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Abstract —The growth of neointima and neointimal smooth muscle cells in baboon polytetrafluoroethylene grafts is regulated by blood flow. Because neointimal smooth muscle cells express both platelet-derived growth factor receptor-α and -β (PDGFR-α and -β), we designed this study to test the hypothesis that inhibiting either PDGFR-α or PDGFR-β with a specific mouse/human chimeric antibody will modulate flow-induced neointimal formation. Bilateral aortoiliac grafts and distal femoral arteriovenous fistulae were placed in 17 baboons. After 8 weeks, 1 arteriovenous fistulae was ligated, normalizing flow through the ipsilateral graft while maintaining high flow in the contralateral graft. The experimental groups received a blocking antibody to PDGFR-α (Ab-PDGFR-α; 10 mg/kg; n=5) or PDGFR-β (Ab-PDGFR-β; 10 mg/kg; n=6) by pulsed intravenous administration 30 minutes before ligation and at 4, 8, 15, and 22 days after ligation. Controls received carrier medium alone (n=8). Serum antibody concentrations were followed. Grafts were harvested after 28 days and analyzed by videomorphometry. Serum Ab-PDGFR-α concentrations fell rapidly after day 7 to 0, whereas serum Ab-PDGFR-β concentrations were maintained at the target levels (>50 μg/mL). Compared with controls (3.7±0.3), the ratio of the intimal areas (normalized flow/high flow) was significantly reduced in Ab-PDGFR-β (1.2±0.2, P P

Purpose: We investigated whether control of constitutive endothelial cell nitric oxide synthase (cNOS) and nitric oxide (NO) by changes in shear stress might be important for the regulation of smooth muscle cell (SMC) growth and vascular diameter. Methods: Bilateral femoral arteriovenous fistulas were placed in baboons to increase the blood flow in the external iliac arteries. At 2 months, the fistula was ligated on one side to restore normal flow (flow switch). Results: In response to flow switch and a decrease in shear stress, iliac artery lumenal area decreased and SMC proliferation was induced. A decline in NO production, cNOS messenger RNA (mRNA), and protein were associated with these biological effects. In a subset of animals with iliac arteries under high flow, infusion of Nω-nitro-l-arginine, an inhibitor of cNOS, did not induce proliferation. Conclusion: Shear stress can regulate cNOS, vasoconstriction, and SMC proliferation. A decrease in nitric oxide may be necessary, but is not sufficient to induce SMC proliferation in response to a decrease in blood flow. (J Vasc Surg 1998:28:514-21.)

Background Heparin is a potent inhibitor of smooth muscle cell (SMC) growth and intimal hyperplasia in animal models but has been ineffective in inhibiting restenosis in humans. This difference may relate to flaws in clinical study design or, alternatively, to interspecies differences in SMC response to heparin. To determine whether heparin could inhibit intimal hyperplasia in a species more closely related to humans, we studied the effect of a low-molecular-weight heparin (LMWH) on baboon SMC proliferation and migration in culture and in arteries subjected to experimental angioplasty. Methods and Results LMWH or saline was infused continuously after experimental angioplasty of baboon peripheral arteries (six animals per group). After 28 days, bromodeoxyuridine (BrdU) was given to label proliferating cells, and balloon-injured arteries were perfusion-fixed in situ and removed for analysis. All arteries had reendothelialized (Evans blue dye exclusion). LMWH increased partial thromboplastin time (LMWH, 81.7±8.4 seconds versus saline, 34.7±0.8 seconds [mean±SEM]; P =.004) but failed to inhibit intimal thickening or SMC proliferation (intimal area: LMWH, 0.19±0.03 mm 2 versus saline, 0.21±0.03 mm 2 ; BrdU labeling: LMWH, 2.9±0.6% versus saline, 2.4±0.4%; P =NS). In culture, LMWH and standard heparin (100 μg/mL) significantly inhibited serum-induced but not platelet-derived growth factor (PDGF-BB)–induced SMC proliferation (% control, serum: LMWH, 60.5±4.0%, P =.0002; standard heparin, 29.4±8.2%, P =.0001; % control, PDGF-BB: LMWH, 117.7±11.3%, P =NS; standard heparin, 90.9±14.4%, P =NS) and SMC migration (% control, serum: LMWH, 15.3±1.9%, P =.0198; standard heparin, 26.4±13.8%, P =.0032; % control, PDGF-BB: LMWH, 98.5±14.3%, P =NS; standard heparin, 100.0±13.5%, P =NS). Conclusions LMWH failed to inhibit intimal hyperplasia in a baboon angioplasty model. Furthermore, LMWH blocked serum-induced but not PDGF-BB–induced SMC proliferation and migration in culture. Thus, heparin-sensitive and -insensitive pathways exist for SMC activation. The relative importance of each pathway induced by injury may vary between species and thus account for different responses to heparin.

Background —We have evaluated the use of a mouse/human chimeric anti–platelet-derived growth factor-β receptor antibody in combination with heparin to inhibit intimal hyperplasia in the saphenous artery of the baboon after balloon angioplasty. Methods and Results —The study evaluated lesion development in sequential injuries made 28 days apart. Each animal received control treatment after the first injury and antibody/heparin therapy after the second injury to the contralateral artery. The antibody was administered by bolus intravenous injections (10 mg/kg) on study days 1, 4, 8, 15, and 22 and heparin coadministered by continuous intravenous infusion at a dose of 0.13 mg/kg per hour. Morphometric analysis of tissue sections showed a 53% decrease in intimal area after antibody/heparin treatment ( P =0.005), corresponding to a 40% decrease in the intima-to-media ratio ( P =0.005). Smooth muscle cell proliferation in the injured wall, measured at both 4 and 29 days after balloon injury, were similar in the control and antibody/heparin-treated animals. Conclusions —These data suggest that platelet-derived growth factor plays a key role in the development of intimal lesions at sites of acute vascular injury in the nonhuman primate.

