Fibroblast Activation Research Articles

HOXC10 promotes hypertrophic scar fibroblast fibrosis through the regulation of STMN2 and the TGF-β/Smad signaling pathway.

The pathophysiology of hypertrophic scar (HS) shares similarities with cancer. HOXC10, a gene significantly involved in cancer development, exhibits higher expression levels in HS than in normal skin (NS), suggesting its potential role in HS regulation. And the precise functions and mechanisms by which HOXC10 influences HS require further clarification. Gene and protein expressions were analyzed using raeal-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques. Cell proliferation and migration were evaluated using EdU proliferation assays, CCK-8 assays, scratch assays, and Transwell assays. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to investigate the interactions between HOXC10 and STMN2. HOXC10 and STMN2 expression levels were significantly higher in HS tissues compared with NS tissues. Silencing HOXC10 led to decreased activation, proliferation, migration, and fibrosis in hypertrophic scar fibroblasts (HSFs). Our findings also indicate that HOXC10 directly targets STMN2. The promotional effects of HOXC10 knockdown on HSF activation, proliferation, migration, and fibrosis were reversed by STMN2 overexpression. We further demonstrated that HOXC10 regulates HSF activity through the TGF-β/Smad signaling pathway. HOXC10 induces the activation and fibrosis of HSFs by promoting the transcriptional activation of STMN2 and engaging the TGF-β/Smad signaling pathway. This study suggests that HOXC10 could be a promising target for developing treatments for HS.

Assessing the Value of 68Ga-FAPI PET/CT in Gastric Mucinous Adenocarcinoma or Signet Ring Cell Carcinoma.

Purpose To investigate the clinical impact and prognostic value of gallium 68 (68Ga)-labeled fibroblast activation protein inhibitor (FAPI) PET/CT in gastric mucinous adenocarcinoma (MAC) and signet ring cell carcinoma (SRCC). Materials and Methods Eighty-six participants with newly diagnosed or recurrent gastric MAC or SRCC were prospectively enrolled from April 2021 to October 2021 and underwent both fluorine 18 (18F) fluorodeoxyglucose (FDG) PET/CT and 68Ga-FAPI PET/CT. The sensitivity, specificity, and accuracy of the two scans in primary and metastatic tumors were evaluated using the McNemar test. Changes of treatment strategies were recorded to compare the treatment management value of the two PET/CT scans. The maximum standardized uptake value (SUVmax) and peritoneal cancer index (PCI) were recorded for survival analysis. Progression-free survival (PFS) was defined as the time interval from the date of PET/CT scans to the date of disease progression. Results Eighty-six participants (median age, 62 years [IQR, 45-78 years]; 49 female) were evaluated. 68Ga-FAPI PET/CT showed higher diagnostic accuracy in detecting involved lymph nodes (87% [212 of 244] vs 71% [173 of 244], P < .001) and peritoneal metastases (96% [70 of 73] vs 55% [40 of 73], P < .001) than 18F-FDG PET/CT. Twenty-six participants (30% [26 of 86]) had treatment changes due to more accurate diagnosis with 68Ga-FAPI PET/CT. Additionally, the 68Ga-FAPI PCI was an independent predictor for PFS (hazard ratio, 6.9; 95% CI: 2.1, 23.1; P = .002). Conclusion 68Ga-FAPI PET/CT had higher accuracy in diagnosis of gastric MAC/SRCC compared with 18F-FDG PET/CT and demonstrated the potential to improve treatment strategies and predict prognosis. Keywords: PET/CT, Mucinous Adenocarcinoma, Signet Ring Cell Carcinoma, Oncology, Abdomen/GI, Molecular Imaging Supplemental material is available for this article. © RSNA, 2024.

Shh regulates M2 microglial polarization and fibrotic scar formation after ischemic stroke

BackgroundFibrotic scar formation is a critical pathological change impacting tissue reconstruction and functional recovery after ischemic stroke. The regulatory mechanisms behind fibrotic scarring in the central nervous system (CNS) remain largely unknown. While macrophages are known to play a role in fibrotic scar formation in peripheral tissues, the involvement of microglia, the resident immune cells of the CNS, in CNS fibrosis requires further exploration. The Sonic Hedgehog (Shh) signaling pathway, pivotal in embryonic development and tissue regeneration, is also crucial in modulating fibrosis in peripheral tissues. However, the impact and regulatory mechanisms of Shh on fibrotic scar formation post-ischemic stroke have not been thoroughly investigated. MethodsThis study explores whether Shh can regulate fibrotic scar formation post-ischemic stroke and its underlying mechanisms through in vivo and in vitro manipulation of Shh expression. ResultsOur results showed that Shh expression was upregulated in the serum of acute ischemic stroke patients, as well as in the serum, CSF, and ischemic regions of MCAO/R mice. Moreover, the upregulation of Shh expression was positively correlated with fibrotic scar formation and M2 microglial polarization. Shh knockdown inhibited fibrotic scar formation and M2 microglial polarization while aggravating neurological deficits in MCAO/R mice. In vitro, adenoviral knockdown or Smoothened Agonist (SAG) activation of Shh expression in BV2 cells following OGD/R regulated their polarization and influenced the expression of TGFβ1 and PDGFA, subsequently affecting fibroblast activation. ConclusionThese results suggest that Shh regulates M2 microglial polarization and fibrotic scar formation after cerebral ischemia.

