The study of calcified atherosclerotic arteries: an alternative to evaluate the composition of a problematic tissue reveals new insight including metakaryotic cells

Background Calcifications of atherosclerotic plaques represent a controversial issue as they either lead to the stabilization or rupture of the lesion. However, the cellular key players involved in the progression of the calcified plaques have not yet been described. The primary reason for this lacuna is that decalcification procedures impair protein and nucleic acids contained in the calcified tissue. The aim of our study was to preserve the cellular content of heavily calcified plaques with a new rapid fixation in order to simplify the study of calcifications. Methods Here we applied a fixation method for fresh calcified tissue using the Carnoy’s solution followed by an enzymatic tissue digestion with type II collagenase. Immunohistochemistry was performed to verify the preservation of nuclear and cytoplasmic antigens. DNA content and RNA preservation was evaluated respectively with Feulgen staining and RT-PCR. A checklist of steps for successful image analysis was provided. To present the basic features of the F-DNA analysis we used descriptive statistics, skewness and kurtosis. Differences in DNA content were analysed with Kruskal-Wallis and Dunn’s post tests. The value of P < 0.05 was considered significant. Results Twenty-four vascular adult tissues, sorted as calcified (14) or uncalcified (10), were processed and 17 fetal tissues were used as controls (9 soft and 8 hard). Cells composing the calcified carotid plaques were positive to Desmin, Vimentin, Osteocalcin or Ki-67; the cellular population included smooth muscle cells, osteoblasts and osteoclasts-like cells and metakaryotic cells. The DNA content of each cell type found in the calcified carotid artery was successfully quantified in 7 selected samples. Notably the protocol revealed that DNA content in osteoblasts in fetal control tissues exhibits about half (3.0 ng) of the normal nuclear DNA content (6.0 ng). Conclusion Together with standard histology, this technique could give additional information on the cellular content of calcified plaques and help clarify the calcification process during atherosclerosis.United Therapeutics Corporatio

The Influence of Medical Therapies on Metalloproteinase-9 Degenerative Activity in Abdominal Aortic aneurysm

Introduzione.Lo sviluppo degli aneurismi dell’aorta addominale (AAA) è da associarsi prevalentemente ad un’eccessiva proteolisi della matrice extracellulare (ECM), mediata da metallo-proteinasi (MMPs). Differenti farmaci hanno mostrato un’attività modulatoria rispetto alla produzione delle MMP-9, principalmente coinvolta nello sviluppo degli AAA, che potenzialmente possono influenzare la crescita dell’aneurisma stesso. Lo scopo dello studio è stato valutare influenza di differenti attività farmacologiche sulla produzione di MMP nei tessuti aortici di aneurismatici. Metodi. Sono stati prelevati segmenti di parete aortica aneurismatica da pazienti portatori di AAA e sottoposti a trattamento chirurgico open. Da tali tessuti sono state ricavate le cellule staminali mesenchimali (CSM – identificate come maggiormente associate alla produzione di MMP-9) e saggiate per l’espressione genica di MMP-9 a livelli basali. Sono stati effettuate valutazioni di vitalità e espressione genica di MMP-9, mediante Real Time PCR in associazione a tre differenti farmaci: Pioglitazone, Sinvastatina e Doxiciclina, a differenti concentrazioni. Risultati. Lo studio è stato approvato dal comitato etico locale e sono stati utilizzati 12 prelievi da pazienti portatori di AAA. Da tutti i segmenti sono state ricavate CSM e saggiate mediante Real Time PCR. Nel campione basale in assenza di farmaco si è riscontrata un’iperespressione di MMP-9, 400 volte superiore rispetto ai controlli sani (P=.0001). Tutti i farmaci testati si sono associati al mantenimento della vitalità cellulare testata e hanno dimostrato una significativa riduzione dell’espressione di MM-9 (P<0,001) con valori risultanti dall’analisi della Real Time mediante il metodo comparativo del 2—ΔCt, di 0,46 per Pioglitazone (10 μM), 0,1 per Doxiciclina (25 μM) e 0,58 per Sinvastatina (10 μM) Conclusioni. L’attività farmacologica testata si è associata a una significativa riduzione dell’espressione di MMP-9 da parte delle CSM mantenendo la vitalità cellulare, questa valutazione pone le basi per un possibile studio in vivo delle differenti attività farmacologiche nel rallentamento dello sviluppo degli AAA.Introduction. The development of abdominal aortic aneurysms (AAA) is associated with excessive proteolysis activity of the extracellular matrix (ECM), mediated by metal-proteinase (MMPs). Different drugs have shown modulatory effect on the production of MMP-9, mainly involved in the development of AAA, which can potentially influence the growth of the aneurysm itself. The aim of the study was to evaluate the influence of different pharmacological activities on the production of MMP in aortic aneurysmal tissues. Methods. Aneurysmal aortic wall segments were taken from AAA patients undergoing open surgical treatment. Mesenchymal stem cells (CSMs - associated with the production of MMP-9) derived from aortic wall were assayed for MMP-9 gene expression at baseline levels. Assessments of viability and gene expression of MMP-9 were than performed, using Real Time PCR in association with three different drugs: Pioglitazone, Sinvastatin and Doxycycline, at different concentrations. Results. The study was approved by the local ethics committee and 12 specimens of aortic wall from different patients were analysed. From all the segments, CSMs were obtained and tested using Real Time PCR. At the baseline sample without medication, an over-expression of MMP-9 was found, 400 times higher than in healthy controls (P = .0001). All the drugs tested were associated with the maintenance of the cellular vitality and showed a significant reduction in the expression of MM-9 (P <0.001) with values resulting from the analysis of Real Time by the comparative method of 2-ΔCt, of 0.46 for Pioglitazone (10 μM), 0.1 for Doxycycline (25 μM) and 0.58 for Sinvastatin (10 μM). Conclusions. The pharmacological activity tested was associated with a significant reduction of MMP-9 expression by the CSMs while maintaining the cell viability, this assessment let speculate a possible in vivo study of the different pharmacological activities in the slowing down of the AAA development

