Human papillomavirus-16-E7 oncoprotein enhances the expression of adhesion molecules in cervical endothelial cells but not in human umbilical vein endothelial cells.

OBJECTIVES: E7 is one of the oncoproteins encoded by human papillomavirus-16 (HPV-16), the major etiologic factor responsible for cervical cancer. Human papillomavirus-16-E7 expressed by human uterine cervix carcinoma cells is also released in the extracellular compartment where it induces immune suppression. We investigated whether E7 was also responsible for the enhanced endothelial adhesiveness required in cancer progression. STUDY DESIGN/METHODS: We treated cervical microvascular endothelial cells (CrMVEn) and human umbilical vein endothelial cells (HUVEC) with E7, tumor necrosis factor-alpha (TNF-alpha), and hydrogen peroxide (H2O2) and measured the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by fluorescent-activated cell sorter analysis. RESULTS: E7 strongly induced the expression of E-selectin, ICAM-1, and VCAM-1 in CrMVEn, but not in HUVEC. Tumor necrosis factor-alpha further increased the endothelial expression of adhesion molecules in CrMVEn. Hydrogen peroxide pre-treatment resulted in an enhanced ICAM-1 and a decreased E-selectin and VCAM-1 expression. We also show indirect effects when endothelial cells were stimulated with the supernatant of E7-pretreated macrophages. CONCLUSIONS: These results show that HPV-16-E7 oncoprotein strongly induces adhesion molecules expression in organ-specific endothelial cells.     

Endothelial and smooth muscle cells express leukocyte adhesion molecules heterogeneously during acute rejection of rabbit cardiac allografts.

Interactions of leukocytes with vascular wall cells figure prominently in acute rejection and development of vascular occlusive disease after cardiac transplantation. To investigate the time course and distribution among different types of vessels of expression of endothelial leukocyte adhesion molecules, issues difficult to address in humans, we studied heterotopic transplants of Dutch-Belted rabbit hearts into New Zealand white recipients without immunosuppression (average time to graft failure 8.2 +/- 0.4 days). We found constitutive expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by coronary arterial endothelium in normal rabbits, whereas myocardial capillaries and the endocardial lining cells showed little or no expression of VCAM-1. VCAM-1 expression increased within 1 day after transplantation on the endothelium of the transplanted aorta and endocardium and on myocardial microvascular endothelial cells. ICAM-1 expression increased remarkably on all endothelia studied from 2 to 8 days after transplantation. Adhesion molecule expression on coronary artery endothelial cells also increased during severe allograft rejection (from a histological score of 1.7 +/- 0.6 pretransplant to 4.8 +/- 0.2 8 days after transplant for VCAM-1 and from 0.9 +/- 0.6 to 4.4 +/- 0.3 for ICAM-1, n = 43 arteries in 5 animals, mean +/- SD). In addition, coronary artery and aortic smooth muscle cells also showed induction of VCAM-1 and ICAM-1 8 days after transplant. We conclude that endothelial activation in a transplanted organ can occur rapidly and varies among microvascular, endocardial, and coronary artery endothelial cells, a point germane to the interpretation of endomyocardial biopsies. Augmented expression of adhesion molecules precedes temporally leukocyte accumulation in vessels. In addition, our finding of activation of coronary artery smooth muscle cells during acute rejection suggests that such episodes may contribute to the development of accelerated coronary arteriosclerosis.     

