Expression of vascular endothelial growth factor receptors in smooth muscle cells.

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial-specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk-1, flt-1, and neuropilin (NP)-1, and respond to VEGF in vitro. In cultured VSMCs, flk-1 and flt-1 expression was inversely related to cell density. The expression of flk-1 was down-regulated with increasing passage numbers. However, NP-1 levels were not affected by cell density or passage numbers. Flk-1, Flt-1, and NP-1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk-1 protein is expressed at low levels in VSMCs, phosphorylation of Flk-1 following VEGF(165) stimulation was still observed. SMCs migrated significantly in response to VEGF(165) and VEGF-E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF-E is a specific activator of flk-1 while PlGF specifically activates only flt-1, SMC migration induced by VEGF(165) is likely to be mediated primarily through the flk-1 receptor. VSMCs did not significantly proliferate in response to VEGF(165), PlGF, and VEGF-E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.

Vascular graft healing. II. FTIR analysis of polyester graft samples from implanted bi-grafts.

FTIR-ATR analysis has shown that the 4-step process for preclotting polyester vascular grafts results in a uniform and reproducible fibrin coating of the polyester fibers. Western blot analyses have shown that FN and VEGF are also present in this fibrin coating. FTIR-ATR analyses of explanted grafts indicate that, while the in vivo healing of these preclotted polyester grafts proceed through the inflammation, proliferation, and remodeling phases of normal wound healing, these phases are modified. Because the fibrin coating provides a nonporous barrier between peri-graft tissue and the flowing blood, these molecular changes are controlled by the interactions of blood-borne constituents with the lumenal surface of the preclotted graft. Also, a well prepared preclotted polyester graft shows a minimal inflammatory response. After implantation, the fibrin preclot is more than 90% gone by the fifth day. However, the proliferation phase, involving synthesis of new protein and polysaccharide materials to replace the fibrin, appears to have begun by the third day. Detection of collagen I in the 5-day explants suggests that the overlapping remodeling phase of healing has begun. Protein and saccharide materials continue to be synthesized and remodeled, and, by the tenth day, collagen IV is detected. By 14-days post-implantation, there is an increase in collagen IV and cellular membrane lipids. Because collagen IV is an indicator of the presence of endothelial cells, some of these cellular membranes must be of endothelial origin. Thus, it appears that FTIR-ATR can be a useful tool in the study of vascular healing.

Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells: evidence for Erk-1/2 activation.

Tissue factor (TF), a transmembrane glycoprotein, initiates the extrinsic coagulation cascade. TF is known to play a major role in mediating thrombosis and thrombotic episodes associated with the progression of atherosclerosis. Macrophages at inflammatory sites, such as atherosclerotic lesions, release numerous cytokines that are capable of modulating TF expression. This study examined the role of oncostatin M (OSM), a macrophage/ T-lymphocyte-restricted cytokine, in the expression of TF in vascular smooth muscle cells (SMCs). It is reported here that OSM stimulated a biphasic and sustained pattern of TF messenger RNA (mRNA). The effect of OSM on TF mRNA expression was regulated at the transcriptional level as determined by nuclear run-offs and transient transfection of a TF promoter-reporter gene construct. OSM-induced TF expression was regulated primarily by the transcription factor NF-kappaB. Activation of NF-kappaB by OSM did not require IkappaB-alpha degradation. Inhibition of MEK activity by U0126 prevented OSM-induced TF expression by suppressing NF-kappaB DNA binding activity as determined by gel-shift analysis. Further, inhibition of Erk-1/2 protein by antisense treatment resulted in suppression of TF mRNA expression, indicating a role for Erk-1/2 in modulating NF-kappaB DNA binding activity. These studies suggest that the induced expression of TF by OSM is primarily through the activation of NF-kappaB and that activation of NF-kappaB is regulated in part by the MEK/Erk-1/2 signal transduction pathway. This study indicates that OSM may play a key role in promoting TF expression in SMCs within atherosclerotic lesions.

