Prussian Blue Staining Research Articles

RAS-Selective Lethal 3-Induced Ferroptosis Promotes the Antitumor Efficiency of Anti-Programmed Cell Death Protein 1 Treatment in Colorectal Cancer.

Anti-programmed cell death protein 1 (PD-1) treatment has exhibited clinical benefits in colorectal cancer (CRC). However, the low response rate of CRC to immunotherapy is an urgent problem that needs to be solved. MC-38 tumor cells was challenged subcutaneously in the flank of 7-week-old male C57BL/6 mice. The mice were randomly divided into 3 groups, and 200µg/mouse anti-PD-1 antibody and 100 mg/kg RAS-Seletive Lethal 3 (RSL) or phosphate buffer saline (PBS) were intraperitoneally injected every 2 days. The expression of oxidative stress and ferroptosis-related genes was measured by Western blotting, real-time reverse transcription-polymerase chain reaction, Prussian blue staining, and enzyme-linked immunosorbent assay. Anti-PD-1 treatment-unresponsive tumors showed stronger immunosuppression than responsive tumors. Notably, the responsive tumors showed higher levels of H2O2 and reactive oxygen species, both of which could impair the antitumor effect of cytotoxic CD8+ T cells. The anti-PD-1 treatment-responsive tumors showed a higher expression of pro-ferroptosis genes and Fe2+ accumulation than those of anti-PD-1 nonresponsive tumors, indicating the potential role of ferroptosis in the efficacy of anti-PD-1 treatment. In MC-38 syngeneic tumor model, (1S, 3R)-RSL3 (RSL), a glutathione peroxidase 4 inhibitor, effectively promoted the antitumor effect of anti-PD-1 treatment in vivo. However, anti-PD-1 treatment did not affect the levels of ferroptosis-related genes in tumor model. Mechanistically, RSL treatment significantly upregulated the frequency of proliferating (ki67+) and cytotoxic (GZMB+) CD8+ T cells. Furthermore, the frequency of tumor neoantigen-specific interferon (IFN)-γ CD8+ T cells showed a significant increase after RSL plus anti-PD-1 treatment. RSL may be a promising drug for potentiating the antitumor efficiency of anti-PD-1 treatment in CRC.

Evaluation of core-shell Fe3O4@Au nanoparticles as radioenhancer in A549 cell lung cancer model

In radiotherapy, metallic nanoparticles are of high interest in the fight against cancer for their radiosensitizing effects. This study aimed to evaluate the ability of core-shell Fe3O4@Au nanoparticles to potentiate the irradiation effects on redox-, pro-inflammatory markers, and cell death of A549 human pulmonary cancer cells. The hybrid Fe3O4@Au nanoparticles were synthesized using green chemistry principles by the sonochemistry method. Their characterization by transmission electron microscopy demonstrated an average size of 8 nm and a homogeneous distribution of gold. The decreased hydrodynamic size of these hybrid nanoparticles compared to magnetite (Fe3O4) nanoparticles showed that gold coating significantly reduced the aggregation of Fe3O4 particles. The internalization and accumulation of the Fe3O4@Au nanoparticles within the cells were demonstrated by Prussian Blue staining. The reactive oxygen species (ROS) levels measured by the fluorescent probe DCFH-DA were up-regulated, as well as mRNA expression of SOD, catalase, GPx antioxidant enzymes, redox-dependent transcription factor Nrf2, and ROS-producing enzymes (Nox2 and Nox4), quantified by RT-qPCR. Furthermore, irradiation coupled with Fe3O4@Au nanoparticles increased the expression of canonical pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6, CXCL8, and CCL5) assessed by RT-qPCR and ELISA. Hybrid nanoparticles did not potentiate the increased DNA damage detected by immunofluorescence following the irradiation. Nevertheless, Fe3O4@Au caused cellular damage, leading to apoptosis through activation of caspase 3/7, secondary necrosis quantified by LDH release, and cell growth arrest evaluated by clonogenic-like assay. This study demonstrated the potential of Fe3O4@Au nanoparticles to potentiate the radiosensitivity of cancerous cells.

Protective effect of astaxanthin on ANCA-associated vasculitis

ObjectiveAnti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease characterized by inflammation and fibrinoid necrosis of medium and small vessels, and its pathogenesis is closely related to inflammation and oxidative stress. Astaxanthin (ATX) is a carotenoid with anti-inflammatory, antioxidant, and immunomodulatory effects. We hypothesized that ATX could play a role in AAV treatment. This study aimed to investigate whether ATX has a protective effect against AAV and to elucidate its regulatory mechanism. MethodsIn vitro experiments, neutrophils isolated from healthy people were treated with ATX or not and cultured with serum from myeloperoxidase (MPO) -ANCA-positive patients and healthy persons. The levels of IL-6 and TNF-α in neutrophil culture supernatant before and after stimulation were measured. Neutrophil extracellular traps (NETs) and intracellular reactive oxygen species (ROS) in neutrophils were detected after stimulation. In vivo study, experimental autoimmune vasculitis (EAV) rat models were established and then treated with ATX via intragastric administration for 6 consecutive weeks. Urinary erythrocytes, urinary proteins, and serum creatinine were detected and HE staining was performed to assess renal injury in rats. Lung hemorrhage was observed by gross dissection and microscopic Prussian blue staining. The level of serum MPO-ANCA was detected. Serum IL-6, TNF-α, superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in rats were measured to explore the effects of ATX on oxidative stress and inflammation in EAV rats. The deposition of MPO in kidney and lung of rats was detected by immunohistochemistry. ResultsATX significantly inhibited neutrophil secretion of inflammatory factors IL-6 and TNF-α. ATX reduced the elevated levels of ROS in neutrophils stimulated by serum from AAV patients and alleviated the release of NETs. ATX administration was observed to reduce the degree of hematuria, proteinuria, and glomerular crescent formation in EAV rats. The degree of pulmonary hemorrhage was significantly reduced. Besides, the serum levels of IL-6 and TNF-α were attenuated, and antioxidant SOD and GSH-px increased in serum. Pathological results showed that MPO deposition was decreased in lung and kidney tissues after ATX treatment. ConclusionATX could ameliorate the organ damages in EAV rats. It could serve as a hopeful therapy for AAV by its anti-inflammatory and anti-oxidative feature as a unique nature carotenoid.

