conclusion, skin cell donor age had major important effect on formation of reconstructed SE. Imperfections in epidermal and dermal structure and composition as well as life span in SE from aged cells can be improved by supplementation with active nutrients

The lysyl oxidase LOX is absent in basal and squamous cell carcinomas and its knockdown induces an invading phenotype in a skin equivalent model.

International audienceLysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity

Lysyl oxidase-like and lysyl oxidase are present in the dermis and epidermis of a skin equivalent and in human skin and are associated to elastic fibers.

International audienceElastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates

Evolutive skin reconstructions: from the dermal collagen-glycosaminoglycan-chitosane substrate to an immunocompetent reconstructed skin.

International audienceThe development of human skin models that have the same properties as genuine human skin is of particular significance. Very promising skin models are the three-dimensional artificial skin constructs, which, similar to genuine skin, consist of an epidermis of differentiated keratinocytes and a dermis. A skin equivalent based on a collagen-glycosaminoglycan-chitosan dermal substrate has been developed to meet the growing demand in tissue engineered skin equivalents. We used this model to investigate whether CD34-generated Langerhans/dendritic cell precursors could be integrated into this skin equivalent model and pursue their differentiation without addition of cytokine and growth factor. To address the issue of dendritic cell (DC) differentiation, an endothelialized skin equivalent coculture model was used to study the behaviour of haematopoietic progenitor cells (HPC) in epidermal and dermal environments. CD34(+) HPC were cultured for 6 days with GM-CSF, TGFbeta1 and TNFalpha and seeded in the endothelialized skin equivalent at different time points to favour dermal or epidermal integration. This integration (after keratinocyte seeding, only and in absence of exogenous GM-CSF, TNFalpha, TGFbeta1) gave rise both cutaneous DC, i.e. epidermal Langerhans cells (CD1a(+), HLA-DR(+)) and dermal DC (DC-SIGN(+), HLA-DR(+)) while endothelial cells are sufficiently activated to acquire HLA-DR expression. For the first time, the presence of a living dermal equivalent could provide a more complex environment integrating vascular components to study the differentiation of interstitial DC in a dermis equivalent. Such sophisticated skin equivalent may clarify some intriguing aspects of the numerous regulatory mechanisms controlling skin homeostasis.The development of human skin models that have the same properties as genuine human skin is of particular significance. Very promising skin models are the three-dimensional artificial skin constructs, which, similar to genuine skin, consist of an epidermis of differentiated keratinocytes and a dermis. A skin equivalent based on a collagen-glycosaminoglycan-chitosan dermal substrate has been developed to meet the growing demand in tissue engineered skin equivalents. We used this model to investigate whether CD34-generated Langerhans/dendritic cell precursors could be integrated into this skin equivalent model and pursue their differentiation without addition of cytokine and growth factor. To address the issue of dendritic cell (DC) differentiation, an endothelialized skin equivalent coculture model was used to study the behaviour of haematopoietic progenitor cells (HPC) in epidermal and dermal environments. CD34(+) HPC were cultured for 6 days with GM-CSF, TGFbeta1 and TNFalpha and seeded in the endothelialized skin equivalent at different time points to favour dermal or epidermal integration. This integration (after keratinocyte seeding, only and in absence of exogenous GM-CSF, TNFalpha, TGFbeta1) gave rise both cutaneous DC, i.e. epidermal Langerhans cells (CD1a(+), HLA-DR(+)) and dermal DC (DC-SIGN(+), HLA-DR(+)) while endothelial cells are sufficiently activated to acquire HLA-DR expression. For the first time, the presence of a living dermal equivalent could provide a more complex environment integrating vascular components to study the differentiation of interstitial DC in a dermis equivalent. Such sophisticated skin equivalent may clarify some intriguing aspects of the numerous regulatory mechanisms controlling skin homeostasis

The Lysyl Oxidase Propeptide Interacts with the Receptor-Type Protein Tyrosine Phosphatase Kappa and Inhibits β-Catenin Transcriptional Activity in Lung Cancer Cells▿

The propeptide region of the lysyl oxidase proenzyme (LOX-PP) has been shown to inhibit Ras signaling in NIH 3T3 and lung cancer cells with activated RAS, but its mechanism of action is poorly understood. Here, a yeast two-hybrid assay of LOX-PP-interacting proteins identified a clone encoding the intracellular phosphatase domains of receptor-type protein tyrosine phosphatase kappa (RPTP-κ), and the interaction of the two proteins in mammalian cells was confirmed. RPTP-κ is proteolytically processed to isoforms that have opposing effects on β-catenin activity. The RPTP-κ transmembrane P subunit interacts with and sequesters β-catenin at the cell membrane, where it can associate with E-cadherin and promote intercellular interactions. At high cell density, further processing of the P subunit yields a phosphatase intracellular portion (PIC) subunit, which chaperones β-catenin to the nucleus, where it can function to activate transcription. Lung cancer cells were found to contain higher PIC levels than untransformed lung epithelial cells. In H1299 lung cancer cells, ectopic LOX-PP expression reduced the nuclear levels of PIC by increasing its turnover in the lysosome, thereby decreasing the nuclear levels and transcriptional activity of β-catenin while increasing β-catenin membrane localization. Thus, LOX-PP is shown to negatively regulate pro-oncogenic β-catenin signaling in lung cancer cells