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Introduction: The nuclear protein HMGB1 can initiate innate immunity as well as sustain autophagy during times of cellular stress. We hypothesize that autophagy, a mechanism of cellular preservation, is a critical process in skeletal muscle biology during ischemia in patients with PAD. We further hypothesize that HMGB1 mobilization is associated with critical myoblast functions such as differentiation, and that this process may be modulated by autophagy. Methods: Human C2C12 myoblasts were cultured on laminin and exposed to hypoxia (1% O2) and serum depletion. Cells were also cultured with 2% serum to induce differentiation over 72hr. C57B6 mice were injected with 3-methyladenine (3MA), an autophagy inhibitor, before femoral artery ligation (FAL) and muscles were harvested after 24hr. Cell fractions from the cultured cells and muscles were probed for HMGB1. Immunoblots were analyzed using Image J. Results: Hypoxia and serum depletion induced complete loss of nuclear HMGB1 after 48hr. In myoblasts cultured in differentiation conditions, there was a 3 fold relative loss of nuclear HMGB1 compared with controls (p=0.014, t-test;N=3). While ischemia resulted in loss of nuclear HMGB1 in vivo, modulation of autophagic signaling with 3MA resulted in the retention of nuclear HMGB1 24 hours after FAL (Figure 1). Conclusion: Loss of nuclear HMGB1 by stressed myoblasts during differentiation suggests that this danger signal may be important in the regulation of muscle repair after injury. Additionally, our data indicate that autophagy may play a role in controlling the mobilization of myocyte HMGB1, affecting its inflammatory and reparative functions in ischemic muscle.

We have previously shown that MyD88 KO mice appear protected from ischemic muscle injury while TRIF KO mice exhibit sustained necrosis after femoral artery ligation (FAL). However, our previous data did not differentiate whether the protective effect of absent MyD88 signaling was secondary to attenuated injury after FAL or quicker recovery from the insult. The purpose of this study was to delineate these different possibilities. On the basis of previous findings, we hypothesized that MyD88 signaling promotes enhanced inflammation while TRIF mediates regeneration after skeletal muscle ischemia. Our results show that after FAL, both MyD88 KO mice and TRIF KO mice have evidence of ischemia, as do their control counterparts. However, MyD88 KO mice had lower levels of serum IL‐6 24 h after FAL, while TRIF KO mice demonstrated sustained serum IL‐6 up to 1 week after injury. Additionally, MyD88 KO mice had higher nuclear content and larger myofibers than control animals 1 week after injury. IL‐6 is known to have differential effects in myoblast function, and can inhibit proliferation and differentiation. In tibialis anterior muscle harvested from injured animals, IL‐6 levels were higher and the proliferative marker MyoD was lower in TRIF KO mice by PCR. Furthermore, expression of MyD88 appeared to be higher in skeletal muscle of TRIF KO mice. In vitro, we showed that myoblast differentiation and proliferation were attenuated in response to IL‐6 treatment giving credence to the finding that low IL‐6 in MyD88 KO mice may be responsible for larger myocyte sizes 1 week after FAL. We conclude that MyD88 and TRIF work in concert to mediate a balanced response to ischemic injury., We describe opposing roles of MyD88 and TRIF, both downstream signaling molecules of TLR4, in the inflammatory and regenerative processes that follow limb ischemia. MyD88 appears to mediate inflammation, while TRIF appears to be required for modulation of MyD88 activity and promoting regeneration. Absence of MyD88 may ultimately have a protective effect in muscle recovery after ischemic injury.

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 × 10 11 particles of an adenoviral vector carrying either the human iNOS (AdiNOS) or β-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 β-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. (J Vasc Surg 2001;34:156-65.)

Atherosclerosis is one of the leading causes of major morbidity and mortality in the United States. Arterial insufficiency resulting from flow-limiting lesions can lead to myocardial, renal, mesenteric, and extremity dysfunction. Treatments for these atherosclerotic arterial lesions include arterial bypass and angioplasty. These therapies are limited by the development of intimal hyperplasia (IH), thus reducing hemodynamic improvement significantly. A number of pharmacological agents with antiplatelet and anticoagulant properties have failed to reduce the incidence and rate of restenosis. Because of the magnitude of the patient population affected by IH, there has been a tremendous need to develop a therapy that will successfully reduce its incidence. Over the last decade, the field of vascular gene therapy has emerged as a viable therapeutic approach, permitting the targeting of genes to produce local and transient effects on the development of IH. This review will discuss the rationale and preliminary data for the different genes that have been evaluated to date. One of the first reports of successful gene transfer to vascular cells was by Nabel et al in 1989.1 These investigators transfected porcine endothelial cells ex vivo with a retrovirus encoding the β-galactosidase gene and reintroduced the cells onto the denuded iliofemoral artery of a syngeneic animal. The arterial segments isolated 2 to 4 weeks later demonstrated endothelial cells that expressed the β-galactosidase gene, thus indicating successful incorporation of the transgene into the transduced cells. Landmark follow-up experiments in which herpes simplex virus thymidine kinase (HSV-tk) was delivered to injured porcine iliac2 or rat carotid3 arteries using an adenoviral vector were published 5 years later (see Table online; http://www.circresaha.org). This approach, which is based on the conversion of coadministered ganciclovir to a toxic metabolite by HSV-tk, decreased the neointima by 86% in the porcine model2 and 46% in the …

Background Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. Methods We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. Results Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. Conclusions We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.

The role nitric oxide (NO) plays in the cardiovascular system is complex and diverse. Even more controversial is the role that the inducible NO synthase enzyme (iNOS) serves in mediating different aspects of cardiovascular pathophysiology. Following arterial injury, NO has been shown to serve many vasoprotective roles, including inhibition of platelet aggregation and adherence to the site of injury, inhibition of leukocyte adherence, inhibition of vascular smooth muscle cell (VSMC) proliferation and migration, and stimulation of endothelial cell (EC) growth. These properties function together to preserve a normal vascular environment following injury. In this review, we discuss what is known about the involvement of iNOS in the vascular injury response. Additionally, we discuss the beneficial role of iNOS gene transfer to the vasculature in preventing the development of neointimal thickening. Lastly, the pathophysiology of transplant vasculopathy is discussed as well as the role of iNOS in this setting.

A wide array of vascular disorders have been shown to benefit from gene therapy. Given the vasoprotective role that nitric oxide plays in the vasculature, it is understandable why gene therapy with the three different isoforms of nitric oxide synthase has been so successful. This review summarizes the current literature pertaining to nitric oxide synthase gene transfer to the vascular wall.

INTRODUCTION: Impaired diabetic wound healing, a source of significant disability, has been attributed to the reduced bioavailability of nitric oxide (NO) and an overabundance of oxidative stress. We demonstrated that xanthine oxidoreductase (XOR), important in purine metabolism and reactive oxygen species production, is highly expressed in regenerating skin. It is also a strong nitrite reductase that can produce NO from nitrite. We hypothesized that XOR can be harnessed to generate NO from nitrite and may improve diabetic wound healing.

Our group recently reported that cultured sheep pulmonary artery endothelial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-induced apoptosis several days after constitutive synthesis of nitric oxide (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso- N-acetylpenicillamine (SNAP) (E. Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova, I. Kovesdi, and T. R. Billiar. Surgery 122: 255–263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNAP) to determine underlying mechanisms of this phenomenon. Continuous endogenous production of NO in itself did not cause apoptosis as assessed by phase-contrast microscopy, nuclear morphology, and internucleosomal DNA fragmentation. Prolonged (72–96 h) synthesis of NO, however, after DFG- or replication-deficient adenovirus (Ad.CMV)-iNOS or SNAP (100 μM, 96 h) inhibited LPS-induced apoptosis. The kinetics of such protection suggested that NO may be inducing other gene products. Ad-mediated transfer of manganese superoxide dismutase (MnSOD) decreased the sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, however, was not induced in an NG-monomethyl-l-arginine (l-NMMA)-sensitive time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant-mediated LPS-induced apoptosis including 70-kDa heat shock protein, heme oxygenase-1, metallothionein, and Bcl-2 also were not elevated in an l-NMMA-sensitive, time-dependent fashion. Although the candidate gene product underlying NO-induced protection remains unclear, we did note that prolonged synthesis of NO inhibited LPS-induced activation of an interleukin-1β-converting enzyme-like cysteine protease (cysteine protease protein-32-like) in a dithiothreitol-sensitive fashion, suggesting that S-nitrosylation of an important downstream target of convergence of apoptotic signals may contribute to the sensitivity of SPAECs to LPS.

Background: Apoptosis limits hepatocyte viability in bioartificial livers in vitro and may contribute to liver dysfunction in vivo. Nitric oxide (NO) inhibits hepatocyte apoptosis; however, methods to deliver NO in a sustained manner to hepatocytes are limited. Here, we tested the feasibility of inducible NO synthase (iNOS) gene transfer as an approach to deliver an intracellular source of NO to inhibit spontaneous and tumor necrosis factor–α(TNF-α)–induced apoptosis in cultured hepatocytes. Methods: An adenoviral vector carrying the human iNOS gene (AdiNOS) was used to overexpress iNOS in cultured rat hepatocytes. Spontaneous apoptosis was induced by prolonged culture (4 days), and stimulated apoptosis was induced by exposure to TNF-α + actinomycin D (TNF-α+ActD). Nitrite (NO 2 – ), cell viability, and cellular caspase-3-like protease activity were measured. Results: AdiNOS gene transfer resulted in sustained NO production and protected hepatocytes from spontaneous and TNF-α+ActD–induced apoptosis. Apoptosis was associated with increases in caspase-3-like protease activity, which was suppressed by iNOS gene transfer in an NO-dependent manner. Dithiothreitol partially reversed the NO-induced suppression of caspase-3-like activity, which is consistent with S-nitrosylation of caspase-3. Conclusions: Adenovirus-mediated iNOS gene transfer effectively blocks spontaneous and TNF-α+ActD–induced cell killing in hepatocytes. iNOS gene transfer could be used to suppress apoptotic hepatocyte death in vitro and possibly in vivo.(Surgery 1998;124:278-83.)

Nitric oxide (NO) is cytostatic for proliferating cells, inhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correlates with established cytostatic activities of NO. In in vitro experiments, various cell types were exposed to NO using either donors or expression of inducible NO synthase (iNOS). The capacity of NO to suppress total protein synthesis, measured by incorporation of 35S-methionine into protein, was correlated with the capacity of NO to suppress cell proliferation, viral replication, or iNOS expression. Phosphorylation of eIF-2α was examined as a possible mechanism for the suppressed protein synthesis by NO. Both NO donors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decreased cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine peritoneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expression in pancreatic islets or RAW264.7 cells almost completely inhibited total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synthesis of specific proteins such as insulin or iNOS itself. This possibility was confirmed in RAW264.7 cells where the inhibition of total protein synthesis correlated with the decreased iNOS protein. The decrease in protein levels occurred without changes in iNOS mRNA levels, implicating an inhibition of translation. Mechanistic studies revealed that iNOS expression in RAW264.7 cells resulted in the phosphorylation of eEF-2α and inhibition of the 80S ribosomal complex formation. These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2α. Furthermore, our observations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of NO.

In cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the development of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibility antigens were used for transplantation. Allografts collected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO suppresses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS expression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon-induced aortic injury. Similarly, CsA inhibited iNOS expression in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arteriosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early immune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this otherwise untreatable disease process.

Background. We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability. Methods. An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated. Results. AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3—like protease activity. Conclusions. Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.

Efficient and organized data storage is an essential aspect of bench research and helps to maintain the integrity of the laboratory. Such processes allow investigators to report scientific data with confidence, accuracy, and transparency. The recording of experimental techniques and protocols also facilitate experimental reproducibility. These elements of scientific record maintenance are key requirements of academic institutions as well as federal funding agencies that support the work. In the modern era, paper laboratory books cannot keep up with the varied forms and volumes of data that can be generated with current investigative tools. The integration of electronic data storage into the laboratory record-keeping process enables investigators to catalog all the complex information derived from scientific investigations. It also allows for accessibility for collaboration. Regardless of the type of laboratory notebook, detail, accountability, and transparency are key elements of maintenance and require diligence on the part of the investigator to consistently achieve these goals.

