Utility of Platelet Endothelial Cell Adhesion Molecule 1 in the Platelet Activity Assessment in Mouse and Human Blood.

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.

CD31 induces inflammatory response by promoting hepatic inflammatory response and cell apoptosis.

OBJECTIVE: To investigate whether CD31 could regulate paracetamol-induced liver injury, thereby providing a new direction for the prevention and treatment of drug-induced hepatitis. MATERIALS AND METHODS: Wild-type (WT) mice were treated with acetaminophen (APAP) (250 mg/kg) or isodose of phosphate-buffered saline (PBS). 1, 3, 6 and 12 h after the treatment, the messenger RNA (mRNA) and protein expression level of CD31 in the liver of mice were determined by Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Once CD31 was confirmed to be involved in APAP-induced liver injury, the acute liver injury model in WT mice and CD31 gene deficient (CD31-/-) mice induced by APAP was established. Serum samples were collected at 8 and 24 h after APAP injection (250 mg/kg), and the activity of serum alanine aminotransferase (ALT) was measured. The liver tissues of mice were isolated and analyzed by hematoxylin and eosin (HE) staining. Meanwhile, mononuclear cells (MNCs) were isolated from the liver tissues of mice. The number of infiltrating macrophages and neutrophils was detected by flow cytometry, and the activation level of these cells was analyzed. The expression levels of proinflammatory cytokines in liver tissues, such as TNF-α, IL-1ß, keratinocyte chemoattractant (KC), MCP-1 and IL-6, were determined by RT-PCR. The expression levels of cytokines in serum were detected by enzyme-linked immunosorbent assay (ELISA). Moreover, the protein expression levels of JNK, Caspase-3, and cytochrome P450 2E1 (CYP2E1) in liver tissues were detected by Western blotting. RESULTS: After APAP treatment, we found that WT mice were more sensitive to APAP-induced liver injury. The level of ALT in WT mice was significantly higher than that of CD31-/- mice, meanwhile, more necrotic or apoptotic cells were found in WT mice. Results also indicated that the expression levels of inflammatory cytokines, including KC, IL-1ß, MCP-1 and IL-6, were significantly higher in WT mice. Meanwhile, the number of infiltrating macrophages and neutrophils in the liver tissues of WT mice were much more than that of CD31-/- mice. CONCLUSIONS: APAP-treated CD31-/- mice exhibited less liver injury when compared with WT mice. We also confirmed that CD31 was greatly involved in APAP-induced inflammatory response by promoting hepatic inflammatory and cell apoptosis, which might provide a new strategy for the prevention and treatment of drug-induced hepatitis.

Molecular mechanisms for enhancement of stromal cell-derived factor 1-induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1).

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1-deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1-regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1-induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1-induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1-induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1-induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1-induced internalization of CXCR4.

The critical role of uterine CD31 as a post-progesterone signal in early pregnancy.

CD31 has been shown to play a role in endothelial cell migration and angiogenesis, which are critical to the formation and function of the endometrium and myometrium in uterine development during early pregnancy. However, the role of CD31 in uterine receptivity during blastocyst implantation is poorly understood. The pregnancy rate in CD31-/- female mice mated with CD31+/+ male mice was higher than that observed in CD31+/+ female mice mated with CD31+/+ male mice. During the receptive phase of implantation, uterine glands were more developed in CD31-/- mice than in CD31+/+ mice, and the uterine weights of CD31-/- mice were increased. Leukemia inhibitory factor (LIF) was highly expressed in the CD31-/- mice during implantation and the expression of LIF was up-regulated by estradiol-17ß (E2 ) + progesterone (P4 ) in ovariectomized CD31-/- mice, compared with CD31+/+ mice at 8 h after hormone treatment. E2 -induced protein synthesis was inhibited by P4 in the CD31+/+ uterus, but not in the uterus of CD31-/- mice. Also, STAT3, HAND2, LIF, and mTOR signals were enhanced in CD31-/- mice. Stromal DNA replication was highly activated in the uterus of CD31-/- mice, manifested by upregulated cyclin series signaling and PCNA expression after E2 + P4 treatment. Collectively, CD31 inhibits E2 -mediated epithelial proliferation via recruitment and phosphorylation of SHP-2 upon receiving P4 signal in early pregnancy.

