Immunofluorescence Research Articles

Introduction: Nearly 1 in 10,000 infants are born with a single ventricular heart, and these children must undergo the Fontan procedure to survive. As of 2020, an estimated 50,000 individuals are alive with a Fontan circulation. Although lifesaving, Fontan procedure is associated with several complications, including Fontan-associated liver disease (FALD). While elevated central venous pressure (CVP) appears to promote FALD, the cellular and molecular mechanisms involved in this unique liver disease are not well understood. Methods: Liver biopsies from Fontan patients were collected at Washington University in St. Louis before heart transplant. Liver tissue was fixed in formalin and embedded in paraffin for H&E and Masson's trichrome staining. Immunohistochemistry, scRNA-seq, and immunofluorescence (IF) were performed. In our preclinical model, mice underwent pIVCL to mimic hepatic congestion and underwent IF, RNA analysis, and snRNA-seq. Results: We recruited 35 patients with a mean age of 18.1 years (SD ±6.6) at the time of liver biopsy. The mean age at the time of the Fontan procedure was 2.4 years (SD ±0.9), with the average duration of Fontan circulation being 13 years. Regarding ventricular morphology, 25 patients (71%) had a single right ventricle, and 10 (29%) had a single left ventricle. The extracardiac conduit was the most common Fontan connection in 23 patients (72%). CVP was elevated in the superior vena cava (SVC) and Fontan conduit, with a mean of 18 mmHg (SD ±3.6). Liver function tests showed a median AST of 30.5 U/L (IQR 29.8), median ALT of 26.5 U/L (IQR 26.9), and median total bilirubin of 1.0 mg/dL (IQR 0.9). The mean APRI score was 0.58 (SD ±1.9), and MELD-XI was 10.2 (SD ±2.9). Fontan liver biopsies and liver sections from pIVCL mice showed sinusoidal dilatation, central hepatocyte dropout, and bridging fibrosis. Multiple areas of CK7-positive biliary metaplastic cells, which originated from HNF4α-positive hepatic progenitor cells, were identified. These areas colocalized with areas of CD68-positive aggregated macrophages and activated hepatic stellate cells (αSMA+). Using snRNA-seq in pIVCL and scRNA-seq in Fontan, we identified the cellular landscape in the cardiogenic liver disease. Conclusions: The interplay between sinusoidal congestion, macrophage activation, and biliary metaplasia in FALD implies a multifactorial pathogenesis that extends beyond passive venous congestion in cardiogenic liver diseases.

Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by progressive vascular remodeling, leading to increased pulmonary arterial pressure and right ventricular failure. Our previous studies identified regulator of G-protein signaling 5 ( Rgs5 ) as a key negative regulator of G protein-coupled receptor ( GPCR ) signaling, uniquely expressed in pulmonary pericytes ( PCs ). Rgs5 has been implicated in the development of pulmonary hypertension ( PH ), and its deficiency has been linked to cardiac fibrosis. However, the specific role of Rgs5 in PC function is underinvestigated. Therefore, we hypothesize that Rgs5 deficiency promotes PC differentiation into contractile protein-enriched cells by activating the GPCR pathway, contributing to vascular remodeling during PH development. Methods: To directly examine the role of Rgs5 in PCs during PH development, novel Rgs5 PC-KO ( Higd1b -CreERT2::Rosa26- Rgs5 fl/fl ) transgenic mice were generated and subjected to hypoxia (FiO 2 :10%) for 3 weeks. PH and right ventricle hypertrophy were evaluated by measuring right ventricle systolic pressure (RVSP) and Fulton index (FI). Vascular remodeling in Rgs5 PC-KO and WT were evaluated by immunofluorescence (IF) staining in precision cut lung slices using a high resolution confocal microscope. RGS5 function in human healthy PCs was evaluated by overexpressing RGS5 using the pcDNA3-RGS5 plasmid. Results: Overexpression of RGS5 in healthy PCs resulted in the downgulation of smooth muscle cell-specific contractile protein expression such as SMA, Vimentin, and SM22, indicating RGS5 negatively regulates contractile protein expression in PCs. Additionally, hypoxic Rgs5 PC-KO mice developed severe PH with RVSP reaching up to 38.4 mmHg ( Fig 1B ) and RVH (FI: 34.1%, Fig 1C ). IF analyses revealed that PCs integrated into muscularized vessels with coexpression of SMA, and exhibited abnormal stromal cell outgrowth along distal pulmonary arterioles in Rgs5 PC-KO under normoxic and hypoxic conditions ( Fig 1D ). Conclusions: Loss of Rgs5 in PCs is a key contributor to PC differentiation into contractile protein-enriched cells, abnormal vascular remodeling, and the PH development ( Fig 1A ). These findings provide novel insights into the role of RGS5 in PCs and could serve as a new treatment strategy for PAH.

