Transendothelial Migration Research Articles

RNA-sequencing of Human Kidney Allografts and Delineation of T-Cell Genes, Gene Sets, and Pathways Associated With Acute T Cell-mediated Rejection.

Delineation of T-cell genes, gene sets, pathways, and T-cell subtypes associated with acute T cell-mediated rejection (TCMR) may improve its management. We performed bulk RNA-sequencing of 34 kidney allograft biopsies (16 Banff TCMR and 18 no rejection [NR] biopsies) from 34 adult recipients of human kidneys. Computational analysis was performed to determine the differential intragraft expression of T-cell genes at the level of single-gene, gene set, and pathways. T-cell signaling pathway gene sets for plenary T-cell activation were overrepresented in TCMR biopsies compared with NR biopsies. Heightened expression of T-cell signaling genes was validated using external TCMR biopsies. Pro- and anti-inflammatory immune gene sets were enriched, and metabolism gene sets were depleted in TCMR biopsies compared with NR biopsies. Gene signatures of regulatory T cells, Th1 cells, Th2 cells, Th17 cells, T follicular helper cells, CD4 tissue-resident memory T cells, and CD8 tissue-resident memory T cells were enriched in TCMR biopsies compared with NR biopsies. T-cell exhaustion and anergy were also molecular attributes of TCMR. Gene sets associated with antigen processing and presentation, and leukocyte transendothelial migration were overexpressed in TCMR biopsies compared with NR biopsies. Cellular deconvolution of graft infiltrating cells by gene expression patterns identified CD8 T cell to be the most abundant T-cell subtype infiltrating the allograft during TCMR. Our delineation of intragraft T-cell gene expression patterns, in addition to yielding new biological insights, may help prioritize T-cell genes and T-cell subtypes for therapeutic targeting.

Abstract 1549: Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer

Abstract Background: CD146, an adhesion molecule involved in inflammatory regulation, is linked to increased aggressiveness in Triple Negative Breast Cancer (TNBC). Yet, the molecular mechanisms of CD1146-driven TNBC metastasis in an inflammatory context remain elusive. We synthesized CD146 nanobody (anti-CD146 #112-2), which degraded CD146 protein specifically, inhibiting IL-6-mediated JAK/STAT3 activation and suppressing TNBC metastasis. This study aims to elucidate the mechanisms of CD146 degradation by 112-2 and the role of CD146 in mediating inflammatory responses leading to TNBC metastasis. Methods: CD146 knockout and overxpression cell models were created using CRISPR/Cas9 or lentiviral techniques. Lung metastasis was evaluated by injecting luciferase-labeled cells into the tail veins of Balb/c-nu mice, monitored with the IVIS Kinetic system. The impact of CD146 on TNBC metastasis in an inflammatory environment was explored through cell-adhesion assays and trans-endothelial migration assays using CD146 knockout models and HUVEC cells. Transwell assays assessed the influence of IL-6 and 112-2 on TNBC. CD146 mutant vectors with specific truncations were generated to confirm the binding sites between CD146 and 112-2. ELISA and immunofluorescence assays examined CD146 protein internalization. CD146 degradation by 112-2 was confirmed using proteasome inhibitor MG132 and lysosome inhibitor Bafilomycin A1, along with western blotting and immunofluorescence experiments. Results: Stimulation of TNBC cells with IL-6 increased CD146 expression and tumor metastasis. Knocking down CD146 significantly reduced IL-6 secretion by TNBC cells, leading to reduced tumor-endothelial cell adhesion and trans-endothelial migration. CD146 knockout or 112-2 treatment inhibited IL-6-mediated JAK/STAT3 activation and tumor metastasis. Experiments with CD146 mutants and molecular docking identified the specific 112-2 binding site at leucine positions 331-332 of CD146 protein. 112-2 treatment led to CD146 protein internalization and cleavage, resulting in an 80 kDa fragment recognized by both 112-2 and C-terminus-specific CD146 antibodies, ultimately inhibiting the activation of IL-6/JAK/STAT3 signaling. Notably, the inhibition of proteasome or lysosome activity failed to reverse the 112-2-induced reduction in CD146 protein levels. However, inhibiting lysosomal acidification resulted in significant co-localization of CD146 with lysosomes, suggesting a potential role of lysosomes in 112-2-mediated cleavage of CD146. Conclusion: CD146 promotes TNBC metastasis by modulating IL-6/JAK/STAT3 activation. The nanobody anti-CD146 #112-2 specifically binds to CD146 extracellular domain induces the proteolysis of CD146 protein, thereby inhibiting the tumor-promoting effect of IL-6. Citation Format: Kaiyuan Huang, Hexing Sun, Chengyu Wu, Danping Lin, Jinling Chen, Yanhan Chen, Xinqiang Fang, Shuzhao Chen, Yuanke Liang, Haoyu Lin. Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1549.

