Pigeon novel-miR-741 targets OTUD1 to inhibit proliferation and promote apoptosis of crop fibroblasts.

Trichomonas gallinae (T. gallinae) is a globally distributed protozoan parasite and could cause serious damage to the pigeon industry. MiRNAs have important roles in regulating parasite infection, but its impacts on T. gallinae resistance have rarely been reported. In the present study, we identified a new miRNA (novel-miR-741) and its predicted target OTU deubiquitinase 1 (OTUD1) that might be associated with immunity to T. gallinae in pigeon. Novel-miR-741 and OTUD1 over-expression vectors and interference vectors were constructed. Results from dual luciferase activity assay demonstrated that OTUD1 was a downstream target of novel-miR-741. The Cell Counting Kit-8 and apoptosis assays showed that novel-miR-741 inhibited the proliferation and promoted apoptosis of pigeon crop fibroblasts. Meanwhile, mRNA levels of OTUD1 were significantly reduced in novel-miR-741 mimic-transfected fibroblasts, while mRNA levels of OTUD1 were significantly increased in the novel-miR-741 inhibitor-transfected fibroblasts. The regulatory roles of si-OTUD1 on fibroblasts proliferation, apoptosis, and migration were similar to novel-miR-741 mimic. Our findings demonstrated that novel-miR-741 inhibited the proliferation, and migration of crop fibroblasts, while OTUD1 promoted the proliferation and migration of crop fibroblasts. Therefore, the regulation of OTUD1 by novel-miR-741 was proposed as a potential therapeutic strategy for T. gallinae.

Generation of induced pluripotent stem cells from rat fibroblasts and optimization of its differentiation into mature functional neurons.

BACKGROUND: Induced pluripotent stem cells (iPSCs) derived neural stem cells (NSCs) provide a potential for autologous neural transplantation therapy following neurological insults. Thus far, in preclinical studies the donor iPSCs-NSCs are mostly of human or mouse origin with concerns centering around graft rejection when applied to rat brain injury models. For better survival and integration of transplanted cells in the injured brain in rat models, use of rat-iPSC-NSCs and in combination with biomaterials is of advantageous. Herein, we report a detailed method in generating rat iPSCs with improved reprogramming efficiency and differentiation into neurons. NEW METHOD: Rat fibroblasts were reprogrammed into iPSCs with polybrene and EF1α-STEMCCA-LoxP lentivirus vector. Pluripotency characterization, differentiation into neuronal linage cells were assessed with RT-qPCR, Western blotting, immunostaining and patch-clamp methods. Cells were cultured in a custom-designed integrin array system as well as in a hydrogel-based 3D condition. RESULTS: We describe a thorough method for the generation of rat-iPSC-NSCs, and identify integrin αvß8 as a substrate for the optimal growth of rat-iPSC-NSCs. Furthermore, with hydrogel as the supporting biomaterial in the 3-D culture, when combined with integrin αvß8 binding peptide, it forms a conducive environment for optimal growth and differentiation of iPSC-NSCs into mature neurons. COMPARISON WITH EXISTING METHODS: Published studies about rat-iPSC-NSCs are rare. This study provides a detailed protocol for the generation of rat iPSC-NSCs and optimal growth conditions for neuronal differentiation. Our method is useable for studies to assess the utility of rat iPSC-NSCs for neural transplantation in rat brain injury models.

Corpora cavernosa fibroblasts mediate penile erection.

Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.

Fibroblasts enable penile erection.

Perivascular fibroblasts may underlie erectile dysfunction.

Translational application of human keratinocyte-fibroblast cell sheets for accelerated wound healing in a clinically relevant type 2 diabetic rat model.

