Vascular Cell Adhesion Protein Research Articles

The Immunohistochemical and Bioinformatics Analysis of the Placental Expressions of Vascular Cell Adhesion Protein 1 (VCAM-1) and High Mobility Group Box 1 (HMGB1) Proteins in Gestational Diabetic Mothers.

We aimed to examine both the expression levels of high mobility group box 1 (HMGB1) and vascular cell adhesion molecule-1 (VCAM-1) proteins in the placentas of pregnant women with gestational diabetes mellitus (GDM) and control groups by immunohistochemical (IHC) method. An experimental case-control study was conducted, including 40 pregnant women complicated with GDM and 40 healthy pregnant women. Placental tissues obtained following cesarean delivery were subjected to routine tissue monitoring. The placental sections were stained with VCAM-1 and HMGB1 immunostains and subjected to IHC examination under a light microscope. H-score (HS) was used to evaluate the results of IHC staining by semi-quantitative analysis. Pathway analysis in Cytoscape software identified GDM-associated proteins within HMGB1 and VCAM-1 interaction networks, followed by GO analysis to explore associated biological processes. Placental HGMB1 expression was significantly increased in the GDM group compared to the control group (p<0.001). However, placental VCAM-1 expression was found to be statistically similar in GDM and control groups (p=0.584). The shared 19 proteins were identified between HMGB1 and GDM, and 13 between VCAM-1 and GDM, with notable GO biological process terms such as immune system activation for HMGB1 and interleukin-6 regulation for VCAM-1 associated with GDM. We consider that GDM-related inflammation and oxidative stress may contribute to tissue damage and inflammation by increasing placental HMGB1 expression. The blockade of HMGB1 and its receptors might represent a promising therapeutic approach to control inflammation in GDM. Understanding the distinct roles of HMGB1 and VCAM-1 may provide valuable insights for the development of targeted therapies aimed at mitigating the inflammatory processes associated with GDM and improving maternal and fetal outcomes.

Epigenetic BET inhibitor apabetalone counters inflammatory and fibrotic processes in activated cardiac fibroblasts providing insight into reduced hospitalizations for heart failure in BETonMACE trial

Abstract Background After myocardial infarction (MI), sustained crosstalk between monocytes and cardiac fibroblasts (CFs) promotes chronic inflammation and interstitial fibrosis that can contribute to heart failure (HF). Resident and myocardium-infiltrating immune cells produce proinflammatory cytokines IL-1β and TNFα that stimulate CFs to produce monocyte chemoattractants. Monocytes secrete TGF-β1 that transforms CFs into contractile myofibroblasts overproducing the extracellular matrix (ECM) responsible for cardiac fibrosis. Epigenetic BET inhibitors (BETi) reduce CF transdifferentiation to prevent cardiac fibrosis and HF in animal models. In patients with cardiovascular disease, type 2 diabetes and recent MI, administration of the BETi apabetalone (APA) resulted in less hospitalization due to HF vs. placebo (BETonMACE phase 3 trial; hazard ratio 0.59, p=0.03). Purpose To examine APA’s in vitro effects on inflammatory and profibrotic processes in CFs associated with cardiac fibrosis and HF. Methods Immortalized and primary human CFs were stimulated with cytokines (10ng/mL IL-1β, TNFα, TGF-β1) or with conditioned media from 10ng/mL IFNγ and 100ng/mL lipopolysaccharide-treated THP-1 macrophages ± 5μM APA on plastic or in 3D collagen gels. Gene expression was analyzed by PCR, protein levels by FACS or ELISA, collagen deposition with picrosirius red, THP-1 monocyte migration in Boyden chambers, and CF contraction with 3D collagen gels. Results IL-1β or TNFα stimulation of CFs upregulated monocyte chemoattractant 1 (CCL2) and vascular cell adhesion protein 1 (VCAM1) expression, promoting THP-1 cell migration and adhesion to CFs. APA treatment reduced cytokine-induced CCL2 (&amp;gt;20%) and VCAM1 (&amp;gt;70%) gene expression as well as THP-1 cell migration and adhesion to stimulated CFs (&amp;gt;60%). TGF-β1 treatment induced CF transdifferentiation into myofibroblasts as shown by increased expression of α smooth muscle actin (α-SMA) protein, and secretion of ECM proteins fibronectin and periostin. APA treatment reduced myofibroblast mRNA and protein expression of α-SMA (~30%), fibronectin (~30%) and periostin (97%). Myofibroblast-mediated collagen deposition was also reduced by APA by ~20%. α-SMA-dependent CF contraction can be visualized in attached 3D collagen gels. TGF-β1, IL-1β, TNFα or macrophage-conditioned media promoted CF-mediated gel contraction by 2.5, 1.4, 2.1, or 1.8-fold, respectively. APA treatment countered gel contraction by 30-60%, thereby reducing the myofibroblast-like behaviour in organotypic cultures. Conclusions In vitro, APA treatment reduced proinflammatory crosstalk between monocytes and CFs, resulting in less monocyte migration, monocyte - CF adhesion and CF contraction. APA also reduced signaling by TGF-β1, thus reducing profibrotic and contractile behaviour of CFs responsible for cardiac fibrosis. Since fibrosis contributes to HF, this data provides insight into the observed reduction in hospitalization due to HF in the BETonMACE trial.

