Aortic Explants Research Articles

Referral Centre Experience with Infected Abdominal Aortic Endograft Explantation

The increasing use of endovascular aneurysm repair (EVAR) appears to be associated with the burden of vascular endograft infections. Complete stent graft explantation is recommended but leads to significant mortality. This study aimed to assess the technical challenges, complications, and mortality rate following infected endograft explantation. Patients who underwent abdominal aortic endograft explantation for infection at the Bordeaux University Hospital from July 2008 to December 2020 were included retrospectively in this single centre observational study. The diagnosis was established based on the MAGIC criteria. The primary endpoint was 30 day mortality. Secondary endpoints were 90 day and in hospital mortality, survival, and re-infection. Thirty-four patients were included, median age 69 years (interquartile range [IQR] 65, 76), with four (12%) treated as an emergency. The median time from EVAR to explantation was 17.5 months (4.5 - 36.3). In situ reconstruction was carried out with prosthetic grafts in 24 patients (71%, including 23 antimicrobial grafts combining silver and triclosan), and biological grafts in 10 (five femoral veins, four arterial allografts, three bovine patches, one biosynthetic graft). Seventeen aorto-enteric fistulae (AEnF) were addressed with direct repair of the intestinal tract (n= 10/17; 59%) or resection and anastomosis (n= 7/17; 41%). The culture was polymicrobial in 12 patients (35%) and remained sterile in four (12%). The 30 day and in hospital mortality rates were 21% (n= 7) and 27% (n= 9). Twenty-five patients (73%) presented with early post-operative complications, requiring 16 revision procedures (47%). Over a median follow up of 16.2 months (IQR 8.3, 33.6), the mortality rate was 35% (n= 12; 11 aortic related; 32%), with two re-infections (6%), both after biological reconstruction (one for an AEnF). Early morbidity and mortality remain high after complete infected endograft explantation, even in a high volume centre. Comparison with other treatment modalities in large multicentre cohorts might be of interest.

Extrapleural approach for thoracoabdominal infected aortic endograft: surgical and circulatory strategies.

Abstract Video description: A 34-year-old male patient presented at the emergency department with fever, dysphagia and thoracic pain. His previous medical history consisted of caustic ingestion 18 years before, resulting in oesophageal-aorta fistula treated by aortic endograft placement and oesophageal exclusion with retrosternal colonic interposition. Diagnosis of thoracic stent graft infection was made based on clinical, microbiological and radiological criteria. After multidisciplinary consultation which involved thoracic surgery unit, the patient was considered eligible for aortic endoprosthesis explantation. The video shows the surgical intervention of removal of the aortic endograft and aortic reconstruction with homograft conduit by extrapleural approach in thoraco-phreno-laparotomy.

Nonwoven spidroin materials as scaffolds for ex vivo cultivation of aortic fragments and dorsal root ganglia

Recombinant spidroins (RS; the analogues of silk proteins of spider’s web) have multiple properties beneficial for bioengineering, including their suitability for electrospinning and thus, for production of materials with oriented fibers. This makes RS-based matrices potentially effective in stimulating regeneration of peripheral nerves. The restoration of injured nerves also depends on prompt regrowth of blood vessels. Therefore, prospective scaffold materials for neuro-regenerative therapy should positively affect both the nerves and the blood vessels. Currently, the experimental models suitable for culturing and quantitative assessment of the vascular and neuronal cells on the same material are lacking. Here, we assessed the suitability of electrospun RS-based matrices for cultivation of the mouse aorta and dorsal root ganglia (DRG) explants. We also quantified the effects of matrix topography upon both types of tissues. The RS-based materials have effectively supported aortic explants survival and sprouting. The cumulative length of endothelial sprouts on rS1/9-coated inserts was significantly higher as compared to type I collagen coatings, suggesting stimulatory effects on angiogenesis in vitro. In contrast to matrices with random fibers, on matrices with parallel fibers the migration of both smooth muscle and endothelial cells was highly oriented. Furthermore, alignment of RS fibers effectively directs the growth of axons and the migration of Schwann cells from DRGs. Thus, the electrospun RS matrices are highly suitable to culture both, the DRGs and aortic explants and to study the effects of matrix topography on cell migration. This model has a high potential for further endeavor into interactions of nerve and vascular cells and tissues.

