Protein Activation Research Articles

Functional crosstalk of sucrose and G protein signaling in maize thermotolerance by modulating osmoregulation system.

Sucrose (SUC) is a signaling molecule with multiple physiological functions. G protein is a kind of receptor that converts extracellular first messenger into intracellular second messenger. However, it is little known that SUC interplays with G protein signaling in maize thermotolerance. In this work, using maize seedlings as materials, the interplay between SUC and G protein signaling in maize thermotolerance was investigated. The results indicate that heat stress-decreased survival percentage and tissue viability of the seedlings was mitigated by SUC. Similarly, heat stress-increased malondialdehyde content and electrolyte leakage also was reduced by SUC. These findings show that SUC can potentially enhance thermotolerance in maize seedlings. Also, SUC-enhanced thermotolerance was abolished by suramin (G protein inhibitor) and N-ethylmaleimide (SUC transport inhibitor), but enhanced by 3-O-methyl-D-glucose (G protein activator), indicating the interplay of SUC and G protein signaling in maize thermotolerance. To investigate the possible mechanism behind SUC-G protein interaction in enhancing maize thermotolerance, osmoregulation in mesocotyls of seedlings were evaluated before and after heat stress. The results suggest that osmolytes (SUC, glucose, fructose, total soluble sugar, proline, and glycine betaine) contents in mesocotyls under non-heat and heat stress were increased by SUC in varying degrees. Likewise, the osmolyte-metabolizing enzymes (sucrose-phosphate synthase, sucrose synthase, pyrroline-5-carboxylate synthase, ornithine aminotransferase, betaine-aldehyde dehydrogenase, and trehalase) activities were enhanced by SUC. Analogously, ZmSPS1, ZmSUS6, ZmP5CS, ZmOAT, ZmBADH, and ZmTRE1 expression in mesocotyls was up-regulated by SUC to different extent. These findings illustrate that the functional crosstalk of sucrose and G protein signaling in maize thermotolerance by modulating osmoregulation system.

Treatment of cancer-associated fibroblast-like cells with celecoxib enhances the anti-cancer T helper 1/Treg responses in breast cancer.

Tumor inflammation, as one of the hallmarks of cancer, has been the target for anti-cancer treatments. Celecoxib is a selective inhibitor of the enzyme cycloxygenase-2 (COX-2) and inhibits the production of PGE2, which is an important mediator of tumor inflammation produced by cancer cells and cells of the tumor microenvironment. In this study, we aimed at inhibiting COX-2 using celecoxib, expressed in cancer-associated fibroblast (CAF)-like cells isolated from breast cancer and evaluated the alterations in their cytokine profile and gene expression. CAF-like cells were isolated by explant culture from 13 breast cancer tissues. Simultaneously, peripheral blood mononuclear cells (PBMCs) were isolated from patients' blood. CAF-like cells were treated with 10µM of celecoxib and expression of genes COX-2, smooth muscle actin-alpha (α-SMA), and production of prostaglandin E2 (PGE2), Interleukin 10 (IL10), and transforming growth factor beta1 (TGF-β1) was evaluated. Next, PBMCs were co-cultured with celecoxib-treated CAF-like cells and the expression of genes T-bet, Foxp3, GATA-3; production of cytokines IFN-ɣ, IL-10, IL-4, TGF-β1, and the mediator PGE2 were assessed by real-time-PCR and ELISA, respectively. Isolated CAF-like cells showed expression of fibroblast activation protein (FAP). Treatment with celecoxib was able to efficiently reduce the production of PGE2 and the expression of α-SMA in isolated CAF-like cells. Furthermore, PBMCs in co-culture with these cells showed enhanced Th1 phenotype including T-bet and IFNγ expression and decreased the phenotypical markers of regulatory T cells such as FoxP3 and IL-10 and TGF-β1 production. Our study shows the important role of COX-2 in CAFs by promoting immune suppression. Our results suggested that high expression of COX-2 in CAFs may serve as a new therapeutic, targeting CAFs in enhancing immune responses in breast cancer treatment.