Abstract 3209: Antibody mediated targeted drug delivery approach for pancreatic cancer

Abstract Pancreatic ductal adenocarcinomas, originating from the epithelial cell lining of ducts, account for approximately 95% of tumors in this category, showcasing a survival rate of less than 5-7%. Unfortunately, little progress has been seen in the outcomes of patients with PDAC as tumor develops high desmoplasia and chemo-resistance to chemotherapeutic drugs, such as gemcitabine (Gem). The therapies are unable to penetrate to the fibrotic tumors leading to insufficient availability of the therapeutic drugs at the tumor site. We and others have shown that MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas, which makes MUC13 as an excellent protein for specifically targeting pancreatic tumors. Herein, we demonstrate a unique ability of our in-house generated mouse and humanized monoclonal antibody of MUC13 to penetrate and target pancreatic cancer. These antibodies have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS) to deliver therapeutics specifically to pancreatic tumors. In this study, we are using curcumin that depletes tumor microenvironment and gemcitabine to investigate the efficacy of the MUC13 conjugated SPION in delivery of therapeutic drugs. Our results demonstrate that enhanced uptake of MUC13-SPION formulation in MUC13 positive (MUC13+) PanCa cells, compared with MUC13 null (MUC13-) cells as demonstrated by immunofluorescence, Prussian blue staining and flow cytometry experiments. Interestingly, the formulation resulted in sustained delivery of curcumin (CUR), enhanced inhibition of cell proliferation, migration and invasion in MUC13+ cells as compared with MUC13- cells, which suggests the targeting efficacy of the formulation. In PanCa orthotopic mice model, MUC13-SPION efficiently targeted pancreatic tumors resulting in significant tumor accumulation. We observed inhibition of tumor volume, metastasis, gem resistance and improved survival in mice treated with the formulation. Additionally, the tumor tissues from treated mice showed extensive downregulation of PCNA and expression of key proteins in SHH pathway, such as SHH, Gli-1, Gli-2, Patched 1, SMO, which has been associated with cancer progression and drug resistance. In conclusion, the results indicate high therapeutic significance of MUC13-SPIONS for achieving pancreatic tumor specific delivery of drugs. Citation Format: Nirnoy Dan, Saini Setua, Poornima Shaji, Sonam Kumari, Murali Mohan Yallapu, Sheema S. Khan, Subhash Chauhan. Antibody mediated targeted drug delivery approach for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3209.

Iron overload promotes hemochromatosis-associated osteoarthritis via the mTORC1-p70S6K/4E-BP1 pathway

BackgroundsJoint iron overload in hemochromatosis induces M1 polarization in synovial macrophages, releasing pro-inflammatory factors and leading to osteoarthritis development. However, the mechanism by which iron overload regulates M1 polarization remains unclear. This study aims to elucidate the mechanism by which synovial iron overload promotes macrophage M1 polarization. MethodsIn vitro, RAW264.7 macrophages were treated with iron and divided into five groups based on the concentration of the iron chelator, desferrioxamine (DFO): Ctrl, Fe, DFO1, DFO2, and DFO3. In vivo, rats were categorized into five groups based on iron overload and intra-articular DFO injection: A-Ctrl, A-Fe, A-DFO1, A-DFO2, and A-DFO3. Osteoarthritis was induced by transecting the left knee anterior cruciate ligament. Macrophage morphology was observed; Prussian Blue staining quantified iron deposition in macrophages, synovium, and liver; serum iron concentration was measured using the ferrozine method; cartilage damage was assessed using H&E and Safranin O-Fast Green staining; qPCR detected iNOS and Arg-1 expression; Western Blot analyzed the protein expression of iNOS, Arg-1, 4E-BP1, phosphorylated 4E-BP1, p70S6K, and phosphorylated p70S6K; ELISA measured TNF-α and IL-6 concentrations in supernatants; and immunohistochemistry examined the protein expression of F4/80, iNOS, Arg-1, 4E-BP1, phosphorylated 4E-BP1, p70S6K, and phosphorylated p70S6K in the synovium. ResultsIn vitro, iron-treated macrophages exhibited Prussian Blue staining indicative of iron overload and morphological changes towards M1 polarization. qPCR and Western Blot revealed increased expression of the M1 polarization markers iNOS and its protein. ELISA showed elevated TNF-α and IL-6 levels in supernatants. In vivo, ferrozine assay indicated significantly increased serum iron concentrations in all groups except A-Ctrl; Prussian Blue staining showed increased liver iron deposition in all groups except A-Ctrl. Iron deposition in rat synovium decreased in a DFO concentration-dependent manner; immunohistochemistry showed a corresponding decrease in iNOS and phosphorylated 4E-BP1 expression, and an increase in Arg-1 expression. ConclusionIntracellular iron overload may exacerbate joint cartilage damage by promoting synovial macrophage M1 polarization through phosphorylation of 4E-BP1 in the mTORC1-p70S6K/4E-BP1 pathway.