CTR-FAPI PET enables precision management of medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) can only be cured through the excision of all metastatic lesions, but 29-60% patients failed to localize the disease in the current clinical practice. Previously, we developed a fibroblast activation protein inhibitor (FAPI)-based covalent targeted radioligand (CTR) for improved detection sensitivity and accuracy. In this first-in-class clinical trial, we head-to-head compared [68Ga]Ga-CTR-FAPI PET-CT and [18F]FDG PET-CT in 50 MTC patients. The primary endpoint was the patient-based detection rate, with [68Ga]Ga-CTR-FAPI exhibiting higher detection than [18F]FDG (98% vs. 66%, p=0.0002). This improved detection was attributed to increased tumor uptake (SUVmax 11.71±9.16 vs. 2.55±1.73, p<0.0001). Diagnostic accuracy, validated on lesions with gold-standard pathology, was greater for [68Ga]Ga-CTR-FAPI compared to [18F]FDG (96.7% vs. 43.3%, p<0.0001). Notably, 32% patients altered management following [68Ga]Ga-CTR-FAPI PET-CT, and 66.7% patients changed their surgical plan. Overall, [68Ga]Ga-CTR-FAPI PET-CT provided superior detection and diagnostic accuracy compared to [18F]FDG PET-CT, enabling precision management of MTC patients.

Fibroblast activation in cardio-oncology

Abstract Introduction Carcinoid heart disease is a rare disease affecting the right sided heart valves (1) while anthracycline cardiotoxicity is a dreaded sequelae of a common chemotherapeutic agent that can occur early or even years after exposure (2). Activated fibroblasts, a key factor in these diseases, can be imaged with 68Gallium-fibroblast activation protein inhibitor (68Ga-FAPI). Method 12 patients with carcinoid syndrome, 12 patients with previous anthracycline exposure and 10 healthy volunteers underwent hybrid 68Ga-FAPI positron emission tomography and magnetic resonance imaging. Areas of fibroblast activation were quantified as mean and maximum target-to-background ratios (TBRmean and TBRmax). Results Carcinoid syndrome: 2 of the 12 patients had carcinoid heart disease [mean age 70, 50% female] (CHD) (Figure 1A). 68Ga-FAPI uptake was seen in the liver lesions of all patients with known carcinoid liver tumours. Of the 10 patients without cardiac involvement, 6 had significant tricuspid valve 68Ga-FAPI uptake (CHD- FAPI+) (Figure 1B) and 4 did not (CHD- FAPI-). Median tricuspid TBRmax was significantly higher in CHD + [2.38 (IQR 2.35-2.42)], than CHD- patients [CHD- FAPI+: 1.68 (IQR 1.65-1.73), CHD- FAPI-: 1.55 (IQR 1.53-1.65)], although this was higher than healthy volunteers [1.45 (IQR 1.43- 1.61; p&amp;lt;0.05). CHD- FAPI+ patients had significantly elevated urinary 5-HIAA levels compared to CHD- FAPI- patients [Median =188 mg/24hr (IQR 151-196) in CHD- FAPI+ vs 32mg/24hr ( IQR 12-73); p=0.03]. Anthracycline chemotherapy: 3 of the 12 patients had cardiotoxicity [mean age 61, 67% female]. Anthracycline patients had higher left ventricular myocardial 68Ga-FAPI uptake than healthy volunteers with cardiotoxicity patients having the highest. [TBRmean: Cardiotoxicity 1.46 (IQR 1.36- 1.53), No cardiotoxicity 1.37 (IQR 1.19- 1.49), Healthy volunteers 0.73 (IQR 0.66 - 0.81); p&amp;lt;0.01 (Figure 1E). Across the cohort a moderate negative correlation was observed between Left ventricular ejection fraction and 68Ga-FAPI activity (LV TBRmean r =-0.46 and TBRmax (r= -0.66) Discussion 68 Ga-FAPI provides us the ability to assess fibroblast activation and for the first time demonstrated the key role of these cells in both these cardio-oncology conditions. Increased fibroblast activation is observed around the tricuspid valve in patients with carcinoid syndrome both in those with and without overt clinical disease. Similarly increased myocardial fibroblast activation is observed in patients exposed to anthracyclines even many years after exposure and where there is no overt evidence of cardiotoxicity. Conclusion 68Ga-FAPI imaging holds promise in evaluating the role of activated fibroblasts in the pathology of both carcinoid heart disease and cardiotoxicity, providing information beyond the current resolution of current clinical approaches.

