Andrew C. Stanley

Fibroblasts cultured from venous ulcers display cellular characteristics of senescence

PURPOSE A well-recognized characteristic of venous ulcers is impaired healing. Fibroblasts cultured from venous ulcers (wound-fb) have been shown to have reduced growth rates and are larger than normal fibroblasts (normal-fb) from the ipsilateral limb. Reduced growth capacity and morphologic changes are 2 well-known traits of cellular senescence. Other molecular changes are overexpression of matrix proteins, such as cellular fibronectin (cFN), and enhanced activity of beta-galactosidase at pH of 6.0 (senescence associated beta-Gal, or SA-beta-Gal). Senescence, an irreversible arrest of cell proliferation with maintenance of metabolic functions, may represent in vivo aging and thus may be related to impaired healing. METHODS Cultured normal-fb and wound-fb from 7 venous ulcer patients (average age, 51 years) were obtained by taking punch biopsies of the perimeter of the ulcer and from the ipsilateral thigh of the same patient. Growth rates, SA-beta-Gal activity, and level of cFN protein (immunoblot) and message (Northern blot) were measured. RESULTS In all patients, wound-fb growth rates were significantly lower than those of normal-fb (P =.006). A higher percentage of SA-beta-Gal positive cells were found in all wound-fb (average, 6.3% vs. 0.21%; P =.016). The level of cFN, was consistently higher in all wound-fb tested. Also, in 4 patients, the level of cFN messenger RNA (mRNA) was increased. CONCLUSION Fibroblasts cultured from venous ulcers exhibited characteristics associated with senescent cells. Accumulation of senescent cell in ulcer environment may be associated with impaired healing.

Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors.

PURPOSE Although the slow healing rate of venous ulcers is well known, the underlying defect in the healing process is not well understood. The purpose of this study was to examine the cellular characteristics of fibroblasts taken from venous ulcers (wound-fb) and compare them with the fibroblasts of normal tissue (normal-fb). METHODS Biopsy specimens were obtained from wound margins and normal tissue of the upper thigh in each patient. Dermal fibroblasts were isolated from explant cultures in Dulbeccos modified Eagles medium supplemented with 10% calf serum. These cells were then plated at 1000 cells per plate, and total cells per plate were counted over time so that growth curves could be generated. In further experimentation, media was supplemented with additional calf serum (20%, 30%, 40%, 50%) and growth factors (epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta) in an attempt to stimulate growth. RESULTS Two major differences were noted: (1) normal-fb replicated more rapidly than wound-fb; and (2) the morphologic features of wound-fb were different. Normal-fb were compact and tapered, with well-defined nuclear morphologic features. Wound-fb were larger and polygonal in shape, with less-uniform nuclear morphologic features. Additional calf serum in tissue culture media enhanced normal-fb growth but had no effect on wound-fb. Supplementation of media with growth factors stimulated the growth of wound-fb. Statistically significant differences were noted at day 10 and 14 with basic fibroblast growth factor supplementation (p = 0.02 and 0.0001, respectively) and at day 14 with epidermal growth factor (p = 0.008). Although interleukin-1 beta stimulated cell growth in five of six patients, the differences observed were not statistically significant. CONCLUSIONS Our data demonstrate that wound-fb proliferate at a slower rate and are morphologically distinct from normal-fb. These characteristics are typical of aged or senescent cells. This decreased growth can be stimulated by growth factors basic fibroblast growth factor, epidermal growth factor, and interleukin-1 beta. Slowed growth may be partially responsible for the defect in healing of venous stasis ulcers. Furthermore, we believe that in some patients ulcer healing may be improved by exogenous provision of specific growth factors.