Impairment in endothelial bioenergetics contributes to diabetes‐induced vascular dysfunction

Diabetes, whether insulin‐dependent or independent is a major cause of endothelial dysfunction and a leading risk factor for cardiovascular disease. While compelling evidence indicates that endothelial dysfunction is a precursor and contributor to cardiovascular disease, the etiology of diabetes‐induced endothelial dysfunction remains ill‐defined. Recently alteration in endothelial cell bioenergetics has emerged as a new contributor to vascular diseases. However, whether alterations in endothelial glycolysis, the main endothelial cell bioenergetic pathway contributes to endothelial dysfunction is unknown. Herein, we tested the hypothesis that diabetes‐associated endothelial dysfunction involved increases in endothelial cells glycolysis and upregulation of its primary regulator, 6‐phosphofructo‐2‐kinase/fructose‐2, 6‐bisphosphatase 3 (PFKFB3). Measurement of glycolytic activity via Seahorse analyzer in primary human aortic endothelial cells (HAEC) from control and diabetic patients revealed a significant increase in basal glycolysis in diabetic subjects (p<0.05). We also employed Akita mice, a mouse model of type 1 diabetes. Male Akita mice showed a diabetic phenotype with 3‐folds increase in blood glucose. Seahorse analysis in intact aortic explants revealed a 1.7‐fold increase in basal glycolysis and a 1.3‐fold increase in maximal glycolysis in Akita mice (P<0.05). The increase in glycolysis was endothelial‐derived as aortic denudation significantly decreased glycolysis in Akita mice and abolished the difference between groups. No significant difference in mitochondrial function was depicted in the mito‐stress test between Akita and WT aortic explants (p>0.05). Additionally, endothelial cells extracted from aortas of Akita mice exhibited a 2‐fold increase in PFKFB3 mRNA expression(P<0.05). Interestingly, inhibition of PFKFB3 using 3PO (20μM) restored endothelial function in Akita mice. Similarly, Non‐obese diabetic females (NOD), a model for type‐1 diabetes, showed 4‐fold increases in PFKFB3 mRNA expression in aortic endothelial cells and impaired EDR which was restored with 3PO (p<0.05). To further evaluate the role of PFKFB3 in vascular endothelial function we overexpressed PFKFB3 in WT aortas with adenovirus (Ad‐PFKFB3), and reported impaired aorta EDR(P<0.05). Transduction of HAEC with Ad‐PFKFB3 showed a significant increase in ROS producing enzyme (NOX1). Also, inhibition of NOX1 using the selective inhibitor GKT771 restored EDR in Akita and Ad‐PFKFB3‐transduced WT aortas. In Conclusion, our data identified for the first time a role for endothelial glycolysis in the control of vascular relaxation and showed a crucial role of PFKFB3‐mediated endothelial glycolysis in vascular endothelial dysfunction associated with Type‐1 diabetes in males and females. The underlying mechanism potentially involves NOX1 as a downstream target for PFKFB3. Thus, Inhibitors of PFKFB3 could be a potential therapeutic target for diabetes‐induced vascular endothelial dysfunction.

PDGFR-β signaling mediates HMGB1 release in mechanically stressed vascular smooth muscle cells

Mechanically stressed vascular smooth muscle cells (VSMCs) have potential roles in the development of vascular complications. However, the underlying mechanisms are unclear. Using VSMCs cultured from rat thoracic aorta explants, we investigated the effects of mechanical stretch (MS) on the cellular secretion of high mobility group box 1 (HMGB1), a major damage-associated molecular pattern that mediates vascular complications in stressed vasculature. Enzyme-linked immunosorbent assay (ELISA) demonstrated an increase in the secretion of HMGB1 in VSMCs stimulated with MS (0-3% strain, 60 cycles/min), and this secretion was markedly and time-dependently increased at 3% MS. The increased secretion of HMGB1 at 3% MS was accompanied by an increased cytosolic translocation of nuclear HMGB1; the acetylated and phosphorylated forms of this protein were significantly increased. Among various inhibitors of membrane receptors mediating mechanical signals, AG1295 (a platelet-derived growth factor receptor (PDGFR) inhibitor) attenuated MS-induced HMGB1 secretion. Inhibitors of other receptors, including epidermal growth factor, insulin-like growth factor, and fibroblast growth factor receptors, did not inhibit this secretion. Additionally, MS-induced HMGB1 secretion was markedly attenuated in PDGFR-β-deficient cells but not in cells transfected with PDGFR-α siRNA. Likewise, PDGF-DD, but not PDGF-AA, directly increased HMGB1 secretion in VSMCs, indicating a pivotal role of PDGFR-β signaling in the secretion of this protein in VSMCs. Thus, targeting PDGFR-β-mediated secretion of HMGB1 in VSMCs might be a promising therapeutic strategy for vascular complications associated with hypertension.

