Fibroblast Activation Research Articles

Angiotensin Receptors and Neprilysin Inhibitors on Myocardial Remodeling in Patients with Acute Myocardial Infarction after Percutaneous Coronary Intervention

Heart failure (HF) is a leading cause of late morbidity and mortality after acute myocardial infarction worldwide. Myocardial remodeling is an important mechanism in the development of chronic heart failure. Development of the HF phenotype in these patients arise from a complex, progressive, molecular and cellular transformation called “ventricular remodeling”. In this paper, the research progress of myocardial remodeling is summarized. Left ventricular remodeling is characterized by a progressive increase in both end-diastolic (LVEDV) and end-systolic volumes (LVESV). Cardium remodeling is due to an increased activity of cardiac fibroblasts in response to different soluble fibrogenic mediators. ARNI can inhibit the biological activity of brain natriuretic peptide enzyme, prevent the deactivation of natriuretic peptide and increase its concentration. The inhibition of brain natriuretic peptidase and the increase of natriuretic peptide level by ARNI is an important means to treat heart failure and fight against ventricular remodeling, and the inhibition of RAAS system over activation is also of great significance to delay myocardial remodeling. much of the observed clinical benefit of sacubitril/valsartan therapy in patients with HFrEF, HFpEF and in the post-myocardial infarction setting is likely related to significant reverse cardiac remodeling.

Adiponectin suppresses stiffness-dependent, profibrotic activation of lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible respiratory disease with limited therapeutic options. A hallmark of IPF is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We performed an unbiased, next-generation sequencing (NGS) screen to identify signaling pathways involved in stiffness-dependent lung fibroblast activation. Adipocytokine signaling was downregulated in primary lung fibroblasts (PFs) cultured on stiff matrices. Re-activating adipocytokine signaling with adiponectin suppressed stiffness-dependent activation of human PFs. Adiponectin signaling depended on CDH13 expression and p38 mitogen-activated protein kinase gamma (p38MAPKγ) activation. CDH13 expression and p38MAPKγ activation were strongly reduced in lungs from IPF donors. Our data suggest that adiponectin-signaling via CDH13 and p38MAPKγ activation suppresses profibrotic activation of fibroblasts in the lung. Targeting of the adiponectin signaling cascade may provide therapeutic benefits in IPF.NEW & NOTEWORTHY A hallmark of idiopathic pulmonary fibrosis (IPF) is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We found that activation of the adipocytokine signaling pathway halts and reverses stiffness-induced, profibrotic fibroblast activation. Specific targeting of this signaling cascade may therefore provide therapeutic benefits in IPF.

Synovial expression of glucocorticoid receptor parallels fibroblast activation in patients with rheumatoid arthritis.

Hypocortisolemia is associated with increased expression of NR3C1 (glucocorticoid receptor, GR) in blood cells. As endogenous cortisol production is decreased in some RA patients, we tested the hypothesis that GR may be aberrantly expressed in rheumatoid synovium. We defined the cellular pattern of NR3C1 synovial expression using human and mouse single-cell RNA-sequencing data. Bulk synovial RNA-sequencing data from early (n = 57) or established (n = 94) RA were compared to osteoarthritis (n = 22) and healthy synovium (n = 28). GR was expressed in all synovial cell types in both human and experimental arthritis. GR synovial expression, as well as 11β-HSD1/11β-HSD2 enzyme ratio, were higher in RA than healthy and osteoarthritic tissue, regardless of disease duration or treatment. Given that GR expression varied across samples, we searched for differences between RA patients with higher versus lower GR expression. Indeed, the synovial transcriptome of RA patients with high versus low GR expression (1st quartile, 30,517 ± 4876 vs. 4th quartile, 19,382 ± 2523 normalized counts) was enriched for proinflammatory gene-sets, including 'inflammatory response', 'IFN-γ response' and 'IL6/JAK/STAT3 signalling'. High synovial GR expression was also associated with increased JAK2 and PTPRK expression, denoting activation of the proinflammatory sublining fibroblasts. In contrast, low GR expression was associated with increased COMP and COL6A2 expression, denoting a resting synovial state. GR is overexpressed in the synovium of some RA patients in association with proinflammatory gene expression and activated sublining fibroblast status. Further studies should examine whether GR overexpression may act as a compensatory mechanism sensitizing synovial tissue to glucocorticoid action in RA.

A comparative review of the application value of FAPI PET/CT and 18F-FDG PET/CT in lung cancer.

