Immunofluorescence Staining Research Articles

Peiminine, an active alkaloid, has been reported to exhibit antitumor properties in several malignancies. This study aims to examine the role and potential mechanism of peiminine in prostate cancer (PCa). CCK-8, wound healing, colony formation, and Transwell assays were employed to evaluate PCa cell phenotypes. Cell cycle progression and apoptosis were examined by flow cytometry. Mitochondrial ROS, mitochondrial membrane potential, and ATP levels were measured to evaluate mitochondrial function in PCa cells. Western blotting was used to assess protein levels associated with apoptosis, EMT, and Wnt/β-catenin signaling. Immunofluorescence staining was performed to detect β-catenin expression. The in vivo effects of peiminine were evaluated using a xenograft mouse model. Peiminine dose-dependently impaired PCa cell viability without significantly affecting non-tumor cells. Peiminine inhibited PCa cell growth and motion and triggered apoptosis, cell cycle arrest, and mitochondrial dysfunction in vitro. Peiminine reduced PCa cell-derived tumor growth in the xenograft mouse model. Peiminine inhibited Wnt/β-catenin signal transduction. Peiminine exhibits an antitumor role in PCa by targeting Wnt/β-catenin signaling.

This study investigated the association between diabetic retinopathy (DR) and histologic features in patients with histologically confirmed diabetic kidney disease (DKD). Of 250 patients who underwent kidney biopsy, 108 patients with biopsy-confirmed DKD were included (DKD with DR, n = 71; DKD without DR, n = 37). DKD was classified into classes I-IV using the Renal Pathology Society Classification. Systemic inflammation was assessed using the red blood cell distribution width/albumin ratio (RAR) and C-reactive protein (CRP) levels. Patients in the DR (+) group were younger and had higher systolic blood pressure. They exhibited significantly lower estimated glomerular filtration rate (36.7 vs. 59.9 mL/min/1.73m², p = 0.001) and serum albumin (3.4 vs. 3.9g/dL, p = 0.001). While CRP levels did not differ between groups, RAR was significantly higher in the DR (+) group (3.935 vs. 3.407, p = 0.003). Histologically, kidney injury was more severe in the DR (+) group, with higher frequencies of class III (54.9% vs. 37.8%) and class IV (35.2% vs. 18.9%) disease. Linear deposition of immunoglobulin (Ig) G and light-chain positivity on immunofluorescence staining were also more common in the DR (+) group (IgG: 66.2% vs. 43.2%; light-chain : 53.5% vs. 29.7%). In biopsy-confirmed DKD, the presence of DR is associated with more severe renal pathology and increased systemic inflammation. These findings support the clinical relevance of DR screening in patients with DKD and highlight the need for prospective validation.

In interphase, 47S pre-rRNA is transcribed by RNA polymerase I (Pol I) and processed to form intermediate pre-rRNAs and finally produce mature rRNAs in the nucleolus. During mitosis, nucleolus disassembles and pre-rRNAs including 45S, 30S and 32S pre-rRNAs relocate in the peri-chromosomal region (PR). Inhibition of pre-rRNA transcription impairs chromosome dispersion in prometaphase. However, how pre-rRNAs regulate mitosis remains elusive. Here, we unravel a novel mechanism for pre-rRNAs to control mitosis. Inhibition of Pol I prolongs the mitotic process and induces defective chromosomal segregation, resulting in mitotic catastrophe. We isolated chromosome and determined the chromosome-binding proteins by mass-spectrometry. Using quantitative proteomics analysis, immunoprecipitation and immunofluorescent staining, we found that AURKA approaches chromosome when Pol I is inhibited. The AURKA-binding proteins on the chromosome were determined by immunoprecipitation and mass-spectrometry after cells were treated with Act D, BMH-21 or CX5461, respectively, and the chromosomal segregation controlling proteins were selected. When Pol I was inhibited, the binding of AURKA with SMC2, the crucial component of Condensin, is significantly enhanced. Importantly, SMC2 is phosphorylated by AURKA only when Pol I was inhibited. Alignment of SMC2 amino acid sequence with substrates of AURKA shows that SMC2 possesses the consensus R/K/N-R-X-S/T-B, and T574 is the only potential AURKA-catalyzed phosphorylation site. Indeed, SMC2 T574 is phosphorylated by AURKA in cell and in vitro. Thereafter, we generated SMC2 T574-P specific antibody, and confirmed that endogenous SMC2 T574 is phosphorylated by AURKA in mitosis in the absence of pre-rRNAs. Consequently, phosphorylation of SMC2 T574 disrupts the SMC2/SMC4 binding and the binding of SMC2 and SMC4 to chromosomal DNA, leading to chromosomal segregation defect. The phosphorylation deficient Flag-SMC2 T574A reverses the mitotic catastrophe caused by Pol I inhibition. Collectively, we demonstrate that pre-rRNAs protect SMC2 from the AURKA-mediated phosphorylation to maintain normal mitosis.

