Michele Marconi

Secreted proteins from carotid endarterectomy: an untargeted approach to disclose molecular clues of plaque progression

BackgroundAtherosclerosis is the main cause of morbidity and mortality in Western countries and carotid plaque rupture is associated to acute events and responsible of 15-20% of all ischemic strokes. Several proteomics approaches have been up to now used to elucidate the molecular mechanisms involved in plaque formation as well as to identify markers of pathology severity for early diagnosis or target of therapy. The aim of this study was to characterize the plaque secretome. The advantage of this approach is that secretome mimics the in vivo condition and implies a reduced complexity compared to the whole tissue proteomics allowing the detection of under-represented potential biomarkers.MethodsSecretomes from carotid endarterectomy specimens of 14 patients were analyzed by a liquid chromatography approach coupled with label free mass spectrometry. Differential expression of proteins released from plaques and from their downstream distal side segments were evaluated in each specimen. Results were validated by Western blot analysis and ELISA assays. Histology and immunohistochemistry were performed to characterize plaques and to localise the molecular factors highlighted by proteomics.ResultsA total of 463 proteins were identified and 31 proteins resulted differentially secreted from plaques and corresponding downstream segments. A clear-cut distinction in the distribution of cellular- and extracellular-derived proteins, evidently related to the higher cellularity of distal side segments, was observed along the longitudinal axis of carotid endarterectomy samples. The expressions of thrombospondin-1, vitamin D binding protein, and vinculin, as examples of extracellular and intracellular proteins, were immunohistologically compared between adjacent segments and validated by antibody assays. ELISA assays of plasma samples from 34 patients and 10 healthy volunteers confirmed a significantly higher concentration of thrombospondin-1 and vitamin D binding protein in atherosclerotic subjects.ConclusionsTaking advantage of the optimized workflow, a detailed protein profile related to carotid plaque secretome has been produced which may assist and improve biomarker discovery of molecular factors in blood. Distinctive signatures of proteins secreted by adjacent segments of carotid plaques were evidenced and they may help discriminating markers of plaque complication from those of plaque growth.

b-Gamma-glutamyltransferase activity in human vulnerable carotid plaques

OBJECTIVE The atherosclerotic plaque that is vulnerable to rupture and to superimposed thrombosis is mainly represented by a thin-cap fibroatheroma with or without ulceration/thrombosis and inflammatory infiltrates. Total serum gamma-glutamyltransferase (GGT) activity is an independent predictor for cardiovascular events. Four GGT fractions have been identified in plasma and only one of them (b-GGT) in atherosclerotic plaques, but the possible role of GGT in plaque pathophysiology has not been assessed yet. We investigated the relationships between plaque b-GGT activity and the histological features of plaque vulnerability. METHODS AND RESULTS Plaque GGT activity was investigated in 65 patients undergoing carotid endarterectomy; plaques were histologically characterized and immunostained for GGT. Intra-plaque total and fractional GGT activity was determined by a cost-effective test of molecular size exclusion chromatography, and compared with histological markers of plaque vulnerability. Plaque cholesterol content was also measured by chromatography. b-GGT was the only fraction detected within the atherosclerotic plaques and intra-plaque b-GGT activity correlated to plaque cholesterol content (r = 0.667, P < 0.0001), plasma b-GGT and f-GGT fractions (r = 0.249; r = 0.298, both P < 0.05). Higher b-GGT activity was found in thin-cap fibroatheromas and it was associated to histological markers of vulnerable plaques, i.e., larger necrotic areas, greater macrophage infiltration and higher cholesterol content (P < 0.05). CONCLUSIONS intra-plaque b-GGT activity correlates with the histological markers of vulnerable plaque and with plasma b-GGT in human carotid atherosclerosis; these data support the possible role of b-GGT in clinically significant atherosclerotic disease.

Preliminary results of endovascular aneurysm sealing from the multicenter Italian Research on Nellix Endoprosthesis (IRENE) study

