Tissue Fibroblasts Research Articles

Ginsenoside Rb1 affects mitochondrial Ca2+ transport and inhibits fat deposition and fibrosis by regulating the wnt signaling pathway to treat rotator cuff tears via docking with SFRP1

BackgroundRotator cuff tears (RCTs) are among the most common musculoskeletal disorders that affect quality of life. This study aimed to investigate the efficacy of ginsenoside Rb1 in RCTs and the mechanisms involved.MethodsFirst, a fibrotic model of FAPs was induced, and FAPs were cultured in media supplemented with different concentrations of ginsenoside Rb1. Next, a rat model of RCTs was constructed and treated with ginsenoside Rb1. Molecular docking was subsequently utilized to detect the binding of ginsenoside Rb1 and SFRP1. Finally, SFRP1 was knocked down and overexpressed in vivo and in vitro to investigate the mechanism of ginsenoside Rb1 and SFRP1 in RCTs.ResultsCompared with the Normal group, FAP viability was decreased, but Collagen II, FN and α-SMA levels were increased in the Control group. After treatment with different concentrations of ginsenoside Rb1, FAP viability increased, but Collagen II, FN and α-SMA levels decreased. Among them, 60 µM ginsenoside Rb1 had the best effect. In vivo experiments revealed that ginsenoside Rb1 improved RCTs in rats. Molecular docking revealed the binding of ginsenoside Rb1 to SFRP1. Additionally, SFRP1 levels were lower in the Control group than in the Normal group. After treatment with ginsenoside Rb1, SFRP1 levels increased. In vivo, overexpressing SFRP1 along with ginsenoside Rb1 treatment further alleviated tendon tissue fibroblast infiltration and fat accumulation and further reduced the expression of Collagen II, FN, and α-SMA. In vitro, overexpressing SFRP1 along with ginsenoside Rb1 treatment further decreased the expression of CaMKII, PLC, PKC, Wnt, and β-catenin, further decreased the Ca2+ fluorescence intensity and mitochondrial length, increased the red/green intensity, and decreased the MitoSOX fluorescence intensity. Additionally, overexpressing SFRP1 along with ginsenoside Rb1 treatment further increased cell proliferation, decreased apoptosis, reduced the protein expression of Collagen II, FN, and α-SMA in muscle tissue, and further reduced the levels of TNF-α, IL-1β, and IL-6 in the cell supernatant.ConclusionsGinsenoside Rb1 inhibited the activation of the Wnt signaling pathway by promoting SFRP1 expression, thereby inhibiting mitochondrial function and Ca2+ absorption to treat fat infiltration and muscle fibrosis caused by RCTs.

Comparison of Biomarkers Playing a Role in Pterygium Development in Pterygium and Recurrent Pterygium Tissues

Background/Objectives: Pterygium is a nonneoplastic elastotic degeneration characterized by subepithelial growth. It manifests as an ocular lesion originating from the bulbar conjunctiva, extending to the corneal surface, and reaching the visual axis in some cases. Although the exact cause is unknown, prolonged exposure to ultraviolet radiation is considered the most significant contributing factor. Chronic irritation and actinic damage are likely responsible for the typical fibrovascular reactions observed in pterygium. Additionally, growth factors, cytokines, and matrix metalloproteinases play roles in the pathogenesis of pterygium. This study compared recurrent and primary pterygium cases at the molecular level to gain new insights into the etiology of pterygium. Methods: Total protein was extracted from surgical samples of patients with primary and recurrent pterygium, and the levels of transforming growth factor beta 1 (TGF-β1), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), IL-8, and IL-10 were analyzed using the enzyme-linked immunosorbent assay technique. Target gene expression levels were analyzed using the ΔΔCt method after cDNA synthesis from isolated RNA, with normalization to GAPDH and quantification performed with SYBR Green PCR Master Mix. Results: Among the studied cytokines, IL-10 levels were higher in primary pterygium than in recurrent pterygium (722.0 ± 600.9/421.4 ± 266.8) (p = 0.0054). Other cytokines (IL-6, IL-8, IL-1β, and TGF-β1) were detected at similar levels in both primary and recurrent pterygium (p = 0.2986). Additionally, the TGF-β1 gene expression was found to be significantly upregulated in recurrent pterygium tissue compared to primary pterygium tissue (p = 0.034). Conclusions: This increase suggests that TGF-β1 may contribute to the recurrence mechanisms of pterygium through processes such as fibroblast activation and tissue remodeling. The higher levels of IL-10 in primary pterygium compared to recurrent pterygium indicate an enhanced early protective response aimed at limiting pterygium progression and controlling the inflammatory process.