Fibroblast activation protein inhibitor (FAPI) positron emission tomography/computed tomography (PET/CT) is a multimodal imaging technique that combines PET and CT, utilizing FAP inhibitors as radiotracers. Fibroblast activation protein, a serine protease highly expressed in many epithelial tumor-associated fibroblasts, plays a crucial role in tumor stroma formation and remodeling. Through the detection of FAP expression, FAPI PET/CT facilitates the diagnosis and staging of both benign and malignant pulmonary tumors. In contrast to traditional fluorine-18-fluorodeoxyglucose (18F-FDG) PET/CT focusing on glucose metabolism, FAPI PET/CT offers benefits such as enhanced specificity, reduced background noise, accelerated imaging speed, and decreased radiation exposure. This review provides an overview of the progress in applying FAPI PET/CT and 18F-FDG PET/CT in pulmonary malignancies and discusses current challenges and future prospects.

Initial clinical experience using 68Ga-FAPI-46 PET/CT for detecting various cancer types.

Numerous studies have shown that gallium-68-labeled fibroblast activation protein inhibitor (68Ga-FAPI) positron emission tomography/computed tomography (PET/CT) scans would yield high intra-tumoral tracer uptake and low uptake in normal tissues as background, thus allowing for excellent visualization of lesions in the cancer microenvironment. This study set out to compare the suitability of novel 68Ga-FAPI-46 PET versus routine fluorine-18-fluorodeoxyglucose (18F-FDG) PET and other few cases of 68Ga-DOTATATE/68Ga-Pentixafor PET/CT for the assessment of different types of cancer. A retrospective analysis of 11 patients (6 males, 5 females; average age: 53 years, range: 10-58 years) with histopathologically confirmed, well-differentiated adenocarcinoma, medullar thyroid cancer (MTC), papillary thyroid carcinoma (PTC), cervical, gastric, glioblastoma multiform (GBM), colon, Ewing's sarcoma, and breast cancer was performed. These patients underwent PET/CT scans using four different radiotracers (9 18F-FDG, 11 68Ga- FAPI, 3 68Ga-DOTATATE, and 1 68Ga-Pentixafor). The patients' PET/CT images were visually evaluated for cancer detection, and analyzed semi-quantitatively through image- derived metrics, such as target-to-background ratio (TBR) and maximum standardized uptake value (SUVmax), for recurrence and metastasis. The study of 11 patients revealed that 68Ga-FAPI-46 was more effective than other tracers for detecting metastases, with 55 vs. 49 metastases in the lymph nodes, 4 vs. 3 in the liver, and 4 vs. 3 in the bones detected in comparison to 18F-FDG. No significant differences were observed in 68Ga-DOTATATE and 68Ga-Pentixafor PET images. In addition, in five patients, the SUVmax and TBR values in 68Ga-FAPI-46 PET images were significantly higher than those in 18F-FDG PET images for lymph nodes and bone metastases. Although the SUVmax in 68Ga-FAPI-46 and 18F-FDG PET images for liver metastases was comparable, 68Ga-FAPI- 46 had a significantly higher TBR than 18F-FDG. Our findings suggest that FAPI PET/CT is not suitable for evaluating GBM and Ewing sarcoma but generally outperforms 18F-FDG PET/CT in various types of breast cancer, gastrointestinal, gynecological, PTC and MTC. However, larger trials are needed to validate these preliminary findings.

Localized in vivo prodrug activation using radionuclides.

