Platelet Endothelial Cell Adhesion Molecule-1 Expression Research Articles

Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.

Role of cellular interaction and cell phenotype in azacytidine to doxorubicin sensitivity in breast cancer cell-derived clones

BackgroundBreast malignancies are the most diverse tumors, having the highest level of cellular heterogeneity at various stages of development. This heterogeneous nature of cells is even observed in a controlled growth environment. Cellular phenotype, expression of cell surface markers, epithelial to mesenchymal transition, and cellular niche play a pivotal role in the heterogeneity of breast cancer cells. Heterogeneity in breast cancer cells is one of the leading causes of drug resistance where only a portion of cells respond to the therapeutic drugs while the other cells escape from therapies. Furthermore, cellular heterogeneity is one of the major barriers to designing therapeutic strategies. Therefore, it is important to design multidimensional strategies that consider various aspects, including cell type, cellular microenvironment, and the type of anticancer drugs to treat breast cancer. AimThis study aimed to investigate the responses of cells from various single-cell-derived breast cancer clones to 5-azacytidine (AzaC) and doxorubicin (Dox). Furthermore, the role of AzaC in the sensitization of breast cancer cells to Dox was studied in breast cancer clones. Materials and methodsSingle cell-derived clones were established from four distinct breast cancer cell lines including MCF7, MDA-MB-231, MCF7-GAPDH-RFP and MDA-GAPDH-RFP. Following one week of clonal development, the clones were subjected to separate or combined treatments of 5 μM AzaC and 500 nM Dox for 48 h. In the sequential group (AzaC/Dox), clones were exposed to 5 μM AzaC for one week, followed by 500 nM Dox for 48 h. Morphological and immunocytochemical analyses were performed by using crystal violet staining and immunolabeling, respectively. Furthermore, the expression levels of specific protein markers, including alpha smooth muscle actin (α-SMA), vimentin (VIM), cytokeratin-8 (CK-8), cytokeratin-19 (CK-19) and platelet endothelial cell adhesion molecule-1 (PECAM-1), were evaluated in response to the drugs. In addition, the expressed target protein markers were quantified in five independent sets of clones from each treatment group (n = 5). ResultsAfter 48 h of treatment, the sizes of the colonies in all treatment groups were markedly decreased as compared to the control group. In the AzaC group, colonies exhibited irregular shapes and scattered cell patterns. In Dox- and AzaC+Dox-treated clones, the cell number in each colony evidently decreased, while in AzaC/Dox-treated clones, a fraction of clonal cells showed a similar phenotype to mesenchymal-like cells. Immunocytochemistry showed a significant decrease in the number of α-SMA- and VIM-expressing cells in the drug-treated groups. Notably, in MCF7- GAPDH-RFP and MDA-GAPDH-RFP clones, VIM expression remained high despite the low cell number. Overall, CK-8 was down-regulated while CK-19 was strongly expressed in AzaC-treated MDA-MB-231 clones. Despite the small cell number, both markers exhibited up-regulation in the drug-treated MCF7- and MDA-GAPDH-overexpressed clones. Likewise, there was a notable increase in the expression of PECAM-1 in drug-treated MCF7- and MDA-RFP clones. ConclusionThis study demonstrates that AzaC-sensitizes breast cancer cells to Dox in single cell-derived clones. Furthermore, the high expression pattern of the target protein markers in drug-treated clones reflects their role in cell survival and drug resistance. Further studies are needed to uncover the detailed molecular mechanism involved in cell survival and drug resistance against AzaC and Dox in breast cancer cell-derived clones.

Porphyromonas gingivalis Gingipains Destroy the Vascular Barrier and Reduce CD99 and CD99L2 Expression To Regulate Transendothelial Migration.

