Reduction of Intimal Hyperplasia and Enhanced Reactivity of Experimental Vein Bypass Grafts with Verapamil Treatment
MOSTAFA N. EL-SANADIKI, M.D.,* K. SIMON CROSS, M. MED. SCI., F.R.C.S.(ED),* JOHN J. MURRAY, M.D., PH.D.,t ROBERT W. SCHUMAN, M.D.,* EILEEN MIKAT, PH.D.,4 RICHARD L. MCCANN, M.D.,* and PER-OTTO HAGEN, PH.D.* § From the Departments of Surgery,* Medicine,t Pathology,t and Biochemistry,§ Duke University Medical Center, Durham, North Carolina
Recent studies have shown that calcium antagonists exert an antiatherogenic effect in animals fed cholesterol. Vein graft intimal hyperplasia is believed to be an early event in atherosclerotic lesion formation, which is a significant cause of graft failure. Altered vasoreactivity has also been postulated in the etiology of vein graft failure. Therefore this study examined the effect of verapamil treatment on the development of intimal hyperplasia and the vasoreactivity of experimental vein bypass grafts. The right external jugular vein was grafted into the right carotid artery of 30 male New Zealand white rabbits fed normal rabbit chow. The left external jugular vein was used as the control vein. Fifteen animals received verapamil (1.25 mg/day for 28 days) via the femoral vein by means of an osmotic pump. In 15 control animals the pump contained saline. Plasma verapamil concentration was 50.9 ± 13.2 ng/mL (x ± SEM), a dose that showed no effect on either blood pressure, total serum cholesterol, or in vitro platelet aggregation to ADP. Fourteen of fifteen grafts were patent in each group, for a patency rate of 93%. Histologic examination using computer morphometry showed significant reduction of intimal hyperplasia at the proximal, middle, and distal graft segments (p < 0.05). In addition in vitro isometric tension studies of the vein grafts,and control veins showed that verapamil causes enhanced reactivity of both vein grafts and control veins in response to norepinephrine and histamine (p < 0.05). Reactivity of vein grafts to serotonin was unaltered. While none of the normal veins in the control group responded to serotonin, normal veins treated with verapamil contracted readily in response to serotonin. Endothelial-dependent relaxation to acetylcholine was absent in both control and verapamil-treated vein grafts, while normal veins from both groups responded to the same extent to acetylcholine. Because we could not demonstrate any difference in platelet or endothelium function between untreated and verapamil-treated animals, we examined the direct effect of verapamil on smooth muscle. Verapamil significantly inhibited [Hj-thymidine incorporation into DNA in vascular smooth muscle cells
in culture in a dose-dependent manner. Verapamil treatment significantly reduces intimal hyperplasia in experimental vein grafts and inhibits smooth muscle cell proliferation in culture. Furthermore the enhanced reactivity to norepinephrine and histamine in the verapamil-treated vessels has no detrimental effect on the patency rate at 4 weeks. Thus by inhibiting intimal hyperplasia, calcium antagonists may improve the long-term patency of vein bypass grafts.
LINICALLY CALCIUM ANTAGONISTS are used in the treatment of a variety of cardiovascular disorders' and frequently are administered to patients who undergo bypass procedures. Studies on various experimental models of atherosclerosis have indicated an antiatherogenic effect of various calcium antagonists such as nifedipine,2 verapamil,3 diltiazem,4 and lanthanum.5 Furthermore we6 and others7-9 have shown that treatment with calcium antagonists significantly reduces the development of intimal hyperplasia after mechanical injury of arteries. The underlying mechanism of this action is still unclear. The reversed autogenous vein has long been the graft material of choice for treatment of occlusive vascular disease. However the reported high failure rates of 10% to 40% at 1 year'0 due mainly to thrombotic events and of 50% to 60% at 10 years" due to progressive intimal hyperplasia are considered to be major clinical problems. The pathogenesis of vein graft occlusion appears to have some similarities to the development and progression of atherosclerosis. Endothelial cell injury is thought to be the precipitating event in both situations.'2 Several investigators have shown that aspirin, dipyridamole,'3 and heparin '4 reduce the development of intimal hyperplasia in vein bypass grafts. C
Supported by National Institute of Health grants HL- 15448 and HL32720. Dr. Murray's present address is Department of Medicine and Pharmacology, Vanderbilt Medical Center, Nashville, TN 37232. JJM is an RJR Nabisco Scholar. Address reprint requests to Dr. Per-Otto Hagen, Atherosclerosis Research Laboratory, P.O. Box 3473, Duke University Medical Center, Durham, NC 27710. Accepted for publication November 8, 1989.
