Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. AIM OF THE STUDY: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. MATERIALS AND METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 µg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 µg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

Inducing Cellular Senescence in Mouse Embryonic Fibroblasts (MEFs).

Inducing cellular senescence in mouse embryonic fibroblasts (MEFs) is a robust tool to study the molecular mechanisms underlying senescence establishment and their heterogeneity. This protocol provides a detailed guide to generate MEFs and routinely induce senescence in MEFs using several DNA damage-dependent and DNA damage-independent induction methods.

Effect of Electromagnetic Field on Proliferation and Migration of Fibroblasts and Keratinocytes: Implications in Wound Healing and Regeneration.

Proliferation and migration of fibroblasts, keratinocytes, and endothelial cells are key events in the physiological process of wound healing. This process includes different but overlapping stages: hemostasis, inflammatory phase, the proliferative phase, and the remodeling phase. Traumatic brain injury (TBI) is defined as a mechanical insult to the brain from external mechanical force (primary injury), usually followed by the secondary injury including edema, inflammation, excitotoxicity, oxidative stress, or mitochondrial dysfunction. The process of tissue repair following TBI is based on the neuronal-glial interactions, where phagocytosis by microglia plays a crucial role. Low-frequency electromagnetic field (LF-EMF) has been shown to enhance tissue repair after TBI, however, there are limited studies investigating the effects of LF-EMF on the proliferation and migration of keratinocytes, fibroblasts, VSMCs, and endothelial cells in the context of wound healing and on neuronal cells and microglia in relation to healing after TBI. Better understanding of the effects of LF-EMF on the proliferation, migration, and differentiation of these cells is important to enhance tissue healing after injury. This review article comprehensively discussed the effect of EMF/LF-EMF on these cells. Results published by different authors are hardly comparable due to different methodological approach and experimental settings. EMF promotes migration and proliferation of fibroblasts, keratinocytes and endothelial cells (EC), and thus could improve wound healing. The pilot study preformed on a large animal model of TBI suggests anti-inflammatory effects of EMF stimulation following TBI. Therefore, EMF is recognized as a potential therapeutic option to accelerate the wound healing and improve cellular recovery and function after TBI. Nonetheless, future studies are needed to define the optimal parameters of EMF stimulation in terms of frequency or duration of exposure.

Obesity-driven changes in breast tissue exhibit a pro-angiogenic extracellular matrix signature.

Obesity has reached epidemic proportions in the United States, emerging as a risk factor for the onset of breast cancer and a harbinger of unfavorable outcomes [1], [2], [3]. Despite limited understanding of the precise mechanisms, both obesity and breast cancer are associated with extracellular matrix (ECM) rewiring [4], [5], [6]. Utilizing total breast tissue proteomics, we analyzed normal-weight (18.5 to < 25 kg/m2), overweight (25 to < 30 kg/m2), and obese (≥30 kg/m2) individuals to identify potential ECM modifying proteins for cancer development and acceleration. Obese individuals exhibited substantial ECM alterations, marked by increased basement membrane deposition, angiogenic signatures, and ECM-modifying proteins. Notably, the collagen IV crosslinking enzyme peroxidasin (PXDN) emerged as a potential mediator of the ECM changes in individuals with an elevated body mass index (BMI), strongly correlating with angiogenic and basement membrane signatures. Furthermore, glycan-binding proteins galectin-1 (LGALS1) and galectin-3 (LGALS3), which play crucial roles in matrix interactions and angiogenesis, also strongly correlate with ECM modifications. In breast cancer, elevated PXDN, LGALS1, and LGALS3 correlate with reduced relapse-free and distant-metastatic-free survival. These proteins were significantly associated with mesenchymal stromal cell markers, indicating adipocytes and fibroblasts may be the primary contributors of the obesity-related ECM changes. Our findings unveil a pro-angiogenic ECM signature in obese breast tissue, offering potential targets to inhibit breast cancer development and progression.

Extracellular vesicles from cancer cell lines of different origins drive the phenotype of normal oral fibroblasts in a CAF-like direction.