Porphyromonas gingivalis is an important periodontal pathogen that can cause vascular injury and invade local tissues through the blood circulation, and its ability to evade leukocyte killing is critical to its distal colonization and survival. Transendothelial migration (TEM) is a series of that enable leukocytes to squeeze through endothelial barriers and migrate into local tissues to perform immune functions. Several studies have shown that P. gingivalis-mediated endothelial damage initiates a series of proinflammatory signals that promote leukocyte adhesion. However, whether P. gingivalis is involved in TEM and thus influences immune cell recruitment remains unknown. In our study, we found that P. gingivalis gingipains could increase vascular permeability and promote Escherichia coli penetration by downregulating platelet/endothelial cell adhesion molecule 1 (PECAM-1) expression in vitro. Furthermore, we demonstrated that although P. gingivalis infection promoted monocyte adhesion, the TEM capacity of monocytes was substantially impaired, which might be due to the reduced CD99 and CD99L2 expression on gingipain-stimulated endothelial cells and leukocytes. Mechanistically, gingipains mediate CD99 and CD99L2 downregulation, possibly through the inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, our in vivo model confirmed the role of P. gingivalis in promoting vascular permeability and bacterial colonization in the liver, kidney, spleen, and lung and in downregulating PECAM-1, CD99, and CD99L2 expression in endothelial cells and leukocytes. IMPORTANCE P. gingivalis is associated with a variety of systemic diseases and colonizes in distal locations in the body. Here, we found that P. gingivalis gingipains degrade PECAM-1 to promote bacterial penetration while simultaneously reducing leukocyte TEM capacity. A similar phenomenon was also observed in a mouse model. These findings established P. gingivalis gingipains as the key virulence factor in modulating the permeability of the vascular barrier and TEM processes, which may provide a new rationale for the distal colonization of P. gingivalis and its associated systemic diseases.

Low dose space radiation-induced effects on the mouse retina and blood-retinal barrier integrity

The health risk of flight condition-triggered ocular injury and neurodegeneration has long been a concern. Spaceflight missions will likely expose the astronauts and experiment payloads to greater radiation levels compared to those encountered on the Earth. Knowledge about the susceptibility to adverse effects from low doses of radiation during space missions is very limited. Our present study aims to investigate and compare the effects of whole-body simulated galactic cosmic rays (GCR) on blood-retinal barrier (BRB) integrity, oxidative stress, and apoptosis in the retina of male and female mouse models. Six-month-old male and female CD1 mice were either sham irradiated or received whole-body 5-ion simulated GCR exposure at a dose of 0.5 Gy. At four months following irradiation, mice were euthanized and ocular tissues were collected for histochemical analysis. BRB integrity was evaluated with biomarkers aquaporin-4 (AQP-4), a water channel protein, tight junction (TJ) Zonula occludens-1 (ZO-1), and adhesive molecule, platelet endothelial cell adhesion molecule-1 (PECAM-1). A significantly increased expression of AQP-4 was observed in the retina following GCR exposure compared to controls (p < 0.05) with more pronounced changes observed in the female mice over males. There was also a significant increase in the expression of PECAM-1 and a decrease in the expression of ZO-1 in the retina of irradiated groups compared to controls. Immunochemical analysis revealed enhanced immunoreactivity for oxidative biomarker, 4-hydroxynonenal (4-HNE) in the retina following radiation exposure. Significant increases in the numbers of apoptotic cells were also documented in the retina of irradiated male and female mice compared to controls (p < 0.05). Our study revealed that exposure to low-dose ionizing radiation induced cellular oxidative damage that may alter retina structure and BRB integrity. The results also demonstrate some sex-related differences in the radiation response. Further studies are needed to investigate the factors that contribute to gender- or genetic related variabilities in ocular changes to radiation exposure for a better understanding of the mechanisms of spaceflight-induced ocular findings.

Evaluating Ocular Response in the Retina and Optic Nerve Head after Single and Fractionated High-Energy Protons.

