Endothelial Cell Chemotaxis Research Articles

The flavonoid hydroxygenkwanin reduces inflammation and neointimal formation

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration play crucial roles in neointimal hyperplasia and restenosis progression in response to stimulation with various inflammatory cytokines, such as platelet-derived growth factor-BB (PDGF-BB) and tumour necrosis factor-α (TNF-α). Hydroxygenkwanin (HGK) exerts remarkable anti-inflammatory, antitumour, antiproliferative and antimigratory effects. The aim of the study was to elucidate the therapeutic effect and regulatory mechanism of HGK on neointimal hyperplasia. The results showed that HGK inhibited the abnormal proliferation, migration, and inflammation of PDGF-BB- or TNF-α-treated VSMCs through regulation of the PDK1/AKT/mTOR pathway. In addition, HGK promoted circulating endothelial progenitor cell (EPC) chemotaxis. In an in vivo assay, HGK dramatically enhanced re-endothelization and reduced neointimal hyperplasia after femoral artery denudation with a guide wire in mice. These results suggest that HGK can serve as a therapeutic target drug or a functional food supplement for the treatment of restenosis.

CCN1 Is a Therapeutic Target for Reperfused Ischemic Brain Injury.

Ischemic stroke can lead to systemic inflammation, which can activate peripheral immune cells, causing neuroinflammation and brain injury. Meningeal lymphatics play a crucial role in transporting solutes and immune cells out of the brain and draining them into cervical lymph nodes (CLNs). However, the role of meningeal lymphatics in regulating systemic inflammation during the reperfusion stage after ischemia is not well understood. In this study, we demonstrated that brain infarct size, neuronal loss, and the effector function of inflammatory macrophage subsets were reduced after ischemia-reperfusion and disruption of meningeal lymphatics. Spatial memory function was improved in the late stage of ischemic stroke following meningeal lymphatic disruption. Brain-infiltrating immune cells, including neutrophils, monocytes, and T and natural killer cells, were reduced after cerebral ischemia-reperfusion and meningeal lymphatic disruption. Single-cell RNA sequencing analysis revealed that meningeal lymphatic disruption reprogrammed the transcriptome profile related to chemotaxis and leukocyte migration in CLN lymphatic endothelial cells (LECs), and it also decreased chemotactic CCN1 expression in floor LECs. Replenishment of CCN1 through intraventricular injection increased brain infarct size and neuronal loss, while restoring numbers of macrophages/microglia in the brains of meningeal lymphatic-disrupted mice after ischemic stroke. Blocking CCN1 in cerebrospinal fluid reduced brain infarcts and improves spatial memory function after ischemia-reperfusion injury. In summary, this study indicates that CCN1-mediated detrimental inflammation was alleviated after cerebral ischemia-reperfusion injury and meningeal lymphatic disruption. CCN1 represents a novel therapeutic target for inhibiting systemic inflammation in the brain-CLN axis after ischemia-reperfusion injury.

Treponema pallidum recombinant protein Tp0768 enhances the ability of HUVECs to promote neutrophil chemotaxis through the TLR2/ER stress signaling pathway.

Neutrophils are essential cells involved in inflammation. However, the specific mechanism of neutrophil chemotaxis induced by Treponema pallidum remains unknown. In this study, human umbilical vein endothelial cells (HUVECs) were utilized as target cells to investigate the expression levels of chemokines when stimulated with different concentrations of Tp0768 (also known as TpN44.5 or TmpA, a T. pallidum infection dependent antigen). The results indicated that Tp0768 treatment enhanced neutrophil chemotaxis in HUVECs, which was closely associated with the expression levels of CXCL1, CXCL2, and CXCL8 (also known as interleukin-8). At the same time, the results show that the Toll-like receptor 2 (TLR2) signaling pathway is activated and that endoplasmic reticulum (ER) stress occurs. Furthermore, the findings revealed that the use of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and immunoglobulin-regulated enhancer 1 (IRE1) inhibitors reduced the expression levels of CXCL1, CXCL2, and CXCL8. Additionally, inhibiting TLR2 significantly decreased the expression levels of ER stress-related proteins (PERK and IRE1), CXCL1, CXCL2, and CXCL8. Consequently, neutrophil chemotaxis was significantly inhibited after treatment with TLR2, PERK, and IRE1 inhibitors. These findings shed light on the role of Tp0768 in enhancing neutrophil chemotaxis in endothelial cells, providing a foundation for further exploration of syphilis pathogenesis and offering a new direction for the diagnosis and treatment of T. pallidum infection.