Introduction: Normal oral fibroblasts (NOFs) are located in the connective tissue of the oral mucosa. The NOFs play an important role in wound healing, tumor progression, and metastasis. They are subjected to influence by external and internal stimuli, among them extracellular vesicles (EVs), that are considered as important players in cell to cell communication, especially in carcinogenesis and metastatic processes. During tumorigenesis, stromal NOFs may undergo activation into cancer-associated fibroblasts (CAFs) that modify their phenotype to provide pro-oncogenic signals that in turn facilitate tumor initiation, progression, and metastasis. The aim of the study was to reveal the effect of EVs derived from local (oral squamous cell carcinoma - OSCC) and distant (pancreatic adenocarcinoma - PDAC; malignant melanoma brain metastasis - MBM) cancer origin on NOFs and their possible change into a CAF-like direction. Methods: The effect of each of the cancer EV types on NOFs proliferation, viability, and migration was tested. Also, changes in gene expression of the well-established CAF biomarkers ACTA2, FAP, PDGFR, and two putative CAF biomarkers, the Ca2+- activated ion channels ANO1 and KCNMA, were studied. Results: Obtained results indicate that NOFs receive and process signals transmitted by EVs originating from both OSCC, PDAC, and MBM. The fibroblast response was dependent on EV origin and concentration, and duration of EV exposure. Conclusion: The present results indicate that the molecular cargo of the EVs direct NOFs towards a pro-tumorigenic phenotype.

In Vitro Effect of Photodynamic Therapy With Curcumin in Combination With Photobiomodulation Therapy by 660 nm on the Viability of Human Gingival Fibroblasts.

Introduction: This study aimed to assess the effect of repeated irradiations of 660 nm photobiomodulation therapy (PBMT) after photodynamic therapy (PDT) with curcumin on the viability of human gingival fibroblasts (HGFs). Methods: In this in vitro, experimental study, HGFs were cultured and assigned to five groups: One control group with no intervention and four experimental groups of PDT with curcumin, PBMT (660 nm laser irradiation) immediately after PDT, PBMT immediately and 24 hours after PDT and PBMT immediately and 24 hours and 48 hours after PDT. Cell viability was assessed after 1, 4, and 7 days using the methyl thiazolyl tetrazolium (MTT) assay. Data were analyzed by one-way ANOVA. Results: On day 1, the control group had no significant difference with one-time (P=1.00), two-time (P=1.00), and three-time (P=0.88) laser irradiation groups. On day 4, the difference between the control and one-time (P<0.001), two-time (P<0.001) and three-time (P=0.02) laser irradiation groups was statistically significant, suggesting more cell viability in test groups. On day 7, the three-time laser irradiation group showed significant cell viability compared to the other two test groups but not with the control group (P=0.98). Conclusion: PBMT with 660 nm laser irradiation after PDT with curcumin would increase the viability of HGFs by increasing the frequency of irradiation.

Resveratrol inhibits pancreatic cancer proliferation and metastasis by depleting senescent tumor-associated fibroblasts.

BACKGROUND: Pancreatic cancer, a formidable gastrointestinal neoplasm, is characterized by its insidious onset, rapid progression, and resistance to treatment, which often lead to a grim prognosis. While the complex pathogenesis of pancreatic cancer is well recognized, recent attention has focused on the oncogenic roles of senescent tumor-associated fibroblasts. However, their precise role in pancreatic cancer remains unknown. Resveratrol is a natural polyphenol known for its multifaceted biological actions, including antioxidative and neuroprotective properties, as well as its potential to inhibit tumor proliferation and migration. Our current investigation builds on prior research and reveals the remarkable ability of resveratrol to inhibit pancreatic cancer proliferation and metastasis. AIM: To explore the potential of resveratrol in inhibiting pancreatic cancer by targeting senescent tumor-associated fibroblasts. METHODS: Immunofluorescence staining of pancreatic cancer tissues revealed prominent coexpression of α-SMA and p16. HP-1 expression was determined using immunohistochemistry. Cells were treated with the senescence-inducing factors known as 3CKs. Long-term growth assays confirmed that 3CKs significantly decreased the CAF growth rate. Western blotting was conducted to assess the expression levels of p16 and p21. Immunofluorescence was performed to assess LaminB1 expression. Quantitative real-time polymerase chain reaction was used to measure the levels of several senescence-associated secretory phenotype factors, including IL-4, IL-6, IL-8, IL-13, MMP-2, MMP-9, CXCL1, and CXCL12. A scratch assay was used to assess the migratory capacity of the cells, whereas Transwell assays were used to evaluate their invasive potential. RESULTS: Specifically, we identified the presence of senescent tumor-associated fibroblasts within pancreatic cancer tissues, linking their abundance to cancer progression. Intriguingly, Resveratrol effectively eradicated these fibroblasts and hindered their senescence, which consequently impeded pancreatic cancer progression. CONCLUSION: This groundbreaking discovery reinforces Resveratrol's stature as a potential antitumor agent and positions senescent tumor-associated fibroblasts as pivotal contenders in future therapeutic strategies against pancreatic cancer.

Ferroptosis-Associated Extracellular Matrix Remodeling in Radiation-Induced Lung Fibrosis Progression.

Background: Radiation-induced lung fibrosis (RILF) is a life-threatening complication of thoracic radiotherapy. Ferroptosis, a recently discovered type of cell death, is believed to contribute to RILF, though the associated mechanisms are unknown. This study aimed to investigate the potential mechanism of ferroptosis in RILF and examine the contribution of different cell types to ferroptosis during RILF progression. Methods: Histopathological changes in RILF lung tissue were assessed through H&E and Masson staining. IHC staining investigated ferroptosis markers (GPX4, ACSL4, NCOA4). Ferroptosis-related genes (FRG) and pathway scores were derived from RILF transcriptome microarray data. The sc-RNAseq analysis detected FRG score dynamics across cell types, validated by IF staining for PDGFR-α and ACSL4. Results: ACSL4 and NCOA4 protein levels were significantly higher and GPX4 lower in IR than control. FRG scores were positively correlated with fibrosis-related pathway scores in the RILF transcriptome data. FRG and ECM scores were concurrently upregulated in myofibroblasts. Enhanced co-staining of PDGFR-α and ACSL4 were observed in the fibrotic areas of RILF lungs. Conclusions: Our research indicated that in RILF, fibroblasts undergoing ferroptosis may release increased levels of ECM, potentially accelerating the progression of lung fibrosis. This finding presents ferroptosis as a potential therapeutic target in RILF.

Paired Transcriptional Analysis of Periodontitis and Peri-Implantitis within same host: a Pilot study.

BACKGROUND: Peri-implantitis, a plaque-associated pathological condition, has been on the rise with the increasing prevalence of dental implants. Despite its similarities to periodontitis, peri-implantitis is difficult to control completely and has high relapse rates. This has sparked interest in exploring the pathogenic differences between the two conditions. METHODS: This cross-sectional study involved 10 participants to concurrently examine periodontitis and peri-implantitis within the same patients, thereby minimizing inter-individual variation. Gingival tissue samples were collected from each participant, comprising 10 periodontitis and 10 peri-implantitis tissues, and RNAs were extracted. Using RNA sequencing and bioinformatics analysis, we investigated complex gene interactions, immune responses, and the role of the extracellular matrix in both conditions. We identified hub genes in each enhanced Protein-Protein Interaction network, providing crucial insights into these diseases' pathogenesis. RESULTS: Our findings highlighted the potential involvement of activated fibroblasts in the pathogenesis of peri-implantitis, identifying three marker genes (ACTA2, FAP, and PDGFRß) overexpressed in peri-implantitis, thus highlighting their potential as disease-specific biomarkers. CONCLUSIONS: Our study uncovered a novel connection between peri-implantitis and activated fibroblasts, examining specific markers and microbial differences between periodontitis and peri-implantitis. These insights improve our understanding of peri-implantitis pathogenesis, encouraging future research for better management and prevention strategies. CLINICAL SIGNIFICANCE: This study identifies key insights into the pathogenesis of peri-implantitis compared to periodontitis. These findings promise to advance clinical approaches for better managing and preventing peri-implantitis, addressing its complexities and high relapse rates effectively.

Anti-Müllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma.

