Normal Endothelial Cells Research Articles

New Pyrrole Derivatives as Promising Biological Agents: Design, Synthesis, Characterization, In Silico, and Cytotoxicity Evaluation.

The current study describes the synthesis, physicochemical characterization and cytotoxicity evaluation of a new series of pyrrole derivatives in order to identify new bioactive molecules. The new pyrroles were obtained by reaction of benzimidazolium bromide derivatives with asymmetrical acetylenes in 1,2-epoxybutane under reflux through the Huisgen [3 + 2] cycloaddition of several ylide intermediates to the corresponding dipolarophiles. The intermediates salts were obtained from corresponding benzimidazole with bromoacetonitrile. The structures of the newly synthesized compounds were confirmed by elemental analysis, spectral techniques (i.e., IR, 1H-NMR and 13C-NMR) and single-crystal X-ray analysis. The cytotoxicity of the synthesized compounds was evaluated on plant cells (i.e., Triticum aestivum L.) and animal cells using aquatic crustaceans (i.e., Artemia franciscana Kellogg and Daphnia magna Straus). The potential antitumor activity of several of the pyrrole derivatives was studied by performing in vitro cytotoxicity assays on human adenocarcinoma-derived cell lines (i.e., LoVo (colon), MCF-7 (breast), and SK-OV-3 (ovary)) and normal human umbilical vein endothelial cells (HUVECs). The obtained results of the cytotoxicity assessment indicated that the tested compounds had nontoxic activity on Triticum aestivum L., while on Artemia franciscana Kellogg nauplii, only compounds 2c and 4c had moderate toxicity. On Daphnia magna, 4b and 4c showed high toxicity; 2a, 2b, and 2c moderate to high toxicity; only 4a and 4d were nontoxic. The compound-mediated cytotoxicity assays showed that several pyrrole compounds demonstrated dose- and time-dependent cytotoxic activity against all tested tumor cell lines, the highest antitumor properties being achieved by 4a and its homologue 4d, especially against LoVo colon cells.

Three-Year Outcomes of Under-flap Stromal Bed CXL for Early Post-LASIK Ectasia.

To investigate the 36-month clinical outcomes of under-flap stromal bed CXL (ufCXL) and report on its ability to stabilize post-laser in situ keratomileusis (LASIK) ectasia. This case series included 20 eyes with diagnosed early post-LASIK ectasia treated with ufCXL. Inclusion criteria were early, mild post-LASIK ectasia, defined as new-onset postoperative manifest refraction cylinder of 1.50 diopters (D) or less, with new topographic inferior steepening consistent with ectasia, uncorrected distance visual acuity of 20/40 or better, and corrected distance visual acuity of 20/25 or better. The existing LASIK flap was lifted, 0.25% isotonic riboflavin was applied directly to the stromal bed, the flap was repositioned, and 18 mW/cm2 ultraviolet light was applied for 5 minutes to the corneal flap surface. Post-ufCXL ophthalmic data were compared to pre-ufCXL baseline measurements. Visual outcomes were maintained pre-ufCXL to 36 months post-ufCXL, with preserved safety index (P = .6545), efficacy index (P = .4980), spherical equivalent accuracy (P = .1536), defocus equivalent accuracy (P = .1032), central corneal thickness (P = .5196), and corneal irregularity indices at 3 mm (P = .8548) and 5 mm (P = .3399). Refractive astigmatism significantly decreased from 0.83 to 0.55 D pre-ufCXL to post-ufCXL (P = .0439), as did maximum keratometry from 42.40 to 42.00 D pre-ufCXL to post-ufCXL (P = .0420). The ufCXL demarcation line depth was 336 ± 21 µm post-ufCXL, with normal endothelial cell density (2,574 ± 203 cells/mm2). Only 1 of 20 eyes showed evidence of progression of 1.00 D in maximum keratometry. The ufCXL procedure shows promise in stabilizing early post-LASIK ectasia. Visual function, refractive astigmatism, maximum keratometry, and corneal irregularity indices were statistically maintained at 36 months postoperatively. [J Refract Surg. 2022;38(8):511-519.].

Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer.

BACKGROUNDThe development of venous thromboembolism (VTE) is associated with high mortality among gastric cancer (GC) patients. Neutrophil extracellular traps (NETs) have been reported to correlate with the prothrombotic state in some diseases, but are rarely reported in GC patients.AIMTo investigate the effect of NETs on the development of cancer-associated thrombosis in GC patients.METHODSThe levels of NETs in blood and tissue samples of patients were analyzed by ELISA, flow cytometry, and immunofluorescence staining. NET generation and hypercoagulation of platelets and endothelial cells (ECs) in vitro were observed by immunofluorescence staining. NET procoagulant activity (PCA) was determined by fibrin formation and thrombin–antithrombin complex (TAT) assays. Thrombosis in vivo was measured in a murine model induced by flow stenosis in the inferior vena cava (IVC).RESULTSNETs were likely to form in blood and tissue samples of GC patients compared with healthy individuals. In vitro studies showed that GC cells and their conditioned medium, but not gastric mucosal epithelial cells, stimulated NET release from neutrophils. In addition, NETs induced a hypercoagulable state of platelets by upregulating the expression of phosphatidylserine and P-selectin on the cells. Furthermore, NETs stimulated the adhesion of normal platelets on glass surfaces. Similarly, NETs triggered the conversion of ECs to hypercoagulable phenotypes by downregulating the expression of their intercellular tight junctions but upregulating that of tissue factor. Treatment of normal platelets or ECs with NETs augmented the level of plasma fibrin formation and the TAT complex. In the models of IVC stenosis, tumor-bearing mice showed a stronger ability to form thrombi, and NETs abundantly accumulated in the thrombi of tumor-bearing mice compared with control mice. Notably, the combination of deoxyribonuclease I, activated protein C, and sivelestat markedly abolished the PCA of NETs.CONCLUSIONGC-induced NETs strongly increased the risk of VTE development both in vitro and in vivo. NETs are potential therapeutic targets in the prevention and treatment of VTE in GC patients.

CXC Motif Chemokine Receptor Type 4 Disrupts Blood-Brain Barrier and Promotes Brain Metastasis Through Activation of the PI3K/AKT Pathway in Lung Cancer

CXC motif chemokine receptor type 4 (CXCR4) is an indispensable factor in the process of lung cancer brain metastasis (LCBM). The PI3K/AKT signal pathway is crucial in affecting cell invasion and metastasis and serves as a pivotal regulator in LCBM. However, the relationship between CXCR4 and the PI3K/AKT signal pathway is unclear. This study aimed to explore the underlying mechanisms of CXCR4 and PI3K/AKT in LCBM. Two lung cancer cells (A549 and H1299) and cells transfected with short hairpin RNA (shRNA)-CXCR4 were cocultured with normal human astrocyte cells and human brain endothelial (hCMEC/D3) cells to establish a blood-brain barrier model invitro. The proliferation, migration, and invasion tight junction proteins (claudin-5, occludin, and ZO-1) were examined. Finally, results were verified in a nude mice model. The abilities of cell proliferation, migration, and invasion were significantly reduced in A549 and H1299 cells transfected with shRNA-CXCR4 compared with the negative control group. The proteins phosphorylated PI3K and phosphorylated AKT were downregulated in lung cancer cells transfected with shRNA-CXCR4. The proteins claudin-5, occludin, and ZO-1 were upregulated in the A549 and H1299 cells transfected with shRNA-CXCR4. Invivo experiment results confirmed that the knockdown of CXCR4 played a protective role in the process of LCBM. Our findings revealed that CXCR4 promotes LCBM by regulating the PI3K/Akt signal pathway. We also demonstrated that inhibiting CXCR4 could lead to prevention of LCBM. This study provides further rationale for clinical therapy that targets CXCR4/PI3K/AKT.

Knocking Out of CEACAM1 Can Reduce Oxidative Stress and Promote Cell Proliferation in the HPMVECs under Hypoxia.

Pulmonary hypertension (PH) induced by hypoxia is common in clinical practice and often suggests a poor prognosis. The oxidative stress and proliferation of pulmonary vascular endothelial cells caused by hypoxia are the major mechanisms involved in the pathophysiology of PH. It has been reported in recent years that the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes angiogenesis. In this study, normal human pulmonary microvascular endothelial cells (HPMVECs) and HPMVECs with stable knockout of CEACAM1 by CRISPR-Cas9 were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) to induce hypoxic conditions. JC-1, ROS, and cell cycle profile were analyzed for each cell line and controls, using flow cytometry. A tube formation assay was used to detect angiogenesis, along with expression levels of CEACAM1, TNF-α, VEGF, VEGFR-2, p-P38/P38, and CyclinD1 proteins (to distinguish profiles of angiogenic growth and cell proliferation). We observed increased expression of CEACAM1 in HPMVECs after OGD/R, while ROS production was reduced and mitochondrial membrane potential was increased after OGD/R in CEACAM1−/− HPMVECs. Furthermore, we observed increased cell division in CEACAM−/− HPMVECs, accompanied by enhanced angiogenesis and reduced TNF-α protein expression and increased VEGF, VEGFR-2, and CyclinD1 expression. Together, these data suggest that upregulation of CEACAM1 in HPMVECs under hypoxic conditions may damage cells by increasing oxidative stress and inhibiting cell proliferation.

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer

BackgroundCrosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME.MethodsTEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell–cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model.ResultsCompared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro.ConclusionsOverall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.Graphical

Abstract 6401: PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

Abstract Introduction & objectives: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA Positron Emission Tomography/Computed Tomography (PET/CT) has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear. Materials & methods: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the conditioned medium. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived conditioned medium using a tube formation assay that mimics angiogenesis. Results: Immunohistochemical positivity of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in four of 33 prostate cancer patients (12.1%). We also found that the 10,000 × g pellet fraction of the LNCaP-derived conditioned medium containing PSMA-positive membranes, including microvesicles, transformed HUVECs from “PSMA-negative” to “PSMA-positive.” Mass spectrometry revealed that the fraction contained an important growth factor. Furthermore, treating HUVECs with the 10,000 × g pellet fraction of the LNCaP-derived conditioned medium significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner. Conclusions: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues. The endocytic process of the PSMA-positive microvesicles in normal endothelial cells may be an attractive target to develop novel anti-angiogenic drugs, which could inhibit transformation of normal endothelial cells to tumor endothelial cells (Watanabe et al., The Prostate. 2021. in press). Citation Format: RYUTA WATANABE, MASASHI MAEKAWA, TAKASHI KIYOI, MIE KURATA, NORIYOSHI MIURA, TADAHIKO KIKUGAWA, SHIGEKI HIGASHIYAMA, TAKASHI SAIKA. PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6401.

Abstract 3802: Micromilieu-dependent transcriptional memory of tumor vessel endothelial cells in colorectal cancer

Abstract Purpose of the study: Tumor microenvironment (TME)-induced plasticity of fibroblasts and immune cells are known to contribute to cancer pathogenesis. In contrast, only little is known about the existence, stability, functions and prognostic potential of different plastic phenotypes of tumor vessel endothelial cells (TECs) in cancer. Recently, transcriptional memory effects have been observed in different cell lines after cytokine stimulation in vitro. From this we developed the hypothesis that isolated TEC may exhibit TME-dependent transcriptional memories, which may provide novel insights into the intratumoral effects on TEC plasticity in cancer. To investigate this, we isolated ultrapure TECs from colorectal cancer (CRC) with prognostically favorable (Th-1-like) or worse (non-Th1-like) immune-micro-environments and analyzed the prescence of TME-associated transcriptional memory patterns. Methods: TECs and as controls normal colon endothelial cells (NECs), PBMCs and tumor cells (isolated by laser-microdissection) from the same CRC patients (n=75) were isolated by FACS-sorting. Ultra high purity (98%) of TECs and NECs was validated by qPCR and immunocytochemistry. Quality validated complete sample sets from 12 patients (Th-1-like, n=6, non-Th1-like, n=6) were compared using exome sequencing, Clariome S chips and EPICmethylation. Integrative bioinformatics was used to identify differential gene sets and to predict their association with prognosis. Results: Differential transcriptional memory patterns were detected in TECs derived from CRC with different prognostic TMEs. Of note, the aggressiveness of the originating tumors was reflected by the functions of the vascular transcriptional memory genes. The transcriptional memory was imprinted by epigenetic DNA methylation and not due to acquired somatic variants in the genome. These results could be successfully confirmed by alternative methods. Most importantly, integrative bioinformatics analyses retrieved seven vascular memory genes (VMG), which were expressed in the TECs in a TME-dependent manner and predicted the outcome of patients in an independent cohort of CRC patients. Conclusion: Our findings demonstrate for the first time a TME-dependent transcriptional memory of TECs, providing a clinically relevant indicator of intra-tumoral TME-dependent vascular plasticity with perspectives for patient stratification in vessel-directed tumor therapies. Citation Format: Michael Stürzl, Maximilian Fuchs, Meik Kunz, Nicholas Dickel, Bernt Popp, Steffen Uebe, Arif B. Ekici, Carol I. Geppert, Susanne Merkel, Vera S. Schellerer, Elisabeth Naschberger. Micromilieu-dependent transcriptional memory of tumor vessel endothelial cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3802.

Activation of CEACAM1 with an agonistic monoclonal antibody results in inhibition of melanoma cells.

Inhibitory receptors (IRs), such as the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), are cell surface molecules expressed on both normal epithelial, endothelial, and hematopoietic cells and on neoplastic cells. IRs are usually used by cancer cells to inhibit immune cell functions. Thus, CEACAM1 positive tumor cells can interact homophilically with CEACAM1 expressed on T and NK cells to inhibit their antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we investigated the effect of agonistic/activating anti-CEACAM1 monoclonal antibody (mAb) on melanoma cell lines in vitro and in vivo, following our hypothesis that activation of CEACAM1 on melanoma cells by distinct mAbs may induce inhibition of cancer cell proliferation and/or their death. To address this, we established an activating anti-CEACAM1 mAb (CCM5.01) and characterized its binding to the CEACAM1 receptor. Using this mAb, we assessed the expression of CEACAM1 on four different human melanoma cell lines by western blot and flow cytometry and determined its effect on cell viability in vitro by MTT assay. Furthermore, we evaluated the mAb mechanism of action and found that binding of CEACAM1 with CCM5.01 induced SHP1 phosphorylation and p53 activation resulting in melanoma cell apoptosis. For in vivo studies, a xenograft model of melanoma was performed by injection of Mel-14 cells subcutaneously (s.c.) in SCID/Beige mice followed by intraperitoneal (i.p.) injection of CCM5.01 or of IgG1 isotype control every other day. CCM5.01 treated mice showed a slight but not significant decrease in tumor weight in comparison to the control group. Based on the obtained data, we suggest that activating CEACAM1 on melanoma cells might be a promising novel approach to fight cancers expressing this IR.

VEGF-A controls the expression of its regulator of angiogenic functions, dopamine D2 receptor, on endothelial cells.

We have previously demonstrated significant upregulation of dopamine D2 (DAD2) receptor (DRD2) expression on tumor endothelial cells. The dopamine D2 receptors, upon activation, inhibit the proangiogenic actions of vascular endothelial growth factor-A (VEGF-A, also known as vascular permeability factor). Interestingly, unlike tumor endothelial cells, normal endothelial cells exhibit very low to no expression of dopamine D2 receptors. Here, for the first time, we demonstrate that through paracrine signaling, VEGF-A can control the expression of dopamine D2 receptors on endothelial cells via Krüppel-like factor 11 (KLF11)-extracellular signal-regulated kinase (ERK) 1/2 pathway. These results thus reveal a novel bidirectional communication between VEGF-A and DAD2 receptors.

Sorafenib-loaded silica-containing redox nanoparticles for oral anti-liver fibrosis therapy.

Liver fibrosis is a chronic disease resulting from repetitive or prolonged liver injury with limited treatment options. Sorafenib has been reported to be a potential antifibrotic agent; however, its therapeutic effect is restricted because of its low bioavailability and severe adverse effects in the gastrointestinal (GI) tract. In this study, we developed sorafenib-loaded silica-containing redox nanoparticles (sora@siRNP) as an oral nanomedicine to treat liver fibrosis. The designed siRNP were prepared by self-assembly of amphiphilic block copolymers, which possess antioxidant nitroxide radicals as a side chain of the hydrophobic segment and porous silica particles in the nanoparticle core. The silica moieties in the core formed a crosslink between the self-assembling block copolymers to afford stable drug absorption, which could be useful in harsh GI conditions after oral drug administration. Based on in vitro evaluation, sora@siRNP exerted antiproliferative and antifibrotic effects against hepatic stellate cells (HSCs) and low toxicity against normal endothelial cells. A pharmacokinetic study showed that siRNP significantly improved the bioavailability and distribution of sorafenib in the liver. In an in vivo study using a mouse model of CCl4-induced liver fibrosis, oral administration of sora@siRNP significantly suppressed the fibrotic area in comparison to free sorafenib administration. In mice with CCl4-induced fibrosis, free sorafenib administration did not suppress the expression of α-smooth muscle actin; however, mice treated with sora@siRNP showed significantly suppressed expression of α-smooth muscle actin, indicating the inhibition of HSC activation, which was confirmed by in vitro experiments. Moreover, oral administration of free sorafenib induced severe intestinal damage and increased leakage into the gut, which can be attributed to the generation of reactive oxygen species (ROS). Our antioxidant nanocarriers, siRNP, reduced the adverse effects of local ROS scavenging in the GI tract. Our results suggest that sora@siRNP could serve as a promising oral nanomedicine for liver fibrosis.

Epigenetic regulation of Anoctamin1 in pulmonary arterial hypertension

Introduction/HypothesisPulmonary arterial hypertension (PAH) is associated with substantial pulmonary vascular remodeling wherein endothelial cells (ECs) within the lumen hyper‐proliferate leading to occlusive plexiform lesions. We previously reported that Anoctamin‐1 (ANO1) expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the mechanism of ANO1 expression regulation in PAH remains unclear. DNA methylation has been recognized as an important mechanism in gene regulation. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically mediated via DNA methylation.Methods/ResultsDNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next‐Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’‐upstream, 5’‐UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. To determine whether decreased DNA methylation changes human ANO1 gene expression, we then exposed normal human pulmonary artery ECs to RG108, which is a DNA methyltransferase inhibitor that broadly reduces DNA methylation. Our data show that cells treated with RG108 for 72 hours have a significant 2‐fold increase in ANO‐1 mRNA expression in comparison to control cells, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. We are developing CRISPR‐based approaches to target these identified CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene.ConclusionsOur results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.

The internal elastic lamina of resistance arteries facilitates vasodilation

In large conduit arteries, smooth muscle cell elastin produces elastic laminae, while in resistance arteries endothelial cell derived elastin is the major contributor to the internal elastic lamina (IEL). The IEL in resistance arteries separates smooth muscle and endothelium and contain distinct holes in the IEL (HIEL) where the two cell types communicate via a structure termed the myoendothelial junction (MEJ). The MEJ is an anatomical hallmark of resistance arteries that pierces through the IEL and facilitates vasodilation by movement of endothelial derived hyperpolarization (EDH) to smooth muscle. In contrast, large arteries dilate via nitric oxide (NO) and do not contain MEJs. To study the role of the IEL in resistance arteries in facilitating MEJ formation and EDH‐based vasodilation, we used a mouse with a Cdh5Cre to constitutively knockout elastin from endothelial cells (elastin ECKO). In en face preparations from third order mesenteric arteries, elastin ECKO mice had a 55.49% reduction in IEL surface area, and, importantly, completely lacked the distinct HIEL where MEJs could form. Regardless of this, en face claudin‐5 staining demonstrated a normal endothelial cell morphology. The lack of distinct HIEL, the canonical MEJ sites, led us to hypothesize vasodilatory mechanisms may be disrupted. To test this hypothesis, we first performed pressure myography on third order mesenteric arteries from elastin ECKO mice. Myogenic tone began to diverge from littermate controls starting at 80 mmHg, with preserved active, but not passive tone. For this reason, to test for changes in EDH between control and elastin ECKO, we used arteries pressurized to 60 mmHg and performed acetylcholine (Ach) dose‐response curves. We found elastin ECKO mice have decreased responses to Ach. Additionally, in the presence of 30 uM L‐NAME, the majority of dilation in elastin ECKO arteries was inhibited, compared to no inhibition with L‐NAME in the littermate controls. This indicated that NO‐mediated dilation was present in elastin ECKO mice and not littermate controls. En face immunohistochemistry demonstrated disruption of proteins localized to MEJs that regulate EDH in resistance arteries: both phosphatidylserine (See Abstract ID:R5515) and alpha globin, which is used to chelate NO. These results indicated that the disruption in the IEL, and thus canonical MEJ sites, affected the polarization of EDH‐associated proteins in the endothelium, supporting the observation of increased NO‐based signaling in elastin ECKO arteries. Overall, these data imply endothelial‐derived elastin is necessary for the functional formation of MEJs, the vasodilatory capacity of the endothelium, and the pathway of vasodilation in resistance arteries.

Expression of ICAM-1 in Blood Vascular Endothelium and Tissues in Human Premalignant Lesion and Gastric/Hepatocellular Carcinomas.

Angiogenesis is essential for the outgrowth and metastasis of tumors. The structure and characteristics of tumor vasculature differ from those of normal vessels. We compared the characteristics of differentially expressed genes in endothelial cells (ECs) isolated from gastric and normal cells. Previously, we had isolated pure tumor ECs (TECs) and normal ECs (NECs) from advanced gastric cancer (AGC) lesions and normal mucosal tissues, respectively. Using the oligomer chip platform of the Affymetrix GeneChip technology, genes that were expressed more than three-fold with a significance of p≤0.001 were measured. The intercellular adhesion molecule 1 (ICAM-1) was found to be overexpressed in the TECs compared to the normal gastric ECs. In this study, the upregulation of ICAM-1 was confirmed in cultured TECs by immunofluorescence. The expression of ICAM-1 was upregulated in the ECs, as well as in the stromal and immune cells, in early human gastric preneoplastic and hepatic fibrotic tissues. Upregulation of ICAM-1 was observed in the TECs, immune cells, and cancer epithelial cells in AGC and hepatocellular carcinoma (HCC). These results suggest that increased ICAM-1 expression in the ECs of the tissue microenvironment progressively contributes to the recruitment of immune cells to promote inflammation, leading to fibrosis and tumorigenesis. Therefore, upregulated ICAM-1 in the tissues in premalignant gastric diseases or hepatic fibrosis and their malignant cancers could be a promising target for disease prevention and treatment.

Insulin like growth factor-1 works synergistically with dopamine to attenuate diabetic retinopathy by downregulating vascular endothelial growth factor

