Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45- cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/-, Plp1+, Cd274+/-, Thy1+, Cdh3+/-) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogeneous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition.

Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation.

Vitiligo is an autoimmune skin disease caused by cutaneous melanocyte loss. Although phototherapy and T cell suppression therapy have been widely used to induce epidermal re-pigmentation, full pigmentation recovery is rarely achieved due to our poor understanding of the cellular and molecular mechanisms governing this process. Here, we identify unique melanocyte stem cell (McSC) epidermal migration rates between male and female mice, which is due to sexually dimorphic cutaneous inflammatory responses generated by ultra-violet B exposure. Using genetically engineered mouse models, and unbiased bulk and single-cell mRNA sequencing approaches, we determine that manipulating the inflammatory response through cyclooxygenase and its downstream prostaglandin product regulates McSC proliferation and epidermal migration in response to UVB exposure. Furthermore, we demonstrate that a combinational therapy that manipulates both macrophages and T cells (or innate and adaptive immunity) significantly promotes epidermal melanocyte re-population. With these findings, we propose a novel therapeutic strategy for repigmentation in patients with depigmentation conditions such as vitiligo.

Mechanical disorders of the cervicocerebral circulation in children and young adults.

Mechanical disorders of the cervicocerebral circulation (MDCC) are conditions in which neurological symptoms result from a disturbance of cerebral blood flow attributable to external mechanical forces exerted on extracranial blood vessels by adjacent musculoskeletal structures during head movement that is presumably within a physiological range. The disease spectrum includes bow hunter's syndrome, carotid-type Eagle syndrome, and various dynamic venous compression syndromes. These conditions have distinct phenotypes in children which differ from those expressed in older adults. In contemporary practice, recognition and diagnostic evaluation is the domain of the neuroendovascular specialist. The diagnostic evaluation of MDCC involves significant technical nuance that can be critical to directing appropriate management, particularly in children. This report aims to provide a comprehensive overview of the pathophysiology, anatomical patterns, diagnosis, and treatment for the full spectrum of MDCC that is commonly encountered in clinical practice.

Dynamic regulation of chromatin accessibility during melanocyte stem cell activation.

Melanocyte stem cells (McSCs) of the hair follicle are necessary for hair pigmentation and can serve as melanoma cells of origin when harboring cancer-driving mutations. McSCs can be released from quiescence, activated, and undergo differentiation into pigment-producing melanocytes during the hair cycle or due to environmental stimuli, such as ultraviolet-B (UVB) exposure. However, our current understanding of the mechanisms regulating McSC stemness, activation, and differentiation remains limited. Here, to capture the differing possible states in which murine McSCs can exist, we sorted melanocyte nuclei from quiescent (telogen) skin, skin actively producing hair shafts (anagen), and skin exposed to UVB. With these sorted nuclei, we then utilized single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) and characterized three melanocyte lineages: quiescent McSCs (qMcSCs), activated McSCs (aMcSCs), and differentiated melanocytes (dMCs) that co-exist in all three skin conditions. Furthermore, we successfully identified differentially accessible genes and enriched transcription factor binding motifs for each melanocyte lineage. Our findings reveal potential gene regulators that determine these melanocyte cell states and provide new insights into how aMcSC chromatin states are regulated differently under divergent intrinsic and extrinsic cues. We also provide a publicly available online tool with a user-friendly interface to explore this comprehensive dataset, which will provide a resource for further studies on McSC regulation upon natural or UVB-mediated stem cell activation.

Outcomes of Pediatric Osteogenesis Imperfecta Patients Requiring Port-a-Cath Placement for Long-Term Vascular Access.