87
88
EL-SANADIKI AND OTHERS
Although vasospasm has been reported as an etiology for vein graft failure,'5 little information is available on vein graft vasomotor function. Calcium has been implicated in a number of the events involved in the etiology of vein graft failure, including aggregation of platelets, release of platelet-derived growth factor (PDGF),16 proliferation'7 and migration'8 of smooth muscle cells, and extracellular matrix formation.'9 Therefore it is reasonable to suppose that interference with intracellular calcium concentration may have a beneficial role in reducing vein graft occlusion rate. Calcium is also involved in the production of endothelium-derived relaxing factor (EDRF) and prostacyclin,20'21 suggesting that the use of calcium antagonists may play a role in modulation of vascular reactivity. In this study we examined the effect of chronic verapamil treatment on vasomotor function and the development of intimal hyperplasia in autogenous experimental vein bypass grafts. To examine other mechanisms by which verapamil may alter intimal hyperplasia, the effects of chronic verapamil treatment on total cholesterol concentration, blood pressure, in vitro platelet function, and DNA synthesis in smooth muscle cells in culture were also investigated. Methods
Experimental Design Male New Zealand white rabbits underwent autogenous vein bypass grafting of the right carotid artery using the right external jugular vein. At surgery an osmotic pump was also placed and verapamil was administered via the femoral vein during the next 28 days. In control animals the pump contained vehicle (saline) only. Plasma verapamil, total serum cholesterol concentration, and blood pressure were determined before and 1 and 3 weeks after initiation of therapy. In vitro platelet aggregation to ADP was tested in both groups. After 4 weeks the vein grafts and the left external jugular vein (control vein) were harvested. Intimal hyperplasia was measured in cross-sections of the proximal (n = 1 1), middle (n = 13), and distal (n = 11) graft segments. Segments of vein grafts (n = 10) and control veins (n = 10) were suspended in an organ bath to assess endothelial cell function and to determine the contractile responses to norepinephrine, histamine, and serotonin. Animal Operations
Thirty male New Zealand white rabbits (weighing 2 to 2.5 kg) were used in this study. All animals were fed normal rabbit chow for the duration of the experiment. Fifteen animals received verapamil hydrochloride (dissolved in saline; Sigma Chemical Co., St. Louis, MO) at a dose of 1.25 mg/day for 28 days via the femoral vein by means
Ann. Surg. * July 1990
of an osmotic pump (the Alzet osmotic pump, model 2ML4, Alza, Palo Alto, CA). In 15 control animals the pump contained sterile saline solution.