Aflatoxin B1 Promotes Pyroptosis in IPEC-J2 Cells by Disrupting Mitochondrial Dynamics through the AMPK/NLRP3 Pathway.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins in food and feed, seriously jeopardizing the intestinal health, while the effects of AFB1 on intestinal damage remain to be well understood. This study aims to evaluate the effect of AFB1 on intestinal injury by regulating AMP-activated protein kinase (AMPK)-mediated pyroptosis in vitro. The present study showed that AFB1 led to the formation of large number of bubble-like protrusions on the cell membrane, releasing lactate dehydrogenase (LDH) and interleukin-1β (IL-1β). Stimulation with AFB1 resulted in the activation of the NOD-like receptor protein 3 (NLRP3) pathway, as indicated by the increased expression of pyroptosis-associated factor mRNAs and proteins, which ultimately led to a significant upregulation of the pyroptosis rate. Meanwhile, AFB1 caused dysfunction of mitochondrial dynamics by activating the AMPK signaling pathway as mainly evidenced by upregulating dynamin-1-like protein 1 (Drp1) mRNA and protein expression. Moreover, inhibition of NLRP3 and AMPK pathways by MCC950 and compound C, respectively, significantly alleviated AFB1-induced damage in IPEC-J2 cells, evidenced by suppressed NLRP3-mediated pyroptosis, and ameliorated AMPK-mediated mitochondrial dynamics imbalance. In conclusion, these results demonstrated that AFB1 promoted pyroptosis of IPEC-J2 cells by interfering with mitochondrial dynamics by activating the AMPK/NRLP3 pathway.

A Vaccine Against Fibroblast Activation Protein Improves Murine Cardiac Fibrosis by Preventing the Accumulation of Myofibroblasts.

Myofibroblasts are primary cells involved in chronic response-induced cardiac fibrosis. Fibroblast activation protein (FAP) is a relatively specific marker of activated myofibroblasts and a potential target molecule. This study aimed to clarify whether a vaccine targeting FAP could eliminate myofibroblasts in chronic cardiac stress model mice and reduce cardiac fibrosis. We coadministered a FAP peptide vaccine with a CpG K3 oligonucleotide adjuvant to male C57/BL6J mice and confirmed an elevation in the anti-FAP antibody titer. After continuous angiotensin II and phenylephrine administration for 28 days, we evaluated the degree of cardiac fibrosis and the number of myofibroblasts in cardiac tissues. We found that cardiac fibrosis was significantly decreased in the FAP-vaccinated mice compared with the angiotensin II and phenylephrine control mice (3.45±1.11% versus 8.62±4.79%; P=4.59×10-3) and that the accumulation of FAP-positive cells was also significantly decreased, as indicated by FAP immunohistochemical staining (4077±1746 versus 7327±1741 cells/mm2; FAP vaccine versus angiotensin II and phenylephrine control; P=6.67×10-3). No systemic or organ-specific inflammation due to antibody-dependent cell cytotoxicity induced by the FAP vaccine was observed. Although the transient activation of myofibroblasts has an important role in maintaining the structural robustness in the process of tissue repair, the FAP vaccine showed no adverse effects in myocardial infarction and skin injury models. Our study demonstrates the FAP vaccine can be a therapeutic tool for cardiac fibrosis.

Evaluating the diagnostic value of 18F-FAPI-04 PET/CT in various malignant tumors: a head-to-head comparison with 18F-FDG PET/CT.

