Normal Endothelial Cells Research Articles

Abstract 2495: Investigating pancreatic cancer-derived extracellular vesicle signaling in the tumor microenvironment

Abstract In many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), the secretion of extracellular vesicles (EVs) has been shown to support tumor progression, chemotherapeutic resistance, and metastasis. Importantly, PDAC tumors are composed of up to 80% non-tumor cells and an extracellular matrix, yet no studies have been performed to determine whether PDAC EVs are imported preferentially by specific non-tumor cell types. To improve our understanding of this signaling, we aim to define the recipient cells of PDAC EVs and evaluate the functional importance of this internalization. Utilizing an in vitro live imaging system, we found that cancer-associated fibroblasts (CAFs) import more PDAC EVs than normal pancreas epithelial, endothelial, or PDAC cells, suggesting PDAC EV trafficking may be preferential to CAFs. We have generated human PDAC and mouse KrasG12D Trp53R172H driven (KPC) PDAC cell lines to express an extracellular particle reporter labeling the inner leaflet of cell membranes with GFP-nLuc. We are currently using this model to characterize recipient cells of the GFP-nLuc-positive EVs in an immunocompetent orthotopic PDAC mouse model. We developed flow cytometry and immunofluorescence panels to assess PDAC EV import by CAFs, endothelial cells, macrophages, B cells, and T cells in developed tumors. Utilizing immunofluorescence staining for a panel of phenotypic markers, we will assess the functional importance of EV internalization. Future studies will begin to determine how EV mRNA cargoes are important for recipient cell phenotypic and functional changes. This study is the first to ascertain the fate of PDAC EVs within the tumor microenvironment in a physiologically relevant model. Citation Format: Jennifer M. Finan, Julie Saugstad, Jonathan Brody. Investigating pancreatic cancer-derived extracellular vesicle signaling in the tumor microenvironment [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 2495.

163 Integrative Genomics Analysis Implicates Decreased FGD6 Expression Underlying Risk of Intracranial Aneurysm Rupture

INTRODUCTION: The genetic determinants of intracranial aneurysm rupture (rIA) are largely unknown. To identify and characterize genetic risk factors for rIA, we perform a genome-wide association study (GWAS) containing 84,353 individuals (7,843 rIA cases and 76,510 controls). We then use functional genomics approaches to delineate the mechanistic consequences of rIA risk loci. METHODS: We perform a meta-analysis across 24 published GWAS of rIA. Logistic regression was used to identify rIA-associated single nucleotide polymorphisms (SNPs). Gene-burden, expression quantitative trait loci (eQTL) mapping, and analysis of publicly-available single-cell RNA sequencing (scRNA-seq) data from normal human brain endothelial and perivascular cells was used to delineate cell-lineage expression of rIA-associated genes. RESULTS: We identify 5 independent genetic loci reaching genome-wide significance (p < 5.0 x 10-8) for rIA: rs73392700 (CDKN2B, OR = 1.12), rs6841581 (EDNRA, OR = 0.77), rs11661542 (RBBP8, OR=0.84), rs62516550 (RP1, OR = 1.20), and rs12310399 (FGD6, OR = 1.16). FGD6, to our knowledge, has not been implicated in prior GWAS of (r)IA. We then quantified gene-level mutation-burden across ∼20,000 genes, and only FGD6 (containing 21 rIA-associated SNPs) reached transcriptome-wide significance. Functional eQTL mapping indicates that rs12310399 causes decreased FGD6 gene expression in arterial tissue. scRNA-seq identified high expression of FGD6 in 1 of 3 arterial lineages, but absent in perivascular cells, identifying the likely cell-lineage underlying FGD6-mediated rIA risk. CONCLUSIONS: We perform the largest genetic study of rIA to date. We identify and characterize a previously unknown risk loci for rIA containing FGD6. Elucidation of high-risk genetic loci may instruct population-genetic screening and clinical-genetic testing strategies to identify patients predisposed to rIA enabling early treatment and improved patient outcomes.

Stromal Senescence following Treatment with the CDK4/6 Inhibitor Palbociclib Alters the Lung Metastatic Niche and Increases Metastasis of Drug-Resistant Mammary Cancer Cells.

CDK4/6 inhibitors (CDKi) have improved disease control in hormone-receptor-positive, HER2-negative metastatic breast cancer, but most patients develop progressive disease. We asked whether host stromal senescence after CDK4/6 inhibition affects metastatic seeding and growth of CDKi-resistant mammary cancer cells by using the p16-INK-ATTAC mouse model of inducible senolysis. Palbociclib pretreatment of naïve mice increased lung seeding of CDKi-resistant syngeneic mammary cancer cells, and this effect was reversed by depletion of host senescent cells. RNA sequencing analyses of lungs from non-tumor-bearing p16-INK-ATTAC mice identified that palbociclib downregulates immune-related gene sets and gene expression related to leukocyte migration. Concomitant senolysis reversed a portion of these effects, including pathway-level enrichment of TGF-β- and senescence-related signaling. CIBERSORTx analysis revealed that palbociclib alters intra-lung macrophage/monocyte populations. Notably, lung metastases from palbociclib-pretreated mice revealed senescent endothelial cells. Palbociclib-treated endothelial cells exhibit hallmark senescent features in vitro, upregulate genes involved with the senescence-associated secretory phenotype, leukocyte migration, and TGF-β-mediated paracrine senescence and induce tumor cell migration and monocyte trans-endothelial invasion in co-culture. These studies shed light on how stromal senescence induced by palbociclib affects lung metastasis, and they describe palbociclib-induced gene expression changes in the normal lung and endothelial cell models that correlate with changes in the tumor microenvironment in the lung metastatic niche.

Modified glucose as a sensor to track the metabolism of individual living endothelial cells - Observation of the 1602 cm−1 band called “Raman spectroscopic signature of life”

A relatively new approach to subcellular research is Raman microscopy with the application of sensors called Raman probes. This paper describes the use of the sensitive and specific Raman probe, 3-O-propargyl-d-glucose (3-OPG), to track metabolic changes in endothelial cells (ECs). ECs play a significant role in a healthy and dysfunctional state, the latter is correlated with a range of lifestyle diseases, particularly with cardiovascular disorders. The metabolism and glucose uptake may reflect the physiopathological conditions and cell activity correlated with energy utilization. To study metabolic changes at the subcellular level the glucose analogue, 3-OPG was used, which shows a characteristic and intense Raman band at 2124 cm−1.3-OPG was applied as a sensor to track both, its accumulation in live and fixed ECs and then metabolism in normal and inflamed ECs, by employing two spectroscopic techniques, i.e. spontaneous and stimulated Raman scattering microscopies. The results indicate that 3-OPG is a sensitive sensor to follow glucose metabolism, manifested by the Raman band of 1602 cm−1. The 1602 cm−1 band has been called the “Raman spectroscopic signature of life” in the cell literature, and here we demonstrate that it is attributed to glucose metabolites. Additionally, we have shown that glucose metabolism and its uptake are slowed down in the cellular inflammation. We showed that Raman spectroscopy can be classified as metabolomics, and its uniqueness lies in the fact that it allows the analysis of the processes of a single living cell. Gaining further knowledge on metabolic changes in the endothelium, especially in pathological conditions, may help in identifying markers of cellular dysfunction, and more broadly in cell phenotyping, better understanding of the mechanism of disease development and searching for new treatments.

Effects of long non-coding RNA MALAT1-targeting miR-570-3p and miR-34a on the invasion, proliferation, and apoptosis of human retinoblastoma cells

Purpose: To investigate the expression of lncRNA MALAT1-targeting miR-570-3p and miR-34a and its effects on the invasion, proliferation, and apoptosis of human retinoblastoma cells.
 Methods: MiR-34a, miR-570-3p, and IncRNA MALAT1 in a human normal retinal vascular endothelial cell line (ACBRI-181), human retinoblastoma cell line (SO-Rb50), human normal retinal tissue and human retinoblastoma tissue were determined. Luciferase assay was used to verify the targeting relationship between LncRNA MALAT1 and miR-570-3p and miR-34a. while cell invasion, cell apoptosis and cell proliferation were assessed by Transwell assay, flow cytometry, and tumor pellet-forming assay, respectively.
 Results: LncRNA MALAT1 in SO-Rb50 cell line and human retinoblastoma tissue line were significantly up-regulated, while the expression levels of miR-34a and miR-570-3p were significantly down-regulated (p < 0.05). Luciferase assay results showed that lncRNA MALAT1 targeted miR-570-3p and miR-34a. The invasion and proliferation of SO-Rb50 cells in the miR-570-3p inhibitor and miR-34a inhibitor groups were significantly increased, while the apoptosis of SO-Rb50 cells was significantly decreased (p < 0.05). However, the invasion and proliferation of SO-Rb50 cells in sh-MALAT1 group were significantly decreased, while apoptosis significantly increased. However, compared with sh-MALAT1 group alone, the invasion and proliferation of SO-Rb50 cells in co-transfected sh-MALAT1+miR-570-3p inhibitor + miR-34a inhibitor group w significantly increased, but apoptosis significantly decreased (p < 0.05).
 Conclusion: LncRNA MALAT1 negatively regulates miR-570-3p and miR-34a to promote the invasion and proliferation of human retinoblastoma SO-Rb50 cells and inhibit apoptosis. These findings may be of significance in developing suitable therapies for human retinoblastoma

Fractionation and Characterization of Triterpenoids from Vaccinium vitis-idaea L. Cuticular Waxes and Their Potential as Anticancer Agents.

Fruit and leaf cuticular waxes are valuable source materials for the isolation of triterpenoids that can be applied as natural antioxidants and anticancer agents. The present study aimed at the semi-preparative fractionation of triterpenoids from cuticular wax extracts of Vaccinium vitis-idaea L. (lingonberry) leaves and fruits and the evaluation of their cytotoxic potential. Qualitative and quantitative characterization of obtained extracts and triterpenoid fractions was performed using HPLC-PDA method, followed by complementary analysis by GC-MS. For each fraction, cytotoxic activities towards the human colon adenocarcinoma cell line (HT-29), malignant melanoma cell line (IGR39), clear renal carcinoma cell line (CaKi-1), and normal endothelial cells (EC) were determined using MTT assay. Furthermore, the effect of the most promising samples on cancer spheroid growth and viability was examined. This study allowed us to confirm that particular triterpenoid mixtures from lingonberry waxes may possess stronger cytotoxic activities than crude unpurified extracts. Fractions containing triterpenoid acids plus fernenol, complexes of oleanolic:ursolic acids, and erythrodiol:uvaol were found to be the most potent therapeutic candidates in the management of cancer diseases. The specificity of cuticular wax extracts of lingonberry leaves and fruits, leading to different purity and anticancer potential of obtained counterpart fractions, was also enclosed. These findings contribute to the profitable utilization of lingonberry cuticular waxes and provide considerable insights into the anticancer effects of particular triterpenoids and pharmacological interactions.

Adaptive fuzzy control of drug delivery in cancer treatment using combination of chemotherapy and antiangiogenic therapy.

This paper introduces the adaptive fuzzy control scheme as a promising control technique for cancer treatment from a theoretical point of view. A mathematical model describing the dynamics of tumor growth under the drug interventions of chemotherapy and antiangiogenic therapy is considered. The model incorporates the effects of normal cells, cancer cells, and endothelial cells. Then, the control goals in cancer treatment are discussed and the desired trajectory for a typical patient is derived using the optimal control theory. Since the dynamic model of tumor growth is not accurate and also varies from patient to patient, an adaptive fuzzy controller is designed to make the outputs of the dynamic model track the desired trajectory. The proposed control system is model-independent and identifies the dynamic model of tumor growth over time. The performance of the designed controller is assessed by several numerical simulations. Finally, a hardware-in-the-loop simulation is conducted to validate the results.

MEG3 sponges miRNA-376a and YBX1 to regulate angiogenesis in ovarian cancer endothelial cells

ObjectivesRecent studies have demonstrated maternally expressed gene 3 (MEG3) as a tumor suppressor across multiple malignancies. Meanwhile, the role of MEG3 in ovarian cancer needs further investigation. We aim to study the effects of MEG3 on angiogenesis in ovarian cancer and the underlying mechanisms. MethodsThe transcript levels of MEG3 in ovarian cancer samples from the GEPIA database were analyzed and compared to those in normal samples. The effect of MEG3 on the tube formation ability was quantified in ovarian carcinoma-derived microvascular endothelial cells (ODMECs). Through sequence analysis, we identified miR-376a as a major candidate to bind to MEG3. A MEG3-miR-376a binding site was identified via genetic modulation methods. RAS p21 protein activator 1 (RASA1) was screened as a middle player to bridge the role of miR-376a and angiogenesis. The regulation between miR-376a and RASA1 was confirmed via a dual-luciferase reporter assay. Finally, the competition was explored between Y-box binding protein 1 (YBX1) and miR-376a in binding to MEG3. ResultsMEG3 was significantly downregulated in ODMECs compared with normal ovarian endothelial cells. Overexpression of MEG3 led to reduced tube formation of ODMECs. The MS2 hairpin assay showed that MEG3 acted as a platform to sponge miR-376a. RASA1, a key suppressor of tube formation, was directly targeted by miR-376a. Further, MEG3 suppressed angiogenesis through the miR-376a/RASA1 axis in ODMECs. Finally, YBX1 and miR-376a were competitively bound to MEG3. ConclusionThis study uncovered a novel mechanism that MEG3 sponged miRNA-376a and YBX1 to regulate the expression of RASA1 and exert an effect on the angiogenesis of ovarian cancer.

Bovine Lactoferrin Suppresses Tumor Angiogenesis through NF-κB Pathway Inhibition by Binding to TRAF6.

Tumor angiogenesis is essential for tumor progression. The inhibition of tumor angiogenesis is a promising therapy for tumors. Bovine lactoferrin (bLF) has been reported as an anti-tumor agent. However, bLF effects on tumor angiogenesis are not well demonstrated. This study evaluated the inhibitory effects of bLF on tumor angiogenesis in vivo and in vitro. Herein, tumor endothelial cells (TECs) and normal endothelial cells (NECs) were used. Proliferation, migration, tube formation assays, RT-PCR, flow cytometry, Western blotting, siRNA experiments and immunoprecipitation were conducted to clarify the mechanisms of bLF-induced effects. CD-31 immunoexpression was examined in tumor tissues of oral squamous cell carcinoma mouse models with or without Liposomal bLF (LbLF)-administration. We confirmed that bLF inhibited proliferation/migration/tube formation and increased apoptosis in TECs but not NECs. TNF receptor-associated factor 6 (TRAF6), p-p65, hypoxia inducible factor-α (HIF-1α) and vascular endothelial growth factor (VEGF) were highly expressed in TECs. In TECs, bLF markedly downregulated VEGF-A, VEGF receptor (VEGFR) and HIF-1α via the inhibition of p-p65 through binding with TRAF6. Since NECs slightly expressed p-p65, bLF-TRAF-6 binding could not induce detectable changes. Moreover, orally administrated LbLF decreased CD31-positive microvascular density only in TECs. Hence, bLF specifically suppressed tumor angiogenesis through p-p65 inhibition by binding to TRAF6 and suppressing HIF-1α activation followed by VEGF/VEGFR down-regulation. Collectively, bLF can be an anti-angiogenic agent for tumors.

Toxicity and efficacy of green tea catechin derivative-based micellar nanocomplexes for anticancer protein delivery.

Toxicity towards non-tumor cells during anticancer therapy can be reduced by using nanoscale systems for anticancer drug delivery. Usually only the loaded drug has anticancer activity. Recently, micellar nanocomplexes (MNCs) comprising green tea catechin derivatives for the delivery of the anticancer proteins, such as Herceptin, have been developed. Herceptin as well as the MNCs without the drug were effective against HER2/neu-overexpressing human tumor cells and had synergistic anticancer effects in vitro and in vivo. It remained unclear which kinds of negative effects the MNCs had on tumor cells exactly, and which of their components mediated them. Also, it was unclear if MNC has any toxicity effects on the normal cells of vital human organ systems. Herein we examined the effects of Herceptin-MNCs and their individual components on human breast cancer cells and on normal primary human endothelial and kidney proximal tubular cells. We applied a novel in vitro model that predicts nephrotoxicity in humans with high accuracy, as well as high-content screening and microfluidic mono- and co-culture models to thoroughly address effects on various cell types. The results showed that MNCs alone were profoundly toxic for breast cancer cells, and induced apoptosis regardless of HER2/neu expression levels. Apoptosis was induced by both green tea catechin derivatives contained within MNCs. In contrast, MNCs were not toxic for normal human cells, and the probability was low that MNCs would be nephrotoxic in humans. Together, the results supported the hypothesis that green tea catechin derivative-based MNCs could improve efficacy and safety of therapies with anticancer proteins.

The treatment efficacy of three-layered functional polymer materials as drug carrier for orthotopic colon cancer

Colorectal cancer (CRC) is a worldwide disease posing serious threats to people’s life. Surgery and postsurgical chemotherapy are still the first choices to control the rapid progression of cancer. However, tumor recurrence and even distant metastasis are prone to occur. As a result, it is in urgent demand to find a new method to control CRC progression while inhibiting distant metastasis. On this basis, this study developed the three-layered functionalized hydrogel-fibrous membrane-hydrogel composite materials. The Chinese traditional drugs 20 (S)-ginsenoside Rg3 (Rg3) and chemotherapeutic agent 5-fluorouracil (5-Fu) were loaded in the inner hydrogel and middle fibrous membrane and could be constantly released at the same time and space. The outer hydrogel was decorated with phenylboronic acid (PA) to interact with sialic acid expressed on the CRC cell surface. The composite materials possessed biocompatibility and showed no toxicity to normal human intestinal mucosa endothelial cells HIEC. According to the results, the cell viability of CT26 could be significantly decreased in vitro. The three-layered functionalized materials inhibited the original tumor progression and distant tumor metastasis to the liver in an orthotopic colon cancer mouse model by increasing the caspase3 expression and inhibiting the expressions of Bcl-2, Ki-67, and VEGF. In addition, the functions of major organs were not significantly damaged. Our study provides a safe and efficacious method of this three-layered functionalized hydrogel-fibrous membrane-hydrogel composite materials for CRC treatment.

Single-cell analysis of multiple cancer types reveals differences in endothelial cells between tumors and normal tissues

Endothelial cells (ECs) play an important role in tumor progression. Currently, the main target of anti-angiogenic therapy is the vascular endothelial growth factor (VEGF) pathway. Some patients do benefit from anti-VEGF/VEGFR therapy; however, a large number of patients do not have response or acquire drug resistance after treatment. Moreover, anti-VEGF/VEGFR therapy may lead to nephrotoxicity and cardiovascular-related side effects due to its action on normal ECs. Therefore, it is necessary to identify targets that are specific to tumor ECs and could be applied to various cancer types. We integrated single-cell RNA sequencing data from six cancer types and constructed a multi-cancer EC atlas to decode the characteristic of tumor ECs. We found that tip-like ECs mainly exist in tumor tissues but barely exist in normal tissues. Tip-like ECs are involved in the promotion of tumor angiogenesis and inhibition on anti-tumor immune responses. Moreover, tumor cells, myeloid cells, and pericytes are the main sources of pro-angiogenic factors. High proportion of tip-like ECs is associated with poor prognosis in multiple cancer types. We also identified that prostate-specific membrane antigen (PSMA) is a specific marker for tip-like ECs in all the cancer types we studied. In summary, we demonstrate that tip-like ECs are the main differential EC subcluster between tumors and normal tissues. Tip-like ECs may promote tumor progression through promoting angiogenesis while inhibiting anti-tumor immune responses. PSMA was a specific marker for tip-like ECs, which could be used as a potential target for the diagnosis and treatment of non-prostate cancers.

Scar-Degrading Endothelial Cells as a Treatment for Advanced Liver Fibrosis.

Deposition of extracellular matrix (ECM) in the liver is an important feature of liver cirrhosis. Recovery from liver cirrhosis is physiologically challenging, partially due to the ECM in scar tissue showing resistance to cell-mediated degradation by secreted matrix metalloproteinases (MMPs). Here, a cell-mediated ECM-degradation screening system (CEDSS) in vitro is constructed for high-throughput searching for cells with tremendous degradation ability. ECM-degrading liver sinusoidal endothelial cells (dLSECs) are screened using CEDSS, which exhibit 17 times the ability to degrade collagen when compared to other cells. The degradation ability of dLSECs is mediated by the upregulation of MMP9. In particular, mRNA expression of MMP9 shows an 833-fold increase in dLSECs compared to normal endothelial cells (nLSECs), and MMP9 is regulated by transcription factor c-Fos. In vivo, single intrasplenic injection of dLSECs alleviates advanced liver fibrosis in mice, while intraperitoneal administration of liver-targeting peptide-modified dLSECs shows enhanced fibrosis-targeting effects. Degradative human umbilical vein endothelial cells (dHUVECs) prove their enhanced potential of clinical translation. Together, these results highlight the potential of ECM-degrading endothelial cells in alleviating advanced liver fibrosis, thus providing remarkable insights in the development of ECM-targeting therapeutics.

O-Halogen-substituted arene linked selenium-N-heterocyclic carbene compounds induce significant cytotoxicity: Crystal structures and molecular docking studies

Synthesis of N-arylated benzimidazolium salts 1–2, as stable N-heterocyclic Carbene (NHC) σ- donor ligands, was carried out by simple, facile and high yielding method. Respective Se-NHC compounds 3–4 were synthesized in water at 100 °C in open air environment using elemental selenium as reactant. Various spectroscopic methods (FT-IR, 1H and 13C NMR) were used for characterization of the products. Single crystal of salt 2 was analyzed by x-rays crystallographic analysis. In-vitro anticancer studies of the products 1–4 were carried out against breast cancer cell line (MDA-MB-231), cervical cancer cell line from Henrietta Lacks (HeLa), human normal endothelial cell line (EA.hy926) and adenocarcinoma cell line (A549) using MTT assay and compared with a standard drug 5-Flourouracil (5FU). The products 1–4 showed IC50 values less than standard drug 5FU against MDA-MB-231. Against HeLa cell line, compound 2 and its complex 4 were more potent with IC50 value of 0.05 µM and 0.082 µM, respectively as compared with 5FU having IC50 value of 4.9 µM. Against A549 cell line the products 1–2 showed good IC50 values while 3 was inactive with very high value of IC50 and 4 also showed good IC50 value of 19.02 µM. Against EA.hy926 cell line, both the salts 1–2 showed more toxicity with lower IC50 values than selenium counterpart 3–4. The docked conformation of VEGFA, EGF, COX1 and HIF with active conformation of selenium compounds 3–4 and 5FU revealed numerous potential interactions. The selenium adducts 3–4 were better with lower binding energies than 5FU, however, both showed almost same values of inhibition constants and binding energies against all proteins. Also, inhibition constants of compounds 3–4 were almost equal but less than 5FU.

Synthesis and Antitumor Activities of Novel Mitochondria-Targeted Dihydroartemisinin Ether Derivatives.

Ten novel mitochondria-targeted dihydroartemisinin ether derivatives were designed, synthesized, and evaluated for antitumor activity against five cancer cell lines in vitro. Profoundly, compound D8-T (IC50 = 56.9 nM) showed the most potent antiproliferative activity against the T24 cells with low cytotoxicity in normal human umbilical vein endothelial cells. High-performance liquid chromatography analysis confirmed that D8-T targeted mitochondria 6.3-fold higher than DHA. ATP content assay demonstrated that D8-T decreased the ATP level of bladder cancer cells. The effect of D8-T on cell apoptosis was determined by flow cytometry and western blot of Bax and Bcl-2. Surprisingly, the results indicated that D8-T did not induce bladder cancer cell apoptosis. In contrast, the cell cycle analysis and western blot of CDK4, CDK6, cyclin D1, and p21 demonstrated that the cancer cell cycle was arrested at the G1 phase after D8-T treatment. Furthermore, the consistent results were received by RNA-seq assay. These promising findings implied that D8-T could serve as a great candidate against bladder cancer for further investigation.

Apelin Receptor Can Act as a Specific Marker and Promising Therapeutic Target for Infantile Hemangioma

Infantile hemangioma (IH), the most common benign tumor in infancy, is generally sensitive to propranolol treatment. However, the challenge remains because resistance or recurrence could occur in some patients, and the mechanism or target of propranolol remains unknown. Therefore, advancement in the drug development is needed. In this study, we explored whether apelin receptor (APJ) can become a candidate target. We found that APJ is expressed only in endothelial cells of IH (HemECs) but not in other vascular anomalies, and its antagonist, ML221, can negatively regulate cellular viability and functions of HemECs. This inhibitory effect could be replicated in a murine hemangioma model. Importantly, invitro experiments also indicated that ML221 failed to affect the proliferation or angiogenesis of normal endothelial cells or APJ-knockout HemECs. Through analysis of the phosphoantibody microarray data, ML221 was revealed to have an inhibitory effect on HemECs by suppressing the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. These results verified the distinctive expression of APJ in IH and specific inhibition of HemEC activity caused by ML221. In addition, APJ was also detected in propranolol-resistant IH. Collectively, we propose that APJ can act as a specific marker and a promising therapeutic target for IH, which will facilitate further drug development.

Investigation of the therapeutic potential of recombinant bispecific bivalent anti-PD-L1/VEGF nanobody in inhibition of angiogenesis

Objective Immunotherapy using monoclonal antibodies targeting programmed death ligand-1 (PD-L1) on cancer cells as a biomarker of escape from response to immune checkpoint has demonstrated efficacy in treating many solid tumors. In addition, some of the signals, such as vascular endothelial growth factor (VEGF), bind to receptors on the surface of normal endothelial cells and encourage angiogenesis, or the formation and survival of new blood vessels. Methods Due to the special features of nanobodies with high specificity and affinity as a powerful new tool in cancer therapy, here, a recombinant bispecific bivalent anti-PD-L1/VEGF nanobody was constructed and its functionality in inhibition of angiogenesis in vitro was investigated. Results Results demonstrated that bivalent anti-PD-L1/VEGF nanobody efficiently inhibited HUVEC and A431 cells proliferation and tube formation. In addition, bivalent anti-PD-L1/VEGF nanobody efficiently inhibited angiogenesis in an ex ovo Chick Chorioallantoic Membrane assay. Discussion The results indicate for the potential of bivalent anti-PD-L1/VEGF nanobody as a novel promising tool for cancer therapy.

