Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.

Arterial homografts.

Arterial allografts, formerly called homografts, came into limited use in the 1940s and 1950s as arterial substitutes. Fresh allografts underwent rapid rejection. Preserved allografts had a longer but still limited clinical life. Allografts demonstrated that arterial replacement was a valid concept and led to the development of synthetic substitutes. Recent renewed interest is based on the need for graft replacements in re-do procedures and in an infected field. Even the best methods of graft procurement and preservation do not preserve normal endothelial and smooth muscle cell functions nor eliminate antigenicity. The biologic and economic costs of immune suppression to obtain a successful allograft for an ischaemic limb are presently unjustifiable. Transplantation between species (xenotransplantation) may be attainable via selective inhibition of the complement system avoiding full immunosuppression now required for organ transplantation. At present allografts may be an acceptable choice for the patient with (1) a critical need for revascularisation and with a life expectancy not exceeding that of the graft, (2) in urgent vascular trauma, and (3) where immunosuppression is contraindicated as in an infected surgical field. Except in most unusual circumstances allografts should not be used for (1) relief of claudication, (2) in the above mid-calf location and (3) anatomic locations where synthetic grafts are superior.

Molecular biology of graft occlusion.

Whatever the method, the biologic price of restitution of arterial flow through or around an obstructed artery is injury to the arterial wall. The artery mounts a healing response that in 30% to 60% of procedures exceeds the need. The result is formation of a lumen-narrowing lesion composed of smooth muscle cells and extracellular matrix with varying amounts of atherosclerotic components. The primary player appears to be the smooth muscle cell. For four decades the standard model for studying these events has been catheter denudation of arterial endothelium in small animals. Molecular biology has provided an enormous amount of new information including growth factors, adhesion molecules, receptor binding sites, and molecules that up- or down-regulate or maintain a steady state of expression of numberless genes in the arterial wall. Predictably, means of inhibiting these molecular events have been found. That their clinical trials have been universally disappointing should not be surprising given that the animal in which the discoveries were made--the rat--is not normally susceptible to atherosclerosis and the histology of the rat artery only approximates that of the human. Nevertheless, reason for optimism exists because of our increasing understanding of the injury response. Recent developments include the use of blocking antisense oligonucleotides, blocking antibodies, gene transfer trials, and early investigations into the role of the immune system.

Halofuginone, a specific collagen type I inhibitor, reduces anastomotic intimal hyperplasia.

OBJECTIVE: To determine if halofuginone hydrobromide, a specific type I collagen inhibitor, could prevent intimal hyperplasia at a vascular anastomosis. DESIGN: Intimal hyperplasia is characterized by smooth muscle cell proliferation and extracellular matrix accumulation. Halofuginone was used to block collagen production and smooth muscle cell proliferation in cell cultures and in a rabbit model of an end-to-end anastomosis of the right common carotid artery. Animals were fed a nontoxic dose of halofuginone. Eighteen rabbits were fed the inhibitor in a randomized blinded fashion and were examined after 4 weeks by harvesting the arteries after perfusion fixation at physiologic pressures. RESULTS: Halofuginone inhibited smooth muscle cell proliferation in vitro and had no effect on cell viability. Morphometric quantification verified that halofuginone treatment significantly attenuated anastomotic intimal thickness. CONCLUSION: Oral administration of halofuginone inhibits intimal hyperplasia at vascular anastomoses. Intimal hyperplasia inhibition by halofuginone may be a therapeutic option for preventing arterial stenosis in vascular surgery.

Vascular permeability factor accelerates endothelial regrowth following balloon angioplasty.

Many failures of vascular reconstructions are due to thrombosis and restenosis and are often attributed to inadequate endothelial regeneration at the site of endothelial denudation. Vascular permeability factor (VPF) is a naturally occurring growth factor responsible for vessel permeability and microvascular angiogenesis. Here, we show that VPF stimulated rabbit endothelial cell proliferation in vitro at concentrations 100 ng/ml. However, VPF had no effect on smooth muscle cell proliferation at these concentrations up to 500 ng/ml. When VPF was administered for 4 weeks (120 micrograms, twice weekly, i.v.) following balloon angioplasty-induced endothelial denudation of rabbit carotid artery, there was a significant increase in the in vivo regeneration of endothelium compared to control (57.5 +/- 6.7% vs. 38.3 +/- 1.9%, P < 0.01). Moreover, 8 weeks of VPF administration resulted in 88.1 +/- 3.1% re-endothelialization compared to control (44.7 +/- 3.8%). Hence, VPF appears to be a specific mitogen for endothelial regeneration.

Inhibition of neointimal hyperplasia by blocking alpha V beta 3 integrin with a small peptide antagonist GpenGRGDSPCA.

PURPOSE: Neointimal hyperplasia is a leading cause of restenosis after vascular procedures. Recent findings showed that smooth muscle cell (SMC) migration from the media into the neointima is a critical step in the development of the hemodynamically compromising neointimal lesion. Moreover, integrins are believed to play a role in SMC motility. Therefore we studied the role of one ubiquitous integrin, alpha V beta 3, in SMC migration. METHODS: Transwell assay was used to study in vitro migration of human and rabbit SMCs after stimulation with platelet-derived growth factor (PDGF). A neutralizing monoclonal antibody to alpha V beta 3, LM609, and a specific arginine-glycine-aspartic acid (RGD) antagonist, GpenGRGDSPCA, were used in the migration assay to inhibit alpha V beta 3-mediated SMC migration. In addition, GpenGRGDSPCA was administered locally to rabbit carotid artery after balloon angioplasty to determine the effect of blocking alpha V beta 3 on neointimal hyperplasia. RESULTS: We showed that PDGF-induced human SMC migration is mediated by the alpha V beta 3 integrin by use of LM609 to inhibit migration and that SMC migration is RGD dependent by use of GpenGRGDSPCA to inhibit migration. We have also inhibited rabbit SMC migration with GpenGRGDSPCA to demonstrate the cross-species preservation of the RGD peptide sequence in SMC mortality. Finally, when we administered GpenGRGDSPCA locally to rabbit carotid artery after balloon angioplasty, there was a statistically significant reduction in neointimal lesion formation compared with arteries administered an inactive peptide or saline solution. CONCLUSIONS: We have demonstrated the important role of the alpha V beta 3 integrin in SMC migration in vitro and in neointimal hyperplasia in vivo.

Cryopreserved aortic homografts contain viable smooth muscle cells capable of expressing transplantation antigens.

Frozen aortic tissue is increasingly used as homografts in reconstructive cardiovascular surgical procedures. The viability of cells within these cryopreserved tissues, their identity, and their potential immunogenicity have been the subject of controversy. We cultured cells from cryopreserved human aortic homografts that reacted with a monoclonal antibody that recognizes muscle actin isoforms, identifying them as smooth muscle cells. Under basal conditions, these smooth muscle cells contained messenger ribonucleic acid for class I human leukocyte antigens detected by northern blotting and expressed class I human leukocyte antigen on their surfaces as measured by enzyme-linked immunoassay and immunohistochemistry. Unstimulated smooth muscle cells contained no class II human leukocyte antigen messenger ribonucleic acid as determined by northern blotting and displayed almost no class II surface antigen as determined by enzyme-linked immunoassay. Interferon gamma (1000 U/ml, 72 hours), a product of activated T lymphocytes, not only increased the expression of class I human leukocyte antigens by smooth muscle cells, but induced class II human leukocyte antigen messenger ribonucleic acid and elevated surface expression from 22 +/- 7 to 819 +/- 35 enzyme-linked immunoassay units (n = 4). Immunohistochemistry revealed few class II-positive smooth muscle cells under basal culture conditions, but all cells showed high levels of DR antigen after exposure to interferon gamma for 3 days. Similar results were obtained in two independent isolates. We conclude that cryopreserved aortic homografts can contain viable smooth muscle cells capable of expressing major histocompatibility antigens that might render them immunogenic and susceptible to rejection by the recipient's immune system.

Healing after arterial dilatation with radiofrequency thermal and nonthermal balloon angioplasty systems.

Thermal balloon angioplasty has been proposed as a means of reducing acute and delayed reclosure of arteries after percutaneous transluminal balloon angioplasty. A radiofrequency (rf) balloon catheter was used to perform thermal balloon angioplasty on canine arteries in vivo. The histologic appearance of rf-treated sites was compared with that of control sites treated by conventional percutaneous transluminal angioplasty. Acutely, rf-treated sites showed a reduced medial cellularity with preservation of internal elastic lamina except at the transitional zone between thermal injury and normal artery, where localized internal elastic lamina disruption was found. Nonthermal sites showed generalized disruption of internal elastic lamina and normal medial cellularity. Both thermal and nonthermal sites displayed a return of intimal cover commencing at 1 to 2 weeks and completed by 4 weeks. Diffuse myointimal hyperplasia appeared by 2 weeks after injury at breaks in the internal elastic lamina along the nonthermal vessels but was localized to the transitional zone in thermal injury sites. In rf-treated vessels, repopulation of the acellular thermally modified media had commenced by 4 weeks, and by 8 weeks the media was diffusely repopulated by spindle-shaped cells resembling smooth muscle cells lying between and aligned with preserved connective tissue laminae. Overall, the distribution and extent of the proliferative response after rf thermal balloon angioplasty were less than those seen after nonthermal balloon angioplasty. Thermal sites, which underwent reintimalization before medial cells returned, were considerably less prone to the development of myointimal hyperplasia. These results suggest that this modality may have beneficial effects on arterial healing after angioplasty.