This study aims to evaluate the diagnostic efficacy of 18F-fibroblast activation protein inhibitor-04 (18F-FAPI-04) PET/CT for various malignant tumors and compare it head-to-head with 18F-FDG PET/CT. This single-center, prospective study continuously recruited patients with suspected or confirmed malignant tumors for concurrent 18F-FDG and 18F-FAPI-04 PET/CT scans from April 2022 to October 2023. The pathological diagnosis or clinical follow-up served as the reference standard. The Z-test for two proportions was used to compare the sensitivity, specificity, and accuracy of tumor diagnosis between the two imaging agents. Wilcoxon signed-rank tests were employed to compare the uptake of the two radiotracers and the tumor-to-background ratio (TBR) differences in tumors. The study involved 15 types of tumors and included 88 patients, comprising 53 males and 39 females, with an average age of 57.7 ± 10.8 years. In patient-based analysis, 18F-FAPI-04 PET/CT demonstrated higher diagnostic accuracy than 18F-FDG PET/CT for both initial staging and restaging patients (77.4% vs 56.6%, p = 0.0389; 94.3% vs 54.3%, p < 0.001), prompting treatment plan adjustments in 17% of restaged patients. The lesion-based analysis revealed comparable diagnostic accuracy of 18F-FAPI-04 PET/CT and 18F-FDG PET/CT for primary tumors (78.9% vs 75.4%, p = 0.8234), while showing superior accuracy for residual/recurrent tumors, lymph node metastases, and distant metastases compared to 18F-FDG PET/CT (100.0% vs 50.0%, p = 0.002; 98.8% vs 86.0%, p < 0.001; 98.3% vs 79.3%, p < 0.001). 18F-FAPI-04 PET/CT exhibits higher uptake and TBR in most tumors demonstrating superior diagnostic efficacy for primary lesions, residual/recurrent disease, and metastases compared to 18F-FDG PET/CT, particularly beneficial for restaging post-treatment patients.

Increased perivascular fibrosis and pro-fibrotic cellular transition in intramyocardial blood vessels in myocardial infarction patients

Background and objectivesStructural and functional changes in the intramyocardial microcirculation increase the risk of myocardial infarction (MI). This study investigated intramyocardial perivascular fibrosis and pro-fibrotic cellular transitions in deceased acute and subacute MI patients to explore their involvement in the pathogenesis of MI. MethodsLeft ventricular tissue (LV) was obtained from the infarction area of autopsied patients with acute-phase MI (3–6 h; n = 24), subacute-phase MI (5–14 days; n = 12), and noninfarcted controls (n = 14). Perivascular fibrosis and fibroblast activation protein (FAP) expression were quantified using (immuno)histochemistry. Fibroblast-like transitioning of vascular smooth muscle cells (VSMC) and endothelial cells (EC) was quantified using immunofluorescent microscopy. ResultsPerivascular fibrosis was elevated in acute-phase (77.69 %) and subacute-phase (72.19 %: border zone 95.18 %: infarct core) MI patients (p < 0.0001) compared to controls (61.03 %). FAP expression was higher in both acute-phase (1.46 %) and subacute-phase (18.01 %: border zone 5.67 %: infarct core) compared to controls (0.46 %) (p < 0.05). VSMC fibroblast-like cellular transition (SMA + S100A4+ vessels fraction) was higher in acute-phase (31.96 %) and subacute-phase (21.90 %: border zone; 37.25 %: infarct core) MI compared to controls (8.95 %) (p < 0.05). Similarly, EC fibroblast-like cellular transition (CD31 + S100A4+ area fraction) was increased in acute-phase MI (10.14 %) and subacute-phase MI (8.31 %: border zone 10.15 %: infarct core) compared to controls (2.67 %) (p < 0.05). ConclusionIncreased perivascular fibrosis, fibroblast activation and vascular cellular transition in intramyocardial blood vessels of MI patients may contribute to MI development. Further increases of FAP expression and perivascular fibrosis, particularly in subacute-phase infarct cores, suggest MI itself exacerbates fibroblast activation and perivascular fibrosis, theoretically increasing reinfarction risk.

Native Valve and Native Neo-Sinus Remodeling Following Transcatheter Aortic Valve Replacement.

