Method for identifying a compound to prevent and / or treat the harmful effects of skin stress

Abstract

TITLE: Method for identifying a compound to prevent and / or treat the deleterious effects of skin stress. The present invention relates to a method for identifying a compound to prevent and / or treat the deleterious effects of skin stress, in particular stress related to pollution and / or UV radiation. The present invention relates to the diagnosis of chronic exposure of a subject to stress, in particular stress affecting the skin. Figure for abstract: none

Description

Title of the invention: Method for identifying a compound for preventing and / or treating the harmful effects of skin stress

[0001] The present invention relates to a method for identifying a compound for preventing and / or treating the deleterious effects of stress on the skin, in particular stress related to pollution and / or UV radiation. The present invention also relates to the diagnosis of chronic exposure of a subject to stress, in particular stress affecting the skin.

[0002] Many factors can cause imperfections in the skin and its complexion. These skin tone imperfections are an increasingly common reason for consultations at aesthetic treatment centers or dermatology clinics.

[0003] Among the stressors that can affect skin tone are sun exposure, exposure to variations in temperature and / or humidity, and exposure to pollutants or cigarette smoke. These various stressors tend to dull the complexion, making it uneven, sallow, waxy, or yellowish, and to cause, or even promote, the presence of pigmentary disorders. Other stressors affecting skin tone include stress, fatigue, hormonal changes, dehydration of the epidermis, impaired skin barrier function, aging, and excessive sebum secretion.

[0004] However, in order to prevent and / or treat the harmful effects on the skin, it may be useful to know the cause, in particular among the different stresses mentioned above.

[0005] Certain urban environments are regularly subjected to pollution peaks. Individuals in their daily environment, and particularly in urban areas, can be subjected to multiple stresses on keratinous materials, especially the skin, by various air pollutants.

[0006] Among the known pollutants, we will first mention exhaust gases which have become a major issue in large cities, heavy metals but also fine particles or polycyclic aromatic hydrocarbons such as benzopyrene or benzoanthracene.

[0007] These pollutants will notably cause the deposition of particles on the surface of the epidermis, and among other consequences, induce a dull skin tone and / or pigmentary disorders.

[0008] The sun emits numerous types of radiation, 10% of which consists of ultraviolet (UV) rays. Only ultraviolet B (UVB, 280 to 320 nm) rays and the Ultraviolet A (UVA, from 320 to 400 nm) reaches the Earth's surface. UVA is subdivided into short UVA (320 to 340 nm) and long UVA (340 to 400 nm).

[0009] The vast majority of UV radiation received on Earth is UVA, as it constitutes on average at least 95% of the UV radiation received; UVB represents only 5%. Among the 95% of UVA received, long-wave UVA predominates, representing at least 77% of the UV radiation received on Earth. Compared to UVB, UVA is more abundant and more persistent throughout the year. UVA can penetrate the epidermis and deep into the dermis. UVA generates reactive oxygen species in skin cells, which are responsible for oxidative stress. Through these mechanisms, UVA induces profound alterations in the dermis, and it has been clearly demonstrated that it is responsible for the signs of photoaging (age spots, wrinkles, dry skin, etc.).

[0010] Identifying exposure to pollution and / or UV radiation as a factor responsible for adverse effects on the skin could make it possible to prevent and / or treat these effects more effectively.

[0011] There is therefore a significant need for diagnostic methods to assess chronic exposure to stress, particularly stress affecting the skin. There is also a significant need to identify compounds that can prevent and / or treat these harmful effects.

[0012] The present invention meets this need.

[0013] The present invention results from the unexpected discovery by the inventors that skin fibroblasts exposed to stress exhibited significantly different biological characteristics, particularly in terms of their proliferation capacity, contractility and the organization of the extracellular matrix formed by them, compared to individuals not exposed to such stress.

[0014] The present invention thus relates to a method for identifying a compound for preventing and / or treating the deleterious effects of stress on the skin, said method comprising the following steps: a. to bring a candidate compound into contact with a biological sample comprising at least one fibroblast, and then to evaluate at least one of the biological characteristics of fibroblasts of said sample; wherein the biological characteristics of fibroblasts are chosen from: the proliferation capacity of fibroblasts, the contractility of fibroblasts and the organization of the extracellular matrix formed by these fibroblasts; b. compare said characteristic assessed in step a) to the same characteristic in a biological sample comprising at least one fibroblast, not brought into contact with said compound; and c. identify said candidate compound as a compound capable of preventing and / or treating the deleterious effects of said stress on the skin, when at least one of the following conditions is met: i. an increase in the proliferative capacity of fibroblasts, ii. a decrease in fibroblast contractility, and / or iii. a decrease in the size and / or surface area of ​​the pores of the extracellular matrix formed by fibroblasts;

[0015] in the biological sample that has been brought into contact with said candidate compound compared to the biological sample that has not been brought into contact with the candidate compound

[0016] said method further comprising a step of exposing said biological samples to stress.

[0017] In a particular embodiment, the step of exposing said biological samples to stress is a step of exposure to polycyclic aromatic hydrocarbons (PAHs) and / or ultraviolet A (UVA).

[0018] The invention also relates to a method for diagnosing chronic exposure of a subject to stress, in particular stress affecting the skin of a subject, comprising measuring at least one of the biological characteristics of fibroblasts from a skin sample of said subject, wherein the biological characteristics of fibroblasts are chosen from among the proliferation capacity of fibroblasts, the contractility of fibroblasts and the organization of the extracellular matrix formed by them.

