Immediate-early gene expression in human saphenous veins harvested during coronary artery bypass graft operations.

Saphenous vein graft occlusion is a common late complication of coronary bypass grafting. Intimal smooth muscle cell hyperplasia is a component of this pathobiology, but the underlying molecular events are poorly understood. Immediate-early genes are activated shortly after growth stimulation and subserve cellular functions, which may contribute to intimal smooth muscle cell accumulation. In the present study, human saphenous vein grafts were harvested with minimal manipulation during coronary bypass and processed for isolation of total ribonucleic acid to examine change in immediate-early gene expression of messenger ribonucleic acid by Northern blotting techniques. Thirty saphenous vein grafts were incubated at 4 degrees C in Dulbecco's modified Eagle media from 30 minutes to 10 hours. The messenger ribonucleic acids for immediate-early genes c-fos and c-myc were weak or undetectable in controls but were increased (> 10 times controls) within 1 hour (c-fos) and persisted for at least 6 hours (c-myc) after harvest. Our results demonstrate, for the first time in human vascular tissue, incipient immediate-early gene induction. This information may lead to molecular therapies to control saphenous vein graft disease.

Localization of Fos and Jun proteins in rat aortic smooth muscle cells after vascular injury.

The availability of specific reagents to measure gene activity has provided important tools and potential new directions for the study of smooth muscle cell (SMC) proliferation in vivo. In this report, we have measured steady-state mRNA levels of several fos and jun family members in aortic tissue by Northern blotting after vascular injury. In addition, protein products of these genes were analyzed by immunocytochemistry. Within 15 minutes of balloon injury, mRNA levels of c-fos, fosB, c-jun, junB, and junD were elevated severalfold. In contrast, fos-related antigen (fra-1) mRNA showed a delayed onset of expression. The expression kinetics of these immediate early genes was similar to those in cultured cells stimulated to undergo proliferation by growth factors, suggesting that such SMC gene activation in vivo reflects permeation of blood-derived growth factors into the vessel wall or intravascular release of preformed growth factors. Translation of fos and jun genes into immunoreactive products was demonstrated 2 hours after balloon injury with antisera to Fos and Jun proteins. Treating rats with cycloheximide abolished this immunoreactivity. The distribution of Fos and Jun products was concentrated in SMC nuclei at the luminal border of the rat aorta. Such focal expression may have consequences for the initiation of SMC DNA synthesis and migration after vascular injury. Furthermore, the expression of Fos and Jun proteins in SMC after vascular balloon injury may be used as an index of SMC activation under a variety of experimental settings.

Smooth muscle cell immediate-early gene and growth factor activation follows vascular injury. A putative in vivo mechanism for autocrine growth.

To understand the molecular events governing smooth muscle cell (SMC) proliferation in vivo, immediate-early gene (IEG) expression was assessed and related to growth factor ligand and receptor mRNA and SMC DNA synthesis after aortic injury. Balloon catheter injury evoked increases in SMC c-myc and thrombospondin (tsp) within 2 hours. The induction of these IEGs was followed by elevated transcripts to platelet-derived growth factor-A (PDGF-A), transforming growth factor-beta 1 (TGF-beta 1) and a basic fibroblast growth factor (bFGF) receptor. Whereas PDGF type-beta receptor mRNA was demonstrated in SMCs from control and balloon-injured aortas, no detectable signal was observed for the PDGF type-alpha receptor. To explore the potential linkage between IEG products and growth factor mRNA expression, cycloheximide was employed to block early protein synthesis after balloon injury. Induction of PDGF-A and TGF-beta 1 was attenuated by cycloheximide, but bFGF induction was unaffected. Moreover, cycloheximide superinduced IEGs and revealed PDGF-B transcripts, which were otherwise undetected. Seven days after aortic injury, a spontaneous increase in c-myc and tsp mRNA was noted. This IEG reactivation was followed 12 hours later by a twofold increase in SMC DNA synthesis. These findings corroborate an autocrine mode of SMC proliferation in vivo and suggest the IEG products may control such growth by stimulating growth factor genes.

Native low density lipoprotein. Endothelial cell recruitment of mononuclear cells.

The effect of native low density lipoprotein (LDL) on human umbilical vein endothelial cell (EC) recruitment of mononuclear cells (Monos) was investigated. ECs were exposed to LDL at atherogenic concentrations (240 mg cholesterol [Chol]/dl) for as long as 4 days (LDL-treated ECs). LDL-treated ECs bound substantially greater amounts of freshly isolated human monocytes and U937 cells than did control ECs. The enhanced Mono binding was time and LDL concentration dependent. LDL-induced binding was reduced to control levels when cycloheximide was added together with LDL, indicating that de novo protein synthesis was required. Furthermore, this LDL effect was not a general feature of apolipoproteins, as high density lipoprotein in physiologically relevant concentrations (45 mg Chol/dl, 4 days) had no effect on EC-Mono binding. Conditioned media from LDL-treated EC cultures did not increase EC binding of Monos. In contrast, minimally modified LDL increased EC-Mono binding more than eightfold. In conclusion, LDL in concentrations associated with the premature development of atherosclerosis increased EC affinity for Monos. Such LDL-induced alterations in EC physiology likely represent a proinflammatory response and an early step in atherogenesis.

Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal.

To study the mechanism(s) of vascular smooth muscle cell proliferation in vivo, mRNA levels of c-fos, c-jun, and c-myc were determined by Northern blot analysis following vascular balloon de-endothelialization (BDE). Medial smooth muscle cells (SMC) were separated and studied by enzymatic digestion of the vessel wall. mRNA levels of c-fos and c-jun from aortic smooth muscle cells (SMC) were simultaneously induced within 30 minutes of BDE and declined to baseline by 1.5 hours, c-myc mRNA did not begin to increase until 1 hour after vascular injury. Levels of c-myc peaked at 2 hours and were sustained for an additional 4 hours before gradually declining. Smooth muscle cells derived from enzyme-treated control aortae that did not undergo BDE expressed c-fos and c-jun, but showed no evidence of c-myc message. In contrast, nonenzymatically treated, non-BDE whole aortae (containing both media and adventitia) demonstrated a prominent c-myc signal, but failed to express c-fos and c-jun. Corresponding examination of adventitia derived from enzyme-treated aortae showed this tissue to be a source of all three proto-oncogenes. The results of this study demonstrate the earliest in vivo molecular markers of vascular injury reported to date and implicate SMC proto-oncogene expression in the initiation of SMC proliferation. Furthermore these findings suggest two avenues for proto-oncogene induction, that are due to (1) vessel wall manipulation and (2) humoral stimulation.

14, 15-Epoxyeicosatrienoic acid promotes endothelial cell dependent adhesion of human monocytic tumor U937 cells.

Arachidonic acid (AA) can be metabolized in endothelial cells (EC) to a series of epoxides via cytochrome P-450 epoxygenase with 14,15 epoxyeicosatrienoic acid (14,15-EET) as the major product. In this communication we report that 14,15-EET significantly enhances U937 cell attachment to EC with maximal cell attachment at 2.5 to 5 x 10(-7) M 14,15-EET. Thus, 14,15-EET may play a substantial role in inflammation and/or atherogenesis by inducing monocyte attachment to EC.