Exploring the anti-Helicobacter pylori activity and mechanism of Shouhui Tongbian through chemical composition analysis and network pharmacology.

CSF1R and IL1R1 inhibitors synergistically attenuate the early pathogenesis of traumatic brain injury in mice.

The LRIG gene family consists of LRIG1-3. While LRIG2 has been described as a tumor promoter, LRIG1 and LRIG3 have been identified as tumor suppressors in previous literature. Because of these contrasting roles, the expression of LRIG1-3 was examined across different grades of glioma, between primary and secondary glioblastoma and with focus on chemotherapy treatment. Human tumor tissue samples were extracted during neurosurgery and grouped among the WHO classification valid at the time of surgery. Quantitative western blot analysis, qPCR and immunofluorescence staining were performed. LRIG1 was less expressed in glioma compared to peritumoral tissue with additional decrease with ascending tumors grade. Further, secondary glioblastoma expressed more LRIG1 protein than primary. On mRNA level, the same was seen for LRIG2, were low grade glioma expressed significantly more LRIG2 than high grade glioma. And on protein level, secondary glioblastoma showed higher expression than primary. LRIG3 mRNA expression, in contrast, was significantly higher in grade II gliomas compared to surrounding control tissue, whereas chemotherapy did not significantly affect expression levels in glioblastoma. Our results reinforce suggestions that LRIG1-3 could function as diagnostic markers and therapeutic targets in the treatment of gliomas.