Aortic graft explants - A single institution analysis of incidence and outcomes.

Explantation of both endovascular endovascular aneurysm repair and open aortic grafts is a procedure associated with high peri-operative risk, and the current study sought to determine the outcomes and trends over time in these patients. This study examined data from all patients undergoing explant of an aortic graft (both open and endovascular) between January 2004 and December 2020 at a single centre. Variables analysed included comorbidities, duration to and indication for explantation, type of revascularization, in-hospital complications and mortality, duration of hospital and ICU stay, and out-patient mortality. Of 688 open and 1352 EVARs performed, 46 patients underwent 48 explants. Five were open grafts and 43 were endografts, equating to an explant rate of 0.73% of open and 3.18% EVARs. Average time to explant was 70 months, with patients presenting electively having a significantly longer duration to representation than those presenting emergently (51 vs 44 months, p=0.003). Indication for explant was endoleak in 70%, infection in 23%, and occlusion in 6%. Of the endoleaks, 61% of were Type 1, 22% Type II, 11% Type IV, and 6% Type V. On representation, 17 patients (35%) were symptomatic. Overall mortality rate was 8.3%, with a trend for higher mortality in emergent than elective presentations (11.8 vs 6.5%, p=0.55). There was no change in explant rate over time. Elective aortic graft explantation is associated with low mortality, despite its complexity and patient comorbidities. Patients presenting with symptoms suffered higher mortality and a longer post-operative course, suggesting that aortic graft explantation should be considered sooner rather than later, rather than persisting with repeated endovascular management.

Chick Embryo Ex Vivo Aortic Sprouting Assays for Cardiovascular Research.

Angiogenesis is the formation of new blood vesicles and is controlled by a dynamic cascade of molecular and cellular activities. The whole procedure can be replicated in vitro under chemically specified conditions by cultivating chick aortic explants in biomatrices. In this technique, angiogenesis is powered by endogenous molecules that the aorta releases to promote its outgrowth. In an ordered series of morphogenetic events, sprouting endothelial cells are strongly associated with macrophages, fibroblasts, and pericytes, recapitulating all the phases of the angiogenic process. The structural, morphologic, and molecular properties of the angiogenic process can be studied and the effectiveness of pro/antiangiogenic drugs can also be evaluated using this aortic culture. We describe in this chapter the basic procedure currently used in our laboratory to measure the angiogenic properties for cardiovascular research.

Aortic valve reintervention in patients with failing transcatheter aortic bioprostheses: A statewide experience

BackgroundDespite the rapid adoption of transcatheter aortic valve replacement since its approval, the frequency and outcomes of aortic valve reintervention after transcatheter aortic valve replacement are poorly understood. MethodsValve reinterventions, either surgical transcatheter aortic valve explantation or repeat transcatheter aortic valve replacement, between 2012 and 2019 were queried using the Society of Thoracic Surgeons Database and the Transcatheter Valve Therapy Registry through the Michigan Statewide quality collaborative. The reintervention frequency and clinical outcomes including observed-to-expected mortality ratio using Society of Thoracic Surgeons Predicted Risk of Mortality were reviewed. ResultsAmong 9694 transcatheter aortic valve replacement recipients, a total of 87 patients (0.90%) received a reintervention, consisting of 34 transcatheter aortic valve explants and 53 repeat transcatheter aortic valve replacement procedures. The transcatheter aortic valve explant group demonstrated a higher Society of Thoracic Surgeons Predicted Risk of Mortality. Reintervention cases increased from 0 in 2012 and 2013 to 26 in 2019. The proportion of transcatheter aortic valve explants among all reinterventions increased and was 65% in 2019. Self-expandable devices had a higher reintervention rate than balloon-expandable devices secondary to a higher transcatheter aortic valve explant frequency (0.58% [23/3957] vs 0.19% [11/5737]; P = .001), whereas repeat transcatheter aortic valve replacement rates were similar (0.61% [24/3957] vs 0.51% [29/5737]; P = .51). Among patients with transcatheter aortic valve explants, contraindications to repeat transcatheter aortic valve replacement included unfavorable anatomy (75%), need for other cardiac surgery (29%), other structural issues by transcatheter aortic valve device (18%), and endocarditis (12%). For transcatheter aortic valve explant and repeat transcatheter aortic valve replacement, the 30-day mortality was 15% and 2% (P = .032) and the observed-to-expected mortality ratio was 1.8 and 0.3 (P = .018), respectively. ConclusionsAortic valve reintervention remains rare but is increasing. The clinical impact of surgical device explantation was substantial, and the proportion of transcatheter aortic valve explants was significantly higher in patients with a self-expandable device.