Polyphyllin B inhibited STAT3/NCOA4 pathway and restored gut microbiota to ameliorate lung tissue injury in cigarette smoke-induced mice

ObjectiveSmoking was a major risk factor for chronic obstructive pulmonary disease (COPD). This study plan to explore the mechanism of Polyphyllin B in lung injury induced by cigarette smoke (CSE) in COPD.MethodsNetwork pharmacology and molecular docking were applied to analyze the potential binding targets for Polyphyllin B and COPD. Commercial unfiltered CSE and LPS were used to construct BEAS-2B cell injury in vitro and COPD mouse models in vivo, respectively, which were treated with Polyphyllin B or fecal microbiota transplantation (FMT). CCK8, LDH and calcein-AM were used to detect the cell proliferation, LDH level and labile iron pool. Lung histopathology, Fe3+ deposition and mitochondrial morphology were observed by hematoxylin–eosin, Prussian blue staining and transmission electron microscope, respectively. ELISA was used to measure inflammation and oxidative stress levels in cells and lung tissues. Immunohistochemistry and immunofluorescence were applied to analyze the 4-HNE, LC3 and Ferritin expression. RT-qPCR was used to detect the expression of FcRn, pIgR, STAT3 and NCOA4. Western blot was used to detect the expression of Ferritin, p-STAT3/STAT3, NCOA4, GPX4, TLR2, TLR4 and P65 proteins. 16S rRNA gene sequencing was applied to detect the gut microbiota.ResultsPolyphyllin B had a good binding affinity with STAT3 protein, which as a target gene in COPD. Polyphyllin B inhibited CS-induced oxidative stress, inflammation, mitochondrial damage, and ferritinophagy in COPD mice. 16S rRNA sequencing and FMT confirmed that Akkermansia and Escherichia_Shigella might be the potential microbiota for Polyphyllin B and FMT to improve CSE and LPS-induced COPD, which were exhausted by the antibiotics in C + L and C + L + P mice. CSE and LPS induced the decrease of cell viability and the ferritin and LC3 expression, and the increase of NCOA4 and p-STAT3 expression in BEAS-2B cells, which were inhibited by Polyphyllin B. Polyphyllin B promoted ferritin and LC3II/I expression, and inhibited p-STAT3 and NCOA4 expression in CSE + LPS-induced BEAS-2B cells.ConclusionPolyphyllin B improved gut microbiota disorder and inhibited STAT3/NCOA4 pathway to ameliorate lung tissue injury in CSE and LPS-induced mice.

Tanshinone Ⅱ_A inhibits ferroptosis via Nrf2 signaling pathway to protect liver in rats of non-alcoholic fatty liver disease

This study investigated the protective effect of tanshinone Ⅱ_A(TSⅡ_A) on the liver in the rat model of non-alcoholic fatty liver disease(NAFLD) and the mechanism of TSⅡ_A in regulating ferroptosis via the nuclear factor E2-related factor 2(Nrf2) signaling pathway. The rat model of NAFLD was established with a high-fat diet for 12 weeks. The successfully modeled rats were assigned into model group, low-and high-dose TSⅡ_A groups, and inhibitor group, and normal control group was set. Enzyme-linked immunosorbent assay was employed to determine the content of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum of rats in each group. A biochemical analyzer was used to measure the content of aspartate aminotransferase(AST), alaninl aminotransferase(ALT), total cholesterol(TC), and triglycerides(TG). Hematoxylin-eosin(HE) staining was used to detect pathological damage in liver tissue. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL) was employed to examine the apoptosis of the liver tissue. Oil red O staining, MitoSOX staining, and Prussian blue staining were conducted to reveal lipid deposition, the content of reactive oxygen species(ROS), and iron deposition in liver tissue. Western blot was employed to determine the expression of Nrf2, heme oxygenase-1(HO-1), glutathione peroxidase 4(GPX4), ferroptosis suppressor protein 1(FSP1), B cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) in the liver tissue. The result showed that TSⅡ_A significantly reduced the content of MDA, AST, ALT, TC, and TG in the serum, increased the activity of SOD, decreased the apoptosis rate, lipid deposition, ROS, and iron deposition in the liver tissue, up-regulated the expression of Nrf2, HO-1, FSP1, GPX, and Bcl-2, and inhibited the expression of Bax in the liver tissue of NAFLD rats. However, ML385 partially reversed the protective effect of TSⅡ_A on the liver tissue. In conclusion, TSⅡ_A could inhibit ferroptosis in the hepatocytes and decrease the ROS and lipid accumulation in the liver tissue of NAFLD rats by activating the Nrf2 signaling pathway.

Evaluation of bronchoalveolar lavage fluid from donkeys using four different cytological stains: A pilot study

Bronchoalveolar lavage fluid (BALF) cytology is used for the diagnosis of non-infectious lower airway inflammation in equids. Discrepancies have been reported in the differential cell count when different staining methods were used both in humans and horses. The objective of this study was to compare the results of BALF cytology in donkeys using four different staining methods: modified May-Grunwald Giemsa (mMGG), Diff-Quick (DQ), Toluidine blue (TB) and Perls Prussian blue (PPB). Nine healthy Amiata female donkeys were enrolled. The BAL procedure was performed as previously described and pairs of cytocentrifuged BALF slides were stained with each method. No differences between mMGG and DQ were found for macrophages, neutrophils, and eosinophils, while differences were found in mast cell count between DQ vs.TB, but not between mMGG vs. DQ or mMGG vs. TB. Finally, no differences were obtained in the differential count for hemosiderophages comparing mMGG, DQ and PPB. The mMGG appears to be an excellent stain for the identification of all possible cell types, including mast cells in the BALF of donkeys. DQ, if used alone, may lead to inappropriate identification of mast cells. These results are consistent with the literature on BALF staining methods in horses.