Revascularisation of Chronic Limb Threatening Ischaemia in Patients with no Pedal Arteries Leads to Lower Midterm Limb Salvage

Objective: Chronic limb threatening ischaemia (CLTI) involving the infragenicular arteries is treated by distal angioplasty or pedal bypass; however, this is not always possible, due to chronically occluded pedal arteries (no patent pedal artery, N-PPA). This pattern represents a hurdle to successful revascularisation, which must be limited to the proximal arteries. The aim of the study was to analyse the outcome of patients with CLTI and N-PPA after a proximal revascularisation.Methods: All patients with CLTI submitted to revascularisation in a single centre (2019 - 2020) were analysed. All angiograms were reviewed to identify N-PPA, defined as total obstruction of all pedal arteries. Revascularisation was performed with proximal surgical, endovascular, and hybrid procedures. Early and midterm survival, wound healing, limb salvage, and patency rates were compared between N-PPA and patients with one or more patent pedal artery (PPA).Results: Two hundred and eighteen procedures were performed. One hundred and forty of 218 (64.2%) patients were male, mean age 73.2 &amp; PLUSMN; 10.6 years. The procedure was surgical in 64/218 (29.4%) cases, endovascular in 138/218 (63.3%), and hybrid in 16/218 (7.3%). N-PPA was present in 60/218 (27.5%) cases. Eleven of 60 (18.3%) cases were treated surgically, 43/60 (71.7%) by endovascular and 6/60 (10%) by hybrid procedures. Technical success was similar in the two groups (N-PPA 85% vs. PPA 82.3%, p = .42). At a mean follow up of 24.5 &amp; PLUSMN; 10.2 months, survival (N-PPA 93.7 &amp; PLUSMN; 3.5% vs. PPA 95.3 &amp; PLUSMN; 2.1%, p = .22) and primary patency (N-PPA 53.1 &amp; PLUSMN; 8.1% vs. PPA 55.2 &amp; PLUSMN; 5%, p = .56) were similar. Limb salvage was significantly lower in N-PPA patients (N-PPA 71.4 &amp; PLUSMN; 6.6% vs. PPA 81.5 &amp; PLUSMN; 3.4%, p = .042); N-PPA was an independent predictor of major amputation (hazard ratio [HR] 2.02, 1.07 - 3.82, p = .038) together with age &gt; 73 years (HR 2.32, 1.17 4.57, p = .012) and haemodialysis (2.84, 1.48 - 5.43, p = .002).Conclusion: N-PPA is not uncommon in patients with CLTI. This condition does not hamper technical success, primary patency, and midterm survival; however, midterm limb salvage is significantly lower than in patients with PPA. This should be considered in the decision making process

Urgent endovascular maneuvers to rescue a failing transplant kidney with a T-stent approach

Renal artery thrombosis (RAT) is a major cause of renal transplant loss and, for this reason, should be treated promptly. We present a case of a 48-year-old man with external iliac thrombosis associated with thrombosis of a transplant renal artery that led to worsening of renal function. Multiple mechanisms have been identified in the literature as risk factors for RAT. In our patient, a combination of anastomotic stenosis, hypercoagulability, and diabetic nephropathy had resulted in RAT, and an unconventional endovascular revascularization technique with a T-stent approach was needed to guarantee patency of the treated vessels. No 30-day perioperative complications occurred, and the postoperative follow-up examination showed patency of the treated vessels; thus, transplant loss was avoided. (J Vasc Surg Cases Innov Tech 202

The Outcome of Technical Intraoperative Complications Occurring in Standard Aortic Endovascular Repair

Background Technical intraoperative complications (TICs) may occur during standard endovascular repair (EVAR) with possible effects on the outcome. This study evaluates the early and midterm effects of TICs on EVARs. Methods All EVARs (from 2012 to 2016) were analyzed to identify all TICs: (1) endoluminal defects (stenosis, dissection, rupture, compression of native arteries, or endograft); (2) type I-III endoleaks; (3) unplanned artery coverage; and (4) surgical access complications. Follow-up was performed by Doppler ultrasound/ontrast enhanced ultrasound/computed tomography scan at yearly intervals. The outcome was compared with that of uneventful cases (UCs) through Fisher's exact test and Kaplan-Maier curve. Results TICs occurred in 68 (18%) of 377 patients undergoing EVAR. Thirty-two endoluminal defects were relined endovascularly; 24 type I-III endoleaks were treated with cuff deployment/forced ballooning (23) and surgical conversion (1); 3 of 8 unplanned artery coverages were revascularized (2 renal and 1 hypogastric); 5 hypogastric coverages had an unsuccessful correction; and 4 access artery injuries were repaired. Although fluoroscopy time and contrast usage were significantly higher in the TIC group than those in the UC group (309 cases), 30-day outcome was similar for death (1.4% TIC vs 0% UC, P = 0.18), reintervention (0% TIC vs 0.3% UC, P = 1), type I-III endoleak (0% TIC vs 0.9% UC, P = 1), steno-occlusions (0% TIC vs 0.3% UC, P = 1), buttock claudication, and renal failure (0% in both groups). At 24 months, TIC and UC groups had similar survival (91.7 ± 8% vs 96.2 ± 2.1%, P = 0.5), freedom from reintervention (81.4 ± 9.9% vs 96 ± 2.2%, P = 0.49), overall complication rate (13.4 ± 7.6% vs 11.4 ± 3.5%, P = 0.49), type I-III endoleak (11.2 ± 7.5% vs 7 ± 2.9%, P = 0.8), buttock claudication (0% vs 2 ± 2% P = 0.6), and hemodialysis (0% in both). Midterm iliac leg occlusion was significantly higher in the TIC group (26.9 ± 12.3% vs 3 ± 2.1%, P = 0.01). Conclusion TICs may affect several aspects during EVAR, leading to the necessity of adjunctive maneuvers, which have no impact on early outcome but may cause an increased rate of midterm iliac leg occlusion

CO2 Angiography in the Standard and Complex Endovascular Repair of the Abdominal Aorta—A Narrative Review of the Literature

Background/Objectives: Carbon dioxide digital-subtraction angiography (CO2-DSA) is an increasingly adopted technique in endovascular aortic repair (EVAR) and fenestrated/branched EVAR (F/B-EVAR); it is used to reduce the amount of iodinate contrast medium (ICM) and prevent postoperative renal function worsening (PO-RFW). Our aim is to report results from the literature on EVAR and F/B-EVAR procedures using CO2-DSA, together with wider applications in aortic endovascular treatment. Methods: We performed a literature review by searching electronic databases for published data on CO2-DSA during EVAR and F/B-EVAR procedures. The endpoints were postoperative renal function worsening (PO-RFW) and efficacy of intraoperative arterial visualization. Further, applications of CO2 for thoracic endovascular aortic repair (TEVAR) were described. Results: Seventeen studies reporting results on CO2-DSA in EVAR (644 patients) were retrieved. Overall, 372 (58%) procedures were performed with CO2 alone, and 272 (42%) were performed with CO2+ICM. Eight studies analyzed the effect of CO2-DSA angiography on PO-RFW; four studies showed a significantly lower rate of PO-RFW compared to ICM. Five studies (153 patients) analyzed intraoperative arterial visualization with CO2-DSA; renal and hypogastric arteries were effectively visualized in 69% and 99% of cases, respectively. The use of CO2-DSA in F/B-EVAR has not been widely investigated. The largest series reported that PO-RFW was lower in the CO2 vs. ICM group. Conclusions: Carbon dioxide is widely applied in modern aortic endovascular treatment. CO2-DSA for EVAR and F/B-EVAR is an efficient technique for reducing PO-RFW while allowing acceptable arterial intraoperative visualization

Diffuse calcifications protect carotid plaques regardless of the amount of neoangiogenesis and related histological complications

