Mechanisms of triple-negative breast cancer extravasation: Impact of the physical environment and endothelial glycocalyx.

Cancer metastasis is the leading cause of death for those afflicted with cancer. In cancer metastasis, the cancer cells break off from the primary tumor, penetrate nearby blood vessels, and attach and extravasate out of the vessels to form secondary tumors at distant organs. This makes extravasation a critical step of the metastatic cascade. Herein, with a focus on triple-negative breast cancer, the role that the prospective secondary tumor microenvironment's mechanical properties play in circulating tumor cells' extravasation is reviewed. Specifically, the effects of the physically regulated vascular endothelial glycocalyx barrier element, vascular flow factors, and subendothelial extracellular matrix mechanical properties on cancer cell extravasation are examined. The ultimate goal of this review is to clarify the physical mechanisms that drive triple-negative breast cancer extravasation, as these mechanisms may be potential new targets for anti-metastasis therapy.

Factors affecting prognosis and need for anti-vascular endothelial growth factor injections in wet age-related macular degeneration.

PURPOSE: To understand factors affecting visual prognosis and the number of intravitreal antivascular endothelial growth factor (anti-VEGF) injections needed to stabilize wet age-related macular degeneration (AMD). METHODS: In this retrospective cohort, 119 treatment-naïve wet AMD patients were followed for two years. In patients with bilateral disease, the eye with worse best-corrected visual acuity (BCVA) or that received more intravitreal injections was recruited as the study eye. In all visits, BCVA was recorded, ophthalmological examination was performed including macular optical coherence tomography imaging. Twenty health status/lifestyle questions were asked to the patients via phone as potential risk factors. All patients received 3 loading doses of intravitreal bevacizumab injections and received repeat injections of aflibercept or ranibizumab when the eye had a new, active neovascular lesion. RESULTS: Patients who took regular micronutrition had similar visual outcome and injection numbers compared to the ones who did not. Patients with bilateral disease needed less intravitreal injections compared to unilateral AMD patients (p = 0.016) and women on hormone replacement therapy (HRT) required less injections compared to the women who were not (p = 0.024). Female patients had a mean gain of 2.7 letters while male patients lost 3.8 letters (p = 0.038). Wet AMD started at an earlier age in smokers (p = 0.002). Patients with a better education level presented earlier with better BCVA (p = 0.037). CONCLUSION: HRT and anti-VEGF injections to the fellow eye improved the prognosis of wet AMD, while male patients had slightly worse prognosis. Estrogen's protective effects and potential contribution in wet AMD needs further attention. Retrospectively registered: 2020/0622.

In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts.

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7-9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

Enhancement of endothelial function and attenuation of portal vein injury using mesenchymal stem cells carrying miRNA-25-3p.

The aims of this study were to determine whether human umbilical cord mesenchymal stem cells (hucMSCs) modified by miRNA-25-3p (miR-25-3p) overexpression could promote venous endothelial cell proliferation and attenuate portal endothelial cell injury. HucMSCs and human umbilical vein endothelial cells (HUVEC) were isolated and cultured from human umbilical cord and characterized. Lentiviral vectors expressing miRNA-25-3p were transfected into hucMSCs and confirmed by PCR. We verified the effect of miR-25-3p-modified hucMSCs on HUVEC by cell co-culture and cell supernatant experiments. Subsequently, exosomes of miR-25-3p-modified hucMSCs were isolated from cell culture supernatants and characterized by WB, NTA and TEM. We verified the effects of miR-25-3p-modified exosomes derived from hucMSCs on HUVEC proliferation, migration, and angiogenesis by in vitro cellular function experiments. Meanwhile, we further examined the downstream target genes and signaling pathways potentially affected by miR-25-3p-modified hucMSC-derived exosomes in HUVEC. Finally, we established a rat portal vein venous thrombosis model by injecting CM-DiR-labeled hucMSCs intravenously into rats and examining the homing of cells in the portal vein by fluorescence microscopy. Histological and immunohistochemical experiments were used to examine the effects of miRNA-25-3p-modified hucMSCs on the proliferation and damage of portal vein endothelial cells. Primary hucMSCs and HUVECs were successfully isolated, cultured and characterized. Primary hucMSCs were modified with a lentiviral vector carrying miR-25-3p at MOI 80. Co-culture and cell supernatant intervention experiments showed that overexpression of miRNA-25-3p in hucMSCs enhanced HUVEC proliferation, migration and tube formation in vitro. We successfully isolated and characterized exosomes of miR-25-3p-modified hucMSCs, and exosome intervention experiments demonstrated that miR-25-3p-modified exosomes derived from hucMSCs similarly enhanced the proliferation, migration, and angiogenesis of HUVECs. Subsequent PCR and WB analyses indicated PTEN/KLF4/AKT/ERK1/2 as potential pathways of action. Analysis in a rat portal vein thrombosis model showed that miR-25-3p-modified hucMSCs could homing to damaged portal veins. Subsequent histological and immunohistochemical examinations demonstrated that intervention with miR-25-3p overexpression-modified hucMSCs significantly reduced damage and attenuated thrombosis in rat portal veins. The above findings indicate suggest that hucMSCs based on miR-25-3p modification may be a promising therapeutic approach for use in venous thrombotic diseases.

