Distinct fibroblast subsets drive inflammation and damage in arthritis.

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. Cancer-associated fibroblasts (CAFs) promote progression of cancer, but their role in cSCC is largely unknown. We examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. We utilized multiplexed fluorescence immunohistochemistry for profiling CAF landscape in metastatic and non-metastatic primary human cSCCs, in metastases, and in premalignant epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and results were correlated with clinical and histopathological parameters including disease-specific mortality. Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression to invasive cSCC. Elevation of FAP and platelet-derived growth factor receptor-ß (PDGFRß) expression was associated with metastasis risk of primary cSCCs. High expression of PDGFRß and periostin correlated with poor prognosis. Multimarker combination defined CAF subset, PDGFRα-/PDGFRß+/FAP+, was associated with invasion and metastasis, and independently predicted poor disease-specific survival. These results identify high PDGFRß expression alone and multimarker combination PDGFRα-/PDGFRß+/FAP+ by CAFs as potential biomarkers for risk of metastasis and poor prognosis.

Cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer predict poor clinical outcome.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. METHODS: Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. RESULTS: We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. CONCLUSION: This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.

Fibroblast activation protein-targeted near-infrared photoimmunotherapy depletes immunosuppressive cancer-associated fibroblasts and remodels local tumor immunity.

BACKGROUND: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a critical role in tumor immunosuppression. However, targeted depletion of CAFs is difficult due to their diverse cells of origin and the resulting lack of specific surface markers. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that leads to rapid cell membrane damage. METHODS: In this study, we used anti-mouse fibroblast activation protein (FAP) antibody to target FAP+ CAFs (FAP-targeted NIR-PIT) and investigated whether this therapy could suppress tumor progression and improve tumor immunity. RESULTS: FAP-targeted NIR-PIT induced specific cell death in CAFs without damaging adjacent normal cells. Furthermore, FAP-targeted NIR-PIT treated mice showed significant tumor regression in the CAF-rich tumor model accompanied by an increase in CD8+ tumor infiltrating lymphocytes (TILs). Moreover, treated tumors showed increased levels of IFN-γ, TNF-α, and IL-2 in CD8+ TILs compared with non-treated tumors, suggesting enhanced antitumor immunity. CONCLUSIONS: Cancers with FAP-positive CAFs in their TME grow rapidly and FAP-targeted NIR-PIT not only suppresses their growth but improves tumor immunosuppression. Thus, FAP-targeted NIR-PIT is a potential therapeutic strategy for selectively targeting the TME of CAF+ tumors.

Development of a transcription factor decoy-nanocarrier system as a successful inhibitor of Enterococcus faecalis virulence in vitro and in vivo.

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens.

Infiltrative pattern of invasion is independently associated with shorter survival and desmoplastic stroma markers FAP and THBS2 in mucinous ovarian carcinoma.

AIMS: Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer histotype with generally good prognosis when diagnosed at an early stage. However, MOC with the infiltrative pattern of invasion has a worse prognosis, although to date studies have not been large enough to control for covariables. Data on reproducibility of classifying the invasion pattern are limited, as are molecular correlates for infiltrative invasion. We hypothesized that the invasion pattern would be associated with an aberrant tumour microenvironment. METHODS AND RESULTS: Four subspecialty pathologists assessed interobserver reproducibility of the pattern of invasion in 134 MOC. Immunohistochemistry on fibroblast activation protein (FAP) and THBS2 was performed on 98 cases. Association with survival was tested using Cox regression. The average interobserver agreement for the infiltrative pattern was moderate (kappa 0.60, agreement 86.3%). After reproducibility review, 24/134 MOC (18%) were determined to have the infiltrative pattern and this was associated with a higher risk of death, independent of FIGO stage, grade, and patient age in a time-dependent manner (hazard ratio [HR] = 10.2, 95% confidence interval [CI] 3.0-34.5). High stromal expression of FAP and THBS2 was more common in infiltrative MOC (FAP: 60%, THBS2: 58%, both P < 0.001) and associated with survival (multivariate HR for FAP: 1.5 [95% CI 1.1-2.1] and THBS2: 1.91 [95% CI 1.1-3.2]). CONCLUSIONS: The pattern of invasion should be included in reporting for MOC due to the strong prognostic implications. We highlight the histological features that should be considered to improve reproducibility. FAP and THBS2 are associated with infiltrative invasion in MOC.

Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer.

PURPOSE: Radiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC). METHODS: Public database was first interrogated to reveal the correlation between CAFs' scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen. RESULTS: CAFs' scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12. CONCLUSION: The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

Enhanced Cellular Delivery of Tildipirosin by Xanthan Gum-Gelatin Composite Nanogels.

Tildipirosin has no significant inhibitory effect on intracellular bacteria because of its poor membrane permeability. To this end, tildipirosin-loaded xanthan gum-gelatin composite nanogels were innovatively prepared to improve the cellular uptake efficiency. The formation of the nanogels via interactions between the positively charged gelatin and the negatively charged xanthan gum was confirmed by powder X-ray diffraction and Fourier transform infrared. The results indicate that the optimal tildipirosin composite nanogels possessed a 3D network structure and were shaped like a uniformly dispersed ellipse, and the particle size, PDI, and ζ potential were 229.4 ± 1.5 nm, 0.26 ± 0.04, and -33.2 ± 2.2 mV, respectively. Interestingly, the nanogels exhibited gelatinase-responsive characteristics, robust cellular uptake via clathrin-mediated endocytosis, and excellent sustained release. With those pharmaceutical properties provided by xanthan gum-gelatin composite nanogels, the anti-Staphylococcus aureus activity of tildipirosin was remarkably amplified. Further, tildipirosin composite nanogels demonstrated good biocompatibility and low in vivo and in vitro toxicities. Therefore, we concluded that tildipirosin-loaded xanthan gum-gelatin composite nanogels might be employed as a potentially effective gelatinase-responsive drug delivery for intracellular bacterial infection.

Progressive changes in the protein expression profile of alveolar septa in early-stage lung adenocarcinoma.

BACKGROUND: Adenocarcinomas show a stepwise progression from atypical adenomatous hyperplasia (AAH) through adenocarcinoma in situ (AIS) to invasive adenocarcinoma (IA). Immunoglobulin superfamily containing leucine-rich repeat (ISLR) is a marker of tumor-restraining cancer-associated fibroblasts (CAFs), which are distinct from conventional, strongly α-smooth muscle actin (αSMA)-positive CAFs. Fibroblast activation protein (FAP) has been focused on as a potential therapeutic and diagnostic target of CAFs. METHODS: We investigated the changes in protein expression during adenocarcinoma progression in the pre-existing alveolar septa by assessing ISLR, αSMA, and FAP expression in normal lung, AAH, AIS, and IA. Fourteen AAH, seventeen AIS, and twenty IA lesions were identified and randomly sampled. Immunohistochemical analysis was performed to evaluate cancer-associated changes and FAP expression in the pre-existing alveolar structures. RESULTS: Normal alveolar septa expressed ISLR. The ISLR level in the alveolar septa decreased in AAH and AIS tissues when compared with that in normal lung tissue. The αSMA-positive area gradually increased from the adjacent lung tissue (13.3% ± 15%) to AIS (87.7% ± 14%), through AAH (70.2% ± 21%). Moreover, the FAP-positive area gradually increased from AAH (1.69% ± 1.4%) to IA (11.8% ± 7.1%), through AIS (6.11% ± 5.3%). Protein expression changes are a feature of CAFs in the pre-existing alveolar septa that begin in AAH. These changes gradually progressed from AAH to IA through AIS. CONCLUSIONS: FAP-positive fibroblasts may contribute to tumor stroma formation in early-stage lung adenocarcinoma, and this could influence the development of therapeutic strategies targeting FAP-positive CAFs for disrupting extracellular matrix formation.