Functional analysis of fibroblasts and macrophages in head and neck paragangliomas.

Head and neck paragangliomas (HNPGN) are tumours that carry significant morbidity The role of the stroma in the pathogenesis of HNPGN is not completely understood. This study explores the profile of fibroblasts and macrophages in HNPGN. Ten patients undergoing HNPGN surgery were recruited. CD68 and CD163 immunohistochemistry was performed for macrophage analysis; CD90 and podoplanin (PDPN) expression was examined to identify fibroblasts. RT-qPCR was performed on HNPGN tissue for macrophage- and fibroblast-associated molecules. Fibroblast cultures were established from HNPGN were analysed by RT-qPCR and flowcytometry. Confocal microscopy for MCT1 and MCT4 was performed in HNPGN. CD68 and CD163 expressing macrophages were noted in HNPGN. CD90 and PDPN expressing cells were present in HNPGN. RT-qPCR analysis showed expression of phenotypic and functional macrophage- and fibroblast-associated molecules in HNPGN. RT-qPCR analysis of fibroblasts cultured from HNPGN confirmed the expression of several molecules including PDPN at comparable levels to healthy tissue fibroblasts. Expression of FAP, MCT-1, insulin receptor (CD220) and insulin growth factor receptor-2 (CD222) was noted on HNPGN derived fibroblasts on flowcytometry. MCT1 and MCT4 were expressed in HNPGN tumour cells and stromal macrophages in-situ. Fibroblasts and macrophages are present in the HNPGN tumour microenvironment, and several macrophage and fibroblast functional markers are expressed in HNPGN. Macrophages in HNPGN tissue express metabolic markers MCT1 and MCT4. Further analysis of the fibroblast and macrophage function in HNPGN will improve our understanding of their potential roles in tumour pathogenesis.

The Presence of Pigment Incontinence in Sinonasal Mucosal Melanoma.

Regression is an immunological phenomenon described in cutaneous melanoma whereby tumor is replaced with tumor-infiltrating lymphocytes, granulation tissue, and mature fibroblasts often accompanied by pigment incontinence (accumulation of melanin in the upper dermis). Pigment incontinence results in grossly pigmented lesions that may be mistaken for viable tumor and has not been described in sinonasal mucosal melanoma (SNMM). This study investigates the presence of regression and pigment incontinence in patients with SNMM. A retrospective chart review was conducted on SNMM patients from 2007 to 2023. Pathology slides from surgical resection were examined by two pathologists blinded to treatment information for the presence and extent of pigment-laden macrophages and other histopathologic features of regression. Seventeen patients with SNMM were included in this study who underwent surgical resection. Three patients received neoadjuvant therapy followed by surgical resection. Regression was present in 94% of patients and pigment incontinence was present in 65% of patients and occurred in both neoadjuvant treated patients and treatment naïve patients. All three patients with neoadjuvant treatment had evidence of pigment incontinence. This study highlights that SNMM often displays characteristics of regression. This study is one of the first to describe the presence of pigment incontinence in patients with SNMM. Pigment incontinence can be a part of the natural tumor life cycle and grossly pigmented lesions could easily be confused for melanoma especially after neoadjuvant therapy. Developing an understanding of regression and pigment incontinence within SNMM is important for diagnosis and clinical management. IV Laryngoscope, 2024.

Abstract 4123639: Tumor Endothelial Marker 1 is Upregulated in Atrial Cardiac Fibroblasts in Atrial Fibrillation and Correlates With Atrial Fibrosis

Background: Tumor endothelial marker 1 (TEM1 or CD248) is a transmembrane protein found in activated mesenchymal cells during embryo development. Its expression level becomes very low or undetectable in adult tissue. Re-expression of TEM1 in fibroblasts has been reported in hepatic, renal, and pulmonary fibrosis. We evaluated atrial TEM1 expression in atrial fibrillation (AF) and its relation with atrial fibrosis. Methods and Results: All data were presented as mean±standard error. Left atrial (LA) tissues were collected from 30 patients (pts) (mean age 64.0±1.6 yrs, male 76.7%) with AF underwent cardiac surgery (mitral and/or tricuspid valve surgery 96.7%) and maze procedure with LA appendage excision. Immunofluorescence (IF) staining showed TEM1 expressed in atrial cardiac fibroblasts (CFs) in the LA tissue of AF pts, but not in normal LA tissue (US Biomax). Primary human CFs were directly isolated from the LA tissue. IF staining showed TEM1 expression in the isolated CFs from AF pts but not in normal human CFs (ScienCell). Western blot could detect TEM1 expression in the LA tissues of all 30 AF pts. Sirius Red staining was used to visualize collagen and atrial fibrosis was quantified using a pixel-based method (Adobe Photoshop) by calculating the total numbers of red pixels and the percentage (%) of red pixels to total pixels. Atrial fibrosis was increased in AF pts compared with normal control (total number of red pixels: 160740±13066 vs. 50863±6357, % of red pixels to total pixels: 11.4±0.8 vs. 3.8±0.2%, all p<0.001). There was a positive correlation between the expression level of TEM1 (ratio of TEM1/actin band density in western blot) with the severity of atrial fibrosis, including the total numbers of red pixels (r = 0.47, p = 0.01) and the % of red pixels to total pixels (r = 0.48, p = 0.01). TEM1 and galectin expression levels had positive correlation (r = 0.60, p < 0.01). There were no correlations of atrial TEM1 expression with the LA diameter and CHA2DS2-VASc score. In vitro study showed TEM1 knockdown in cultured CFs with siRNA reduced cell proliferation (scrambled- vs TEM1-siRNA, BrdU absorbance: 1.84±0.05 vs 1.50±0.07, p<0.05), increased starvation-induced cell apoptosis (cell death ELISA absorbance: 0.69±0.11 vs 2.88±0.05, p<0.05) and decreased cell migration. Conclusions: There is increased TEM1 expression in atrial CFs in AF and its expression level is associated with atrial fibrosis. TEM1 expression changes the cell behaviors of CFs.

3D-environment and muscle contraction regulate the heterogeneity of myonuclei

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation.

Immune responses in checkpoint myocarditis across heart, blood and tumour.

Immune checkpoint inhibitors are widely used anticancer therapies1 that can cause morbid and potentially fatal immune-related adverse events such as immune-related myocarditis (irMyocarditis)2-5. The pathogenesis of irMyocarditis and its relationship to antitumour immunity remain poorly understood. Here we sought to define immune responses in heart, tumour and blood in patients with irMyocarditis by leveraging single-cell RNA sequencing coupled with T cell receptor (TCR) sequencing, microscopy and proteomics analyses of samples from 28 patients with irMyocarditis and 41 unaffected individuals. Analyses of 84,576 cardiac cells by single-cell RNA sequencing combined with multiplexed microscopy demonstrated increased frequencies and co-localization of cytotoxic T cells, conventional dendritic cells and inflammatory fibroblasts in irMyocarditis heart tissue. Analyses of 366,066 blood cells revealed decreased frequencies of plasmacytoid dendritic cells, conventional dendritic cells and B lineage cells but an increased frequency of other mononuclear phagocytes in irMyocarditis. Fifty-two heart-expanded TCR clones from eight patients did not recognize the putative cardiac autoantigens α-myosin, troponin I or troponin T. Additionally, TCRs enriched in heart tissue were largely nonoverlapping with those enriched in paired tumour tissue. The presence of heart-expanded TCRs in a cycling blood CD8 T cell population was associated with fatal irMyocarditis case status. Collectively, these findings highlight crucial biology driving irMyocarditis and identify putative biomarkers.

Eosinophilic Esophagitis Drives Tissue Fibroblast Regenerative Programs Towards Pathologic Dysfunction

BackgroundPathologic tissue remodeling with scarring and tissue rigidity has been demonstrated in inflammatory, autoimmune, and allergic diseases. Eosinophilic esophagitis (EoE) is an allergic disease that is diagnosed and managed by repeated biopsy procurement, allowing an understanding of tissue fibroblast dysfunction. While EoE associated tissue remodeling causes clinical dysphagia, food impactions and esophageal rigidity and strictures, molecular mechanisms driving these complications remain under investigation. ObjectiveWe hypothesized that chronic EoE inflammation induces pathogenic fibroblasts with dysfunctional tissue regeneration and motility. MethodsWe used single cell RNA sequence (scRNA-Seq), fluorescence activated cell sorting analysis, and fibroblast differentiation and migration assays to decipher the induced and retained pathogenic dysfunctions in EoE versus healthy esophageal fibroblasts. ResultsDifferentiation assays demonstrated that active EoE fibroblasts retain regenerative programs for rigid cells such as chondrocytes (p<0.05) but lose healthy fibroblast capacity for soft cells such as adipocytes (p<0.01) which was reflected in biopsy immunostaining (p<0.01). EoE, but not healthy, fibroblasts have pro-inflammatory and pro-rigidity transcriptional programs on scRNA-Seq. In vivo, regenerative fibroblasts reside in perivascular regions and near the epithelial junction and, during EoE, have significantly increased migration (p<0.01). Flow analysis and functional assays demonstrated that regenerative EoE fibroblasts have decreased surface CD73 expression and activity (both p<0.05) compared to healthy, indicating aberrant ATP handling. EoE fibroblast dysfunctions were induced in healthy fibroblasts by reducing CD73 activity and rescued in EoE using adenosine repletion. ConclusionA normalization of perturbed extracellular ATP handling and CD73 could improve pathogenic fibroblast dysfunction and tissue regeneration in type 2 inflammatory diseases.

