Platelet Endothelial Cell Adhesion Molecule-1 Research Articles

Novel Locally Acting Dual Antiplatelet and Anticoagulant (APAC) Targets Multiple Sites of Vascular Injury in an Experimental Porcine Model

Vascular binding of dual antiplatelet and anticoagulant (APAC) was assessed in surgically created femoral arteriovenous fistula (AVF) and iliac and carotid artery injury in porcine models. Three models of collagen exposing injury were used: 1) femoral AVF, 2) invivo iliac and carotid artery balloon angioplasty injury, and 3) invitro femoral artery endothelial denudation injury. Biotinylated APAC (0.5mg/mL) was incubated with the injury site before releasing blood flow. APAC, von Willebrand factor (vWF), laminin, platelet endothelial cell adhesion molecule 1 (PECAM-1), and podocalyxin were detected in histological sections using immunofluorescence and confocal microscopy and Manders' co-localisation coefficient (M1). APAC bound to AVF at anastomosis and to both invivo and invitro injured arteries. APAC co-localised with matrix vWF (M1≥0.66) and laminin (M1≥0.60), but less so if endothelial PECAM-1 or podocalyxin was present (M1≤0.25). APAC targeted and penetrated the injured vessel wall, especially the AVF vein. APAC, compatible with its high negative charge, rapidly targets injured vessels co-localizing with matrix vWF and laminin, but not with endothelial PECAM-1 and podocalyxin. This localising feature may have potential antithrombotic implications for vascular interventions.

Hyperglycemia-induced ubiquitination and degradation of β-catenin with the loss of platelet endothelial cell adhesion molecule-1 in retinal endothelial cells.

Increased retinal vascular permeability is one of the earliest manifestations of diabetic retinopathy. The aim of this study was to investigate the role of hyperglycemia-induced platelet endothelial cell adhesion molecule-1 loss on retinal vascular permeability via the β-catenin pathway. Type I diabetes was induced in male Wistar rats using streptozotocin injections, with age-matched non-diabetic rats as controls. Rat retinal microvascular endothelial cells were grown under normal or high glucose conditions for 6days. Small interfering Ribonucleic Acid was used to knock down platelet endothelial cell adhesion molecule-1 in rat retinal microvascular endothelial cells for loss-of-function studies. Retinas and rat retinal microvascular endothelial cells were subjected to Western blot, immunofluorescence labeling, and co-immunoprecipitation analyses to assess protein levels and interactions. A biotinylated gelatin and fluorescein isothiocyanate-avidin assay was used for retinal endothelial cell permeability studies. β-catenin, β-catenin/platelet endothelial cell adhesion molecule-1 interaction, active Src homology 2 domain-containing protein tyrosine phosphatasewere significantly decreased, while β-catenin ubiquitination levels and endothelial permeability were significantly increased, in hyperglycemic retinal endothelial cells. Similar results were observed with platelet endothelial cell adhesion molecule-1 partial knockdown, where β-catenin and active Src homology 2 domain-containing protein tyrosine phosphatase levels were decreased, while phospho-β-catenin and retinal endothelial cell permeability were increased. Platelet endothelial cell adhesion molecule-1 loss may contribute to increased retinal endothelial cell permeability by attenuating β-catenin levels under hyperglycemic conditions.

Adenosine Inhibits Ovarian Cancer Growth Through Regulating RhoGDI2 Protein Expression.

