Significant Calcification Research Articles

High-sensitivity cardiac troponin I is a biomarker for occult coronary plaque burden and cardiovascular events in patients with rheumatoid arthritis.

Patients with RA display greater occult coronary atherosclerosis burden and experience higher cardiovascular morbidity and mortality compared with controls. We here explored whether pro-inflammatory cytokines and high-sensitivity cardiac troponin I (hs-cTnI), a biomarker of myocardial injury, correlated with plaque burden and cardiovascular events (CVEs) in RA. We evaluated 150 patients with 64-slice coronary CT angiography. Coronary artery calcium, number of segments with plaque (segment involvement score), stenotic severity and plaque burden were assessed. Lesions were described as non-calcified, mixed or fully calcified. Blood levels of hs-cTnI and pro-inflammatory cytokines were assessed during coronary CT angiography. Subjects were followed over 60 (s.d. 26) months for both ischaemic [cardiac death, non-fatal myocardial infarction (MI), stroke, peripheral arterial ischaemia] and non-ischaemic (new-onset heart failure hospitalization) CVEs. Plasma hs-cTnI correlated with all coronary plaque outcomes (P < 0.01). Elevated hs-cTnI (⩾1.5 pg/ml) further associated with significant calcification, extensive atherosclerosis, obstructive plaque and any advanced mixed or calcified plaques after adjustments for cardiac risk factors or Framingham D'Agostino scores (all P < 0.05). Eleven patients suffered a CVE (1.54/100 patient-years), eight ischaemic and three non-ischaemic. Elevated hs-cTnI predicted all CVE risk independent of demographics, cardiac risk factors and prednisone use (P = 0.03). Conversely, low hs-cTnI presaged a lower risk for both extensive atherosclerosis (P < 0.05) and incident CVEs (P = 0.037). Plasma hs-cTnI independently associated with occult coronary plaque burden, composition and long-term incident CVEs in patients with RA. Low hs-cTnI forecasted a lower risk for both extensive atherosclerosis as well as CVEs. hs-cTnI may therefore optimize cardiovascular risk stratification in RA.

Degree of calcification and cyst activity in hepatic cystic echinococcosis in humans

To evaluate the relationship between cyst activity and calcification degree in cystic echinococcosis (CE) in humans, 99 hepatic cysts at successive stages of involution, surgically excised from 72 Sardinian patients, have been analyzed. Cysts were classified into 4 groups according to calcification extent: CALC 0 (no calcification); CALC 1 (scattered punctate calcifications); CALC 2 (large coarse segmental/partial calcifications); CALC 3 (complete or nearly complete circumferential ring of calcification up to thick wall of osseous consistency/calcified content of cyst). In addition the possible correlation with antibody response has been explored analyzing IgG1, IgG4 and IgE produced against somatic PSCAg. Results showed that calcification is not restricted to the inactive WHO cyst types CE4 and CE5, but occurs to a varying extent in all morphotypes of metacestode, from active classic unilocular or multivesicular cysts to the more complicated and highly degenerate stages, where cyst wall appears massively calcified. Prevalence of calcification increases with progression of cyst degenerative process, but is not synonymous with parasite inactivity and can be misleading as signs of calcification may coexist with still metabolically active cysts. On the contrary, detection of entirely firmly solidified content seems a reliable indication of cyst inactivity. IgG4 is the dominant isotype associated particularly with the evolutive phase. Positive rates and OD levels, higher in active vs inactive stages, are stable or increase slightly in weakly and moderately calcified cysts (CALC 1/CALC 2), compared to non-calcified ones (CALC 0), strongly decreasing in highly calcified forms (CALC 3). In conclusion, evaluation of calcification extent may be pertinent for staging CE, and immunological tests, particularly for IgG4, and IgE may help to better define cyst activity.

Fibroblast growth factor-23 in patients with homozygous familial hypercholesterolemia

Patients with homozygous familial hypercholesterolemia (HoFH) develop significant vascular calcification early in life, the cause of which is not yet fully understood. Patients with chronic kidney disease have similar vascular calcification, with fibroblast growth factor-23 (FGF23) implicated in these patients. To determine whether there was a difference in FGF23 between patients with HoFH and age- and gender-matched controls and whether there is a correlation between FGF23 and serum low-density lipoprotein, total cholesterol, and carotid intima-media thickness in patients with HoFH. The study was a cross-sectional review involving 30 patients with HoFH attending the Charlotte Maxeke Johannesburg Academic Hospital Lipid Clinic in Parktown, South Africa, as well as 30 age- and gender-matched healthy controls. FGF23, fasting lipid profiles, calcium, and phosphate were measured. B-mode ultrasonography of the carotid arteries was done to assess the extent and severity of arterial calcification. There was no difference in mean FGF23 between the patient and control groups (62.07±26.42 pg/mL vs 63.69±19.84 pg/mL; P=.4621) nor was there any correlation between FGF23 and low-density lipoprotein cholesterol (P=.9483 and .8474) or total cholesterol (P=.9261 and .859). In the HoFH patients, FGF23 did not correlate significantly with any cardiovascular disease. Serum FGF23 is not elevated in patients with HoFH when compared to non-familial hypercholesterolemia age- and gender-matched controls, and there is no correlation between serum FGF23 and cardiovascular disease in patients with HoFH. FGF23 does not appear to be a major factor for arterial calcification in HoFH.