Evaluating chronic venous disease with a new venous severity scoring system

BACKGROUND The Venous Clinical Severity Score (VCSS) has been proposed by the American Venous Forum as an objective means to clinically assess venous disease more completely than with the clinical CEAP classification. However, validation of the VCSS against an objective test is lacking. The purpose of this study was to test the VCSS against abnormalities found on venous ultrasound (US) scans. METHODS As part of a screening project in a large kindred population with protein C deficiency, VCSS and venous US scanning were performed in 210 patients (420 limbs). A single examiner scored the VCSS (0-3) clinically for pain, varicose veins, edema, skin pigmentation, inflammation, induration, ulcer duration and size, and compressive therapy. Another experienced examiner, blinded to the subjects medical history, performed a US examination of the deep and superficial venous system, with a hand-carried US system. The relationship between US and VCSS scores was analyzed by calculating an odds ratio (OR) and its 95% confidence interval (CI). RESULTS Of the 420 limbs screened, VCSS was 0 in 283 limbs, and VCSS was 1 or greater in the following categories: pain, 63 limbs; varicose veins, 70 limbs; edema, 51 limbs; skin pigmentation, 17 limbs; inflammation, 2 limbs; induration, 8 limbs; and compressive therapy, 9 limbs. The highest total score in any limb was 8. A clear association was seen with the VCSS and abnormalities found on US scans. When the score was dichotomized (0 = normal, 1 = any abnormality), it was a strong predictor of US scan abnormalities; limbs with VCSS greater than 0 had a 26-fold greater chance of US scan abnormalities than did limbs with VCSS = 0 (OR, 26.5; 95% CI, 11-64). With ultrasonography as the standard, sensitivity of VCSS compared with US scans was 89.3%, and specificity was 76.1%. Negative predictive value of VCSS = 0 was 97.9%, and positive predictive value for any positive score was 36.5% CONCLUSIONS The results of this study are based on a large kindred population with a higher risk for venous disease than found in the general population. Though the VCSS was devised to quantify the severity of chronic venous disease, this study found it a useful screening tool. The VCSS showed good association with abnormalities on US scans, and when VCSS = 0 there is a high likelihood that the patient does not have venous disease. This simple test may prove valuable in clinical practice.

Factors associated with amputation or graft occlusion one year after lower extremity bypass in northern New England

BACKGROUND Optimal patient selection for lower extremity bypass surgery requires surgeons to predict which patients will have durable functional outcomes following revascularization. Therefore, we examined risk factors that predict amputation or graft occlusion within the first year following lower extremity bypass. METHODS Using our regional quality-improvement initiative in 11 hospitals in northern New England, we studied 2,306 lower extremity bypass procedures performed in 2,031 patients between January 2003 and December 2007. Sixty surgeons contributed to our database, and over 100 demographic and clinical variables were abstracted by trained researchers. Cox proportional hazards models were used to generate hazard ratios and surrounding 95% confidence intervals (CIs) for our combined outcome measure of major amputation (above-knee or below-knee) or permanent graft occlusion (loss of secondary patency) occurring within the first year postoperatively. RESULTS We found that within our cohort of 2,306 bypass procedures 17% resulted in an amputation or graft occlusion within 1 year of surgery. Of the 143 amputations performed (8% of all limbs undergoing bypasses), 17% occurred in the setting of a patent graft. Similarly, of the 277 graft occlusions (12% of all bypasses), 42% resulted in a major amputation. We identified eight preoperative patient characteristics associated with amputation or graft occlusion in multivariate analysis: age <50, nonambulatory status preoperatively, dialysis dependence, diabetes, critical limb ischemia, need for venovenostomy, tarsal target, and living preoperatively in a nursing home. While patients with no risk factors had 1-year amputation/occlusion rates that were <1%, patients with three or more risk factors had a nearly 30% chance of suffering amputation or graft occlusion by 1 year postoperatively. When we compared risk-adjusted rates of amputation/occlusion across centers, we found that one center in our region performed significantly better than expected (observed/expected ratio 0.7, 95% CI 0.6-0.9, p < 0.04). CONCLUSION Preoperative risk factors allow surgeons to predict the risk of amputation or graft occlusion following lower extremity bypass and to more precisely inform patients about their operative risk and functional outcomes. Additionally, our model facilitates comparison of risk-adjusted outcomes across our region. We believe quality-improvement measures such as these will allow surgeons to identify best practices and thereby improve outcomes across centers.