ObjectiveThis study aimed to investigate the effect of adenosine (Ado) on the growth of ovarian cancer and to explore the related mechanisms.MethodsThe effect of Ado on the proliferation of A2780 human ovarian cancer cells was examined according to the MTT method. Moreover, the nude mouse model of subcutaneous A2780 xenograft was constructed, and then, Ado and cisplatin were administered intraperitoneally to investigate the effect of Ado on tumor growth in vivo. Immunohistochemistry (IHC) was carried out to study the effect of Ado on the expression of Rho-specific guanine nucleotide dissociation inhibitor 2 (RhoGDI2) in the subcutaneous xenografts. Afterwards, the commercially constructed RhoGDI2 siRNA plasmid was transfected into A2780 cells, and tube formation assay was conducted to determine the effect of down-regulating RhoGDI2 expression on the regulation of angiogenesis in ovarian cancer by Ado. Besides, Western blotting was performed to detect the effect of RhoGDI2 down-regulation on the regulation of matrix metalloproteinase 2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), tumor necrosis factor (TNF-α), and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) expression in ovarian cancer cells by Ado.ResultsThe relative viability of cells subsequent to Ado treatment proved to be both concentration- and time dependent. IHC results showed that Ado evidently enhanced the RhoGDI2 protein expression. In addition, interference with RhoGDI2 outstandingly attenuated the ability of Ado to suppress tumor cell invasion and induce angiogenesis in vitro. Furthermore, molecular mechanism studies indicated that Ado remarkably inhibited the expression of MMP-2, MMP-9, VEGF, TGF-β, TNF-α, and CD31, while interference with RhoGDI2 restored the expression of the above-mentioned angiogenic factors.ConclusionAdo inhibits the growth of A2780 human ovarian cancer cells through inhibiting tumor cell invasion and angiogenesis in a RhoGDI2-dependent manner.

Coxsackievirus and adenovirus receptor mediates the responses of endothelial cells to fluid shear stress

Endothelial mechanotransduction by fluid shear stress (FSS) modulates endothelial function and vascular pathophysiology through mechanosensors on the cell membrane. The coxsackievirus and adenovirus receptor (CAR) is not only a viral receptor but also a component of tight junctions and plays an important role in tissue homeostasis. Here, we demonstrate the expression, regulatory mechanism, and role of CAR in vascular endothelial cells (ECs) under FSS conditions. Disturbed flow increased, whereas unidirectional laminar shear stress (LSS) decreased, CAR expression in ECs through the Krüppel-like factor 2 (KLF2)/activator protein 1 (AP-1) axis. Deletion of CAR reduced the expression of proinflammatory genes and endothelial inflammation induced by disturbed flow via the suppression of NF-κB activation. Consistently, disturbed flow-induced atherosclerosis was reduced in EC-specific CAR KO mice. CAR was found to be involved in endothelial mechanotransduction through the regulation of platelet endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation. Our results demonstrate that endothelial CAR is regulated by FSS and that this regulated CAR acts as an important modulator of endothelial mechanotransduction by FSS.

Role of Erythropoietin in Cerebral Glioma: An Innovative Target in Neuro-Oncology

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation. Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation. In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats. The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells

To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone. SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days. The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 μm) and SHED (7 734.3 μm) was higher than DPSC (5 541.0 μm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm). SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.

Delayed neutrophil apoptosis in granulomatosis with polyangiitis: dysregulation of neutrophil gene signature and circulating apoptosis-related proteins