AimLevels of Insulin-like growth factor-1 (IGF-1), a proangiogenic growth factor is elevated and dopamine downregulated in proliferative diabetic retinopathy (PDR). This study aims to investigate whether IGF-1 with dopamine can together modulate vascular endothelial growth factor (VEGF) to prevent proliferative diabetic retinopathy while also attenuating angiogenic effects of IGF-1. MethodsEffect of combination of levodopa L-Dopa with IGF-1 was tested on normal retinal pigment epithelium cells (ARPE-19) and human umbilical vein endothelial cells (HUVEC), followed by tube formation. Invivo analysis of anti-angiogenic potential assessed by chick chorioallantoic membrane (CAM) assay. Diabetes induction in wistar rats at two time points, 12 and 16 weeks, treated with L-Dopa+IGF-1 and analysed for morphological variations, serum and tissue dopamine levels, gene expression by real-time PCR and western blot assay. ResultsL-Dopa+IGF-1 on ARPE-19 cells caused no toxicity and worked synergistically. Reduced number of vessels observed. Significant improvement in inner retina thickness (*p < 0.05) was observed when L-Dopa was given alone and/or with IGF-1. Dopamine levels improved significantly in both serum and tissue (*p < 0.05). Levels of VEGF and IGF-1 receptors reduced significantly in 12 weeks. Western studies suggest that L-Dopa+IGF-1 modulates its effects via Akt/ERK dependent pathway. ConclusionFirst ever report on synergistic effect of L-Dopa+IGF-1 in a rat model of diabetic retinopathy. Even though the effect of L-Dopa in combination with IGF-1 is comparable to levels of L-Dopa alone, this study presents an interesting finding of neuroprotective function of IGF-1, which has been studied in disease models of Parkinson’s but not diabetes.

Hsa_circ_0001017 promotes cell proliferation, migration and invasion in osteosarcoma by sponging miR-145-5p

BackgroundCircular RNAs (circRNAs) have displayed important roles in the development and progression of various cancers. However, the functions of the majority of circRNAs in osteosarcoma (OS) remain unknown.MethodsCircular RNA microarray analysis was performed in three OS cell lines (Saos-2, U2OS and MG63) and normal vascular endothelial cells. The co-differentially expressed circRNAs (CDECs) were identified in OS cell lines with the criterion of FDR (false discovery rate) < 0.05 and |fold change (FC)|> 2. Quantitative real-time PCR was used to validate the expression levels of selected CDECs. A series of functional assays, including MTT assay, flow cytometry and transwell assay were conducted in OS cells. The interaction between circRNA and miRNAs was confirmed by luciferase reporter assay and RNA immunoprecipitation assay.ResultsA total of 241 CDECs, including 75 upregulated and 166 downregulated CDECs, were identified in three OS cell lines compared with normal vascular endothelial cells. PCR validation showed that hsa_circ_0000704, hsa_circ_0001017 and hsa_circ_0005035 were all highly expression in the three OS cell lines, compared with osteoblast cell lines (HECC, hFOB1.19 and HFF-1). Functionally, overexpression of circ_0001017 significantly promoted the cell proliferation, migration and invasion and decreased apoptosis in U2OS cells. Knockdown of circ_0001017 obtained the opposite results. Circ_0001017 may downregulate miR-145-5p through direct binding. Furthermore, the expression of miR-145-5p was negatively regulated by circ_0001017 in OS cells. In addition, further functional studies indicated that miR-145-5p inhibitor eliminated the effects caused by si-circ_0001017 in OS cells.ConclusionsIn conclusion, our study suggested that circ_0001017 may be a novel oncogenic factor during the progression and development of OS by targeting miR-145-5p.

Tumor endothelial cell-induced CD8+ T-cell exhaustion via GPNMB in hepatocellular carcinoma.

Tumor endothelial cells (TECs) promote tumor angiogenesis and regulate cytotoxic T cells in the tumor microenvironment. However, the roles of TECs for tumor‐infiltrating T‐cell in hepatocellular carcinoma (HCC) is still unknown. Here, we aimed to investigate how TECs influenced tumor growth and immune responses of HCC focusing on CD8+ T‐cell infiltration and exhaustion. First, TECs were isolated from subcutaneous HCC tumors with murine HCC cell lines (BNL‐T) with magnetic selection of CD31+ cells, and normal endothelial cells (NECs) were isolated from normal liver. Second, immunocompetent mice were injected with BNL‐T alone, BNL‐T + NECs, or BNL‐T + TECs for tumor formation, and the functions and exhaustion of tumor‐infiltrating CD8+ T cells were evaluated. The mice injected with BNL‐T + TEC showed rapid tumorigenesis and a decrease in the number of infiltrating CD8+ T cells. In addition, the percentage of CD8+ T‐cell exhaustion was significantly higher in tumors from the administration of BNL‐T + TEC. Third, the next‐generation sequencing on TECs was performed to identify mRNAs that might be a novel treatment target. The molecule of glycoprotein nonmetastatic melanoma protein B (GPNMB) was identified and the functions of GPNMB was analyzed by silencing of GPNMB expression using small interfering RNAs. The silencing of GPNMB expression in TECs induced the suppression of tumor growth and T‐cell exhaustion. In conclusion, TECs induced tumor‐infiltrating T‐cell exhaustion via GPNMB expression and GPNMB might be a novel therapeutic target in HCC.

Nectin cell adhesion molecule 4 regulates angiogenesis through Src signaling and serves as a novel therapeutic target in angiosarcoma

Angiosarcoma is a rare, life-threatening soft tissue sarcoma with malignant endothelial cells that is mainly found in the skin. Multidisciplinary approaches are used to treat patients with unresectable metastasized lesions; considering the cellular origin of angiosarcoma, anti-angiogenic therapy has also been used recently. However, these treatments have limited efficacy, and the survival rate remains low. Thus, more effective treatments need to be developed. Nectin cell adhesion molecule 4 (NECTIN4) is highly expressed in malignant tumors and promotes tumor progression. Thus, NECTIN4 is expected to be a novel therapeutic target for cancer. However, the significance of NECTIN4 in angiosarcoma remains unknown. Using immunohistochemistry, we investigated NECTIN4 expression in 74 tissue samples from angiosarcoma patients, finding variable NECTIN4 expression. In addition, we investigated NECTIN4 expression and function in human angiosarcoma cell lines. NECTIN4 expression was higher in angiosarcoma cells than normal endothelial cells, and angiosarcoma cells were sensitive to monomethyl auristatin E, the cytotoxic part of a NECTIN4-targetting antibody–drug conjugate. NECTIN4 knockdown inhibited the proliferation and angiogenesis of angiosarcoma cells, and Src kinase signaling was shown to be involved in NECTIN4 function, at least in part. NECTIN4-targeted therapy has the potential to be a novel treatment strategy for angiosarcoma.

Tumour endothelial cells for translational research and therapeutics

AbstractTumour vascularization plays an important role in tumour development and progression. Tumour endothelial cells (TECs) form the inner lining of tumour blood vessels. TECs are mainly derived from endothelial progenitor cells (EPCs); however, it is increasingly evident that in some tumours, TECs are also transformed from cancer stem cells. TECs are different from normal endothelial cells (NECs) in many aspects. On the one hand, the hierarchy of TECs is disorganized, leading to perturbed blood flow, and creating a hypoxic microenvironment in the tumour tissues, which stimulates tumour blood vessels to grow into the tumour tissues. However, the normal endothelial structure is intact, and blood flow is smooth. On the other hand, TECs are more dependent on glycolysis, mitochondrial respiration, and fatty acid metabolism, so there are many potential targets for anti‐angiogenesis therapy. TECs and NECs differ in their gene expressions; biological function experiments have demonstrated that TECs and NECs have different phenotypes. This is essentially caused by gene mutations in TECs. Previous reports mostly described TECs as endothelial cells covering the inner surface of tumour blood vessels. Up to now, there is no clear definition of a group of cells as TECs. Based on this, we summarize the characteristics of TECs as follows. (1) They are derived from EPCs or tumour stem cells. (2) They have up‐regulation of glycolysis, mitochondrial respiration, and fatty acid metabolism. (3) They secrete different cytokines. (4) Some TECs highly express epidermal growth factor receptor and interleukin‐4 receptor. (5) They are distributed around or in tumours and play an important role in the processes of tumour growth, development, and metastasis.