Preferential Crosstalk between Perifollicular Capillary Vessels and Dermal Papilla Cells during Hair Cycling Homeostasis

Preferential Crosstalk between Perifollicular Capillary Vessels and Dermal Papilla Cells during Hair Cycling Homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preferential Crosstalk between Perifollicular Capillary Vessels and Dermal Papilla Cells during Hair Cycling Homeostasis Ying Zeng, Akinari Abe, Satsuki Takashima, Miyu Kono, Reina Kagiyama, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7791533/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 3 You are reading this latest preprint version Abstract The tissue-specific capillary system supports the unique function of each organ and dynamically remodels in response to local requirements. In this study, we found that perifollicular vascularization flexibly adjusts to accommodate physiological changes. Notably, not all capillary vessels responded and migrated equally. However, those around the dermal papilla (DP) exhibited preferential mobilization. Treatment with minoxidil, a hair growth agent, significantly increased perifollicular vessel mobilization around the DP, whereas it was inhibited in experimental models of tissue aging, such as those involving vascular endothelial growth factor-neutralizing antibody or testosterone treatment, similar to the physiological tissue aging process. Furthermore, vascular endothelial cells triggered the expression of angiogenic chemokine molecules, including CC chemokine ligand 2 (CCL2), in DP cells, and signaling improved crosstalk between perifollicular vessels and the DP. CCL2 expression changed cyclically in the DP vicinity and significantly decreased in aged skin, and treatment with CCL2-neutralizing antibody decreased perifollicular vascularization and suppressed DP function. These findings indicate that the crosstalk between perifollicular vessels and the DP plays a critical role in hair cycling homeostasis and aging, providing a potential target for the treatment of hair loss and other degenerative skin disorders. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Physiology Perifollicular capillary vessels Vascular remodeling Dermal papilla Vascular niche Hair follicle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Adult mammalian skin and hair follicles contain multiple stem and progenitor cell populations, which are crucial for tissue regeneration. The coordinated interaction between epithelial hair follicle stem cells residing in the bulge (Bg) [ 1 ] and mesenchymal cells located at the base of the hair follicle, known as the dermal papilla (DP), plays an important role in the morphogenesis and regeneration of hair follicles. The hair growth cycle includes three phases: anagen (rapid growth), catagen (regression), and telogen (resting period). The number of DP cells (DPCs) fluctuates throughout the cycle. However, the mechanisms underlying the maintenance of the number of DPCs in healthy follicles remain unclear. DPCs stimulate anagen phase initiation by providing instructive signals required to induce epithelial Bg cell proliferation [ 2 ]. When the number of DPCs decreases, the initiation of the anagen phase is delayed, and the telogen phase is maintained [ 3 ]. Hair thinning and loss have been reported to be partly related to the number of DPCs [ 4 ], which are essential for initiating the anagen phase and preventing hair loss. Angiocrine and angiogenic factors mediate the communication between vascular endothelial cells (ECs) and their surrounding cells during tissue development and repair. The hair follicle is surrounded by capillary vessels arising from a small set of capillaries near the DP [ 5 ]. These vessels nourish the hair follicle by delivering nutrients and oxygen, removing waste products from tissues, and establishing spatiotemporal vascular niches during development, homeostasis, and regeneration. However, these functions are impaired with aging [ 6 – 9 ]. Recently, perifollicular angiogenesis has been reported to be associated with hair cycle homeostasis [ 10 – 14 ]. Vascular endothelial growth factor (VEGF) plays an important role in regulating hair follicle vascularization and is expressed in DPCs, outer root sheath keratinocytes, and ECs. VEGF expression is upregulated during the anagen phase, and transgenic mice with increased cutaneous VEGF levels have been reported to have larger hair follicles than their wild-type littermates [ 11 ]. The vascular annulus surrounds the upper Bg throughout the hair cycle and forms a specialized perivascular niche for hair follicle stem cells [ 13 ]. Skin vasculature dysplasia is associated with several human diseases characterized by hair loss [ 15 , 16 ]. Alopecia can arise from a shortened hair cycle, resulting in thinner and shorter new hairs. Additionally, it is characterized by decreased perifollicular vascularization and downregulation of vascularization-related genes in balding scalps [ 17 ]. A recent study has shown a reciprocal interaction between ECs and DPCs, where ECs release signaling molecules to activate DPCs, which induce hair regeneration, whereas DPCs emit a signal to activate ECs and promote angiogenesis [ 18 ]. However, these interactions decrease in aged skin. The primary mechanism of action of minoxidil (MD), a hair growth-promoting drug, is likely mediated by the induction of VEGF production, which stimulates hair follicle vascularization [ 19 ]. These findings indicate that effective crosstalk between hair follicles and perifollicular vessels plays a crucial role in the morphogenesis and growth of hair follicles. However, the physiological role of spatiotemporal perifollicular vascular remodeling remains unknown. Therefore, this study aimed to examine how perifollicular vascularization flexibly changes in a spatiotemporally restricted manner to adapt to physiological changes. Particularly, this study investigated the vascular remodeling around DPCs during the hair cycle, aging, and treatment with a VEGF-neutralizing antibody or MD and testosterone (TST) and the physiological significance of crosstalk between DPCs and ECs in proper skin homeostasis. Results Changes in the distribution of capillaries around the DP based on hair cycle and aging Blood vessel localization during the hair cycle and aging was carefully observed to clarify the role of perifollicular capillary vessels around the DP. First, dorsal skin sections of VEGR1 -tandem dsRed ( Flt1 -tdsRed) mice were analyzed during the anagen (postnatal day 33 [P33]) and telogen (P44) phases of the physiological postnatal natural hair cycle to investigate the localization of capillary vessels, with vessels marked by DsRed and the panvascular marker CD31 (also known as PECAM1). During the natural hair cycle, the skin vasculature was remarkably remodeled during the anagen phase, and capillary vessels were abundant and located close to the entire hair follicle (Fig. 1 a arrowhead and Supplementary Fig. 1a). Conversely, the number of perifollicular capillary vessels surrounding the DP decreased during the telogen phase. However, other capillaries remained in the upper Bg (Fig. 1 a and Supplementary Fig. 1a). Similar findings were observed during depilation-induced synchronized adult hair cycling (Supplementary Fig. 1c). Furthermore, the distribution of capillaries was quantified (Supplementary Fig. 1d) to further characterize capillary vessel localization in relation to hair follicles (Fig. 1 b and c). The distributed capillaries were preferentially concentrated around the DP during the anagen phase (layer 3). In this analysis to evaluate capillary distribution within the tissue, vascular area (pixel) was used. However, when comparing the anagen phase and telogen phase, the tissue area undergoes drastic changes, potentially leading to overestimation of the quantitative values. Therefore, vascular percentage (%) was also quantified (see Materials and methods for details). Results similarly confirmed increased distribution in layer 3 during the anagen phase (Supplementary Fig. 1b). Second, the vascular networks of young (2 months old) and older tissues (15 months old) were compared using the dorsal skin of VEGFR1 -tdsRed mice (Fig. 1 d and Supplementary Fig. 2a) to investigate the aging of capillaries around hair follicles. Quantification data showed that the total capillary density slightly decreased in older mice. However, no statistically significant difference was observed (Fig. 1 e and f). Nevertheless, the localization of the regular vascular patterning was altered in the hair follicles of older mice. Notably, vascular density was significantly reduced in layer 3 in the older group (Fig. 1 d arrowheads) compared with that in the younger group, as determined by VEGFR1- and CD31-positive areas (Fig. 1 e and f). However, the capillary densities of layers 1 and 2 did not exert a significant effect. Furthermore, the distribution of capillaries around the DP was investigated, and the vascular density within 100 µm of the DP was analyzed. The distribution of blood vessels near the DP was significantly reduced in the older group (Fig. 1 g). Such selective changes in the capillary vessels surrounding the DP indicate that a biologically responsive population within perifollicular capillary vessels plays an important role in maintaining hair follicle tissue homeostasis. VEGF signaling regulates perifollicular niche VEGF signaling is crucial for physiological and pathological angiogenesis [ 20 , 21 ], and VEGF signaling insufficiency affects aging in various tissues [ 9 ]. During the natural hair cycle, abundant VEGF proteins were found in the DP at the anagen phase (Fig. 2 a, arrowheads), although the expression was dramatically attenuated and weak VEGF expression was maintained mainly around the Bg at the telogen phase (Fig. 2 a, arrows). Therefore, the influence of VEGF impairment on perifollicular vessel homeostasis was investigated. VEGFR1 -tdsRed mice were treated with the VEGF-neutralizing antibody bevacizumab (Bev) daily for 6 days, and immunostaining was performed (Fig. 2 b and c). Bev is a humanized monoclonal antibody against human VEGF-A that is extensively used to inhibit angiogenesis [ 22 ]. Bev treatment significantly decreased the capillary density of layer 3 (Fig. 2 b arrowhead, 2d and e). However, it did not exert a significant effect on the capillary density of layers 1 and 2 (Fig. 2 d and e). Furthermore, the vascular density within 100 µm of the DP was analyzed. The results confirmed that Bev treatment significantly reduced the distribution of blood vessels near the DP (Fig. 2 f). Therefore, VEGF signaling downregulation showed a preferential decrease in capillary vessels in the DP vicinity. The perivascular microenvironment contributes to the maintenance of stem cells. Stem and progenitor cells preferentially localize near vessels in various tissues, including the bone marrow [ 23 ], testes [ 24 ], and brain [ 25 , 26 ]. Immunofluorescence analysis was performed using Ki67 (Fig. 2 c) to test whether perivascular reduction can cause the loss of stem and progenitor cell functions in the hair follicle as in several tissues [ 27 – 29 ]. The ratio of Ki67-positive cells in the vicinity of these vessels in Bev-treated tissues decreased dramatically in VEGFR1- and CD31-positive vessels (Fig. 2 g). On the other hand, the older dorsal skin exhibited a significantly decreased number of Ki67-positive cells in the hair follicle tissue in both anagen (around the DP) and telogen (around the Bg) phases (Supplementary Fig. 2b) in parallel with a decrease of periventricular vessels. Given that the vascular environment is strongly associated with hair follicle function, the ability of the vascular