Nitric oxide (NO) is an intra- and inter-signaling molecule that regulates vessel dilatation, neuronal transmission, cardiac contraction, immunomodulation, and stem cell differentiation and proliferation. NO plays an important protective role in the cardiovascular system. NO inhibits smooth muscle cells proliferation and migration; enhances proliferation and migration of endothelial cell and inhibits apoptosis; suppresses platelet aggregation; and prevents platelet, leukocyte and monocyte adhesion to endothelium. NO exerts an inhibitory effect on the development of intimal hyperplasia in mechanically or immunologically injured vessel. New therapeutic approaches aimed at enhancing NO bioavailability or assisting delivery of NO locally may help patients with cardiovascular disease.

Introduction MyD88 is an adapter protein involved in sensing tissue damage by innate immune receptors. We have previously shown that the absence of MyD88 protected against ischemia induced muscle injury in mice. This dramatic finding could be due to resistance to ischemia and/or accelerated regeneration. We hypothesize that MyD88 promotes ischemic muscle injury through increased inflammation and cell necrosis. Methods MyD88KO (KO) and WT littermates underwent femoral artery ligation (FAL) on the right hindlimb. Serum TNFα and IL6 were measured by ELISA at 24 hrs. After 1 wk, hindlimb muscle was examined for inflammatory cell infiltration, muscle necrosis, fat replacement, regenerating myocytes, and myofiber area (1 wk). Results After 24 h, serum TNFα levels were increased in mice undergoing FAL but were similar in KO and WT mice. Serum IL6, a myokine that inhibits muscle growth, was higher in WT than KO mice (79.3 ± 18.4 vs. 22.9 ± 11.8 pg/ml; p2 vs. 77.3 ± 23.5 μm 2 , N=4-6 each; p=0.03; Fig 1). Discussion Ischemia promotes muscle inflammation and necrosis independent of MyD88. Instead, the absence of MyD88 enhanced regeneration, potentially through a reduction in IL6 production. Thus, contrary to our hypothesis, MyD88 appears to increase muscle injury following ischemia by inhibiting muscle regeneration and not through increased inflammation and necrosis.

Objective Abdominal aortic aneurysms (AAA) affect up to 9% of men over 65 years of age with increasing incidence with further aging. Little is still known about the pathogenesis of AAA but inflammation appears to be one of the main contributing factors. Carbon monoxide (CO) is an endogenously produced molecule that has been shown to have significant anti-inflammatory and cytoprotective properties. Thus, we hypothesize that exogenous CO may attenuate AAA growth. Methods Eight week-old C57Bl6 mice were anesthetized and infrarenal aorta were exposed, measured in 3 locations, and treated with topical porcine pancreatic elastase (30 ul). Mice were either maintained in room air for the duration of the experiment or treated with inhaled CO (250 PPM) for 1 hr daily beginning on day 1. Animals were sacrificed on day 14 when aortic diameters were measured and tissues collected for morphometric analysis. Results Topical elastase treatment resulted in marked aortic enlargement with true aneurysm formation (253% of baseline, N=8). However, CO treatment reduced aneurysm size significantly (222%, N=8; p-0.039) with noticeable reduction in adhesion formation and scarring around the aorta. Histologically, CO treated animals maintained a larger medial area than controls (0.092 mm 2 vs 0.057 mm 2 ; p = 0.023) (Figure 1). Conclusion Inhaled CO appears to mitigate aneurysmal degeneration of the abdominal aorta induced by topical elastase. There was a clear reduction in adhesions to the aorta, strongly supporting the anti-inflammatory mechanism involved. The maintenance of a thicker medial component of the aorta in CO treated mice also suggests protection of the smooth muscle cells.

Background Nonhealing wounds are a major source of morbidity in diabetic patients. These wounds persist due to a persistent inflammatory state and poor resistance to infection. Carbon monoxide (CO) is a naturally occurring byproduct of heme degradation that possesses both anti-inflammatory and pro-angiogenic properties. Thus, we hypothesize that inhaled CO will improve cutaneous wound healing in the setting of diabetes. Methods Excisional wounds (1.5 x 1.5 cm) were created in db/db mice. The mice were treated with inhaled CO at 250 ppm for 1 hr daily starting immediately after wounding or starting on day 8. Control mice remained in room air. Wounds were measured by planimetry. At day 20, mice were sacrificed and the residual wounds were collected for immunohistochemistry. Results Inhaled CO initiated immediately after wounding did not improve healing with less granulation tissue formation. However, when CO treatment was delayed until day 8, wound healing was greatly improved compared to controls (residual wounds measuring 27.2 ± 5.3% of original vs 52.2 ± 5.8% on day 20; N=10-14 per group; p < 0.005). CD31 staining showed increased angiogenesis in the delayed CO treated animals (Figure 1). Conclusions Early inhaled CO was detrimental to wound healing while delaying CO treatment accelerated healing, potentially through increased angiogenesis. Early administration of CO likely inhibits inflammatory responses and cellular recruitment essential for wound healing. Delayed CO may inhibit the persistent inflammation that leads to chronic non-healing wounds, allowing for improved wound healing in diabetes.

