Antiproliferative Effect of Heparin on Human Smooth Muscle Cells Cultured from Intimal Hyperplastic Lesions of Vein Grafts Jeffrey J. Gilbertson, MD, Olive S. Pettengill, PhD, Jack L. Cronenwett, MD, Lebanon, New Hampshire
The antiproliferative effect of heparin on cultured smooth muscle cells in proliferating human smooth muscle cells derived from clinical lesions of intimal hyperplasia was tested. Smooth muscle cells were obtained from stenotic segments excised from failing in situ saphenous vein bypass grafts in three patients. The nonadventitial portion of the excised tissue was explanted into cell culture using standard techniques without the addition of exogenous growth factors. Under these conditions, rapid cell outgrowth was observed from these explants, in contrast to minimal growth of smooth muscle cells from normal veins from the same patients. Immunohistochemical staining with antiactin antibody confirmed that the cells cultured from the stenotic lesions were smooth muscle cells. Incubation of these cells with porcine mucosai heparin revealed a significant (p < .01) dose-dependent inhibition of cell proliferation as measured by radioactive thymidine incorporation. Mean inhibition of six subcultures tested ranged from 3 to 46%, at heparin concentrations of 1 to 1,000 /~g/ml. The magnitude of heparin's antiproliferative effect varied among the cell lines from different patients, but 10-30% inhibition was consistently observed at heparin concentrations usually attained in vivo. The maximal inhibition achieved was 65% in one cell line at the highest heparin dose. We conclude that heparin exerts a significant antiproliferative effect on human smooth muscle cells cultured from intimal hyperplastic lesions from in situ saphenous vein bypass grafts. (Ann Vasc Surg 1992;6:265-271). KEY WORDS: intimal hyperplasia; heparin; smooth muscle cells; saphenous vein grafts; antiproliferative effect.
Although autogenous vein grafts are used successfully for inffainguinal arterial reconstruction, a significant number occlude due to the development From the Section of Vascular Surgery, the Department of Surgery, and the Department of Pathology, DartmouthHitchcock Medical Center, Lebanon, New Hampshire. Presented at the 5th Annual Meeting of the Eastern Vascular Society, May 4, 1991, Pittsburgh, Pennsylvania. Reprint requests: Jack L. Cronenwett, MD, Section of Vascular Surgery, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, New Hampshire 03756.
of intimai hyperplasia within the graft. Szilagyi and associates found that 10% of lower extremity saphenous vein grafts developed focal stenoses and 8% developed diffuse thickening detected by arteriography after one to two years of follow-up [1]. When examined histologically, these changes in the vein grafts consist of proliferating smooth muscle cells (SMCs) in both the media and intima, with associated increased cellular matrix. All arterialized vein grafts demonstrate these changes to some degree as an adaptation to arterial conditions [2]. However, when this response becomes excessive, either diffusely or focally, luminal encroachment occurs, and
265
A N T I P R O L I F E R A TIVE EFFECT OF HEPAR1N
266
ANNALS OF VASCULAR SURGERY
graft thrombosis may result. The etiology of intimal hyperplasia is not completely understood, although platelets, endothelial cells, macrophages, and SMCs themselves likely play a role in regulating cellular proliferation. Because of the clinical significance of intimal hyperplasia, substantial research has focused on methods to prevent this problem. In 1977, Karnovsky and Clowes first reported that heparin exerted an antiproliferative effect on rat SMCs in vivo [3]. Subsequent studies have verified this observation in normal animal and human SMCs in culture [4--6]. To date, this inhibitory effect has not been documented in human cells cultured from lesions of intimal hyperplasia, the most likely disease process for which clinical administration of heparin might be appropriate. This is an important distinction, since human SMCs from intimal hyperplastic lesions have been shown to have markedly upregulated cell growth [7], which may render them unresponsive to heparin or other inhibitors of normal SMC proliferation. This study was undertaken to assess the effect of heparin on the growth of human SMCs cultured from lesions of intimal hyperplasia in failing lower extremity saphenous vein bypass grafts.
MATERIALS AND METHODS Tissue source
Fig. 1. Photomicrograph of representative intimal hyperplastic lesion from vein graft of Patient C. Marked intimal thickening with increased cellularity (bracket) separated from adjacent medial fibrosis by the internal elastic lamina (*) (2250X).
