The statin-iron nexus: anti-inflammatory intervention for arterial disease prevention.

OBJECTIVES: We postulated the existence of a statin-iron nexus by which statins improve cardiovascular disease outcomes at least partially by countering proinflammatory effects of excess iron stores. METHODS: Using data from a clinical trial of iron (ferritin) reduction in advanced peripheral arterial disease, the Iron and Atherosclerosis Study, we compared effects of ferritin levels versus high-density lipoprotein to low-density lipoprotein ratios (both were randomization variables) on clinical outcomes in participants receiving and not receiving statins. RESULTS: Statins increased high-density lipoprotein to low-density lipoprotein ratios and reduced ferritin levels by noninteracting mechanisms. Improved clinical outcomes were associated with lower ferritin levels but not with improved lipid status. CONCLUSIONS: There are commonalities between the clinical benefits of statins and the maintenance of physiologic iron levels. Iron reduction may be a safe and low-cost alternative to statins.

Reduction of iron stores and clinical outcomes in peripheral arterial disease: outcome comparisons in smokers and non-smokers.

A prospective randomized trial suggested that iron (ferritin) reduction improved outcomes in smokers. The present study reanalyzed the trial results in smokers compared with non-smokers. Randomization of 1262 men with peripheral arterial disease (540 smokers and 722 non-smokers) to iron reduction (phlebotomy) or control groups permitted analysis of the effects of iron reduction and smoking on primary (all-cause mortality) and secondary (death plus non-fatal myocardial infarction or stroke) endpoints. Iron reduction resulted in significant improvement in the primary (hazard ratio [HR] 0.661, 95% confidence interval [CI] 0.45, 0.97; P = 0.036) and secondary (HR 0.64, 95% CI 0.46, 0.88; P = 0.006) endpoints compared with controls in smokers but not in non-smokers. Smokers required removal of a greater volume of blood to attain targeted ferritin reduction as compared with non-smokers (P = 0.003) and also exhibited differing characteristics from non-smokers, including significantly less statin use. Phlebotomy-related outcomes favored smokers over non-smokers. Biological linkages responsible for this unique effect offer promising lines for future iron reduction studies (ClinicalTrial.Gov Identifier: NCT00032357).

Effect of controlled reduction of body iron stores on clinical outcomes in peripheral arterial disease.

BACKGROUND: Published results from a controlled clinical trial in patients with peripheral arterial disease found improved outcomes with iron (ferritin) reduction among middle-aged subjects but not the entire cohort. The mechanism of the age-specific effect was explored. METHODS: Randomization to iron reduction (phlebotomy, n = 636) or control (n = 641) stratified by prognostic variables permitted analysis of effects of age and ferritin on primary (all-cause mortality) and secondary (death, nonfatal myocardial infarction, and stroke) outcomes. RESULTS: Iron reduction improved outcomes in youngest age quartile patients (primary outcome hazard ratio [HR] 0.44, 95% CI 0.21-0.92, P = .028; secondary outcome HR 0.34, 95% CI 0.19-0.61, P < .001). Mean follow-up ferritin levels (MFFL) declined with increasing entry age in controls. Older age (P = .035) and higher ferritin (P < .001) at entry predicted poorer compliance with phlebotomy and rising MFFL in iron-reduction patients. Intervention produced greater ferritin reduction in younger patients. Improved outcomes with lower MFFL were found in iron-reduction patients (primary outcome HR 1.11, 95% CI 1.01-1.23, P = .028; secondary outcome HR 1.10, 95% CI 1.0-1.20, P = .044) and the entire cohort (primary outcome HR 1.11, 95% CI 1.01-1.23, P = .037). Improved outcomes occurred with MFFL below versus above the median of the entire cohort means (primary outcome HR 1.48, 95% CI 1.14-1.92, P = .003; secondary outcome HR 1.22, 95% CI 0.99-1.50, P = .067). CONCLUSIONS: Lower iron burden predicted improved outcomes overall and was enhanced by phlebotomy. Controlling iron burden may improve survival and prevent or delay nonfatal myocardial infarction and stroke.

Prohemostatic therapy: the rise and fall of aprotinin.

