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Human skin vasculature features a unique anatomy in close proximity to the skin appendages and acts as a gatekeeper for constitutive lymphocyte trafficking to the skin. Approximating such structural complexity and functionality in 3D skin models is an outstanding tissue engineering challenge. In this study, we leverage the capabilities of the digital-light-processing bioprinting to generate an anatomically-relevant and miniaturized 3D skin-on-a-chip (3D-SoC) model in the size of a 6 mm punch biopsy. The 3D-SoC contains a perfusable vascular network resembling the superficial vascular plexus of the skin and closely surrounding bioengineered hair follicles. The perfusion capabilities of the 3D-SoC enables the circulation of immune cells, and high-resolution imaging of the immune cell-endothelial cell interactions, namely tethering, rolling, and extravasation in real-time. Moreover, the vascular pattern in 3D-SoC captures the physiological range of shear rates found in cutaneous blood vessels and allows for studying the effect of shear rate on T cell trafficking. In 3D-SoC, as expected, in vitro -polarized T helper 1 (Th1) cells show a stronger attachment on the vasculature compared to naïve T cells. Both naïve and T cells exhibit higher retention in the low-shear zones in the early stages (<5 min) of T cell attachment. Interestingly, at later stages T cell retention rate becomes independent of the shear rate. The attached Th1 cells further transmigrate from the vessel walls to the extracellular space and migrate toward the bioengineered hair follicles and interfollicular epidermis. When the epidermis is not present, Th1 cell migration toward the epidermis is significantly hindered, underscoring the role of epidermal signals on T cell infiltration. Our data validates the capabilities of 3D-SoC model to study the interactions between immune cells and skin vasculature in the context of epidermal signals. The biopsy-sized 3D-SoC model in this study represents a new level of anatomical and cellular complexity, and brings us a step closer to generating a truly functional human skin with its tissue-specific vasculature and appendages in the presence of circulating immune cells., (Creative Commons Attribution license.)

Central centrifugal cicatricial alopecia (CCCA) is a scarring alopecia that primarily affects women of African descent. Although histopathological features of CCCA have been described, the pathophysiology of this disease remains unclear. To better understand the components of CCCA pathophysiology, we evaluated the composition of the inflammatory infiltrate, the distribution of Langerhans cells (LCs), and the relationship between fibrosis and perifollicular vessel distribution. Our data indicate that CCCA is associated with a CD4-predominant T-cell infiltrate with increased LCs extending into the lower hair follicle. Fibroplasia associated with follicular scarring displaces blood vessels away from the outer root sheath epithelium. These data indicate that CCCA is an inflammatory scarring alopecia with unique pathophysiologic features that differentiate it from other lymphocytic scarring processes., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dermal sheath (DS) is located at the outermost border of hair follicles, comprising the connective tissue sheath of these follicles; DS cells are known to contribute to hair cycling and follicle neogenesis. However, the mechanisms by which DS cells contribute to hair formation are currently unclear. We investigated the global transcriptional profile of human DS cells in early passaged culture, compared with those of human dermal papilla cells (DP cells) and dermal fibroblasts. Vascular related genes were highly expressed in DS cells, and expression of the multi-ligand receptor, CD36, was significantly higher in DS cells than in DP cells. Further analyses with whole-mount imaging technique showed that dense networks of blood capillaries were formed in the DS of human anagen hair follicles, whereas regression of blood capillaries was observed in telogen and catagen hair follicles. We found that CD36-expressing cells were present in populations of DS cells, but were rarely observed in populations of DP cells and fibroblasts. Furthermore, our results indicated that CD36-expressing DS cells may participate in angiogenesis. Therefore, we concluded that CD36-expressing DS cells may modulate blood capillaries in hair follicles, in association with hair cycling., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Because of the shortage of human skin for research purposes, porcine skin has been used as a model of human skin. The aim of this study was to identify the region of German Landrace pig skin that could be used as the best possible substitute for human abdominal skin. Porcine samples were collected from the ear, flank, back and caudal abdomen; human abdominal skin samples were excised during plastic surgery. Histological and ultrastructural assessments were carried out on the epidermis and dermis, with emphasis on the dermo-epidermal interface length, dermo-epidermal thickness ratio as well as densities of; hair follicles, arrector pili muscles, blood vessels and sweat glands. In the pig, the barrier function of the four anatomical regions was assessed. Results showed that both histologically and ultrastructurally, all four regions of porcine skin were similar to human skin. These include the shapes of keratinocytes, structure of cell contacts and presence of Weibel Palade bodies in endothelial cells. Other parameters such as the thickness of epidermis, the thickness of stratum basale, spinosum and granulosum and the number of cell layers in the stratum corneum were similar in human abdominal and in all four regions of porcine skin. However, there were also significant differences especially in the thickness of the stratum corneum, the dermo-epidermal interface length and the blood vessel density., (© 2019 Blackwell Verlag GmbH.)

