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J Thorac Cardiovasc Surg 1995;110:209-213
© 1995 Mosby, Inc.

SURGERY FOR ACQUIRED HEART DISEASE

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

Valhalla N.Y.

From the Departments of Surgery, Experimental Pathology, and Medicine, New York Medical College, Valhalla, N.Y.

Address for reprints: Richard A. Moggio, MD, Division of Cardiothoracic Surgery, Westchester Medical Center, Valhalla, NY 10595.

Abstract

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 changes in immediate-early gene expression of messenger ribonucleic acid by Northern blotting techniques. Thirty saphenous vein grafts were incubated at 4º 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. (J THORACCARDIOVASCSURG1995;110:209-13)

The mechanisms leading to saphenous vein graft occlusion, and atherosclerosis in general, are complex. Important events include vascular injury, reactivity of circulating cells and lipoproteins, remodeling of the extracellular matrix, and smooth muscle migration and hyperplasia. ¹ Migration of smooth muscle cells (SMCs) may be stimulated by platelet-derived growth factor from circulating cells ² or fromother factors released from the vessel wall. ³ Proliferation of SMCs appears to initially involve the modulation or dedifferentiation of cells from an "adult" contractile phenotype to that which resembles an embryonic "synthetic" proliferative phenotype. ⁴ SMC hyperplasia can be accelerated by endothelial loss, which may occur during surgical harvesting and manipulation of saphenous vein conduits. ^5-7 This growth may also occur in the vessel wall with an intact endothelial barrier. ⁸ Both mechanical and humoral factors activate variousgrowth-related genes in SMCs. ¹ Included among these genes are immediate-early genes, genes that are activated within minutes to hours of stimulation. ⁹ Animal studies have demonstrated the rapid induction of immediate-early genes after intimal injury from balloon deendothelialization. ^10-12 Our present study seeks to demonstrate induction of messenger ribonucleic acid (RNA) of two immediate-early genes, c-fos and c-myc, in human saphenous veins removed at the time of coronary artery bypass grafting.

METHODS

Saphenous vein graft preparation
Segments of saphenous vein grafts were obtained at the time of vein harvesting during coronary artery bypass grafting, according to a protocol approved by the Internal Review Boards of New York Medical College and the Westchester County Medical Center. The vein segments (2 to 5 cm in length) were harvested with minimal manipulation and no distention. After the vessel was gently rinsed in physiologic saline solution, the control, "time zero," segments were snap-frozen in liquid nitrogen. In the case of experimental segments, the rinsed vessel was placed in a Petri dish containing sterile Dulbecco's minimally modified Eagle's medium (DMEM; GIBCO-BRL, Gaithersburg, Md.) and incubated at 4º C for various times before the vein was frozen in liquid nitrogen. The time points studied after vein excision were 30 minutes and 1, 2, 4, 6, 8, and 10 hours. Because of the limited amount of material available from a single patient, each time point represents material from a different patient. RNA samples from four to six different patients were collected for the time zero through 6-hour points; thigh segments from two different patients were divided in half for the 8-hour and 10-hour time points. In several cases, a large tissue sample was cut in half so that two time points could be studied. Vein specimens were obtained for this study from five women (age range 49 to 79 years) and 21 men (mean 69 years, range 31 to 80 years). Twenty vessel segments were harvested from the thigh (17 men; 3 women) and six from the calf.

