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The effect of proteolytic enzymes on hair follicles of transgenic mice expressing the lac Z‐protein in cells of the bulge region
Abstract
To study the effects of proteolytic enzymes on mice hair follicles, particularly on cells of the bulge area regarded as follicle stem cells.
Previous application by iontophoresis of proteolytic enzymes on guinea pig skin resulted in degenerative effects on hair follicles and the hypothesis was proposed that some of the affected cells could be stem cells.
To mark putative stem cells transgenic mice were produced carrying the lac-Z gene fused to the Upstream Regulatory Region (URR) of Human Papilloma Virus 11 (HPV11), as they express this gene specifically in the cells of the bulge area. Chymotrypsin and papain were applied on skin by iontophoresis, trypsin in the form of liposomes.
Enzyme application, both by electrophoresis and as liposomes, led to intense degenerative effects of the hair follicle, such as detachment of the inner root sheath, cystic dilation of the hair shaft and presence of epithelial cells within the lumen. Some of these cells represent hair follicle stem cells expressing beta-galactosidase (beta-gal), having been detached from the bulge area as a result of enzyme treatment, implying impairment of their function.
- ... Proteolytic enzymes applied by iontophoresis to skin of experimental animals have shown depilatory effects (Protopapa et al., 1999). As the depilatory action is long-lasting, the hypothesis had been put forward that hair follicle stem cells could be among the cells affected by the proteolytic enzyme. ...Cited By (since 1996):1, Export Date: 24 February 2014, Source: Scopus, References: Aubin, G., Experimentação biológica dos produtos cosméticos (1994) Manual de Cosmetologia Dermatológica, pp. 233-243. , PRUNIÉRAS, M. São Paulo: Organização Andrei;
- ... Proteolytic enzymes applied by iontophoresis to skin of experimental animals have shown depilatory effects (Protopapa et al., 1999). As the depilatory action is long-lasting, the hypothesis had been put forward that hair follicle stem cells could be among the cells affected by the proteolytic enzyme. ...Histological alterations in the skin and hair follicle of mice were evaluated as a result of the application of gel and cream formulas containing papain as a harmless treatment for hirsutism. Papain is a proteolytic enzyme and it has been used in pharmaceutical, cosmetic and nutrition areas. The purpose of this study was to evaluate the efficacy of a depilatory product, through histological analysis using light microscopy. Gel and cream formulas containing papain were developed and daily applied on the back of two groups of mice for 31 days. The depilatory effect of the gel formula applied on the first group was less evident. The second group treated with the cream formula presented an intensive depilatory effect; the morphometrical analysis showed dilation of about 55% of the hair follicle lumen and an increase of the thickness of epidermis. Papain cream had a significantly higher depilatory effect than the papain gel.
- Although the loss of scalp hair is distressing and many medical treatments focus on its restoration, the removal of body hair has been adopted since ancient times. Beauty standards, which r eflect the culture of each society, have been presenting the depilated body as absolutely desirable. Through the ages various methods of hair removal have been used depending on the requirements of the individuals. In recent years, Laser and Intense Pulse Light devices have been considered as the most promising solution for excess hair growth, without excluding the efficacy of other methods to induce satisfactory epilatory results. The enzyme-based hair removal method has received little recognition even though experimental and clinical data support its efficacy to provide long term or even permanent epilation. The present review presents these data and examines the likelihood of considering the aforementioned method as ideal.
- The preparation of lecithin organogels and their possible application as drug carriers was examined. Organogels have been prepared from solutions of soyabeen lecithin in the organic solvents isooctane, isopropyl palmitate and isopropyl myristate, by addition of a critical amount of water. The organanogels are transparent, with a high viscosity reaching 1000 Pas. Their water content varies from 0.15 to 1.15% v/v and depends on the nature of the organic solvent and the lecithin concentration, as shown by the phase diagram study. The capacity of the organogels to incorporate molecules with pharmacological action, such as xanthines, can be studied spectroscopically.
- Transdermal drug delivery has been intensively studied over the last two decades because of the many advantages offered by this route of administration. However, the number of drugs used in transdermal drug delivery systems has been somewhat limited, in part resulting from the formidable barrier to drug permeation presented by the upper layer of the skin, the stratum corneum. In order to overcome this, different strategies have been implemented to render the skin more permeable to drugs. These strategies include both chemical and physical approaches. In this review, we outline the enhancing activities and mechanisms of action of some of the more extensively studied chemical penetration enhancers (oxazolidinones, propylene glycol, and epidermal enzymes). In addition, we discuss novel physical strategies, such as the use of microneedles or electroporation.
- Cultured hair follicles lacking sebaceous glands do not appear to degrade the inner root sheath (IRS), suggesting that the gland may be involved in this process. To examine this supposition in cultured hair follicles. Pilosebaceous units were isolated from hair follicles cultured in vitro, and IRS degradation was studied by histology. When grown in culture, the fibres of follicles lacking sebaceous glands were encased in a layer of translucent tissue. During hair growth in vitro this tissue remained intact at the distal end of the follicle but disappeared further down towards the bulb and then reappeared towards the proximal end. Transection within the region lacking this tissue resulted in the release of a naked hair fibre and the production of hair with no attached tissue upon subsequent hair growth. The translucent tissue represented the IRS, thereby demonstrating that this tissue is indeed degraded in vitro. Histological comparison with freshly isolated pilosebaceous units indicated that IRS degradation in vitro strongly resembled the process that occurs in vivo. These data suggest that the sebaceous gland does not itself participate in IRS degradation. Indeed, this phenomenon appears to be a function of the follicle itself and is probably intimately linked with the processes of cellular proliferation, differentiation and death that occur during hair biogenesis.