Background: Cilostazol, a phosphodiesterase 3 (PDE3) inhibitor, increases the intracellular level of cyclic adenosine monophosphate to cause vasodilation. Topical application of cilostazol is reported to improve local blood flow and enhance wound healing; however, its effect on human hair follicles is unknown., Objective: The purpose of this study was to determine the effect of cilostazol on hair growth., Methods: We investigated the expression of PDE3 in human dermal papilla cells (DPCs), outer root sheath cells (ORSCs), and hair follicles. The effects of cilostazol on DPC and ORSC proliferation were evaluated using BrdU and WST-1 assays. The expression of various growth factors in DPCs was investigated by growth factor antibody array. Additionally, hair shaft elongation was measured using ex vivo hair follicle organ cultures, and anagen induction was evaluated in C57BL/6 mice. Finally, the effects of cilostazol on vessel formation and activation of the mitogen-activated protein kinase pathway were evaluated., Results: We confirmed high mRNA and protein expression of PDE3 in human DPCs. Cilostazol not only enhanced the proliferation of human DPCs but also regulated the secretion of several growth factors responsible for hair growth. Furthermore, it promoted hair shaft elongation ex vivo, with increased proliferation of matrix keratinocytes. Cilostazol also accelerated anagen induction by stimulating vessel formation and upregulating the levels of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase, and P38 after its topical application in C57BL/6 mice., Conclusion: Our results show that cilostazol promotes hair growth and may serve as a therapeutic agent for the treatment of alopecia., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Platelet-rich plasma (PRP) is an autologous concentration of human platelets in plasma. In this paper, we aimed to investigate the effect of PRP on hair growth., Methods: Platelet-rich plasma and platelet-poor plasma were prepared by sterile centrifugation and injected into shaved dorsal skin of mice (n = 10). Saline injection was used in the control group. The length of randomly plucked hairs was measured at 8, 13, 18 days after PRP injection. Histological examination was preformed to observe the histologic changes of skins. The immunohistochemistry analysis of CD31 was performed to detect the changes of hair length and formation of new vessels., Results: At 13 and 18 days after the last injection, the hair length of mice in PRP group (4.24 ± 0.60 and 8.29 ± 0.48 mm, respectively) was significantly longer compared with the control group (3.70 ± 0.52 and 7.21 ± 0.64 mm, p < 0.05). No significant difference in the hair length was found between the PPP group and the control (p > 0.05). In addition, the number of CD31-positive vessel in the PRP group (9.90 ± 0.60) was more than that in the control group (8.60 ± 2.34, p < 0.05)., Conclusion: Platelet-rich plasma might promote hair length growth and increase the number of hair follicles by inducing angiogenesis.

Follicular unit transplantation is the most commonly performed technique in modern restorative hair transplantation surgery. It relies on the acquisition of intact follicular units from microdissected scalp skin strips and their subsequent transplantation into the recipient regions affected by alopecia. Ideally, the translocation of follicular units from the balding-resistant areas of the scalp (usually the occipital region) to the recipient site should not result in any morphological change in the grafts. Nevertheless, the insults associated with surgical intervention present grafted follicles to mechanical and chemical cues differently from those of the physiological steady-state conditions in undamaged skin. This disruption of the normal follicular microenvironment might alter important aspects of hair biology in grafts, for example, hair cycle and pigmentation, and, in turn, could lead to differences in hair appearance, eventually culminating in a diminished esthetical outcome of the surgery. In this study, the authors analyzed native and grafted scalp hair follicles (HFs) from 2 patients who had undergone follicular unit transplantation surgeries formerly. Scanning electron microscopy and light microscopy-based histomorphometry revealed a marked enlargement of follicular structures in the grafts with a concomitant increase in hair shaft diameter. Immunohistological staining confirmed a thickening of the dermal sheath in transplanted HFs that also harbored a denser vascular network. Taken together, these results show that the grafted HFs analyzed were subjected to marked morphological changes during their residence in the recipient site and that this phenomenon is associated with a modulation of follicular vascularization.

Although thymosin beta 4 (Tβ4) is known to play a role in hair growth, its mechanism of action is unclear. We examined the levels of key genes in a Tβ4 epidermal-specific over-expressing mouse model and Tβ4 global knockout mouse model to explore how Tβ4 affects hair growth. By depilation and histological examination of the skin, we confirmed the effect of Tβ4 on hair growth, the number of hair shafts and hair follicle (HF) structure. The mRNA and protein expression of several genes involved in hair growth were detected by real-time PCR and western blotting, respectively. Changes in the expression of β-catenin and Lef-1, the two key molecules in the Wnt signaling pathway, were similar to the changes observed in Tβ4 expression. We also found that compared to the control mice, the mRNA and protein expression of MMP-2 and VEGF were increased in the Tβ4 over-expressing mice, while the level of E-cadherin (E-cad) remained the same. Further, in the Tβ4 global knockout mice, the mRNA and protein levels of MMP-2 and VEGF decreased dramatically and the level of E-cad was stable. Based on the above results, we believe that Tβ4 may regulate the levels of VEGF and MMP-2 via the Wnt/β-catenin/Lef-1 signaling pathway to influence the growth of blood vessels around HFs and to activate cell migration. Tβ4 may have potential for the treatment of hair growth problems in adults, and its effects should be further confirmed in future studies.