Abrupt reductions in fluid shear stress induce subendothelial smooth muscle cells (SMCs) to proliferate in experimental prosthetic grafts. Platelet-derived growth factor (PDGF), an important SMC mitogen, is expressed by cultured endothelial cells and modulated by shear stress. We hypothesized that this growth factor would be modulated by changes in shear stress in vivo. Bilateral aortoiliac prosthetic grafts were implanted into five baboons. High flow was generated by construction of femoral arteriovenous fistulas on both sides. Two months later, one of the fistulas was ligated, reducing shear stress in the upstream graft by 78±6%. Four days after fistula ligation, all grafts were removed and analyzed. As previously reported, SMC proliferation in low-flow grafts exceeded that in high-flow grafts, although the neointimal area was similar. mRNA levels for PDGF-A were significantly increased in low-flow grafts compared with high-flow grafts. In situ hybridization and immunohistochemical studies localized the increased PDGF-A mRNA and protein to the luminal endothelium and subjacent SMCs. Abrupt reductions in blood flow and fluid shear stress may induce accelerated neointimal thickening by a PDGF-A–mediated mechanism, since endothelial expression of this gene is temporally and anatomically associated with neointimal SMC proliferation.

Prosthetic vascular grafts containing retrovirally transduced autologous vascular smooth muscle cells were studied as a model for introduction of human genes into baboons. Retroviral vectors encoding beta-galactosidase (beta-Gal) (LNPoZ) or human purine nucleoside phosphorylase (LPNSN-2), a control gene, were used for ex vivo transduction of autologous baboon smooth muscle cells obtained from vein biopsies. Transduced cells were placed into a collagen solution and seeded into the interstices of polytetrafluoroethylene vascular grafts. Endothelial cells were then seeded onto the luminal surface of the grafts to reduce thrombus formation. One LNPoZ-seeded graft and one LPNSN-2-seeded control graft were implanted bilaterally into the aorto-iliac circulation of each of 4 animals. All grafts remained patent until they were removed after 3-5 weeks and examined histochemically for vector-expressing cells. All histological cross-sections from the beta-Gal vector seeded grafts contained cells staining blue with the X-Gal chromogen. For the four grafts, the mean fraction of LNPoZ expressing cells was 10%, with a range of 2-20%, while no sections from the control grafts contained stainable cells. Smooth muscle cells expressing the reporter gene were localized within the graft wall but not in the newly forming intima or outer capsule of fibrous tissue. Implantation of transduced cells within this type of vascular graft may provide a useful approach for long-term local and systemic gene therapy.

Polytetrafluoroethylene (PTFE) grafts placed into the arterial circulation of baboons for 8 weeks under high blood flow (HF) conditions develop a thin intima composed of smooth muscle cells (SMCs) and extracellular matrix beneath an endothelial monolayer. When these grafts are returned abruptly to normal flow (NF), they develop marked intimal thickening within 1 month. The mechanisms underlying this thickening are unclear. We studied the SMC response to altered flow by placing bilateral aortoiliac PTFE grafts into baboons with bilateral femoral arteriovenous fistulas. After 8 weeks, one fistula was closed, returning the graft flow on that side to NF. The opposite graft remained under HF conditions. Flow differences were monitored with duplex ultrasound (for all grafts: NF, 135 +/- 21 [mean +/- SEM] mL/min; HF, 507 +/- 35 mL/min; P < .001). Grafts were removed 2, 4, 7, 14, or 28 days later (five animals per group). Endothelial coverage, as assessed by scanning electron microscopy, was intact in each graft. Intimal area and SMC number increased progressively in NF grafts through 28 days (for area: NF, 3.0 +/- 0.3 mm2; HF, 0.6 +/- 0.2 mm2; P < .001; and for SMCs per cross section: NF, 11.8 +/- 1.1 x 10(3); HF, 2.6 +/- 1.0 x 10(3); P < .002). Intimal SMC proliferation (thymidine labeling) was increased significantly in NF grafts at 4 and 7 days (at 4 days: NF, 5.9 +/- 1.5%; HF, 1.4 +/- 0.6%; P < .05). Extracellular matrix accounted for an equal proportion of intimal mass in NF and HF grafts (percent matrix at 28 days: NF, 62.9 +/- 1.6%; HF, 63.7 +/- 4.7%; P = NS). We conclude that intimal thickening in this model of flow-induced vascular remodeling is due to increased SMC proliferation and accumulation of SMCs with a proportionate amount of extracellular matrix.

Both heparin and the angiotensin converting enzyme inhibitor cilazapril inhibit intimal thickening in rat carotid arteries injured by the passage of a balloon catheter. The purpose of this study was to determine if combinations of the two drugs were more effective than either drug alone and whether the effect could be accounted for by inhibition of smooth muscle cell proliferation. Heparin (0.1-0.3 mg/kg/hr) administered by continuous intravenous infusion with or without cilazapril (0-25 mg/kg/day p.o.) produced a dose-dependent inhibition of smooth muscle accumulation at 14 days after rat carotid ballooning. At the lower doses, the inhibitory effects of heparin and cilazapril were additive when the drugs were used together. This overall effect on growth was reflected in decreased smooth muscle cell proliferation at 2 and 7 days. A 7-day course of heparin combined with cilazapril, a regimen that might be applicable in the clinical setting, produced an 80% inhibition of intimal thickening at 28 days. These results provide evidence that heparin and cilazapril together might prove to be more effective than either drug alone in the control of intimal hyperplasia after arterial injury.