Albiflorin Attenuates Neuroinflammation and Improves Functional Recovery After Spinal Cord Injury Through Regulating LSD1-Mediated Microglial Activation and Ferroptosis.

Spinal cord injury (SCI) is a serious, prolonged, and irreversible injury with few therapeutic options. Albiflorin (AF) possesses powerful pharmacodynamic properties and exerts protective effects against neuroinflammation. However, no research has examined the neuroprotective effect of AF following SCI.Rats were received laminectomy to establish SCI animal model and treated with AF (20mg/kg and 40mg/kg). Behavioral experiments were conducted to assess the impacts of AF on motor function after SCI in rats. Hematoxylin-eosin (HE) staining, Nissl staining, and Prussian Blue staining were performed to observe histological changes, neuronal damage, and iron deposition, respectively. Transmission electron microscope was adopted to observe the ultrastructure of spinal cord tissues. Immunofluorescence assay was performed to examine neurons and microglia. ELISA assay was used to examine the production of cytokines. Western blot assay was used to detect the expression level of ferroptosis-related proteins. Microglia BV-2 cells were induced by LPS to mimic the neuroinflammatory condition. Cell viability was assessed by CCK-8 assay, and lipid peroxidase level was measured by C11 BODIPY 581/591 staining. Molecular docking technology was utilized to confirm the relationship between AF and LSD1.AF improved the motor functional recovery after SCI in rats. Meanwhile, AF attenuated neuron apoptosis and microglia activation, reduced the production of pro-inflammatory cytokines and iron accumulation, and inhibited spinal cord ferroptosis following SCI in rats. LSD1 was verified to be a target protein of AF, and AF could concentration-dependently downregulate LSD1 expression in injured spinal cordsin vivoand LPS-induced BV-2 cellsin vitro. In addition, AF not only inhibited ferroptosis through reducing lipid peroxidase and iron levels and regulating ferroptosis-related proteins, but also inhibited microglial activation and reduced pro-inflammatory cytokines production in LPS-induced BV-2 cells; however, these changes were partly counteracted by LSD1 overexpression.AF could reduce microglial activation and ferroptosis, attenuate neuroinflammation, and improve functional recovery following SCI by downregulating LSD1, providing novel therapeutic strategies for the treatment of SCI.

LGR5 as a Therapeutic Target of Antibody-Functionalized Biomimetic Magnetoliposomes for Colon Cancer Therapy.

The lack of specificity of conventional chemotherapy is one of the main difficulties to be solved in cancer therapy. Biomimetic magnetoliposomes are successful chemotherapy controlled-release systems, hyperthermia, and active targeting agents by functionalization of their surface with monoclonal antibodies. The membrane receptor Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) stands out as colorectal cancer (CRC) biomarker and appears to be related to treatment resistance and the development of metastasis. The aim of this study was to assess the effectiveness and safety of LGR5-targeted biomimetic magnetoliposomes loaded with oxaliplatin (OXA) or 5-fluorouracil (5-FU) in the selective treatment of CRC and their possible application in hyperthermia. Synthesis, characterization and determination of heating capacity of magnetoliposomes transporting OXA or 5-FU (with and without LGR5 functionalization) were conducted. In vitro antitumoral activity was assayed in multiple colorectal cell lines at different times of exposition. In addition to this, cell internalization was studied by Prussian Blue staining, flow cytometry and fluorescence microscopy. In vivo acute toxicity of magnetoliposomes was performed to evaluate iron-related toxicity. OXA and 5-FU loaded magnetoliposomes functionalized with LGR5 antibody showed higher cellular uptake than non-targeted nanoformulation with a reduction of the percentage of proliferation in colon cancer cell lines up to 3.2-fold of the IC50 value compared to that of free drug. The differences between non-targeted and targeted nanoformulations were more evident after short exposure times (4 and 8 hours). Interestingly, assays in the MC38 transduced cells with reduced LGR5 expression (MC38-L(-)), showed lower cell internalization of LGR5-targeted magnetoliposomes compared to non-transduced MC38 cell line. In addition, magnetoliposomes showed an in vitro favorable heating response under magnetic excitation and great iron-related biocompatibility data in vivo. Drug-loaded magnetoliposomes functionalized with anti-LGR5 antibodies could be a promising CRC treatment strategy for LGR5+ targeted chemotherapy, magnetic hyperthermia, and both in combination.

Electroacupuncture Inhibits Neural Ferroptosis in Rat Model of Traumatic Brain Injury via Activating System Xc-/GSH/GPX4 Axis.

