Introduction: Current guidelines recommend a diet rich in fish or n-3 polyunsaturated fatty acid (PUFA) supplementation in patients with cardiovascular disease including PAD. The omega-3 index represents the red blood cell (RBC) content of the two major long-chain n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with less within-patient variation compared to plasma levels of n-3 PUFA. We aimed to quantify the change in the omega-3 index with high-dose n-3 PUFA supplementation in a PAD cohort. Methods: This study was a secondary analysis of the OMEGA-PAD I Trial (NCT01310270), a randomized, double-blinded, placebo-controlled trial addressing the hypothesis that short-duration, high-dose n-3 PUFA oral supplementation improves endothelial function and inflammation in subjects with PAD. Eighty patients with stable claudication received 4.4g of fish oil (2.6g of EPA + 1.8g of DHA) daily (n=40) or placebo capsules (n=40) for 1 month. The RBC fatty acids (FA) content was measured by gas chromatography (OmegaQuant Analytics, Sioux Falls, SD) and expressed as a percent of total FA. Results: There was no difference in baseline PUFA content between the two groups. There was a shift in the n-6:n-3 ratio in RBC from 5 ± 1 to 3 ± 1 (p<0.0001) and the omega-3 index increased from 5 ± 1 to 9 ± 2 (p<0.0001) in the fish but not in the placebo group (Table). There was a significant increase in EPA, DHA, and n-3 docosapentaenoic acid and a significant decrease in all n-6 PUFA content of RBC in the fish oil but not in the placebo group. Following treatment, there was also a significant reduction in plasma triglycerides (-34 ± 46, p<0.001) in the fish oil group. The change in triglycerides was significantly correlated with the change in omega-3 index (p=0.03). Conclusions: High-dose fish oil supplementation increases the omega-3 index and reduces n-6 PUFA levels. The implications of these changes for patients with PAD will continue to be investigated in a follow-up trial (OMEGA-PAD II).

Introduction: Current knowledge supports significant associations between stressful life events including post-traumatic stress disorder (PTSD) and increases in cardiovascular disease (CVD) and mortality. Despite strong evidence supporting the association of PTSD and CVD, the underlying mechanisms remain poorly understood. Hypothesis: We hypothesized that patients with PTSD would demonstrate endothelial dysfunction, a key factor in the development and progression of CVD. Methods: Two hundred sixty one outpatients treated at the Veterans Affairs Medical Center (VAMC) underwent endothelial function (EF) testing as part of the OMEGA-PAD Study or the Mind Your Heart Study. Flow-mediated brachial artery vasodilation (FMD) was performed to assess EF and current PTSD symptoms were assessed with the PTSD Checklist for DSM-IV. Multivariate linear regression models were used to determine the association between PTSD and FMD. Results: Patients with PTSD (n=69) had a significantly lower brachial artery FMD (6.2 ± 3.4% vs 7.4 ± 3.8%; p=0.03). They also had higher systolic blood pressure (p=0.04), diastolic blood pressure (p=0.01) but were also less likely to be on an ace inhibitor (p=0.04) or a beta-blocker (p=0.05) (See Table). Furthermore, patients with PTSD were more likely to have depressive symptoms (p=<0.0001). Univariate analysis demonstrated that PTSD (p=0.02), age (p=0.04), renal function as measured by eGFR (p=0.02), and hypertension (p=0.009) were associated with FMD. After adjusting for these factors, PTSD remained significantly associated with worse EF (coefficient -1.62, CI -2.93, -0.31, p=0.02). Conclusions: PTSD is associated with worse EF in an outpatient population treated at the VAMC San Francisco. Since current literature supports a heightened risk of CVD with a decrease in FMD, our data suggest that treatment of mental health issues has the potential to improve vascular function and subsequently the morbid burden of CVD.