Induction of the urokinase plasminogen activator system by oncostatin M promotes endothelial migration.

Oncostatin M (OSM) is an inflammatory cytokine produced by activated macrophages and T-lymphocytes. We have previously demonstrated that OSM-induced endothelial cell migration, unlike endothelial cell proliferation and spindle formation, is independent of basic fibroblast growth factor expression (Wijelath et al. [1997] J. Cell. Sci. 110:871-879). To better understand the mechanism of OSM-induced endothelial cell migration, this study examined the potential role of the plasminogen activator system in promoting OSM mediated endothelial cell migration. OSM stimulated increased mRNA levels of urokinase-plasminogen activator (uPA) and urokinase-plasminogen activator receptor (uPAR) in a time and dose-dependent manner. Transcriptional run-off and mRNA stability analysis demonstrated that the increase in uPA and uPAR mRNA levels was due to both increased gene transcription and mRNA stability. The increase in mRNA correlated with increased protein levels of both uPA and uPAR. This increase was reflected in elevated levels of membrane-bound plasmin activity. OSM-induced endothelial cell migration was only partially dependent on plasmin activity since incubating endothelial cells without plasminogen or, in the presence of aprotinin, resulted in suppression of endothelial cell migration, indicating that OSM promoted endothelial cell migration through both a plasmin-dependent and -independent mechanism. Our results imply a role for OSM in promoting endothelial cell migration via a plasmin-dependent pathway and a uPAR-mediated pathway. Together, these and other recent studies support a role for OSM in modulating the different phases of angiogenesis.

Genetic tracing of arterial graft flow surface endothelialization in allogeneic marrow transplanted dogs.

In order to trace genetically the source of fallout endothelialization on arterial grafts, six beagle dogs with successful autologous bone marrow transplantation received composite tandem aortic grafts with an isolated, totally impervious Dacron graft and a porous Dacron graft for 12 weeks. For impervious segments, five of 12 fresh tissue samples were Factor VIII/von Willebrand factor + (FVIII/vWF) and seven had faint or negative signals; three of the FVIII/vWF + samples had alpha-actin + smooth muscle cells. Polymerase chain reaction (PCR) study showed eight had a pure donor DNA genotype and four had donor/host mixed, with the donor predominant. Of 12 AgNO3-stained samples, 11 showed pure donor type and one had donor/host mixed, with the donor predominant. For porous segments, all 12 fresh samples had positive flow surface FVIII/vWF and alpha-actin cells. PCR showed all these samples and all 12 AgNO3-stained samples had donor/host mixed type, but the host pattern was predominant. Porous graft healing appears to involve both cellular fallout and tissue ingrowth, and bone-marrow-derived cells may be a source for fallout.

Evidence for circulating bone marrow-derived endothelial cells.

It has been proposed that hematopoietic and endothelial cells are derived from a common cell, the hemangioblast. In this study, we demonstrate that a subset of CD34(+) cells have the capacity to differentiate into endothelial cells in vitro in the presence of basic fibroblast growth factor, insulin-like growth factor-1, and vascular endothelial growth factor. These differentiated endothelial cells are CD34(+), stain for von Willebrand factor (vWF), and incorporate acetylated low-density lipoprotein (LDL). This suggests the possible existence of a bone marrow-derived precursor endothelial cell. To demonstrate this phenomenon in vivo, we used a canine bone marrow transplantation model, in which the marrow cells from the donor and recipient are genetically distinct. Between 6 to 8 months after transplantation, a Dacron graft, made impervious to prevent capillary ingrowth from the surrounding perigraft tissue, was implanted in the descending thoracic aorta. After 12 weeks, the graft was retrieved, and cells with endothelial morphology were identified by silver nitrate staining. Using the di(CA)n and tetranucleotide (GAAA)n repeat polymorphisms to distinguish between the donor and recipient DNA, we observed that only donor alleles were detected in DNA from positively stained cells on the impervious Dacron graft. These results strongly suggest that a subset of CD34+ cells localized in the bone marrow can be mobilized to the peripheral circulation and can colonize endothelial flow surfaces of vascular prostheses.

