Expression In Microvascular Endothelial Cells Research Articles

Aspirin Inhibits Brain Metastasis of Lung Cancer via Upregulation of Tight Junction Protein Expression in Microvascular Endothelial Cells.

The brain is one of the most vulnerable metastasis sites in lung cancer; approximately 40-50% of lung cancer patients develop brain metastasis during the disease course, contributing to the poor prognosis and high mortality of lung cancer patients. Therefore, it is important to clarify the molecular mechanism underlying brain metastasis of lung cancer for improving the overall survival of lung cancer patients. The present study aimed to investigate the potential role of blood-brain barrier (BBB) permeability in the development of brain metastasis of lung cancer and explore the effect of aspirin in an in-vitro BBB model. An in-vitro BBB model was established. The expression of heat shock protein 70 (HSP 70), zonula occludens-1 (ZO-1), and occludin in rat brain microvascular endothelial cells was detected using Western blot at different time points following the administration of aspirin. HSP70, ZO-1, and occludin expressions did not show significant changes before aspirin administration, but increased noticeably after aspirin administration. Tumor necrosis factor-α (TNF-α) could significantly attenuate the increased expression of these proteins induced by aspirin. Additionally, TNF-α also significantly reversed the aspirin-induced decrease of BBB permeability. Aspirin may inhibit brain metastasis of lung cancer in a time-dependent manner via upregulating tight junction proteins to reduce BBB permeability, and this effect can be reversed by TNF-α.

Imbalance of Pulmonary Microvascular Endothelial Cell-Expression of Metalloproteinases and Their Endogenous Inhibitors Promotes Septic Barrier Dysfunction.

Sepsis is a life-threatening disease characterized by excessive inflammation leading to organ dysfunction. During sepsis, pulmonary microvascular endothelial cells (PMVEC) lose barrier function associated with inter-PMVEC junction disruption. Matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases (ADAM), which are regulated by tissue inhibitors of metalloproteinases (TIMPs), can cleave cell-cell junctional proteins, suggesting a role in PMVEC barrier dysfunction. We hypothesize that septic PMVEC barrier dysfunction is due to a disruption in the balance between PMVEC-specific metalloproteinases and TIMPs leading to increased metalloproteinase activity. The effects of sepsis on TIMPs and metalloproteinases were assessed ex vivo in PMVEC from healthy (sham) and septic (cecal ligation and perforation) mice, as well as in vitro in isolated PMVEC stimulated with cytomix, lipopolysaccharide (LPS), and cytomix + LPS vs. PBS. PMVEC had high basal Timp expression and lower metalloproteinase expression, and septic stimulation shifted expression in favour of metalloproteinases. Septic stimulation increased MMP13 and ADAM17 activity associated with a loss of inter-PMVEC junctional proteins and barrier dysfunction, which was rescued by treatment with metalloproteinase inhibitors. Collectively, our studies support a role for metalloproteinase-TIMP imbalance in septic PMVEC barrier dysfunction, and suggest that inhibition of specific metalloproteinases may be a therapeutic avenue for septic patients.

Cognitive impairment of MRL mice is related to NMDA receptor-mediated inflammatory response and production of adhesion molecules in MRL/lpr mice-derived micro-vascular endothelial cells.