BACKGROUND AIMS: Despite advancements in wound care, wound healing remains a challenge, especially in individuals with type 2 diabetes. Cell sheet technology has emerged as an efficient and promising therapy for tissue regeneration and wound repair. Among these, bilayered human keratinocyte-fibroblast cell sheets constructed using temperature-responsive culture surfaces have been shown to mimic a normal tissue-like structure and secrete essential cytokines and growth factors that regulate the wound healing process. METHODS: This study aimed to evaluate the safety and therapeutic potential of human skin cell sheets to treat full-thickness skin defects in a rat model of type 2 diabetes. RESULTS: Our findings demonstrate that diabetic wounds transplanted with bilayered cell sheets resulted in accelerated re-epithelialization, increased angiogenesis, enhanced macrophage polarization and regeneration of tissue that closely resembled healthy skin. In contrast, the control group that did not receive cell sheet transplantation presented characteristic symptoms of impaired and delayed wound healing associated with type 2 diabetes. CONCLUSIONS: The secretory cytokines and the upregulation of Nrf2 expression in response to cell sheet transplantation are believed to have played a key role in the improved wound healing observed in diabetic rats. Our study suggests that human keratinocyte-fibroblast cell sheets hold great potential as a therapeutic alternative for diabetic ulcers.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis.

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with "secretory", "contractile" and "vascular" transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Treatment with a red-laser-based wound therapy device exerts positive effects in models of delayed keratinocyte and fibroblast wound healing.

BACKGROUND: Light therapy is widely used in medicine. Specifically, photobiomodulation has been shown to exert beneficial effects in wound healing disorders, which present a major challenge in health care. The study's aim was providing information on the effect of a novel, red-laser-based wound therapy device (WTD) on keratinocytes and fibroblasts during wound healing under optimal and non-optimal conditions. METHODS: The scratch wound assay was employed as a wound healing model for mechanical damage with readjustment of specific cell milieus, explicitly chronic TH1 inflammation and TH2-dominant conditions. Furthermore, gene expression analysis of pro-inflammatory cytokines (IL1A, IL6, CXCL8), growth factors (TGFB1, PDGFC), transcription factors (NFKB1, TP53) and heat shock proteins (HSP90AA1, HSPA1A, HSPD1) as well as desmogleins (DSG1, DSG3) in keratinocytes and collagen (COL1A1, COL3A1) in fibroblasts was performed after WTD treatment. RESULTS: It was shown that WTD treatment is biocompatible and supports scratch wound closure under non-optimal conditions. A distinct enhancement of desmoglein and collagen gene expression as well as induction of early growth factor gene expression was observed under chronic inflammatory conditions. Moreover, WTD increased HSPD1 transcript levels in keratinocytes and augmented collagen expression in fibroblasts during wound healing under TH2 conditions. WTD treatment also alleviated the inflammatory response in keratinocytes and induced early growth factor gene expression in fibroblasts under physiological conditions. CONCLUSION: Positive effects described for wound treatment with WTD could be replicated in vitro and seem to be to be conferred by a direct influence on cellular processes taking place in keratinocytes and fibroblasts during wound healing.

Fibroblasts in heart scar tissue directly regulate cardiac excitability and arrhythmogenesis.

After heart injury, dead heart muscle is replaced by scar tissue. Fibroblasts can electrically couple with myocytes, and changes in fibroblast membrane potential can lead to myocyte excitability, which suggests that fibroblast-myocyte coupling in scar tissue may be responsible for arrhythmogenesis. However, the physiologic relevance of electrical coupling of myocytes and fibroblasts and its impact on cardiac excitability in vivo have never been demonstrated. We genetically engineered a mouse that expresses the optogenetic cationic channel ChR2 (H134R) exclusively in cardiac fibroblasts. After myocardial infarction, optical stimulation of scar tissue elicited organ-wide cardiac excitation and induced arrhythmias in these animals. Complementing computational modeling with experimental approaches, we showed that gap junctional and ephaptic coupling, in a synergistic yet functionally redundant manner, excited myocytes coupled to fibroblasts.

Fibroblasts generate topographical cues that steer cancer cell migration.

Fibroblasts play a fundamental role in tumor development. Among other functions, they regulate cancer cells' migration through rearranging the extracellular matrix, secreting soluble factors, and establishing direct physical contacts with cancer cells. Here, we report that migrating fibroblasts deposit on the substrate a network of tubular structures that serves as a guidance cue for cancer cell migration. Such membranous tubular network, hereafter called tracks, is stably anchored to the substrate in a ß5-integrin-dependent manner. We found that cancer cells specifically adhere to tracks by using clathrin-coated structures that pinch and engulf tracks. Tracks thus represent a spatial memory of fibroblast migration paths that is read and erased by cancer cells directionally migrating along them. We propose that fibroblast tracks represent a topography-based intercellular communication system capable of steering cancer cell migration.