Mouse models of various types of inflammatory skin disease are often accompanied by increased dermal angiogenesis. The C3H/HeJ inbred strain spontaneously develops alopecia areata (AA), a cell mediated autoimmune disorder that can be controllably expanded using full thickness skin grafts to young unaffected mice. This provides a reproducible and progressive model for AA in which the vascularization of the skin can be examined. Mice receiving skin grafts from AA or normal mice were evaluated at 5, 10, 15, and 20 weeks after engraftment. Lymphatics are often overlooked as they are small slit-like structures above the hair follicle that resemble artifact-like separation of collagen bundles with some fixatives. Lymphatics are easily detected using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) by immunohistochemistry to label their endothelial cells. Using LYVE1, there were no changes in distribution or numbers of lymphatics although they were more prominent (dilated) in the mice with AA. Lyve1 transcripts were not significantly upregulated except at 10 weeks after skin grafting when clinical signs of AA first become apparent. Other genes involved with vascular growth and dilation or movement of immune cells were dysregulated, mostly upregulated. These findings emphasize aspects of AA not commonly considered and provide potential targets for therapeutic intervention., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

Aims: Recently, immature adipocyte lineage cells have been suggested as a potential hair-growth stimulator. Diverse studies have been attempted to find methods for the preconditioning of immature adipocyte lineage cells. The present study investigates the effect of conditioned medium (CM) from vitamin D3 (Vd3) pre-activated preadipocytes on hair-growth ability., Main Methods: To test the effect of CM from Vd3 pre-activated preadipocytes on hair-growth efficiency in mice, we compared the differences in hair regenerated after injecting CM from mouse preadipocytes pre-activated with or without Vd3. Next, to determine the regulating factors, the VEGF level was measured by ELISA and angiogenesis level was evaluated by IHC. Finally, the signaling mechanism was investigated by inhibitor kinase assay and western blotting., Key Findings: The CM from Vd3 pre-activated preadipocyte injection markedly promoted the ability of hair regeneration in mice. The VEGF levels were increased by Vd3 treatment in vitro and the CM from Vd3 pre-activated preadipocytes significantly increased the angiogenesis in vivo, suggesting the involvement of angiognensis in the hair regeneration induced by CM from pre-activated preadipocytes. In signaling study, Vd3-enhanced VEGF production was reduced by an ERK1/2 inhibitor and the level of ERK1/2 phosphorylation was increased by treatment with Vd3., Significance: This has been the first report on CM from Vd3 pre-activated preadipocyte displaying stimulatory effects on hair growth via the enhancement of angiogenesis in a hairless-induced C57BL/6 mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Chronic telogen effluvium (CTE), a poorly understood condition, can be confused with or may be a prodrome to female pattern hair loss (FPHL). The pathogenesis of both is related to follicle cycle shortening and possibly to blood supply changes., Aim: To analyze a number of histomorphometric and immunohistochemical findings through vascular endothelial growth factor (VEGF), Ki-67, and CD31 immunostaining in scalp biopsies of 20 patients with CTE, 17 patients with mild FPHL and 9 controls., Methods: Ki-67 index and VEGF optical density were analyzed at the follicular outer sheath using ImageJ software. CD31 microvessel density was assessed by a Chalkley grid., Results: Significant follicle miniaturization and higher density of nonanagen follicles were found in FPHL, compared with patients with CTE and controls. Ki-67+ index correlated positively with FPHL histological features. The FPHL group showed the highest VEGF optical density, followed by the CTE and control groups. No differences were found in CD31 microvessel density between the three groups., Conclusions: Histomorphometric results establish CTE as a distinct disorder, separate from FPHL from its outset. Its pathogenic mechanisms are also distinct. These findings support the proposed mechanism of 'immediate telogen release' for CTE, leading to cycle synchronization. For FPHL, accelerated anagen follicular mitotic rates and, thus, higher Ki-67 and VEGF values, would leave less time for differentiation, resulting in hair miniaturization., (© 2014 British Association of Dermatologists.)

The skin protects mammals from insults, infection and dehydration and enables thermoregulation and sensory perception. Various skin-resident cells carry out these diverse functions. Constant turnover of cells and healing upon injury necessitate multiple reservoirs of stem cells. Thus, the skin provides a model for studying interactions between stem cells and their microenvironments, or niches. Advances in genetic and imaging tools have brought new findings about the lineage relationships between skin stem cells and their progeny and about the mutual influences between skin stem cells and their niches. Such knowledge may offer novel avenues for therapeutics and regenerative medicine.

Vascular endothelial growth factors (VEGFs) play important roles in neovascularization, tissue development, and angiogenesis. In this study, changes in VEGF expression patterns and microvessel density (MVD), and their correlations, were investigated during hair follicle development in epidermal appendages of Liaoning cashmere goats. Polyclonal antibodies to VEGF and microvessels were used for monthly immunohistochemical examinations of normal skin specimens from adult female goats for one year. VEGF was expressed in the hair bulb of primary and secondary hair follicles, the outer and inner root sheaths, sebaceous glands (ductal and secretory portions), eccrine sweat glands (ductal and secretory portions), and the epidermis. Abundant expression of VEGF was observed in the follicular basement membrane zone surrounding the bulb matrix and in ductal and secretory portions of eccrine sweat glands. The change in VEGFs in primary hair follicles showed a bimodal pattern, with the first peak observed from March to May, and the second in August. Maximal expression in secondary hair follicles occurred in May and August. Therefore, VEGF expression in primary and secondary hair follicles is synchronized throughout the year, and is correlated to hair development. In the later telogen and anagen phases, VEGF expression was higher in the secondary, compared to the primary, hair follicle. Changes in MVD also showed a bimodal pattern with peaks in May and August. VEGF expression and MVD showed moderate and strongly positive correlation in the primary and secondary hair follicles, respectively. Therefore, MVD and VEGF are closely related to the processes involved in hair cycle regulation.