Tissue-specific reprogramming leads to angiogenic neutrophil specialization and tumor vascularization in colorectal cancer.

Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during the transition from inflammatory ulceration to CRC we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their proximity to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA data set and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in patients with UC. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings demonstrate a niche-directed PMN functional specialization and identify TAN contributions to tumor vascularization, delineating what we believe to be a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.

Abstract 1956 CMG2 interaction with actin is required for growth factor-induced chemotaxis in endothelial cells

Abstract 1956 CMG2 interaction with actin is required for growth factor-induced chemotaxis in endothelial cells

ABHD1 Facilitates Intermediate Filament–Mediated Endothelial Cell Chemotaxis by Regulating KRT1 and KRT2 in Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most common complications of diabetes and induces severe visual impairment worldwide. Endothelial cell dysfunction plays an important role in the pathogenesis of DR. Here, we keep a watchful eye on α/β‐hydrolase domain–containing 1 (ABHD1), a potential regulator in lipid metabolism and neovascularization. Results revealed that ABHD1 expression increased both in retina tissues of DR patients and in high‐glucose–treated human retina endothelial cells. Inhibition of ABHD1 remitted endothelial cell proliferation and migration. And GSEA uncovered that ABHD1 knockdown remits endothelial cell chemotaxis and intermediate filament (IF) might be mediated in the progress by regulating keratin 1 (KRT1) and keratin 2 (KRT2). Therefore, we assume that ABHD1 is concerned with endothelial cell proliferation and migration in DR, consequently leading to pathological neovascularization. The findings may provide a potential therapeutic target for DR.

Abstract 1350: Kupffer cells secrete CXCL5 to promote liver cancer

Abstract Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leading cause of cancer related deaths. In addition to the genomic and transcriptomic heterogeneity of liver tumor cells which is recognized as a major driver in liver cancer progression, the liver immune system is also fundamental to liver carcinogenesis and presents a promising target for therapy. The liver immune response is orchestrated by cytokines and chemokines. Recent studies suggest that chemokines not only recruit immune cells but also regulate various liver functions. In partial hepatectomy, CXCL2 has been shown to promote hepatocyte proliferation. CXCL1, 2, 5 and 8 can induce endothelial cells chemotaxis to promote angiogenesis through binding to CXCR2. These diverse functions suggest that chemokines could play multifaceted roles in liver cancer development. However, chemokines that are commonly associated with liver cancer is still unknown.We analyzed HCC patient data from the GEO database, and we categorized the datasets based on HCC etiologies including HBV, HCV, alcoholic and NASH. We identified CXCL5 as the only chemokine consistently upregulated in HCC with different etiologies compared to healthy or cirrhotic livers. Immunohistochemistry (IHC) analysis reveals that CXCL5 was produced by immune cells but not tumor cells in human HCC tissues. To further study HCC associated CXCL5 expression, the liver-specific Pten deletion mouse model (PM mice) that recapitulates NAFLD-NASH-HCC progression was used. A gradual increase of hepatic CXCL5 expression is observed during HCC development, reaching nearly 100-fold upregulation of CXCL5 mRNA expression in 12-month-old PM mice livers carrying tumors. Examination of liver immune cell populations showed that macrophages were significantly enriched in Pten deleted livers bearing tumors than wild type livers without tumors. Flow cytometry and IHC analysis further identified Kupffer cells (KCs), the liver resident macrophages as the source of CXCL5 in tumor bearing livers using these mice. Since increased LPS is a prominent feature in most chronic liver diseases, we isolated and treated mouse KCs with LPS and found that LPS treatment robustly increased CXCL5 expression by nearly 20-fold. Interestingly, neither murine macrophage cell lines nor primary peritoneal macrophages displayed induced CXCL5 expression in response to LPS. These data suggest that induction of CXCL5 in KCs is likely a unique function of the KCs but not of other macrophages. To explore the function of CXCL5 in HCC development, we treated mouse hepatocytes and HCC cells with CXCL5 and showed that CXCL5 induces the proliferation of these cells. This effect is further blocked by the inhibition of CXCR2, the receptor of CXCL5, demonstrating the specificity for CXCL5 mediated effects. Together we show here for the first time that CXCL5 expression is a unique property of Kupffer cells and the induction of CXCL5 promotes HCC progression. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Curtis T. Okamoto, Bangyan L. Stiles. Kupffer cells secrete CXCL5 to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1350.

The Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitor Empagliflozin Reverses Hyperglycemia-Induced Monocyte and Endothelial Dysfunction Primarily through Glucose Transport-Independent but Redox-Dependent Mechanisms

Hyperglycaemia-induced oxidative stress and inflammation contribute to vascular cell dysfunction and subsequent cardiovascular events in T2DM. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin significantly improves cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is known to be expressed on cells other than the kidney cells, we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate hyperglycaemia-induced dysfunction of these cells. Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs), and fetoplacental endothelial cells (HPECs) were used as the EC model cells. Cells were exposed to hyperglycaemic conditions in vitro in 40 ng/mL or 100 ng/mL empagliflozin. The expression levels of the relevant molecules were analysed by RT-qPCR and confirmed by FACS. Glucose uptake assays were carried out with a fluorescent derivative of glucose, 2-NBDG. Reactive oxygen species (ROS) accumulation was measured using the H2DFFDA method. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. Both primary human monocytes and endothelial cells express SGLT-2. Hyperglycaemic conditions did not significantly alter the SGLT-2 levels in monocytes and ECs in vitro or in T2DM conditions. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly, but not significantly, suppressed glucose uptake by monocytes and endothelial cells. However, we detected the significant suppression of hyperglycaemia-induced ROS accumulation in monocytes and ECs when empagliflozin was used to inhibit SGLT-2 function. Hyperglycaemic monocytes and endothelial cells readily exhibited impaired chemotaxis behaviour. The co-treatment with empagliflozin reversed the PlGF-1 resistance phenotype of hyperglycaemic monocytes. Similarly, the blunted VEGF-A responses of hyperglycaemic ECs were also restored by empagliflozin, which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. The induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by hyperglycaemic monocytes and endothelial cells, and a general antioxidant N-acetyl-L-cysteine (NAC) was able to mimic the effects of empagliflozin. This study provides data indicating the beneficial role of empagliflozin in reversing hyperglycaemia-induced vascular cell dysfunction. Even though both monocytes and endothelial cells express functional SGLT-2, SGLT-2 is not the primary glucose transporter in these cells. Therefore, it seems likely that empagliflozin does not directly prevent hyperglycaemia-mediated enhanced glucotoxicity in these cells by inhibiting glucose uptake. We identified the reduction of oxidative stress by empagliflozin as a primary reason for the improved function of monocytes and endothelial cells in hyperglycaemic conditions. In conclusion, empagliflozin reverses vascular cell dysfunction independent of glucose transport but could partially contribute to its beneficial cardiovascular effects.

Proteomic aptamer analysis reveals serum markers that characterize preclinical systemic sclerosis (SSc) patients at risk for progression toward definite SSc

BackgroundThe study of molecular mechanisms characterizing disease progression may be relevant to get insights into systemic sclerosis (SSc) pathogenesis and to intercept patients at very early stage. We aimed at investigating the proteomic profile of preclinical systemic sclerosis (PreSSc) via a discovery/validation two-step approach.MethodsSOMAcan aptamer-based analysis was performed on a serum sample of 13 PreSSc (discovery cohort) according to 2001 LeRoy and Medsger criteria (characterized solely by Raynaud phenomenon plus a positive nailfold capillaroscopy and SSc-specific antibodies without any other sign of definite disease) and 8 healthy controls (HCs) age, gender, and ethnicity matched. Prospective data were available up to 4±0.6 years to determine the progression to definite SSc according to the EULAR/ACR 2013 classification criteria. In proteins with relative fluorescence units (RFU) > |1.5|-fold vs HCs values, univariate analysis was conducted via bootstrap aggregating models to determine the predicting accuracy (progression vs non-progression) of categorized baseline protein values. Gene Ontologies (GO terms) and Reactome terms of significant proteins at the adjusted 0.05 threshold were explored. Significant proteins from the discovery cohort were finally validated via ELISAs in an independent validation cohort of 50 PreSSc with clinical prospective data up to 5 years. Time-to-event analysis for interval-censored data was used to evaluate disease progression.ResultsIn the discovery cohort, 286 out of 1306 proteins analyzed via SomaScan, were differentially expressed versus HCs. Ten proteins were significantly associated with disease progression; analysis through GO and Reactome showed differentially enriched pathways involving angiogenesis, endothelial cell chemotaxis, and endothelial cell chemotaxis to fibroblast growth factor (FGF). In the validation cohort, endostatin (HR=10.23, CI95=2.2–47.59, p=0.003) was strongly associated with disease progression, as well as bFGF (HR=0.84, CI95=0.709-0.996, p=0.045) and PAF-AHβ (HR=0.372, CI95=0.171–0.809, p=0.013)ConclusionsA distinct protein profile characterized PreSSc from HCs and proteins associated with hypoxia, vasculopathy, and fibrosis regulation are linked with the progression from preclinical to definite SSc. These proteins, in particular endostatin, can be regarded both as markers of severity and molecules with pathogenetic significance as well as therapeutic targets.

Evaluating material-driven regeneration in a tissue engineered human in vitro bone defect model.

Advanced in vitro human bone defect models can contribute to the evaluation of materials for in situ bone regeneration, addressing both translational and ethical concerns regarding animal models. In this study, we attempted to develop such a model to study material-driven regeneration, using a tissue engineering approach. By co-culturing human umbilical vein endothelial cells (HUVECs) with human bone marrow-derived mesenchymal stromal cells (hBMSCs) on silk fibroin scaffolds with in vitro critically sized defects, the growth of vascular-like networks and three-dimensional bone-like tissue was facilitated. After a model build-up phase of 28days, materials were artificially implanted and HUVEC and hBMSC migration, cell-material interactions, and osteoinduction were evaluated 14days after implantation. The materials physiologically relevant for bone regeneration included a platelet gel as blood clot mimic, cartilage spheres as soft callus mimics, and a fibrin gel as control. Although the in vitro model was limited in the evaluation of immune responses, hallmarks of physiological bone regeneration were observed in vitro. These included the endothelial cell chemotaxis induced by the blood clot mimic and the mineralization of the soft callus mimic. Therefore, the present in vitro model could contribute to an improved pre-clinical evaluation of biomaterials while reducing the need for animal experiments.

Mucocutaneous manifestations of Behçet's disease: Pathogenesis and management from perspectives of vasculitis.

Behçet's disease (BD) is a systemic inflammatory disorder characterized by vasculitis affecting blood vessels of any caliber or type. It can present with a wide spectrum of vasculitic lesions, including erythema nodosum-like lesions and retinal vasculitis, and may also lead to larger vessel diseases, such as aortic aneurysm and deep vein thrombosis. The full etiology of BD remains unclear, but it is considered a polygenetic disease with multiple genetic risk factors that promote immune dysregulation and thrombophilia. Inflammation can be triggered by environmental factors, such as bacteria or viruses, and the dysregulation of innate and adaptive immune cell subsets. Neutrophils and lymphocytes are the primary players involved in BD pathogenesis, with specific innate (i.e., neutrophil-derived reactive oxygen species and neutrophil extracellular traps) and adaptive (i.e., anti-endothelial cell antibodies) processes inducing endothelial cell activation and chemotaxis of inflammatory cells, leading to coagulation and vasculitis. These inflammation-induced vasculitic or vasculopathic features are observed in most mucocutaneous BD lesions, although vasculitis per se is often pathologically evident only during a brief period of the disease process. Due to the multifactorial nature of BD-associated inflammation, broad-spectrum anti-inflammatory medications, including glucocorticoids and immunosuppressive drugs, have been the mainstay for managing BD. In addition, inhibitors of interleukin (IL)-1, tumor necrosis factor (TNF)-α, and IL-17, which target innate and adaptive immune functions dysregulated in BD, have emerged as promising new therapeutics. In this review, we discuss the muco-cutaneous manifestations of BD by focusing on the underlying vasculitic components in their pathologies, as well as the current array of treatment options.

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with α5β1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma.

Monocyte and endothelial dysfunction induced by hyperglycemia is reversed by Empagliflozin mainly through glucose-transport independent mechanisms

Abstract Type II Diabetes mellitus (T2DM) leads via hyperglycemia (HG)-associated inflammation and oxidative stress to vascular cell dysfunction. The EMPA-REG trial revealed a reduction of cardiovascular mortality in T2DM patients by Empagliflozin, a sodium-glucose co-transporter-2 (SGLT-2) inhibitor. SGLT-2 is expressed on monocytes and endothelial cells (EC), therefore the aim of this study was to investigate the potential role of Empagliflozin for glucose transport regulation and improvement of HG-induced dysfunction of monocytes and EC. HUVEC and HCAEC were used as EC model cells. Primary human monocytes were isolated from T2DM patients and healthy controls. The cells were exposed to HG conditions in vitro in presence of 40 or 100 ng/ml Empagliflozin. Using RT-qPCR and FACS, expression levels of SGLT-2 were analysed. Glucose uptake assays were performed with a fluorescent derivative of glucose, 2-NBDG. To measure reactive oxygen species (ROS) accumulation, we used the H2DFFDA method via FACS and fluorescence microscopy. Monocyte and EC chemotaxis was analysed using modified Boyden chamber assays. We could prove an expression of SGLT-2 in primary human monocytes and EC. The transcripts of SGLT-2 in T2DM monocytes did not show a difference. SGLT2-levels in monocytes and EC were not altered by HG. In presence of GLUT inhibitor in vitro glucose uptake assays revealed the ability of SGLT-2 inhibition to mildly supress the glucose uptake, whereas uptake by monocytes and EC remained comparable. Nevertheless, in presence of Empagliflozin HG-induced ROS accumulation in monocytes (1.3-fold, p=0.021) and ECs (1.5-fold, p=0.003) was reduced. HG monocytes and EC readily exhibited an impaired chemotaxis behaviour. Treating the cells in HG with Empagliflozin reversed the PlGF-1 resistance phenotype of HG monocytes (1.4-fold, p=0.002). Comparably, Empagliflozin was able to rescued the blunted VEGF-A responses of HG ECs (1.35-fold, p=0.028), which could be ascribed to restoration of VEGFR-2 receptor levels on the EC surface. Most of the aberrant phenotypes exhibited by HG monocytes and EC were entirely recapitulated by induction of oxidative stress. We could mimic these effects of Empagliflozin using the general antioxidant N-acetyl-L-cysteine (NAC). Our study reveals a beneficial role of Empagliflozin for reversing HG-induced vascular cell dysfunction. The expressed SGLT-2 on monocytes and EC does not serve as primary glucose transporter. So it is highly to speculate that HG-mediated enhanced glucotoxicity in those cells is not prevented by Empagliflozin through inhibition of glucose uptake itself. Reduction of oxidative stress by Empagliflozin was revealed as central mechanism for an improved function of HG monocytes and EC in vitro. Finally, we conclude that antioxidant properties of Empagliflozin reverse vascular cell dysfunction independently of glucose transport. This is likely to support the beneficial effect of Empagliflozin in cardiovascular disease. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Medical Faculty, University of Münster, Germany

Highly elastic and bioactive bone biomimetic scaffolds based on platelet lysate and biomineralized cellulose nanocrystals

Bone is a vascularized organic-inorganic composite tissue that shows a heavily-mineralized extracellular matrix (ECM) on the nanoscale. Herein, the nucleation of calcium phosphates during the biomineralization process was mimicked using negatively-charged cellulose nanocrystals (CNCs). These mineralized-CNCs were combined with platelet lysate to produce nanocomposite scaffolds through cryogelation to mimic bone ECM protein-mineral composite nature and take advantage of the bioactivity steaming from platelet-derived biomolecules. The nanocomposite scaffolds showed high microporosity (94–95%), high elasticity (recover from 75% strain cycles), injectability, and modulated platelet-derived growth factors sequestration and release. Furthermore, they increased alkaline phosphatase activity (up to 10-fold) and up-regulated the expression of bone-related markers (up to 2-fold), without osteogenic supplementation, demonstrating their osteoinductive properties. Also, the scaffolds promoted the chemotaxis of endothelial cells and enhanced the expression of endothelial markers, showing proangiogenic potential. These results suggest that the mineralized nanocomposite scaffolds can enhance bone regeneration by simultaneously promoting osteogenesis and angiogenesis.

Single-cell transcriptome profile of mouse skin undergoing antigen-driven allergic inflammation recapitulates findings in atopic dermatitis skin lesions

Allergic skin inflammation elicited in mice by epicutaneous (EC) sensitization with antigen shares characteristics with human atopic dermatitis (AD). We characterized gene expression by single cells in mouse skin undergoing antigen-driven allergic inflammation and compared the results with findings in AD skin lesions. Mice were EC sensitized by application of ovalbumin (OVA) or saline to tape-stripped skin. Single-cell RNA sequencing was performed on skin cells 12 days later. Flow cytometry analysis was performed to validate results. Sequencing identified 7 nonhematopoietic and 6 hematopoietic cell subsets in EC-sensitized mouse skin. OVA sensitization resulted in the expansion in the skin of T cells, dendritic cells, macrophages, mast cells/basophils, fibroblasts, and myocytes cell clusters, and in upregulation of TH2 cytokine gene expression in CD4+ T cells and mast cells/basophils. Genes differentially expressed in OVA-sensitized skin included genes important for inflammation in dendritic cells and macrophages, collagen deposition, and leukocyte migration in fibroblasts, chemotaxis in endothelial cells and skin barrier integrity, and differentiation in KCs-findings that recapitulate those in AD skin lesions. Unexpectedly, mast cells/basophils, rather than T cells, were the major source of Il4 and ll13 in OVA-sensitized mouse skin. In addition, our results suggest novel pathways in fibroblast and endothelial cells that may contribute to allergic skin inflammation. The gene expression profile of single cells in mouse skin undergoing antigen-driven shares many features with that in AD skin lesions and unveils novel pathways that may be involved in allergic skin inflammation.

Capillary morphogenesis gene 2 (CMG2) mediates growth factor-induced angiogenesis by regulating endothelial cell chemotaxis.

Anthrax protective antigen (PA) is a potent inhibitor of pathological angiogenesis with an unknown mechanism. In anthrax intoxication, PA interacts with capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8). Here, we show that CMG2 mediates the antiangiogenic effects of PA and is required for growth-factor-induced chemotaxis. Using specific inhibitors of CMG2 and TEM8 interaction with natural ligand, as well as mice with the CMG2 or TEM8 transmembrane and intracellular domains disrupted, we demonstrate that inhibiting CMG2, but not TEM8 reduces growth-factor-induced angiogenesis in the cornea. Furthermore, the antiangiogenic effect of PA was abolished when the CMG2, but not the TEM8, gene was disrupted. Binding experiments demonstrated a broad ligand specificity for CMG2 among extracellular matrix (ECM) proteins. Ex vivo experiments demonstrated that CMG2 (but not TEM8) is required for PA activity in human dermal microvascular endothelial cell (HMVEC-d) network formation assays. Remarkably, blocking CMG2-ligand binding with PA or CRISPR knockout abolishes endothelial cell chemotaxis but not chemokinesis in microfluidic migration assays. These effects are phenocopied by Rho inhibition. Because CMG2 mediates the chemotactic response of endothelial cells to peptide growth factors in an ECM-dependent fashion, CMG2 is well-placed to integrate growth factor and ECM signals. Thus, CMG2 targeting is a novel way to inhibit angiogenesis.

Identification of the Relationship between Hub Genes and Immune Cell Infiltration in Vascular Endothelial Cells of Proliferative Diabetic Retinopathy Using Bioinformatics Methods.

Background Diabetic retinopathy (DR) is a serious ophthalmopathy that causes blindness, especially in the proliferative stage. However, the pathogenesis of its effect on endothelial cells, especially its relationship with immune cell infiltration, remains unclear. Methods The dataset GSE94019 was downloaded from the Gene Expression Omnibus (GEO) database to obtain DEGs. Through aggregate analyses such as Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis, a protein-protein interaction (PPI) network was constructed to analyze the potential function of DEGs. Weighted gene coexpression network analysis (WGCNA) and Cytoscape software including molecular complex detection (MCODE) and cytoHubba plug-ins were used to comprehensively analyze and determine the hub genes. ImmuCellAI analysis was performed to further study the relationship between samples, hub genes, and 24 types of immune cell infiltration. Finally, gene-set enrichment analysis (GSEA) was employed to identify the enrichment of immune cell infiltration and endothelial cell phenotype modifications in GO biological processes (BP) based on the expression level of hub genes. Results 2393 DEGs were identified, of which 800 genes were downregulated, and 1593 genes were upregulated. The results of functional enrichment revealed that 1398 BP terms were significantly enriched in DEGs. Three hub genes, EEF1A1, RPL11, and RPS27A, which were identified by conjoint analysis using WGCNA and Cytoscape software, were positively correlated with the number of CD4 naive T cells and negatively correlated with the numbers of B cells. The number of CD4 naive T cells, T helper 2 (Th2) cells, and effector memory T (Tem) cells were significantly higher while CD8 naive T cells and B cells significantly were lower in the diabetic group than in the nondiabetic group. Conclusions We unearthed the DEGs and Hub genes of endothelial cells related to the pathogenesis of PDR: EEF1A1, RPL11, and RPS27A, which are highly related to each other and participate in the specific biological process of inflammation-related immune cell infiltration and endothelial cell development, chemotaxis, and proliferation, thus providing new perspectives into the diagnosis of and potential “killing two birds with one stone” targeted therapy for PDR.

Feasibility of Growth Factor Agent Therapy in Repairing Motor Injury.

Growth factors (GF), with the activity of stimulating cell growth, play a significant role in biology, medicine, and exercise physiology. In the process of exercise, human tissues are impacted, making cells suffer damage. Growth factor can accelerate the repair of damaged cells and regulate the synthesis of protein, so biological preparations of growth factors can be added to traditional therapies. A combination of growth factor biologics and conventional therapies may improve the efficiency of injury repair, but growth factor biologics may not produce any results. The feasibility of growth factor biologics in the treatment of motor injury was discussed. The research have shown that: 1) GF biological agent therapy is a very promising treatment for motor injury, which is based on the power of autologous growth factor (GFs) to accelerate tissue healing, promote muscle regeneration, increase angiogenesis, reduce fibrosis, and make the muscle injury rapid recovery. 2) There are various methods for delivering the higher dose of GF to the injured tissue, but most of them depend on the platelet release of GF. At the site of injury, there are several ways to deliver higher doses of GF to the injured tissue. 3) At present, the inhibition of GF is mainly through signal transduction inhibitors and inhibition of transcription factor production. 4) Pattern of GF during wound repair: GF directly regulates many key steps of normal wound repair, including inflammatory cell chemotaxis, division and proliferation of fibroblasts, keratinocytes and vascular endothelial cells, formation of new blood vessels, and synthesis and degradation of intercellular substances. 5) When GF promotes chronic wound healing, in most cases, certain GF can be used targeted only when in vivo regulation still cannot meet the need for repair.

