Adhesion Molecule-1 sVCAM-1 Research Articles

P1174 Engineering therapeutic Tregs to overexpress GPR15 improves functional fitness for in vivo gut homing

Abstract Background Inflammatory bowel diseases (IBD) like Crohn’s disease and ulcerative colitis, are marked by an imbalance of pro- and anti-inflammatory T cells in the lamina propria of the intestinal tract. Only subgroups of patients respond to current therapeutic approaches designed to restrain pro-inflammatory cells or signalling. Adoptive transfer of autologous ex vivo expanded regulatory T cells (Tregs) has previously been suggested as a promising future therapeutic approach to resolve chronic intestinal inflammation by promoting anti-inflammatory pathways. Methods Using a previously established protocol for the ex vivo expansion of Tregs (Investigational Medicinal Product-Tregs; IMP-Tregs), we characterized the migration and homing capabilities as well as the immunosuppressive function of non-expanded and IMP-Tregs using 3D-motility, in vivo gut homing and T cell suppression assays. Furthermore, via electroporation with GPR15 mRNA, IMP-Tregs were engineered to express the gut homing marker GPR15 and tested for their functional fitness in dynamic adhesion, T cell suppression assays as well as in vivo gut homing assays. Results Our data show that while the expansion protocol generates highly suppressive Tregs, only a fraction of them is equipped with surface molecules for gut homing. In order to overcome this limitation, we successfully engineered IMP-Tregs to express the gut homing marker GPR15. On a functional level, this led to improved adhesion to the cell adhesion molecules MAdCAM-1 and VCAM-1, which are expressed on the intestinal endothelium, as well as to increased gut homing in a humanized in vivo mouse model without affecting the immunosuppressive character of the engineered IMP-Tregs. Conclusion In conclusion, our data indicate superior functional fitness of GPR15-engineered IMP-Tregs for homing to the inflamed gut nominating them a promising further development of autologous Treg transfer therapy for IBD.

CD93 blockade promotes effector T-cell infiltration and facilitates adoptive cell therapy in solid tumors

BackgroundAdaptive cellular therapy (ACT), particularly chimeric antigen receptor (CAR)-T cell therapy, has been successful in the treatment of hemopoietic malignancies. However, poor trafficking of administered effector T cells to the...

Single-cell transcriptomics identifies the common perturbations of monocyte/macrophage lineage cells in inflammaging of bone marrow.

Bone marrow inflammaging is a low-grade chronic inflammation that induces bone marrow aging. Multiple age-related and inflammatory diseases involve bone marrow inflammaging. Whether common pathological pathways exist in bone marrow inflammaging remains unclear. We collected bone marrow from telomerase-deficient mice (telomerase RNA component, TERCko/ko), 5×FAD mice and Dmp1 Cre -DTA ki/wt mice and High-fat diet-fed mice (HFD), and lumbar 5 nerve compression mice. We performed scRNA-Seq analysis on bone marrow obtained from these mouse models to investigate the potential shared pathway of bone marrow inflammation. We identified the monocyte/macrophage lineage was activated via the App-Cd74 axis in multiple aging and inflammatory mouse models. Increased expression of CD38 and Ly6a, and decreased expression of Col1a and Lif in macrophages serve as shared changes in different mouse models. The activated macrophages, interacting with other cells, control the expansion of B cells via the CD52-Siglec-G axis. The Ccl6-Ccr2 and Ccl9-Ccr1 ligand-receptor pairs, along with Fn1 and C3-related pathways in macrophages, were associated with immune cell activation and the recruitment of lymphocytes. Interactions with mesenchymal cells were enriched for integrins (Itga4), Fn1, and adhesion molecules (Vcam1). Our study demonstrates that monocyte/macrophage lineage stimulation is a key event in bone marrow inflammaging. We identified common differentially expressed genes and activated pathways in this lineage, suggesting potential targets for future interventions. Our study revealed shared genes and ligand-receptor pairs in the activated monocyte/macrophage lineage within inflammaging bone marrow. These findings offer potential therapeutic targets for cell-specific anti-inflammatory treatments.

