Targeting the nuclear long noncoding transcript LSP1P5 abrogates extracellular matrix deposition by trans-upregulating CEBPA in keloids.

Keloids are characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix (ECM) and are a major global health care burden among cutaneous diseases. However, the function of long noncoding RNA (lncRNA)-mediated ECM remodeling during the pathogenesis of keloids is still unclear. Herein, we identified a long noncoding transcript, namely, lymphocyte-specific protein 1 pseudogene 5 (LSP1P5), that modulates ECM component deposition in keloids. First, high-throughput transcriptome analysis showed that LSP1P5 was selectively upregulated in keloids and correlated with more severe disease in a clinical keloid cohort. Therapeutically, the attenuation of LSP1P5 significantly decreased the expression of ECM markers (COL1, COL3, and FN1) both in vitro and in vivo. Intriguingly, an antifibrotic gene, CCAAT enhancer binding protein alpha (CEBPA), is a functional downstream candidate of LSP1P5. Mechanistically, LSP1P5 represses CEBPA expression by hijacking Suppressor of Zeste 12 to the promoter of CEBPA, thereby enhancing the polycomb repressive complex 2-mediated H3K27me3 and changing the chromosomal opening status of CEBPA. Taken together, these findings indicate that targeting LSP1P5 abrogates fibrosis in keloids through epigenetic regulation of CEBPA, revealing a novel antifibrotic therapeutic strategy that bridges our current understanding of lncRNA regulation, histone modification and ECM remodeling in keloids.

Salvaging Complex Lower Extremity Injury with Laparoscopically Harvested Omental Flaps.

Background: Traumatic injuries to the lower extremities are frequently accompanied by extensive soft tissue loss, combined with vascular damage or exposure of bony tissues, making it difficult to reconstruct; consequently, patients are commonly at risk of amputation. Due to its superior anatomical and biochemical properties, the omental flap has been used to reconstruct soft tissue defects for decades. However, few studies have reported the omental flap's effectiveness in treating severe and complex lower extremity deformities. We attempted to use a laparoscopically harvested omental flap in conjunction with a second-stage skin graft to reduce infections during limb preservation, increase flap survival probability, and restore the aesthetic and functional integrity of the affected extremity. Methods: Seventeen patients with severe lower extremity wounds underwent omental flap transplantation and were followed up for 6 to 12 months to check for surgical complications, evaluate cosmetic results, and ensure proper limb function. Results: There were no complications, such as intestinal adhesion, intestinal volvulus, and peritonitis, with any of the omental grafts. The affected extremities were well-functioning and aesthetically pleasing. Conclusion: Laparoscopically harvested omental flap transplantation with skin grafting is an alternative reconstruction technique for severe lower extremity injuries with massive soft tissue loss and exposed bones and tendons.

ALKBH5-mediated m6A demethylation fuels cutaneous wound re-epithelialization by enhancing PELI2 mRNA stability.

BACKGROUND: Impaired wound re-epithelialization contributes to cutaneous barrier reconstruction dysfunction. Recently, N6-methyladenosine (m6A) RNA modification has been shown to participate in the determination of RNA fate, and its aberration triggers the pathogenesis of numerous diseases. Howbeit, the function of m6A in wound re-epithelialization remains enigmatic. METHODS: Alkbh5‒/‒ mouse was constructed to study the rate of wound re-epithelialization after ALKBH5 ablation. Integrated high-throughput analysis combining methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq was used to identify the downstream target of ALKBH5. In vitro and in vivo rescue experiments were conducted to verify the role of the downstream target on the functional phenotype of ALKBH5-deficient cells or animals. Furthermore, the interacting reader protein and regulatory mechanisms were determined through RIP-qPCR, RNA pull-down, and RNA stability assays. RESULTS: ALKBH5 was specifically upregulated in the wound edge epidermis. Ablation of ALKBH5 suppressed keratinocyte migration and resulted in delayed wound re-epithelialization in Alkbh5‒/‒ mouse. Integrated high-throughput analysis revealed that PELI2, an E3 ubiquitin protein ligase, serves as the downstream target of ALKBH5. Concordantly, exogenous PELI2 supplementation partially rescued keratinocyte migration and accelerated re-epithelialization in ALKBH5-deficient cells, both in vitro and in vivo. In terms of its mechanism, ALKBH5 promoted PELI2 expression by removing the m6A modification from PELI2 mRNA and enhancing its stability in a YTHDF2-dependent manner. CONCLUSIONS: This study identifies ALKBH5 as an endogenous accelerator of wound re-epithelialization, thereby benefiting the development of a reprogrammed m6A targeted therapy for refractory wounds.