Clustering-aided prediction of outcomes in patients with idiopathic pulmonary fibrosis

BackgroundBlood biomarkers predictive of the progression of idiopathic pulmonary fibrosis (IPF) would be of value for research and clinical practice. We used data from the IPF-PRO Registry to investigate whether the addition of “omics” data to risk prediction models based on demographic and clinical characteristics improved prediction of the progression of IPF.MethodsThe IPF-PRO Registry enrolled patients with IPF at 46 sites across the US. Patients were followed prospectively. Median follow-up was 27.2 months. Prediction models for disease progression included omics data (proteins and microRNAs [miRNAs]), demographic factors and clinical factors, all assessed at enrollment. Data on proteins and miRNAs were included in the models either as raw values or based on clusters in various combinations. Least absolute shrinkage and selection operator (Lasso) Cox regression was applied for time-to-event composite outcomes and logistic regression with L1 penalty was applied for binary outcomes assessed at 1 year. Model performance was assessed using Harrell’s C-index (for time-to-event outcomes) or area under the curve (for binary outcomes).ResultsData were analyzed from 231 patients. The models based on demographic and clinical factors, with or without omics data, were the top-performing models for prediction of all the time-to-event outcomes. Relative changes in average C-index after incorporating omics data into models based on demographic and clinical factors ranged from 1.7 to 3.2%. Of the blood biomarkers, surfactant protein-D, serine protease inhibitor A7 and matrix metalloproteinase-9 (MMP-9) were among the top predictors of the outcomes. For the binary outcomes, models based on demographics alone and models based on demographics plus omics data had similar performances. Of the blood biomarkers, CC motif chemokine 11, vascular cell adhesion protein-1, adiponectin, carcinoembryonic antigen and MMP-9 were the most important predictors of the binary outcomes.ConclusionsWe identified circulating protein and miRNA biomarkers associated with the progression of IPF. However, the integration of omics data into prediction models that included demographic and clinical factors did not materially improve the performance of the models.Trial registrationClinicalTrials.gov; No: NCT01915511; registered August 5, 2013; URL: www.clinicaltrials.gov.

Inflammatory proteins associated with Alzheimer’s disease reduced by a GLP1 receptor agonist: a post hoc analysis of the EXSCEL randomized placebo controlled trial

BackgroundGlucagon-like peptide-1 receptor agonists are a viable option for the prevention of Alzheimer’s disease (AD) but the mechanisms of this potential disease modifying action are unclear. We investigated the effects of once-weekly exenatide (EQW) on AD associated proteomic clusters.MethodsThe Exenatide Study of Cardiovascular Event Lowering study compared the cardiovascular effects of EQW 2 mg with placebo in 13,752 people with type 2 diabetes mellitus. 4,979 proteins were measured (Somascan V0.4) on baseline and 1-year plasma samples of 3,973 participants. C-reactive protein (CRP), ficolin-2 (FCN2), plasminogen activator inhibitor 1 (PAI-1), soluble vascular cell adhesion protein 1 (sVCAM1) and 4 protein clusters were tested in multivariable mixed models.ResultsEQW affected FCN2 (Cohen’s d -0.019), PAI-1 (Cohen’s d -0.033), sVCAM-1 (Cohen’s d 0.035) and a cytokine-cytokine cluster (Cohen’s d 0.037) significantly compared with placebo. These effects were sustained in individuals over the age of 65 but not in those under 65.ConclusionsEQW treatment was associated with significant change in inflammatory proteins associated with AD.Trial RegistrationEXSCEL is registered on ClinicalTrials.gov: NCT01144338 on 10th of June 2010.

VCAM-1 mediates proximal tubule-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Studies in animal models have suggested a linkage between the inflammatory response to injury and subsequent nephron loss during the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Failure of normal repair during the CKD transition correlates with de novo expression of vascular cell adhesion protein-1 (VCAM-1) by a subset of injured proximal tubule cells. This study identified the role of VCAM-1 expression in promoting the failed repair state. Single-cell transcriptome analysis of patients with AKI and CKD and whole kidney RNA and protein analyses of mouse models of CKD confirmed a marked increase of VCAM-1 expression in the proximal tubules of injured kidneys. In immortalized mouse proximal tubular cells and primary cultured renal cells (PCRCs), VCAM-1 expression was induced by proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Analyses of bulk RNA sequencing of TNF-α-treated primary cultured renal cells or pseudo-bulk RNA sequencing of biopsies from Kidney Precision Medicine Project datasets indicated activation of NF-κB and an enrichment of inflammatory response and cell adhesion pathways in VCAM-1-positive cells. Pharmacological inhibition of NF-κB signaling or genetic deletion of myeloid differentiation factor 88 and TIR domain-containing adapter-inducing interferon-β suppressed TNF-α- and IL-1β-induced VCAM-1 expression in vitro. TNF-α stimulation or overexpression of VCAM-1 significantly increased splenocyte adhesion to the mouse proximal tubular monolayer in culture. These results demonstrate that persistence of proinflammatory cytokines after AKI can induce NF-κB-dependent VCAM-1 expression by proximal tubule cells, mediating increased immune cell adhesion to the tubule and thus promoting further tubule injury and greater risk of progression from AKI to CKD.NEW & NOTEWORTHY We demonstrated the induction of VCAM-1 and its biological function in proximal tubules. We found that proinflammatory cytokines (TNF-α and IL-1β) significantly induced VCAM-1 expression via NF-κB signaling pathway. TNF-α treatment or overexpression of VCAM-1 in immortalized MPT cells increased CD45+ splenocyte adhesion. Pharmacological inhibition of NF-κB or genetic deletion of Vcam1 suppressed TNF-α-induced splenocyte adhesion in vitro, suggesting that VCAM-1 mediates proximal tubular-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

5-Hydroxytryptamine 1F Receptor Agonist Lasmiditan Differentially Regulates Successful Repair and Failed Repair Genes in a Mouse Model of Acute Kidney Injury.