Valvular and myocardial fibroblast activation in aortic stenosis: a prospective positron emission tomography study

Abstract Introduction In patients with aortic stenosis, fibrosis is a common pathophysiological feature of leaflet thickening as well as left ventricular decompensation as it transitions from hypertrophy to heart failure. Valvular fibrosis can be quantified by contrast-enhanced computed tomography, and myocardial fibrosis by late gadolinium enhancement on magnetic resonance imaging, but neither can detect the activity of fibrosis and extracellular matrix remodelling. Gallium-68 Fibroblast Activation Protein Inhibitor (68Ga-FAPI) binds to activated fibroblasts, the key effector cell of fibrogenesis, and provides a measure of fibroblast activation and fibrosis activity. We aimed to measure fibrosis activity in the aortic valve and left ventricular myocardium of patients with aortic stenosis using 68Ga-FAPI uptake. Methods In a prospective observational cohort, patients with aortic stenosis and control subjects with normal aortic valves underwent transthoracic echocardiography, 68Ga-FAPI positron emission tomography, computed tomography, and magnetic resonance imaging. Valvular and myocardial 68Ga-FAPI uptake was quantified using the target-to-background ratio (TBRmax). Valvular non-calcific volume was quantified on computed tomography and replacement myocardial fibrosis was quantified by late gadolinium enhancement on magnetic resonance imaging. Results Fifty patients with aortic valve disease (9 with aortic sclerosis, and 7 with mild, 23 with moderate and 11 with severe aortic stenosis; 72±8 years, 70% male) and 4 control subjects (66±7 years, 50% male) participated. Increased 68Ga-FAPI uptake was observed in the aortic valves of patients with aortic sclerosis and stenosis and peaked in those with moderate disease (Figure 1). TBRmax of 68Ga-FAPI uptake correlated with the aortic valve non-calcific volume (r=0.354, p&amp;lt;0.05) on computed tomography. Thirty-one patients (62%) had myocardial 68Ga-FAPI uptake, of whom half (n=17; 34%) also had late gadolinium enhancement corresponding to the region of 68Ga-FAPI uptake (Figure 2). Myocardial 68Ga-FAPI uptake correlated with increased indexed left ventricular mass (r=0.333, p&amp;lt;0.05) and left ventricular wall thickness (r=0.385, p&amp;lt;0.05). Conclusions For the first time, we have identified both valvular and myocardial fibrosis activity in patients with aortic stenosis. Valvular fibroblast activation is increased in patients with aortic sclerosis and stenosis, peaking in patients with moderate disease and correlating with leaflet thickening. Myocardial fibroblast activation is seen in the majority of patients with aortic stenosis both in areas with and without established fibrosis, and is associated with indices of adverse left ventricular remodelling. Activated fibroblasts are thus an important potential treatment target in the valve and myocardium, with 68Ga-FAPI positron emission tomography holding major promise in better understanding the pathophysiology, progression and consequences of aortic stenosis.

An Update on Molecular Mechanisms of Scarring—A Narrative Review

Fibroblasts, the principal cellular mediators of connective tissue remodeling, play a crucial role in the formation of physiological and pathological scars. Understanding the intricate interplay between fibroblasts and other cellular and molecular components is essential for elucidating the underlying mechanisms driving scar formation. Hypertrophic scars, keloids and atrophic scars arise from dysregulated wound healing processes characterized by persistent inflammation, aberrant collagen deposition, and impaired extracellular matrix remodeling. Fibroblasts play a central role in the pathogenesis of such pathological scars, driving aberrant extracellular matrix remodeling, subsequently contributing to the formation of raised or depressed fibrotic lesions. The investigation of complex interactions between fibroblasts and the microenvironment is crucial for developing targeted therapeutic interventions aimed at modulating fibroblast activity and improving clinical outcomes in patients with pathological scars. Further research into the molecular pathways governing fibroblast behavior and their heterogeneity holds promise for advancing scar management strategies. This narrative review was performed to shed light on the mechanisms behind scar formation, with a special focus on the role of fibroblasts in the formation of different types of scars, providing insights into the pathophysiology of these conditions. Through the analysis of current knowledge, this review seeks to identify the key cellular and molecular mechanisms involved in fibroblast activation, collagen synthesis, and extracellular matrix remodeling in hypertrophic scar, keloid, or atrophic scar formation.

Integrating single-cell sequencing and genetic lineage tracing reveals endothelial plasticity during atrial remodeling

Abstract Background Atrial fibrillation (AF), as the most burdensome cardiac arrhythmia, could lead to the significant mortality and morbidity. Atrial fibrosis is a well-recognized pathophysiological factor of the development and progression of AF, which also hampers its treatment. However, the underlying mechanisms of this intricate process remain largely unknown. Purpose This study sought to reveal the cellular composition of AF substrate and investigate the vital contributors to atrial structural remodeling. Methods Left atrial appendages from three patients with AF undergoing surgical ablation and three individuals with sinus rhythm undergoing heart transplantation were subjected to 10X single-cell RNA sequencing (scRNA-seq). Another five public scRNA-seq or snRNA-seq datasets of potentially remodeled right or left appendages were retrieved across three publications. The vital contributors of atrial structural remodeling and their characteristics were identified by intercellular communication analysis and trajectory inference. Transverse aortic constriction (TAC) was used to induce atrial remodeling in Cdh5-CreERT2;RFP mice, followed by scRNA-seq of endothelial cells (ECs). Human primary atrial ECs and fibroblasts were isolated for cell-cell interaction experiments. ECs-specific mouse models of Transforming growth factor beta 1 (TGFB1) (knockout and overexpression) were generated. Electrophysiological and histology analyses were performed to determine the consequences of ECs-derived TGFB1 gain and loss of function in the atrium. Results The comprehensive analysis of different scRNA-seq datasets revealed that ECs played an important regulatory role in the homeostasis of atrial substrate and displayed remarkable mesenchymal activation during atrial remodeling, which was further confirmed by TAC mice. Immunofluorescence staining and high-content screening suggested that the complete transition from ECs to mesenchymal cells slightly contributed to atrial remodeling. However, TGFB1 secreted from mesenchymal activated ECs significantly promoted activation, proliferation, and collagen secretion of fibroblasts. We discovered that overexpression of TGFB1 in ECs of mice significantly increased atrial fibrosis, and thus prolonging AF duration. Besides, knockout of TGFB1 in ECs of mice underwent TAC significantly attenuated the atrial fibrosis and decreased the rate of AF. Conclusion Our work demonstrates that mesenchymal ECs-derived Tgfb1 plays an important role in atrial remodeling by regulating the fibroblast function. EC-specific interventions were suggested to facilitate the development of novel strategies for mitigating AF substrate remodeling.

Dysfunctional atrial fibroblast processing of filamin A in atrial fibrillation

Abstract Background/Introduction Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with atrial structural remodelling, hallmarked by fibrosis. Atrial fibrosis is instigated by atrial fibroblasts (AFBs). It was previously uncovered the hormone calcitonin (CT) is produced in the heart and, via binding to the CT-receptors (CTR), inhibits atrial fibrogenesis and fibroblast profibrotic activity. [1] In AF, abnormally increased CTR internalisation causes loss of physiological surface CTR and prevents fibroblast activation by CT. The molecular mechanisms of this phenomenon in ACFs are unknown. Aim To explore the role of filamin A (FLNa), an actin cross-linking protein that was previously shown to interact with CTR, [2] in the regulation of CTR trafficking. Methods AFBs were enzymatically isolated from left atrial appendage biopsies collected from 30 patients in persistent AF or normal sinus rhythm (SR, controls) undergoing elective heart surgery. Immunostaining assessed the localisation of CTR and its overlap with the nucleus (stained with DAPI) and FLNa in AFBs. Co-localisation was quantified by the JACoP BIOP plugin for ImageJ. Target mRNA expression was assessed by RT-qPCR and protein levels by immunoblot. Immunoprecipitation (IP) then immunoblot (IB) validated protein-protein interactions. Calpain activity was assessed with a fluorescence-based assay. The effects of calpains on FLNa proteolytic processing was examined with calpain inhibitor calpeptin (10nM–100nM vs DMSO loading control). Results Immunostaining revealed aberrant, intracellular localisation of CTR in AF-AFBs (A) with increased nuclear localisation (p=0.03) and reduced co-localisation with FLNa (p=0.03). FLNa gene expression showed a trend towards reduction in AF (by 36%, p=0.06; B). Meanwhile, although full-length FLNa protein was unaltered in AF, there was a striking 67% reduction (p=0.02) in the expression of FLNa’s c-terminal fragment (FLNaCT), which contains the CTR binding domain (C). Co-immunoprecipitation confirmed a physical interaction between CTR and FLNaCT (D) in both SR and AF AFBs. FLNaCT is typically generated when FLNa is cleaved by the protease calpain 1, whose protein (F) but not mRNA (E), was significantly downregulated (71%, p&amp;lt;0.001) in AF. Puzzlingly, a ~70% loss of calpain 1 protein was associated with unchanged calpain activity (G) and FLNaCT levels in AFBs treated with calpain inhibitor calpeptin (H). Conclusions Persistent AF is associated with an aberrant CTR localisation and FLNa proteolytic processing, not attributable to altered calpain activity. Further characterisation of the molecular determinants of the altered CTR localisation may uncover novel and potentially druggable facets of structural remodelling in AF.