Intraoperative Indocyanine Green Angiography Facilitates Flap Fenestration and Facial Organ Fabrication in Total Facial Restoration.

BACKGROUND: As effective measures to visualize flap vasculature and perfusion were unavailable, flap fenestration and facial organ fabrication could not be performed safely, preventing the transition from 2-D coverage to the restoration of the 3-D structure of facial organs. This study aims to evaluate the efficacy of indocyanine green angiography (ICGA) in guiding flap fenestration and facial organ fabrication in total facial restoration. METHODS: Ten patients with total facial scarring following burn injury were enrolled in the study. They were treated with pre-expanded, prefabricated monoblock flaps for total face restoration. The opening of nostrils, oral and palpebral orifices, together with organ fabrication, were conducted under the guidance of intraoperative ICGA via hemodynamic evaluation of flap perfusion. Postoperative follow-up parameters include vascular crisis, infection, flap necrosis and patients' aesthetic and functional recovery. RESULTS: The opening of facial organ orifices was performed at the stage of flap transfer in nine patients. To avoid damaging the major nourishing vessels, the left palpebral orifice was opened eight days after the flap transfer in one patient, as observed by ICGA. Based on ICGA evaluation, the decision to perform additional vascular anastomosis before flap fenestration was made in six patients. Hemodynamic analysis of flap perfusion following fenestration revealed no significant change. Follow-up showed satisfactory aesthetic recovery and well-restored 3-D structures of facial organs. CONCLUSION: This pilot study demonstrates how intraoperative ICGA can enhance the safety of flap fenestration, thereby transforming full facial restoration from the 2-D to the 3-D realm by facilitating facial organ fabrication.

Lean adipose tissue macrophage derived exosome confers immunoregulation to improve wound healing in diabetes.

Chronic non-healing wounds, a prevalent complication of diabetes, are associated with increased mortality in diabetic patients. Excessive accumulation of M1 macrophages in diabetic wounds promotes inflammation and results in dysregulated tissue repair. Adipose tissue macrophages (ATMs) derived from healthy lean donors have the ability to improve glucose tolerance and insulin sensitivity, as well as modulate inflammation. MicroRNAs (miRs), which can be packaged into exosomes (Exos) and secreted from cells, serve as essential regulators of macrophage polarization. Here, we revealed that ATMs isolated from lean mice secrete miRs-containing Exos, which modulate macrophage polarization and promote rapid diabetic wound healing when administered to diabetes-prone db/db mice. The miRs sequence of tissue samples from wounds treated with Exos secreted by lean ATMs (ExosLean) revealed that miR-222-3p was up-regulated. Further analyses showed that inhibiting miR-222-3p using a miR inhibitor impaired the macrophage-reprogramming effect of ExosLean. In the excisional skin wound mouse model, locally inhibiting miR-222-3p disrupted healing dynamics and failed to modulate macrophage polarization. Mechanistic studies revealed a connection between miR-222-3p, Bcl2l11/Bim, an inflammatory response effector, macrophage polarization, and diabetic wound healing. In summary, ExosLean act as positive regulators of macrophage polarization by regulating miR levels in wounds and accelerating wound healing, and thus have important implications for wound management in diabetes.

CD39+ Fibroblasts Enhance Myofibroblast Activation by Promoting IL-11 Secretion in Hypertrophic Scars.