Increasing evidence substantiates the role of mitochondrial dysfunction, inflammation, fibrosis, and cell senescence in the onset and progression of acute kidney injury (AKI) to chronic kidney disease . The underlying governing cellular and transcriptional events, however, are not fully understood. Recently, the key factors that regulate successful and failed repair states in the proximal tubule have been identified at a single-cell resolution following bilateral ischemia-reperfusion (I/R) in a mouse model of AKI. Previously, our group showed that treatment with the FDA-approved selective 5-hydroxytryptamine receptor 1F agonist lasmiditan following AKI induces mitochondrial biogenesis , restores renal mitochondrial function, and increases renal and vascular recovery in vivo. Here, we assessed the effect of lasmiditan on transcriptional and translational changes that are responsible for successful repair, injury, and failed repair states in the renal cortex following I/R-induced AKI. Increased levels of successful repair genes such as acyl-coA synthase medium-chain family member 2a, low-density lipoprotein receptor-related protein 2, solute carrier family 5 member 12, and hepatocyte nuclear factor 4 alpha were observed with 6 and 12 days of lasmiditan treatment following AKI compared to vehicle control. While 6 days of lasmiditan treatment had no effect on failed repair genes, the administration of lasmiditan for 12 days decreased the levels of vascular cell adhesion protein 1, tumor necrosis factor α, and interleukin-1β, which drive maladaptive repair. These data reveal that lasmiditan treatment post-AKI differentially regulates successful and failed repair gene expression in the renal cortex, likely contributing to the restoration of renal function and providing a potential targeted therapeutic pathway for the treatment of AKI.

Exploring biomarkers for diagnosing and predicting organ dysfunction in patients with perioperative sepsis: a preliminary investigation

ObjectiveEarly diagnosis and prediction of organ dysfunction are critical for intervening and improving the outcomes of septic patients. The study aimed to find novel diagnostic and predictive biomarkers of organ dysfunction for perioperative septic patients.MethodThis is a prospective, controlled, preliminary, and single-center study of emergency surgery patients. Mass spectrometry, Gene Ontology (GO) functional analysis, and the protein-protein interaction (PPI) network were performed to identify the differentially expressed proteins (DEPs) from sepsis patients, which were selected for further verification via enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was used to estimate the relative correlation of selected serum protein levels and clinical outcomes of septic patients. Calibration curves were plotted to assess the calibration of the models.ResultsFive randomized serum samples per group were analyzed via mass spectrometry, and 146 DEPs were identified. GO functional analysis and the PPI network were performed to evaluate the molecular mechanisms of the DEPs. Six DEPs were selected for further verification via ELISA. Cathepsin B (CatB), vascular cell adhesion protein 1 (VCAM-1), neutrophil gelatinase-associated lipocalin (NGAL), protein S100-A9, prosaposin, and thrombospondin-1 levels were significantly increased in the patients with sepsis compared with those of the controls (p < 0.001). Logistic regression analysis showed that CatB, S100-A9, VCAM-1, prosaposin, and NGAL could be used for preoperative diagnosis and postoperative prediction of organ dysfunction. CatB and S100-A9 were possible predictive factors for preoperative diagnosis of renal failure in septic patients. Internal validation was assessed using the bootstrapping validation. The preoperative diagnosis of renal failure model displayed good discrimination with a C-index of 0.898 (95% confidence interval 0.843–0.954) and good calibration.ConclusionSerum CatB, S100-A9, VCAM-1, prosaposin, and NGAL may be novel markers for preoperative diagnosis and postoperative prediction of organ dysfunction. Specifically, S100-A9 and CatB were indicators of preoperative renal dysfunction in septic patients. Combining these two biomarkers may improve the accuracy of predicting preoperative septic renal dysfunction.Trial registrationThe study was registered at the Chinese Clinical Trials Registry (ChiCTR2200060418) on June 1, 2022.

