Howard P. Greisler

Outcome of moderate carotid artery stenosis in patients who are asymptomatic

PURPOSE The incidence rate of disease progression and stroke after the diagnosis of a moderate (50% to 79%) carotid stenosis was determined by means of color-flow duplex scanning. METHODS During a 4-year period, 344 male veterans with moderate internal carotid artery stenoses, on one or both sides, were examined at regular intervals for a mean period of 25 months. Carotid color-flow scans were obtained semiannually. Clinical follow-up was performed to determine the incidence rate of amaurosis fugax, transient ischemic attacks, nonhemispheric symptoms, and strokes. RESULTS New neurologic symptoms developed in 75 patients (21.8%). Fifty-one (14.8%) had ipsilateral symptoms during follow-up: 18 amaurosis fugax (5.2%), 14 transient ischemic attacks (4%), 5 nonhemispheric symptoms (1.4%), and 14 strokes (4%). Twenty-four patients (6.9%) had contralateral symptoms: 20 strokes (5.8%) and 4 transient ischemic attacks (1.2%). Life-table analysis showed that the annual rate of ipsilateral neurologic events was 8.1%, and the annual rate of stroke was 2.1%. Seventy-five patients (22%) died in the follow-up period. Disease progression to 80% to 99% stenosis or occlusion occurred in 71 of 458 vessels (15.5%). The internal carotid arteries that showed evidence of disease progression had a significantly higher initial peak systolic velocity (251 vs 190 cm/s; P <.0001) and end diastolic velocity (74 vs 52 cm/s; P < 0.0001). Black patients and patients with ischemic heart disease were at a higher risk for disease progression. We could not identify any atherosclerotic risk factors that reliably predicted patients in whom future ipsilateral neurologic symptoms were more likely to develop. However, there was an increased risk of stroke associated with progression of disease. CONCLUSION Patients who are asymptomatic and who have moderate carotid stenoses are at significant risk for neurologic symptoms and death, but have a relatively low incidence rate of ipsilateral events. The initial flow characteristics in the stenotic vessel are predictive of future disease progression, but they are not helpful in identifying patients in whom symptoms will develop.

The S130K fibroblast growth factor–1 mutant induces heparin-independent proliferation and is resistant to thrombin degradation in fibrin glue

OBJECTIVE Site-directed mutagenesis is an important technique that can alter cytokine function, thereby eliciting desired responses. S130K is a mutation of fibroblast growth factor-1 (FGF-1), with lysine replacing serine in the heparin-binding site. We measured molecular stability and mitogenic activity of FGF-1 and S130K, both in the media and when suspended in fibrin glue (FG), on smooth muscle cells (SMCs) and endothelial cells (ECs) to determine if the mutation altered the function and potential clinical applicability. METHODS EC and SMC proliferation of soluble FGF-1 or S130K at 0, 0. 1, 1, 10, or 100 ng/mL with heparin at 0, 5, 50, or 500 units (U)/mL was measured on growth-arrested cells in serum-free media. EC and SMC proliferation assays with cells on FG containing either FGF-1 or S130K at 0, 1, 10, 100, or 1000 ng/mL in combination with heparin at 0, 5, 50 or 500 U/mL were also performed during the exponential growth phase. Molecular degradation by thrombin was measured by sodium dodecylsulfate-polyacrylamide gel electrophoresis. RESULTS S130K induces greater EC and SMC proliferation in the absence of heparin than FGF-1 does (P <.0001 for both the 10 and 100 ng/mL doses). S130K is also significantly more potent than FGF-1 in the presence of heparin. Heparin in the media enhances cytokine-induced SMC and EC proliferation at doses of 5 U/mL, but inhibits SMC proliferation at concentrations of 500 U/mL. For the FG data, unlike FGF-1, S130K induces EC and SMC proliferation in the absence of heparin. The addition of 5 U/mL of heparin enhances the proliferation induced by S130K. For ECs, as the heparin dose increases to 50 U/mL, proliferation decreases, as compared with the 5 U/mL concentration when either FGF-1 or S130K in the FG was compared at concentrations of 10, 100, and 1000 ng/mL (P <.01). S130K is more potent in FG than is FGF-1 both with and without heparin and exhibits maximal EC and SMC proliferation at 10 ng/mL, whereas FGF-1 activity is maximal at 100 ng/mL. Gel electrophoresis demonstrated that S130K was relatively more resistant to thrombin degradation than FGF-1. CONCLUSIONS Site-directed mutagenesis changed the potency and the heparin dependency on cellular proliferation of FGF-1 in vitro. These techniques should allow the delivery of mutant growth factors to areas of vascular intervention to induce specific, desired responses. We believe that these studies will enhance our knowledge of the function of various regions of the FGF-1 molecule, allowing us to more precisely design increasingly more useful FGF-1 mutants.

Modulation of vascular cell growth kinetics by local cytokine delivery from fibrin glue suspensions

PURPOSE Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplasty sites. Reports from two different institutions suggest that heparin concentrations of 500 U/mL in FG inhibit smooth muscle cell (SMC) proliferation, but do not effect endothelial cell (EC) proliferation. The purposes of this study were to (1) quantify the diffusive release of fibroblast growth factor-1 (FGF-1) and heparin from FG; (2) determine the effect of heparin and FGF-1 on SMC proliferation when the cells are immediately plated on the FG; and (3) by means of the diffusive release data, design a new in vitro model that may differentiate the effect of FG-incorporated FGF-1 and heparin, rather than the released, solubilized components of these two factors, on SMC and EC proliferation. METHODS 125I-FGF-1 or 3H-heparin release from FG into the overlying media was measured serially in a 96-hour period, either with or without cells. SMCs were immediately plated on FG containing various concentrations of FGF-1 and heparin. SMCs or ECs were plated on identical groups of FG containing FGF-1 and heparin 24 hours after the FG was made to exclude the effect on cell growth of the initial release of FGF-1 into the media. RESULTS In the first 24 hours, 70% +/- 1% of the FGF-1 and 59% +/- 2% of the heparin in the FG was released into the overlying media, with minimal release occurring thereafter. The cell type or absence of cells did not affect release, but there was five times more FGF-1 and four times more heparin in the media at 72 hours for the immediate plating versus the delayed plating because of a diffusive release primarily in the first 24 hours. A heparin concentration of 500 U/mL inhibited SMC proliferation, as compared with 5 U/mL heparin, only when immediate plating of SMCs was used. Comparing immediate versus delayed SMC plating, at equivalent FGF-1 and heparin doses, immediate plating induced greater proliferation than delayed plating; this was likely caused by the higher soluble FGF-1 concentration. Heparin doses as high as 500 U/mL had little effect on SMC proliferation. In contrast, ECs died with delayed plating on FG containing 500 U/mL heparin, and their growth was inhibited at 50 U/mL heparin, as compared with 5 U/mL heparin. CONCLUSION The differences in SMC proliferation when comparing immediate versus delayed plating are likely caused by diffusive release of heparin and FGF-1 into the media. Our ongoing work uses an optimized in vitro FG system that minimizes the effects of soluble factors. This is an important distinction, because the cytokines that are released in vivo will be removed by blood flow and, thus, may not exert an effect unless they are contained within the FG.

Modeling the transmural stress distribution during healing of bioresorbable vascular prostheses

Little attention has been given to the stresses within the wall of bioresorbable vascular prostheses and how they might affect the resorption process. We modeled the graft “complex” (inner tissue capsule, residual graft, and outer tissue capsule) as a three-layered compound tube under internal pressure. Using this biomechanical model, we studied the effects of alterations in the geometry (i. e., radius and thickness) and mechanical properties of each stratum on the overall transmural stress distribution. Hypothetical simulations were performed to investigate the possible-sequence of and alterations in the radial and circumferential stresses during the resorption process. Our results suggest that early in the resorption phase, the inner tissue capsule is subjected to compressive hoop stresses and concentrated, largemagnitude compressive radial stresses. This distribution gives way to the more typical distribution for a thick-walled tube when equilibration (i.e., complete resorption) is approached. The prediction of the compressive stresses in the pseudo-intima during early resorption parallels findings of an elevated mitotic index in that region at that time. This leads to a new hypothesis, namely, that compressive stresses, both in-plane and out-of-plane with respect to the regenerated vascular cells, participate in the resorption process of bioresorbable vascular grafts by modulating elevated cellular proliferative activity and may play an important role in other aspects of vascular cell biology. Results of recent experimentation support this hypothesis.

Timing and frequency of perioperative carotid color-flow duplex scanning: A preliminary report.

PURPOSE The results of intraoperative and early postoperative carotid color-flow duplex scanning (CFS) after endarterectomy were reviewed to determine whether any perioperative studies could be eliminated. METHODS Patients undergoing carotid endarterectomy with intraoperative CFS between 1986 and 1997 were identified. Early postoperative CFS was performed between 1 day and 3 weeks postoperatively, then it was performed again at 6 months postoperatively. RESULTS During the study period, 560 patients, 325 men and 235 women, underwent 621 carotid endarterectomies. A satisfactory intraoperative carotid CFS was completed in 611 (98.4%) patients. There were 20 (3.2%) vessels with a major defect that required revision for fronds or flaps (n = 11), retained atheroma (n = 5), low flow (n = 2), high velocity or turbulence (n = 1), or dissection (n = 1). Another 146 vessels (23.5%) had minor defects, such as retained proximal atheromas or small (less than 3 mm) fronds, but were not revised. The remaining 445 vessels were normal. The first postoperative CFS was normal in all the revised carotids and in 138 (94.5%) vessels with minor intraoperative defects. At 6 months, recurrent stenosis (more than 75% area reduction) was identified in 1 of 18 revised carotids (5.5%), 4 of 138 vessels (2. 9%) with minor defects, and 17 of 406 vessels (4.2%) that were normal intraoperatively. The incidence of recurrent stenosis was not significantly different in the three groups (P =.7). CONCLUSION Intraoperative CFS is useful because major unsuspected defects can be corrected immediately, thus avoiding potential neurologic morbidity. However, the postoperative day 1 CFS can be eliminated in most cases, because it does not provide any relevant clinical information.

Analyzer-based phase-contrast x-ray imaging of carotid plaque microstructure

BACKGROUND Plaque vulnerability depends, in part, on composition. Imaging techniques are needed that can aid the prediction of plaque stability. High-contrast images of soft-tissue structure have been obtained with x-ray phase-contrast (PC) imaging. This research investigates multiple image radiography (MIR), an x-ray PC imaging technique, for evaluation of human carotid artery plaques. METHODS Carotid plaques were imaged with ultrasound and subsequently excised and formalin fixed. MIR imaging was performed. By using synchrotron radiation, conventional radiographs were acquired for comparison. Image texture measures were computed for soft-tissue regions of the plaques. RESULTS Ultrasound evaluation identified plaques as homogeneous without calcifications. MIR images revealed complex heterogeneous structure with multiple microcalcifications consistent with histology, and possessed more image texture in specific regions than conventional radiographs (P < .05). MIR refraction images allowed imaging of the geometric structure of tissue interfaces within the plaques, while scatter images contained more texture in soft-tissue regions than absorption or refraction images. CONCLUSIONS X-ray PC imaging better depicts plaque soft-tissue heterogeneity than ultrasound or conventional radiographs. MIR imaging technique should be investigated further as a viable imaging technique to identify high-risk plaques.