Transcatheter aortic valve replacement (TAVR) pushes aside the diseased native aortic valve and creates a native neo-sinus bordered by the aortic root wall and the displaced native valve. There are limited data on the progression of native valve disease post-TAVR and no previous analysis of the native neo-sinus. Native aortic valves and native neo-sinus explants obtained post-TAVR were evaluated histologically (hematoxylin and eosin, Movat pentachrome, and Martius Scarlet Blue stains) and by immunohistochemistry (TGF-β1 [transforming growth factor-beta 1], FAP [fibroblast activation protein], and ALP [alkaline phosphatase]) to assess disease mechanisms. Native aortic valves were obtained from 20 patients from 0 to 2583 days (7.08 years) post-TAVR. Native leaflets showed persistent calcific aortic stenosis-like disease activity with positivity for ALP and FAP. Native valve remodeling was observed as changes in architecture evident in explants >1.5 years, which was observed as crumpling of the leaflets. Disease activity was also present in native neo-sinuses with transcatheter heart valve implant durations >1 year with positive staining for TGF-β1, FAP, and ALP. Extensive native neo-sinus remodeling occurred with replacement and filling-in of this space with contiguous extracellular matrix, calcific deposits, and microvessels. Following TAVR, there is ongoing calcific aortic stenosis-like disease with architectural changes to native leaflets and extensive remodeling of the native neo-sinus, evidenced by replacement and contiguous filling-in of the native neo-sinus blood pool space with increasing implant duration. The dynamic nature of these tissues has potential implications for neo-sinus flow, valve degeneration, and re-intervention.

Targeting fibroblast activation protein with chimeric antigen receptor macrophages

Under the rapid advancement of chimeric antigen receptor T cell (CAR-T) technology, CAR-macrophages (CAR-Ms) are also being developed currently in the pre-clinical stage and have been shown to inhibit tumor growth in several mouse tumor models. Fibroblast activation protein (FAP) is a type II transmembrane serine protease, which is expressed in stromal fibroblasts of over 90 % of common human epithelial cancers and is upregulated in fibrotic diseases of the liver, lung and colon, etc. In this study, we firstly constructed FAP-CAR macrophages to target FAP+ cells through in vitro phagocytosis assays. In subsequent in vivo assays, we discovered that FAP-CAR-ΔZETA bone marrow-derived macrophages (BMDMs) rather than FAP-CAR BMDMs, exhibited a pronounced anti-tumor effect in mouse subcutaneous MC38 colon cancer model. In addition, FAP-CAR and FAP-CAR-ΔZETA BMDMs therapy could effectively improve CCl4-induced liver fibrosis in mice. Collectively CAR-Ms targeting FAP demonstrated great therapeutic potential in cancer and liver fibrosis therapy.

Inhibition of lncEPS by TLR4/NF-κB pathway induces ventilator-induced lung injury by decreasing its binding to and upregulating Hspa5

Whether LincRNA erythroid prosurvival (LncEPS) reduces VILI remains unclear. A GSE200932 microarray was used to screen differentially expressed genes (DEGs). A VILI mouse model was constructed by mechanical ventilation (MV), with or without TAK242 or SN50 pretreatment. Airway transfection with adeno-associated virus (AAV) was used to overexpress lncEPS in alveolar macrophages (AMs). Lung tissues were collected to assess pathological injury and macrophage polarization. NLRP3 inflammasome, TLR4/ NF-κB pathway activation and heat shock protein family A member 5 (Hspa5) in lung tissue and AMs wre evaluated. LncEPS localization and regulatory changes were assessed using in situ hybridization and RT–PCR. Lung tissues after lncEPS overexpression were subjected to transcriptomics. Chromatin isolation and mass spectrometry (MS) were performed to identify proteins interacting with lncEPS. GSE200932 microarray showed that the DEGs were related to NF-κB pathway, Toll-like receptor pathway and NOD-like receptor pathway. TAK242 or SN50 treatment increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation by inhibiting TLR4/NF-κB pathway in VILI. Inhibition of TLR4/NF-κB pathway upregulated lncEPS expression in AMs. Overexpression of lncEPS increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation, eventually alleviating VILI in mice. Mechanistically, lncEPS bound to Hspa5 and downregulated its expression to inhibit inflammatory response.