niche to regulate paracrine factors that activate DPCs was investigated. Conditioned media (CM) were collected from cultured human umbilical vein endothelial cells (HUVECs), and the effects of CM (EC-CM) on cultured human follicle dermal papilla cells (HFDPCs) were investigated (Supplementary Fig. 2c-f). Incubation with EC-CM significantly increased DPC proliferation and Ki67-positive cell percentage (Supplementary Fig. 2d and e) compared with incubation with equivalent amounts of control-CM. In addition, DPCs with EC-CM increased VEGF and ALP mRNA levels (Supplementary Fig. 2f) [ 30 ]. Therefore, vascular ECs may preferentially activate DPCs. These findings indicate that the appropriate localization of perifollicular capillary vessels influences the vascular niche that regulates DP function. MD enhances vascular remodeling near the DP The potential of MD to directly enhance vascular mobilization and remodeling around hair follicles was investigated in vivo . MD was continuously topically applied in 7-week-old mice with synchronized depilation. Briefly, after 3 days of depilation, 5% MD was topically applied daily for 27 days, thick sections of the dorsal skin were prepared, and capillary vessel localization was examined by immunofluorescence staining. After continuous MD treatment, the capillary vessels were dramatically mobilized around the hair follicles (Fig. 3 a arrowhead). Particularly, the characteristic horizontal capillaries were adjacent to the DP (Fig. 3 a). Quantification data showed that the MD treatment significantly increased the capillary density of layer 3. However, no significant effect was observed on layers 1 and 2 (Fig. 3 b and c). Additionally, the vascular density within 100 µm of the DP was analyzed. MD treatment significantly increased the number of blood vessels near the DP (Fig. 3 d). These findings indicate that MD treatment significantly induces vascular localization around the DP. Next, 5% MD was topically applied daily for 8 days, and the localization of both Ki67-positive cells and capillary vessels around the DP at the anagen phase was investigated. Interestingly, MD treatment significantly increased the number of Ki67-positive cells in the vicinity of vessels near the DP (Fig. 3 e arrowhead, 3f). However, no such changes were observed around the Bg (Fig. 3 f). Additionally, MD treatment strongly increased VEGF expression at the hair follicle tip during the anagen phase, predominantly in the DP (Fig. 3 g arrowhead), where induced angiogenesis was most prominent. These findings indicate that MD directly enhances vascular mobilization around the hair follicle tip and confers an appropriate vascular niche, thereby regulating DPCs. TST treatment suppresses vascular remodeling near the DP Androgens, such as TST and dihydrotestosterone, exert their effect on human hair follicles either directly or after conversion by 5α-reductase into dihydrotestosterone, inhibiting the proliferation of DPCs, resulting in shorter hair cycle and hair loss [ 31 ]. However, the influence of androgenic alopecia on perifollicular vessels remains unknown. Therefore, the influence of TST on vascular mobilization and remodeling in hair follicles was investigated in vivo . TST, with or without MD, was topically applied daily for 25 days in mice. The TST-treated group demonstrated approximately a 10% reduction in vascular density in layer 3 (Fig. 4 a arrowhead) compared with the control group, as determined by VEGFR1- and CD31-positive areas (Fig. 4 a-c). Furthermore, the DP tissue area after TST treatment shrank significantly compared with that after the control treatment (Fig. 4 d arrowhead). However, MD treatment recovered the TST-induced reduction (Fig. 4 d, e). Therefore, TST treatment preferentially influenced the capillary vessels near the DPCs, resulting in abnormal DP growth and maintenance. However, topical MD administration partially improved TST-induced vascular abnormalities of the stem cell niche. The involvement of vascular mobilization after MD treatment helped determine whether MD directly regulates the angiogenic potential of vascular ECs. The angiogenic activities of MD and TST were investigated at the cellular level. The in vitro angiogenesis assay results showed that MD treatment significantly increased the formation and branching of tube-like structures on Matrigel (Fig. 4 f), with an associated increment in the tube length and master segments. Conversely, TST treatment significantly inhibited angiogenesis. Furthermore, MD treatment reversed TST-induced angiogenesis abnormalities (Fig. 4 g). These findings highlight the importance of further clarifying the interaction between blood vessels and DPCs, as perifollicular capillary vessels near the DP are the most responsive. Vascular EC-derived secretion factors promote DPCs function The transcriptional changes in the HFDPC culture induced by EC-CM were investigated using RNA sequencing to explore the molecular mechanism underlying the crosstalk between ECs and DPCs (Fig. 5 a). The enrichment analysis revealed that the significantly upregulated genes in the EC-CM-treated group were enriched for Gene Ontology terms, including chemokine activity, chemokine-mediated signaling pathway, and cellular response to chemokine (Fig. 5 b). Additionally, the top 10 genes significantly associated with EC-CM-treated HFDPCs were curated (Fig. 5 c). Some of these key changes were verified by quantitative polymerase chain reaction. Collectively, changes in several chemokine molecules, including CC chemokine ligand 2 (CCL2), were identified in EC-CM-treated HFDPCs (Fig. 5 d). EC-CM treatment combined with MD (ECMD-CM) significantly increased the mRNA expression level of CCL2 compared with CM treatment alone or control treatment (Fig. 5 d). Furthermore, the expression of CCL2 was specifically localized in hair follicles including DPCs, and its expression level was significantly promoted by MD treatment (Fig. 5 e). The CC family of chemokines has been implicated in the cell mobilization ability of stem cells [ 32 , 33 ]. However, their functions in DPCs remain poorly understood. Therefore, HFDPCs were cultured in the presence of CCL2 recombinant protein, and the proliferation and expression levels of DP marker genes were determined. The CCL2-containing medium significantly improved HFDPC proliferation (Fig. 5 f) and the expression levels of some marker genes, such as VEGF, ALP, and BMP2 (Fig. 5 g). Furthermore, TST treatment significantly decreased angiogenesis, as shown in the tube formation assay (Fig. 4 g), whereas CCL2 treatment reversed TST-induced angiogenesis abnormalities (Fig. 5 h, Supplementary Fig. 3a). These findings indicate that the accumulation of vascular ECs induces CCL2 expression in DPCs and that this signal transduction promotes DP function and contributes to promoting angiogenesis by acting on surrounding ECs. CCL2 regulates the crosstalk between the DP and the perifollicular vessels CCL2 expression levels in tissues subjected to hair cycle changes, aging changes, and Bev administration were investigated to clarify the importance of CCL2 in hair follicles. CCL2 expression in hair follicles increased during the anagen phase and decreased during the telogen phase (Fig. 6 a). Additionally, CCL2 expression in aged hair follicles was preferentially downregulated in the DP vicinity (Fig. 6 b). Furthermore, Bev-treated hair follicle tissues demonstrated a significant reduction in CCL2 expression near the DP (Supplementary Fig. 3b). Finally, the effect of CCL2 impairment on both perifollicular vessels and DP function was investigated. CCL2-neutralizing antibodies (CCL2nab) were intradermally injected into 7-week-old mice daily for 27 days, and immunostaining was performed (Fig. 6 c). The continuous administration of CCL2nab reduced the density of perifollicular capillary vessels (Fig. 6 c, 6 d and Supplementary Fig. 3c). The capillaries surrounding the DP were most affected (Fig. 6 e). Additionally, CCL2nab significantly decreased the DP tissue area (Fig. 6 f arrowhead and 6g), consistent with the findings indicating decreased DPC proliferation by CCL2 inhibitor (Fig. 5 f). These findings indicate that blood vessels localized near the hair follicle tip are closely related to functional changes in the DP, and CCL2 plays an important role in regulating their crosstalk. Discussion Although the tissue microenvironment is important for stem cell function, the molecular signals controlling the dynamics of skin vasculature and mechanisms underlying the changes in these vessel growth patterns during the hair cycle, aging, and MD treatment remain unknown. This study showed that the spatiotemporal localization of capillary vessels created perifollicular-specific niches that DPCs used for proper skin homeostasis. This study focused on the spatiotemporal variation of VEGF expression levels in hair follicle tissues. VEGF and its receptors are highly pleiotropic signaling pathway that acts on both vascular [ 9 ] and nonvascular cells such as DP [ 10 , 11 , 34 ]. In addition to its angiogenic activity, VEGF acts as a survival factor for newly formed blood vessels, maintaining organ-specific vascular traits and inducing certain organ-specific angiocrine factors. In our recent study, the spatiotemporal localization of VEGF during brain development constructed the microenvironment of neural stem cells from the growth state to the differentiation state [ 25 ]. VEGF mRNA expression levels in hair follicles have been reported to be temporally upregulated during the anagen growth phase in the induced adult hair cycle and the physiological first postnatal hair cycle [ 10 ]. This study confirmed that VEGF expression is spatiotemporally controlled between the anagen phase (abundant VEGF protein predominantly in the DP) and the telogen phase (faint VEGF protein predominantly in the Bg). Additionally, continuous MD treatment did not influence the whole perifollicular ECs but enhanced angiogenesis near the hair follicle tip, consistent with VEGF expression localization. Therefore, the vascular network surrounding the hair follicle tip may preferentially exhibit remodeling and reconstruction in response to various environmental changes, and capillary mobilization to the DP may regulate DPC functions. DPCs are specialized mesenchymal cells located in the skin that regulate hair follicle growth and serve as a reservoir of multipotent stem cells [ 35 ]. DPCs regulate the hair cycle by secreting growth factors and cytokines. However, the molecular mechanism underlying the crosstalk between DPCs and ECs remains unknown. Recent reports based on single-cell RNA-seq analysis have shown that the reciprocal interaction between DPCs and ECs regulates hair regeneration and angiogenesis [ 18 ]. This study showed that perifollicular mobilization near the DP sensitively responded to physiological changes. Additionally, both aging and treatment with a VEGF-neutralizing antibody or TST decreased capillary vessels near the DP. However, continuous MD application significantly increased them, even during the telogen phase. Furthermore, endothelial vessels supplied signals to strengthen chemokine expression in the DP, thereby stimulating DPC proliferation and function, and MD treatment greatly increased this expression. Vascular endothelial niches play an extensive role. Tissue-specific ECs mastermind these complex tasks by providing the repopulating cells with stimulatory and inhibitory growth factors, morphogenesis, extracellular matrix, and chemokines. The CC chemokine family has been implicated in stem cell mobilization processes, such as migration, homing, and retention [ 36 , 37 ]. CCL2 has been reported to enhance pluripotency and improve