Factors associated with death 1 year after lower extremity bypass in Northern New England

BACKGROUND Using 30-day operative mortality reported with lower extremity bypass (LEB) in preoperative decision making may underestimate the actual death rate encountered before patients have truly recovered from surgery, especially in elderly, debilitated patients with significant tissue loss. Therefore, we examined preoperative, patient-level risk factors that predict survival within the first year following LEB. METHODS Using our regional quality improvement initiative in 11 hospitals in Northern New England, we studied 2306 LEB procedures performed in 2031 patients between January 2003 and December 2007. Sixty surgeons contributed to our database, and over 100 demographic and clinical variables were abstracted by trained researchers. Cox proportional hazards models were used to generate hazard ratios (HR) and surrounding 95% confidence intervals (CI) for our combined outcome measure of death occurring within the first year postoperatively. RESULTS We found that within our cohort of 2306 bypass procedures, 11% of patients died within 1 year of surgery (2% prior to discharge, 9% prior to 1-year follow-up). We identified six preoperative patient characteristics associated with higher risk of death in multivariate analysis: congestive heart failure (HR 1.3, 95% CI 1.0-1.8), diabetes (HR 1.5, 95% CI 1.1-2.1), critical limb ischemia (CLI) (HR 1.7, 95% CI 1.3-2.4), lack of single-segment saphenous vein (HR 1.9, 95% CI 1.5-2/5), age over 80 (HR 2.0, 95% CI 1.5-2.7), dialysis dependence (HR 2.7, 95% CI 1.9-3.6), and emergent nature of the procedure (HR 3.4, 95% CI 1.7-6.8). While patients with no risk factors had 1-year death rates that were less than 5%: patients with three or more risk factors had a 28% chance of dying before 1 year postoperatively. When we compared risk-adjusted survival across centers, we found that one center in our region performed significantly better than expected (observed-to-expected outcome ratio 0.7, 95% CI 0.6-0.9, P = .04). CONCLUSIONS Preoperative risk factors allow surgeons to predict survival in the first year following LEB, and to more precisely inform patients about their operative risk with LEB. Additionally, our model facilitates benchmarking comparison of risk-adjusted outcomes across our region. We believe quality improvement measures such as these will allow surgeons to identify best practices and thereby improve outcomes with LEB across centers.

Variation in smoking cessation after vascular operations

OBJECTIVE Smoking is the most important modifiable risk factor for patients with vascular disease. The purpose of this study was to examine smoking cessation rates after vascular procedures and delineate factors predictive of postoperative smoking cessation. METHODS The Vascular Study Group of New England registry was used to analyze smoking status preoperatively and at 1 year after carotid endarterectomy, carotid artery stenting, lower extremity bypass, and open and endovascular abdominal aortic aneurysm repair between 2003 and 2009. Of 10,734 surviving patients after one of these procedures, 1755 (16%) were lost to follow-up and 1172 (11%) lacked documentation of their smoking status at follow-up. The remaining 7807 patients (73%) were available for analysis. Patient factors independently associated with smoking cessation were determined using multivariate analysis. The relative contribution of patient and procedure factors including treatment center were measured by χ-pie analysis. Variation between treatment centers was further evaluated by calculating expected rates of cessation and by analysis of means. Vascular Study Group of New England surgeons were surveyed regarding their smoking cessation techniques (85% response rate). RESULTS At the time of their procedure, 2606 of 7807 patients (33%) were self-reported current smokers. Of these, 1177 (45%) quit within the first year of surgery, with significant variation by procedure type (open abdominal aortic aneurysm repair, 50%; endovascular repair, 49%; lower extremity bypass, 46%; carotid endarterectomy, 43%; carotid artery stenting, 27%). In addition to higher smoking cessation rates with more invasive procedures, age >70 years (odds ratio [OR], 1.90; 95% confidence interval [CI], 1.30-2.76; P < .001) and dialysis dependence (OR, 2.38; 95% CI, 1.04-5.43; P = .04) were independently associated with smoking cessation, whereas hypertension (OR, 1.23; 95% CI, 1.00-1.51; P = .051) demonstrated a trend toward significance. Treatment center was the greatest contributor to smoking cessation, and there was broad variation in smoking cessation rates, from 28% to 62%, between treatment centers. Cessation rates were higher than expected in three centers and significantly lower than expected in two centers. Among survey respondents, 78% offered pharmacologic therapy or referral to a smoking cessation specialist, or both. The smoking cessation rate for patients of these surgeons was 48% compared with 33% in those who did not offer medications or referral (P < .001). CONCLUSIONS Patients frequently quit smoking after vascular surgery, and multiple patient-related and procedure-related factors contribute to cessation. However, we note significant influence of treatment center on cessation as well as broad variation in cessation rates between treatment centers. This variation indicates an opportunity for vascular surgeons to impact smoking cessation at the time of surgery.

The impact of diabetes on postoperative outcomes following lower-extremity bypass surgery.

OBJECTIVE The effect of diabetes type (noninsulin dependent vs insulin dependent) on outcomes after lower-extremity bypass (LEB) has not been clearly defined. Therefore, we analyzed associations between diabetes type and outcomes after LEB in patients with critical limb ischemia. METHODS We performed a retrospective analysis of 1977 infrainguinal LEB operations done for critical limb ischemia between 2003 and 2010 within the Vascular Study Group of New England. Patients were categorized as nondiabetic (ND), noninsulin-dependent diabetic (NIDD), or insulin-dependent diabetic (IDD) based on their preoperative medication regimen. Our main outcome measures were in-hospital mortality and major adverse events (MAEs)--a composite outcome, including myocardial infarction, dysrhythmia, congestive heart failure, wound infection, renal insufficiency, and major amputation. We compared crude and adjusted rates of mortality and MAEs using logistic regression across diabetes categories. RESULTS Overall, 41% of patients were ND, 28% were NIDD, and 31% were IDD. Crude rates of in-hospital mortality were similar across these groups (1.7% vs 3.1% vs 2.1%; P = .211). Adjusted analyses accounting for differences in patient characteristics showed that diabetes is not associated with increased risk of in-hospital mortality. However, type of diabetes was associated with a higher risk of MAEs in both crude (15.1% for ND; 21.1% for NIDD; and 25.2% for IDD; P < .001) and adjusted analyses (odds ratio for NIDD, 1.41; 95% confidence interval, 1.2-1.7; odds ratio for IDD, 1.53; 95% confidence interval, 1.3-1.8). CONCLUSIONS Diabetes is a significant contributor to the risk of postoperative complications after LEB surgery, and insulin dependence is associated with higher risk. Quality measures aimed at limiting complications after LEB may have the most impact if these initiatives are focused on patients who are IDD.

Evaluation of Simulation-Based Training Model on Vascular Anastomotic Skills for Surgical Residents

Introduction Reduced work hours and concerns over patient safety have encouraged surgical educators to find methods to advance resident skills more efficiently. Simulation provides the opportunity to improve technical surgical skills outside the operating room. We hypothesized that practice on surgical task simulators would improve residents’ technical performance of vascular anastomotic technique. Methods Senior general surgery residents at an academic medical center completed pretests and posttests on 3 vascular surgery simulators: femoral-popliteal bypass, carotid endarterectomy, and abdominal aortic aneurysm repair. The initial training sessions began with a 15-minute instructional video on how to perform the procedures, followed by supervised sessions in anastomotic technique with attending vascular surgeons. Initial individual sessions were videotaped as a pretest, and the final attempt was videotaped as the posttest. Each test was evaluated by a single experienced attending vascular surgeon blinded to the examinees. Anastomoses were graded using a performance rating and a modified objective structured assessment of technical skill rating. Results were analyzed using mixed model P values. Results The residents showed statistically significant improvement between the pretest and the posttest in both their performance rating (1.9 vs. 2.4, P = 0.02) and the objective structured assessment of technical skill (2.6 vs. 3.1, P = 0.01), as well as in most subsets of each assessment scale. Conclusions We conclude that practice using simulated anastomotic models leads to measurable improvement in vascular anastomotic technique in senior general surgery residents.

Impact of a critical pathway on postoperative length of stay and outcomes after infrainguinal bypass.