Objective: Because of advances in technology and experience of the operator, endovascular aneurysm repair (EVAR) has supplanted open repair to treat abdominal aortic aneurysm (AAA). The low 30‐day mortality and morbidity of EVAR make the endovascular approach particularly suitable for patients at high surgical risk. However, endoleak or endograft migration requiring secondary intervention or open surgical conversion is a limitation of EVAR. The Nellix system (Endologix, Inc, Irvine, Calif) has been designed to seal the entire AAA to overcome these limitations with EVAR. We report the results of a retrospective, multicenter study with endovascular aneurysm sealing (EVAS) aimed to assess technical success, procedure‐related mortality, complications, and reinterventions. Methods: This study included patients selected for elective treatment with the Nellix device per the endovascular repair protocol at 16 Italian vascular centers. All patients were enrolled in a postoperative surveillance imaging program including duplex ultrasound investigations, computed tomography, and magnetic resonance controls following local standards of care. Results: From 2013 to 2015, there were 335 patients (age, 75.5 ± 7.4 years; 316 men) who underwent elective EVAS. In 295 cases (88.0%), EVAS was performed under standard instructions for use of the Nellix system. Preoperative aneurysm diameter was 55.5 ± 9.4 mm (range, 46‐65 mm). The inferior mesenteric artery and lumbar arteries emerging from the AAA were patent in 61.8% and 81.3% of cases, respectively. Chimney grafts were electively carried out in eight cases (2.4%). One (0.3%) intraprocedural type IB endoleak was observed and promptly corrected. Device deployment was successful in all patients, with no perioperative mortality. Early (≤30 days) complications included 1 (0.3%) type IA endoleak, 2 (0.6%) type II endoleaks (0.6%), 2 (0.6%) stent occlusions (0.6%), 3 (0.9%) distal embolizations, and 2 (0.2%) femoral artery dissections. Six (2.9%) patients underwent reinterventions. At 1‐year follow‐up, complications included 3 (1.1%) type II endoleaks, 4 (1.4%) type IA endoleaks, 1 (0.3%) type IB endoleak, 2 (0.7%) distal stent migrations, 5 (1.8%) distal embolizations, and 1 (0.3%) stent occlusion. Twelve patients (3.7%) underwent reinterventions, including four (1.4%) surgical conversions due to aortoduodenal fistula (1), endograft infection (1), and type IA endoleak that was unsuccessfully treated percutaneously (2). Two AAA‐related deaths occurred. Freedom from aneurysm‐related reintervention was 98.3% at 1‐month and 94.7% at 12‐month follow‐up. Conclusions: The preliminary results of this real‐world multicenter study showed that EVAS with Nellix for the management of AAAs appears feasible. This device platform is associated with acceptable procedure‐related mortality and low overall complication and reintervention rates. Definitive conclusions on the value of this novel device await long‐term follow‐up data.

Basic Endovascular Skills Trainer: A surgical simulator for the training of novice practitioners of endovascular procedures.

In recent years the clinical interest for structured training in endovascular procedures has increased. Such procedures respect the physical integrity of the patient and at the same time ensure good therapeutic results. This study describes the development and testing of the B.E.S.T. (Basic Endovascular Skills Trainer) simulator. The B.E.S.T is an innovative physical endovascular simulator to learn basic skills of endovascular surgery. The simulator was tested by 25 clinicians with different levels of experience: novices, intermediates, and experts. All clinicians agree on affirming the importance of training in endovascular surgery; in particular they consider the B.E.S.T a valid simulator to learn specific basic skills of vascular surgery.

Extracellular matrix characterization in plaques from carotid endarterectomy by a proteomics approach

Extracellular matrix (ECM) is a fundamental component of multicellular organisms. Alteration of its structure and/or molecular composition are associated with several pathologies, among the others with atherosclerosis. To determine how the overall ECM architecture of a tissue as complex as the atheroma may change under varying pathological conditions constitutes a great technical challenge. This entails removing cell components and solubilisation of fibrillar proteins in order to allow enzymatic digestion and mass spectrometry analyses. This work aimed at testing and assessing an easy to use, standardized and reproducible proteomics protocol to map ECM proteins in carotid plaque specimens. To this end, plaques from endarterectomies were incubated in different buffers and the resulting solutions and tissue homogenates after incubation were processed for mass spectrometry. Comparison of the different workflows showed that 4M Guanidine treatment (following 0.5M NaCl and 0.08% SDS incubations) is the most promising in terms of ECM enrichment. The protocol can also be used to identify different and complementary classes of proteins both in plaque extracts and homogenates.

A tele-ultrasonographic platform to collect specialist second opinion in less specialized hospitals

In non-urban scenarios: rural areas or small cities, there is often a limited access to specialistic healthcare due to the inherent challenges associated with recruitment, retention, and access to healthcare professionals. Telemedicine is an economical and effective way to address this problem. In this research, we developed a framework for real-time communication during ultrasound examination that combines interaction via standard video conference protocols and basic AR functionalities (commercial) and a custom-developed application. The tele-ultrasonographic platform has been installed in a rural hospital in the Tuscan Apennines, and was tested on 12 patients. The study explores the utility of the system from the local and remote clinician perspectives. The results obtained provide valuable insight: the platform and the telemedicine paradigm can reduce the costs related to the necessity to move critical patients when there is a need for a specialist second opinion. Moreover, the possibility of having an expert guiding and commenting on the fly the diagnostic examination has also a didactic power, and thus allows the local less specialized clinicians to grow in competencies over time.

