Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30 days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7 days, while human keratinocytes were seeded on the scaffold epidermal side for 10 days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30 days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7 days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7 days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10 days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Hyaluronan metabolism in human keratinocytes and atopic dermatitis skin is driven by a balance of hyaluronan synthases 1 and 3.

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Reconstruction of normal and pathological human epidermis on polycarbonate filter.

This chapter provides methods suitable for the culture of primary human keratinocytes in serum-free culture conditions, starting from very small skin biopsies. It also explains procedures required for reconstruction of a stratified epidermis on polycarbonate filter, starting from keratinocytes cultured in serum-free conditions. Tissues reconstructed according to this method have been proven suitable for characterization of epidermal morphogenesis and for in vitro studies of the epidermal barrier. Utilization of the same method for successful isolation of keratinocytes from a patient suffering from Darier's disease and the reconstruction of a pathological epidermis which displays the same histological features as in vivo are also presented.

Ophiobolin A, a sesterterpenoid fungal phytotoxin, displays higher in vitro growth-inhibitory effects in mammalian than in plant cells and displays in vivo antitumor activity.

Ophiobolin A, a sesterterpenoid produced by plant pathogenic fungi, was purified from the culture extract of Drechslera gigantea and tested for its growth-inhibitory activity in both plant and mammalian cells. Ophiobolin A induced cell death in Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells at concentrations ≥10 µM, with the TBY-2 cells showing typical features of apoptosis-like cell death. At a concentration of 5 µM, ophiobolin A did not affect plant cell viability but prevented cell proliferation. When tested on eight cancer cell lines, concentrations <1 µM of ophiobolin A inhibited growth by 50% after 3 days of culture irrespective of their multidrug resistance (MDR) phenotypes and their resistance levels to pro-apoptotic stimuli. It is, thus, unlikely that ophiobolin A exerts these in vitro growth-inhibitory effects in cancer cells by activating pro-apoptotic processes. Highly proliferative human keratinocytes appeared more sensitive to the growth-inhibitory effects of ophiobolin A than slowly proliferating ones. Ophiobolin A also displayed significant antitumor activity at the level of mouse survival when assayed at 10 mg/kg in the B16F10 mouse melanoma model with lung pseudometastases. Ophiobolin A could, thus, represent a novel scaffold to combat cancer types that display various levels of resistance to pro-apoptotic stimuli and/or various MDR phenotypes.

TMEM45A is essential for hypoxia-induced chemoresistance in breast and liver cancer cells.

BACKGROUND: Hypoxia is a common characteristic of solid tumors associated with reduced response to radio- and chemotherapy, therefore increasing the probability of tumor recurrence. The aim of this study was to identify new mechanisms responsible for hypoxia-induced resistance in breast cancer cells. METHODS: MDA-MB-231 and HepG2 cells were incubated in the presence of taxol or etoposide respectively under normoxia and hypoxia and apoptosis was analysed. A whole transcriptome analysis was performed in order to identify genes whose expression profile was correlated with apoptosis. The effect of gene invalidation using siRNA was studied on drug-induced apoptosis. RESULTS: MDA-MB-231 cells incubated in the presence of taxol were protected from apoptosis and cell death by hypoxia. We demonstrated that TMEM45A expression was associated with taxol resistance. TMEM45A expression was increased both in MDA-MB-231 human breast cancer cells and in HepG2 human hepatoma cells in conditions where protection of cells against apoptosis induced by chemotherapeutic agents was observed, i.e. under hypoxia in the presence of taxol or etoposide. Moreover, this resistance was suppressed by siRNA-mediated silencing of TMEM45A. Kaplan Meier curve showed an association between high TMEM45A expression and poor prognostic in breast cancer patients. Finally, TMEM45 is highly expressed in normal differentiated keratinocytes both in vitro and in vivo, suggesting that this protein is involved in epithelial functions. CONCLUSION: Altogether, our results unravel a new mechanism for taxol and etoposide resistance mediated by TMEM45A. High levels of TMEM45A expression in tumors may be indicative of potential resistance to cancer therapy, making TMEM45A an interesting biomarker for resistance.

Studies of cell signaling in a reconstructed human epidermis exposed to sensitizers: IL-8 synthesis and release depend on EGFR activation.

Models of reconstructed human epidermis (RHE) holding proliferating and fully differentiated cultured keratinocytes allow in vitro investigation of early molecular and cellular epidermal events during the complex response of keratinocytes at the onset of allergic contact dermatitis (ACD) or sensitization. In this study, data collected on RHE exposed to well-characterized sensitizing chemicals, such as dinitrofluorobenzene, oxazolone, cinnamaldehyde and isoeugenol, revealed a transient expression of IL-8 mRNA in association with abundant IL-8 cell release. Investigations of keratinocyte signaling illustrate transient activation by tissue exposure to sensitizing chemicals of the epidermal growth factor receptor (EGFR). This activation of EGFR tyrosine kinase is involved in the expression and release of IL-8. The IL-8 release appears also to be partially dependent on p38 and ERK 1/2 MAPK activation. Moreover, data suggest that heparin-binding EGF-like growth factor (HB-EGF) expression and release induced after exposure of RHE to sensitizing chemicals are also under the control of EGFR tyrosine kinase activity, independently of the IL-8 expression and release. Mechanistic approach of keratinocyte responses in the context of RHE underlying regulation of expression and release of epidermal cytokines and growth factors after topical application of sensitizing chemicals is proposed to identify biomarkers which could then be analysed for in vitro toxicological screening of new or undefined compounds.

Development of a chitosan nanofibrillar scaffold for skin repair and regeneration.

The final goal of the present study was the development of a 3-D chitosan dressing that would shorten the healing time of skin wounds by stimulating migration, invasion, and proliferation of the relevant cutaneous resident cells. Three-dimensional chitosan nanofibrillar scaffolds produced by electrospinning were compared with evaporated films and freeze-dried sponges for their biological properties. The nanofibrillar structure strongly improved cell adhesion and proliferation in vitro. When implanted in mice, the nanofibrillar scaffold was colonized by mesenchymal cells and blood vessels. Accumulation of collagen fibrils was also observed. In contrast, sponges induced a foreign body granuloma. When used as a dressing covering full-thickness skin wounds in mice, chitosan nanofibrils induced a faster regeneration of both the epidermis and dermis compartments. Altogether our data illustrate the critical importance of the nanofibrillar structure of chitosan devices for their full biocompatibility and demonstrate the significant beneficial effect of chitosan as a wound-healing biomaterial.

In vitro anticancer activity, toxicity and structure-activity relationships of phyllostictine A, a natural oxazatricycloalkenone produced by the fungus Phyllosticta cirsii.

The in vitro anticancer activity and toxicity of phyllostictine A, a novel oxazatricycloalkenone recently isolated from a plant-pathogenic fungus (Phyllosticta cirsii) was characterized in six normal and five cancer cell lines. Phyllostictine A displays in vitro growth-inhibitory activity both in normal and cancer cells without actual bioselectivity, while proliferating cells appear significantly more sensitive to phyllostictine A than non-proliferating ones. The main mechanism of action by which phyllostictine displays cytotoxic effects in cancer cells does not seem to relate to a direct activation of apoptosis. In the same manner, phyllostictine A seems not to bind or bond with DNA as part of its mechanism of action. In contrast, phyllostictine A strongly reacts with GSH, which is a bionucleophile. The experimental data from the present study are in favor of a bonding process between GSH and phyllostictine A to form a complex though Michael attack at C=C bond at the acrylamide-like system. Considering the data obtained, two new hemisynthesized phyllostictine A derivatives together with three other natural phyllostictines (B, C and D) were also tested in vitro in five cancer cell lines. Compared to phyllostictine A, the two derivatives displayed a higher, phyllostictines B and D a lower, and phyllostictine C an almost equal, growth-inhibitory activity, respectively. These results led us to propose preliminary conclusions in terms of the structure-activity relationship (SAR) analyses for the anticancer activity of phyllostictine A and its related compounds, at least in vitro.

HB-EGF synthesis and release induced by cholesterol depletion of human epidermal keratinocytes is controlled by extracellular ATP and involves both p38 and ERK1/2 signaling pathways.

The heparin-binding EGF-like growth factor (HB-EGF) is an autocrine/paracrine keratinocyte growth factor, which binds to the epidermal growth factor (EGF) receptor family and plays a critical role during the re-epithelialization of cutaneous wound by stimulating the keratinocytes proliferation and migration. In this study, cellular stressing condition in autocrine cultures of human keratinocytes was induced by cholesterol depletion using methyl-beta-cyclodextrin (MßCD). MßCD treatment induces the expression and the release of HB-EGF. By analysis of the culture media, large amounts of cellular ATP were measured particularly after 1 h of MßCD treatment. To investigate whether ATP contributes to the expression of HB-EGF, the nonhydrolyzable ATP analogue, ATP-γ-S, was used to mimic the extracellular ATP released. We report that keratinocytes stimulated with ATP-γ-S induce HB-EGF expression and activate EGFR and ERK1/2. Using an antagonist of P2 purinergic receptors, we demonstrate that HB-EGF synthesis induced by lipid rafts disruption is dependent on ATP interaction with P2 purinergic receptors. Moreover, our data suggest that both MAPKs p38 and ERK1/2 are involved together or independently in the regulation of HB-EGF gene expression. These findings provide new insight into the signaling pathway by which HB-EGF is expressed after lipid rafts disruption. In summary, after lipid raft disruption, keratinocytes release large amount of extracellular ATP. ATP induces HB-EGF synthesis and release by interacting with the P2 purinergic receptor and through p38 and ERK1/2 signaling in response to a challenging environment. A release of ATP acts as an early stress response in keratinocytes.