Abstract 829: Phthalate exposure results in circadian rhythm-induced BBB dysfunction increasing the risk for breast cancer brain metastasis

Abstract Phthalates, classified as plasticizers, represent a group of chemicals pervasive in the general U.S. population, with notably higher levels in non-Hispanic Blacks compared to non-Hispanic Whites. These persistent organic pollutants (POPs) have been identified for their impact on the circadian rhythms of the brain and vascular system. Several studies indicate that phthalates can elevate neuroinflammation and reduce levels of the junctional protein ZO-1 in the hypothalamus, home to the master circadian clock known as the suprachiasmatic nucleus (SCN). The SCN regulates cellular and tissue circadian rhythms, including tight junction proteins essential for the blood-brain barrier's (BBB) main barrier function. Interestingly, experimental studies reveal that prolonged chronic circadian rhythm disruption (CRD) in mice leads to increased breast cancer metastasis. However, the effects of phthalates on CRD concerning brain cancer and metastasis remain poorly understood. Given their significant prevalence and biological impact, we hypothesize that phthalate exposure induces CRD through hypothalamic dysregulation, resulting in BBB dysfunction and an elevated risk of brain metastasis. Results. We developed an in vitro BBB model using endothelial cells and exposed them to an environmentally relevant mixture of five phthalates simulating chronic low-grade exposure. Analysis showed dysregulation of critical clock genes (Bmal1 and Clock), along with a substantial decrease in BBB tight junction proteins (claudin-5 and occludin) at the protein and mRNA levels. The tumor trans-endothelial migration assay using breast adenocarcinoma cells along with endothelial cells demonstrated an increased metastatic potential following phthalate exposure. Conclusion. These findings offer crucial insights into the molecular mechanisms underlying phthalate exposure, suggesting potential targets for future brain metastasis treatments. Additionally, they hold implications for the future regulation and management of phthalate usage and policy. This work was supported by the University of Miami U-LINK Resilience Challenge grant. Citation Format: Destiny Tiburcio, Sarah Adolphe, Ijeoma B. Akpu, Timea Teglas, Oandy Naranjo, Sophia H. George, Michal J. Toborek. Phthalate exposure results in circadian rhythm-induced BBB dysfunction increasing the risk for breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 829.

Abstract 6284: Multimoics reveal aberrant pathways and regulation of castration resistant and metastatic prostate cancer

Abstract Prostate cancer is a highly heterogeneous disease and in the end, 30% of patients will experience metastatic and fatal castration resistant prostate cancer (mCRPC). In spite of the vigorous efforts to search the causes of metastatic CRPC (mCRPC), the mechanisms are still largely unresolved. To understand the progression of mCRPC, we utilized scRNA-seq to profile global expression patterns in LNCaP, LNCaP-Abl and PC3 representing castration sensitive and resistant states. ScRNA-seq revealed 335 and 2393 differentially expressed (DE) genes in early and advanced castration resistant states enriched in 43 and 163 aberrant pathways. These included focal adhesion, adherens junction, leukocyte transendothelial migration, ECM-receptor interaction, regulation of actins, gap junction, TGFb, WNT, Hippo, Ephrin and other pathways. Upregulation of these pathways predicted worst disease free/overall survival of The Cancer Genomes Atlas (TCGA) PC cohort using Kaplan-Meir estimation. Further scRNA-seq also indicated most of the pathways were also enriched in ex vivo circulating tumor cells derived from biochemical recurrent or mCRPC patients. Small molecule perturbation verified function of 9 pathways involved in cell proliferation, migration, invasion, and organoid formation of mCRPC cell lines and CTCs. We then delved into how these genes are regulated by the global chromatin landscape through Assay for Transposase-Accessible Chromatin and high throughput DNA-seq (ATAC-seq). In castration sensitive LNCaP, active chromatins are associated with genes but most of them are located in distal intergenic regions. Conversely, the majority (51.9-57.6%) of open chromatins in castration resistant PC cells are situated in promoters that lie within 1kb of transcription start sites. KEGG pathway enrichment analysis uncovered Hippo, Wnt, tight junction, adherens junction, circadian and other pathways that corroborate the scRNA-seq findings. GRO-seq data indicated that nascent transcription supports gene body transcription in castration resistant cells but leads to massive pausing in castration sensitive cells. Citation Format: Aaron M. Horning, Chia-Nung Hung, Brandon Liebermann, Chih-Wei Chou, Che-Kuang Lin, Yao Vickie Wang, Victor X. Jin, Michael A. Liss, Chiou-Miin Wang, Chun- Lin, Meizhen Chen, Tim H-M Huang, Chun-Liang Chen. Multimoics reveal aberrant pathways and regulation of castration resistant and metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6284.