Is there a relationship between the mitochondrial activity and the meiotic progression of oocytes from germinal vesicle (GV) to metaphase II (MII) stages in young and advanced maternal age (AMA) women? Poor mitochondrial metabolism impairs the meiotic progression of human GV oocytes, contributing to a lower oocyte maturation capacity of AMA oocytes. AMA oocytes are characterized by diminished quality, mostly due to the higher rates of chromosomal segregation errors occurring during meiosis I. Another hallmark of AMA oocytes is impaired mitochondrial metabolism. Studies in mice have suggested a link between metabolic dysfunction and meiotic failure, but this relationship has not been fully elucidated in humans. Metabolic dynamics can be visualized by indirect measurements through mitochondrial staining and quantified more directly using fluorescence lifetime imaging microscopy (FLIM). This live-imaging approach can generate metabolic timelapse profiles of oocytes throughout meiosis. In the present study, we explored mitochondrial distribution and functionality in human oocytes at the GV and MII stages, obtained from young and AMA women, to establish the role of mitochondrial metabolism in meiosis progression. A total of 340 GV oocytes from young (≤34 years) and AMA (>37 years) women were included in the study. Denuded GVs were matured in vitro in G2-plus medium for 30 h. Maturation was determined by the presence of the extruded first polar body (PB1). The collected oocytes were processed for mitochondrial protein imaging (n = 80), or for live imaging (n = 171). Moreover, 89 oocytes were used for loss-of-function analysis by treating young GVs with 1 μM trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 min before in vitro maturation. The proteins dihydrolipoamide-S-acetyltransferase (D-LAT) and translocase-of-outer mitochondrial-membrane (TOMM20) were analyzed in young and AMA oocytes by immunofluorescence to assess mitochondrial activity and localization, respectively. Fluorescence mean intensities (arbitrary-unit, AU) were quantified with ImageJ and compared by t-test; maturation rates were compared by chi-squared test. FLIM comprehensive metabolism (NAD(P)H; FAD+) was taken at GV stage. Different FLIM parameters (fluorescence intensity, fraction bound, short/long lifetime) and the Redox ratio (NAD(P)H intensity/FAD+ intensity) were evaluated. The findings revealed that active mitochondria are specifically localized in the subcortical area, while mitochondria in general are distributed across the whole oocyte. This pattern was substantially maintained in AMA oocytes, which were in turn characterized by a lower mitochondrial activity (D-LAT intensity of 78614 ± 58534 AU in young, 12517 ± 10187 AU in AMA, P = 0.003), while a lower number of mitochondria was observed In AMA patients but the difference did not reach statistical significance (TOMM20 intensity of 61674 ± 24322 AU in young, 32186 ± 33414 AU in AMA, P = 0.195). Using non-invasive FLIM, we assessed the metabolic dynamics of maturing oocytes (Redox ratio in young 2e + 00 ± 0.15, in AMA 1e + 00 ± 0.16, P = 2.969e-05), confirming a similar pattern observed by immunofluorescence. Specifically, FLIM microscopy revealed that GV oocytes from young women slightly increased their metabolism, by 4% on average, after the GV breakdown, and the increase was very consistent across different oocytes. On the contrary, in AMA maturing oocytes, little to no increase in metabolism was observed; they were characterized instead by higher variability, and more AMA oocytes failed to successfully reach the MII stage [AMA oocytes (62.3%; 38/61) compared with young oocytes (86.3%; 63/73; P = 0.002). These differential trends observed in AMA oocytes compared to the young oocytes suggest that impaired metabolic activity significantly compromises maturation capacity, revealing a functional link between adequate metabolic levels and successful meiosis progression. Maturation rates were assessed by the presence of an extruded PB1 and variations in spindle assembly timings may have been overlooked. The quantification of mitochondrial activity in loss-of-function studies was assessed only by immunofluorescence staining. Additionally, the oocytes included in the present study were collected from women who underwent ovarian stimulation and may not faithfully recapitulate physiological maturation. Our findings demonstrate the presence of a functional link between oocyte mitochondrial metabolism and meiosis progression, which may contribute to the decline of oocyte quality with aging. Overall, we provided evidence to understand the biological mechanisms in mitochondrial metabolism that might contribute to driving the decay in oocyte quality in AMA women. This project received intramural funding from the Eugin Group and funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 860960. T.S. is a former owner and former stock owner of Optiva Fertility Inc (company closed) and filed two patents for Optiva Fertility Inc (both abandoned). D.S.: Presenter EMD Senoro and Dep. Editor of Human Reproduction. All of the other authors (S.P., M.M., M.B., E.I., M.P., R.V., and F.Z.) have no conflicts of interest to declare. All of the authors contributed substantially to the manuscript and approve its submission. N/A.

Siglec-10 is a cell-surface lectin that belongs to the sialic acid-binding immunoglobulin-like lectins (Siglecs) receptor involved in immune tolerance, but its role in reproduction remains unknown. Because immune regulation is essential for sperm survival and fertilization, Siglec-10 may represent an unexplored factor in camel fertility. Here, we investigated its expression and localization in the male reproductive tract and spermatozoa of dromedary camels during the rutting season. Testis, epididymis, and vas deferens were analyzed by immunohistochemistry and quantitative PCR, while spermatozoa from fresh ejaculates, frozen–thawed semen, and epididymal samples were examined using chromogenic and fluorescent immunostaining. Siglec-10 was strongly expressed in the testis and epididymis but absent in the vas deferens. In spermatozoa, the signal was localized mainly to the head and midpiece, with consistent patterns in fresh and frozen–thawed semen but absent in epididymal sperm. These findings provide the first descriptive evidence of Siglec-10 in camel reproductive tissues and ejaculated spermatozoa, suggesting a possible involvement in mechanisms that support sperm integrity under stress conditions; however, further studies are needed to clarify its functional role in semen preservation, and highlighting its relevance as a fertility biomarker in seasonal breeders.