[0019] Another object of the invention relates to a method of exposing a model, preferably a skin or dermis model, to chronic stress comprising exposing said model: a. to at least one polycyclic aromatic hydrocarbon, preferably to benzo-α-pyrene (BaP), and / or b. to UVA irradiation.

[0020] Thus the invention also relates to a model of skin or dermis of chronic exposure to stress that can be obtained by this method.

[0021] All the methods described in the present invention are preferably in vitro methods. Detailed description of the invention

[0022] Fibroblasts

[0023] The dermis provides the epidermis with a solid support. It is also its nourishing element. It is mainly composed of fibroblasts and an extracellular matrix.

[0024] The term "fibroblasts" here refers to "dermal fibroblasts," that is, any fibroblast originating from the dermis. More precisely, the dermis contains two distinct layers: the superficial papillary layer (300-400 µm, which is in contact with the epidermis) and the underlying reticular layer (which extends into the hypodermis). Connective tissue trabeculae of the dermis may also extend into the hypodermis.

[0025] Preferably, the fibroblasts according to the invention are selected from papillary fibroblasts, reticular fibroblasts, dermo-hypodermal junction fibroblasts, and mixtures thereof. Particularly preferred, the fibroblasts according to the invention are papillary fibroblasts.

[0026] The term "papillary fibroblast" here refers to a fibroblast of the papillary dermis, the papillary dermis being characterized by a relatively thin extracellular matrix and a high cell density. Cultured papillary fibroblasts typically have a thin, spindle-shaped morphology.

[0027] The term "reticular fibroblast" here refers to a fibroblast of the reticular dermis, the reticular dermis being characterized by a very dense network of matrix fibers and a low cell density. Cultured reticular fibroblasts typically have an extended and more stellate appearance.

[0028] The term “dermo-hypodermal junction fibroblast” or “DHJF” refers to a fibroblast located at the level of the connective tissue trabeculae extending from the dermis into the hypodermis (see Haydont V, et al., Fibroblasts from the Human Skin Dermo-Hypodermal Junction are Distinct from Dermal Papillary and Reticular Fibroblasts and from Mesenchymal Stem Cells and Exhibit a Specimen Molecular Profile Related to Extracellular Matrix Organization and Modeling. Cells. 2020 Feb 5;9(2):368). Cultured DHJFs typically exhibit a highly heterogeneous morphology. Thus, a wide variety of shapes can be observed in the cell mat, ranging from very small tricuspid cells to very large multipolar cells with a strongly marked intracellular trabecular network (visible under light microscopy).

[0029] The type of fibroblast (papillary, reticular or other) can be determined from its morphology, origin or detected biomarkers.

[0030] By "biological characteristics of fibroblasts" is meant phenotypic characteristics of fibroblasts that can be demonstrated by their culture and / or by tests. Preferably, the biological characteristics according to the invention include the capacity for proliferation, contractility, and / or the organization of the extracellular matrix formed by the fibroblasts.

[0031] The proliferation capacity of fibroblasts is the ability of these cells to multiply. Measuring cell proliferation is a routine procedure for those skilled in the art. In the case of fibroblasts, it can be performed, in particular, by estimating the doubling time of the cell population during fibroblast culture or by estimating the number of population doublings observed over a given time, for example between each passage.

[0032] The fibroblast population doubling number (or DP) is calculated according to the following formula: DP = log (number of cells counted at the end of the expansion / number of cells seeded) / log2. Then, the cumulative fibroblast population doubling number (or cumulative DP) is obtained by adding the DPs of each fibroblast passage, for example: cumulative DP = DP passage 1 + DP passage 2 + DP passage 3. A passage corresponds to obtaining a confluence of cells in their culture medium of approximately 80%, then detaching the cells from the culture medium, notably with the help of trypsin, then diluting and reseeding them in a new culture medium.

[0033] Fibroblast contractility is their ability to contract a collagen gel. This characteristic of fibroblasts is well known, and several techniques are used to measure it. Gel contraction can be induced by various means, such as stimulation by TGF-3, lysophosphatidic acid (LPA), or by detachment of the gel from its container. Agents that block this contraction include compounds from the ROCK (Rho-associated protein kinase) inhibitor family, such as Y-27632.

[0034] The contractility of fibroblasts can be estimated from the measured diameter of a dermis equivalent formed by culturing the fibroblasts to be tested in a collagen solution, particularly type I collagen, and then, after polymerization of the collagen, inducing contraction of the resulting gel. Preferably, 50,000, 75,000, or 100,000 fibroblasts are added to a collagen solution with a weight / volume ratio of between 15 and 30%, preferably between 20 and 27%. Preferably, 50,000, 75,000, or 100,000 fibroblasts are added to a collagen solution with a final volume of between 3 and 7 ml, preferably between 4 and 6 ml.

[0035] Dermal fibroblasts are known to generate an extracellular matrix that supports the epidermis. This extracellular matrix is ​​composed primarily of collagen. The "organization of the extracellular matrix" formed by fibroblasts refers here to the collagen network, and more specifically to the type I collagen network they produce. Indeed, the molecular network of type I collagen consists of an organization of fibers that are more or less dense and interwoven. These fibers sometimes form the outline of pores (where fibers are absent). The number, average size, and surface area covered by these pores can vary depending on the stress conditions to which the fibroblasts have been previously exposed. The number, surface area, and average size of these characteristic pores can be studied by analyzing images acquired by microscopy. extracellular matrix. Preferably by "the organization of the extracellular matrix formed by fibroblasts" is meant the size and / or area of ​​the surface of the pores, preferably the average size and / or average of the surface areas of the pores, preferably the surface area of ​​the pores greater than 0.04 mm2.

[0036] By "pore size" we mean the diameter of the pores when the surface of the pores is circular, the length and width being identical; or the length and / or the width when the surface of the pores is oval or elliptical, the length and width corresponding respectively to the major axis and the minor axis of an ellipse.

[0037] By "pore surface" is meant a set of points created by the interlocking of collagen fibers, particularly type I collagen fibers, delimiting a two-dimensional space, this space is also called a pore. More preferably, the pore surface is created by the intersection of at least two collagen fibers; even better, the pore surface is created by the intersection of at least three collagen fibers.

[0038] Preferably, the surface of the pores is flat.

[0039] The surface area of ​​the pores can be measured using Image J software, which is a multi-platform, free and open source image processing and analysis software developed by the National Institutes of Health in 1997.

[0040] The surface area of ​​the pores is preferably expressed in mm2.

[0041] Samples and equivalents

[0042] The skin sample from a subject according to the invention is an ex vivo skin sample. Typically, the subject's skin sample can be obtained by biopsy or by sampling, the latter preferably being performed non-invasively and, in particular, not requiring local anesthesia. Preferably, the subject's skin sample is chosen from a human skin expiry, a skin biopsy, and a micro-biopsy.

[0043] The term "subject" here refers to a human being, aged 18 to 80 years, preferably aged 20 to 40 years. Preferably, the subject is a woman. Preferably, the subject is of Caucasian descent. In a preferred embodiment, the subject does not suffer from any skin pathologies.

[0044] In a particular embodiment, the subject is between 20 and 40 years old.

[0045] In the present application, the skin equivalent and the dermis equivalent are also called skin model and dermis model, respectively.

[0046] The skin or epidermis model is any in vitro model of reconstructed skin or reconstructed dermis comprising at least one fibroblast.

[0047] The skin equivalent comprises a dermis equivalent and an epidermis equivalent. In particular, the skin equivalent according to the invention comprises, on the dermis equivalent, an epidermis equivalent comprising at least keratinocytes.

[0048] The dermis equivalent includes at least fibroblasts; it can be a cellular model (also called a 2D model) comprising at least one cultured fibroblast.

[0049] Keratinocytes and fibroblasts can be of any origin but are preferably of human origin. They can be prepared by any method well known to those skilled in the art. Preferably, the keratinocytes and / or fibroblasts are normal human skin cells.

[0050] Stress

[0051] The term "stress" here refers to environmental pressure or constraint. Stresses typically have deleterious effects on the body. Preferably, the stresses according to the invention induce deleterious effects on the skin such as a dull complexion, pigmentary disorders, age spots, wrinkles, and dry skin.

[0052] Preferably, a stress according to the invention is an exposure, preferably chronic, to pollution and / or UV radiation, particularly to polycyclic aromatic hydrocarbons (PAHs) and / or UVA radiation.

[0053] By "chronic exposure" of a subject, we mean here regular exposure, in particular daily, over a given period, preferably a period of more than one year, possibly interrupted on one or more occasions for short periods, typically periods of less than one month, preferably less than one week.

[0054] By "pollution" we mean here exposure to polluting agents, in particular to particles of matter, and preferably to polycyclic aromatic hydrocarbons (PAHs).

[0055] As is well known to those skilled in the art, PAHs are a subfamily of aromatic hydrocarbons whose structure comprises at least two condensed aromatic rings. They are produced by the incomplete combustion of organic matter (coal, oil, gas, waste and biomass, cigarettes) and released as environmental contaminants.

[0056] PAHs typically include acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chryzene, benzo-b-fluoranthene, benzo-k-fluoranthene, benzo[a]pyrene, indenol,2,3-c( / )pyrene, dibenz-ah-anthracene, benzo-ghi-perylene.

[0057] In a particular embodiment, the PAHs are selected from acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, the pyrene, benz[a]anthracene and benzo[a]pyrene (BaP). In a particular embodiment, the PAH is benzo[a]pyrene (BaP).

[0058] By "UVs" we mean here radiation with a wavelength ranging from 400 to 100 nm. When the stress according to the invention is exposure to UV, this is preferably due to solar exposure or to so-called artificial UV, for example in a tanning booth.

[0059] By definition, long UVA refers to UV radiation with wavelengths ranging from 340 to 400 nm, short UVA refers to UV radiation with wavelengths ranging from 315 to 339 nm.

[0060] "Radiation including long UVA" therefore means radiation including radiation with wavelengths ranging from 340 to 400 nm.

[0061] Radiation including long UVA can be radiation consisting of, or essentially comprising, radiation with wavelengths from 320 nm to 450 nm (total UVA and a portion of radiation in the visible range from 400 to 450 nm). This type of radiation can be obtained, for example, using a solar simulator equipped with a xenon lamp and a WG 335 / 3 mm filter delivering a spectrum between 320 and 450 nm.

[0062] Radiation including long UVA can be radiation consisting of, or essentially comprising, radiation with wavelengths from 340 nm to 450 nm (long UVA and a portion of radiation in the visible range from 400 to 450 nm). This type of radiation can be obtained, for example, using a solar simulator equipped with a xenon lamp and a WG 360 / 2 mm filter that suppresses wavelengths below 340 nm.