Hypoxia-Induced Differences in the Expression of Pyruvate Dehydrogenase Kinase 1-Related Factors in the Renal Tissues and Renal Interstitial Fibroblast-like Cells of Yak (Bos Grunniens).

Hypoxia is one of the factors severely affect renal function, and, in severe cases, it can lead to renal fibrosis. Although much progress has been made in identifying the molecular mediators of fibrosis, the mechanisms that govern renal fibrosis remain unclear, and there have been no effective therapeutic anti-fibrotic strategies to date. Mammals exposed to low oxygen in the plateau environment for a long time are prone to high-altitude disease, while yaks have been living in the plateau for generations do not develop kidney fibrosis caused by low oxygen. It has been suggested that metabolic reprogramming occurs in renal fibrosis and that pyruvate dehydrogenase kinase 1 (PDK1) plays a crucial role in metabolic reprogramming as an important node between glycolysis and the tricarboxylic acid cycle. The aim of this study was to investigate the effects of hypoxia on the renal tissues and renal interstitial fibroblasts of yaks. We found that, at the tissue level, HIF-1α, PDK1, TGF-β1, Smad2, Smad3, and α-SMA were mainly distributed and expressed in tubular epithelial cells but were barely present in the renal mesenchymal fibroblasts of healthy cattle and yak kidneys. Anoptical density analysis showed that in healthy cattle kidneys, TGF-β1, Smad2, and Smad3 expression was significantly higher than in yak kidneys (p < 0.05), and HIF-1α and PDK1 expression was significantly lower than in yak kidneys (p < 0.05). The results at the protein and gene levels showed the same trend. At the cellular level, prolonged hypoxia significantly elevated PDK1 expression in the renal mesangial fibroblasts of cattle and yak kidneys compared with normoxia (p < 0.05) and was proportional to the degree of cellular fibrosis. However, PDK1 expression remained stable in yaks compared with renal interstitial fibroblast-like cells in cattle during the same hypoxic time period. At the same time, prolonged hypoxia also promoted changes in cellular phenotype, promoting the proliferation, activation, glucose consumption, lactate production, and anti-apoptosis in the both of cattle and yaks renal interstitial fibroblasts The differences in kidney structure and expression of PDK1 and HIF-1α in kidney tissue and renal interstitial fibroblasts induced by different oxygen concentrations suggest that there may be a regulatory relationship between yak kidney adaptation and hypoxic environment at high altitude. This provides strong support for the elucidation of the regulatory relationship between PDK1 and HIF-1α, as well as a new direction for the treatment or delay of hypoxic renal fibrosis; additionally, these findings provide a basis for further analysis of the molecular mechanism of hypoxia adaptation-related factors and the adaptation of yaks to plateau hypoxia.

The regenerative effect of exosomes isolated from mouse embryonic fibroblasts in mice created as a sciatic nerve crush injury model.

Exosomes (Exos) are candidates for functional recovery and regeneration following sciatic nerve crushed (SNC) injury due to their composition which can accelerate tissue regeneration. Therefore, mouse embryonic fibroblast-derived exosomes were evaluated for their regenerative capacity in SNC injury. In the study, 40Balb/c males (20 ± 5g) and two pregnant mice (for embryonic fibroblast tissue) were used and crushed injury was induced in the left sciatic nerve with an aneurysm clamp. Sciatic nerve model mice were randomly divided into 5 groups (n = 8; control, n = 8; sham, n = 8; SNC, n = 8; Mouse embryonic fibroblast exosome (mExo), n = 8; SNC + Mouse embryonic fibroblast exosome (SNC + mExo). Rotarod tests for motor functions and hot plate and von Frey tests for sensory functions were analyzed in the groups. Expression changes of exosome genes (RARRES1, NAGS, HOXA13, and MEIS1) immunohistochemical analysis of these gene proteins, and structural exosome NF-200 and S100 proteins were evaluated by confocal microscopy. Behavioral analyses showed that the damage in SNC was significant between groups on day14 and day28 (P < 0.05). In behavioral analyses, it was determined that motor functions and mechanical sensitivity lost in SNC were regained after mExo treatment. While expression of all genes was detected in MEF-derived exosomes, the high expression was MESI1 and the low expression was HOXA13. NF200, an indicator of axon number and neurofilament density, was found to decrease in SNC (P < 0.001) and increase after treatment, but not significantly. The decreased S100 protein levels in SNC and the increase detected after treatment were not significant. The expression of four mRNAs in mExos indicates that these genes may have an effect on regenerative processes after SNC injury. The regenerative process supported by tissue protein expressions demonstrates the therapeutic potential of mExo treatment.