Intravenous (iv) bisphosphonates are widely used to treat the skeletal manifestations of osteogenesis imperfecta (OI). Obtaining peripheral iv access in pediatric patients with OI is often difficult and traumatic. Although this may be mitigated with surgically placed iv ports (port-a-caths), surgeons may be hesitant to perform this procedure on these children because of the lack of safety data. This study aims to gain better insight into the safety and efficacy of port-a-cath use in this population and identify risk factors for port-a-cath complications. In the present study, we conducted a retrospective cohort analysis of patient characteristics and the incidence of port-a-cath-related complications in children with OI. Fifty-three port-a-caths were placed in 29 children (21 males and 8 females). Of the 29 patients, most are OI type III (n = 18), followed by type I (n = 4), type IV (n = 4), and type V (n = 3). At the time of initial port-a-cath placement, the median age was 52 months (10-191 months), and the median weight was 7.9 kg (5.1-41.1 kg). Most patients (n = 20) weighed less than 10 kg during initial placement. Weight correlated significantly with OI type (p = 0.048), sex (p = 0.03), and vessel used (p = 0.02). Median initial port-a-cath longevity was 43 months (1-113 months), and we found no significant difference in port-a-cath longevity between sexes, OI types, or vessels used. Most patients (n = 19) required multiple port-a-cath placements. There is a significant difference (p = 0.02) between the number of placements and OI type, with type IV having more than type III. Port-a-cath removal was almost always due to mechanical complications (n = 19) but also for infection (n = 1) and malposition (n = 1). Eight patients still had their initial port-a-caths in place at the conclusion of this study. These findings indicate that complications associated with port-a-cath placement are mild and can be used to safely deliver iv bisphosphonates to pediatric OI patients. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation.

Vitiligo is an autoimmune skin disease caused by cutaneous melanocyte loss. Although phototherapy and T cell suppression therapy have been widely used to induce epidermal repigmentation, full pigmentation recovery is rarely achieved due to our poor understanding of the cellular and molecular mechanisms governing this process. Here, we identify unique melanocyte stem cell (McSC) epidermal migration rates between male and female mice, which is due to sexually dimorphic cutaneous inflammatory responses generated by ultra-violet B exposure. Using genetically engineered mouse models, and unbiased bulk and single-cell mRNA sequencing approaches, we determine that manipulating the inflammatory response through cyclooxygenase and its downstream prostaglandin product regulates McSC proliferation and epidermal migration in response to UVB exposure. Furthermore, we demonstrate that a combinational therapy that manipulates both macrophages and T cells (or innate and adaptive immunity) significantly promotes epidermal melanocyte re-population. With these findings, we propose a novel therapeutic strategy for repigmentation in patients with depigmentation conditions such as vitiligo.

Ccr2+ Monocyte-Derived Macrophages Influence Trajectories of Acquired Therapy Resistance in Braf-Mutant Melanoma.

Therapies targeting oncogene addiction have had a tremendous impact on tumor growth and patient outcome, but drug resistance continues to be problematic. One approach to deal with the challenge of resistance entails extending anticancer treatments beyond targeting cancer cells by additionally altering the tumor microenvironment. Understanding how the tumor microenvironment contributes to the evolution of diverse resistance pathways could aid in the design of sequential treatments that can elicit and take advantage of a predictable resistance trajectory. Tumor-associated macrophages often support neoplastic growth and are frequently the most abundant immune cell found in tumors. Here, we used clinically relevant in vivo Braf-mutant melanoma models with fluorescent markers to track the stage-specific changes in macrophages under targeted therapy with Braf/Mek inhibitors and assessed the dynamic evolution of the macrophage population generated by therapy pressure-induced stress. During the onset of a drug-tolerant persister state, Ccr2+ monocyte-derived macrophage infiltration rose, suggesting that macrophage influx at this point could facilitate the onset of stable drug resistance that melanoma cells show after several weeks of treatment. Comparison of melanomas that develop in a Ccr2-proficient or -deficient microenvironment demonstrated that lack of melanoma infiltrating Ccr2+ macrophages delayed onset of resistance and shifted melanoma cell evolution towards unstable resistance. Unstable resistance was characterized by sensitivity to targeted therapy when factors from the microenvironment were lost. Importantly, this phenotype was reversed by coculturing melanoma cells with Ccr2+ macrophages. Overall, this study demonstrates that the development of resistance may be directed by altering the tumor microenvironment to improve treatment timing and the probability of relapse. SIGNIFICANCE: Ccr2+ melanoma macrophages that are active in tumors during the drug-tolerant persister state following targeted therapy-induced regression are key contributors directing melanoma cell reprogramming toward specific therapeutic resistance trajectories.

Safety and efficacy of the Pipeline Flex embolization device with Shield Technology for the acute treatment of ruptured internal carotid artery pseudoaneurysms: a multi-institution case series.