Diagnostic Performances of PET/CT Using Fibroblast Activation Protein Inhibitors in Patients with Primary and Metastatic Liver Tumors: A Comprehensive Literature Review.

PET/CT using radiolabeled fibroblast activation protein inhibitors (FAPIs) is a promising diagnostic tool in oncology, especially when non-increased and/or physiologically high [18F]FDG uptake (as in liver parenchyma) is observed. We aimed to review the role of PET/CT using radiolabeled FAPIs in primary and/or metastatic liver lesions, and to compare their performances with more "conventional" radiopharmaceuticals. A search algorithm based on the terms "FAPI" AND ("hepatic" OR "liver") was applied, with the last update on 1st January 2024. Out of 177 articles retrieved, 76 studies reporting on the diagnostic application of radiolabeled FAPI PET/CT in at least one patient harboring primary or metastatic liver lesion(s) were fully analyzed. Although there was some heterogeneity in clinical conditions and/or study methodology, PET/CT with radiolabeled FAPIs showed an excellent performance in common primary liver malignancies (hepatocarcinoma, intrahepatic cholangiocarcinoma) and liver metastases (mostly from the gastrointestinal tract and lungs). A higher tumor-to-background ratio for FAPIs than for [18F]FDG was found in primary and metastatic liver lesions, due to lower background activity. Despite limited clinical evidence, radiolabeled FAPIs may be used to assess the suitability and effectiveness of FAPI-derived therapeutic agents such as [177Lu]Lu-FAPI. However, future prospective research on a wider population is needed to confirm the excellent performance.

Tailoring Fibroblast-Activation Protein Targeting for Theranostics: A Comparative Preclinical Evaluation of the 68Ga- and 177Lu-Labeled Monomeric and Dimeric Fibroblast-Activation Protein Inhibitors DOTA.SA.FAPi and DOTAGA.(SA.FAPi)2.

BACKGROUND: FAP radiopharmaceuticals show promise for cancer diagnosis; however, their limited tumor residency hinders treatment. This study compared two FAPi derivatives, DOTA.SA.FAPi and DOTAGA.(SA.FAPi)2, labeled with gallium-68 and lutetium-177, aiming to determine an optimum combination for creating theranostic pairs. METHODS: The radiotracers were studied for lipophilicity, binding to human serum proteins, and binding to human cancer-associated fibroblasts (CAFs) in vitro, including saturation and internalization/externalization studies. PET/SPECT/CT and biodistribution studies were conducted in PC3 and U87MG xenografts for [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTAGA.(SA.FAPi)2. [177Lu]Lu-DOTA.SA.FAPi and [177Lu]Lu-DOTAGA.(SA.FAPi)2, were evaluated in PC3 xenografts. Biodistribution studies of [68Ga]Ga-DOTA.SA.FAPi were performed in healthy male and female mice. RESULTS: All radiotracers exhibited strong binding to FAP. Their internalization rate was fast while only [177Lu]Lu-DOTAGA.(SA.FAPi)2 was retained longer in CAFs. [68Ga]Ga-DOTAGA.(SA.FAPi)2 and [177Lu]Lu-DOTAGA.(SA.FAPi)2 displayed elevated lipophilicity and affinity for human serum proteins compared to [68Ga]Ga-DOTA.SA.FAPi and [177Lu]Lu-DOTA.SA.FAPi. In vivo studies revealed slower washout of [68Ga]Ga-DOTAGA.(SA.FAPi)2 within 3 h compared to [68Ga]Ga-DOTA.SA.FAPi. The tumor-to-tissue ratios of [68Ga]Ga-DOTAGA.(SA.FAPi)2 versus [68Ga]Ga-DOTA.SA.FAPi did not exhibit any significant differences. [177Lu]Lu-DOTAGA.(SA.FAPi)2 maintained a significant tumor uptake even after 96 h p.i. compared to [177Lu]Lu-DOTA.SA.FAPi. CONCLUSIONS: Dimeric compounds hold promise for therapy, while monomers are better suited for diagnostics. Finding the right combination is essential for effective disease management.