Fibroblasts (Fbs) are critical to hypertrophic scar (HTS) formation and were recently shown to be highly heterogeneous. However, Fb heterogeneity in HTSs has not been fully elucidated. In this study, we observed an increased fraction of CD39+ Fbs in HTS after screening four Fb subtypes (CD26+, CD36+, FAP+, and CD39+). CD39+ Fbs, enriched in the upper dermis, were positively correlated with scar severity. The transcriptional analysis of CD39+ and CD39- Fbs sorted from HTS revealed that IL-11 was more highly expressed in CD39+ Fbs. We then showed that IL-11 was upregulated in HTSs and that its expression was induced by TGFß1 in vitro. TGFß1 also stimulated the expression of CD39 at the transcriptional and protein levels, mediating the maintenance of the CD39+ phenotype. Furthermore, IL-11 facilitated myofibroblast activation and extracellular matrix production in both CD39+ and CD39- Fbs. Interestingly, CD39+ Fbs secreted more IL-11 on TGFß1 treatment and were less responsive to IL-11 than CD39- Fbs. Notably, a CD39 inhibitor effectively reduced stretch-induced scar formation and attenuated bleomycin-induced skin fibrosis, suggesting an antiscarring approach by targeting CD39+ Fbs.

Identification and functional analysis of a three-miRNA ceRNA network in hypertrophic scars.

BACKGROUND: Hypertrophic scar (HTS) is a fibrotic disorder of skins and may have repercussions on the appearance as well as functions of patients. Recent studies related have shown that competitive endogenous RNA (ceRNA) networks centering around miRNAs may play an influential role in HTS formation. This study aimed to construct and validate a three-miRNA (miR-422a, miR-2116-3p, and miR-3187-3p) ceRNA network, and explore its potential functions. METHODS: Quantitative real­time PCR (qRT­PCR) was used to compare expression levels of miRNAs, lncRNAs, and genes between HTS and normal skin. Target lncRNAs and genes of each miRNA were predicted using starBase as well as TargetScan database to construct a distinct ceRNA network; overlapping target lncRNAs and genes of the three miRNAs were utilized to develop a three-miRNA ceRNA network. For every network, protein-protein interaction (PPI) network analysis was performed to identify its hub genes. For each network and its hub genes, Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted to explore their possible functions. RESULTS: MiR-422a, miR-2116-3p, and miR-3187-3p were all downregulated in HTS tissues and fibroblasts. MiR-422a-based ceRNA network consisted of 101 lncRNAs with 133 genes; miR-2116-3p-centered ceRNA network comprised 85 lncRNAs and 978 genes; miR-3187-3p-derived ceRNA network encompassed 84 lncRNAs as well as 1128 genes. The three-miRNA ceRNA network included 2 lncRNAs with 9 genes, where MAPK1, FOSL2, ABI2, KPNA6, CBL, lncRNA-KCNQ1OT1, and lncRNA-EBLN3P were upregulated. According to GO and KEGG analysis, these networks were consistently related to ubiquitination. Three ubiquitination-related genes (CBL, SMURF2, and USP4) were upregulated and negatively correlated with the expression levels of the three miRNAs in HTS tissues. CONCLUSIONS: This study identified a three-miRNA ceRNA network, which might take part in HTS formation and correlate with ubiquitination.

Targeting Dermal Fibroblast Subtypes in Antifibrotic Therapy: Surface Marker as a Cellular Identity or a Functional Entity?

Fibroblasts are the chief effector cells in fibrotic diseases and have been discovered to be highly heterogeneous. Recently, fibroblast heterogeneity in human skin has been studied extensively and several surface markers for dermal fibroblast subtypes have been identified, holding promise for future antifibrotic therapies. However, it has yet to be confirmed whether surface markers should be looked upon as merely lineage landmarks or as functional entities of fibroblast subtypes, which may further complicate the interpretation of cellular function of these fibroblast subtypes. This review aims to provide an update on current evidence on fibroblast surface markers in fibrotic disorders of skin as well as of other organ systems. Specifically, studies where surface markers were treated as lineage markers and manipulated as functional membrane proteins are both evaluated in parallel, hoping to reveal the underlying mechanism behind the pathogenesis of tissue fibrosis contributed by various fibroblast subtypes from multiple angles, shedding lights on future translational researches.

Inhibition of CUB and sushi multiple domains 1 (CSMD1) expression by miRNA-190a-3p enhances hypertrophic scar-derived fibroblast migration in vitro.