FAP+ gastric cancer mesenchymal stromal cells via paracrining INHBA and remodeling ECM promote tumor progression

Gastric cancer (GC) mesenchymal stromal cells (GCMSCs) are the predominant components of the tumor microenvironment (TME) and play a role in the occurrence, development, and metastasis of tumors. However, GCMSCs exhibit phenotypic and functional heterogeneity. The key population of GCMSCs which are vital to tumor progression remains elusive. The expression of fibroblast activation protein (FAP) in gastric cancer was analyzed and verified using clinical pathology data and single-cell RNA sequencing database of gastric cancer patients. FAP positive GCMSCs (FAP+ GCMSCs) were isolated via flow cytometry and characterized through transcriptomic sequencing. The impact of conditioned medium from FAP+ GCMSCs on gastric cancer cell lines was assessed using Enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Additionally, immunohistochemistry (IHC) and Masson’s trichrome staining were employed to explore the association between FAP+ GCMSCs and extracellular matrix (ECM) deposition in gastric cancer tissues. Our study demonstrates that FAP is predominantly expressed in the mesenchymal stromal cells within the gastric cancer milieu. FAP+ GCMSCs exhibited enhanced proliferation, migration, contraction, and tumor-promoting capabilities compared to their FAP− counterparts. These cells significantly increased proliferation and migration of gastric cancer cells through the paracrine secretion of Inhibin Subunit Beta A (INHBA) and activation of the SMAD2/3 signaling pathway. Moreover, FAP+ GCMSCs also induced collagen deposition in ECM and then up-regulated invasion and stemness of GC cells. Mechanistically, this process was mediated by the interaction of collagen with Integrin Subunit Beta 1 (ITGB1), triggering the phosphorylation of Focal Adhesion Kinase (FAK) and Yes Associated Transcriptional Regulator (YAP). Our findings reveal that FAP+ GCSMCs enhanced the GC progression via releasing cytokine INHBA and remodeling ECM providing a theoretical basis for further exploration of tumor stromal-targeting therapy of gastric cancer.

High-affinity ELR+ chemokine ligands show G protein bias over β-arrestin recruitment and receptor internalization in CXCR1 signalling

The human CXC chemokine receptor 1 (CXCR1), a G protein-coupled receptor (GPCR), plays significant roles in inflammatory diseases and cancer. While CXCL8 is a well-established high-affinity ligand for CXCR1, there is no consensus regarding the binding ability of the other ELR+ chemokines (CXCL1-3 and CXCL5-8). Since research has predominantly focused on CXCL8-mediated CXCR1 signalling, insight into potential signalling bias induced by different CXCR1 ligands is lacking. Therefore, in this study we first compared and clarified the binding ability of all ELR+ chemokines using a competition binding assay. In this assay CXCL1-3 and CXCL5 behaved as low-affinity ligands while CXCL6-8 were high affinity ligands. We further investigated potential ligand bias within the CXCR1 signalling system. Using NanoBRET-based assays heterotrimeric G protein dissociation, β-arrestin recruitment and receptor internalisation induced by chemokine binding to CXCR1 were investigated. A quantitative and qualitative investigation of ligand bias showed that the high-affinity ELR+ chemokines were biased towards G protein activation over β-arrestin recruitment and receptor internalisation, when CXCL8 was used as a reference ligand.

A Chemically Defined Culture for Tooth Reconstitution.

It is known for decades that dental epithelium and mesenchyme can reconstitute and regenerate a functional tooth. However, the mechanism of tooth reconstitution remains largely unknown due to the lack of an efficient in vitro model. Here, a chemically defined culture system is established that supports tooth reconstitution, further development with normal anatomy, and prompt response to chemical interference in key developmental signaling pathways, termed as toothoids. By using such a system, it is discovered that, during reconstitution, instead of resetting the developmental clock, dental cells reorganized and restarted from the respective developmental stage where they are originally isolated. Moreover, co-stimulation of Activin A and Hedgehog/Smoothened agonist (SAG) sustained the initial induction of tooth fate from the first branchial arch, which would be otherwise quickly lost in culture. Furthermore, activation of Bone Morphogenetic Protein (BMP) signaling triggered efficient enamel formation in the late-stage toothoids, without affecting the normal development of ameloblasts. Together, these data highlight the toothoid culture as a powerful tool to dissect the molecular mechanisms of tooth reconstitution and regeneration.

Comparison of [18F]FAPI-42 and [18F]FDG PET/CT in the evaluation of systemic vasculitis.

