Thomas R. Kirkman

Increased blood flow inhibits neointimal hyperplasia in endothelialized vascular grafts.

Intimal hyperplasia is a primary cause of failure after vascular reconstruction and may be affected by blood flow. We have studied the effects of increased blood flow on intimal hyperplasia in porous polytetrafluoroethylene grafts implanted in baboons. These grafts develop an endothelial lining by 2 weeks and neointimal thickening due to proliferation of underlying smooth muscle cells by 1 month. Creation of a distal arteriovenous fistula increased flow (from 230 +/- 35 to 785 +/- 101 ml/min, p less than 0.001) and mean shear (from 26 +/- 4 to 78 +/- 10 dynes/cm2, p less than 0.001) without causing a drop in pressure across the grafts. Fistula flow did not alter the pattern of endothelial coverage but did cause a marked reduction in the cross-sectional area of the neointima (from 2.60 +/- 0.52 to 0.42 +/- 0.07 mm2 at 3 months, p less than 0.01). Detailed morphometric analysis revealed an equivalent percentage decrease in smooth muscle cells and matrix content, suggesting that the primary effect of increased flow was to reduce smooth muscle cell number without affecting the amount of matrix produced by individual cells. The neointima remained sensitive to changes in flow at late times; ligation of the fistula after 2 months resulted in a rapid increase in neointimal thickness (from 0.60 +/- 0.03 mm2 after 2 months of fistula flow to 3.88 +/- 0.55 mm2 1 month after ligation of fistula, p less than 0.01). These results support the hypothesis that changes in blood flow affect the structure of diseased as well as normal vessels.

Healing of polytetrafluoroethylene arterial grafts is influenced by graft porosity

The importance of porosity in synthetic arterial graft healing has not been adequately defined. To determine the effect of porosity on graft healing, we measured the extent of cellular response at late times in 4 mm internal diameter polytetrafluoroethylene grafts of varying porosity (between 10 and 90 microns internodal distance) inserted into the arterial system of baboons. After 1 and 3 months the grafts were retrieved and examined for endothelial coverage, intimal thickening, and endothelial cell and smooth muscle cell proliferation. The pattern of intimal healing with endothelial cells and smooth muscle cells was only related to porosity in the sense that there was an abrupt switch in the pattern of healing as porosity was increased from 30 to 60 microns. In low porosity grafts (10 and 30 microns internodal distances) endothelial coverage of the luminal surface was incomplete and, along with intimal thickening, was limited to graft near the anastomosis. In high porosity grafts (60 and 90 microns internodal distances) luminal endothelial coverage was complete, and intimal thickening was uniformly distributed throughout the graft. The highest porosity graft studied (90 microns) developed areas of focal loss of endothelial cells at late time periods. In this limited series there does appear to be an optimal porosity for polytetrafluoroethylene grafts near 60 microns, since 10 and 30 micron grafts fail to achieve luminal endothelial cell coverage, and 90 micron grafts exhibit instability of the intima with focal endothelial cell loss.

Mechanisms of arterial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses

In a previous study of arterial bypass grafts (4 mm polytetrafluoroethylene [PTFE]) in baboons we observed that endothelial and smooth muscle cells (SMCs) formed the neointima and were derived from the cut edges of adjacent artery. The purpose of this study was to determine at late times whether endothelial cells would continue to migrate and to proliferate to cover the graft surface and whether the underlying proliferating SMCs would produce a progressively thickened intima, graft stenosis, and eventual thrombosis. At 6 and 12 months after grafts were placed, endothelial coverage by ingrowth from the anastomoses was more advanced than at 3 months, and by 12 months 60% of grafts (7 to 9 cm in length) were covered. Endothelial cells proliferated in association with the growing edge and focally in other regions. Underlying SMCs proliferated in the region of the growing edge of the endothelial cells and also at anastomoses. Intimal cross-sectional area was greatest at anastomoses and at late times was principally due to an increase in connective tissue; actual SMC mass remained constant after 3 months. These results demonstrated slow but progressive healing of the grafts by ingrowth of endothelium. There was also an increased turnover rate of SMCs and endothelial cells in established intima at late times, which might be the consequence of chronic endothelial injury. This condition represents a stable state since it does not produce further intimal thickening and accumulation of SMCs and does not lead to a high rate of thrombosis.