Systemic lupus erythematosus (SLE) is a chronic recurrent autoimmune disease affecting almost all organs. This study was conducted to investigate cognitive impairment of SLE mice (MRL/lpr mice), and explore associated pathological mechanism. Behavior tests (open-field test, elevated plus-maze test, forced swimming test, sucrose preference test, and Morris water maze test) were conducted in MRL/MPJ and MRL/lpr mice. ELISA test was performed to determine levels of antibodies (anti-dsDNA, anti-RPA, anti-ACA, and anti-NR2a/b) and inflammatory factors [tumour necrosis factor a (TNF-a), interleukin (IL)-6, IL-8, and IL-10]. Micro-vascular endothelial cells (MVECs) were isolated, identified, and divided into MVECs (NC), anti-NR2a/2b, memantine, glycine, dexamethasone, and IL-1b groups. Cell proliferation was measured using cell counting kit-8 (CCK-8) assay, and Western blotting was applied to evaluate ELAM-1, VCAM-1, ICAM-1, IKBa, p-IKBa expression. MRL/lpr mice demonstrated lower locomotion/exploration ability, higher anxiety, obvious depression symptoms, lower learning/memory capability compared with MRL/MPJ mice. MRL/lpr mice demonstrated high levels of anti-NR2a/b antibody and auto-antibodies. NMDA receptor antagonist (memantine) significantly increased, and NMDA receptor agonist (glycine) significantly decreased MVECs proliferation compared with NC group ( p < 0.05). Memantine significantly reduced and glycine predominantly enhanced TNF-a, IL-6, IL-8, and IL-10 levels compared with NC group ( p < 0.05). NMDA receptor antagonist and agonist modulated adhesion molecules expression in MVECs. ELAM-1, VCAM-1, and ICAM-1 expressions were significantly down-modulated in memantine group, and remarkably up-modulated in glycine group compared with NC group ( p < 0.05). NMDA receptor antagonist and agonist regulated phosphorylation of p-IKBa. The above effects of memantine evenly equaled to dexamethasone, and glycine evenly equaled to IL-1b. In conclusion, cognitive impairment of MRL mice might be associated with NMDA receptor-mediated inflammatory response and production of adhesion molecules in MRL/lpr mice-derived MVECs.

Benzo[b]fluoranthene (B[b]F) affects apoptosis, oxidative stress, mitochondrial membrane potential and expressions of blood-brain barrier markers in microvascular endothelial cells

Exposure to polycyclic aromatic hydrocarbons (PAHs) contributes to the damage of blood-brain barrier. While a number of studies were focused on benzo[a]pyrene, direct effects and mechanisms of benzo[b]fluoranthene (B[b]F), another main component of PAHs, on blood-brain barrier (BBB) are not documented. Here, we investigated if B[b]F at concentrations of environmental relevance could affect apoptosis, oxidative stress, mitochondrial membrane potential (MMP) and BBB marker expression in mouse brain microvascular endothelial (bEnd.3) cells, an in vitro model typically used to study BBB toxicology. Cells were treated with varying concentrations of B[b]F (0, 10, 20 and 40 μM) for 48 h. Cell proliferation, cell cycle, apoptosis, oxidative stress, MMP and BBB marker expressions were evaluated by label-free real-time cell analysis, flow cytometry, immunofluorescence and Western-blot. The proliferation of bEnd.3 cells was inhibited by B[b]F in a concentration dependent manner. B[b]F treatment significantly affected cell cycle, induced apoptosis, increased levels of reactive oxygen species (ROS) and disputed MMP. Expressions of BBB marker Occludin and Claudin-5 were decreased in the presence of 40 μM B[b]F. In conclusion, B[b]F might damage BBB by affecting proliferation, apoptosis, ROS level and Occludin and Claudin-5 expressions in microvascular endothelial cells.

Loss of lung microvascular endothelial Piezo2 expression impairs NO synthesis, induces EndMT, and is associated with pulmonary hypertension.

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFβ stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT.

Abstract 12771: HDAC Inhibition With Quisinostat Reduces Pulmonary Vascular Remodeling in Experimentally Induced Pulmonary Arterial Hypertension

Introduction: Histone deacetylases (HDACs) expression and activity are increased in pulmonary arterial hypertension (PAH). Although inhibiting HDACs has been promoted as a treatment for PAH, results from preclinical studies were equivocal and raised concerns regarding safety. Quisinostat is a “second generation” HDAC inhibitor, which is highly potent against HDAC1 and elicits a prolonged pharmacodynamic response. Hypothesis: Quisinostat may exert therapeutic effects in experimentally-induced PAH. Methods: HDAC1 expression was measured in lungs and microvascular endothelial cells (MVECs) from control donors and PAH patients. HDAC activity, proliferation and inflammation were measured after quisinostat treatment of PAH MVECs. Chronic quisinostat treatment was tested in three PAH rat models: the Sugen + hypoxia (SuHx), the Monocrotaline shunt flow (MF) and pulmonary artery banding (PAB) model. Results: HDAC1 expression is increased in lungs of PAH patients. Quisinostat decreased proliferation and mitigated inflammatory cytokine expression in MVECs. Conditioned medium from quisinostat treated PAH MVECs inhibited the growth of healthy pulmonary artery smooth muscle cells. In vivo , quisinostat treatment reduced right ventricular (RV) systolic pressure, RV afterload and total pulmonary resistance in SuHx rats. Further analysis by histology revealed that quisinostat reduced pulmonary vascular remodeling in both SuHx and MF rat models by reducing proliferation and perivascular inflammation. The PAB rat model demonstrated that quisinostat had no independent effect on RV function or remodeling. Conclusions: Quisinostat can partly reverse RV afterload and pulmonary vascular remodeling in established experimental PAH and may be a promising intervention for PAH.