[0063] By radiation comprising "essentially" radiation of specified wavelengths, it is understood that the spectral irradiance (expressed in mW.cm⁻¹ as a function of the wavelength in nm) between the specified wavelengths represents at least 80%, preferably at least 90%, of the total spectral irradiance.

[0064] Preferably, the radiation including long UVA is mainly composed of long UVA, that is to say that the spectral irradiance (expressed in mW.cm 2.nm1 as a function of the wavelength in nm) for wavelengths from 340 to 400 nm represents at least 50%, preferably at least 60%, of the total spectral irradiance.

[0065] Exposing a sample to stress according to the invention can be achieved by various methods known to those skilled in the art. Preferably, the sample is exposed to pollution and / or UV radiation, more particularly to polycyclic aromatic hydrocarbons (PAHs) and / or UVA radiation. This exposure can be achieved by culturing fibroblasts from the biological sample: a. in the presence of at least one polycyclic aromatic hydrocarbon, preferably in the presence of benzo-α-pyrene (BaP), and / or b. with exposure of fibroblasts to UVA irradiation.

[0066] Preferably, said at least one polycyclic aromatic hydrocarbon is present at a concentration between 0.1 nM and 100 nM, more particularly between 0.5 nM and 50 nM, between 1 nM and 100 nM, and more particularly between 3 nM and 8 nM, for at least 7 days, at least 14 days, at least 21 days, at least 28 days. Said at least one polycyclic aromatic hydrocarbon may be added directly to the culture medium, preferably after dilution in dimethyl sulfoxide (DMSO).

[0067] Preferably, irradiation is carried out with radiation including UVA, and more particularly including long UVA, for at least 10 minutes, at least 20 minutes, and preferably at least 30 minutes. This exposure is preferably carried out at regular intervals, preferably at least once a week, twice a week, or three times a week, over a total duration of at least 7 days, at least 14 days, at least 21 days, and at least 28 days. During irradiation, the culture medium is preferably replaced so as not to contain polycyclic aromatic hydrocarbons.

[0068] Method for identifying a compound

[0069] The present invention thus relates to a method for identifying a compound for preventing and / or treating the deleterious effects of stress on the skin, said method comprising the following steps: a. to bring a candidate compound into contact with a biological sample comprising at least one fibroblast, and then to evaluate at least one of the biological characteristics of fibroblasts of said sample; wherein the biological characteristics of fibroblasts are chosen from: the proliferation capacity of fibroblasts, the contractility of fibroblasts and the organization of the extracellular matrix formed by these fibroblasts; b. compare said characteristic assessed in step a) to the same characteristic in a biological sample comprising at least one fibroblast, not brought into contact with said compound; and c. identify said candidate compound as a compound capable of preventing and / or treating the deleterious effects of said stress on the skin, when at least one of the following conditions is met: i. an increase in the proliferative capacity of fibroblasts, ii. a decrease in fibroblast contractility, and / or iii. a decrease in the size and / or surface area of ​​the pores of the extracellular matrix formed by fibroblasts;

[0070] in the biological sample that has been brought into contact with said candidate compound compared to the biological sample that has not been brought into contact with the candidate compound, said method further comprising a step of exposing said biological samples to stress.

[0071] The identification method of the invention is preferably implemented ex vivo.

[0072] The compound or candidate compound for identification can be any molecule or active ingredient, for example of an organic, mineral or particulate nature.

[0073] The biological sample according to the invention comprises at least one dermal fibroblast, as defined in the "Fibroblasts" section above.

[0074] The biological sample may in particular be an in vitro culture of dermal fibroblasts or a mixture of dermal fibroblasts, a skin sample from a subject, or a sample from a dermis or skin equivalent.

[0075] Step c) includes identifying said candidate compound as a compound capable of preventing and / or treating the deleterious effects of stress on the skin, when at least one of the following conditions is met: i. an increase in the proliferative capacity of fibroblasts, ii. a decrease in fibroblast contractility, and / or iii. a decrease in the size and / or surface area of ​​the pores of the extracellular matrix formed by fibroblasts;

[0076] Preferably said increases or decreases are significant, that is to say the variation is statistically significant, typically with a p-value less than 0.01, more preferably less than 0.001, after application of the appropriate statistical test.

[0077] Preferably, in the method of identifying a compound according to the invention, if the contractility ratio of the gel is reduced by more than 10% compared to the contractility obtained with the biological sample that has not been in contact with said candidate compound (also called control sample), preferably reduced by more than 12%, even better reduced by more than 15%, then the compound is identified as enabling the prevention and / or treatment of the deleterious effects of stress on the skin.

[0078] The contractility ratio is preferably calculated from the measured diameter of a dermis equivalent formed by culturing fibroblasts in a collagen solution, in particular type I collagen, obtained with the biological sample that has been in contact with said candidate compound divided by the measured diameter of a dermis equivalent formed by culturing fibroblasts in a collagen solution, in particular type I collagen, obtained with the biological sample that has not been in contact with said candidate compound (also called the control sample),

[0079] Preferably, in the method of identifying a compound according to the invention, if the cumulative population doubling number of fibroblasts is increased by at least 1.5 compared to the cumulative population doubling number of fibroblasts obtained with the biological sample that has not been in contact with said candidate compound (also called control sample), preferably increased by at least 2, even better, increased by at least 2.4, then the compound is identified as enabling the prevention and / or treatment of the deleterious effects of stress on the skin.