STUDY QUESTION: What is the involvement of ovarian stroma in the anti-Müllerian hormone (AMH) signaling pathway and which stromal cells are involved? SUMMARY ANSWER: Mouse and human ovaries show high expression of AMH receptor II (AMHR2) in the stromal fibroblasts surrounding the follicles and activation of the post-AMHR2 pathway by recombinant AMH was evidenced by increased phosphorylation of SMAD1,5 and 9, increased expression AMHR2 and upregulation of αSMA, suggesting fibroblast activation to initiate myofibroblast differentiation. WHAT IS KNOWN ALREADY: AMH secreted by small growing follicles, regulates ovarian activity. It suppresses initial primordial follicle (PMF) recruitment and FSH-dependent growth. AMH signal transduction is mediated by AMHR2, activating intracellular SMAD proteins and other signaling cascades to induce target-gene expression. Although AMHR2 expression has been reported within the follicle unit, there is evidence suggesting it may be identified in the stroma as well. STUDY DESIGN, SIZE, DURATION: Fresh murine ovaries were extracted from BALB/c mice (6 weeks old; n = 12 and 21 days old; n = 56). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18-35, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (6 weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1α/αSMA/VCAM1, with additional vimentin staining in mice. Murine (21 days old) and human donor ovaries were used for fibroblast isolation and subsequent 7-day cultures. Prior to assessing AMH effects on isolated fibroblast culture, purity validation tests were implemented to ensure the absence of epithelial, immune, endothel, granulosa, and theca ovarian cell populations. The fibroblast culture's homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for αSMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200 ng/ml) for 0-72 h, with an additional time point of 96 h for human samples, followed by RT-qPCR, western blot, and immunocytochemistry (ICC) for AMHR2 expression. AMHR2 post-receptor signaling was examined by pSMAD1,5,9 levels via western blot. Activated fibroblast marker, αSMA, was assessed via western blot and ICC. MAIN RESULTS AND THE ROLE OF CHANCE: Immunostaining of mouse and human ovarian tissue showed that stromal cells around follicles at all developmental stages exhibit high AMHR2 expression, while granulosa cells of growing follicles show considerably lower levels. The majority of these AMHR2-positive stromal cells were identified as fibroblasts (Collagen1α in mice and human; vimentin in mice). RT-qPCR, western blot, and immunostaining were performed on cultured mouse and human fibroblasts, confirming that they consisted of a pure fibroblast population (αSMA/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94 ± 1.34 cells/field in mice) and 100% (average 19.20 ± 1.39 cells/field in human samples) of these fibroblasts expressed AMHR2 (ICC). rAMH treated cultured fibroblasts showed increased pSMAD1,5 and 9 levels, demonstrating the effects of AMH on its downstream signaling pathway. pSMAD1,5 and 9 expression increased, as detected by western blot: 1.92-fold in mice (48 h, P = 0.026) and 2.37-fold in human samples (48 h, P = 0.0002). In addition, rAMH treatment increased AMHR2 protein expression, as observed in ICC (human): a 2.57-fold upregulation of AMHR2 Mean Fluorescence Intensity (MFI) (96 h, P = 0.00036), and western blot, showing a 4.2-fold time-dependent increase (48 h, P = 0.026) in mice and 2.4-fold change (48 h, P = 0.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72 h, P = 0.0137) in mice and a 7.87-fold change (72 h, P < 0.0001) in humans. rAMH treatment induced fibroblast activation (αSMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. αSMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96 h, P = 0.000067), and in western blot with a 5.12-fold increase in mice (48 h, P = 0.0345) and a 2.69-fold increase in humans (48 h, P ≤ 0.0001). Activated AMHR2-positive stained fibroblast fractions were solely located around growing follicles, in both human and mice. In addition, a small population of AMHR2-positive stained theca cells (VCAM1 positive) was observed. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ex vivo, fibroblast gene expression might be changed by adhesion to the tissue-culture plate. Nevertheless, cultured fibroblasts (with and without rAMH) are subjected to the same conditions. Observations or significant differences can therefore be considered reliable. In addition, the presented effect of rAMH on fibroblasts is not directly linked to the known inhibitory effect of AMH on follicle activation. WIDER IMPLICATIONS OF THE FINDINGS: Clarifying the populations of AMH-responsive cells in the ovary provides a foundation for further investigation of the complex AMH signaling across the ovary. The composition of AMH-releasing and -responsive cells can shed light on the communication network between follicles and their environment, which may elucidate the mechanisms behind the AMH inhibitory effect on PMF activation. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study. TRIAL REGISTRATION NUMBER: N/A.

Transcriptomic profiling of lung fibroblasts in silicosis: Regulatory roles of Nrf2 agonists in a mouse model.

Silicosis is an occupational disease caused by long-term inhalation of free silica, resulting in a significant global health burden. Its pathogenesis remains unclear, and there is no effective treatment. Proliferative and activated myofibroblasts play a key role in the development of silicosis. Traditional studies have focused on fibroblast proliferation and collagen secretion, neglecting their functional heterogeneity. With the advancement of omics research, more pathogenic fibroblast subgroups and their functions have been identified. In this study, we applied transcriptomics to analyze gene changes in primary lung fibroblasts during silicosis development using a mouse model. Our results indicate that DEGs are enriched in collagen secretion, ECM synthesis, leukocyte migration, and chemotaxis functions. Altered core genes are associated with immune cell recruitment and cell migration. Nrf2 agonists, known for anti-inflammatory and antioxidant properties, have shown potential therapeutic effects in fibrotic diseases. However, their effects on fibroblasts in silicosis are not fully understood. We used four common Nrf2 agonists to study gene expression changes in lung fibroblasts at the transcriptome level, combined with histopathological and biochemical methods, to investigate their effects on silicosis in mice. Results show that Nrf2 agonists can exert anti-silicosis fibrosis functions by downregulating genes like Fos and Egr1, involved in cell differentiation, proliferation, and inflammation. In conclusion, this study suggests that inflammation-related co-functions of fibroblasts may be a potential mechanism in silicosis pathogenesis. Targeting Nrf2 may be a promising strategy to alleviate oxidative stress and inflammation in silicosis.

Umbelliferone alleviates impaired wound healing and skin barrier dysfunction in high glucose-exposed dermal fibroblasts and diabetic skins.

Skin wound healing is a complex process involving various cellular and molecular events. However, chronic wounds, particularly in individuals with diabetes, often experience delayed wound healing, potentially leading to diabetic skin complications. In this study, we examined the effects of umbelliferone on skin wound healing using dermal fibroblasts and skin tissues from a type 2 diabetic mouse model. Our results demonstrate that umbelliferone enhances several crucial aspects of wound healing. It increases the synthesis of key extracellular matrix components such as collagen I and fibronectin, as well as proteins involved in cell migration like EVL and Fascin-1. Additionally, umbelliferone boosts the secretion of angiogenesis factors VEGF and HIF-1α, enhances the expression of cell adhesion proteins including E-cadherin, ZO-1, and Occludin, and elevates levels of skin hydration-related proteins like HAS2 and AQP3. Notably, umbelliferone reduces the expression of HYAL, thereby potentially decreasing tissue permeability. As a result, it promotes extracellular matrix deposition, activates cell migration and proliferation, and stimulates pro-angiogenic factors while maintaining skin barrier functions. In summary, these findings underscore the therapeutic potential of umbelliferone in diabetic wound care, suggesting its promise as a treatment for diabetic skin complications. KEY MESSAGES: Umbelliferone suppressed the breakdown of extracellular matrix components in the skin dermis while promoting their synthesis. Umbelliferone augmented the migratory and proliferative capacities of fibroblasts. Umbelliferone activated the release of angiogenic factors in diabetic wounds, leading to accelerated wound healing. Umbelliferone bolstered intercellular adhesion and reinforced the skin barrier by preventing moisture loss and preserving skin hydration.

Cell and transcriptomic diversity of infrapatellar fat pad during knee osteoarthritis.

OBJECTIVES: In this study, we employ a multiomic approach to identify major cell types and subsets, and their transcriptomic profiles within the infrapatellar fat pad (IFP), and to determine differences in the IFP based on knee osteoarthritis (KOA), sex and obesity status. METHODS: Single-nucleus RNA sequencing of 82 924 nuclei from 21 IFPs (n=6 healthy control and n=15 KOA donors), spatial transcriptomics and bioinformatic analyses were used to identify contributions of the IFP to KOA. We mapped cell subclusters from other white adipose tissues using publicly available literature. The diversity of fibroblasts within the IFP was investigated by bioinformatic analyses, comparing by KOA, sex and obesity status. Metabolomics was used to further explore differences in fibroblasts by obesity status. RESULTS: We identified multiple subclusters of fibroblasts, macrophages, adipocytes and endothelial cells with unique transcriptomic profiles. Using spatial transcriptomics, we resolved distributions of cell types and their transcriptomic profiles and computationally identified putative cell-cell communication networks. Furthermore, we identified transcriptomic differences in fibroblasts from KOA versus healthy control donor IFPs, female versus male KOA-IFPs and obese versus normal body mass index (BMI) KOA-IFPs. Finally, using metabolomics, we defined differences in metabolite levels in supernatants of naïve, profibrotic stimuli-treated and proinflammatory stimuli-treated fibroblasts from obese compared to normal BMI KOA-IFPs. CONCLUSIONS: Overall, by employing a multiomic approach, this study provides the first comprehensive map of the cellular and transcriptomic diversity of human IFP and identifies IFP fibroblasts as key cells contributing to transcriptomic and metabolic differences related to KOA disease, sex or obesity.

RNA-seq and bulk RNA-seq data analysis of cancer-related fibroblasts (CAF) in LUAD to construct a CAF-based risk signature.

Angiogenesis, metastasis, and resistance to therapy are all facilitated by cancer-associated fibroblasts (CAFs). A CAF-based risk signature can be used to predict patients' prognoses for Lung adenocarcinoma (LUAD) based on CAF characteristics. The Gene Expression Omnibus (GEO) database was used to gather signal-cell RNA sequencing (scRNA-seq) data for this investigation. The GEO and TCGA databases were used to gather bulk RNA-seq and microarray data for LUAD. The scRNA-seq data were analyzed using the Seurat R program based on the CAF markers. Our goal was to use differential expression analysis to discover differentially expressed genes (DEGs) across normal and tumor samples in the TCGA dataset. Pearson correlation analysis was utilized to discover prognostic genes related with CAF, followed by univariate Cox regression analysis. Using Lasso regression, a risk signature based on CAF-related prognostic genes was created. A nomogram model was created based on the clinical and pathological aspects. 5 CAF clusters were identified in LUAD, 4 of which were associated with prognosis. From 2811 DEGs, 1002 genes were found to be significantly correlated with CAF clusters, which led to the creation of a risk signature with 8 genes. These 8 genes were primarily connected with 41 pathways, such as antigen paocessing and presentation, apoptosis, and cell cycle. Meanwhile, the risk signature was significantly associated with stromal and immune scores, as well as some immune cells. Multivariate analysis revealed that risk signature was an independent prognostic factor for LUAD, and its value in predicting immunotherapeutic outcomes was confirmed. A novel nomogram integrating the stage and CAF-based risk signature was constructed, which exhibited favorable predictability and reliability in the prognosis prediction of LUAD. CAF-based risk signatures can be effective in predicting the prognosis of LUAD, and they may provide new strategies for cancer treatments by interpreting the response of LUAD to immunotherapy.

SPP1 induces idiopathic pulmonary fibrosis and NSCLC progression via the PI3K/Akt/mTOR pathway.

BACKGROUND: The prevalence of non-small cell lung cancer (NSCLC) is notably elevated in individuals diagnosed with idiopathic pulmonary fibrosis (IPF). Secreted phosphoprotein 1 (SPP1), known for its involvement in diverse physiological processes, including oncogenesis and organ fibrosis, has an ambiguous role at the intersection of IPF and NSCLC. Our study sought to elucidate the function of SPP1 within the pathogenesis of IPF and its subsequent impact on NSCLC progression. METHODS: Four GEO datasets was analyzed for common differential genes and TCGA database was used to analyze the prognosis. The immune infiltration was analyzed by TIMER database. SPP1 expression was examined in human lung tissues, the IPF fibroblasts and the BLM-induced mouse lung fibrosis model. Combined with SPP1 gene gain- and loss-of-function, qRT-PCR, Western blot, EdU and CCK-8 experiments were performed to evaluate the effects and mechanisms of SPP1 in IPF progression. Effect of SPP1 on NSCLC was detected by co-cultured IPF fibroblasts and NSCLC cells. RESULTS: Through bioinformatics analysis, we observed a significant overexpression of SPP1 in both IPF and NSCLC patient datasets, correlating with enhanced immune infiltration of cancer-associated fibroblasts in NSCLC. Elevated levels of SPP1 were detected in lung tissue samples from IPF patients and bleomycin-induced mouse models, with partial colocalization observed with α-smooth muscle actin. Knockdown of SPP1 inhibits TGF-ß1-induced differentiation of fibroblasts to myofibroblasts and the proliferation of IPF fibroblasts. Conversely, SPP1 overexpression promoted IPF fibroblast proliferation via PI3K/Akt/mTOR pathway. Furthermore, IPF fibroblasts promoted NSCLC cell proliferation and activated the PI3K/Akt/mTOR pathway; these effects were attenuated by SPP1 knockdown in IPF fibroblasts. CONCLUSIONS: Our findings suggest that SPP1 functions as a molecule promoting both fibrosis and tumorigenesis, positioning it as a prospective therapeutic target for managing the co-occurrence of IPF and NSCLC.