We screened the ability of radionuclides to chemically activate a model radiation-activated prodrug consisting of the microtubule destabilizing monomethyl auristatin E caged by a radiation-responsive phenyl azide ("caged-MMAE") and interpreted experimental results using the radiobiology computational simulation suite TOPAS-nBio. RAiDER was evaluated in syngeneic mouse models of cancer using fibroblast activation protein inhibitor (FAPI) agents 99m Tc-FAPI-34 and 177 Lu-FAPI-04, the prostate-specific membrane antigen (PSMA) agent 177 Lu-PSMA-617, combined with caged-MMAE or caged-exatecan. Biodistribution in mice, combined with clinical dosimetry, estimated the relationship between radiopharmaceutical uptake in patients and anticipated concentrations of activated prodrug using RAiDER. RAiDER efficiency varied by 250-fold across radionuclides ( 99m Tc> 177 Lu> 64 Cu> 68 Ga> 223 Ra> 18 F), yielding up to 1.22µM prodrug activation per Gy of exposure from 99m Tc. Computational simulations implicated low-energy electron-mediated free radical formation as driving prodrug activation. Clinically relevant radionuclide concentrations chemically activated caged-MMAE restored its ability to destabilize microtubules and increased its cytotoxicity by up to 600-fold compared to non-irradiated prodrug. Mice treated with 99m Tc-FAPI-34 and caged-MMAE accumulated up to 3000× greater concentrations of activated MMAE in tumors compared to other tissues. RAiDER with 99m Tc-FAPI-34 or 177 Lu-FAPI-04 delayed tumor growth, while monotherapies did not ( P <0.03). Clinically-guided dosimetry suggests sufficient radiation doses can be delivered to activate therapeutically meaningful levels of prodrug. This proof-of-concept study shows that RAiDER is compatible with multiple radionuclides commonly used in nuclear medicine and has the potential to improve the efficacy of radiopharmaceutical therapies to treat cancer safely. RAiDER thus shows promise as an effective strategy to treat disseminated malignancies and broadens the capability of radiopharmaceuticals to trigger diverse biological and therapeutic responses.

Integrated single-cell and bulk RNA sequencing analyses identify an immunotherapy nonresponse-related fibroblast signature in gastric cancer.

Factors that determine nonresponse to immune checkpoint inhibitor (ICI) remain unclear. The protumor activities of cancer-associated fibroblasts (CAFs) suggest that they are potential therapeutic targets for cancer treatment. There is, however, a lack of CAF-related signature in predicting response to immunotherapy in gastric cancer (GC). Single-cell RNA sequencing (scRNA-seq) and RNA sequencing (RNA-seq) data of GC immunotherapy were downloaded from the Gene Expression Omnibus database. Bulk RNA-seq data were obtained from The Cancer Genome Atlas. The R package 'Seurat' was used for scRNA-seq data processing. Cellular infiltration, receptor-ligand interactions, and evolutionary trajectory analysis were further explored. Differentially expressed genes affecting overall survival were obtained using the limma package. Weighted Gene Correlation Network Analysis was used to identify key modules of immunotherapy nonresponder. Prognostic model was constructed by univariate Cox and least absolute contraction and selection operator analysis using the intersection of activated fibroblast genes (AFGs) with key module genes. The differences in clinicopathological features, immune microenvironment, immunotherapy prediction, and sensitivity to small molecule agents between the high- and low-risk groups were further investigated. Based on scRNA-seq, we finally identified 20 AFGs associations with the prognosis of GC patients. AFGs' high expression levels were correlated with both poor prognosis and tumor progression. Three genes (FRZB, SPARC, and FKBP10) were identified as immunotherapy nonresponse-related fibroblast genes and used to construct the prognostic signature. This signature is an independent significant risk factor affecting the clinical outcomes of GC patients. Remarkably, there were more CD4 memory T cells, resting mast cells, and M2 macrophages infiltrating in the high-risk group, which was characterized by higher tumor immune exclusion. Moreover, patients with higher risk scores were more prone to not respond to immunotherapy but were more sensitive to various small molecule agents, such as memantine. In conclusion, this study constructed a fibroblast-associated ICI nonresponse gene signature, which could predict the response to immunotherapy. This study potentially revealed a novel way to overcome immune resistance in GC.

Evening primrose and its compounds of 1-Oxohederagenin and remangilone C ameliorate bleomycin-induced pulmonary fibrosis by regulating β-catenin signaling

BackgroundPulmonary fibrosis (PF) is a progressive and severe respiratory disease for which there is still a lack of satisfactory treatment methods other than lung transplantation. Evening primrose (EP) is widely used in Chinese folk medicinal herbs, especially for the treatment of lung-related diseases. However, the protective effect of evening primrose against PF has yet to be reported. PurposeThis study explores the pharmacological effect of EP and its possible active components against PF from the perspectives of lung function, histopathological staining, and molecular biology assays. MethodsEstablishing a rat pulmonary fibrosis model using bleomycin to detect lung function, pathological changes, and collagen deposition. TGF-β1 was used to establish an in vitro model of PF in BEAS-2B cells, and the active ingredients in evening primrose were screened. Then, the therapeutic effects of 1-Oxohederagenin (C1) and remangilone C (C2) derived from EP were observed in an in vivo model of bleomycin-induced PF, and the differentially expressed genes between the C1 and C2 treatment groups and the model group were screened with transcriptome sequencing. Finally, TGF-β1-induced damage to HFL1 cell was used to explore the specific mechanisms by which C1 and C2 alleviate PF and the involvement of β-catenin signaling. ResultsEvening primrose extract showed some ameliorative effects on bleomycin-induced PF in rats, manifested as reduced pathological damage and reduced collagen deposition. The chemical components of C1 and C2 potently ameliorated BLM-induced PF in animals and effectively inhibited fibroblast activation by interfering with β-catenin signaling. ConclusionEvening primrose extract has certain ameliorative effects on PF. In addation, C1 and C2 might be related with the suppression of fibroblast activation by inhibiting β-catenin signaling.