Generation and characterization of two immortalized dermal fibroblast cell lines from the spiny mouse (Acomys).

The spiny mouse (Acomys) is gaining popularity as a research organism due to its phenomenal regenerative capabilities. Acomys recovers from injuries to several organs without fibrosis. For example, Acomys heals full thickness skin injuries with rapid re-epithelialization of the wound and regeneration of hair follicles, sebaceous glands, erector pili muscles, adipocytes, and dermis without scarring. Understanding mechanisms of Acomys regeneration may uncover potential therapeutics for wound healing in humans. However, access to Acomys colonies is limited and primary fibroblasts can only be maintained in culture for a limited time. To address these obstacles, we generated immortalized Acomys dermal fibroblast cell lines using two methods: transfection with the SV40 large T antigen and spontaneous immortalization. The two cell lines (AcoSV40 and AcoSI-1) maintained the morphological and functional characteristics of primary Acomys fibroblasts, including maintenance of key fibroblast markers and ECM deposition. The availability of these cells will lower the barrier to working with Acomys as a model research organism, increasing the pace at which new discoveries to promote regeneration in humans can be made.

Transcriptional space-time mapping identifies concerted immune and stromal cell patterns and gene programs in wound healing and cancer.

Tissue repair responses in metazoans are highly coordinated by different cell types over space and time. However, comprehensive single-cell-based characterization covering this coordination is lacking. Here, we captured transcriptional states of single cells over space and time during skin wound closure, revealing choreographed gene-expression profiles. We identified shared space-time patterns of cellular and gene program enrichment, which we call multicellular "movements" spanning multiple cell types. We validated some of the discovered space-time movements using large-volume imaging of cleared wounds and demonstrated the value of this analysis to predict "sender" and "receiver" gene programs in macrophages and fibroblasts. Finally, we tested the hypothesis that tumors are like "wounds that never heal" and found conserved wound healing movements in mouse melanoma and colorectal tumor models, as well as human tumor samples, revealing fundamental multicellular units of tissue biology for integrative studies.

Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration.

Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.

Lost in HELLS: Disentangling the mystery of SALNR existence in senescence cellular models.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular senescence by transcriptionally and post-transcriptionally modulating the expression of many important genes involved in senescence-associated pathways and processes. Among the different lncRNAs associated to senescence, Senescence Associated Long Non-coding RNA (SALNR) was found to be down-regulated in different cellular models of senescence. Since its release in 2015, SALNR has not been annotated in any database or public repository, and no other experimental data have been published. The SALNR sequence is located on the long arm of chromosome 10, at band 10q23.33, and it overlaps the 3' end of the HELLS gene. This investigation helped to unravel the mystery of the existence of SALNR by analyzing publicly available short- and long-read RNA sequencing data sets and RT-PCR analysis in human tissues and cell lines. Additionally, the expression of HELLS has been studied in cellular models of replicative senescence, both in silico and in vitro. Our findings, while not supporting the actual existence of SALNR as an independent transcript in the analyzed experimental models, demonstrate the expression of a predicted HELLS isoform entirely covering the SALNR genomic region. Furthermore, we observed a strong down-regulation of HELLS in senescent cells versus proliferating cells, supporting its role in the senescence and aging process.

Advanced co-culture model: Soft tissue cell and bacteria interactions at the transgingival dental implant interface.