Background. Previously we demonstrated that the heat shock response (HSR) inhibits cytokine-stimulated nitric oxide (NO) synthesis and inducible NO synthase (NOS2) expression in hepatocytes. In this study we sought to determine the molecular basis of this inhibition using a human liver cell line. Methods. After induction of the HSR by sodium arsenite or hyperthermia, the AKN-1 human liver cell line was treated with cytokines to stimulate NOS2 expression and NO production. Western blot analysis for hsp 70 was performed, and NOS2 mRNA and 24-hour NO synthesis were quantitated. Cytokine-induced NOS2 promoter activity of AKN-1 cells transfected with a 7.0 kilobase NOS2 promoter luciferase construct and NO production of AKN-1 cells transduced with the NOS2 gene were measured. Results. Sodium arsenite or hyperthermia induced the synthesis of hsp 70 protein in AKN-1 cells, indicating activation of the HSR. Cytokines stimulated high levels of NOS2 mRNA and NO production. However, prior induction of the HSR significantly inhibited NOS2 expression and NO synthesis. Cytokine-stimulated NOS2 promoter activity of transfected AKN-1 cells was decreased by 77%, but the HSR did not affect NOS2 enzyme activity in transduced AKN-1 cells. Conclusions. These findings indicate that the HSR inhibits cytokine-induced NOS2 expression and NO synthesis in AKN-1 cells by preventing NOS2 promoter activation. Effects on NOS2 protein translation or stability were not observed. These data suggest that the HSR, which is expressed in the liver after trauma, shock, or ischemia-reperfusion, blocks NOS2 gene expression at the transcriptional level.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Classically, osteoarthritis (OA) has been considered a noninflammatory disease. However, the detection of selected inflammatory mediators in osteoarthritic fluid, in the absence of significant inflammatory cell infiltrate, is increasingly appreciated. We sought to identify the inflammatory component in human OA-affected cartilage that may be involved in cartilage damage/destruction. Using Western blot analysis and an antibody to the conserved region of nitric oxide synthase (NOS), we have observed up-regulation of NOS, one of the "key players" of inflammation, in chondrocytes of OA-affected patients. Remarkably, none of the cartilage samples examined from normal joints demonstrated detectable amounts of this NOS. Western blot analysis using the same alpha-NOS antibody indicated that this NOS from OA-affected cartilage (OA-NOS) was larger in size than (and distinct from) transfected human hepatocyte or murine inducible NOS (iNOS) (150 versus 133 kD) and similar in size to neuronal constitutive NOS (ncNOS). Antibodies specific for iNOS showed binding to murine and human iNOS but not to OA-NOS, endothelial constitutive NOS, or ncNOS. Antibodies specific for ncNOS bound to ncNOS and also to OA-NOS, but not to murine or human iNOS or endothelial constitutive NOS. Incubation of OA cartilage in serum-free medium resulted in spontaneous release, for up to 72 h, of substantial amounts of nitrite (up to approximately 80 microM/100 mg wet tissue), which could be inhibited by at least 80% with various inhibitors of iNOS, including inhibitors of protein synthesis and transcription factor NF-kappa B, but which (unlike murine macrophage iNOS) was not sensitive to hydrocortisone or TGF-beta. Exposure of OA-affected cartilage to interleukin 1 beta, tumor necrosis factor-alpha, and lipopolysaccharide resulted in approximately 20-50% augmentation of nitrite accumulation, which was also sensitive to cycloheximide and pyrrolidine dithiocarbamate. Hence, our data indicate that OA-NOS (based on immunoreactivity and molecular weight) is similar to ncNOS and that it releases nitric oxide, which may contribute to the inflammation and pathogenesis of cartilage destruction in OA.

Importance Because of the similarity in clinical outcomes after elective open and endovascular repair of abdominal aortic aneurysm (AAA), cost may be an important factor in choosing a procedure. Objective To compare total and AAA-related use of health care services, costs, and cost-effectiveness between groups randomized to open or endovascular repair. Design, Setting, and Participants This unblinded randomized clinical trial enrolled 881 patients undergoing planned elective repair of AAA who were candidates for open and endovascular repair procedures. Patients were randomized from October 15, 2002, to April 15, 2008, at 42 Veterans Affairs medical centers. Follow-up was completed on October 15, 2011, and data were analyzed from April 15, 2013, to April 15, 2016, based on intention to treat. Main Outcomes and Measures Mean total and AAA-related health care cost per life-year and per quality-adjusted life-year (QALY). Results A total of 881 patients (876 men [99.4%]; 5 women [0.6%]; mean [SD] age, 70 [7.8] years) were included in the analysis. After a mean of 5.2 years of follow-up, mean life-years were 4.89 in the endovascular group and 4.84 in the open repair group ( P = .68), and mean QALYs were 3.72 in the endovascular group and 3.70 in the open repair group ( P = .82). Total mean health care costs did not differ significantly between the 2 groups (endovascular group, $142 745; open repair group, $153 533; difference, −$10 788; 95% CI, −$29 796 to $5825; P = .25). Costs related to AAA, including the initial repair, constituted nearly 40% of total costs and did not differ significantly between the 2 groups (endovascular group, $57 501; open repair group, $57 893; difference, −$393; 95% CI, −$12 071 to $7928; P = .94). Lower costs due to shorter hospitalization for initial endovascular repair were offset by increased costs from AAA-related secondary procedures and imaging studies. The probability of endovascular repair being less costly and more effective was 56.8% when effectiveness was measured in life-years and 55.4% when effectiveness was measured in QALYs for total costs and 31.3% and 34.3%, respectively, for AAA-related costs. Conclusions and Relevance In this multicenter randomized clinical trial with follow-up to 9 years, survival, quality of life, costs, and cost-effectiveness did not differ between elective open and endovascular repair of AAA. Trial Registration clinicaltrials.gov Identifier:NCT00094575

In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.

BackgroundWe have previously shown that the danger signal high-mobility group box 1 (HMGB1) promotes angiogenesis when administered to ischemic muscle. HMGB1 signals through Toll-like receptor 4 (TLR4) as well as the receptor for advanced glycation end-products (RAGE). However, the actions of these receptors in ischemic injury and muscle recovery are not known. We hypothesize that TLR4 mediates tissue recovery and angiogenesis in response to ischemia.MethodsFemoral artery ligation was performed in control, TLR4 competent (C3H/HeOuJ) and incompetent (C3H/HeJ) mice, as well as RAGE knockout mice and their C57B6 control counterparts. In other experiments, control mice were pretreated with anti-HMGB1 neutralizing antibody before femoral artery ligation. After 2 weeks, limb perfusion was evaluated using laser Doppler perfusion imaging and reported as the ratio of blood flow in the ischemic to nonischemic limb. Muscle necrosis, fat replacement, and vascular density in the anterior tibialis muscle were quantified histologically. In vitro, TLR4 and RAGE expression was evaluated in human dermal microvascular endothelial cells in response to hypoxia. Human dermal microvascular endothelial cells treated with HMGB1 alone and in the presence of anti-TLR4 antibody were probed for phosphorylated extracellular signal-regulated kinase (ERK), a signaling molecule critical to endothelial cell (EC) angiogenic behavior.ResultsBoth anti-HMGB1 antibody as well as defective TLR4 signaling in HeJ mice resulted in prominent muscle necrosis 2 weeks after femoral artery ligation. Control HeOuJ mice had less necrosis than TLR4 incompetent HeJ mice, but a greater amount of fat replacement. In contrast to control C3H mice, control C57B6 mice demonstrated prominent muscle regeneration with very little necrosis. Muscle regeneration was not dependent on RAGE. While vascular density did not differ between strains, mice with intact RAGE and TLR4 signaling had less blood flow in ischemic limbs compared with mutant strains. In vitro, EC TLR4 expression increased in response to hypoxia while TLR4 antagonism decreased HMGB1-induced activation of extracellular signal-regulated kinase.ConclusionsBoth HMGB1 and TLR4 protect against muscle necrosis after hindlimb ischemia. However, muscle regeneration does not appear to be tied to vascular density. HMGB1 likely activates angiogenic behavior in ECs in vitro, and this activation may be modulated by TLR4. The improvement in blood flow seen in mice with absent TLR4 and RAGE signaling may suggest anti-angiogenic roles for both receptors, or vasoconstriction induced by TLR4 and RAGE mediated inflammatory pathways.Clinical RelevanceNonreconstructable peripheral artery disease causes significant functional disability and is associated with a high risk of limb loss. The mechanisms that govern muscle recovery and angiogenesis after ischemia are important to understand to improve medical therapy for patients who cannot have an intervention. This article evaluates the role of high-mobility group box 1 and the innate immune receptor Toll-like receptor 4 in mediating muscle recovery after ischemia. While high-mobility group box 1 has been shown to mediate end-organ damage in other clinical scenarios, it may also play an important role in regenerative processes such as myocyte regeneration and angiogenesis.