Abstract We108: Adipocyte Enhancer Binding Protein 1 (AEBP1) Inhibition as a Potential Anti-Fibrotic Therapy in Heart Failure

Introduction: Cardiac fibrosis is one of the major hallmarks of heart failure (HF) progression. Fibrosis is initiated as a repair mechanism following any kind of cardiac injury including a heart attack, inflammation, hypertension, etc. Briefly, fibroblasts are activated to form myofibroblasts that secrete collagen. Persistent fibroblast activation leads to excess collagen deposition leading to tissue stiffening and impaired contractility and thereby HF. To date, there is no anti-fibrotic therapy used in HF patients. Using bulk and snRNA sequencing data performed on myocardial tissue from HF patients, we identified adipocyte enhancer binding protein (AEBP1) to be significantly upregulated in activated fibroblasts. AEBP1 has been shown to play a crucial role in fibroblast activation following injury in the kidney, liver, and lungs. AEBP1 inhibition resulted in fibrosis attenuation in these organs. However, its role in cardiac fibrosis is not well understood. Hypothesis: We hypothesize that AEBP1 is crucial for cardiac fibroblast activation and AEBP1 inhibition will prevent collagen secretion and lead to improved cardiac function. Methods: Human cardiac fibroblasts (HCF) from non-failing donors were used for in vitro experiments. AdV-CMV-AEBP1 was used for AEBP1 overexpression (OE) and AdV-CMV-shAEBP1 was used to knockdown (KD) AEBP1 in vitro. AAV9-CMV-shAEBP1 was used for in vivo KD of AEBP1 in mice following myocardial infarction (MI). Human myocardial slices from n=6 HF patients were used and AdV-CMV-shAEBP1 was used to knockdown AEBP1 in human samples. Results and Discussion: AEBP1 OE in HCF resulted in fibroblast activation and collagen secretion (n=6, p=0.002). Contrarily, loss of AEBP1 significantly prevented fibroblast activation, evident from a reduction in transgelin (SM22) (n=6, p=0.02) and collagen (COL1A1) protein levels (n=6, p=0.01). AEBP1 KD in a mouse model of acute cardiac fibrosis resulted in reduced fibrosis and improved cardiac function (n=6, 12% absolute improvement compared to untreated mice, relative improvement in ejection fraction, p=0.02). Tissue culture studies of human HF myocardium showed a significant improvement in cardiac structure evident from reduced cardiomyocyte hypertrophy (n=6 HF patients, p=0.05) and reduced fibrosis (n=6, p&lt;0.0001) following AEBP1 KD. Overall, we show that AEBP1 plays a critical role in fibrogenesis, and its inhibition has the potential to attenuate fibrosis in acute and chronic HF.

Abstract We145: δ-catenin mRNA m 6 A Methylation Promotes Cardiac Fibrosis following Acute Myocardial Infarction