Cryo-Temperature Pretreatment Increases the Pro-Angiogenic Capacity of Three-Dimensional Mesenchymal Stem Cells via the PI3K-AKT Pathway

To increase the potential and effectiveness of three-dimensional (3D) mesenchymal stem cells (MSCs) for clinical applications, this study explored the effects of short cryo-temperature pretreatment on MSC function. Adipose-derived MSCs (A-MSCs) were cultured via the ordinary monolayer method and 3D hanging drop spheroid method. When the cells adhered to the wall or formed a spheroid, they were subjected to hypothermic stress at 4°C for 1 h and then divided into three recovery periods at 37°C, specifically 0, 12, and 24 h. The control group was not subjected to any treatment throughout the study. Monolayer and 3D spheroid A-MSCs were analyzed via RNA sequencing after hypothermic stress at 4°C for 1 h. Subsequently, each group of cells was collected and subjected to phenotype identification via flow cytometry, and mRNA expression was detected via reverse transcription–quantitative polymerase chain reaction analysis. Western blot analysis was performed to analyze the PI3K-AKT signaling pathway in A-MSCs. The effects of A-MSCs on angiogenesis in vivo were examined using a chick chorioallantoic membrane assay. Transwell assays were performed to determine whether the culture supernatant from each group could induce the chemotaxis of human umbilical vein endothelial cells (HUVECs). Three-dimensional spheroid culture did not change the phenotype of A-MSCs. The expression of fibroblast growth factors, hepatocyte growth factors, and other angiogenesis-related factors in A-MSCs was upregulated. A-MSCs subjected to hypothermic stress promoted angiogenesis under both monolayer and 3D spheroid cultures. Moreover, the chemotaxis of HUVECs to the 3D spheroid culture supernatant increased substantially. Short cryo-temperature pretreatment could stimulate 3D spheroid A-MSCs and activate the PI3K-AKT pathway. This approach has the advantages of promoting angiogenesis and maintaining cell viability.

STING inhibitor ameliorates LPS-induced ALI by preventing vascular endothelial cells-mediated immune cells chemotaxis and adhesion.

Acute lung injury (ALI) is a common and devastating clinical disorder featured by excessive inflammatory responses. Stimulator of interferon genes (STING) is an indispensable molecule for regulating inflammation and immune response in multiple diseases, but the role of STING in the ALI pathogenesis is not well elucidated. In this study, we explored the molecular mechanisms of STING in regulating lipopolysaccharide (LPS)-induced lung injury. Mice were pretreated with a STING inhibitor C-176 (15, 30 mg/kg, i.p.) before LPS inhalation to induce ALI. We showed that LPS inhalation significantly increased STING expression in the lung tissues, whereas C-176 pretreatment dose-dependently suppressed the expression of STING, decreased the production of inflammatory cytokines including TNF-α, IL-6, IL-12, and IL-1β, and restrained the expression of chemokines and adhesion molecule vascular cell adhesion protein-1 (VCAM-1) in the lung tissues. Consistently, in vitro experiments conducted in TNF-α-stimulated HMEC-1cells (common and classic vascular endothelial cells) revealed that human STING inhibitor H-151 or STING siRNA downregulated the expression levels of adhesion molecule and chemokines in HMEC-1cells, accompanied by decreased adhesive ability and chemotaxis of immunocytes upon TNF-α stimulation. We further revealed that STING inhibitor H-151 or STING knockdown significantly decreased the phosphorylation of transcription factor STAT1, which subsequently influenced its binding to chemokine CCL2 and adhesive molecule VCAM-1 gene promoter. Collectively, STING inhibitor can alleviate LPS-induced ALI in mice by preventing vascular endothelial cells-mediated immune cell chemotaxis and adhesion, suggesting that STING may be a promising therapeutic target for the treatment of ALI.