Periostin regulates the activity of keloid fibroblasts by activating the JAK/STAT signaling pathway

A keloid is secondary to skin trauma or has spontaneously manifested as an overgrowth and occurs when the skin heals abnormally after an injury. The main pathological manifestations are abnormal proliferation of keloid fibroblasts (KEL-FIB). This study researched periostin (POSTN) on keloid fibroblasts (KEL-FIB) and the associated mechanism, aiming to provide a reference for the targeted therapy of keloid. We got tissues from Second People's Hospital of Guangxi Zhuang Autonomous Region between June 2022 and March 2023. POSTN expression was increased in keloid skin tissue and KEL-FIB than normal skin tissue and normal fibroblasts. We collected and inoculated KEL-FIB cells, transfection of si-NC (Silencing of POSTN negative control), si-POSTN (Silencing of POSTN), pcDNA-NC (Overexpression of POSTN negative control), and POSTN (Overexpression of POSTN) (Thermo Fisher Scientific) used Lipofectamine 2000 transfection reagent. Wound closure, cell proliferation viability, migrated cell numbers, and POSTN, p-JAK2, p-STAT3 protein levels were reduced in the si-POSTN group. Wound closure, cell proliferation viability, migrated cell numbers, and POSTN, p-JAK2, p-STAT3 protein levels were elevated in the POSTN group. POSTN protein levels did not changed and wound closure, cell proliferation viability, migrated cell numbers, were reduced in the POSTN + S-Ruxolitinib group. The study results indicated that POSTN promotes cell migration and proliferation by activating the JAK/STAT pathway, promoting KEL-FIB development.

Meflin/ISLR is a marker of adipose stem and progenitor cells in mice and humans that suppresses white adipose tissue remodeling and fibrosis.

Identifying specific markers of adipose stem and progenitor cells (ASPCs) in vivo is crucial for understanding the biology of white adipose tissues (WAT). PDGFRα-positive perivascular stromal cells represent the best candidates for ASPCs. This cell lineage differentiates into myofibroblasts that contribute to the impairment of WAT function. However, ASPC marker protein(s) that are functionally crucial for maintaining WAT homeostasis are unknown. We previously identified Meflin as a marker of mesenchymal stem cells (MSCs) in bone marrow and tissue-resident perivascular fibroblasts in various tissues. We also demonstrated that Meflin maintains the undifferentiated status of MSCs/fibroblasts. Here, we show that Meflin is expressed in WAT ASPCs. A lineage-tracing experiment showed that Meflin+ ASPCs proliferate in the WAT of obese mice induced by a high-fat diet (HFD), while some of them differentiate into myofibroblasts or mature adipocytes. Meflin knockout mice fed an HFD exhibited a significant fibrotic response as well as increases in adipocyte cell size and the number of crown-like structures in WAT, accompanied by impaired glucose tolerance. These data suggested that Meflin expressed by ASPCs may have a role in reducing disease progression associated with WAT dysfunction.

Comparative study of the histology and Micro-CT scan of the goldfish hood

The hood (also known as head growth or wen) is a distinctive feature of goldfish characterized by skin tissue proliferation. The classification of goldfish hood types and the study of the underlying skeletal structure still have substantial gaps in research. This study aimed to investigate the structural differences in goldfish hood tissues using histological and Micro-CT comparisons, based on traditional Chinese classifications. Histological observations revealed two distinct types: the one-piece type (Crown Pearlscale), characterized by a uniformly thickened epidermis, and the fused type (Oranda, Red Cap Oranda, and Ranchu), which showed irregular epidermal extensions into the dermis. Micro-CT scans further demonstrated that hooded goldfish exhibited skull protrusions, bone spurs, and widening of the cranial region. These findings indicate that fibroblast activity, tissue edema, and bone structural changes may be key drivers of hood formation in these goldfish varieties.