Abstract 9717: Reduction in Arnt/hif1 Beta Mediates Vascular Dysfunction in Mice with Type 2 Diabetes Mellitus

Introduction: Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood Hypothesis: Aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes Methods and Results: We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation (ArntΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia (n=8-12, p&lt;0.01 vs Control group), delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow (n=10, P&lt;0.01vs Control) In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in HUVECs cultured under high-glucose conditions. The ArntΔEC, ERT2 mutation also reduced measures of cell viability while increasing the production of reactive oxygen species (ROS) in MVECs isolated from mouse skeletal muscle, and the viability of ArntΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor. Conclusions: Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice, and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.

DETERMINING THE ROLE OF VON WILLEBRAND FACTOR IN THROMBOTIC POST-TRANSPLANTATION COMPLICATIONS USING EX VIVO LUNG PERFUSION SYSTEM

BACKGROUND von Willebrand factor (VWF) is a pro-coagulant glycoprotein expressed only in endothelial cells and megakaryocytes. It plays a critical role in hemostasis and thrombus formation. It mediates the adhesion of platelets to the endothelium/sub-endothelium surfaces, which is the primary step in thrombogenesis. Our previous analyses demonstrated that hypoxia induced an increase in levels, and alteration in vascular tree expression pattern, of the VWF specifically in the lungs of mice. Alteration in expression pattern corresponded to a significant increase in the proportion of lung microvascular endothelial cells that exhibited de novo activation of VWF expression. Hypoxia-induced elevated and altered VWF expression was correlated with a significant increase in platelet aggregates formation in the lung vasculature. Since during organ-transplantation donor organs are under hypoxic conditions, we explored whether this may lead to alterations in VWF expression and whether modification of transplantation procedure to reduce hypoxic exposure could prevent such alterations. METHODS AND RESULTS Procured pig's lungs that were maintained in static cold storage “SCS” or exposed to ex vivo lung perfusion (before and after perfusion) were used. Lung tissue biopsies were obtained immediately after organ harvest, 12 hours post cold storage, or post warm perfusion. VWF RNA and protein expression levels were analyzed using RT-PCR and western blot. In addition, we proceeded to determine whether SCS preservation also alters the proportion of pig's lung vascular endothelial cells that exhibit VWF expression. For these analyses, we performed double stained immunofluorescence (IF) analyses using CD31 (endothelial cell marker) and VWF antibodies on samples of lungs that were preserved and transplanted. Results demonstrated that VWF mRNA and protein levels were similar after 12 hours of cold storage compared to immediate harvest, but were significantly reduced in lungs that were perfused under ex vivo lung perfusion “EVLP” for 12 hours. Furthermore, IF analysis demonstrated that preservation of lungs under cold storage conditions prior to transplantation altered VWF expression pattern leading to an increasing number of microvascular (indicated by CD31 staining) endothelial cells that express VWF. CONCLUSION Increased VWF expression in microvascular endothelial cells under SCS may contribute to transplant associated thrombogenicity. Reduction of VWF expression through ex vivo normothermic perfusion may have a significant effect in reducing potential thrombogenic consequences, which is a major complication of organ transplantation. von Willebrand factor (VWF) is a pro-coagulant glycoprotein expressed only in endothelial cells and megakaryocytes. It plays a critical role in hemostasis and thrombus formation. It mediates the adhesion of platelets to the endothelium/sub-endothelium surfaces, which is the primary step in thrombogenesis. Our previous analyses demonstrated that hypoxia induced an increase in levels, and alteration in vascular tree expression pattern, of the VWF specifically in the lungs of mice. Alteration in expression pattern corresponded to a significant increase in the proportion of lung microvascular endothelial cells that exhibited de novo activation of VWF expression. Hypoxia-induced elevated and altered VWF expression was correlated with a significant increase in platelet aggregates formation in the lung vasculature. Since during organ-transplantation donor organs are under hypoxic conditions, we explored whether this may lead to alterations in VWF expression and whether modification of transplantation procedure to reduce hypoxic exposure could prevent such alterations. Procured pig's lungs that were maintained in static cold storage “SCS” or exposed to ex vivo lung perfusion (before and after perfusion) were used. Lung tissue biopsies were obtained immediately after organ harvest, 12 hours post cold storage, or post warm perfusion. VWF RNA and protein expression levels were analyzed using RT-PCR and western blot. In addition, we proceeded to determine whether SCS preservation also alters the proportion of pig's lung vascular endothelial cells that exhibit VWF expression. For these analyses, we performed double stained immunofluorescence (IF) analyses using CD31 (endothelial cell marker) and VWF antibodies on samples of lungs that were preserved and transplanted. Results demonstrated that VWF mRNA and protein levels were similar after 12 hours of cold storage compared to immediate harvest, but were significantly reduced in lungs that were perfused under ex vivo lung perfusion “EVLP” for 12 hours. Furthermore, IF analysis demonstrated that preservation of lungs under cold storage conditions prior to transplantation altered VWF expression pattern leading to an increasing number of microvascular (indicated by CD31 staining) endothelial cells that express VWF. Increased VWF expression in microvascular endothelial cells under SCS may contribute to transplant associated thrombogenicity. Reduction of VWF expression through ex vivo normothermic perfusion may have a significant effect in reducing potential thrombogenic consequences, which is a major complication of organ transplantation.