the culture of mouse and human induced pluripotent stem cells [ 38 , 39 ]. Additionally, this chemokine factor regulates the self-renewal and proliferation of neural stem cells [ 40 ]. Furthermore, CCL2 mediates the crosstalk between cancer cells and stromal fibroblasts to control breast cancer stem cells [ 41 ]. CCL2 recombinant protein has been reported to increase multipotency and regenerative potential in the skin organoid culture system [ 42 ]. Therefore, CCL2 participates in regulating the multipotency of various tissue stem cells. Furthermore, the CC chemokine family is involved in angiogenesis [ 43 ]. CCL2 has been considered an angiogenic and angiocrine chemokine [ 36 , 37 , 44 – 46 ], and VEGF mediates CCL2-induced angiogenesis [ 47 ]. This study showed that EC-CM stimulated CCL2 expression in DPCs. Incubation with EC-CM significantly increased DPC proliferation and VEGF and ALP expression levels. Therefore, incubation of DPCs with EC-CM treated with MD further increased CCL2 expression in DPCs. These findings indicate that CCL2 in the DP is an autocrine regulator of DPC proliferation and function and serves as a paracrine mediator of communication with perifollicular capillary vessels to control angiogenesis. The findings of this study deepen our understanding of vascular remodeling around the hair follicle and provide valuable insights into the treatment of skin disorders. Materials and methods Chemicals MD and TST were purchased from Tokyo Chemical Industry Co., Ltd. and FUJIFILM Wako Pure Chemical Corp., respectively. Five % of MD solution was obtained by dissolving MD powder in a mixture of water, ethanol, phosphoric acid, and 1,3-butylene glycol at room temperature for 30 min using a magnetic stirrer. The vehicle consists of all the ingredients solution except for MD. The VEGF-A monoclonal antibody bevacizumab (#HY-P9906, Avastin), anti-CCL2-neutralizing antibody (A2132), and recombinant human CCL2 (#AF-300-04) were purchased from MedChemExpress Co., Ltd., Selleck Inc., and Pepro Tech, Inc., respectively. Mice All experimental procedures involving mice and their care were conducted in accordance with the ARRIVE guidelines and approved by the Committee on the Ethics of Animal Experiments in Kobe Gakuin University (A23-31). Every effort was made to minimize the suffering of the mice. VEGFR1 ( Flt1 )-tdsRed BAC Tg mouse had been developed previously [ 48 ]. All mice were crossed with C57BL/6J mice more than 10× and maintained. Wild-type mice were purchased from Japan SLC (Shizuoka, Japan). Immunohistochemistry The ketamine and xylazine at 50 and 10 mg / kg were used, and scarification of the mice via cervical dislocation, and the dorsal skin was removed, cleansed with ice-cold phosphate-buffered saline (PBS). Dorsal skin samples were treated with a microwave oven 700 w for 30 s, followed by 30 min on ice in 4% paraformaldehyde. After cryoprotection in 30% sucrose, we embedded fixed tissue in OCT compound (Sakura Tissue-Tek) and prepared sections on a cryostat. Cryostat sections (150 µm) were treated with blocking buffer (10% donkey serum and 0.1% Triton X-100, pH 7.4) for 1 h at room temperature, followed by incubation with primary antibodies diluted in the same buffer overnight at 4°C. Furthermore, the sections were washed thrice with 0.1% phosphate-buffered saline with Tween® detergent (PBST) for 10 min and incubated for 1 h at room temperature with secondary antibodies. Next, we washed them again in PBST for 10 min thrice at room temperature and mounted them under a cover glass with a mounting medium. We used the following primary antibodies: GFP (rabbit, 1/1000; MBL #598), GFP (rat, 1/1000; nacalai tesque #04404-8), VEGF (rabbit, 1/500; Abcam #ab46154), Ki67/MKI67 (rabbit, 1/500; Novus Biologicals #NB110-89717), Keratin 15 (Chicken, 1/500; Biolegend #833904), CD31 (rat, 1/500; BD Biosciences #557355), CD31 (hamster, 1/200; merckmillipore #MAB1398Z), and CCL2 (hamster, 1/100; Thermo Fisher Scientific #14-7096-81). Images were acquired on a confocal microscope (FV3000, Olympus) or a fluorescent microscope (IX81, Olympus). CellSens and Metamorph software suites were used to acquire all confocal and fluorescent microscope images, respectively. Images were processed using Adobe Photoshop. The capillary density was quantified based on previous reports [ 49 ]. VEGFR1 -DsRed + and CD31 + vasculature area was defined in ImageJ as shown in Supplementary Fig. 1d, and subsequently quantified. Briefly, using DAPI staining as a morphological guide, we divided the perifollicular vessels in each image into three regions: Layer 1 (vicinity of infundibulum and isthmus) was defined as the upper area of the superior border of the bulge; Layer 2 (vicinity of Bg) was defined as characteristic slight protrusion, and further confirmed by a dense aggregation of small and intense cell nuclei; Layer 3 (vicinity of DP) was defined as the region extending from the inferior border of the bulge downward to the base of the hair follicle, encompassing the DP. Each layer was enclosed into a region of interest (ROI) by the freehand drawing tool of ImageJ. 8-bit grayscale images were generated for each channel, and a uniform threshold was applied to distinguish specific staining from background. Following threshold application, a binary mask was created where the area (pixel) of the perifollicular vessels above the threshold were calculated in total area and each layer. Because the hair cycle is accompanied by marked changes in skin area, quantification based solely on area was insufficient. Therefore, we also evaluated the perifollicular vessel ratio (%) of each layer to account for hair cycle-dependent changes, as shown in Supplementary Fig. 1b. Multiple fields of view were analyzed for each replicate during measurement. To further characterize changes in capillary vessels surrounding the DP, we analyzed the vasculature within a 100 µm diameter region centered on the DP. The DP was identified based on its characteristic morphology based on DAPI staining. Using the oval selection tool, a circular ROI with a 100 µm diameter was created and saved. Within this ROI, the area occupied by VEGFR1-DsRed + and CD31 + capillary vessels were quantified. To define “the ratio of Ki67 + cells in the vicinity of vessels,” we analyzed the Z-projected images where each frame had a resolution of 512 × 512 pixels. If the distance between the Ki67 + cells and blood vessels was below 60 µm in the overlaid in Z-projected images, the Ki67 + cells were defined as “Ki67 + cells in the vicinity of vessels.” The Ki67 + cells were quantified using ImageJ on 40× or 60× skin fields. Dermal papilla size was defined by the K15-negative area and quantified using ImageJ, after the skin sections were stained with DAPI and K15. Cell culture ECs-derived conditioned medium (EC-CM) preparation HUVECs were purchased from Lonza Japan and cultured at 37°C under a 5% CO 2 atmosphere, and were maintained in EGM-2 medium (#CC-3162, Lonza) with additive factor kit. HFDPCs were obtained from PromoCell and cultivated in Follicle Dermal Papilla Cell Basal Medium (PromoCell, Heidelberg, Germany). All experiments were conducted using cells at passages 4–6. For HUVEC subculture, trypsin/EDTA (0.025%/0.01 mM) was used. Meanwhile, HFDPCs were detached using the PromoCell Detach Kit according to the manufacturer’s instructions. To prepare ECs-derived conditioned medium (EC-CM) [ 50 ], passage 4 (P4) HUVECs were cultured to 50%-60% confluency in a 100-mm cell culture dish in 10 mL of EGM-2 medium with or without MD for 24 h. Then, the medium was replaced with 10 mL of fresh EGM-2 medium for 24 h, and the EC-CM was harvested. After centrifugation at 3000 min-1for 10 min to remove the cell debris, the EC-CM was filtered through a 0.22 µm filter (Millipore) and directly used for HFDPC culture (P4). The EGM-2 medium (10 mL) incubated for 24 h in a culture dish without cells was used as the control. Cell proliferation assay Before the experiments, cells were seeded in 96-well plates and incubated for 24 h at 37°C under a 5% CO 2 atmosphere. Cells were then treated with EC-CM, ECMD-CM, or CCL2 recombinant protein for 24 h under the same culture conditions. The proliferation rate was measured using the Cell Counting Kit-8 (#343–07623, DOJINDO) and an absorbance meter according to the manufacturer’s protocol. Real-time qPCR (RT-qPCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen) in line with the manufacturer’s instructions. In addition, we synthesized cDNA from 250 ng of RNA by using a QuantiTect Retrotranscriptase reaction kit (Qiagen) and conducted qPCR by using SYBR green labeling (SYBR Premix Ex TaqII, Takara) and a TP850 Real-Time PCR System (Takara), with glyceraldehyde-3-phosphate dehydrogenase expression as the internal control. All individual sample reactions were conducted thrice. The relative fold change in target gene expression was then calculated according to the ∆∆Ct method. Supplementary Table 1 lists the qPCR primer pairs. RNA sequencing and data analysis We used the Direct-Zol RNA miniprep kit (Zymo Research) and the Agilent 2200 TapeStation to extract total RNA and determine its quality, respectively. Sequencing libraries were prepared using the SMART-Seq v4 Ultra Low Input Kit for Sequencing (TaKaRa) and sequenced on an Illumina Novaseq 6000 to generate 150 bp paired-end reads. Reads were aligned using DRAGEN with the human reference genome (GENCODE/GRCh38 [hg38]). We used differentially expressed genes in GO term analysis, which then employed Expression Miner 2.0 to find enriched functional annotations. Tube formation assay The procedures were performed as previously described [ 49 ]. Briefly, 48-well plates were coated with Matrigel (Corning) and incubated for 30 min at 37°C under a 5% CO 2 atmosphere. HUVECs were plated on the gel and cultured in the medium with or without MD, TST, and CCL2 recombinant protein. After 6 h of additional incubation, we acquired microscopic images of the tubes by using a phase-contrast microscope. Five images were then captured per well and analyzed using ImageJ software. HUVECs from passage 4 were used for this assay. All experimental conditions were replicated thrice. Topical treatment with MD or TST Seven-week-old female VEGFR1 -dsRed mice in the telogen stages were used, and the dorsal areas of each mouse were synchronized to the anagen stage by depilation. After 3 days, we then randomly grouped the animals. From this time point, one group was treated with vehicle or 5% MD topically for 27 days to evaluate MD application, and another group received vehicle or 0.05% TST or 0.05% TST plus 5% MD topical treatment at 9 AM for 25 days to evaluate TST application. Thereafter, the dorsal skin samples were dissected and used for immunostaining analyses. Administration of VEGF- and CCL2-neutralizing antibody The 5mg/kg Bevacizumab, VEGF-neutralizing antibody, was intraperitoneally injected during the telogen phase (P38) for 6 consecutive days, after which the back skin was collected for immunostaining. To evaluate the effect of CCL2, mice were treated topically with vehicle or 5% MD on back skin for 25 consecutive days, with the intradermal administration of vehicle or 60 µg anti-CCL2-neutralizing antibody every three days. Quantification and statistical analysis Differences between two groups were evaluated using an unpaired Student’s t-test. For comparisons among three or more groups, statistical analysis was performed using one-way analysis of variance (ANOVA). When a significant difference was detected, pairwise comparisons between groups were conducted using the Least Significant Difference (LSD) post hoc test. Data are presented as mean ± Standard Error of the Mean (SEM), and p < 0.05 was considered statistically significant. Declarations Acknowledgments: The authors thank Ryohei Arai, Akiko Takaoka, and Toru Nagahama for their helpful discussions. We also thank Yoshimi Abe, Sae Asayama, Ayaka Iwasaki, and Sakiho Koyama for excellent technical assistance. Author Contributions Statement: Conceptualization: K.M.; Methodology: Y.Z., M.M., H.M., M.I., and K.M.; Investigation: Y.Z., S.T., M.K., R.K., M.K-S., and K.M.; Writing—Original Draft: K.M.; Writing—Review & Editing: all the authors.; Supervision: T.O., M.T., M.I., M.E., and K.M. Funding: None declared. Data availability statement: Sequence data that support the findings of this study have been deposited in the NCBI with the primary accession code GSE282648. Declaration of interest: The author Akinari Abe is regular employee of Taisho Pharmaceutical Co., Ltd. However, the funder did not have any additional role in the study design, data analysis, decision to publish, or manuscript preparation. 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F., Kang, S., Tumbar T. Skin vasculature and hair follicle cross-talking associated with stem cell activation and tissue homeostasis. eLife 19 , e45977 (2019). Asayama, S., Igarashi. T., Abe, Y., Iwasaki A., Kubo, M., Ikeda, A., Akiyama, K., Okamoto, T., Yagi, M., Niki, Y., Ando, H., Ichihashi, M., Mizutani, K. Rosae multiflorae fructus extracts regulate the differentiation and vascular endothelial cell-mediated proliferation of keratinocytes. Bioscience, Biotechnology, and Biochemistry 89 , 750-760 (2025). Additional Declarations Competing interest reported. The author Akinari Abe is regular employee of Taisho Pharmaceutical Co., Ltd. However, the funder did not have any additional role in the study design, data analysis, decision to publish, or manuscript preparation. This does not alter our adherence to Nature Portfolio policies on sharing data and materials. All other authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Supplementary Files SupplementaryFigureslegends.docx SupplementaryFigure1.pdf SupplementaryFigure2.pdf SupplementaryFigure3.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 Oct, 2025 Submission checks completed at journal 09 Oct, 2025 First submitted to journal 09 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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07:28:40","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1938629,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/42705ffb39282ef9f0f94949.pdf"},{"id":93466964,"identity":"41163afb-1cf8-47cd-9c54-abaabd619e8e","added_by":"auto","created_at":"2025-10-14 07:36:40","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128254,"visible":true,"origin":"","legend":"","description":"","filename":"118bb068ee9a481196a5d1dac3cc25761structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/97163eb87b65d2e1a3ef9743.xml"},{"id":93465578,"identity":"d6366db4-99ae-4d64-b926-78f3828af8d9","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143738,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/69669057c1bb6ac5215d5a0b.html"},{"id":93465558,"identity":"f44ebf1a-1406-46f9-868d-7971529d06ac","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3987036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe capillaries surrounding the DP undergo preferential changes during the hair cycle and aging. \u003c/strong\u003e(a) Immunofluorescence of CD31 in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin. The vascular pattern of at least 20 murine tissues analyzed during the anagen and telogen phases was typical. Higher magnification is shown in Supplemental Fig. 1a. (b, c) Quantification of vascular distribution as detected by VEGFR1- (b) and CD31- (c) positive cells within the epidermis and dermis tissues divided into three regions from the epidermal surface to the dermal papilla (DP) base at both anagen (A) and telogen (T) phases (layer 1 [L1]: vicinity of infundibulum and isthmus; layer 2 [L2]: vicinity of bulge (Bg); layer 3 [L3]: vicinity of DP); 10 independent sections from 3 individual dorsal skin samples. (d) Analysis of the vascular pattern in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin at 2 and 15 months of age. The arrowhead indicates the preferential decreased vascularization in aged tissue around the DP. (e, f) Quantification of vascular distribution as detected by VEGFR1- (e) and CD31- (f) positive cells within the epidermis and dermis tissues divided into three regions from the epidermal surface to the DP base (layer 1 [L1]: vicinity of infundibulum and isthmus; layer 2 [L2]: vicinity of Bg; layer 3 [L3]: vicinity of DP); 10 independent sections from 3 individual dorsal skin samples. (g) Comparison of VEGFR1- or CD31-positive cells distributed within 100 µm of the DP between 2 and 15 months of age.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/665485a874c01b8596404e88.png"},{"id":93466543,"identity":"0689d45d-e8c0-409e-8b7e-5ae15bbdf5e5","added_by":"auto","created_at":"2025-10-14 07:28:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5221253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe capillaries surrounding the DP undergo preferential regulation by treatment with a VEGF-neutralizing antibody. \u003c/strong\u003e(a) Immunofluorescence of VEGF in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin. Distinct vascularization around the dermal papilla (DP) dependent on the hair cycle, consistent with the VEGF expression level. The arrowhead indicates the strong VEGF expression around the DP at the anagen phase, whereas the arrow denotes the weak expression level around the bulge (Bg) during the telogen phase.\u003cstrong\u003e \u003c/strong\u003e(b, c) Treatment of \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice with VEGF-neutralizing antibody (bevacizumab; Bev) daily for 10 days, followed by immunostaining with CD31 (b, c) and Ki67 (c) during the telogen phase.\u003cstrong\u003e \u003c/strong\u003e(d, e) Quantification of vascular distribution as detected by VEGFR1- (d) and CD31- (e) positive cells within the epidermis and dermis tissues divided into three regions from the epidermal surface to the DP base (layer 1 [L1]: vicinity of infundibulum and isthmus; layer 2 [L2]: vicinity of Bg; layer 3 [L3]: vicinity of DP); 10 independent sections from 3 individual dorsal skin samples. (f) Analysis of VEGFR1- or CD31-positive cells distributed within 100 µm of the DP in Bev-treated dorsal skin tissues.\u003cstrong\u003e \u003c/strong\u003e(g) Quantification of the ratio of Ki67-positive cells in the vicinity of capillary vessels, calculated from eight independent sections from each dorsal skin sample.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/78261c430797d96160e90739.png"},{"id":93465565,"identity":"29337cf6-9845-40d5-95b9-c2d1426fc7ee","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5691617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTopical minoxidil (MD) application enhances vascular remodeling near the DP. \u003c/strong\u003e(a) Treatment of \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice with 5% MD solution daily for 27 days, followed by immunostaining with CD31 during the telogen phase. Higher magnification in the lower panel (merge view). (b, c) Quantification of vascular distribution as detected by VEGFR1- (b) and CD31- (c) positive cells within the epidermis and dermis tissues divided into three regions from the epidermal surface to the dermal papilla (DP) base (layer 1 [L1]: vicinity of infundibulum and isthmus; layer 2 [L2]: vicinity of bulge (Bg); layer 3 [L3]: vicinity of DP); 10 independent sections from 3 individual dorsal skin samples. (d) Analysis of VEGFR1- or CD31-positive cells distributed within 100 µm of the DP in MD-treated dorsal skin tissues. (e) Immunofluorescence of Ki67 in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin after continuous treatment with 5% MD solution for 8 days. (f) Quantification of Ki67-positive cells in the vicinity of capillary vessels around the DP and Bg, calculated from eight independent sections from each individual dorsal skin sample. (g) Immunofluorescence of VEGF in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin after treatment with continuous 5% MD solution for 8 days (upper panel; anagen phase) or for 27 days (lower panel; telogen phase).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/d41c5ace6ea66a35e060811c.png"},{"id":93465563,"identity":"09f205f4-3bbe-4a3a-a643-2e74b3f61a8c","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2601773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTopical TST application suppresses vascular remodeling near the DP. \u003c/strong\u003e(a) Treatment of \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice with 0.05% testosterone (TST) or 0.05% TST with 5% MD daily for 25 days, followed by immunostaining with CD31 during the telogen phase. Quantification of VEGFR1- (b) or CD31-positive (c) vascular distribution in dorsal skin tissues within the epidermis and dermis tissues divided into three regions from the epidermal surface to the dermal papilla (DP) base (layer 1 [L1]: vicinity of infundibulum and isthmus; layer 2 [L2]: vicinity of bulge (Bg); layer 3 [L3]: vicinity of DP); 10 independent sections from 3 individual dorsal skin samples. (d) Immunofluorescence of K15 after continuous treatment with 0.05% TST or 0.05% TST with 5% MD daily for 25 days to determine the tissue areas (e) of hair germ (HG) and DP. (f,g) Treatment of human umbilical vein endothelial cells (HUVECs) on Matrigel with MD in the presence or absence of TST for 6 h, followed by a tube formation assay. Representative images (f) of the untreated and treated groups, quantified tube length, and number of branch points and junctions.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/7b9fd469990aa0d455ced35e.png"},{"id":93465568,"identity":"8c1d8988-ddca-4309-ba09-22d4064d2cae","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3127700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPC-derived chemokine regulates DPCs and the ECs. \u003c/strong\u003e(a) RNA sequencing analysis of expression profiles of HUVEC-derived conditioned medium (EC-CM)-treated human follicle dermal papilla cells (HFDPCs) and control medium-treated HFDPCs (control HFDPCs). (b) Gene Ontology analysis indicated signaling pathways enriched in HFDPCs treated with HUVEC-CM. (c) Changes in some chemokine molecules. (d) Comparison of candidate chemokine gene expression among EC-CM-treated HFDPCs, EC in the presence of MD (ECMD)-CM-treated HFDPCs, and control HFDPCs by quantitative polymerase chain reaction (qPCR); n = 3 independent experiments. (e) Immunofluorescence of CCL2 and CCR2 in the dorsal skin of \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice during the telogen phase after continuous treatment with 5% MD solution daily for 27 days. (f) Treatment of HUVECs with CCL2 recombinant protein for 24 h, followed by a cell proliferation assay. (g) Treatment of HUVECs with CCL2 recombinant protein for 24 h, followed by qPCR analysis of dermal papilla (DP) function markers. (h) Treatment of HUVECs on Matrigel with CCL2 recombinant protein in the presence or absence of TST for 6 h, followed by a tube formation assay. Representative images of the untreated and treated groups, quantified tube length, and number of branch points and junctions were shown in Supplemental Fig. 3a.