Michael B. Freeman

Activation of the AMP-activated Protein Kinase by the Anti-diabetic Drug Metformin in Vivo ROLE OF MITOCHONDRIAL REACTIVE NITROGEN SPECIES

Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-α that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.

Covered stents for injuries of subclavian and axillary arteries.

INTRODUCTION Injury to the subclavian and axillary arteries is uncommon. Exposure of these vessels is associated with significant morbidity, and mortality ranges from 5% to 30%. Endovascular methods may offer an alternative approach to these technically challenging injuries. METHODS We retrospectively studied patients with blunt or penetrating (including iatrogenic) injuries to the subclavian or axillary artery between January 1, 1996 and July 30, 2002. Demographic data, mechanism of injury, concomitant injuries, angiographic findings, and treatment method and outcome were recorded. RESULTS Twenty-seven patients with injury to the subclavian or axillary artery were seen at our institution during the study. Twenty-three patients underwent interventions. Eleven patients required open repair; 12 patients had lesions amenable to endovascular repair. Depending on the preference of the surgeon, 5 patients with injuries amenable to endovascular repair underwent open repair, and 7 underwent endovascular repair. A Wallgraft endoprosthesis was used in all patients; two grafts were required in 1 patient. Endovascular repair was associated with shorter operative time (P =.04) and less blood loss (P =.01). One-year patency was similar between the two groups. CONCLUSION Covered stents are a feasible alternative to open repair in properly selected patients with subclavian or axillary artery injury, resulting in shorter procedure time and less blood loss.

Iliac artery stenting versus surgical reconstruction for TASC (transatlantic inter-society consensus) type B and type C iliac lesions

OBJECTIVE The TransAtlantic Inter-Society Consensus (TASC) document did not define the best treatment for moderately severe iliac artery lesions, ie, TASC type B and type C iliac lesions, because of insufficient solid evidence to make firm recommendations. The purpose of this study was to evaluate the influence of risk factors on outcome of iliac stenting and operative procedures used to treat TASC type B and type C lesions. METHODS Over the 5 years from 1996 to 2001, 188 endovascular and direct aortoiliac surgical reconstruction procedures were performed in 87 women and 101 men with TASC type B and type C iliac lesions and chronic limb ischemia. The criteria prepared by the Ad Hoc Committee on Reporting Standards (Society for Vascular Surgery/International Society for Cardiovascular Surgery (SVS/ISCVS) were followed to define variables. Both univariate analysis (Kaplan-Meier method) and multivariate analysis (Cox proportional hazards model) were used to determine the association between variables, cumulative patency rate, limb salvage, and survival. RESULTS Indications for revascularization were disabling claudication (73%), limb salvage (25%), and blue toe syndrome (2%). Patients in the surgery group (n = 52) had significantly higher primary patency rates compared with patients in the stent group (n = 136) at univariate analysis (Kaplan-Meier method, log-rank test; P =.015). Primary patency rates at 1, 3, and 5 years were 85%, 72%, and 64% after iliac stenting, and 89%, 86%, and 86% after surgical reconstruction, respectively. Univariate and multivariate Cox regression analysis enabled identification of poor runoff (ie, runoff score >5 for unilateral procedures or >2.5 for bilateral outflow procedures; relative risk, 2.5; 95% confidence interval [CI], 1.4-4.2; P =.001) as the only independent predictor of decreased primary patency in all patients. However, stratified analysis including only patients with poor runoff revealed that patients undergoing iliac stenting had significantly lower primary patency rates compared with those undergoing surgical reconstruction (Kaplan-Meier method, log-rank test; P =.05). External iliac artery disease and female gender were also identified as independent predictors of decreased primary stent patency. CONCLUSIONS Poor infrainguinal runoff is the main risk factor for decreased primary patency after surgical reconstruction and iliac stenting to treat TASC type B and type C iliac lesions. However, primary patency is less affected by poor runoff in patients undergoing surgical procedures. The presence of poor runoff, external iliac artery disease, and female gender are independent predictors of poor outcome after iliac stenting, and therefore these risk factors should determine the need for surgical reconstruction.

Vascular endothelial growth factor and heparin in a biologic glue promotes human aortic endothelial cell proliferation with aortic smooth muscle cell inhibition.