Comparative transcriptome of normal and cancer-associated fibroblasts.

BACKGROUND: The characteristics of a tumor are largely determined by its interaction with the surrounding micro-environment (TME). TME consists of both cellular and non-cellular components. Cancer-associated fibroblasts (CAFs) are a major component of the TME. They are a source of many secreted factors that influence the survival and progression of tumors as well as their response to drugs. Identification of markers either overexpressed in CAFs or unique to CAFs would pave the way for novel therapeutic strategies that in combination with conventional chemotherapy are likely to have better patient outcome. METHODS: Fibroblasts have been derived from Benign Prostatic Hyperplasia (BPH) and prostate cancer. RNA from these has been used to perform a transcriptome analysis in order to get a comparative profile of normal and cancer-associated fibroblasts. RESULTS: The study has identified 818 differentially expressed mRNAs and 17 lincRNAs between normal and cancer-associated fibroblasts. Also, 15 potential lincRNA-miRNA-mRNA combinations have been identified which may be potential biomarkers. CONCLUSIONS: This study identified differentially expressed markers between normal and cancer-associated fibroblasts that would help in targeted therapy against CAFs/derived factors, in combination with conventional therapy. However, this would in future need more experimental validation.

The role and mechanism of CRISPLD2 in skin fibrosis of systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune connective tissue disease involving multiple organs. The most common clinical symptom of SSc is progressive fibrosis of the skin, and the pathologically manifestations of skin were activation and proliferation of fibroblasts and continuous proliferation of extracellular matrix. Transforming growth factor ß (TGFß) can promote the proliferation and activation of fibroblasts, causing excessive deposition of collagen and structural proteins. Therefore, exploring the specific mechanism of TGFß-related pathway on fibrosis is of great significance for improving skin fibrosis in SSc. METHODS: Genes related to TGFß pathway were screened through bioinformatics analysis, and SSc phenotypes were verified in vivo and vitro. The relevant molecular mechanisms were preliminarily discussed in combination with transcriptome sequencing. RESULTS: Human cysteine-rich secreted protein LCCL domain protein 2 (CRISPLD2) was found increased reactivity in TGFß induced fibroblasts, and the expression of ACTA2 (ɑ-SMA) decreased significantly in TGFß-mediated fibroblasts with up-regulation of CRISPLD2. CONCLUSION: CRISPLD2 was found increased reactivity in TGFß induced fibroblasts, and we further confirmed that CRISPLD2 can participate in TGFß induced fibroblast fibrosis from multiple perspectives and levels in negative feedback regulation, and investigated the mechanism of CRISPLD2 in fibrosis.

Bulk integrated single-cell-spatial transcriptomics reveals the impact of preoperative chemotherapy on cancer-associated fibroblasts and tumor cells in colorectal cancer, and construction of related predictive models using machine learning.