ICAM-1 and VCAM-1 expression by endothelial cells grown on fibronectin-coated TCPS and PS

Small-diameter vascular grafts rapidly fail as a result of blood coagulation and platelet deposition. Endothelial cells lining the inner side of blood vessels can provide the graft lumen with an antithrombogenic surface. One of the remaining problems is cell detachment after restoration of blood flow, because of infiltration of leukocytes that respond to an inflammatory-like activation of the endothelial cells. This endothelial activation is possibly caused by the surface characteristics of the underlying polymer. To get more insight into the effects of the polymer surface on endothelial cell activation, we seeded human umbilical vein endothelial cells (HUVECs) in various densities and subsequently grew them on tissue culture polystyrene (TCPS; hydrophilic) and polystyrene (PS; hydrophobic) surfaces. To improve cell adhesion, surfaces were coated with purified fibronectin prior to cell seeding. During proliferation, the expressions of the leukocyte adhesion molecules ICAM-1 and VCAM-1 were determined. Results indicate that ICAM-1 expression is not influenced by the character of the polymer surface, and that VCAM-1 expression is slightly higher on the TCPS surface. Expressions of both adhesion molecules are influenced by the seeding density and time of proliferation. At low seeding densities (< or = 10,000 cells/cm(2)), a relatively low percentage of nonexogenously activated cells expressed ICAM-1 during the first 3 days of proliferation compared to higher seeding densities. Although less pronounced, this was also observed for the percentage of cells expressing VCAM-1. During proliferation, the amount of ICAM-1 per endothelial cell increased, whereas the expression of VCAM-1 remained low. The absence of large differences in leukocyte adhesion molecule expression by endothelial cells grown on TCPS or PS is possibly caused by coating of the surfaces with fibronectin. It is known that surface hydrophilicity influences protein adsorption. Although this had no or little effect on leukocyte adhesion molecule expression, endothelial cell growth was affected, because proliferation was slower on the hydrophobic PS.     

Expression of vascular cell adhesion molecule (VCAM-1) in liver and pancreas allograft rejection.

VCAM-1, a leukocyte adhesion molecule expressed by cytokine-activated endothelial cells in culture, may mediate mononuclear leukocyte infiltration in vessels and interstitium in solid organ allograft rejection. Using the avidin-biotin immunoperoxidase technique and two different antibodies--4B9, a murine monoclonal antibody, and rabbit polyclonal antisera to recombinant human VCAM (rVCAM Ab), which work in methacarn-fixed tissues--we studied the expression of this molecule in biopsies of transplanted liver and pancreas with and without features of rejection as well as nontransplant control tissues. The rVCAM Ab, but not 4B9, showed a population of reactive endothelial cells limited to sites of prominent subendothelial leukocytic cell infiltration in arteries and veins in rejecting allografts. VCAM-1 expression by sinusoidal endothelium in rejecting liver allografts was also observed. In addition, a population of cells (DC) with dendritic morphology was identified by rVCAM Ab within sites of lymphoid cell aggregations in both liver and pancreas allografts. Further evidence that these cells represent true DC was obtained by identification of VCAM-1 positive morphologically similar cells in both germinal centers and interfollicular areas of reactive lymph nodes; and by similar staining of these cells in allograft organs by a monoclonal antibody to nerve growth factor receptor, previously shown to recognize DC. DCs were generally not seen in normal control organs or portions of allografts uninvolved by lymphoid aggregates. This study provides evidence that 1) endothelial cell expression of VCAM-1 may be important in transplant rejection, 2) different epitopes of VCAM-1 may be preserved in tissue sections and recognized by different antibodies, and 3) there is probably a population of VCAM-1 expressing DC that participates in the cellular rejection progress.     

Vascular cell adhesion molecule-1 expression and signaling during disease: regulation by reactive oxygen species and antioxidants