Aprotinin has been used clinically to enhance hemostasis for decades and was approved in the United States by the Food and Drug Administration in 1993 to reduce the transfusion requirement during coronary artery bypass surgery. Marketing of aprotinin ceased recently when observational studies and a randomized clinical trial reported increased cardiovascular toxicity in patients receiving this drug. The importance of prohemostatic therapy is reviewed in light of new information on long-term deleterious effects of blood transfusion, including increased risk of cardiovascular disease, malignancy, and infection possibly attributable to delivery of a load of red cell-derived redox-active iron. Weaknesses in design of clinical trials that failed to control adequately for such alternative mechanisms of toxicity complicate interpretation of risks versus benefits in clinical trials of aprotinin given to reduce transfusion requirement in the acute surgical setting. Properties and applications of aprotinin that may not have received sufficient attention in the decision to remove this drug from the therapeutic armamentarium are reviewed. Potential application of prohemostatic drugs, including aprotinin to special populations at risk for operative blood loss requiring transfusion, is illustrated by the description of nine patients with coagulopathies whose operative bleeding was managed effectively with aprotinin. This drug may remain safe and effective in patients at risk of bleeding with surgery. Beneficial effects of aprotinin seemingly unrelated to its prohemostatic properties, especially its apparent striking antineoplastic effects, warrant further study.

Ferritin levels, inflammatory biomarkers, and mortality in peripheral arterial disease: a substudy of the Iron (Fe) and Atherosclerosis Study (FeAST) Trial.

BACKGROUND: This study delineated correlations between ferritin, inflammatory biomarkers, and mortality in a cohort of 100 cancer-free patients with peripheral arterial disease (PAD) participating in the Veterans Affairs (VA) Cooperative Study #410, the Iron (Fe) and Atherosclerosis Study (FeAST). FeAST, a prospective, randomized, single-blind clinical trial, tested the hypothesis that reduction of iron stores using phlebotomy would influence clinical outcomes in 1227 PAD patients randomized to iron reduction or control groups. The effects of statin administration were also examined in the Sierra Nevada Health Care (SNHC) cohort by measuring serum ferritin levels at entry and during the 6-year study period. No difference was documented between treatment groups in all-cause mortality and secondary outcomes of death plus nonfatal myocardial infarction and stroke. Iron reduction in the main study caused a significant age-related improvement in cardiovascular disease outcomes, new cancer diagnoses, and cancer-specific death. METHODS: Tumor necrosis factor (TNF)-alpha, TNF-alpha receptors 1 and 2, interleukin (IL)-2, IL-6, IL-10, and high-sensitivity C reactive protein (hs-CRP) were measured at entry and at 6-month intervals for 6 years. Average levels of ferritin and lipids at entry and at the end of the study were compared. The clinical course and ferritin levels of 23 participants who died during the study were reviewed. RESULTS: At entry, mean age of entry was 67 +/- 9 years for the SNHCS cohort, comparable to FeAST and clinical and laboratory parameters were equivalent in substudy participants randomized to iron reduction (n = 51) or control (n = 49). At baseline, 53 participants on statins had slightly lower mean entry-level ferritin values (114.06 ng/mL; 95% confidence interval [CI] 93.43-134.69) vs the 47 off statins (127.62 ng/mL; 95% CI, 103.21-152.02). Longitudinal analysis of follow-up data, after adjusting for the phlebotomy treatment effect, showed that statin use was associated with significantly lower ferritin levels (-29.78 ng/mL; Cohen effect size, -0.47 [t(df, 134) = 2.33, P = .02]). Mean follow-up average ferritin levels were higher in 23 participants who died (132.5 ng/mL; 95% CI, 79.36-185.66) vs 77 survivors (83.6 ng/mL; 95% CI, 70.34-96.90; Wilcoxon P = .05). Mean follow-up IL-6 levels were higher in dead participants (21.68 ng/mL; 95% CI, 13.71-29.66) vs survivors (12.61 ng/mL; 95% CI, 10.72-14.50; Wilcoxon P = .018). Ferritin levels correlated (Pearson) with average IL-6 levels (r = 0.1845; P = .002) and hsCRP levels (r = .1175; P = .04) during the study. CONCLUSION: These data demonstrate statistical correlations between levels of ferritin, inflammatory biomarkers, and mortality in this subset of patients with PAD.

