Nina Klingman

Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts

A possible approach to the low seeding efficiency of endothelial cells into prosthetic grafts is to increase the number of cells to be seeded in cell culture and improve seeding efficiency by graft precoating with fibronectin. The effect of cell culture on cell adhesion is unknown, however, and fibronectin also binds fibrin, which may increase the thrombogenicity of the graft luminal surface. To investigate these questions, freshly harvested canine jugular vein endothelial cells from six animals and similar cells harvested from six primary and eight secondary cell cultures were labeled with 111Indium and seeded into 5 cm, 4 mm PTFE grafts coated with fibronectin, using similar uncoated PTFE grafts as controls. Platelet accumulation and distribution on six similar coated and uncoated grafts placed in canine carotid, external jugular arterial venous shunts for 2 hr were also determined using autogenous 111Indium-labeled platelets. Significant differences between group means were determined using the paired Students t test. Results reveal that seeding efficiency is significantly better in all groups of coated grafts compared to uncoated grafts (P less than 0.01). Cells derived from cell culture also had significantly higher seeding efficiencies than freshly harvested cells when seeded into coated grafts (P less than 0.05) and tended to have higher seeding efficiencies than harvested cells when seeded into uncoated grafts (P = 0.53). Fibronectin coating increased mean platelet accumulation on the entire graft luminal surface, but not to a statistically significant degree (P greater than 0.1). Whether this increased seeding efficiency will improve graft endothelialization remains to be investigated.

Improved in vivo endothelialization of prosthetic grafts by surface modification with fibronectin

Endothelial cell growth in vitro is enhanced by coating with fibronectin the surface on which cells grow. Similar coating of prosthetic arterial grafts may promote in vivo graft endothelialization if graft patency is not adversely affected. In each of 15 dogs, two fibronectin-coated polytetrafluoroethylene grafts and two grafts that were not coated were implanted. One graft in each pair was seeded with autologous endothelial cells, so that four different grafts were studied in each animal: a coated, seeded graft; a coated graft that was not seeded; a seeded graft that was not coated; a graft that was neither coated nor seeded. At 2, 4, and 8 weeks, grafts from five animals were examined for patency, surface endothelialization, and indium 111 platelet reactivity. After seeding, surface coverage by endothelium of coated grafts was more complete and more rapid than in uncoated grafts (64% +/- 23% vs 31% +/- 13% at 4 weeks, p less than 0.05). Without seeding, coated grafts also appeared to have increased endothelial cell ingrowth compared with plain grafts (48.8% +/- 15.1% vs 37.6% +/- 1.5% at 8 weeks). Early (2-week) platelet reactivity of coated grafts was increased (p = 0.06), but patency was not adversely affected. Thus fibronectin coating of prosthetic grafts promotes surface endothelialization in vivo without altering graft patency.

Hydrophilic surface modification of metallic endoluminal stents.

PURPOSE Stainless steel endovascular stents are inherently thrombogenic so that thrombus accumulates on these devices, leading to acute vessel occlusion. A potential solution to this problem is stent surface modification with hydrophilic polymers, which might limit platelet adhesion and reactivity. METHODS N-vinylpyrrolidone (NVP) and potassium sulfopropyl acrylate (KSPA) hydrophilic monomers were gamma graft polymerized onto 1 cm2 stainless steel slabs and 4 mm Palmaz stainless steel stents. Surface characteristics of modified and plain stainless steel stents were then investigated with contact angle and x-ray photoelectron spectroscopy measurements, and in vitro and in vivo platelet reactivity was assessed as 111Indium platelet accumulation expressed as counts/min/cm2. RESULTS Surface modification of stainless steel slabs and stents with both NVP and KSPA hydrophilic polymers significantly reduced in vitro platelet adhesion (plain = 2249 +/- 723 counts/min/cm2, NVP = 428 +/- 156 counts/min/cm2, KSPA = 958 +/- 223 counts/min/cm2) and in vivo platelet accumulation after 1 hour of blood flow exposure (plain = 1407 +/- 796 counts/min/cm2, NVP = 426 +/- 175 counts/min/cm2, KSPA = 399 +/- 124 counts/min/cm2. In addition, platelet accumulation on modified stents indexed to plain stents was lowest in KSPA-modified stents (NVP = 79.3% +/- 31.7% of plain, KSPA = 51.2% +/- 36.2% of plain). Surface analysis confirmed surface grafting with both monomers, and SEM documented smoothing of the irregular surfaces of the stainless steel stents after grafting. CONCLUSION Hydrophilic polymer surface modification of stainless steel stents decreases initial stent surface platelet accumulation, which may decrease the risk of vessel thrombosis associated with the use of these devices.