Surgical Procedures Anesthesia was induced with Ketamine hydrochloride (60 mg/kg, Ketaset, Aveco Co., Inc., Fort Dodge, IA), xylazine (6 mg/kg, Rompun, Mobay Corp., Shawnee, KA), and acepromazine maleate (1.5 mg/kg, PromAce, Aveco Co., Inc., Fort Dodge, IA). Antibiotic prophylaxis was provided by giving one intramuscular dose of benzathine and procaine penicillin (30,000 units/kg, Flo-cillin, Aveco Co., Inc.) at induction of anesthesia. The surgical procedures were performed using clean conditions and an operating microscope (JKH 1420, Edward Weck, Inc., Research Triangle Park, NC). Through a small longitudinal inguinal incision, the right femoral vein was dissected. An EP-60 catheter (Intramedic, polyethylene tube, Clay Adams, Parsippany, NJ) attached to the osmotic pump was inserted via a small transverse venotomy and fixed to the vein. The pump was placed in the subcutaneous tissue and the wound was closed. After exposure through a right longitudinal neck incision, the right external jugular vein was carefully dissected, excised, and maintained in heparinized saline (5 units/mL, ElkinsSinn, Inc., Cherry Hill, NJ) for approximately 15 minutes while the right common carotid artery was exposed and mobilized. Heparin (200 units/kg) was administered intravenously. After proximal and distal control ofthe right common carotid artery, a proximal longitudinal arteriotomy was made. The reversed external jugular vein was then anastomosed to the artery in an end-to-side manner using a continuous 10-0 microvascular monofilament nylon suture (Ethilon, Ethicon, Inc., NJ). The distal anastomosis was performed in a similar manner. Care was taken throughout the procedure to avoid unnecessary instrumentation of the vein segment. The right common carotid artery was ligated and divided between the two anastomoses. The artery was then unclamped, hemostasis was obtained, and the wound was closed. Four weeks after grafting, each rabbit was anesthetized and the graft was harvested without perfusion fixation because we have previously determined that this is unnecessary for histologic examination of the grafts (unpublished data). The previously unmanipulated left external jugular vein was excised and used as a control vein for the in vitro isometric tension studies. Animal care complied with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1985). Plasma Levels and Physiologic Effect of Verapamil Blood samples were drawn from the ear vein for determination of plasma verapamil and total serum choles-
Vol. 212 No. I
INTIMAL HYPERPLASIA AND EXPEFUIMENTAL VEIN BYPASS GRAFTS
terol concentrations. Plasma samples for verapamil were assayed by high-pressure liquid chromatography (HPLC, Pumpsystem:LCD, Riviera Beach, FL) based on the method of Harapat.22 Verapamil was extracted in ethyl ether and after evaporation the drug residue was reconstituted in the HPLC solvent and injected onto the column (Chromanetics Corp., Kensington, MD). Verapamil was quantitated using a fluorescence detector (FS 970, Schoeffel Instruments, Westwood, NJ). Total serum cholesterol was determined by the method ofChiamori and Henry,23 using ferric chloride and glacial acetic acid. Blood pressure was measured before and after treatment via the middle ear artery of the rabbit using a Millar Micro-Tip pressure transducer (Millar Instruments, Inc., Houston, TX). In vitro platelet aggregation was tested in both groups. Citrated blood samples were collected and ADP at a concentration from 0.2 ,umol/L (micromolar) to 2.0 ,tmol/L was added to platelet-rich plasma adjusted to 300,000 platelets/,uL with platelet-poor plasma.24 Aggregation was measured by light transmission using a Chrono-log aggregometer (Chrono-log model 335, Havertown, PA). In Vitro Isometric Tension Studies
Vessel rings, 4 to 5 mm in length, were cut from both the vein graft and the control vein (two rings of tissue from each vessel). The vessel rings were suspended by stainless steel hooks in an organ bath containing Krebs solution of the following millimolar composition: NaCl 122; KC1 4.7; MgCl2 1.2; CaCl2 2.5; NaHCO3 15.4; KH2PO4 1.2; glucose 5.5, bubbled with 95% 02 and 5% CO2 (pH 7.4) and maintained at 37 C. One hook was fixed to the bottom of the bath and the other hook was connected to a force transducer (Myograph F-60, Narco Bio Systems, Houston, TX). Tension was recorded on a multichannel chart recorder (Physiograph MKIII-S, Narco Bio Systems, Houston, TX). The vessel rings were allowed to equilibrate at a constant tension of 0.5 g for 1 hour. During the equilibration period, the Krebs' solution in the organ bath was replaced every 30 minutes. Cumulative addition of norepinephrine (dissolved in 10-3 mol/L [molar] HC1, Sigma Chemical Co.) to the organ bath resulted in sigmoid dose-response curves. After a washout and re-equilibration period of 30 minutes, endothelium-dependent relaxation was studied by adding acetylcholine (10-8 mol/L to 10-5 mol/L) to the vessel rings precontracted with the ED50 dose (the concentration of agonist required to elicit 50% of the maximal response) of norepinephrine. After further washouts and re-equilibration periods, dose-response curves were obtained in response to histamine and serotonin (dissolved in distilled water; Sigma Chemical Co.). These curves were normalized in terms of the percentage of maximal contraction, and Probit
89
analysis (SAS, SAS Institute, Inc., Cary, NC) was used to calculate the ED50 value for each ring of tissue. After further washouts, normal veins from the verapamil-treated animals were incubated with phentolamine [10-6 mol/L] for 30 minutes and then the ED50 dose of serotonin was added to the organ bath.