There are serious concerns about possible late radiation damage to ocular tissue from prolonged space radiation exposure, and occupational and medical procedures. This study aimed to investigate the effects of whole-body high-energy proton exposure at a single dose on apoptosis, oxidative stress, and blood-retina barrier (BRB) integrity in the retina and optic nerve head (ONH) region and to compare these radiation-induced effects with those produced by fractionated dose. Six-month-old C57BL/6 male mice were either sham irradiated or received whole-body high energy proton irradiation at an acute single dose of 0.5 Gy or 12 equal dose fractions for a total dose of 0.5 Gy over twenty-five days. At four months following irradiation, mice were euthanized and ocular tissues were collected for histochemical analysis. Significant increases in the number of apoptotic cells were documented in the mouse retinas and ONHs that received proton radiation with a single or fractionated dose (p < 0.05). Immunochemical analysis revealed enhanced immunoreactivity for oxidative biomarker, 4-hydroxynonenal (4-HNE) in the retina and ONH following single or fractionated protons with more pronounced changes observed with a single dose of 0.5 Gy. BRB integrity was also evaluated with biomarkers of aquaporin-4 (AQP-4), a water channel protein, a tight junction (TJ) protein, Zonula occludens-1 (ZO-1), and an adhesion molecule, the platelet endothelial cell adhesion molecule-1 (PECAM-1). A significantly increased expression of AQP-4 was observed in the retina following a single dose exposure compared to controls. There was also a significant increase in the expression of PECAM-1 and a decrease in the expression of ZO-1 in the retina. These changes give a strong indication of disturbance to BRB integrity in the retina. Interestingly, there was very limited immunoreactivity of AQP-4 and ZO-1 seen in the ONH region, pointing to possible lack of BRB properties as previously reported. Our data demonstrated that exposure to proton radiation of 0.5 Gy induced oxidative stress-associated apoptosis in the retina and ONH, and changes in BRB integrity in the retina. Our study also revealed the differences in BRB biomarker distribution between these two regions. In response to radiation insults, the cellular response in the retina and ONH may be differentially regulated in acute or hyperfractionated dose schedules.

Effect of Vitamin D3 to Platelet Activation Mediated by Tumor Cell Culture Medium

To investigate the effect of vitamin D3 to platelet activation by tumor cell culture medium. The peripheral blood platelets of BALB/c mice were isolated. The platelets were activated in 4T1 culture fluid for 24 h. The platelets were divided into 7 groups: control group, activation group, 1 nmol/L vitamin D3 group, 10 nmol/L vitamin D3 group, 50 nmol/L vitamin D3 group, 100 nmol/L vitamin D3 group, and positive drug (0.1 μmol/L eptifibatide) group. CCK-8 assay was used to detect the platelet proliferation at 24, 48 and 72 h. Flow cytometry was used to detect the expression of CD61 and CD62p and receptor for advanced glycation end products (RAGE) at 24, 48 and 72 h. ELISA was used to detect the level of platelet-endothelial cell adhesion molecule-1 (PECAM-1) at 24, 48 and 72 h. The CD41+/CD61+ and CD41+/CD62p+ ratio, PECAM-1 content and RAGE expression of platelets in activated group were all significantly increased as compared with those in control group (P<0.05). Compared with the activated group, the platelet proliferation, proportion of CD41+/CD61+ and CD41+/CD62p+, content of PECAM-1 and RAGE expression in vitamin D3 groups were all decreased (P<0.05). Vitamin D3 shows antiplatelet effect and can inhibit platelet proliferation and activation.

The exosome of platelet endothelial cell adhesion molecule-1 (PECAM1) protein: A potential risking star in high blood pressure patients (HBPP).