Iron reduction response and demographic differences between diabetics and non-diabetics with cardiovascular disease entered into a controlled clinical trial.

Iron-catalyzed oxygen-free radical-induced oxidative stress mediates the pathogenesis of diabetes and cardiovascular disease (CVD). Diabetics (n = 473) and non-diabetics (n = 804) with CVD entered into a randomized trial of iron (ferritin) reduction by calibrated phlebotomy (, Identifier NCT00032357) had comparable iron measures at entry but diabetics had a greater burden of CVD and comorbidities, lower hemoglobin and hematocrit levels, and higher glucose levels than non-diabetics. Entry iron measures were lower in diabetics on hypoglycemic therapy compared to previously untreated diabetics. Diabetics and non-diabetics had comparable iron measures during follow-up. The Loess analysis of paired ferritin and hemoglobin, and paired ferritin and glucose levels in diabetics randomized to phlebotomy showed higher ferritin levels associated with lower hemoglobin and higher glucose levels. Progressive ferritin reduction in diabetics correlated with increasing hemoglobin and decreasing glucose levels, neither of which reached levels observed in non-diabetics. We postulate that phlebotomy-triggered autophagy (ferritinophagy) released redox-active iron sequestered intracellularly, worsening anemia and glucose utilization that corrected partially with ferritin reduction. Intracellular redox-active iron levels contributory to disease, not reflected in peripheral iron measures, may persist because of glycation of iron transport proteins in diabetes. These findings suggest novel strategies for disease prevention and improving outcomes in diabetes and CVD.

Reduction of iron stores and cardiovascular outcomes in patients with peripheral arterial disease: a randomized controlled trial.

CONTEXT: Accumulation of iron in excess of physiologic requirements has been implicated in risk of cardiovascular disease because of increased iron-catalyzed free radical-mediated oxidative stress. OBJECTIVE: To test the hypothesis that reducing body iron stores through phlebotomy will influence clinical outcomes in a cohort of patients with symptomatic peripheral arterial disease (PAD). DESIGN, SETTING, AND PATIENTS: Multicenter, randomized, controlled, single-blinded clinical trial based on the Iron (Fe) and Atherosclerosis Study (FeAST) (VA Cooperative Study #410) and conducted between May 1, 1999, and April 30, 2005, within the Department of Veterans Affairs Cooperative Studies Program and enrolling 1277 patients with symptomatic but stable PAD. Those with conditions likely to cause acute-phase increase of the ferritin level or with a diagnosis of visceral malignancy within the preceding 5 years were excluded. Analysis was by intent-to-treat. INTERVENTION: Patients were assigned to a control group (n = 641) or to a group undergoing reduction of iron stores by phlebotomy with removal of defined volumes of blood at 6-month intervals (avoiding iron deficiency) (n = 636), stratified by hospital, age, and baseline smoking status, diagnosis of diabetes mellitus, ratio of high-density to low-density lipoprotein cholesterol level, and ferritin level. MAIN OUTCOME MEASURES: The primary end point was all-cause mortality; the secondary end point was death plus nonfatal myocardial infarction and stroke. RESULTS: There were no significant differences between treatment groups for the primary or secondary study end points. All-cause deaths occurred in 148 patients (23%) in the control group and in 125 (20%) in the iron-reduction group (hazard ratio (HR), 0.85; 95% confidence interval (CI), 0.67-1.08; P = .17). Death plus nonfatal myocardial infarction and stroke occurred in 205 patients (32%) in the control group and in 180 (28%) in the iron-reduction group (HR, 0.88; 95% CI, 0.72-1.07; P = .20). CONCLUSION: Reduction of body iron stores in patients with symptomatic PAD did not significantly decrease all-cause mortality or death plus nonfatal myocardial infarction and stroke. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT00032357.

Ferritin and Percent Transferrin Saturation Levels Predict Type 2 Diabetes Risk and Cardiovascular Disease Outcomes.

