**1. Introduction**

A hair follicle (HF) is a real mini-organ that makes hair growth possible. The hair shaft extends under the skin into the HF. Histology of a HF shows that it is organized into two compartments [1]. The first compartment with a dermal origin is composed of connective tissue sheath (CTS) and dermal papillae (DP). The second compartment is of epithelial origin and comprises the hair matrix, the outer (ORS) and inner (IRS) root sheaths, and the hair shaft. The bulb is comprised, at the bottom, of the DP surrounded by the germinative hair matrix, and at the top, of the differentiation zone of the matrix.

**Citation:** Colin-Pierre, C.; Untereiner, V.; Sockalingum, G.D.; Berthélémy, N.; Danoux, L.; Bardey, V.; Mine, S.; Jeanmaire, C.; Ramont, L.; Brézillon, S. Hair Histology and Glycosaminoglycans Distribution Probed by Infrared Spectral Imaging: Focus on Heparan Sulfate Proteoglycan and Glypican-1 during Hair Growth Cycle. *Biomolecules* **2021**, *11*, 192. https://doi.org/10.3390/biom11020192

Academic Editors: Dragana Nikitovic and Serge Perez Received: 1 December 2020 Accepted: 22 January 2021 Published: 30 January 2021

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In addition, it has appendages, the sebaceous gland, and the arrector pili muscle. The sebaceous gland associated with the HFs protects the hair by sebum secretion, a substance rich in lipids [2].

A HF undergoes cyclic changes over the course of its life [3] allowing the renewal of lost hair (40 to 100 lost hairs per day). One cycle is comprised of three main phases: anagen, catagen, and telogen. The exogen phase is a phase of the hair growth cycle that is controlled separately leading to hair shaft loss [4,5]. The anagen phase is characterized by intense proliferation allowing the generation of new hair shafts [6] and lasts on the average from three to six years. It is divided into six stages during which the morphology of the HF undergoes major remodeling due to the activation of different cell types at the end of the telogen phase [7,8]. As soon as the hair growth is complete, its degeneration begins. The catagen phase corresponds to the cessation of hair shaft growth and a regression in the size of the HF [8]. This phase is characterized by apoptosis of the keratinocytes separating the secondary hair germ (SHG) from the DP. In contrast to the anagen phase, the catagen phase is more rapid, lasting on the average between 15 and 20 days; it is divided into eight stages [8]. The telogen phase is a resting phase during which the hair shaft remains anchored in the hair follicle [6]. At this stage the DP in the dormant state is in contact with the SHG [8]. This phase may last several months until a stimulus causes the HF to return to the anagen phase.

The morphological changes in the HF observed during the hair cycle involve many cell types: keratinocytes, fibroblasts of the DP, endothelial, fat, and immune cells. The presence of these different cell types makes the study of the regulation of the hair cycle complex. It is also known that a fine regulation of growth factors involved in the hair shaft growth is essential for the passage between the different phases of the hair cycle [9].

The mechanisms involved in the regulation of these growth factors are still poorly understood, but it is highly probable that heparan sulfate proteoglycans (HSPGs) are involved because of their capacity to sequester growth factors [10]. HSPGs are composed of linear chains of heparan sulfate glycosaminoglycans (GAGs) covalently attached to a core protein [11]. They are classified according to their localization, either in the cell membrane or secreted in the extracellular matrix (ECM). The secreted HSPGs interact with the macromolecules of the ECM and growth factors and thus play a pivotal role in cell growth, survival, proliferation, adhesion, migration, and differentiation [12]. The cell membrane HSPGs are divided into two major families: syndecans (SDCs), which are transmembrane PGs, and glypicans (GPCs), which are attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Syndecans interact with the ECM and growth factors and have a role in embryonic development [13]. GPCs are essential for the formation or regeneration of many tissues and organs by regulating many pathways involved in development. For example, they regulate the Hedgehog (Hh) pathway during the embryonic development or long bone formation [14,15], the Wnt pathway during the embryonic development or regeneration of intestinal crypts [16,17], and the bone morphogenetic protein (BMP) pathway involved in osteogenesis [18]. The HF is a true mini-organ regulated by Wnt, BMP, and Hh pathways [19,20]. Therefore, it appears highly likely that HSPGs and particularly GPCs also play a key role in the growth of a new hair shaft.

Previous studies have shown modifications in the distribution of various ECM HSPGs [21] or membrane HSPGs such as syndecan-1 [22] during the hair cycle. This phase-dependent change seems to indicate a role of HSPGs in the regulation of hair shaft growth. Coulson– Thomas et al. showed that complex control of HSPG sulfation is necessary for correct morphogenesis of the hair shaft [23]. However, expression of GPCs in different HF compartments and their potential roles during hair shaft formation are still poorly understood.

HSPG distribution, localization, and quantification in HFs are classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach based on infrared spectral imaging (IRSI) is proposed to assess, in the first part, the HSPG distribution in the HF, and in the second part, to compare HSPG variations

in HFs at different phases of hair cycle. Fourier-transform infrared (FTIR) spectroscopy is a vibrational method based on the principle of interaction between mid-IR radiation and matter, which is used to analyze pure samples but also more complex systems such as cells, tissues, or biofluids. It is non-invasive, label- and chemical-free, very sensitive, and requires no specific preparation [24]. The spectral signature of a sample contains vibrations of molecular bonds that are related to its molecular structure and composition. Today, IRSI is a proven technique that is intensively used for cell (spectral cytology) and tissue (spectral histology) characterization [25–28]. In an infrared image, each pixel is associated with an entire IR spectrum and thus both molecular and spatial information can be obtained. Recently, our group has reported studies on vibrational spectroscopic analyses of standard GAGs [29] and of GAGs in cells and tissues [30,31]. These studies permitted identifying specific spectral signatures. As we reported previously, two major spectral ranges were used to characterize HSPG distribution: the spectral window 1800–900 cm−<sup>1</sup> , also known as the fingerprint region, shown to be the most specific range for GAG studies [24], and the spectral window 1350–1190 cm−<sup>1</sup> , centered at 1248 cm−<sup>1</sup> , that is specific for GAG sulfation [29].

Based on this knowledge, we propose in this original study to probe the distribution of HSPGs in HFs at different phases of the hair growth cycle by IRSI using sulfation as a spectral marker. IRSI might constitute a promising technique for early diagnosis and prevention in alopecia. The goal was to study HSPG, GAG, and sulfated GAG distribution and variation in HFs without any staining or labeling. It allows investigating these changes during the hair growth cycle. In parallel, our data were supported by Western immunoblotting and immunohistochemistry assays, more specifically by analyzing the GPC1 expression and distribution in HFs at different phases of the cycle.