Atherosclerosis is a primary contributor to cardiovascular mortality. The pathological proliferation, migration to the intima, and phenotypic switching of Smooth Muscle Cells (SMCs) leads to vascular remodeling and plaque development. MicroRNAs (miRs) have emerged as a promising target for modulating SMC activity while maintaining the proper function of Endothelial Cells (ECs). This study investigates whether modulating miR-331-3p can therapeutically target SMCs in atherosclerosis and how this affects ECs. MiR-331-3p levels in SMCs, ECs, and murine tissue were measured via qRT-PCR. Pre- and anti-miR transfection was used to assess its upregulation or inhibition on proliferation, migration, metabolism, and apoptosis. Morphological changes were analyzed by immunofluorescence microscopy. In silico predicted targets were validated at the mRNA level, and protein-level validation is ongoing. Initial investigations revealed a significant increase in miR-331-3p expression during disease progression (p<0.0001) in ApoE--/- mice, a commonly used atherosclerosis model. Further research uncovered that miR-331-3p is highly conserved across species, suggesting its relevance for subsequent in vitro experiments with human SMCs and ECs. Cytokines such as IFNγ, IL-1β, and TNFα were found to influence miR-331-3p expression in SMCs. Following pre- or anti-miRNA transfection, the cellular morphology of SMCs and ECs remained unchanged. Microscopically, the increase in SMC cell area observed 24 hours after miR-331-3p overexpression (p<0.0277) was linked to enhanced proliferation, as validated by BrdU assay results (p<0.05). In contrast, inhibition of miR-331-3p inhibited SMC migration (p<0.0178) and revealed divergent effects on SMC and EC function. Cell death assays showed that miR-331-3p causes anti-apoptotic effects in SMCs 48 hours after regulation. In silico analyses, utilizing literature research, target prediction tools, and public sequencing data, identified several potential targets, including KLF16, BAK1, PHLPP1, SOCS1, TNFα, TGFBR1, and DUSP5. Their upregulation in SMCs was confirmed 24 and 48 hours after miR-331-3p overexpression (p<0.05), while ECs showed remarkably few, if any, target changes. In conclusion, inhibiting miR-331-3p efficiently modulates vascular remodeling in SMCs without impairing EC function, highlighting its potential as a valuable target for cardiovascular treatment strategies potentially by majorly regulating the identified targets DUSP5 and PHLPP1.