Hsa-miR-134-5p predicts cardiovascular risk in circulating mononuclear cells and improves angiogenic action of senescent endothelial progenitor cells.

This research explores the role of microRNA in senescence of human endothelial progenitor cells (EPCs) induced by replication. Hsa-miR-134-5p was found up-regulated in senescent EPCs where overexpression improved angiogenic activity. Hsa-miR-134-5p, which targeted transforming growth factor ß-activated kinase 1-binding protein 1 (TAB1) gene, down-regulated TAB1 protein, and inhibited phosphorylation of p38 mitogen-activated protein kinase (p38) in hsa-miR-134-5p-overexpressed senescent EPCs. Treatment with siRNA specific to TAB1 (TAB1si) down-regulated TAB1 protein and subsequently inhibited p38 activation in senescent EPCs. Treatment with TAB1si and p38 inhibitor, respectively, showed angiogenic improvement. In parallel, transforming growth factor Beta 1 (TGF-ß1) was down-regulated in hsa-miR-134-5p-overexpressed senescent EPCs and addition of TGF-ß1 suppressed the angiogenic improvement. Analysis of peripheral blood mononuclear cells (PBMCs) disclosed expression levels of hsa-miR-134-5p altered in adult life, reaching a peak before 65 years, and then falling in advanced age. Calculation of the Framingham risk score showed the score inversely correlates with the hsa-miR-134-5p expression level. In summary, hsa-miR-134-5p is involved in the regulation of senescence-related change of angiogenic activity via TAB1-p38 signalling and via TGF-ß1 reduction. Hsa-miR-134-5p has a potential cellular rejuvenation effect in human senescent EPCs. Detection of human PBMC-derived hsa-miR-134-5p predicts cardiovascular risk.

Selective mural cell recruitment of pericytes to networks of assembling endothelial cell-lined tubes.

Mural cells are critically important for the development, maturation, and maintenance of the blood vasculature. Pericytes are predominantly observed in capillaries and venules, while vascular smooth muscle cells (VSMCs) are found in arterioles, arteries, and veins. In this study, we have investigated functional differences between human pericytes and human coronary artery smooth muscle cells (CASMCs) as a model VSMC type. We compared the ability of these two mural cells to invade three-dimensional (3D) collagen matrices, recruit to developing human endothelial cell (EC)-lined tubes in 3D matrices and induce vascular basement membrane matrix assembly around these tubes. Here, we show that pericytes selectively invade, recruit, and induce basement membrane deposition on EC tubes under defined conditions, while CASMCs fail to respond equivalently. Pericytes dramatically invade 3D collagen matrices in response to the EC-derived factors, platelet-derived growth factor (PDGF)-BB, PDGF-DD, and endothelin-1, while minimal invasion occurs with CASMCs. Furthermore, pericytes recruit to EC tube networks, and induce basement membrane deposition around assembling EC tubes (narrow and elongated tubes) when these cells are co-cultured. In contrast, CASMCs are markedly less able to perform these functions showing minimal recruitment, little to no basement membrane deposition, with wider and shorter tubes. Our new findings suggest that pericytes demonstrate much greater functional ability to invade 3D matrix environments, recruit to EC-lined tubes and induce vascular basement membrane matrix deposition in response to and in conjunction with ECs.

Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Interleukin-7-based identification of liver lymphatic endothelial cells reveals their unique structural features.

Background & Aims: The lymphatic system plays crucial roles in maintaining fluid balance and immune regulation. Studying the liver lymphatics has been considered challenging, as common lymphatic endothelial cell (LyEC) markers are expressed by other liver cells. Additionally, isolation of sufficient numbers of LyECs has been challenging because of their extremely low abundance (<0.01% of entire liver cell population) in a normal liver. Methods: Potential LyEC markers was identified using our published single-cell RNA sequencing (scRNA-seq) dataset (GSE147581) in mouse livers. Interleukin-7 (IL7) promoter-driven green fluorescent protein knock-in heterozygous mice were used for the validation of IL7 expression in LyECs in the liver, for the development of liver LyEC isolation protocol, and generating liver ischemia/reperfusion (I/R) injury. Scanning electron microscopy was used for the structural analysis of LyECs. Changes in LyEC phenotypes in livers of mice with I/R were determined by RNA-seq analysis. Results: Through scRNA-seq analysis, we have identified IL7 as an exclusive marker for liver LyECs, with no overlap with other liver cell types. Based on IL7 expression in liver LyECs, we have established an LyEC isolation method and observed distinct cell surface structures of LyECs with fenestrae and cellular pores (ranging from 100 to 400 nm in diameter). Furthermore, we identified LyEC genes that undergo alterations during I/R liver injuries. Conclusions: This study not only identified IL7 as an exclusively expressed gene in liver LyECs, but also enhanced our understanding of LyEC structures and demonstrated transcriptomic changes in injured livers. Impact and implications: Understanding the lymphatic system in the liver is challenging because of the absence of specific markers for liver LyEC. This study has identified IL7 as a reliable marker for LyECs, enabling the development of an effective method for their isolation, elucidating their unique cell surface structure, and identifying LyEC genes that undergo changes during liver damage. The development of IL7 antibodies for detecting it in human liver specimens will further advance our understanding of the liver lymphatic system in the future.

IGF1R and FLT1 in female endothelial cells and CHD2 in male microglia play important roles in Alzheimer's disease based on gender difference analysis.

OBJECTIVE: This study investigated sex-specific pathogenesis mechanisms in Alzheimer's disease (AD) using single-nucleus RNA sequencing (snRNA-seq) data. METHODS: Data from the Gene Expression Omnibus (GEO) were searched using terms "Alzheimer's Disease", "single cell", and "Homo sapiens". Studies excluding APOE E4 and including comprehensive gender information with 10× sequencing methods were selected, resulting in GSE157827 and GSE174367 datasets from human prefrontal cortex samples. Sex-stratified analyses were conducted on these datasets, and the outcomes of the analysis for GSE157827 were compared with those of GSE174367. The findings were validated using expression profiling from the mouse dataset GSE85162. Furthermore, real-time PCR experiments in mice further confirmed these findings. The Seurat R package was used to identify cell types, and batch effects were mitigated using the Harmony R package. Cell proportions by sex were compared using the Mann-Whitney-Wilcoxon test, and gene expression variability was displayed with an empirical cumulative distribution plot. Differentially expressed genes were identified using the FindMarkers function with the MAST test. Transcription factors were analyzed using the RcisTarget R package. RESULTS: Seven cell types were identified: astrocytes, endothelial cells, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, and oligodendrocyte progenitor cells. Additionally, five distinct subpopulations of both endothelial and microglial cells were also identified, respectively. Key findings included: (1) In endothelial cells, genes involved in synapse organization, such as Insulin Like Growth Factor 1 Receptor (IGF1R) and Fms Related Receptor Tyrosine Kinase 1(FLT1), showed higher expression in females with AD. (2) In microglial cells, genes in the ribosome pathway exhibited higher expression in males without AD compared to females (with or without AD) and males with AD. (3) Chromodomain Helicase DNA Binding Protein 2 (CHD2) negatively regulated gene expression in the ribosome pathway in male microglia, suppressing AD, this finding was further validated in mice. (4) Differences between Asians and Caucasians were observed based on sex and disease status stratification. CONCLUSIONS: IGF1R and FLT1 in endothelial cells contribute to AD in females, while CHD2 negatively regulates ribosome pathway gene expression in male microglia, suppressing AD in humans and mice.

Modeling lung endothelial dysfunction in sepsis-associated ARDS using a microphysiological system.

Endothelial dysfunction is a critical feature of acute respiratory distress syndrome (ARDS) associated with higher disease severity and worse outcomes. Preclinical in vivo models of sepsis and ARDS have failed to yield useful therapies in humans, perhaps due to interspecies differences in inflammatory responses and heterogeneity of human host responses. Use of microphysiological systems (MPS) to investigate lung endothelial function may shed light on underlying mechanisms and targeted treatments for ARDS. We assessed the response to plasma from critically ill sepsis patients in our lung endothelial MPS through measurement of endothelial permeability, expression of adhesion molecules, and inflammatory cytokine secretion. Sepsis plasma induced areas of endothelial cell (EC) contraction, loss of cellular coverage, and luminal defects. EC barrier function was significantly worse following incubation with sepsis plasma compared to healthy plasma. EC ICAM-1 expression, IL-6 and soluble ICAM-1 secretion increased significantly more after incubation with sepsis plasma compared with healthy plasma. Plasma from sepsis patients who developed ARDS further increased IL-6 and sICAM-1 compared to plasma from sepsis patients without ARDS and healthy plasma. Our results demonstrate the proof of concept that lung endothelial MPS can enable interrogation of specific mechanisms of endothelial dysfunction that promote ARDS in sepsis patients.

A pilot study of nintedanib in molecularly selected patients with advanced non-small cell lung cancer.

Background: Nintedanib is a small molecule tyrosine kinase inhibitor (TKI) targeting vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). The purpose of the study was to evaluate the response rate for patients with advanced non-small cell lung cancer (NSCLC) with mutations in TP53, VEGFR1-3, PDGFR-A, PDGFR-B, and FGFR1-3 treated with nintedanib as part of an open-label, single-arm pilot study. Methods: Patients with advanced NSCLC previously treated with platinum-doublet chemotherapy with the above mutations were enrolled. Exclusion criteria included necrotic tumors with invasion of blood vessels, history of recent thromboembolic events, increased risk of bleeding or thrombosis, myocardial infarction, and weight loss >10% within past 6 months. Nintedanib was administered at a dose of 200 mg orally twice daily until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Secondary endpoints included progression-free survival (PFS) and correlating outcomes with specific mutations. This study was registered with ClinicalTrials.gov, number NCT02299141. Results: Between 2015 and 2019, 20 patients were enrolled with a median age was 66 years, 15 (75%) were females, 15 (75%) had adenocarcinoma, and 17 patients had a TP53 mutation (85%). Seventeen (85%) had received prior immunotherapy and 11 (55%) had received at least three prior lines of systemic therapy. The ORR was 15% with three partial responses (PR), while 12 patients had stable disease (SD), with disease control rate (DCR) consisting of a PR and SD greater than or equal to 16 weeks of 65% (n=13). Median PFS was 4.3 months [95% confidence interval (CI): 1.8-7.9] and median overall survival (OS) was 11.3 months (95% CI: 3.5-44.2). Three patients experienced prolonged clinical benefit from nintedanib, remaining on treatment for over 1 year and all three had a TP53 mutation and received prior immunotherapy. The most common adverse events of any grade included nausea (80%), fatigue (70%), diarrhea (60%), and anorexia (60%). Conclusions: In this pilot study in heavily pretreated and molecularly selected patients with metastatic NSCLC, nintedanib showed modest activity.

Late-onset corneal edema after customized crosslinking for progressive keratoconus.

Purpose: We describe a patient after customized crosslinking (CXL) for progressive keratoconus who developed corneal edema with spontaneous resolution. Observations: A 24-year-old male with progressive keratoconus of the left eye underwent a customized CXL procedure with a total energy of 10 J/cm2 for 16.4 minutes. Preoperative corrected distance visual acuity (CDVA) was 20/30 with a maximum keratometry (K)-value of 58.6 diopter (D) and the thinnest point measured 414 µm. The preoperative endothelial cell density (ECD) was 2414 cells/mm2. During treatment, corneal thickness was 325 µm after epithelial debridement and 375 µm after the application of 0.1 % riboflavin containing HPMC. After the treatment, antibiotic and steroid drops were prescribed for 5 days and 3 weeks, respectively. At the 1-month post-CXL visit the patient had no complaints, visual acuity and clinical examination showed no irregularities. At the 4-months post-CXL visit the patient complained of blurry vision. The CDVA was 20/100 and slit-lamp examination showed microcystic corneal edema. The corneal thickness at the thinnest point measured 440 µm. One month later the edema had resolved spontaneously and CDVA had restored to 20/25. Corneal thickness at the thinnest point measured 415 µm, the ECD was 1514 cells/mm2 and confocal microscopy showed normal structural changes in the anterior stroma after CXL, with the demarcation line located at a depth of 414 µm, just above the corneal endothelium. Conclusions and importance: We report a case of corneal edema following customized CXL with endothelial cell loss that resolved spontaneously. We recommend either adhering to a minimal stromal thickness of 400 µm before administering UV-A irradiation, using a contact lens or adjusting the irradiation to prevent this complication.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Shear-Sensitive circRNA-LONP2 Promotes Endothelial Inflammation and Atherosclerosis by Targeting NRF2/HO1 Signaling.

Hemodynamic shear stress is a frictional force that acts on vascular endothelial cells and is essential for endothelial homeostasis. Physiological laminar shear stress (LSS) suppresses endothelial inflammation and protects arteries from atherosclerosis. Herein, we screened differentially expressed circular RNAs (circRNAs) that were significantly altered in LSS-stimulated endothelial cells and found that circRNA-LONP2 was involved in modulating the flow-dependent inflammatory response. Furthermore, endothelial circRNA-LONP2 overexpression promoted endothelial inflammation and atherosclerosis in vitro and in vivo. Mechanistically, circRNA-LONP2 competitively sponged miR-200a-3p and subsequently promoted Kelch-like ECH-associated protein 1, Yes-associated protein 1, and enhancer of zeste homolog 2 expression, thereby inactivating nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling, promoting oxidative stress and endothelial inflammation, and accelerating atherosclerosis. LSS-induced down-regulation of circRNA-LONP2 suppresses endothelial inflammation, at least in part, by activating the miR-200a-3p-mediated nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway. CircRNA-LONP2 may serve as a new therapeutic target for atherosclerosis.

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1ß). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1ß (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Elevated tumor necrosis factor alpha and vascular endothelial growth factor in intermediate age-related macular degeneration and geographic atrophy.

Introduction: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine implicated in pathological changes to the retinal pigment epithelium that are similar to changes in geographic atrophy (GA), an advanced form of age related macular degeneration (AMD). TNF-α also modulates expression of other cytokines including vascular endothelial growth factor (VEGF), leading to choroidal atrophy in models of AMD. The purpose of this study was to investigate systemic TNF-α and VEGF in patients with GA and intermediate AMD (iAMD) compared to controls without AMD. Methods: We examined plasma levels of TNF-α and VEGF in patients with GA, iAMD, and controls without AMD from the University of Colorado AMD registry (2014 to 2021). Cases and controls were characterized by multimodal imaging. TNF-α and VEGF were measured via multiplex immunoassay and data were analyzed using a non-parametric rank based linear regression model fit to plasma biomarkers. Results: There were 97 GA, 199 iAMD patients and 139 controls. TNF-α was significantly increased in GA (Median:9.9pg/ml, IQR:7.3-11.8) compared to iAMD (Median:7.4, IQR:5.3-9.1) and in both GA and iAMD compared to controls (Median:6.4, IQR:5.3-7.8), p<0.01 for all comparisons. VEGF was significantly increased in iAMD (Median:8.9, IQR:4.8-14.3) compared to controls (Median:7.7, IQR:4.6-11.1), p<0.01. There was a significant positive correlation between TNF-α and VEGF in GA (0.46, p<0.01), and iAMD (0.20, p=0.01) with no significant interaction between TNF-α and VEGF in any group. Discussion: These findings suggest TNF-α and VEGF may contribute to systemic inflammatory processes associated with iAMD and GA. TNF-α and VEGF may function as systemic biomarkers for disease development.

Identification of key biomarkers of endothelial dysfunction in hypertension with carotid atherosclerosis based on WGCNA and the LASSO algorithm.

Background: Endothelial dysfunction is the early stage of carotid atherosclerosis (CAS) in patients with hypertension. It is worth identifying the potential hub genes of endothelial dysfunction to elucidate pathological mechanism in the progression of the disease. Method: We obtained gene expression profiles of GSE43292 from the Gene Expression Omnibus (GEO) database. Hub genes associated with CAS were identified through weighted gene correlation network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) regression. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore potential biological mechanisms and signaling pathways. Finally, in vitro experiments on human umbilical vein endothelial cells (HUVECs) were conducted to validate these hub genes. Results: The microarray dataset GSE43292 included 32 CAS plaques samples and corresponding macroscopically intact tissues from patients with hypertension. A total of 161 differentially expressed genes were discovered. Through WGCNA analysis, the gray60 module emerged as the most significant module associated with clinical features. The GO and KEGG enrichment analyses of genes in the gray60 module highlighted the substantial involvement of immune response-related signaling pathways. Two key hub genes (CCR1 and NCKAP1L) were pinpointed via LASSO regression. We found a significant increase in the mRNA expression level of the hub genes in oxidized low density lipoprotein (ox-LDL) treated HUVECs. Conclusions: Our study indicated that the hub genes related to immune responses are involved in the development of CAS. Two hub genes (CCR1 and NCKAP1L) of endothelial dysfunction were identified. These genes may provide a valuable therapeutic target of CAS in patients with hypertension.

Epigenetic regulation by hypoxia, N-acetylcysteine and hydrogen sulphide of the fetal vasculature in growth restricted offspring: A study in humans and chicken embryos.

Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3 methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.

Age and sex differences in the effects of short- and long-term exposure to air pollution on endothelial dysfunction.

BACKGROUND: The effects of air pollution on endothelial function remain unclear across populations. We aimed to use brachial artery flow-mediated dilatation (FMD) to identify demographic differences in the effects of air pollution exposure on endothelial dysfunction. METHODS: We measured FMD in 850 participants from October 2016 to January 2020. Location-specific concentrations of fine particulate matter < 2.5 µm aerodynamic diameter (PM2.5), inhalable particulate matter < 10 µm aerodynamic diameter (PM10), sulfur dioxide (SO2), nitrogen dioxide (NO2), carbon monoxide (CO), and ozone (O3) measured by fixed ambient air monitoring stations were collected for short- and long-term exposure assessment. Multiple linear regression models and restricted cubic splines were used to assess the associations before and after stratification by age and sex. RESULTS: This study eventually included 828 participants [551 (66.5%) younger than 65 years and 553 (66.8%) men]. Each 10 µg/m3 increase in 7-day exposure to PM2.5 and PM10 was significantly linearly associated with a 0.07% (ß = -0.07, 95% CI: -0.13 to -0.004) and 0.05% (ß = -0.05, 95% CI: -0.10 to -0.004) decrease in FMD in the fully adjusted model. After full adjustment, long-term exposure to all air pollutants was significantly associated with impaired FMD. Each 10 µg/m3 increase in long-term exposure to PM2.5 and PM10 was significantly associated with a -0.18% (95% CI: -0.34 to -0.03) and - 0.23% (95% CI: -0.40 to -0.06) change in FMD, respectively. After stratification, the associations of lower FMD with long-term exposure to PM2.5, PM10, SO2, NO2, and CO significantly persisted in men and participants younger than 65 years instead of women or older participants. For short-term exposure, we observed differences consistent with long-term exposure and a stronger effect of 7-day exposure to SO2 in men due to a significant interaction effect. CONCLUSION: Short- and long-term exposure to different air pollutants are strongly associated with decreased endothelial function, and susceptibility to air pollution varies significantly with age and sex.