HDAC6 Negatively Regulates miR-155-5p Expression to Elicit Proliferation by Targeting RHEB in Microvascular Endothelial Cells under Mechanical Unloading.

Mechanical unloading contributes to significant cardiovascular deconditioning. Endothelial dysfunction in the sites of microcirculation may be one of the causes of the cardiovascular degeneration induced by unloading, but the detailed mechanism is still unclear. Here, we first demonstrated that mechanical unloading inhibited brain microvascular endothelial cell proliferation and downregulated histone deacetylase 6 (HDAC6) expression. Furthermore, HDAC6 promoted microvascular endothelial cell proliferation and attenuated the inhibition of proliferation caused by clinorotation unloading. To comprehensively identify microRNAs (miRNAs) that are regulated by HDAC6, we analyzed differential miRNA expression in microvascular endothelial cells after transfection with HDAC6 siRNA and selected miR-155-5p, which was the miRNA with the most significantly increased expression. The ectopic expression of miR-155-5p inhibited microvascular endothelial cell proliferation and directly downregulated Ras homolog enriched in brain (RHEB) expression. Moreover, RHEB expression was downregulated under mechanical unloading and was essential for the miR-155-5p-mediated promotion of microvascular endothelial cell proliferation. Taken together, these results are the first to elucidate the role of HDAC6 in unloading-induced cell growth inhibition through the miR-155-5p/RHEB axis, suggesting that the HDAC6/miR-155-5p/RHEB pathway is a specific target for the preventative treatment of cardiovascular deconditioning.

Abstract MP250: Reduction In Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator (arnt) Mediates Vascular Dysfunction In Mice With Type 2 Diabetes Mellitus

Background: Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood. Results and method: We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation ( Arnt ΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia, delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow. In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in HUVECs cultured under high-glucose conditions. The Arnt ΔEC, ERT2 mutation also reduced measures of cell viability while increasing the production of reactive oxygen species (ROS) in MVECs isolated from mouse skeletal muscle, and the viability of Arnt ΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor. Conclusion: Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.