Objectives: Neutrophil apoptosis is mandatory for resolving inflammation and is regulated by expression of pro- and anti-apoptotic genes. We studied neutrophils isolated from patients with granulomatosis with polyangiitis (GPA) to investigate apoptosis alterations and to identify transcriptional and circulating factors affecting this process.Method: We enrolled 36 patients (18 in active stage, 18 in remission) and 18 healthy controls. Circulating levels of tumour necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF), macrophage migration inhibitory factor, plasminogen activator inhibitor-1, interferon-γ, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, soluble Fas (sFas), sFas ligand, survivin, and pentraxin-3 (PTX3) were evaluated by enzyme-linked immunosorbent assay/Luminex; circulating apoptotic neutrophils by flow cytometry; and apoptosis-related gene transcripts by real-time polymerase chain reaction.Results: Patients had decreased fractions of circulating apoptotic neutrophils and delayed neutrophil apoptosis was present in vitro. Circulating levels of TNF-α, GM-CSF, sFas, and PTX3 were higher in GPA. Delayed neutrophil apoptosis was accompanied by decreased mRNA of pro-apoptotic genes and transcription factors (DIABLO, PMAIP1, BAX, CASP3, CASP7, RUNX3, E2F1, TP53) and increased anti-apoptotic CFLAR and BCL2A1 mRNA. TNF-α and sFas levels correlated with circulating apoptotic neutrophils and expression of apoptosis genes. Stimulation with TNF-α of neutrophils from controls significantly down-regulated E2F1 and CASP3 expression.Conclusions: Circulating neutrophils in GPA have anti-apoptotic phenotype involving both intrinsic and extrinsic pathways of apoptosis. This is accompanied by increased levels of circulating pro-survival factors (GM-CSF, TNF-α, sFas), independent of disease activity. Anti-apoptotic phenotype of neutrophils in GPA is reproduced by exposure to low concentrations of TNF-α.

Houshiheisan promotes angiogenesis via HIF-1α/VEGF and SDF-1/CXCR4 pathways: in vivo and in vitro.

Rationale: Houshiheisan (HSHS), a classic prescription in traditional Chinese medicine (TCM), has remarkable efficacy in the treatment of ischemic stroke.Objective: To investigate the pro-angiogenic effect and molecular mechanism of HSHS for stroke recovery.Methods and results: The rat permanent middle cerebral artery occlusion (pMCAO) model was constructed by suture method, HSHS (5.25 or 10.5 g/kg) and Ginaton (28 mg/kg) treatment was intragastrically administrated at 6 h after modeling which remained for 7 consecutive days. Pathological evaluation conducted by Hematoxylin–Eosin (HE) staining and the results showed that HSHS alleviated blood vessel edema, reduced the damage to blood vessels and neurons in the ischemic areas. Immunostaining, quantitative real-time fluorescence PCR results showed that HSHS up-regulated pro-angiogenic factors including platelet endothelial cell adhesion molecule-1 (cluster of differentiation 31 (CD31)), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGFA), VEGF receptor 2 (VEGFR2), angiopoietin-1 (Ang-1), while down-regulated angiopoietin-2 (Ang-2), stromal cell derived factor-1 (SDF-1), and cxc chemokine receptor 4 (CXCR4) expression in infarct rat cortex, and similar results were obtained in subsequent Western blot experiment. Furthermore, CCK8 assay and transwell migration assay were performed to assess cell proliferation, migration, and tube formation. The medicated serum (MS) of HSHS appeared to have beneficial effects for immortalized human umbilical vein cells (Im-HUVECs) on proliferation and migration after persistence hypoxia. Western blot analysis revealed that the expression of hypoxia inducible factor-1α (HIF-1α), VEGFA, Ang-1, Ang-2, and CXCR4 were significantly up-regulated while Ang-2 was down-regulated by HSHS MS treatment compared with vehicle group in vitro.Conclusion: The present study suggests a novel application of HSHS as an effective angiogenic formula for stroke recovery.

Interval Exercise Lowers Circulating CD105 Extracellular Vesicles in Prediabetes.