CAR-Macrophage Therapy Alleviates Myocardial Ischemia-Reperfusion Injury.

Given the growing acknowledgment of the detrimental effects of excessive myocardial fibrosis on pathological remodeling after myocardial ischemia-reperfusion injury (I/R), targeting the modulation of myocardial fibrosis may offer protective and therapeutic advantages. However, effective clinical interventions and therapies that target myocardial fibrosis remain limited. As a promising chimeric antigen receptor (CAR) cell therapy, whether CAR macrophages (CAR-Ms) can be used to treat I/R remains unclear. The expression of FAP (fibroblast activation protein) was studied in mouse hearts after I/R. FAP CAR-Ms were generated to target FAP-expressing cardiac fibroblasts in mouse hearts after I/R. The phagocytosis activity of FAP CAR-Ms was tested in vitro. The efficacy and safety of FAP CAR-Ms in treating I/R were evaluated in vivo. FAP was significantly upregulated in activated cardiac fibroblasts as early as 3 days after I/R. Upon demonstrating their ability to engulf FAP-overexpressing fibroblasts, we intravenously administered FAP CAR-Ms to mice at 3 days after I/R and found that FAP CAR-Ms significantly improved cardiac function and reduced myocardial fibrosis in mice after I/R. No toxicities associated with FAP CAR-Ms were detected in the heart or other organs at 2 weeks after I/R. Finally, we found that FAP CAR-Ms conferred long-term cardioprotection against I/R. Our proof-of-concept study demonstrates the therapeutic potential of FAP CAR-Ms in alleviating myocardial I/R and potentially opens new avenues for the treatment of a range of heart diseases that include a fibrotic phenotype.

Anti-fibrotic effects of the sirt6-activator mdl-800 in cardiac stromal cells

Abstract Background Epigenetic changes are among the molecular substrates of cellular stress responses and tissue remodeling in heart failure progression. The sirtuin family of NAD+-dependent histone and non-histone protein deacetylases exert a protective anti-fibrotic role by negatively modulating the inflammatory response and by positive regulation of autophagy-related genes. Sirtuin6 (SIRT6) stands out as a key cardiac protector that can mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. Cardiac stromal cells (CSCs) play a pivotal role in fibrosis and remodeling, regulating the composition and stiffness of the extracellular matrix (ECM). Autophagy enhancement has anti-fibrotic effects in CSCs, but the specific role of SIRT6 modulation in CSCs remains unexplored. Purpose To investigate the biological effects of MDL-800, a synthetic allosteric SIRT6 activator, on the stress response and fibrotic activation of CSCs. Methods CSCs were isolated from 4-week-old C57Bl6J mice by explant culture and stimulated with 10ng/ml of TGF-beta1 (TGFb1) up to 72h to induce fibrotic activation. CSCs were treated with MDL-800 up to 10µM and up to 72h, analyzed for cell viability, gene and protein expression levels. Results TGFb1 treatment led to a 0.64-fold reduction in SIRT6 gene expression after 24h. The MTS assay was used to assess a viability dose-response to MDL-800 up to 72h of treatment. The non-toxic concentrations of 2.5µM and 5µM were selected for further experiments (2.5µM vs CTR 0.97-fold and 5µM vs CTR 0.92-fold, normalized OD vs t0). Co-treatment of 5µM MDL-800 with TGFb1 for 72h resulted in a dose-dependent decrease in the expression of TGFb1-induced fibroblast activation markers, as confirmed by immunofluorescence staining for aSMA (TGFb1 vs CTR 2.21-fold, p&amp;lt;0.05; 5µM vs TGFb1 0.46-fold, p&amp;lt;0.05, N=4; mean fluorescence intensity). The anti-fibrotic effect mediated by MDL-800 was further validated by real-time PCR indicating a reduction in the mRNA expression of multiple fibrotic markers, including ACTA2 (TGFb1 vs CTR 1.67-fold, 5µM vs TGFB 0.29-fold; N=2), COL1a1 (TGFb1 vs CTR 1.32-fold, 5µM vs TGFb1 0.87-fold; N=2) COL3a1 (TGFb1 vs CTR 1.06-fold, 5µM vs TGFb1 0.43-fold; N=2) and POSTN (TGFb1 vs CTR 6.37-fold, 5µM vs TGFb1 0.23-fold; N=2). Treatment with 5µM MDL-800 demonstrated an increase in SIRT6 gene expression, suggesting a transcriptional modulation (TGFb1 vs CTR 0.95-fold, 5µM vs TGFb1 1.68-fold; N=2). Western Blot analyses showed an increase in protein levels of the autophagosome marker LC3-II following treatment with 5µM MDL-800 (TGFb1 vs CTR 0.95-fold; 5µM vs TGFb1 3.01-fold), suggesting that autophagy enhancement may be a potential mechanism for the observed anti-fibrotic effects. Conclusion MDL-800 treatment in CSCs is associated with a dose-dependent reduction of TGFb1-induced fibrotic markers and to increased levels of the autophagy marker, suggesting its potential use as an anti-fibrotic molecule for the heart.

