Lipocalin-2 and Calprotectin Potential Prognosis Biomarkers in Peripheral Arterial Disease.

OBJECTIVE: Peripheral arterial disease (PAD) is the most prevalent cardiovascular (CV) condition globally. Despite the high CV risk of PAD patients, no reliable predictors of adverse clinical evolution are yet available. In this regard, previous transcriptomic analyses revealed increased expression of calprotectin (S100A8/A9) and lipocalin-2 (LCN2) in circulating extracellular vesicles (EVs) of patients with PAD. The aim of this study was to determine the prognostic value of LCN2 and calprotectin for CV risk assessment in PAD. METHODS: LCN2 and the S100A9 subunit of calprotectin were examined in human femoral plaques by immunohistochemistry and qPCR. LCN2 and calprotectin were determined by ELISA in PAD (CHN cohort, n = 331, Fontaine II-IV, serum), and PAD diagnosed by population based screening (VIVA trial, n = 413, the majority Fontaine 0-I, plasma). Patients were followed up for a mean of four years, recording the primary outcomes; CV death or amputation in the CHN cohort and CV death or major lower limb events (MALE) in the VIVA population. Secondary outcomes were all cause death or amputation, and all cause death or MALE, respectively. RESULTS: LCN2 and S100A9 were detected in human plaques in regions rich in inflammatory cells. LCN2 and calprotectin levels were 70% and 64% lower in plasma than in serum. In the CHN cohort, high serum levels of LCN2 and calprotectin increased the risk of primary and secondary outcomes 5.6 fold (p < .001) and 1.8 fold (p = .034), respectively, after covariable adjustment. Similarly, elevated plasma levels of LCN2 and calprotectin increased by three fold the risk of primary and secondary outcomes (p < .001) in the VIVA cohort. Moreover, addition of the combined variable to basal models, considering clinically relevant risk factors, improved reclassification for the primary outcome in both cohorts (p ≤ .024). CONCLUSION: Combined assessment of the inflammatory biomarkers LCN2 and calprotectin might be useful for risk stratification in advanced and early PAD.

The Role of Circulating Biomarkers in Peripheral Arterial Disease.

Peripheral arterial disease (PAD) of the lower extremities is a chronic illness predominantly of atherosclerotic aetiology, associated to traditional cardiovascular (CV) risk factors. It is one of the most prevalent CV conditions worldwide in subjects >65 years, estimated to increase greatly with the aging of the population, becoming a severe socioeconomic problem in the future. The narrowing and thrombotic occlusion of the lower limb arteries impairs the walking function as the disease progresses, increasing the risk of CV events (myocardial infarction and stroke), amputation and death. Despite its poor prognosis, PAD patients are scarcely identified until the disease is advanced, highlighting the need for reliable biomarkers for PAD patient stratification, that might also contribute to define more personalized medical treatments. In this review, we will discuss the usefulness of inflammatory molecules, matrix metalloproteinases (MMPs), and cardiac damage markers, as well as novel components of the liquid biopsy, extracellular vesicles (EVs), and non-coding RNAs for lower limb PAD identification, stratification, and outcome assessment. We will also explore the potential of machine learning methods to build prediction models to refine PAD assessment. In this line, the usefulness of multimarker approaches to evaluate this complex multifactorial disease will be also discussed.

Fenestrated and Scalloped Endovascular Grafts in Zone 0 and Zone 1 for Aortic Arch Disease.