Disease Evolution-based Specificity Target Discovery (DESTD) by analyzing Jiawei-Maxing-Shigan Decoctions against COVID-19

IntroductionDiverse disease courses, each corresponding to a unique pathophysiological mechanism, characterize the coronavirus 2019 disease (COVID-19). Intensive research on discovering specificity targets for treating COVID-19 has significant implications for understanding pathogenesis mechanisms in disease evolution. Traditional Chinese medicine (TCM) has shown distinctive advantages in treating COVID-19 in different periods, which may provide new clues for target discovery. In order to accomplish the goal of providing a Disease Evolution-based Specificity Target Discovery (DESTD), the purpose of this paper was to conduct an analysis of the difference in target weighted value during each cycle of COVID-19. MethodsOur study employed a comprehensive methodology combining TCM pharmacology with modern biological techniques. Three Jiawei-Maxing-Shigan Decoctions (MS Decoction), were selected as samples, including Hanshi-Yufei decoction (HY Decoction), Shidu-Yufei decoction (SY Decoction), and Yidu-Bifei decoction (YB Decoction). The core components of these decoctions are the same, while the differences correspond to different periods of COVID-19. The DESTD approach consists of several parts: network pharmacology analysis to identify potential targets, molecular docking simulation to investigate TCM components acting on potential targets, and biological validation. This study employed several cell line models, including RAW264.7 (Mouse Mononuclear Macrophages Cells), 16HBE (Human Bronchial Epithelial Cells), and HUVECC (Human Umbilical Vein Endothelial Cells), to explore new approaches for key target discovery. ResultsDuring the mild phase of COVID-19, HY Decoction exhibited anti-inflammatory effects by specifically down-regulating the expression of heat shock protein HSP 90-alpha (HSP90AA1), transcription factor JUN (JUN) and other five genes in RAW264.7. In the common phrase, inflammation and fibrosis could be specifically alleviated by down-regulating the expression of interleukin-10 (IL10) and mitogen-activated protein kinase 14 (MAPK14) by SY Decoction respectively acting on RAW264.7 and 16HBE cell lines. In severe cases, YB Decoction specifically down-regulated the expression of prostaglandin endoperoxide synthase-2 (PTGS2), caspase-3 (CASP3), and three other genes to protect vascular endothelial cells from oxidative damage. Moreover, YB also played a role in anti-fibrosis by specifically down-regulating the expression of estrogen receptor beta (ESR2), vascular cell adhesion protein 1 (VCAM1), and other three genes, and up-regulating the expression of prostaglandin endoperoxide synthase-1 (PTGS1) in the severe period. These targeted actions align with a holistic treatment approach and provide tailored therapies for different stages of COVID-19. ConclusionThis research proposed a novel strategy for Disease Evolution-based Specificity Target Discovery (DESTD) to identify specific targets of different stages of disease and specific targets and mechanisms throughout the evolution of COVID-19.

The Evolving Role of Bruton's Tyrosine Kinase Inhibitors in B Cell Lymphomas.

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase crucial for B cell development and function, acts downstream of the B cell receptor (BCR) in the BCR pathway. Other kinases involved downstream of the BCR besides BTK such as Syk, Lyn, PI3K, and Mitogen-activated protein (MAP) kinases also play roles in relaying signals from the BCR to provide pro-survival, activation, and proliferation cues. BTK signaling is implicated in various B-cell lymphomas such as mantle cell lymphoma, Waldenström Macroglobulinemia, follicular lymphoma, and diffuse large B cell lymphoma, leading to the development of transformative treatments like ibrutinib, the first-in-class covalent BTK inhibitor, and pirtobrutinib, the first-in-class noncovalent BTK inhibitor. However, kinase-deficient mutations C481F, C481Y, C481R, and L528W in the BTK gene confer resistance to both covalent and non-covalent BTK inhibitors, facilitating B cell survival and lymphomagenesis despite kinase inactivation. Further studies have revealed BTK's non-catalytic scaffolding function, mediating the assembly and activation of proteins including Toll-like receptor 9 (TLR9), vascular cell adhesion protein 1 (VCAM-1), hematopoietic cell kinase (HCK), and integrin-linked kinase (ILK). This non-enzymatic role promotes cell survival and proliferation independently of kinase activity. Understanding BTK's dual roles unveils opportunities for therapeutics targeting its scaffolding function, promising advancements in disrupting lymphomagenesis and refining B cell lymphoma treatments.

Diagnostic and Prognostic Value of Machine Perfusion Biomarkers in Kidney Graft Evaluation

BackgroundWith the rising prevalence of end-stage kidney disease, the use of expanded criteria donor allografts, seen as essential for meeting organ demand, still proves challenging due to their higher risk of graft loss, delayed function, and rejection. Machine perfusion, a technique in preserving allografts, offers improved allograft outcomes compared to static cold storage while allowing for the noninvasive measurement of kidney injury biomarkers in the perfusate solution. This offers an objective method to assess graft function at various preservation stages. Materials and MethodsWe conducted a narrative review of the databases PubMed and Scopus, including studies written in the English language and published after 2010. ResultsIn this narrative review, we identified biomarkers, like 4-hydroxyproline, taurine, and glutathione transferase, as predictive markers of delayed graft function. Additionally, biomarkers, like extracellular histone h3, vascular cell adhesion protein, and matrix metalloprotease protein, have shown correlation with decreased graft function, although their predictive ability remains inconclusive. DiscussionThe review outlines various suggestions for potential areas of research focus to enhance future expanded criteria donor allograft utilization. However, limitations exist, including the absence of a singular reliable biomarker and the challenges of validating biomarker effectiveness across diverse outcomes.

Early Effects of Modern Radiotherapy for Lung Cancer on Endothelial Damage and Myocardial Fibrosis: A Prospective Single-Center Study.