OBJECTIVE: Ruptured blister, dissecting, and iatrogenic pseudoaneurysms are rare pathologies that pose significant challenges from a treatment standpoint. Endovascular treatment via flow diversion represents an increasingly popular option; however, drawbacks include the requirement for dual antiplatelet therapy and the potential for thromboembolic complications, particularly acute complications in the ruptured setting. The Pipeline Flex embolization device with Shield Technology (PED-Shield) offers reduced material thrombogenicity, which may aid in the treatment of ruptured internal carotid artery pseudoaneurysms. METHODS: The authors conducted a multi-institution, retrospective case series to determine the safety and efficacy of PED-Shield for the treatment of ruptured blister, dissecting, and iatrogenic pseudoaneurysms of the internal carotid artery. Clinical, radiographic, treatment, and outcomes data were collected. RESULTS: Thirty-three patients were included in the final analysis. Seventeen underwent placement of a single device, and 16 underwent placement of two devices. No thromboembolic complications occurred. Four patients were maintained on aspirin alone, and all others were treated with long-term dual antiplatelet therapy. Among patients with 3-month follow-up, 93.8% had a modified Rankin Scale score of 0-2. Complete occlusion at follow-up was observed in 82.6% of patients. CONCLUSIONS: PED-Shield represents a new option for the treatment of ruptured blister, dissecting, and iatrogenic pseudoaneurysms of the internal carotid artery. The reduced material thrombogenicity appeared to improve the safety of the PED-Shield device, as this series demonstrated no thromboembolic complications even among patients treated with only single antiplatelet therapy. The efficacy of PED-Shield reported in this series, particularly with placement of two devices, demonstrates its potential as a first-line treatment option for these pathologies.

Technical Nuances and Outcomes of Telescoping Pipeline Flow Diverters: A Multicenter Study.

BACKGROUND: "Telescoping" multiple overlapping Pipeline Embolization Devices (PEDs; Medtronic) has increased their utility by allowing for more impermeable coverage and providing the ability to off-set landing zone sites and extend treatment constructs. OBJECTIVE: To consider the technical nuances and challenges of telescoping PEDs for the treatment of intracranial aneurysms. METHODS: Databases from 3 U.S. academic neurovascular centers were retrospectively queried to identify patients with intracranial aneurysms treated with multiple PED constructs. Data on patient and aneurysm characteristics, as well as outcomes including Raymond-Roy occlusion classification, modified Rankin Scale score, and complications, were gathered. RESULTS: Forty-six patients had 48 intracranial aneurysms treated, including 16 (33%) in whom placement of telescoping PEDs was planned. Fourteen (30%) patients presented with a ruptured aneurysm. Twenty-one aneurysms (44%) were treated with proximal extension, 13 (27%) with distal extension, and 14 (29%) with PED placement inside one another. Thirty (70%) patients had complete aneurysm occlusion at follow-up. Two (4%) patients had to be retreated. Three patients with unruptured and 1 with ruptured aneurysm had a permanent intraprocedural complication. We present descriptive cases illustrating PEDs that were placed inside one another, proximally, distally, and to improve wall apposition because of vessel tortuosity. CONCLUSION: Our data indicate a higher than expected complication rate that is likely because of the technical complexity of these cases. The case illustrations presented demonstrate the indications and challenging aspects of telescoping PEDs.

Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45-cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/- , Plp1+, Cd274+/-, Thy1+, Cdh3+/- ) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogenous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition. Significance: Single cell transcriptomics has revolutionized our ability to interrogate the dynamic nature of tissues. Here we provide a high-resolution map of the melanocyte stem cell population during quiescence. This map provides one of few examples highlighting broad heterogeneity in stem cells during the quiescent cell state. The map also unifies previous observations using other cell, molecular and functional analyses to define the unique features of the quiescent melanocyte stem cell population. This data provides a valuable resource to individuals interested in further evaluating aspects of cellular quiescence in stem cells broadly or melanocyte stem cells specifically.

PLEKHA4 Promotes Wnt/ß-Catenin Signaling-Mediated G1-S Transition and Proliferation in Melanoma.

Despite recent promising advances in targeted therapies and immunotherapies, patients with melanoma incur substantial mortality. In particular, inhibitors targeting BRAF-mutant melanoma can lead to resistance, and no targeted therapies exist for NRAS-mutant melanoma, motivating the search for additional therapeutic targets and vulnerable pathways. Here we identify a regulator of Wnt/ß-catenin signaling, PLEKHA4, as a factor required for melanoma proliferation and survival. PLEKHA4 knockdown in vitro decreased Dishevelled levels, attenuated Wnt/ß-catenin signaling, and blocked progression through the G1-S cell-cycle transition. In mouse xenograft and allograft models, inducible PLEKHA4 knockdown attenuated tumor growth in BRAF- and NRAS-mutant melanomas and exhibited an additive effect with the clinically used inhibitor encorafenib in a BRAF-mutant model. As an E3 ubiquitin ligase regulator with both lipid- and protein-binding partners, PLEKHA4 presents several opportunities for targeting with small molecules. Our work identifies PLEKHA4 as a promising drug target for melanoma and clarifies a controversial role for Wnt/ß-catenin signaling in the control of melanoma proliferation. SIGNIFICANCE: This study establishes that melanoma cell proliferation requires the protein PLEKHA4 to promote pathologic Wnt signaling for proliferation, highlighting PLEKHA4 inhibition as a new avenue for the development of targeted therapies.

Woven Endobridge device for treatment of dissection-related PICA aneurysm.

Ruptured vertebrobasilar dissecting aneurysms require urgent, often challenging treatment as they have with a high re-hemorrhage rate within the first 24 hours. The patient is a 57-year-old woman who presented with severe-sudden onset headache. Further work up showed a ruptured dissecting aneurysm of the caudal loop of the posterior inferior cerebellar artery (PICA) with associated narrowing distally, in the ascending limb. The aneurysm was immediately occluded with a Woven Endobridge (WEB) device (MicroVention, Tustin, CA, USA) while flow diversion treatment of the diseased ascending limb was postponed. Follow-up angiography three months later showed complete occlusion of the aneurysm, as well as healing of the diseased distal vessel, obviating the need for further intervention. WEB embolization of a ruptured dissecting posterior circulation aneurysm provided an excellent outcome for this patient.

Minimally Invasive Endoscopy for Acute Subdural Hematomas: A Report of 3 Cases.

BACKGROUND: Acute subdural hematomas (aSDHs) occur in approximately 10% to 20% of all closed head injury and represent a significant cause of morbidity and mortality in traumatic brain injury patients. Conventional craniotomy is an invasive intervention with the potential for excess blood loss and prolonged postoperative recovery time. OBJECTIVE: To evaluate the outcomes of minimally invasive endoscopy for evacuation of aSDHs in a pilot feasibility study. METHODS: We retrospectively reviewed the records of consecutive patients with aSDHs who underwent surgical treatment at our institution with minimally invasive endoscopy using the Apollo/Artemis Neuro Evacuation Device (Penumbra, Alameda, California) between April 2015 and July 2018. RESULTS: The study cohort comprised three patients. The Glasgow Coma Scale on admission was 15 for all 3 patients, median preoperative hematoma volume was 49.5 cm3 (range 44-67.8 cm3), median postoperative degree of hematoma evacuation was 88% (range 84%-89%), and median modified Rankin Scale at discharge was 1 (range 0-3). CONCLUSION: Endoscopic evacuation of aSDHs can be a safe and effective alternative to craniotomy in appropriately selected patients. Further studies are needed to refine the selection criteria for endoscopic aSDH evacuation and evaluate its long-term outcomes.

New insights into the functions of Cox-2 in skin and esophageal malignancies.

Understanding the cellular and molecular mechanisms of tumor initiation and progression for each cancer type is central to making improvements in both prevention and therapy. Identifying the cancer cells of origin and the necessary and sufficient mechanisms of transformation and progression provide opportunities for improved specific clinical interventions. In the last few decades, advanced genetic manipulation techniques have facilitated rapid progress in defining the etiologies of cancers and their cells of origin. Recent studies driven by various groups have provided experimental evidence indicating the cellular origins for each type of skin and esophageal cancer and have identified underlying mechanisms that stem/progenitor cells use to initiate tumor development. Specifically, cyclooxygenase-2 (Cox-2) is associated with tumor initiation and progression in many cancer types. Recent studies provide data demonstrating the roles of Cox-2 in skin and esophageal malignancies, especially in squamous cell carcinomas (SCCs) occurring in both sites. Here, we review experimental evidence aiming to define the origins of skin and esophageal cancers and discuss how Cox-2 contributes to tumorigenesis and differentiation.

Comparative Analysis of Unruptured Cerebral Aneurysm Treatment Outcomes and Complications with the Classic versus Flex Pipeline Embolization Devices and Phenom versus Marksman Microcatheter Delivery System: The Role of Microcatheter Choice on Complication Rate.

OBJECTIVE: The second-generation pipeline embolization device (PED), flex, has improved opening and resheathing ability compared to the first-generation classic PED device. A previously reported single-institutional study suggests that the PED flex devices are associated with lower rates of complications. However, there was limited discussion regarding the complication rate with respect to microcatheter choice for PED delivery and deployment. The present study aims to evaluate outcomes of aneurysm treatment with PED flex versus classic along with the Phenom microcatheter versus Marksman microcatheter. METHODS: A retrospective, IRB-approved database of all patients who received a PED classic or PED flex device between January 2012 and July 2018 was analyzed. Microcatheter choice, patient demographics, medical comorbidities, aneurysm characteristics, treatment information, and outcome data were analyzed using univariate analyses. RESULTS: A total of 75 PED procedures were analyzed. There was no significant difference in major complications between the PED classic and PED flex. However, those treated using the Marksman microcatheter were more likely to have a major complication (periprocedural hemorrhage or ischemic event; 16.6% vs. 0%, p = 0.0248) than those treated with the Phenom microcatheter. Within the PED flex cohort, all major complications were associated with the Marksman microcatheter (p = 0.0289). CONCLUSIONS: The present study does not replicate significantly fewer complications with PED flex but demonstrates a significant reduction in complications with the Phenom microcatheter. Ultimately, this suggests multiple factors are involved in achieving positive outcomes and low complication rates in PED treated unruptured cerebral aneurysms.

Intraosseous Cannulation of a Calvarial Diploic Vein for Embolization of a Symptomatic Dural Arteriovenous Fistula: A Technical Case Report.

BACKGROUND AND IMPORTANCE: Dural arteriovenous fistulas (DAVFs) may present unique challenges for treatment depending on the anatomy and pattern of venous drainage. If endovascular techniques are to be employed, the DAVF must be amenable to transvenous or transarterial therapy. When access of peripheral vasculature does not provide a straightforward path, less conventional options may be available. This case highlights a novel, technically simple, and effective approach for the treatment of a subset of DAVFs, with venous drainage through calvarial diploic veins, that would make endovascular treatment otherwise challenging or impossible. CLINICAL PRESENTATION: We present a case of a 66-yr-old female patient who was diagnosed with a symptomatic DAVF located along the sphenoid ridge with a large intraosseous channel containing the draining vein of the fistula. This lesion was successfully treated with transcranial endovascular embolization via direct intraosseous cannulation of the calvarial diploic vein. This novel approach obviated the need for a full-thickness craniotomy, afforded only minimal bone loss, and preserved the integrity of the dura. A 3-mo follow-up angiogram confirmed complete cure of the DAVF with no residual arteriovenous shunt. At 20 mo postembolization, the patient was symptom free, with no reported neurologic deficits. Complete diagnostic work-up, treatment planning in a multidisciplinary environment, and a novel approach for endovascular embolization utilizing a hybrid operating suite played key roles in the successful implementation of this technique. CONCLUSION: This is the first report of direct intraosseous cannulation of a calvarial diploic vein for successful transcranial endovascular embolization of a symptomatic DAVF.

Heparin Treatment in Aneurysmal Subarachnoid Hemorrhage: A Review of Human Studies.

Aneurysmal subarachnoid hemorrhage (aSAH) remains a significant cause of stroke disability despite gradual reductions in physical morbidity and mortality. Heparin is an effective anti-inflammatory agent and may potentially prevent delayed neurological injury in the days to weeks after the hemorrhage. Various human studies have shown the safety of a continuous infusion of low-dose unfractionated heparin in the setting of subarachnoid hemorrhage as well as its efficacy in minimizing delayed neurological deficits including symptomatic cerebral vasospasm, vasospasm-related infarction, and cognitive dysfunction. Studies have also shown mixed results with low-molecular-weight heparin usage in this patient population. Heparin treatment is not associated with significant hemorrhagic complications; however, vigilance is essential for early detection of heparin-induced thrombocytopenia in order to prevent devastating sequelae. Multicenter randomized controlled trials are necessary for objective characterization of the effects of heparin.

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells' activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.