Stenotic segments from surgically revised in situ saphenous vein bypass grafts were obtained from three patients. All segments represented focal stenoses within the body of the grafts (not including the anastomoses), that were detected by duplex ultrasound surveillance and replaced from three to 14 months after initial graft implantation to prevent culture and replaced with a segment of saphenous anticipated graft failure. vein. Patient A was a 61-year-old man who required Patient C was an 82-year-old man who required two vein graft revisions within five months of his five graft revisions within a 13 month period. original femoral-dorsalis pedis in situ saphenous Each of these was secondary to sequential focal vein bypass. The specimen used for tissue culture stenoses which developed at several points along was obtained from an area of focal stenosis in the the graft, including the distal anastomosis. At least mid-thigh portion of this graft, three months after two were felt to be due to hyperplasia at the sites of implantation. previously lysed venous valves. The tissue used for Patient B was a 77-year-old woman who required three graft revisions for thrombosis or impending explant was derived from the graft near, but not thrombosis within a 14 month interval after her including, the strictured distal anastomosis at the below-knee femoropopliteal in situ saphenous vein time of a revision 13 months after the original graft was constructed. Initially, a mid-graft stenosis bypass. In each patient the vein segment removed was repaired with a local vein patch eight months appeared focally narrowed, fibrotic, pearly white, after implantation. This same area soon required and demonstrated severe intimal hyperplasia by replacement with an interposition saphenous vein light microscopy (Fig. 1). Use of normally dissegment. Later, at her third revision, a 2 cm seg- carded human tissue in this study was approved by ment of focally stenotic vein graft (proximal to the the Committee for the Protection of Human Subprevious revision) was excised, used for tissue jects of the Dartmouth-Hitchcock Medical Center.
VOLUME 6 N o 3 - 1992
ANT1PROLIFERAT1VE EFFECT OF H E P A R I N
Culture of human smooth muscle cells
Vein segments removed from the three revised grafts, as well as segments of normal vein from the same or similar patients were transported to the laboratory in buffered serum-free media [Dulbecco's modified Eagle's medium*, plus 15 mmol/1 4-(2-hydroxyethyl)- 1-piperazineethansulfonic acid (HEPES)], at which time they were opened longitudinally under a dissecting microscope. The luminal surface of the vein was scraped with a scalpel blade and washed with fresh media to remove endothelial cells. The inner one-half to two-thirds of the remaining vein wall was then sharply dissected away from the adventitial portion of the vessel. This intimal/medial tissue was then cut into 2 mm discs with a disposable skin biopsy punch and placed into 25 cm 2 culture flasks with a drop of buffered media [(DMEM/HEPES/10% newborn calf serum (NBCS)*]. After allowing a period of six to 12 hours for attachment, the explant discs were covered with media and incubated undisturbed for seven days (37°C, humidified 5% CO2). Examination at this time with phase-contrast microscopy revealed outgrowth of viable cells at the periphery of the tissue discs derived from the vein grafts. The tissue discs were then removed, leaving the cellular outgrowth intact and the medium was replaced twice weekly thereafter. Cells were subcultured when confluent by treatment with trypsin/ethylenediamineteraacetic acid (0.25%/0.05%) and reseeded at a concentration of 105 cells per 25 cm 2 culture flask. Immunohistochemistry
Cultured cells from the explants described above, in various subcultures, were also seeded on sterile microscope slides. After attachment and growth they were fixed in anhydrous acetone for 10 minutes and air dried. After incubation with equine serum to decrease nonspecific binding, the cells were incubated overnight at 25°C with HHF-35 ~, an actinspecific monoclonal antibody used to identify cells of smooth muscle origin [81. Bound actin-specific antibody was then detected with an avidin-biotin immunoperoxidase kit**. Established lines of human skeletal muscle cells, rabbit SMCs, and human skin fibroblasts, similarly seeded on sterile microscope slides, served as positive and negative controls. Smooth muscle cell proliferation assay
267
three patients were then studied to determine the effect of heparin on proliferation rate. Due to limitations in the number of cells available for study, the proliferation assay was carried out in a variable number of subcultures for each patient tested. Cells from patient A were tested in subculture 1, those from patient B in subcultures 2 and 4, and those from patient C in subcultures 1, 2, and 4. Cells from a given subculture were seeded into 96-weU tissue culture plates at a density of 2 x 104/cm2 in DMEM/15 mmol HEPES/10% NBCS. After 24 hours the cells were growth arrested by serum deprivation (0.5% NBCS) for 72 hours. The cells were then released from growth arrest by addition of fresh medium containing 10% serum. The effect of heparin on the subsequent SMC proliferation was then tested by the addition of either 0 (control), 1, 10, 100, or 1,000 /xg/ml of sodiumheparin (pork mucosal, 178 U/mg, Sigma) at the time of release from growth arrest. Eight replicate wells were used at each heparin concentration. Twenty-four hours after release the cells were treated with methyl-3H-thymidine (5 Ci/mmol thymidine, 1 mCi/ml, 1/xCi/well t*) and incubated for 24 hours. After a freeze/thaw cycle, the cells were harvested from the culture plates with a cell harvester (Model 7000~~), which collected the water insoluble cellular components (nuclei) from each well on a separate filter. After addition of 5 ml of scintillation fluid (Beta Max***), 3H-thymidine uptake, as an indicator of cellular DNA synthesis, was measured through liquid scintillation spectroscopy (Beckman 5000TD***). Uptake was recorded as counts per minute per well and reported as the mean of eight replicate wells per heparin concentration. Data were also expressed as the percent of control (no heparin) growth for each subculture in order to normalize different baseline labeling efficiencies and allow comparison between subcultures.
Statistical methods
Effects of heparin dosage on SMC proliferation was assessed by one way analysis of variance (ANOVA) for each smooth muscle cell subculture tested. Differences between control and individual heparin concentrations were analyzed by the Tukey-HSD test for multiple comparisons.
Smooth muscle cells (verified by immunohistochemistry and culture appearance) from each of the *Hyclone Laboratories, Logan, Utah. *Sigma Chemical Co., St. Louis, Missouri. "~Enzo Biochemicals, New York, New York. **Vector Laboratories, Burtingame, California.
**Amersham Corp., Arlington Heights, Illinois. ~Skatron Inc., Sterling, Virginia. ***ICN Radiochemicals, Irvine, California. ***Beckman Bioanalytical Systems, Wakefield, Massachusetts.
ANTIPROL1FERATIVE EFFECT OF H E P A R I N
268
ANNALS OF VASCULAR SURGERY
Fibroblast cultures exposed to the same antibody failed to stain, while cultures of human skeletal muscle and rabbit aortic smooth muscle stained with the same cabled appearance. Cells were subcultured at regular intervals, but after three to five passages would not undergo further cell division, even though they remained viable as judged by phase-contrast microscopy. Attempts at exploration of normal vein segments from the same elderly patients under the culture conditions used in this study resulted in too few cells to perform the proliferation assay. The tissues discs derived from these segments of normal vein showed no evidence of growth at seven days. After 10 to 14 days most of these explants showed outgrowth of a few viable cells, but these cells did not proliferate. Fig. 2. Photomicrograph of smooth muscle cell outgrowth from tissue explant after seven days in primary culture (Phase contrast, 260X).
RESULTS Characteristics of cultured cells
When first inspected seven days after placement into primary culture, radial cell outgrowth was observed in approximately 75% of the tissue discs from each of the three hyperplastic vein segments (Fig. 2). By 14 days, when the tissue discs were removed, a "hill and valley" morphology characteristic of cultured vascular SMCs was apparent. Cells from both primary and subsequent cultures were characterized by their positive reaction with HHF-35, an antiactin monoclonal antibody indicative of cells of muscular origin. Positively staining cells had the typical actin-cable appearance (Fig. 3).
Effect of heparin on smooth muscle growth
Heparin significantly inhibited SMC growth in all of the patients and subcultures tested (p < 0.01, ANOVA, Table I). The mean percentage inhibition compared to control (untreated) cells for all subcultures was 3%, 18%, 36%, and 43% for heparin concentrations of 1, 10, I00, and 1,000 ~g/ml, respectively. Cells from the three individual patients showed some difference in their response to heparin, but none was significantly different (p > . 12, Fig. 4). The slope and shape of the growth inhibition curves were similar for each of the different cell strains, although SMCs originating from patient B demonstrated the greatest degree of heparin inhibition. When analyzed at individual dosages, heparin concentrations of 10, 100, and 1,000 tzg/ml each caused statistically significant SMC growth inhibition overall (p < .01, Table I). Only the first subculture from Patient C demonstrated an inhibitory effect of heparin at a concentration of 1 /~g/ml. At a heparin concentration of 10 /~g/ml, significant inhibition was seen in cells from patient B, and in the first subculture from patient C. As the heparin concentration was increased to 100 p~g/ml, significant inhibition ranging from 22% to 50% was noted in all cell strains except the first subculture from patient A. At the highest dose tested, 1,000 ~g/ml, inhibition varied from 27% to 65% and was significant in all subcultures tested. Phase contrast microscopy of all cultures at all dosage levels confirmed cell viability and revealed no evidence of cell death due to toxicity.