Background: While statin therapy effectively lowers plasma LDL cholesterol (LDL-C), residual cardiovascular risk remains. Emerging evidence suggests that LDL-C may exert deleterious effects beyond its role in plaque development. Even moderately elevated or borderline LDL-C levels can act as nutrient signals that chronically activate maladaptive pathways in immune cells. In particular, LDL-C can stimulate mTORC1 signaling in monocytes and macrophages — key immune cells involved in atherosclerosis — leading to autophagy suppression, unresolved inflammation, and plaque progression. Methods: In a translational study, we compared monocyte mTORC1 signaling and autophagic markers in hypercholesterolemic patients (LDL-C >160 mg/dL, n=10) and healthy controls (LDL-C <100 mg/dL, n=10). Monocyte mTORC1 activity was assessed via its downstream target, phosphorylated S6 (pS6), and mTOR–LAMP2 colocalization (lysosomal mTOR recruitment). Autophagy was measured by LC3 intensity using immunofluorescence microscopy. Follow-up samples were collected after 12 weeks of statin therapy, administered as part of routine clinical care. Mechanistic insights were supported by in vitro LDL-C stimulation in human monocyte-derived macrophages (HMDMs) and in vivo studies in ApoE-/- mice. Results: Hypercholesterolemic patients exhibited a ~3.5-fold increase in monocyte pS6/Total S6 levels and ~1.5-fold higher mTOR–LAMP2 colocalization compared to controls, consistent with elevated mTORC1 activity. LC3 intensity was reduced by approximately 50%, indicating suppressed autophagy. In vitro, LDL-C triggered mTORC1 activation, reactive oxygen species (ROS) production, and apoptosis in HMDMs. In mice, dietary cholesterol increased monocyte mTORC1 signaling, which reversed after a switch to chow. Preliminary follow-up data suggests partial normalization of mTOR activity after statin treatment. Conclusions: These findings show that elevated LDL-C is linked to increased mTORC1 activity and autophagy suppression in monocytes. Using both pS6 and lysosomal mTOR localization provided complementary evidence of pathway activation. Targeting the cholesterol nutrient-sensing mTOR–autophagy axis may offer new strategies to reduce atherosclerotic risk beyond standard lipid-lowering. This approach could support precision cardiovascular medicine by tailoring treatment to individual cellular and metabolic profiles, rather than relying solely on population-level LDL thresholds.

Background: Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation, plays a key role in neuronal injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Glutathione peroxidase 4 (GPX4) is a critical inhibitor of ferroptosis, but mechanisms driving its degradation in post-CA brain injury are unclear. TRIM3, an E3 ubiquitin ligase implicated in neurodegeneration, may regulate GPX4 turnover. Objective: We investigated whether TRIM3 promotes ferroptosis via ubiquitination-dependent degradation of GPX4, contributing to hippocampal damage after CA-CPR, and whether TRIM3 suppression offers neuroprotection. Methods: A rat model of CA-CPR was used to assess hippocampal injury. Serum LDH and NSE levels quantified acute neuronal damage. Time-specific inhibition of necrosis, apoptosis, and ferroptosis helped define cell death dynamics. Immunoblotting, qPCR, immunofluorescence, and MDA assays evaluated ferroptotic markers. TRIM3–GPX4 interaction was confirmed via co-immunoprecipitation and mass spectrometry. AAV9-mediated TRIM3 knockdown assessed therapeutic potential. Neurological function was tested with NDS, Y-maze, Morris water maze, and open-field assays. PBMCs from rats and human CA-CPR patients were analyzed for TRIM3 expression. Results: Necrosis and apoptosis contributed to early injury (4 h post-CPR), while ferroptosis dominated at 24 h. GPX4 levels declined, with increased MDA in hippocampal CA1. TRIM3 expression rose in neurons and directly bound GPX4, promoting its K48-linked ubiquitination and degradation. TRIM3 knockdown preserved GPX4, suppressed ferroptosis, and improved cognitive outcomes. TRIM3 was also elevated in PBMCs from rats and patients post-CA. Conclusion: TRIM3 drives ferroptosis by mediating GPX4 degradation, worsening neuronal injury and cognitive decline after CA. Targeting TRIM3 may offer a novel therapeutic approach for post-resuscitation brain injury.

