Platelet Endothelial Cell Adhesion Research Articles

PECAM-1-Dependent Regulation of Heme Oxygenase-1 via the Antioxidant Nrf2 Pathway in the Endothelium

Heme oxygenase-1 (HO-1) is the inducible isoform of the first and rate-limiting enzyme of heme degradation and has major anti-inflammatory and immunomodulatory functions. Overexpression of HO-1 in endothelial cells (EC) provides major protection against graft rejection in experimental animal transplantation models. Platelet EC adhesion molecule (PECAM)-1 is a multifunctional cell adhesion receptor that belongs to the immunoglobulin-like (Ig) superfamily and is highly expressed on EC. In addition to its role in modulating leukocyte transmigration through the endothelial monolayer, PECAM-1 has more recently been shown to be a major regulator of intracellular signaling. To investigate whether PECAM-1 might be involved in endothelial regulation of HO-1, we performed a knockdown in human umbilical vein EC (HUVEC) with an adenoviral-based vector approach encoding specific PECAM-1 shRNA sequences. Knockdown of PECAM-1 caused a marked up-regulation of HO-1 expression, but not that of two other inducible genes, Bcl-2 and cyclooxygenase-2, as determined by real time PCR and immunofluorescence studies. PECAM-1 deficiency in HUVEC led to increased intracellular levels of reactive oxygen species (ROS) and to nuclear translocation of the transcription factor Nrf2, which is a key regulator of the cellular antioxidant response. In addition, overexpression of PECAM-1 in two stably transfected cell lines (Chinese hamster ovary cells and L-cells) led to a marked down-regulation of HO-1 gene expression. Similarly, reporter gene activity of a HO-1 promoter gene construct with a specific Nrf2 target sequence was down-regulated in PECAM-1 overexpressing cells. In conclusion, these findings show a regulatory link of the signaling receptor PECAM-1 to HO-1 gene expression in the endothelium and may afford novel therapeutic approaches in organ transplantation.

PECAM-1-Dependent Regulation of Heme Oxygenase-1 via the Antioxidant Nrf2 Pathway in the Endothelium

Heme oxygenase-1 (HO-1) is the inducible isoform of the first and rate-limiting enzyme of heme degradation and has major anti-inflammatory and immunomodulatory functions. Overexpression of HO-1 in endothelial cells (EC) provides major protection against graft rejection in experimental animal transplantation models. Platelet EC adhesion molecule (PECAM)-1 is a multifunctional cell adhesion receptor that belongs to the immunoglobulin-like (Ig) superfamily and is highly expressed on EC. In addition to its role in modulating leukocyte transmigration through the endothelial monolayer, PECAM-1 has more recently been shown to be a major regulator of intracellular signaling. To investigate whether PECAM-1 might be involved in endothelial regulation of HO-1, we performed a knockdown in human umbilical vein EC (HUVEC) with an adenoviral-based vector approach encoding specific PECAM-1 shRNA sequences. Knockdown of PECAM-1 caused a marked up-regulation of HO-1 expression, but not that of two other inducible genes, Bcl-2 and cyclooxygenase-2, as determined by real time PCR and immunofluorescence studies. PECAM-1 deficiency in HUVEC led to increased intracellular levels of reactive oxygen species (ROS) and to nuclear translocation of the transcription factor Nrf2, which is a key regulator of the cellular antioxidant response. In addition, overexpression of PECAM-1 in two stably transfected cell lines (Chinese hamster ovary cells and L-cells) led to a marked down-regulation of HO-1 gene expression. Similarly, reporter gene activity of a HO-1 promoter gene construct with a specific Nrf2 target sequence was down-regulated in PECAM-1 overexpressing cells. In conclusion, these findings show a regulatory link of the signaling receptor PECAM-1 to HO-1 gene expression in the endothelium and may afford novel therapeutic approaches in organ transplantation.