DISCUSSION Fig. 3. Human smooth muscle cell (subculture 2) derived from vein graft intimal hyperplastic lesion. Immunohistochemical staining with antiactin monoclonal antibody HHF-35 demonstrating actin cables (4750X).
In this study, stenotic segments from in situ saphenous vein grafts in three patients were excised and replaced with segments of normal vein to prevent impending graft failure. The lesions respon-
VOLUME 6
ANTIPROLIFERATIVE EFFECT OF HEPARIN
N o 3 - 1992
269
TABLE I.mEffect of heparin on smooth muscle cell proliferation
Patient A SC 1 Patient B SC 2 Patient B SC 4 Patient C SC 1 Patient C SC 2 Patient C SC 4 All (mean)
Heparin concentration (/~g/ml) 10
0 (control)
1
100
1000
832 -- 73
829 +- 51
629 -+ 61
644 -+ 68
*549 +-- 49
1404 -+ 103
1276 -+ 111
t1006 - 61
t718 _+ 31
t526 _+ 24
981 -+ 62
1002 _+ 74
*730 -- 61
t488 -+ 27
t351 _ 12
1397 -+ 52
t1151 +-- 53
t1139 - 32
t925 -+ 32
t941 - 39
947 -+ 32
904 _+ 29
915 _+ 32
t724 -+ 23
t688 + 20
1766 +- 68
1635 -+ 89
1522 -- 83
t967 -+ 28
t986 -+ 20
1245 -+ 56
1152 _+ 50
*1008 _+ 49
t749 -+ 29
t679 _+ 37
3H Thymidine uptake in counts per minute. Each value is the mean of 8 replicate well -+ standard *p
The antiproliferative effect of heparin on cultured smooth muscle cells in proliferating human smooth muscle cells derived from clinical lesions of intimal hyperplasia was tested. Smooth muscle cells were obtained from stenotic segments excised from failing in situ saphenous vein bypass grafts in three patients. The nonadventitial portion of the excised tissue was explanted into cell culture using standard techniques without the addition of exogenous growth factors. Under these conditions, rapid cell outgrowth was observed from these explants, in contrast to minimal growth of smooth muscle cells from normal veins from the same patients. Immunohistochemical staining with antiactin antibody confirmed that the cells cultured from the stenotic lesions were smooth muscle cells. Incubation of these cells with porcine mucosai heparin revealed a significant (p < .01) dose-dependent inhibition of cell proliferation as measured by radioactive thymidine incorporation. Mean inhibition of six subcultures tested ranged from 3 to 46%, at heparin concentrations of 1 to 1,000 /~g/ml. The magnitude of heparin's antiproliferative effect varied among the cell lines from different patients, but 10-30% inhibition was consistently observed at heparin concentrations usually attained in vivo. The maximal inhibition achieved was 65% in one cell line at the highest heparin dose. We conclude that heparin exerts a significant antiproliferative effect on human smooth muscle cells cultured from intimal hyperplastic lesions from in situ saphenous vein bypass grafts. (Ann Vasc Surg 1992;6:265-271). KEY WORDS: intimal hyperplasia; heparin; smooth muscle cells; saphenous vein grafts; antiproliferative effect.