A number of studies have demonstrated that exosomes were involved in important physiological and pathological processes through cell-to-cell communication in cardiovascular disease, which contained nucleic acids, proteins, and lipid contents. In our study, we found that the protein platelet endothelial cell adhesion molecule-1 (PECAM1) was an extracellular vesicle in the blood of high blood pressure patients (HBPP).Isolated the vesicles from the blood of HBPP and health examiners and detected its size and morphology with nanoparticle tracking analysis, then we identified its surface protein CD63, CD81, and the protein expression of PECAM1 in the exosome with western blot. Furthermore, we analyzed the correlation between the expression of PECAM1 and the high blood degree with linear regression analysis.Our results showed that the morphology of extracellular vesicles was more evident in high blood pressure groups than healthy controls, and the protein expression of PECAM1 was also abundant in the vesicles of HBPP, however, there were no extracellular vesicles in the blood samples of healthy controls. Besides, linear regression showed the linear correlation coefficient R = 0.901, P < .01 between the expression of PECAM1 and the systolic blood pressure of the high blood patients. Therefore, the exosome of protein of PECAM1 was a potential risking star in HBPP.

PECAM1 Combines With CXCR4 to Trigger Inflammatory Cell Infiltration and Pulpitis Progression Through Activating the NF-κB Signaling Pathway.

Pulpitis is a frequent bacterially driven inflammation featured with the local accumulation of inflammatory products in human dental pulps. A GEO dataset GSE16134 comprising data of inflamed dental pulp tissues was used for bioinformatics analyses. A protein-protein interaction (PPI) analysis suggested that chemokine receptor 4 (CXCR4) owned a high correlation with platelet endothelial cell adhesion molecule-1 (PECAM1). A rat model with pulpitis was established, and lipopolysaccharide (LPS)-induced human dental pulp fibroblasts (HDPFs) were used for in vitro experiments. Then, high expression of PECAM1 and CXCR4 was validated in the inflamed dental pulp tissues in rats and in LPS-induced HDPFs. Either downregulation of PECAM1 or CXCR4 suppressed inflammatory cell infiltration in inflamed tissues as well as the inflammation and apoptosis of HDPFs. A transcription factor myocyte-enhancer factor 2 (MEF2C) was predicted and validated as a positive regulator of either PECAM1 or CXCR4, which activated the NF-κB signaling pathway and promoted pulpitis progression. To sum up, this study suggested that MEF2C transcriptionally activates PECAM1 and CXCR4 to activate the B-cell and NF-κB signaling pathways, leading to inflammatory cell infiltration and pulpitis progression.

Radix Paeoniae Rubra Ameliorates Lupus Nephritis in Lupus-Like Symptoms of Mrl Mice by Reducing Intercellular Cell Adhesion Molecule-1, Vascular Cell Adhesion Molecule-1, and Platelet Endothelial Cell Adhesion Molecule-1 Expression.

Vasculitis is the basic pathological change of systemic lupus erythematosus (SLE). Radix Paeoniae Rubra (RPR), a traditional Chinese herb with the function of reducing blood stasis, has anti-inflammatory and immunoregulatory properties. This study explored the effects of RPR on the kidneys of lupus-like symptoms of mrl (MRL/lpr) mice from the perspective of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1). Eighteen MRL/lpr lupus model mice were randomly divided into three groups, the model control group, prednisone-treated group, and RPR-treated group, and 6 C57BL/ 6 mice were classified as a control group. After the mice had been treated for 12 weeks, the expression of ICAM-1, VCAM-1 and PECAM-1in the kidney was determined by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). After 12 weeks, there were significant differences in body weight in the model, prednisone and RPR groups compared with the normal group (P <0.05). Pathological observation: Compared with the model group, the proliferation of inflammatory cells infiltrated glomeruli and interstitial cells in prednisone and RPR groups were reduced, and renal pathological damage was reduced. Compared with the model group, urine protein level of prednisone and RPR groups were reduced with no significance (P> 0.05). The mRNA expression levels of ICAM-1 and VCAM-1 were significantly reduced in the prednisone group and RPR group compared with the model group (P <0.05 or P <0.01). Meanwhile, the immunohistochemistry expressions of ICAM-1 and VCAM- 1 expressed in the kidney were significantly reduced in the prednisone group and RPR group (P <0.01 or P <0.05). However, The mRNA expression level and the immunohistochemistry expressions of PECAM-1 expressed in the kidney were reduced in each treatment group (prednisone group and RPR group), but these differences were not significant (P>0.05). ICAM-1, VCAM-1 and PECAM-1 expression in the model group was found to be significantly increased. In addition, RPR could reduce the expression of ICAM-1, VCAM-1 and PECAM-1 in MRL/lpr lupus mice as effectively as prednisone, which may result in the dosage reduction of prednisone, thus decreasing the toxicity and improving the efficacy of prednisone - based treatment of SLE.