Angiotensin II induces microtubule reorganization mediated by a deacetylase SIRT2 in endothelial cells

Angiotensin II has been implicated in vascular remodeling. Microtubule composed of tubulins regulates cell shape, migration and survival. Tubulin acetylation has an important role in the control of microtubule structure and microtubule-based cellular functions. In this study, angiotensin II induced disassembly and deacetylation of α-tubulin, which were blocked by pretreatment with an angiotensin II type 1 receptor blocker losartan and a sirtuin class deacetylase inhibitor sirtinol, and by depletion of a deacetylase SIRT2 using RNA interference. We investigated the involvement of SIRT2 in angiotensin II-induced endothelial cell migration using the Boyden chamber method. Angiotensin II caused a significant increase in cell migration, which was blocked by pretreatment with sirtinol and SIRT2 depletion. It has been reported that angiotensin II is involved in cytoskeletal reorganization stimulated by mechanical stretch in endothelial cells. We also demonstrated that endothelial cells subjected to a 10% uniaxial stretch showed vertical alignment to the direction of tension and tubulin deacetylation in the peripheral side of cells, in comparison with control static cells. The mechanical stretch-induced changes of microtubules were blocked by pretreatment with sirtinol and SIRT2 depletion. Immunofluorescence microscopy showed that acetylated tubulin was decreased in platelet-endothelial cell adhesion molecule-1-positive cells in the intima of the aortic walls in mice loaded with angiotensin II, in comparison with mice loaded with control vehicle. These data show that angiotensin II and mechanical stretch stimulate microtubule redistribution and deacetylation via SIRT2 in endothelial cells, suggesting the emerging role of SIRT2 in hypertension-induced vascular remodeling.

Influence of Polyclonal Antithymocyte Globulins on the Expression of Adhesion Molecules of Isolated Human Umbilical Vein Endothelial Cells

Objectives Polyclonal antithymocyte globulins (ATGs) are immunosuppressive agents applied for the treatment and prevention of organ rejection after transplantation. ATGs induce complement-mediated cell death in T lymphocytes and decrease leukocyte adhesion. However, little is known about the effects of ATGs on endothelial cells (EC). Our aim was to study the influence of ATGs upon the expression of adhesion molecules on human umbilical vein endothelial cells (HUVECs) after stimulation with tumor necrosis factor (TNF)-α. Material and Methods HUVECs obtained from umbilical cords were incubated with ATGs before and after 6-hour stimulation with TNF-α. The group incubated without ATG served as the controls. Another group was not stimulated with TNF-α. By flow cytometry, we analyzed the expression of several adhesion molecules: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM), platelet EC adhesion molecule (PECAM), and CD62E. Statistical analysis used analysis of variance. Results After TNF-α stimulation, the EC surface expression of ICAM-1 and CD62E was reduced, although not significantly, in treated as compared with untreated cells. The expression of ICAM-1 and CD62E was similar in the unstimulated groups. The expression of VCAM, PECAM, CD55, and CD58 was not modified by ATG treatment. Conclusion Our results demonstrated that ATGs insignificantly reduced the expression of adhesion molecules in HUVECs. The effect of ATGs on stimulated HUVECs remains unclear, probably due to the lack of effector cells.

Microvascular Responses to Cardiovascular Risk Factors

Hypertension, hypercholesterolemia, diabetes, and obesity are among a growing list of conditions that have been designated as major risk factors for cardiovascular disease (CVD). While CVD risk factors are well known to enhance the development of atherosclerotic lesions in large arteries, there is also evidence that the structure and function of microscopic blood vessels can be profoundly altered by these conditions. The diverse responses of the microvasculature to CVD risk factors include oxidative stress, enhanced leukocyte- and platelet-endothelial cell adhesion, impaired endothelial barrier function, altered capillary proliferation, enhanced thrombosis, and vasomotor dysfunction. Emerging evidence indicates that a low-grade systemic inflammatory response that results from risk factor-induced cell activation and cell-cell interactions may underlie the phenotypic changes induced by risk factor exposure. A consequence of the altered microvascular phenotype and systemic inflammatory response is an enhanced vulnerability of tissues to the deleterious effects of secondary oxidative and inflammatory stresses, such as ischemia and reperfusion. Future efforts to develop therapies that prevent the harmful effects of risk factor-induced inflammation should focus on the microcirculation.