OBJECTIVES: To better simulate and understand the clinical situation in which tissue cells and bacteria compete for settlement on an implant surface, the aim was to develop an improved transgingival co-culture model. METHODS: For this model human gingival fibroblasts (HGF) were seeded on different titanium surfaces in the presence of the early colonizer Streptococcus gordonii or mixed oral bacteria. Subsequently adhesion and viability of HGF cells was analyzed. RESULTS: Simultaneous co-culture showed no decrease in the viability of HGF cells at early stages compared to the control group. However, a moderate impact on HGF viability (76 ± 23 %) was observed after 4 h of co-culture, which then significantly decreased after 5 h (21 ± 2 %) of co-cultivation, resulting in cell death and detachment from the surface. Further experiments including saliva pre-treatment of smooth and structured titanium surfaces with Streptococcus gordonii or mixed oral bacteria suggested a cell-protective property of saliva. SIGNIFICANCE: Our study revealed that during simultaneous co-culture of cells and bacteria, which resembles the clinical situation the closest, the viability of gingival cells is considerably high in the early phase, suggesting that increasing initial cell adhesion rather than antibacterial functionality is a major goal and a relevant aspect in the development and testing of transgingival implant and abutment surface modifications.

Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

Effects of substrate topography on the regulation of human fibroblasts and capsule formation via modulating macrophage polarization.

The host-material interface is critical in determining the successful integration of medical devices into human tissue. The surface topography can regulate the fibrous capsule formation around implants through macrophage polarization, but the exact mechanism remains unclear. In this study, four types of microgrooves (10 or 50 µm in groove depths and 50 or 200 µm in groove widths) were fabricated on polydimethylsiloxane (PDMS) using lithography. The microgroove surfaces were characterized using the laser scanning confocal microscopy and fourier transform infrared spectroscopy. The effect of surface topography on macrophage phenotypes and conditioned medium (CM) collected from macrophages on human foreskin fibroblast 1 (HFF-1) were investigated. The result revealed that a deeper and narrower microgroove structure means a rougher surface. Macrophages tended to adhere and aggregate on group 50-50 surface (groove depths and widths of 50 µm). THP-1 cell polarized toward both inflammatory M1 and anti-inflammatory M2 macrophages on the surface of each group. Meanwhile, CM from macrophages culture on PDMS differentially up-regulated the proliferation, migration and fibrosis of HFF-1. Among them, the group 50-50 had the strongest promoting effect. In vivo, the inflammatory response and fibrotic capsule around the implants were observed at 1 week and 4 weeks. As time passed, the inflammatory response decreased, while the capsule thickness continued to increase. The rough material surface was more inclined to develop a severe fibrotic encapsulation. In conclusion, this finding further suggested a potential immunomodulatory effect of macrophages in mediating the fibrotic response to implants and facilitated the design of biomaterial interfaces for improving tissue integration.

Distinct fibroblast progenitor subpopulation expedites regenerative mucosal healing by immunomodulation.

Injuries that heal by fibrosis can compromise organ function and increase patient morbidity. The oral mucosal barrier has a high regenerative capacity with minimal scarring, but the cellular mechanisms remain elusive. Here, we identify distinct postnatal paired-related homeobox-1+ (Prx1+) cells as a critical fibroblast subpopulation that expedites mucosal healing by facilitating early immune response. Using transplantation and genetic ablation model in mice, we show that oral mucosa enriched with Prx1+ cells heals faster than those that lack Prx1+ cells. Lineage tracing and scRNA-seq reveal that Prx1+ fibroblasts exhibit progenitor signatures in physiologic and injured conditions. Mechanistically, Prx1+ progenitors accelerate wound healing by differentiating into immunomodulatory SCA1+ fibroblasts, which prime macrophage recruitment through CCL2 as a key part of pro-wound healing response. Furthermore, human Prx1+ fibroblasts share similar gene and spatial profiles compared to their murine counterpart. Thus, our data suggest that Prx1+ fibroblasts may provide a valuable source in regenerative procedures for the treatment of corneal wounds and enteropathic fibrosis.

Fibronectin 1 derived from tumor-associated macrophages and fibroblasts promotes metastasis through the JUN pathway in hepatocellular carcinoma.