The role of fibroblast activation protein (FAP)-targeted imaging in systemic vasculitis is currently unclear. We aimed to evaluate the clinical value of fluorine-18-labeled FAP inhibitor 42 ([18F]FAPI-42) in patients with systemic vasculitis and to compare with [18F]fluorodeoxyglucose (FDG) imaging. Patients with systemic vasculitis who underwent dual-tracer PET/CT([18F]FDG and [18F]FAPI) imaging from September 2020 to March 2022 were retrospectively analyzed. Positive lesions are defined as vascular/extravascular lesions with increased tracer uptake above surrounding background, which cannot be attributed to the physiologic biodistribution of the radiotracer. The vascular/extravascular lesion detection rate and semiquantitative values (SUVmax, TBRblood and TBRliver) of [18F]FAPI and [18F]FDG were compared, and the correlation between the extent and range of tracer uptake and levels of inflammatory markers was investigated. Thirty patients (13 males and 17 females; mean age, 52.5 ± 17.2 years) with systemic vasculitis were included (17 large vessel vasculitis, 10 anti-neutrophil cytoplasmic antibody-associated vasculitis, 2 Behcet's disease and 1 polyarteritis nodosa). [18F]FDG PET/CT had positive findings in 93.3% (28/30) of patients, while [18F]FAPI PET/CT had positive findings in all patients (100%, P = 0.500). Compared with [18F]FDG PET/CT, [18F]FAPI PET/CT detected more lesions (161/168 vs. 145/168, P = 0.005), and more extensive vascular involvement in 60% (18/30) of patients. Although SUVmax did not differ significantly between [18F]FAPI and [18F]FDG (median, 5.94 vs. 5.46, P = 0.517), [18F]FAPI had higher TBRliver (median, 9.59 vs. 3.15, P < 0.001) and TBRblood (median, 5.45 vs. 4.20, P = 0.006). The total number of positive lesions in FAPI PET/CT show a moderate correlation with erythrocyte sedimentation rate (rs =0.478, P = 0.008) and C-reactive protein (rs =0.486, P = 0.006). After treatment, follow-up FAPI PET/CT of 6 patients showed decreased SUVmax, TBR and number of detected lesions, paralleling the clinical remission. [18F]FAPI PET/CT imaging is a promising imaging modality for the diagnosis and therapeutic monitoring of systemic vasculitis.

Multiomics approaches disclose very-early molecular and cellular switches during insect-venom allergen-specific immunotherapy: an observational study

Allergen-specific immunotherapy (AIT) induces immune tolerance, showing the highest success rate (>95%) for insect venom while a much lower chance for pollen allergy. However, the molecular switches leading to successful durable tolerance restoration remain elusive. The primary outcome of this observational study is the comprehensive immunological cellular characterization during the AIT initiation phase, whereas the secondary outcomes are the serological and Th2-cell-type-specific transcriptomic analyses. Here we apply a multilayer-omics approach to reveal dynamic peripheral immune landscapes during the AIT-initiation phase in venom allergy patients (VAP) versus pollen-allergic and healthy controls. Already at baseline, VAP exhibit altered abundances of several cell types, including classical monocytes (cMono), CD4+ hybrid type 1-type 17 cells (Th1-Th17 or Th1/17) and CD8+ counterparts (Tc1-Tc17 or Tc1/17). At 8-24 h following AIT launch in VAP, we identify a uniform AIT-elicited pulse of late-transitional/IL-10-producing B cells, IL-6 signaling within Th2 cells and non-inflammatory serum-IL-6 levels. Sequential induction of activation and survival protein markers also immediately occur. A disequilibrium between serum IL-6 and cMono in VAP baseline is restored at day seven following AIT launch. Our longitudinal analysis discovers molecular switches during initiation-phase insect-venom AIT that secure long-term outcomes. Trial number: NCT02931955.