INTRODUCTION: Type 2 diabetes (T2D) and cardiovascular disease (CVD) risk associate with ferritin and percent transferrin saturation (%TS) levels. However, increased risk has been observed at levels considered within the "normal range" for these markers. OBJECTIVE: To define normative ferritin and %TS levels associated with T2D and CVD risk. METHODS: Six-monthly ferritin, %TS and hemoglobin levels from 1,277 iron reduction clinical trial participants with CVD (peripheral arterial disease, 37% diabetic) permitted pair-wise analysis using Loess Locally Weighted Smoothing plots. Curves showed continuous quantitative ferritin, hemoglobin (reflecting physiologic iron requirements), and %TS (reflecting iron transport and sequestration) levels over a wide range of values. Inflection points in the curves were compared to ferritin and %TS levels indicating increased T2D and CVD risk in epidemiologic and intervention studies. RESULTS: Increasing ferritin up to about 80 ng/mL and %TS up to about 25% TS corresponded to increasing hemoglobin levels, and minimal T2D and CVD risk. Displaced Loess trajectories reflected lower hemoglobin levels in diabetics compared to non-diabetics. Ferritin levels up to about 100 ng/mL paralleled proportionately increasing %TS levels up to about 55%TS corresponding to further limitation of T2D and CVD risk. Ferritin levels over 100 ng/mL did not associate with hemoglobin levels and coincided with increased T2D and CVD risk. CONCLUSIONS: Recognition of modified normal ranges for ferritin from about 15 ng/mL up to about 80- 100 ng/mL and %TS from about 15% up to about 25-55% may improve the value of iron biomarkers to assess and possibly lower T2D and CVD risk.

Comparison of the serum ferritin and percentage of transferrin saturation as exposure markers of iron-driven oxidative stress-related disease outcomes.

BACKGROUND: Iron-catalyzed oxidative stress may be the primary mechanism for the pathogenesis of diseases related to iron excess. We hypothesized previously that certain markers of iron in bound form that are commonly used in epidemiologic studies might be inappropriate for investigating iron-related adverse health effects because oxidative stress requires iron in redox-active form. METHODS: To study aspects of this hypothesis, we examined the association between levels of serum ferritin or the percentage of transferrin saturation (%TS) and levels of serum antioxidant vitamins and C-reactive protein (CRP). This cross-sectional analysis included 11245 adults aged 20 years or older who participated in the Third National Health and Nutrition Examination Survey. RESULTS: Adjusted concentrations of serum alpha-carotene, beta-carotene, beta-cryptoxanthin, and lycopene were inversely correlated with the serum ferritin concentration (P for trend < .01), even within the lower deciles of the serum ferritin. In contrast, the %TS was significantly and positively associated with beta-cryptoxanthin, vitamin C, and vitamin E. In addition, the serum ferritin was positively associated but the %TS was strongly and inversely associated with the serum CRP (P for trend < .01). CONCLUSIONS: The serum ferritin and %TS showed contrasting associations with serum antioxidant vitamin levels and CRP although they have been used interchangeably in epidemiologic studies as markers of body iron. These results suggest that the %TS may not be a valid marker of exposure to iron-related oxidative stress. It appears that the serum ferritin is the preferred marker for assessment of clinical outcomes presumed to be caused by iron-related oxidative stress.

Anticoagulation, ferrotoxicity and the future of translational lung cancer research.

Numerous studies have shown that elements of coagulation reactions mediate tumor cell proliferation, motility (invasiveness), tissue remodeling and metastasis. Coagulation activation is virtually a universal feature of human malignancy that differs from the clotting response to injury in that it is self-perpetuating rather than self-attenuating. Coagulation activation participates in tumor matrix deposition and local inflammation, and predicts subsequent cancer risk and adverse cancer outcomes. Several clinical trials of anticoagulants have shown improved outcomes in small cell carcinoma of the lung (SCCL) that have been correlated with assembly on the tumor cells of an intact coagulation pathway. However, variable efficacy of anticoagulant therapy has raised doubts about the coagulation hypothesis. Recently, initiators of coagulation and fibrinolytic pathways have been identified that mediate tumor inception and progression. Notable among these is oxidative stress driven by iron-catalyzed reactive oxygen species that may be the basis for local coagulation activation, tumor matrix deposition, inflammation and aberrant properties characteristic of the malignant phenotype. Recognition of important biological characteristics of individual tumor types, disease stage, choice of standard therapy including chemotherapy and the iron status of the host may clarify mechanisms. All of these are subject to modification based on controlled clinical trial design. Further tests of the coagulation hypothesis may lead to novel, low cost and relatively non-toxic approaches to treatment of malignancy including lung cancer that contrast with certain current cancer treatment paradigms.

Racial Health Disparities, and Variant Red Cell and Iron Homeostasis.

Oxidative stress from excess iron may contribute to racial health disparities. Previously we reported improved clinical outcomes with lower ferritin and higher percent transferrin saturation (%TS) levels in White but not Black participants with peripheral arterial disease entered to a clinical trial. This report demonstrates racially variant interactions between hemoglobin, ferritin, and %TS levels. Lower hemoglobin and %TS levels, and higher ferritin levels were documented in Black compared with White participants within cardiovascular disease risk categories. Ferritin levels near 80 ng/mL related to higher hemoglobin levels in White but not Black participants. Higher %TS levels with ferritin levels above 80 ng/mL in White participants were blunted in Black participants. Ferritin/%TS ratios were significantly higher in Black than White participants. Iron incorporation into hemoglobin and scavenging by transferrin may buffer iron toxicity more effectively in White than in Black individuals. Metabolic vulnerability to iron excess may explain, in part, racial health disparities.

Racial differences in iron measures and outcomes observed during an iron reduction trial in peripheral arterial disease.

Elevated body iron (ferritin) levels may contribute to adverse health outcomes. Racial differences in iron measures and clinical outcomes were observed during an iron reduction trial in peripheral arterial disease. At entry, Black compared with White participants had higher ferritin and lower red cell measures, as well as differing ferritin and percent transferrin saturation (%TS) responses, and HDL/LDL ratios associated with statin use. Lower hematocrit levels during follow-up resulted in fewer phlebotomies, less iron unloading (ferritin reduction, p=.035) and 32% less iron removed in Black compared with White participants randomized to iron reduction. Improved primary (all-cause mortality) and secondary (death plus non-fatal myocardial infarction and stroke) outcomes among White participants correlated with lower ferritin levels (p=.005 and p=.053, respectively) and higher %TS levels (p<.001 and p=.001 respectively), associations not observed in Black participants. Variant iron homeostasis contributory to racial health disparities warrants personalized intervention strategies and race-specific clinical trial design.

Implementation of an iron reduction protocol in patients with peripheral vascular disease: VA cooperative study no. 410: the Iron (Fe) and Atherosclerosis Study (FeAST).

BACKGROUND: Iron accumulates imperceptibly over time in adults because intake exceeds loss and because no physiologic mechanism exists for excreting levels that may be toxic. Levels of stored iron represented by the serum ferritin concentration have been implicated in the pathogenesis of vascular (and other) diseases, but the role of such stored iron remains controversial. Our hypothesis was that reduction in body iron stores to levels typical of children and premenopausal women (corresponding to ferritin levels of approximately 25 ng/mL) would alter morbidity and mortality rates in patients with advanced peripheral vascular disease. METHODS AND RESULTS: A randomized, single-blinded, clinical trial of graded reduction of iron stores by controlled phlebotomy was undertaken in patients with advanced peripheral vascular disease. Details of implementation of the protocol for testing this unusual experimental intervention are reported. CONCLUSIONS: A methodology is described for testing the concept that reduction of body iron stores (while avoiding iron deficiency) may alter disease outcomes. This methodology appears to be suitable for further testing to determine whether levels of iron stores presumed to be pathologic contribute to disease initiation or progression.

Anticoagulants in cancer treatment: malignancy as a solid phase coagulopathy.