Transcriptional profiling after lipid raft disruption in keratinocytes identifies critical mediators of atopic dermatitis pathways.

Lipid rafts are cholesterol-rich cell signaling platforms, and their physiological role can be explored by cholesterol depletion. To characterize transcriptional changes ongoing after lipid raft disruption in epidermal keratinocytes, a cell type that synthesizes its cholesterol in situ, we performed whole-genome expression profiling. Microarray results show that over 3,000 genes are differentially regulated. In particular, IL-8, urokinase-like plasminogen activator receptor, and metalloproteinases are highly upregulated after cholesterol extraction. Quantitative reverse transcriptase PCR validation and protein release measurements demonstrate the physiological relevance of microarray data. Major enriched terms and functions, determined by Ingenuity Pathways Analysis, identify cholesterol biosynthesis as a major function, illustrating the specificity of keratinocyte response toward cholesterol depletion. Moreover, the inflammatory skin disorder atopic dermatitis (AD) is identified as the disease most closely associated with the profile of lipid raft-disrupted keratinocytes. This finding is confirmed in skin of AD patients, in whom transcript levels of major lipid raft target genes are similarly regulated in lesional atopic skin, compared with non-lesional and normal skin. Thus, lipid raft disruption evokes typical features of AD, thereby suggesting that lipid raft organization and signaling could be perturbed in atopic keratinocytes.

Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes.

Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Raft disruption was achieved in normal human keratinocytes and precancerous (HaCaT) or transformed (A431) keratinocytes by cholesterol extraction or inactivation with methyl-beta-cyclodextrin, filipin III, or 5-cholestene-5-beta-ol. Lipid raft disruption did not affect PI3K binding to its main target, the epidermal growth factor receptor, nor its ability to convert phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate but impaired Akt phosphorylation at the regulatory sites Thr(308) and Ser(473). Diminished Akt activity resulted in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid rafts is required for the activity of Akt and cell survival and may serve as a potential pharmacological target in the treatment of epidermal cancers.

p38 MAPK-regulated EGFR internalization takes place in keratinocyte monolayer during stress conditions.

The epidermis is the outermost protection of the organism. As so, defence program has to be initiated in stress situation in order to protect keratinocytes. The EGF receptor (EGFR) controls cell proliferation and migration in keratinocytes, being a major regulator of keratinocyte homeostasis within the epidermis. The EGFR is known to be internalized without addition of ligand under the control of p38 MAPK during stress conditions in HeLa cells, but also following lipid rafts disruption in keratinocytes. This could represent an alternative internalization process that removes the EGFR from cell surface. Here, we investigated whether other stress conditions such as scratch wounding keratinocyte monolayer or incubation with a sensitizer chemical (i.e. DNFB), could also induce this peculiar mechanism of EGFR internalization. Our results show that both stressing conditions induce p38 MAPK activation concomitantly with EGFR internalization, independently of ligand binding to the EGFR. Inhibition of p38 MAPK activity during scratch wound blocks EGFR internalization at the margin of the wound while cell migration is impeded. Our results show thus that the p38 MAPK-dependent EGFR internalization is a process shared by keratinocytes when submitted to challenging conditions.

Repeated exposures to UVB induce differentiation rather than senescence of human keratinocytes lacking p16(INK-4A).

Skin cancers and extrinsic aging are delayed consequences of cumulative UV radiation insults. Exposure of human keratinocytes to UVB has been previously shown to trigger premature senescence. In order to explore the involvement of the cyclin-dependent kinase inhibitor p16(INK-4a) in UVB-induced premature senescence, we developed an original model of repeated sublethal exposures of human keratinocytes deficient in p16(INK-4a). We did not observe any significant increase of senescence-associated beta-galactosidase activity positive cells following UVB exposure in this cell line in contrast to primary keratinocytes, suggesting a role for p16(INK-4a) in UVB-induced senescence. However, we detected sustained DNA damage, prolonged cell cycle arrest, and induction of markers of epidermal differentiation like involucrin and filaggrin as consequences of the repeated exposures. Keratinocytes exposed to the same dose of UVB in a single exposure died. Furthermore, the abundance of the keratins 6, 16 and 17 was increased in keratinocytes exposed repeatedly to UVB suggesting an alternative differentiation. This model allows the induction of a state of differentiation observed in vivo with differentiation uncoupled from premature senescence.