Skeletal muscle development depends on the directed differentiation of myoblasts and their fusion into myotubes. Elucidating the mechanisms governing myoblast differentiation is essential for understanding muscle formation. Although suppressor of cytokine signaling 2 (SOCS2) has been implicated in this process, its precise regulatory role remains unclear. Here, the Cytosine Base Editor (CBE) system, offers a powerful approach for studying gene-specific functions, was used to investigate SOCS2 specific functions. sgRNAs targeting the murine SOCS2 gene were designed and expression plasmids were constructed. In C2C12 myoblasts, one sgRNA (sg1) mediated efficient base editing (53.0%), introducing a point mutation at amino acid 19 that generated a premature stop codon. Monoclonal cell lines with this mutation were established using limiting dilution. Western blot (WB) analysis confirmed a significant (P < 0.01) reduction in SOCS2 protein expression in the edited cells, accompanied by elevated levels of Growth Hormone Receptor (GHR). Immunofluorescence (IF) staining further validated increased GHR expression following SOCS2 knockdown. Differentiation assays indicated that SOCS2 knockout promoted C2C12 differentiation, with significantly (P < 0.01) upregulated expression of the myogenic markers MyoD1, MyoG and MYH1. Proteomic sequencing revealed enrichment of differentially expressed proteins in the PI3K/AKT and mTOR signaling pathways. Correspondingly, WB results showed that SOCS2 knockout significantly (P < 0.05) increased the expression of AKT, mTOR, and the phosphorylated forms of PI3K, AKT, and mTOR. Together, these findings demonstrate that CBE-mediated SOCS2 knockout enhances C2C12 differentiation and activates the PI3K/AKT/mTOR signaling pathway, thereby contributing new insights into the molecular regulation of skeletal muscle development.

Triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to confer immunosuppressive effects when expressed on tumor associated macrophages and thus has become a prominent focus of cancer research in recent years. The primary aims of this study were to explore the distribution of TREM2-expressing macrophages in early-stage ER+ breast cancer, specifically asking if there is a correlation with tumor aggressiveness and/or metabolic syndrome. To address these aims, we performed immunofluorescent staining for TREM2, CD68, and DAPI in 95 early-stage breast cancer samples from patients who underwent surgery at Vanderbilt University Medical Center. We assessed associations between TREM2+CD68+ cell density in three distinct regions of the tumor and multiple tumor characteristics, prognostic factors, and metabolic syndrome criteria. Although some analyses, including associations with menopausal status, hormone receptor expression, and histological subtype, reached statistical significance, the overall data revealed no significant associations between TREM2-expressing macrophages and tumor prognostic factors or metabolic syndrome criteria in early-stage ER+ breast cancer. Consequently, our results indicate that TREM2 likely does not serve as a reliable biomarker for ER+ breast cancer. However, TREM2 may still hold prognostic value in other subtypes such as triple negative breast cancer.

Cerebellar vermis and somatosensory-motor cortex differentially contribute to sex differences in acute pain perception in rats.

&#xD;Conductive materials play a crucial role in enhancing functional performance in muscle tissue engi-neering. This study investigates the impact of the conductive polymer polyaniline (PANi) in Polycapro-lactone-Collagen Type I (PCL-collagen I) nanofiber scaffolds designed to support the coculture of hu-man adipose-derived stem cells (ADSCs) and myoblasts. &#xD;Objectives:&#xD;The effect of varying PANi concentrations (0%, 2%, 4%, 6%) in PCL-collagen I nanofiber scaffolds was evaluated concerning the cell alignment, differentiation and gene expression of cocultured my-oblasts and ADSC.&#xD;Methods:&#xD;Nanofiber scaffolds with different PANi concentrations were analyzed. Acetatic acid was used as a non-toxic and biocompatible solvent for electrospinning the nanofibers. In vitro experiments involved a 1:1 coculture of myoblasts and ADSCs for up to 28 days on the scaffolds. The cell viability, differentia-tion and myotube morphology were assessed using live-dead-assay, CCK-8-assay, immunofluores-cence staining and gene expression analysis.&#xD;Results:&#xD;Scaffolds with 2% and 4% PANi showed a higher percentage of live cells compared to the control at both 7 and 28 days. The nanofibers with 2%, 4% and 6% PANi concentration showed promising re-sults in terms of cell differentiation and myotube morphology. After 14 days, the scaffolds with 4% PANi showed superior cell differentiation with strong myotube alignment along the nanofibers. At high-er PANi concentrations (6%), only the myotube width increased significantly, whereas 4% PANi re-sulted in a markedly higher myotube number.&#xD;Conclusion:&#xD;PCL-collagen I nanofibers incorporating PANi enhance myoblast alignment and differentiation com-pared to the control group, showing promise for muscle tissue engineering applications. The non-toxic solvent makes the nanofibers suitable for translational purposes. Further in vivo studies are needed to explore the full impact on cellular function and regeneration.&#xD;&#xD.

Emerging evidence suggests that TBI triggers ferroptosis, and dexmedetomidine (Dex) has a neuroprotective effect. This study aimed to explore the underlying mechanism of function of Dex in ferroptosisi after TBI. TBI model was established using the modified Feeney's weight drop injury method. Our experiment included the assessment of lesion volume by hematoxylin and eosin (HE) staining, the evaluation of the expression levels of ferroptosis-related proteins NRF2, HO-1, GPX4, FPN1, and TRFC by Western blotting (WB), the morphological changes via transmission electron microscopy (TEM), the increase in reactive oxygen species (ROS) through the measurement of malondialdehyde (MDA), the expression of HO-1 and GPX4 in the hippocampal tissues by immunofluorescence staining (IF), the behavioral assay by the Morris water maze (MWM) test and the open field test (OFT). Dex could alleviate the cognitive impairment in TBI mice and reduce ferroptosis after TBI. Dex could promote the nuclear translocation of NRF2 and enhance the expression of downstream HO-1, xCT, and GPX4, thereby inhibiting ferroptosis of neuronal cells. In addition, ML385 inhibited the expression of NRF2 and then reversed the neuroprotective effect of Dex. Dex alleviates ferroptosis and oxidative stress responses after TBI in mice through the NRF2/HO-1/GPX4 pathway, thus relieving the cognitive impairment in mice after TBI.

Anaesthetics temporarily inhibit neural activity by acting on voltage-gated sodium channels and GABA receptors. Although their neurological mechanisms are well-defined, their wider cellular effects, especially in non-neuronal systems, are inadequately understood. This study utilized Solanum lycopersicum plant's root apex cells as a transparent model to examine anaesthetic-induced subcellular alterations via live-cell fluorescence imaging, immunostaining, and super-resolution microscopy. These findings demonstrate the hierarchical cascade of organelle dysfunction, such as mitochondria, lysosomes, vesicle trafficking, and nuclear architectures under anaesthesia in plants. The nucleus is identified as the main controller of recovery potential and cellular fate. In a time dependent experiment, it is found that plant cells exposed to lidocaine for up to 4 h can still recover mitochondrial potential, lysosomal function, and nuclear integrity when anaesthesia is removed. However, beyond 4 h the damage, especially to the nucleus, is irreversible, and cells proceeded to cell death. The data further demonstrate that organelles can recover after brief exposure, but prolonged exposure stops recovery, resulting in the irreversible degradation of the nucleus leading to complete cell death. The results may help to uncover organelle-related dysfunction under anaesthetic toxicity and provide a clearer understanding for minimizing or reversing such damage.

Modeling cardiac disease has been a central goal in tissue engineering. Engineered human cardiac tissues now serve as valuable platforms for studying heart tissue replacement, disease modeling, and advancing therapeutic discovery. However, we are yet to recreate the complex and functional human myocardium in vitro , particularly with respect to integrating perfused vasculature and immune components. The lack of vasculature continues to significantly limit the ability to study conditions that involve disruptions in blood flow, such as coronary heart disease and ischemia-reperfusion injury. To this end, we engineered an in vitro model of human myocardium from a single iPSC line encompassing the different cellular landscape of the native myocardium to more faithfully recapitulate the functionality of adult human heart. The vascularized cardiac tissues were generated using WTC11-hiPSCs differentiated into cardiomyocytes (iCM), cardiac fibroblasts (iCF), endothelial cells (iEC) and resident macrophages (irMf) and assessed for the appropriate cell-identity markers via immunofluorescent staining. Tissues were generated by encapsulating cells in fibrin hydrogel and culturing the resulting cell-hydrogel constructs stretched between two elastic pillars. The tissues were cultured for 7 days and electrically stimulated for another 7 days. Immunofluorescent staining was performed to assess capillary formation. We demonstrate that hiPSC-derived endothelial cells have the capacity to form dense vascular networks that are aligned with hiPSC-cardiomyocytes in the direction of tissue contraction. After 7 days of culture, the tissues were highly vascularized with an interconnected network of capillaries demonstrating active angiogenic sprouting. We further observed a close interaction between iCF and iECs with fibroblasts adopting a perivascular-like morphology and wrapping around capillaries with open lumens. Finally, the incorporation of hiPSC-derived macrophages and application of electrical stimulation enabled long-term survival of vascular networks and increased the network complexity. This work paves the way for the development of autologous vascularized cardiac tissues, to support patient-specific studies of cardiovascular diseases.

Introduction: Myocardial infarction (MI) is a leading cause of death worldwide, and endothelial cells (ECs) are central to the repair process by coordinating angiogenesis, immune cell recruitment, and endothelial-to-mesenchymal transition (EndMT). ADAM17, a membrane-bound protease elevated in cardiovascular disease, regulates multiple cellular processes through ectodomain shedding of different molecules. However, the causal role of endothelial ADAM17 in post-MI recovery remains poorly defined. Methods: Male and female mice with inducible endothelial-specific ADAM17 knockdown ( Adam17 f/f / Cdhr5 Cre ERT ; Adam17 EC-KD ) and control cohorts ( Adam17 f/f , Cdhr5 Cre ERT , WT) underwent left anterior descending coronary artery ligation to induce experimental MI. Cardiac structure and function were evaluated (echocardiography), histological analyses (Trichrome staining), molecular analyses (immunofluorescent staining (IF), western blotting, and single-nucleus RNA sequencing (snRNA-seq)) at various post-MI time points. Results: Adam17 EC-KD mice exhibited reduced post-MI survival (increased LV rupture), increased left ventricular rupture, and progressive decline in cardiac function with reduced ejection fraction. Adam17 EC-KD mice exhibited increased neutrophil infiltration, NETosis, and cytotoxic T cell accumulation at 1 day post-MI; however, depletion of any of these immune cells further exacerbated the post-MI mortality (due to LV rupture), highlighting their potential protective contribution. Loss of endothelial ADAM17 resulted in decreased coronary density in the infarct myocardium (CD31 IF; 3-D micro-CT imaging), with reduced pVEGFR2 signaling, suggesting impaired angiogenesis. In addition, SnRNA-seq confirmed the suppressed pro-angiogenic pathways, and identified an endothelial cell subpopulation enriched for necroptotic markers, displaying increased ligand-receptor interactions with inflammatory macrophages. Mechanistically, endothelial ADAM17 deficiency enhanced necroptotic cell death via activation of the TNFR1-RIP3K-RIP1K-MLKL axis. Adam17 EC-KD hearts also showed impaired collagen crosslinking and reduced activation of the SMAD pathway (pSMAD2/3), lysyl oxidase, and Fibronectin expression, indicating defective EndMT, supported by reduced EndMT gene signatures in snRNA-seq. Conclusion: Endothelial ADAM17 is essential for effective post-MI cardiac repair by regulating endothelial survival, angiogenesis, immune cell infiltration, and infarct formation.