Extracellular vesicles (EV) are purported to mediate type 2 diabetes and CVD risk and development. Physical activity and a balanced diet reduce disease risk, but no study has tested the hypothesis that short-term interval (INT) training would reduce EV compared with continuous (CONT) exercise in adults with prediabetes. Eighteen obese adults (age, 63.8 ± 1.5 yr; body mass index, 31.0 ± 1.3 kg·m) were screened for prediabetes using American Diabetes Association criteria (75 g oral glucose tolerance test). Subjects were randomized to INT (n = 10, alternating 3-min intervals at 90% and 50% HRpeak, respectively) or CONT (n = 8, 70% HRpeak) training for 12 supervised sessions over 13 d for 60 min·d. Cardiorespiratory fitness (V˙ O2peak), weight (kg), as well as ad libitum dietary intake were assessed and arterial stiffness (augmentation index via applanation tonometry) was calculated using total AUC during a 75-g oral glucose tolerance test performed 24 h after the last exercise bout. Total EV, platelet EV (CD31/CD41), endothelial EV (CD105; CD31/ CD41), platelet endothelial cell adhesion molecule (PECAM) (CD31), and leukocyte EV (CD45; CD45/CD41) were analyzed via imaging flow cytometry preintervention/postintervention. The INT exercise increased V˙O2peak (P = 0.04) compared with CONT training. Although training had no effect on platelet or leukocyte EV, INT decreased Annexin V- endothelial EV CD105 compared with CONT (P = 0.04). However, after accounting for dietary sugar intake, the intensity effect was lost (P = 0.18). Increased ad libitum dietary sugar intake after training was linked to elevated AV+ CD105 (r = 0.49, P = 0.06) and AV- CD45 (r = 0.59, P = 0.01). Nonetheless, increased V˙O2peak correlated with decreased AV+ CD105 (r = -0.60, P = 0.01). Interval exercise training decreases endothelial-derived EV in adults with prediabetes. Although increased sugar consumption may alter EV after a short-term exercise intervention, fitness modifies EV count.

Alteration of Tear Cytokine Expressions in Primary Acquired Nasolacrimal Duct Obstruction – Potential Insights into the Etiopathogenesis

ABSTRACTPurpose: To investigate the presence and level of 35 distinct cytokines in the tear fluid obtained from patients with primary acquired nasolacrimal duct obstruction (PANDO) and compare it with controls in an effort to understand the disease etiopathogenesis.Methods: Standard protocols were used for collecting tears from 60 eyes (20 diseased eyes and 20 healthy fellow eyes of unilateral PANDO, 20 control eyes of healthy subjects). A total of 35 analytes involved in inflammation, angiogenesis and wound healing were assessed by multiplex ELISA. Alterations in the tear levels of cytokines in PANDO and their comparison with the levels in the non-diseased fellow eye and healthy volunteers were noted. STRING analysis was used to assess the involved biological pathways of the altered cytokines. Linear mixed effect model was used for statistical analysis. A P value of <0.05 was considered significant.Results: There was significant upregulation of 10 pro-inflammatory cytokines in tears from diseased eyes of PANDO patients in comparison with the non-diseased controls and include matrix metalloproteinase 9 (MMP 9), serpin E1, Interleukin-6 (IL-6), hepatocyte growth factor (HGF), vascular endothelial growth factor-A and R2 (VEGF-A, VEGF R2), platelet-endothelial cell adhesion molecule (PECAM-1), c-reactive protein (CRP), chemokine ligand 2 (CCL2) and platelet-derived growth factor- AA (PDGF-AA). Amongst the anti-inflammatory cytokines, three were significantly upregulated in diseased eyes of PANDO patients in comparison with the non-diseased controls and include granulocyte-colony stimulating factor (G-CSF), retinol binding protein 4 (RBP4) and tissue inhibitor of metalloproteinases −1 (TIMP-1). There were no significant differences between the control eyes of the diseased patient and control eyes of healthy subjects. Based on the significantly altered cytokines, string analysis revealed that the biological pathways involved in the etiopathogenesis of PANDO include inflammation, angiogenesis, negative regulation of apoptosis, cellular proliferation and hormonal regulation.Conclusions: In cases of PANDO, dysregulation of certain cytokines was disease specific. Biological pathways reflect a possible link and interaction between the inflammatory cytokines with vasculature and hormonal microenvironments of the lacrimal drainage system, which in a way is bringing three promising candidates in the PANDO etiopathogenesis on a common ground.

Maternal Diet-Induced Obesity Compromises Oxidative Stress Status and Angiogenesis in the Porcine Placenta by Upregulating Nox2 Expression.