Peritumoral stroma and systemic inflammatory response in cervical cancer.

OBJECTIVE: To compare cervical stroma in advanced cervical cancer with the control group; to compare, in the pre-treatment period, hemogram parameters in patients with advanced cervical cancer with the same parameters as the control group; and to verify if there is an association of stromal markers with prognostic factors in cervical cancer. MATERIALS AND METHODS: We prospectively evaluated 16 patients diagnosed with advanced invasive cervical cancer. A control group of 22 patients was used (uterine leiomyoma). Immunohistochemistry was performed to verify the stromal immunostaining of alpha-smooth muscle actin (SMA) and fibroblast activation protein alpha (FAP). Immunostainings and hemogram parameters were compared using Fisher's exact and Mann-Whitney Test, respectively. RESULTS: Strong FAP immunostaining was more frequent in patients with cervical cancer when compared with patients with leiomyoma (P = 0.0002). Regarding SMA, strong immunostaining was also found more in the group of cancer patients compared to the control group (P < 0.00001). The neutrophil-lymphocyte ratio (NLR) values were higher in the cancer patient group compared to the control group (P = 0.0019). There was no association of the parameters studied with prognostic factors. CONCLUSIONS: Strong FAP and SMA immunostaining was found more in patients with cervical cancer when compared to the control group. NLR values were also higher in cervical cancer.

Fibroblast heterogeneity in solid tumors: From single cell analysis to whole-body imaging.

Cancer-Associated Fibroblasts (CAFs) represent the most prominent component of the tumor microenvironment (TME). Recent studies demonstrated that CAF are heterogeneous and composed of different subpopulations exerting distinct functions in cancer. CAF populations differentially modulate various aspects of tumor growth, including cancer cell proliferation, extra-cellular matrix remodeling, metastatic dissemination, immunosuppression and resistance to treatment. Among other markers, the Fibroblast Activation Protein (FAP) led to the identification of a specific CAF subpopulation involved in metastatic spread and immunosuppression. Expression of FAP at the surface of CAF is detected in many different cancer types of poor prognosis. Thus, FAP recently appears as an appealing target for therapeutic and molecular imaging applications. In that context, 68Ga-labeled radiopharmaceutical-FAP-inhibitors (FAPI) have been recently developed and validated for quantitatively mapping FAP expression over the whole-body using Positron Emission Tomography (PET/CT). In this review, we describe the main current knowledge on CAF subpopulations and their distinct functions in solid tumors, as well as the promising diagnostic and therapeutic implications of radionuclides targeting FAP.

Cinnamaldehyde inhibits Enterococcus faecalis biofilm formation and promotes clearance of its colonization by modulation of phagocytes in vitro.

The nosocomial pathogen, Enterococcus faecalis plays a crucial role in the pathogenesis of variety of infections including endocarditis, urinary tract, and recurrent root canal infections. Primary virulence factors of E. faecalis such as biofilm formation, gelatinase production and suppression of host innate immune response can severely harm host tissue. Thus, novel treatments are needed to prevent E. faecalis biofilm development and pathogenicity due to the worrisome rise in enterococcal resistance to antibiotics. The primary phytochemical in cinnamon essential oils, cinnamaldehyde, has shown promising efficacy against a variety of infections. Here, we looked into how cinnamaldehyde affected the growth of biofilms, the activity of the enzyme gelatinase, and gene expression in E. faecalis. In addition, we looked at the influence of cinnamaldehyde on RAW264.7 macrophages' interaction with biofilm and planktonic E. faecalis in terms of intracellular bacterial clearance, NO generation, and macrophage migration in vitro. According to our research, cinnamaldehyde attenuated the biofilm formation potential of planktonic E. faecalis and gelatinase activity of the biofilm at non-lethal concentrations. The expression of the quorum sensing fsr locus and its downstream gene gelE in biofilms were also found to be significantly downregulated by cinnamaldehyde. Results also demonstrated that cinnamaldehyde treatment increased NO production, intracellular bacterial clearance, and migration of RAW264.7 macrophages in presence of both biofilm and planktonic E. faecalis. Overall these results suggest that cinnamaldehyde has the ability to inhibit E. faecalis biofilm formation and modulate host innate immune response for better clearance of bacterial colonization.