5‐Fluorouracil Induces Vascular Calcification in Rat Aortic Smooth Muscle Cells

Background 5-Fluorouracil (5-FU) is a commonly prescribed chemotherapy used to treat malignancies of the gastrointestinal tract, breast, and head/neck. Despite its high clinical efficacy in treating cancer, it is associated with late occurring cardiotoxicity, specifically increases in arterial stiffness along with a heightened risk for developing heart failure and cardiovascular disease. Because increases in arterial stiffness and vessel calcification can occur concurrently and are associated with cardiovascular disease outcomes, we determined if 5-FU treated aortic smooth muscle cells adopted a calcified phenotype. We hypothesized 1) aortic smooth muscle cells resected from 5-FU treated rats would have a faster calcification onset when challenged with inorganic phosphate media and 2) a single in vitro dose of 5-FU in healthy cells would result in a calcified phenotype. Methods Primary aortic smooth muscle cell culture (ASMC) was performed via resection of rat thoracic aorta from 6-month-old Sprague Dawley rats (n= 8) that were treated with a clinically relevant continuous infusion of 5-FU (50mg/kg and 270mg/kg) or saline equivalent volume. After treatment and resection, ASMC were cultured using explant methods until cells were identifiable via smooth muscle actin staining. Cells derived from each group were cultured with normal media (NM) or media including a pathological concentration (3.2 mmol) of inorganic phosphate media (PI) for 5, 8, or 10 days to determine differences in calcification onset between conditions. In a second experiment, healthy rat aortic smooth muscle cells were incubated in NM or media containing 7x10-3 5-FU (NM + 5-FU) to determine if a single dose, in vitro, directly results in calcification. Calcification was quantified using an Alizarin Red staining assay and colorimetric calcium assay. Data are expressed as Alizarin Red Stain concentration (mM) and total calcium (mg/dL). Results In experiment 1, 5-FU+PI presented with significantly higher concentration of Alizarin Red at days 8 (.043 vs .031; p=.048) and 10 (.068 vs .017mM; p<0.0001) compared to 5-FU+NM whereas no differences were found between CON+PI and CON+NM at any timepoint (all p>0.05). These data were confirmed via Calcium assay in both 5-FU (.346 vs.235 mg/dl; p =0.0039) and CON (.293 vs .269 mg/dl; p=0.69) at the 8-day timepoint. Experiment 2 indicated that healthy rat aortic smooth muscle cells treated directly with 5-FU for 72 h expressed significantly greater calcium absorbance compared to CON (5-FU = .3347 mg/dl, CON = .2362 mg/dl; p=0.02). Conclusion After treatment with 5-FU, ASMCs from rat thoracic aorta explants are prone to a calcification phenotype compared to saline control treated cells. This predisposition with treatment with 5-FU is an ongoing investigation into the possible molecular mechanisms involved in oxidative stress signaling.