Tumor necrosis factor alpha expression is increased in maternal microvascular endothelial cells in preeclampsia

ABSTRACT Objective: The aim of this study was to explore the expression and effects of tumor necrosis factor alpha (TNFα) in maternal endothelial cells in preeclampsia (PE). Methods: Expression levels in primary microvascular endothelial cells (MVEC) isolated from patients with severe preeclampsia (PE) and normal pregnancies were determined by RT-qPCR with or without treatment of TNFα and inhibitors for downstream signaling. Results: PE MVEC exhibited increased basal TNFα expression. TNFα treatment increased TNFα, VCAM, and endocan expression in MVEC. Conclusion: TNFα expression is increased in PE MVEC and the treatment of these cells with exogenous TNFα modifies their gene expression.

Inflammatory Cytokine IL-1β Downregulates Endothelial LRP1 via MicroRNA-mediated Gene Silencing

Effective clearance of neurotoxic amyloid-beta (Aβ) from the brain is a critical process to prevent Alzheimer’s disease (AD). One major clearance mechanism is Aβ transcytosis mediated by low-density lipoprotein receptor-related protein 1 (LRP1) in capillary endothelial cells. A marked loss of endothelial LRP1 is found in AD brains and is believed to significantly impair Aβ clearance. Recently, we demonstrated that pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, significantly down-regulated LRP1 in human primary microvascular endothelial cells (MVECs). In this study, we sought to determine the underlying molecular mechanism by which IL-1β led to LRP1 loss in MVECs. Reduced LRP1 protein and transcript were detected up to 24 h post-exposure and returned to the baseline levels after 48 h post-exposure with 1 ng/ml IL-1β. This reduction was in part mediated by microRNA-205-5p, -200b-3p, and -200c-3p, as these microRNAs were concomitantly upregulated in MVECs exposed to IL-1β. Synthetic microRNA-205-5p, -200b-3p, and -200c-3p mimics recapitulated LRP1 loss in MVECs without IL-1β, and their synthetic antagomirs effectively reversed IL-1β-mediated LRP1 loss. Importantly, we found that the expression of these three microRNAs was controlled by NF-κB as pharmacological NF-κB inhibitor, BMS-345541, inhibited the IL-1β-mediated upregulation of these microRNAs and rescued LRP1 expression. siRNA-mediated silencing of IκB in MVECs elevated microRNA-200b-3p and decreased LRP1 transcript, partially confirming our overall findings. In conclusion, our study provides a mechanism by which pro-inflammatory IL-1β instigates the suppression of LRP1 expression in MVECs. Our findings could implicate spatiotemporal loss of LRP1 and impairment of the LRP1-mediated clearance mechanism by endothelial cells.

Abstract P5-09-02: Tβ4 expression in cancer-associated endothelial cells enhances progression of invasive breast cancer

Abstract Background: Invasive breast cancer is a highly aggressive primary breast tumour with poor prognosis. As tumour angiogenesis is exhibited by invasive breast cancer, anti-angiogenic therapy has been intensively evaluated over the past decade. However, clinical studies were disappointing. Thymosin β4 (Tβ4), a multi-functional peptide, is associated with induction of angiogenesis, but the role of Tβ4 in tumorigenesis of invasive breast cancer is unknown. We tested whether Tβ4 inhibition could be a new target involved in tumour development and tumour angiogenesis for treatment of invasive breast cancer. Methods: We have adapted a 3D co-culture system to acquire invasive breast cancer cells-associated endothelial cells (ECs), which is followed by characterising the differential gene expressions by PCR-based subtraction analysis. We performed Western blot analysis on the protein samples obtained from ECs-stimulated by invasive breast cancer cells. Expression of Tβ4 in invasive breast cancer was assessed by qPCR in the breast tissues (tumour, n=119; background, n=55), and protein expression confirmed by immunohistochemical examination in an invasive breast cancer tissue microarray. Finally, mice breast cancer xenografts plus non-invasively photoacoustic microscopy were used to analyse the effect of Tβ4 knockdown on the tumorigenesis of invasive breast cancer. Results: We found that invasive breast cancer cells could stimulate an increase in Tβ4 expression in microvascular ECs. High Tβ4 levels were strongly associated with high malignant invasive breast cancer and poor clinical outcome. In vivo study showed that siRNA targeting Tβ4 blocked growth of invasive breast cancer in mice. Photoacoustic imaging revealed that knockdown of Tβ4 elicited anti-breast cancer growth, in part, through disruption of tumour vasculature. Our results demonstrate that endothelial cell-derived Tβ4 enhances the progression of breast cancer by up-regulating tumour angiogenesis and it indicates that a high level of tumour stromal Tβ4 is an independent predictor of poor outcome. Conclusions: Inhibiting endothelial Tβ4 could be a new therapeutic target in anti-angiogenic strategy for treatment of invasive breast cancer. Citation Format: Cai J, Jiang W. Tβ4 expression in cancer-associated endothelial cells enhances progression of invasive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-09-02.