Abstract 6763: A human-relevant vascularized organ-on-a chip platform for anti-tumor drug efficacy and liver injury assessment

Abstract Cancer global burden accounts for 19.3 million new cases and 10 million deaths in 2020. Despite advances on the understanding of disease progression and efforts to find successful therapeutic strategies, most drugs fail in clinical stages. Lack of models with clinical mimicry recapitulating the in vivo pathophysiology and the organ microenvironment where the tumor forms is a key driver reducing the success rate of anti-cancer drugs. Animal models remain the gold standard for preclinical research but fail to recapitulate the complex human tumor microenvironment, a critical component determining tumor development, progression, and response to treatments. Organ-on-a-chip models allow to control the environment and organization of cells in an in vivo-like arrangement by culturing multiple cell types to better replicate human conditions, and by implementing different biochemical and physical stimuli, such as fluid shear stress, chemical/nutrient gradients, and perfusion/flow to recapitulate the human vascular system physiology. We leverage our 3D bioprinting technologies to develop complex vascularized organ-on-a-chip models aimed to recapitulate the tumor and liver niche. Both organ-systems are interconnected by our h-VIOS™ platform, allowing dynamic perfusion for 21 days and enabling the simultaneous assessment of anti-tumor therapeutic efficacy and drug-induced liver injury. Our hydrogels feature a complex perfusable vasculature that interfaces with an interstitial chamber where tissues of interest are grown. The vasculature allows for continued and controlled delivery of nutrients, oxygen, and small molecule drugs. Dextran diffusion studies revealed diffusion coefficients ranging from 43.2 μm2/s (4kDa) to 8 μm2/s (70kDa). Vascular architectures are lined with endothelial cells forming a functional barrier expressing the junctional markers ZO-1 and PECAM-1. Moreover, we can modulate the permeability of our vasculature architecture, allowing the extravasation of immune and cancer cells. PBMC-derived T cells and Jurkat cells undergo transendothelial migration in response to a CXCL12 gradient, highlighting the applicability of our model for immunotherapeutic efficacy studies. The tumor interstitial chamber incorporates co-culture of cancer spheroids or tumoroids, fibroblasts, endothelial and immune cells. Colon carcinoma spheroids effectively integrate into the tumor niche with no growth arrest. The liver model consists of an interstitial chamber with a lobular microstructure supporting the monoculture of primary human hepatocytes, and sustaining their viability and function for 9 days as demonstrated by high albumin production, urea synthesis and CYP activity. Our organ-on-a-chip models are fully customizable and anticipated to contribute to advance personalized medicine and improve clinical translation rates. Citation Format: Juliana Navarro-Yepes, Queeny Dasgupta, Purboja Purkayastha, Kevin Janson, Cassio Mello, Ameya Narkar, Soon Seng Ng, Taci Pereira. A human-relevant vascularized organ-on-a chip platform for anti-tumor drug efficacy and liver injury assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6763.

Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

Infiltrative Vessel Co-optive Growth Pattern Induced by IQGAP3 Overexpression Promotes Microvascular Invasion in Hepatocellular Carcinoma.