The relationship between carotid plaque composition and neurologic symptoms.

Variations in plaque composition, particularly an increased lipid concentration, could make carotid plaques unstable and prone to embolization. To investigate this hypothesis, 35 carotid bifurcation plaques from 31 patients undergoing carotid endarterectomy (20 symptomatic, 11 asymptomatic) were prospectively analyzed. Plaque total lipid, cholesterol, collagen, and Ca2+ content were determined, and the plaque collagen was fractionated into pepsin-soluble collagen (PSC) (indicative of less crosslinked, more recently synthesized collagen) and pepsin-insoluble collagen. Preoperative serum lipid and plasma lipoprotein levels were also obtained in all patients. Results revealed that plaques removed from symptomatic patients contained significantly more total lipid and cholesterol (P less than 0.001) than those removed from asymptomatic patients. There was also an increased amount of pepsin-soluble collagen in symptomatic plaques, potentially indicating increased metabolic activity (P less than 0.05). In addition, mean plasma low-density lipoprotein cholesterol was higher in symptomatic patients than in asymptomatic patients (P less than 0.05). Since lipid-laden plaques can potentially be identified by B-mode ultrasound, it may be important to remove these plaques in asymptomatic patients or, alternatively, to attempt to lower the plaque cholesterol by lipid-lowering maneuvers.

External-Beam Radiation Therapy for Improved Dialysis Access Patency: Feasibility and Early Safety

Abstract Rectenwald, J. E., Pretus, H. A., Seeger, J. M., Huber, T. S., Mendenhall, N. P., Zlotecki, R. A., Palta, J. R., Li, Z. F., Hook, S. Y., Sarac, T. P., Welborn, M. B., Klingman, N. V., Abouhamze, Z. S. and Ozaki, C. K. External-Beam Radiation Therapy for Improved Dialysis Access Patency: Feasibility and Early Safety. Radiat. Res. 156, 53–60 (2001). Prosthetic dialysis access grafts fail secondary to neointimal hyperplasia at the venous anastomosis. We hypothesized that postoperative single-fraction external-beam radiation therapy to the venous anastomosis of hemodialysis grafts can be used safely in an effort to improve access patency. Dogs (n = 8) underwent placement of expanded polytetrafluoroethylene grafts from the right carotid artery to the left jugular vein. Five dogs received single-fraction external-beam photon irradiation (8 Gy) to the venous anastomosis after surgery. Controls were not irradiated. Shunt angiograms were completed 3 and 6 months postoperatively. Anastomoses, mid-graft, and the surrounding tissues were analyzed. Immunohistochemistry for smooth muscle cell α-actin, proliferating cellular nuclear antigen (PCNA), and apoptosis was performed. Incisions healed well, though all animals developed wound seromas. One control suffered graft thrombosis 4 months postoperatively. Angiography/histology confirmed severe neointimal hyperplasia at the venous anastomosis. The remaining seven dogs developed similar amounts of neointimal hyperplasia. PCNA studies showed no accelerated fibroproliferative response at irradiated anastomoses compared to controls. Skin incisions and soft tissues over irradiated anastomoses revealed no radiation-induced changes or increase in apoptosis. Thus we conclude that postoperative single-fraction external-beam irradiation of the venous anastomosis of a prosthetic arteriovenous graft that mimics the situation in humans is feasible and safe with regard to early wound healing.