BACKGROUND: Hypertrophic scar (HTS) is a fibroproliferative skin disorder characterized by excessive cell proliferation, migration, and extracellular matrix (ECM) deposition. The CUB and Sushi multiple domains 1 (CSMD1) has previously been identified as the key regulatory gene of hypertrophic scar by a large sample GWAS study. However, further research has not yet been conducted to verify this finding in other HTS patients and to determine the underlying mechanism. RESULTS: In this study, we verified that CSMD1 was downregulated in both HTS tissue and HTS-derived fibroblasts. The knockdown of CSMD1 resulted in enhanced migration and fibronectin1 (FN1) secretion in fibroblasts in vitro. In addition, the upstream and downstream regulatory mechanisms of CSMD1 were also investigated through microRNA (miRNA) databases screening and RNA-sequencing (RNA-seq) respectively. The screening of four common microRNA (miRNA) databases suggested that miR-190a-3p binds to the CSMD1 and may regulate its expression. We confirmed that miR-190a-3p directly targeted the CSMD1-3'-UTR using luciferase reporter assays. Furthermore, the overexpression of miR-190a-3p showed promotion of migratory activity and FN1 secretion in fibroblasts, resembling the effect of CSMD1 knockdown; whereas the knockdown of miR-190a-3p exerted the opposite effect. Finally, transcriptomic analysis showed activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway in the CSMD1 knockdown fibroblasts. CONCLUSIONS: This study has validated the conclusions of the previous GWAS study conducted in Chinese population. In vitro experiments have provided further evidence on the function of CSMD1 in the development of HTS, and have also revealed the underlying upstream and downstream regulating mechanisms. Additionally, the JAK/STAT signaling pathway identified using RNA-seq might provide a potential treatment approach, especially for HTS.

Augmentation of Perforator Flap Blood Supply with Vascular Supercharge or Flap Prefabrication: Evaluation in a Rat Model.

BACKGROUND: Vascular supercharge and flap prefabrication are two surgical maneuvers to improve flap blood supply. Although these techniques have been studied intensively, few studies have focused on the differences between supercharge and prefabricated flaps regarding their flap survival areas, vasculatures, and hemodynamics. METHODS: In this study, 21 male Sprague-Dawley rats were divided into three groups as follows: group A, single perforator flap; group B, supercharge flap; and group C, prefabricated flap. Flap survival was measured 1 week after flap elevation. Indocyanine green angiography was applied to visualize flap vascularity and to analyze flap hemodynamics. Von Willebrand factor immunohistochemical staining was applied to assess the number of microvessels in the choke zone of the abdominal wall. RESULTS: The flap survival areas were expanded significantly in the arteriovenous supercharge group and the vascular bundle prefabricated group compared with that in the single-perforator group (81.34 ± 8.12 percent and 75.51 ± 8.08 percent versus 46.27 ± 10.01 percent, respectively; p < 0.05). Hemodynamic analysis suggested that although a significant increase in arterial infusion could be achieved with flap prefabrication, the venous effusion of the prefabricated flap was the worst among the three groups, indicating greater susceptibility to compromised venous return. Active neovascularization was confirmed by an increased number of microvessels in group C. Specifically, the dilatation of choke vessels and the newly formed vessels of the prefabricated pedicle could be appreciated by indocyanine green angiographic mapping. CONCLUSIONS: Both vascular supercharge and flap prefabrication can augment the blood supply of the perforator flap but by means of different mechanisms. Because a supercharge flap is less susceptible to venous compromise, it is suggested to first consider the use of vascular supercharging when feasible.

Facial cleft? The first case of manitoba-oculo-tricho-anal syndrome with novel mutations in China: a case report.

BACKGROUND: Manitoba-oculo-tricho-anal (MOTA) syndrome is a rare syndrome with only 27 cases reported worldwide so far, but none was reported in the population of Eastern Asia. Such extremely low prevalence might be contributed by misdiagnosis due to its similarities in ocular manifestations with facial cleft. In our study, we discovered the first case of MOTA syndrome in the population of China, with 2 novel FRAS1 related extracellular matrix 1 (FREM1) gene stop-gain mutations confirmed by whole exome sequencing. CASE PRESENTATION: A 12-year-old Chinese girl presented with facial cleft-like deformities including aberrant hairline, blepharon-coloboma and broad bifid nose since birth. Whole exome sequencing resulted in the identification of 2 novel stop-gain mutations in the FREM1 gene. Diagnosis of MOTA syndrome was then established. CONCLUSIONS: We discovered the first sporadic case of MOTA syndrome according to clinical manifestations and genetic etiology in the Chinese population. We have identified 2 novel stop-gain mutations in FREM1 gene which further expands the spectrum of mutational seen in the MOTA syndrome. Further research should be conducted for better understanding of its mechanism, establishment of an accurate diagnosis, and eventually the exploitation of a more effective and comprehensive therapeutic intervention for MOTA syndrome.

microRNA-646 inhibits angiogenesis of endothelial progenitor cells in pre-eclamptic pregnancy by targeting the VEGF-A/HIF-1α axis.