OBJECTIVE: Intercellular communication in the tumor microenvironment is a potential regulator of metastasis. To explore the specific mechanism, we performed a multi-omics analysis of hepatocellular carcinoma. MATERIALS AND METHODS: Multiple omics data including scRNA-seq, ATAC-seq, RNA-seq, and methylation data were obtained from GEO and TCGA databases. SCENIC was used to identify key transcription factors and their Regulatory networks. ScMLnet was used to explore the mechanism of intercellular communication in the microenvironment. Multiple omics studies based on RNA-seq, ATAC-seq, and methylation data were used to explore downstream mechanisms of key transcription factors. Based on the analysis of cell differentiation trajectory and transcription subtypes, the regulation of cell communication on tumor subtypes was studied, and possible therapeutic compounds were explored. The universality of this mechanism was investigated by post-Pan-cancer analysis. RESULTS: JUN and its regulatory network play a key role in HCC, which was mainly positively correlated with tumor-associated macrophages and fibroblasts. Intercellular communication analysis showed that macrophage and fibroblast-derived FN1 could increase JUN by TNFRSF11B/SMAD3. Multiomics analysis showed that KIF13A was a key downstream gene of JUN, which was involved in the activation of the hippo pathway. Analysis of cell differentiation trajectory, transcriptome subtypes, and neural network modeling showed that intercellular communication in the microenvironment can regulate the transcriptome characterization of HCC. Pan-cancer analysis indicates that this mechanism may be universal. CONCLUSION: FN1 derived from tumor-associated macrophages and fibroblasts promotes metastasis and alters transcriptome subtypes through the JUN-Hippo signaling pathway in HCC, which may be universal in cancers.

Glutaminase inhibitors rejuvenate human skin via clearance of senescent cells: a study using a mouse/human chimeric model.

Skin aging caused by various endogenous and exogenous factors results in structural and functional changes to skin components. However, the role of senescent cells in skin aging has not been clarified. To elucidate the function of senescent cells in skin aging, we evaluated the effects of the glutaminase inhibitor BPTES (bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl)ethyl sulfide) on human senescent dermal fibroblasts and aged human skin. Here, primary human dermal fibroblasts (HDFs) were induced to senescence by long-term passaging, ionizing radiation, and treatment with doxorubicin, an anticancer drug. Cell viability of HDFs was assessed after BPTES treatment. A mouse/human chimeric model was created by subcutaneously transplanting whole skin grafts from aged humans into nude mice. The model was treated intraperitoneally with BPTES or vehicle for 30 days. Skin samples were collected and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and histological analysis. BPTES selectively eliminated senescent dermal fibroblasts regardless of the method used to induce senescence; aged human skin grafts treated with BPTES exhibited increased collagen density, increased cell proliferation in the dermis, and decreased aging-related secretory phenotypes, such as matrix metalloprotease and interleukin. These effects were maintained in the grafts 1 month after termination of the treatment. In conclusion, selective removal of senescent dermal fibroblasts can improve the skin aging phenotype, indicating that BPTES may be an effective novel therapeutic agent for skin aging.

Sulfated Galactans from Gracilaria fisheri with Supplementation of Octanoyl Promote Wound Healing Activity In Vitro and In Vivo.

Sulfated galactans (SG) isolated from Gracilaria fisheri is partially degraded (DSG), and subsequentially supplemented with octanoyl (DSGO) and sulfate (DSGS) groups. The molecular weights of DSG, DSGO, and DSGS are 7.87, 152.79, and 97.07 kDa, respectively. The modification is confirmed using FTIR and NMR, while in vitro wound healing activity is assessed using scratched wound fibroblasts. The results reveal that DSGO exhibits highest percentage of wound closure in scratched fibroblast L929 cells. Furthermore, DSGO is able to promote proliferation and accelerate migration of scratched fibroblasts, which correspond to the regulation of proteins and mRNA (Ki67, p-FAK, vimentin, and E-cadherin) determined by Western blotting and qPCR analysis. The superior wound healing activity of DSGO is also confirmed in excision wound of rats. The results demonstrate that DSGO significantly enhances the percentage of wound closure, re-epithelialization, and collagen arrangement, increases α-smoth muscle actin (α-SMA) and vimentin expression, and decreases that of tumor necrosis factor-α (TNF-α) at the wound site. The results suggest that degraded SG supplemented with medium-chain fatty acids of octanoyl group may pass through the membrane, subsequently activating the mediators associated with proliferation and migration of fibroblasts, which can potentially lead to the promotion of wound healing activity.