Endothelial Cell Retention on a Viscoelastic Nanocomposite Vascular Conduit Is Improved by Exposure to Shear Stress Preconditioning Prior to Physiological Flow

In this study, endothelial cell (EC)-seeded nanocomposite grafts were preconditioned with 1-2 dynes/cm(2) in vitro to establish whether low shear stress resulted in improved cell adherence prior to physiological shear stress (15 dynes/cm(2)). Alamar blue cell viability was assessed. Polymerase chain reaction was conducted for glyceraldehyde-3-phosphate dehydrogenase, transforming growth factor beta-1 (TGFbeta-1), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet EC adhesion molecule-1, and vascular endothelial growth factor receptor-2 (VEGFR-2). The Alamar blue results demonstrated improved cellular retention following preconditioning (P < 0.001). VEGFR-2 and TGFbeta-1 expression was up-regulated, and VEGFR-1 down-regulated following preconditioning. This investigation confirms previous findings regarding the potential benefits of preconditioning, and demonstrates that these benefits can be applied to ECs seeded on the nanocomposite employed. It also demonstrates further the suitability and potential of nanocomposite for future use in tissue-engineered cardiovascular devices.

Mechanotransduction in an extracted cell model: Fyn drives stretch- and flow-elicited PECAM-1 phosphorylation

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1–based mechanosensory complex in ECs.

ORIGINAL ARTICLE: Trophoblasts and Shear Stress Induce an Asymmetric Distribution of ICAM‐1 in Uterine Endothelial Cells

Problem We have used an in vitro co‐culture system consisting of early gestation macaque trophoblasts cultured on top of human uterine microvascular endothelial cells (UtMVECs) to investigate the inflammatory response of endothelial cells to trophoblasts under shear stress conditions.Method of study Uterine microvascular endothelial cells and trophoblasts were co‐cultured in a parallel plate chamber under shear stress (15 dyn/cm2) conditions. The distribution and expression of endothelial intercellular adhesion molecule‐1 (ICAM‐1) was quantified by immunofluorescence image analysis and flow cytometry. Endothelial regulated upon activation normal T‐cell expressed and secreted (RANTES) secretion was measured by enzyme‐linked immunosorbent assay and permeability was assessed using fluorescein isothiocyanate‐dextran.Results Intercellular adhesion molecule‐1, but not vascular cell adhesion molecule‐1 or platelet endothelial cell adhesion molecule‐1, was re‐distributed towards the downstream edge of endothelial cells when the cells were co‐cultured with trophoblasts under shear stress conditions. Changes in ICAM‐1 distribution were also observed when UtMVECs were co‐cultured with trophoblast‐conditioned medium under shear stress conditions. Incubation of UtMVECs with trophoblast‐conditioned medium increased endothelial permeability, RANTES secretion, and trophoblast adhesion.Conclusion These data support the idea that trophoblasts induce an inflammatory response in uterine endothelial cells that could enhance trophoblast invasion and transmigration.

Characterization of Porcine Circulating Progenitor Cells: Toward a Functional Endothelium