Fibroblast growth factor 8 (FGF8) up-regulates gelatinase expression in chondrocytes through nuclear factor-κB p65.

INTRODUCTION: Gelatinases, namely MMP2 and MMP9, are involved in the natural turnover of articular cartilage, as well as the loss of the cartilage matrix in osteoarthritis (OA). Studies have reported that fibroblast growth factor 8 (FGF8) promoted the degradation of cartilage in OA. In the present study, we predicted that FGF8 promoted chondrocyte expression and secretion of gelatinases by activating NF-κB p65 signaling. MATERIALS AND METHODS: Primary chondrocytes from C57 mice were cultured with recombinant FGF8. RNA sequencing was employed to explore the gene expression changes of gelatinases. Gelatin zymography was used to determine the activation of gelatinases. Western blot was used to investigate the expression of the gelatinases and NF-κB p65 signaling pathways, and immunofluorescence staining and NF-κB inhibitor assays were performed to confirm the activation of NF-κB p65 signaling. RESULTS: FGF8 could increase the expression and activity of gelatinases in primary chondrocytes. And FGF8-induced expression of gelatinases was regulated through activation of NF-κB signaling with acetylated p65 accumulating in the cell nucleus. We further found that the NF-κB inhibitor, BAY 11-7082, could suppress up-regulation of gelatinase induced by FGF8. CONCLUSION: FGF8 enhanced the expression and activity of MMP2 and MMP9 in chondrocytes via NF-κB p65 signaling.

Cellular-scale sex differences in extracellular matrix remodeling by valvular interstitial cells.

Males acquire calcific aortic valve disease (CAVD) twice as often as females, yet stenotic valves from females display significantly higher levels of fibrosis compared to males with similar extent of disease. Fibrosis occurs as an imbalance between the production and degradation of the extracellular matrix (ECM), specifically type I collagen. This work characterizes ECM production and remodeling by male and female valvular interstitial cells (VICs) to better understand the fibrocalcific divergence between sexes evident in CAVD. Male and female VICs were assessed for gene and protein expression of myofibroblastic markers, ECM components, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) via qRT-PCR and western blot. Overall metabolic activity was also measured. Activity assays for collagenase and gelatinase were performed to examine degradation behavior. Male VICs produced greater levels of myofibroblastic markers while female VICs showed greater metabolic activity and collagen production. In general, females displayed a greater level of MMP expression and production than males, but no sex differences were observed in TIMP production. Male VICs also displayed a greater level of collagenase and gelatinase activity than female VICs. This work displays sex differences in ECM remodeling by VICs that could be related to the sexual dimorphism in ECM structure seen in clinical CAVD.

An Overview of the Factors Involved in Biofilm Production by the Enterococcus Genus.

Enterococcus species are known for their ability to form biofilms, which contributes to their survival in extreme environments and involvement in persistent bacterial infections, especially in the case of multi-drug-resistant strains. This review aims to provide a comprehensive understanding of the mechanisms underlying biofilm formation in clinically important species such as Enterococcus faecalis and the less studied but increasingly multi-drug-resistant Enterococcus faecium, and explores potential strategies for their eradication. Biofilm formation in Enterococcus involves a complex interplay of genes and virulence factors, including gelatinase, cytolysin, Secreted antigen A, pili, microbial surface components that recognize adhesive matrix molecules (MSCRAMMs), and DNA release. Quorum sensing, a process of intercellular communication, mediated by peptide pheromones such as Cob, Ccf, and Cpd, plays a crucial role in coordinating biofilm development by targeting gene expression and regulation. Additionally, the regulation of extracellular DNA (eDNA) release has emerged as a fundamental component in biofilm formation. In E. faecalis, the autolysin N-acetylglucosaminidase and proteases such as gelatinase and serin protease are key players in this process, influencing biofilm development and virulence. Targeting eDNA may offer a promising avenue for intervention in biofilm-producing E. faecalis infections. Overall, gaining insights into the intricate mechanisms of biofilm formation in Enterococcus may provide directions for anti-biofilm therapeutic research, with the purpose of reducing the burden of Enterococcus-associated infections.