Maternal obesity is associated with placental oxidative stress. However, the mechanism underlying this association remains poorly understood. In the present study, a gilt obesity model was developed by exposure to different energy diets and used to investigate the role of NADPH oxidase 2 (Nox2) in the placenta. Specifically, 99 gilts (Guangdong Small-ear Spotted pig) at day 60 of gestation were randomly assigned to one of the following three treatments: low-energy group (L, DE = 11.50 MJ/kg), medium-energy group (M, DE = 12.41 MJ/kg), and high-energy group (H, DE = 13.42 MJ/kg), with 11 replicate pens per treatment and 3 gilts per pen. At the start of the study, maternal body weight and backfat thickness were not significantly different in the three treatments. After the study, data indicated that the H group had higher body weight and backfat thickness gain for gilts during gestation and lower piglet birth weight compared with the other two groups. Additionally, the H group showed glucolipid metabolic disorders and increased triglyceride and nonesterified fatty acid contents in the placenta of gilts. Compared with the L group, the H group exhibited lower mitochondrial biogenesis and increased oxidative damage in the placenta. Importantly, increased mRNA expression and protein abundance of Nox2 were observed for the first time in H group placentae. Furthermore, compared with the L group, the H group showed a decrease in the density of placental vessels and the protein levels of vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor A (VEGF-A), and phosphorylation of vascular endothelial growth factor receptor 2 (p-VEGFR2) as well as the immunostaining intensity of platelet endothelial cell adhesion molecule-1 (CD31). Our findings suggest that maternal high-energy diet-induced obesity increases placental oxidative stress and decreases placental angiogenesis possibly through the upregulation of Nox2.

Aloe Vera/Collagen Mixture Induces Integrin α1β1 and PECAM-1 Genes Expression in Human Adipose-Derived Stem Cells.

Purpose: Natural biomaterials are a key base in tissue engineering, and collagen, as the main content of the extracellular matrix (ECM), is frequently used in tissue engineering. Aloe vera has some therapeutic effects on ulcers, therefore, the use of this natural resource has always been considered for improving collagen function. We aimed to evaluate the effect of Aloe vera/ Collagen blended on cell viability, cell attachment, and angiogenic potential by determining of integrin α1β1 and platelet endothelial cell adhesion molecule (PECAM-1) genes expression in human adipose-derived stem cells (hASCs). Methods: In this study, hASCs after harvesting of adipose tissues from abdominal subcutaneous adipose tissue and isolation, were cultured in four groups of control, collagen gel, Aloe vera gel, and Aloe vera/collagen blended in vitro environment at 24h and then cell viability was assessed by MTT (3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium) assay. Integrin α1β1 and PECAM-1 genes expression were evaluated by real-time RT-PCR. Results: The results of MTT showed that the combination of Aloe vera/collagen was retained the cell viability at the normal range and improved it. In real-time RT-PCR results, integrin α1β1 and PECAM-1 gene expression were increased in the Aloe vera/collagen blended group compared to the control group. Conclusion: For tissue engineering purposes, Aloe vera improves collagen properties in the culture of hASCs by increasing the expression of the integrin α1β1 and PECAM-1 genes.

Effect of Bifidobacterium-induced changes in tumor tissue acoustic properties on efficacy of high-intensity focused ultrasound ablation