BACKGROUND Incomplete luminal endothelialization may contribute to small diameter vascular graft failure. Vascular endothelial growth factor (VEGF) can be used to stimulate endothelialization without provoking smooth muscle cell (SMC) proliferation. Heparin and VEGF in a fibrin glue (FG) were investigated for their ability to promote selective human aortic endothelial cell (HAEC) proliferation and human aortic smooth muscle cell (HASMC) inhibition. METHODS HAECs and HASMCs were seeded on FG containing VEGF (2.5, 10, 30, 100 ng/ml) or VEGF and heparin (5, 50, 500 units/ml). Proliferation assays were performed with tritiated thymidine on days 1 and 3. Results were analyzed by ANOVA, with p < or = 0.05 significant. RESULTS HAEC proliferation on FG with 10, 30, and 100 ng/ml VEGF was significantly greater than FG alone at days 1 and 3. The addition of 50 units/ml heparin to VEGF significantly increased HAEC proliferation to greater than FG with VEGF alone at day 1. Human aortic SMC proliferation was not stimulated by the addition of VEGF. The addition of 5, 50, and 500 units/ml heparin significantly inhibited HASMC proliferation regardless of VEGF concentration. DISCUSSION VEGF at 10 ng/ml combined with heparin at 50 units/ml exhibited maximal stimulation of HAECs with inhibition of HASMCs. VEGF and heparin in a biologic glue may improve patency by selectively promoting HAEC proliferation without HASMC growth on synthetic vascular bypass grafts.

Regulation of vascular smooth muscle cell expression and function of matrix metalloproteinases is mediated by estrogen and progesterone exposure

OBJECTIVE Postmenopausal women receiving hormone replacement therapy (HRT) have been reported to have more adverse outcomes after vascular reconstructions, including increased intimal hyperplasia development and bypass graft failure. HRT may be affecting the pathway contributing to intimal hyperplasia. An important component of this pathway involves matrix metalloproteinases (MMPs), implicated in vascular remodeling due to their ability to degrade components of the extracellular matrix. We hypothesize that estrogen (Est) and progesterone (Prog) upregulate the MMP pathway in vascular smooth muscle cells (VSMCs) thereby increasing MMP activity and function. METHODS AND RESULTS VSMCs were incubated with Est (5 ng/mL), Prog (50 ng/mL), Est + Prog combination (Est/Prog), and/or doxycycline (40 microg/mL; Doxy). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis we have previously shown membrane type 1-MMP (MT1-MMP) messenger ribonucleic acid (mRNA) levels are significantly increased by Est. Here, Western blot analyses indicated MT1-MMP and MMP-2 protein levels, not tissue inhibitor of MMP-2 (TIMP-2), were increased in response to Est and Est/Prog (P < .05 vs control). In-gel zymography revealed that Est and Est/Prog resulted in increased MMP-2 activity (hormone groups, P < .05 vs control) with no significant difference among the hormone groups. VSMC migration was increased by 45 +/- 14% in response to Est (P < .05 vs control), as measured using a modified Boyden chamber assay. Doxycycline significantly inhibited basal and Est/Prog-stimulated increases in MMP-2 activity (P < .05 vs control; P < .05 vs hormone groups), and partially blocked basal and hormonally stimulated migration (P < .05 vs control and Est). CONCLUSION Estrogen and progesterone affects the MMP pathway by increasing MMP-2 enzymatic activity, possibly via the upregulation of MT1-MMP expression without a corresponding increase in TIMP expression. This increased collagenase activity increases VSMC motility and their ability to migrate through a collagen type IV lattice. Est/Prog upregulation of MT1-MMP may contribute to the adverse effect of HRT on vascular interventions.

Endotension Distribution and the Role of Thrombus following Endovascular AAA Exclusion

PURPOSE To determine the pattern of strain and pressure transmitted to an aortic aneurysm wall before and after endovascular exclusion and to evaluate the role of sac thrombus on the conduction of pressure and wall strain. METHODS Three canine thoracic aortas were used to create abdominal aortic aneurysms (AAA). The segments were placed on a pulsatile pump system, and 8 strain transducers were positioned in the aneurysm sac. Baseline strain/pressure (S/P) was recorded in 1 animal, then the AAA was excluded with a stent-graft. Thrombin was injected into the sac, and strain/pressure was recorded at 7 systemic pressures (35 to 120 mmHg) over 6 hours. The thrombus was replaced with fibrin glue, and S/P was recorded over 4 hours. Additional trials using whole and 50% diluted unclotted blood were performed prior to sac thrombosis. Computed tomography and angiography were performed before and after aneurysm exclusion. RESULTS Pressure transmitted to the aneurysm wall decreased following stent-graft placement (p<or=0.001). Strain/pressure was not distributed evenly in the sac (p<or=0.05), and varying systemic pressures did not affect this distribution. Pressures near the stent-graft were higher than those laterally (p<or=0.001) in all trials with interposed fresh thrombus and fibrin thrombus. The fibrin group had elevated baseline measurements, but correction for the elevated values did not influence the statistical significance (p<or=0.001). Blood and fibrin thrombus reduced transmitted wall pressure to a similar degree. Overall S/P in the fluid-filled nonclotted sac was significantly lower (p<or=0.001) than in the thrombus groups. CONCLUSIONS Endovascular AAA exclusion reduced strain and pressure conducted to the aneurysm wall, and the distribution of transmitted pressure in the excluded sac without endoleak differed regardless of the sac contents. Fresh thrombus reduced transmittedS/P in all trials at all systemic pressures, as did fibrin thrombus but in a less predictable fashion.