[0080] Preferably, in the method of identifying a compound according to the invention, if the pore surface area, in particular the pore surface area greater than 0.04 mm2, is reduced by a factor of at least 1.1 compared to the pore surface area obtained with the biological sample that has not been in contact with said candidate compound (also called the control sample), preferably reduced by a factor between 1.1 and 3, even better reduced by a factor between 1.1 and 2.9, then the compound is identified as enabling the prevention and / or treatment of the deleterious effects of stress on the skin.

[0081] Preferably, in the method of identifying a compound according to the invention, if the pore surface area, in particular the pore surface area greater than 0.04 mm2, is reduced by a factor of between 1.2 and 2.7 compared to the pore surface area obtained with the biological sample that has not been in contact with said candidate compound (also called the control sample), preferably reduced by a factor of between 1.2 and 2.69, then the compound is identified as enabling the prevention and / or treatment of the deleterious effects of stress on the skin such as pollution, preferably exposure to polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene.

[0082] Preferably, in the method of identifying a compound according to the invention, if the pore surface area, in particular the pore surface area greater than 0.04 mm2, is reduced by a factor of between 1.1 and 2.9 compared to the pore surface area obtained with the biological sample that has not been in contact with said candidate compound (also called the control sample), preferably reduced by a factor of between 1.1 and 2.87, then the compound is identified as enabling the prevention and / or treatment of the deleterious effects of stress on the skin such as pollution and UV, preferably exposure to polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene and UVA.

[0083] The identification method includes a step of exposing said biological samples to stress, and more particularly a step of exposure to pollution and / or UV, even more particularly to polycyclic aromatic hydrocarbons (PAHs) and / or UVA.

[0084] Exposure of the samples to stress according to the invention can be achieved: - before the contact is made in step a); and / or - after initial contact but before evaluating characteristics biological fibroblasts of said sample.

[0085] Diagnostic method

[0086] The present invention relates to a method for diagnosing chronic exposure of a subject to stress, in particular stress affecting the skin of a subject, comprising measuring at least one of the biological characteristics of fibroblasts from a skin sample of said subject, wherein the biological characteristics of fibroblasts are chosen from among the proliferation capacity of fibroblasts, the contractility of fibroblasts and the organization of the extracellular matrix formed by them.

[0087] Preferably, this method further includes the steps of: a. compare the level of the measured biological characteristic with a control, and b. Based on the comparison from step (a), determine whether the subject, in particular the subject's skin, has undergone chronic exposure to stress.

[0088] By "control" or "control level", we mean here a reference value corresponding preferably to the level of the biological characteristic measured in a skin sample known not to be exposed to stress and in particular to chronic stress according to the invention.

[0089] The control level is typically the level of the biological characteristic measured in a subject living in a city with low pollution, in particular a city with an air quality index for fine (< 10 microns) and ultrafine (< 2.5 microns) particles of less than 40, preferably an air quality index of less than 20, for at least 100 days, in particular at least 85 days, over a year and / or in a subject not regularly exposed to UV, and / or on a part of the body of a subject with low UV exposure.

[0090] In a particular embodiment, the control is a reference value.

[0091] In a particular embodiment, the reference value is determined by the average value of the level of the biological characteristic in a given population, for example a population in a given age group, and / or having a defined skin type.

[0092] In a particular embodiment, the reference value is the average value of the level of said biological characteristic in a population of subjects living in a city with low pollution, in particular a city with an air quality index for fine (< 10 microns) and ultrafine (< 2.5 microns) particles of less than 40, preferably an air quality index of less than 20, for at least 100 days, in particular at least 85 days, over a year and / or in a population not regularly exposed to UV radiation, and / or on a part of the body of a population with low UV exposure.

[0093] In a particular embodiment, the reference value is determined by an ROC study (“The ROC (receiver operating characteristic) curve: principles and main applications in clinical biology”, H. Delacour et al., Ann Biol Clin 2005; 63 (2): 145-54).

[0094] In a particular embodiment of the invention, the method is a method for diagnosing exposure of a subject to UV radiation, comprising measuring at least one of the biological characteristics of fibroblasts from a skin sample exposed to UV radiation from said subject and wherein the control is a skin sample from said subject on a part of the body with low UV radiation exposure.

[0095] Step b) includes determining the subject, and in particular the subject's skin, as having undergone chronic exposure to stress, when at least one of the following conditions is met: i. a decrease in the proliferative capacity of fibroblasts, ii. an increase in fibroblast contractility, and / or iii. an increase in the size and / or surface area of ​​the pores of the extracellular matrix formed by fibroblasts;

[0096] is observed in relation to the control.

[0097] Preferably said increases or decreases are significant, that is to say the variation is statistically significant, typically with a p-value less than 0.01, more preferably less than 0.001, after application of the appropriate statistical test.

[0098] Preferably, in the diagnostic method according to the invention, if the contractility ratio of the gel is increased by more than 10% compared to the control, preferably increased by more than 12%, even better increased by more than 15%, then the subject is determined to have undergone chronic exposure to stress.

[0099] The contractility ratio is preferably calculated from the measured diameter of a dermis equivalent formed by culturing fibroblasts in a collagen solution, in particular type I collagen, obtained from the subject's skin sample,

[0100] Preferably, in the diagnostic method according to the invention, if the cumulative population doubling number of fibroblasts is decreased by at least 1.5 compared to the control, preferably decreased by at least 2, even better decreased by at least 2.4, then the subject is determined to have undergone chronic exposure to stress.

[0101] Preferably, in the diagnostic method according to the invention, if the pore surface area, in particular the pore surface area greater than 0.04 mm2, is increased by a factor of at least 1.1 compared to the control, preferably increased by a factor between 1.1 and 3, even better increased by a factor between 1.1 and 2.9, then the subject is determined to have undergone chronic exposure to stress.