PECAM-1 protects against DIC by dampening inflammatory responses via inhibiting macrophage pyroptosis and restoring vascular barrier integrity

Disseminated intravascular coagulation (DIC) is a frequent complication of sepsis that affects patient outcomes due to accompanying thrombo-inflammation and microvascular permeability changes. Platelet endothelial cell adhesion molecule-1 (PECAM-1), a cellular adhesion and signaling receptor that is expressed on both hematopoietic and endothelial cells, plays an important anti-inflammatory role in acute and chronic inflammatory disease models. Little is known, however, about role and mechanism of PECAM-1 in septic DIC. Here, we investigated whether PECAM-1 might play a protective role in hindering the development of septic DIC. Plasma levels of soluble PECAM-1 were markedly elevated in septic patients that developed DIC, with a correspondingly poorer outcome. PECAM-1 knockout exhibited more severe DIC and poorer outcome in the LPS induced- and cecal ligation and puncture-induced DIC model, which could be alleviated by tissue factor inhibitor. This phenomenon seemed to be equally linked to PECAM-1 expression by both endothelial and blood cells. Furthermore, PECAM-1 was found to exert its protective effect on developing septic DIC by the following 2 distinct mechanisms: the inhibition of macrophage pyroptosis and the acceleration of the restoration of the endothelial cell barrier. Taken together, these results implicate PECAM-1 as a potentially attractive target for the development of novel therapeutics to manage and treat septic DIC.

Low shear stress induces endothelial cell apoptosis and monocyte adhesion by upregulating PECAM‑1 expression.

Low shear stress serves an important role in the initiation and progression of atherosclerotic lesions, with an impact on progression, but its detailed mechanisms are .not yet fully known. The present study aimed to investigate endothelial cell (EC) apoptosis, as well as monocyte adhesion induced by low shear stress and the potential underlying mechanisms. The expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) was demonstrated to be enhanced in human umbilical vascular ECs with a trend that was associated with time when stimulated by low shear stress compared with unstimulated cells. EC apoptosis was increased under low shear stress compared with unstimulated cells, and knockdown of PECAM-1 inhibited this process. Furthermore, downregulation of PECAM-1 reduced monocyte adhesion induced by low shear stress compared with that in the negative control cells. Mechanistically, PECAM-1 small interfering RNA transfection increased Akt and forkhead box O1 phosphorylation under low shear stress conditions compared with that in the negative control cells. Collectively, the findings of the present study revealed that low shear stress induced EC apoptosis and monocyte adhesion by upregulating PECAM-1 expression, which suggested that PECAM-1 may be a potential therapeutic target for atherosclerosis.

Syk/PECAM-1 Signaling Pathway Mediates Low Shear Stress-Induced Inflammatory&amp;nbsp;in Atherosclerosis