Although autogenous vein grafts are used successfully for inffainguinal arterial reconstruction, a significant number occlude due to the development From the Section of Vascular Surgery, the Department of Surgery, and the Department of Pathology, DartmouthHitchcock Medical Center, Lebanon, New Hampshire. Presented at the 5th Annual Meeting of the Eastern Vascular Society, May 4, 1991, Pittsburgh, Pennsylvania. Reprint requests: Jack L. Cronenwett, MD, Section of Vascular Surgery, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, New Hampshire 03756.
of intimai hyperplasia within the graft. Szilagyi and associates found that 10% of lower extremity saphenous vein grafts developed focal stenoses and 8% developed diffuse thickening detected by arteriography after one to two years of follow-up [1]. When examined histologically, these changes in the vein grafts consist of proliferating smooth muscle cells (SMCs) in both the media and intima, with associated increased cellular matrix. All arterialized vein grafts demonstrate these changes to some degree as an adaptation to arterial conditions [2]. However, when this response becomes excessive, either diffusely or focally, luminal encroachment occurs, and
265
A N T I P R O L I F E R A TIVE EFFECT OF HEPAR1N
266
ANNALS OF VASCULAR SURGERY
graft thrombosis may result. The etiology of intimal hyperplasia is not completely understood, although platelets, endothelial cells, macrophages, and SMCs themselves likely play a role in regulating cellular proliferation. Because of the clinical significance of intimal hyperplasia, substantial research has focused on methods to prevent this problem. In 1977, Karnovsky and Clowes first reported that heparin exerted an antiproliferative effect on rat SMCs in vivo [3]. Subsequent studies have verified this observation in normal animal and human SMCs in culture [4--6]. To date, this inhibitory effect has not been documented in human cells cultured from lesions of intimal hyperplasia, the most likely disease process for which clinical administration of heparin might be appropriate. This is an important distinction, since human SMCs from intimal hyperplastic lesions have been shown to have markedly upregulated cell growth [7], which may render them unresponsive to heparin or other inhibitors of normal SMC proliferation. This study was undertaken to assess the effect of heparin on the growth of human SMCs cultured from lesions of intimal hyperplasia in failing lower extremity saphenous vein bypass grafts.
MATERIALS AND METHODS Tissue source
Fig. 1. Photomicrograph of representative intimal hyperplastic lesion from vein graft of Patient C. Marked intimal thickening with increased cellularity (bracket) separated from adjacent medial fibrosis by the internal elastic lamina (*) (2250X).
Stenotic segments from surgically revised in situ saphenous vein bypass grafts were obtained from three patients. All segments represented focal stenoses within the body of the grafts (not including the anastomoses), that were detected by duplex ultrasound surveillance and replaced from three to 14 months after initial graft implantation to prevent culture and replaced with a segment of saphenous anticipated graft failure. vein. Patient A was a 61-year-old man who required Patient C was an 82-year-old man who required two vein graft revisions within five months of his five graft revisions within a 13 month period. original femoral-dorsalis pedis in situ saphenous Each of these was secondary to sequential focal vein bypass. The specimen used for tissue culture stenoses which developed at several points along was obtained from an area of focal stenosis in the the graft, including the distal anastomosis. At least mid-thigh portion of this graft, three months after two were felt to be due to hyperplasia at the sites of implantation. previously lysed venous valves. The tissue used for Patient B was a 77-year-old woman who required three graft revisions for thrombosis or impending explant was derived from the graft near, but not thrombosis within a 14 month interval after her including, the strictured distal anastomosis at the below-knee femoropopliteal in situ saphenous vein time of a revision 13 months after the original graft was constructed. Initially, a mid-graft stenosis bypass. In each patient the vein segment removed was repaired with a local vein patch eight months appeared focally narrowed, fibrotic, pearly white, after implantation. This same area soon required and demonstrated severe intimal hyperplasia by replacement with an interposition saphenous vein light microscopy (Fig. 1). Use of normally dissegment. Later, at her third revision, a 2 cm seg- carded human tissue in this study was approved by ment of focally stenotic vein graft (proximal to the the Committee for the Protection of Human Subprevious revision) was excised, used for tissue jects of the Dartmouth-Hitchcock Medical Center.