Mineralocorticoid Receptor in Myeloid Cells Mediates Angiotensin II-Induced Vascular Dysfunction in Female Mice

Enhanced mineralocorticoid receptor (MR) signaling is critical to the development of endothelial dysfunction and arterial stiffening. However, there is a lack of knowledge about the role of MR-induced adipose tissue inflammation in the genesis of vascular dysfunction in women. In this study, we hypothesize that MR activation in myeloid cells contributes to angiotensin II (Ang II)-induced aortic stiffening and endothelial dysfunction in females via increased pro-inflammatory (M1) macrophage polarization. Female mice lacking MR in myeloid cells (MyMRKO) were infused with Ang II (500 ng/kg/min) for 4 weeks. This was followed by determinations of aortic stiffness and vasomotor responses, as well as measurements of markers of inflammation and macrophage infiltration/polarization in different adipose tissue compartments. MyMRKO mice were protected against Ang II-induced aortic endothelial stiffening, as assessed via atomic force microscopy in aortic explants, and vasorelaxation dysfunction, as measured by aortic wire myography. In alignment, MyMRKO mice were protected against Ang II-induced macrophage infiltration and M1 polarization in visceral adipose tissue (VAT) and thoracic perivascular adipose tissue (tPVAT). Collectively, this study demonstrates a critical role of MR activation in myeloid cells in the pathogenesis of vascular dysfunction in females associated with pro-inflammatory macrophage polarization in VAT and tPVAT. Our data have potential clinical implications for the prevention and management of cardiovascular disease in women, who are disproportionally at higher risk for poor outcomes.

Intravascular Ultrasound-Guided Contrast-Free Transcatheter Aortic Valve Implantation: A Porcine Feasibility Study.

Patients undergoing transcatheter aortic valve implantation (TAVI) frequently present with chronic kidney disease and are therefore particularly susceptible to nephrotoxic influences like iodinated contrast media. Acute kidney injury after TAVI is a severe complication that independently predicts short- and long-term mortality. The present study investigates the feasibility of a contrast-free approach by using intravascular ultrasound (IVUS) in conjunction with fluoroscopy. Six domestic pigs (60 ± 5 kg) were anesthetized and underwent transapical implantation of a balloon-expandable transcatheter heart valve. In the control group (n = 3), the procedures were guided by fluoroscopy/angiography. In the study group (n = 3), the procedures were guided by IVUS for preimplantation evaluation, intra-procedural guidance, and post-implantation evaluation, in conjunction with fluoroscopy without contrast. The procedures were evaluated by IVUS, fluoroscopy, aortic root angiography, and explantation and dissection of the hearts. Relevant anatomical landmarks for correct implantation were assessed by IVUS. The following annulus measurements were obtained: area (359.67 ± 29.58 mm2), perimeter (68.28 ± 2.63 mm), maximum diameter (22.20 ± 1.22 mm), minimum diameter (20.43 ± 1.12 mm), mean diameter (21.32 ± 0.70 mm), ellipticity index (1.09 ± 0.10), and area-derived diameter (21.39 ± 0.87 mm). IVUS-guided valve deployment resulted in correct expansion within the aortic annulus without signs of paravalvular leak, compromised mitral valve, or coronary obstruction. IVUS-guided post-implantation assessment confirmed circular expansion (25.88 ± 0.30 mm) of the valves. IVUS-guided, contrast-free transapical TAVI is feasible in a porcine model.

A Canadian multicenter experience describing outcomes after endovascular abdominal aortic aneurysm repair stent graft explantation

BackgroundMost studies describing the outcomes after endovascular abdominal aortic aneurysm repair (EVAR) explantation have been from single, high-volume, centers. We performed a multicenter cross-Canadian study of outcomes after EVAR stent graft explantation. Our objectives were to describe the outcomes after late open conversion and EVAR graft explantation at various Canadian centers and the techniques and outcomes stratified by the indication for explant. MethodsThe Canadian Vascular Surgery Research Group performed a retrospective multicenter study of all cases of EVAR graft explantation at participating centers from 2003 to 2018. Data were collected using a standardized, secure, online platform (RedCap [Research Electronic Data Capture]). Univariate statistical analysis was used to compare the techniques and outcomes stratified the indication for graft explantation. ResultsPatient data from 111 EVAR explants collected from 13 participating centers were analyzed. The mean age at explantation was 74 years, the average aneurysm size was 7.5 cm, and 28% had had at least one instructions for use violation at EVAR. The average time between EVAR and explantation was 42.5 months. The most common indication for explantation was endoleak (n = 66; type Ia, 46; type Ib, 2; type II, 9; type III, 2; type V, 7), followed by infection in 20 patients; rupture in 18 patients (due to type Ia endoleak in 10 patients, type Ib in 1, type II in 1, type III in 2, and type V in 1), and graft thrombosis in 7 patients. The overall 30-day mortality was 11%, and 45% of the patients had experienced at least one major perioperative complication. Mortality was significantly greater for patients with rupture (33.3%) and those with infection (15%) compared with patients undergoing elective explantation for endoleak (4.5%; P = .003). The average center volume during the previous 15 years was 8 cases with a wide range (2-19 cases). A trend was seen toward greater mortality for patients treated at centers with fewer than eight cases compared with those with eight or more cases (19% vs 9%). However, the difference did not reach statistical significance (P = .23). Overall, 41% of patients had undergone at least one attempt at endovascular salvage before explantation, with the highest proportion among patients who had undergone EVAR explantation for endoleak (51%). Only 22% of patients with rupture had undergone an attempt at endovascular salvage before explantation. ConclusionsThe performance of EVAR graft explantation has increasing in Canada. Patients who had undergone elective explantation for endoleak had lower mortality than those treated for either infection or rupture. Thus, patients with an indication for explanation should be offered surgery before symptoms or rupture has occurred. A trend was seen toward greater mortality for patients treated at centers with lower volumes