BACKGROUND: The aim of this study is to present midterm results of thoracic endovascular aortic repair (TEVAR) using scalloped or fenestrated custom-made endovascular grafts (CMEGs) in aortic arch Zones 0 and 1. METHODS: A retrospective review of prospectively collected data involving consecutive patients with aortic arch disease treated by scalloped or fenestrated Relay Plus stent grafts (Terumo Aortic, Sunrise, FL) landed in Zones 0 and 1. Patient demographics, operative details, clinical outcomes, and complications were analyzed. RESULTS: Between February 2014 and February 2020, 14 patients (9 male and 5 female) with a median age of 66 years (range 48-84) underwent scalloped or fenestrated TEVAR to preserve flow to the supra-aortic trunks (SATs). In 6 cases the landing zone was Zone 0 and in 8, Zone 1. Target vessels for the scallops were left common carotid artery in 8 cases (Zone 1) and innominate artery (IA) in 1 (Zone 0). All 5 fenestrations were designed to preserve the IA (Zone 0). Technical success was 100% with no endoleaks on completion angiography. One fatal perioperative stroke (7%) occurred in a patient with a fenestration for the IA and atherosclerotic plaques in the arch. During median follow-up of 37.5 (3-72) months, no other patient died, and all the target vessels and cervical revascularizations remained patent. There was no paraplegia, no retrograde dissection, and no other complication. Two patients (14%) with scallops in Zone 1 developed late endoleak: 1 type Ib at 6 months and 1 type Ia endoleak at 12 months. There were no endoleaks at all in the group of fenestrated endografts (Zone 0). CONCLUSIONS: When anatomy allows, endovascular treatment using scalloped or fenestrated CMEGs in Zones 0 and 1 is a feasible technique to treat patients with aortic arch disease involving the SATs.

Reduced high-density lipoprotein cholesterol: A valuable, independent prognostic marker in peripheral arterial disease.

OBJECTIVE: The prognosis of patients with peripheral arterial disease (PAD) is characterized by an exceptionally high risk for myocardial infarction, ischemic stroke, and death; however, studies in search of new prognostic biomarkers in PAD are scarce. Even though low levels of high-density lipoprotein cholesterol (HDL-C) have been associated with higher risk of cardiovascular (CV) complications and death in different atherosclerotic diseases, recent epidemiologic studies have challenged its prognostic utility. The aim of this study was to test the predictive value of HDL-C as a risk factor for ischemic events or death in symptomatic PAD patients. METHODS: Clinical and demographic parameters of 254 symptomatic PAD patients were recorded. Amputation, ischemic coronary disease, cerebrovascular disease, and all-cause mortality were recorded during a mean follow-up of 2.7 years. RESULTS: Multivariate analyses showed that disease severity (critical limb ischemia) was significantly reduced in patients with normal HDL-C levels compared with the group with low HDL-C levels (multivariate analysis odds ratio, 0.09; 95% confidence interval [CI], 0.03-0.24). A decreased risk for mortality (hazard ratio, 0.46; 95% CI, 0.21-0.99) and major adverse CV events (hazard ratio, 0.38; 95% CI, 0.16-0.86) was also found in patients with normal vs reduced levels of HDL-C in both Cox proportional hazards models and Kaplan-Meier estimates, after adjustment for confounding factors. CONCLUSIONS: Reduced HDL-C levels were significantly associated with higher risk for development of CV complications as well as with mortality in PAD patients. These findings highlight the usefulness of this simple test for early identification of PAD patients at high risk for development of major CV events.

Functional MMP-10 is required for efficient tissue repair after experimental hind limb ischemia.

We studied the role of matrix metalloproteinase-10 (MMP-10) during skeletal muscle repair after ischemia using a model of femoral artery excision in wild-type (WT) and MMP-10 deficient (Mmp10(-/-)) mice. Functional changes were analyzed by small animal positron emission tomography and tissue morphology by immunohistochemistry. Gene expression and protein analysis were used to study the molecular mechanisms governed by MMP-10 in hypoxia. Early after ischemia, MMP-10 deficiency resulted in delayed tissue reperfusion (10%, P < 0.01) and in increased necrosis (2-fold, P < 0.01), neutrophil (4-fold, P < 0.01), and macrophage (1.5-fold, P < 0.01) infiltration. These differences at early time points resulted in delayed myotube regeneration in Mmp10(-/-) soleus at later stages (regenerating myofibers: 30 ± 9% WT vs. 68 ± 10% Mmp10(-/-), P < 0.01). The injection of MMP-10 into Mmp10(-/-) mice rescued the observed phenotype. A molecular analysis revealed higher levels of Cxcl1 mRNA (10-fold, P < 0.05) and protein (30%) in the ischemic Mmp10(-/-) muscle resulting from a lack of transcriptional inhibition by MMP-10. This was further confirmed using siRNA against MMP-10 in vivo. Our results demonstrate an important role of MMP-10 for proper muscle repair after ischemia, and suggest that chemokine regulation such as Cxcl1 by MMP-10 is involved in muscle regeneration.

Matrix metalloproteinase 10 is associated with disease severity and mortality in patients with peripheral arterial disease.

OBJECTIVE: Peripheral arterial disease (PAD) is associated with poor prognosis in terms of cardiovascular (CV) morbidity and mortality. Matrix metalloproteinases (MMPs) contribute to vascular remodeling by degrading extracellular matrix components and play a role in atherosclerosis as demonstrated for MMP-10 (stromelysin-2). This study analyzed MMP-10 levels in PAD patients according to disease severity and CV risk factors and evaluated the prognostic value of MMP-10 for CV events and mortality in lower limb arterial disease after a follow-up period of 2 years. METHODS: MMP-10 was measured by enzyme-linked immunosorbent assay in 187 PAD patients and 200 sex-matched controls. RESULTS: PAD patients presented with increased levels of MMP-10 (702 ± 326 pg/mL control vs 946 ± 473 pg/mL PAD; P < .001) and decreased levels of tissue inhibitor of matrix metalloproteinase 1 (312 ± 117 ng/mL control vs 235 ± 110 ng/mL PAD; P < .001) compared with controls. Among PAD patients, those with critical limb ischemia (n = 88) showed higher levels of MMP-10 (1086 ± 478 pg/mL vs 822 ± 436 pg/mL; P < .001) compared with those with intermittent claudication (n = 99), whereas the MMP-10/tissue inhibitor of matrix metalloproteinase 1 ratio remained similar. The univariate analysis showed an association between MMP-10, age (P = .015), hypertension (P = .021), and ankle-brachial index (P = .006) in PAD patients that remained significantly associated with PAD severity after adjustment for other CV risk factors. Patients with the highest MMP-10 tertile had an increased incidence of all-cause mortality and CV mortality (P < .03). CONCLUSIONS: Our results suggest that MMP-10 is associated with severity and poor outcome in PAD.

Hemostatic Biomarkers and Volumetry Help to Identify High-Risk Abdominal Aortic Aneurysms.

Predicting the progression of small aneurysms is a main challenge in abdominal aortic aneurysm (AAA) management. The combination of circulating biomarkers and image techniques might provide an alternative for risk stratification. We evaluated the association of plasma TAT complexes (TAT) and D-dimer with AAA severity in 3 groups of patients: group 1, without AAA (n = 52), group 2, AAA 40−50 mm (n = 51) and group 3, AAA > 50 mm (n = 50). TAT (p < 0.001) and D-dimer (p < 0.001) were increased in patients with AAA (groups 2 and 3) vs. group 1. To assess the association between baseline TAT and D-dimer concentrations, and AAA growth, aortic diameter and volume (volumetry) were measured by computed tomography angiography (CTA) in group 2 at recruitment (baseline) and 1-year after inclusion. Baseline D-dimer and TAT levels were associated with AAA diameter and volume variations at 1-year independently of confounding factors (p ≤ 0.044). Additionally, surgery incidence, recorded during a 4-year follow-up in group 2, was associated with larger aneurysms, assessed by aortic diameter and volumetry (p ≤ 0.036), and with elevated TAT levels (sub-hazard ratio 1.3, p ≤ 0.029), while no association was found for D-dimer. The combination of hemostatic parameters and image techniques might provide valuable tools to evaluate AAA growth and worse evolution.

Technique for planning and implantation of a branched endograft as a proximal extension of a previous fenestrated endovascular aneurysm repair.