GPR68 Mediates Lung Endothelial Dysfunction Caused by Bacterial Inflammation and Tissue Acidification.

Tissue acidification resulting from dysregulated cellular bioenergetics accompanies various inflammatory states. GPR68, along with other members of proton-sensing G protein-coupled receptors, responds to extracellular acidification and has been implicated in chronic inflammation-related diseases such as ischemia, cancer, and colitis. The present study examined the role of extracellular acidification on human pulmonary endothelial cell (EC) permeability and inflammatory status per se and investigated potential synergistic effects of acidosis on endothelial dysfunction caused by bacterial lipopolysaccharide (LPS, Klebsiella pneumoniae). Results showed that medium acidification to pH 6.5 caused a delayed increase in EC permeability illustrated by a decrease in transendothelial electrical resistance and loss of continuous VE-cadherin immunostaining at cell junctions. Likewise, acidic pH induced endothelial inflammation reflected by increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1, upregulated mRNA transcripts of tumor necrosis factor-α, IL-6, IL-8, IL-1β, and CXCL5, and increased secretion of ICAM-1, IL-6, and IL-8 in culture medium monitored by ELISA. Among the GPCRs tested, acidic pH selectively increased mRNA and protein expression of GPR68, and only the GPR68-specific small molecule inhibitor OGM-8345 rescued acidosis-induced endothelial permeability and inflammation. Furthermore, acidic pH exacerbated LPS-induced endothelial permeability and inflammatory response in cultured lung macrovascular as well as microvascular endothelial cells. These effects were suppressed by OGM-8345 in both EC types. Altogether, these results suggest that GPR68 is a critical mediator of acidic pH-induced dysfunction of human pulmonary vascular endothelial cells and mediates the augmenting effect of tissue acidification on LPS-induced endothelial cell injury.

Reversal of Endothelial Cell Anergy by T Cell-Engaging Bispecific Antibodies.

Objectives: Reduced expression of adhesion molecules in tumor vasculature can limit infiltration of effector T cells. To improve T cell adhesion to tumor endothelial cell (EC) antigens and enhance transendothelial migration, we developed bispecific, T-cell engaging antibodies (bsAb) that activate T cells after cross-linking with EC cell surface antigens. Methods: Recombinant T-cell stimulatory anti-VEGFR2-anti-CD3 and costimulatory anti-TIE2-anti-CD28 or anti-PD-L1-anti-CD28 bsAb were engineered and expressed. Primary lines of human umbilical vein endothelial cells (HUVEC) that constitutively express VEGFR2 and TIE2 growth factor receptors and PD-L1, but very low levels of adhesion molecules, served as models for anergic tumor EC. Results: In cocultures with HUVEC, anti-VEGFR2-anti-CD3 bsAb increased T cell binding and elicited rapid T cell activation. The release of proinflammatory cytokines TNF-α, IFN-γ, and IL-6 was greatly augmented by the addition of anti-TIE2-anti-CD28 or anti-PD-L1-anti-CD28 costimulatory bsAb. Concomitantly, T cell-released cytokines upregulated E-selectin, ICAM1, and VCAM1 adhesion molecules on HUVEC. HUVEC cultured in breast cancer cell-conditioned medium to mimic the influence of tumor-secreted factors were similarly activated by T cell-engaging bsAb. Migration of T cells in transwell assays was significantly increased by anti-VEGFR2-anti-CD3 bsAb. The combination with costimulatory anti-TIE2-anti-CD28 bsAb augmented activation and proliferation of migrated T cells and their cytotoxic capacity against spheroids of the MCF-7 breast cancer cell line seeded in the lower transwell chamber. Conclusions: T cells activated by anti-VEGFR2-anti-CD3 and costimulatory EC-targeting bsAb can reverse the energy of quiescent EC in vitro, resulting in improved T cell migration through an EC layer.

Endothelial dysfunction in combination of occupational pathology of the respiratory system and atherosclerosis in coal industry workers

Introduction. The increasing prevalence of cardiovascular pathology among industrial workers determines the importance of studying not only occupational diseases, but also their combination with diseases of the circulatory system. Endothelial dysfunction is a key link in the pathogenesis of atherosclerosis. Evaluation of endothelial function indicators in occupational respiratory diseases in combination with atherosclerosis in workers of the coal mining industry is an urgent task. The study aims to consider the indicators of endothelial function in miners with occupational respiratory diseases in combination with atherosclerosis. Materials and methods. The authors have examined 88 miners with previously diagnosed occupational respiratory diseases (chronic dust bronchitis, anthracosilicosis, chronic obstructive pulmonary disease), 45 of them showed signs of atherosclerosis. There were two comparison groups without signs of atherosclerosis: 44 miners who work long-term in harmful working conditions, without lung pathology (control group) and 42 residents of Novokuznetsk who have never worked in harmful working conditions, and without lung pathology (unexposed group). Results. We found that miners with occupational respiratory diseases in combination with atherosclerosis had several forms of endothelial dysfunction: vasomotor (decreased synthesis of nitric oxide, increased endothelin-1 levels), hemostatic (increased levels of Willebrand factor), adhesive (increased levels of endothelial adhesion molecules sVCAM-1). The development of respiratory insufficiency in miners with occupational respiratory diseases increases endothelial dysfunction: miners with respiratory insufficiency have lower levels of nitric oxide (NO), higher levels of endothelin-1 and endothelial adhesion molecules sVCAM-1 than miners without respiratory insufficiency. There are correlations between indicators of endothelial function and production factors: with work experience in harmful working conditions, the level of the average concentration of coal-rock dust in the workplace and the frequency of exceeding the maximum permissible concentration of coal-rock dust. Limitations. The study is limited to a sample of workers in the main professions of coal mines who were examined at the Research Institute of Complex Hygiene Problems and Occupational Diseases, aged 40 to 54 years, with more than 15 years of experience working in underground dust conditions. Conclusion. Miners with occupational respiratory diseases in combination with atherosclerosis develop several forms of endothelial dysfunction: vasomotor, hemostatic, adhesive. The development of respiratory failure increases endothelial dysfunction. Ethics. This medical study involving a human as a subject was carried out in compliance with the Ethical principles presented in the latest version of the Helsinki Declaration, developed by the World Medical Association.

Non-HLA Autoantibodies Against Angiotensin II Receptor 1 (AT1R) and Endothelin A Receptor (ETAR) in Pediatric Kidney Transplantation.

Antibody-mediated rejection (AMR) is the leading cause of premature kidney transplant failure. The role of alloantibodies against Human Leukocyte Antigens (HLA) has been a primary focus in AMR. More recently autoantibodies and alloantibodies against the angiotensin II receptor type 1 (AT1R) and the endothelin A receptor (ETAR) have been linked to poor allograft outcomes in kidney transplantation. Nevertheless, evidence supporting routine testing remains insufficient. ELISA testing for anti-AT1R and anti-ETAR antibodies was performed in a pediatric renal transplant cohort. We selected 12 pediatric recipients who had undergone protocol biopsies and antibody measurements at 6 and 24 months post-transplant. Immunohistochemistry was performed on biopsies for AT1R and ETAR as well as the adhesion molecules ICAM-1 and VCAM-1. The analysis showed that ICAM-1 and VCAM-1 expression was significantly increased, along with the presence of circulating antibodies, in patients at 24 months post-transplant compared to patients without circulating antibodies. The presence of anti-AT1R and anti-ETAR antibodies does not seem to influence the expression of their receptors in the transplanted organ. Instead, the increase in adhesion molecules may precede the development of histological damage. Therefore, enlarging the cohort and extending long-term observation would help to understand the impact of anti-AT1R and anti-ETAR antibodies after transplantation.

Cholesterol binding to VCAM-1 promotes vascular inflammation.

Hypercholesterolemia has long been implicated in endothelial cell (EC) dysfunction, but the mechanisms by which excess cholesterol causes vascular pathology are incompletely understood. Here we used a cholesterol-mimetic probe to map cholesterol-protein interactions in primary human ECs and discovered that cholesterol binds to and stabilizes the adhesion molecule VCAM-1. We show that accessible plasma membrane (PM) cholesterol in ECs is acutely responsive to inflammatory stimuli and that the nonvesicular cholesterol transporter Aster-A regulates VCAM-1 stability in activated ECs by controlling the size of this pool. Deletion of Aster-A in ECs increases VCAM-1 protein, promotes immune cell recruitment to vessels, and impairs pulmonary immune homeostasis. Conversely, depleting cholesterol from the endothelium in vivo dampens VCAM-1 induction in response to inflammatory stimuli. These findings identify cholesterol binding to VCAM-1 as a key step during EC activation and provide a biochemical explanation for the ability of excess membrane cholesterol to promote immune cell recruitment to the endothelium.

Brain ischemia causes systemic Notch1 activity in endothelial cells to drive atherosclerosis

Stroke leads to persistently high risk for recurrent vascular events caused by systemic atheroprogression that is driven by endothelial cell (EC) activation. However, whether and how stroke induces sustained pro-inflammatory and proatherogenic endothelial alterations in systemic vessels remain poorly understood. We showed that brain ischemia induces persistent activation, the upregulation of adhesion molecule VCAM1, and increased senescence in peripheral ECs until 4weeks after stroke onset. This aberrant EC activity resulted from sustained Notch1 signaling, which was triggered by increased circulating Notch1 ligands DLL1 and Jagged1 after stroke in mice and humans. Consequently, this led to increased myeloid cell adhesion and atheroprogression by generating a senescent, pro-inflammatory endothelium. Notch1- or VCAM1-blocking antibodies and the genetic ablation of endothelial Notch1 reduced atheroprogression after stroke. Our findings revealed a systemic machinery that induces the persistent activation of peripheral ECs after stroke, which paves the way for therapeutic interventions or the prevention of recurrent vascular events following stroke.

C-Geranylated flavanone diplacone enhances in vitro antiproliferative and anti-inflammatory effects in its copper(II) complexes

Two copper(II) complexes containing diplacone (H4dipl), a naturally occurring C-geranylated flavanone derivative, in combination with bathophenanthroline (bphen) or 1,10-phenanthroline (phen) with the composition [Cu3(bphen)3(Hdipl)2]⋅2H2O (1) and {[Cu(phen)(H2dipl)2]⋅1.25H2O}n (2) were prepared and characterized. As compared to diplacone, the complexes enhanced in vitro cytotoxicity against A2780 and A2780R human ovarian cancer cells (IC50 ≈ 0.4–1.2 μM), human lung carcinoma (A549, with IC50 ≈ 2 μM) and osteosarcoma (HOS, with IC50 ≈ 3 μM). Cellular effects of the complexes in A2780 cells were studied using flow cytometry, covering studies concerning cell-cycle arrest, induction of cell death and autophagy and induction of intracellular ROS/superoxide production. These results uncovered a possible mechanism of action characterized by the G2/M cell cycle arrest. The studies on human endothelial cells revealed that complexes 1 and 2, as well as their parental compound diplacone, do possess anti-inflammatory activity in terms of NF-κB inhibition. As for the effects on PPARα and/or PPARγ, complex 2 reduced the expression of leukocyte adhesion molecules VCAM-1 and E-selectin suggesting its dual anti-inflammatory capacity. A wide variety of Cu-containing coordination species and free diplacone ligand were proved by mass spectrometry studies in water-containing media, which might be responsible for multimodal effect of the complexes.

MXRA7 is involved in monocyte-to-macrophage differentiation

Macrophages are critical in mediating immune and inflammatory responses, while monocyte-to-macrophage differentiation is one of the main macrophage resources that involves various matrix proteins. Matrix remodeling associated 7 (MXRA7) was recently discovered to affect a variety of physiological and pathological processes related to matrix biology. In the present study, we investigated the role of MXRA7 in monocyte-to-macrophage differentiation in vitro. We found that knockdown of MXRA7 inhibited the proliferation of THP-1 human monocytic cells. Knockdown of MXRA7 increased the adhesion ability of THP-1 cells through upregulation the expression of adhesion molecules VCAM-1 and ICAM1. Knockdown of MXRA7 alone could promoted the differentiation of THP-1 cells to macrophages. Furthermore, the MXRA7-knockdown THP-1 cells produced a more significant upregulation pattern with M1-type cytokines (TNF-α, IL-1β and IL-6) than with those M2-type molecules (TGF-β1 and IL-1RA) upon PMA stimulation, indicating that knockdown of MXRA7 facilitated THP-1 cells differentiation toward M1 macrophages. RNA sequencing analysis revealed the potential biological roles of MXRA7 in cell adhesion, macrophage and monocyte differentiation. Moreover, MXRA7 knockdown promoted the expression of NF-κB p52/p100, while PMA stimulation could increase the expression of NF-κB p52/p100 and activating MAPK signaling pathways in MXRA7 knockdown cells. In conclusion, MXRA7 affected the differentiation of THP-1 cells toward macrophages possibly through NF-κB signaling pathways.

Abstract 3144: Mitochondrial Rnas Regulate Endothelial Function Through Nuclear Transcription

Mitochondrial-nuclear communication is vital for cellular homeostasis and response to environmental stress. The known messengers for mitochondria to communicate their functional status to the nucleus include ATP, ROS, and Ca 2+ . It remains unclear whether the immediate output of mitochondrial transcription, i.e., the mitochondrial RNAs (mtRNAs), are a type of messenger to communicate to the nucleus. Herein, we show that mt RNAs are attached to the nuclear genome and thus constitute a subset of the c hromatin- a ssociated RNA (mt-caRNA). Mt-caRNAs preferentially attach to promoter regions and the attachment levels change in response to cellular stress, i.e. high glucose and TNFα (HT). We identify the mitochondrial non-coding RNA SncmtRNA as a mt-caRNA. In human endothelial cells (ECs), suppression of SncmtRNA attenuates HT stress induction of nascent RNA transcribed from the nuclear genome, including the cell adhesion molecules ICAM1 and VCAM1, and abolishes stress-induced monocyte-EC adhesion. In addition to SncmtRNA, we show nuclear localization of other mtRNAs such as mt-CYB and mt-ND5, which is increased in the diabetic human condition. Collectively, our findings suggest the potential involvement of many mtRNAs in mitochondrial-nuclear communications and that mt-caRNAs may regulate nuclear transcription and thus play a role in mediating cellular response in health and disease.

The TNF superfamily factor TWEAK promotes colonic inflammatory fibroblast differentiation and fibroblast-monocyte interaction

Background and Aims: Colonic fibroblasts have emerged as key players in inflammatory bowel disease (IBD) for their potential to acquire a pro-inflammatory phenotype, but the factors driving this process and how these fibroblasts interact with the immune system is incompletely understood. The TNF superfamily factor 12 (TNFSF12/TWEAK) has gained interest as a potential mediator of chronic inflammation and tissue remodelling in IBD. Here we explore its role as a driver of inflammatory fibroblast differentiation and as a modulator of fibroblast-monocyte interaction. Methods: Human primary colonic fibroblasts were stimulated with TWEAK or vehicle for 24 hours. fibroblasts were co-cultured with THP1 cells or primary monocytes for an additional 24 hours to investigate their interaction. RNA sequencing, immunoblotting, ELISA and flow cytometry were used for analysis. Results: TWEAK up-regulated the expression of >600 and down-regulated the expression of >500 genes in primary colonic fibroblasts (pAdj<0.05, fold>1.5). The up-regulated transcriptome was characterised by a pro-inflammatory programme including numerous cytokines, chemokines and immune receptors, which resembled an inflammatory stroma subpopulation previously identified in in ulcerative colitis (29% transcriptional alignment). The shared transcriptome included a marked signature of non-canonical NF-κB genes. At protein level, TWEAK inflicted changes in the expression of stromal markers (PDPN, PDGFRα); stimulated the secretion of IL-6, CCL2 and CXCL10; And enhanced the expression of surface adhesion molecules ICAM-1 and VCAM-1. Co-culture of THP1 cells with fibroblasts induced their adhesion and promoted a CD14high/ICAM-1high phenotype in THP1 cells, both of which were further enhanced when fibroblasts were pre-stimulated with TWEAK, while TWEAK did not directly affect adhesion or differentiation of THP1 cultured alone. Moreover, treatment of fibroblasts with a small molecule inhibitor of non-canonical NF-κB partially abrogated the fibroblast-mediated increase in CD14 expression on THP1 cells. In primary monocytes extracted from whole blood of 7 human donors, co-culture with TWEAK-treated fibroblasts promoted differentiation to a CD16intermediate, TREM-1high phenotype. Moreover, media from TWEAK-treated fibroblast-monocyte co-cultures displayed elevated levels of CXCL1 and IL-8 compared to vehicle-treated co-cultures, or to either cell type cultured alone, pointing towards reciprocal pro-inflammatory crosstalk. Conclusions: Our results indicate that TWEAK can promote pro-inflammatory polarisation in colonic fibroblasts and positions TWEAK as a mediator in the pro-inflammatory interaction between colonic fibroblasts and monocytes. This work is supported by funding from Science Foundation Ireland and The Irish Research Council under the Pathway program (21/PATH-S/9621), Enterprise Ireland, and the Conway Institute Director’s Strategic Award. 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.

Deciphering the Role of LIGHT/TNFSF14 in Driving Inflammatory Activity in Human Lung Fibroblasts

Abstract LIGHT/ tumor necrosis factor superfamily member 14 (TNFSF14), is a protein that we previously found was central to lung inflammation and tissue fibrosis in mouse models of severe asthma and systemic sclerosis. We have hypothesized that one of its cellular targets involved in airway remodelling is the lung fibroblast given that both of its receptors, LTβR and HVEM are strongly expressed by these cells. With RNA-seq, we investigated the activity of LIGHT in these cells compared to other important inflammatory cytokines involved in remodeling in lung diseases, namely IL13, IL17A, and TGFβ. LIGHT induced a distinct set of inflammatory gene transcripts in human lung fibroblasts, including the adhesion molecules ICAM-1 and VCAM-1; cytokines IL32, IL33 and IL34; chemokines CXCL1, CXCL5, CXCL6 and CXCL10; and matrix metalloproteinases MMP9 and ADAM8. LIGHT with IL13 synergistically induced CCL2, CCL26, IL32 and ADAM8. LIGHT with IL17A also strongly boosted expression of several chemokines CXCL1, CXCL2, CXCL5, and CXCL6. Lastly, in fibroblasts differentiated into myofibroblasts by TGFβ pre-treatment, LIGHT enhanced the expression of many inflammatory and profibrotic transcripts such as ITGA1, ITGA11, MYH11, and Col1A1. This study highlights the unique as well as synergistic activities of LIGHT on these cells and suggests it could drive strongly inflammatory and remodeling effects in these cells relevant for several diseases dependent on the cytokine environment found in the lungs.

Abstract 1117: Il-1 Signaling On Vascular Smooth Muscle Cells Accelerates Atherosclerosis After Murine Kawasaki Disease Vasculitis

Objective: We have previously shown that Lactobacillus casei cell wall extract (LCWE)-induced Kawasaki disease (KD) vasculitis significantly accelerates atherosclerosis in hypercholesterolemic mice on high fat diet. IL-1 signaling is known to play a key role in both KD vasculitis and in the development of atherosclerosis. Here, we investigated the contribution of IL-1 signaling on vascular smooth muscle cells (VSMCs) in KD vasculitis-induced acceleration of atherosclerosis. Methods: Tamoxifen-inducible VSMC-specific IL-1 receptor ( Il1r1) knockout ( Myh11 Cre -ERT2 Il1r1 Δ/Δ ) mice and Il1r1 fl/fl littermate controls, all on ApoE -/- background, were injected with either PBS or LCWE. Following induction of KD vasculitis for 2 weeks, mice were fed a tamoxifen diet for 2 weeks to induce Il1r1 deletion on VSMCs, before being exposed to 8 weeks of Western diet to promote atherosclerosis. Results: KD vasculitis was associated with a significant acceleration of atherosclerosis, as expected. Myh11 Cre -ERT2 Il1r1 Δ/Δ mice had significantly diminished atherosclerotic plaque size, lipid composition, macrophage infiltration and necrotic core formation in aortic root as well as diminished lipid accumulation in aorta en face measurements compared with Il1r1 fl/fl control mice despite similar cholesterol levels. We also observed that Myh11 Cre -ERT2 Il1r1 Δ/Δ mice had significantly diminished endothelial adhesion molecules VCAM-1 and ICAM-1 expression in the lesion area and serum monocyte chemotaxis protein-1 (MCP-1) level compared with Il1r1 fl/fl control mice, consistent with the reduction of macrophage infiltration that we observed. Conclusions: Our results suggest an important pathophysiologic link between IL-1 signaling, specifically on VSMCs, and subsequent acceleration of atherosclerosis in hypercholesterolemic mice following KD vasculitis. Thus, further studies are warranted on the role of IL-1 signaling not only in acute KD, but also in the subsequent vascular remodeling and complications following acute KD vasculitis including atherosclerosis.

Abstract 3988: Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics

Abstract The most well-described mechanism of immune evasion in PDAC is exclusion of immune cells by a desmoplastic stroma, but such immune exclusion is not uniform. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of patient tumors, we previously reported marked heterogeneity in the immune composition of PDAC. Here, we developed a novel multi-omics workflow integrating single cell spatial proteomics (96-plex, Akoya Phenocycler®-Fusion) and transcriptomics (990-plex, Nanostring CosMx) in primary patient PDAC tumors to systematically study spatial relationships and functional states of malignant, stromal, and immune cells. Consecutive sections from four independent tumors enabled whole slide proteomic assessment of >3.5M cells, and transcriptomic assessment of >200K cells. Strikingly, we observed an abundance of immune cell aggregates in all tissue sections, up to 57 within a 1 cm2 tumor section. Aggregates were diverse in composition with representation of myeloid cells, CD8+ T cells, and stromal cells, including fibroblasts and nerves. B/T lymphoid aggregates were well vascularized with co-localized CD31+CD34+ endothelial cells and appeared at various phases of maturation towards putative tertiary lymphoid structures (TLSs) containing CD38+ B cells, CD4+ T cells, and CD21+ follicular DCs. These tissues were enriched for interferon stimulated genes in T cells and type 1 interferon response signatures in malignant cells and cancer associated fibroblasts (CAFs) forming inflammatory multicellular hubs that have been associated with improved response to immune checkpoint blockade. Surprisingly, B cells, in addition to macrophages and CD8+ T cells, were major producers of interferon gamma (B cells: 13.7%, macrophages: 36.6%, CD8+ T cells: 12.1% of IFNG+ cells). Finally, to gain insight into the necessary factors for TLS formation in PDAC, we quantified the expression of chemokines and adhesion molecules associated with TLS initiation. Activated dendritic cells expressed CCL19 and CCL21, endothelial cells expressed CCL21, and in concert with inflammatory CAFs (iCAFs), CXCL12. Malignant cells expressed CXCL13 and CCL20 and displayed high levels of cell adhesion molecules ICAM1, VCAM1, and ICAM2. Together, our data suggests that the functions of lymphoid tissue organizer cells can be substituted by endothelial cells, dendritic cells, iCAFs, and, surprisingly, malignant cells. Our spatial immunophenotyping study of pancreatic cancer thus highlights the marked immune heterogeneity of the disease and suggests a central role for malignant and stromal cells in mediating diverse mechanisms of immune evasion and engagement. Functional studies will be conducted to investigate these mechanisms of tumor-immune crosstalk and identify novel immunotherapeutic vulnerabilities for this deadly disease. Citation Format: Dennis Gong, Jingyi Cao, Peter Wang, Niyati Jhaveri, Yue Hou, Bassem B. Cheikh, HaYeun Ji, Mark Gregory, Jason Reeves, Michael Patrick, Carina Shiau, Jennifer Su, Jimmy A. Guo, Joseph M. Beechem, Martin Hemberg, William L. Hwang, Ryan Park. Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics [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 3988.