OR17-06 Transglutaminase 2 Inhibition Reduces Aortic Stiffness in Western Diet-Fed Female Mice

Widespread consumption of diets high in fat, sugars and salt (Western diet, WD) is associated arterial stiffening, which is a major independent risk factor for cardiovascular disease (CVD). Notably, while WD feeding increases the risk of CVD in both males and females, the latter are more prone to develop arterial stiffening. However, the mechanisms underlying WD-induced arterial stiffening are poorly understood, particularly in females, and there are currently no specific treatments targeted at vascular stiffening.Tissue transglutaminase 2 (TG2) is an enzyme that mediates the cross-linking and stabilization of extracellular matrix proteins such as collagen, and promotes the polymerization of actin stress fibers of the cytoskeleton. It is ubiquitously expressed and abundantly present in the vasculature. Mounting evidence implicates TG2 activation in the pathogenesis of arterial stiffening and vascular fibrosis. Herein we propose that TG2 activation is central to WD-induced arterial stiffening and sought to determine the efficacy of cystamine (a non-specific competitive inhibitor of TG2) for reducing arterial stiffening in the setting of WD consumption. Accordingly, we fed 20 female mice (4 weeks old) a WD (4.65 kcal/g of food, fat 46% kcals, high-fructose corn syrup 17.5%, sucrose 17.5%, protein 17.6%, salt 1.6%) for 43 weeks. Ten of these mice received cystamine (40 mg/Kg/d in the drinking water) during their last 8 weeks on the WD. Another group of female mice (n=10) fed regular chow was used as reference controls. Aortic stiffness was measured in vivo via ultrasound-based pulse wave velocity and ex vivo by aortic explant atomic force microscopy. Vasomotor responses were assessed in isolated aortic rings via wire myography.Cystamine did not influence glucose homeostasis (intraperitoneal glucose tolerance test) or blood pressure (tail-cuff) (control 77.208±2.229 mm Hg versus WD 77.208±6.077 versus WD+Cystamine 76.297±7.894), but it was associated with increased body weight (control 26.860±2.215 grams versus WD 25.320±2.889 versus WD+Cystamine 33.220±4.848, p<0.05). Notably, cystamine reduced aortic stiffness in WD-fed mice both in vivo and ex vivo such that differences between chow-fed and WD-fed mice were normalized (control 5.294±1.713 versus WD 11.735±5.962 p≤0.05, control 5.294±1.713 versus WD+Cystamine 3.940±0.378 KPa, p<0.05). In addition, WD-induced impairments in endothelium-independent vasorelaxation (i.e. responses to sodium nitroprusside) were restored with cystamine. Collectively, our data show that cystamine reduces aortic stiffness and improves endothelium-independent vasorelaxation in female mice chronically exposed to WD, and that these effects occur despite an increase in weight gain. These findings implicate TG2 as a promising therapeutic target for reducing arterial stiffening in the context of chronic over-nutrition in females.

Optimisation of cell and ex vivo culture conditions to study vascular calcification.

Medial vascular calcification (MVC) is a highly prevalent disease associated with a high risk of severe, potentially lethal, complications. While animal studies may not systematically be circumvented, in vitro systems have been proven useful to study disease physiopathology. In the context of MVC, the absence of a clinically relevant standardized in vitro method prevents the appropriate comparison and overall interpretation of results originating from different experiments. The aim of our study is to establish in vitro models mimicking in vivo vascular calcification and to select the best methods to unravel the mechanisms involved in MVC. Human aortic smooth muscle cells and rat aortic rings were cultured in different conditions. The influence of fetal calf serum (FCS), alkaline phosphatase, phosphate and calcium concentrations in the medium were evaluated. We identified culture conditions, including the herein reported Aorta Calcifying Medium (ACM), which allowed a reproducible and specific medial calcification of aortic explants. Studying cells and aortic explants cultured, the involvement of bone morphogenetic protein 2 (BMP2) pathway, fibrosis and apoptosis processes in in vitro MVC were demonstrated. Expression of osteoblastic markers was also observed suggesting the occurrence of transdifferentiation of smooth muscle cells to osteoblasts in our models. The use of these models will help researchers in the field of vascular calcification to achieve reproducible results and allow result comparison in a more consistent way.

Experimental In Situ Laser Fenestration Creates Immediate Substantial Textile Damages

Introduction - Commercially available fenestrated and branched endografts (FBE) using reinforced nitinol ring fenestrations represent the endovascular treatment of choice for thoraco-abdominal aortic aneurysm (TAAA). In situ laser fenestration (INLF) emerges as an innovative and immediately available alternative, which could be less deleterious than mechanical in situ fenestration. However, biomechanical features of INLF are poorly known. Therefore, the aim of this study was to experimentally evaluate the textile damages secondary to INLF. Methods - In a first step, an in vitro bench study consisted in a microscopic textile analysis of textile damages following INLF with various standard endografts. We compared INLF to mechanical fenestration using an 18G needle in two clinical scenarios (with or without tension applied to the fabric, corresponding to a 90mmHg blood pressure). The subsequent hole was dilated using a non-compliant balloon followed by a cutting balloon and stabilized by a balloon-expandable covered stent. Tear and burst strength tests were performed to determine the loss of textile mechanical strength. Leak tests at the stent-fenestration junctions were implemented using a water column. In a second step, an in vivo study evaluating the feasibility and the damages secondary to INLF was performed at the level of the thoraco-abdominal aorta in sheep. Results - In total, 24 stent-graft textile samples (8 Cook, 8 Medtronic and 8 Vascutek) were analyzed in vitro. Primary fenestrated holes morphology differed between mechanical and INLF with all types of textiles, in both clinical scenarios. Following mechanical fenestration, the hole created, resulted in spacing fabric filaments whereas one circular with cauterized edges were observed after INLF. Tear and burst strength tests results, highlighted a 26±4% textile mechanical strength loss after in situ fenestration. A non-significant decrease of 7% of mechanical resistance was observed following INLF compared to mechanical fenestration (p=0.21). For each in situ fenestration technique, a substantial leak rate persisted at the stent-fenestration junction, even after ballooning. This was found both in in vitro and in vivo analyses. Regarding the in vivo study, 14 INLF were performed in 10 sheep using the Vascutek Anaconda, the Cook Zenith Alpha, the Medtronic Endurant II and the Gore Excluder stent-graft textiles in 4, 3, 2 and 1 cases respectively. Technical success of INLF was 80% but all INLF had a satisfactory final morphology once the first hole was successfully created. Mean visceral and renal ischemia durations were 10±2 and 15±3 minutes, respectively. Although sheep aortas were not aneurysmal, no endoleak was detected at completion angiography. Aortic explants demonstrated that failed INLF led to substantial damages of the textile and the native arterial wall. Conclusion - This study demonstrates that in situ fenestration creates substantial textile damages with all available stent-graft textiles, whatever the technique used. However, initial technical success can be obtained in vivo, perhaps thanks to blood viscosity. Complementary fatigue tests are needed to conclude on the long-term effects of INLF, and its non-inferiority to reinforced fenestrations used in commercially available FBE.

Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.

Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-β1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-β dependent, underlie its effects.

