R. Anthony Carabasi

Human adult endothelial cell growth in culture

The purpose of the present study was to culture human adult endothelial cells (HAECs) on a long-term basis in the laboratory. Previous inability to accomplish this has been the major impediment to the in vitro study of endothelialization of prosthetic grafts with human cells, a problem of significant clinical relevance. We have been successful in developing a technique that allows HAECs from human adult arteries, veins, and capillaries to proliferate vigorously in culture for up to 80 population doublings. HAECs are grown on a gelatin surface (medium 199 containing 20% fetal calf serum). Heparin and endothelial cell growth factor (ECGF) are required for optimal growth. With this technique, which will be described in detail, over 10(23) HAECs can be produced from each 1 cm2 of vascular tissue. This makes large numbers of HAECs available for high-density seeding on prosthetic grafts prior to implantation. It also permits for the first time with human cells the in vitro study of prosthetic grafts--HAEC interactions and the factors that enhance optimal growth and adherence to prosthetic materials. It is hoped that identification of the factors promoting graft endothelialization in combination with high-density seeding will lower graft thrombogenicity and therefore result in greater graft longevity than has been possible heretofore.

Complications encountered with the use of the Greenfield filter

The Greenfield filter can be used with a low complication rate provided one adheres to certain principles. First, preoperative venography to define the inferior vena caval anatomy will help avoid difficulties associated with anatomic variations. At the time the study is carried out, it would be extremely useful if the radiologist places a radiopaque marker at the level of the renal veins. This will ensure that filters will be placed in the infrarenal position when appropriate, thus preventing occasional inadvertent discharge, particularly into the right renal vein. Second, use of a guide wire greatly facilitates passage of the introducer and accurate intracaval positioning. Third, intraoperative technical errors must be recognized and promptly corrected. Finally, meticulous postoperative follow-up is essential, and recurrent embolism or any change in filter position requires repeat roentgenography of the vena cava to guide appropriate corrective treatment.

Treatment of iatrogenic femoral artery injuries with ultrasound-guided compression

Iatrogenic injuries of the groin are becoming more common after increasingly sophisticated vascular intervention. These injuries are accurately detected by duplex and color Doppler ultrasonography. Recent treatment of these lesions by ultrasound-guided compression repair (UGCR) has been described. During a 1-year period we identified 18 femoral artery injuries, including 17 pseudoaneurysms and one arteriovenous fistula. Three of the pseudoaneurysms thrombosed spontaneously before attempted treatment. The remaining 15 lesions underwent a trial of UGCR. Successful closure was accomplished in 10 patients (56%). Seven of these lesions were successfully treated during the initial session, and thrombosis was accomplished after repeat compression in three additional lesions. Three patients who were given anticoagulants had a failed UGCR, but their pseudoaneurysms thrombosed after administration of anticoagulants was discontinued. Two patients had failed UGCR and required operation. Seven (88%) of eight patients who were not given anticoagulants were successfully treated. In contrast only two (29%) of seven patients given therapeutic doses of anticoagulant medication were successfully treated by the technique. There was no statistical difference between mean pseudoaneurysm diameter, mean width and length of pseudoaneurysm neck, or depth of pseudoaneurysm neck from skin surface in patients in whom successful initial closure was achieved when compared with those patients in whom the initial attempt failed. UGCR is a safe, simple, noninvasive technique that can be used to treat many femoral artery injuries that traditionally were treated with surgery. The technique can be applied by any laboratory that has the necessary ultrasonography equipment and is currently the method of choice for treating uncomplicated iatrogenic femoral artery injuries at our institution.

Adult human endothelial cell compatibility with prosthetic graft material

We have developed a system for the in vitro evaluation of the interaction of human adult endothelial cells (HAEC) with prosthetic vascular graft material. HAEC, isolated from adult human iliac veins, proliferated vigorously in culture for approximately 70 population doublings. The large number of HAECs produced permitted high-density seeding of prosthetic grafts. Samples of prosthetic material were immobilized on a plastic ring and were used either untreated or coated with extracellular matrix, fibronectin, or plasma. HAEC were seeded at high density and adherence was evaluated by light and electron microscopy after a 2-hr incubation. While essentially no HAEC adhered to untreated grafts, treatment of grafts with either extracellular matrix, plasma, or fibronectin resulted in dramatic adherence of HAEC. The highest density of HAEC adherence was observed on collagen-coated Dacron grafts, and was equal to the cell density observed in confluent monolayers of HAEC grown on gelatin-coated tissue culture plastic. This study demonstrates a method capable of determining HAEC-graft biocompatibility prior to the use of an in vivo system.

Kinetics of endothelial cell-surface attachment forces.

Physical and biochemical forces exist that are necessary for the persistent attachment and function of ECs on native and prosthetic blood vessels. The optimization of conditions that permit regeneration of these attachment forces may allow rapid establishment of a durable, biocompatible EC monolayer. We examined the effects of three major factors, protein substrate, EC incubation time, and shear stress, on the attachment kinetics of human adult ECs to two different polymers. ECs were incubated up to 30 minutes on polymers (PS or PET) coated with extracellular matrix proteins: collagen I/III, fibronectin, collagen IV/V, laminin, gelatin, or saline control. After incubation, continued attachment in the presence of shear stress (created in a rotating disc device) between zero and 90 dynes/cm2 for 30 minutes was evaluated. Maximal adherence was observed on all substrates by 30 minutes. Therefore, after a 30-minute incubation, the percentage of cells attached (postshear ECs/preshear ECs/preshear ECs X 100) was measured as a function of shear stress. ECs attached to a matrix of fibronectin or collagen I/III demonstrated shear-resistant adherence after as little as 5 minutes of static incubation before initial shear exposure. By 30 minutes, more than 90% of the ECs on both matrices demonstrated the ability to remain attached in the presence of 90 dynes/cm2 of shear stress. We conclude that forces that attach ECs to surfaces are affected by temporal factors (incubation time) and substrate composition and may be quantified with a defined shear stress detachment assay. Understanding and manipulating these temporal physiochemical parameters should allow one to re-create an optimal EC monolayer on a blood-contacting surface.

Initial adherence of human capillary endothelial cells to dacron

Successful endothelialization of vascular grafts by seeding with endothelial cells (EC) at implantation is related to the number of EC which initially adhere to the graft. Using an in vitro system we examined the initial adherence of EC from human perinephric fat capillaries to woven Dacron that was either unmodified or precoated with several substrates. We studied capillary EC because they have not been investigated as a source of EC for graft seeding, although transinterstitial capillary ingrowth is one possible mechanism for spontaneous graft endothelialization. EC were isolated using collagenase and characterized morphologically and functionally including positive factor VIII-related antigen staining. EC were studied at three phases in culture: (A) primary EC with no subcultivations (EC-0); (B) EC after two subcultivations with trypsin (EC-2); and (C) EC after 10 subcultivations with trypsin (EC-10). EC were seeded onto graft material at a density of 10(5) cells/cm2 (100% confluence) and examined for cell counts and morphology after one day in culture by light and electron microscopy. Results are as follows: (table; see text) The conclusions are: (1) All capillary EC demonstrated adherence to Dacron, but this initial adherence was strongly influenced by graft pretreatment with collagen or plasma. (2) Serially subcultivated EC (EC-2 and EC-10) had significantly higher initial adherence to pretreated Dacron compared to the primary cells (EC-0) (P less than .05). This suggests that briefly cultured and subcultivated EC have superior initial adherence characteristics to treated dacron compared to primary EC with no subcultivations. (3) Fat capillary EC are easily procured and cultured and provide a rich source of human EC for endothelializing vascular prostheses.

Thrombus-free, human endothelial surface in the midregion of a Dacron vascular graft in the splanchnic venous circuit--observations after nine months of implantation.

The addition of an endothelial cell lining to a prosthetic vascular graft may reduce the thrombogenicity of the blood-contacting surface. An endothelialized mesoatrial graft was implanted in a patient with Budd-Chiari syndrome caused by a primary inferior vena caval leiomyosarcoma. During the initial surgery a Dacron vascular graft was preclotted with plasma and then lined with microvascular endothelial cells derived from the patients subcutaneous adipose tissue. The patient did well initially but 9 months later required resection of a mechanical stricture of the graft that occurred as it passed beneath the costochondral junction. Grossly, the luminal surface of the resected graft was free of thrombus, with a smooth, glistening, white surface. Light microscopy demonstrated a surface layer of cells morphologically consistent with an endothelial cell monolayer, a subendothelial layer composed of extracellular matrix and spindle-shaped cells, and granulation tissue around the Dacron fabric. Immunohistochemistry and electron microscopy confirmed the presence of vascular endothelium on the luminal surface. This report documents the successful achievement of a human endothelial cell monolayer that persisted for 9 months in the midportion of a Dacron vascular graft.