Face, content, and construct validity of a simulator for training in endovascular procedures.

Abstract Aim: In recent years the interest in structured training programs in endovascular surgical procedures has increased. In this study we assess face, content, and construct validity of a simulator to teach basic skills of endovascular surgery. Material and methods: A cohort of 21 medical students, 26 residents, and 14 expert surgeons participated in the study. Experts assessed face and content validity. Then, they executed four tasks once, while medical students and residents were allowed two attempts to reach a five-minute threshold under expert supervision. Medical students and residents repeated the same exercises during a second session plus three new additional ones, without expert supervision. Results: The simulator was rated as good by experts (four out of five on a Likert scale) in terms of realism (face validity) and usefulness as training tool for the training of basic skills (content). For construct validity, experts outperformed with a statistically significant difference (p < .05) medical students and residents in all tasks, except cannulation of upper mesenteric (p = .053). Differences between novices and intermediates persisted in the second session in the same four tasks and in the three additional ones, with statistically significant difference (p < .05) in the last four exercises. Conclusions: This study showed face, content, and construct validity of BEST simulator.

Colonic Ischemia after Standard Endovascular Abdominal Aortic Aneurysm Repair, a Rare but Dangerous Complication

Colonic ischemia (CI) after abdominal aortic aneurysm repair, although rare, is associated with severe prognosis. Endovascular aneurysm repair (EVAR) is becoming the standard of practice in most vascular centers, and it also may reduce CI incidence in comparison with conventional open repair. We report 2 cases of fatal CI after 636 standard EVAR procedures performed at our institution, from January 1998 to December 2017. Both patients were electively treated by highly skilled operators. In one patient, presenting early CI, EVAR procedure was complicated by intraoperative common iliac artery rupture. The other one, presenting CI in seventh postoperative day, had a history of previous left hemicolectomy. In both patients, CI with leakage of fecal material in the abdominal cavity was confirmed by surgical exploration. Only few cases of CI after EVAR have been reported in literature, and the etiology of this complication remains uncertain. While saving the inferior mesenteric artery is almost impossible during standard EVAR, the preservation of hypogastric arteries could play an important role, especially after colonic surgery, but other factors should also be considered. Our preliminary, although limited experience, seems to suggest that in CI developing, intraoperative persistent hypotension and hypogastric branches distal embolization have both a role that should be better addressed.

Prophylactic Use of Silver-acetate-coated Polyester Dacron Graft in Aortic Disease for Prevention of Graft Infection

From January 2000 to December 2009, in the Division of Vascular Surgery of the hospitals of Pisa and Naples (Monaldi Hospital), InterGard silver (InterVascular, La Ciotat, France) grafts were implanted in 1,424 patients (93.6% male and 6.4% female) for the treatment of aorto-iliac diseases. The mean age of patients was 71.4 ± 7.4 years. Some patient characteristics were considered as increased risk factors for graft infection; these include diabetes, age >80 years, mycotic aneurysms, and ruptured aneurysms. Hyperleukocytosis, fever >38°C, and wound infections were also recorded. The primary patency at 30 days was 99.7%. At 3, 5, and 10 years it was 95.6 ± 2.2%, 94.3 ± 2.3%, and 93.8% ± 2.7%, respectively. Secondary patency at 10 years was 98.6 ± 1.7%. During hospital stay, 41 patients experienced leukocytosis and 32 had peaks of hyperpyrexia >38%C. Early postoperative complications requiring reintervention occurred in 56 (4%) patients. In the perioperative period (30 days from surgery), 49 wound infections were recorded. Among all 1,424 silver grafts, 5 (0.35%) cases of graft infection have been found: three in the first four months (early infections: 0.21 %) and two later (late infections: 0.14%). Clinical findings at onset of graft infection have been: one case of graft-enteric fistula (0.07%), treated by total graft excision, proximal aortic stump closure, and axillobifemoral bypass; two others cases (0.14%) presented with severe bleeding due to anastomotic disruption that led to patient death. These data, compared to the numbers published in the literature, seem to support the hypothesis that extensive use of silver-acetate-coated Dacron graft could reduce Dacron graft infection, impoving both the immediate and long-term outcome in patients. Nevertheless, accurate selection of patients for elective aorto-iliac surgery, accurate operative technique, and meticulous respect for any perioperative prophylactic measures, are of paramount importance for the prevention of early and late graft infection.