Ferroptosis is an iron-dependent regulating programmed cell death discovered recently that has been receiving much attention in traumatic brain injury (TBI). xCT, a major functional subunit of Cystine/glutamic acid reverse transporter (System Xc-), promotes cystine intake and glutathione biosynthesis, thereby protecting against oxidative stress and ferroptosis. The intention of this research was to verify the hypothesis that electroacupuncture (EA) exerted an anti-ferroptosis effect via an increase in the expression of xCT and activation of the System Xc-/GSH/GPX4 axis in cortical neurons of TBI rats. After the TBI rat model was prepared, animals received EA treatment at GV20, GV26, ST36 and PC6, for 15 min. The xCT inhibitor Sulfasalazine (SSZ) was administered 2h prior to model being prepared. The degree of neurological impairment was evaluated by means of TUNEL staining and the modified neurological severity score (mNSS). Specific indicators of ferroptosis (Ultrastructure of mitochondria, Iron and ROS) were detected by transmission electron microscopy (TEM), Prussian blue staining (Perls stain) and flow cytometry (FCM), respectively. GSH synthesis and metabolism-related factors in the content of the cerebral cortex were detected by an assay kit. Real-time quantitative PCR (RT-QPCR), Western blot (WB), and immunofluorescence (IF) were used for detecting the expression of System Xc-/GSH/GPX4 axisrelated proteins in injured cerebral cortex tissues. EA successfully relieved nerve damage within 7 days after TBI, significantly inhibited neuronal ferroptosis, upregulated the expression of xCT and System Xc-/GSH/GPX4 axis forward protein and promoted glutathione (GSH) synthesis and metabolism in the injured area of the cerebral cortex. However, aggravation of nerve damage and increased ferroptosis effect were found in TBI rats injected with xCT inhibitors. EA inhibits neuronal ferroptosis by up-regulated xCT expression and by activating System Xc-/GSH/GPX4 axis after TBI, confirming the relevant theories regarding the EA effect in treating TBI and providing theoretical support for clinical practice.

Abstract 116: Increased Collagen I/Collagen III Ratio is Associated With Hemorrhage in Brain Arteriovenous Malformations in Human and Mouse

Background and purpose: Brain arteriovenous malformation (bAVM) is an important risk factor for hemorrhagic stroke in young adults, but the underlying mechanisms are unclear. Increase of Col I/Col III ratio enhances vessel wall stiffness and renders vessel less resistant to changes of blood flow and pressure. Hypothesis: Col I/Col III ratio is increased in bAVM vessels, which is associated with bAVM hemorrhage. Method: Human bAVMs were collected from HHT and sporadic bAVM patients. Temporal lobe specimens from epilepsy patients were used as controls. Three mouse models were used: 1) PdgfbicreER; Alk1 f/f ; 2) PdgfbicreER; Eng f/f ; 3) Alk 1 f/f . Mice in models 1 and 2 have Pdgfb promoter driving, tamoxifen (TM) inducible cre expression in endothelial cells (ECs). BAVMs were induced by intra-brain injection of AAV-VEGF to induce angiogenesis and i.p. injection of TM to delete Alk1 or Eng in ECs. In model 3, bAVMs were by intra-brain injection of AAV-VEGF and an adenoviral vector expressing Cre to delete Alk1 gene focally. Colony stimulating factor receptor inhibitor (PLX5622) was given to Alk1 f/f mice 8 week after model induction when bAVMs have fully developed to transiently deplete microglia. Vascular Col I and Col III levels were analyzed by immunostaining using anti- CD31 and anti-Col I or Col II antibodies. Brain AVM bleeding was detected by Prussian blue staining, and RNA-Sequencing used to study gene profiles. Results: Col I and Col III levels and the Col I/Col III ratio were higher in the vessels of human HHT and sporadic bAVM than controls. Col I/Col III ratio was higher in ruptured than unruptured sporadic bAVM. RNA-seq data shown that Col I and Co III mRNA levels were higher in Eng deficient bAVMs than normal brain angiogenic regions. Col I and Col III levels were higher on Alk1 and Eng deficient bAVMs compared to normal vessels. Col I/Col III ratio was also increased in the Alk1 deficient bAVMs. Depletion of microglia by PLX5622 reduced Col I and Col III levels and reduced microhemorrhage of Alk1 deficient bAVMs. Conclusion: Col I, Col III and Col I/Col III ratio are increased on human and mouse bAVM vessels, which is associated with bAVM hemorrhage. Depletion of microglia reduces Col I and Col III and hemorrhage in bAVMs in mice.

Inhibition of p62-Keap1-Nrf2 Pathway Activation by Realgar Promotes the Inhibition of Esophageal Cancer Cell Proliferation, Migration, and Ferroptosis.

Realgar, a Chinese herbal decoction, has been used to treat various types of tumors with positive outcomes; however, there is a lack of convincing evidence on its use for the treatment of esophageal cancer (EC). In this study, the role of the p62-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the regulation of EC cell proliferation, migration, and ferroptosis in response to realgar was assessed. Different concentrations of realgar (0, 10, 20, 40, 60, 80, and 100 μmol/L) were applied to the EC cell lines Eca109 and KYSE150. The inhibition rate and half-inhibitory concentration (IC50) were determined using the Cell Counting Kit-8 (CCK-8) method. Subsequently, the cells were treated with realgar (1/2IC50, IC50, 2IC50). Cell migration was measured using the scratch assay, and cell invasion was measured using the transwell assay. The mRNA expression of p62, Keap1, and Nrf2 was measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expression of p62, Keap1, Nrf2, matrix metalloproteinase (MMP)-2, MMP-9, E-cadherin, Slug, N-cadherin, and vimentin was measured by Western blot. The control, 2IC50, shRNA-NC, shRNA-p62, 2IC50 + shRNA-NC, 2IC50 + shRNA-p62, shRNA-Keap1, 2IC50 + shRNA-Keap1, and 2IC50 + shRNA-p62 + shRNA-Keap1 groups were defined. The CCK-8 method was used to measure the cell inhibition rate, and the clone formation assay was used to measure the clone formation ability. Moreover, the scratch assay was used to detect the cell migration ability, and the transwell assay was used to detect the cell invasion ability. Transmission electron microscopy was used to observe the mitochondrial morphology, Prussian blue staining was used to observe the intracellular iron particle distribution, and flow cytometry was used to detect changes in intracellular reactive oxygen species. In addition, qRT-PCR was performed to detect p62, Keap1, Nrf2, and glutathione peroxidase 4 (GPX4) mRNA expression, and Western blot was performed to detect p62, Keap1, Nrf2, E-cadherin, Slug, N-cadherin, and GPX4 protein expression. Realgar inhibited Eca109 and KYSE150 cell proliferation in a time- and concentrationdependent manner. It also significantly inhibited the migration and invasion of Eca109 and KYSE150 cells and affected the mRNA and protein expression of p62, Keap1, and Nrf2. In response to realgar, low p62 expression inhibited the proliferation, migration, and invasion of Eca109 and KYSE150 cells, as well as ferroptosis induction. The findings demonstrate that inhibiting the p62-Keap1-Nrf2 signaling pathway promotes the inhibitory effects of realgar on EC cells.