Isolation of RNA and Northern blotting
Total RNA was isolated as previously described ¹⁰ according to method of Chirgwin and associates. ¹³ Control and experimental vein segments were removed from the liquid nitrogen and disrupted by homogenization (Polytron homogenizer; Brinkman Instruments, Westbury, N.Y.) in guanidine thiocyanate 4 mol/L (Boehringer Mannheim; Indianapolis, Ind.) containing 2% ß-mercaptoethanol. After addition of N-lauroylsarcosine (0.5%; Fluka, Ronkonkoma, N.Y.), homogenates were sheared three times through a 23-gauge needle and clarified by centrifugation (10º C, 10,000 g, 10 minutes). The resulting supernatants were layered over a cushion of cesium chloride 5.7 mol/L (Boehringer Mannheim) and RNA was pelleted by centrifugation. ¹⁴ Equivalent amounts of total RNA (15 µg/lane) were separated on formaldehyde-containing 1% agarose gels. After electrophoresis, comparable RNA loading between gel samples was verified by visualizing the ethidium bromide-stained gel under ultraviolet illumination. Lack of RNA degradation in all samples was also confirmed by the greater staining intensity of the 28S as compared with the 18S ribosomal RNA.

The RNA was transferred onto a nylon membrane (BioRad Zeta-probe; Rockville Centre, NY.) by an overnight capillary transfer as described. ¹⁰ After ultraviolet crosslinking (Stratagene; La Jolla, Calif.) of the RNA to the membrane, blots were hybridized with radiolabeled probes as described. ^{10, 11} Complementary deoxyribonucleic acid (DNA) probes were labeled to a specific activity of 10 ⁹ cpm/ µg by random priming (Ambion, Austin, Tex.). Blots were washed at 55º C for 30 minutes each ¹⁰ in 2 x sodium chloride/sodium citrate (SSC)/1% sodium dodecylsulfate (SDS), 1 x SSC/0.5% SDS and 0.5 x SSC/0.25% SDS, then exposed to Kodak XAR-5 x-ray film in the presence of enhancing screens at -80º C for 1 day (c-fos and glyceraldehyde-3-phosphodehydrogenase) to 7 days (c-myc). Unless replicate blots were prepared, the weakest probe was used first (e.g., c-myc); then blots were stripped and reprobed with additional cDNAs. Glyceraldehyde-3-phosphodehydrogenase served as a reference gene to normalize for RNA loading and transfer efficiency in all blots. Autoradiographic signals of mRNA levels on light film exposures were quantified by scanning densitometry on an LKB Ultrascan scanner (LKB Instruments, Inc., Rockville, Md.) as described and normalized with that of glyceraldehyde-3-phosphodehydrogenase. ¹⁰ Eleven different Northern blots were prepared; three were replicates of the majority of respective time points containing the same patients' RNA (i.e., blots 16 = 17 = 18 or 11 = 12). Replicate blots served to confirm relative mRNA signals as a function of time.

cDNA probes
The murine c-fos probe was a 1.8 kb EcoRI-SalI fragment kindly provided by Dr. T. Curran (Roche Institute of Molecular Biology, Nutley, N.J.). The human third exon c-myc probe was from Oncor (Gaithersburg, Md.) and human glyceraldehyde-3-phosphodehydrogenase 1.1 kb probe was from Clonetech (Palo Alto, Calif.).

RESULTS

Fig. 1 illustrates the induction of c-fos mRNA in human saphenous vein grafts at several times after vessel excision and subsequent incubation of the vessel in cold DMEM (Methods). In one of the control (time zero) vessels (Fig. 1), trace levels of c-fos were detected by Northern blots of total saphenous vein graft RNA. The most robust (10 to 100-fold) and consistent elevations in this immediate-early gene were observed between 30 minutes and 2 hours; these levels tended to decrease between 4 and 6 hours. In two patients a strong signal was also observed at 8 and 10 hours. The changes in c-fos levels cannot be explained by differential RNA loading efficiency because the level of glyceraldehyde-3-phosphodehydrogenase, a reference mRNA, was comparable among all samples.

