Mechanical factors predisposing to intimal hyperplasia and medial thickening in autogenous vein grafts

Autogenous veins undergo intimal hyperplasia and medial thickening when used as arterial bypass grafts. Exposing veins to arterial pressure and flow subjects them to three static deformations, three static stresses, increased pulsatile deformations, pulsatile stresses, and altered shear stress at the blood-intima interface. All of these occur simultaneously; thus it is unclear which of these nine mechanical factors predispose to the histologic changes that occur in the vein wall. Three sequential experiments were performed in 38 dogs to determine the role of each of these factors. Results showed that intimal hyperplasia is best associated with low flow velocity, a factor correlated with low blood-artery shear stress. By contrast, medial thickening is best associated with increased deformation of the vein wall in the circumferential direction (increased diameter). These findings correlate with clinical responses of vein grafts.     

Significance of the endothelial lining in prevention of intimal thickening of autogenous vein grafts in dogs

To better comprehend the role of the endothelial lining in occurrence and development of intimal thickening in arterially implanted autogenous vein grafts, two models of canine femoral arteries were prepared. In the Group I model, the implanted autogenous vein graft was kept under a normal flow condition for 2 to 4 weeks after implantation, then was exposed to an abnormal flow (poor run-off). In case of a 3- to 4-week normal flow, intimal thickening was practically nil. Scanning electron microscopic studies showed that this 3- to 4-week period corresponded to that of re-endothelialization of the autogenous vein grafts. Immunohistochemical studies of fibrinogen distribution in the autogenous vein graft wall in the Group II model revealed that the permeation of fibrinogen was enhanced in case of an abnormal flow condition for about 2 weeks after the implantation. We interpret these observations to mean that intimal thickening was induced by an accelerated permeability in the presence of abnormal flow conditions until full re-endothelialization after the implantation.     

Chronic ACE inhibition reduces intimal hyperplasia in experimental vein grafts.

Intimal hyperplasia is an important factor in the pathophysiology of vein graft failure. Local renin-angiotensin systems recently have been shown to modulate the development of intimal hyperplasia in arteries after intimal injury. The effect of chronic angiotensin-converting enzyme (ACE) inhibition on the development of intimal hyperplasia in experimental vein grafts was examined in this study. Ten New Zealand White rabbits received 10 mg/kg of captopril daily in their drinking water. One week later the right carotid artery was divided and bypassed with the reversed right external jugular vein in these rabbits and in 10 matched controls. Captopril was continued for 28 days after operation, when all the grafts were harvested. Five grafts from each group were perfusion fixed, and the intimal thickness in the proximal, middle, and distal segments was determined. Rings from the remaining grafts (n = 20 in each group) were studied in vitro under isometric tension, and their responses to norepinephrine (NE), histamine (HIST), serotonin (5-HT), angiotensin I (AI), and angiotensin II (AII) was measured. The intimal thickness of the proximal, middle, and distal segments of the captopril-treated grafts were significantly less than controls, being reduced in all segments by approximately 40% (p less than 0.0001). With regard to vasoreactivity, the captopril-treated grafts were hypersensitive to 5-HT (control ED50 5.5 +/- 0.5 X 10(-7) mol/L vs. captopril-treated 1.1 +/- 0.2 X 10(-6) mol/L; p less than 0.005) although the maximal response was significantly reduced (control 1.6 +/- 0.3 g vs. captopril-treated 0.8 +/- 0.1 g; p less than 0.05). There were no differences in sensitivity between control and captopril-treated rings with respect to NE, HIST, AI, or AII. Four of the ten captopril-treated segments, however, failed to respond to AI, and the maximal active tension of the responders was significantly reduced (control 0.47 +/- 0.06 g vs. 0.20 +/- 0.05 g; p less than 0.02). These results suggest that ACE is involved in the modulation of vein graft intimal hyperplasia, and that ACE inhibitors may have therapeutic applications in patients undergoing vein bypass procedures.     