The involvement of HDAC3 in the pathogenesis of lung injury and pulmonary fibrosis.

Acute lung injury (ALI) and its severe counterpart, acute respiratory distress syndrome (ARDS), are critical respiratory conditions with high mortality rates due primarily to acute and intense pulmonary inflammation. Despite significant research advances, effective pharmacological treatments for ALI and ARDS remain unavailable, highlighting an urgent need for therapeutic innovation. Notably, idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease characterized by the irreversible progression of fibrosis, which is initiated by repeated damage to the alveolar epithelium and leads to excessive extracellular matrix deposition. This condition is further complicated by dysregulated tissue repair and fibroblast dysfunction, exacerbating tissue remodeling processes and promoting progression to terminal pulmonary fibrosis. Similar to that noted for ALI and ARDS, treatment options for IPF are currently limited, with no specific drug therapy providing a cure. Histone deacetylase 3 (HDAC3), a notable member of the HDAC family with four splice variants (HD3α, -β, -γ, and -δ), plays multiple roles. HDAC3 regulates gene transcription through histone acetylation and adjusts nonhistone proteins posttranslationally, affecting certain mitochondrial and cytoplasmic proteins. Given its unique structure, HDAC3 impacts various physiological processes, such as inflammation, apoptosis, mitochondrial homeostasis, and macrophage polarization. This article explores the intricate role of HDAC3 in ALI/ARDS and IPF and evaluates its therapeutic potential the treatment of these severe pulmonary conditions.

Abstract C120: Characterization of the inflammatory tumor microenvironment and its association with cancer health disparities

Abstract Black persons have the highest mortality rates of both cervical cancer (CC) and pancreatic ductal adenocarcinoma (PDAC) in the United States. Differences in access to quality healthcare and socioeconomic status contribute to these cancer health disparities as well as ancestry and biology. In other cancer types, differences in the inflammatory tumor landscape across races and ancestral populations have been reported. However, the inflammatory tumor microenvironment (TME) as it relates to CC and PDAC health disparities is not well defined. Black patients are often underrepresented in PDAC studies. Meanwhile, CC health disparities studies primarily investigate socioeconomic status, screening, and vaccination rates. Therefore, we aim to characterize the inflammatory TME in these two cancer types with the long-term goal of informing prevention, prognostic, and therapeutic strategies. Clinicopathological characteristics were compared between Black (n = 144) and White (n = 198) patients with CC who received care at Johns Hopkins Medicine between 1995 and 2019. Surgically resected formalin-fixed paraffin embedded (FFPE) CC tissue samples were obtained from Non-Hispanic Black (n = 10), Non-Hispanic White (n = 10), Hispanic Black (n = 5), and Hispanic White (n = 5) women matched on grade, age, and subtype. Tissue microarrays (TMAs) were constructed using archival surgically resected FFPE PDAC tissues from Black (n = 45) and White (n = 45) patients, matched on sex, age, stage, and time of surgery. Multiplexing immunohistochemistry (mIHC) was used to assess markers for i) T cells (CD3, CD8, CD4, FoxP3), M2 macrophages (CD163), mast cells (Tryptase), and fibroblasts (FAP) in PDAC tissues and ii) B cells (CD79A), T cells (CD3, CD8, CD4, FoxP3), plasma cells (CD138/syndecan-1), programmed death-ligand 1 protein (PD-L1), macrophages (CD68, CD163), natural killer cells (NKP46/CD56/CD16), neutrophils (CD66ce), and mast cells (Tryptase) in CC tissues. HALO Image Analysis Platform (version 3.6.4134.137; Indica Labs) was used to quantify marker expression. GraphPad Prism Software (version 10.0.03), was used for graphing and statistical analysis. Squamous cell CC was the most frequent histological subtype in both Black (68%) and White (44%) women. Progression through treatment was more common among Black women (24%) compared to White women (7%). CD163+ M2 macrophages were significantly increased in PDAC tumors compared to benign tissues among Black patients (p = 0.0435), and in tumors from Black women compared to White women (p = 0.0067). FAP+ fibroblasts were also significantly increased in PDAC tumors compared to benign tissues among Black patients (p = 0.0001) and in tumors from Black women compared to White women (p = 0.0013). CD8+ cytotoxic T cells and FoxP3+ regulatory T cells were comparable between races. We observed differences in macrophage and fibroblast distribution between races supporting the effort for future studies that examine the significance of characterizing the TME to address cancer health disparities. Citation Format: Jelani Jarrett, Jae W Lee, Ali Dbouk, Vahinipriya Manoharan, Kennedy Rains, Terri Mason, Kimberly Levinson, Laura D Wood, Michael G Goggins, Janielle P Maynard. Characterization of the inflammatory tumor microenvironment and its association with cancer health disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C120.