Introduction: Vascular calcification is a major contributor to cardiovascular morbidity and mortality, yet the molecular mechanisms of calcification remain poorly defined. Research Question: Here, we aimed to identify novel differentially expressed genes (DEGs) and altered signaling pathways associated with small and large arterial calcification, as modeled by calciphylaxis and coronary artery disease. Methods/Results: Transcriptomic profiling of human calciphylaxis lesions revealed 783 DEGs enriched in pathways related to mineralization, inflammation, and thrombosis, with fibronectin type III domain containing 1 ( FNDC1 ) among the most significantly upregulated (FDR-adjusted p-value ≤ 1.2 x10 -7 ) ( Fig. 1A ). FNDC1 protein expression was also significantly upregulated on immunofluorescent staining of dermal arterioles in calciphylaxis compared to chronic kidney disease-matched controls ( Fig. 1B, left panel ). Similarly, among the top upregulated DEGs in calciphylaxis that were validated at the protein level on immunofluorescent staining, FNDC1 was most significantly upregulated in atherosclerotic coronaries at the transcriptomic (FDR-adjusted p-value = 5.87 x 10 -5 ) and protein level (p = 0.0016) ( Fig. 1B, right panel ). In human vascular smooth muscle cells (VSMCs), FNDC1 promoted osteogenic phenotype switch, proliferation, and migration via PI3K/AKT signaling and upregulated nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD salvage pathway. In murine models, genetic deletion of Fndc1 ( Fig. 2A ) or pharmacologic NAMPT inhibition ( Fig. 2B ) attenuated arterial calcification and improved survival. Clinically, circulating FNDC1 levels were elevated in patients with calciphylaxis and coronary artery disease, and independently predicted coronary risk in over 43,000 UK Biobank participants. Conclusions: These findings position FNDC1 as a central regulator of vascular calcification and highlight the FNDC1–NAMPT axis as a previously unrecognized mechanistic link between vascular signaling and metabolic reprogramming and a tractable target for therapeutic intervention.

Background: Cardiac allograft vasculopathy (CAV) is the leading cause of morbidity and mortality one-year post heart transplant. It is characterized by diffuse neointimal thickening of the coronary arteries, leading to lumen narrowing and graft failure. CAV extending beyond the coronary arteries and to the donor aorta remains largely unexplored. The presence of CAV in aortic tissue would further inform the pathophysiology of the disease. Hypothesis: We hypothesize that CAV is a transplant-specific disease defined by the pathologic fate specification of donor vascular smooth muscle cells (SMC). As such, the disease expands beyond the coronary arteries to involve all donor arteries, specifically the aorta. Methods: The University of Colorado curates one of the world’s largest adult tissue cardiac biobanks with over 1700 explanted hearts cryopreserved and well-phenotyped. Human explanted hearts were collected at the time of transplant from recipients with CAV and non-ischemic cardiomyopathy (NICM). The human CAV cohort (n= 4, 2 males, 2 females) included patients with a diagnosis of CAV for an average of 6.5 years, and tissue was collected at the time of retransplant. In parallel, our group established a novel mouse heart technique to include a longer segment of aortic tissue. During donor harvest in our mouse model, an extended segment of the donor aorta was transplanted into the recipient mouse. Human and mouse aortic tissues were formalin-fixed, paraffin-embedded, and sectioned for histology and immunofluorescence. Results: The human CAV aortic tissue revealed the same morphology in coronary arteries, including a modulated SMC phenotype within the neointima. Similarly, the CAV mouse model's extended aorta and coronary arteries exhibited neointimal hyperplasia continuously in the vascular bed and stopping at the anastomosis site to the recipient. Based on histologic and immunofluorescence staining, the neointima of the mouse recapitulates the human neointima. NICM human aortas and the syngeneic mouse model aortas lacked CAV morphology, indicating unique CAV pathology throughout the donor vascular bed. Conclusion: We identify that CAV expands beyond the donor coronary arteries to include the donor aorta and verify a novel mouse heart transplant model that phenocopies human pathobiology. Ultimately, this model acts as a rich source of additional tissue to ask mechanistic questions involving the source of CAV and can inform novel therapeutic targets.