Accumulation of Iron Oxide-Based Contrast Agents in Rabbit Atherosclerotic Plaques in Relation to Plaque Age and Vulnerability Features.

In this study, a detailed characterization of a rabbit model of atherosclerosis was performed to assess the optimal time frame for evaluating plaque vulnerability using superparamagnetic iron oxide nanoparticle (SPION)-enhanced magnetic resonance imaging (MRI). The progression of atherosclerosis induced by ballooning and a high-cholesterol diet was monitored using angiography, and the resulting plaques were characterized using immunohistochemistry and histology. Morphometric analyses were performed to evaluate plaque size and vulnerability features. The accumulation of SPIONs (novel dextran-coated SPIONDex and ferumoxytol) in atherosclerotic plaques was investigated by histology and MRI and correlated with plaque age and vulnerability. Toxicity of SPIONDex was evaluated in rats. Weak positive correlations were detected between plaque age and intima thickness, and total macrophage load. A strong negative correlation was observed between the minimum fibrous cap thickness and plaque age as well as the mean macrophage load. The accumulation of SPION in the atherosclerotic plaques was detected by MRI 24 h after administration and was subsequently confirmed by Prussian blue staining of histological specimens. Positive correlations between Prussian blue signal in atherosclerotic plaques, plaque age, and macrophage load were detected. Very little iron was observed in the histological sections of the heart and kidney, whereas strong staining of SPIONDex and ferumoxytol was detected in the spleen and liver. In contrast to ferumoxytol, SPIONDex administration in rabbits was well tolerated without inducing hypersensitivity. The maximum tolerated dose in rat model was higher than 100 mg Fe/kg. Older atherosclerotic plaques with vulnerable features in rabbits are a useful tool for investigating iron oxide-based contrast agents for MRI. Based on the experimental data, SPIONDex particles constitute a promising candidate for further clinical translation as a safe formulation that offers the possibility of repeated administration free from the risks associated with other types of magnetic contrast agents.

Abstract TMP5: Mutation of Pdgfrβ Enhances the Severity of Brain Arteriovenous Malformation on Endoglin Mutant Mice Through Exacerbation of Angiogenesis and Inflammation

Background and Purpose: Brain arteriovenous malformations (bAVMs) are tangles of abnormal vessels shunting blood directly from arteries to veins. Reduction of pericytes is correlated with bAVM hemorrhage. The PDGFB/PDGFRβ signaling plays important roles in regulating pericyte recruitment during angiogenesis. Hypothesis: Mutation of PDGFRβ causes cerebrovascular malformation and enhances bAVM severity in endoglin ( Eng ) deficient mice. Methods: Three mouse models were used: (1) Pdgfrβ F7 (F7) mice that have mutations disrupting most Pdgfrβ signaling, (2) Pdgfb icreER; Eng f/f mice that have Pdgfb promoter driving, tamoxifen (TM) inducible cre expression in endothelial cells (ECs) and a floxed Eng gene; and (3) PdgfbicreER; Eng f/f ;F7 +/- mice were used. Brain angiogenesis was induced by intra-parenchymal injection of AAV-VEGF. BAVMs were induced in PdgfbicreER; Eng f/f and PdgfbicreER; Eng f/f ;F7 +/- mice by inducting EC Eng deletion through TM treatment and brain angiogenesis through intra-brain injection of AAV-VEGF. BAVM phenotypes were analyzed 8-weeks after model induction by latex vascular cast to detect arteriovenous shunts, immunostaining to analyze vessel morphology, Prussian blue staining to quantify microhemorrhage and RNAseq to investigate gene expression profiles. Results: Dysplastic vessels with reduced pericyte coverage were detected in the brain and brain angiogenic region of F7 +/- and F7 +/+ mice. AVM like vessels were detected in the brain of 16% F7 +/- and 66% of F7 +/+ mice. F7 mutation increases bAVM penetrance in Eng deficient mice (71% vs 50%), dysplastic vessels and microhemorrhage. RNAseq data revealed that mutation of Pdgfrb increased the expression of pro-angiogenic and endothelial cell proliferation genes and genes promote cell migration and inflammation in the bAVMs of Eng mutant mice. Conclusion: Pdgfrβ mutation exacerbates the severity of bAVM in Eng mutant mice by enhancing angiogenesis and inflammation.

