Arterio-venous Anastomoses of the Sucquet-Hoyer Type: Complexity and Distribution in the Human Dermis.

Our study aims at providing detailed information on numbers, form, and spatial distribution of arterio-venous anastomoses of the Sucquet-Hoyer type in the dermis of the nail bed, nail fold corner, thumb pad, arm, nose, glabella, lip, and ear. It further aims at providing a system, which relies on objective morphologic criteria for classifying Sucquet-Hoyer canals (SHCs). Using high-resolution episcopic microscopy (HREM), digital volume data of eight samples of each skin region were produced. Virtual three-dimensional (3D) models of the dermally located SHCs were created, and their 3D tortuosity (τ) values were determined. Dermal SHCs were identified in all 24 finger samples and in 1 lip sample. Beneath a field of 2 × 2 mm2, an average of four were located in the nail bed, three in the dermis of the thumb pad, and one in the dermis of the nail fold corner. Only a single dermal SHC was found in one lip sample. No SHCs were observed in the dermis of the other samples. The τ values of the SHCs ranged from 1.11 to 10. Building on these values, a classification system was designed, which distinguishes four SHC classes. The dermal distribution of the SHCs of different classes was similar in all specimens.

Three-Dimensional Skin CT Based on Intelligent Algorithm in the Analysis of Skin Lesion Sites Features in Children with Psoriasis.

This research was to explore the application value of three-dimensional computed tomography (CT) based on artificial intelligent algorithm in analyzing the characteristics of skin lesions in children with psoriasis. In this study, 15 children with psoriasis were selected as the observation group, and 15 children with other skin diseases were selected as the control group. The CT images were optimized, and the feature selection was carried out based on artificial intelligent algorithm. Firstly, the results were compared with the results of simple skin three-dimensional CT to determine the effectiveness. Then, the two groups of three-dimensional skin CT image features of skin psoriasis-like hyperplasia, Munro microabscess, dermal papillary vascular dilation, and squamous epithelium based on intelligent algorithms were compared. After comparison, the detection rate of psoriasis-like hyperplasia, Munro microabscess, dermal papillary vascular dilation, and squamous epithelium in the observation group was higher than that in the control group, with significant difference and statistical significance (P < 0.05). In addition, the sensitivity of psoriasis-like hyperplasia, Munro microabscess, dermal papilla vascular dilatation, and squamous epithelium in children with psoriasis was 80.0%, 86.7%, 80.0%, and 93.3%, respectively. The specificity of psoriasis-like hyperplasia, Munro microabscess, dermal papilla vascular dilatation, and squamous epithelium in children with psoriasis was 86.7%, 93.3%, 60.0%, and 73.3%, respectively. The results showed that Munro microabscess and psoriasis-like hyperplasia had high sensitivity and specificity in all diagnostic items, which could be used as important features of skin lesion sites in the diagnosis of psoriasis in children. The research provides a basis for the clinical diagnosis of psoriasis in children, which is worthy of clinical promotion.

A comparative analysis of deferoxamine treatment modalities for dermal radiation-induced fibrosis.

The iron chelator, deferoxamine (DFO), has been shown to potentially improve dermal radiation-induced fibrosis (RIF) in mice through increased angiogenesis and reduced oxidative damage. This preclinical study evaluated the efficacy of two DFO administration modalities, transdermal delivery and direct injection, as well as temporal treatment strategies in relation to radiation therapy to address collateral soft tissue fibrosis. The dorsum of CD-1 nude mice received 30 Gy radiation, and DFO (3 mg) was administered daily via patch or injection. Treatment regimens were prophylactic, during acute recovery, post-recovery, or continuously throughout the experiment (n = 5 per condition). Measures included ROS-detection, histology, biomechanics and vascularity changes. Compared with irradiated control skin, DFO treatment decreased oxidative damage, dermal thickness and collagen content, and increased skin elasticity and vascularity. Metrics of improvement in irradiated skin were most pronounced with continuous transdermal delivery of DFO. In summary, DFO administration reduces dermal fibrosis induced by radiation. Although both treatment modalities were efficacious, the transdermal delivery showed greater effect than injection for each temporal treatment strategy. Interestingly, the continuous patch group was more similar to normal skin than to irradiated control skin by most measures, highlighting a promising approach to address detrimental collateral soft tissue injury following radiation therapy.

Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils.

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.

Single-cell transcriptome profiling reveals vascular endothelial cell heterogeneity in human skin.

Vascular endothelial cells (ECs) are increasingly recognized as active players in intercellular crosstalk more than passive linings of a conduit for nutrition delivery. Yet, their functional roles and heterogeneity in skin remain uncharacterized. We have used single-cell RNA sequencing (scRNA-seq) as a profiling strategy to investigate the tissue-specific features and intra-tissue heterogeneity in dermal ECs at single-cell level. Methods: Skin tissues collected from 10 donors were subjected to scRNA-seq. Human dermal EC atlas of over 23,000 single-cell transcriptomes was obtained and further analyzed. Arteriovenous markers discovered in scRNA-seq were validated in human skin samples via immunofluorescence. To illustrate tissue-specific characteristics of dermal ECs, ECs from other human tissues were extracted from previously reported data and compared with our transcriptomic data. Results: In comparison with ECs from other human tissues, dermal ECs possess unique characteristics in metabolism, cytokine signaling, chemotaxis, and cell adhesions. Within dermal ECs, 5 major subtypes were identified, which varied in molecular signatures and biological activities. Metabolic transcriptome analysis revealed a preference for oxidative phosphorylation in arteriole ECs when compared to capillary and venule ECs. Capillary ECs abundantly expressed HLA-II molecules, suggesting its immune-surveillance role. Post-capillary venule ECs, with high levels of adhesion molecules, were equipped with the capacity in immune cell arrest, adhesion, and infiltration. Conclusion: Our study provides a comprehensive characterization of EC features and heterogeneity in human dermis and sets the stage for future research in identifying disease-specific alterations of dermal ECs in various dermatoses.

Coexistence of three nevus lipomatosus cutaneus superficialis with typical, cutaneous adenolipoma-like, and dermal spindle cell lipoma-like histopathological features in a patient.

We report an unique case of a patient who showed coexistence of three nevus lipomatosus cutaneus superficialis (NLCS) with typical, cutaneous adenolipoma (AL)-like, and dermal spindle cell lipoma (SCL)-like histopathological features. A 53-year-old woman presented with a 20-year history of skin-colored and slightly elevated nodules. These lesions were separately located on the lateral side (lesion 1) and medial side (lesion 2) of her left buttock and on her right thigh (lesion 3). Microscopically, all were ill-defined dermal lesions with some subcutaneous involvement and were mostly composed of mature adipocytes. The adipocytes formed small aggregates around blood vessels in the upper dermis. Lesions 1, 2, and 3 were diagnosed as NLCS, and additional features were recognized in lesions 2 and 3. Lesion 2 revealed eccrine glands and ducts amongst the lipomatous component, as seen in cutaneous AL. Lesion 3 had scattered CD34-positive spindle cells, which is representative of dermal SCL. These appearances were considered to be on the morphological spectrum of NLCS. In all three lesions, CD34-positive cells proliferated between the upper dermal blood vessels and their peripheral mature adipocytes. This pathological finding could be principal in NLCS and might be associated with its pathogenesis.

Linalool inhibits the angiogenic activity of endothelial cells by downregulating intracellular ATP levels and activating TRPM8.

Angiogenesis crucially contributes to various diseases, such as cancer and diabetic retinopathy. Hence, anti-angiogenic therapy is considered as a powerful strategy against these diseases. Previous studies reported that the acyclic monoterpene linalool exhibits anticancer, anti-inflammatory and anti-oxidative activity. However, the effects of linalool on angiogenesis still remain elusive. Therefore, we investigated the action of (3R)-(-)-linalool, a main enantiomer of linalool, on the angiogenic activity of human dermal microvascular endothelial cells (HDMECs) by a panel of angiogenesis assays. Non-cytotoxic doses of linalool significantly inhibited HDMEC proliferation, migration, tube formation and spheroid sprouting. Linalool also suppressed the vascular sprouting from rat aortic rings. In addition, Matrigel plugs containing linalool exhibited a significantly reduced microvessel density 7 days after implantation into BALB/c mice. Mechanistic analyses revealed that linalool promotes the phosphorylation of extracellular signal-regulated kinase (ERK), downregulates the intracellular level of adenosine triphosphate (ATP) and activates the transient receptor potential cation channel subfamily M (melastatin) member (TRPM)8 in HDMECs. Inhibition of ERK signaling, supplementation of ATP and blockade of TRPM8 significantly counteracted linalool-suppressed HDMEC spheroid sprouting. Moreover, ATP supplementation completely reversed linalool-induced ERK phosphorylation. In addition, linalool-induced ERK phosphorylation inhibited the expression of bone morphogenetic protein (BMP)-2 and linalool-induced TRPM8 activation caused the inhibition of ß1 integrin/focal adhesion kinase (FAK) signaling. These findings indicate an anti-angiogenic effect of linalool, which is mediated by downregulating intracellular ATP levels and activating TRPM8.

Subdermal Dissection for Elevation of Pure Skin Perforator Flaps and Superthin Flaps: The Dermis as a Landmark for the Most Superficial Dissection Plane.

BACKGROUND: Pure skin perforator and superthin flaps have been reported and are becoming popular, as they allow one-stage thin skin reconstruction even from a thick donor site. However, currently reported elevation procedures use proximal-to-distal dissection requiring free-style perforator selection and primary thinning procedures. With distal-to-proximal dissection using the dermis as a landmark for dissection plane, it is expected that elevation of pure skin perforator or superthin flaps can be simplified. METHODS: Patients who underwent pure skin perforator or superthin flap transfers with the subdermal dissection technique were included. Flaps were designed based on location of pure skin perforators visualized on color Doppler ultrasound, and elevated just below the dermis under an operating microscope. Medical charts were reviewed to obtain clinical and intraoperative findings. Characteristics of the patients, flaps, and postoperative courses were evaluated. RESULTS: Thirty-six flaps were transferred in 34 patients, all of which were elevated as true perforator flaps (superficial circumflex iliac artery perforator flap in 29 cases, other perforator flaps in seven cases). Mean ± SD flap thickness was 2.24 ± 0.77 mm (range, 1.0 to 4.0 mm). Skin flap size ranged from 3.5 × 2 cm to 27 × 8 cm. Time for flap elevation was 27.4 ± 11.6 minutes. All flaps survived without flap atrophy/contracture 6 months after surgery, except for two cases of partial necrosis. CONCLUSION: The subdermal elevation technique allows straightforward and direct elevation of a pure skin perforator or superthin flap within 30 minutes on average without the necessity of primary thinning. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

MiR-200c-3p increased HDMEC proliferation through the notch signaling pathway.

Excessive proliferation of vascular endothelial cells can cause hemangioma. Although typically benign, hemangiomas can become life-threatening. The microRNA miR-200c-3p is abnormally expressed in some types of tumors, but its expression, biological role, and mechanism of action in infantile hemangioma remain to be fully elucidated. The expression levels of miR-200c-3p in hemangioma tissue were compared with those in adjacent healthy tissue by using bioinformatics analyses and TargetScan. Western blot, enzyme-linked immunosorbent assay, and Cell Counting Kit 8 analyses were used to determine the biological function and site of action of miR-200c-3p in human dermal microvascular endothelial cells (HDMECs). MiR-200c-3p was one of the top 10 differentially expressed genes between healthy tissue, and hemangiomas tissues, having markedly decreased expression in hemangioma tissue. Reduction of miR-200c-3p expression in HDMECs through the transfection of a miR-200c-3p inhibitor significantly increased HDMEC proliferation. The addition of the Notch signaling pathway inhibitor DAPT to HDMECs transfected with the miR-200c-3p inhibitor eliminated the inhibitor-induced enhancement of proliferation in HDMECs. These findings indicate that miR-200c-3p targets the Notch signaling pathway to promote the proliferation of vascular endothelial cells, suggesting that miR-200c-3p plays an important role in the pathogenesis of hemangioma.

The long noncoding RNA PDK1-AS/miR-125b-5p/VEGFA axis modulates human dermal microvascular endothelial cell and human umbilical vein endothelial cell angiogenesis after thermal injury.

Our previous study confirmed the critical role of miR-125b and vascular endothelial growth factor (VEGF) in burn wound repair., The present study was aimed to identify the role of long noncoding RNAs (lncRNAs) related to the function of miR-125b and VEGF in burn wound repair and the underlying mechanism. First, we found that lncRNA PDK1-AS and VEGFA expression was significantly increased in heat-denatured dermal tissue samples and in human dermal microvascular endothelial cells (HDMECs) and human umbilical vein endothelial cells (HUVECs) after thermal injury. PDK1-AS knockdown significantly inhibited cell viability, cumulative tube length, cell migratory ability, and cell invasion of thermally injured HDMECs and HUVECs. PDK1-AS knockdown decreased VEGFA protein levels in HDMECs and HUVECs. While overexpression of PDK1-AS showed the opposite effects. Online tools prediction and luciferase assay confirmed that miR-125b-5p targeted PDK1-AS and VEGFA 3'-untranslated region. miR-125b-5p inhibition significantly increased VEGFA protein levels and enhanced viability, cumulative tube length, migratory ability, and invasion of HUVECs and HDMECs. Furthermore, the effects of PDK1-AS knockdown on VEGFA protein levels in the two cell lines were partially reversed by miR-125b-5p inhibition. Finally, in the tissue samples, PDK1-AS and VEGFA expression was increased, while miR-125b-5p expression was decreased in heat-denatured dermal tissues; the expression of miR-125b-5p had a negative correlation with PDK1-AS and VEGFA, respectively, and PDK1-AS and VEGFA were positively correlated with each other in tissue samples. In conclusion, PDK1-AS relieves miR-125b-5p-induced inhibition on VEGFA by acting as a endogenous RNA, therefore modulating HDMEC and HUVEC angiogenesis after thermal injury.

Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins.

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.

Co-culture Model for Cutaneous Wound Healing to Assess a Porous Fiber-Based Drug Delivery System.

In vitro tissue-engineered cell culture models are an essential instrument to investigate physiological and pathophysiological wound healing mechanisms and to evaluate new beneficial wound dressing materials and therapeutics to identify possible drug targets and to improve regeneration processes in nonhealing and chronic wounds. In this study, the authors established an in vitro model for cutaneous wound healing, based on primary human dermal microvascular endothelial cells (HDMEC) and primary human dermal fibroblasts (HDF) to study wound healing-associated processes. Co-cultivation of HDMEC and HDF results in the formation of microvessel-like structures in long-term co-cultures. The proposed in vitro co-culture model can be easily modified by adding macrophages to simulate the process of inflammation, thus allowing in vitro investigation of pathophysiological wound healing processes present in nonhealing wounds. Furthermore, the beneficial in vitro wound healing model was used to evaluate a porous fiber-based drug delivery dressing material consisting of melt-spun porous fibers that were filled with a hydrogel carrier (gellan gum) containing vascular endothelial growth factor (VEGF). Angiogenic capability was chosen as functional parameter for improved wound healing, and release of deposited VEGF from the dressing material was evaluated up to 7 days of cultivation. The experiments demonstrated that the porous fiber-based drug delivery dressing material for dermal wound healing with incorporated VEGF strongly enhances the process of angiogenesis in the in vitro co-culture model through a release of VEGF over 7 days of cultivation. In conclusion, tissue-engineered human skin equivalents could contribute significantly to the understanding and improvement of drug releasing dressing materials in the context of treating chronic wounds.