The endothelium is immunoregulatory in that inhibiting the function of vascular adhesion molecules blocks leukocyte recruitment and thus tissue inflammation. The function of endothelial cells during leukocyte recruitment is regulated by reactive oxygen species (ROS) and antioxidants. In inflammatory sites and lymph nodes, the endothelium is stimulated to express adhesion molecules that mediate leukocyte binding. Upon leukocyte binding, these adhesion molecules activate endothelial cell signal transduction that then alters endothelial cell shape for the opening of passageways through which leukocytes can migrate. If the stimulation of this opening is blocked, inflammation is blocked. In this review, we focus on the endothelial cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1). Expression of VCAM-1 is induced on endothelial cells during inflammatory diseases by several mediators, including ROS. Then, VCAM-1 on the endothelium functions as both a scaffold for leukocyte migration and a trigger of endothelial signaling through NADPH oxidase-generated ROS. These ROS induce signals for the opening of intercellular passageways through which leukocytes migrate. In several inflammatory diseases, inflammation is blocked by inhibition of leukocyte binding to VCAM-1 or by inhibition of VCAM-1 signal transduction. VCAM-1 signal transduction and VCAM-1-dependent inflammation are blocked by antioxidants. Thus, VCAM-1 signaling is a target for intervention by pharmacological agents and by antioxidants during inflammatory diseases. This review discusses ROS and antioxidant functions during activation of VCAM-1 expression and VCAM-1 signaling in inflammatory diseases.     

VCAM-1 signals during lymphocyte migration: role of reactive oxygen species

Vascular cell adhesion molecule-1 (VCAM-1) regulates leukocyte migration from the blood into tissues. VCAM-1 expression is induced on endothelial cells during inflammatory bowel disease, atherosclerosis, allograft rejection, infection, and asthmatic responses. During these responses, VCAM-1 forms a scaffold for leukocyte migration. VCAM-1 also activates signals within endothelial cells resulting in the opening of an "endothelial cell gate" through which leukocytes migrate. Immediately following this migration, the endothelial cell-endothelial cell contact is re-established. VCAM-1 outside-in signals are mediated by NADPH oxidase production of reactive oxygen species and subsequently activation of matrix metalloproteinases. These signals are required for endothelial cell shape changes and leukocyte migration. In addition, VCAM-1-activated signals in endothelial cells are regulated by cytokines indicating that it is important to consider both endothelial cell adhesion molecule expression and function during inflammatory processes.     

Expression of leukocyte adhesion molecules by endothelial cells seeded on various polymer surfaces

Although endothelial cell seeding in small-diameter vascular prostheses significantly improves graft survival, the detachment of adherent endothelial cells after the restoration of circulation remains one of the major obstacles. Because in vivo experiments indicate that leukocyte infiltration is involved in endothelial cell loss, we hypothesize that seeded endothelial cells become activated and express leukocyte adhesion molecules and cytokines because of an interaction with the underlying polymer surface. The aim of this study was to investigate the expression of the leukocyte adhesion molecules ICAM-1, VCAM-1, PECAM-1, and E-selectin by cultured human umbilical vein endothelial cells (HUVECs) and human adipose microvascular endothelial cells (HAMVECs). The cells were seeded on tissue culture poly(styrene) and the vascular graft materials Dacron and Teflon. The results of this study indicate that the expression of leukocyte adhesion molecules by cultured endothelial cells is mainly affected by the endothelial cell origin, that is, umbilical vein or adipose tissue. Expressions of both ICAM-1 and E-selectin by HUVECs and HAMVECs are characterized by the presence of two cell populations with distinct levels of expression. With respect to endothelial cell seeding in vascular prostheses, the increased expression of E-selectin by microvascular endothelial cells deserves further attention.     

Adhesive Interactions between Human NK Cells and Porcine Endothelial Cells

Human natural killer (NK) cells are able to adhere to xenogeneic porcine endothelial cells (EC) and evidence from in vitro studies as well as animal models suggests a potential role for NK cells in the cellular recognition and damage of porcine xenogeneic tissues. One possible explanation for the observed NK cell-mediated xenogeneic cytotoxicity against porcine EC is the molecular incompatibility between porcine major histocompatibility complex (MHC) class I molecules and MHC-specific inhibitory receptors on human NK cells. In this review we attempt to summarize the current knowledge concerning adhesive interactions between human NK cells and porcine EC under special considerations of the cross-species receptor-ligand interactions. Methodological differences in assessing adhesion between various studies are reviewed and comparisons to the syngeneic/allogeneic adhesion mechanisms are made. Finally, the therapeutic potential of blocking antibodies and transgenic HLA expression in preventing NK-cell adhesion and xenogeneic cytotoxicity is discussed.     

Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.

Vascular cell adhesion molecule-1 (VCAM-1), a mononuclear leukocyte adhesion molecule, is expressed in cultured vascular endothelial cells activated by cytokines and is induced in rabbit aortic endothelium in vivo within 1 week after initiation of an atherogenic diet. We now demonstrate that vascular smooth muscle cells can also express VCAM-1 in rabbit atherosclerotic lesions in vivo and in response to cytokines in vitro. Immunohistochemical staining of aortas from rabbits fed a 0.3% cholesterol-containing diet revealed that a portion of smooth muscle cells within intimal foam cell-rich lesions expressed VCAM-1. The intimal VCAM-1-expressing cells localized predominantly in regions above the internal elastic lamina. These VCAM-1-positive cells had the typical spindle shape of smooth muscle cells but had reduced alpha-actin expression in comparison to normal medial smooth muscle cells, and did not bear markers for endothelium, macrophages, and T cells. In culture, rabbit aortic smooth muscle cells expressed VCAM-1 mRNA and protein in a time- and concentration-dependent fashion when exposed to interferon-gamma or Gram-negative bacterial lipopolysaccharide. Cultured human vascular smooth muscle cells also expressed VCAM-1 mRNA and protein in response to lipopolysaccharide, interferon-gamma, and interleukin-4. The monokines interleukin-1 alpha and tumor necrosis factor-alpha did not induce VCAM-1 expression in either rabbit or human vascular smooth muscle cells. Inducible VCAM-1 expression by vascular smooth muscle cells in vivo during hypercholesterolemia and in vitro in response to certain cytokines suggests a broader range of VCAM-1 functions in vascular biology than heretofore appreciated.     

Estradiol reduces basal and cytokine induced monocyte adhesion to endothelial cells

OBJECTIVE: To investigate the effect of 17beta-estradiol (E2) on binding of monocytes to human aortic endothelial cells (HAECs) with or without cytokine induction. METHODS: Confluent monolayers of HAECs were incubated with or without E2 for 48 h prior to the monocyte adhesion assay. In studies with cytokines, 1 ng/ml tumor necrosis factor-alpha (TNF-alpha), 20 U/ml interleukin-1beta (IL-1beta) or both were added to the culture medium for the final 24 or 4 h. For the measurement of monocyte adhesion, 3H-thymidine labeled human THP-1 monocytes (4 x 10(5) cells per well) were added to the confluent monolayer of HAECs and incubated at 37 degrees C for 90 min. The unbound THP-1 cells were removed by gentle washing, and bound cells were digested with NaOH and quantified by measuring radioactivity. RESULTS: When HAECs were pretreated for 48 h with E2 the basal adhesion of THP-1 cells was reduced by an average of 28%. Estrogen significantly reduced cytokine-induced adhesion by 30-35% when the cytokines were added for 4 h. When the cytokine treatment was prolonged to 24 h, pretreatment of HAECs with E2 had no effect on THP-1 cell adhesion. CONCLUSIONS: E2 reduces basal and short-term cytokine induced monocyte binding to HAECs. Since monocyte adhesion to vascular endothelial cells is one of the initial steps in the pathogenesis of atherosclerosis, E2 may mediate vascular protection by reducing monocyte-endothelial cell binding in the early stages of atherogenesis.     

Direct interaction with primed CD4+ CD45R0+ memory T lymphocytes induces expression of endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of vascular endothelial cells.

The process of recruitment of leukocytes at sites of inflammation involves direct cell-to-cell interactions between leukocytes and vascular endothelial cells (EC) mediated by various adhesion receptors on leukocytes and their inducible endothelial ligands. In this study we have examined the induction on EC of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) upon their interaction with subpopulations of human T cells. When co-cultured with EC both resting CD4+ T and CD8+ T cells caused a modest increase in the expression of endothelial ICAM-1. Moreover, resting CD4+ but not CD8+ T cells induced expression of ELAM-1 and VCAM-1 on a small fraction of unstimulated EC. Prior activation with phorbol 12-myristate 13-acetate (PMA) significantly increased the ability of T cells to up-regulate endothelial ICAM-1 and also induced the expression of both ELAM-1 and VCAM-1. PMA-primed CD4+ T cells induced both VCAM-1 and ELAM-1 on EC more efficiently than CD8+ T cells. Furthermore, the ability to induce the expression of ELAM-1 and VCAM-1 was confined to the CD4+ CD45R0+ memory/primed subpopulation of T cells. This induction of various endothelial adhesion ligands could also be mediated by antigen-primed CD4+ T cell lines. The CD4+ T cell-mediated induction of adhesion ligands required direct intercellular contact with EC because neither cultures of EC and PMA-primed CD4+ T cells separated by a microporous membrane insert nor the conditioned medium of PMA-primed T cells induced expression of ELAM-1 and VCAM-1 on EC. Cyclosporin A significantly inhibited the activation of T cells with PMA but had no effect on the ability of PMA-primed T cells to up-regulate endothelial CAM. Thus, CD4+CD45R0+ T cells via as yet unknown mechanism can significantly enhance the expression of each of the three endothelial adhesion ligands and, thereby, may facilitate the process of recruitment of additional leukocytes to exacerbate inflammation.     

Anti-metastatic prostacyclins inhibit the adhesion of colon carcinoma to endothelial cells by blocking E-selectin expression

Prostacyclins have long been shown to have anti-metastatic activity. One hypothesis is their modulation of cell adhesion molecule (CAM) expression by target organ endothelial cells. We have postulated that prostacyclin, its analogs, and mechanistic mimics decrease colon carcinoma adhesion to cytokine-stimulated endothelial cells by blocking endothelial expression of the adhesion molecule E-selectin, but not the vascular cell adhesion molecule-1 (VCAM-1). Cultured human microvascular endothelial cells (HDMEC) were pre-incubated with prostacyclin (PGI₂), dibutyrl-CAMP (dbcAMP), forskolin (FOR), and/or iso-methylbutylxanthine (IBMX) for 15 min, then co-incubated with the cytokine tumor necrosis factor (TNF) for 4 h. HDMEC surface expression of E-selectin and VCAM-1 was evaluated by flow cytometry and ELISA. Adherence of ⁵¹Cr-labeled colon carcinoma cells to HDMEC monolayers was then determined. In parallel assays, HDMECs were incubated with anti-E-selectin and anti-VCAM-1 monoclonal antibody (1:100) prior to the addition of tumor cells. Prostacyclins, its analogs, and mimics significantly reduced E-selectin expression by HDMEC, while the reduction of VCAM-1 expression was much less pronounced. Prostacyclins also significantly decreased colon carcinoma adherence to stimulated HDMECs. The inhibition of E-selectin expression, but not VCAM-1 expression, corresponded to the reduction of tumor cell adherence. Prostacyclin's effects on tumor adhesion were nullified by pre-incubation with E-selectin antibody. The inhibition of colon carcinoma adherence to cytokine-stimulated endothelial cells treated with prostacyclin, its analogs, and mimics appears to result from blocking endothelial E-selectin, but not VCAM-1, expression. These data support the hypothesis that prostacyclins may exert their anti-metastatic effect, in part, by inhibiting CAM-mediated adherence of colon carcinoma to endothelial cells in metastatic target organs.     

Endothelial cells upregulate eosinophil superoxide generation via VCAM-1 expression

BACKGROUND: In vitro eosinophil (EOS) adhesion to recombinant human (rh)-vascular cell adhesion molecule (VCAM)-1 stimulates superoxide anion (O2-) generation and enhances formyl-methionyl-leucyl phenylalanine (FMLP)-activated O2- generation. Therefore, EOS adhesion via VLA-4 to VCAM-1 expressed on endothelium may be instrumental in the selective recruitment and function of EOS in airway inflammation. OBJECTIVE: We hypothesized that EOS interaction with endothelial cells expressing VCAM-1 will undergo an enhancement in inflammatory function. METHODS: To determine this possibility, human umbilical vein endothelial cells (HUVEC) were stimulated with either a combination of interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha (100 pM) or medium alone for 24 h; the expression of adhesion proteins on HUVEC and their effect on EOS O2- generation was subsequently determined. RESULTS: As determined by both enzyme-linked immunosorbent assay and flow cytometry, IL-4 and TNFalpha acted synergistically to induce VCAM-1 expression on HUVEC. Treating HUVEC with IL-4/TNFalpha also increased EOS adhesion and primed subsequent FMLP (0.1 microM) activated EOS O2- generation. Although EOS adhesion was partially inhibited by both antialpha4 and antibeta2 monoclonal antibodies (MoAbs), O2- generation was completely inhibited by either antialpha4 integrin MoAb (HP1/2) or anti-VCAM MoAb (BBIG-V1). Furthermore, enhanced O2- generation, but not adhesion, associated with IL-4 + TNFalpha-treatment of HUVEC was inhibited when EOS were treated with the platelet activating factor (PAF)-antagonist WEB 2086 (20 microM), thus suggesting an involvement of PAF in priming EOS. However, paraformaldehyde fixation of IL-4/TFN-alpha treated HUVEC did not significantly alter EOS function. CONCLUSIONS: These results suggest EOS adhesion to endothelial cells via an VLA-4/VCAM-1 interaction may be important in the development of the function of this cell. Furthermore, our results suggest that modulation of EOS function involves two priming factors: EOS adhesion to HUVEC expressing VCAM-1 and PAF.     

Xenogeneic interaction between human CD40L and porcine CD40 activates porcine endothelial cells through NF-kappaB signaling

Xenotransplantation is a promising alternative to overcome donor shortage in transplantation. CD40 molecule plays an important role in the interaction of T cells with antigen-presenting cells and in the activation of vascular endothelial cells. We investigated whether the xenogeneic interaction between human CD40L (hCD40L) on T cells and porcine endothelial CD40 (pCD40) can activate porcine endothelial cells (PECs). The interaction between hCD40L and pCD40 induced the expression of chemokines on PECs as well as MHC and adhesion molecules. Furthermore, NF-kappaB signaling was activated in HEK 293 cells expressing pCD40 and PECs by stimulation with hCD40L+ Jurkat T clones. Both anti-CD40L neutralizing antibodies and NF-kappaB signal inhibitors interfered with immune activation of PECs. Overall, this study shows that xenogeneic interaction between hCD40L and pCD40 can activate PECs through NF-kappaB signaling, and therefore may contribute to acute vascular rejection in xenotransplantation.     

Activation of naive xenogeneic but not allogeneic endothelial cells by human naive neutrophils: a potential occult barrier to xenotransplantation.

Here we demonstrate that human neutrophils, the predominant circulating leukocytes in intimate contact with endothelial cells lining the vasculature, directly recognize xenogeneic endothelium independently of xenoreactive natural antibody and complement. A rapid and calcium-dependent activation of native (unstimulated) xenogenic endothelial cells by human neutrophils leads to 1) translocation of P-selectin from the Wiebel-Palade bodies to the surface of xenogeneic endothelial cells, 2) increased synthesis and expression of vascular cell adhesion molecule-1 on the xenogeneic endothelial cells, and 3) enhanced killing of the xenogeneic endothelium by natural killer cells. Our data directly implicate naive neutrophils as major early participants in xenograft recognition and endothelial activation independent of xenoreactive natural antibodies and complement.