Fluid shear stress combined with shear stress spatial gradients regulates vascular endothelial morphology.

High shear stress (SS) causes local changes around arterial bifurcations, which are common sites for cerebral aneurysms. High SS and SS spatial gradient (SSG) are thought to play important roles in the pathology of cerebral aneurysms. However, whether SS and SSG independently affect the function and morphology of vascular endothelial cells (ECs) exposed to fluid flow remains unclear. This study evaluated the morphology of ECs exposed to various SS and SSG combinations. Confluent ECs were exposed to a SS of 2-60 Pa and a uniform SSG of 0, 5, 10, or 15 Pa mm-1 for 24 h. Although ECs exposed to lower levels of SS/SSG were not oriented or elongated in the direction of flow, they began to exhibit orientation, elongation, and development of actin stress fibers under the conditions of SS with a SSG when the SS exceeded a threshold value depending on the magnitude of SSG. Using a simplified computational model, we found that the presence of a SSG affects the strain field in ECs, resulting in a morphological response. SS combined with a SSG can alter the localization of SS mechano-sensing proteins along the strain field as a result of shear flow. Our results suggest that the magnitude of the relationship between SS and SSG plays an important role in regulating morphological changes in ECs in response to fluid flow by regulating EC polarity.

Proliferation, differentiation and migration of SCA1-/CD31- cardiac side population cells in vitro and in vivo.

BACKGROUND: Side-population (SP) cells, identified by their capacity to efflux Hoechst dye, are highly enriched for stem/progenitor cell activity. They are found in many mammalian tissues, including mouse heart. Studies suggest that cardiac SP (CSP) cells can be divided into SCA1+/CD31-, SCA1+/CD31+ and SCA1-/CD31- CSP subpopulations. SCA1+/CD31- were shown to be cardiac and endothelial stem/progenitors while SCA1+/CD31+ CSP cells are endothelial progenitors. SCA1-/CD31- CSP cells remain to be fully characterized. In this study, we characterized SCA1-/CD31- CSP cells in the adult mouse heart, and investigated their abilities to proliferate, differentiate and migrate in vitro and in vivo. METHODS AND RESULTS: Using fluorescence-activated cell sorting, reverse transcriptase/polymerase chain reaction, assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction we show that SCA1-/CD31- CSP cells are located in the heart mesenchyme and express genes characteristic of stem cells and endothelial progenitors. These cells were capable of proliferation, differentiation, migration and vascularization in vitro and in vivo. Following experimental myocardial infarction, the SCA1-/CD31- CSP cells migrated from non-infarcted areas to the infarcted region within the myocardium where they differentiated into endothelial cells forming vascular (tube-like) structures. We further demonstrated that the SDF-1α/CXCR4 pathway may play an important role in migration of these cells after myocardial infarction. CONCLUSIONS: Based on their gene expression profile, localization and ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we conclude that SCA1-/CD31- CSP cells may serve as endothelial progenitor cells in the adult mouse heart.

Targeted Recycling of the Lateral Border Recycling Compartment Precedes Adherens Junction Dissociation during Transendothelial Migration.

Leukocyte transendothelial migration (TEM) requires two major events: local dissociation of adherens junctions manifested as gaps in vascular endothelial (VE)-cadherin staining at the site of TEM and targeted trafficking of the lateral border recycling compartment (LBRC) to the site of TEM. However, the association between LBRC recycling and VE-cadherin gaps remains unknown. We found that when targeting of the LBRC is selectively inhibited using established methods, such as a function blocking anti-platelet endothelial cell adhesion molecule 1 antibody, depolymerizing microtubules, or microinjection of an antibody that inhibits kinesin, VE-cadherin gaps do not form around the blocked leukocyte. This is the first time that the LBRC has been implicated in this process. We obtained similar results for neutrophils and monocytes and in studies using live cell imaging microscopy conducted under fluid shear conditions. Depolymerizing microtubules did not affect the ability of leukocytes to induce tyrosine phosphorylation of VE-cadherin. A VE-cadherin double mutant (Y658F, Y731F) expressed in endothelial cells acted as a dominant negative and inhibited VE-cadherin gap formation and TEM, yet targeting of the LBRC still occurred. These data suggest that targeting of the LBRC to the site of TEM precedes VE-cadherin clearance. Recruitment of the LBRC may play a role in clearing VE-cadherin from the site of TEM.

TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response.

Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca(2+)]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca(2+)]i regulates TEM and the channels mediating this ↑[Ca(2+)]i are unknown. Buffering ↑[Ca(2+)]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca(2+)]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca(2+) channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca(2+)]i required for TEM at a step downstream of PECAM homophilic interactions.

Altered CD31 expression and activity in helper T cells of acute coronary syndrome patients.

In acute coronary syndrome (ACS), T cell abnormalities are associated to a worse outcome. Loss of inhibitory activity of CD31, an Ig-like adhesion molecule, on peripheral leukocytes has been found to enhance atherosclerosis in experimental models. In this study, we examined the expression of CD31 on T cells, and its role on TCR signaling in 35 patients with non-ST elevation ACS, in 35 patients with stable angina (SA), and in 35 controls. Furthermore, 10 ACS and 10 SA patients were re-analyzed at 1-year follow-up. Flow-cytometry analysis showed that in ACS patients, CD31 expression was reduced on total CD4(+) and CD4(+)CD28(null) (P < 0.001, ACS vs. SA), on naïve (P < 0.001, ACS vs. SA) and on central-memory and effector-memory CD4(+) T cells (P < 0.05, ACS vs. SA and controls). The immunomodulatory effect of CD31 on TCR signaling of CD4(+) and CD4(+)CD28(null) T cells, was lower in ACS than SA patients (P < 0.05, for both comparisons). At 1-year follow-up, CD31 expression and function increased in ACS becoming similar to that found in SA. CD31 recruitment in the immunological synapse was lower in ACS than controls (P = 0.012). Moreover, CD31 modulated MAPK signaling and reduced the expression of T bet and Rorγ-t, necessary for Th1 and Th17 differentiation. Finally, we studied TCR signaling in CD31(+) naïve and primed T cell subsets observing a different pattern of protein phosphorylation. A CD31-mediated regulatory pathway is enhanced in SA and temporarily downregulated in ACS. As CD31 modulates both T cell activation, by increasing the threshold for TCR stimulation, and T cell differentiation, it might represent a novel molecular target to treat T cell abnormalities in ACS.

Cultured human bone marrow-derived CD31(+) cells are effective for cardiac and vascular repair through enhanced angiogenic, adhesion, and anti-inflammatory effects.

BACKGROUND: Cell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31(+) cells are a promising cell source owing to their high angiovasculogenic and paracrine activities. OBJECTIVES: This study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31(+) cells, and to determine whether these cultured CD31(+) cells are effective for cardiac and vascular repair. METHODS: CD31(+) cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms. RESULTS: The CD31(+) cells cultured in endothelial cell medium (EC-CD31(+) cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31(+) cells. When implanted into mouse MI or HLI models, EC-CD31(+) cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31(+) cells. Histologically, injected EC-CD31(+) cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year. CONCLUSIONS: Short-term cultured EC-CD31(+) cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31(+) cells may be a promising source for treating ischemic cardiovascular diseases.

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbß3.

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbß3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of ß3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3ß Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbß3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

Vascular targeting of nanocarriers: perplexing aspects of the seemingly straightforward paradigm.

Targeted nanomedicine holds promise to find clinical use in many medical areas. Endothelial cells that line the luminal surface of blood vessels represent a key target for treatment of inflammation, ischemia, thrombosis, stroke, and other neurological, cardiovascular, pulmonary, and oncological conditions. In other cases, the endothelium is a barrier for tissue penetration or a victim of adverse effects. Several endothelial surface markers including peptidases (e.g., ACE, APP, and APN) and adhesion molecules (e.g., ICAM-1 and PECAM) have been identified as key targets. Binding of nanocarriers to these molecules enables drug targeting and subsequent penetration into or across the endothelium, offering therapeutic effects that are unattainable by their nontargeted counterparts. We analyze diverse aspects of endothelial nanomedicine including (i) circulation and targeting of carriers with diverse geometries, (ii) multivalent interactions of carrier with endothelium, (iii) anchoring to multiple determinants, (iv) accessibility of binding sites and cellular response to their engagement, (v) role of cell phenotype and microenvironment in targeting, (vi) optimization of targeting by lowering carrier avidity, (vii) endocytosis of multivalent carriers via molecules not implicated in internalization of their ligands, and (viii) modulation of cellular uptake and trafficking by selection of specific epitopes on the target determinant, carrier geometry, and hydrodynamic factors. Refinement of these aspects and improving our understanding of vascular biology and pathology is likely to enable the clinical translation of vascular endothelial targeting of nanocarriers.