Radiotherapy (RT) may have a cardiotoxic effect on the heart and cardiovascular system. Postulated mechanisms mediating these complications include vascular endothelium damage and myocardial fibrosis. The aim of our study was to assess endothelial damage and myocardial fibrosis in the early period after RT on the basis of cardiac biomarkers and in relation to the radiation dose applied to individual heart structures in patients treated for non-small-cell lung cancer. This single-center prospective study included consecutive patients with lung cancer (LC) who were referred for treatment with radiochemotherapy (study group) or chemotherapy (control group). The study protocol included performing an echocardiographic examination, a standard ECG examination, and collecting blood samples for laboratory tests before starting treatment for lung cancer in the first week after completing RT (after four cycles of chemotherapy in the control group) and after 12 weeks from the end of treatment. The study included 23 patients in the study group and 20 patients in the control group. Compared to the baseline values, there was a significant increase in total cholesterol concentration in the study group immediately after the end of RT, which persisted for three months after the end of therapy. After taking into account the use of statins in the analysis, it was found that an increase in total cholesterol concentration after oncological treatment was observed only among patients who did not use statins. Taking into account the assessment of myocardial fibrosis markers, there were no significant changes in the concentration of matrix metallopeptidase 9 (MMP-9) and tissue inhibitors of metalloproteinases 1 (TIMP-1) in the study group. In patients treated with radiochemotherapy, there was a significant increase in the concentration of intercellular adhesion molecule 1 (ICAM-1) immediately after RT, when compared to the baseline. After taking into account the use of statins, an increase in ICAM-1 concentration immediately after RT was observed only in patients who did not use statins. There was also a significant correlation between the radiation dose received by the left anterior descending coronary artery (LAD) and left circumferential coronary artery, and vascular cell adhesion protein 1 (VCAM-1) concentration measured at three months after the end of RT. Immediately after completion of radiotherapy, a significant increase in the level of ICAM-1 is observed indicating endothelial damage. The radiation dose to coronary arteries should be minimized, as it correlates with the concentration of VCAM-1. The use of statins may prevent the increase in total cholesterol and ICAM-1 concentration after irradiation for lung cancer; however, further studies designed for this specific purpose are necessary to confirm the effectiveness of statins in this area.

Effects of antagonism of thromboxane-prostanoid receptor on alcohol-associated liver disease

Backgrounds: Alcohol-associated liver disease (ALD) is one of the leading causes of liver diseases. Thromboxane-prostanoid receptor (TP-R) is widely expressed in the liver and is activated by thromboxane A2 (TXA2). A previous study reported an increase in TP-R mRNA level in liver after ethanol feeding. Preclinical studies have documented that TP-R antagonists exert a protective effect against cardiovascular and liver diseases. In particular, TP-R antagonist is reported to attenuate ALD in rodents. However, the mechanism(s) by which antagonizing TP-R attenuates ALD remains unclear. Methods: Herein, we used the chronic plus binge ethanol feeding model. Briefly, male, C57BL/6 wild-type mice (8-week) were fed a Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by a single binge administration of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8) by oral administration. Results: Western blot analysis showed that the protein level of thromboxane A2 synthase 1 ( P&lt;0.05), responsible for TXA2 production, was upregulated in the mouse liver upon ethanol exposure. Of note, ethanol feeding significantly increased plasma aspartate transferase (AST) level ( P&lt;0.05), which was abolished by SQ administration ( P&lt;0.05). Moreover, compared with CON mice, ET mice showed an increase in liver protein levels of pro-inflammatory markers, including TNFa ( P&lt;0.05), cluster of differentiation 68 (CD68, P&lt;0.001), and vascular cell adhesion protein 1 ( P&lt;0.05). In addition, the pro-fibrogenic markers including collagen type 1 alpha 1 chain (COL1A1, P&lt;0.05) and matrix metalloproteinase 9 (MMP9, P&lt;0.01) were also increased in ET mice. However, these alterations were not found in ET+SQ mice compared to their controls. In particular, ET+SQ mice exhibited a marked reduction in hepatic protein levels of CD68 ( P&lt;0.001), COL1A1 ( P&lt;0.001), and MMP9 ( P&lt;0.01) versus ET mice. Interestingly, we noted that ethanol consumption decreases the protein level of NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), a component of mitochondrial complex I, in mouse liver ( P&lt;0.05), which was reversed by SQ administration( P&lt;0.05). Conclusion: These findings suggest that blockade of TP-R activity attenuates ethanol-induced liver inflammation and fibrosis possibly through improving the function of mitochondrial complex I. This study was supported by the NIH-NIAAA P50 award-Alcohol Center of Research-Nebraska (P50AA030407-5130). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Rapamycin on Endothelial Function in Systemic Lupus Erythematosus (SLE)