- Effects of proteolytic enzymes papain, chymotrypsin and tryp-sin on the guinea pig skin, with special reference to the hair follicle
- A Protopapa Ee
- Stavrianeas
- Ng
Protopapa EE, Tsigonia-Primikiriou A, Stavrianeas NG. Effects of proteolytic enzymes papain, chymotrypsin and tryp-sin on the guinea pig skin, with special reference to the hair follicle. Rev Clinical Pharmacol Pharmacokin Internat Ed 1994;8:84. - Regulation of HPV11 gene expression in transgenic mice
- H Gaissert
- L Gissman
Gaissert H, Gissman L, et al. Regulation of HPV11 gene expression in transgenic mice. In: O'Donoghue PN, editor. Proceedings of the Sixth FELASA Symposium, London: The Royal Society of Medicine Press, 1997. pp. 54±55. - Mouse skin treated with iontophoresis in the presence of chymotrypsin and papain. For experimental details see Section 2. Staining as in legend to Fig
- Fig
Fig. 7. Mouse skin treated with iontophoresis in the presence of chymotrypsin and papain. For experimental details see Section 2. Staining as in legend to Fig. 1 (£450). - Transgenic mice carrying the regulatory region of the human papilloma virus type 11 (HPV11): their use in studying the effect of proteolytic enzymes on mouse skin hair follicles
- H Gaissert
- E E Protopapa
Gaissert, H., Protopapa, E.E., et al., Transgenic mice carrying the regulatory region of the human papilloma virus type 11 (HPV11): their use in studying the effect of proteolytic enzymes on mouse skin hair follicles. 35. Wissenchaftliche Tagung der Gesellschaft fur Versuchstierkunde, Jena P6. - We have evaluated, at the histological level, the in vitro effects of three proteolytic enzymes, trypsin, chymotrypsin and papain on guinea pig skin. After mechanical epilation with wax, pieces of guinea pig skin were soaked in 1 mg/ml enzyme solutions at 37°C for different time periods, subsequently embedded in paraffin, cut and the sections stained with hematoxylin-eosin. All three enzymes led to intense histological effects such as the presence of epithelial cells with hyperchromatic nuclei in the center of the shaft of the hair follicles, detachment of the keratin layer from the granular layer and acantholytic changes on the skin. Trypsin, in addition, leads to pemphygous like defects at the borderline of the epithelium. Chymotrypsin shows the most intense effects among the three enzymes. The observed reported in vivo action of the three enzymes, support their application as biological epilatory agents.
- The conditions under which lecithin can effectively mix oil and water into homogeneous solution are discussed. Lecithin is slightly too lipophilic in water-oil systems to spontaneously form the zero mean curvature amphiphile layers needed for balanced microemulsions, but by adjusting the polarity of the polar solvent balanced microemulsions may be prepared. It is found that, by adding a short-chain alcohol as cosolvent, lecithin forms microemulsions at low amphiphile concentrations and over wide ranges of solvent compositions. An investigation of the phase behavior of the system lecithin-1-propanol-water-n-hexadecane is presented. At 1 wt % of lecithin, three liquid phases, i.e., aqueous alcohol solution, oil, and microemulsion, form over wide ranges of solvent ratios. About 2.3% lecithin is the minimum amount needed to form microemulsions at all mixing ratios. The propanol concentration in the microemulsions is in the range 10-15 wt % of the aqueous solvent, the propanol concentration slightly decreasing with increasing oil content. Solution microstructure was investigated from molecular self-diffusion coefficients, and it was found that, depending on the propanol concentration, either discrete droplet or bicontinuous microemulsions may form. With decreasing propanol concentration there is a gradual transition from oil droplets in a water continuum, to bicontinuous structure, and then to a structure of water droplets in oil. This is compared with the analogous sequence of structures of other systems on varying some other parameter (temperature, salinity, surfactant composition).
- Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.
- Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.
- Directed expression of SV40 large T antigen (TAg) in transgenic mice can induce tissue-specific tumorigenesis and useful cell lines exhibiting differentiated characteristics can be established from resultant tumor cells. In an attempt to produce an immortalised mouse hair follicle cortical cell line for the study of hair keratin gene control, SV40 TAg expression was targeted to the hair follicles of transgenic mice using a sheep hair gene promoter. Expression of SV40 TAg in the follicle cortex disrupted normal fiber ultrastructure, producing a marked phenotypic effect. Affected hairs were wavy or severely kinked (depending on the severity of the phenotype) producing an appearance ranging from a ruffled coat to a stubble covering the back of the mouse. The transgenic hairs appeared to be weakened at the base of the fibers, leading to premature hair-loss and a thinner pelage, or regions of temporary nudity. No follicle tumors or neoplasia were apparent and immortalisation of cortical cells could not be established in culture. In situ hybridisation studies in the hair follicle using histone H3 as a cell proliferation marker suggested that cell proliferation had ceased prior to commencement of K2.10-TAg expression and was not re-established in the differentiating cortical cells. Hence, TAg was unable to induce cell immortalisation at that stage of cortical cell differentiation. However, transgenic mice developed various other abnormalities including vertebral abnormalities and bladder, liver and intestinal tumors, which resulted in reduced life expectancy.
- The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease.