Our patient had undergone a previous three-fenestration Anaconda (Terumo Medical Corp, Tokyo, Japan) fenestrated endovascular aneurysm repair (EVAR) to treat a juxtarenal aortic aneurysm. At 10 years postoperatively, distal migration of the prosthesis, a proximal type I endoleak, and aortic sac enlargement of 10 mm in 6 months was observed. Because of the short length of the Anaconda's bifurcated body, we chose to use a Zenith custom-made endograft with four branches and a bifurcated body with an inverted contralateral limb. We have also described the issues that can arise during branched EVAR after fenestrated EVAR and some of the bailout techniques we performed to successfully perform the treatment.

Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD).

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.

Meta-analysis of randomized, controlled clinical trials in angiogenesis: gene and cell therapy in peripheral arterial disease.

We aim to determine the efficacy and safety of gene and cell angiogenic therapies in the treatment of peripheral arterial disease (PAD) and evaluate them for the first time by a meta-analysis. We include in the formal meta-analysis only the randomized placebo-controlled phase 2 studies with any angiogenic gene or cell therapy modality to treat patients with PAD (intermittent claudication, ulcer or critical ischemia) identified by electronic search in MEDLINE and EMBASE databases (1980 to date). Altogether, 543 patients are analyzed from six randomized, controlled trials that are comparable with regard to patient selection, study design, and endpoints. We perform the meta-analysis regarding clinical improvement (improvement of peak walk time, relief in rest pain, ulcer healing or limb salvage) and rate of adverse events. At the end of treatment, therapeutic angiogenesis shows a significantly clinical improvement as compared to placebo in patients with PAD (odds ratio [OR] = 1.437; 95% confidence interval [CI] = 1.03-2.00; P = 0.033). The response rate (improvement of peak walk time) of the pooled groups according to clinical severity does not significantly differ for gene therapy as compared with placebo in the treatment of claudicating patients (OR = 1.304; 95% CI = 0.90-1.89; P = 0.16). Otherwise, we find significant efficacy of the treatment in critical ischemia (OR = 2.20; 95% CI = 1.01-4.79; P = 0.046). The adverse events rates show a slightly significantly higher risk of potential nonserious adverse events (edema, hypotension, proteinuria) in the treated group (OR = 1.81; 95% CI = 1.01-3.38; P = 0.045). We find no differences in mortality from any cause, malignancy, or retinopathy. The patients with PAD, and particularly those with critical ischemia, improve their symptoms when treated with angiogenic gene and cell therapy with acceptable tolerability.

Trimethylamine-N-Oxide (TMAO) Predicts Cardiovascular Mortality in Peripheral Artery Disease.

Peripheral artery disease (PAD) is a major cause of acute and chronic illness, with extremely poor prognosis that remains underdiagnosed and undertreated. Trimethylamine-N-Oxide (TMAO), a gut derived metabolite, has been associated with atherosclerotic burden. We determined plasma levels of TMAO by mass spectrometry and evaluated their association with PAD severity and prognosis. 262 symptomatic PAD patients (mean age 70 years, 87% men) categorized in intermittent claudication (IC, n = 147) and critical limb ischemia (CLI, n = 115) were followed-up for a mean average of 4 years (min 1-max 102 months). TMAO levels were increased in CLI compared to IC (P < 0.001). Receiver operating characteristic (ROC) curves for severity (CLI) rendered a cutoff of 2.26 µmol/L for TMAO (62% sensitivity, 76% specificity). Patients with TMAO > 2.26 µmol/L exhibited higher risk of cardiovascular death (sub-hazard ratios ≥2, P < 0.05) that remained significant after adjustment for confounding factors. TMAO levels were associated to disease severity and CV-mortality in our cohort, suggesting an improvement of PAD prognosis with the measurement of TMAO. Overall, our results indicate that the intestinal bacterial function, together with the activity of key hepatic enzymes for TMA oxidation (FMO3) and renal function, should be considered when designing therapeutic strategies to control gut-derived metabolites in vascular patients.

Endovascular Treatment of Aortic Arch Lesions Using Scalloped Endografts.