VOLUME 6 N o 3 - 1992
ANT1PROLIFERAT1VE EFFECT OF H E P A R I N
Culture of human smooth muscle cells
Vein segments removed from the three revised grafts, as well as segments of normal vein from the same or similar patients were transported to the laboratory in buffered serum-free media [Dulbecco's modified Eagle's medium*, plus 15 mmol/1 4-(2-hydroxyethyl)- 1-piperazineethansulfonic acid (HEPES)], at which time they were opened longitudinally under a dissecting microscope. The luminal surface of the vein was scraped with a scalpel blade and washed with fresh media to remove endothelial cells. The inner one-half to two-thirds of the remaining vein wall was then sharply dissected away from the adventitial portion of the vessel. This intimal/medial tissue was then cut into 2 mm discs with a disposable skin biopsy punch and placed into 25 cm 2 culture flasks with a drop of buffered media [(DMEM/HEPES/10% newborn calf serum (NBCS)*]. After allowing a period of six to 12 hours for attachment, the explant discs were covered with media and incubated undisturbed for seven days (37°C, humidified 5% CO2). Examination at this time with phase-contrast microscopy revealed outgrowth of viable cells at the periphery of the tissue discs derived from the vein grafts. The tissue discs were then removed, leaving the cellular outgrowth intact and the medium was replaced twice weekly thereafter. Cells were subcultured when confluent by treatment with trypsin/ethylenediamineteraacetic acid (0.25%/0.05%) and reseeded at a concentration of 105 cells per 25 cm 2 culture flask. Immunohistochemistry
Cultured cells from the explants described above, in various subcultures, were also seeded on sterile microscope slides. After attachment and growth they were fixed in anhydrous acetone for 10 minutes and air dried. After incubation with equine serum to decrease nonspecific binding, the cells were incubated overnight at 25°C with HHF-35 ~, an actinspecific monoclonal antibody used to identify cells of smooth muscle origin [81. Bound actin-specific antibody was then detected with an avidin-biotin immunoperoxidase kit**. Established lines of human skeletal muscle cells, rabbit SMCs, and human skin fibroblasts, similarly seeded on sterile microscope slides, served as positive and negative controls. Smooth muscle cell proliferation assay
267
three patients were then studied to determine the effect of heparin on proliferation rate. Due to limitations in the number of cells available for study, the proliferation assay was carried out in a variable number of subcultures for each patient tested. Cells from patient A were tested in subculture 1, those from patient B in subcultures 2 and 4, and those from patient C in subcultures 1, 2, and 4. Cells from a given subculture were seeded into 96-weU tissue culture plates at a density of 2 x 104/cm2 in DMEM/15 mmol HEPES/10% NBCS. After 24 hours the cells were growth arrested by serum deprivation (0.5% NBCS) for 72 hours. The cells were then released from growth arrest by addition of fresh medium containing 10% serum. The effect of heparin on the subsequent SMC proliferation was then tested by the addition of either 0 (control), 1, 10, 100, or 1,000 /xg/ml of sodiumheparin (pork mucosal, 178 U/mg, Sigma) at the time of release from growth arrest. Eight replicate wells were used at each heparin concentration. Twenty-four hours after release the cells were treated with methyl-3H-thymidine (5 Ci/mmol thymidine, 1 mCi/ml, 1/xCi/well t*) and incubated for 24 hours. After a freeze/thaw cycle, the cells were harvested from the culture plates with a cell harvester (Model 7000~~), which collected the water insoluble cellular components (nuclei) from each well on a separate filter. After addition of 5 ml of scintillation fluid (Beta Max***), 3H-thymidine uptake, as an indicator of cellular DNA synthesis, was measured through liquid scintillation spectroscopy (Beckman 5000TD***). Uptake was recorded as counts per minute per well and reported as the mean of eight replicate wells per heparin concentration. Data were also expressed as the percent of control (no heparin) growth for each subculture in order to normalize different baseline labeling efficiencies and allow comparison between subcultures.
Statistical methods
Effects of heparin dosage on SMC proliferation was assessed by one way analysis of variance (ANOVA) for each smooth muscle cell subculture tested. Differences between control and individual heparin concentrations were analyzed by the Tukey-HSD test for multiple comparisons.
Smooth muscle cells (verified by immunohistochemistry and culture appearance) from each of the *Hyclone Laboratories, Logan, Utah. *Sigma Chemical Co., St. Louis, Missouri. "~Enzo Biochemicals, New York, New York. **Vector Laboratories, Burtingame, California.
**Amersham Corp., Arlington Heights, Illinois. ~Skatron Inc., Sterling, Virginia. ***ICN Radiochemicals, Irvine, California. ***Beckman Bioanalytical Systems, Wakefield, Massachusetts.