The perivascular microenvironment helps in maintaining stem cells in many tissues. We sought to determine whether there is a perivascular niche for hair follicle stem cells. The association of vessels and follicle progenitor cells began by embryonic day 14.5, when nascent hair placodes had blood vessels approaching them. By birth, a vascular annulus stereotypically surrounded the keratin 15 negative (K15-) stem cells in the upper bulge and remained associated with the K15- upper bulge throughout the hair cycle. The angiogenic factor Egfl6 was expressed by the K15- bulge and was localized adjacent to the vascular annulus, which comprised post-capillary venules. Although denervation altered the phenotype of upper bulge stem cells, the vascular annulus persisted in surgically denervated mouse skin. The importance of the perivascular niche was further suggested by the fact that vascular annuli formed around the upper bulge of de novo-reconstituted hair follicles before their innervation. Together, these findings demonstrate that the upper bulge is associated with a perivascular niche during the establishment and maintenance of this specialized region of hair follicle stem cells.

Excellent surgical results and high patient satisfaction with hair transplantation depend on attaining optimal growth. Unfortunately, even experienced surgeons acknowledge that graft survival often is not as high as is commonly stated. Hair transplant surgeons should be thoroughly familiar with the many variables that affect graft survival and refine their surgical techniques accordingly. This article provides a brief overview of the key factors that most significantly influence graft survival, including graft trauma, vascular/oxygenation factors, and biochemical injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Anecdotical experiences indicate that Koebner phenomenon (KP) can also be observed in alopecia areata (AA). The present short report gives an account of what has been observed in some patients with remitting-relapsing AA in multiple patches, in whom the phenomenon was accidentally caused by perilesional Trichogram. The almost immediate appearance of relapses of the disease (1-7 days) and their evolution can be useful to understand the physiopathology of AA and and emphasize the compelling need for a rapid and appropriate diagnosis and treatment in the acute phase of AA, even with the active participation of the patient adequately trained. The method of trichogram should be reserved for cases in which non-invasive methods such as the Pull Test or Trichoscopy are not sufficient to verify the activity of the disease.

Using nestin-driven green fluorescent protein (ND-GFP) transgenic mice, we previously demonstrated an inter-hair-follicle blood vessel network that expresses ND-GFP and appears to originate from ND-GFP expressing hair-follicle stem cells. We report here that angiogenesis of transplanted skin or healing wounds originates from this ND-GFP-expressing microvasculature network. ND-GFP-expressing blood vessels were visualized growing from the ND-GFP-expressing hair-follicle stem cell area and re-establishing the dermal microvasculature network after skin transplantation or wound healing. When the ND-GFP stem cell area from the vibrissa (whisker) from ND-GFP mice was transplanted to transgenic mice ubiquitously expressing RFP, we observed chimeric ND-GFP-RFP blood vessels, suggesting the joining of inter-follicular blood vessel networks from the transplant and host. These observations suggest that the inter-hair-follicle blood-vessel network contributes to skin transplant survival and wound healing., ((c) 2010 Wiley-Liss, Inc.)

The perifollicular vasculature undergoes hair-cycle dependent expansion and degeneration. Multiple soluble factors derived from dermal papilla cells (DPCs) may act on surrounding blood vessels to influence angiogenesis, growth and differentiation, and thereby regulate cyclic hair growth. The goal of this study was to examine the expression of angiogenin, a potent angiogenic factor, in human DPCs, and to determine its role in hair growth. Reverse transcription polymerase chain reaction (RT-PCR), western blotting, immunofluorescence and ELISA analyzes were used to investigate the expression of angiogenin in human DPCs, while semi-quantitative RT-PCR was used to assess angiogenin mRNA expression in murine skin phased at different stages of the hair cycle. We detected angiogenin expression in DPCs, where it was found to be localized to the cytoplasm. Angiogenin mRNA was expressed in murine skin in a hair-cycle dependent manner, with maximum levels observed at the late anagen. Local injection of angiogenin promoted skin angiogenesis and induced anagen VI. In vitro studies showed that angiogenin significantly enhanced the elongation of hair follicles, and stimulated DPCs and ORS keratinocytes to proliferate. Taken together, these findings show that angiogenin is expressed in human DPCs, where it might contribute to hair growth directly, by stimulating DPCs and ORS keratinocytes to proliferate, or indirectly, by inducing local vascularization.

Betacellulin (BTC) belongs to the EGF family, whose members play important roles in skin morphogenesis, homeostasis, and repair. However, the role of BTC in skin biology is still unknown. We employed transgenic mice overexpressing BTC ubiquitously to study its role in skin physiology. Immunohistochemistry revealed increased levels of BTC especially in the hair follicles and in the epidermis of transgenic animals. Expression of key markers of epithelial differentiation was unaltered, but keratinocyte proliferation was significantly increased. At post-natal day 1 (P1), transgenic mice displayed a significant retardation of hair follicle morphogenesis. At P17, when most follicles in control mice had initiated hair follicle cycling and had already entered into their first late catagen or telogen phase, all follicles of transgenic mice were still at the mid- to late catagen phases, indicating retarded initiation of hair follicle cycling. Healing of full-thickness excisional wounds and bursting strength of incisional wounds were similar in control and transgenic mice. However, an increase in the area covered by blood vessels at the wound site was detected in transgenic animals. These results provide evidence for a role of BTC in the regulation of epidermal homeostasis, hair follicle morphogenesis and cycling, and wound angiogenesis.