To investigate the effects of Bifidobacterium on the acoustic characteristics of tumor tissue and how such acoustic changes affect the efficacy of high-intensity focused ultrasound (HIFU) ablation in nude mice. Forty mice bearing human breast cancer cell (MDA-MB-231) xenograft were randomized into experimental group (n=20) and control group (n=20) for intravenous injection of Bifidobacterium suspension (200 μL, 4 × 108 cfu/mL) and PBS (200 μL) for 3 consecutive days, respectively. Before and at 3 and 7 days after the first injection, shear wave elastography was used to evaluate the hardness of the tumor tissue. On day 7 after the first injection, 10 mice from each group were sacrificed and the sound velocity and sound attenuation of the tumor tissues were measured. The changes in the collagen fibers in the tumors were evaluated using Masson staining, and neovascularization in the tumor was assessed with immunohistochemistry for platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). The remaining 10 tumor-bearing mice in each group were subjected to HIFU ablation, and the ablation efficiency was evaluated by assessing the changes in irradiation gray values, coagulative necrosis volume, energy efficiency factor (EEF) and irradiation area and by pathological examination with HE staining. In the experimental group, the collagen fibers in the tumor tissues were strong and densely aligned, and the tumors contained fewer new blood vessels showing strip-or spot-like morphologies. In the control group, the collagen fibers in the tumors were thin and loosely arranged, and the tumors showed abundant elongated or round new blood vessels. Bifidobacterium colonized in the tumor 7 days after the injection, and the tumor hardness was significantly greater in the experimental group than in the control group (P=0.01); the acoustic velocity (P=0.001) and the acoustic attenuation (P=0.000) of the tumor tissues were also greater in the experimental group. HIFU irradiation resulted in significantly greater changes in the gray scale of tumor (P=0.0006) and larger coagulative necrosis volume (P=0.0045) in the experimental group than in the control group, and the EEF was significantly smaller in the experimental group (P=0.0134). Bifidobacterium can cause changes in collagen fiber content, acoustic velocity and attenuation in the tumor tissue and reduce the EEF of HIFU irradiation, thereby improving the efficacy of HIFU irradiation.

Mechanoregulation of p38 activity enhances endoplasmic reticulum stress-mediated inflammation by arterial endothelium.

Endothelial up-regulation of VCAM-1 at susceptible sites in arteries modulates the recruitment efficiency of inflammatory monocytes that initiates atherosclerotic lesion formation. We reported that hydrodynamic shear stress (SS) mechanoregulates inflammation in human aortic endothelial cells through endoplasmic reticulum (ER) stress via activation of the transcription factor x-box binding protein 1 (XBP1). Here, a microfluidic flow channel that produces a linear gradient of SS along a continuous monolayer of endothelium was used to delve the mechanisms underlying transcriptional regulation of TNF-α-stimulated VCAM-1 expression. High-resolution immunofluorescence imaging enabled continuous detection of platelet endothelial cell adhesion molecule 1 (PECAM-1)-dependent, outside-in signaling as a function of SS magnitude. Differential expression of VCAM-1 and intercellular adhesion molecule 1 (ICAM-1) was regulated by the spatiotemporal activation of MAPKs, ER stress markers, and transcription factors, which was dependent on the mechanosensing of SS through PECAM-1 and PI3K. Inhibition of p38 specifically abrogated the rise to peak VCAM-1 at low SS (2 dyn/cm2), whereas inhibition of ERK1/2 attenuated peak ICAM-1 at high SS (12 dyn/cm2). A shear stress-regulated temporal rise in p38 phosphorylation activated the nuclear translocation of XBP1, which together with the transcription factor IFN regulatory factor 1, promoted maximum VCAM-1 expression. These data reveal a mechanism by which SS sensitizes the endothelium to a cytokine-induced ER stress response to spatially regulate inflammation promoting atherosclerosis.-Bailey, K. A., Moreno, E., Haj, F. G., Simon, S. I., Passerini, A. G. Mechanoregulation of p38 activity enhances endoplasmic reticulum stress-mediated inflammation by arterial endothelium.