Effect of endothelin-1 infusion on the development of intimal hyperplasia after balloon catheter injury.

Previous studies have shown endothelin-1 (ET-1) to be mitogenic for smooth-muscle cells. We explored in vivo the ability of high ET-1 levels to worsen angioplasty restenosis. Left carotid artery balloon endothelial denudation was performed on 14 rats. ET-1 was delivered via osmotic pump at a rate of 5 pmol/kg/min. Intimal development and plasma ET-1 levels were assessed at 2 weeks. Blood pressure and heart rate were measured throughout the study. For analysis, the animals were divided into three groups based on ET-1 levels at harvest: control, 4.3 +/- 0.5 pmol/ml (n = 6); low ET-1, 5.2 +/- 0.9 pmol/ml (n = 4); and high ET-1, 23.1 +/- 5.9 pmol/ml (n = 4). Although ET-1 infusion caused blood pressure elevation in both ET-1 groups, this was more marked and prolonged in the group with a high ET-1 level at study conclusion. Evaluation of the intimal:medial area ratio showed a marked increase in intimal thickness in the high ET-1 group versus control (1.13 +/- 0.23 versus 0.35 +/- 0.11; p < 0.05). We conclude that ET-1 infusion in responsive animals can cause worsening of the intimal hyperplastic response after mechanical injury. Further study is required to elucidate whether this is entirely caused by a direct effect of ET-1 on smooth-muscle cell mitogenesis or is also by the hemodynamic effects of ET-1-induced hypertension, or an effect of another mediator released in response to the ET-1 (e.g., angiotensin II).

PEO enhancement of platelet deposition, fibrinogen deposition, and complement C3 activation.

Whereas it has been commonly thought that adding polyethylene oxide PEO to a surface would diminish the capacity of the surface to cause deposition of platelets and of fibrinogen, and to activate complement C3, we present data showing exactly the opposite. These unexpected results are obtained with low molecular weight (2000) PEO, and are not found with higher molecular weight (20,000) PEO.

The synergistic effect of hypercholesterolemia and mechanical injury on intimal hyperplasia.

In an attempt to clarify data obtained from animal models of intimal hyperplasia, we used New Zealand white rabbits, a standardized balloon catheter injury model, and a 0.25% cholesterol supplemented diet. The effects of mechanical injury and hypercholesterolemia separately and combined were determined at the carotid and iliac positions at 12 weeks. En-face planimetry of lesioned intima and measurement of transverse intima-to-media thickness were taken as indices of intimal hyperplasia. No animals received antiplatelet agents or postoperative anticoagulation and all vessels remained patent. Neither procedure alone resulted in statistically significant lesion increase. However, combinations of injury and cholesterol resulted in statistically significant and synergistic lesion enhancement. The quantitative data, coupled with distinctive features noted on scanning electron microscopy and transmission electron microscopy, showed separate and synergistic effects of mechanical injury and cholesterol diet on intimal lesions in this model. Additionally, these effects must be considered in evaluation of animal models of intimal hyperplasia and atherosclerosis. Furthermore, this may help dissect mechanisms of failed revascularizations.

Increased in-vitro incubation time of endothelial cells on fibronectin-treated ePTFE increases cell retention in blood flow.

Endothelial cell (EC) seeding is postulated as a mechanism of improving patency of small calibre vascular grafts. However, the majority of seeded cells are lost within hours following restoration of blood flow. We postulated that incubating EC in-vitro on a graft will improve adherence and resistance to the sheer stresses of pulsatile blood flow. Fibronectin-treated ePTFE (5 cm x 4 mm ID) seeded with Indium-111-labelled autologous canine EC (1.5 x 10(5) cells/cm2) were incubated for four different time periods; 90 min, 24 h, 72 h and 6 days. Incubated grafts were subjected to blood flow of 75 ml/min for 6 h, in a canine ex-vivo arteriovenous shunt circuit. EC retention during perfusion was studied by measuring gamma activity emitted by the grafts. Cell morphology of non-perfused control groups and perfused groups was compared using scanning electron microscopy (SEM). SEM of control grafts showed progressive EC spreading on the ePTFE surface for up to 72 h incubation. Gamma activity was significantly higher at 6 h perfusion in grafts incubated for 72 h (82 +/- 4%) and 24 h (63 +/- 6%) vs. 90 min (34 +/- 13%, p less than 0.05), and between grafts incubated for 72 h vs. 6 days (55 +/- 7%, p less than 0.05). Perfused grafts incubated for 72 h showed unaltered EC morphology on SEM, few cells remained on 90 min incubated grafts. We conclude that incubating EC on fibronectin-treated ePTFE for 72 h in-vitro after seeding improves cell retention during blood flow.

Complement activation by vascular sutures both alone and in combination with synthetic vascular prostheses.

Polymer surfaces activate complement pathways resulting in platelet and leucocyte deposition as well as possible release of growth factors. A consequence of these interactions may be early graft failure or intimal hyperplasia leading to late graft failure. C5a generation in human plasma by vascular sutures, both alone and in combination with synthetic vascular prostheses was measured by radioimmunoassay to determine the influence of suture materials on C5a activation. Prolene and ePTFE suture material caused significant activation of C5a (p less than 0.01), while Novafil did not. Both Dacron and ePTFE graft material caused significant activation (p less than 0.01) of C5a. The addition of the suture materials to the ePTFE did not increase the C5a levels above the ePTFE material alone. In contrast, the addition of either Prolene or Novafil suture to Dacron material elevated C5a levels significantly over Dacron alone (p less than 0.01). The combination of Dacron material with ePTFE suture did not increase C5a levels over Dacron alone. The pattern of C5a activation by Prolene, ePTFE and Novafil sutures parallels the relative degree of in-vivo platelet accumulation on these suture materials as previously reported by our group. Since these experiments demonstrate that vascular suture material influences human complement activation, it may be that this interaction contributes to either early or late graft failure by enhancing platelet reactivity or neointimal proliferation, respectively.

Optimum results of the surgical treatment of carotid territory ischemia.

Continuing controversy over the role of carotid endarterectomy in stroke prevention is based largely on reports in which high perioperative morbidity and mortality rates obviate possible long-term benefit from the procedure. The purpose of this review is to examine optimal results of carotid surgery in order to describe the potential for the procedure in stroke prevention. Optimal surgical results are compared with optimal medical results in the therapy of symptomatic patients and with optimal nonsurgical results in the therapy of asymptomatic patients. Factors common to series with excellent results, such as patient selection and operative technique, are examined, and problems such as recurrent carotid stenosis and coexisting coronary disease, which continue to plague even the best surgical series, are discussed.

Long-term follow-up of patients operated on for recurrent carotid stenosis.

We reviewed our experience with 29 operations for recurrent carotid stenosis in 27 patients who underwent both their primary carotid endarterectomy and their reoperations at our institution. These 27 patients represent 4% of the 667 patients who underwent primary carotid endarterectomies at our institution and who are included in our carotid follow-up registry. Reoperation was prompted by recurrent symptoms in 19/29 (65.5%) cases. Comparison of long-term stroke prevention in those patients who did (84% at 5 years, 78.6% at 10 years) and did not (90.3% at 5 years, 83.6% at 10 years) develop recurrent stenosis requiring reoperation revealed no statistically significant difference (p = 0.48) when measured from the time of primary operation. The perioperative stroke and death rates for reoperation (3.4% and 0%) were acceptable. We conclude that with our acceptably low perioperative stroke morbidity (3.4%), surgery for recurrent carotid stenosis in symptomatic patients or in asymptomatic patients with high-grade (greater than or equal to 75%) stenosis maintains the durable stroke prevention offered by primary carotid endarterectomy.

Carotid endarterectomy contralateral to an occluded carotid artery: perioperative risk and late results.

To define better the short-term risk and long-term benefit of carotid endarterectomy opposite an occluded carotid artery, we reviewed our experience since 1961. Angiographic data are available for 598 of 670 (89.3%) patients in our carotid registry. In 63 (10.5%) patients the internal or common carotid artery on the side opposite the endarterectomy was occluded. All operations were carried out under general anesthesia with selective shunting based on electroencephalographic criteria. Shunting was required in 29 of 63 (46.0%) patients with contralateral occlusion and 72 of 535 (13.5%) control subjects (p less than 0.0001). Perioperative strokes occurred in 3 of 63 (4.8%) patients with contralateral occlusion and 14 of 535 control subjects (2.6%) (p = 0.23). Perioperative death occurred in 0 of 63 patients with contralateral occlusion and 6 of 535 (1.1%) control subjects (p = 0.40). Life-table cumulative stroke-free rates at 1, 5, and 10 years were 95.2%, 91.0%, and 76.2% in the group with contralateral occlusion and 96.0%, 89.4%, and 84.1% in control subjects (p = 0.25). Life-table cumulative survival rates at 1, 5, and 10 years were 93.1%, 80.8%, and 75.4% in the group with contralateral occlusion and 94.8%, 77.0%, and 57.9% in control subjects (p = 0.58). Carotid endarterectomy contralateral to an occluded carotid artery may be carried out with acceptable risk and late stroke-free and survival rates comparable to those seen in other patients who have undergone carotid endarterectomy.