Members of the EGF receptor family in normal and pathological epidermis.

Epidermal growth factor (EGF) and analogs bind to transmembrane receptors that exhibit tyrosine kinase activity in their cytoplasmic domain and which belong to the EGF receptor family. These growth factors and relevant receptors were named after initial identification of the functions of their founding members in the epidermis. However, since the EGF receptor was recognized as an oncogene, it has been mainly analyzed in cancer; the members of the receptor's family were also found and characterized initially in tumors and cancer cells. The present article reviews mainly the expression and function of EGF receptor family members in normal epidermis, together with the expression and function of their respective ligands, and we extend the review to potential involvement of these systems in epidermal disease.

Development of a procedure to simultaneously isolate RNA, DNA, and proteins for [corrected] characterizing cells invading or cultured on chitosan scaffolds.

For many years, chitosan and its derivatives have been considered to be promising biomaterials for tissue engineering and repair. However, information regarding their biological effect on cell phenotype is usually limited to evaluation of cell proliferation and survival, overlooking proteomic and transcriptomic analysis. This is largely related to the lack of efficient and quantitative procedures for protein and nucleic acid purification from cells cultured on, or inside, chitosan scaffold. Here we describe an ultracentrifugation procedure enabling the simultaneous and quantitative recovery of high quality RNA, DNA and proteins from cells growing in close contact of biomaterial matrices containing chitosan.

Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation.

The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests the processing and localization of EGFR following lipid raft disruption. Here, we report the dimerization and the slow internalization of the receptor accompanied by the delayed phosphorylation of tyrosine 1068 and its degradation by the proteasome. We also demonstrate the involvement of p38 MAPK during the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption.

The inhibition of the expression of the small Rho GTPase Rac1 induces differentiation with no effect on cell proliferation in growing human adult keratinocytes.

Rac1 is a Rho subfamily small GTPase which is highly expressed in epidermal keratinocytes. In mice the significance of Rac1 for the maintenance of the epidermis has been controversial. In keratinocytes from human origin, the role of Rac1 in the control of growth/differentiation is still obscure. In this study we used RNA interference to induce specific inhibition of Rac1 expression in cultured human keratinocytes and analyzed the consequences on proliferation and differentiation. We found that the autocrine proliferation of keratinocytes is unaltered by Rac1 silencing. However, the suppression of Rac1 induced premature differentiation as revealed by the expression of markers (keratin 10, involucrin), but the involved mechanism is independent of the activity of p38 mitogen-activated protein kinase. Rather, we found that the effects of Rac1 silencing on keratinocytes differentiation are concomitant with negative regulation of the Ser62/Thr58 phosphorylation on the transcription factor c-myc, a mechanism known to control post-translational stability of the c-myc protein. Thus, in growing human keratinocytes, Rac1 could impede the expression of premature differentiation markers, probably by exerting positive control on c-myc activity and its binding to specific promoters.

Effects of the CDK-inhibitor CYC202 on p38 MAPK, ERK1/2 and c-Myc activities in papillomavirus type 16 E6- and E7-transformed human keratinocytes.

In the present study, we have investigated the effect of the chemical CDK-inhibitor CYC202 on E6 and E7-transformed keratinocytes, in which the function of the cellular cell cycle inhibitor p21Cip1 is abrogated by the viral genes. The cyto-toxicity and the inhibition of the cell growth were analysed by MTT assay and analysis of DNA synthesis respectively. The effect on some signalling molecules was tested by Western blot analysis. CYC202 effectively inhibited the proliferation of E6 and E7 keratinocytes in a dose-dependent manner. Treatment with CYC202 strongly increased the activity of p38 MAP kinase. Furthermore, it inhibited ERK1/2 at the highest concentration used and had no effect on the activity of JNK1/2. CYC202 also increased the phosphorylation of HSP27 and decreased the phosphorylation and DNA-binding activity of the transcriptional regulator c-Myc, in correlation with the corresponding upstream kinases p38 MAPK and ERK1/2. Our results provide additional data for the anti-proliferative actions and potency of the chemical CDK-inhibitor CYC202.

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc.

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity.