Human EC Research Articles

Modeling the processes of transendothelial transport of LDL and macrophage migration

Atherosclerosis is a multifactorial process involving various pathological mechanisms: inflammation, lipoprotein modification, cholesterol accumulation, endothelial dysfunction, oxidative stress and formation of atherosclerotic plaque. The main participants in the development of this disease are endothelial cells, leukocytes and smooth muscle cells of intima. The adult endothelium contains heterogeneous cells, including typical endothelial cells (TEC) and giant multinucleated EC variants (MVEC). The process of passing molecules is called transendothelial transport (TET). The purpose of this work is to model the processes of TET of low-density lipoproteins (LDL) and monocyte migration using various EC variants. In the study, a human EC line (EA.hy926) was used. Multicore variants of EC (MVEC) were obtained by treating EC with a 50% solution of polyethylene glycol 6000. To study LDL transcytosis and monocyte migration, tablets with inserts (0.4 or 3.0 micron pore diameter, respectively) forming a two-level system were used. The LDL transmission rate was assessed by measuring the amount of cholesterol in the upper and lower chambers every 2, 5 and 24 hours. A modified Folch method was used to measure the amount of cholesterol. The cholesterol content was adjusted according to the total protein level in the well, measured by the Lowry method. The migration of macrophages was estimated by direct counting of cells. The content of internalized cholesterol in the MVEC was statistically significantly higher than in the EC. The rate of LDL transport did not differ. The rate of passage of the endothelial barrier formed by MVEC by macrophages was higher at points 2 and 5 hours. After 24 hours the number of migrated cells did not differ. The rate of transendothelial transport for typical and multinucleated variants of EC did not statistically differ. Although the presence of MBEC does not affect the transport of lipoproteins into the subendothelial layer, the fact that multinucleated cells accumulate lipid droplets more actively than typical ECS may indicate an important role in the pathogenesis of atherosclerosis. The rate of macrophage migration after 2 and 5 hours was higher for MVEC than for TEC, whereas immature macrophages did not migrate through the endothelial barrier for 24 hours.

Sesamolin serves as an MYH14 inhibitor to sensitize endometrial cancer to chemotherapy and endocrine therapy via suppressing MYH9/GSK3β/β-catenin signaling

BackgroundEndometrial cancer (EC) is one of the most common gynecological cancers. Herein, we aimed to define the role of specific myosin family members in EC because this protein family is involved in the progression of various cancers.MethodsBioinformatics analyses were performed to reveal EC patients’ prognosis-associated genes in patients with EC. Furthermore, colony formation, immunofluorescence, cell counting kit 8, wound healing, and transwell assays as well as coimmunoprecipitation, cycloheximide chase, luciferase reporter, and cellular thermal shift assays were performed to functionally and mechanistically analyze human EC samples, cell lines, and a mouse model, respectively.ResultsMachine learning techniques identified MYH14, a member of the myosin family, as the prognosis-associated gene in patients with EC. Furthermore, bioinformatics analyses based on public databases showed that MYH14 was associated with EC chemoresistance. Moreover, immunohistochemistry validated MYH14 upregulation in EC cases compared with that in normal controls and confirmed that MYH14 was an independent and unfavorable prognostic indicator of EC. MYH14 impaired cell sensitivity to carboplatin, paclitaxel, and progesterone, and increased cell proliferation and metastasis in EC. The mechanistic study showed that MYH14 interacted with MYH9 and impaired GSK3β-mediated β-catenin ubiquitination and degradation, thus facilitating the Wnt/β-catenin signaling pathway and epithelial–mesenchymal transition. Sesamolin, a natural compound extracted from Sesamum indicum (L.), directly targeted MYH14 and attenuated EC progression. Additionally, the compound disrupted the interplay between MYH14 and MYH9 and repressed MYH9-regulated Wnt/β-catenin signaling. The in vivo study further verified sesamolin as a therapeutic drug without side effects.ConclusionsHerein, we identified that EC prognosis-associated MYH14 was independently responsible for poor overall survival time of patients, and it augmented EC progression by activating Wnt/β-catenin signaling. Targeting MYH14 by sesamolin, a cytotoxicity-based approach, can be applied synergistically with chemotherapy and endocrine therapy to eventually mitigate EC development. This study emphasizes MYH14 as a potential target and sesamolin as a valuable natural drug for EC therapy.Graphical

Abstract 3122: The Role Of Oct4 In Endothelial Cell Sexual Dimorphism And Angiogenesis

Background: Biological sex differences play a critical role in vascular diseases. Using endothelial ( EC ) lineage tracing atherosclerotic mice and cultured mouse and human aortic EC, we recently found that female EC have lower angiogenic potential, higher expression of pro-inflammatory genes and intracellular ROS, and worse mitochondria functions than male EC. However, the mechanisms responsible for this EC sexual dimorphism are still unknown. We previously identified the pluripotency factor OCT4 as a critical regulator of EC characteristics, including angiogenic potential, ROS accumulation, and mitochondrial functions. Therefore, this study aimed to test whether OCT4 is responsible for sex-dependent differences in EC in vivo and in vitro . Methods: We used male and female EC-specific tamoxifen-inducible OCT4 knockout and EC-lineage tracing (EC-OCT4 KO) mice to study the role of OCT4 in non-atherosclerotic conditions leading to pro-inflammatory angiogenesis, such as hind-limb ischemia and skin injury models. Male and female mouse aortic EC (MAEC) were isolated from EC-OCT4 KO and wild-type littermate mice for in vitro experiments. We performed BrdU assay, EC tube formation in Matrigel (in vitro angiogenesis), wound scratch assay, and angiogenic proteome profile array to evaluate angiogenic potential. Results: Compared to wild-type mice, EC-OCT4 KO male mice exhibited accelerated blood flow recovery and wound healing after hind-limb ischemia and skin injury. However, EC-OCT4 KO female mice showed no difference in blood flow recovery and delayed skin wound healing compared to control mice. In vitro , although male and female OCT4-KO MAEC had higher angiogenic potential than control, the phenotype in female EC was less pronounced. Mechanistically, we found that male OCT4 KO MAEC express higher levels of monocyte cytokine protein 1 ( MCP1 ). Furthermore, the inhibition of MCP1 in male OCT4 KO MAEC, but not in female cells, led to significant decreases in cell migration, proliferation, and endothelial tube formation in Matrigel. Conclusions: Our data indicate that OCT4-dependent mechanisms contribute to the sex differences observed in EC, suggesting a novel biological role for pluripotency signaling in vascular pathologies and homeostasis.

Angiotensin II induces endothelial dysfunction and vascular remodeling by downregulating TRPV4 channels

Angiotensin II (Ang II) is a potent vasoconstrictor of vascular smooth muscle cells (VSMC) and is implicated in hypertension, but it's role in the regulation of endothelial function is not well known. We and others have previously shown that mechanically activated ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4) mediates flow- and/or receptor-dependent vasodilation via nitric oxide (NO) production in endothelial cells. Ang II was demonstrated to crosstalk with TRPV4 via angiotensin 1 receptor (AT1R) and β-arrestin signaling in epithelial and immortalized cells, however, the role of this crosstalk in endothelial cell function is not fully explored. Ang II treatment significantly downregulated TRPV4 protein expression and TRPV4-mediated Ca2+ influx in human EC without altering TRPV4 mRNA levels. Further, TRPV4-induced eNOS phosphorylation and NO production were significantly reduced in Ang II-treated human EC. Importantly, Ang II infusion in mice revealed that, TRPV4/p-eNOS expression and colocalization was reduced in endothelium in vivo. Finally, Ang II infusion induced vascular remodeling as evidenced by decreased lumen to wall ratio in resistant mesenteric arteries. These findings suggest that Ang II induces endothelial dysfunction and vascular remodeling via downregulation of TRPV4/eNOS pathway and may contribute to hypertension, independent of or in addition to its effect on vascular smooth muscle contraction.

Loss of TIMP3, but not TIMP4, exacerbates thoracic and abdominal aortic aneurysm

AimsAorta exhibits regional heterogeneity (structural and functional), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. Region-dependent functions have been reported for TIMP3 and TIMP4 in vascular pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. Methods & resultsTAA or AAA was induced in male and female mice lacking TIMP3 (Timp3−/−), TIMP4 (Timp4−/−) or in wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females, with a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA, while increased inflammation was detected in the media post-AAA, but in the adventitia post-TAA. Timp3−/− mice showed impaired intimal barrier integrity post-AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in AAA versus TAA. Severity of TAA and AAA in Timp4−/− mice was similar to WT mice. In vitro, Timp3 knockdown more severely compromised the permeability of human aortic EC monolayer compared to Timp4 knockdown or the control group. In aneurysmal aorta specimens from patients, TIMP3 expression decreased in the media in AAA, and in adventitial in TAA specimens, consistent with the impact of its loss in AAA versus TAA in mice. ConclusionTIMP3 loss exacerbates inflammation, adverse remodeling and aortic dilation, but triggers different patterns of remodeling in AAA versus TAA, and through different mechanisms.

Association of anti-TPM4 autoantibodies with vasculopathic cutaneous manifestations in juvenile dermatomyositis.

AECAs are detected in multiple forms of vasculitis or vasculopathy, including JDM. High levels of tropomyosin alpha-4 chain (TPM4) gene expression in cutaneous lesions and TPM4 protein expression in some endothelial cells (ECs) have been proven. Furthermore, the presence of autoantibodies to tropomyosin proteins have been discovered in DM. We therefore investigated whether anti-TPM4 autoantibodies are an AECA in JDM and are correlated with clinical features of JDM. The expression of TPM4 protein in cultured normal human dermal microvascular ECs was investigated by Western blotting. Plasma samples from 63 children with JDM, 50 children with polyarticular JIA (pJIA) and 40 healthy children (HC) were tested for the presence of anti-TPM4 autoantibodies using an ELISA. Clinical features were compared between JDM patients with and without anti-TPM4 autoantibodies. Autoantibodies to TPM4 were detected in the plasma of 30% of JDM, 2% of pJIA (P < 0.0001) and 0% of HC (P < 0.0001). In JDM, anti-TPM4 autoantibodies were associated with the presence of cutaneous ulcers (53%; P = 0.02), shawl sign rash (47%; P = 0.03), mucous membrane lesions (84%; P = 0.04) and subcutaneous edema (42%; P < 0.05). Anti-TPM4 autoantibodies significantly correlated with the use of intravenous steroids and IVIG therapy in JDM (both P = 0.01). The total number of medications received was higher in patients with anti-TPM4 autoantibodies (P = 0.02). Anti-TPM4 autoantibodies are detected frequently in children with JDM and are novel myositis-associated autoantibodies. Their presence correlates with vasculopathic and other cutaneous manifestations of JDM that may be indicative of more refractory disease.

Loss of tissue inhibitor of proteinase 3 (TIMP3), but not TIMP4, worsens abdominal and thoracic aortic aneurysm

Background: Aorta exhibits regional heterogeneity (structural and mechanical), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. TIMP3 and TIMP4 have shown region-dependent functions in aortic pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. Methods and Results: TAA or AAA was induced in male or female mice lacking TIMP3 ( Timp3 -/- ), TIMP4 ( Timp4 -/- ) or wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females. Timp3 -/- mice exhibited a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA whereas inflammation increased in the media post-AAA, but in the adventitia post-TAA. Timp3 -/- mice showed impaired intimal barrier integrity ( in vivo) following AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in TAA versus AAA. Timp4 -/- mice responded similarly to TAA and AAA induction as the WT. In vitro, Timp3 knockdown in human aortic EC more severely compromised the EC monolayer permeability compared to Timp4 knockdown and control groups following elastase treatment. In human aorta specimens, TIMP3 showed a region-specific expression pattern in TAA versus AAA consistent with the impact of its loss in the impaired intimal barrier in AAA, and the adventitial region in TAA. Conclusions: TIMP3 plays a protective role in both AAA and TAA, whereas loss of TIMP4 has minimal impact on the severity of aneurysm formation. TIMP3 loss in the aneurysmal aorta could be responsible for the inflammation, adverse remodeling and aortic dilation in AAA and TAA, through different mechanisms. Support: CIHR, Heart and Stroke. CIHR, Heart and Stroke. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

A Network Pharmacological Elucidation of the Systematic Treatment Activities and Mechanisms of the Herbal Drug FDY003 Against Esophageal Cancer

Despite accumulating evidence for the value of herbal drugs for cancer treatment, the mechanisms underlying their effects have not been fully elucidated in a systematic manner. In this study, we performed a network pharmacological analysis to elucidate the anti-esophageal cancer (EC) properties of the herbal drug FDY003, a mixture of Artemisia capillaris Thunberg (AcT), Cordyceps militaris (Linnaeus) Link (Cm), and Lonicera japonica Thunberg (LjT). FDY003 reduced human EC cell viability and increased the pharmacological effects of chemotherapeutic drugs. There were 15 active pharmacological chemicals targeting 61 EC-associated genes and proteins in FDY003. The FDY003 targets were key regulators of major oncogenic EC-associated signaling pathways, such as phosphoinositide 3-kinase (PI3K)-Akt, hypoxia-inducible factor (HIF)-1, mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF), p53, Janus kinase (JAK)-signal transducer and activator of transcription (STAT), erythroblastic leukemia viral oncogene homolog (ErbB), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B), and vascular endothelial growth factor (VEGF) cascades. These EC-associated genes, proteins, and pathways targeted by FDY003 determine the malignant behaviors of EC cells, including cell death, survival, division, proliferation, and growth. This network pharmacological analysis provides an integrative view of the mechanisms by which FDY003 contributes to EC treatment.

Dysfunctional Mitochondria and Hemolysis are Linked by Impaired Maturation of Reticulocytes: Possible Role in Pulmonary Hypertension

IntroductionWe previously demonstrated that significantly elevated levels of circulating free heme (CFH) is associated with preclinical (Sugen/Hypoxia in rats) pulmonary hypertension (PH) and clinical pulmonary arterial hypertension (PAH). However, the pathogenic mechanisms of free heme release in PAH patients and animal models in the absence of hemolytic disease are yet to be elucidated.HypothesisThe mitochondria play a crucial role in reticulocyte maturation (precursors to red blood cells). Our hypothesis tests if mitochondrial dysfunction (MD) impaired reticulocyte maturation increases its fragility and causes rupture, elevating CFH levels that promote PAH.MethodsRecently reported genetic PH rat models carrying the NFU1G206C mutation spontaneously develop PH with severe MD. Erythropoiesis activation, reticulocyte counts, and CFH levels were monitored in this model. ChemiDoc imaging system was used to visualize FITC‐dextran extravasation in fresh transversely cut pulmonary slices 24 hours after FITC‐dextran injection (60 mg/kg. i.v.). Changes in right ventricle (RV) hemodynamics were used to determine the level of disease severity. Pulmonary artery smooth muscle cells (PASMCs) isolated from wild‐type (WT) and NFU1G206C rats were used to evaluate effects of free heme in vitro. Matrigel assay was used to evaluate the tube formation potential of endothelial cells (EC’s).ResultsThe NFU1G206C rats showed significantly higher erythropoietin levels and immature reticulocyte counts compared to WTs. Correspondingly, significantly elevated plasma free heme and extravasation of FITC‐dextran into lung parenchyma were observed, indicating pulmonary endothelial barrier dysfunction, previously demonstrated as an essential contributor to PH pathobiology. Notably, treatment of human pulmonary artery EC’s with 50µM heme completely abolished EC tube formation, indicating that CFH could trigger a cellular environment leading to PH. Additionally, exposing isolated PASMC from NFU1G206C to 50µM heme increased the proliferation rate by ~1.5 and ~2.9 folds compared to untreated NFU1G206C or WT PASMC. Chronic lipoic acid (LA) supplementation to restore mitochondrial function (1.7mM orally), normalized total/immature reticulocyte counts in NFU1G206C rats (WT/NFU1G206C/NFU1G206C+LA for total and immature reticulocyte, p&lt;0.002 and p&lt;0.001) and fully reversed the PAH phenotype. Alternatively, the RV systolic pressure of NFU1G206C rats was significantly (p&lt;0.0001) attenuated by blocking the heme transporter (HCP1) with sulfasalazine (SL, 20mg/ml/kg, i.p.).ConclusionWe show that mitochondrial dysfunction impaired reticulocyte maturation promotes hemolysis resulting in increased CFH. Elevated CFH can trigger pathogenic pathways leading to PH/PAH via EC barrier dysfunction and increased PASMC proliferation. Treatment approaches that improve mitochondrial metabolism (LA) or inhibit free heme entrance to the cell (SL) abrogate the PH phenotype, highlighting the critical role of free heme‐induced signaling in disease pathobiology.

Diane-35 and Metformin Induce Autophagy and Apoptosis in Polycystic Ovary Syndrome Women with Early-Stage Endometrial Carcinoma.

Objective: Women with polycystic ovary syndrome (PCOS) are at increased risk ofendometrial carcinoma (EC). Previous studies indicated that the combined therapy of Diane-35 and metformin significantly suppresses disease progression in PCOS patients with early EC; however, the mechanisms remain unclear. Methods: An established murine model of PCOS with early EC, clinical specimens, and human EC cells was used in this study. The levels of protein and mRNA were measured with Western blotting and RT-PCR, respectively. Cell proliferation was determined with MTT, colony formation, and flow cytometry. Proteins were analyzed with immunofluorescence and immunohistochemistry. Results: Diane-35 and metformin significantly inhibited proliferative activity and promoted apoptosis in EC cells. Additionally, cell autophagy was induced by the combined therapy. Quantitive PCR revealed that Diane-35 and metformin decreased androgen receptor (AR) expression but elevated GLUT4 expression. AR was found to repress GLUT4 expression by binding to the promoter of GLUT4. Moreover, the combined treatment mediated the onset of cellular autophagy by regulating the mTORC pathway via the suppression of IGF-1 and inhibited the development of EC by the activation of the PI3K/mTORC pathway. Conclusion: The results and previous clinical evidence support the use of Diane-35 and metformin combination therapy for patients with PCOS and early EC.

Structural connectivity-based segmentation of the human entorhinal cortex

The medial (MEC) and lateral entorhinal cortex (LEC), widely studied in rodents, are well defined and characterized. In humans, however, the exact locations of their homologues remain uncertain. Previous functional magnetic resonance imaging (fMRI) studies have subdivided the human EC into posteromedial (pmEC) and anterolateral (alEC) parts, but uncertainty remains about the choice of imaging modality and seed regions, in particular in light of a substantial revision of the classical model of EC connectivity based on novel insights from rodent anatomy. Here, we used structural, not functional imaging, namely diffusion tensor imaging (DTI) and probabilistic tractography to segment the human EC based on differential connectivity to other brain regions known to project selectively to MEC or LEC. We defined MEC as more strongly connected with presubiculum and retrosplenial cortex (RSC), and LEC as more strongly connected with distal CA1 and proximal subiculum (dCA1pSub) and lateral orbitofrontal cortex (OFC). Although our DTI segmentation had a larger medial-lateral component than in the previous fMRI studies, our results show that the human MEC and LEC homologues have a border oriented both towards the posterior-anterior and medial-lateral axes, supporting the differentiation between pmEC and alEC.

Novel SARS-CoV-2 variants induce higher toxicity in cardiovascular cells

Abstract Objective SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19) and has spawned a global health crisis. Virus infection can lead to elevated markers of cardiac injury and inflammation associated with a higher risk of mortality. However, it is so far unclear whether cardiovascular damage is caused by direct virus infection or is mainly secondary due to inflammation. Recently, additional novel SARS-CoV-2 variants have emerged accounting for more than 70% of all cases in Germany. To what extend these variants differ from the original strain in their pathology remains to be elucidated. Here, we investigated the effect of the novel SARS-CoV-2 variants on cardiovascular cells. Results To study whether cardiovascular cells are permissive for SARS-CoV-2, we inoculated human iPS-derived cardiomyocytes and endothelial cells from five different origins, including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells, in vitro with SARS-CoV-2 isolates (G614 (original strain), B.1.1.7 (British variant), B.1.351 (South African variant) and P.1 (Brazilian variant)). While the original virus strain infected iPS-cardiomyocytes and induced cell toxicity 96h post infection (290±10 cells vs. 130±10 cells; p=0.00045), preliminary data suggest a more severe infection by the novel variants. To what extend the response to the novel variants differ from the original strain is currently investigated by phosphoproteom analysis. Of the five endothelial cells studied, only human coronary artery EC took up the original virus strain, without showing viral replication and cell toxicity. Spike protein was only detected in the perinuclear region and was co-localized with calnexin-positive endosomes, which was accompanied by elevated ER-stress marker genes, such as EDEM1 (1.5±0.2-fold change; p=0.04). Infection with the novel SARS-CoV-2 variants resulted in significant higher levels of viral spike compared to the current strain. Surprisingly, viral up-take was also seen in other endothelial cell types (e.g. HUVEC). Although no viral replication was observed (850±158 viral RNA copies at day 0 vs. 197±43 viral RNA copies at day 3; p=0.01), the British SARS-CoV-2 variant B.1.1.7 reduced endothelial cell numbers (0.63±0.03-fold change; p=0.0001). Conclusion Endothelial cells and cardiomyocytes showed a distinct response to SARS-CoV-2. Whereas cardiomyocytes were permissively infected, endothelial cells took up the virus, but were resistant to viral replication. However, both cell types showed signs of increased toxicity induced by the British SARS-CoV-2 variant. These data suggest that cardiac complications observed in COVID-19 patients might at least in part be based on direct infection of cardiovascular cells. The more severe cytotoxic effects of the novel variants implicate that patients infected with the new variants should be even more closely monitored. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): DFG and Willy-Pitzer Foundation

P–288 Changes in gene and protein expression in human endometrial cancer cell lines after low dose metformin treatment over time

Abstract Study question Does metformin treatment lead to temporal expression changes of specific genes and proteins in endometrial cancer cell lines? Summary answer The expression of three different genes and proteins was investigated, of which all displayed changes over time. What is known already Endometrial cancer (EC) is one of the most common malignancies among postmenopausal women. A long-term estrogen effect on the endometrium often seen in women with obesity or the polycystic ovary syndrome (PCOS), as well as type II diabetes mellitus (T2DM) are well-known risk factors for the development and progression of EC. Metformin is a biguanide used in the treatment of T2DM patients and off label in women with PCOS. Moreover, metformin displays anti-tumor and anti-proliferative effects in various cancer types, including EC. In that regards BCL2L11, CDH1 and CDKN1A play an important role in apoptotic pathways, proliferation and invasion processes. Study design, size, duration The EC cells were cultured in normal (5.5 mmol/L) or high (17 mmol/L) glucose medium supplemented with 10 nmol/L ß-estradiol. The cells were treated with low dose metformin (1.0 mmol/L) for 2, 6, 24, 48 and 168 h (7 d). In addition, EC cells were treated with a combination of metformin and insulin (100 ng/mL) or remained untreated. Five independent experiments were fulfilled and untreated cells served as controls. Participants/materials, setting, methods The study was accomplished using two different human EC cell lines. HEC–1A represents an estrogen-independent EC, whereas Ishikawa represents the more common, estrogen-dependent EC. Proteins were extracted, quantified with a BCA assay, and the protein expression of BCL2L11, CDH1 and CDKN1A was analyzed by western blots. Furthermore, total RNA was extracted, transcribed to cDNA and Taqman real-time PCR was carried out to measure the expression of the associated genes, using fold change (FC) as parameter. Main results and the role of chance The expression of the selected genes, analyzed by RT-PCR, changed in both cell lines over time as follows: After 6 h, metformin induced a decrease in the expression of BCL2L11 (FC = 0.7) and CDH1 (FC = 0.75), whereas the expression of CDKN1A slightly increased (FC = 0.95–1.35). After 24 h, BCL2L11 expression increased in normal glucose groups (FC = 1.3, high glucose: FC = 0.93) and CDH1 expression decreased in combination with metformin and high glucose (FC = 0.7, normal glucose: FC = 1.1). CDKN1A expression was increased by metformin in both cell lines after 24 h (FC = 1.2–1.8). After 48 h of metformin treatment, expression for all three genes was only slightly changed (FC = 0.9–1.0). After 7 d it was observed that the combination of high glucose and metformin (i.e. like obese T2DM patients) led to an increased expression of BCL2L11, CDH1 and CDKN1A (FC = 1.4–2.9) in the presence and absence of insulin, whereas metformin induced a decreased expression of CDH1 and CDKN1A (FC = 0.5–0.75) in normal glucose medium. BCL2L11, CDH1 and CDKN1A expression was investigated at the protein level as well. Limitations, reasons for caution The results cannot be directly transferred to metformin treatment of patients, since the study was carried out in vitro. Additionally, further studies including more timepoints would indicate a more precisely gene and protein expression over time. Wider implications of the findings: This is the first in vitro study showing the temporal changes of BCL2L11, CDH1 and CDKN1A expression, genes related to tumorigenesis due to low dose metformin over time, suggesting differentially pathways in long term metformin treatment using physiologically achievable metformin levels. Trial registration number Not applicable

TRPV4 regulates matrix stiffness‐dependent activation of YAP/VEGFR2 signaling via Rho/Rho kinase/LATS1/2 pathway

TRPV4 is a mechanosensitive ion channel in endothelial cells and shown to regulate physiological and pathological angiogenesis. We have recently shown that TRPV4 deletion and/or down regulation activates vascular endothelial growth factor receptor-2 (VEGFR2) via yes-associated protein (YAP) in human EC. However, the molecular mechanisms of TRPV4-dependant YAP/VEGFR2 activation are unknown. We have employed human microvascular endothelial cells (HMEC-1; EC) on ECM gels of varying stiffness that mimic normal and failing hearts (8 and 50 kPa) and examined nuclear translocation of YAP and disappearance of perinuclear VEGFR2, after siRNA knockdown of TRPV4. Next, we investigated the role of upstream and downstream regulators of YAP translocation by focusing on LATS-1, CYR61 and Rho/Rho Kinase, using phospho-specific antibodies (pYAP and pLATS) and pharmacological inhibitor of Rho kinase (Y-27632). siRNA knockdown of TRPV4 significantly increased nuclear translocation of YAP with concomitant disappearance of perinuclear-VEGFR2 in matrix stiffness-dependent manner. Further, we found increased Rho-GTP and decreased LATS-1 phosphorylation in TRPV4 knockdown EC compared to control siRNA treated EC. Furthermore, pharmacological inhibition of Rho kinase with Y-27632, increased phosphorylation of LATS1/2 kinase, inhibited nuclear translocation of YAP and VEGFR2 redistribution. Consistent with above results, YAP target genes CYR61 and CTGF mRNA expression levels were increased in a matrix stiffness-dependent manner in TRPV4 knockdown EC than the control siRNA treated EC. Our results suggest that TRPV4 regulates YAP/VEGFR2 activation in a matrix stiffness dependent manner during angiogenesis via Rho/Rho kinase/LATS1/2 pathway.

Purification of Gekko Small Peptide Fraction and Its Effect of Inducing Apoptosis of EC 9706 Esophageal Cancer Cells by Inhibiting PI3K/Akt/GLUT1 Signaling Pathway.

This study aimed to isolate and purify a cytotoxic extraction from Gekko japonicus, identify its components and determine its cytotoxic activity in vitro. We isolated and identified the most potent cytotoxic Gekko small peptide LH-20-15. The identification and analysis of peptide sequences of LH-20-15 were performed by de novo peptide sequencing, and two new peptides were found. LH-20-15 significantly inhibited the proliferation of human esophageal squamous carcinoma EC 9706 cells in a dose-dependent manner. Furthermore, LH-20-15 induced apoptosis in esophageal cancer cells by activating the mitochondrial apoptotic pathway. Further research showed that LH-20-15 inhibited the PI3 K/Akt/GLUT1 signaling pathway. In conclusion, LH-20-15 from Gekko japonicus is a peptide mixture and may inhibit EC 9706 cell proliferation and induce apoptosis by activating the mitochondrial apoptotic pathway. It also regulates glucose metabolism by targeting the PI3 K/Akt/GLUT1 signaling pathway. These small peptides could be new sources of natural cytotoxic ingredients against esophageal cancer with potential drug values.

LncRNA HOTAIR-mediated MTHFR methylation inhibits 5-fluorouracil sensitivity in esophageal cancer cells

BackgroundEsophageal cancer (EC) represents one of the most aggressive digestive neoplasms globally, with marked geographical variations in morbidity and mortality. Chemoprevention is a promising approach for cancer therapy, while acquired chemoresistance is a major obstacle impeding the success of 5-fluorouracil (5-FU)-based chemotherapy in EC, with the mechanisms underlying resistance not well-understood. In the present study, we focus on exploring the role of long non-coding RNA (lncRNA) HOTAIR in EC progression and sensitivity of EC cells to 5-FU.MethodsPaired cancerous and pre-cancerous tissues surgically resected from EC patients were collected in this study. Promoter methylation of the MTHFR was assessed by methylation-specific PCR. RIP and ChIP assays were adopted to examine the interaction of DNA methyltransferases (DNMTs) with lncRNA HOTAIR and MTHFR, respectively. EC cells resistant to 5-FU were induced by step-wise continuous increasing concentrations of 5-FU. The sensitivity of EC cells to 5-FU in vivo was evaluated in nude mice treated with xenografts of EC cells followed by injection with 5-FU (i.p.).ResultsWe found reciprocal expression patterns of lncRNA HOTAIR and MTHFR in EC tissues and human EC cells. Interference with lncRNA HOTAIR enhanced 5-FU-induced apoptosis, exhibited anti-proliferative activity, and reduced promoter methylation of the MTHFR in EC cells. Besides, overexpression of MTHFR attenuated the acquired chemoresistance induced by overexpression of lncRNA HOTAIR in EC cells. At last, enhanced chemosensitivity was observed in vivo once nude mice xenografted with lncRNA HOTAIR-depleted EC cells.ConclusionTogether, our study proposes that pharmacologic targeting of lncRNA HOTAIR sensitizes EC cells to 5-FU-based chemotherapy by attenuating the promoter hypermethylation of the MTHFR in EC.

Actin‑like protein 8 executes a promoting function in the malignant progression of endometrial cancer: identification of a promising biomarker.

Endometrial cancer (EC) is generally considered as a disease that affects older women. We attempt to explore the role of actin‑like protein 8 (ACTL8) in EC and how it achieves its function. Based on the data from The Cancer Genome Atlas (TCGA), we found that ACTL8 expression was up-regulated in EC tissues and correlated with shorter overall survival of EC patients. ACTL8 expression was significantly associated with age, clinical-stage, or grade. Cox proportional hazards model analysis revealed that ACTL8 expression, grade, and clinical-stage were promising independent prognostic factors of EC. Knockdown of ACTL8 repressed the proliferative, migrating and invading capabilities of human EC cell lines KLE and Ishikawa. Silencing ACTL8 up-regulated the negative cell cycle regulator p21 and epithelial marker E-cadherin, and down-regulated the positive cell cycle regulator Cyclin A, mesenchymal markers MMP-9 and N-cadherin in KLE cells. Collectively, these outcomes illustrated that ACTL8 might act as a tumor facilitator during EC progression.

P4146The six-transmembrane protein Stamp2 protects from hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension via actions in mononuclear cells and CXCL12

Abstract Background The six-transmembrane protein of prostate (Stamp2) is a potent anti-inflammatory player in adipocytes and also in macrophages. Stamp2's actions in these cells protects from diet-induced diabetes and from atherosclerosis mice. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PH), we sought to investigate the role of Stamp2 in PH. Methods and results Morphometric analyses of small pulmonary arteries after 3 weeks of chronic hypoxia (10% O2) showed aggravated pulmonary vascular remodeling in Stamp2−/− mice as compared to WT, demonstrated by a significantly reduced number of non-muscularized vessels and higher extent of fully-muscularized vessels. Consequently, right ventricular systolic pressure (RVSP, Millar catheter via right jugular vein) was significantly higher in Stamp2−/− mice (33.4±0.7 mmHg vs. 30.3±1.4, p&lt;0.05). As endothelial (EC) and smooth muscle cells (PASMC) are critical for remodeling processes in PH, the role of Stamp2 in these cells was explored. However, siRNA-mediated knock-down of Stamp2 in human microvascular EC had no effect on apoptotic susceptibility (CellDeath Detection ELISA), or release of IL-6 (qPCR). Furthermore, Stamp2-deficiency in isolated primary PASMC had no effect on proliferation (BrdU incorporation) and chemotaxis (modified Boyden chamber). As Stamp2 deficiency promotes higher expression of inflammatory cytokines (IL6, IL1b, MCP1, TNFa, CXCL12, qPCR) and increased numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages are potentially driving vascular remodeling in PH. To test this hypothesis, PASMC proliferation and chemotaxis were assessed in response to treatment with supernatants from primary thioglycolate-elicited peritoneal Stamp2−/− or WT-macrophages. These experiments revealed that supernatants from Stamp2−/− macrophages induced PASMC proliferation and chemotaxis significantly stronger, thus providing a link between inflammatory actions in Stamp2 deficiency and vascular remodeling. To gain further insights, a cytokine array was performed with supernatants from Stamp2−/− and WT-macrophages, revealing CXCL12 as the most relevant candidate. Experiments with neutralizing antibodies confirmed the role of CXCL12 in driving Stamp2's actions on vascular remodelling processes in PASMC. Importantly, Stamp2 expression (qPCR, western blot analyses) was significantly lower in the lung of humans with idiopathic PAH (IPAH), as well as in experimental PH in rats (monocrotalin, sugen/hypoxia) and in mice (hypoxia). Conclusions Stamp2 deficiency aggravates hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension in mice. On the cellular level, actions of Stamp2 in macrophages drive vascular remodelling processes in smooth muscle cells via secreted factors such as CXCL12. The finding of decreased expression of Stamp2 in human and various experimental forms of PH points towards a general protective role of Stamp2.

Abstract 783: miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal cancer

Abstract Esophageal cancer (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), is one of the deadliest cancers with extremely aggressive nature and poor survival rate. Diagnostic methods for EC mainly include endoscopy, barium swallow and ultrasound, which are either invasive or lack of sensitivity and specificity at early stages. Therefore, the development of a non-invasive and reliable diagnostic method with high sensitivity and specificity is crucial in improving early detection, diagnosis and treatment. microRNAs (miRNAs) are small RNA molecules that regulate the expression of protein-coding genes by directly binding to target mRNAs in a sequence-specific manner. The aim of this study is to identify novel miRNA biomarkers for EC diagnosis. By cross-referencing our data with others’ from NCBI, we identified a list of dysregulated miRNAs in EC, including miR-375. We assessed the expression of miR-375 in EC cell lines and tissues using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). We found that miR-375 expression is significantly decreased in human EC cell lines (KEYS-70, KEYS-180, FLO-1 and JHU-ad1) compared to the normal esophageal squamous cell line, Het-1A. Using microdissection technique, we isolated normal epithelium, dysplasia, and invasive EC components from formalin-fixed, paraffin-embedded (FFPE) tissue followed by qRT-PCR analysis. Decreased expression of miR-375 was detected in 12 of 14 EC tissue samples (86%) compared to the matched normal tissues. MTT and Matrigel invasion assay demonstrated that forced expression of miR-375 suppressed the proliferation and invasion in EC cell lines. TargetScanS and miRanda were used to identify potential target genes of miR-375, which includes epidermal growth factor receptor 2 (HER2) and YES-associated protein (YAP1), both of them act as oncogenes in EC development. Interestingly, forced expression of miR-375 resulted in significantly reduced expression of human vascular endothelial growth factor-A (VEGF-A) gene, which is a downstream target gene of YAP1/ERBB2 pathway involving in tumor angiogenesis in EC cell lines. More functional analysis is under way. These findings suggest that miR-375 may act as a tumor suppressor, and could serve as a marker for EC diagnosis and management. Citation Format: Shuchang Ren, Xiaohui Tan, Xiaoling Wu, Tao Chen, Patricia Latham, Sidney W. Fu. miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 783.