Modification of Cardiovascular Risk Factors and the Evolution of The Cardiovascular Phenotype of Liver Transplant Recipients in the Long -Term Postoperative Period.

To evaluate the contribution of traditional and additional cardiovascular risk factors (CVRFs) to the development of chronic ischemic heart disease (CIHD) in liver transplant recipients during the long-term postoperative period. A single-center prospective cohort study was conducted. The study included 740 patients with chronic end-stage liver disease (CESLD) and cirrhotic cardiomyopathy (CCMP). During the observation period (5.4±2.29 years), patients were divided into two groups: liver transplant recipients (n=420) and patients with CESLD on the waiting list who did not receive a donor organ (n=320). In patients enrolled to the study upon inclusion in the waiting list, CVRFs, history, clinical and laboratory and instrumental data were studied at all stages of the hepato-cardiac continuum. During the long-term postoperative period, liver transplant recipients belonged to the group of high cardiovascular risk: over a 5-year observation period, 35.7% (n=150) of them developed metabolic syndrome (MS), 9.8% developed verified CIHD associated with MS. The incidence of traditional CVRFs was high (arterial hypertension, 88.6%; obesity, 36.6%; hypercholesterolemia, 77.8%; hypertriglyceridemia, 43.6%; reduced concentration of high-density lipoprotein cholesterol, 35.4%; increased concentrations of low-density lipoprotein cholesterol, 66.8% and very low-density lipoprotein cholesterol, 51.2%; increased atherogenic index, 61.5%). During the long-term postoperative period as compared to the period when patients were on the waiting list, additional CVRFs appeared: increases in body mass index, calcium index, nitric oxide metabolites, endothelin-1, homocysteine, intercellular adhesion molecules VCAM-1 and ICAM-1, and decreases in endothelium-dependent vasodilation and glomerular filtration rate to less than 60 ml/min/1.73 m2. A model for the development of CIHD was created. The model uses a complex of independent risk factors and demonstrates a predictive accuracy of 84.6%. The study results indicate a modification of CVRFs and a dynamic change in the cardiovascular phenotype of liver transplant recipients: progression of CCMP during their stay on the waiting list, regression of CCMP manifestations during the first 12 months after orthotopic liver transplantation, and increases in the total cardiovascular risk and likelihood of CIHD in the long-term postoperative period.

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia.

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo-angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C-HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD-HUVECs), leading to a permanent pro-inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti-inflammatory and pro-angiogenic properties of OA are still under investigation. We tested OA on C- and GD-HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA-pre-treated C- and GD-HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD-HUVEC endothelium under OA pre-treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD-HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C- and GD-HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.