Long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits

BACKGROUND: Rho kinase plays an important role in vascular smooth muscle cell (VSMC) contraction and other cellular functions, such as proliferation, migration, and apoptosis. Recent studies have demonstrated that long-term inhibition of Rho kinase suppresses coronary artery spasm and vascular lesion formation after arterial injury. In the cardiovascular surgery field, intimal thickening in vein grafts is the major cause of late graft failure, for which no effective treatment has yet been developed. In this study, we examined whether long-term inhibition of Rho kinase suppresses intimal thickening in autologous vein grafts in rabbits. METHODS: Male rabbits were randomly divided into two groups and received normal chow (control group) or a special chow containing 0.09% fasudil (fasudil group). After oral administration, fasudil is metabolized to a specific Rho kinase inhibitor, hydroxyfasudil. Each group underwent reversed autologous vein graft surgery with the internal jugular vein into the left common carotid artery. At 1, 2, and 4 weeks after the operation, we examined the extent of intimal thickening of the graft and VSMC proliferation and apoptosis. RESULTS: The intimal thickening was significantly suppressed in the fasudil group compared with the control group at 2 and 4 weeks after the operation. In the fasudil group, VSMC proliferation was suppressed at 1 and 2 weeks after the operation, whereas VSMC apoptosis was enhanced at 2 weeks after the procedure. CONCLUSIONS: These results indicate that Rho kinase is substantially involved in the pathogenesis of intimal thickening of vein grafts and that it is an important therapeutic target for the prevention of graft failure.     

Non-penetrating vascular clips anastomosis inhibited intimal thickening under poor runoff conditions in canine autogenous vein grafts.

OBJECTIVE: Late graft failure is still a significant problem, particularly in cases with poor runoff vessels. The main cause of late graft failure is intimal thickening of the anastomotic region. Vascular closure system (VCS) clips may provide ideal anastomosis, since they do not penetrate the wall. Therefore, we examined whether the VCS clips affect intimal thickening under poor runoff conditions in the canine autogenous vein grafts. METHODS: A canine poor runoff model was prepared at both femoral veins. Four weeks after the first surgical procedure, two groups were established according to the two different methods of anastomosis employed. The right femoral vein graft was performed using polypropylene sutures, conventional surgical anastomosis (control group), while the left femoral vein graft was performed using VCS clips anastomosis (VCS group). Four weeks after grafting, the vein grafts were removed and the intimal thickening of proximal, distal anastomosis and midportion of the vein grafts were examined histologically. RESULTS: In the control group, flow rate and variation were 26+/-8 ml/min and 51+/-10 dynes/cm(2), respectively. In the VCS group, the flow rate and variation were 23+/-11 ml/min and 44+/-14 dynes/cm(2), respectively. There were no significant differences between the two groups. The average value of intimal thickening of both the anastomotic region and the midportion of the vein graft in the VCS group was significantly inhibited compared to that of the control group. The number of positive cells of masson trichrome stain in the VCS group was significantly less than that of the control group. CONCLUSIONS: These experiments indicate that VCS clips significantly inhibit intimal thickening under poor runoff conditions in canine autogenous vein grafts to a greater extent compared to suture-constructed anastomosis. One mechanism that may account for the decreased intimal thickening is the inhibition of the expression of transforming growth factor-beta (TGF-beta), because the number of positive cells of masson trichrome stain in the VCS group was significantly less than that of the control group.     

Reduction of intimal and medial thickening in sheathed vein grafts.

BACKGROUND: Arterial pressures are described as an important factor in the development of graft degeneration and in reduced patency rate in vein bypass grafts. Sheathing of the graft with a pressure resistant mesh tubing might slow down this development. METHODS: Saphenous vein grafts were implanted into the carotid arteries of five pigs in order to evaluate the influence on myointimal hyperplasia of a compliant Phynox mesh tubing (a wrought Cobalt-Chromium-Nickel-Molybdenum-Iron Alloy), which surrounded autologous vein grafts that were exposed to arterial pressure. Each pig was operated on using a sheathed vein graft (biocompound-graft, a hybrid vascular prosthesis) on one side and an untreated saphenous vein on the other. RESULTS: After 4 weeks intimal hyperplastic changes were found in all histological sections. The wall thickness (medial and intimal layer) varied from 351 microm to 432 microm in the biocompound-graft and from 391 microm to 1196 microm in the native vein grafts (p < 0.05, n = 5). Severe myocytial and fibroblast proliferation was only found in the control grafts. Cellularity of the medial layer differed at sites of maximal cellular density and ranged from 11 to 12 cells in the biocompound-graft and from 17 to 18 cells per counting field in the native vein grafts (p < 0.05, n = 5). CONCLUSIONS: External support of vein grafts reduces intimal and medial layer proliferation. The findings of this study are in accordance with the results reported by other research groups.     

Heparin reduces the intimal hyperplasia seen in microvascular vein grafts.

The influence of heparin on microvascular vein graft intimal hyperplasia was studied in a rat model. The iliolumbar vein was grafted into the iliac artery in 80 rats. Heparin was delivered via a subcutaneous miniosmotic pump, starting either 2 days before grafting (early heparin group, n = 20) or immediately after grafting (heparin group, n = 30). Saline-containing pumps were placed in the control group (n = 30). Heparin activity was measured at 24 h, and again 3 weeks later when the animals were sacrificed. The grafts were harvested and prepared for histological examination. The intimal thickness was measured at the anastomoses and in the mid-graft region using an eye-piece graticule set at right angles to the graft internal elastic lamina. Heparin significantly reduced the intimal thickness at the anastomoses, from a median of 38 microns (range: 10-100 microns) in the control group to a median of 20 microns (range: 10-150 microns) in the heparin group. A similar reduction was seen in the mid-graft region. Although intimal thickening was reduced in the early heparin group, this reduction failed to reach statistical significance. The possible clinical application is discussed.     

Therapeutic approach against intimal hyperplasia of vein grafts through endothelial nitric oxide synthase/nitric oxide (eNOS/NO) and the Rho/Rho-kinase pathway

Late graft failure of autologous vein grafts is associated with intimal hyperplasia resulting from the migration and proliferation of vascular smooth muscle cells (VSMCs). Endothelial nitric oxide synthase (eNOS) is an enzyme that synthesizes nitric oxide (NO). An impairment of NO-mediated vasorelaxation and increases in cell proliferation occurs in vein grafts after the surgery and these pathophysiological changes cause intimal thickening. The Rho/Rho-kinase pathway negatively regulates eNOS and is involved in intimal hyperplasia. Several studies have been conducted with the goal of controlling intimal hyperplasia targeting eNOS/NO and the Rho/Rho-kinase pathway. The oral administration of drugs, such as Rho-kinase inhibitor, l-arginine, beta-blocker and statins, significantly suppressed intimal thickening in animal models. This study revealed that statins upregulate eNOS through Rho-kinase inhibition to suppress intimal hyperplasia. The intraluminal gene transfer of eNOS inhibited intimal hyperplasia, thereby reducing the cell proliferation. These approaches are thus considered to be potentially promising therapeutic modalities for graft failure.     

Pre- vs postoperative pharmacologic inhibition of platelets: effect on intimal hyperplasia in canine autogenous vein grafts.

Several clinical studies have shown that pharmacologic inhibition of platelets can increase the patency of vascular grafts, but only if platelet-inhibition is initiated before surgery. This study was performed to compare the efficacy of pre- vs postoperative platelet-inhibition on the development of intimal hyperplasia in canine autogenous vein grafts. Reversed femoral veins were used to bypass the ligated femoral arteries in 15 dogs. End-to-side anastomoses were constructed. Eleven dogs were treated with aspirin (325 mg QD) and dipyridamole (25 mg BID). In six dogs treatment was begun 48 hours preoperatively and continued for 3 months. In five other dogs treatment was begun 48 hours after surgery and was continued for 3 months. In 4 control dogs no antiplatelet treatment was given. Excision of the vein grafts 3 months after surgery disclosed reduced intimal hyperplasia (p < 0.05) in the grafts excised from all of the treated animals as compared with those obtained from the control animals. However, there was no difference in intimal hyperplasia observed in the dogs treated both pre- and postoperatively (11 grafts) as compared with those treated only postoperatively (9 grafts). These data demonstrate that it is not necessary to begin antiplatelet therapy preoperatively in order to inhibit intimal hyperplasia. They also suggest that preoperative antiplatelet therapy may improve early graft patency by directly preventing thrombosis, not by inhibiting the development of intimal hyperplasia.     

Effect of thromboxane synthetase inhibition on canine autogenous vein grafts

This study examined the effect of an orally active thromboxane (TXA2) synthetase inhibitor (TSI) on the patency, TXA2 production, and platelet accumulation of reversed autogenous vein grafts. Ten dogs received TSI (U-63557A) 10 mg/kg po q8 hr for 6 weeks, beginning 24 hr prior to surgery, while 15 control dogs were untreated. One jugular vein was harvested and stored in 37 degrees C saline for 1 hr to induce mild endothelial injury (stored). Normal and stored jugular vein grafts (8 cm) were then implanted in opposite femoral arteries while 3-cm segments of the same veins were implanted in the carotid arteries. Femoral graft flow was restricted with a 5 Fr distal arterial stenosis and patency determined by arteriography at 1, 2, 4, and 6 weeks. Vein graft endothelial surface TXB2 production was measured by RIA at graft implantation and in carotid grafts harvested at 1 week. 111In-labeled platelets were given iv 24 hr prior to carotid graft harvest to determine graft-platelet deposition. TSI treatment improved early (1 week) femoral vein graft patency from 63 to 89% (P less than 0.05), a trend that persisted for 6 weeks. Warm saline storage reduced 1-week graft patency from 83 to 63% (P less than 0.05), a difference that decreased with time. TSI treatment resulted in a marked decrease in TXB2 production, but was not associated with decreased 111In-labeled platelet deposition in carotid vein grafts. Warm saline storage increased graft-platelet deposition which was predominant at the arterial anastomoses. TSI treatment may improve early vein graft patency during the transient period of endothelial injury.     

Development and regression of intimal thickening of arterially transplanted autologous vein grafts in dogs

To investigate the influence of hemodynamic conditions on intimal thickening of arterially transplanted autologous vein grafts, two experimental models of canine femoral arteries were prepared. In group I, in which an autologous vein graft was transplanted under abnormal flow conditions (distal poor runoff), intimal thickening gradually developed and reached 358 +/- 33 microns at 8 months, whereas no thickening was observed under normal flow conditions at any time throughout the observation period. In group II the thickened intima, which developed under abnormal flow conditions for 1 month, was reimplanted into the contralateral leg under normal flow conditions. The thickness of the intima markedly regressed to about 66% at 1 month, 50% at 3 months, and 25% at 8 months, respectively, whereas no regression of the thickened intima was observed under continued abnormal flow conditions. Electron microscopic studies revealed that the thickened intima in group I was composed of proliferation of transformed smooth muscle cells with a marked increase in the mitochondria, the rough endoplasmic reticulum, and an abundant fibrous matrix, whereas with the regressed thickness of the intima of group II, the smooth muscle cells were spindle-shaped with distinct myofibrillae. These results provide pertinent data on the process involved in the intimal thickening in cases of graft placement.     

Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia.

OBJECTIVE: The aim of this study is to determine whether adenoviral inducible nitric oxide synthase (iNOS) gene transfer could inhibit intimal hyperplasia (IH) in porcine internal jugular veins interposed into the carotid artery circulation. METHODS: Porcine internal jugular veins were transduced passively with 1 x 10(11) particles of an adenoviral vector carrying either the human iNOS (AdiNOS) or beta-galactosidase (AdlacZ) cDNA for 30 minutes and then interposed into the carotid artery circulation. Segments of each vein graft were maintained in an ex vivo organ culture to measure nitrite accumulation, a marker of nitric oxide synthesis. The grafts were analyzed immunohistochemically for the presence of neutrophils, macrophages, and leukocytes by staining for myeloperoxidase, ED1, and CD45, respectively, at 3 (n = 4) and 7 (n = 4) days. Morphometric analyses and cellular proliferation (Ki67 staining) were assessed at 3 (n = 4), 7 (n = 4), and 21 days (n = 8). RESULTS: AdlacZ-treated vein grafts demonstrated high levels of beta-galactosidase expression at 3 days with a gradual decline thereafter. Nitrite production from AdiNOS-treated vein grafts was approximately fivefold greater than AdlacZ-treated grafts (P =.00001). AdiNOS or AdlacZ treatment was associated with minimal graft inflammation. Cellular proliferation rates were significantly reduced in AdiNOS-treated grafts as compared with controls at both 3 (41%, P =.000004) and 7 days (32%, P =.0001) after bypass. This early antiproliferative effect was most pronounced at the distal anastomosis (65%, P =.0005). The iNOS gene transfer reduced the intimal/medial area ratio in vein grafts at 7 (36%, P =.009) and 21 days (30%, P =.007) versus controls. This inhibition of IH was again more prominent in the distal segments of the grafts (P =.01). CONCLUSION: Adenovirus-mediated iNOS gene transfer to porcine internal jugular vein grafts effectively reduced cellular proliferation and IH. Although iNOS gene transfer reduced IH throughout the entire vein graft, the most pronounced effect was measured at the distal anastomosis. These results suggest potential for iNOS-based genetic modification of vein grafts to prolong graft patency.     

Cod-liver oil in the prevention of intimal hyperplasia in autogenous vein grafts used for arterial bypass

Cod-liver oil, rich in eicosapentaenoic acid, an unsaturated fatty acid, was administered to 14 mongrel dogs to determine if this acid would prevent platelet-mediated intimal hyperplasia. Twenty-eight 1 cm segments of undistended jugular vein were interposed between bilaterally divided femoral arteries. Seven control animals were fed a 2% cholesterol diet 1 week before and for 6 weeks after the operation. A further seven animals received cod-liver oil capsules containing 1.8 gm of eicosapentaenoic acid daily 1 week before and for 6 weeks after autogenous vein implantation, in addition to the lipid-supplemented diet. Baseline serum cholesterol was 4.6 +/- 0.4 mmol/L. The rise in serum cholesterol was similar in the two groups and increased to 7.4 +/- 0.6 mmol/L (control group) and to 6.8 +/- 0.2 mmol/L (eicosapentaenoic acid group) (p less than 0.001). Prothrombin time, partial thromboplastin time, bleeding time, and platelet counts were unchanged in the two groups. Vein grafts, harvested at 6 weeks, were fixed in formaldehyde. Mean intimal thickness was measured from multiple vein graft cross sections with a Zeiss computerized interactive image analyzing system. A mean of 140 +/- 11 measurements were computed from each graft. Marked intimal hyperplasia occurred in the control group and increased from 4.3 +/- 0.3 to 86.4 +/- 14 micron. In contrast, a high eicosapentaenoic acid diet inhibited intimal hyperplasia, with intimal thickness only increasing from 4.0 +/- 0.4 to 24.8 +/- 2.7 micron (p less than 0.001). These data indicate that eicosapentaenoic acid inhibits platelet-mediated intimal hyperplasia and suggest that cod-liver oil could be used to prevent intimal hyperplasia in vein grafts used for myocardial revascularization.     

Intimal thickening in autogenous vein grafts in rabbits: influence of aspirin and dipyridamole.

The effects of three different platelet modifying regimens on the degree of intimal thickening of autogenous vein grafts in rabbits were measured one month after operation. Dipyridamole alone (2 mg/kg/6 h) had little effect on the intimal thickness of these rabbits compared with that of controls (mean (1 SEM) for treated animals: 72.0 (7.9) micron; controls: 63.6 (6.0) micron). High dose acetylsalicylic acid (40 mg/kg/24 h) plus dipyridamole (2 mg/kg/6 h) increased intimal thickening significantly (85.8 (6.0) v 63.6 (6.2) micron; p = 0.05, n = 17). Low dose acetylsalicylic acid (0.5 mg/kg/24 h) plus dipyridamole (2 mg/kg/6 h) also increased intimal thickening (79.0 (6.1) v 63.6 (6.0] micron but not to a significant degree. It has previously been shown in vitro that acetylsalicylic acid increases the area of an exposed subendothelial arterial surface covered by platelets. Such an effect may explain our finding of increased intimal thickening with high dose acetylsalicylic acid plus dipyridamole.     

Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts

OBJECTIVES: Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. METHODS: Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. RESULTS: Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet-derived growth factor-induced phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase. PTEN-treated vascular smooth muscle cells demonstrated decreased basal, platelet-derived growth factor-stimulated, and serum-stimulated proliferation. CONCLUSION: This study demonstrates that PTEN overexpression in aortocoronary saphenous vein grafts reduces intimal hyperplasia. The mechanism of this antiproliferative effect in vascular smooth muscle cells is likely due to inhibition of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in vascular smooth muscle cell growth and survival. Therefore modulation of the phosphatidylinositol 3-kinase pathway through PTEN overexpression might represent a novel therapy to prevent saphenous vein graft intimal hyperplasia after coronary artery bypass grafting.     

Isotopic study of the effects of platelets on development of intimal thickening in autologous vein grafts in dogs

The effects of platelets on the development of intimal thickening of arterially transplanted autologous vein grafts was investigated using canine poor run-off models. A new apparatus consisting of a shielding lead block to measure in vivo platelet adherence only on the intima of the vein graft was developed. In 23 dogs, 51Cr-labelled platelets (20 microCi/kg) were injected and isotope emission over the grafts was counted. Platelet adherence was expressed as the thrombocyte accumulation index (TAI), i.e. the ratio of counts over the graft under an abnormal flow condition in the poor run-off model to those over the graft under normal flow conditions of the contralateral leg. The TAI of the total graft (TTAI) was significantly high immediately and on the first and third days after implantation. The TAIs of the proximal (PTAI) and distal (DTAI) portions of the vein graft were also high at these same times. At 7, 10, 14 and 21 days, the TAI was almost equal to 1.0, a time at which endothelial regeneration was complete as confirmed by scanning electron microscopy. Thus, the prominent intimal thickening of arterially transplanted autologous vein grafts in dogs, induced under an abnormal flow condition, correlates well with the enhanced platelet adherence on the intima of the vein grafts in an early period after implantation.     

Vaccinia virus-induced inhibition of nitric oxide production

BACKGROUND: The role of nitric oxide (NO) in the host defense against viruses has not been well defined. Several studies have implicated NO as responsible for the destruction of a variety of viruses. However, others have reported that certain viruses can impair the ability of macrophages to produce NO. This study was initiated to determine the ability of macrophages to produce NO in response to vaccinia virus infection. METHODS: RAW 264.7 murine macrophages in minimum essential medium were exposed to virus-containing supernatants for 1 h before stimulation with Escherichia coli lipopolysaccharide (LPS, 0.001 and 1.0 microg/ml). After further 24-h incubations, nitrite concentration, cell viability, and inducible nitric oxide synthase (iNOS) were quantitated. RESULTS: The viral preparation alone did not stimulate nitric oxide synthesis (measured as nitrite) by macrophages. However, macrophages exposed to 0.001 and 1.0 microg/ml LPS produced 7.7 +/- 0.6 and 16.6 +/- 0.8 nmole/1.1 x 10(6) cells/24-h nitrite, respectively. Production of nitrite caused cell death. Macrophages incubated with vaccinia virus prior to exposure to LPS resulted in a dose-dependent decrease in nitrite production. An 80% inhibition of nitrite was noted when macrophages were exposed to vaccinia virus (m.o.i. 10(-4)) plus LPS (1.0 microg/ml) (P < 0.05). Further study showed that this inhibition was not associated with changes in cell viability or substrate availability, but was associated with a marked reduction in iNOS protein. When the virus was inactivated with UV-irradiation, the same incubation caused a 46% inhibition of nitrite production (P < 0.05 vs active virus). However, this effect occurred without altering the quantity of iNOS protein. CONCLUSION: These results indicate that active vaccinia virus inhibits the ability of stimulated macrophages to produce NO by hindering iNOS protein expression. Because live viral particles were not entirely required for this inhibition, it is possible that by products of viral infection, such as soluble viral proteins, may also be responsible for this effect.