T1-weighted MRI of targeting atherosclerotic plaque based on CD40 expression on engulfed USPIO’s cell surface

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of cholesterol within the arterial wall. Its progression can be monitored via magnetic resonance imaging (MRI). Ultrasmall Superparamagnetic Particles of Iron Oxide (USPIO) (<5 nm) have been employed as T1 contrast agents for MRI applications. In this study, we synthesized USPIO with an average surface carboxylation of approximately 5.28 nm and a zeta potential of −47.8 mV. These particles were phagocytosed by mouse aortic endothelial cells (USPIO-MAECs) and endothelial progenitor cells (USPIO-EPCs), suggesting that they can be utilized as potential contrast agent and delivery vehicle for the early detection of atherosclerosis. However, the mechanism by which this contrast agent is delivered to the plaque remains undetermined. Our results demonstrated that with increasing USPIO concentration during 10–100 μg ml−1, consistent change appeared in signal enhancement on T1-weighted MRI. Similarly, T1-weighted MRI of MAECs and EPCs treated with these concentrations exhibited a regular change in signal enhancement. Prussian blue staining of USPIO revealed substantial absorption into MAECs and EPCs after treatment with 50 μg ml−1 USPIO for 24 h. The iron content in USPIO-EPCs was much higher (5 pg Fe/cell) than in USPIO-MAECs (0.8 pg Fe/cell). In order to substantiate our hypothesis that CD40 protein on the cell surface facilitates migration towards inflammatory cells, we utilized AuNPs-PEI (gold nanoparticles-polyethylenimine) carrying siRNACD40 to knockout CD40 expression in MAECs. It has been documented that gold nanoparticle-oligonucleotide complexes could be employed as intracellular gene regulation agents for the control of protein level in cells. Our results confirmed that macrophages are more likely to bind to MAECs treated with AuNPs-PEI-siRNANC (control) for 72 h than to MAECs treated with AuNPs-PEI-siRNACD40 (reduced CD40 expression), thus confirming CD40 targeting at the cellular level. When USPIO-MAECs and MAECs (control) were delivered to mice (high-fat-fed) via tail vein injection respectively, we observed a higher iron accumulation in plaques on blood vessels in high-fat-fed mice treated with USPIO-MAECs. We also demonstrated that USPIO-EPCs, when delivered to high-fat-fed mice via tail vein injection, could indeed label plaques by generating higher T1-weighted MRI signals 72 h post injection compared to controls (PBS, USPIO and EPCs alone). In conclusion, we synthesized a USPIO suitable for T1-weighted MRI. Our results have confirmed separately at the cellular and tissue and in vivo level, that USPIO-MAECs or USPIO-EPCs are more accessible to atherosclerotic plaques in a mouse model. Furthermore, the high expression of CD40 on the cell surface is a key factor for targeting and USPIO-EPCs may have potential therapeutic effects.

FGF12 restrains mitochondria-dependent ferroptosis in doxorubicin-induced cardiomyocytes through the activation of FGFR1/AMPK/NRF2 signaling.

Fibroblast growth factor-12 (FGF12) has been reported to play important role in regulating heart diseases. We aimed to explore the role of FGF12 in doxorubicin (DOX)-induced myocardial injury. DOX-induced mice and DOX-induced HL-1 cells were used as the myocardial injury in vivo and in vitro. Then, FGF12, Anp, Bnp, and Myh7 expression was detected. The pathological injury in myocardium tissue was observed by H&E staining. The levels of markers related to myocardial damage and oxidative stress were assessed. Then, immunohistochemistry and immunofluorescence staining were used to detect FGF12 and 4-HNE expression. Ferroptosis were detected by Prussian blue staining and western blot. The FGFR1/AMPK/NRF2 signaling was measured by western blot. FGF12 expression was downregulated in DOX-induced mice myocardium tissues. FGF12 overexpression alleviated DOX-induced myocardial tissue pathological injury and reduced Anp, Bnp, and Myh7 expression. Additionally, the levels of CK-MB, LDH and cTnT in serum were decreased after FGF12 upregulation in DOX-induced mice. Moreover, FGF12 overexpression reduced the levels of ROS, MDA, and 4-HNE but increased SOD and GSH-Px activities. Meanwhile, FGF12 led to less deposition of iron ion, decreased ACSL4, PTGS2 and increased GPX4, FTH1 expression. Additionally, FGF12 activated the expressions of FGFR1, p-AMPK, and NRF2. Moreover, FGFR1 silencing reversed the protective effects of FGF12 overexpression on cell viability, oxidative stress, ferroptosis, and FGFR1/AMPK/NRF2 pathway. To sum up, FGF12 inhibited mitochondria-dependent ferroptosis in cardiomyocytes induced by DOX through activation of FGFR1/AMPK/NRF2 signaling. These findings clarify a new mechanism of DOX-induced cardiac injury and provide a promising target to limit the disease development.

ADSCs labeled with SPIONs tracked in corpus cavernosum of rat and miniature pig by MR imaging and histological examination