Skeletal Muscle Satellite Cells Co-Opt the Tenogenic Gene Scleraxis to Instruct Regeneration.

Skeletal muscles connect bones and tendons for locomotion and posture. Understanding the regenerative processes of muscle, bone and tendon is of importance to basic research and clinical applications. Despite their interconnections, distinct transcription factors have been reported to orchestrate each tissue's developmental and regenerative processes. Here we show that Scx expression is not detectable in adult muscle stem cells (also known as satellite cells, SCs) during quiescence. Scx expression begins in activated SCs and continues throughout regenerative myogenesis after injury. By SC-specific Scx gene inactivation (ScxcKO), we show that Scx function is required for SC expansion/renewal and robust new myofiber formation after injury. We combined single-cell RNA-sequencing and CUT&RUN to identify direct Scx target genes during muscle regeneration. These target genes help explain the muscle regeneration defects of ScxcKO, and are not overlapping with Scx -target genes identified in tendon development. Together with a recent finding of a subpopulation of Scx -expressing connective tissue fibroblasts with myogenic potential during early embryogenesis, we propose that regenerative and developmental myogenesis co-opt the Scx gene via different mechanisms.

The expression of signal regulatory protein alpha (SIRPα) in periodontal cells and tissue.

Signal regulatory protein alpha (SIRPα) is mainly expressed by cells of myeloid origin. This membrane glycoprotein is shown to be involved in regulation of different inflammatory conditions, such as colitis and arthritis. However, SIRPα has not been investigated in relationship to periodontitis, an inflammatory condition affecting the tooth supporting tissues. We aim to investigate if resident cells in the periodontium express SIRPα and whether a possible expression is affected by inflammatory conditions. Primary human keratinocytes, fibroblasts, periodontal ligament cells, and osteoblasts were cultured with or without the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) or interleukin-1-beta (IL-1β). All different periodontal cell types showed a basal mRNA expression of SIRPα. Pro-inflammatory cytokines induced a 2-3-fold significant increase in SIRPα expression in both cultured human gingival fibroblasts and osteoblasts but neither in keratinocytes nor in periodontal ligament cells. Tissue sections from human gingival tissue biopsies were histochemically stained for SIRPα. Epithelial keratinocytes and gingival fibroblasts stained positive in sections from periodontally healthy as well as in sections from periodontitis. In periodontitis sections, infiltrating leukocytes stained positive for SIRPα. We highlight our finding that oral keratinocytes, gingival fibroblasts, and periodontal ligament cells do express SIRPα, as this has not been presented before. The fact that inflammatory stimulation of gingival fibroblasts increased the expression of SIRPα, while an increased expression by gingival fibroblasts in periodontitis tissue in situ could not be detected, is indeed contradictory.

Investigating the Effect of Olive Leaf Extract on Antioxidant Activity, and its Regulatory Role on RBP4 Inflammatory Gene Expression in 3T3-L1 Cell Line

Background: The leaves of the olive plant (Olea europaea) contain significantly higher levels of phenolic compounds than the fruit and olive oil, acting as a natural supplement for antioxidant activities. Objectives: This research aimed to investigate the effect of this extract on certain inflammatory factors (TNF-α, IL-1, IL-6) and its regulatory role on tissue gene (RBP4) in fibroblast cell lines (3T3-L1). Methods: After culturing adipose tissue fibroblast cells (3T3-L1) under sterile and standard conditions, the cells were exposed to different logarithmic concentrations of the extract. The production of inflammatory cytokines TNF-α, IL-1, and IL-6 was measured using ELISA methods, and the expression level of the RBP4 gene was assessed using a real-time PCR technique. Results: The results showed dose-dependent effects of the extract on the 3T3-L1 cells. The MTT assay indicated that the hydroalcoholic extract of olive plant leaves has a concentration-dependent effect on cell proliferation (∗∗P ≤ 0.05). The DPPH test results showed a significant decrease in the level of active oxygen in the presence of Olive leaf extract (OLE). The catalase and MDA test results also indicated an increase in antioxidant activity in cells treated with the extract. Conversely, the expression levels of inflammatory cytokines IL-1 and IL-6 increased by 0.05 and 0.01 (pg/mL), respectively, while changes in TNF-α and RBP4 gene expression were insignificant (P &gt; 0.05). Conclusions: The results demonstrated that OLE can enhance the efficiency of antioxidant and inflammatory pathways in the cell and significantly reduce the level of active oxygen in the cell.