Discovery of selective HDAC6 inhibitors capped by flavonoid or flavonoid-analogous moieties as anti-cancer therapeutics simultaneously harboring anti-proliferative and immunomodulatory activities

Specific HDAC6 inhibitors (HDAC6is) simultaneously harboring anti-proliferative and immunomodulatory properties may prohibit tumor progression via intrinsic and immune driven effects. Herein, built upon the structurally novel lead TFH-7, structure–activity relationship study culminated in the identification of azaflavone-capped compound 20, which exhibited comparable HDAC6 inhibitory activity (IC50 = 8.5 nM) to that of Tubastatin A, a highly selective HDAC6i, as well as favorable subtype specificity. Importantly, concurrent with its impressive anti-proliferative efficacy against several solid tumor cell lines, 20 remarkably alleviated the transduction of immune-related STAT3 signaling and attenuated the expression of immunosuppressive checkpoint PD-L1 at submicromolar concentration, highlighting the immunomodulatory properties. Moreover, consistent with its favorable subtype selectivity, 20 displayed low cytotoxicity against normal human umbilical vein endothelial cells, revealing a promising safety profile. Following the intravenous administration, it demonstrated acceptable elimination half-life and exposure in Sprague-Dawley rats. Hence, the extensive functional investigation or structural modification of 20 is valuable.

Recent advances in molecular biology of metabolic syndrome pathophysiology: endothelial dysfunction as a potential therapeutic target

Current advances in molecular pathobiology of endotheliocytes dysfunctions are promising in finding the pathogenetic links to the emergence of insulin resistance syndrome. Physiologically, human organism homeostasis is strictly controlled to maintain metabolic processes at the acquainted level. Many factors are involved in maintaining these physiological processes in the organism and any deviation is undoubtedly accompanied by specific pathologies related to the affected process. Fortunately, the body's defense system can solve and compensate for the impaired function through its multi-level defense mechanisms. The endothelium is essential in maintaining this homeostasis through its ability to modulate the metabolic processes of the organism. Pathological activity or impairment of physiological endothelium function seems directly correlated to the emergence of metabolic syndrome. The most accepted hypothesis is that endothelium distribution is due to endoplasmic reticulum stress and unfolded protein response development, which includes inhibition of long non-coding RNAs expression, cytokines disbalance, Apelin dysregulation, glycocalyx degradation, and specific microparticles. Clinically, the enhancement or restoration of normal endothelial cells can be a target for novel therapeutic strategies since the distribution of its physiological activity impairs homeostasis and results in the progression of metabolic syndrome, and induction of its physiological activity can ameliorate insulin resistance syndrome. Novel insights on the molecular mechanisms of endothelial cell dysfunction are concisely represented in this paper to enhance the present therapeutic tactics and advance the research forward to find new therapeutic targets.

Hypoxia Promotes Angiogenic Effect in Extracranial Arteriovenous Malformation Endothelial Cells.

Arteriovenous malformation (AVM) is characterized by high-flow blood vessels connecting arteries and veins without capillaries. This disease shows increased angiogenesis and a pathophysiological hypoxic environment in proximal tissues. Here, we analyzed the effects of hypoxia on angiogenesis in the endothelial cells (ECs) of AVM and normal tissues. ECs from human normal and AVM tissues were evaluated using immunocytochemistry with CD31. In vitro tube formation under hypoxia was tested in both ECs using Matrigel. The relative expression of angiogenesis-related genes was measured using real-time PCR. Under normoxia, CD31 was significantly higher in AVM ECs (79.23 ± 0.65%) than in normal ECs (74.15 ± 0.70%). Similar results were observed under hypoxia in AVM ECs (63.85 ± 1.84%) and normal ECs (60.52 ± 0.51%). In the tube formation test under normoxic and hypoxic conditions, the junction count and total vessel length were significantly greater in AVM ECs than normal ECs. Under both normoxia and hypoxia, the angiogenesis-related gene FSTL1 showed a significantly higher expression in AVM ECs than in normal ECs. Under hypoxia, CSPG4 expression was significantly lower in AVM ECs than in normal ECs. Accordingly, the angiogenic effect was increased in AVM ECs compared with that in normal ECs. These results provide a basic knowledge for an AVM treatment strategy.