Time course of flow-induced smooth muscle cell proliferation and intimal thickening in endothelialized baboon vascular grafts.

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.

Shear stress regulates smooth muscle proliferation and neointimal thickening in porous polytetrafluoroethylene grafts.

High shear stress appears to decrease wall thickening in diseased arteries and vascular grafts. To determine if increased shear stress diminishes smooth muscle (SMC) proliferation, we studied the effect of increased blood flow on neointimal thickening in porous polytetrafluoroethylene grafts implanted in baboons. An aorto-aortic 5-mm graft was placed in tandem with a pair of aorto-iliac 5-mm grafts, so that the proximal graft supplied all flow to both distal grafts. At 12 weeks, calculated luminal shear stress in proximal grafts was twice that in distal grafts (24 +/- 8 versus 11 +/- 5 dynes/cm2; p less than 0.05). All grafts were completely endothelialized. The neointimal cross-sectional area in proximal grafts was about half as large as in distal grafts (3.36 +/- 1.61 versus 5.93 +/- 0.61 mm2; p less than 0.05). Proximal grafts also had significantly less SMC proliferation (0.14 +/- 0.05% versus 0.24 +/- 0.10%; p less than 0.05) and SMC volume (6.1 +/- 4.0 versus 12.4 +/- 2.6 mm3/cm graft; p less than 0.01) when compared with distal grafts. We conclude that the elevation in shear stress in the proximal graft, which remained within the physiological range, inhibits SMC proliferation and neointimal thickening in these grafts.

PDGFβ Receptor Blockade Inhibits Intimal Hyperplasia in the Baboon

BACKGROUND We have evaluated the use of a mouse/human chimeric anti-platelet-derived growth factor-beta 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.

Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. Association in vivo of platelet-derived growth factor mRNA and protein with cellular proliferation.

In a baboon graft model of arterial intimal thickening, smooth muscle cells (SMC) have been observed to proliferate underneath an intact monolayer of endothelium and in the absence of platelet adherence. Because platelets are not present and therefore cannot be a major source of growth stimulus, we have proposed that the vascular wall cells in the graft intima express mitogens and regulate SMC proliferation. To test this hypothesis, we assayed the grafts for mitogenic activity and expression of growth factor genes. Segments of healing graft and of normal artery, when perfused ex vivo, released mitogenic activity into the perfusate. The graft released more mitogen than the normal arterial segment, and some of the activity was inhibitable with an antibody to human platelet-derived growth factor (PDGF). In addition, Northern analysis of total RNA demonstrated higher expression of PDGF-A chain mRNA in the graft intima compared to normal artery. PDGF-B chain mRNA was barely detectable in both tissues. PDGF mRNA levels within the graft interstices were not measured. In situ hybridization of 7.5- or 12-wk grafts indicated that some luminal endothelial cells and adjacent intimal SMC contained PDGF-A chain mRNA. By thymidine autoradiography, intimal SMC were observed to be proliferating in the inner third of the intima. These data demonstrate a difference in the pattern of PDGF transcript expression and luminal perfusate activity in graft as compared with control arteries. The association of intimal smooth muscle cell proliferation with intimal PDGF mRNA expression and release of PDGF-like protein supports the hypothesis that factors from cells that have grown into the graft or populated its surface rather than platelets may regulate intimal smooth muscle cell proliferation in this model.