Introduction: Acute plaque rupture followed by luminal thrombosis is a major cause of myocardial infarction and sudden cardiac death. Vulnerable plaques are characterized by a thin fibrous cap and reduced numbers of smooth muscle cells (SMCs). SMCs are essential for plaque stability, as they synthesize collagen and support fibrous cap structure. Our previous work demonstrated that insulin-like growth factor-1 enhances plaque stability by upregulating La ribonucleoprotein domain family member 6 (Larp6), a collagen mRNA-binding protein in SMCs. However, the specific role of Larp6 in atherosclerotic plaque stability remains undefined. Hypothesis: We hypothesize that Larp6 enhances atherosclerotic plaque stability by increasing collagen synthesis and promoting SMC survival. Methods and Results: Using the Myh11 promotor we generated SMC specific Larp6 overexpression mice on an ApoE -/- background (SMC-Larp6) to assess effects on plaque stability. 8-wk-old SMC-Larp6 mice and littermate controls (n=20/group) were fed a Western diet for 12 wks. Tissues were harvested for assessment of atherosclerotic burden and plaque phenotype. Histological analysis showed plaques from SMC-Larp6 mice exhibited a significantly higher collagen content (22.9 ± 9.2%, P&lt;0.05), accompanied by a thicker fibrous cap (40.6 ± 15.8%, P&lt;0.05) and a smaller necrotic core (28.1 ± 13.2%, P&lt;0.05), without significant changes in overall plaque burden compared to controls. Immunofluorescence staining on aortic root sections revealed elevated α-smooth muscle actin expression within plaques (80.0 ± 26.9%, P&lt;0.01), suggesting increased SMC presence. Mechanistically, adenoviral LARP6 overexpression in human aortic SMCs significantly increased cell proliferation and survival in the presence of oxidant stress (100 μg/mL oxidized LDL). Additionally, LARP6 overexpression promoted collagen accumulation by enhancing collagen synthesis, as demonstrated by increased hydroxyproline production both in vitro and in vivo. Finally, RNA sequencing analysis of human atherosclerotic plaques (GSE120521) revealed reduced expression of LARP6 and downregulation of extracellular matrix-related pathways in unstable plaques, underscoring the clinical relevance of these findings. Conclusions: Larp6 overexpression promotes collagen synthesis and increases smooth muscle cell survival under atherosclerotic conditions. These findings suggest that Larp6 is a potential therapeutic target for stabilization of atherosclerotic plaques.

Introduction: Myxomatous mitral valve degeneration (MMVD) is a prevalent cause of mitral valve regurgitation in developed countries, yet effective medical therapies targeting this condition remain lacking. Although the inflammatory processes associated with mitral valve degeneration are well established, the specific mechanisms by which immune cells contribute to extracellular matrix (ECM) remodeling in MMVD remain poorly understood. Methods: Single-cell RNA sequencing performed on human myxomatous mitral valves was analyzed to map the immune cell landscape of MMVD. Both human and mouse specimens were analyzed to confirm macrophage-specific alterations and the expression of legumain (LGMN), a protease implicated in ECM remodeling. Additionally, in vivo studies using macrophage-specific LGMN-deficient mice were conducted to further investigate the functional role of LGMN in macrophage-mediated ECM changes. A macrophage and valvular interstitial cell co-culture system was employed to examine cellular interactions in vitro. Results: An association of MMVD with Marfan syndrome resulting from pathogenic FBN1 variants supports the use of fibrillin-1 deficient mice to investigate mechanism for MMVD. Fbn1 C1041G/+ mice developed severe myxomatous mitral valve disease. Using single-cell RNA sequencing, we mapped the immune cell landscape in myxomatous mitral valves (Figure 1A). Our findings revealed a significant increase in a subset of Lgmn+ macrophages in the myxomatous mitral valves (Figure 1B), which was confirmed by immunofluorescence staining in both mouse and human specimens (Figure 2). Analysis of cell-cell interactions revealed that macrophages exhibit strong interactions with valvular interstitial cells (Figure 3A). Additionally, LGMN amplified the TGF-β-mediated transition of fibroblasts into myofibroblasts (Figure 3B). Furthermore, macrophage-specific deficiency of legumain (Lgmn) resulted in reduced ECM remodeling and stabilization of mitral valve degeneration (Figure 3C). Conclusion: This study demonstrated that macrophage derived LGMN promote myxomatous mitral valve degeneration by enhancing the extracellular matrix remodelling via macrophage-valvular interstitial cell interaction and provided a novel target for the treatment of MMVD.