Background: Cardiac fibrosis is pivotal in heart failure progression, where excessive extracellular matrix (ECM) secretion by activated fibroblasts leads to adverse remodeling and dysfunction. While Wnt/β-catenin signaling influences fibroblast activation and cardiac fibrosis post-MI, its precise regulation remains unclear. Emerging evidence suggests N6-methyladenosine (m 6 A) mRNA methylation's role in disease pathology, yet its specific contribution to post-MI cardiac fibrosis is not well understood. Thus, we hypothesized that “ MI-induced METTL3 (a Key m6A mRNA methyltransferase) activation stabilizes δ-catenin mRNA, facilitating cardiac fibrosis and adverse remodeling ”. Methods: The mice underwent sham/MI surgeries for 4 weeks, after which heart tissues were collected for biochemical and histological analysis following heart function measurements. Additionally, methyl-immunoprecipitation followed by RNA sequencing (MeRIP-sequencing) was performed on the heart tissue post-surgery, and the data were analyzed to identify fibrosis-associated targets. Results: Ischemic injury significant increase in m6A mRNA methylation in heart tissues. This increase in m 6 A RNA was also observed in adult cardiac fibroblasts (AMFs) following TGFb treatment. Interestingly, METTL3 inhibition (METTL3 siRNA) resulted in a significant reduction in TGFb-induced periostin and fibronectin gene expression, while METTL3 overexpression enhanced the expression of fibrotic genes in AMFs. To identify differentially regulated m6A target genes, MeRIP-seq was performed on RNA isolated from mice hearts post-MI. The sequencing data suggested hypermethylation of fibrosis-associated genes, including δ-catenin, post-MI. Notably, TGFb-induced increased δ-catenin mRNA methylation led to δ-catenin mRNA stabilization. In contrast, METTL3 inhibition using siMETTL3 in AMFs and in METTL3 KO mice significantly reduced fibroblast activation and cardiac fibrosis. Conclusion: Our data suggests that hypermethylation of δ-catenin mRNA plays a significant role in the progression of cardiac fibrosis following AMI. Therefore, regulating METTL3 could be a potential therapeutic target for attenuating cardiac fibrosis.

Potential of 68Ga-FAPI-04 PET/MR to predict worsening renal function after acute ST-elevation myocardial infarction

PurposeThe fibroblast activation protein inhibitor-04 (FAPI-04) specifically binds to the FAP of activated myocardial fibroblasts, which makes 68Ga-labelled FAPI-04 (68Ga-FAPI-04) positron emission tomography (PET)/magnetic resonance (MR) a new potential imaging technique for the evaluation of myocardial fibrosis. This study aimed to evaluate the potential value of 68Ga-FAPI-04 PET/MR in assessing and predicting changes in renal function in patients with acute ST-elevation myocardial infarction (STEMI). MethodsThirty-three patients with STEMI were included in this study. 68Ga-FAPI-04 PET/MR and cardiac magnetic resonance were performed before discharge in all patients. Worsening renal function(WRF) was defined as ≥20% decrease in estimated glomerular filtration rate(eGFR) from baseline to 12 months. ResultsThe WRF group demonstrated higher 68Ga-FAPI-04 uptake volume (UV) at baseline than the non-WRF group(P = 0.009). 68Ga-FAPI-04 UV at baseline was correlated with follow-up eGFR (r = −0.493, P = 0.004). 68Ga-FAPI-04 UV at baseline was a significant predictor of WRF (OR = 1.014, P = 0.029) at 12 months after STEMI. ConclusionsAs an effective tool to non-invasively quantify myocardial fibroblast activation, 68Ga-FAPI-04 PET/MR has potential value for assessing and predicting worsening renal function in patients with STEMI.

Abstract Tu088: Fibroblast Activation, Collagen Secretion, and Migration Occurs Independently of Has2 Expression

Background: Hyaluronan (HA) accumulates after tissue injury and may regulate the wound-healing process. We found that HA is increased in and around the scar following myocardial infarction (MI). Fibroblasts are the main producers of the collagen that comprises the scar, and we have shown that they are responsible for HA accumulation. Given that these two extraordinarily high-volume synthetic processes occur in the same place and simultaneously, we queried whether alterations in Has2 -dependent HA synthesis impacted collagen secretion and other fibroblast functions. Goal: To determine the impact of HA synthesis on fibroblast function. Methods and Results: We isolated cardiac fibroblasts from Has2 fl/fl mice and treated with Cre adenovirus to delete Has2 expression or Has2 adenovirus to overexpress Has2. We assessed cell number and found no differences when Has2 was deleted or overexpressed in both naïve fibroblasts (n=6); activation with TGF-b did not change this response . We used a scratch assay to assess migration and found no change between treatments in naïve fibroblasts (n=6). Hydroxyproline assessment of deposited collagen and immunoblotting for soluble collagen secretion in the media indicated no differences between treatments in naïve fibroblasts (n=3). Conclusion: Genetic manipulation of HA production did not impact proliferation, migration, or collagen secretion in fibroblasts. Considering the enormous metabolic requirement of fibroblasts to produce HA and collagen, these results suggest that the regulation of these processes may be distinct or that the substrate is not limiting in our experimental conditions. In the intact heart, substrate could become limiting and a relationship between these two biosynthetic processes may emerge

Abstract We101: Targeted gene silencing with small interfering RNA-loaded lipid nanoparticles to treat cardiac fibrosis