View larger version (66K): [in this window] [in a new window]   Fig. 1. Northern blots of c-fos mRNA expression in total RNA isolated from human saphenous veins. Vessels were excised and either immediately frozen in liquid nitrogen (time zero) or incubated at 4º C in DMEM for the indicated times before freezing the tissue. Total saphenous vein graft RNA was isolated by cesium chloride centrifugation and 15 µg of RNA was separated on agarose gels for transfer to nylon membranes (blots). The blots were hybridized with a murine probe for c-fos or human glyceraldehyde-3-phosphodehydrogenase (GAPDH) (#16); The size of the c-fos human mRNA detected was about 2.2 kb. (Although not shown here, blots #10 and 14 were also probed for GAPDH with results similar to those of blot #16.) One-day film exposures of three representative blots are illustrated. Patient characteristics for each blot are as follows for #10, #14, and #16, respectively (F, female, M, male, T, thigh, L, leg). Control time zero: F56T, M54L, M80T/F50T; 0.5 hour, M66L, M66L, F50T; 1 hour, M77T, M63T, F50T; 2 hour, M77T, M63T, M63T; 4 hour, M45T, M45T, M62T; 6 hour, M45T, F49L, M62T; 8 hour and 10 hour (blot #14, #16); M62T, F64T.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Northern blots of c-myc mRNA expression in total RNA isolated from human saphenous veins. A human c-myc cDNA was used to detect c-myc mRNA (about 2.5 kb). Patient characteristics are indicated in the legend to Fig. 1 because blot #18 contains the replicate 15 µg RNA samples of those used in #16. This blot was also probed with glyceraldehyde-3-phosphodehydrogenase to confirm comparable RNA loading as in blot #16 (data not shown).

Animal studies have demonstrated induction of c-fos and c-myc expression in rat or rabbit aortas after balloon deendothelialization. ^10-12 In either model, these mRNAs increase within 15 to 30 minutes (c-fos) to 2 hours (c-myc) after vascular injury, and the protein products are detectable in rat medial SMCs by 2 hours. ¹⁵ These events precede SMC hyperplasia, ¹ and there is substantial evidence from in vitro cell culture studies that these immediate-early genes play key roles in the proliferative process. ^{16, 17} This report demonstrates, for the first time, induction of these genes directly from in vivo human vascular tissue before the onset of intimal SMC proliferation. This study does not localize the gene response to the smooth muscle or endothelium of the vein, because the entire vein was processed. Our previous animal work, however, has localized these genes in the smooth muscle layer of the aorta. ^{10, 15}

Immediate-early genes are defined as genes whose transcripts are transiently increased early after growth factor stimulation without requisite protein synthesis. ⁹ The products of immediate-early genes presumably subserve protean functions within the cell related to growth and differentiation, as in the case of c-fos. The immediate-early gene c-fos is a transcription factor which, in a heterodimeric complex with members of the c-jun family, binds to and activates AP-1 sites in the promoters of genes. ⁹ In the case of SMC growth responses aftervascular injury, ¹ the activation of c-fos could lead to activation of growth factors such as transforming growth factor ß₁ . ¹⁶ In neural systems, c-fos is rapidly activated by cellular depolarization or ischemia. ⁹

The transcription factor c-myc seems to be more closely linked to cell proliferation than to differentiation. Various human cancers, including hematologic malignancies, exhibit elevated expression of the c-myc gene. ¹⁷ When various growth-quiescent cell lines are stimulated with serum or growth factors, c-myc expression is enhanced and precedes DNA synthesis. ¹⁸

The demonstration of these immediate-early genes in human vascular tissue is not unexpected, given their presence in many cells. With the lack of other tissue as a control, it is possible that the immediate-early gene response may be part of a total body response to trauma. Further, these results are semiquantitative, given the limited number of samples and the use of different patients for each time point. Signal strength, however, is increased approximately 100-fold from barely detectable control levels to maximum exposure. The activation of these genes, however, was initiated by minimal gross disturbance of the tissue itself. The saphenous veins were freshly harvested, not flushed, distended, or manipulated in any manner common to preparation of veins for coronary artery bypass grafting. "Excessive" manipulation and nonphysiologic distention of vein grafts leads to greater endothelial loss and consequent acceleration of SMC hyperplasia. ^5-7 This may explain the positive signals in the "time zero" control vessels. The temperature and DMEM incubation media may also have some secondary effect on the expression of immediate-early genes, because the time frame for the activation of these genes is consistent with parallel manipulations of the rat thoracic aorta (results not shown; R. A. Moggio, J. Ding and C. J. Smith, preliminary data).