The purpose of this review is to demonstrate the potential for development of coagulation-reactive drugs for experimental cancer treatment. Improved cancer outcome has been reported in several tumor types with drugs that interfere with thrombin-generating or urokinase-initiated pathways expressed by tumor cells. These pathways participate in the response to injury and their inappropriate expression by malignant cells supports tumor growth. A proposed classification of tumor types based on the differential occurrence of these pathways may account for variation in drug responsiveness and guide clinical trial design. Among drugs available for testing, aprotinin and low molecular weight heparin have particular appeal. The former blocks a pathway of tumor cell growth, invasion and metastasis while the latter blocks growth factor activity, angiogenesis, and other tumor growth mechanisms as well as coagulation activation. Conceiving of malignancy as a solid phase coagulopathy may facilitate development of effective but non-toxic cancer treatments and encourage inquiry into the putative 'injury' responsible for both coagulation activation and carcinogenesis.

Low-molecular-weight heparin in oncology.

Venous thromboembolism (VTE) is a wellknown complication of malignant disease and multiple risk factors contribute to the hypercoagulability that commonly accompanies malignancy. Thromboprophylaxis with antithrombotic drugs such as the low-molecular-weight heparins (LMWHs) can be used to control the hypercoagulable state and to reduce the incidence of VTE in patients with cancer. Clinical and biochemical data suggest that LMWHs may also inhibit tumour growth, leading to a survival benefit in these patients. This review addresses the efficacy and safety of LMWHs for prophylaxis and treatment of VTE in patients with cancer. Outstanding issues relating to thromboprophylaxis and treatment of VTE in patients with cancer and the clinical trials that may resolve these issues are also highlighted.

Rationale for clinical trials of coagulation: reactive drugs in hepatocellular carcinoma.

Evidence for the regulation of cancer growth by components of the blood coagulation mechanism provides abundant opportunity for the development of novel hypotheses for the experimental treatment of malignancy. Information available on the heterogeneity in mechanisms of interaction between various cancer cell types, and procoagulant and fibrinolytic pathways, platelets, glycosaminoglycan-regulated growth factors and cell-adhesion molecules indicates that insightful clinical trial design may allow targeting of individual cancer cell types with agents capable of intercepting mechanisms of growth control that are relevant to specific tumor types. This paper reviews the evidence that the common anticoagulant, heparin, inhibits hepatocellular carcinoma cell proliferation and hepatocellular carcinoma tumor dissemination in experimental animals. Clinical trials of heparin performed to date have shown increased tumor response rates and survival in other tumor types. Expression of urokinase-type plasminogen activator by hepatocellular carcinoma cells enhances tumor cell proliferation, motility, invasiveness and metastatic dissemination. Inhibition of the urokinase-type plasminogen activator/plasmin system by protease inhibitors such as aprotinin (Trasylol, Bayer) have shown improvement in the clinical course of certain tumor types. These data suggest that drugs that are well-known in the field of vascular medicine may find a role in the treatment of hepatocellular carcinoma, a common tumor type that has resisted containment by other means.

Cancer and venous thromboembolism: a multidisciplinary approach.

In this paper, we briefly review the relation between cancer and venous thromboembolism (VTE), an association that has been recognized for more than a century. In particular we focus on VTE as predictor and prognostic factor for cancer and the antineoplastic potential of antithrombotic treatment. Cancer may cause disturbances in the haemostatic system by numerous mechanisms that can lead to an increased risk of VTE. Patients with known cancer are at increased risk of VTE; however, VTE may also be a predictor of subsequent cancer in the years afterwards. Furthermore, cancer patients with VTE are more likely to have advanced disease and a worse prognosis than cancer patients without a VTE episode. These findings may have implications for the clinical care of patients with VTE in terms of screening for cancer. However, extensive cancer screening programs are not recommended in general for patients with VTE. A wide range of antithrombotic drugs including heparin, oral anticoagulants, and platelet inhibitors have been examined in order to study the effect on the prevention and treatment of various cancers. These efforts have resulted in a considerable amount of useful data from animal models and a number of promising reports from retrospective analyses and small-scale trials linking antithrombotic treatment with an increased survival in cancer patients and even a lower risk of primary cancer. However, the clinical implications of these findings must await properly designed and conducted randomized clinical trials.