Adam S. Asch

G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.

The Kaposis sarcoma-associated herpesvirus (KSHV/HHV8) is a γ-2 herpesvirus that is implicated in the pathogenesis of Kaposis sarcoma, and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposis sarcoma and PEL,,, it encodes putative oncogenes,, and genes that may cause Kaposis sarcoma pathogenesis by stimulating angiogenesis,,,. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposis sarcoma lesions and in PEL, and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis, and Kaposis-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposis sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.

Isolation of the thrombospondin membrane receptor.

Thrombospondin (TSP), a 450-kD multifunctional glycoprotein with a broad tissue distribution, is secreted upon platelet stimulation, binds to the activated platelet surface, and supports platelet aggregation. We have identified and isolated an 88-kd membrane glycoprotein present in platelets, endothelial cells, monocytes, and a variety of human tumor cell lines that is the membrane binding site for TSP. Endogenous platelet TSP binding to thrombin- and ionophore-stimulated human platelets was inhibited in the presence of the monoclonal antibody OKM5. TSP binding to C32 melanoma cells and HT1080 fibrosarcoma cells was specific and also inhibitable with OKM5 Mab. Cell labeling followed by specific immunoprecipitation demonstrated biosynthesis of a single 88-kD glycoprotein. Binding of TSP to the isolated membrane protein was specific and saturable. These studies identify an 88-kD membrane glycoprotein that reacts with the monoclonal antibody, OKM5, and may function as the cellular TSP receptor.

A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IE) specifically adhere to vascular endothelium in vivo and to human endothelial cells, some human melanoma cell lines, and human monocytes in vitro. The tissue cell receptor for a ligand on the surface of the infected erythrocytes is an Mr 88,000 glycoprotein (GP88) recognized by the MAb OKM5, which also blocks cytoadherence of IE. Isolated, affinity-purified GP88 (CD36) competitively blocks cytoadherence and when absorbed to plastic surfaces, specifically binds P. falciparum IE. Additionally, monoclonal and polyclonal antibodies to GP88 block cytoadherence to both target cells and immobilized GP88. Binding to GP88 by IE is unaffected by the absence of calcium or the absence of thrombospondin, a putative mediator for cytoadherence of P. falciparum IE. Thus, GP88 (CD36), which has been demonstrated to be the same as platelet glycoprotein IV, interacts directly with P. falciparum IE, presumably via a parasite-induced ligand exposed on the surface of the infected erythrocytes. CD36 is shown to be present on brain endothelium in both individuals without malaria and individuals with cerebral malaria. This would suggest that factors other than just cerebral sequestration of IE play an initiating role in the genesis of cerebral malaria.

A link between diabetes and atherosclerosis: Glucose regulates expression of CD36 at the level of translation

Both the risk and the rate of development of atherosclerosis are increased in diabetics, but the mechanisms involved are unknown. Here we report a glucose-mediated increase in CD36 mRNA translation efficiency that results in increased expression of the macrophage scavenger receptor CD36. Expression of CD36 was increased in endarterectomy lesions from patients with a history of hyperglycemia. Macrophages that were differentiated from human peripheral blood monocytes in the presence of high glucose concentrations showed increased expression of cell-surface CD36 secondary to an increase in translational efficiency of CD36 mRNA. We obtained similar data from primary cells isolated from human vascular lesions, and we found that glucose sensitivity is a function of ribosomal reinitiation following translation of an upstream open reading frame (uORF). Increased translation of macrophage CD36 transcript under high glucose conditions provides a mechanism for accelerated atherosclerosis in diabetics.

Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.

Glycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 cell adhesion.

An adhesive interaction between activated platelets and mononuclear phagocytes may contribute to the role these cells play in regulating inflammation, thrombosis, and atherosclerosis. We have previously shown that this adhesive interaction is mediated by the expression of the glycoprotein thrombospondin (TSP) on the surface of activated platelets. We now show that TSP-dependent platelet-monocyte interactions are mediated by glycoprotein IV (GPIV), an intrinsic membrane protein recently identified as a cell surface TSP receptor. Monoclonal antibodies to GPIV bound to cells of the human monocytoid line U937 as assessed by flow cytometry and inhibited the binding of 125I-TSP to the cell surface by 83%. U937 cells preincubated with anti-GPIV were not rosetted by thrombin-stimulated platelets (72% inhibition compared with control anti-monocyte antibodies). In addition, when platelets were stimulated in the presence of saturating concentrations of monoclonal antibodies to GPIV, only 18% of U937 cells were rosetted (78% inhibition). Control antibodies including anti-GPIb did not inhibit rosette formation. These data suggest that TSP can cross-link platelets and monocytes via an interaction with GPIV on the surface of both cells. This molecular bridge may mediate platelet-macrophage communication in various pathophysiologic settings.