OBJECTIVE: To present our early and midterm results using thoracic endovascular aortic repair (TEVAR) with a custom-made proximal scalloped stent graft to accommodate left common carotid artery (LCCA) and innominate artery (IA) in treating aortic lesions involving the arch. MATERIALS AND METHODS: Between February 2014 and April 2017, select patients presenting with aortic arch lesions and short proximal landing zone were treated by proximal scalloped Relay Plus stent grafts. Patient demographics, operative details, clinical outcomes, and complications were analyzed. RESULTS: Six patients (50% male) with a median age of 71 years (range, 60-82) underwent scalloped TEVAR using thoracic custom-made Relay Plus stent graft to preserve flow in the proximal supra-aortic trunks. Target vessels for the scallop were LCCA in 5 cases and IA in 1 case. The technical success rate was 100%, and proximal seal was achieved in all cases with no type I endoleaks on completion angiography. The median follow-up period was 20 (7-32) months. No conversion to open surgical repair and no aortic rupture occurred. One patient had a distal type I endoleak on the 6-month computed tomography (CT) scan, and 1 patient had a proximal type I endoleak on the 12-month CT scan. There was no stroke, paraplegia, retrograde type A dissection, or other aortic-related complication. We routinely used temporary rapid right ventricular pacing to obtain a near-zero blood pressure level during the graft deployment. No complications were observed related to the use of rapid pacing. CONCLUSION: When anatomy allows, proximal scalloped stent graft to accommodate LCCA and IA is a viable therapeutic option in treating aortic lesions involving the arch with short proximal landing zones. In addition, these findings represent a strong argument for the use of temporary rapid pacing during graft deployment.

Nitric oxide: link between endothelial dysfunction and inflammation in patients with peripheral arterial disease of the lower limbs.

OBJECTIVES: To analyse the role of nitric oxide (NO) in peripheral arterial disease (PAD) and its association with inflammation and brachial artery flow-mediated dilation (BAFMD) as an estimation of endothelial dysfunction. MATERIAL AND METHODS: Cross-sectional study of 82 patients with ischaemia (50 with Fontaine stage II and 32 with Fontaine stage III-IV) in whom BAFMD, hsCRP and nitrite levels in plasma were determined by colorimetric assay using the Griess reaction. They were compared with a control group of healthy subjects (n=41) with ABI >0.9, under 30 years of age. RESULTS: No significant differences were found between the different stages of ischaemia in relation to risk factors or concomitant treatments. The patients with PAD had significantly higher NO levels in plasma than the control group (23.92+/-23.27 microM vs. 12.77+/-11.12 microM, P=0.001). However, no statistically significant differences were observed in the NO levels between the two groups of patients with PAD (25.24+/-24.47 microM vs. 21.86+/-19.86 microM, P=0.38). Neither were differences found between the two in BAFMD (4.7+/-4.2 vs. 4.3+/-2.8, P=0.1). The hsCRP values were statistically higher in PAD stage III-IV (8.2+/-13.5 vs. 29.2+/-33.2, P=0.0001). CONCLUSIONS: The presence of elevated NO values in PAD, in conjunction with elevated CRP levels, reinforces the theory that atherosclerosis has an inflammatory nature. Its lack of correlation with the clinical severity, also occurring in BAFMD, lends weight to the hypothesis that endothelial dysfunction is an event which takes place in the first stages of the disease.

[Repair of secondary aortoenteric fistulas. A systematic review]. / Reparación de las fístulas aortoentéricas secundarias. Revisión sistemática.

We performed a systematic review of the literature on the diagnosis and treatment of secondary aortoenteric fistulas (AEF). A MEDLINE search was performed of articles published in English or Spanish between January 1991 and August 2006. Diagnostic methods, treatment modalities and the results of surgical treatment were analyzed. The most frequent first aortic surgery associated with AEF was repair of abdominal aortic aneurysm (54.31%). The most common form of presentation was gastrointestinal bleeding. Repair through in situ prosthetic replacement had the lowest early mortality rates (8-13.3%) compared with graft excision and extraanatomic revascularization (18.2-44%). AEF is a serious entity and diagnosis requires a high index of suspicion based on clinical findings and indirect data from imaging techniques (computed tomography). The most appropriate therapeutic option continues to be controversial.