Hypoxia upregulates integrin gene expression in microvascular endothelial cells and promotes their migration and capillary-like tube formation.

Tissue hypoxia affects gene expression through the hypoxia-inducible transcription factors, HIF-1 and HIF-2, in both physiological and pathological angiogenesis. Angiogenesis is a complex response of endothelial cells integrating cell proliferation, migration, tube formation, and their interaction with the extracellular matrix through integrin receptors. In this report, we studied the effect of hypoxia on the angiogenic functions of human microvascular endothelial cells (HMEC-1) as well as on expression of the angiogenic integrins αν β3 , αν β5 , and α5 β1 . Exposure of HMEC-1 to hypoxia (1% O2 ) or to DMOG, a prolyl-4-hydroxylase inhibitor, caused significant reduction to their proliferation rate, whereas their migration ability toward laminin-1 or collagen IV and capillary-like tube formation were significantly enhanced. In addition, αv , β1 , β3 , and β5 integrins expression was increased under hypoxia in HMEC-1, while α5 integrin was not affected. Both HIF-1 and HIF-2 protein expression and transcriptional activity were induced under hypoxia in HMEC-1. The knockdown of either HIF-1α or HIF-2α inhibited integrin β3 hypoxic stimulation, suggesting a HIF-dependent induction of β3 integrin in HMEC-1. Taken together, our results indicate that hypoxia transcriptionally up-regulates angiogenic integrins in microvascular endothelial cells along with promoting migration and tube formation of HMEC-1.

Vascular endothelial cell Toll-like receptor pathways in sepsis.

The endothelium forms a vast network that dynamically regulates vascular barrier function, coagulation pathways and vasomotor tone. Microvascular endothelial cells are uniquely situated to play key roles during infection and injury, owing to their widespread distribution throughout the body and their constant interaction with circulating blood. While not viewed as classical immune cells, endothelial cells express innate immune receptors, including the Toll-like receptors (TLRs), which activate intracellular inflammatory pathways mediated through NF-κB and the MAP kinases. TLR agonists, including LPS and bacterial lipopeptides, directly upregulate microvascular endothelial cell expression of inflammatory mediators. Intriguingly, TLR activation also modulates microvascular endothelial cell permeability and the expression of coagulation pathway intermediaries. Microvascular thrombi have been hypothesized to trap microorganisms thereby limiting the spread of infection. However, dysregulated activation of endothelial inflammatory pathways is also believed to lead to coagulopathy and increased vascular permeability, which together promote sepsis-induced organ failure. This article reviews vascular endothelial cell innate immune pathways mediated through the TLRs as they pertain to sepsis, highlighting links between TLRs and coagulation and permeability pathways, and their role in healthy and pathologic responses to infection and sepsis.

Chinese medicine Tongxinluo increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells.