Adipose tissue-derived stem cells (ADSCs) have been shown to improve erectile function in animal models of erectile dysfunction. However, few studies have been carried out using a reliable in vivo imaging method to trace transplanted cells in real time, which is necessary for systematic investigation of cell therapy. The study aims to explore the feasibility of non-invasively monitoring intracavernous injection of ADSCs in rat and miniature pig corpus cavernosum using in vivo magnetic resonance (MR) imaging. Thirty-six male Sprague Dawley rats (10 weeks old) and six healthy, sexually mature male miniature pigs (20 kg weight) were obtained. ADSCs were isolated from paratesticular fat of donor rats and cultured. Then ADSCs were labeled with superparamagnetic iron oxide nanoparticles (SPIONs), a type of MR imaging contrast agent, before transplantation into rats and pigs. After intracavernous injection, all rats and pigs underwent and were analyzed by MR imaging at the day of ADSC transplantation and follow-up at 1, 2 and 4 weeks after transplantation. In addition, penile histological examination was performed on all rats and pigs before (n = 6) and at 1 day (n = 6), 1 week (n = 6), 2 weeks (n = 6) or 4 weeks (n = 12) after ADSC transplantation. SPION-labeled ADSCs demonstrated a strong decreased signal intensity compared with distilled water, unlabeled ADSCs or agarose gel. SPION-labeled ADSCs showed a hypointense signal at all concentrations, and the greatest hypointense signal was observed at the concentration of 1 × 106. MR images of the corpus cavernosum showed a hypointense signal located at the injection site. T2*-weighted signal intensity increased over the course of 1 week after ADSCs transplantation, and demonstrated a similar MR signal with that before ADSCs transplantation. After SPION-labeled ADSC injection, T2*-weighted MR imaging clearly demonstrated a marked hypointense signal in pig corpus cavernosum. The T2*-weighted signal faded over time, similar to the MR imaging results in rats. Obvious acute inflammatory exudation was induced by intracavernous injection, and the T2*-weighted signal intensity of these exudation was higher than that of the injection site. The presence of iron was detected by Prussian blue staining, which demonstrated ADSC retention in rat corpus cavernosum. Lack of cellular infiltrations were demonstrated by H&E staining before and 4 weeks after transplantation, which indicated no negative immune response by rats. Prussian blue staining was positive for iron oxide nanoparticles at 2 weeks after transplantation. SPION-labeled ADSCs showed a clear hypointense signal on T2-weight MRI in vitro and in vivo. The MR signal intensity in the corpus cavernosum of the rats and miniature pigs faded and disappeared over time after ADSC transplantation. These findings suggested that MR imaging could trace transplanted ADSCs in the short term in the corpus cavernosum of animals.

Early detection of cardiac fibrosis in diabetic mice by targeting myocardiopathy and matrix metalloproteinase 2

Early detection of myocardial fibrosis in diabetic cardiomyopathy (DCM) has significant clinical implications for diabetes management. In this study, we identified matrix metalloproteinase 2 (MMP2) as a potential biomarker for early fibrosis detection. Based on this finding, we designed a dual-targeting nanoparticle CHP-SPIO-ab MMP2 to specifically target myocardiopathy and MMP2, enabling sensitive fibrosis detection using magnetic resonance imaging (MRI). Our results demonstrate that collagen hyperplasia (early fiber formation) begins to develop in diabetic mice at 12 weeks old, with observable fibrosis occurring at 16 weeks old. Additionally, MMP2 expression significantly up-regulates around collagen starting from 12 weeks of age. T2 MRI analysis revealed significant T2% enhancement in the hearts of 12-week-old diabetic mice following administration of the CHP-SPIO-ab MMP2 probe, indicating noninvasive detection of fiber formation. Furthermore, after fibrosis treatment, a reduction in T2% signal was observed in the hearts of 16-week-old diabetic mice. These findings were supported by Sirius red and Prussian blue staining techniques. Overall, our study presents a promising strategy for early identification of myocardial fibrosis. Statement of significanceMyocardial damage typically exhibits irreversibility, underscoring the paramount importance of early fibrosis diagnosis. However, the clinical used T1 mapping for fibrosis detection still exhibits limitations in terms of sensitivity. Therefore, it is imperative to develop highly sensitive strategies for early cardiac fibrosis detection. Here, we investigated the development of myocardial fibrosis in diabetic mice, and designed a highly sensitive probe that specifically targets cardiomyopathy and high expression of MMP2 for the early diagnosis of fibrosis. The probe enables non-invasive detection of abnormalities through MRI imaging as soon as fiber deposition appear, which can be detected earlier than T1 mapping. This advancement holds great potential for clinical diagnosis of myocardial fibrosis using cardiac magnetic resonance.

Non-targeted metabolomics strategy reveals the role of Geng-Nian-Shu in regulating ferroptosis in perimenopausal syndrome

Ovariectomy (OVX) is usually accompanied by the occurrence of metabolic syndrome. Previous studies have shown that Geng-Nian-Shu (GNS) plays an important regulatory role in perimenopausal syndrome (PMS) rats. GNS is a traditional Chinese medicine (TCM) prescription which composed of Suanzaoren Decoction and Ganmai Dazao Decoction in "Jingui Yaolue" and Siwu Decoction in "Heji Jufang". Recently, metabolomics analysis has been used to identify slight changes in the metabolic profile and to help understand disease progression and therapeutic interventions in PMS. However, the mechanism of GNS in the treatment of PMS is still unknown. We purposed to study the metabolic characteristics of PMS by serum and fecal metabolomics, and revealed the internal mechanism of GNS regulating ferroptosis against PMS. The PMS model was established by surgical removal of 4/5 ovaries of rats. HPLC-Q-TOF/MS was used to analyze the metabolomics of rat plasma and feces to explore the potential mechanism of GNS in PMS. The expression of ferroptosis-related proteins in rat ovaries was detected by tissue Prussian blue staining, Elisa kit and Western blotting. Cluster analysis of differential metabolites in plasma and feces between the control group and the model group showed that organic acids and their derivatives, lipids and lipid molecules were mainly disturbed during PMS in rats. After GNS administration, 17 differential metabolites were adjusted, involving several major pathways, such as the tricarboxylic acid (TCA) cycle, biosynthesis of amino acids and biosynthesis of unsaturated fatty acids. Further, we found that GNS affected ferroptosis in ovarian cells by regulating endogenous substances in OVX rats. Our study provides new insights into the mechanism of OVX-induced metabolic syndrome based on non-targeted metabolomics. It provides new ideas for the development and application of GNS and the diagnosis and treatment of PMS.