ANTIPROL1FERATIVE EFFECT OF H E P A R I N
268
ANNALS OF VASCULAR SURGERY
Fibroblast cultures exposed to the same antibody failed to stain, while cultures of human skeletal muscle and rabbit aortic smooth muscle stained with the same cabled appearance. Cells were subcultured at regular intervals, but after three to five passages would not undergo further cell division, even though they remained viable as judged by phase-contrast microscopy. Attempts at exploration of normal vein segments from the same elderly patients under the culture conditions used in this study resulted in too few cells to perform the proliferation assay. The tissues discs derived from these segments of normal vein showed no evidence of growth at seven days. After 10 to 14 days most of these explants showed outgrowth of a few viable cells, but these cells did not proliferate. Fig. 2. Photomicrograph of smooth muscle cell outgrowth from tissue explant after seven days in primary culture (Phase contrast, 260X).
RESULTS Characteristics of cultured cells
When first inspected seven days after placement into primary culture, radial cell outgrowth was observed in approximately 75% of the tissue discs from each of the three hyperplastic vein segments (Fig. 2). By 14 days, when the tissue discs were removed, a "hill and valley" morphology characteristic of cultured vascular SMCs was apparent. Cells from both primary and subsequent cultures were characterized by their positive reaction with HHF-35, an antiactin monoclonal antibody indicative of cells of muscular origin. Positively staining cells had the typical actin-cable appearance (Fig. 3).
Effect of heparin on smooth muscle growth
Heparin significantly inhibited SMC growth in all of the patients and subcultures tested (p < 0.01, ANOVA, Table I). The mean percentage inhibition compared to control (untreated) cells for all subcultures was 3%, 18%, 36%, and 43% for heparin concentrations of 1, 10, I00, and 1,000 ~g/ml, respectively. Cells from the three individual patients showed some difference in their response to heparin, but none was significantly different (p > . 12, Fig. 4). The slope and shape of the growth inhibition curves were similar for each of the different cell strains, although SMCs originating from patient B demonstrated the greatest degree of heparin inhibition. When analyzed at individual dosages, heparin concentrations of 10, 100, and 1,000 tzg/ml each caused statistically significant SMC growth inhibition overall (p < .01, Table I). Only the first subculture from Patient C demonstrated an inhibitory effect of heparin at a concentration of 1 /~g/ml. At a heparin concentration of 10 /~g/ml, significant inhibition was seen in cells from patient B, and in the first subculture from patient C. As the heparin concentration was increased to 100 p~g/ml, significant inhibition ranging from 22% to 50% was noted in all cell strains except the first subculture from patient A. At the highest dose tested, 1,000 ~g/ml, inhibition varied from 27% to 65% and was significant in all subcultures tested. Phase contrast microscopy of all cultures at all dosage levels confirmed cell viability and revealed no evidence of cell death due to toxicity.
DISCUSSION Fig. 3. Human smooth muscle cell (subculture 2) derived from vein graft intimal hyperplastic lesion. Immunohistochemical staining with antiactin monoclonal antibody HHF-35 demonstrating actin cables (4750X).
In this study, stenotic segments from in situ saphenous vein grafts in three patients were excised and replaced with segments of normal vein to prevent impending graft failure. The lesions respon-
VOLUME 6
ANTIPROLIFERATIVE EFFECT OF HEPARIN
N o 3 - 1992
269
TABLE I.mEffect of heparin on smooth muscle cell proliferation
Patient A SC 1 Patient B SC 2 Patient B SC 4 Patient C SC 1 Patient C SC 2 Patient C SC 4 All (mean)
Heparin concentration (/~g/ml) 10
0 (control)
1
100
1000
832 -- 73
829 +- 51
629 -+ 61
644 -+ 68
*549 +-- 49
1404 -+ 103
1276 -+ 111
t1006 - 61
t718 _+ 31
t526 _+ 24
981 -+ 62
1002 _+ 74
*730 -- 61
t488 -+ 27
t351 _ 12
1397 -+ 52
t1151 +-- 53
t1139 - 32
t925 -+ 32
t941 - 39
947 -+ 32
904 _+ 29
915 _+ 32
t724 -+ 23
t688 + 20
1766 +- 68
1635 -+ 89
1522 -- 83
t967 -+ 28
t986 -+ 20
1245 -+ 56
1152 _+ 50
*1008 _+ 49
t749 -+ 29
t679 _+ 37
3H Thymidine uptake in counts per minute. Each value is the mean of 8 replicate well -+ standard *p