Tongxinluo (TXL) is a compound prescription formulated according to the meridian theory of traditional Chinese medicine. It may play an important role in cardiovascular protection by improving endothelial cell function. The aim of present study was to investigate whether endothelial protection with TXL is related to its regulation of tight junction protein expression. Human cardiac microvascular endothelial cells (HCMECs) were cultured and treated with 10(-7) mol l(-1) angiotensin II (Ang II) and the different doses of TXL; the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin was determined by Western blotting and real-time PCR. Gain-of-function and loss-of-function of Krüppel-like factor 5 (KLF5) were carried out in HCMEC transfected with either KLF5 adenovirus pAd-KLF5 or siRNA specific for KLF5. Angiotensinogen transgenic mice were treated with TXL by oral administration of TXL of 0.75 g kg(-1) day(-1) , and immunohistochemical staining was performed with antioccludin, anticlaudin, anti-VE-cadherin, antibeta-catenin and anti-KLF5 antibodies. Ang II treatment significantly reduced the expression of tight junction proteins occludin, claudin, VE-cadherin and beta-catenin in cultured HCMECs. TXL pretreatment could abrogate the down-regulation of these tight junction proteins induced by Ang II. Ang II treatment also decreased KLF5 expression at the mRNA and protein levels; TXL pretreatment markedly reversed the inhibitory effect of Ang II on KLF5 expression. Gain-of-function and loss-of-function of KLF5 showed that KLF5 mediated the expression of tight junction proteins in HCMECs. TXL-enhanced expression of the tight junction proteins was mediated by KLF5. In angiotensinogen transgenic mice, TXL also increased the tight junction protein levels by inducing KLF5 expression. Chinese medicine TXL increases tight junction protein levels by inducing KLF5 expression in microvascular endothelial cells.

633: Altered gene expression in microvascular endothelial cells isolated from preeclamptic patients

633: Altered gene expression in microvascular endothelial cells isolated from preeclamptic patients

Abstract 565: LPA-PKD-1-HDAC7/NCoR1-FoxO1 Signaling Axis Regulates Endothelial Cell CD36 Transcription and Stimulates Arteriogenic Responses

Lysophosphatidic acid (LPA), a bioactive signaling phospholipid, down-regulates CD36 expression in microvascular endothelial cells (MVECs) via protein kinase PKD-1 signaling, thereby abolishing endothelial cell responses to its antiangiogenic ligand thrombospondin-1. However, little is known regarding mechanisms by which MVEC-specific CD36 transcription is regulated. We describe that in MVECs LPA represses CD36 transcription by activating a PKD-1 signaling that induces formation of a HDAC7/NCoR1/FoxO1 complex in the nucleus. Promoter analysis first identified FoxO1 as a transcription factor responsible for the CD36 transcription, which was confirmed by a chromatin-immunoprecipitation assay. Using a combination of PKD-1 gene transduction with co-immmunoprecipitation assay, we showed an increased interaction of HDAC7/NCoR1 with FoxO1 in response to LPA. However, HDAC7 and FoxO1 interaction was attenuated with PKD-1 silencing. Furthermore, based on results from an angiogenesis profiling with real time qPCR, doxycycline inducible constitutively active PKD-1 plasmids were transduced into tumor associated endothelial cells using a Lentiviral system to induce the PKD-1 expression. The results showed that turning off CD36 transcription reprograms by PKD-1 signaling was accompanied by an induced expression of ephrin B2 and activation of MAPK/ERK1/2 signaling, which are two critical “molecular signatures” involved in arteriogenesis. Moreover, three dimensional spheroid assay, a modified Boyden Chamber assay and in vivo Matrigel assay revealed that turning off CD36 transcription promoted angiogenesis in vitro and in vivo in a PKD-1-dependent manner. Immunofluorescence microscopy also showed the presence of this signaling pathway in the vasculature of Lewis lung carcinomas grown in cd36 deficient mice. In summary, our data suggest that a LPA-PKD-1-HDAC7/NCoR1-FoxO1 signaling axis is critical for transcriptional regulation of CD36 and mediates silencing of this antiangiogenic switch. This subsequently results in MVEC reprogramming for proangiogenic and arteriogenic responses. Therefore, targeting this signaling cascade could be a novel approach for malignant tumors, cardiovascular ischemia and other thrombotic diseases.

C0230: Monocytes Through Wnt Signaling Trigger Tissue Factor Expression in Microvascular Endothelial Cells During Tube Formation

C0230: Monocytes Through Wnt Signaling Trigger Tissue Factor Expression in Microvascular Endothelial Cells During Tube Formation

Dynamic Changes in Endothelial Cell Adhesion Molecule Nepmucin/CD300LG Expression under Physiological and Pathological Conditions

Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4+ T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.