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A significant portion of patients undergoing lower extremity bypass surgery (LEB) for peripheral arterial disease (PAD) will have cardiovascular or graft-related events. It has been previously demonstrated that systemic inflammation is associated with PAD and its clinical outcomes. We hypothesized that serum biomarkers of insulin resistance and inflammation would identify a sub-group at elevated risk for graft failure, limb loss, and mortality.
This was a prospective longitudinal study of patients (N=225) undergoing LEB using autogenous vein. Baseline blood samples were obtained prior to surgery in the fasting state. High-sensitivity C-reactive protein (hsCRP) and the adipokines resistin and high-molecular weight adiponectin (HMWA) were measured by ELISA. Median follow-up was 893 days. The major endpoints of primary patency (PP) and amputation free survival (AFS) were examined using multivariable methods. Endpoints were screened against biomarkers and patient characteristics for univariate associations. Promising explanatory variables (P < 0.1) were included in multivariable Cox proportional hazard models.
The mean age of subjects was 67.6 years; 71.6% were male and 87.1% were Caucasian. One-hundred and thirty-three (59.1%) subjects underwent bypass for critical limb ischemia (CLI) and 73 (32.4%) had tissue loss. Patients with CLI and diabetes demonstrated elevated resistin and hsCRP levels. HMWA levels correlated with CLI and with a measure of insulin resistance (HOMA-IR) but not with clinical diabetes. Baseline biomarkers were higher in those presenting with tissue loss and in patients with post-operative events (mortality, limb loss). After multivariable analysis (including CLI, diabetes, age, estimated glomerular filtration rate (eGFR), adiponectin, resistin, and CRP), resistin (HR 1.75, 95% CI 1.07 to 2.85, P=0.025) and CRP (HR 2.39, 95% CI 1.30 to 4.39, P=0.005) were independently predictive of reduced AFS. However, only resistin maintained its significance when restricted to the diabetic cohort (HR 2.10, 95% CI 1.10 to 3.99, P=0.025). Higher levels of HMWA were found to be associated with primary graft patency (HR 0.73 for graft failure, 95% CI 0.55 to 0.97, P=0.031) in a multivariable model adjusting for diabetes, CRP, African-American race, CLI, high-risk conduits, and redo bypass procedures.
These findings suggest that serum biomarkers of insulin resistance and inflammation may be predictive of clinical outcomes following LEB. Improving the systemic milieu of insulin resistance and inflammation in these high-risk patients may lead to reduced morbidity and mortality.
The incidence of peripheral artery disease (PAD), a manifestation of atherosclerosis, is widespread and is only expected to increase as the aging population grows and the prevalence of diabetes mellitus (DM) continues to increase. Patients undergoing lower extremity bypass (LEB) surgery for severe PAD demonstrate a profound inflammatory phenotype, based on plasma high-sensitive C-reactive protein (hsCRP) concentrations.(1) Although inflammatory biomarkers such as hsCRP have been linked to adverse cardiovascular events such as myocardial infarction, death, and stroke, in PAD patients,(2) their specific relationship to the outcomes of surgical revascularization are not clearly established.(1) LEB using autogenous vein is commonly employed and often effective, however factors responsible for the variability in outcome are poorly understood.(3) For example, conventional cardiovascular risk factors such as hypertension, hypercholesterolemia, and diabetes mellitus (DM) are not predictive for lower extremity vein graft failure.(3) Specifically, DM, characterized by insulin resistance, central obesity, and inflammation, adversely affects morbidity and mortality following vascular surgery, but, counterintuitively, is not directly associated with lower extremity vein graft failure.(3)
Investigations exploring the intersection of inflammation and insulin resistance, with respect to outcomes following lower extremity revascularization, have been lacking even though both are linked to atherosclerosis. These investigations seem especially pertinent given the burgeoning epidemic of obesity and DM in Western populations.(4) The relationship between central obesity and adverse cardiovascular outcomes is well known.(5) We have now come to recognize that differentiated adipose tissue, far from being a simple fat depot, can function as an endocrine organ.(6) As adipocytes enlarge during obesity, macrophages are recruited and pro-inflammatory cytokines are produced.(7) This may lead to vascular inflammation, endothelial dysfunction, and neointimal thickening, further promoting atherosclerotic disease. In particular, two novel adipokines, adiponectin and resistin, have garnered interest for their potential roles in inflammation and insulin resistance. Adiponectin, a relatively abundant protein found in adult adipocytes from which it is mostly made, has many isoforms of which the high-molecular weight adiponectin (HMWA) has been found to be the most sensitive as a clinically useful biomarker in cardiovascular disease.(8–10) In general, adiponectin can be thought of as a vascular protective hormone having anti-atherogenic and anti-diabetic effects.(11) By contrast, considerably less clinical information is available for resistin. As its name implies, resistin has been shown to provide a link between obesity and diabetes in mice by impairing glucose tolerance and antagonizing insulin.(12) In humans, however, its role is not as straightforward, although it has been implicated in inflammatory processes.(13)
In this study we sought to assess the association of these novel adipokines with the clinical outcomes of amputation-free survival (AFS) and vein graft patency in patients undergoing lower extremity bypass surgery with autogenous vein.