Objectives: Arteriogenesis is a process by which small resistance vessels mature into large caliber collateral arteries in response to arterial occlusive disease. The forces stimulating arteriogenesis nclude shear stress and circumferential wall stress. Nucleotides are released from cells in response to mechanical stimuli and may activate nucleotide receptors. Thus, we hypothesize that the P2Y 2 nucleotide receptor, expressed by vascular cells, mediates arteriogenesis. Methods: C57Bl6 NJ (WT) and P2Y 2 R KO mice (n=8/group) underwent unilateral femoral artery ligation (FAL). Perfusion was measured immediately after ligation and at 3, 7, 14, 21, and 28 days using laser Doppler perfusion imaging (LDPI). Mice were sacrificed at 28 days and tibialis anterior collected for histology. Results: LDPI of the ischemic hindlimb showed a return to baseline perfusion in WT mice (Figure). P2Y 2 KO mice recovered to 2 KO mice developed wounds of the ischemic limbs vs. 0/8 of WT mice. Histological analysis of tibialis anterior demonstrated regenerating myocytes in 3/4 P2Y 2 KO vs. 1/4 WT animals. By immunohistochemistry, angiogenesis was significantly increased in P2Y 2 KO with a CD31+cell/myocyte ratio of 1.974 vs. 1.314 of WT (p=0.05). There was also greater inflammation in P2Y 2 KO with CD45+ cell infiltration over 3-fold that of WT (pConclusion: The P2Y 2 nucleotide receptor is important for blood flow recovery in a murine model of FAL. P2Y 2 KO mice also demonstrated delayed muscle regeneration with greater angiogenesis and inflammation in ischemic limbs. Further molecular analyses are needed to elucidate the mechanisms involved.

Intro: Diabetic wounds heal poorly leading to major morbidity, mortality, and financial cost. Nitric oxide (NO), produced in wounds by NO synthases (NOSs), is essential for normal wound healing and is reduced in diabetic wounds, contributing to poor healing. It is known that NO can also be regenerated from nitrite through xanthine oxidoreductase (XOR). Thus, we hypothesize that dietary nitrite may be an important source of NO in diabetic wound healing. Methods: Diabetic Db/Db mice were fed normal chow, nitrite depleted chow, or nitrite depleted chow with 1.5g/L nitrite supplemented water starting 1 week prior to wounding. Excisional wounds were created on each mouse and wound areas were measured every other day. Skin and wound samples from a separate group of Db/Db mice were obtained prior to and 5 days after wounding. Tissues were used for Western blots and XOR activity assay. Plasma was used for nitrite measurements. Results: Nitrite depletion lowered plasma nitrite by 50-60% vs mice on chow or on nitrite water. XOR was highly expressed in the skin, less so at the wound edge, and minimally in granulation tissue by Western. This was confirmed by XOR enzyme assay showing 5 fold more activity in the skin compared to the wound tissues. Wound healing was delayed in nitrite depleted mice as compared to the chow fed mice (Figure). Nitrite supplementation showed a trend in improved healing rates vs nitrite depleted mice. Conclusion: Diabetic skin and wound edge expresses XOR. Nitrite does contribute to wound healing in Db/Db mice. Dietary nitrite increased plasma nitrite levels and increased the wound healing rate. This suggests a potential therapeutic role for dietary nitrite in diabetic wound care.

Introduction: We have shown that TLR2 mediates muscle regeneration and angiogenesis after muscle ischemia, independent of TLR4. Downstream signaling of TLR2 and TLR4 include MyD88 (TLR2 and 4) and TRIF (TLR4 and 3) pathways. We hypothesize that MyD88 is required for normal angiogenesis and muscle regeneration following ischemia while TRIF is not. Methods: MyD88 -/- (KO), TRIF KO and control C57B6 mice (WT) (N=4-7) underwent femoral artery ligation on the right hindlimb, and sham operation on the left. LDPI was performed at day 14 and anterior tibialis muscle was collected to quantify muscle necrosis and regeneration. Vascular structures were identified with CD31 staining. Results: MyD88 KO and WT mice demonstrated partial perfusion recovery by 14 days while TRIF KO had minimal recovery. Despite this, there were significantly fewer vascular structures in MyD88 KO mice while TRIF KO had the greatest (17.9±1.5 MyD88 KO; 55.3±6.8 WT; 69.1±5.2 TRIF KO; p Conclusions: Contrary to our hypothesis, TRIF appears to be required for normal muscle regeneration and functional angiogenesis after ischemia, while MyD88 is an important mediator of hypoxic injury. These results, along with our prior findings that muscle regeneration is independent of TLR4, suggest that TLR3 may play a key role in ischemic muscle recovery independent of angiogenesis.

Vascular intimal hyperplasia (IH) limits the long term efficacy of current surgical and percutaneous therapies for atherosclerotic disease. There are extensive changes in gene expression and cell signaling in response to vascular therapies, including changes in nitric oxide (NO) signaling. NO is well recognized for its vasoregulatory properties and has been investigated as a therapeutic treatment for its vasoprotective abilities. The circulating molecules nitrite ( NO 2 - ) and nitrate ( NO 3 - ), once thought to be stable products of NO metabolism, are now recognized as important circulating reservoirs of NO and represent a complementary source of NO in contrast to the classic l -arginine–NO–synthase pathway. Here we review the background of IH, its relationship with the NO and nitrite/nitrate pathways, and current and future therapeutic opportunities for these molecules.