Bromodomain-containing protein 4 (BRD4) is an effector of transforming growth factor-β (TGF-β) signaling. Upon injury, BRD4 stimulates the differentiation of quiescent cardiac fibroblasts into activated fibroblasts. These activated fibroblasts unfavorably remodel the myocardium by depositing excess extracellular matrix. Here, we reported lipid nanoparticles (LNPs) loaded with small interference RNA against BRD4 (siBRD4) to deactivate fibroblasts after cardiac injury. Because fibroblast activation protein (FAP) is a biomarker of activated fibroblasts, we conjugated anti-FAP antibodies on the LNPs for targeted delivery to activated fibroblasts. Systemic administration of these targeted siBRD4-loaded LNPs inhibited fibroblast activation, reduced fibrosis, and improved cardiac functions in rodent models of myocardial infarction and cardiac fibrosis. Single-nucleus RNA sequencing and cytokine analysis revealed that this therapy further converted the pro-fibrotic and pro-inflammatory microenvironment to a pro-resolution and pro-regeneration one. The safety and toxicity of this nanomedicine were tested in nonhuman primates.

Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p&gt;0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.

Increased imaging ligand hydrophilicity and improved pharmacokinetic properties provides enhanced in vivo targeting of fibroblast activation protein

In this work, the impact of physicochemical modifications on pharmacokinetics and in vivo targeting of a small molecule fibroblast activation protein inhibitor (FAPI) imaging ligand in a murine model of rheumatoid arthritis was evaluated. While similar ligands have been well-reported in oncology for molecular imaging and radiotherapy, there are limited reports of FAPI derivatives in targeted applications in immunology. As inflammation may increase both specific and non-specific delivery of targeted agents in general, we sought to identify the optimal targeted molecular imaging probe characteristics for efficient cell surface engagement. A series of FAPI derivatives were synthesized and their physicochemical properties modified via conjugation of fluorescent dyes and/or an albumin-binding small molecule. The impact of these modifications on cell surface binding affinity was assessed using an overexpressing cell line. Additionally, a thorough mechanistic characterization of fibroblast activation protein (FAP) cell surface internalization was evaluated in both overexpressing and endogenously expressing cells. Lastly, the pharmacokinetics and in vivo uptake in inflamed arthritic paws were characterized via near-infrared (NIR) imaging. All targeted molecular imaging agents tested maintained strong nanomolar binding affinity to cell surface FAP independent of chemical modification. The murine fibroblast-like synoviocytes expressed lower absolute cell-surface FAP compared to a transfected line, and the net internalization half-life measured for the transfected cells via flow cytometry was 7.2 h. The unmodified FAPI ligand exhibited the poorest in vivo targeting, likely resulting from its large apparent volume of distribution (62.7 ml) and rapid systemic clearance (t1/2 = 0.5 h). Conjugation of a charged, hydrophilic AF647 fluorophore decreased systemic clearance (t1/2 = 2.1 h) and demonstrated a 2-fold improvement in blocking FAPI-800CW engagement of FAP in vivo when compared to blocking of FAPI-800CW with FAPI with up to 2.8-fold improvements noted for the equivalent albumin binding construct comparison.

Abstract Mo087: A shared gene regulatory network underlies atrial pathophysiology in atrial fibrillation and heart failure mouse models

Atrial fibrillation and heart failure have a strong epidemiologic link, however the mechanisms underlying their mutual risk remain unresolved. We revealed a remarkable correlation of both gene expression and genomic features in the atria of atrial fibrillation (AF, Tbx5 deletion) and heart failure (HF, TAC banding) mouse models. Tbx5 - and TAC-dependent gene expression in the left atria demonstrated a correlation coefficient of 0.8. Remarkably, 118 transcription factors were dysregulated in both models and 109 shared directionality in both cases, suggesting a shared transcriptional response. Differential non-coding transcriptional profiling to identify altered cis -regulatory regions demonstrated a correlation coefficient of 0.81 between the models, including ~1,800 shared differentially expressed ncRNAs, of which ~400 emanated from accessible candidate regulatory elements. Integration with chromatin architecture allowed identification of lineage-specific shared AF and HF gene regulatory networks. A TBX5-driven ncRNA-defined regulatory element drives expression of Klf15, an inhibitor of cardiac hypertrophy, in the wild-type cardiomyocytes that is downregulated in both mouse and human heart failure. Whereas in mouse HF and AF a disease-specific ncRNA-defined regulatory element is upregulated with Sox9 , an important transcriptional regulator of fibroblast activation. This work indicates that AF and HF initiate a shared atrial pathological genomic response, including downregulation of a wild-type GRN that normally prevents injury-based disease signaling and upregulation of a disease GRN that promotes atrial remodeling, uncovering a common genomic underpinning of atrial disease.