Astragaloside IV Promotes Antiphotoaging by Enhancing the Proliferation and Paracrine Activity of Adipose-Derived Stem Cells.

Photoaging is a degenerative biological process. As a kind of pluripotent stem cells, adipose-derived stem cells (ADSCs) are widely used in the treatment of photoaging. Therefore, we aimed to find an effective way to improve the antiaging ability of ADSCs. In this study, we isolated ADSCs and assessed multilineage differentiation ability and markers. Cultured ADSCs were preconditioned with astragaloside IV (ASI) at 10-7, 10-6, and 10-5 M. Cell proliferation was assessed by CCK-8 assay and cytokine secretion by enzyme-linked immunosorbent assay (ELISA). A fibroblast photoaging model was established and cocultured with normal ADSCs or ASI-treated ADSCs. Matrix metalloproteinase-1 (MMP1) and type I procollagen (PC-I) secreted by human dermal fibroblasts were measured by ELISA. The effects of ASI-treated ADSCs on skin texture, including dermal thickness, collagen content, and microvessel density, in a photoaging animal model were analyzed using H&E staining, Masson staining, and CD31 immunohistochemistry, respectively. We found that 10-6 M ASI could significantly promote cell proliferation and stimulate robust secretion of growth factors in ADSCs. Furthermore, our data showed that ASI-treated ADSCs could markedly reverse the ultraviolet B-induced decrease of PC-I secretion and increase of MMP-1 release in fibroblasts. Moreover, in photoaged skin of nude mice, ASI-treated ADSCs significantly increased dermal thickness, collagen content, and microvessel density.

Characterization of dermal skin innervation in fibromyalgia syndrome.

INTRODUCTION: We characterized dermal innervation in patients with fibromyalgia syndrome (FMS) as potential contribution to small fiber pathology. METHODS: Skin biopsies of the calf were collected (86 FMS patients, 35 healthy controls). Skin was immunoreacted with antibodies against protein gene product 9.5, calcitonine gene-related peptide, substance P, CD31, and neurofilament 200 for small fiber subtypes. We assessed two skin sections per patient; on each skin section, two dermal areas (150 x 700 µm each) were investigated for dermal nerve fiber length (DNFL). RESULTS: In FMS patients we found reduced DNFL of fibers with vessel contact compared to healthy controls (p<0.05). There were no differences for the other nerve fiber subtypes. DISCUSSION: We found less dermal nerve fibers in contact with blood vessels in FMS patients than in controls. The pathophysiological relevance of this finding is unclear, but we suggest the possibility of a relationship with impaired thermal tolerance commonly reported by FMS patients.

Effect of Hypoxia on Gene Expression in Cell Populations Involved in Wound Healing.

Wound healing is a complex process regulated by multiple signals and consisting of several phases known as haemostasis, inflammation, proliferation, and remodelling. Keratinocytes, endothelial cells, macrophages, and fibroblasts are the major cell populations involved in wound healing process. Hypoxia plays a critical role in this process since cells sense and respond to hypoxic conditions by changing gene expression. This study assessed the in vitro expression of 77 genes involved in angiogenesis, metabolism, cell growth, proliferation and apoptosis in human keratinocytes (HaCaT), microvascular endothelial cells (HMEC-1), differentiated macrophages (THP-1), and dermal fibroblasts (HDF). Results indicated that the gene expression profiles induced by hypoxia were cell-type specific. In HMEC-1 and differentiated THP-1, most of the genes modulated by hypoxia encode proteins involved in angiogenesis or belonging to cytokines and growth factors. In HaCaT and HDF, hypoxia mainly affected the expression of genes encoding proteins involved in cell metabolism. This work can help to enlarge the current knowledge about the mechanisms through which a hypoxic environment influences wound healing processes at the molecular level.