PURPOSE To determine the effect of a critical pathway on postoperative length of stay and outcomes after infrainguinal bypass. METHODS A critical pathway for care of patients after infrainguinal bypass was introduced in December 1995 to coordinate postoperative care at our institution. We compared care of 67 consecutively treated patients before institution of the pathway with care of 69 consecutively treated patients with the critical pathway in place. Data collection was done by means of chart review. Univariate analyses were used to identify differences between prepathway and postpathway patients and to identify factors influencing postoperative length of stay. Multivariate analysis was used to identify factors that influenced length of stay and to examine the effect of use of the pathway after adjusting for other factors. RESULTS Patients on the pathway were similar to prepathway controls with respect to comorbid illnesses, vascular risk factors, indications for surgical treatment, type of conduit, and type of operation. Factors associated with longer postoperative stays included distal anastomoses to tibial rather than popliteal vessels (p = 0.02), preexisting cardiac disease (p = 0.005), postoperative complications (p = 0.0003), lower preoperative hematocrit (p = 0.01), and elevated preoperative creatinine level (p = 0.006). Overall, pathway patients had somewhat shorter postoperative lengths of stay (median value 7 days; range 2 to 29 days) than prepathway patients (median value 6 days; range 2 to 35; p = 0.01), and the two groups had similar frequencies of postoperative complications, readmission, and 6-month mortality. However, patients on the pathway were more likely to be discharged to an intermediate-care facility rather than directly home. After 12 patients with extraordinarily prolonged postoperative stays were excluded, multivariate analysis indicated that pathway patients had significantly shorter postoperative stays (p = 0.001). However, the difference was not significant if patients with extraordinarily long postoperative stays were included in the analysis (p = 0.28). CONCLUSION Use of a critical pathway was associated with a modest decrease in postoperative length of stay for most patients. This was accomplished without an adverse effect on readmission, complication, or mortality rates. However, the decrease in stay may have been achieved primarily by discharging more patients to intermediate-care facilities. The pathway did not appear to have any effect when the subset of patients with extraordinarily long stays because of complex medical problems was included.

A new in vitro model of venous hypertension: the effect of pressure on dermal fibroblasts

PURPOSE Venous hypertension leads to venous stasis ulcers. White cell activation, protein leakage from pressurized capillaries, and cytokine imbalances have all been implicated as indirect effects of venous hypertension that contribute to dermal changes seen in chronic venous insufficiency. The direct effect of increased tissue pressures on dermal elements has not been investigated. Prior studies have shown that fibroblasts isolated from venous ulcers have altered growth rates, morphologies, and protein production similar to senescent or aged fibroblasts. We hypothesize that neonatal fibroblasts (NNFs) cultured in conditions of increased atmospheric pressure will demonstrate altered cell function when compared with those grown at normal atmospheric pressure (ATM). METHODS A pressure incubator was used to culture populations of NNFs at ATM, 60 mm Hg over ATM (ATM + 60 mm Hg), and 120 mm Hg over ATM (ATM + 120 mm Hg). NNF population growth rates were determined by periodic flow cytometry analysis over a 2-week period. Light microscopy and digital imaging were used to evaluate cell morphology. Senescence-associated B-galactosidase (SA-beta-Gal) activity was determined using the X-Gal stain. Fibronectin production was assessed by exposing cells sequentially to anti-fibronectin antibodies and Oregon Green-conjugated goat anti-mouse secondary antibodies. Flow cytometry then was used to determine relative proportions of cells staining positively for fibronectin. Statistical analysis was accomplished with analysis of variance. RESULTS Populations of cells grown under increased pressures (both ATM + 60 and ATM + 120) showed reduced growth rates (P <.001). Similarly, morphologies of cells grown under pressure had increased cytoplasm to nuclear ratios with abnormal nuclear shapes. Populations of cells grown under pressure had higher percentages of cells staining positive for fibronectin (ATM = 45%, ATM + 60 = 59%, ATM + 120 = 79%). After 14 days of growth under pressure, fibroblast populations did not demonstrate augmented productions of the senescence marker SA-beta-Gal (ATM =.5%, ATM + 60 =.25%, ATM + 120 =.75%). CONCLUSIONS This study demonstrated that NNFs grown in culture under increased pressures undergo a transformation not seen in cells grown at atmospheric pressure. Cells grown under pressure demonstrated reduced growth rates, increased fibronectin production, and abnormal morphologies similar to fibroblasts isolated from venous ulcers. This study suggests that pressure elevations (like venous hypertension) can directly result in altered cell function and morphology that may contribute to the delayed wound healing seen in patients with venous ulcers. This model uses a pressurized incubator that may prove to be a valuable adjunct in studying the effects of venous hypertension.