Roles of Matrix Metalloproteinases and Their Natural Inhibitors in Metabolism: Insights into Health and Disease.

Matrix metalloproteinases (MMPs) are a family of zinc-activated peptidases that can be classified into six major classes, including gelatinases, collagenases, stromelysins, matrilysins, membrane type metalloproteinases, and other unclassified MMPs. The activity of MMPs is regulated by natural inhibitors called tissue inhibitors of metalloproteinases (TIMPs). MMPs are involved in a wide range of biological processes, both in normal physiological conditions and pathological states. While some of these functions occur during development, others occur in postnatal life. Although the roles of several MMPs have been extensively studied in cancer and inflammation, their function in metabolism and metabolic diseases have only recently begun to be uncovered, particularly over the last two decades. This review aims to summarize the current knowledge regarding the metabolic roles of metalloproteinases in physiology, with a strong emphasis on adipose tissue homeostasis, and to highlight the consequences of impaired or exacerbated MMP actions in the development of metabolic disorders such as obesity, fatty liver disease, and type 2 diabetes.

Probiotic Potential and Safety Assessment of Lactiplantibacillus plantarum cqf-43 and Whole-Genome Sequence Analysis.

This study reports the whole-genome sequence of Lactiplantibacillus plantarum cqf-43 isolated from healthy sow feces. Based on genomic analysis, we performed a comprehensive safety assessment of strain cqf-43, using both in vitro and in vivo experiments, and explored its probiotic potential. The total genome length measures 3,169,201 bp, boasting a GC content of 44.59%. Through phylogenetic analyses, leveraging both 16S rRNA gene and whole-genome sequences, we confidently categorize strain cqf-43 as a member of Lactiplantibacillus. Genome annotation using Prokka unveiled a total of 3141 genes, encompassing 2990 protein-coding sequences, 71 tRNAs, 16 rRNAs, and 1 tmRNA. Functional annotations derived from COG and KEGG databases highlighted a significant abundance of genes related to metabolism, with a notable emphasis on carbohydrate utilization. The genome also revealed the presence of prophage regions and CRISPR-Cas regions while lacking virulence and toxin genes. Screening for antibiotic resistance genes via the CARD database yielded no detectable transferable resistance genes, effectively eliminating the potential for harmful gene transfer. It is worth highlighting that the virulence factors identified via the VFDB database primarily contribute to bolstering pathogen resilience in hostile environments. This characteristic is particularly advantageous for probiotics. Furthermore, the genome is devoid of menacing genes such as hemolysin, gelatinase, and biogenic amine-producing genes. Our investigation also unveiled the presence of three unannotated secondary metabolite biosynthetic gene clusters, as detected by the online tool antiSMASH, suggesting a great deal of unknown potential for this strain. Rigorous in vitro experiments confirmed tolerance of strain cqf-43 in the intestinal environment, its antimicrobial efficacy, sensitivity to antibiotics, absence of hemolysis and gelatinase activity, and its inability to produce biogenic amines. In addition, a 28-day oral toxicity test showed that the strain cqf-43 did not pose a health hazard in mice, further establishing it as a safe strain.

The Phosphatase Bph and Peptidyl-Prolyl Isomerase PrsA Are Required for Gelatinase Expression and Activity in Enterococcus faecalis.

Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities, such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis. IMPORTANCE In Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.