A possible implication of our study is that even the most meticulous process of preparing and "arterializing" a vein segment is a sufficient stimulus to activate growth-related genes. Whether this physical manipulation ¹⁰ in concert with humoral factors such as platelet-derived growth factor ¹⁹ or other mitogens leads to a synergistic SMC growth response is unclear. The relative importance of SMC proliferation and secretion and extracellular matrix formation and scarring in human atherosclerotic processes is also unknown. ²⁰ The results reported here, however, are consistent with the hypothesis that early induction of the immediate-early genes c-fos and c-myc in human SMCs may be part of a pathway that leads to intimal hyperplasia. Further elucidation of these mechanisms may lead to more fundamental therapeutic strategies to limit the early atherogenic response.

Appendix: DISCUSSION

Dr. Andrew S. Wechsler (Richmond, Va.).
Did you consider doing something like in situ hybridization to see if you could figure out which cell moieties were responsible for the immediate-early gene response?

Dr. Moggio.
We do not have that technique for human tissue. In related histochemical techniques we have identified these proteins in the medial SMC layer of the rat aorta after injury. In a second experiment we have made a medial SMC-enriched preparation by removal of adventitial tissue and have demonstrated the mRNA precursor of the protein products.

Dr. Pedro J. del Nido (Pittsburgh, Pa.).
Correct me if I am wrong, but it is my understanding that one of the early triggers to this early gene expression is protein kinase C activation or translocation, and there are ways to inhibit that. Wouldn't it be simpler to tackle the early transduction signal rather than trying to inject or prevent new gene expression? Do you have any information about the early signal transduction?

Dr. Moggio.
This study did not look at this system, but we do have preliminary data in a rat aortic preparation that inhibition of protein kinase C can prevent some balloon injury induced expression of c-fos and other immediate-early genes. However, most inhibitors that we have used are not specific for selected PK isoforms, such as the zeta form.

Dr. John C. Alexander (Evanston, Ill.).
This paper shows a genetic reason for some of the things that we see in bypass grafts.

Did you use a pulse duplicator model in this study? I have made the clinical observation, as have many others, that atherosclerosis in vein grafts seems to surround valves The pressure changes that can be seen in association with bypass grafts and valves may be an interesting local area of this gene expression leading to atherosclerosis and thrombosis.

Dr. Moggio.
I think that is possible. We have not examined endothelial changes or pulsatile flow situations, but these genes are quite sensitive to mechanical and stretch stimuli.

Our technique requires the maintenance of high-quality mRNA, which we preserve in vitro with buffer solution. Careful incubation and processing are required to bring out the RNA signal. Cells not preserved in this manner will start to die and the mRNA will begin to deteriorate.

Dr. Andrew S. Wechsler (Richmond, Va.).
These early genes are sometimes just a manifestation of tissue response to injury. Would you accept that as another possibility, that it isn't really a precursor to development?

Dr. Moggio.
That is possible because the amount of manipulation to the vein was minimal, much less that is done during meticulous vein graft harvest and preparation. We are continuing to examine whether later genes that are stimulated are more predictive of cellular proliferation.

Footnotes

Read at the Seventy-fourth Annual Meeting of The American Association for Thoracic Surgery, New York, N.Y., April 24-27, 1994.

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Richard A. Moggio George E. Reed Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moggio, R. A. Articles by Reed, G. E. Search for Related Content PubMed PubMed Citation Articles by Moggio, R. A. Articles by Reed, G. E.