Effect of Hormones on Matrix Metalloproteinases Gene Regulation in Human Aortic Smooth Muscle Cells

BACKGROUND Postmenopausal women receiving hormone replacement therapy have more adverse outcomes after vascular reconstructions. Estrogen-binding receptors have been identified on vascular smooth muscle cells (VSMCs), indicating that vascular function may be under direct hormonal control. A key group of enzymes involved in vascular remodeling are matrix metalloproteinases (MMPs). Here we studied the effect of estrogen (Est) and progesterone (Prog) on MMP gene expression in human VSMCs. METHODS AND RESULTS VSMCs were incubated with Est (5 ng/mL), Prog (50 ng/mL), Est+Prog combination (Est/Prog), and interleukin-1beta (100 U/mL; IL-1beta). Gene array analysis indicated Est+IL-1beta increased the expression of MMP-3. Reverse transcriptase-polymer chain reaction (RT-PCR) analyses revealed MMP-3 mRNA levels were significantly increased by Est/Prog+IL-1beta treatment. However, Western blot and further RT-PCR analyses indicated no change in MMP-3 in response to hormones alone. RT-PCR analyses revealed membrane type 1 (MT1)-MMP mRNA levels, not MMP-2 or tissue inhibitor of MMP (TIMP), were significantly increased by Est/Prog+IL-1beta, and Western blot analyses confirmed a significant increase in MT1-MMP protein in response to Est alone. CONCLUSION Estrogen and progesterone affect the MMP pathway of VSMCs via isoform specific mechanisms and may lead to unbalanced MMP regulation. Estrogen up-regulates MT1-MMP without a corresponding increase in TIMP-2, known activator and inhibitor of MMP-2, respectively. Additionally, estrogen up-regulates MMP-3 only in the presence of IL-1beta. This differential regulation, combined with case-specific variations in degree of inflammatory response, may explain why some women receiving exogenous hormone therapy at the time of vascular interventions are more susceptible to complications.

Hepatic triglyceride lipase deficiency in liver disease

The activity of post-heparin lipases in patients with alcoholic hepatitis and viral hepatitis was evaluated. Lipoprotein lipase and hepatic triglyceride lipase were differentiated by assay under high and low salt conditions and also by separation on heparin-agarose affinity chromatography columns. The mean activity of hepatic triglyceride lipase in the sera of liver disease patients was only 21–24% of the mean of controls, but lipoprotein lipase in patients’ sera was not different from normal levels. Hepatic triglyceride lipase deficiency may partially account for the accumulation of a triglyceride-rich low density lipoprotein in liver disease.

Synthetic vascular grafts seeded with genetically modified endothelium in the dog: Evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) preclotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 x 10(3) to 1.5 x 10(5) cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 micrograms/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced ECs, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

Effect of anesthetic technique on cardiac morbidity following carotid artery surgery

PURPOSE To investigate the effect of anesthetic technique on cardiac morbidity after carotid artery surgery. PATIENTS AND METHODS From 1991 to 1994, 266 consecutive carotid endarterectomies were performed under local/regional (n=140) or general anesthesia (n=126). The effects of anesthetic technique on postoperative adverse cardiac events were assessed retrospectively. RESULTS Preoperative cardiac testing was performed in all patients undergoing general or local/regional anesthesia. Medical characteristics were similar among patients in both groups. Forty-seven adverse cardiac events (4 myocardial infarction, 9 congestive heart failure, 7 angina, and 27 new ventricular dysrhythmias) occurred postoperatively in 38 patients (14.3%). There were no deaths. The relative risks of general anesthesia for dysrhythmias, myocardial infarction, angina, congestive heart failure, and total adverse cardiac events were 2.22, 0.37, 0.83, 1.38, and 1.5, respectively. The only statistically significant differential was the increased risk of postoperative dysrhythmias after general anesthesia (P<0.03). CONCLUSIONS Major cardiac morbidity following carotid endarterectomy is independent of anesthetic technique.