Microvascular invasion (MVI) is a major unfavorable prognostic factor for intrahepatic metastasis and postoperative recurrence of hepatocellular carcinoma (HCC). However, the intervention and preoperative prediction for MVI remain clinical challenges due to the absent precise mechanism and molecular marker(s). Herein, we aimed to investigate the mechanisms underlying vascular invasion that can be applied to clinical intervention for MVI in HCC. The histopathologic characteristics of clinical MVI+/HCC specimens were analyzed using multiplex immunofluorescence staining. The liver orthotopic xenograft mouse model and mechanistic experiments on human patient-derived HCC cell lines, including coculture modeling, RNA-sequencing, and proteomic analysis, were used to investigate MVI-related genes and mechanisms. IQGAP3 overexpression was correlated significantly with MVI status and reduced survival in HCC. Upregulation of IQGAP3 promoted MVI+-HCC cells to adopt an infiltrative vessel co-optive growth pattern and accessed blood capillaries by inducing detachment of activated hepatic stellate cells (HSC) from the endothelium. Mechanically, IQGAP3 overexpression contributed to HCC vascular invasion via a dual mechanism, in which IQGAP3 induced HSC activation and disruption of the HSC-endothelial interaction via upregulation of multiple cytokines and enhanced the trans-endothelial migration of MVI+-HCC cells by remodeling the cytoskeleton by sustaining GTPase Rac1 activity. Importantly, systemic delivery of IQGAP3-targeting small-interfering RNA nanoparticles disrupted the infiltrative vessel co-optive growth pattern and reduced the MVI of HCC. Our results revealed a plausible mechanism underlying IQGAP3-mediated microvascular invasion in HCC, and provided a potential target to develop therapeutic strategies to treat HCC with MVI.

Inhibition of leukocyte migration after ischemic stroke by VE-cadherin mutation in a mouse model leads to reduced infarct volumes and improved motor skills.

To distinguish between the genuine cellular impact of the ischemic cascade by leukocytes and unspecific effects of edema and humoral components, two knock-in mouse lines were utilized. Mouse lines Y731F and Y685F possess point mutations in VE-cadherin, which lead to a selective inhibition of transendothelial leukocyte migration or impaired vascular permeability. Ischemic stroke was induced by a model of middle cerebral artery occlusion. Analysis contained structural outcomes (infarct volume and extent of brain edema), functional outcomes (survival analysis, rotarod test, and neuroscore), and the extent and spatial distribution of leukocyte migration (heatmaps and fluorescence-activated cell sorting (FACS) analysis). Inhibition of transendothelial leukocyte migration as in Y731F mice leads to smaller infarct volumes (52.33±4719 vs. 70.43±6483mm3 , p=.0252) and improved motor skills (rotarod test: 85.52±13.24s vs. 43.06±15.32s, p=.0285). An impaired vascular permeability as in Y685F mice showed no effect on structural or functional outcomes. Both VE-cadherin mutations did not influence the total immune cell count or spatial distribution in ischemic brain parenchyma. Selective inhibition of transendothelial leukocyte migration by VE-cadherin mutation after ischemic stroke in a mouse model leads to smaller infarct volumes and improved motor skills.

Proteomic analysis of plasma proteins from patients with cardiac rupture after acute myocardial infarction using TMT-based quantitative proteomics approach

BackgroundCardiac rupture (CR) is a rare but catastrophic mechanical complication of acute myocardial infarction (AMI) that seriously threatens human health. However, the reliable biomarkers for clinical diagnosis and the underlying signaling pathways insights of CR has yet to be elucidated.MethodsIn the present study, a quantitative approach with tandem mass tag (TMT) labeling and liquid chromatography–tandem mass spectrometry was used to characterize the differential protein expression profiles of patients with CR. Plasma samples were collected from patients with CR (n = 37), patients with AMI (n = 47), and healthy controls (n = 47). Candidate proteins were selected for validation by multiple reaction monitoring (MRM) and enzyme-linked immunosorbent assay (ELISA).ResultsIn total, 1208 proteins were quantified and 958 differentially expressed proteins (DEPs) were identified. The difference in the expression levels of the DEPs was more noticeable between the CR and Con groups than between the AMI and Con groups. Bioinformatics analysis showed most of the DEPs to be involved in numerous crucial biological processes and signaling pathways, such as RNA transport, ribosome, proteasome, and protein processing in the endoplasmic reticulum, as well as necroptosis and leukocyte transendothelial migration, which might play essential roles in the complex pathological processes associated with CR. MRM analysis confirmed the accuracy of the proteomic analysis results. Four proteins i.e., C-reactive protein (CRP), heat shock protein beta-1 (HSPB1), vinculin (VINC) and growth/differentiation factor 15 (GDF15), were further validated via ELISA. By receiver operating characteristic (ROC) analysis, combinations of these four proteins distinguished CR patients from AMI patients with a high area under the curve (AUC) value (0.895, 95% CI, 0.802–0.988, p < 0.001).ConclusionsOur study highlights the value of comprehensive proteomic characterization for identifying plasma proteome changes in patients with CR. This pilot study could serve as a valid foundation and initiation point for elucidation of the mechanisms of CR, which might aid in identifying effective diagnostic biomarkers in the future.

The Mechanism of Elizabethkingia miricola Infection of the Black Spotted Frog as Revealed by Multi-Omics Analysis

Elizabethkingia miricola (E. miricola) is a significant pathogen that causes the crooked head disease in black spotted frogs. This disease has plagued numerous frog farms in China and has resulted in substantial losses to the frog farming industry. Nonetheless, the exact mechanism that causes the disease in frogs remains unknown. In this study, transcriptomic and microbiomic analyses were conducted to analyze frog samples infected with E. miricola to reveal the infection mechanism of the pathogen. Liver transcriptomic analysis indicated that the livers of infected frogs had 1469 differentially expressed genes when compared with an uninfected group. These DEGs are mainly involved in immunity and metabolism, including neutrophil extracellular trap formation, the NOD-like receptor signaling pathway, leukocyte transendothelial migration, chemokine signaling pathway, Fc gamma R-mediated phagocytosis, and “metabolism”-related pathways such as the pentose phosphate pathway, carbon metabolism, glycerophospholipid metabolism, and glycerolipid metabolism. Similarly, 4737 DEGs were found in the kidney of infected frogs. These DEGs are mainly involved in immunity, including neutrophil extracellular trap formation, the NOD-like receptor signaling pathway, B cell receptor signaling pathway, C-type lectin receptor signaling pathway, complement and coagulation cascade, and Toll-like receptor signaling pathway. Ten immune-associated DEGs were screened in liver and kidney DEGs, respectively. And it was hypothesized that E. miricola infection could influence the host immune response. Microbiome analysis results showed that some opportunistic pathogens such as Citrobacter, Shigella, and Providencia were significantly elevated (p &lt; 0.05) in infected frogs. Additionally, functional prediction confirmed that most of the microbiota in infected frogs were linked to metabolism-related KEGG pathways. In this study, the screened genes linked to immunity showed an association with the gut microbiome. The majority of these genes were found to be linked with the abundance of opportunistic pathogens. The results showed that E. miricola infection led to the downregulation of immune and metabolic-related genes, which led to the inhibition of immune function and metabolic disorder, and then increased the abundance of opportunistic pathogens in the gut microbiota. The findings of this study offer a preliminary foundation for comprehending the pathogenic processes of E. miricola infection in black spotted frogs.

Discrete-state models identify pathway specific B cell states across diseases and infections at single-cell resolution

Oxygen (O2) regulated pathways modulate B cell activation, migration and proliferation during infection, vaccination, and other diseases. Modeling these pathways in health and disease is critical to understand B cell states and ways to mediate them. To characterize B cells by their activation of O2 regulated pathways we develop pathway specific discrete state models using previously published single-cell RNA-sequencing (scRNA-seq) datasets from isolated B cells. Specifically, Single Cell Boolean Omics Network Invariant-Time Analysis (scBONITA) was used to infer logic gates for known pathway topologies. The simplest inferred set of logic gates that maximized the number of “OR” interactions between genes was used to simulate B cell networks involved in oxygen sensing until they reached steady network states (attractors). By focusing on the attractors that best represented sequenced cells, we identified genes critical in determining pathway specific cellular states that corresponded to diseased and healthy B cell phenotypes. Specifically, we investigate the transendothelial migration, regulation of actin cytoskeleton, HIF1A, and Citrate Cycle pathways. Our analysis revealed attractors that resembled the state of B cell exhaustion in HIV+ patients as well as attractors that promoted anerobic metabolism, angiogenesis, and tumorigenesis in breast cancer patients, which were eliminated after neoadjuvant chemotherapy (NACT). Finally, we investigated the attractors to which the Azimuth-annotated B cells mapped and found that attractors resembling B cells from HIV+ patients encompassed a significantly larger number of atypical memory B cells than HIV− attractors. Meanwhile, attractors resembling B cells from breast cancer patients post NACT encompassed a reduced number of atypical memory B cells compared to pre-NACT attractors.

Endothelial dysfunction of syphilis: Pathogenesis.

Treponema pallidum is the causative factor of syphilis, a sexually transmitted disease (STD) characterized by perivascular infiltration of inflammatory cells, vascular leakage, swelling and proliferation of endothelial cells (ECs). The endothelium lining blood and lymphatic vessels is a key barrier separating body fluids from host tissues and is a major target of T. pallidum. In this review, we focus on how T. pallidum establish intimate interactions with ECs, triggering endothelial dysfunction such as endothelial inflammation, abnormal repairment and damage of ECs. In addition, we summarize that migration and invasion of T. pallidum across vascular ECs may occur through two pathways. These two mechanisms of transendothelial migration are paracellular and cholesterol-dependent, respectively. Herein, clarifying the relationship between T. pallidum and endothelial dysfunction is of great significance to provide novel strategies for diagnosis and prevention of syphilis, and has a great potential prospect of clinical application.

Human Breast Cancer Cell Lines Differentially Modulate Signaling from Distant Microenvironments, Which Reflects Their Metastatic Potential.

Metastasis is the stage at which the prognosis substantially decreases for many types of cancer. The ability of tumor cells to metastasize is dependent upon the characteristics of the tumor cells, and the conditioning of distant tissues that support colonization by metastatic cells. In this report, we investigated the systemic alterations in distant tissues caused by multiple human breast cancer cell lines and the impact of these alterations on the tumor cell phenotype. We observed that the niche within the lung, a common metastatic site, was significantly altered by MDA-MB-231, MCF7, and T47 tumors, and that the lung microenvironment stimulated, to differing extents, an epithelial-to-mesenchymal transition (EMT), reducing proliferation, increasing transendothelial migration and senescence, with no significant impact on cell death. We also investigated the ability of an implantable scaffold, which supports the formation of a distant tissue, to serve as a surrogate for the lung to identify systemic alterations. The scaffolds are conditioned by the primary tumor similarly to the lung for each tumor type, evidenced by promoting a pro-EMT profile. Collectively, we demonstrate that metastatic and non-metastatic breast cancers condition distant tissues, with distinct effects on tumor cell responses, and that a surrogate tissue can distinguish the metastatic potential of human breast cancer cell lines in an accessible site that avoids biopsy of a vital organ.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Abstract 16: Integrin α9β1 and VCAM-1 Interaction Promotes Neutrophil Hyperactivation and Cognitive Decline Following Subarachnoid Hemorrhage

Background: Integrin α9β1 is a transmembrane glycoprotein that is highly expressed on neutrophils and mediates transendothelial migration via vascular cell adhesion molecule-1 (VCAM-1). Neutrophils are major contributors to cerebral thrombosis and oxidative stress following subarachnoid hemorrhage (SAH); however, the mechanism by which neutrophil integrin α9 contributes to SAH pathogenesis is unclear. Here, we evaluated the mechanistic role of neutrophil integrin α9 in the post-SAH sensorimotor and cognitive recovery. Methods: SAH was induced by filament perforation in neutrophil-specific α9 -/- (α9 fl/fl Mrp8Cre +/- ) mice and littermate controls. Cognitive recovery (Y-maze, Barnes maze, and novel object recognition test) and sensorimotor recovery (modified neurological severity score, corner, and cylinder test) were performed for up to 4-weeks. Unbiased RNA sequencing (RNA-seq) was used to evaluate the potential mechanisms by which neutrophil integrin α9-VCAM-1 interactions promote SAH. Computational modeling, docking, virtual screening, and cell-free assays were used to identify small-molecule inhibitors of the integrin α9-VCAM-1 interactions. Results: Experimental SAH resulted in increased levels of neutrophil integrin α9 which was concomitant with neutrophil hyperactivation. Post-SAH neutrophil-specific α9 -/- mice exhibited improved sensorimotor and cognitive recovery, along with reduced plasma elastase and MPO levels and brain neutrophil infiltration. Mechanistically, integrin α9/VCAM-1 interactions promote neutrophil hyperactivation, extracellular signal-regulated kinase (ERK) phosphorylation, and endothelial cell apoptosis. RNA-seq data revealed that α9/VCAM-1 interactions promotes gene expression related to neutrophil inflammation, exocytosis, NF-κB signaling, and chemotaxis. Virtual screening and cell-free assay identified macitentan as a potent inhibitor of integrin α9-VCAM-1 interactions. In vitro, macitentan treatment decreased neutrophil adhesion to VCAM-1 and macitentan treatment at a dose of 5 mg/kg resulted in improved SAH outcomes. Conclusion: The results of this study describe a previously unrecognized pathway involving integrin α9 and VCAM-1 in the pathogenesis of SAH.

PKM2 is a Novel Osteoporosis-Associated Protein in Chinese

ABSTRACT Purpose: Osteoporosis is characterized by low bone mineral density (BMD) and high risk of osteoporotic fracture (OF). Peripheral blood monocytes (PBM) can differentiate into osteoclasts to resorb bone. This study was to identify PBM-expressed proteins significant for osteoporosis in Chinese Han elderly population (>65 years), and focused on two phenotypes of osteoporosis: low BMD and OF. Methods: Label-free quantitative proteomics was employed to profile PBM proteome and to identify differentially expressed proteins (DEPs) between OF (N=27) vs. non-fractured (NF, N=24) subjects and between low BMD (N=12) vs. high BMD (N=12) subjects in women. Western blotting (WB) was conducted to validate differential expression, and ELISA to evaluate translational value for secretory protein of interest. Results: We discovered 59 DEPs with fold change (FC)>1.3 (P<1×10-5), and validated the significant up-regulation of pyruvate kinase isozyme 2 (PKM2) with osteoporosis (P<0.001). PKM2 protein upregulation with OF was replicated with PBM in men (P=0.04). Plasma PKM2 protein level was significantly elevated with OF in an independent sample (N=100, FC=1.68, P=0.01). Pursuant functional assays showed that extracellular PKM2 protein supplement not only promoted monocyte trans-endothelial migration, growth, and osteoclast differentiation (marker gene expression), but also inhibited osteoblast growth, differentiation (ALP gene expression), and activity. Conclusion: The above findings suggest that PKM2 protein is a novel osteoporosis-associated functional protein in Chinese Han elderly population. It may serve as a risk biomarker and drug target for osteoporosis.

Abstract 102: Microanatomy of Intracranial Human Thrombi by Single Nuclei Transcriptomics

Introduction: Microvascular dysfunction and inflammation are thought to underlie reperfusion failure following endovascular thrombectomy. Here we present the first study to produce a transcriptome-based cellular landscape of human intracranial thrombi highlighting the importance of cellular plasticity and intercellular communication at the site of occlusion. Methods: We performed single nuclei RNA-sequencing (snRNA-seq) on intracranial human thrombi harvested during revascularization. Unsupervised clustering, data visualization, differential expression and functional gene ontology/pathway analysis were utilized to deconvolve expression data into cell type-specific expression profiles. Thrombi snRNA-seq data were integrated with single cell RNA-seq data from symptomatic and asymptomatic human carotid plaques and healthy brain vasculature to contextualize thrombi in atherosclerotic disease, perform diffusion coefficient analysis, and identify ligand-receptor interactions. Results: Unbiased clustering identified six cellular populations in distinct effector states within the thrombus microenvironment. Functional gene ontology and pathway analysis revealed a monocyte subpopulation in a distinct endothelial-activation and neuroplasticity state, supporting the notion of cellular trans-differentiation and interconversion. Integration with carotid plaque data and diffusion coefficient analysis revealed shared transcriptional programs existing in a continuum, progressing from asymptomatic plaque to embolized thrombus. Ligand-receptor analysis identified macrophage mediated T-cell activation, pro-inflammatory response, immunological synapse stabilization, neuroplasticity, and monocyte trans-endothelial migration. Conclusion: Our novel application of snRNA-seq to thrombectomy specimens provides the highest resolution exploration of cellular differences and a best understanding of individual contextualized cell function to date. This detailed cellular landscape will facilitate mapping novel interventional targets with direct functional relevance and allow for greater personalization of post-reperfusion therapy.