Recent advances in nuclear medicine and their role in inflammatory arthritis: focus on the emerging role of FAPI PET/CT

AbstractImaging molecular processes associated with inflammatory disease has been revolutionized by hybrid imaging using positron emission tomography/computed tomography (PET/CT). PET/CT visualizes metabolic activity as well as protein expression and provides a comprehensive whole-body evaluation. It has the potential to reveal inflammation prior to detection of structural changes in inflammatory joint diseases. FAP is a type II transmembrane glycoprotein overexpressed not only in the stroma of tumors but also in the fibrotic processes of certain immune-mediated disorders. The recent introduction of fibroblast activation protein inhibitors (FAPI) labeled by positron emitters and thus suitable for PET/CT allows to investigate FAP expression in vivo. This review will focus on the use of FAPI-PET/CT for the diagnosis and evaluation of treatment response in inflammatory joint diseases.

Unveiling the Tumor Microenvironment Through Fibroblast Activation Protein Targeting in Diagnostic Nuclear Medicine: A Didactic Review on Biological Rationales and Key Imaging Agents

The tumor microenvironment (TME) is a dynamic and complex medium that plays a central role in cancer progression, metastasis, and treatment resistance. Among the key elements of the TME, cancer-associated fibroblasts (CAFs) are particularly important for their ability to remodel the extracellular matrix, promote angiogenesis, and suppress anti-tumor immune responses. Fibroblast activation protein (FAP), predominantly expressed by CAFs, has emerged as a promising target in both cancer diagnostics and therapeutics. In nuclear medicine, targeting FAP offers new opportunities for non-invasive imaging using radiolabeled fibroblast activation protein inhibitors (FAPIs). These FAP-specific radiotracers have demonstrated excellent tumor detection properties compared to traditional radiopharmaceuticals such as [18F]FDG, especially in cancers with low metabolic activity, like liver and biliary tract tumors. The most recent FAPI derivatives not only enhance the accuracy of positron emission tomography (PET) imaging but also hold potential for theranostic applications by delivering targeted radionuclide therapies. This review examines the biological underpinnings of FAP in the TME, the design of FAPI-based imaging agents, and their evolving role in cancer diagnostics, highlighting the potential of FAP as a target for precision oncology.

The cell adhesion molecule CD44 acts as a modulator of 5-HT7 receptor functions

BackgroundHomo- and heteromerization of G protein-coupled receptors (GPCRs) plays an important role in the regulation of receptor functions. Recently, we demonstrated an interaction between the serotonin receptor 7 (5-HT7R), a class A GPCR, and the cell adhesion molecule CD44. However, the functional consequences of this interaction on 5-HT7R-mediated signaling remained enigmatic.MethodsUsing a quantitative FRET (Förster resonance energy transfer) approach, we determined the affinities for the formation of homo- and heteromeric complexes of 5-HT7R and CD44. The impact of heteromerization on 5-HT7R-mediated cAMP signaling was assessed using a cAMP responsive luciferase assay and a FRET-based cAMP biosensor under basal conditions as well as upon pharmacological modulation of the 5-HT7R and/or CD44 with specific ligands. We also investigated receptor-mediated G protein activation using BRET (bioluminescence resonance energy transfer)-based biosensors in both, homo- and heteromeric conditions. Finally, we analyzed expression profiles for 5-HT7R and CD44 in the brain during development.ResultsWe found that homo- and heteromerization of the 5-HT7R and CD44 occur at similar extent. Functionally, heteromerization increased 5-HT7R-mediated cAMP production under basal conditions. In contrast, agonist-mediated cAMP production was decreased in the presence of CD44. Mechanistically, this might be explained by increased Gαs and decreased GαoB activation by 5-HT7R/CD44 heteromers. Unexpectedly, treatment of the heteromeric complex with the CD44 ligand hyaluronic acid boosted constitutive 5-HT7R-mediated cAMP signaling and receptor-mediated transcription, suggesting the existence of a transactivation mechanism.ConclusionsInteraction with the hyaluronan receptor CD44 modulates both the constitutive activity of 5-HT7R as well as its agonist-mediated signaling. Heteromerization also results in the transactivation of 5-HT7R-mediated signaling via CD44 ligand.

YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease

Renal tubule cells act as a primary site of injury in diabetic kidney disease (DKD), with dysfunctional mitochondrial quality control (MQC) closely associated with progressive kidney dysfunction in this context. Our investigation delves into the observed inactivation of yes-associated protein 1 (YAP1) and consequential dysregulation of MQC within renal tubule cells among DKD subjects through bioinformatic analysis of transcriptomics data from the Gene Expression Omnibus (GEO) dataset. Receiver operating characteristic curve analysis unequivocally underscores the robust diagnostic accuracy of YAP1 and MQC-related genes for DKD. Furthermore, we observed YAP1 inactivation, accompanied by perturbed MQC, within cultured tubule cells exposed to high glucose (HG) and palmitic acid (PA). This pattern was also evident in the tubulointerstitial compartment of kidney sections from biopsy-approved DKD patients. Additionally, renal tubule cell-specific Yap1 deletion exacerbated kidney injury in diabetic mice. Mechanistically, Yap1 deletion disrupted MQC, leading to mitochondrial aberrations in mitobiogenesis and mitophagy within tubule cells, ultimately culminating in histologic tubular injury. Notably, Yap1 deletion-induced renal tubule injury promoted the secretion of C-X-C motif chemokine ligand 1 (CXCL1), potentially augmenting M1 macrophage infiltration within the renal microenvironment. These multifaceted events were significantly ameliorated by administrating the YAP1 activator XMU-MP-1 in DKD mice. Consistently, bioinformatic analysis of transcriptomics data from the GEO dataset revealed a noteworthy upregulation of tubule cells-derived chemokine CXCL1 associated with macrophage infiltration among DKD patients. Crucially, overexpression of YAP1 via adenovirus transfection sustained mitochondrial membrane potential, mtDNA copy number, oxygen consumption rate, and activity of mitochondrial respiratory chain complex, but attenuated mitochondrial ROS production, thereby maintaining MQC and subsequently suppressing CXCL1 generation within cultured tubule cells exposed to HG and PA. Collectively, our study establishes a pivotal role of tubule YAP1 inactivation-mediated MQC dysfunction in driving DKD progression, at least in part, facilitated by promoting M1 macrophage polarization through a paracrine-dependent mechanism.

Overexpression of Egr1 Transcription Regulator Contributes to Schwann Cell Differentiation Defects in Neural Crest-Specific Adar1 Knockout Mice.

Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects.

68Ga-FAPI and 18F-NaF PET/CT in psoriatic arthritis: a comparative study.

As fibroblast-like synoviocyte activation and bone formation are associated with psoriatic arthritis (PsA), positron emission tomography (PET) using the tracers of 68Ga-fibroblast activation protein inhibitor (FAPI) and 18F-sodium fluoride (NaF) may sensitively detect the disease. In this prospective study, we aimed to evaluate the performance of 68Ga-FAPI PET/CT in PsA and to compare it with 18F-NaF PET/CT. Sixteen participants (female 7/16, age 42.31 ± 10.66 years) with PsA were prospectively enrolled and underwent dual-tracer PET/CT, clinical assessment and ultrasonography. PET/CT images were scored for PET-positive lesions at the peripheral joints, entheses, and axial joints. The positivity rate of 68Ga-FAPI in peripheral joints is higher than that in entheses and axial joints (21.84% vs 12.15% vs 0%), whereas high positivity rates of 18F-NaF in peripheral joints, entheses, and axial joints were observed (85.23%, 78.13%, and 75%, respectively). The disease activity score 28 was higher in the PET-positive than in the PET-negative group with 68Ga-FAPI (5.25 ± 1.84 vs 2.55 ± 0.94, p= 0.037), but not with 18F-NaF. Besides, the PET joint count at 68Ga-FAPI PET/CT was positively correlated with the TJC (r = 0.604, p= 0.017), SJC (r = 0.773, p= 0.001), DAS28-CRP (r = 0.556, p= 0.032), PASDAS (r = 0.540, p= 0.038) and PsASon13 (r = 0.701, p= 0.005), while no correlation was observed in 18F-NaF PET/CT. The positivity rates of 68Ga-FAPI- and 18F-NaF PET/CT were different in patients with PsA in peripheral joints, entheses, and axial joints. The extent of joint involvement at 68Ga-FAPI PET/CT correlated with clinical and ultrasound variables as well as disease activity.