Characterization of hematopoietic cells arising on the textured surface of left ventricular assist devices

BACKGROUND Textured biomaterial surfaces in implantable left ventricular assist devices induce development of a nonthrombotic neointimal surface and allow elimination of anticoagulation therapy in device recipients. Characterization of the hematopoietic cells formed within the neointimal surfaces of these devices will contribute to our understanding of this unique neointima. METHODS The blood-contacting surface of seven ThermoCardiosystems left ventricular assist devices was removed, washed with phosphate-buffered saline solution, and digested with 0.1% collagenase for 15 to 20 minutes. The hematopoietic cells released from the explants were isolated and analyzed by flow cytometry and immuno-histochemical staining. RESULTS More than 80% +/- 6% of hematopoietic cells isolated in this fashion are of myelomonocytic origin and express CD14, CD15, and CD33 surface molecules. Four percent of cells express the CD34 surface marker, which suggests that the neointima is colonized by pluripotent hematopoietic stem cells. Continuous culture of these hematopoietic cells in the presence of the cytokines interleukin-3, c-kit ligand, granulocyte colony-stimulating factor resulted in tenfold expansion by day 7 and 25-fold expansion by day 14. CONCLUSIONS Pluripotent hematopoietic cells with a high proliferative capacity colonize textured surfaces of left ventricular assist devices and may contribute to the development of a biologically nonthrombogenic neointima.

Enhanced HIV-1 replication in Vβ12 T cells due to human cytomegalovirus in monocytes: Evidence for a putative herpesvirus superantigen

HIV-1 replicates more efficiently in cultured IL-2-dependent CD4 T cells expressing V beta 12 T cell receptors (TCRs) rather than other TCRs (Laurence et al., 1992). A viral reservoir is frequently established in V beta 12 T cells in HIV-1-infected patients. Here we show that cytomegalovirus (CMV) is responsible for V beta 12-selective HIV-1 replication that is indistinguishable from the effect of known superantigens (SAGs). This effect is dependent on direct contact of T cells with CMV-infected monocytes. CMV infection, but not ie1 or ie2 transfection, reproduces this effect in a monocytoid cell line (U937). In HIV-infected patients, the presence of CMV antibodies correlates with an HIV-1 viral load preferentially skewed to the V beta 12 subset. Together, these data suggest that a CMV gene product is responsible for a SAG-driven V beta 12-selective HIV-1 reservoir in vivo.

Histidine-rich glycoprotein inhibits the antiangiogenic effect of thrombospondin-1

Angiogenesis is critical for the growth and proliferation of tumors as well as for normal development. We now describe a novel role for histidine-rich glycoprotein (HRGP) in the modulation of angiogenesis. HRGP is a plasma protein that circulates in relatively high concentrations (1.5 microM), but has no known function in vivo. We have shown previously that HRGP binds with high affinity to thrombospondin-1 (TSP-1), a homotrimeric glycoprotein that is a potent inhibitor of angiogenesis. The antiangiogenic activity of TSP-1 is mediated by the binding of properdin-like type I repeats to the receptor CD36. We found that binding of HRGP to TSP-1 was similarly mediated by TSP type I repeats. HRGP colocalized with TSP-1 in the stroma of human breast cancer specimens, and this interaction masked the antiangiogenic epitope of TSP-1. In assays performed in vitro of endothelial cell migration and tube formation, and in vivo corneal angiogenesis assays, HRGP inhibited the antiangiogenic effect of TSP-1. These studies suggest that HRGP can modulate the antiangiogenic activity of TSP-1, and identify a potential mechanism of resistance to the antiangiogenic effect of TSP-1.

Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2′,5′-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a Gi signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the Gs/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells.