Correction to: VEIN STEP: A Prospective, Observational, International Study to Assess Effectiveness of Conservative Treatments in Chronic Venous Disease.

Video Abstract.

Reduction of lower-limb edema in patients with chronic venous disease by micronized purified flavonoid fraction: a systematic literature review and meta-analysis.

INTRODUCTION: The prevalence of lower limb edema is high among patients with chronic venous disease (CVD). Several clinical studies with various designs have assessed the effect of micronized purified flavonoid fraction (MPFF) on edema. The aim of this work was to provide a comprehensive and accurate evaluation of the reduction in ankle and calf circumference as an indicator of lower limb edema reduction in patients with CVD treated with MPFF by combining studies that use different designs in a single group meta-analysis. EVIDENCE ACQUISITION: We conducted a systematic literature review in April 2022 based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) criteria to identify prospective studies investigating the effect of oral MPFF treatment 1000 mg/day on ankle and calf circumference in patients with CVD. Studies with population including at least one patient with an ulcer were excluded. All prospective studies irrespectively of design (i.e., interventional and non-interventional studies, randomized controlled trials (RCTs), non-randomized studies, studies without a control or reference treatment) were eligible. The Medline, Embase and Cochrane databases were searched. Endpoints were ankle and calf circumference measurements and their overall mean change from baseline estimated with random-effects meta-analysis methods. The evaluation criterion feeling of swelling was also analyzed as a standardized mean change (SMC) with 95% confidence intervals after combination of quantitative scales. EVIDENCE SYNTHESIS: Among 861 articles identified, eight studies (five RCTs including one placebo-controlled, three non-comparative studies) met the criteria. The overall population consisted of 1635 patients, predominantly female (89% ranging from 64% to 94%) with a mean age of 47 years ranging from 41 to 48 years. Mean reduction in ankle circumference was 6.0 mm (95%CI: 3.6 to 8.4; P<0.001) and 7.0 mm (95%CI: 0.9 to 13.1; P=0.024) after two and at least six months of treatment respectively. The results were similar when considering the study type RCTs and non-RCTs. Mean reduction in calf circumference was 5.7 mm (95%CI: 2.8 to 8.6; P<0.001) and 6.7 mm (95%CI: 5.2 to 8.1; P<0.001), at two months and at the last post-baseline evaluation respectively. Heterogeneity among studies was statistically significant (degree of consistency I2=93.5%; P<0.001 and I2=81.1%, P<0.01 for ankle and calf circumference, respectively). In the three studies reporting the effect on feeling of swelling a significant standardized mean change (SMC) reduction of 2.2 (95%CI: 0.2 to 4.2; P=0.028) on a quantitative scale was observed after two months of treatment with MPFF. CONCLUSIONS: MPFF appeared to be effective in reducing ankle and calf circumference as well as feeling of swelling irrespective of study design. The circumference reduction is present at short and long term, suggesting that benefit occurs early and is maintained overtime. Despite the observed heterogeneity among included studies, this meta-analysis supports the significant therapeutic efficacy of MPFF in reducing lower-limb edema in patients with CVD. The complete video presentation of the work is available online at www.minervamedica.it (Supplementary Digital Material 1: Supplementary Video 1, 5 min, 192 MB).

VEIN STEP: A Prospective, Observational, International Study to Assess Effectiveness of Conservative Treatments in Chronic Venous Disease.

INTRODUCTION: VEIN STEP was conducted to collect international data on the management of chronic venous disease (CVD) and to assess the effectiveness of conservative treatments for the relief of CVD signs and symptoms. METHODS: This international, observational, prospective, longitudinal, cohort study recruited adult outpatients consulting for symptomatic CVD. The primary objective was the effectiveness of conservative treatments on symptoms, signs and quality of life in a real-life setting assessed using a range of patient-reported outcome measures: 10-cm Visual Analog and Patient Global Impression of Change scales for symptoms; Venous Clinical Severity Score for physician assessment of signs; and 14-item ChronIc Venous Insufficiency Questionnaire (CIVIQ-14) for quality of life. At inclusion, patients were prescribed conservative treatment according to the physicians' usual practice. Follow-up visits took place at weeks 2 and 4, with an optional week 8 visit. RESULTS: The analysis set comprised 6084 subjects (78% female) from nine countries with a mean age of 50.6 ± 13.8 years and BMI of 28.0 ± 4.9 kg/m2. The most common CEAP classifications were C1 (23.0%), C2 (31.6%), and C3 (30.7%). Conservative therapy consisted of oral venoactive drugs (VADs; 95.8% of subjects) including micronized purified flavonoid fraction (MPFF 75.5%) and diosmin (18.8%), compression (52.0%), and topicals (31.5%). Conservative therapy led to global symptom improvement in 89% of patients after 2 weeks and 96% at 4 weeks. Pain, leg heaviness, cramps, and sensation of swelling were improved in 82%, 71%, 45.5%, and 46% of patients, respectively. Conservative therapy was associated with a decrease over time in patient-assessed global symptom intensity: - 2.37 ± 1.73 (P < 0.001) and physician-assessed disease severity - 1.83 ± 2.82 (P < 0.001). Among the VADs, MPFF-based conservative therapy was associated with the greatest reduction in symptom and sign intensity. Improvements in CIVIQ-14 were observed with all treatments but were greatest for MPFF. CONCLUSION: In this prospective study conducted in the real-world setting, treatment with conservative therapy, in particular MPFF, was associated with meaningful improvements in the clinical signs and symptoms of disease as well as in quality of life in patients with CVD. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04574375.

STUDY AIM: The VEIN STEP study aimed to gather global data on managing chronic venous disease (CVD) and evaluate the usefulness of conservative (non-surgical) treatments for improving CVD signs and symptoms. METHODS: Persons included in the study group had symptomatic CVD and were visiting outpatient clinics. The main aim was to measure how well treatments improved symptoms, physical signs of the illness, and quality of life. Different methods were used to measure these aspects, such as rating symptoms on a 10-point scale and using questionnaires completed by patients and doctors. STUDY FINDINGS: 6084 participants from nine countries joined the study. They were mostly women (78%) with an average age of around 50. Common symptoms included leg pain and leg heaviness. Treatments consisted mainly of drugs active on vein function, like MPFF and diosmin, along with compression stockings and creams. Conservative treatment led to symptom improvement in 89% of patients after 2 weeks and 96% at 4 weeks. Pain and leg heaviness improved in most patients (82% and 71% over the same period) while cramps, and swelling showed improvement in 45.5% and 46% of patients, respectively. Patients reported a significant decrease in symptom intensity, and doctors observed a reduction in disease severity. MPFF was associated with the highest reduction in symptom intensity. Improvements in quality of life were observed with all treatments but were greatest for MPFF. CONCLUSION: The study highlights that conservative treatments, especially MPFF, are associated with significant improvements in the clinical signs and symptoms of patients with CVD as well as in their quality of life.

Sex differences in the generalizability of randomized clinical trials in heart failure with reduced ejection fraction.

AIMS: In order to understand how sex differences impact the generalizability of randomized clinical trials (RCTs) in patients with heart failure (HF) and reduced ejection fraction (HFrEF), we sought to compare clinical characteristics and clinical outcomes between RCTs and HF observational registries stratified by sex. METHODS AND RESULTS: Data from two HF registries and five HFrEF RCTs were used to create three subpopulations: one RCT population (n = 16 917; 21.7% females), registry patients eligible for RCT inclusion (n = 26 104; 31.8% females), and registry patients ineligible for RCT inclusion (n = 20 810; 30.2% females). Clinical endpoints included all-cause mortality, cardiovascular mortality, and first HF hospitalization at 1 year. Males and females were equally eligible for trial enrolment (56.9% of females and 55.1% of males in the registries). One-year mortality rates were 5.6%, 14.0%, and 28.6% for females and 6.9%, 10.7%, and 24.6% for males in the RCT, RCT-eligible, and RCT-ineligible groups, respectively. After adjusting for 11 HF prognostic variables, RCT females showed higher survival compared to RCT-eligible females (standardized mortality ratio [SMR] 0.72; 95% confidence interval [CI] 0.62-0.83), while RCT males showed higher adjusted mortality rates compared to RCT-eligible males (SMR 1.16; 95% CI 1.09-1.24). Similar results were also found for cardiovascular mortality (SMR 0.89; 95% CI 0.76-1.03 for females, SMR 1.43; 95% CI 1.33-1.53 for males). CONCLUSION: Generalizability of HFrEF RCTs differed substantially between the sexes, with females having lower trial participation and female trial participants having lower mortality rates compared to similar females in the registries, while males had higher than expected cardiovascular mortality rates in RCTs compared to similar males in registries.

Generalizability of randomized controlled trials in heart failure with reduced ejection fraction.

BACKGROUND: Heart failure (HF) trials have stringent inclusion and exclusion criteria, but limited data exist regarding generalizability of trials. We compared patient characteristics and outcomes between patients with HF and reduced ejection fraction (HFrEF) in trials and observational registries. METHODS AND RESULTS: Individual patient data for 16 922 patients from five randomized clinical trials and 46 914 patients from two HF registries were included. The registry patients were categorized into trial-eligible and non-eligible groups using the most commonly used inclusion and exclusion criteria. A total of 26 104 (56%) registry patients fulfilled the eligibility criteria. Unadjusted all-cause mortality rates at 1 year were lowest in the trial population (7%), followed by trial-eligible patients (12%) and trial-non-eligible registry patients (26%). After adjustment for age and sex, all-cause mortality rates were similar between trial participants and trial-eligible registry patients [standardized mortality ratio (SMR) 0.97; 95% confidence interval (CI) 0.92-1.03] but cardiovascular mortality was higher in trial participants (SMR 1.19; 1.12-1.27). After full case-mix adjustment, the SMR for cardiovascular mortality remained higher in the trials at 1.28 (1.20-1.37) compared to RCT-eligible registry patients. CONCLUSION: In contemporary HF registries, over half of HFrEF patients would have been eligible for trial enrolment. Crude clinical event rates were lower in the trials, but, after adjustment for case-mix, trial participants had similar rates of survival as registries. Despite this, they had about 30% higher cardiovascular mortality rates. Age and sex were the main drivers of differences in clinical outcomes between HF trials and observational HF registries.

Carboxypeptidase U (TAFIa) Is Rapidly Activated and Deactivated Following Thrombolysis and Thrombectomy in Stroke Patients.

The antifibrinolytic enzyme carboxypeptidase U (CPU, TAFIa, CPB2) is an appealing target for the treatment of acute ischemic stroke (AIS). Increased insights in CPU activation and inactivation during thrombolysis (rtPA) with or without endovascular thrombectomy (EVT) are required to develop CPU inhibitors as profibrinolytic agents with optimal benefits/risks. Therefore, CPU kinetics during ischemic stroke treatment were evaluated. AIS patients with documented cerebral artery occlusion receiving rtPA (N = 20) or rtPA + EVT (N = 16) were included. CPU activation during thrombolysis was measured by an ultrasensitive HPLC-based CPU activity method and by an ELISA measuring both CPU and inactivated CPU (CPU + CPUi). Intravenous blood samples were collected at admission and throughout the first 24 h. Additional in situ blood samples were collected in the rtPA + EVT cohort proximal from the thrombus. The NIHSS score was determined at baseline and 24 h. CPU activity and CPU + CPUi levels increased upon rtPA administration and reached peak values at the end of thrombolysis (1 h). High inter-individual variability was observed in both groups. CPU activity decreased rapidly within 3 h, while CPU + CPUi levels were still elevated at 7 h. CPU activity or CPU + CPUi levels were similar in in situ and peripheral samples. No correlation between CPU or CPU + CPUi and NIHSS or thrombus localization was found. The CPU system was rapidly activated and deactivated following thrombolysis and thrombectomy in stroke patients, suggesting that a CPU inhibitor would have to be administered during rtPA infusion and over the next few hours. The high CPU generation variability suggests that some patients may not respond to the treatment. EudraCT number 2017-002760-41.

Time course of microvalve pathophysiology in high pressure low flow model of venous insufficiency and the role of micronized purified flavonoid fraction.