Chemotherapy-induced alopecia is a major problem in clinical oncology. Doxorubicin, a widely used cancer chemotherapy drug, induces disruption of the hair cycle and subsequent alopecia. We show in this report that doxorubicin causes disruption of the hair-follicle-associated blood vessel network resulting in a greatly reduced density of these blood vessels. Dystrophic hair follicles were also observed with abnormal melanogenesis in the mice treated with doxorubicin. Visualization of the effect of doxorubicin on hair-follicle angiogenesis was made possible by the use of transgenic mice in which green fluorescent protein was driven by regulatory elements of the nestin gene (ND-GFP). In these transgenic mice, the hair-follicle stem cells and the follicle structure as well as the blood vessels associated with the hair follicles express ND-GFP. The hair-follicle stem cells did not appear to be affected by doxorubicin, which may explain why hair regrows after chemotherapy. These results suggest that inhibition of hair-follicle-associated angiogenesis by doxorubicin may be an important factor in hair-follicle dystrophy associated with chemotherapy-induced alopecia. The ND-GFP mouse model is thus useful for the study of the role of angiogenesis in the hair-follicle cycle and the effect of drugs on processes associated with chemotherapy-induced alopecia.

In developing organs, parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor (PPR) signaling inhibits proliferation and differentiation of mesenchyme-derived cell types resulting in control of morphogenic events. Previous studies using PPR agonists and antagonists as well as transgenic overexpression of the PPR ligand PTHrP have suggested that this ligand receptor combination might regulate the anagen to catagen transition of the hair cycle. To further understand the precise role of PTHrP and the PPR in the hair cycle, we have evaluated hair growth in the traditional K14-PTHrP (KrP) and an inducible bitransgenic PTHrP mice. High levels of PTHrP trangene expression limited to the adult hair cycle resulted in the production of shorter hair shafts. Morphometric analysis indicated that reduced proliferation in the matrix preceded the appearance of thinner hair follicles and shafts during late anagen. CD31 staining revealed that the late anagen hair follicles of the KrP mice were surrounded by reduced numbers of smaller diameter capillaries as compared to controls. Moreover, the fetal skins of the PTHrP and PPR knockouts (KOs) had reciprocal increases in the length, diameter, and density of capillaries. Finally, crossing the KrP transgene onto a thrombospondin-1 KO background reversed the vascular changes as well as the delayed catagen exhibited by these mice. Taken together, these findings suggest that PTHrP's influence on the hair cycle is mediated in part by its effects on angiogenesis.

Vibrissal follicle-sinus complexes (F-SCs) are sensory receptors of the mammalian integument system. They are best developed within Pinnipedia. The objective of this study was to investigate the F-SCs of bearded seals (Erignathus barbatus) for benthic foraging adaptations. Bearded seals possessed approximately 244 mystacial F-SCs. In this species, F-SCs consisted of an outer dermal capsule (DC) surrounding a blood sinus system [upper cavernous sinus (UCS), ring sinus (RS), and lower cavernous sinus (LCS)] and concentric rings of epidermal tissue. The UCS comprised up to 62% of the F-SC length and may function as thermal protection for mechanoreceptors. A large asymmetrical ringwulst was located in the RS. A deep vibrissal nerve penetrated the DC at its base and terminated on mechanoreceptors in the epidermal tissues of the LCS and RS. The mean number of myelinated axons per F-SC was 1,314 (range, 811-1,650) and was among the highest number of axons per F-SC reported to date. An estimated mean number of 320,616 myelinated axons innervate the entire mystacial vibrissal array. Merkel-Neurite complexes (MNCs) and small simple laminated corpuscles were found in the region of the LCS. Myelinated axons also terminated on MNCs and lanceolate endings apical to the ringwulst. The number of F-SCs, their geometry in the mystacial region, the number of myelinated axons per F-SC, and the distribution of mechanoreceptors support the premise that pinniped vibrissae are sensitive active-touch receptor systems, and that structural differences in bearded seals, relative to other phocids, may be adaptations for benthic foraging.

This study aimed to describe the impressive diversity of vascular plexiform structures of the hypodermal layer of human skin. We chose the human body site with the highest concentration of dermal corpuscles, the human digit, and processed it with the corrosion casting technique and scanning electron microscopy analysis (SEM). This approach proved to be the best tool to study these microvascular architectures, free from any interference by surrounding tissues. We took high-definition pictures of the vascular network of sweat glands, thermoreceptorial and tactile corpuscles, the vessels constituting the glomic bodies and those feeding the hair follicles. We observed that the three-dimensional disposition of these vessels strictly depends on the shape of the corpuscles supplied. We could see the tubular vascularization of the excretory duct of sweat glands and the ovoid one feeding their bodies, sometimes made up of two lobes. In some cases, knowledge of these morphological data regarding the normal disposition in space and intrinsic vascularization structure of the dermal corpuscles can help to explain many of the physiopathological changes occurring during chronic microangiopathic diseases.