BACKGROUND: Preoperative chemotherapy (PC) is an important component of Colorectal cancer (CRC) treatment, but its effects on the biological functions of fibroblasts and epithelial cells in CRC are unclear. METHODS: This study utilized bulk, single-cell, and spatial transcriptomic sequencing data from 22 independent cohorts of CRC. Through bioinformatics analysis and in vitro experiments, the research investigated the impact of PC on fibroblast and epithelial cells in CRC. Subpopulations associated with PC and CRC prognosis were identified, and a predictive model was constructed using machine learning. RESULTS: PC significantly attenuated the pathways related to tumor progression in fibroblasts and epithelial cells. NOTCH3 + Fibroblast (NOTCH3 + Fib), TNNT1 + Epithelial (TNNT1 + Epi), and HSPA1A + Epithelial (HSPA1A + Epi) subpopulations were identified in the adjacent spatial region and were associated with poor prognosis in CRC. PC effectively diminished the presence of these subpopulations, concurrently inhibiting pathway activity and intercellular crosstalk. A risk signature model, named the Preoperative Chemotherapy Risk Signature Model (PCRSM), was constructed using machine learning. PCRSM emerged as an independent prognostic indicator for CRC, impacting both overall survival (OS) and recurrence-free survival (RFS), surpassing the performance of 89 previously published CRC risk signatures. Additionally, patients with a high PCRSM risk score showed sensitivity to fluorouracil-based adjuvant chemotherapy (FOLFOX) but resistance to single chemotherapy drugs (such as Bevacizumab and Oxaliplatin). Furthermore, this study predicted that patients with high PCRSM were resistant to anti-PD1therapy. CONCLUSION: In conclusion, this study identified three cell subpopulations (NOTCH3 + Fib, TNNT1 + Epi, and HSPA1A + Epi) associated with PC, which can be targeted to improve the prognosis of CRC patients. The PCRSM model shows promise in enhancing the survival and treatment of CRC patients.

The axis of tumor-associated macrophages, extracellular matrix proteins, and cancer-associated fibroblasts in oncogenesis.

The extracellular matrix (ECM) is a complex, dynamic network of multiple macromolecules that serve as a crucial structural and physical scaffold for neighboring cells. In the tumor microenvironment (TME), ECM proteins play a significant role in mediating cellular communication between cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Revealing the ECM modification of the TME necessitates the intricate signaling cascades that transpire among diverse cell populations and ECM proteins. The advent of single-cell sequencing has enabled the identification and refinement of specific cellular subpopulations, which has substantially enhanced our comprehension of the intricate milieu and given us a high-resolution perspective on the diversity of ECM proteins. However, it is essential to integrate single-cell data and establish a coherent framework. In this regard, we present a comprehensive review of the relationships among ECM, TAMs, and CAFs. This encompasses insights into the ECM proteins released by TAMs and CAFs, signaling integration in the TAM-ECM-CAF axis, and the potential applications and limitations of targeted therapies for CAFs. This review serves as a reliable resource for focused therapeutic strategies while highlighting the crucial role of ECM proteins as intermediates in the TME.

The mechanism of MMP14-positive tumor-associated fibroblast subsets in inhibiting PD-1 immunotherapy for esophageal cancer through exosomal tsRNA-10522.

Esophageal cancer (EC) continues to pose a significant health risk. Cancer-associated fibroblasts (CAFs), an essential part of the tumor microenvironment (TME), are viewed as potential therapeutic targets. However, their role in tumor mechanisms specific to esophageal cancer remains to be elucidated. This study identified MMP14+ CAFs and MMP14- CAFs using immunofluorescence staining. The cytotoxic activity of CD8 T cells was assessed via western blot and ELISA. Using a transwell test, the migratory potential of MMP14+ CAFs was evaluated. Using flow cytometry, apoptosis was found in the esophageal squamous cell carcinoma cell line KYSE30. To determine the important tsRNAs released by MMP14+ CAFs, tsRNA-seq was used. Two subgroups of EC receiving PD-1 immunotherapy were identified by our research: MMP14+ CAFs and MMP14- CAFs. MMP14+ CAFs showed improved migratory capacity and released more inflammatory factors linked to cancer. Through exosomes, these CAFs may prevent anti-PD-1-treated CD8 T cells from being cytotoxic. Furthermore, exosomal tsRNA from MMP14+ CAFs primarily targeted signaling pathways connected with cancer. Notably, it was discovered that tsRNA-10522 plays a critical role within inhibiting CD8 T cell tumor cell death. The tumor cell killing of CD8 T cells by exosomal tsRNA-10522 is inhibited by a subgroup of cells called MMP14+ CAFs inside the EC microenvironment during PD-1 immunotherapy. This reduces the effectiveness of PD-1 immunotherapy for EC. Our findings demonstrate the inhibitory function of MMP14+ CAFs within EC receiving PD-1 immunotherapy, raising the prospect that MMP14+ CAFs might serve as predictive indicators in EC receiving PD-1 immunotherapy.