[0102] Preferably, in the diagnostic method according to the invention, if the pore surface area, in particular the pore surface area greater than 0.04 mm2, is increased by a factor of between 1.2 and 2.7 compared to the control, preferably increased by a factor of between 1.2 and 2.69, then the subject is determined to have undergone chronic exposure to skin stress such as pollution, preferably exposure to polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene.

[0103] Preferably, in the diagnostic method according to the invention, if the pore surface area, in particular the pore surface area greater than 0.04 mm2, is increased by a factor of between 1.1 and 2.9 compared to the control, preferably increased by a factor of between 1.1 and 2.87, then the subject is determined to have undergone chronic exposure to skin stress such as pollution and UV, preferably exposure to polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene and UVA.

[0104] Method of exposing a model

[0105] The invention relates to a method of exposing a model, preferably a skin or dermis model, to chronic stress comprising the exposure of said model: a. to at least one polycyclic aromatic hydrocarbon, preferably to benzo-α-pyrene (BaP), and / or b. to UVA irradiation.

[0106] Preferably, said at least one polycyclic aromatic hydrocarbon is present at a concentration between 0.1 nM and 100 nM, more particularly between 0.5 nM and 50 nM, between 1 nM and 100 nM, and more particularly between 3 nM and 8 nM, for at least 7 days, at least 14 days, at least 21 days, at least 28 days. Said at least one polycyclic aromatic hydrocarbon may be added directly to the skin or dermis model, preferably after dilution in dimethyl sulfoxide (DMSO).

[0107] Preferably, the irradiation is carried out with radiation including UV radiation, and more particularly including long UVA radiation, for at least 10 minutes, at least 20 minutes, preferably at least 30 minutes. This exposure is preferably carried out at regular intervals, preferably at least once a week, twice a week or three times a week, over a total duration of at least 7 days, at least 14 days, at least 21 days, at least 28 days. During irradiation, the model is preferably not exposed to said at least one polycyclic aromatic hydrocarbon.

[0108] The invention also relates to the skin or dermis model of chronic exposure to stress, preferably obtained by the method of exposing a skin or dermis model to chronic stress according to the invention.

[0109] Throughout the above description, unless otherwise stated, the term "between x and y" or "ranging from x to y" corresponds to an inclusive range, that is to say that the values ​​x and y are included in the range.

[0110] The present invention will be illustrated in more detail by the example below. [YES] Figure:

[0112] [Fig.l] [Fig.l]: Schematic representation of the chronic stress protocol applied to papillary fibroblast cultures from young donors.

[0113] [Fig. 2] [Fig. 2]: Left: Representative image of a collagen network produced by fibroblasts, acquired using a Leica transmission optical microscope coupled with a camera. The complexity of the collagen network (white filament) and the presence of pores (black) are visible. Right: Example of an image generated after applying image segmentation software to the image on the left. The categorization of the pores is shown: the pores are colored in different shades of gray according to their surface area.

[0114] [Fig.3] [Fig.3]: Histogram representing on the ordinate the cumulative population doubling number after 3 weeks of culture and exposure to stress with or without application of vitamin C. Cell preparation

[0115] Papillary fibroblasts (Fp) were isolated from undefatted human skin. These samples were collected after breast reduction for cosmetic reasons. Four donors (aged 22, 22, 25, and 31) were included.

[0116] Isolation of Fp is carried out on dermatomed tissue at 300 pm then de-epithelialized after dispase action for 16h at 4°C.

[0117] After laceration, the dermis fragments are digested under the action of type II collagenase at 0.2% at 37 °C.

[0118] The cells are then amplified in minimum essential medium (MEM) - 10% fetal bovine serum (FBS) supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin and fungizone, under a humid atmosphere, at 37 °C and 5% CO2.

[0119] The isolated cells were validated as papillary fibroblasts based on their relative expression levels of signature genes. For this purpose, mRNAs were extracted using a QIAgen column according to the manufacturer's instructions after an expansion phase (between 7 and 10 population doublings). Then, the expression levels of the signature genes were measured by PCR. exposure to chronic stress in vitro

[0120] After cell expansion (between 7 and 10 population doublings), the Fp cells are cultured in the presence of pollutants (BaP). The pollutant is added to the culture medium at a concentration of 6 nM after dilution in DMSO. The medium is changed daily except on weekends. In parallel, the cells are exposed to long-wave UVA irradiation (340 / 400 nm - 7.5 J / cm² - 35 min exposure) on average twice a week (for a total of 6 cumulative irradiations), see [Fig. 1]. During irradiation, the culture medium is replaced with phosphate-buffered saline (PBS) that does not contain the pollutant.

[0121] The cells are maintained in culture under these conditions for 21 days. When the cells reach 80% confluence, they are trypsinized and reseeded under the same stress conditions.

[0122] Since BaP was diluted in DMSO, control cultures receiving the same concentration of DMSO as the cultures in the presence of BaP were prepared. Thus, according to the tests carried out, four culture conditions were established:

[0123] Control: stress-free

[0124] DMSO: culture in the presence of the content of a small volume of DMSO equivalent to that allowing the addition of BaP (1 / 2000).

[0125] BaP 6 nM

[0126] UV cells irradiated twice a week + BaP 6 nM I / - Monitoring of proliferation

[0127] The cumulative population doubling calculation was used to estimate the proliferation capacity of fibroblasts. To do this, the population doubling number (PD) for each passage was calculated using the formula:

[0128] DP = log (number of cells counted at the end of the expansion / number of cells seeded) / log2.