Introduction: The centrosome is a key regulator of cell division, structure, and function. While it acts as the microtubule-organizing center (MTOC) in proliferating cells, differentiated cells often repurpose it for tissue-specific roles. In cardiomyocytes (CMs), the loss of proliferative capacity after birth is well established, but how centrosome remodeling contributes to CM division and maturation is not well understood. Hypothesis: We hypothesize that centrosome structural dynamics—specifically, the disassembly and relocalization of pericentriolar material (PCM) components—regulate the balance between CM division and maturation. Aims: This study aims to define centrosome reorganization during CM development, assess PCM remodeling outcomes, and identify upstream regulators of centrosome disassembly. Approach: We utilized developing mouse hearts and human iPSC-derived CMs to track the centrosome throughout differentiation. CRISPR-Cas9 was used to deplete PCM1 and PCNT. Immunofluorescence, live imaging, and functional assays were used to assess centrosome positioning, sarcomere structure, microtubules, and mitochondria. Single-cell transcriptomics profiled gene expression during remodeling and maturation. Results: Upon CM differentiation, centrosomes disassemble in a stepwise manner, with PCM1 relocating to the nuclear envelope before PCNT. This relocalization shifts MTOC activity to the nuclear periphery. Depletion of key PCM components, PCM1 or PCNT, disrupts sarcomere organization, impairs contractility, and alters mitochondrial dynamics. Single-cell transcriptomic analysis revealed that PCM inactivation leads to impaired transcriptional maturation of CMs, reinforcing the importance of centrosome remodeling in coordinating structural and molecular aspects of CM development. These results suggest that PCM1 translocation is essential for establishing a nuclear envelope-centered microtubule scaffold that supports sarcomere organization and promotes cardiomyocyte maturation. Conclusion: Centrosome remodeling, via PCM translocation to the nuclear envelope, regulates the transition from CM division to maturation. PCM1 plays a central role in this process by establishing a perinuclear microtubule network that facilitates sarcomere assembly. Together, these findings highlight the importance of centrosome structural dynamics in suppressing mitotic apparatus formation and enabling cardiomyocyte maturation via a nuclear envelope-anchored cytoskeletal architecture.

Background: Atherosclerosis occurs preferentially in regions of disturbed or low fluid shear stress (FSS), whereas physiological laminar FSS protects against disease by suppressing endothelial inflammation. Pro- vs anti-inflammatory programs are associated with glycolysis vs mitochondrial metabolism, respectively. Endothelial cells (ECs) sensing FSS from blood flow regulates these responses, but the underlying mechanisms are poorly understood. The transcription factor Forkhead box protein O1 (FOXO1) is known to regulate endothelial metabolism, yet its role in FSS-regulated endothelial inflammation remains largely unclear. Methods: In vitro, ECs were subjected to defined flow patterns using a parallel plate flow chamber. Immunofluorescence, RNA sequencing, and biochemical assays were used to evaluate FOXO1 localization, gene expression, and post-translational modifications. In vivo experiments used FOXO1 floxed mice crossed with Bmx-CreERT2 for artery ECs-specific FOXO1 knockout. Hyperlipidemia was induced via injection of PCSK9 adeno-associated virus and high-cholesterol/high-fat diet (HCHFD) to assess atherosclerosis. Results: Oscillatory FSS and inflammatory cytokines induce, whereas physiological FSS inhibits FOXO1 nuclear translocation. RNA sequencing revealed that FOXO1 depletion in ECs upregulates the protective flow-responsive transcription factors KLF2/4 and reduces oscillatory FSS-induced inflammatory genes. Inhibition of FOXO1 nuclear translocation by physiological FSS is mediated via a KLF2-CDK2 pathway, with the latter phosphorylating FOXO1 at S249. Artery ECs-specific deletion of FOXO1 significantly reduces atherosclerotic plaques formation in hyperlipidemic mice. Inhibition of glycolysis attenuates OSS-induced FOXO1 nucleus translocation, suggesting metabolic regulation. Notably, treatment with lactate promotes FOXO1 nuclear localization and lactylation, which is mediated by a lactyltransferase AARS1 (Alanyl-tRNA synthetase). Conclusions: These findings identify FOXO1 as a key mediator linking atheroprone flow and endothelial inflammation via lactate-driven nuclear translocation and lactylation, promoting atherosclerosis. Conversely, physiological FSS suppresses FOXO1 via KLF2-CDK2 signaling. These complementary pathways suggest potential new therapeutic targets for treating atherosclerotic cardiovascular disease.