Abstract We138: Single-nucleus and bulk multiomic analyses reveal the dynamics of individual cardiac cell types after myocardial infarction and the underlying gene regulatory networks

Cardiovascular diseases are the leading causes of death in the world, among which myocardial infarction (MI) is the most fatal form. Following MI, the death of cardiomyocytes induces cardiac remodeling, which is mainly mediated by cardiac fibroblasts but also involves other cardiac cell types. A deeper understanding of the dynamics of individual cell types among different stages after myocardial infarction may help identify novel therapeutic strategies. Here, through single-nucleus multiomic (snRNAseq and snATACseq) analysis of uninjured mouse hearts and mouse hearts at different time points (2 h, 1 d, 3 d, 7 d, and 4 w) after MI, we identified multiple cardiac cell types, including cardiomyocyte, fibroblast, epicardial cell, endothelial cell, mural cell, and immune cell, and their dynamic gene expression and chromatin accessibility after MI. Features specific to the acute and chronic responses of different cell types to MI were identified. Cell type-specific single-nucleus gene regulatory networks (snGRNs) constructed through integrated analysis of snRNAseq and snATACseq data revealed transcription factors possibly responsible for the gene expression changes in individual cell types. The cardiac fibroblast GRN was further enriched using bulk RNAseq and ATACseq data collected at more time points (uninjured, and 2 h, 12 h, 1 d, 3 d, 7 d, 2 w, and 4 w after MI) and non-coding RNA transcriptomics. Promoter and enhancer activities and chromatin interactions in cardiac fibroblasts were further verified by CUT&amp;Tag targeting corresponding histone markers and Hi-C, respectively. The refined cardiac fibroblast GRN identified multiple key transcription factors that likely govern cardiac fibroblast activation and differentiation after MI.

Abstract Tu096: The Aryl Hydrocarbon Receptor Agonist, L-Kynurenine, Modulates Cardiac Fibroblast Activation and Antigen Presentation

Background: The Aryl Hydrocarbon Receptor (AHR) is a ubiquitously expressed transcription factor that regulates the cellular response to external stimuli through the binding of ligands including tryptophan metabolites such as L-Kynurenine (L-Kyn). Alterations in tryptophan metabolic pathways and downregulation of myocardial AHR are associated with cardiac inflammation and fibrosis in experimental heart failure (HF) induced by transverse aortic constriction (TAC). We hypothesized cardiac fibroblast (CFB) AHR and its agonism by L-Kyn prevent the pro-fibrotic and pro-inflammatory activity of CFBs in experimental HF. Methods and Results: We performed TAC or sham surgery on Ahr -/- mice and WT littermates for 4 weeks and found that Ahr-/- mice showed significantly decreased contractile function measured by echocardiography, and increased collagen deposition in left ventricular histological sections after TAC compared to WT mice. We performed bulk RNA sequencing in sorted CFB (CD31-CD45-MEFSK4+) from Ahr -/- and WT mice. Gene set enrichment analysis showed Ahr -/- CFB had increased expression in pathways for fibroblast activation, chemokine production, and immune cell recruitment. To investigate the role of L-Kyn agonism of CFB AHR in transformation and contractile function, murine CFB were treated with Transforming Growth Factor β (TGFβ) (100ng/ml) or seeded in collagen discs, in the presence or absence of L-Kyn(100ug/ml). L-Kyn attenuated TGFβ mediated induction of αSMA and collagen 1 protein expression by immunofluorescence and western blot, as well as decreased disc contraction 72h after mechanical activation and release. The role of AHR agonism by L-Kyn on the pro-inflammatory function of CFB was determined in WT CFB treated with IFNγ (100U/ml) in the presence or absence of L-Kyn. IFNγ induction of chemokines Cxcl9 and Cxcl10, as well as MHC-II surface expression were attenuated by L-Kyn, so was T cell adhesion to CFB in co-culture assays. In contrast, L-Kyn had no effect on the ability of CFB to take up and process antigens, as determined by DQ-Ovalbumin internalization quantified by flow cytometry. Conclusions: Our results demonstrate that AHR regulates activation and pro-inflammatory signatures in CFB at baseline, and that stimulation of CFB transformation and pro-inflammatory activity is dampened by agonism with L-Kyn. Current studies are aimed at investigating AHR agonism in the context of pressure overload induced HF in CFB reporter mice.