OBJECTIVES: Patients with peripheral artery disease (PAD) experience significant morbidity and mortality, at least partially related to vascular inflammation and endothelial dysfunction. The OMEGA-PAD I Trial (NCT01310270), a randomized, double-blinded, placebo-controlled trial addressed the hypothesis that short-duration, high-dose n-3 polyunsaturated fatty acids (n-3 PUFA) oral supplementation improves endothelial function (EF) and inflammation in subjects with PAD. METHODS: Eighty patients with stable, mild-severe claudication and ABI<0.9 received 4.1gm of fish oil (FISH) vs placebo capsules (CTL) for 1 month. The primary endpoint was EF as measured by brachial artery flow-mediated vasodilation (FMD). Secondary endpoints included biomarkers of inflammation, generation of n-3 fatty acid-derived lipid metabolites, lipid profile and walking impairment questionnaires. RESULTS: The FISH and CTL group were no different with regards to age, baseline EF, inflammation and lipid profiles. Following treatment, there was a significant reduction in triglycerides (-34 ± 46, p=0.0001) and an improvement in HDL (+2 ± 6, p=0.03) in the FISH group. These changes were accompanied by an increase in the omega-3 index of 4 ± 1% (p<0.00001). We observed a significant increase in the production of downstream metabolites of n-3 fatty acids including 18-, 15- and 5-hydroxy eicosapentaenoic acids and 4-hydroxy docosahexaenoic acid in the FISH group. n-3 PUFA led to a significant improvement in FMD in the FISH group (+0.7 ± 4.0%, p=0.04) and a non-significant improvement in the CTL (+0.6 ± 2.5, p=0.18) group. There were no significant differences between groups in pro-inflammatory markers or walking parameters post-treatment. CONCLUSIONS: High-dose, short-duration n-3 PUFA supplementation significantly improves the metabolo-lipidomic profile of patients with PAD. Longer studies are needed to assess the effects of n-3 PUFA on inflammation, vascular function, and clinical endpoints in patients with established PAD and to determine whether generation of n-3 fatty acid-derived bioactive lipid mediators is related to clinical outcomes.

Background Patients with peripheral artery disease (PAD) have an increased risk for cardiovascular events, likely related to inflammation and impaired endothelial function. n-3 polyunsaturated fatty acids (n3-PUFAs) have been shown to improve endothelial function and reduce inflammation in other cohorts. We hypothesized that n3-PUFAs would improve endothelial function and the inflammatory profile in patients with PAD. Methods This is a randomized, double-blind, placebo-controlled trial: the OMEGA-PAD trial ( NCT01310270 ). Eighty patients aged 50 and more with intermitted claudication and an ankle-brachial index (ABI) of <0.9 presenting to vascular surgery clinic at the Veterans Affairs Medical Center in San Francisco will be randomized to n3-PUFAs 2.2 g orally twice daily (total of 4.4gm/day) or a matched placebo for 1 month. Outcome measurements are done at baseline and after 1 month. The primary endpoint is a change in endothelial function measured by brachial artery flow mediated, endothelium-dependent vasodilation (FMD). Secondary endpoints include a change in inflammatory markers (C-reactive protein, IL-6, sICAM-1 and TNF-α), improvement in lipid profile (LDL, triglycerides, HDL), blood pressure and walking distance by questionnaire. The omega-3 index will be measured to ensure physiological treatment effect. Results Recruitment for the OMEGA-PAD trial started in April 2011. Fifty male veterans have been enrolled and randomized. The mean age is 67 ± 9 years. Mean index limb ABI is 0.8 ± 0.2. CAD is present in 36%, hypertension in 92%, and diabetes mellitus in 32%. 47% are current smokers and 45% are former smokers. Mean HgA1c is 8±1, LDL 93±37 mg/dL, triglycerides 160±98 mg/dL and HDL 43 ±12 mg/dL. Baseline brachial FMD is 7±4% indicating an overall impairment of endothelial function in this cohort. The inflammatory burden of this cohort is substantial as evidenced by hsCRP 5±5 mg/L. The baseline omega-3 index is 5±2%. Conclusions The OMEGA-PAD trial will test the novel hypothesis that n3-PUFAs supplementation improves functional and inflammatory parameters in a cohort at high vascular risk. The results of this study will provide valuable mechanistic insight into PUFAs as well as inform on this class of agents for cardiovascular outcome trials.