BACKGROUND: The aim of this study was to characterize, in an experimental model, the mechanisms involved in the initiation of venous insufficiency at the level of microvenous valve and whether they can be influenced by early treatment with micronized purified flavonoid fraction (MPFF). METHODS: The external right iliac vein of 78 male golden Syrian hamsters was ligated to induce chronic venous insufficiency. Internal venular diameter as well as leukocyte-endothelium-interaction (leukocytes sticking after staining with rhodamine 6G), were assessed using an intravital microscope. In the second part of the study 30 animals were divided into three groups and underwent: ligation plus MPFF, ligation plus 10% lactose solution (vehicle), or sham operation. Treatment with MPFF 100 mg/kg/day or vehicle started 2 days before ligation and lasted for 7 days. Venular diameter and number of adherent leukocytes were assessed 5 days post-ligature. RESULTS: Venular diameter increased immediately after ligature and reached a maximum at 4 hours (P<0.001 vs. baseline), followed by a plateau before gradually returning to baseline dimensions. The increase in the number of adherent leukocytes was also immediate but attained maximal number at 3 days (P<0.0001), followed by a plateau and then gradual return to baseline numbers. In MPFF-treated animals, leukocyte adhesion to the microvalves was prevented compared with vehicle-treated animals (P<0.0001) and venular diameter was also significantly reduced (P<0.05). CONCLUSIONS: Venous hypertension induced immediate venular dilatation followed by an increase in the number of adherent leukocytes at microvalve level. Treatment with MPFF prevented the initiation of microvalve inflammation and may play a protective role in the progression of chronic venous insufficiency.

Protective Effects of Micronized Purified Flavonoid Fraction (MPFF) on a Novel Experimental Model of Chronic Venous Hypertension.

OBJECTIVES: To assess protective effects of micronized purified flavonoid fraction (MPFF) on microcirculation in an original chronic model of hind limb venous hypertension with low blood flow in small animals. METHODS: Vein ligatures were performed on male hamsters, as follows: A-right femoral vein; A + B-right femoral vein and its right branch; A + C-right femoral vein and its left branch; A + B + C-right femoral and its right and left branches; D-external right iliac vein. In sham operated groups, similar vascular dissections were performed without ligatures. Superficial (epigastric) and central (jugular) venous pressure evaluations were made during a 10 week period. Hamsters subjected to A + B + C and D ligatures were selected for leukocyte rolling and sticking, functional capillary density (FCD), and venular and arteriolar diameter observations. D ligature was selected to evaluate pharmacological treatment efficacy. MPFF (100 mg/kg), concomitant active flavonoids of MPFF (diosmetin, hesperidin, linarin, and isorhoifolin) (10 mg/kg), diosmin (100 mg/kg) or drug vehicle were administered orally during 2 weeks before vein ligature and 6 weeks thereafter. RESULTS: A, A + B and A + C models maintained venous return through collaterals. From the 2nd to the 10th weeks after vein ligatures, A + B + C and D models elicited a progressive increase of superficial venous pressure (3.83 ± 0.65 vs. 8.56 ± 0.72 mmHg, p < .001 and 4.13 ± 0.65 vs. 9.35 ± 0.65 mmHg, p < .001, respectively) with significant changes to the microcirculation. As D model significantly increased superficial venous pressure without affecting central venous pressure, it was used to evaluate the long-term effects of treatment. Compared with vehicle, MPFF, concomitant active flavonoids of MPFF, and diosmin, significantly decreased leukocyte-endothelium interaction and prevented FCD reduction. Only MPFF significantly prevented venular enlargement as observed in the vehicle treated group. CONCLUSION: MPFF was more effective than diosmin in improving all microvascular variables. The superiority of MPFF over diosmin alone can be explained by the synergistic beneficial effects of the association between diosmin and active flavonoids of MPFF.

Effects of a novel NADPH oxidase inhibitor (S42909) on wound healing in an experimental ischemic excisional skin model.

Chronic wounds are a serious healthcare problem. As non-healing wounds involve continuous pathologic inflammatory stage, research is focused on anti-inflammatory treatments. Our objective was to analyze the effect of S42909, a potent NADPH oxidase inhibitor activity, with vascular anti-inflammatory properties. An ischemic rabbit ear ulcer model (24 New Zealand white rabbits) was used to evaluate the reepithelialization/contraction areas, anti-/pro-inflammatory cytokines mRNA (TGF-ß1/IL-10/IFN-γ/VEGF) by qRT-PCR, collagen I/III deposition, and neovascularization (TGF-ß1/VEGF) by morphological and immunohistochemical analyses. Three different doses were administered by gavage for 2 weeks: 10 and 30 mg/kg/d in self-microemulsion drug delivery system (SMEDDS) and 100 mg/kg/d in arabic gum. Each vehicle was used as control. No signs of infection or necrosis were found. Reepithelialization was almost complete whatever the groups reaching 95% at the dose of 100 mg/kg. Wound contraction was significantly reduced in all S42909-treated groups. A significant increase in anti-inflammatory cytokines TGF-ß1 mRNA and IL-10 mRNA was observed at the dose of 100 and 30 mg/kg/d, respectively. No changes were observed in pro-inflammatory factors INF-γ and VEGF mRNA. Ischemic skin wound areas had scarce expression of collagen I/III and showed rich glycosaminoglycans content. Treatment increased the collagen deposition and TGF-ß1 protein expression and decreased glycosaminoglycan content dose dependently; however, no effect in VEGF was appreciated. Therefore, our results indicate that S42909 improved healing process by dampening excessive inflammation and facilitating collagen deposition without wound contraction phenomena. S42909 might be a promising therapy to treat chronic wounds as venous leg ulcers.

Terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, prevents hypertensive vascular hypertrophy and fibrosis.

Thromboxane A(2) and other eicosanoids such as isoprostanes contribute to vascular proliferation and atherosclerosis by binding to the thromboxane/prostaglandin endoperoxide receptors. The effects of terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, on aorta remodeling were evaluated in spontaneously hypertensive stroke-prone rats (SHRSPs), a model of severe hypertension, endothelial dysfunction, vascular inflammation, and cerebrovascular diseases. Male SHRSPs were allocated to three groups receiving a standard diet (n = 5) or a high-sodium permissive diet plus vehicle (n = 6) or plus terutroban (30 mg · kg(-1) · day(-1); n = 6). After 6 wk of dietary treatment, all of the animals were injected with bromodeoxyuridine and simultaneously euthanized for aorta collection. The aortic media thickness-to-lumen ratio significantly (P < 0.0001) increased in the salt-loaded rats compared with the rats fed a standard diet, whereas terutroban treatment completely prevented media thickening (P < 0.001). When compared with vehicle, terutroban was also effective in preventing cell proliferation in the media, as indicated by the reduced number of bromodeoxyuridine-positive (P < 0.0001) and proliferating cell nuclear antigen-positive cells (P < 0.0001). Severe fibrosis characterized by a significant accumulation of collagen and fibronectin in the vascular wall was observed in the vehicle-treated rats (P < 0.01) but was completely prevented by terutroban (P < 0.001). The latter also inhibited heat shock protein-47 (P < 0.01) and TGF-1ß expression (P < 0.001), which were significantly increased by the high-salt diet. In conclusion, terutroban prevents the development of aorta hyperplasia and has beneficial effects on fibrotic processes by affecting TGF-ß and heat shock protein-47 expression in SHRSPs. These findings provide mechanistic data supporting the beneficial effects of terutroban in preventing or retarding atherogenesis.

Terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, increases survival in stroke-prone rats by preventing systemic inflammation and endothelial dysfunction: comparison with aspirin and rosuvastatin.

This study investigated the efficacy of terutroban, a specific thromboxane/prostaglandin endoperoxide receptor antagonist, on stroke incidence in spontaneously hypertensive stroke-prone rats (SHRSP). The effects of terutroban were compared with those of aspirin, another antiplatelet agent, and rosuvastatin, known to exert end-organ protection in SHRSP. Salt-loaded male SHRSP were treated orally once a day with vehicle, terutroban (30 mg/kg/day), aspirin (60 mg/kg/day), or rosuvastatin (10 mg/kg/day). Compared with vehicle, and regardless of any effect on blood pressure or serum thromboxane B(2) levels, terutroban significantly increased survival (p < 0.001) as a consequence of a delayed brain lesion occurrence monitored by magnetic resonance imaging (p < 0.001), and a delayed increase of proteinuria (p < 0.001). Terutroban decreased cerebral mRNA transcription of interleukin-1beta, transforming growth factor-beta, and monocyte chemoattractant protein-1 after 6 weeks of dietary treatment. Terutroban also prevented the accumulation of urinary acute-phase proteins at high molecular weight, identified as markers of systemic inflammation, and assessed longitudinally by one-dimensional electrophoresis. Terutroban also has protective effects on the vasculature as suggested by the preservation of endothelial function and endothelial nitric-oxide synthase expression in isolated carotid arteries. These effects are similar to those obtained with rosuvastatin, and superior to those of aspirin. Terutroban increases survival in SHRSP by reducing systemic inflammation as well as preserving endothelial function. These data support clinical development of terutroban in the prevention of cerebrovascular and cardiovascular complications of atherothrombosis.