[0129] The cumulative number of DP is obtained by adding the DP of each pass:

[0130] Cumulative DP = DP passage 1 + DP passage 2 + DP passage 3

[0131] [Tables 1] Control DMSO UVA + BaP 6 nM Average cumulative DP 15.64 15.56 13.22 Standard deviation 2.26 2.23 2.13 SEM 1.13 1.12 1.07

[0132] The table above shows the cumulative population doubling number calculated for each condition after 21 days of exposure to stress. Dual exposure to BaP and UVA affects fibroblast proliferation potential with a mean reduction of 2.42 ± 0.33 doublings (n=7, p<0.001 - paired Student's t-test). II / - Cellular Contractility Test

[0133] Dermal equivalents (lattices) are produced by adding 100,000 fibroblasts to a solution of type I bovine collagen diluted in culture medium (MEM 10% FBS) at a ratio of 26% by weight / volume. The resulting mixture, with a final volume of 5 ml, is poured into a 34 mm diameter Petri dish. Spontaneous polymerization of the collagen occurs within the first few hours. After 48 hours in an incubator, the latticees are detached from their support to make them float. This detachment induces contraction of the latticees, which is manifested by a decrease in their diameter. The progress of this process is monitored over time by measuring the lattice diameter with a ruler. The smaller the lattice diameter, the greater the contractile properties of the cells.

[0134] Three lattice samples are prepared for each condition. The average of these three diameters establishes the reference value for the condition.

[0135] The variations in percentage of contractility are calculated by relating the values ​​measured under the conditions exposed to stress to those obtained under the control condition.

[0136] 5 donors were analyzed.

[0137] [Table 2] Average of the values ​​measured on the 5 donors after normalization to the control DMSO BaP 6 nM UVA + BaP 6 nM Control Contractility Ratio 1.00 1.00 0.85 0.84 Standard Deviation 0.00 0.00 0.12 0.11 SEM 0.00 0.00 0.05 0.05

[0138] The table above shows the results of the evaluation of the contractile properties of papillary fibroblasts previously exposed to various chronic stresses for 21 days. Values ​​normalized to the control condition. The cells having been exposed to BaP have increased contractile capacity (+15.4% with BaP alone, +16% with UVA + BaP, n=5, p<0.05 - Student's test on paired values).

[0139] III / - Analysis of the organization of the type I collagen network; Preparation of extracellular matrices

[0140] After 21 days of stress exposure, fibroblasts are seeded in plain medium (used for expansion). The culture is maintained for 48 hours post-confluence. Then the cells are lysed using a buffer containing 0.5% Triton X100 and 20 mM NH4OH diluted in PBS. Immunostaining of extracellular matrices

[0141] The presence of type I collagen is demonstrated by immunostaining of the extracellular matrices without prior fixation. For this purpose, the extracellular matrices are saturated with PBS / 3% bovine serum albumin (BSA) for 15 minutes at room temperature. They are then exposed to an anti-type I collagen antibody for 30 minutes at room temperature. The samples are subsequently washed twice in PBS / 0.5% BSA. The staining is revealed by a secondary anti-goat antibody coupled to Alexa 488 diluted 1 / 250 in PBS / 3% BSA. After three washes, the samples are mounted between a slide and coverslip. Images are acquired using a Leica transmission optical microscope coupled to an ORCA-Flash4.0 LT+ camera (Hamamatsu Photonics KK).

[0142] Analysis of the organization of the type I collagen network

[0143] To define the surface area occupied by pores under a given condition, image segmentation software was developed (see [Fig. 2]). This software is based on an anti-granulometry principle. Five pore categories were thus defined. Each category corresponds to a range of surface area, which can be defined as follows:

[0144] Less than 0.004 mm², between 0.004 and 0.02 mm², between 0.02 and 0.04 mm², between 0.04 and 0.07 mm², greater than 0.07 mm². It is then possible to know the surface area occupied for each pore size category or the sum of the surface areas occupied by all the pores present in a sample.

[0145] After measuring the surface areas covered by the pores for each category, it is possible to compare the evolution of distributions by donor.

[0146] The ratios of the mean pore surface areas (mean pore surface area obtained under the control condition / mean pore surface area obtained under the stress condition), established between the different pre-stress cell exposure conditions, were calculated where possible. They are presented in the table below. When, the difference between the two If the difference between the two compared conditions is significantly increased (p<0.05 - Mann-Whitney / Wilcoxon test), the value is marked with an asterisk. When the difference between the two compared conditions is significantly decreased (p<0.05 - Wilcoxon test), the value is underlined. NC means that the ratio value cannot be calculated because one of the two values ​​is zero.

[0147] [Tables3] Pre-exposure to stress Donor Pore surface area < 0.004 mm² 0.004 to 0.02 mm² 0.02 to 0.04 mm² 0.04 to 0.07 mm² > 0.07 mm² Sum Control / BaP Donor A 1.13 1.20* 1.24* 0.73 0.64 0.98 Control / BaP Donor B 0.99 1.08 1.36 1.36 2.08* 1.35* Control / BaP Donor C 1st trial 1.07 1.38* 1.42 2.69* NC 1.23* Control / BaP Donor C 2nd trial 0.92 1.17 1.83* 2.65* NC 1.23* Control / UV A + BaP Donor A 1.22* 1.10 1.04 0.88 0.73 0.99 Control / UV A + BaP Donor B 1.22* 1.39* 1.09 1.46 0.98 1.22* Control / UV A + BaP Donor C 1st trial 0.97 0.97 0.78 2.87* NC 1.02 Control / UV A + BaP Donor C 2nd trial 0.90 0.85 1.26 1.51 NC 0.96

[0148] A significant change in pore surface area is observed for all donors when the cells have been previously exposed to BaP. Depending on the categories and the donors, the pore surface area is increased by a factor ranging from 1.2 to 2.69 times compared to those measured in cells not previously exposed.

[0149] With regard to double pre-exposure to UVA and BaP, a slight increase in the pore surface area is observed whether compared with the control condition (1.2 < ratio < 2.87).

[0150] These observations show that each of the applied stresses induces a specific modification in the ability of fibroblasts to organize type I collagen fibers.

[0151] IV / - Effect of a reference molecule: vitamin C

[0152] Vitamin C (L-ascorbic acid) is a water-soluble antioxidant. It plays a role in several important metabolic pathways, notably as a cofactor for the hydroxylation of prolyl and lysyl residues present on procollagen chains. It thus helps to stabilize its tertiary structure.

[0153] In this protocol, vitamin C was used as a curative treatment. Proliferation

[0154] Proliferation (see [Fig. 3]), synthesis, and collagen I network organization assays were deployed to visualize the impact of vitamin C on cells previously exposed to stress. The protocols used for these assessments are the same as those described above. Quantification of pore surface area

[0155] Comparison of values ​​obtained for matrices synthesized by fibroblasts previously exposed to stress for 21 days + / - vitamin C used during the extracellular matrix synthesis phase.

[0156] The ratios of the mean pore surface areas, established between the vitamin C-treated and untreated conditions, were calculated. They are presented in the table below. Where the difference between the two compared conditions is significantly reduced (*** = p<0.001 - Mann-Whitney / Wilcoxon test), the box is underlined.

[0157] [Tables4] Pre-exposure to very high S + / - Vitamin C Pore surface area < 0.004 mm² 0.004 to 0.02 mm² 0.02 to 0.04 mm² 0.04 to 0.07 mm² > 0.07 mm² Control 1.15 0.80 0.63 0.39 0.09 BaP 0.87 0.58 0.84 0.74 0.17 UVA + BaP 0.99 0.89 0.83 0.47 0.15

[0158] Post-treatment with vitamin C significantly reduces pore surface area, regardless of the stress to which the cells were previously exposed. Thus, the larger the pore size, the more visible the surface area reduction effect will be.

[0159] Vitamin C therefore appears to be effective in reducing the effects produced by stress such as BaP, and BaP with UVA. Statistical test

[0160] For the proliferation and contractility tests, the test used is a paired-value Student's t-test.

[0161] For the analyses of pore surface areas, anisotropy and watershed circularity index, a Mann-Whitney-Wilcoxon test was used.

Claims

1.

2.

3. Demands Method for identifying a compound for preventing and / or treating the deleterious effects of stress on the skin in a subject, said method comprising the following steps: a. to bring a candidate compound into contact with a biological sample comprising at least one fibroblast, and then to evaluate at least one of the biological characteristics of fibroblasts of said sample; wherein the biological characteristics of fibroblasts are chosen from: the proliferation capacity of fibroblasts, the contractility of fibroblasts and the organization of the extracellular matrix formed by these fibroblasts; b. compare said characteristic assessed in step a) to the same characteristic in a biological sample comprising at least one fibroblast, not brought into contact with said compound; and c. identify said candidate compound as a compound capable of preventing and / or treating the deleterious effects of said stress on the skin, when at least one of the following conditions is met: i. an increase in the proliferative capacity of fibroblasts, ii. a decrease in fibroblast contractility, and / or iii. a decrease in the size and / or surface area of ​​the pores of the extracellular matrix formed by fibroblasts; in the biological sample that has been in contact with said candidate compound compared to the biological sample that has not been in contact with the candidate compound said method further comprising a step of exposing said biological samples to stress. Method according to claim 1, wherein the biological samples are skin samples from a subject. A method according to claim 1 or 2, wherein the step of exposing said biological samples to stress is a step exposure to polycyclic aromatic hydrocarbons (PAHs) and / or ultraviolet A (UVA).

4. Method according to claim 3, said method further comprising the following step: culturing fibroblasts from biological samples, a. in the presence of at least one polycyclic aromatic hydrocarbon, in a concentration between 0.1 and 100 nM, and / or b. with exposure of the fibroblasts to UVA irradiation for at least 10 minutes, at least once a week.

5. A method for diagnosing chronic exposure of a subject to stress, in particular stress affecting the skin of a subject, comprising measuring at least one of the biological characteristics of fibroblasts from a skin sample of said subject, wherein the biological characteristics of fibroblasts are chosen from among the proliferation capacity of fibroblasts, the contractility of fibroblasts and the organization of the extracellular matrix formed by them.

6. Method according to any one of claims 2 to 5, wherein said skin samples are selected from: a human skin expiant, a skin biopsy, and a micro-biopsy.

7. Diagnostic method according to claim 5 or 6, the method further comprising the steps of: a. comparing the level of the measured biological characteristic to a control, and b. on the basis of the comparison in step (a), determining whether the subject, in particular the subject's skin, has undergone chronic exposure to stress.

8. Method according to claim 5 or 7, wherein the stress is due to chronic exposure to polycyclic aromatic hydrocarbons (PAHs) and / or UVA.

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