Mitophagy is regulated by multiple molecular pathways, including the mechanistic target of rapamycin (mTOR), which inhibits mitophagy. Elevated activation of mTOR complex 1 (mTORC1) is reported in humans and mice with SLE, suggesting that impaired mitophagy may contribute to the pathogenesis of SLE. As previously studied in genetically modified mice, mTORC1 also is proposed to be a critical regulator of endothelial function. However, it is unclear if pathologically elevated mTORC1, as in SLE, causes impaired mitophagy and endothelial dysfunction. Therefore, the study aimed to determine the effect of inhibition of mTORC1 on mitophagy, endothelial function, and inflammatory responses in SLE. We hypothesize that inhibiting mTORC1 with rapamycin will increase mitophagy and attenuate endothelial dysfunction and inflammatory responses in SLE. Nine-week-old female lupus-prone (MRL/lpr) and healthy control (MRL/MpJ) mice were randomly assigned into one of two groups, rapamycin treatment (lpr_Rapa and MpJ_Rapa) or control (lpr_Con and MpJ_Con). Rapamycin was injected i.p. three days a week for 8 weeks. After 8 weeks of treatment, endothelium-dependent vasorelaxation to acetylcholine (Ach) and endothelium-independent vasorelaxation to sodium nitroprusside (SNP) were measured in thoracic aortas using a wire myograph. Maximal responses to Ach were significantly impaired in lpr_Con (51.7 ± 6.6%, n = 11) compared to MpJ_Con (86.7 ± 3.6%, n = 11) (p &lt; 0.0001). Rapamycin prevented endothelial dysfunction in the thoracic aorta from lupus mice (lpr_Rapa) (79.6 ± 4.2%, n = 10) compared to lpr_Con (p = 0.002). Maximal responses to SNP were not significantly different across the groups. Thoracic aortas were used to measure the content of mitophagy, inflammation, and endothelial function related proteins (n = 4-6/group). We assessed markers downstream of mTORC1 to confirm the inhibition of the mTORC1 activity. As expected, inhibition of mTORC1 activity decreased the phosphorylation of s6rp (p-s6rp/s6rp) in lpr_Rapa (0.39 ± 0.07) compared to lpr_Con (1.47 ± 0.16) (p &lt; 0.0001). Phosphorylation of endothelial nitric oxide synthase (p-eNOS/eNOS) was 42% lower in lpr_Con (0.69 ± 0.14) than MpJ_Con (1.00 ± 0.12). Phosphorylated eNOS was 46% higher in lpr_Rapa (1.28 ± 0.15) than lpr_Con. Vascular cell adhesion protein 1 (Vcam 1), is an indicator of inflammation and is associated with atherosclerosis susceptibility. Protein content of Vcam 1 was significantly higher in lpr_Con (1.36 ± 0.06) compared to MpJ_Con (1.00 ± 0.01) (p = 0.001). The protein content of LC3II was measured as a marker of mitophagy. Though it was not significant, LC3II level was 2-fold higher in lpr_Con (2.12 ± 1.13) compared to MpJ_Con (1.00 ± 0.17), which may imply the accumulation of autophagosome resulted from inhibition of autophagic degradation. Collectively, these results demonstrate the beneficial effects of inhibiting mTORC1 on endothelial function in SLE mice and suggest inflammation and altered mitophagy contributing to endothelial dysfunction in SLE. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Procyanidin B2 alleviates oxidized low-density lipoprotein-induced cell injury, inflammation, monocyte chemotaxis, and oxidative stress by inhibiting the nuclear factor kappa-B pathway in human umbilical vein endothelial cells

BackgroundOxidized low-density lipoprotein (ox-LDL) can initiate and affect almost all atherosclerotic events including endothelial dysfunction. In this text, the role and underlying molecular basis of procyanidin B2 (PCB2) with potential anti-oxidant and anti-inflammatory activities in ox-LDL-induced HUVEC injury were examined.MethodsHUVECs were treated with ox-LDL in the presence or absence of PCB2. Cell viability and apoptotic rate were examined by CCK-8 assay and flow cytometry, respectively. The mRNA and protein levels of genes were tested by RT-qPCR and western blot assays, respectively. Potential downstream targets and pathways of apple procyanidin oligomers were examined by bioinformatics analysis for the GSE9647 dataset. The effect of PCB2 on THP-1 cell migration was examined by recruitment assay. The effect of PCB2 on oxidative stress was assessed by reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and mitochondrial membrane potential (MMP).Resultsox-LDL reduced cell viability, induced cell apoptosis, and facilitated the expression of oxidized low-density lipoprotein receptor 1 (LOX-1), C-C motif chemokine ligand 2 (MCP-1), vascular cell adhesion protein 1 (VCAM-1) in HUVECs. PCB2 alleviated ox-LDL-induced cell injury in HUVECs. Apple procyanidin oligomers triggered the differential expression of 592 genes in HUVECs (|log2fold-change| > 0.58 and adjusted p-value < 0.05). These dysregulated genes might be implicated in apoptosis, endothelial cell proliferation, inflammation, and monocyte chemotaxis. PCB2 inhibited C-X-C motif chemokine ligand 1/8 (CXCL1/8) expression and THP-1 cell recruitment in ox-LDL-stimulated HUVECs. PCB2 inhibited ox-LDL-induced oxidative stress and nuclear factor kappa-B (NF-κB) activation in HUVECs.ConclusionPCB2 weakened ox-LDL-induced cell injury, inflammation, monocyte recruitment, and oxidative stress by inhibiting the NF-κB pathway in HUVECs.

Endothelium-related biomarkers enhanced prediction of kidney support therapy in critically ill patients with non-oliguric acute kidney injury

Acute kidney injury (AKI) is a common condition in hospitalized patients who often requires kidney support therapy (KST). However, predicting the need for KST in critically ill patients remains challenging. This study aimed to analyze endothelium-related biomarkers as predictors of KST need in critically ill patients with stage 2 AKI. A prospective observational study was conducted on 127 adult ICU patients with stage 2 AKI by serum creatinine only. Endothelium-related biomarkers, including vascular cell adhesion protein-1 (VCAM-1), angiopoietin (AGPT) 1 and 2, and syndecan-1, were measured. Clinical parameters and outcomes were recorded. Logistic regression models, receiver operating characteristic (ROC) curves, continuous net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were used for analysis. Among the patients, 22 (17.2%) required KST within 72 h. AGPT2 and syndecan-1 levels were significantly greater in patients who progressed to the KST. Multivariate analysis revealed that AGPT2 and syndecan-1 were independently associated with the need for KST. The area under the ROC curve (AUC-ROC) for AGPT2 and syndecan-1 performed better than did the constructed clinical model in predicting KST. The combination of AGPT2 and syndecan-1 improved the discrimination capacity of predicting KST beyond that of the clinical model alone. Additionally, this combination improved the classification accuracy of the NRI and IDI. AGPT2 and syndecan-1 demonstrated predictive value for the need for KST in critically ill patients with stage 2 AKI. The combination of AGPT2 and syndecan-1 alone enhanced the predictive capacity of predicting KST beyond clinical variables alone. These findings may contribute to the early identification of patients who will benefit from KST and aid in the management of AKI in critically ill patients.

Th1 cells contribute to retinal ganglion cell loss in glaucoma in a VCAM-1-dependent manner.

Glaucoma is a complex neurodegenerative disorder characterized by the progressive loss of retinal ganglion cells (RGC) and optic nerve axons, leading to irreversible visual impairment. Despite its clinical significance, the underlying mechanisms of glaucoma pathogenesis remain poorly understood. In this study, we aimed to unravel the multifaceted nature of glaucoma by investigating the interaction between T cells and retinas. By utilizing clinical samples, murine glaucoma models, and T cell transfer models, we made several key findings. Firstly, we observed that CD4+ T cells from glaucoma patients displayed enhanced activation and a bias towards T helper (Th) 1 responses, which correlated with visual impairment. Secondly, we identified the infiltration of Th1 cells into the retina, where they targeted RGC and integrated into the pro-inflammatory glial network, contributing to progressive RGC loss. Thirdly, we discovered that circulating Th1 cells upregulated vascular cell adhesion protein 1 (VCAM-1) on retinal microvessels, facilitating their entry into the neural retina. Lastly, we found that Th1 cells underwent functional reprogramming before reaching the retina, acquiring a phenotype associated with lymphocyte migration and neurodegenerative diseases. Our study provides novel insights into the role of peripheral CD4+ T cells in glaucoma pathogenesis, shedding light on the mechanisms underlying their infiltration into the retina and offering potential avenues for innovative therapeutic interventions in this sight-threatening disease.

Long non-coding RNA H19X as a regulator of mononuclear cell adhesion to the endothelium in systemic sclerosis.

To define the functional relevance of H19 X-linked (H19X) co-expressed long non-coding RNA (lncRNA) in endothelial cell (EC) activation as a key process in SSc vasculopathy. H19X expression in SSc skin biopsies was analysed from single-cell RNA sequencing (scRNA-seq) data. Differential expression and pathway enrichment analysis between cells expressing (H19Xpos) and non-expressing H19X (H19Xneg) cells was performed. H19X function was investigated in human dermal microvascular ECs (HDMECs) by silencing. H19X and EC adhesion molecule levels were analysed by real-time quantitative PCR and western blot after stimulation with pro-inflammatory cytokines. Cytoskeletal rearrangements were analysed by fluorescent staining. Endothelial adhesion was evaluated by co-culture of HDMECs and fluorescent-labelled peripheral blood mononuclear cells (PBMCs). Shedding vascular cell adhesion protein 1 (VCAM1) was evaluated by ELISA on HDMEC supernatant. The scRNA-seq data showed significant upregulation of H19X in SSc compared with healthy ECs. In HDMECs, H19X was consistently induced by IFN type I and II. H19X knockdown lead to a significant decrease in the mRNA of several adhesion molecules. In particular, VCAM1 was significantly reduced at the protein and mRNA levels. Co-expression analysis of the scRNA-seq data confirmed higher expression of VCAM1 in H19Xpos ECs. ECs were also strongly associated with the 'cell adhesion molecule' pathway. Moreover, the VCAM1 downstream pathway displayed less activation following H19X knockdown. Contractility of HDMECs, PBMC adhesion to HDMECs and VCAM1 shedding were also reduced following H19X knockdown. lncRNA H19X may contribute to EC activation in SSc vasculopathy, acting as a regulator of expression of adhesion molecules in ECs.

FSH Is Responsible for Androgen Deprivation Therapy-Associated Atherosclerosis in Mice by Exaggerating Endothelial Inflammation and Monocyte Adhesion.

Androgen deprivation therapy (ADT) is the mainstay treatment for advanced prostate cancer. But ADTs with orchiectomy and gonadotropin-releasing hormone (GnRH) agonist are associated with increased risk of cardiovascular diseases, which appears less significant with GnRH antagonist. The difference of follicle-stimulating hormone (FSH) in ADT modalities is hypothesized to be responsible for ADT-associated cardiovascular diseases. We administered orchiectomy, GnRH agonist, or GnRH antagonist in male ApoE-/- mice fed with Western diet and manipulated FSH levels by testosterone and FSH supplementation or FSH antibody to investigate the role of FSH elevation on atherosclerosis. By combining lipidomics, in vitro study, and intraluminal FSHR (FSH receptor) inhibition, we delineated the effects of FSH on endothelium and monocytes and the underlying mechanisms. Orchiectomy and GnRH agonist, but not GnRH antagonist, induced long- or short-term FSH elevation and significantly accelerated atherogenesis. In orchiectomized and testosterone-supplemented mice, FSH exposure increased atherosclerosis. In GnRH agonist-treated mice, blocking of short FSH surge by anti-FSHβ antibody greatly alleviated endothelial inflammation and delayed atherogenesis. In GnRH antagonist-treated mice, FSH supplementation aggravated atherogenesis. Mechanistically, FSH, synergizing with TNF-α (tumor necrosis factor alpha), exacerbated endothelial inflammation by elevating VCAM-1 (vascular cell adhesion protein 1) expression through the cAMP/PKA (protein kinase A)/CREB (cAMP response element-binding protein)/c-Jun and PI3K (phosphatidylinositol 3 kinase)/AKT (protein kinase B)/GSK-3β (glycogen synthase kinase 3 beta)/GATA-6 (GATA-binding protein 6) pathways. In monocytes, FSH upregulated CD29 (cluster of differentiation 29) expression via the PI3K/AKT/GSK-3β/SP1 (specificity protein 1) pathway and promoted monocyte-endothelial adhesion both in vitro and in vivo. Importantly, FSHR knockdown by shRNA in endothelium of carotid arteries markedly reduced GnRH agonist-induced endothelial inflammation and atherosclerosis in mice. FSH is responsible for ADT-associated atherosclerosis by exaggerating endothelial inflammation and promoting monocyte-endothelial adhesion.

CU06-1004 alleviates oxidative stress and inflammation on folic acid-induced acute kidney injury in mice

PurposeAcute kidney injury (AKI) is characterized by reduced renal function, oxidative stress, inflammation, and renal fibrosis. CU06-1004, an endothelial cell dysfunction blocker, exhibits anti-inflammatory effects by reducing vascular permeability in pathological conditions. However, the potential effects of CU06-1004 on AKI have not been investigated. We investigated the renoprotective effect of CU06-1004 against oxidative stress, inflammation, and fibrotic changes in a folic acid-induced AKI model. MethodsAKI was induced by intraperitoneal injection of high dose (250 mg/kg) folic acid in mice. CU06-1004 was orally administered a low (10 mg/kg) or high dose (20 mg/kg). ResultsCU06-1004 ameliorated folic acid-induced AKI by decreasing serum blood urea nitrogen and creatinine levels, mitigating histological abnormalities, and decreasing tubular injury markers such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in folic acid-induced AKI mice. Additionally, CU06-1004 alleviated folic acid-induced oxidative stress by reducing 4-hydroxynonenal and malondialdehyde levels. Furthermore, it attenuated macrophage infiltration and suppressed the expression of the proinflammatory factors, including tumor necrosis factor-α, intercellular adhesion molecule-1, and vascular cell adhesion protein-1. Moreover, CU06-1004 mitigated folic acid-induced tubulointerstitial fibrosis by decreasing α-smooth muscle actin and transforming growth factor-β expression. ConclusionThese findings suggest CU06-1004 as a potential therapeutic agent for folic acid-induced AKI.

Abstract 18426: Secreted Frizzled-Related Protein 1 Mediates Adipogenic Differentiation of Vascular Cell Adhesion Protein 1 Fibroadipogenic Progenitors in the Ischemic Limb

Introduction: Chronic limb threatening ischemia (CLTI) patients display a skeletal muscle myopathy that is hallmarked by fatty infiltration. The replacement of functional muscle fibers with adipocytes impairs ambulatory function and is associated with poor clinical outcomes. The cell type responsible for fatty infiltration is a mesenchymal stromal cellular population, term fibroadipogenic progenitors (FAPs). We previously demonstrated that Vcam1+ FAPs represent a sub-population with increased adipogenic potential. However, the molecular mechanisms which govern Vcam1+ FAP mediated adipogenesis remain unknown. Herein, we sought to identify the intracellular mechanisms that drive Vcam1+ FAPs into an adipogenic cellular fate. Methods: We used single cell and bulk RNA-sequencing to identify Sfrp1 as an enriched gene in Vcam1+ FAPs. Next, to determine the sufficiency and necessity of Sfrp1, we overexpress Sfrp1 with a recombinant protein and inhibit Sfrp1 with a siRNA and assess adipogenesis. To assess the clinical significance of this FAP subpopulation, we apply single-cell RNA sequencing to CLTI patients. Results: We show in a CLTI murine model that Vcam1+ FAPs are enriched in Sfrp1. Next, we demonstrate that Sfrp1 is both sufficient and necessary for the differentiation of Vcam1+ FAPs into adipocytes. Finally, we demonstrate that human CLTI patients also have a pathologic FAP population that is enriched in Vcam1 and Sfrp1Conclusions: Our results reveal a novel mechanism for fatty infiltration, a histologic hallmark of CLTI patients, in the ischemic limb. We also validate the clinical significance of Vcam1+ Sfrp1+ FAPs by demonstrating the presence of this population in human CLTI patients. This work lays the foundation for future studies aimed at inhibiting fatty infiltration and promoting skeletal muscle regeneration in CLTI patients.