We have recently shown that the neural-stem cell marker nestin is expressed in hair follicle stem cells and the blood vessel network interconnecting hair follicles in the skin of transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). The hair follicles were shown to give rise to the nestin-expressing blood vessels in the skin. In the present study, we visualized tumor angiogenesis by dual-color fluorescence imaging in ND-GFP transgenic mice after transplantation of the murine melanoma cell line B16F10 expressing red fluorescent protein. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that the blood vessel-specific antigen CD31 was expressed in ND-GFP-expressing nascent blood vessels. ND-GFP expression was diminished in the vessels with increased blood flow. Progressive angiogenesis during tumor growth was readily visualized during tumor growth by GFP expression. Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors.

Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a follicle-linking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFP-expressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening.

We have generated homozygous transgenic mice (hpa-tg) overexpressing human heparanase (endo-beta-D-glucuronidase) in all tissues and characterized the involvement of the enzyme in tissue morphogenesis, vascularization, and energy metabolism. Biochemical analysis of heparan sulfate (HS) isolated from newborn mice and adult tissues revealed a profound decrease in the size of HS chains derived from hpa-tg vs. control mice. Despite this, the mice appeared normal, were fertile, and exhibited a normal life span. A significant increase in the number of implanted embryos was noted in the hpa-tg vs. control mice. Overexpression of heparanase resulted in increased levels of urinary protein and creatinine, suggesting an effect on kidney function, reflected also by electron microscopy examination of the kidney tissue. The hpa-tg mice exhibited a reduced food consumption and body weight compared with control mice. The effect of heparanase on tissue remodeling and morphogenesis was best demonstrated by the phenotype of the hpa-tg mammary glands, showing excess branching and widening of ducts associated with enhanced neovascularization and disruption of the epithelial basement membrane. The hpa-tg mice exhibited an accelerated rate of hair growth, correlated with high expression of heparanase in hair follicle keratinocytes and increased vascularization. Altogether, characterization of the hpa-tg mice emphasizes the involvement of heparanase and HS in processes such as embryonic implantation, food consumption, tissue remodeling, and vascularization.

This study is an investigation to evaluate how the controlled release of different growth factors modifies the hair follicle growth of mice. For the controlled release, basic fibroblast growth factor or hepatocyte growth factor was incorporated into a biodegradable gelatin hydrogel, while vascular endothelial growth factor was incorporated into a biodegradable collagen hydrogel. After subcutaneous implantation of the two different hydrogels incorporating growth factors into the backs of mice, hair follicle growth was evaluated photometrically and histologically 10 days later. The darkness of the reverse side of skin implanted with every hydrogel incorporating growth factor was significantly higher than that of skin injected with the corresponding growth factor in the solution. Implantation of the hydrogel incorporating growth factor increased the area of the hair follicles to a significantly greater extent than other control groups, whereas no effect on the skin thickness was observed. The length of hair shaft elongated was significantly high by the hydrogel incorporating every growth factor. Neither empty gelatin nor collagen hydrogels affected hair follicle growth. These findings indicate that the controlled release enabled the growth factor to positively act on the hair growth cycle of mice.

Hair growth is associated with pronounced vascular-endothelial-growth-factor-induced perifollicular angiogenesis, whereas the catagen regression phase is characterized by apoptosis-driven blood vessel regression. The biologic relevance of endogenous inhibitors of angiogenesis in the control of hair cycling, however, has remained unknown. We studied the expression and biologic role of the angiogenesis inhibitor thrombospondin-1 (TSP-1) during the induced adult hair follicle cycle in wild-type, TSP-1 deficient, and TSP-1 overexpressing transgenic mice. TSP-1 expression was absent from hair bulb and dermal papilla cells during early to mid-anagen but was highly upregulated throughout the catagen involution phase. In TSP-1 deficient mice, the follicle growth phase was significantly prolonged, associated with increased perifollicular vascularization and vascular proliferation. Conversely, hair follicle growth was delayed in K14/TSP-1 transgenic mice that expressed high levels of TSP-1 in outer root sheath keratinocytes, associated with reduced perifollicular vascularization. These effects were most probably mediated via its antiangiogenic effects because TSP-1 did not affect the growth of cultured murine vibrissae in the absence of a functional vascular system. These results identify a critical role of TSP-1 in the induction of anagen follicle involution, with potential implications for the therapeutic modulation of hair follicle growth.

Background: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches., Methods: We used two-photon microscopy and advanced imaging software to render three-dimensional (3D) images of in situ nerves, blood vessels, and hair follicles labeled with various fluorescent markers. Archived frozen human skin biopsy specimens were cryosectioned up to 150 micro m in thickness and fluorescently stained with rhodamine- or fluorescein-labeled antibodies or lectins. Optical sections were collected by two-photon microscopy and the resulting data sets were analyzed in three dimensions using Voxx software., Results: Reconstructed image volumes demonstrated the complex 3D morphology of nerves, blood vessels and adnexal structures in normal mucocutaneous tissue., Conclusion: Two-photon microscopy and Voxx rendering software allow for detailed 3D visualization of structures within human mucocutaneous biopsy specimens, as they appear in situ, and facilitate objective interpretation of variations in their morphology. These techniques may be used to investigate disorders involving cutaneous structures that are difficult to visualize by means of traditional microscopy.