APC/Cdh1 targets PECAM-1 for ubiquitination and degradation in endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily and is expressed by hematopoietic and endothelial cells (ECs). Recent studies have shown that PECAM-1 plays a crucial role in promoting the development of the EC inflammatory response in the context of disturbed flow. However, the mechanistic pathways that control PECAM-1 protein stability remain largely unclear. Here, we identified PECAM-1 as a novel substrate of the APC/Cdh1 E3 ubiquitin ligase. Specifically, lentivirus-mediated Cdh1 depletion stabilized PECAM-1 in ECs. Conversely, overexpression of Cdh1 destabilized PECAM-1. The proteasome inhibitor MG132 blocked Cdh1-mediated PECAM-1 degradation. In addition, Cdh1 promoted K48-linked polyubiquitination of PECAM-1 in a destruction box-dependent manner. Furthermore, we demonstrated that compared with pulsatile shear stress (PS), oscillatory shear stress decreased the expression of Cdh1 and the ubiquitination of PECAM-1, therefore stabilizing PECAM-1 to promote inflammation in ECs. Hence, our study revealed a novel mechanism by which fluid flow patterns regulate EC homeostasis via Cdh1-dependent ubiquitination and subsequent degradation of PECAM-1.

Escin: a review of its anti-edematous, anti-inflammatory, and venotonic properties.

This review discusses historical and recent pharmacological and clinical data on the anti-edematous, anti-inflammatory, and venotonic properties of escin (Reparil®). Escin, the active component of Aesculus hippocastanum, or horse chestnut, is available as orally absorbable dragées and as a transdermal gel. The anti-inflammatory and anti-edematous effects of escin have been studied over many years in pre-clinical models. More recent data confirm the anti-inflammatory properties of escin in reducing vascular permeability in inflamed tissues, thereby inhibiting edema formation. The venotonic effects of escin have been demonstrated primarily by in vitro studies of isolated human saphenous veins. The ability of escin to prevent hypoxia-induced disruption to the normal expression and distribution of platelet endothelial cell-adhesion molecule-1 may help explain its protective effect on blood vessel permeability. Escin oral dragées and transdermal gel have both demonstrated efficacy in blunt trauma injuries and in chronic venous insufficiency. Both oral escin and the transdermal gel are well tolerated.

Development of A New Mouse Model for Intrahepatic Cholangiocellular Carcinoma: Accelerating Functions of Pecam-1.

Due to the lack of suitable in-vivo models, the etiology of intrahepatic cholangiocellular carcinoma (ICC) is poorly understood. We previously showed the involvement of platelet endothelial cell adhesion molecule-1 (Pecam-1/CD31) in acute liver damage. Here, we developed a model of ICC using thioacetamide (TAA) in drinking water of wild-type (WT)-mice and Pecam-1-knock-out (KO)-mice. Gross inspection and microscopy revealed liver-cirrhosis and ICC in both groups after 22 weeks of TAA. The severity of cirrhosis and ICC (Ck-19-positive) was reduced in Pecam-1 KO mice (stage-4 cirrhosis in WT vs. stage-3 in KO mice). Tumor networks (accompanied by neutrophils) were predominantly located in portal areas, with signs of epithelial-to-mesenchymal transition (EMT). In serum, TAA induced an increase in hepatic damage markers, with lower levels in Pecam-1 null mice. With qPCR of liver, elevated expression of Pecam-1 mRNA was noted in WT mice, in addition to Icam-1, EpCam, cytokines, cMyc, and Mmp2. Thereby, levels of EpCAM, cytokines, cMyc, and Mmp2 were significantly lower in Pecam-1 null mice. Lipocalin-2 and Ccl5 were elevated significantly in both WT and Pecam-1 null mice after TAA administration. Also, EMT marker Wnt5a (not Twist-1) was increased in both groups after TAA. We present a highly reproducible mouse model for ICC and show protective effects of Pecam-1 deficiency.

Increased Lymphangiogenesis and Lymphangiogenic Growth Factor Expression in Perivascular Adipose Tissue of Patients with Coronary Artery Disease.