Objective Secondary interventions after endovascular aneurysm repair (EVAR) remain a concern. Most are simple catheter-based procedures, but in some instances, open conversions (OCs) are required and carry a worse outcome. We reviewed our experience to characterize these OCs. Methods A retrospective review was conducted of all patients who underwent an OC after a previous EVAR for an aneurysm-related indication from 2001 to 2010. Clinical outcomes are reported. Results Data were reviewed for 44 patients (77% men) with a mean age of 74 years (range, 55-90 years). The average time from EVAR to the first OC was 45 months (range, 2-190 months). In six patients (14%), the initial EVAR was at another institution. The endografts used were Ancure in 16, Excluder in 13, AneuRx in eight, Zenith in three, Lifepath in one, Renu in one, and undetermined in two. Twenty-two patients had previously undergone a total of 32 endovascular reinterventions before their index OC. Indications for OC were aneurysm expansion in 28 (64%), rupture in 12 (27%), and infection in four (9%). The endograft was preserved in situ in 10 patients (23%). Explantation was partial in 18 (41%) or complete in 16 (36%). Endograft preservation was used for type II endoleak in all but one patient by selective ligation of the culprit arteries (lumbar in four, inferior mesenteric artery in five, and middle sacral in one). Proximal neck banding was performed in one type Ia endoleak. Overall morbidity was 55%, and mortality was 18%. No deaths occurred in a subgroup of patients who underwent endograft preservation with selective ligation of culprit vessels for type II endoleak. Intraoperative complications included bowel injury in two, bleeding in two, splenectomy in one, and ureteral injury in one. At a mean follow-up of 20 months, two patients underwent additional procedures after the index OC: one after endograft preservation and one after partial explantation. None of the patients who underwent elective OC with endograft preservation required subsequent endograft explantation. Conclusions Most OCs after EVAR are associated with significant morbidity and mortality, except when electively treating an isolated type II endoleak with ligation of branches and preservation of the endograft.

RESULTS: This study included 294 open and 165 FEVAR repairs. FEVAR patients were older (76 9 yrs. vs 71 9; p

Veins are still the best conduits available for arterial bypass surgery. When these arterialized vein grafts fail, it is often due to the development of intimal hyperplasia (IH). We investigated the feasibility and efficacy of the ex vivo pre-treatment of vein grafts with soluble carbon monoxide (CO) in the inhibition of IH.The inferior vena cava was excised from donor rats and placed as an interposition graft into the abdominal aorta of syngeneic rats. Prior to implantation, vein grafts were stored in cold Lactated Ringer (LR) solution with or without CO saturation (bubbling of 100% CO) for 2 h. Three and 6 weeks following grafting, vein grafts treated with cold LR for 2 h developed IH, whereas grafts implanted immediately after harvest demonstrated significantly less IH. Treatment in CO-saturated LR significantly inhibited IH and reduced vascular endothelial cell (VEC) apoptosis. Electron microscopy revealed improved VEC integrity with less platelet/white blood cell aggregation in CO-treated grafts. The effects of CO in preventing IH were associated with activation of hypoxia inducible factor-1α (HIF-1α) and an increase in vascular endothelial growth factor (VEGF) expression at 3-6 h after grafting. Treatment with a HIF-1α inhibitor completely abrogated the induction of VEGF by CO and reversed the protective effects of CO on prevention of IH.Ex vivo treatment of vein grafts in CO-saturated LR preserved VEC integrity perioperatively and significantly reduced neointima formation. These effects appear to be mediated through the activation of the HIF1α/VEGF pathway.

Background— Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty–induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated. Methods and Results— Exposure to CO has diametrically opposite effects on endothelial cell (EC) and vascular smooth muscle cell proliferation in rodent models of carotid injury. In contrast to its effect of blocking vascular smooth muscle cell growth, CO administered as a gas or as a CO-releasing molecule enhances proliferation and motility of ECs in vitro by >50% versus air controls, and in vivo, it accelerates reendothelialization of the denuded artery by day 4 after injury versus day 6 in air-treated animals. CO enhanced EC proliferation via rapid activation of RhoA (Ras homolog gene family, member A), followed by downstream phosphorylation of Akt, endothelial nitric oxide (NO) synthase phosphorylation, and a 60% increase in NO generation by ECs. CO drives cell cycle progression through phosphorylation of retinoblastoma, which is dependent in part on endothelial NO synthase–generated NO. Similarly, endothelial repair in vivo requires NO-dependent mobilization of bone marrow–derived EC progenitors, and CO yielded a 4-fold increase in the number of mobilized green fluorescent protein–Tie2–positive endothelial progenitor cells versus controls, with a corresponding accelerated deposition of differentiated green fluorescent protein–Tie2–positive ECs at the site of injury. CO was ineffective in augmenting EC repair and the ensuing development of intimal hyperplasia in eNOS −/− mice. Conclusions— Collectively, the present data demonstrate that CO accelerates EC proliferation and vessel repair in a manner dependent on NO generation and enhanced recruitment of bone marrow–derived endothelial progenitor cells.

INTRODUCTION: Impaired diabetic wound healing, a source of significant disability, has been attributed to the reduced bioavailability of nitric oxide (NO) and an overabundance of oxidative stress. We demonstrated that xanthine oxidoreductase (XOR), important in purine metabolism and reactive oxygen species production, is highly expressed in regenerating skin. It is also a strong nitrite reductase that can produce NO from nitrite. We hypothesized that XOR can be harnessed to generate NO from nitrite and may improve diabetic wound healing.