In androgenetic alopecia, overactivation of the androgen hormone cascade in genetically predisposed persons leads to miniaturization of the dermal papilla of the hair follicle and to reduction in the number of papilla cells in the scalp, but the mechanisms explaining this miniaturization have remained unclear. According to our hypothesis, the increase of dihydrotestosterone (DHT) production in the overactive androgen state inhibits cell mitosis in the dermal papilla and contributes to the induction of programmed cell death (apoptosis). Normally, DNA molecules have a negative charge, which doubles in every cell mitosis. In the catagen and telogen phases, the sulphur-rich hair moves upwards, dehydrates and develops an increasing positive charge. In a normal hair-growth cycle, the epithelial column shortens and the secondary germ is formed and it invaginates the dermal papilla by electromagnetic attraction. In the mitotic inhibition state induced by DHT, the negative charge decreases, leading to a weakening of the electromagnetic adhesion forces and weaker electrical attraction between the undifferentiated germ cells and the dermal papilla. Insulin resistance has an additional pathogenic role in the excessive miniaturization of the hair follicle. The vasoactive substances associated with endothelial dysfunction in insulin resistance induce microcirculatory disturbance, perifollicular vasoconstriction and stimulation of smooth muscle cell proliferation in the vascular wall. This leads to microvascular insufficiency and local tissue hypoxia and progressive miniaturization of hair follicles., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

The length and size of hair are depend on the anagen term in its hair cycle. It has been reported that the some cell growth factors, such as VEGF, FGF-5S, IGF-1 and KGF, induce the proliferation of cells in the matrix, dermal papilla and dermal papillary vascular system and increase the amount of extra cellular matrix in dermal papilla and then maintain follicles in the anagen phase. On the other hand, negative factors, like FGF-5, thrombospondin, or still unknown ones, terminate the anagen phase. If the negative factors become dominant against cell proliferation factors according to fulfilling some time set by the biological clock for hair follicles, TGF beta induced in the matrix tissues evokes apoptosis of matrix cells and shifts the follicles from anagen to catagen. Androgenetic alopecia is caused by miniaturizing of hair follicles located in the frontal or crown part of scalp and are hereditarily more sensitive to androgen. In their hair cycles, the androgen shortens the anagen phase of follicles and shifts them to the catagen phase earlier than usual. The mode of action of hair growth effect of minoxidil is not completely elucidated, but the most plausible explanation proposed here is that minoxidil works as a sulfonylurea receptor (SUR) activator and prolongs the anagen phase of hair follicles in the following manner: minoxidil (1) induces cell growth factors such as VEGF, HGF, IGF-1 and potentiates HGF and IGF-1 actions by the activation of uncoupled SUR on the plasma membrane of dermal papilla cells, (2) inhibits of TGF beta induced apoptosis of hair matrix cells by opening the Kir 6.0 channel pore coupled with SUR on the mitochondrial inner membrane, and (3) dilates hair follicle arteries and increases blood flow in dermal papilla by opening the Kir 6.0 channel pore coupled with SUR on the plasma membrane of vascular smooth muscle cells.

The study demonstrates tissue kallikrein with the help of a very sensitive immunohistochemical B-SA method in smooth muscle cells and myocytes / pericytes of the wall of subepidermal arterioles and capillaries of 17 mammalian species from 6 different orders. Positive immunoreactions could also be detected in the wall of blood vessels near to and between hair follicles and skin glands. The reaction staining in subepidermal blood vessels was particularly distinct in animals with a vital epidermis that was significantly thicker than 20 microm. The results obtained are discussed with special regard to the general skin biology of the wild mammals studied, considering comparative hair coat density and normal hazards to the integument.

Purpose: Evaluation of the healing and persistence of a meshed composite skin graft applied without immunosuppression., Methods: The contraction of wounds grafted with 9:1 split-thickness autograft/1.5:1 allodermal mesh composite skin grafts (auto/allo MCSGs) was investigated. No immunosuppressive agent was applied. Male ACI rats and female Lewis rats reciprocally served as allodermis graft donors and recipients. Autograft/dermal autograft and allograft/dermal allograft MCSGs were the controls., Results: AT 3 months after grafting, when epithelized auto/allo MCSG wounds were measured by computerized morphometric analysis, the silver nylon (SN) dressing group displayed less contraction than the Vaseline (petroleum jelly) dressing group (p < 0.003), and direct current treatment (SNDC) was more effective than SN (p < 0.005). The histologic structures of the hair follicles appear to confine the rejection process to the allogeneic follicles of the graft. The focal nature of the rejection process and the relatively low antigenicity of the dermal matrix allowed the survival of the allodermis layer. Although direct current significantly enhanced MCSG healing, SN and SNDC were not the immunosuppressive agents that were confirmed., Conclusion: This type of MCSG can heal without immunosuppressive treatment.

The thermal characteristics of the mystacial vibrissae of harbour seals (Phoca vitulina) and of the follicle crypts on the rostrum of the dolphin Sotalia fluviatilis guianensis were measured using an infrared imaging system. Thermograms demonstrate that, in both species, single vibrissal follicles are clearly defined units of high thermal radiation, indicating a separate blood supply to these cutaneous structures. It is suggested that the high surface temperatures measured in the area of the mouth of the follicles is a function of the sinus system. In seals and dolphins, surface temperature gradually decreased with increasing distance from the centre of a follicle, indicating heat conduction from the sinus system via the follicle capsule to adjacent tissues. It is suggested that the follicular sinus system is a thermoregulatory structure responsible for the maintenance of high tactile sensitivity at the extremely low ambient temperatures demonstrated for the vibrissal system of seals. The vibrissal follicles of odontocetes have been described as vestigial structures, but the thermograms obtained in the present study provide the first evidence that, in Sotalia fluviatilis, the follicles possess a well-developed sinus system, suggesting that they are part of a functional mechanosensory system.

The known role of steroids on the hair follicle leads us to investigate their effects on hair follicle cell angiogenic responses in vitro. We verified, using the immunohistochemical technique, whether human occipital scalp follicle cells express steroid receptors in vitro. We showed that androgen and estrogen receptors were expressed by dermal papilla cells (DPC) and keratinocytes from the outer root sheath in vitro. With regard to steroidal enzymes (type I and II 5alpha-reductases and Cytochrome-p-450-aromatase), the type I 5alpha-reductase gene is much more expressed in DPC than in dermal fibroblasts; however, the type II 5a-reductase gene is transcribed more in dermal fibroblasts than in DPC. The transcription of the two 5alpha-reductase isoform genes in cultured DPC is regulated by a 5alpha-reductase inhibitor. We also demonstrated that DPC, dermal fibroblasts, and outer root shealth keratinocytes expressed cytochrome-p-450-aromatase. Using ELISA and reverse transcriptase-polymerase chain reaction, we investigated the role played by some steroids (estrogens, androgens, antiandrogens) in the modulation of vascular endothelial growth factor (VEGF) expression by DPC. The association of different treatments of DPC (5alpha-reductase inhibitor and androgen receptor antagonist) shows a great stimulation of VEGF and aromatase expression. Strong stimulation of VEGF protein and gene expression is observed in the presence of 17beta-estradiol. Also, the concentration-dependent inhibition of VEGF expression by DPC using the cytochrome-p-450-aromatase inhibitor, confirms the involvement of this estrogenic pathway in the regulation of VEGF expression in vitro.

We report a case of postoperative pressure induced alopecia in a 21-year-old black female after multiple intraoperative procedures. The histopathology is distinctive and demonstrated features in common with trichotillomania and alopecia areata, including the presence of pigment casts, catagen follicles, melanophages and apoptotic bodies. External hair manipulation is considered the primary event in the etiology of pigment casts, however, our present case demonstrated numerous pigment casts despite a complete lack of evidence of external hair manipulation. We performed pattern analysis and in situ end-labeling in 19 cases of non-scarring alopecia. Pigment casts were seen in postoperative alopecia (1 case), alopecia areata (1 case) and trichotillomania (5 cases). These forms of alopecia have in common the sudden termination of the anagen phase of the hair cycle. When the anagen portion of the hair cycle is prematurely disrupted hairs enter into catagen. Pigment casts may represent a non-specific reaction pattern of follicles that are suddenly transformed from anagen to catagen. We therefore propose that hair manipulation is not uniquely responsible for the formation of pigment casts. The primary pathophysiology resulting in the formation of pigment casts more correctly reflects the sudden termination of the anagen phase of the hair cycle.

We report the induction by topical minoxidil of increased fenestration in the perifollicular capillary wall. Male 30-day-old Wistar rats were divided into two groups, i.e. an experimental group which received 4% minoxidil solution topically on the dorsal skin, and a control group which received only vehicle solution topically. Using transmission electron microscopy, follicular and subepidermal capillaries and dermal fibres were compared between both groups. There were no obvious differences in subepidermal capillaries or dermal fibres between the two groups. However, topically applied minoxidil increased fenestration in follicular capillary walls around anagen hair bulbs.

Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.

The authors prospectively encountered skin biopsies from 20 patients which demonstrated a neutrophilic or suppurative and granulomatous folliculitis accompanied by a folliculocentric neutrophilic vascular reaction of Sweet's-like or leukocytoclastic vasculitis subtypes. While in each case the histomorphology raised diagnostic consideration of bacterial folliculitis, patients frequently expressed systemic complaints such as arthritis, fever, and malaise, and special stains for micro-organisms were negative. Among the clinical presentations were folliculitis, vasculitis, acneiform eruptions, vesiculopustular lesions, and erythema nodosum-like lesions, with the legs, arms, and upper back being the most commonly involved sites. Nineteen patients were found to have specific underlying systemic diseases, namely, inflammatory bowel disease, Reiter's disease, Behçet's disease, hepatitis B, connective tissue disease including mixed connective tissue disease and rheumatoid arthritis, scrofuloderma, and hematologic dyscrasias. The other patient had antecedent bacterial sinusitis in the setting of atopy. The folliculocentric nature of these lesions may reflect preferential processing of antigens through the hair follicle and/or homology between bacterial and follicular heat shock proteins in the susceptible host, namely, one who responds excessively to exogenous antigenic triggers. Folliculitis with folliculocentric vasculopathy may be a clue to underlying systemic disease and/or an extracutaneous infection. Certain light microscopic features in concert with the clinical presentation may distinguish such cases from conventional infectious folliculitis.