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/1faf5331db0c9ecc84f8591e.png"},{"id":93466963,"identity":"2ce19a15-ff4e-4661-835a-0eb78eb40645","added_by":"auto","created_at":"2025-10-14 07:36:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6723094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL2 impairment influences EC and DP function. (\u003c/strong\u003ea) Immunofluorescence of CCL2 in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin at the anagen and telogen phases. (b) Immunofluorescence of CCL2 in \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed dorsal skin at the anagen phase at 2 and 15 months of age. (c) Topical application of vehicle or 5% MD on the back skin of \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice daily for 25 days, with or without intradermal administration of CCL2-neutralizing antibody (CCL2nab), followed by immunostaining with CD31. (d) Quantification of VEGFR1-positive vascular distribution in dorsal skin tissues within the epidermis and dermis tissues divided into three regions from the epidermal surface to the dermal papilla (DP) base (layer 1 [L1]: vicinity of infundibulum and isthmus; layer 2 [L2]: vicinity of bulge (Bg); layer 3 [L3]: vicinity of DP); 10 independent sections from 3 individual dorsal skin samples. (e) Analysis of VEGFR1- or CD31-positive cells distributed within 100 µm of the DP in CCL2nab-treated dorsal skin tissues. (f) Immunofluorescence of K15 after continuous treatment with CCL2nab daily for 25 days to determine the HG and DP tissue areas (g).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/b50732d76f1713f9bce8f03c.png"},{"id":106344320,"identity":"f1d8e59f-3418-4756-92be-a698f2ce1565","added_by":"auto","created_at":"2026-04-07 16:13:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30919950,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/4467b3a2-fd3a-4832-8397-a47d460c9e64.pdf"},{"id":93465557,"identity":"2a4010ed-3f04-471c-bf34-b66a721c8656","added_by":"auto","created_at":"2025-10-14 07:20:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16738,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureslegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/a5962369e7f3efc9cc7ac80d.docx"},{"id":93465562,"identity":"b5ff4ca5-fd2c-4fb0-a528-4e7861b6edcc","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1899291,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/ae2c8a1f4be64ef8b7c863fa.pdf"},{"id":93465561,"identity":"f6c45a2f-f455-47da-ad86-1645c4ef7ba1","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":896112,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/c98d46fe592daaea2c656ab2.pdf"},{"id":93465564,"identity":"fe412673-7a97-47a9-a56c-ff1d63da624d","added_by":"auto","created_at":"2025-10-14 07:20:40","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":596504,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7791533/v1/215bc858114753c8866237a4.pdf"}],"financialInterests":"Competing interest reported. The author Akinari Abe is regular employee of Taisho Pharmaceutical Co., Ltd. However, the funder did not have any additional role in the study design, data analysis, decision to publish, or manuscript preparation. This does not alter our adherence to Nature Portfolio policies on sharing data and materials. All other authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.","formattedTitle":"Preferential Crosstalk between Perifollicular Capillary Vessels and Dermal Papilla Cells during Hair Cycling Homeostasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdult mammalian skin and hair follicles contain multiple stem and progenitor cell populations, which are crucial for tissue regeneration. The coordinated interaction between epithelial hair follicle stem cells residing in the bulge (Bg) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and mesenchymal cells located at the base of the hair follicle, known as the dermal papilla (DP), plays an important role in the morphogenesis and regeneration of hair follicles. The hair growth cycle includes three phases: anagen (rapid growth), catagen (regression), and telogen (resting period). The number of DP cells (DPCs) fluctuates throughout the cycle. However, the mechanisms underlying the maintenance of the number of DPCs in healthy follicles remain unclear. DPCs stimulate anagen phase initiation by providing instructive signals required to induce epithelial Bg cell proliferation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When the number of DPCs decreases, the initiation of the anagen phase is delayed, and the telogen phase is maintained [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hair thinning and loss have been reported to be partly related to the number of DPCs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which are essential for initiating the anagen phase and preventing hair loss.\u003c/p\u003e\u003cp\u003eAngiocrine and angiogenic factors mediate the communication between vascular endothelial cells (ECs) and their surrounding cells during tissue development and repair. The hair follicle is surrounded by capillary vessels arising from a small set of capillaries near the DP [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These vessels nourish the hair follicle by delivering nutrients and oxygen, removing waste products from tissues, and establishing spatiotemporal vascular niches during development, homeostasis, and regeneration. However, these functions are impaired with aging [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, perifollicular angiogenesis has been reported to be associated with hair cycle homeostasis [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Vascular endothelial growth factor (VEGF) plays an important role in regulating hair follicle vascularization and is expressed in DPCs, outer root sheath keratinocytes, and ECs. VEGF expression is upregulated during the anagen phase, and transgenic mice with increased cutaneous VEGF levels have been reported to have larger hair follicles than their wild-type littermates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The vascular annulus surrounds the upper Bg throughout the hair cycle and forms a specialized perivascular niche for hair follicle stem cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Skin vasculature dysplasia is associated with several human diseases characterized by hair loss [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Alopecia can arise from a shortened hair cycle, resulting in thinner and shorter new hairs. Additionally, it is characterized by decreased perifollicular vascularization and downregulation of vascularization-related genes in balding scalps [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A recent study has shown a reciprocal interaction between ECs and DPCs, where ECs release signaling molecules to activate DPCs, which induce hair regeneration, whereas DPCs emit a signal to activate ECs and promote angiogenesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, these interactions decrease in aged skin. The primary mechanism of action of minoxidil (MD), a hair growth-promoting drug, is likely mediated by the induction of VEGF production, which stimulates hair follicle vascularization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings indicate that effective crosstalk between hair follicles and perifollicular vessels plays a crucial role in the morphogenesis and growth of hair follicles. However, the physiological role of spatiotemporal perifollicular vascular remodeling remains unknown.\u003c/p\u003e\u003cp\u003eTherefore, this study aimed to examine how perifollicular vascularization flexibly changes in a spatiotemporally restricted manner to adapt to physiological changes. Particularly, this study investigated the vascular remodeling around DPCs during the hair cycle, aging, and treatment with a VEGF-neutralizing antibody or MD and testosterone (TST) and the physiological significance of crosstalk between DPCs and ECs in proper skin homeostasis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChanges in the distribution of capillaries around the DP based on hair cycle and aging\u003c/h2\u003e\u003cp\u003eBlood vessel localization during the hair cycle and aging was carefully observed to clarify the role of perifollicular capillary vessels around the DP.\u003c/p\u003e\u003cp\u003eFirst, dorsal skin sections of \u003cem\u003eVEGR1\u003c/em\u003e-tandem dsRed (\u003cem\u003eFlt1\u003c/em\u003e-tdsRed) mice were analyzed during the anagen (postnatal day 33 [P33]) and telogen (P44) phases of the physiological postnatal natural hair cycle to investigate the localization of capillary vessels, with vessels marked by DsRed and the panvascular marker CD31 (also known as PECAM1). During the natural hair cycle, the skin vasculature was remarkably remodeled during the anagen phase, and capillary vessels were abundant and located close to the entire hair follicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea arrowhead and Supplementary Fig.\u0026nbsp;1a). Conversely, the number of perifollicular capillary vessels surrounding the DP decreased during the telogen phase. However, other capillaries remained in the upper Bg (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;1a). Similar findings were observed during depilation-induced synchronized adult hair cycling (Supplementary Fig.\u0026nbsp;1c). Furthermore, the distribution of capillaries was quantified (Supplementary Fig.\u0026nbsp;1d) to further characterize capillary vessel localization in relation to hair follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c). The distributed capillaries were preferentially concentrated around the DP during the anagen phase (layer 3). In this analysis to evaluate capillary distribution within the tissue, vascular area (pixel) was used. However, when comparing the anagen phase and telogen phase, the tissue area undergoes drastic changes, potentially leading to overestimation of the quantitative values. Therefore, vascular percentage (%) was also quantified (see Materials and methods for details). Results similarly confirmed increased distribution in layer 3 during the anagen phase (Supplementary Fig.\u0026nbsp;1b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSecond, the vascular networks of young (2 months old) and older tissues (15 months old) were compared using the dorsal skin of \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;2a) to investigate the aging of capillaries around hair follicles. Quantification data showed that the total capillary density slightly decreased in older mice. However, no statistically significant difference was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and f). Nevertheless, the localization of the regular vascular patterning was altered in the hair follicles of older mice. Notably, vascular density was significantly reduced in layer 3 in the older group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed arrowheads) compared with that in the younger group, as determined by VEGFR1- and CD31-positive areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and f). However, the capillary densities of layers 1 and 2 did not exert a significant effect. Furthermore, the distribution of capillaries around the DP was investigated, and the vascular density within 100 \u0026micro;m of the DP was analyzed. The distribution of blood vessels near the DP was significantly reduced in the older group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Such selective changes in the capillary vessels surrounding the DP indicate that a biologically responsive population within perifollicular capillary vessels plays an important role in maintaining hair follicle tissue homeostasis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVEGF signaling regulates perifollicular niche\u003c/h3\u003e\n\u003cp\u003eVEGF signaling is crucial for physiological and pathological angiogenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and VEGF signaling insufficiency affects aging in various