The prognostic value of platelet endothelial cell adhesion molecule-1 in non-small-cell lung cancer patients.

Our previous studies have shown that platelet endothelial cell adhesion molecule-1 (PECAM-1), a member of the immunoglobulin superfamily, is a critical mediator of anchorage-independent growth and anoikis resistance in lung carcinoma cells. The purpose of this study was to analyze the protein expression of PECAM-1 in non-small-cell lung carcinoma (NSCLC) tissues and its clinical significance in NSCLC patients. By immunohistochemical analysis, high microvessel density (MVD) of PECAM-1 was detected in the stromal tissues of NSCLC. The MVD of PECAM-1 was strongly correlated with the N stage (p = 0.029), M stage (p = 0.001) and clinical stage (p = 0.001) of NSCLC patients. Survival analysis revealed high MVD of PECAM-1 in both primary NSCLC lesions and metastatic lymph node tissues, and these results were found to be significantly correlated with poor overall survival in NSCLC patients (p < 0.001 and p = 0.021, respectively). Moreover, patients with high PECAM-1 MVD had worse overall survival in either adenocarcinoma or EGFR mutation subgroups. Multivariate analysis revealed that the MVD of PECAM-1 was an independent prognostic factor for NSCLC patients. The MVD of PECAM-1 is also a potential predictor for NSCLC patients treated with first-line platinum-based doublet chemotherapy, as high PECAM-1 MVD correlated with worse overall survival. Our results demonstrated that MVD of PECAM-1 could be a potential prognostic factor and therapeutic target in NSCLC.

RhoA goes GLOBAL.

Mechanical forces influence nearly all aspects of biology. Cells are equipped with numerous mechanosensitive proteins that activate various signaling cascades in response to mechanical cues from their environment. Much interest lies in understanding how cells respond to external stresses. A number of studies have highlighted the coordination of mechanical and chemical signaling cascades downstream of integrins. In recent years, the study of mechanotransduction has expanded to other mechanosensitive adhesion receptors, such as platelet endothelial cell adhesion molecule-1 (PECAM-1). This commentary will highlight our current understanding of integrin and PECAM-1-mediated mechanotransduction and expand on the observation that a localized mechanical stress can elicit a global mechanosignaling response.

Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway.

BACKGROUND: Mechanical forces regulate cell behavior and function during development, differentiation, and tissue morphogenesis. In the vascular system, forces produced by blood flow are critical determinants not only of morphogenesis and function, but also of pathological states such as atherosclerosis. Endothelial cells (ECs) have numerous mechanotransducers, including platelet endothelial cell adhesion molecule-1 (PECAM-1) at cell-cell junctions and integrins at cell-matrix adhesions. However, the processes by which forces are transduced to biochemical signals and subsequently translated into downstream effects are poorly understood. RESULTS: Here, we examine mechanochemical signaling in response to direct force application on PECAM-1. We demonstrate that localized tensional forces on PECAM-1 result in, surprisingly, global signaling responses. Specifically, force-dependent activation of phosphatidylinositol 3-kinase (PI3K) downstream of PECAM-1 promotes cell-wide activation of integrins and the small GTPase RhoA. These signaling events facilitate changes in cytoskeletal architecture, including growth of focal adhesions and adaptive cytoskeletal stiffening. CONCLUSIONS: Taken together, our work provides the first evidence of a global signaling event in response to a localized mechanical stress. In addition, these data provide a possible mechanism for the differential stiffness of vessels exposed to distinct hemodynamic force patterns in vivo.