Experimental and human autopsy studies have associated adventitial lymphangiogenesis with atherosclerosis. An analysis of perivascular lymphangiogenesis in patients with coronary artery disease is lacking. Here, we examined lymphangiogenesis and its potential regulators in perivascular adipose tissue (PVAT) surrounding the heart (C-PVAT) and compared it with PVAT of the internal mammary artery (IMA-PVAT). Forty-six patients undergoing coronary artery bypass graft surgery were included. Perioperatively collected C-PVAT and IMA-PVAT were analyzed using histology, immunohistochemistry, real time PCR, and PVAT-conditioned medium using cytokine arrays. C-PVAT exhibited increased PECAM-1 (platelet endothelial cell adhesion molecule 1)-positive vessel density. The number of lymphatic vessels expressing lymphatic vessel endothelial hyaluronan receptor-1 or podoplanin was also elevated in C-PVAT and associated with higher inflammatory cell numbers, increased intercellular adhesion molecule 1 (ICAM1) expression, and fibrosis. Significantly higher expression of regulators of lymphangiogenesis such as vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor-3 was observed in C-PVAT compared to IMA-PVAT. Cytokine arrays identified angiopoietin-2 as more highly expressed in C-PVAT vs. IMA-PVAT. Findings were confirmed histologically and at the mRNA level. Stimulation of human lymphatic endothelial cells with recombinant angiopoietin-2 in combination with VEGF-C enhanced sprout formation. Our study shows that PVAT surrounding atherosclerotic arteries exhibits more extensive lymphangiogenesis, inflammation, and fibrosis compared to PVAT surrounding a non-diseased vessel, possibly due to local angiopoietin-2, VEGF-C, and VEGF-D overexpression.

Effect of quinolinic acid – A uremic toxin from tryptophan metabolism – On hemostatic profile in rat and mouse thrombosis models

PurposeWe aimed to determine the effect of quinolinic acid (QA) on hemostasis in rat and mouse models of thrombosis. Material and methodsWistar rats (male, n = 72) received QA dissolved in drinking water in doses of 3, 10, 30 mg/kg or pure drinking water (vehicle control group -VEH) for 14 days. On the 14th day of the experiment the effect of QA on hemostasis was evaluated using electrically induced arterial thrombosis model. The following parameters were measured: thrombus weight, hematology, thromboelastometric (ROTEM) parameters, TXA2 and 6-keto-PGF1α concentration, coagulation and fibrinolytic markers activity and concentration.GFP mice (male, n = 30) were assigned to the group receiving QA (30 mg/kg) or VEH for 14 days and to the group receiving: single intravenous dose of QA (30 mg/kg) or VEH or the same dose of QA and anti-CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1) antibody conjugated with Alexa Fluor 647. The effect of QA on hemostasis was evaluated in the model of laser-induced injury of mesentery vein using intravital confocal microscopy. ResultsAdministering QA for 14 days resulted in a divergent, depending on dose, increase in concentration of active form of tPA and PAI-1 and concentration of total PAI-1 and PAP complexes in rats’ plasma. In turn, administering QA for 14 days in mice revealed its prothrombotic activity, while single-dose IV administration revealed its antithrombotic activity, through the up-regulation of PECAM-1 expression. ConclusionsWe demonstrated the first evidence for the opposite biological effects of QA on hemostasis in rat and mouse thrombosis models.

Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations

The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/− 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4–19.3 mmHg vs 16.3–20.3 mmHg) (p < 0.05) for the left eye. Flight group had significant apoptosis in the retina and retinal vascular endothelial cells compared to control groups (p < 0.05). Immunohistochemical analysis of the retina revealed that an increased expression of aquaporin-4 (AQP-4) in the flight mice compared to controls gave strong indication of disturbance of BRB integrity. There were also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BRB-related tight junction protein, Zonula occludens-1 (ZO-1). Proteomic analysis showed that many key proteins and pathways responsible for cell death, cell cycle, immune response, mitochondrial function and metabolic stress were significantly altered in the flight mice compared to ground control animals. These data indicate a complex cellular response that may alter retina structure and BRB integrity following long-term spaceflight.