tissues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. During the natural hair cycle, abundant VEGF proteins were found in the DP at the anagen phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, arrowheads), although the expression was dramatically attenuated and weak VEGF expression was maintained mainly around the Bg at the telogen phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, arrows). Therefore, the influence of VEGF impairment on perifollicular vessel homeostasis was investigated. \u003cem\u003eVEGFR1\u003c/em\u003e-tdsRed mice were treated with the VEGF-neutralizing antibody bevacizumab (Bev) daily for 6 days, and immunostaining was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and c). Bev is a humanized monoclonal antibody against human VEGF-A that is extensively used to inhibit angiogenesis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Bev treatment significantly decreased the capillary density of layer 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb arrowhead, 2d and e). However, it did not exert a significant effect on the capillary density of layers 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and e). Furthermore, the vascular density within 100 \u0026micro;m of the DP was analyzed. The results confirmed that Bev treatment significantly reduced the distribution of blood vessels near the DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Therefore, VEGF signaling downregulation showed a preferential decrease in capillary vessels in the DP vicinity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe perivascular microenvironment contributes to the maintenance of stem cells. Stem and progenitor cells preferentially localize near vessels in various tissues, including the bone marrow [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], testes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and brain [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Immunofluorescence analysis was performed using Ki67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) to test whether perivascular reduction can cause the loss of stem and progenitor cell functions in the hair follicle as in several tissues [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The ratio of Ki67-positive cells in the vicinity of these vessels in Bev-treated tissues decreased dramatically in VEGFR1- and CD31-positive vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). On the other hand, the older dorsal skin exhibited a significantly decreased number of Ki67-positive cells in the hair follicle tissue in both anagen (around the DP) and telogen (around the Bg) phases (Supplementary Fig.\u0026nbsp;2b) in parallel with a decrease of periventricular vessels. Given that the vascular environment is strongly associated with hair follicle function, the ability of the vascular niche to regulate paracrine factors that activate DPCs was investigated. Conditioned media (CM) were collected from cultured human umbilical vein endothelial cells (HUVECs), and the effects of CM (EC-CM) on cultured human follicle dermal papilla cells (HFDPCs) were investigated (Supplementary Fig.\u0026nbsp;2c-f). Incubation with EC-CM significantly increased DPC proliferation and Ki67-positive cell percentage (Supplementary Fig.\u0026nbsp;2d and e) compared with incubation with equivalent amounts of control-CM. In addition, DPCs with EC-CM increased VEGF and ALP mRNA levels (Supplementary Fig.\u0026nbsp;2f) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, vascular ECs may preferentially activate DPCs. These findings indicate that the appropriate localization of perifollicular capillary vessels influences the vascular niche that regulates DP function.\u003c/p\u003e\n\u003ch3\u003eMD enhances vascular remodeling near the DP\u003c/h3\u003e\n\u003cp\u003eThe potential of MD to directly enhance vascular mobilization and remodeling around hair follicles was investigated \u003cem\u003ein vivo\u003c/em\u003e. MD was continuously topically applied in 7-week-old mice with synchronized depilation. Briefly, after 3 days of depilation, 5% MD was topically applied daily for 27 days, thick sections of the dorsal skin were prepared, and capillary vessel localization was examined by immunofluorescence staining. After continuous MD treatment, the capillary vessels were dramatically mobilized around the hair follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea arrowhead). Particularly, the characteristic horizontal capillaries were adjacent to the DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Quantification data showed that the MD treatment significantly increased the capillary density of layer 3. However, no significant effect was observed on layers 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c). Additionally, the vascular density within 100 \u0026micro;m of the DP was analyzed. MD treatment significantly increased the number of blood vessels near the DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These findings indicate that MD treatment significantly induces vascular localization around the DP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, 5% MD was topically applied daily for 8 days, and the localization of both Ki67-positive cells and capillary vessels around the DP at the anagen phase was investigated. Interestingly, MD treatment significantly increased the number of Ki67-positive cells in the vicinity of vessels near the DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee arrowhead, 3f). However, no such changes were observed around the Bg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Additionally, MD treatment strongly increased VEGF expression at the hair follicle tip during the anagen phase, predominantly in the DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg arrowhead), where induced angiogenesis was most prominent. These findings indicate that MD directly enhances vascular mobilization around the hair follicle tip and confers an appropriate vascular niche, thereby regulating DPCs.\u003c/p\u003e\n\u003ch3\u003eTST treatment suppresses vascular remodeling near the DP\u003c/h3\u003e\n\u003cp\u003eAndrogens, such as TST and dihydrotestosterone, exert their effect on human hair follicles either directly or after conversion by 5α-reductase into dihydrotestosterone, inhibiting the proliferation of DPCs, resulting in shorter hair cycle and hair loss [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, the influence of androgenic alopecia on perifollicular vessels remains unknown. Therefore, the influence of TST on vascular mobilization and remodeling in hair follicles was investigated \u003cem\u003ein vivo\u003c/em\u003e. TST, with or without MD, was topically applied daily for 25 days in mice. The TST-treated group demonstrated approximately a 10% reduction in vascular density in layer 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea arrowhead) compared with the control group, as determined by VEGFR1- and CD31-positive areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). Furthermore, the DP tissue area after TST treatment shrank significantly compared with that after the control treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed arrowhead). However, MD treatment recovered the TST-induced reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). Therefore, TST treatment preferentially influenced the capillary vessels near the DPCs, resulting in abnormal DP growth and maintenance. However, topical MD administration partially improved TST-induced vascular abnormalities of the stem cell niche.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe involvement of vascular mobilization after MD treatment helped determine whether MD directly regulates the angiogenic potential of vascular ECs. The angiogenic activities of MD and TST were investigated at the cellular level. The in vitro angiogenesis assay results showed that MD treatment significantly increased the formation and branching of tube-like structures on Matrigel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), with an associated increment in the tube length and master segments. Conversely, TST treatment significantly inhibited angiogenesis. Furthermore, MD treatment reversed TST-induced angiogenesis abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg).\u003c/p\u003e\u003cp\u003eThese findings highlight the importance of further clarifying the interaction between blood vessels and DPCs, as perifollicular capillary vessels near the DP are the most responsive.\u003c/p\u003e\n\u003ch3\u003eVascular EC-derived secretion factors promote DPCs function\u003c/h3\u003e\n\u003cp\u003eThe transcriptional changes in the HFDPC culture induced by EC-CM were investigated using RNA sequencing to explore the molecular mechanism underlying the crosstalk between ECs and DPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The enrichment analysis revealed that the significantly upregulated genes in the EC-CM-treated group were enriched for Gene Ontology terms, including chemokine activity, chemokine-mediated signaling pathway, and cellular response to chemokine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Additionally, the top 10 genes significantly associated with EC-CM-treated HFDPCs were curated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Some of these key changes were verified by quantitative polymerase chain reaction. Collectively, changes in several chemokine molecules, including CC chemokine ligand 2 (CCL2), were identified in EC-CM-treated HFDPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). EC-CM treatment combined with MD (ECMD-CM) significantly increased the mRNA expression level of \u003cem\u003eCCL2\u003c/em\u003e compared with CM treatment alone or control treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Furthermore, the expression of CCL2 was specifically localized in hair follicles including DPCs, and its expression level was significantly promoted by MD treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe CC family of chemokines has been implicated in the cell mobilization ability of stem cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, their functions in DPCs remain poorly understood. Therefore, HFDPCs were cultured in the presence of CCL2 recombinant protein, and the proliferation and expression levels of DP marker genes were determined. The CCL2-containing medium significantly improved HFDPC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) and the expression levels of some marker genes, such as VEGF, ALP, and BMP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Furthermore, TST treatment significantly decreased angiogenesis, as shown in the tube formation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), whereas CCL2 treatment reversed TST-induced angiogenesis abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh, Supplementary Fig.\u0026nbsp;3a). These findings indicate that the accumulation of vascular ECs induces CCL2 expression in DPCs and that this signal transduction promotes DP function and contributes to promoting angiogenesis by acting on surrounding ECs.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCCL2 regulates the crosstalk between the DP and the perifollicular vessels\u003c/h2\u003e\u003cp\u003eCCL2 expression levels in tissues subjected to hair cycle changes, aging changes, and Bev administration were investigated to clarify the importance of CCL2 in hair follicles. CCL2 expression in hair follicles increased during the anagen phase and decreased during the telogen phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Additionally, CCL2 expression in aged hair follicles was preferentially downregulated in the DP vicinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Furthermore, Bev-treated hair follicle tissues demonstrated a significant reduction in CCL2 expression near the DP (Supplementary Fig.