[Role of mechanosensitive protein Piezo1 in human age-dependent changes in the number of fibroblasts and blood vessels in human skin.]

The aim of this work was to examine the content of Piezo1 in fibroblasts and blood vessels of human dermis from the development until deep aging (from 20 weeks of pregnancy until 85 years old), and defining of a role of Piezo1 in age-dependent changes in the number of fibroblasts and blood vessels in the dermis. Piezo1, proliferating cells nuclear antigen (PCNA), endothelial cells marker CD31 were detected with indirect immunohistochemical technique. Results showed that a portion of fibroblasts with positive staining for Piezo1 in the dermis is decreased from 20 weeks of pregnancy to 40 years old. Percent of Piezo1 positive fibroblasts in dermis is increased sufficiently since 41 years old until 60-85 years old group. The content of Piezo1 in blood vessels in the human dermis is decreased sufficiently from 20 weeks of pregnancy until 40 years old. Age-related changes in the content of Piezo1 in fibroblasts and blood vessels is not associated with an age-related decrease in total number and percent of PCNA positive fibroblasts, the number of blood vessels in the dermis.

Characterization of human dermal sheath cells reveals CD36-expressing perivascular cells associated with capillary blood vessel formation in hair follicles.

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.

Characterization of Laser-Resistant Port Wine Stain Blood Vessels Using In Vivo Reflectance Confocal Microscopy.

BACKGROUND AND OBJECTIVES: Port wine stain (PWS) is a congenital vascular malformation of the human skin. Laser is the treatment of choice for PWS. Laser-resistant PWS is one crucial factor accounting for inadequate treatment outcome, which needs to be fully characterized. This study aims to quantitatively characterize the morphology of laser-resistant PWS blood vessels in the upper papillary dermis using in vivo reflectance confocal microscopy (RCM). STUDY DESIGN/MATERIALS AND METHODS: A total of 42 PWS subjects receiving laser treatment from August 2016 through July 2018 were enrolled into this study. Thirty-three subjects had facial PWS; nine had extremity PWS. All subject's PWS received multiplex 585/1,064 nm laser treatment. RCM images were taken before and after treatment. The density, diameter, blood flow, and depth of PWS blood vessels were analyzed. RESULTS: We found 44.4% PWS on the extremities (four out of nine subjects) were laser-resistant, which was significantly higher (P < 0.001) when compared with those PWS on the face (15.2%, 5 out of 33 subjects). The laser-resistant facial PWS blood vessels had significantly higher blood flow (1.35 ± 0.26 U vs. 0.89 ± 0.22 U, P < 0.001), larger blood vessel diameters (109.60 ± 18.24 µm vs. 84.36 ± 24.04 µm, P = 0.033) and were located deeper in the skin (106.01 ± 13.87 µm vs. 87.82 ± 12.57 µm, P < 0.001) in the skin when compared with laser-responsive PWS on the face. The average PWS blood vessel density (17.01 ± 4.63/mm2 vs. 16.61 ± 4.44/mm2 , P = 0.857) was not correlated to the laser resistance. CONCLUSIONS: Laser-resistant PWS blood vessels had significantly higher blood flow, larger diameters, and were located deeper in the skin. RCM can be a valuable tool for a prognostic evaluation on laser-resistant lesions before treatment, thereby providing guidance for tailored laser treatment protocols, which may improve the therapeutic outcome. The limitations for this study include relative small sample size and acquisitions of different blood vessels before and after 2 months of treatment. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.