This was a National Institutes of Health sponsored prospective cohort study examining the relationship of metabolic factors, inflammation, and outcomes following LEB with autogenous vein. The study was undertaken at three Boston teaching hospitals. All participating institutions underwent independent review of the study and received approval from the Institutional Review Board. Each patient provided written informed consent. Enrollment began in February 2004 and ended in May 2008.
Details of patient selection/inclusion and exclusion criteria have been published previously.(1, 14) In brief, subjects were excluded if they were treated with a prosthetic or other non-autolgous vein material. Other exclusion criteria included history of diagnosed hypercoagulable state, evidence of active infection, and other concurrent significant illness within 30 days (i.e. myocardial infarction, stroke, or other major surgery, etc.). While patients with small ulcers or small areas of dry gangrene were accepted into the study, deep space infections of the foot, those with large areas of ulceration, or ulceration or gangrene requiring operative debridement were excluded from participation. In total, 225 patients were enrolled.
Plasma was collected with the patient in the fasting state on the morning of their bypass procedure. Blood was collected into EDTA and citrate vacutainer tubes and immediately iced. Tubes were spun at 3000 revolutions per minute for 20 minutes at 4 degrees C in a refrigerated centrifuge. All samples were stored in a −80 degree C freezer until the time of analysis. All analysis was conducted in batch at a core lab to avoid variation. The hsCRP level was determined using an immunoturbidimetric assay on a Hitachi 917 analyzer (Roche-Diagnostics, Indianapolis, IN) using reagents and calibrators from Denka Seiken (Niigata, Japan). This assay has a sensitivity of 0.03 mg/L and the day-to-day variations of the assay at differing concentrations are less than 3%. Adiponectin was analyzed using an ELISA method from ALPCO Diagnostics Inc. (Salem, NH). The day to day variation of the assay at various concentrations of adiponectin is less than 15% and has a sensitivity of 0.386 µg/mL. Resistin was analyzed using an ELISA assay (R & D Systems, Minneapolis, MN) and it has a sensitivity of 0.26 ng/mL.
Insulin, glucose, lipids, hemoglobin A1c (hgbA1c), and creatinine were all analyzed at the clinical laboratory of each site using standard techniques.
The homeostasis model of insulin resistance (HOMA-IR) was calculated from the product of [insulin (microunits/mL) and glucose (mmoles/L)] divided by 22.5. The HOMA-IR correlates well with insulin sensitivity derived from the glucose clamp technique, the gold standard measure of insulin sensitivity.(15) Subjects taking insulin (N = 79) were excluded from HOMA-IR measurements.
The Modification of Diet in Renal Disease Study equation was used to estimate glomerular filtration rate (eGFR) from serum creatinine (SCr): eGFR (ml·min−1·1.73 m−2) = 175·(SCr)−1.154·(age)−0.203 (0.742 if female)·(1.210 if black).
Patients underwent lower extremity bypass surgery utilizing autogenous vein under the direction of the attending surgeon. Ipsilateral great saphenous vein was the conduit of first choice but in its absence, contralateral great saphenous or arm vein was used. Details of the operative procedure were recorded, as well as any adjunctive procedures and the results of completion studies. It is the standard practice at all participating centers to perform completion duplex ultrasound and/or completion angiography. Any flow disturbances or intrinsic vein disease would be identified during this time.
Patients were monitored for postoperative complications during their initial hospitalization and subsequent visits. All patients enrolled were followed postoperatively by the attending vascular surgeon and underwent duplex ultrasound surveillance at 1, 3, 6, 9, and 12 months. The duplex ultrasound was performed in an Intersocietal Commission for the Accreditation of Vascular Laboratories (ICAVL) vascular laboratory at each of the three enrolling facilities. After 12 months, the follow up visits occurred every 6 months. Indications for graft re-intervention were left at the discretion of the operating surgeon. All patients underwent a complete vascular examination at these follow up assessments and any clinical or graft related events were recorded. In addition to the scheduled appointments, research coordinators from each participating site monitored events and outcomes through contact with the patients, their primary care providers, and the electronic medical record. Any patient without contact with study personnel for 18 months were considered lost to follow up and censored from their last point of contact.
