Modulating vascular intimal hyperplasia using HSV-1 mutant requires activated MEK.

Outcomes of cardiovascular procedures, such as angioplasty and stent or bypass grafting are limited by failure, predominantly caused by pathological smooth muscle cell (SMC) proliferation, known as intimal hyperplasia. Local delivery of a genetically engineered herpes simplex virus (HSV) is known to block vascular SMC proliferation while allowing for re-endothelialization. However, the mechanism this mutant virus uses to prevent SMC hyperplasia is unknown. The Ras signaling cascade is activated in SMCs undergoing hyperplasia leading to phosphorylation of the mitogen-activated protein kinase (MAPK). In this study we tested the hypothesis that MAPK kinase (MEK) activity is the molecular basis by which SMCs are susceptible to mutant HSV. We show that genetically engineered herpes simplex-1 viruses (HSV-1) can target proliferating SMCs. We demonstrate that the molecular basis of this HSV-1 anti-proliferative effect is MEK activation in SMCs. We demonstrate efficacy and practicality of the MEK-dependent HSV-1 for the treatment of intimal hyperplasia in a clinically relevant in vivo model. Important to this strategy is the ability to modulate the effects by controlling viral dose. These results propel genetically engineered HSV-1 therapy towards clinical evaluation in treatment of intimal hyperplasia.

Long-term results justify autogenous infrainguinal bypass grafting in patients with end-stage renal failure.

INTRODUCTION: Infrainguinal bypass grafting for limb-threatening ischemia in patients with end-stage renal disease is generally thought to be associated with increased operative risk and poor long-term outcome. This retrospective study was undertaken to examine the modern-era, long-term results of infrainguinal bypass grafting in dialysis-dependent patients. METHODS: Over the past 5 years in a single institution, 425 lower extremities (368 consecutive patients) were revascularized for the indication of limb salvage. Sixty-four patients (82 limbs) were dialysis-dependent at the time of revascularization, and this group was analyzed separately. They exhibited statistically significant higher incidences of diabetes (83% vs 56%; P <.001), hypertension (91% vs 74%; P <.001), and more distal vascular disease, which required a greater proportion of proximal anastomoses at the popliteal level (24% vs 11%; P <.01) and distal anastomoses at the infrapopliteal level (75% vs 65%; P <.05). RESULTS: Despite the higher prevalence of comorbid conditions and distal disease in patients with renal failure, their perioperative 30-day mortality rate remained low (4.9%) and was not significantly different from that in patients with functioning kidneys (2.9%; P = not significant). After a median follow-up of 11 months (range, 0-60 months), the 3-year autogenous conduit secondary graft patency in patients with renal failure was no different than in patients with functioning kidneys (67% +/- 9% vs 64% +/- 5%; P = not significant). Nonautogenous conduits in dialysis-dependent patients exhibited a significantly poorer outcome with only 27% +/- 12% remaining secondarily patent at 2 years. As expected, both limb salvage and patient survival were significantly less in patients with renal faiture, although both exceeded 50% at 3 years (limb salvage 59% +/- 8% vs 68% +/- 5%; P <.05; patient survival 60% +/- 8% vs 86% +/- 4%; P <.001). The often-quoted phenomenon of limb loss, despite a patent bypass graft, occurred infrequently in this study (n = 3 of 82 limbs). CONCLUSION: Infrainguinal revascularization can be performed in dialysis-dependent patients with acceptable perioperative and long-term results, especially in patients in whom adequate autologous conduit is available.

The effects of extremely low shear stress on cellular proliferation and neointimal thickening in the failing bypass graft.

OBJECTIVE: Previous studies demonstrating a correlation between low shear stress (tau = 5-15 dyne/cm(2)) and experimental vein graft neointimal thickening (NIT) support the role of low tau in vein graft failure. However, a simple linear relationship between low tau and NIT would underestimate the degree of NIT evident in high-grade occlusive lesions of failing human vein grafts. In this study we used a new experimental model that maintains patency at low tau (< 2 dyne/cm(2)), to delineate possible deviations from linearity in the low tau --> NIT hypothesis. METHODS: Thirty-two New Zealand White rabbits underwent creation of a common carotid vein patch with a segment of ipsilateral external jugular vein. Very low tau was created in 13 patches by ligation of the distal common carotid artery, leaving the only outflow through a small muscular branch. Normal tau was created in 11 patches by leaving the common carotid artery outflow intact. High tau was created in eight patches by ligation of the contralateral common carotid artery. Six patches were harvested after 2 weeks for measurement of cell cycle entry by proliferating cell nuclear antigen (PCNA) immunohistochemistry. The remaining 26 patches were harvested after 4 weeks, perfusion fixed, and excised for morphometric analysis. RESULTS: Mean blood flow and tau at implantation ranged from 0.5 to 41 mL/min and 0.07 to 15 dyne/cm(2), respectively. At the time of harvest, 30 of 32 patches remained patent, and the artificially created aberrations in blood flow were maintained (range, 0.7-41 mL/min). After 2 weeks PCNA immunohistochemistry showed a significantly higher level of cell cycling in patches exposed to low tau (40 +/- 5 vs 1.6 +/- 0.3 PCNA-positive cells per high-power field; P <.001), which is equivalent to approximately 20% of the total cells present. In patches harvested after 4 weeks, NIT ranged from 42 to 328 microm and significantly correlated with mean tau at implantation. Patches with very low tau exhibited histologic characteristics similar to those of failing human bypass grafts, including laminar thrombus and flow-limiting luminal stenosis. The relationship between tau and NIT was nonlinear in that extremely low tau (< 2 dyne/cm(2)) resulted in NIT beyond that predicted by a simple linear correlation (P =.003). CONCLUSION: Extremely low tau (< 2 dyne/cm(2)) stimulates high rates of smooth muscle cellular proliferation in arterialized vein patches. NIT is accelerated in these regions of low tau far beyond that predicted by a simple linear model. The nonlinear nature of the cellular proliferative response and NIT at tau less than 2 dyne/cm(2) may explain the rapid progression of neointimal lesions in failing bypass grafts.

Adenoviral-mediated gene transfer in human and animal vein grafts using clinically relevant exposure times, pressures, and viral concentrations.

This study examined the efficiency of adenoviral-mediated gene transfer in experimental vein grafts and cultured human saphenous vein under physiologic conditions using clinically relevant exposure times, pressures, and viral concentrations. The external jugular veins of 25 male New Zealand White rabbits were exposed to 0.5 mL of replication-deficient adenovirus vectors encoding beta-galactosidase (AdlacZ), control adenovirus (AdBg/II), or vehicle at pressures ranging from 0 to 120 mmHg for 10 min. Veins were excised and grafted into the carotid circulation. After 5 days, the vessels were reexposed, excised, and stained with X-gal chromagen for beta-galactosidase (beta-gal) activity. Gene transfer was also performed in 13 segments of human saphenous vein discarded at the time of bypass grafting. The veins were cultured for 0-21 days and assayed for beta-gal activity as above. Rabbit vein grafts exposed to high-pressure AdlacZ transfection showed significant transgene expression in 100% of grafts (39 +/- 2% positive cells/hpf) while only 60% of those transfected at low pressure expressed beta-gal (9 +/- 3% positive cells/hpf). All human veins exposed to AdlacZ expressed beta-gal to a variable degree (range 10-50% positive cells/hpf). No control grafts or veins expressed the transgene. Efficient adenoviral-mediated gene transfer in experimental vein grafts and human saphenous vein segments can be achieved using clinically feasible parameters of exposure time, pressure, and viral concentration.

Patency and limb salvage after infrainguinal bypass with severely compromised ("blind") outflow.

HYPOTHESIS: Infrainguinal graft patency and limb salvage are adversely affected by severely compromised outflow. DESIGN: Retrospective review of all infrainguinal bypass procedures performed at a single institution during a 5-year period. SETTING: University teaching hospital. PATIENTS: Two hundred seventy-four patients underwent infrainguinal bypass for limb salvage (351 grafts in 307 limbs). INTERVENTIONS: All infrainguinal bypasses originated from a femoral artery. The distal anastomosis in 279 grafts was located in an artery with at least 1 patent outflow vessel with anatomically normal end-artery runoff (Society for Vascular Surgery/International Society for Cardiovascular Surgery ad hoc committee runoff score, 1-9). The distal anastomosis of 72 grafts was located in an artery with only collateral outflow ("blind bypass"; runoff score, 10). MAIN OUTCOME MEASURES: Perioperative morbidity and mortality, primary-assisted and secondary graft patency, limb salvage, and survival. RESULTS: All data are presented as mean +/- SEM. Patients undergoing blind bypass were older (age, 70 +/- 2 vs. 66 +/- 1 years; P <.05) and had a higher incidence of hypertension (90% vs 70%; P <.05) and end-stage renal disease (24% vs. 13%; P <.05). Comparing patients undergoing blind bypass to bypass with at least 1 patent outflow vessel, there were no differences in the use of nonautogenous conduits (50% vs 59%; P =.21) or postoperative warfarin (30% vs 32%; P =.69), or in perioperative mortality rates (2.7% vs 3.2%; P =.79). After a median follow-up of 13 months (range, 0-60 months), 2-year secondary graft patency for the entire group was 63% +/- 4%. The secondary patency rate of blind bypass grafts was no different from that of grafts with at least 1 patent outflow vessel (67% +/- 7% vs. 64% +/- 4%; P was not significant). However, the 2-year limb salvage rate in limbs with blind outflow was significantly worse than in limbs with at least 1 patent outflow vessel (67% +/- 7% vs. 76% +/- 3%; P =.04). CONCLUSION: Acceptable long-term patency rates can be achieved in infrainguinal bypass grafts with blind outflow, although blind outflow remains a marker for subsequent limb loss in the chronically ischemic leg.

The hemodynamics of vein grafts: measurement and meaning.

The long-term patency of infrainguinal vein grafts appears to depend primarily on the size and quality of the venous conduit. Therefore, those quantities which directly relate to the conduit's ability to act as a transporter of blood, namely internal diameter and longitudinal impedance (Z(L)), have predictive value for patency. Autologous grafts of good quality frequently remain patent even with compromised outflow. Therefore, those quantities that are outflow dependent, including deltaP, flow, velocity, shear stress, and resistance, carry less predictive value for long-term performance.

Prevention of restenosis by a herpes simplex virus mutant capable of controlled long-term expression in vascular tissue in vivo.

Neointimal hyperplasia resulting from vascular smooth muscle cell (SMC) proliferation and luminal migration is the major cause of autologous vein graft failure following vascular coronary or peripheral bypass surgery. Strategies to attenuate SMC proliferation by the delivery of oligonucleotides or genes controlling cell division rely on the use of high concentrations of vectors, and require pre-emptive disruption of the endothelial cell layer. We report a genetically engineered herpes simplex virus (HSV-1) mutant that, in an in vivo rabbit model system, infects all vascular layers without prior injury to the endothelium; expresses a reporter gene driven by a viral promoter with high efficiency for at least 4 weeks; exhibits no systemic toxicity; can be eliminated at will by administration of the antiviral drug acyclovir; and significantly reduces SMC proliferation and restenosis in vein grafts in immunocompetent hosts.

Estimating the contemporary in-hospital costs of carotid endarterectomy.

Carotid endarterectomy (CEA) is the treatment of choice for symptomatic carotid stenosis and selective asymptomatic lesions. Alternative approaches have recently been championed under the guise of increased efficacy and decreased cost. The purpose of this study was to determine the results and in-hospital costs of CEA in a university hospital in the modern era. A retrospective chart review was undertaken for all patients undergoing CEA between January 1995 and December 1997. This corresponded to the implementation of a clinical path and extended efforts toward cost reduction. Patients undergoing combined CEA and cardiopulmonary bypass were excluded (n = 3). Cost was analyzed by the hospital Office of Program Planning using TSI (Transition Systems, Inc.) software. Direct costs are related to the utilization of clinical resources and are therefore manageable by clinicians (bed, room, supplies, nursing staff, OR staff, radiology, pharmacy, etc.). Total costs additionally include administration and overhead costs not directly chargeable to patient accounts. The results of this study showed that CEA can be safely performed with brief hospital stays and reasonable hospital costs. Results of alternative interventions for the treatment of carotid stenosis should be compared to these contemporary data.

Effective hemodynamic diameter: an intrinsic property of vein grafts with predictive value for patency.

INTRODUCTION: Conduit size and quality are major determinants of the long-term success of infrainguinal autologous vein grafting. However, accurate measurement of the internal diameter of vein grafts is difficult given their variable wall thickness and taper. The purpose of this study was to define the "effective" internal diameter of a vein graft according to its hemodynamic properties and to determine its significance for graft patency. METHODS: Sixty infrainguinal bypass grafts performed on 57 patients were evaluated intraoperatively. Proximal and distal graft pressure and blood flow (Q(meas)) were measured with fluid-filled catheter transduction and ultrasonic transit-time flowimetry, respectively, after unclamping. Waveforms were recorded digitally at 200 Hz under baseline conditions and after stimulation with 60 mg of papaverine. According to Fourier transformation of the measured pressure gradient (DeltaP), the Womersley solution for fluid flow in a straight rigid tube was used to calculate theoretical flow waveforms (Q(calc)) for a range of graft diameters. The theoretical waveforms were then compared with the measured flow waveforms and the best-fit diameter chosen as the "effective hemodynamic diameter" (EHD). Only grafts in which the correlation coefficient of Q(calc) versus Q(meas) was more than 0.90 were accepted (n = 47) to assure validity of the hemodynamic model. After a mean follow-up of 12.5 months (range, 0.1-43.9 months), patency was determined by the life table method. Hemodynamic and clinical variables were tabulated, and their effect on patency determined the use of univariate and multivariate Cox regression. RESULTS: Mean EHD was 4.1 +/- 0.1 mm with a range of 2.5 to 5.7 mm. Administration of papaverine caused profound changes in DeltaP (+78% +/- 17%) and Q(meas) (+71% +/- 12%) as expected, but had no effect on EHD (+0.05% +/- 0.1%). Univariate regression identified five variables associated with decreased secondary patency (P <.10): low EHD, conduit source other than the greater saphenous vein, high baseline DeltaP(mean), female sex, and redo operation. Of these, only low EHD was significant after multivariate analysis (P =.03). Patency of small diameter grafts (EHD < 3.6 mm; n = 11) was compared with patency of larger grafts (EHD > 3.6 mm; n = 36) to test a frequently espoused clinical guideline. Grafts with an EHD less than 3.6 mm exhibited significantly lower secondary patency compared with larger grafts (P =.0001). The positive and negative predictive values for an EHD less than 3.6 mm for secondary graft failure for grafts with at least 1 year follow-up were 86% and 88%, respectively. CONCLUSION: An EHD is a unique parameter that quantifies conduit size and has a significant impact on vein graft patency. An EHD less than 3.6 mm portends graft failure.