Oncostatin M induces basic fibroblast growth factor expression in endothelial cells and promotes endothelial cell proliferation, migration and spindle morphology.

Oncostatin M (OSM), a pleiotropic cytokine originally isolated from supernatants of the U937 histiocytic lymphoma cell line, has been shown to have regulatory effects on a wide variety of cultured and tumor cells. We investigated the effects of OSM on basic fibroblast growth factor (bFGF) gene expression in bovine arterial endothelial (BAE) cells. Levels of bFGF mRNA transcripts were low in uninduced BAE cells, were maximal at 8 hours of exposure to OSM, and returned to control levels by 24 hours. Induction of bFGF mRNA transcripts by OSM was dose-dependent. Nuclear transcriptional run-on analysis demonstrated that exposure of BAE cells to OSM stimulated bFGF gene transcription. OSM treatment of BAE cells enhanced the synthesis of bFGF protein as determined by ELISA assays. Immunocytochemistry studies demonstrated the presence of low levels of bFGF protein within the cytoplasm in uninduced cells. After stimulation for 8 hours with OSM there was significant staining for bFGF in the cytoplasm. However, 24 hours after exposure to OSM, bFGF antigen was located only within the nuclei. Western blot analysis demonstrated that OSM stimulated predominantly the synthesis of a 22 kDa form of bFGF. In addition, OSM stimulated endothelial cell proliferation and migration as well as acquisition of a spindle shape. Phosphorothioate antisense oligonucleotide directed against bFGF inhibited OSM induced BAE cell proliferation and spindle shape formation but had only a minimal effect on migration. The levels of the 22 kDa form of bFGF were reduced by antisense treatment indicating that OSM induced proliferation and morphology change is likely to be regulated by intracellular bFGF. Our studies suggest that OSM released at sites of vascular injury could stimulate angiogenesis by inducing bFGF synthesis, endothelial cell proliferation and migration.

Interleukin-one induced arachidonic acid turnover in macrophages.

Recombinant Interleukin-1 beta (rIL-1 beta) induced dose-dependent synthesis of PGE2 and activation of both cell-associated and soluble phospholipase A2 (PLA2) in cultured mouse peritoneal macrophages. Kinetic studies showed that rIL-1 beta was able to induce a 2-fold increase in membrane bound PLA2 activity in the cells within 1 hour. Elevated levels of PGE2 were however observed only after 4 hours stimulation of the cells while significant levels of soluble PLA2 activity were observed only after 16 hours stimulation of the cells. Stimulation with rIL-1 beta of macrophage cultures pre-labelled with [14C]arachidonic acid resulted in a loss of label from both phosphatidyl-choline and phosphatidylinositol in only 10 min, which was not observed in control (unstimulated) cultures. These results are indicative that the two phospholipids are the principal source of arachidonic acid for IL-1 induced prostaglandin synthesis.

Interleukin-one induced inositol phospholipid breakdown in murine macrophages: possible mechanism of receptor activation.

Stimulation of mouse peritoneal macrophages by Interleukin-one (IL-1) provoked rapid increases in the levels of inositol mono, bis, tris and tetrakisphosphates (IP1, IP2, IP3 and IP4 respectively). IP3 was by far the major metabolite formed and time course studies revealed that IP2 and IP3 were formed more rapidly than IP1 and IP4 in response to IL-1 stimulation. The IP2 and IP3 levels peaked at five seconds while there was a time lag of five seconds in the IP4 response and the IP1 levels increased relatively steadily over the time course of the experiments. Levels of IP2, IP3 and IP4 all returned almost to control levels by 60s. The rapid formation of the inositol phosphate metabolites was concomitant with a decrease (84%) in the levels of phosphatidyl-inositol 4,5-bisphosphate (PIP2) in the macrophages. These results suggest that the mechanism of IL-1 receptor activation is by the rapid hydrolysis of phosphoinositides and generation of the second messenger IP3.