Background. Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group showed that Nestin-positive intraplaque neovessels correlated with histological complications. The aim of the present work is to evaluate the relationship between neoangiogenesis, plaque morphology, and clinical instability of the plaque. Materials and Methods. Seventy-three patients (53 males and 20 females, mean age 71 years) were consecutively enrolled. Clinical data and 14 histological variables, including intraplaque hemorrhage and calcifications, were collected. Immunohistochemistry for CD34 and Nestin was performed. RT-PCR was performed to evaluate Nestin mRNA (including 5 healthy arteries as controls). Results. Diffusely calcified plaques (13/73) were found predominantly in females (P=0.017), with a significantly lower incidence of symptoms (TIA/stroke (P=0.019) than noncalcified plaques but with the same incidence of histological complications (P=0.156)). Accordingly, calcified and noncalcified plaques showed similar mean densities of positivity for CD34 and Nestin. Nestin density, but not CD34, correlated with the occurrence of intraplaque hemorrhage. Conclusions. Plaques with massive calcifications show the same incidence of histological complications but without influencing symptomatology, especially in female patients, and regardless of the amount of neoangiogenesis. These results can be applied in a future presurgical identification of patients at major risk of developing symptoms

Diffuse Calcifications Protect Carotid Plaques regardless of the Amount of Neoangiogenesis and Related Histological Complications

Background. Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group showed that Nestin-positive intraplaque neovessels correlated with histological complications. The aim of the present work is to evaluate the relationship between neoangiogenesis, plaque morphology, and clinical instability of the plaque. Materials and Methods. Seventy-three patients (53 males and 20 females, mean age 71 years) were consecutively enrolled. Clinical data and 14 histological variables, including intraplaque hemorrhage and calcifications, were collected. Immunohistochemistry for CD34 and Nestin was performed. RT-PCR was performed to evaluate Nestin mRNA (including 5 healthy arteries as controls). Results. Diffusely calcified plaques (13/73) were found predominantly in females (P=0.017), with a significantly lower incidence of symptoms (TIA/stroke (P=0.019) than noncalcified plaques but with the same incidence of histological complications (P=0.156)). Accordingly, calcified and noncalcified plaques showed similar mean densities of positivity for CD34 and Nestin. Nestin density, but not CD34, correlated with the occurrence of intraplaque hemorrhage. Conclusions. Plaques with massive calcifications show the same incidence of histological complications but without influencing symptomatology, especially in female patients, and regardless of the amount of neoangiogenesis. These results can be applied in a future presurgical identification of patients at major risk of developing symptoms

Circulating CXCL9, monocyte percentage, albumin, and C-reactive protein as a potential, non-invasive, molecular signature of carotid artery disease in 65+ patients with multimorbidity: a pilot study in Age.It

Background: Carotid endarterectomy (CEA) for the prevention of upcoming vascular and cerebral events is necessary in patients with high-grade stenosis (≥70%). In the framework of the Italian National project Age.It, a pilot study was proposed aiming at the discovery of a molecular signature with predictive potential of carotid stenosis comparing 65+ asymptomatic and symptomatic inpatients. Methods: A total of 42 inpatients have been enrolled, including 26 men and 16 women, with a mean age of 74 ± 6 years. Sixteen symptomatic and 26 asymptomatic inpatients with ≥70% carotid stenosis underwent CEA, according to the recommendations of the European Society for Vascular Surgery and the Society for Vascular Surgeons. Plaque biopsies and peripheral blood samples from the same individuals were obtained. Hematobiochemical analyses were conducted on all inpatients, and plasma cytokines/molecules, such as microRNAs (miRs), IL-6, sIL-6Ralpha, sgp130, myostatin (GDF8), follistatin, activin A, CXCL9, FGF21, and fibronectin, were measured using the ELISA standard technique. MiR profiles were obtained in the discovery phase including four symptomatic and four asymptomatic inpatients (both plasma and plaque samples), testing 734 miRs. MiRs emerging from the profiling comparison were validated through RT-qPCR analysis in the total cohort. Results and conclusion: The two groups of inpatients differ in the expression levels of blood c-miRs-126-5p and -1271-5p (but not in their plaques), which are more expressed in symptomatic subjects. Three cytokines were significant between the two groups: IL-6, GDF8, and CXCL9. Using receiver operating characteristic (ROC) analysis with a machine learning-based approach, the most significant blood molecular signature encompasses albumin, C-reactive protein (CRP), the percentage of monocytes, and CXCL9, allowing for the distinction of the two groups (AUC = 0.83, 95% c.i. [0.85, 0.81], p = 0.0028). The potential of the molecular signature will be tested in a second cohort of monitored patients, allowing the application of a predictive model and the final evaluation of cost/benefit for an assessable screening test