Mechanistic investigation of quercetin in the management of diabetic foot ulcer by network pharmacology and molecular docking

BackgroundQuercetin (QUE), being a strong antioxidant, has been a part of Chinese traditional medicine for ages for the management of a large number of ailments including type 2 diabetes mellitus and its complications, primarily, Diabetic Foot Ulcer (DFU), but it is under lying mechanisms remain elusive. The introduction of network pharmacology and molecular docking has made it facile to investigate the mechanism of QUE-containing different plants and to validate the binding affinity of QUE with DFU protein respectively. AimThis study is aimed to investigate the mechanism of QUE in protecting against DFU through modern approaches viz. network pharmacology and molecular docking. Materials and methodsThe traditional Chinese medicine system pharmacology analysis platform (TCMSP) has been used to retrieve the active ingredients in various Chinese formulations. QUE was found to be a bioactive component of various Chinese formulations. The core therapeutic targets of QUE against DFU were identified using a protein–protein interaction (PPI) network. QUE-associated targets and genes related to DFU were obtained from different comprehensive databases (Pubchem, Swiss target prediction database, SEA database, String database, DisGeNET database, Molegro virtual docker version 6.0, ADMET lab2.0, SwissADME database) were intersected, and analyzed to obtain mapping targets and ranked to obtain key targets. ResultsThe Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway shows major pathways were obtained from for QUE was matrix metallopeptidase 9 (MMP9). Receptor-ligand interaction between QUE with MMP9 were evaluated via MVD 6.0 software. A total of 10 interactions of quercetin with the following moldock score, rerank score, and H-bond interactions were observed as -110.912, -37.33, and -12.11 respectively. Candidate targets MMP9 in diabetic complications were ascertained and found associated with QUE against the management of DFU. Molecular docking models showed that QUE could closely bind to the key targets MMP9 (PDB id: 2OVX). ADME/T Analysis is performed to check the pharmacokinetic and toxicity profile which complies with the ideal values. ConclusionsThis study proposed MMP9, as the key target of bioactive QUE in mechanistic control of DFU by lowering the activity of the IL-17 signalling pathway, Leukocyte trans-endothelial migration, Fluid shear stress, and atherosclerosis respectively. Future aspectsThe explication of mode of action of bioactive constituent QUE against DFU provide a theoretical basis for designing more promising agents further for treatment of DFU in diabetic patients.

INFLAMED INTESTINAL ENDOTHELIAL CELLS ESTABLISH SELF-REGULATORY NICHES VIA IL16 DEPENDENT RECRUITMENT OF CD4 EXPRESSING VESSEL-ASSOCIATED MACROPHAGES

Abstract Macrophages, play important roles in initiation and resolution of inflammation. Notably, a subset of tissue macrophages strategically localizes to the blood vessels, exerting essential regulatory functions at the blood-tissue interface, including regulation of vascular permeability and neutrophil (PMN) transendothelial migration (TEM). Although the number of vessel-associated macrophages (VAMs) is increased in inflamed tissue, the underlying mechanisms of VAMs recruitment are unknown. Our current study uncovers a novel mechanism whereby inflamed intestinal vascular endothelial cells (ECs) release IL-16 to selectively recruit CD4+ tissue macrophages to become VAMs. Whole-mount confocal microscopy studies in CX3CR1-EGFP macrophages reporter mice confirmed significant increases in VAMs numbers in several murine models of intestinal inflammation. Surprisingly, the majority of VAMs were found to express a classical T cell marker, CD4 and to be of an inflammatory phenotype, enriched for ICAM-1 and TNFα expression. VAMs-derived TNFα was previously found to prime ECs to form PMN TEM hot spots. Whole genome mRNA sequencing, and flow cytometry protein expression analyses revealed heightened expression of the CD4 ligand, IL-16 by inflamed intestinal lamina propria ECs. In vitro, using transwell assays, recombinant IL-16 and supernatants derived from murine (bEnds.3) endothelial cells induced robust migration of murine, LPS/IFNγ-stimulated bone marrow-derived macrophages. Whereas antibody-mediated IL-16 or CD4 neutralization in cultures and in vivo reduced VAMs recruitment. Collectively, our findings establish CD4 as a novel marker of VAMs and identify the regulatory role of ECs IL-16 and macrophages CD4+ axis in VAMs recruitment in inflamed mucosal tissues.

Diacylglycerols and Lysophosphatidic Acid, Enriched on Lipoprotein(a), Contribute to Monocyte Inflammation.

Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1β after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.