Histologic Methods The morphology of the control and grafted vessels from both groups was examined by light microscopy. Four weeks after surgery, the proximal, middle, and distal graft segments were excised and fixed in 10% formalin. Crosssections were dehydrated and embedded in paraffin. Sixmicron sections were cut and stained with a modified Masson's trichrome and Verhoeffs elastic tissue stain. Intimal thickening was measured by computerized videometric analysis (Innovision 100, American Innovision Inc., San Diego, CA). The average intimal thickness was calculated from 20 measurements of intimal thickness taken at even intervals around the circumference of each section.
[3H] Thymidine Incorporation in Vascular Smooth Muscle Cells Vascular smooth muscle cells from the media of the thoracic aorta of male New Zealand white rabbits were isolated and grown in tissue culture. Cells in the 6th through 12th passage were used for these experiments. Confluent cells in 75 cm2 culture flasks (Falcon Plastics, Lincoln Park, NJ) were washed twice with 8 mL phosphate-buffered saline (Gibco Laboratories, Grand Island, NY) followed by trypsinization for 7 minutes at 37 C with 8 mL trypsin/EDTA solution (Gibco). Three milliliters of Dulbecco's minimal essential medium (Gibco) with 10% fetal calf serum (Gibco) was then added to inactivate the trypsin. The cells were isolated by centrifugation (250g, for 10 minutes) and resuspended in Dulbecco's minimal essential medium with 10% fetal calf serum containing penicillin (100 ,u/mL) (growth medium, Gibco). Culture wells (6-well cluster dishes, 35 mm diameter; Falcon Plastics) were innoculated with 2 mL growth medium containing 5 X 10' cells. The growth medium was replaced with new medium every third day until the cells reached confluency as judged by microscopic examination. The cells grew in hills and valleys typical of vascular smooth muscle cells. On reaching confluency, the medium was not changed for 4 days. This regimen produced a quiescent state in the cells similar to that achieved by incubating the cells in minimal essential medium containing 0. 1% fetal calf serum. On the day of the experiment, new growth medium was exchanged 4 hours and again 2 hours before the initiation of the experiment. The experiments were initiated by exchanging the growth medium with new medium contain-
*J -.:
EL-SANADIKI AND OTHERS
90
ing 4 ,umCi [3H]-thymidine (New England Nuclear, Boston MA) and various concentrations of verapamil. The concentration of verapamil used ranged from 0 to 100 ,umol/L. Incorporation of [3H]-thymidine into acid-precipitable material was measured after a 24-hour incubation period (air plus 5% CO2 at 37 C). At harvest the dishes were placed on ice and each well was washed twice with 2 mL of ice-cold Dulbecco's phosphate-buffered saline (Gibco). Two milliliters ice-cold 5% trichloracetic acid was added. After 5 minutes the acid was removed and each well washed with 3 mL ice-cold water. Sodium hydroxide (1 mL, 0.3N) was then added and the dishes were incubated for 10 minutes at 37 C. A 100-,uL aliquot of the sodium hydroxide solution was counted in 10 mL Biofluor (New England Nuclear, Boston, MA) by a Packhard Tri-Carb 4530 scintillation counter (Packard Instruments Inc., Downers Grove, IL). Results are expressed as percentage of control.
Ann. Surg. * July 1990
TABLE 1. Effect of Verapamil on Vein Graft Intimal Thickness
Intimal Hyperplasia Group
Proximal
Middle
Distal
Control Verapamil p value
252 ± 19 195 ± 7
MOSTAFA N. EL-SANADIKI, M.D.,* K. SIMON CROSS, M. MED. SCI., F.R.C.S.(ED),* JOHN J. MURRAY, M.D., PH.D.,t ROBERT W. SCHUMAN, M.D.,* EILEEN MIKAT, PH.D.,4 RICHARD L. MCCANN, M.D.,* and PER-OTTO HAGEN, PH.D.* § From the Departments of Surgery,* Medicine,t Pathology,t and Biochemistry,§ Duke University Medical Center, Durham, North Carolina
Recent studies have shown that calcium antagonists exert an antiatherogenic effect in animals fed cholesterol. Vein graft intimal hyperplasia is believed to be an early event in atherosclerotic lesion formation, which is a significant cause of graft failure. Altered vasoreactivity has also been postulated in the etiology of vein graft failure. Therefore this study examined the effect of verapamil treatment on the development of intimal hyperplasia and the vasoreactivity of experimental vein bypass grafts. The right external jugular vein was grafted into the right carotid artery of 30 male New Zealand white rabbits fed normal rabbit chow. The left external jugular vein was used as the control vein. Fifteen animals received verapamil (1.25 mg/day for 28 days) via the femoral vein by means of an osmotic pump. In 15 control animals the pump contained saline. Plasma verapamil concentration was 50.9 ± 13.2 ng/mL (x ± SEM), a dose that showed no effect on either blood pressure, total serum cholesterol, or in vitro platelet aggregation to ADP. Fourteen of fifteen grafts were patent in each group, for a patency rate of 93%. Histologic examination using computer morphometry showed significant reduction of intimal hyperplasia at the proximal, middle, and distal graft segments (p < 0.05). In addition in vitro isometric tension studies of the vein grafts,and control veins showed that verapamil causes enhanced reactivity of both vein grafts and control veins in response to norepinephrine and histamine (p < 0.05). Reactivity of vein grafts to serotonin was unaltered. While none of the normal veins in the control group responded to serotonin, normal veins treated with verapamil contracted readily in response to serotonin. Endothelial-dependent relaxation to acetylcholine was absent in both control and verapamil-treated vein grafts, while normal veins from both groups responded to the same extent to acetylcholine. Because we could not demonstrate any difference in platelet or endothelium function between untreated and verapamil-treated animals, we examined the direct effect of verapamil on smooth muscle. Verapamil significantly inhibited [Hj-thymidine incorporation into DNA in vascular smooth muscle cells
in culture in a dose-dependent manner. Verapamil treatment significantly reduces intimal hyperplasia in experimental vein grafts and inhibits smooth muscle cell proliferation in culture. Furthermore the enhanced reactivity to norepinephrine and histamine in the verapamil-treated vessels has no detrimental effect on the patency rate at 4 weeks. Thus by inhibiting intimal hyperplasia, calcium antagonists may improve the long-term patency of vein bypass grafts.
LINICALLY CALCIUM ANTAGONISTS are used in the treatment of a variety of cardiovascular disorders' and frequently are administered to patients who undergo bypass procedures. Studies on various experimental models of atherosclerosis have indicated an antiatherogenic effect of various calcium antagonists such as nifedipine,2 verapamil,3 diltiazem,4 and lanthanum.5 Furthermore we6 and others7-9 have shown that treatment with calcium antagonists significantly reduces the development of intimal hyperplasia after mechanical injury of arteries. The underlying mechanism of this action is still unclear. The reversed autogenous vein has long been the graft material of choice for treatment of occlusive vascular disease. However the reported high failure rates of 10% to 40% at 1 year'0 due mainly to thrombotic events and of 50% to 60% at 10 years" due to progressive intimal hyperplasia are considered to be major clinical problems. The pathogenesis of vein graft occlusion appears to have some similarities to the development and progression of atherosclerosis. Endothelial cell injury is thought to be the precipitating event in both situations.'2 Several investigators have shown that aspirin, dipyridamole,'3 and heparin '4 reduce the development of intimal hyperplasia in vein bypass grafts. C
Supported by National Institute of Health grants HL- 15448 and HL32720. Dr. Murray's present address is Department of Medicine and Pharmacology, Vanderbilt Medical Center, Nashville, TN 37232. JJM is an RJR Nabisco Scholar. Address reprint requests to Dr. Per-Otto Hagen, Atherosclerosis Research Laboratory, P.O. Box 3473, Duke University Medical Center, Durham, NC 27710. Accepted for publication November 8, 1989.
87
88
EL-SANADIKI AND OTHERS
Although vasospasm has been reported as an etiology for vein graft failure,'5 little information is available on vein graft vasomotor function. Calcium has been implicated in a number of the events involved in the etiology of vein graft failure, including aggregation of platelets, release of platelet-derived growth factor (PDGF),16 proliferation'7 and migration'8 of smooth muscle cells, and extracellular matrix formation.'9 Therefore it is reasonable to suppose that interference with intracellular calcium concentration may have a beneficial role in reducing vein graft occlusion rate. Calcium is also involved in the production of endothelium-derived relaxing factor (EDRF) and prostacyclin,20'21 suggesting that the use of calcium antagonists may play a role in modulation of vascular reactivity. In this study we examined the effect of chronic verapamil treatment on vasomotor function and the development of intimal hyperplasia in autogenous experimental vein bypass grafts. To examine other mechanisms by which verapamil may alter intimal hyperplasia, the effects of chronic verapamil treatment on total cholesterol concentration, blood pressure, in vitro platelet function, and DNA synthesis in smooth muscle cells in culture were also investigated. Methods
Experimental Design Male New Zealand white rabbits underwent autogenous vein bypass grafting of the right carotid artery using the right external jugular vein. At surgery an osmotic pump was also placed and verapamil was administered via the femoral vein during the next 28 days. In control animals the pump contained vehicle (saline) only. Plasma verapamil, total serum cholesterol concentration, and blood pressure were determined before and 1 and 3 weeks after initiation of therapy. In vitro platelet aggregation to ADP was tested in both groups. After 4 weeks the vein grafts and the left external jugular vein (control vein) were harvested. Intimal hyperplasia was measured in cross-sections of the proximal (n = 1 1), middle (n = 13), and distal (n = 11) graft segments. Segments of vein grafts (n = 10) and control veins (n = 10) were suspended in an organ bath to assess endothelial cell function and to determine the contractile responses to norepinephrine, histamine, and serotonin. Animal Operations
Thirty male New Zealand white rabbits (weighing 2 to 2.5 kg) were used in this study. All animals were fed normal rabbit chow for the duration of the experiment. Fifteen animals received verapamil hydrochloride (dissolved in saline; Sigma Chemical Co., St. Louis, MO) at a dose of 1.25 mg/day for 28 days via the femoral vein by means
Ann. Surg. * July 1990
of an osmotic pump (the Alzet osmotic pump, model 2ML4, Alza, Palo Alto, CA). In 15 control animals the pump contained sterile saline solution.
Surgical Procedures Anesthesia was induced with Ketamine hydrochloride (60 mg/kg, Ketaset, Aveco Co., Inc., Fort Dodge, IA), xylazine (6 mg/kg, Rompun, Mobay Corp., Shawnee, KA), and acepromazine maleate (1.5 mg/kg, PromAce, Aveco Co., Inc., Fort Dodge, IA). Antibiotic prophylaxis was provided by giving one intramuscular dose of benzathine and procaine penicillin (30,000 units/kg, Flo-cillin, Aveco Co., Inc.) at induction of anesthesia. The surgical procedures were performed using clean conditions and an operating microscope (JKH 1420, Edward Weck, Inc., Research Triangle Park, NC). Through a small longitudinal inguinal incision, the right femoral vein was dissected. An EP-60 catheter (Intramedic, polyethylene tube, Clay Adams, Parsippany, NJ) attached to the osmotic pump was inserted via a small transverse venotomy and fixed to the vein. The pump was placed in the subcutaneous tissue and the wound was closed. After exposure through a right longitudinal neck incision, the right external jugular vein was carefully dissected, excised, and maintained in heparinized saline (5 units/mL, ElkinsSinn, Inc., Cherry Hill, NJ) for approximately 15 minutes while the right common carotid artery was exposed and mobilized. Heparin (200 units/kg) was administered intravenously. After proximal and distal control ofthe right common carotid artery, a proximal longitudinal arteriotomy was made. The reversed external jugular vein was then anastomosed to the artery in an end-to-side manner using a continuous 10-0 microvascular monofilament nylon suture (Ethilon, Ethicon, Inc., NJ). The distal anastomosis was performed in a similar manner. Care was taken throughout the procedure to avoid unnecessary instrumentation of the vein segment. The right common carotid artery was ligated and divided between the two anastomoses. The artery was then unclamped, hemostasis was obtained, and the wound was closed. Four weeks after grafting, each rabbit was anesthetized and the graft was harvested without perfusion fixation because we have previously determined that this is unnecessary for histologic examination of the grafts (unpublished data). The previously unmanipulated left external jugular vein was excised and used as a control vein for the in vitro isometric tension studies. Animal care complied with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1985). Plasma Levels and Physiologic Effect of Verapamil Blood samples were drawn from the ear vein for determination of plasma verapamil and total serum choles-
Vol. 212 No. I
INTIMAL HYPERPLASIA AND EXPEFUIMENTAL VEIN BYPASS GRAFTS
terol concentrations. Plasma samples for verapamil were assayed by high-pressure liquid chromatography (HPLC, Pumpsystem:LCD, Riviera Beach, FL) based on the method of Harapat.22 Verapamil was extracted in ethyl ether and after evaporation the drug residue was reconstituted in the HPLC solvent and injected onto the column (Chromanetics Corp., Kensington, MD). Verapamil was quantitated using a fluorescence detector (FS 970, Schoeffel Instruments, Westwood, NJ). Total serum cholesterol was determined by the method ofChiamori and Henry,23 using ferric chloride and glacial acetic acid. Blood pressure was measured before and after treatment via the middle ear artery of the rabbit using a Millar Micro-Tip pressure transducer (Millar Instruments, Inc., Houston, TX). In vitro platelet aggregation was tested in both groups. Citrated blood samples were collected and ADP at a concentration from 0.2 ,umol/L (micromolar) to 2.0 ,tmol/L was added to platelet-rich plasma adjusted to 300,000 platelets/,uL with platelet-poor plasma.24 Aggregation was measured by light transmission using a Chrono-log aggregometer (Chrono-log model 335, Havertown, PA). In Vitro Isometric Tension Studies
Vessel rings, 4 to 5 mm in length, were cut from both the vein graft and the control vein (two rings of tissue from each vessel). The vessel rings were suspended by stainless steel hooks in an organ bath containing Krebs solution of the following millimolar composition: NaCl 122; KC1 4.7; MgCl2 1.2; CaCl2 2.5; NaHCO3 15.4; KH2PO4 1.2; glucose 5.5, bubbled with 95% 02 and 5% CO2 (pH 7.4) and maintained at 37 C. One hook was fixed to the bottom of the bath and the other hook was connected to a force transducer (Myograph F-60, Narco Bio Systems, Houston, TX). Tension was recorded on a multichannel chart recorder (Physiograph MKIII-S, Narco Bio Systems, Houston, TX). The vessel rings were allowed to equilibrate at a constant tension of 0.5 g for 1 hour. During the equilibration period, the Krebs' solution in the organ bath was replaced every 30 minutes. Cumulative addition of norepinephrine (dissolved in 10-3 mol/L [molar] HC1, Sigma Chemical Co.) to the organ bath resulted in sigmoid dose-response curves. After a washout and re-equilibration period of 30 minutes, endothelium-dependent relaxation was studied by adding acetylcholine (10-8 mol/L to 10-5 mol/L) to the vessel rings precontracted with the ED50 dose (the concentration of agonist required to elicit 50% of the maximal response) of norepinephrine. After further washouts and re-equilibration periods, dose-response curves were obtained in response to histamine and serotonin (dissolved in distilled water; Sigma Chemical Co.). These curves were normalized in terms of the percentage of maximal contraction, and Probit
89
analysis (SAS, SAS Institute, Inc., Cary, NC) was used to calculate the ED50 value for each ring of tissue. After further washouts, normal veins from the verapamil-treated animals were incubated with phentolamine [10-6 mol/L] for 30 minutes and then the ED50 dose of serotonin was added to the organ bath.