Although blood flow shear stress has been demonstrated to alter the physiology of vascular endothelial cells, little is known about the role of Syk in PECAM-1 signaling pathway exposed to low shear stress (LSS). Methods: In vitro, primary human umbilical vein endothelial cells (HUVECs) were stimulated with parallel plate flow chamber system for 12h under normal shear stress (NSS, 15dyne/cm2), LSS (5dyne/cm2) and high shear stress (HSS, 25dyne/cm2), respectively, followed by inflammatory response analysis. In vivo, atherosclerotic animal models of rat and rabbit were created by feeding with high cholesterol diets. The animals were applied with LSS stimulation by constricting left carotid artery with a blunted needle (0.8mm in diameter). Small molecule R406, a well-demonstrated Syk inhibitor, was applied to the atherosclerotic animal models as well as HUVEC cells. Results: LSS influenced carotid atherosclerosis in the LSS model of rabbits; LSS promoted expression of PECAM-1 and Syk in LSS-treated atherosclerotic rat model; LSS influenced the morphology of abdominal aortic endothelial cells in rats; LSS increased the expression of pPECAM-1, Syk and NF-κB in HUVECs, and inhibition of Syk attenuated LSSinduced inflammatory response. Conclusion: Activation of spleen tyrosine kinase (Syk)/platelet endothelial cell adhesion molecule-1(PECAM-1) signaling pathway was accompanied with inflammatory response induced by LSS stimulation both in vitro and in vivo. This work demonstrated the important role Syk/PECAM1/NF-κB signaling pathway plays in LSS-induced inflammatory response. Funding Statement: This work was supported by Pudong New Area Health Bureau of Shanghai (grant no.PWZbr2017-09), and the National Natural Science Foundation of China (grant no.81401428, 81571693 and 81871361). Declaration of Interests: The authors have declared that no competing interest exists. Ethics Approval Statement: Human umbilical vein from normal full-term newborns was obtained with informed consent and approved in accordance with the procedures of the Medical Ethics Committee of Shanghai East Hospital Affiliated to Tongji University (China).

Allicin improves the function of cardiac microvascular endothelial cells by increasing PECAM-1 in rats with cardiac hypertrophy

ObjectiveCardiac microvascular damage is significantly associated with the development of cardiac hypertrophy (CH). Researchers found that allicin could inhibit CH, but the relationship between cardiac microvessel and the inhibition of allicin on CH has not been reported. We aimed to investigate the effect of allicin on the function of cardiac microvascular endothelial cells (CMECs) in CH rat. Materials and MethodsThe hemodynamic parameters were measured by BL-420F biological function experimental system and the indicators of the ventricular structure and function were measured by echocardiographic system. MTT assay was performed to assess the cell viability. Nitrite detection was performed to detect nitric oxide content. The morphology and molecular characteristics were detected by electron micrographs, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), western blot. Wound healing experiment, analysis of tube formation and shear adaptation were performed to assess CMECs migration ability, angiogenesis and shear-responsiveness respectively. ResultOur findings have identified that microvascular density was decreased by observing the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) in CH rats. Interestingly, allicin improved the distribution and expression of PECAM-1. Meanwhile, allicin enhanced the migration and angiogenesis ability of CMECs, activated PECAM-1-PI3K-AKT-eNOS signaling pathway, however, the role of allicin was disappear after PECAM-1 was silenced. Allicin decreased the expression of caspase-3 and receptor interacting protein 3 (RIP3), inhibited necroptosis, and increased the levels of Angiopoietin-2 (Ang-2) and platelet-derived growth factor receptor-β (PDGFR-β). Under 10 dyn/cm2 condition, allicin advanced the modification ability of CMECs's shear-adaptation by activating PECAM-1. ConclusionAllicin provided cardioprotection for CH rats by improving the function of CMECs through increasing the expression of PECAM-1.

Glycosylation controls cooperative PECAM-VEGFR2-β3 integrin functions at the endothelial surface for tumor angiogenesis.

Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1-/- mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1-/- endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin β3 was disturbed in St6gal1-/- mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-β3, indicating that the reduction of cell surface integrin-β3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.

Research on influence mechanism of G protein coupled receptor kinase interacting protein 1 on differentiation of bone marrow mesenchymal stem cells into endothelial cells

To investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice. Male and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry. Western blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 ( P<0.05), and in group B1 than in groups A and B ( P<0.05); but no significant difference was found between groups A and B ( P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ ( P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ ( P>0.05). GIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.

AMP-activated protein kinase mediates the effects of lipoprotein-associated phospholipase A2 on endothelial dysfunction in atherosclerosis.

The present study aimed to investigate the effects of lipoprotein-associated phospholipase A2 (Lp-PLA2) on endothelial dysfunction in an in vitro cell model of atherosclerosis, and to determine whether AMP-activated protein kinase (AMPK) mediates the effects of Lp-PLA2 on endothelial dysfunction. A total of 392 patients with coronary artery disease (CAD), including various sub-conditions, were recruited, and the plasma concentrations of Lp-PLA2 were evaluated. In addition, an in vitro model of atherosclerosis was established by exposing human umbilical vein endothelial cells (HUVECs) to oxidized low-density lipoprotein (oxLDL). SB-435495 was used to inhibit Lp-PLA2, and compound C was used to suppress AMPK expression. Lp-PLA2, AMPKα and phosphorylated-AMPKα (T172) expression in HUVECs were evaluated using western blot analysis. The concentrations of nitric oxide (NO), endothelin 1 (ET-1), intercellular adhesion molecule 1 (ICAM-1) and platelet/endothelial cell adhesion molecule 1 (PECAM-1) in cell culture supernatant were determined using commercially available ELISA kits. MTT assays were employed to indicate changes in cell viability. The current study found the plasma Lp-PLA2 levels were elevated in the CAD patients with stable angina pectoris, unstable angina pectoris, acute coronary syndromes and acute myocardial infarction, compared with a healthy control population. In addition, the in vitro results showed that Lp-PLA2 expression levels were elevated in oxLDL-exposed HUVECs. Lp-PLA2 suppression could increase cell viability, induce the production of NO and decrease the secretion of ET-1, in addition to suppressing the expression of cell adhesion molecules, including ICAM-1 and PECAM-1 in oxLDL-exposed HUVECs. The expression of AMPKα and phosphorylated-AMPKα (T172) was regulated by Lp-PLA2, and AMPK suppression was able to reverse the effects of Lp-PLA2 with regard to cell viability, endothelial vasorelaxation capacity and the secretion of adhesion molecules in oxLDL-exposed HUVECs. In conclusion, the present study provides initial evidence that Lp-PLA2 is able to cause endothelial dysfunction in an in vitro model of atherosclerosis, and the effects of Lp-PLA2 on endothelial dysfunction was at least partially a result of the downregulation of AMPKα, thus contributing to the progression of atherosclerosis.

The Short Term Response of PECAM-1 to Mechanical Loading: Cyclooxygenase-2 and Nitric Oxide Production with AFM Pulling and Shear Stress

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a transmembrane protein located at the intercellular junction of endothelial cells (EC). PECAM-1 in adjacent EC bind to each other through homophilic interactions and regulate endothelial function. Recent studies have identified the role of PECAM-1 in mechanotransduction, sensing luminal shear stress and coordinating the production of both prostacyclin/prostaglandin I2 (PGI2) and nitric oxide. While most of these studies use inhibition methods, such as PECAM-1 knockdown cells or knockout animals, our group has implemented novel methods to selectively activate PECAM-1 on the EC surface for 10-30 minutes. Using AFM pulling with PECAM-1 antibody coated cantilevers on rat fat pad endothelial cell (RFPEC) monolayers, we have found significant activation of cyclooxygenase-2 (COX-2), the mediating enzyme of prostacyclin (PGI2) production. Conversely, AFM pulling with PECAM-1 coated probes did not induce any significant change in NO production. These data suggest that fluid shear stress acts directly on PECAM-1 to induce PGI2 production but indirectly to induce NO production. We have shown that pulling with probes targeting the heparan sulfate proteoglycan glypican-1 initiates NO production, suggesting that glypican-1 is upstream of PECAM-1 in the mechanotransduction cascade that produces NO. We are currently investigating the potential interaction between glypican-1 and PECAM-1 in shear-induced NO production. Heparin and surfen were used to inhibit extracellular heparan sulfate-to-PECAM-1 binding and shear-induced NO production was studied at both 10 and 30 minutes. The inhibition of PECAM-1 expression and intermediate intracellular signaling pathways may further illuminate the role of PECAM-1 in glycocalyx-dependent shear-induced NO production. This work aims to identify critical mechanosensing structures on the surface of endothelial cells that mediate the production of vasoregulatory and anti-atherogenic substances for the treatment of vascular disease.