Race, ethnicity, and gender were assessed by self-report. Subjects were considered to have hypertension if they were taking prescription medications for hypertension, if they had a systolic blood pressure > 140 mmHg or a diastolic blood pressure > 90 mmHg. A diagnosis of hypercholesterolemia was present if they were taking prescription medications for cholesterol or if they self-report a prior diagnosis. Subjects were considered to have DM if the subject was taking prescription medications (oral or insulin) for diabetes, or if they self-reported a prior diagnosis. Active smoking was determined by self-report (a positive answer to the question, Do you now smoke cigarettes?”). Former smokers were defined as individuals who had smoked more than 100 cigarettes in their life but had not in 30 days and never smokers were defined as persons who had never smoked. Body mass index (BMI) was calculated as the quotient of weight divided by height in (kg/m2).
Primary patency was defined according to accepted published clinical reporting standards.(16) Loss of primary patency includes any graft revision (patch angioplasty, interposition graft, or balloon angioplasty) based on detection of vein graft stenosis by duplex ultrasound or documentation of graft stenosis by an imaging procedure in the appropriate clinical setting. Loss of primary patency was also noted if there was documentation of occlusion of the vein graft without revision. Amputation was defined as any supra-malleolar amputation of the index leg and a loss of amputation-free survival was defined as either amputation or death.
Baseline characteristics and biomarker values are presented as mean +/− SD or median and inter-quartile (IQR) range depending on the normality of their distribution. Proportions between groups were compared with chi2 test. Correlations were assessed with Pearson’s correlation coefficients on log-normalized marker values. Univariate differences in plasma levels of markers between sub-groups of patients were analyzed with one-way nonparametric Wilcoxon rank sum (Mann-Whitney U) tests. Multivariable Cox proportional hazard modeling was performed on clinical survival outcomes. Log-transformed biomarker values were used as predictors along with other clinical variables having univariate associations with P-values ≤ 0.10. For all time-to-event analysis, subjects not experiencing an adverse event were censored at the last known contact. Statistical analyses were performed on Stata/SE 10.0 (StataCorp, College station, TX) and with SAS 9.2 (SAS Institute, Cary, NC).
The demographic summary of the study population is presented in Table 1. There were 117 (52%) patients with DM, of which 79 were taking insulin and 52 were taking an oral hypoglycemic agent (14 were taking both insulin and an oral hypoglycemic agent). Chronic kidney disease (CKD), defined as the presence of the estimated glomerular filtration rate (eGFR) of < 60 ml ·min−1·1.73 m−2, was present in 74 (33%) patients and 10 were on hemodialysis. The indication for bypass surgery was critical limb ischemia (CLI) in 133 (59.1%) patients, 73 of which had distal tissue loss (32.4%). Twenty-two of the patients had a previous failed ipsilateral bypass graft. A single segment great saphenous vein was used for construction of the bypass in 190 (84.4%) of the subjects, while composite constructions of great saphenous vein or arm vein were used in the remainder. The mean follow up from LEB was 925 days (median 893; IQR 539–1315 days). No surviving individual was followed less than 365 days.
Biomarkers of insulin resistance and inflammation, and other selected laboratory values, are presented in Table II. Baseline markers were strongly associated with PAD clinical severity. Both HMWA (2.2 vs. 1.6 µg/mL, P = 0.0206) and resistin (15.1 vs. 10.2 ng/mL, P < .00001) were higher in patients presenting with CLI vs. claudication. There were also marked differences in the metabolic and inflammatory profile of subjects with clinical diabetes. Patients with DM had higher levels of hsCRP, resistin, body mass index (BMI) and hemoglobin A1c (hgbA1c); and lower hematocrit, eGFR, total cholesterol and HDL (all P < .05). Interestingly, both HMWA values (1.9 vs. 2.0 µg/mL, P = 0.8807) and HOMA-IR scores (1.7 vs. 2.1, P = 0.4161) were not different between the diabetic and non-diabetic subjects, suggesting the underlying prevalence of insulin resistance in the overall study population.
A summary of correlations among the biomarkers is presented in Table III. HMWA was positively correlated with HDL cholesterol, and more weakly with hsCRP. There was an inverse correlation of HMWA and HOMA-IR indicating the association between HMWA and insulin resistance in this patient population (r = − 0.2508, P = 0.0165, Figure 1a).
Resistin was positively correlated with hsCRP (r = 0.40, P < .0001, Figure 1b). Resistin was independently associated with CLI as an indication for bypass after controlling for hsCRP and DM (odds ratio 2.20; 1.30–3.71, P = 0.003).
There were 78 (34.7%) bypass grafts which met the definition of loss of primary patency. The 2-year primary patency rate was 61.4 ± 3.6% for the entire cohort. After adjusting for high risk conduits (composite and arm vein grafts), redo surgery, CLI, elevated hsCRP (≥ 5 mg/L), the presence of DM, and African American (AA) race, HMWA (HR 0.731, 95% CI (0.551–0.972), P = 0.031) and AA race (HR 2.70, 95% CI (1.39−5.24), P = 0.003) were the only significant risk factors associated with loss of primary patency (Figure 2).
There were 60 (26.7%) subjects who either died or underwent amputation during the follow up period. The 2-year AFS in the overall cohort was 79.3 ± 2.8%. Both HMWA and resistin, in addition to hsCRP, age, eGFR, DM, and CLI were associated with AFS on univariate testing. However, only resistin (HR 1.75; 1.07–2.85, P = 0.025; Figure 3) and hsCRP (HR 2.39; 1.30–4.39; P=0.005) were independently associated with loss of AFS after multivariable analysis. Resistin and hsCRP appeared to have additive ability to discriminate AFS outcomes. For example, in patients with hsCRP < 5 mg/L and in the lowest tertile of resistin, there were only 3/55 (5.5%) patients who either died or underwent major amputation, as compared with 28/43 (65.1%) for individuals with both hsCRP ≥ 5 mg/L and resistin values in the highest tertile. When restricting the cohort to only subjects with DM, the effect size of hsCRP was attenuated leaving only resistin as an independent predictor of AFS (HR 2.10; 1.100–3.993, P=.025).
The present study demonstrates that the adipokines, HMWA and resistin, further characterize the inflammatory phenotype of LEB patients and are associated with clinical outcomes. Levels of resistin were markedly elevated and strongly correlated with hsCRP, however resistin was independent of, and added predictive value to hsCRP with respect to AFS. Importantly, resistin was the only baseline variable that maintained its predictive ability for AFS within the DM-restricted cohort. By contrast, plasma HMWA concentrations were notably low in patients undergoing LEB for advanced PAD in comparison to patients with coronary artery disease(17) suggesting the breadth of the insulin-resistant phenotype in this population. A striking finding was the association of lower levels of HMWA with increased risk for vein graft failure. To our knowledge, this represents the first report of a circulating biomarker that may be associated with a protective effect on vein graft primary patency.
Adiponectin exerts its biological action on the vascular wall through one of two receptors that have been found in the liver and on human endothelial and smooth muscle cells.(18) Its biological activity is dependent, in part, on its plasma oligerimization state, with the HMWA species being the most biologically sensitive and relevant to clinical outcomes.(19) Low levels of adiponectin have been associated with impaired vascular reactivity,(20) symptomatic atherosclerotic peripheral arterial disease (PAD),(21) and coronary artery disease (CAD). Similar to previous investigations, HMWA was significantly correlated with insulin resistance in our study (estimated by HOMA-IR).