Obesity is associated with enteric dysfunctions, including gut dysmotility and neurodegeneration, which may involve Toll-like receptor 4 (TLR4) signaling. To investigate this relationship, we examined the impact of TLR4 deficiency on the enteric nervous system (ENS) of the small intestine in a mouse model of high-fat diet (HFD)-induced obesity. Male TLR4−/− and wild-type (WT) C57BL/6J mice were fed either a standard diet (SD; 18% kcal fat) or an HFD (60% kcal fat) for 8 weeks. ENS alterations were evaluated using real-time qPCR and confocal immunofluorescence microscopy on longitudinal muscle–myenteric plexus (LMMP) whole-mount preparations. Alterations in gut motility were evaluated by assessing stool frequency, transit of a fluorescent-labeled marker, and isometric motor responses of ileal preparations to receptor- and non-receptor-mediated stimuli. In WT mice, HFD induced delayed gastrointestinal transit, impaired cholinergic and nitrergic responses, and altered 5-HT-mediated concentration–response curves. These functional deficits were accompanied by neuroglial network disruption, myenteric neurodegeneration, loss of ChAT+ and nNOS+ neurons, and increased 5-HT ileal tissue levels. In contrast, TLR4 deficiency mitigated body weight gain and largely prevented HFD-induced structural and functional alterations. Overall, our findings highlight a key role for TLR4 signaling in modulating small intestine inflammation and ENS remodeling associated with obesity.

Traumatic brain injury (TBI) affects millions of people globally each year, yet effective treatments remain limited. A major challenge is the complexity of cellular and molecular responses to brain injury, many of which overlap with those seen in aging. A key hallmark of aging is nucleolar enlargement in brain and other tissues, reflecting increased ribosome biogenesis. Nucleolar size is regulated by the target of rapamycin (TOR) signaling pathway, which during aging is aberrantly activated. Inhibiting TOR reduces nucleolar size and extends lifespan in several model organisms. Using a Drosophila melanogaster model of closed-head TBI, we investigated whether injury influences nucleolar dynamics. Immunofluorescence microscopy of fibrillarin, a major nucleolar protein, revealed that brains of young, injured flies had substantially larger nucleoli than uninjured controls within one day of injury. Over the following weeks, the difference gradually diminished as nucleolar size increased in uninjured flies, eventually matching that of injured flies, which remained relatively stable. Additionally, heterogeneity in nucleolar size across cells became more pronounced with injury and aging. Finally, injury of older flies resulted in little or no nucleolar enlargement and even shrinkage within a few days of injury. These results suggest that TBI and aging converge on shared mechanisms that regulate nucleolar size, which may reach a maximal limit through either process. Consistent with this, mortality at 24 hours post-injury in young flies was significantly reduced by pharmacological inhibition of TOR with rapamycin or RapaLink-1, indicating that nucleolar enlargement contributes to TBI-induced damage. Overall, our results suggest that TBI accelerates the aging-associated increase in nucleolar size, implicating elevated ribosome biogenesis in TBI pathogenesis and highlighting TOR inhibition as a promising therapeutic approach.

Characterization of a novel B-cell epitope in the structural protein of goose astrovirus 1 and its application in serological detection.

Surface display of the protective antigen FlaB of Vibrio anguillarum in Lactobacillus plantarum PO23 and its immune response in Paralichthys olivaceus.

SGK1 inhibits oxidative injury and extracellular matrix degradation by activating the GSK-3β (Ser9)/Fyn/NRF2 pathway in pelvic organ prolapse.

Melatonin alleviates retinal damage and visual function impairment by suppressing ROS/TXNIP/NLRP3 signalling and inhibiting microglial activation in rats with optic nerve crush injury.

Preparation and characterization of monoclonal antibodies against porcine caspase-7.

Discovery of novel salidroside derivatives as potent hypoxia inducible factor 1α (HIF-1α) signaling inhibitors to treat high altitude cerebral edema.

Chitosan-tripolyphosphate/Eudragit® S100 nanoparticles containing quinic acid and ferulic acid ameliorate ulcerative colitis in rats via modulating Th17 cells and pro-inflammatory cytokines.

Intermittent heat stress facilitates the autophagy and apoptosis of the vascular endothelium in spontaneously hypertensive rats via the AMPK/mTOR/ULK1 pathway.

Use of R848, a TLR 7/8 agonist to separate X and Y spermatozoa in buffalo: effects on cleavage rates, blastocyst production, and the ratio of male to female embryos.

Huashi Baidu granules alleviate LPS-induced endothelial injury by modulating the AKT1-FOXO3a signaling pathway.

Dihydroartemisinin inhibits histone lactylation through YAP1 to act as a 'hot' switch for 'cold' tumor in hepatocellular carcinoma.