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1, PI3K, FAK, and p38.

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2 Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5β1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5β1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1-/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals.NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).

Increased Cell Adhesion Molecules, PECAM-1, ICAM-3, or VCAM-1, Predict Increased Risk for Flare in Patients With Quiescent Inflammatory Bowel Disease.

Predicting the risk of flare-ups for patients with inflammatory bowel disease (IBD) is difficult. Alterations in gut endothelial regulation of mucosal immune homeostasis might be early events leading to flares in IBD. Cell adhesion molecules (CAMs), in particular, are important in maintaining endothelial integrity and regulating the migration of leukocytes into the gut. We evaluated the mRNA expression of various tight junction proteins, with an emphasis on CAMs, in 40 patients with IBD in clinical remission. Patients were retrospectively assessed at 6, 12, and 24 months after baseline colonoscopy, and at the end of all available follow-up (maximum 65 mo), for flare events to determine whether baseline mRNA expression was associated with subsequent flares. At all follow-up points, the baseline expression of platelet endothelial cell adhesion molecule-1 (PECAM-1), ICAM-3, and VCAM-1 was significantly higher in patients who flared than in those who did not (2.4-fold elevation, P=0.012 for PECAM-1; 1.9-fold increased, P=0.03 for ICAM-3; and 1.4-fold increased, P=0.02 for VCAM-1). PECAM-1 and ICAM-3 expression was significantly increased in patients who flared as early as 6 months after baseline colonoscopy. In contrast, there were no significant differences between patients with and without flares in baseline expression of other CAMs (ESAM, ICAM-1, ICAM-2, E-selectin, P-selectin, and MadCAM1). Increased expression of PECAM-1, ICAM-3, and VCAM-1 in colonic biopsies from patients with IBD in clinical remission is associated with subsequent flares. This suggests that increases in the expression of these proteins may be early events that lead to flares in patients with IBD.

Expression of pro-angiogenic genes in mesenchymal stem cells derived from dermis of patients with psoriasis.

Recent experimental studies revealed that angiogenesis and lymphangiogenesis are closely related to psoriasis. Our microarray analysis suggested that the pro-angiogenic genes platelet endothelial cell adhesion molecule-1 (PECAM1), facio-genital dysplasia-5 (FGD5), prostaglandin-endoperoxide synthase-1 (PTGS1), melanoma cell adhesion molecule (MCAM), vasohibin-2 (VASH2), and stabilin-1 (STAB1) are differentially expressed in dermal mesenchymal stem cells in psoriasis. The aim of this study was to investigate the mRNA and protein expression of PECAM1, FGD5, PTGS1, MCAM, VASH2, and STAB1 for angiogenesis and the possible mechanisms in psoriasis. We studied 12 patients with plaque psoriasis and 14 healthy controls matched for age and sex. Dermal mesenchymal stem cells were expanded, passaged, and identified by cellular morphology, immunophenotyping, and multipotential differentiation. The mRNA and protein expression of the above-mentioned six genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. The significantly decreased expression of PECAM1, PTGS1, FGD5, and MCAM at both mRNA and protein level (except VASH2 and STAB1) were demonstrated in mesenchymal stem cells from psoriatic skin lesions compared with non-lesional from healthy controls. We provide the first report that pro-angiogenic genes PECAM1, PTGS1, FGD5, and MCAM rather than VASH2 and STAB1 may be play a vital role in pathological dermal angiogenesis disorders of psoriasis. Therefore, anti-angiogenesis is attractive and offers future potential for application in patients with psoriasis.