Mechanisms of healing in synthetic grafts

In previous baboon studies we have shown that porous (60 micron mean internodal distance) polytetrafluoroethylene (PTFE) grafts heal by ingrowth of endothelium and smooth muscle cells from the adjacent artery and from capillaries penetrating through the interstices of the graft. However, porous grafts (principally made of Dacron) in humans do not heal. This has been attributed to a wound healing deficiency in humans; however, it might be due to an inhibitory effect of the Dacron itself. To examine the latter possibility, we undertook this study to compare the healing of 4 mm internal diameter porous Dacron grafts (USCI, Sauvage Filamentous Knitted) with that of Gore-Tex 60 micron PTFE grafts in baboons (the latter graft not available for clinical use). The grafts were harvested at 2, 4, and 12 weeks and assessed for (1) percentage of endothelial coverage, (2) endothelial cell (EC) proliferation (thymidine labeling index), (3) intimal area, and (4) smooth muscle cell (SMC) proliferation (thymidine labeling index). The PTFE grafts at all three time points were fully covered, whereas only one of five Dacron grafts was completely covered at 12 weeks. The intima of the PTFE grafts consisted of ECs and SMCs, whereas that of the Dacron grafts contained ECs and SMCs as well as focal accumulations of thrombus. The intimal cross-sectional areas in the Dacron grafts (3.0 +/- 1.2 mm2) were significantly greater than in the PTFE grafts (0.8 +/- 0.6 mm2) at 4 weeks; there was no difference at 12 weeks (Dacron, 2.6 +/- 2.3 mm2 and PTFE, 3.0 +/- 2.5 mm2).(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental Arterial Thromboembolism in Baboons: MECHANISM, QUANTITATION, AND PHARMACOLOGIC PREVENTION

A quantitative primate model of arterial thromboembolism has been characterized with respect to mechanism and usefulness in evaluating modifying variables. The model involved the kinetic measurements of (51)Cr-platelets and (125)I-fibrinogen consumption by femoral arteriovenous cannulae in chaired baboons. Cannula platelet consumption correlated directly with exposed cannular area for irradiated Silastic and polyurethane (correlation coefficients of 0.940 and 0.901, respectively; P < 0.001) and remained steady state for months. Nonirradiated Silastic was only minimally reactive with platelets. Despite increased rates of platelet consumption circulating fibrinogen was not measurably destroyed by any of the cannulae tested. Cannula platelet consumption was independent of cannula flow rate, platelet count, heparin anti-coagulation, and ancrod defibrinogenation.(111)In-platelet imaging of irradiated Silastic cannulae demonstrated luminal accumulation and subsequent embolization of irregular platelet masses. When irradiated Silastic cannulae were inserted as extension segments in the renal arteries of four animals the glomerular vessels became progressively occluded with nonfibrin-containing platelet thromboemboli. Nonirradiated Silastic cannulae in control arteries produced no significant vascular occlusion. Because the survival of platelets from animals with consumptive cannulae was not shortened in normal recipient animals we concluded that platelets were either irreversibly removed through thromboembolic consumption or unaffected in their viability. Oral administration of dipyridamole and sulfinpyrazone decreased cannula platelet consumption in a dose-dependent manner with complete interruption at 20 and 250 mumol/kg body wt per d (in three divided doses), respectively, whereas oral acetylsalicylic acid (10-330 mumol/kg per d) had no measurable effect on cannula platelet consumption. We conclude that this primate model simulates arterial thrombotic processes in man and that this model is suitable for the in vivo evaluation of biomaterials and of drugs that modify platelet behavior.

Heparin inhibits the expression of tissue-type plasminogen activator by smooth muscle cells in injured rat carotid artery.

Smooth muscle cells (SMCs) in balloon-injured rat carotid artery express tissue-type plasminogen activator (t-PA) at a time when they are migrating from the media to the intima. Since heparin inhibits SMC migration and intimal thickening, we have examined the possibility that heparin might also inhibit t-PA expression. Heparin (nonanticoagulant fraction; molecular weight, approximately 6,000) was administered by continuous intravenous infusion (1.0 mg/kg per hour) to Sprague-Dawley rats subjected to balloon injury of the left common carotid artery. At various times up to 14 days after injury, plasminogen activator expression was analyzed by zymography, plasmin generation, enzyme-linked immunosorbent assay, Northern blotting, and in situ hybridization. This dose of heparin inhibited SMC accumulation at 14 days by 60%. Both urokinase plasminogen activator (u-PA) and t-PA activity increased in injured arteries and reached a maximum at 7 days. Heparin treatment decreased t-PA, but not u-PA, activity. Total t-PA protein was decreased by treatment with heparin but not chondroitin sulfate, and the decrease in t-PA protein was associated with decreased t-PA mRNA in the media. These results in the injured rat carotid artery agree with our earlier observations that heparin inhibits t-PA gene expression in cultured baboon aortic SMCs. They also provide support for the hypothesis that heparin interferes with the expression of certain proteases required for SMC migration and proliferation.