\u0026nbsp;3b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFinally, the effect of CCL2 impairment on both perifollicular vessels and DP function was investigated. CCL2-neutralizing antibodies (CCL2nab) were intradermally injected into 7-week-old mice daily for 27 days, and immunostaining was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The continuous administration of CCL2nab reduced the density of perifollicular capillary vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;3c). The capillaries surrounding the DP were most affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Additionally, CCL2nab significantly decreased the DP tissue area (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef arrowhead and 6g), consistent with the findings indicating decreased DPC proliferation by CCL2 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). These findings indicate that blood vessels localized near the hair follicle tip are closely related to functional changes in the DP, and CCL2 plays an important role in regulating their crosstalk.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough the tissue microenvironment is important for stem cell function, the molecular signals controlling the dynamics of skin vasculature and mechanisms underlying the changes in these vessel growth patterns during the hair cycle, aging, and MD treatment remain unknown. This study showed that the spatiotemporal localization of capillary vessels created perifollicular-specific niches that DPCs used for proper skin homeostasis.\u003c/p\u003e\u003cp\u003eThis study focused on the spatiotemporal variation of VEGF expression levels in hair follicle tissues. VEGF and its receptors are highly pleiotropic signaling pathway that acts on both vascular [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and nonvascular cells such as DP [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition to its angiogenic activity, VEGF acts as a survival factor for newly formed blood vessels, maintaining organ-specific vascular traits and inducing certain organ-specific angiocrine factors. In our recent study, the spatiotemporal localization of VEGF during brain development constructed the microenvironment of neural stem cells from the growth state to the differentiation state [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. VEGF mRNA expression levels in hair follicles have been reported to be temporally upregulated during the anagen growth phase in the induced adult hair cycle and the physiological first postnatal hair cycle [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This study confirmed that VEGF expression is spatiotemporally controlled between the anagen phase (abundant VEGF protein predominantly in the DP) and the telogen phase (faint VEGF protein predominantly in the Bg). Additionally, continuous MD treatment did not influence the whole perifollicular ECs but enhanced angiogenesis near the hair follicle tip, consistent with VEGF expression localization. Therefore, the vascular network surrounding the hair follicle tip may preferentially exhibit remodeling and reconstruction in response to various environmental changes, and capillary mobilization to the DP may regulate DPC functions.\u003c/p\u003e\u003cp\u003eDPCs are specialized mesenchymal cells located in the skin that regulate hair follicle growth and serve as a reservoir of multipotent stem cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. DPCs regulate the hair cycle by secreting growth factors and cytokines. However, the molecular mechanism underlying the crosstalk between DPCs and ECs remains unknown. Recent reports based on single-cell RNA-seq analysis have shown that the reciprocal interaction between DPCs and ECs regulates hair regeneration and angiogenesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This study showed that perifollicular mobilization near the DP sensitively responded to physiological changes. Additionally, both aging and treatment with a VEGF-neutralizing antibody or TST decreased capillary vessels near the DP. However, continuous MD application significantly increased them, even during the telogen phase. Furthermore, endothelial vessels supplied signals to strengthen chemokine expression in the DP, thereby stimulating DPC proliferation and function, and MD treatment greatly increased this expression.\u003c/p\u003e\u003cp\u003eVascular endothelial niches play an extensive role. Tissue-specific ECs mastermind these complex tasks by providing the repopulating cells with stimulatory and inhibitory growth factors, morphogenesis, extracellular matrix, and chemokines. The CC chemokine family has been implicated in stem cell mobilization processes, such as migration, homing, and retention [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. CCL2 has been reported to enhance pluripotency and improve the culture of mouse and human induced pluripotent stem cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, this chemokine factor regulates the self-renewal and proliferation of neural stem cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, CCL2 mediates the crosstalk between cancer cells and stromal fibroblasts to control breast cancer stem cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. CCL2 recombinant protein has been reported to increase multipotency and regenerative potential in the skin organoid culture system [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, CCL2 participates in regulating the multipotency of various tissue stem cells. Furthermore, the CC chemokine family is involved in angiogenesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. CCL2 has been considered an angiogenic and angiocrine chemokine [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and VEGF mediates CCL2-induced angiogenesis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This study showed that EC-CM stimulated CCL2 expression in DPCs. Incubation with EC-CM significantly increased DPC proliferation and VEGF and ALP expression levels. Therefore, incubation of DPCs with EC-CM treated with MD further increased CCL2 expression in DPCs. These findings indicate that CCL2 in the DP is an autocrine regulator of DPC proliferation and function and serves as a paracrine mediator of communication with perifollicular capillary vessels to control angiogenesis. The findings of this study deepen our understanding of vascular remodeling around the hair follicle and provide valuable insights into the treatment of skin disorders.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eChemicals\u003c/h2\u003e\u003cp\u003eMD and TST were purchased from Tokyo Chemical Industry Co., Ltd. and FUJIFILM Wako Pure Chemical Corp., respectively. Five % of MD solution was obtained by dissolving MD powder in a mixture of water, ethanol, phosphoric acid, and 1,3-butylene glycol at room temperature for 30 min using a magnetic stirrer. The vehicle consists of all the ingredients solution except for MD. The VEGF-A monoclonal antibody bevacizumab (#HY-P9906, Avastin), anti-CCL2-neutralizing antibody (A2132), and recombinant human CCL2 (#AF-300-04) were purchased from MedChemExpress Co., Ltd., Selleck Inc., and Pepro Tech, Inc., respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003e All experimental procedures involving mice and their care were conducted in accordance with the ARRIVE guidelines and approved by the Committee on the Ethics of Animal Experiments in Kobe Gakuin University (A23-31). Every effort was made to minimize the suffering of the mice. \u003cem\u003eVEGFR1\u003c/em\u003e (\u003cem\u003eFlt1\u003c/em\u003e)-tdsRed BAC Tg mouse had been developed previously [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. All mice were crossed with C57BL/6J mice more than 10\u0026times; and maintained. Wild-type mice were purchased from Japan SLC (Shizuoka, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eThe ketamine and xylazine at 50 and 10 mg / kg were used, and scarification of the mice via cervical dislocation, and the dorsal skin was removed, cleansed with ice-cold phosphate-buffered saline (PBS). Dorsal skin samples were treated with a microwave oven 700 w for 30 s, followed by 30 min on ice in 4% paraformaldehyde. After cryoprotection in 30% sucrose, we embedded fixed tissue in OCT compound (Sakura Tissue-Tek) and prepared sections on a cryostat. Cryostat sections (150 \u0026micro;m) were treated with blocking buffer (10% donkey serum and 0.1% Triton X-100, pH 7.4) for 1 h at room temperature, followed by incubation with primary antibodies diluted in the same buffer overnight at 4\u0026deg;C. Furthermore, the sections were washed thrice with 0.1% phosphate-buffered saline with Tween\u0026reg; detergent (PBST) for 10 min and incubated for 1 h at room temperature with secondary antibodies. Next, we washed them again in PBST for 10 min thrice at room temperature and mounted them under a cover glass with a mounting medium. We used the following primary antibodies: GFP (rabbit, 1/1000; MBL #598), GFP (rat, 1/1000; nacalai tesque #04404-8), VEGF (rabbit, 1/500; Abcam #ab46154), Ki67/MKI67 (rabbit, 1/500; Novus Biologicals #NB110-89717), Keratin 15 (Chicken, 1/500; Biolegend #833904), CD31 (rat, 1/500; BD Biosciences #557355), CD31 (hamster, 1/200; merckmillipore #MAB1398Z), and CCL2 (hamster, 1/100; Thermo Fisher Scientific #14-7096-81). Images were acquired on a confocal microscope (FV3000, Olympus) or a fluorescent microscope (IX81, Olympus). CellSens and Metamorph software suites were used to acquire all confocal and fluorescent microscope images, respectively. Images were processed using Adobe Photoshop.\u003c/p\u003e\u003cp\u003eThe capillary density was quantified based on previous reports [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. \u003cem\u003eVEGFR1\u003c/em\u003e-DsRed\u003csup\u003e+\u003c/sup\u003e and CD31\u003csup\u003e+\u003c/sup\u003e vasculature area was defined in ImageJ as shown in Supplementary Fig.\u0026nbsp;1d, and subsequently quantified. Briefly, using DAPI staining as a morphological guide, we divided the perifollicular vessels in each image into three regions: Layer 1 (vicinity of infundibulum and isthmus) was defined as the upper area of the superior border of the bulge; Layer 2 (vicinity of Bg) was defined as characteristic slight protrusion, and further confirmed by a dense aggregation of small and intense cell nuclei; Layer 3 (vicinity of DP) was defined as the region extending from the inferior border of the bulge downward to the base of the hair follicle, encompassing the DP. Each layer was enclosed into a region of interest (ROI) by the freehand drawing tool of ImageJ. 8-bit grayscale images were generated for each channel, and a uniform threshold was applied to distinguish specific staining from background. Following threshold application, a binary mask was created where the area (pixel) of the perifollicular vessels above the threshold were calculated in total area and each layer. Because the hair cycle is accompanied by marked changes in skin area, quantification based solely on area was insufficient. Therefore, we also evaluated the perifollicular vessel ratio (%) of each layer to account for hair cycle-dependent changes, as shown in Supplementary Fig.\u0026nbsp;1b. Multiple fields of view were analyzed for each replicate during measurement.\u003c/p\u003e\u003cp\u003eTo further characterize changes in capillary vessels surrounding the DP, we analyzed the vasculature within a 100 \u0026micro;m diameter region centered on the DP. The DP was identified based on its characteristic morphology based on DAPI staining. Using the oval selection tool, a circular ROI with a 100 \u0026micro;m diameter was created and saved. Within this ROI, the area occupied by VEGFR1-DsRed\u003csup\u003e+\u003c/sup\u003e and CD31\u003csup\u003e+\u003c/sup\u003e capillary vessels were quantified.\u003c/p\u003e\u003cp\u003eTo define \u0026ldquo;the ratio of Ki67\u003csup\u003e+\u003c/sup\u003e cells in the vicinity of vessels,\u0026rdquo; we analyzed the Z-projected images where each frame had a resolution of 512 \u0026times; 512 pixels. If the distance between the Ki67\u003csup\u003e+\u003c/sup\u003e cells and blood vessels was below 60 \u0026micro;m in the overlaid in Z-projected images, the Ki67\u003csup\u003e+\u003c/sup\u003e cells were defined as \u0026ldquo;Ki67\u003csup\u003e+\u003c/sup\u003e cells in the vicinity of vessels.\u0026rdquo; The Ki67\u003csup\u003e+\u003c/sup\u003e cells were quantified using ImageJ on 40\u0026times; or 60\u0026times; skin fields. Dermal papilla size was defined by the K15-negative area and quantified using ImageJ, after the skin sections were stained with DAPI and K15.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCell culture ECs-derived conditioned medium (EC-CM) preparation\u003c/h2\u003e\u003cp\u003eHUVECs were purchased from Lonza Japan and cultured at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, and were maintained in EGM-2 medium (#CC-3162, Lonza) with additive factor kit. HFDPCs were obtained from PromoCell and cultivated in Follicle Dermal Papilla Cell Basal Medium (PromoCell, Heidelberg, Germany). All experiments were conducted using cells at passages 4\u0026ndash;6. For HUVEC subculture, trypsin/EDTA (0.025%/0.01 mM) was used. Meanwhile, HFDPCs were detached using the PromoCell Detach Kit according to the manufacturer\u0026rsquo;s instructions. To prepare ECs-derived conditioned medium (EC-CM) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], passage 4 (P4) HUVECs were cultured to 50%-60% confluency in a 100-mm cell culture dish in 10 mL of EGM-2 medium with or without MD for 24 h. Then, the medium was replaced with 10 mL of fresh EGM-2 medium for 24 h, and the EC-CM was harvested. After centrifugation at 3000 min-1for 10 min to remove the cell debris, the EC-CM was filtered through a 0.22 \u0026micro;m filter (Millipore) and directly used for HFDPC culture (P4). The EGM-2 medium (10 mL) incubated for 24 h in a culture dish without cells was used as the control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCell proliferation assay\u003c/h2\u003e\u003cp\u003eBefore the experiments, cells were seeded in 96-well plates and incubated for 24 h at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Cells were then treated with EC-CM, ECMD-CM, or CCL2 recombinant protein for 24 h under the same culture conditions. The proliferation rate was measured using the Cell Counting Kit-8 (#343\u0026ndash;07623, DOJINDO) and an absorbance meter according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eReal-time qPCR (RT-qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated using the RNeasy Mini Kit (Qiagen) in line with the manufacturer\u0026rsquo;s instructions. In addition, we synthesized cDNA from 250 ng of RNA by using a QuantiTect Retrotranscriptase reaction kit (Qiagen) and conducted qPCR by using SYBR green labeling (SYBR Premix Ex TaqII, Takara) and a TP850 Real-Time PCR System (Takara), with glyceraldehyde-3-phosphate dehydrogenase expression as the internal control. All individual sample reactions were conducted thrice. The relative fold change in target gene expression was then calculated according to the ∆∆Ct method. Supplementary Table\u0026nbsp;1 lists the qPCR primer pairs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing and data analysis\u003c/h2\u003e\u003cp\u003eWe used the Direct-Zol RNA miniprep kit (Zymo Research) and the Agilent 2200 TapeStation to extract total RNA and determine its quality, respectively. Sequencing libraries were prepared using the SMART-Seq v4 Ultra Low Input Kit for Sequencing (TaKaRa) and sequenced on an Illumina Novaseq 6000 to generate 150 bp paired-end reads. Reads were aligned using DRAGEN with the human reference genome (GENCODE/GRCh38 [hg38]). We used differentially expressed genes in GO term analysis, which then employed Expression Miner 2.0 to find enriched functional annotations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eTube formation assay\u003c/h2\u003e\u003cp\u003eThe procedures were performed as previously described [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Briefly, 48-well plates were coated with Matrigel (Corning) and incubated for 30 min at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. HUVECs were plated on the gel and cultured in the medium with or without MD, TST, and CCL2 recombinant protein. After 6 h of additional incubation, we acquired microscopic images of the tubes by using a phase-contrast microscope. Five images were then captured per well and analyzed using ImageJ software. HUVECs from passage 4 were used for this assay. All experimental conditions were replicated thrice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTopical treatment with MD or TST\u003c/h2\u003e\u003cp\u003eSeven-week-old female \u003cem\u003eVEGFR1\u003c/em\u003e-dsRed mice in the telogen stages were used, and the dorsal areas of each mouse were synchronized to the anagen stage by depilation. After 3 days, we then randomly grouped the animals. From this time point, one group was treated with vehicle or 5% MD topically for 27 days to evaluate MD application, and another group received vehicle or 0.05% TST or 0.05% TST plus 5% MD topical treatment at 9 AM for 25 days to evaluate TST application. Thereafter, the dorsal skin samples were dissected and used for immunostaining analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAdministration of VEGF- and CCL2-neutralizing antibody\u003c/h2\u003e\u003cp\u003eThe 5mg/kg Bevacizumab, VEGF-neutralizing antibody, was intraperitoneally injected during the telogen phase (P38) for 6 consecutive days, after which the back skin was collected for immunostaining. To evaluate the effect of CCL2, mice were treated topically with vehicle or 5% MD on back skin for 25 consecutive days, with the intradermal administration of vehicle or 60 \u0026micro;g anti-CCL2-neutralizing antibody every three days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e\u003cp\u003eDifferences between two groups were evaluated using an unpaired Student\u0026rsquo;s t-test. For comparisons among three or more groups, statistical analysis was performed using one-way analysis of variance (ANOVA). When a significant difference was detected, pairwise comparisons between groups were conducted using the Least Significant Difference (LSD) post hoc test. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of the Mean (SEM), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Ryohei Arai, Akiko Takaoka, and Toru Nagahama for their helpful discussions. We also thank Yoshimi Abe, Sae Asayama, Ayaka Iwasaki, and Sakiho Koyama for excellent technical assistance.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: K.M.; Methodology: Y.Z., M.M., H.M., M.I., and K.M.; Investigation: Y.Z., S.T., M.K., R.K., M.K-S., and K.M.; Writing\u0026mdash;Original Draft: K.M.; Writing\u0026mdash;Review \u0026amp; Editing: all the authors.; Supervision: T.O., M.T., M.I., M.E., and K.M.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the NCBI with the primary accession code GSE282648.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author Akinari Abe is regular employee of Taisho Pharmaceutical Co., Ltd. However, the funder did not have any additional role in the study design, data analysis, decision to publish, or manuscript preparation. This does not alter our adherence to Nature Portfolio policies on sharing data and materials. All other authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eC. Blanpain, W.E. Lowry, A. Geoghegan, L. Polak, E. Fuchs. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 635-648 (2004).\u003c/li\u003e\n \u003cli\u003eRompolas, P., Deschene, E.R., Zito, G., Gonzalez, D.G., Saotome, I., Haberman, A.M., Greco, V. 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T., Abe, Y., Iwasaki A., Kubo, M., Ikeda, A., Akiyama, K., Okamoto, T., Yagi, M., Niki, Y., Ando, H., Ichihashi, M., Mizutani, K. \u003cem\u003eRosae multiflorae \u003c/em\u003efructus extracts regulate the differentiation and vascular endothelial cell-mediated proliferation of keratinocytes. \u003cem\u003eBioscience, Biotechnology, and Biochemistry\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e, 750-760 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Perifollicular capillary vessels, Vascular remodeling, Dermal papilla, Vascular niche, Hair follicle","lastPublishedDoi":"10.21203/rs.3.rs-7791533/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7791533/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe tissue-specific capillary system supports the unique function of each organ and dynamically remodels in response to local requirements. In this study, we found that perifollicular vascularization flexibly adjusts to accommodate physiological changes. Notably, not all capillary vessels responded and migrated equally. However, those around the dermal papilla (DP) exhibited preferential mobilization. Treatment with minoxidil, a hair growth agent, significantly increased perifollicular vessel mobilization around the DP, whereas it was inhibited in experimental models of tissue aging, such as those involving vascular endothelial growth factor-neutralizing antibody or testosterone treatment, similar to the physiological tissue aging process. Furthermore, vascular endothelial cells triggered the expression of angiogenic chemokine molecules, including CC chemokine ligand 2 (CCL2), in DP cells, and signaling improved crosstalk between perifollicular vessels and the DP. CCL2 expression changed cyclically in the DP vicinity and significantly decreased in aged skin, and treatment with CCL2-neutralizing antibody decreased perifollicular vascularization and suppressed DP function. These findings indicate that the crosstalk between perifollicular vessels and the DP plays a critical role in hair cycling homeostasis and aging, providing a potential target for the treatment of hair loss and other degenerative skin disorders.\u003c/p\u003e","manuscriptTitle":"Preferential Crosstalk between Perifollicular Capillary Vessels and Dermal Papilla Cells during Hair Cycling Homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 07:20:35","doi":"10.21203/rs.3.rs-7791533/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T10:03:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-09T14:21:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-09T12:50:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c8f59c7-ffb3-4fe7-9775-68d7730288f9","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56194139,"name":"Biological sciences/Cell biology"},{"id":56194140,"name":"Health sciences/Diseases"},{"id":56194141,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-07T16:06:58+00:00","versionOfRecord":{"articleIdentity":"rs-7791533","link":"https://doi.org/10.1038/s41598-026-46001-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-01 15:58:46","publishedOnDateReadable":"April 1st, 2026"},"versionCreatedAt":"2025-10-14 07:20:35","video":"","vorDoi":"10.1038/s41598-026-46001-2","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46001-2","workflowStages":[]},"version":"v1","identity":"rs-7791533","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7791533","identity":"rs-7791533","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}