Rapamycin-based drug delivery platforms in peripheral arteries have proven to be safe and feasible but not effective in reducing intimal hyperplasia (IH).We sought to determine whether peri-adventitial delivery of rapamycin would be effective in reducing luminal stenosis in a porcine balloon injury model. Femoral artery injury sites were created with a pressure-controlled endothelial denudation. Two weeks later,a 20-30% overstretch of the injury sites was performed. Under fluoroscopy, nanoparticle albumin-bound (NAB) rapamycin (Abraxis Bioscience) (5 or 500 μg) or vehicle was delivered to the adventitia using a micro-infusion catheter (Mercator MedSystems). After 28 days,the arteries were harvested. The primary outcome was histomorphometric evidence of luminal stenosis and remodeling. Vessel injury was scored on a validated ordinal scale. There was 100% procedural success in 16 injury sites treated with adventitial injections. NAB rapamycin treated vessels had significantly larger lumen (p=0.01, ANOVA) and total vessel areas (p=0.005) indicating less negative remodeling. Percent luminal stenosis and maximal intimal width decreased in a dose-dependent fashion.Control vessels had significantly more fibrosis (p<0.001). These data suggest that adventitial delivery of rapamycin decreases luminal stenosis by inhibiting IH and preventing negative remodeling.The decreased fibrosis in treated vessels suggests a mechanism by which rapamycin may affect vessel remodeling.Tabled 1Mean values for measured and calculated metrics for 16 injured femoral segments treated with adventitial injection of vehicle or NAB rapamycin.vehicle5 μg rapamycin500 μg rapamycinp-valueLumen area (mm2)3.355.18*3.81*p=0.01Total vessel area (mm2)7.369.90*8.33*p=0.005Max initimal width (mm)0.230.200.12NSIntima/Media ratio0.170.160.11NSPercent luminal stenosis19.5%17.3%*11.4%p=.01Fibrosis score1.781.76*0.68*p<0.0001Inflammation score1.091.211.13NS Open table in a new tab

Renal efferent sympathetic nerve overactivity is contributory to the hypertensive state. Guanethidine is known to induce an autonomic denervation through an immune mediated pathway.We hypothesized that guanethidine could be safely delivered into the renal peri-vascular space to produceautonomic denervation and reduction in renal norepinephrine (NE) content. A micro-infusion catheter (Mercator MedSystems) was introduced via the femoral artery into the renal arteries of (N=11) female swin.Two to three ml of guanethidine (20-30 mg) or vehicle, each with 20% dilution of contrast, was infused into the adventitial space under fluoroscopic guidance. The primary efficacy endpoint in this study was kidney NE tissue content measured by high-performance liquid chromatography.Secondary outcomes included histological evidence of renal artery denervation to corroborate efficacy and renal artery injury and neural inflammation scores. There were 18 treated renal arteries and 4 vehicle controls in this study.There was 100% procedural success in the delivery of guanethidine or vehicle to the renal artery adventitial space.The mean renal artery injury scores were not different between treated and control pigs (P=NS), and in all cases the endothelial integrity was maintained.However,in all guanethidine-treated cases there was histological evidence of peri-neural inflammation and nerve destruction whereas the nerves appeared normal in all control animals.There was an overall decrease in renal cortex NE content in a dose dependent fashion, control 245±34 ng/g tissue, 20 mg dose 161±18 ng/g tissue and 30 mg dose 133±16 ng/g tissue, P=.038 (Figure). These data support the hypothesis that guanethidine can be delivered safely and efficiently into the renal peri-adventitial space through a novel minimally invasive technique.Further, renal NE content is significantly reduced suggesting that this could be a viable procedure in the treatment of refractory sympathetic-driven hypertension.

Resistance to apoptosis is a salient feature of neoplasia and neointimal hyperplasia. In cancer, a mitochondrial chaperone network consisting of heat shock proteins (HSP)75 and HSP90 mediates apoptosis-resistance. We hypothesize that these mitochondrial proteins regulate survival in venous smooth muscle cells (VSMC) after arterialization, and may be critical in the hyperplastic response. Primary cultured VSMC from human saphenous veins were stimulated with platelet-derived growth factors (PDGF)-BB (100 ng/mL) or tumor necrosis factor-α (TNF-α; 10 ng/mL) for 24 hours. Cells were fractionated for immunoblotting and cytosolic and mitochondrial fractions probed for HSP75 and HSP90. Excised segments of diseased human vein grafts (n = 12) and control saphenous veins (n = 10) were obtained from discarded specimens. Rabbits (n = 9) underwent carotid interposition vein grafting and grafts were harvested on day 5 or 28. Specimens were stained for HSP75, HSP90, or the inducible isoform HSP90β. Immunoblotting showed that HSP90 and HSP75 were expressed primarily in the cytosol and mitochondria of VSMC, respectively. Mitochondrial HSP75 was significantly increased after either cytokine or growth factor stimulation in vitro. After arterialization, HSP75 expression was notably increased in all vein grafts. In the rabbit, both early (5 days, n = 2) and late (28 days, n = 7) vein grafts had significantly greater HSP75 staining throughout the intima and media as compared to absent or minimal expression in control jugular veins (n = 9; analysis of variance [ANOVA]; P < .05). Human vein grafts (age 84-1330 days) showed significantly increased HSP75 expression in the intima and media as compared to controls (P < .05). Early vein grafts (<365 days, n = 3) may have greater relative HSP75 expression than late vein grafts (>365 days; n = 9) (ANOVA; P < .05). Both saphenous veins and human vein grafts had positive staining for HSP90 and weaker staining for HSP90β in the intima and media. Upregulation of the mitochondrial chaperone protein HSP75 after venous arterialization may promote a state of relative apoptosis-resistance within VSMC in the healing graft wall. Further studies are underway to determine the specific role of these proteins in vein graft remodeling.

Successful adaptation of a vein graft to an arterial environment is incompletely understood. We sought to investigate whether early vein graft remodeling is predictive of subsequent patency. A prospective longitudinal study of patients undergoing LEB with autologous vein (n=98). Preoperative blood samples were drawn for biomarkers. At the bypass operation, a 5 cm index segment of the graft was registered for serial lumen diameter measurements (1, 3, 6, 9, and 12 months) using duplex ultrasound. Index segment lumen diameter at 1 month was predictive of primary graft patency (p=0.013, Cox model) with a median follow-up of 28 months. Percent change in vein diameter from 0-1 month showed a trend of positive correlation with primary graft patency (p=0.1). On multivariate regression, larger initial vein diameter (p<0.001), baseline high-sensitivity C-reactive protein (hsCRP) level (p=0.007) and non-white race (p=0.035) were negatively correlated with 0-1 month graft remodeling, while statin use (p=0.049) was positively correlated with remodeling. Similarly, initial diameter (p<0.001), hsCRP (p=0.03) and non-white race were negatively correlated with diameter achieved at 1 month. Early remodeling of the arterialized vein, measured from a representative mid-graft segment, appears to predict mid-term bypass graft patency. In addition to baseline diameter, race, inflammation (hsCRP) and statin use are associated with early adaptive remodeling, but the mechanism for these observations are not understood.Tabled 1Index segment diameter at 1 month (by quartiles).Quartile1 month diameterPercent diameter change 0-1 monthshsCRP (mg/L)Primary patency rate at 2 years10.37 (0.34-0.39)11%4.0 (1.5-23.8)39%20.43 (0.41-0.44)13%5.1 (1.2-19.3)44%30.47 (0.47-0.5)18%3.3 (1.8-5.5)69%40.59 (0.54-0.65)45%2.9 (1.2-4.6)80%All values are median (IQR). Open table in a new tab

Intradialytic hypotension and hypertension are both independently associated with mortality among persons with end-stage renal disease on hemodialysis. Endothelial dysfunction and arterial stiffness are two possible mechanisms underlying these phenomena, but their association with hemodynamic instability during dialysis has not been evaluated. Thirty patients were recruited from chronic dialysis units at San Francisco General Hospital and San Francisco Veterans Affairs Medical Center. Endothelial dysfunction was assessed with flow-mediated dilation of the brachial artery after upper arm occlusion. Arterial stiffness was assessed using carotid-femoral pulse wave velocity measured by tonometry. Intradialytic hypotension and hypertension were defined as the average decrease in systolic blood pressure (SBP) over 1 week, as well as the frequency over 1 month of hypotension or hypertension. Every 5% decrease in flow-mediated dilation was associated with a 7.5 mmHg decrease in SBP after adjustment for phosphorus, body mass index, atherosclerosis, and ultrafiltration (P=0.02). Every 5 m/s increase in pulse wave velocity was associated with an 8 mmHg increase in SBP after adjustment for predialysis SBP and ultrafiltration (P=0.03). Over 1 month, every 5% lower flow-mediated dilation was associated with a 10% higher frequency of hypotension (P=0.09), and every 5 m/s increase in pulse wave velocity was associated with an 15% higher frequency of hypertension (P=0.02). In a cross-sectional analysis of 30 dialysis patients, endothelial dysfunction and arterial stiffness were independently associated with intradialytic hypotension and intradialytic hypertension, respectively. Elucidating these potential mechanisms of hemodynamic instability during dialysis may facilitate development of treatment strategies specific to this pathophysiology.