Pre-eclampsia is a complication that occurs during pregnancy, the pathological feature of which is a change in vascular endothelial homeostasis. microRNA (miR)-646 is an anti-angiogenic miRNA that has been indicated to exhibit potential anti-angiogenic effects in endothelial cells cultured in vitro and in ischemia-induced angiogenesis. However, whether miR-646 has therapeutic potential in placental angiogenesis in pre-eclampsia remains to be determined. In the current study, human peripheral blood-derived endothelial progenitor cells (EPCs) were isolated to study the coordination between miR-646, vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor (HIF)-1α expression in preeclampsia EPCs. EPCs were isolated from human peripheral blood to demonstrate a potential interaction between miR-646 and targets (VEGF-A) in vitro. The number of EPCs and the expression of miR-646 in patients with preeclampsia was detected, and the effects of miR-646 on EPC function and preeclampsia angiogenesis was assessed. Clinical specimens demonstrated that miR-646 expression was enhanced in pregnancy with preeclampsia. The results indicated that miR-646 suppressed EPCs multiplication, differentiation and migration. miR-646 was observed to exert an anti-angiogenic function by suppressing the expression of angiogenic cytokines VEGF-A and HIF-1α. Additionally, luciferase results displayed that miR-646 downregulated VEGF-A expression by directly binding to a specific sequence in its 3'-untranslated region. The results of the current study demonstrated that the miR-646/VEGF-A/HIF-1α axis is significant for angiogenic properties of EPCs in vitro and in vivo placental vasculogenesis. The results of the present study provide a new insight into microRNA regulation of vessel homeostasis and angiogenesis, and a basis for alternative treatments for patients with pre-eclampsia.

Augmentation of Perforator Flap Blood Supply with Sole or Combined Vascular Supercharge and Flap Prefabrication for Difficult Head and Neck Reconstruction.

Importance: The strategic option between vascular supercharge and flap prefabrication in the fabrication of multipedicle flaps for difficult head and neck reconstruction has not been reported. Objectives: To summarize our 13-year experience in the design of multipedicle pre-expanded perforator flaps for extensive head and neck defect reconstruction, with vascular supercharge and flap prefabrication, either solely or in combination. To discuss the strategy for judicious selection between these techniques in the design of multipedicle flaps at different donor sites. Design, Setting, and Participants: A retrospective study was performed in patients with severe head and neck deformities and treated with multipedicle pre-expanded perforator flaps between May 2005 and May 2018. Intraoperative indocyanine green angiography was utilized for visualization of flap perfusion and analysis of the hemodynamics of pre-existing perforator and prefabricated vessels. Main Outcomes and Measures: The main outcomes measured were (1) features of deformities, (2) multipedicle flap designs, and (3) postoperative complications and long-term functional and aesthetic outcomes. Results: Seventy-five multipedicle pre-expanded perforator flaps were harvested, including 44 supercharged, 26 prefabricated, and 5 tripedicle, combining both techniques, with sizes ranging from 22 × 12 to 45 × 27 cm2. Hemodynamic analysis demonstrated slower arterial inflow (0.60 ± 0.29 U/s vs. 2.65 ± 1.29 U/s, p < 0.05) and venous outflow (0.10 U/s vs. 0.23 ± 0.11 U/s) in prefabricated vessels, compared to the pre-existing perforator, namely the internal mammary arterial perforator. Partial necroses were observed in three patients with only one requiring skin grafting. The aesthetic and functional outcomes were satisfying after reconstruction. Conclusions and Relevance: Multipedicle pre-expanded perforator flaps fabricated by vascular supercharge and flap prefabrication, either solely or in combination in various donor sites, are powerful reconstructive tools for extensive head and neck defects. For a more reliable design, vascular supercharging should be considered a priority strategy, and flap prefabrication an alternative when suitable supercharging vessels are unavailable.