Histologic Methods The morphology of the control and grafted vessels from both groups was examined by light microscopy. Four weeks after surgery, the proximal, middle, and distal graft segments were excised and fixed in 10% formalin. Crosssections were dehydrated and embedded in paraffin. Sixmicron sections were cut and stained with a modified Masson's trichrome and Verhoeffs elastic tissue stain. Intimal thickening was measured by computerized videometric analysis (Innovision 100, American Innovision Inc., San Diego, CA). The average intimal thickness was calculated from 20 measurements of intimal thickness taken at even intervals around the circumference of each section.
[3H] Thymidine Incorporation in Vascular Smooth Muscle Cells Vascular smooth muscle cells from the media of the thoracic aorta of male New Zealand white rabbits were isolated and grown in tissue culture. Cells in the 6th through 12th passage were used for these experiments. Confluent cells in 75 cm2 culture flasks (Falcon Plastics, Lincoln Park, NJ) were washed twice with 8 mL phosphate-buffered saline (Gibco Laboratories, Grand Island, NY) followed by trypsinization for 7 minutes at 37 C with 8 mL trypsin/EDTA solution (Gibco). Three milliliters of Dulbecco's minimal essential medium (Gibco) with 10% fetal calf serum (Gibco) was then added to inactivate the trypsin. The cells were isolated by centrifugation (250g, for 10 minutes) and resuspended in Dulbecco's minimal essential medium with 10% fetal calf serum containing penicillin (100 ,u/mL) (growth medium, Gibco). Culture wells (6-well cluster dishes, 35 mm diameter; Falcon Plastics) were innoculated with 2 mL growth medium containing 5 X 10' cells. The growth medium was replaced with new medium every third day until the cells reached confluency as judged by microscopic examination. The cells grew in hills and valleys typical of vascular smooth muscle cells. On reaching confluency, the medium was not changed for 4 days. This regimen produced a quiescent state in the cells similar to that achieved by incubating the cells in minimal essential medium containing 0. 1% fetal calf serum. On the day of the experiment, new growth medium was exchanged 4 hours and again 2 hours before the initiation of the experiment. The experiments were initiated by exchanging the growth medium with new medium contain-
*J -.:
EL-SANADIKI AND OTHERS
90
ing 4 ,umCi [3H]-thymidine (New England Nuclear, Boston MA) and various concentrations of verapamil. The concentration of verapamil used ranged from 0 to 100 ,umol/L. Incorporation of [3H]-thymidine into acid-precipitable material was measured after a 24-hour incubation period (air plus 5% CO2 at 37 C). At harvest the dishes were placed on ice and each well was washed twice with 2 mL of ice-cold Dulbecco's phosphate-buffered saline (Gibco). Two milliliters ice-cold 5% trichloracetic acid was added. After 5 minutes the acid was removed and each well washed with 3 mL ice-cold water. Sodium hydroxide (1 mL, 0.3N) was then added and the dishes were incubated for 10 minutes at 37 C. A 100-,uL aliquot of the sodium hydroxide solution was counted in 10 mL Biofluor (New England Nuclear, Boston, MA) by a Packhard Tri-Carb 4530 scintillation counter (Packard Instruments Inc., Downers Grove, IL). Results are expressed as percentage of control.
Ann. Surg. * July 1990
TABLE 1. Effect of Verapamil on Vein Graft Intimal Thickness
Intimal Hyperplasia Group
Proximal
Middle
Distal
Control Verapamil p value
252 ± 19 195 ± 7