CD31 exhibits multiple roles in regulating T lymphocyte trafficking in vivo.

The role of CD31, an Ig-like molecule expressed by leukocytes and endothelial cells (ECs), in the regulation of T lymphocyte trafficking remains contentious. Using CD31-deficient mice, we show that CD31 regulates both constitutive and inflammation-induced T cell migration in vivo. Specifically, T cell:EC interactions mediated by CD31 molecules are required for efficient localization of naive T lymphocytes to secondary lymphoid tissue and constitutive recirculation of primed T cells to nonlymphoid tissues. In inflammatory conditions, T cell:EC CD31-mediated interactions facilitate T cell recruitment to Ag-rich sites. However, endothelial CD31 also provides a gate-keeping mechanism to limit the rate of Ag-driven T cell extravasation. This event contributes to the formation of Ag-specific effector T cell infiltrates and is induced by recognition of Ag on the endothelium. In this context, CD31 engagement is required for restoring endothelial continuity, which is temporarily lost upon MHC molecule ligation by migrating cognate T cells. We propose that integrated adhesive and signaling functions of CD31 molecules exert a complex regulation of T cell trafficking, a process that is differentially adapted depending on cell-specific expression, the presence of inflammatory conditions and the molecular mechanism facilitating T cell extravasation.

CD31 angiogenesis and combined expression of HIF-1α and HIF-2α are prognostic in primary clear-cell renal cell carcinoma (CC-RCC), but HIFα transcriptional products are not: implications for antiangiogenic trials and HIFα biomarker studies in primary CC-RCC.

Hypoxia-inducible factors, HIF-1α and HIF-2α, are expressed in the majority of clear-cell renal cell carcinoma (CC-RCC). In vitro, HIFα isoforms regulate a differential set of genes, and their effects in vivo within CC-RCC tumours may affect outcome. The role of angiogenesis and HIFα transcriptional products, including those involved in cell metabolism and morphological dedifferentiation have not been extensively investigated and might have relevance to the development of antiangiogenic or anti-HIFα trials in primary CC-RCC, either before or after radical nephrectomy. We analysed 168 consecutive clear-cell renal tumours from 1983 to 1999 within tissue microarrays and assessed expression of HIF-1α and HIF-2α together with the protein expression of seven of their target genes (BNIP3, CA9, Cyclin D1, GLUT-1, LDH5, Oct-4 and VEGF). The expression of these factors was compared with patient overall survival and CD31 angiogenesis. We found that HIFα antigenicity deteriorated with the age of the paraffin block (P < 0.0001) and in tumours from 1983 to 1992 was deemed not to be reliable. Similar findings were found in aged archival osteosarcoma samples. This might have important implications for retrospective biomarker studies that rely on archival tissue material. HIF-1α(HIGH)/HIF-2α(LOW) tumours had a worse overall survival compared with HIF-1α(LOW)/HIF-2α(LOW) tumours (P = 0.04). Surprisingly, on multivariate analysis, high levels of CD31(+) angiogenesis was shown to be an independent prognostic marker of increased overall survival (P = 0.003). We propose that better differentiation of vascular endothelium may be a reflection of a greater production of vessel stabilization factors versus pro-angiogenic factors, and therefore a less aggressive phenotype.

Crosstalk between reticular adherens junctions and platelet endothelial cell adhesion molecule-1 regulates endothelial barrier function.

OBJECTIVE: Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. METHODS AND RESULTS: In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins ß-catenin and p120-catenin. ß-catenin binds to α-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-α increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-α. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-α-induced alterations of barrier function, including increased ß-catenin phosphorylation, without altering the actomyosin cytoskeleton. CONCLUSIONS: We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-α.

Homeward bound: how do skin dendritic cells find their way into the lymph system?

The success of the adaptive immune system relies upon the transport of antigens by dendritic cells (DCs) from skin via lymphatic vessels to lymph nodes where DCs present antigen to T cells. Little is known about the requirements for so-called "reverse transmigration" (RT). Torzicky et al. in this issue demonstrate that CD31, CD99, and CXCR4 play key roles in RT, suggesting that adhesion occurs in a defined sequence during the passage of DC into lymphatics.