Objectives: Primary closure after carotidendarterectomy (CEA) has been much maligned as an inferior technique with worse outcomes than patch closure. Our purpose was to compare perioperative and long-term results of different CEA closure techniques in a large institutional experience. Methods: A consecutive cohort of CEAs between January 1, 2000 and December 31, 2010 was retrospectively analyzed. Closure technique was used to divide patients into three groups: primary longitudinal arteriotomy closure (PRC), patch closure (PAC), and eversion closure (EVC). End points were perioperative events, long-term strokes, and restenosis $70%. Multivariate regression models were used to assess the effect of baseline predictors. Results: During the study period, 1737 CEAs (bilateral, 143; mean age, 71.4 6 9.3 years; 56.2% male; 35.3% symptomatic) were performed, with a mean clinical followup of 49.8 6 36.4 months (0-155 months). More men had primary closure, but other demographic and baseline symptomswere similar between the groups.Half the patients had patching, with the rest evenly distributed between PRC and EVC. The rate of nerve injury was 2.7%, reintervention for hematoma was 1.5%, and hospital length of stay was 2.4 6 3.0 days, with no significant differences among groups. Periprocedural stroke and death rates and longterm results were not different among the groups (Table). Multivariate analysis showed baseline symptoms (OR, 2.4; P 1⁄4 .007) and heart failure (OR, 3.1; P 1⁄4 .003) were

Objective Prior analysis in the Open vs Endovascular Repair Veterans Affairs (VA) Cooperative Study (CSP #498) demonstrated that survival, quality of life, and total health care costs are not significantly different between the open and endovascular methods of repair of abdominal aortic aneurysm. The device is a major cost of this method of repair, and the objective of this study was to evaluate the costs of the device, abdominal aortic aneurysm repair, and total health care costs when different endograft systems are selected for the endovascular repair (EVR). Within each selected system, EVR costs are compared with open repair costs. Methods The study randomized 881 patients to open (n = 437) or EVR (n = 444). Device selection was recorded before randomization; therefore, open repair controls were matched to each device cohort. Data were excluded for two low-volume devices, implanted in only 13 individuals, leaving 423 control and 431 endovascular patients: 166 Zenith (Cook Medical, Bloomington, Ind), 177 Excluder (W. L. Gore & Associates, Flagstaff, Ariz), and 88 AneuRx (Medtronic, Minneapolis, Minn). Mean device, hospitalization, and total health care costs from randomization to 2 years were compared. Health care utilization data were obtained from patients and national VA and Medicare data sources. VA costs were determined using methods previously developed by the VA Health Economics Resource Center. Non-VA costs were obtained from Medicare claims data and billing data from the patient's health care providers. Results Implant costs were 38% of initial hospitalization costs. Mean device (range, $13,600-$14,400), initial hospitalization (range, $34,800-$38,900), and total health care costs at 2 years in the endovascular (range, $72,400-$78,200) and open repair groups (range, $75,600-$82,100) were not significantly different among device systems. Differences between endovascular and corresponding open repair cohorts showed lower mean costs for EVR (range, $3200-$8300), but these were not statistically different. Conclusions The implant costs of endovascular aneurysm repair are substantial. When evaluating total health care system expenditures, there is large individual variability in costs, and there is no significant difference at 2 years among systems or when an individual system is compared with open repair.

Several findings on computed tomography (CT) scans of intact aneurysms have been taken to suggest “imminent” or “impending” aneurysm rupture. Often these are identified incidentally in asymptomatic patients when an urgent operation was not planned and may even be ill advised. The authors evaluated whether these signs can truly predict short-term aneurysm rupture. A computerized medical archival system was reviewed from August 1994 to August 2004. Patients with aortic aneurysms and official CT scan reports of “impending rupture” were reviewed. CT films and reports were reviewed for aneurysm characteristics, while computerized medical records were reviewed for patient demographics, comorbidities, symptoms, documented subsequent rupture, and operative findings. Signs of “impending rupture” included the crescent sign, discontinuous circumaortic calcification, aortic bulges or blebs, aortic draping, and aortic wall irregularity. Rupture occurring within 2 weeks of the index CT was defined as supporting the “imminent” label. Forty-five patients with aortic aneurysms and CT stigmata of “impending rupture” were identified. Five patients with additional signs of suspicious leak and 1 with an infected previously repaired aneurysm were excluded. Of 39 intact aneurysms, 26 (67%) were infrarenal, 2 (5%) were suprarenal, and the remaining 11 (28%) were thoracoabdominal. The patient group had more women than expected (19/39, 49%) and larger aneurysms (mean diameter, 6.8 ±1.4 cm). Mean age was 74 years. Ten patients underwent elective repair within the first 2 weeks after the index CT scan (mean, 4 days), precluding adequate observation for early rupture. None had intraoperative signs of rupture. Early rupture: 2 of the 29 remaining patients ruptured within 72 hours of the CT scan, for a positive predictive value of 6.9%. One additional patient ruptured 7 months later after declining an early intervention. No Rupture: 26 patients were observed an average of 246 days (range, 14 days to 3 years) without evidence of rupture. Fourteen were repaired electively 2 weeks to 3 years after the index CT scan, and 12 never underwent repair, mostly because of severe associated comorbidities, and were observed a mean of 394 days without rupture. Although they should be taken seriously, CT signs of “impending rupture” alone are poor predictors of short-term aortic aneurysm rupture, and alternative terminology is needed until better predictors can be identified.

Vascular bypass surgery involves the use of a vascular conduit to circumvent a site of vascular compromise. Vascular graft failure continues to plague both the patients receiving and the surgeons performing these interventions. Demand for the development of a therapy to reduce intimal hyperplasia--the most common cause of bypass graft failure--is significant and has been the goal of many biotechnology groups. The development of gene therapy as a feasible clinical intervention has allowed for novel methods of inhibiting intimal hyperplasia to be conceived. This review describes the evolution of gene transfer of the inducible nitric oxide synthase (iNOS) gene, one of the most successful preclinical interventions to date for vascular disease.

Nitric oxide (NO) is a molecule that has gained recognition as a crucial modulator of vascular disease. NO has a number of intracellular effects that lead to vasorelaxation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet adhesion. Its role in vascular disease has been intensively investigated and further elucidated over the past two decades. It is important in the pathogenesis of many cardiovascular diseases, including atherosclerosis, intimal hyperplasia, and aneurysmal disease. In addition, NO has been used as a therapeutic tool to treat diseases that range from recurrent stenosis to inhibiting thrombotic events. Many commonly used medications have their therapeutic actions through the production of NO. This review highlights the vascular biologic characteristics of NO, its role in the pathogenesis of cardiovascular disease processes, and its potential therapeutic applications.

Vascular injury with endothelial dysfunction results in an imbalance between the production of vasoprotective molecules such as nitric oxide (NO) and deleterious reactive oxygen species (ROS). The purpose of this work was to test the hypothesis that inhibition of geranylgeranyltransferase I (GG Tase I) reduces vascular injury by increasing vascular NO production while decreasing ROS generation.GGTI-298 decreased the formation of intimal hyperplasia at 14 days following balloon injury. GGTI-298 (10 microm) inhibited activation of RhoA and Rac1 as well as inhibited SMC proliferation. GGTI increased SMC-inducible NO synthase (iNOS) levels and NO production in vitro. Additionally, the activation of NAD(P)H oxidase subunits was decreased by GGTI in vitro. This correlated with a decrease in TNF-alpha- or angiotensin-II-induced ROS production assayed by DCF fluorescence. In vivo, GGTI treatment increased endothelial NOS (eNOS) expression in uninjured arteries and iNOS expression in balloon-injured arteries. Furthermore, GGTI treatment attenuated balloon-injury-induced superoxide generation assayed by MCLA luminescence.GGTI decreases the production of ROS and increases the production of NO both in vitro and in vivo. These effects may be mediated via the inhibition of activation of the small GTPases Rac1 and RhoA. Pharmacological inhibition of GGTase I may prove to be a useful clinical adjunct in the treatment of cardiovascular diseases.

The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

ObjectivesPoor outcomes have been reported with endovascular aneurysm repair (EVAR) in patients with hostile neck anatomy. Unsupported endografts with active fixation may offer certain advantages in this situation. We compared EVAR results using the Ancure (Guidant) endograft in patients with and without hostile neck anatomy.MethodsRecords of EVAR patients from October 1999 to July 2002 at a tertiary care hospital were retrospectively reviewed from a division database. Patients with elective open abdominal aortic aneurysm (AAA) repair during the same period were reviewed to determine those unsuitable for EVAR. Hostile neck anatomy, assessed by computer tomography (CT) scans and angiograms, was defined as one or more of the following: (1) neck length ≤10 mm, (2) focal bulge in the neck >3 mm, (3) >2-mm reverse taper within 1 cm below the renal arteries, (4) neck thrombus ≥50% of circumference, and (5) angulation ≥60 degrees within 3 cm below renals.ResultsThree hundred and twenty-two patients underwent EVAR with an average follow-up of 18 months. Patients in Phase II trials (n = 41), repaired with other graft types (n = 48), or without complete anatomic records (n = 27) were excluded. Demographics and co-morbidities were similar in the 115 good-neck (GN) and 91 bad-neck (BN) patients except for age (mean, 72.9 years GN vs 75.7 BN; P = 0.13), gender (11% female GN vs 22% BN; P =.04); neck length (mean, 21.8 mm GN vs 14.4 mm BN: P < .001), and angulation (mean, 22 degrees GN vs 40 degrees BN; (P < .001). Perioperative mortality (0 GN vs 1.1% BN), late mortality (5.2% GN vs 4.4% BN), all endoleaks (19.1% GN vs 17.6% BN), proximal endoleaks (0.8% GN vs 2.1% BN), and graft migration (0 for both groups) did not reach statistical significance. Neck anatomy precluded EVAR in 106 of 165 (64%) patients with open AAA.ConclusionsUnsupported endografts with active fixation can yield excellent results in treating many medically compromised patients with hostile neck anatomy. Nonetheless, an unsuitable neck remains the most frequent cause for open abdominal AAA.

Vascular injury and inflammation are associated with elaboration of a number of cytokines that signal through multiple pathways to act as smooth muscle cell (SMC) mitogens. Activation of the nuclear factor-kappa B (NF-kappaB) transcription factor is essential for SMC proliferation in vitro and is activated by vascular injury in vivo. Activation of NF-kappaB is controlled by several upstream regulators, including the inhibitors of kappa B (IkappaB). These proteins bind to and keep NF-kappaB inactivated. The purpose of this study was to determine whether adenoviral gene transfer of a mutated IkappaBalpha super-repressor (AdIkappaBalphaSR) could inhibit development of intimal hyperplasia in vivo and to investigate how over-expression of this construct influences in vitro SMC proliferation and cell cycle regulatory proteins.A rat carotid injury model was used to study prevention of intimal hyperplasia. Arteries were assayed 14 days after injury and infection with AdIkappaBalphaSR or adenoviral beta-galactosidase (AdLacZ). Untreated SMC or SMC infected with AdLacZ or AdIkappaBalphaSR were stimulated with 10% fetal bovine serum, interleukin-1beta, or tumor necrosis factor-alpha. Electrophoretic mobility shift assays were used to assay for NF-kappaB activation. Protein levels of IkappaBalpha and cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1) were determined with Western blot analysis. Proliferation was measured with (3)H-thymidine incorporation assays.AdIkappaBalphaSR inhibited the development of intimal hyperplasia by 49% (P.05). Infection with AdIkappaBalphaSR significantly suppressed in vitro SMC proliferation when stimulated with serum, interleukin 1, or tumor necrosis factor alpha, and did not result in cell death. Inhibition of proliferation was associated with increased p21(Cip1/Waf1) and p27(Kip1) protein levels.Gene transfer of IkappaBalpha super-repressor inhibited development of intimal hyperplasia in vivo and SMC proliferation in vitro. The antiproliferative activity may be related to cell cycle arrest through upregulation of the cyclin-dependent kinase inhibitors p21 and p27. Overexpression of IkappaBalpha may be a future therapeutic option in treatment of vascular diseases.

Background. Pregnancy during general surgery residency has traditionally been discouraged. Methods. In 2001, using an approved protocol, we anonymously surveyed 25 residents (PGY3 level or greater) concerning their experiences working with each other during episodes of resident pregnancy and maternity leave. Results. From 1995 to 2001, 13 of 59 residents in general surgery were female (22%). While training, 6 of 13 residents reported 8 pregnancies with 2 miscarriages. Five residents (39%) gave birth to 6 children and adopted 1 child. Residents worked until the day of term delivery in 5 of 6 cases; 1 pregnancy was complicated by placental abruption at 33 weeks. Residents were off work postpartum for a median of 6 weeks (range 2-6). Nursing was universal for ≥ 3 months but at-work problems with privacy and stress were frequent. On survey, all resident mothers believed they had been treated very fairly, and 94% of surveyed male peers stated that the coworker's status had no effect or a positive effect on their own work life. Fatherhood was reported to occur during residency by 42% of male respondents. Conclusions. Parenthood during residency is frequent. The complexities of resident maternity can be handled with mutual safety, equity, and satisfaction by the residents and faculty of a surgical training program. (Surgery 2002;132:682-8).