Mapping and Analysis of Protein and Gene Profile Identification of the Important Role of Transforming Growth Factor Beta in Synovial Invasion in Patients With Pigmented Villonodular Synovitis.

Pigmented villonodular synovitis (PVNS) is a rare benign proliferative disease affecting the soft-tissue lining the synovial joints and tendons. Its etiology is poorly understood, largely limiting the availability of current therapeutic options. Here, we mapped the synovial gene and protein profiles of patients with PVNS, revealed a link between synovial inflammation and invasion, and elucidated the potential molecular mechanism involved. The expression of synovial genes from 6 control individuals, 7 patients with osteoarthritis (OA), and 19 patients with PVNS was analyzed via RNA sequencing. Protein profiles from 5 control individuals, 10 patients with OA, and 32 patients with PVNS were analyzed using label-free proteomics. Microarray and reverse transcription-polymerase chain reaction analyses and immunohistochemical staining were used to evaluate inflammatory cytokine and target gene expression levels in synovial tissue, epithelial cells, and synovial fibroblasts (FLSs) derived from tissue of patients with PVNS. Various signaling pathway inhibitors, small interfering RNAs, and Western blots were used for molecular mechanism studies. Transwell migration and invasion assays were subsequently performed. In total, 522 differentially expressed proteins were identified in the tissues of patients with PVNS. By integrating RNA sequencing and microarray analyses, significant changes in the expression of epithelial-mesenchymal transition (EMT)-related genes, including transforming growth factor TGF-b induced, neural cadherin, epithelial cadherin, SNAIL, and TWIST, were confirmed in the tissue of patients with PVNS compared to the control tissue. In vitro, TGFβ induced EMT and increased epithelial cell migration and invasion. Moreover, TGFβ not only promoted interactions between epithelial cells and FLSs but also directly increased the migration and invasion abilities of FLSs by activating the classical Smad2/3 and nonclassical JNK/AKT signaling pathways. This study provides overall protein and gene profiles of PVNS and identifies the crucial role of TGFβ in synovial invasion pathology. Exploring the related molecular mechanism may also reveal a new strategy or target for PVNS therapy.

Investigating the effect of Fusobacterium nucleatum on the aggressive behavior of cancer-associated fibroblasts in colorectal cancer

Fusobacterium nucleatum, (F. nucleatum) as a known factor in inducing oncogenic, invasive, and inflammatory responses, can lead to an increase in the incidence and progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAF) are also one of the key components of the tumor microenvironment (TME), which lead to resistance to treatment, metastasis, and disease recurrence with their markers, secretions, and functions. This study aimed to investigate the effect of F. nucleatum on the invasive phenotype and function of fibroblast cells isolated from normal and cancerous colorectal tissue. F. nucleatum bacteria were isolated from deep periodontal pockets and confirmed by various tests. CAF cells from tumor tissue and normal fibroblasts (NF) from a distance of 10 cm of tumor tissue were isolated from 5 patients by the explant method and were exposed to secretions and ghosts of F. nucleatum. The expression level of two markers, fibroblast activation protein (FAP), and α-smooth muscle actin (α-SMA), and the amount of production of two cytokines TGF-β and IL-6 from fibroblast cells were measured by flow cytometry and ELISA test, respectively before and after exposure to different bacterial components. The expression of the FAP marker was significantly higher in CAF cells compared to NF cells (P < 0.05). Also, the expression of IL-6 in CAF cells was higher than that of NF cells. In investigating the effect of bacterial components on the function of fibroblastic cells, after comparing the amount of IL-6 produced between the normal tissue of each patient and his tumoral tissue under 4 treated conditions, it was found that the amount of IL-6 production from the CAF cells of patients in the control group, treated with heat-killed ghosts and treated with paraformaldehyde-fixed ghosts had a significant increase compared to NF cells (P < 0.05). Due to the significant increase in FAP marker expression in fibroblast cells of tumor tissue compared to normal tissue, it seems that FAP can be used as a very good therapeutic marker, especially in patients with high levels of CAF cells. Various components of F. nucleatum could affect fibroblast cells differentially and at least part of the effect of this bacterium in the TME is mediated by CAF cells.