Abstract Tu102: The Role of the β IV -spectrin/STAT3 Complex in Regulating Ischemic Cardiac Remodeling

Adverse fibrotic remodeling contributes to high morbidity and mortality in patients following myocardial infarction (MI). The healing process after MI requires necrotic tissue to be cleared and replaced with fibrosis, which is driven by the coordinated effort of several distinct cardiac cell populations, including macrophages and cardiac fibroblasts (CFs). While previous studies have focused on identifying the stimuli that drives fibrotic remodeling in the infarcted myocardium, the contribution of specific cell populations to the signaling cascade and the manner of how these communication networks are tuned in response to chronic stress remains unclear. Prior work has identified a role for the cytoskeletal protein, β IV -spectrin, in coordinating the activity of the transcription factor, STAT3, with implications for CF activation and profibrotic signaling. Specifically, it was found that mice expressing truncated β IV -spectrin ( qv 3J ) are resistant to cardiac fibrosis in the setting of chronic pressure overload, with maintained β IV -spectrin expression and STAT3 localization/activity in CFs. Further, β IV -spectrin deficiency causes increased secretion of profibrotic and proinflammatory factors, including matrix metallopeptidases (MMPs). Therefore, we hypothesized that stress-induced loss of β IV -spectrin and subsequent dysregulation of STAT3 signaling in CFs proximal to the infarct promotes fibroblast activation, an important first step in repairing the infarct region. In this study, we subjected WT and qv 3J mice to permanent ligation of the left anterior descending artery and assessed cardiac function and survival through 28 days. A subset of mice were sacrificed at 7 days for histology, qPCR, and flow cytometry. Compared to WT, qv 3J mice showed a significant decrease in 28-day survival with increased mortality in the first week post-MI. Autopsy identified increased incidence of cardiac rupture in qv 3J animals. Immunohistochemistry revealed impaired scar formation at 7 days post-MI in qv 3J compared to WT. Quantitative PCR showed altered expression of matrix metallopeptidases (MMPs 3, 8, 9, and 14) in the qv 3J hearts at day 7. MMP activity was assessed in whole heart lysates using gelatin-zymography. Collagen uptake was assessed in CFs and bone marrow-derived macrophages isolated from WT and qv 3J mice. Our work identifies a novel role of the spectrin-based pathway in facilitating fibrotic remodeling in response to ischemic stress.

Dynamically visualizing profibrotic maladaptive repair after acute kidney injury by fibroblast activation protein imaging

A major challenge in prevention and early treatment of organ fibrosis is the lack of valuable tools to assess the evolving profibrotic maladaptive repair after injury invivo in a non-invasive way. Here, using acute kidney injury (AKI) as an example, we tested the utility of fibroblast activation protein (FAP) imaging for dynamic assessment of maladaptive repair after injury. The temporospatial pattern of kidney FAP expression after injury was first characterized. Single-cell RNA sequencing and immunostaining analysis of patient biopsies were combined to show that FAP was specifically upregulated in kidney fibroblasts after AKI and was associated with fibroblast activation and chronic kidney disease (CKD) progression. This was corroborated in AKI mouse models, where a sustained and exaggerated kidney FAP upregulation was coupled to persistent fibroblast activation and a fibrotic outcome, linking kidney FAP level to post-insult maladaptive repair. Furthermore, using positron emission tomography (PET)/CT scanning with FAP-inhibitor tracers ([18F]FAPI-42, [18F]FAPT) targeting FAP, we demonstrated the feasibility of non-invasively tracking of maladaptive repair evolution toward kidney fibrosis. Importantly, a sustained increase in kidney [18F]FAPT (less hepatobiliary metabolized than [18F]FAPI-42) uptake reflected persistent kidney upregulation of FAP and characterized maladaptive repair after AKI. Kidney [18F]FAPT uptake at hour 2-day 7 correlated with kidney fibrosis 14 days after AKI. Similar changes in [18F]FAPI-42 PET/CT imaging were observed in patients with AKI and CKD progression. Thus, persistent kidney FAP upregulation after AKI was associated with maladaptive repair and a fibrotic outcome. Hence, FAP-specific PET/CT imaging enables dynamic visualization of maladaptive repair after AKI and prediction of kidney fibrosis within a clinically actionable window.