Enhancing wound healing through innovative technologies: microneedle patches and iontophoresis

IntroductionWound healing is a complex process involving multiple stages, including inflammation, proliferation, and remodeling. Effective wound management strategies are essential for accelerating healing and improving outcomes. The CELLADEEP patch, incorporating iontophoresis therapy and microneedle technology, was evaluated for its potential to enhance the wound healing process.MethodsThis study utilized a full-thickness skin defect model in Sprague-Dawley rats, researchers compared wound healing outcomes between rats treated with the CELLADEEP Patch and those left untreated. Various histological staining techniques were employed to examine and assess the wound healing process, such as H&amp;amp;E, MT and immunofluorescence staining. Furthermore, the anti-inflammatory and proliferative capabilities were further investigated using biochemical assays.ResultsMacroscopic and microscopic analyses revealed that the CELLADEEP patch significantly accelerated wound closure, reduced wound width, and increased epidermal thickness and collagen deposition compared to an untreated group. The CELLADEEP patch decreased nitric oxide and reactive oxygen species levels, as well as pro-inflammatory cytokines IL-6 and TNF-α, indicating effective modulation of the inflammatory response. Immunofluorescence staining showed reduced markers of macrophage activity (CD68, F4/80, MCP-1) in the patch group, suggesting a controlled inflammation process. Increased levels of vimentin, α-SMA, VEGF, collagen I, and TGF-β1 were observed, indicating enhanced fibroblast activity, angiogenesis, and extracellular matrix production.DiscussionThe CELLADEEP patch demonstrated potential in promoting effective wound healing by accelerating wound closure, modulating the inflammatory response, and enhancing tissue proliferation and remodeling. The CELLADEEP patch offers a promising non-invasive treatment option for improving wound healing outcomes.

Evaluation of Targeted Alpha Therapy Using [211At]FAPI1 in Triple-Negative Breast Cancer Xenograft Models

Triple-negative breast cancer (TNBC) presents limited therapeutic options and is associated with poor prognosis. Early detection and the development of novel therapeutic agents are therefore imperative. Fibroblast activation protein (FAP) is a membrane protein expressed on cancer-associated fibroblasts (CAFs) that plays an essential role in TNBC proliferation, migration, and invasion. Consequently, it is hypothesized that the Astatine (211At)-labeled FAP inhibitor (FAPI) selectively exerts anti-tumor effects through alpha-particle emission. In this study, we aimed to assess its theranostic capabilities by integrating [18F]FAPI-74 PET imaging with targeted alpha therapy using [211At]FAPI1 in TNBC models. Mice xenografts were established by transplanting MDA-MB-231 and HT1080 cells (control). As a parallel diagnostic method, [18F]FAPI-74 was administered for PET imaging to validate FAP expression. A single dose of [211At]FAPI1 (1.04 ± 0.10 MBq) was administered to evaluate the therapeutic efficacy. [18F]FAPI-74 exhibited high accumulation in MDA-MB-231 xenografts, and FAP expression was pathologically confirmed via immunostaining. The group that received [211At]FAPI1 (n = 11) demonstrated a significantly enhanced anti-tumor effect compared with the control group (n = 7) (p = 0.002). In conclusion, [18F]FAPI-74 PET imaging was successfully used to diagnose FAP expression, and as [211At]FAPI1 showed promising therapeutic efficacy in TNBC models, it is expected to be a viable therapeutic option.

Same-Day Positron Emission Tomography/Computed Tomography with 68Ga-Radiolabeled Fibroblast Activation Protein Inhibitors and 18F-Fluorodeoxyglucose Imaging for Gastrointestinal Cancers.

Objective: We investigated the clinical practicability of same-day 68Ga-radiolabeled fibroblast activation protein inhibitors (68Ga-FAPI)-first and 18F-fluorodeoxyglucose (18F-FDG) imaging and compared it with same-day 18F-FDG-first or 2-day procedures in diagnosing gastrointestinal cancers. Materials and Methods: Sixty-five patients with confirmed gastrointestinal cancers were divided into same-day 68Ga-FAPI-first group (Group A), same-day 18F-FDG-first group (Group B), and 2-day group (Group C). Low-dose CT and low injection activity were performed on 68Ga-FAPI positron emission tomography/computed tomography (PET/CT). Interval times, radiation dose, diagnostic performance, and detectability were assessed among groups. Additionally, the uptake, tumor-to-liver ratio (TLR), diagnostic efficacy, and TNM stage were compared between the two modalities. Results: The total waiting time for Group C was significantly longer than that for Group A or B (both p < 0.001). The total dose-length product and effective dose decreased in all groups. There were comparable detectability and diagnostic performance among groups (all p > 0.05). The within-group analysis in Group B indicated that 68Ga-FAPI PET/CT had higher uptake in the primary and recurrent lesions than 18F-FDG without differences in TLR, due to higher liver background on 68Ga-FAPI PET than Group A or C (both p < 0.001).68Ga-FAPI PET/CT possessed higher accuracy than 18F-FDG and changed staging in 14 patients (14/65, 21.54%). Conclusions: The same-day 68Ga-FAPI-first and 18F-FDG imaging reduced examination waiting time without increased radiation dose, simultaneously achieving excellent diagnostic performance and improving clinical staging in diagnosing gastrointestinal cancers.

Ca2+ Signaling in Cardiovascular Fibroblasts

Fibrogenesis is a physiological process required for wound healing and tissue repair. It is induced by activation of quiescent fibroblasts, which first proliferate and then change their phenotype into migratory, contractile myofibroblasts. Myofibroblasts secrete extracellular matrix proteins, such as collagen, to form a scar. Once the healing process is terminated, most myofibroblasts undergo apoptosis. However, in some tissues, such as the heart, myofibroblasts remain active and sensitive to neurohumoral factors and inflammatory mediators, which lead eventually to excessive organ fibrosis. Many cellular processes involved in fibroblast activation, including cell proliferation, protein secretion and cell contraction, are highly regulated by intracellular Ca2+ signals. This review summarizes current research on Ca2+ signaling pathways underlying fibroblast activation. We present receptor- and ion channel-mediated Ca2+ signaling pathways, discuss how localized Ca2+ signals of the cell nucleus may be involved in fibroblast activation and present Ca2+-sensitive transcription pathways relevant for fibroblast biology. When investigated, we highlight how the function of Ca2+-handling proteins changes during cardiac and pulmonary fibrosis. Many aspects of Ca2+ signaling remain unexplored in different types of cardiovascular fibroblasts in relation to pathologies, and a better understanding of Ca2+ signaling in fibroblasts will help to design targeted therapies against fibrosis.

Targeting cardiac fibrosis with Chimeric Antigen Receptor-Engineered Cells.

Cardiac fibrosis poses a significant challenge in cardiovascular diseases due to its intricate pathogenesis, and there is currently no standardized and effective treatment approach. The fibrotic process entails the involvement of various cell types and molecular mechanisms, such as fibroblast activation and proliferation, increased collagen synthesis, and extracellular matrix rearrangement. Traditional therapies often fall short in efficacy or carry substantial side effects. However, recent studies have shown that Chimeric Antigen Receptor T (CAR-T) cells can selectively target and eliminate activated cardiac fibroblasts (CFs) in mice, leading to reduced cardiac fibrosis and improved myocardial tissue compliance. This breakthrough presents a new and promising avenue for treating cardiac fibrosis. Currently, CAR-T cell-based therapy for cardiac fibrosis is undergoing animal experimentation, indicating ample scope for enhancement. Future investigations could explore the application of CAR cell therapy in cardiac fibrosis treatment, including the potential of CAR-natural killer (CAR-NK) cells and CAR macrophages (CAR-M), offering novel insights and strategies for combating cardiac fibrosis.

A Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

Targeting FAP-positive chondrocytes in osteoarthritis: a novel lipid nanoparticle siRNA approach to mitigate cartilage degeneration

BackgroundOsteoarthritis (OA) is a common joint disease that leads to chronic pain and functional limitations. Recent research has revealed soluble fibroblast activation protein (FAP) secreted from OA synovium could degrade type II collagen (Col2) in cartilage to promote the progression of OA. This study aimed to reveal the role of FAP from chondrocytes in OA and develop a novel lipid nanoparticle (LNP)-FAP siRNA delivery system for OA treatment.MethodsThe expression of FAP in the cartilage of knee OA patients was investigated using [68 Ga]Ga-FAPI-04 PET in vivo and immunofluorescence, western blotting, and RT-qPCR in vitro. Cell senescence was determined by senescence-associated β-galactosidase (SA-β-Gal) assay after FAP overexpressing or knockdown in chondrocytes. An OA model with chondrocyte-specific FAP knockout mice was applied to investigate the role of FAP in chondrocyte senescence and OA development. The therapeutic effects of lipid nanoparticle (LNP) @FAP siRNA on cartilage degeneration were evaluated in the rat OA model.ResultsOur study found that higher [68 Ga]Ga-FAPI-04 uptake was detected in knee OA patients by PET/CT scan. FAP mRNA and protein levels were highly expressed in OA-damaged cartilage. Moreover, we found that overexpression of FAP promotes chondrocyte senescence, and the genetic knockout of FAP in chondrocytes alleviates OA. Knockdown FAP by siRNA could alleviate chondrocyte senescence and suppress the NF-κB pathway to reduce the senescence-associated secretory phenotype (SASP). In the rat model of OA, intraarticular injection of LNP@FAP siRNA can reduce senescent cells and ameliorate cartilage destruction.ConclusionFAP-positive chondrocytes play a significant role in the pathogenesis of OA. Targeting these cells selectively has the potential to mitigate the progression of the disease. Our study provides valuable insights into the intraarticular injection of LNP@FAP siRNA as a promising strategy for the treatment of OA.Graphical abstract

Acute contact with profibrotic macrophages mechanically activates fibroblasts via αvβ3 integrin-mediated engagement of Piezo1.

Fibrosis-excessive scarring after injury-causes >40% of disease-related deaths worldwide. In this misguided repair process, activated fibroblasts drive the destruction of organ architecture by accumulating and contracting extracellular matrix. The resulting stiff scar tissue, in turn, enhances fibroblast contraction-bearing the question of how this positive feedback loop begins. We show that direct contact with profibrotic but not proinflammatory macrophages triggers acute fibroblast contractions. The contractile response depends on αvβ3 integrin expression on macrophages and Piezo1 expression on fibroblasts. The touch of macrophages elevates fibroblast cytosolic calcium within seconds, followed by translocation of the transcription cofactors nuclear factor of activated T cells 1 and Yes-associated protein, which drive fibroblast activation within hours. Intriguingly, macrophages induce mechanical stress in fibroblasts on soft matrix that alone suppresses their spontaneous activation. We propose that acute contact with suitable macrophages mechanically kick-starts fibroblast activation in an otherwise nonpermissive soft environment. The molecular components mediating macrophage-fibroblast mechanotransduction are potential targets for antifibrosis strategies.

Immunohistochemical Localization of Fibroblast Activation Protein in Coronary Arteries with Different Forms of Atherosclerosis

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblasts in areas of active tissue remodeling. It plays a potentially important role in cardiac remodeling, atherosclerotic plaque formation, and plaque rupture. Given the distinct pathophysiology and morphology of different forms of atherosclerosis, we analyzed FAP expression in human coronary vessels with no coronary artery disease, atherosclerotic plaques at different levels of progression, and other distinct forms of coronary disease in post bypass vein grafting and cardiac allograft vasculopathy after a heart transplant. Methods: Immunohistochemical staining with monoclonal F19 mouse anti-human FAP antibody was performed to identify FAP in human atherosclerotic plaques, coronary bypass atherosclerosis, and post-transplant arteriosclerosis. The presence and distribution of FAP in different types and stages of human atherosclerosis were compared. Results: There was no FAP staining in patients with no significant coronary disease. All different types of human atherosclerotic lesioning lesions showed the presence of FAP expression, with different staining patterns in advanced atherosclerotic plaque, vein graft atherosclerosis lesions, and arteriosclerosis after a heart transplant. Conclusions: These data suggest that FAP may be a potential diagnostic marker and target for interventions, not only in coronary atherosclerotic plaque, but also in other forms of coronary disease, which have distinct pathophysiologies and currently limited treatment options.