Ex vivo model for studying endothelial tip cells: Revisiting the classical aortic-ring assay

A drug undergoes several in silico, in vitro, ex vivo and in vivo assays before entering into the clinical trials. In 2014, it was reported that only 32% of drugs are likely to make it to Phase-3 trials, and overall, only one in 10 drugs makes it to the market. Therefore, enhancing the precision of pre-clinical trial models could reduce the number of failed clinical trials and eventually time and financial burden in health sciences. In order to attempt the above, in the present study, we have shown that aortic ex-plants isolated from different stages of chick embryo and different regions of the aorta (pulmonary and systemic) have differential sprouting potential and response to angiogenesis modulatory drugs. Aorta isolated from HH37 staged chick embryo showed 16% (p < 0.001) and 11% (p < 0.001) increase in the number of tip cells at 72 h of culture compared to that of HH35 and HH29 respectively. The ascending order of the number of tip cells was found as central (Gen II), proximal (Gen I) and distal (Gen III) in a virtual zonal segmentation of endothelial sprouting. The HH37 staged aortas displayed differential responses to pro- and anti-angiogenic drugs like Vascular endothelial growth factor (VEGF), nitric oxide donor (spNO), and bevacizumab (avastin), thalidomide respectively. The human placenta tissue-culture however evinced endothelial sprouting only on day 12, with a gradual decrease in the number of tip cells until 21 days. In summary, this study provides an avant-garde angiogenic model emphasized on tip cells that would enhance the precision to test next-generation angiogenic drugs.

Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis.

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.

Contribution of transcription factor EB to adipoRon-induced inhibition of arterial smooth muscle cell proliferation and migration.

Abnormal vascular smooth muscle cell (SMC) dedifferentiation with increased proliferation and migration during pathological vascular remodeling is associated with vascular disorders, such as atherosclerosis and in-stent restenosis. AdipoRon, a selective agonist of adiponectin receptor, has been shown to protect against vascular remodeling by preventing SMC dedifferentiation. However, the molecular mechanisms that mediate adipoRon-induced SMC differentiation are not well understood. The present study aimed to elucidate the role of transcription factor EB (TFEB), a master regulator of autophagy, in mediating adipoRon's effect on SMCs. In cultured arterial SMCs, adipoRon dose-dependently increased TFEB activation, which is accompanied by upregulated transcription of genes involved in autophagy pathway and enhanced autophagic flux. In parallel, adipoRon suppressed serum-induced cell proliferation and caused cell cycle arrest. Moreover, adipoRon inhibited SMC migration as characterized by wound-healing retardation, F-actin reorganization, and matrix metalloproteinase-9 downregulation. These inhibitory effects of adipoRon on proliferation and migration were attenuated by TFEB gene silencing. Mechanistically, activation of TFEB by adipoRon is dependent on intracellular calcium, but it is not associated with changes in AMPK, ERK1/2, Akt, or molecular target of rapamycin complex 1 activation. Using ex vivo aortic explants, we demonstrated that adipoRon inhibited sprouts that had outgrown from aortic rings, whereas lentiviral TFEB shRNA transduction significantly reversed this effect of adipoRon on aortic rings. Taken together, our results indicate that adipoRon activates TFEB signaling that helps maintain the quiescent and differentiated status of arterial SMCs, preventing abnormal SMC dedifferentiation. This study provides novel mechanistic insights into understanding the therapeutic effects of adipoRon on TFEB signaling and pathological vascular remodeling.

Vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic stress

The intermediate filament (IF) cytoskeleton has been proposed to regulate morphogenic processes by integrating the cell fate signaling machinery with mechanical cues. Signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) through the Notch pathway regulates arterial remodeling in response to changes in blood flow. Here we show that the IF-protein vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic forces. Vimentin is important for Notch transactivation by ECs and vimentin knockout mice (VimKO) display disrupted VSMC differentiation and adverse remodeling in aortic explants and in vivo. Shear stress increases Jagged1 levels and Notch activation in a vimentin-dependent manner. Shear stress induces phosphorylation of vimentin at serine 38 and phosphorylated vimentin interacts with Jagged1 and increases Notch activation potential. Reduced Jagged1-Notch transactivation strength disrupts lateral signal induction through the arterial wall leading to adverse remodeling. Taken together we demonstrate that vimentin forms a central part of a mechanochemical transduction pathway that regulates multilayer communication and structural homeostasis of the arterial wall.