The seemingly paradoxical finding that HMWA levels were higher in patients with CLI than in those with claudication merits discussion. Several studies have demonstrated that the beneficial effects of adiponectin are not a straight forward dose-response relationship, indicating that other factors are involved. For example, higher adiponectin values were associated with increased mortality in patients with congestive heart failure (CHF).(22) This observation may be explained, in part, by a positive association of adiponectin and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in CHF patients.(22) It has recently been shown that NT-proBNP is associated with adverse cardiovascular events and mortality in patients undergoing vascular surgery.(23) Another intriguing possibility is that higher HMWA in some patients might be indicative of a wasting process.(24) It has been shown that circulating adiponectin levels are higher in patients undergoing weight loss.(25) Therefore, patients with advanced CHF or or severe CLI have may have higher baseline energy expenditure.(26) This is consistent with the present investigation, as patients with CLI had significantly lower total cholesterol, lower albumin, and lower body weight, suggesting a relative wasting state over claudication counterparts. A recent study by Komei et al, reported that total plasma adiponectin levels were associated with decreased survival following lower extremity bypass surgery.(27) However, several noteworthy differences exist from the present study. While there was a weak univariate association of HMWA with decreased AFS in the present investigation, this result was attenuated after controlling for disease severity and inflammation. In addition, we elected to measure HMWA rather than total adiponectin as several previous investigations suggests that this is the more biologically active species and most closely correlates with clinical outcomes. Finally, there were only 12 deaths in the study by Komei et al, which significantly limits the ability to construct reliable event-driven models and therefore limits the conclusions.(27)
Another noteworthy finding of the present investigation was that the low density lipoprotein (LDL) fraction of cholesterol was markedly low in this patient population, even among subjects not taking a statin, (mean LDL on statin, 60.9 mg/dL; mean LDL not on statin, 74.5 mg/dL). These values are well below the National Cholesterol Education Program Adult Treatment Plan III (NCEP ATP III) recommendation of targeting LDL to below 100 mg/dL with an option to decrease LDL to 70 mg/dL in patients with established cardiovascular disease.(28) Despite this, the median hsCRP was 3.0 mg/L which places these patients in the American Heart Association’s highest risk category.(29) Therefore, this combination of low LDL cholesterol and high levels of inflammatory biomarkers potentially makes this an ideal population to test the hypothesis that lowering inflammation per se will be efficacious in the secondary prevention of cardiovascular events as well as vein graft related outcomes.
In this investigation, resistin was independently associated with the clinical outcome, AFS, after controlling for hsCRP and other comorbidities. Further, in the diabetic-restricted cohort, resistin was the only significant predictor of AFS after the effect of high hsCRP and CLI were attenuated. Therefore, we believe that plasma resistin values represent a potentially useful marker for adverse outcomes following LEB surgery, particularly in patients with DM.
Another novel finding of the present study is that resistin was independently associated with CLI after controlling for other inflammatory markers and DM. How does resistin contribute to the inflammatory state of the PAD patient? Unlike rodents where high levels of resistin mRNA transcripts are found in adipose tissue, the majority of resistin in humans can be found in inflammatory and mononuclear cells.(30) Thus, resistin appears to be produced in the periphery unlike the acute phase reactant, hsCRP, which is predominantly produced in the liver.(31)
It should also be emphasized that the values of resistin observed in this patient population were extremely high. In the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA), a study of apparently asymptomatic middle aged men and women, the median resistin values were about 5.6 ng/ml which compares to patients with DM, where the resistin levels were about 5.8 ng/ml.(32)This is in stark contrast to the median values in the present study which were 12.9 ng/ml (15.1 ng/ml for CLI; 10.2 ng/ml for claudicants) and only patients in the lowest 10 percentile had values as low as those seen in the SIRCA study. It is not currently known why these patients had such high values but it may relate to resistin production by vascular wall resident macrophages within atherosclerotic plaques.(33) Resistin, unlike hsCRP, has been shown to be correlated with coronary artery calcification, a well known marker of the burden of atherosclerosis.(32) Hence, we surmise that resistin may reflect the total burden of atherosclerosis whereas CRP is most closely associated with the inflammatory activity of the plaque.
One limitation of the present study is that we elected to measure only one species of adiponectin, HMWA, rather than considering the complex distribution or ratios of specific fractions. However, recent studies have reported that the HMWA is more relevant to the pathogenesis of DM, obesity, metabolic syndrome, and vascular disease than total serum adiponectin concentrations.(34, 35) Others report a ratio of HMWA to total adiponectin as having a stronger predictive ability in some populations.(36) Further investigation is required to determine the more accurate species with regards to vein graft related outcomes. In addition, the association between HMWA and vein graft outcomes is rather modest. HMWA was significant in the full multivariate model as well in models exhibiting more parsimony. Nevertheless, in a sample size of 225 with 78 graft-related events, the potential for erroneous association exists and therefore these results must be interpreted with caution. Clearly larger investigations are needed to corroborate the present findings. As with hsCRP and other biomarkers, a potential causal relationship between plasma adipokine levels and graft or other vascular events remains speculative at the present time.
HMWA and resistin are novel adipokines that have apparent clinical implications in patients undergoing peripheral vascular reconstruction. HMWA is associated with insulin resistance and to the development of vein graft stenosis whereas resistin is a marker of inflammation with clinical predictive ability for AFS in both diabetics and non diabetics, independent of hsCRP.
Supported by funding from the National Heart, Lung, and Blood Institute R01 HL 75771
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Presented at the 2009 Western Vascular Society Meeting, Sept 19–22, Tucson, AZ