The lack of available healthy vessels, significant patient morbidity, and high costs hinders the successful clinical utilization of autologous endothelial cells (ECs). Herein we assess the feasibility of using endothelial progenitor cells (EPC) found in circulating blood to engineer a functional endothelium on poly(1,8-octanediol-co-citrate) (POC), a hemocompatible and biodegradable elastomer used in vascular tissue engineering. EPCs were isolated from porcine blood and biochemically differentiated into porcine endothelial (PE)-like cells in vitro. Once differentiated, EC phenotype and function on POC were assessed according to the presence of the EC-specific markers von Willebrand factor, platelet EC adhesion molecule, and vascular endothelial cadherin; metabolism of acetylated low-density lipoprotein; secretion of the anti-thrombogenic factors nitric oxide and prostacyclin; and inhibition of platelet adhesion and clotting processes in vitro. The effects of PE-like cells on porcine aortic smooth muscle cells (PASMCs) were also investigated via co-culture. PE-like cells on POC had phenotype, function, and clotting responses similar to those of primary aortic ECs. The presence of PE-like cells resulted in a 71 +/- 20% decrease in PASMC proliferation; a 52 +/- 2% decrease in the protein:deoxyribonucleic acid ratio; and an elongated, spindle-shaped morphology indicative of a shift from the proliferative to the contractile phenotype. These data suggest that EPCs and POC can provide the basis for a functional tissue-engineered endothelium.

Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury.

The forkhead box (Fox) proteins are a growing family of transcription factors that have important roles in cellular proliferation and differentiation and in organ morphogenesis. The Fox family members hepatocyte nuclear factor (HNF)-3beta (Foxa2) and HNF-3/forkhead homolog (HFH)-8 (FREAC-1, Foxf1) are expressed in adult pulmonary epithelial and mesenchymal cells, respectively, but these cells display only low expression levels of the proliferation-specific HFH-11B gene (Trident, Foxm1b). The regulation of these Fox transcription factors in response to acute lung injury, however, has yet to be determined. We report here on the use of butylated hydroxytoluene (BHT)-mediated lung injury to demonstrate that HFH-11 protein and RNA levels were markedly increased throughout the period of lung repair. The maximum levels of HFH-11 were observed by day 2 following BHT injury when both bronchiolar and alveolar epithelial cells were undergoing extensive proliferation. Although BHT lung injury did not alter epithelial cell expression of HNF-3beta, a 65% reduction in HFH-8 mRNA levels was observed during the period of mesenchymal cell proliferation. HFH-8-expressing cells were colocalized with platelet endothelial cell adhesion molecule-1-positive alveolar endothelial cells and with alpha-smooth muscle actin-positive peribronchiolar smooth muscle cells.

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Modulation of PECAM-1 (CD31) Expression in Human Endothelial Cells: Effect of IFNγ and IL-10

Intercellular contacts formed between endothelial cells (EC) permit the formation of a confluent monolayer and play a major role in the recruitment and the migration of leukocytes in the inflammatory response. It is currently accepted that cytokines are responsible for the signals involved in the induction of such endothelial alterations. The platelet EC adhesion molecule (PECAM-1), a specific component of EC junctions, is one of many molecules which participate in the regulation of EC interaction with blood components. Given that the regulatory mechanisms which affect the expression of this adhesion molecule are only partially understood, the aim of the present study was to compare the effects of two antagonistic inflammatory cytokines, interferon (IFN)-γ and interleukin (IL)-10, on the expression of PECAM-1. Human umbilical vein EC grown to subconfluence were stimulated with IL-10 (10 ng/ml) and/or IFNγ (100 U/ml) for 24 h. PECAM-1 expression was determined by FACScan and immunofluorescence. Morphological analysis of the cell cultures was performed by optical and scanning electron microscopy. In the presence of IL-10, no changes in cell growth and morphology or in the intensity of PECAM-1 expression were observed. However, when the cultures were treated with IFNγ, the EC acquired a fibroblast-like appearance, growth was disorganized and PECAM-1 disappeared from cell junctions. The mean intensity expression and the percentage of EC expression of this antigen were analyzed by flow cytometry and significantly decreased after culture in the presence of IFNγ. The simultaneous addition of IFNγ and IL-10 to the EC cultures induced modifications similar to those observed in the presence of IFNγ alone. Regulation of the expression of PECAM-1 with the subsequent functional implications seems to be dependent upon the IFNγ signal and it is unaffected by IL-10. The different effects shown by IL-10 and IFNγ on the expression of PECAM-1 in EC could reflect opposite regulatory actions of the inflammatory response induced by these cytokines.