Comparison of Doppler US and CT angiography for evaluation of renal artery stenosis

Rationale and Objectives. The authors compared Doppler ultrasound (US) with computed tomographic (CT) angiography in the evaluation of stenosis of the main renal artery. Materials and Methods. Fifty-six patients who had undergone conventional angiography of the renal arteries participated in a prospective comparison of Doppler US (45 patients) and CT angiography (52 patients). US evaluation included both the main renal artery and segmental renal arteries. Results. There were 27 main renal arteries with at least 50% stenosis in 20 patients. In 36 patients, there was no significant stenosis. All cases of main renal artery stenosis detected with Doppler US of the segmental arteries were also identified with Doppler US of the main renal artery. The by-artery sensitivity (63%) of US of the main renal artery was greater than that (33%) of US of the segmental arteries. CT angiography was more sensitive (96%) than Doppler US (63%) in the detection of stenosis, but the specificity of CT (88%) was similar to that of US (89%). The difference in the area under the receiver operating characteristic curve (AUC) between CT (AUC = 0.94) and US (AUC = 0.82) was statistically significant (P = .038). Conclusion. Doppler US of the main renal artery is more sensitive than Doppler US of segmental arteries in the detection of stenosis. CT angiography is more accurate than Doppler US in the evaluation of renal artery stenosis.

Long-term follow-up of reoperative carotid surgery

BACKGROUND We examined our long-term results of carotid reoperation to identify risk factors for morbidity and secondary recurrence. METHODS Medical record review revealed 27 patients had reoperative surgery for recurrent stenosis. Demographics, operative details, pathology, clinical outcome, and follow-up imaging results were reviewed. RESULTS No neurologic deficits and no mortalities were noted perioperatively. Long-term follow-up (average 54 months) revealed an 85% 5-year and 29% 10-year estimated survival. The 5- and 10-year estimated neurologic event rates were 15% and 35%, respectively. These included 3 ipsilateral strokes and 1 ipsilateral TIA; only the TIA involved secondary restenosis. Follow-up imaging revealed a 21% incidence of secondary restenosis, occurring more frequently in patients with hyperlipidemia (P < 0.05) and previous contralateral endarterectomy (P < 0.05). CONCLUSIONS (1) Reoperation provides long-term protection from stroke due to recurrent stenosis. (2) Secondary restenosis rates appear higher than those for primary surgery. (3) Hyperlipidemia and contralateral endarterectomy are risk factors for secondary restenosis.

Role of thrombin in endothelial cell monolayer repair in vitro

PURPOSE We examined the effect of thrombin on human iliac artery endothelial cell monolayer repair and proliferation after denuding vascular injury. METHODS Human iliac artery endothelial cell monolayer repair was determined by scrape wounding confluent monolayers and measuring the advancement of the cells into the wounded area for 3 days. Proliferation studies involved plating human iliac artery endothelial cells at one tenth confluence and counting the increase in cell number every 2 days for a 2-week period. Proliferation during monolayer repair was examined by determining bromodeoxyuridine uptake in cells located at the leading edge of a scrape-wounded monolayer. RESULTS Thrombin (1 to 8 U/ml) inhibited human iliac artery endothelial cell monolayer repair in a concentration-related, reversible manner. The effect was augmented by decreasing serum concentration and was independent of the presence of endothelial cell growth supplement. Inactivation of thrombins proteolytic site with diisopropylfluorophosphate eliminated its effect on monolayer repair. Thrombin (0.5 to 8 U/ml) inhibited human iliac artery endothelial cell proliferation in a dose-related manner. This effect was augmented by decreasing serum concentration. Finally, thrombin (4 U/ml) inhibited the proliferative response of cells located at the leading edge of wounded monolayers compared with control groups. CONCLUSION Thrombin inhibits human arterial endothelial cell monolayer repair and proliferation after denuding vascular injury.