CRT-100.50 Outcomes Of Patients With Left Main Disease Who Underwent Rotational Atherectomy

Significant coronary calcification is a challenge for percutaneous coronary intervention (PCI). Rotational atherectomy is useful for plaque modification prior to stent delivery. In the present study, we report our experience with left main disease and rotational atherectomy. From January 2008 to

One-year experience of a regional service model of teleconsultation for planning and treatment of complex thoracoabdominal aortic disease

ObjectiveThe objective of this study was to report the methodology and 1-year experience of a regional service model of teleconsultation for planning and treatment of complex thoracoabdominal aortic disease (TAAD). MethodsComplex TAADs without a feasible conventional surgical repair were prospectively evaluated by vascular surgeons of the same public health service (National Health System) located in a huge area of 22,994 km2 with 3.7 million inhabitants and 11 tertiary hospitals. Surgeons evaluated computed tomography scans and clinical details that were placed on a web platform (Google Drive; Google, Mountain View, Calif) and shared by all surgeons. Patients gave informed consent for the teleconsultation. The surgeon who submits a case discusses in detail his or her case and proposes a possible therapeutic strategy. The other surgeons suggest other solutions and options in terms of grafts, techniques, or access to be used. Computed tomography angiography, angiography, and clinical outcomes of cases are then presented at the following telemeetings, and a final agreement of the operative strategy is evaluated. Teleconsultation is performed using a web conference service (WebConference.com; Avaya Inc, Basking Ridge, NJ) every month. An inter-rater agreement statistic was calculated, and the κ value was interpreted according to Altman's criteria for computed tomography angiography measurements. ResultsThe rate of participation was constant (mean number of surgeons, 11; range, 9-15). Twenty-four complex TAAD cases were discussed for planning and operation during the study period. The interobserver reliability recorded was moderate (κ = 0.41-0.60) to good (κ = 0.61-0.80) for measurements of proximal and distal sealing and very good (κ = 0.81-1) for detection of any target vessel angulation >60 degrees, significant calcification (circumferential), and thrombus presence (>50%). The concordance for planning and therapeutic strategy among all participants was complete in 16 cases. In one case, the consultation was decisive for creating an innovative therapeutic strategy; in the remaining seven cases, the strategy proposed by the patient's surgeon was changed completely after the discussion. Technical success was the same (100%) if concordance in planning was present initially or not. Overall 6-month mortality was 4%, 0% for those patients with initial concordance in planning vs 12% for those without initial concordance (P = .33). Surgery was always performed in a tertiary hospital by local surgeons, and in two cases (8%) external surgeons joined the local surgical team. ConclusionsSuch a regional service of teleconsultation may be of value in standardizing the treatment and derived costs of complex TAADs in a huge region under the same health provider. The shared decision-making strategy may be of medical-legal value as well.

Deletion of IκB-Kinase β in Smooth Muscle Cells Induces Vascular Calcification Through β-Catenin-Runt-Related Transcription Factor 2 Signaling.

BackgroundVascular calcification was previously considered as an advanced phase of atherosclerosis; however, recent studies have indicated that such calcification can appear in different situations. Nevertheless, there has been a lack of mechanistic insight to explain the difference. For example, the roles of nuclear factor‐κB, a major regulator of inflammation, in vascular calcification are poorly explored, although its roles in atherosclerosis were well documented. Herein, we investigated the roles of nuclear factor‐κB signaling in vascular calcification.Methods and ResultsWe produced mice with deletion of IKKβ, an essential kinase for nuclear factor‐κB activation, in vascular smooth muscle cells (VSMCs; KO mice) and subjected them to the CaCl2‐induced aorta injury model. Unexpectedly, KO mice showed more calcification of the aorta than their wild‐type littermates, despite the former's suppressed nuclear factor‐κB activity. Cultured VSMCs from the aorta of KO mice also showed significant calcification in vitro. In the molecular analysis, we found that Runt‐related transcription factor 2, a transcriptional factor accelerating bone formation, was upregulated in cultured VSMCs from KO mice, and its regulator β‐catenin was more activated with suppressed ubiquitination in KO VSMCs. Furthermore, we examined VSMCs from mice in which kinase‐active or kinase‐dead IKKβ was overexpressed in VSMCs. We found that kinase‐independent function of IKKβ is involved in suppression of calcification via inactivation of β‐catenin, which leads to suppression of Runt‐related transcription factor 2 and osteoblast marker genes.Conclusions IKKβ negatively regulates VSMC calcification through β‐catenin–Runt‐related transcription factor 2 signaling, which revealed a novel function of IKKβ on vascular calcification.

Severe Hypocalcemia and Extreme Elevation of Serum Creatinkinase in a 16-Year Old Boy with Pseudohypoparathyroidism Type Ib

Calcium is essential for proper muscular function and metabolism. Myopathy with high creatinkinase activity can be a rare manifestation of hypocalcemia of various origin, such as vitamin D deficiency, hypoparathyroidism, pseudohypoparathyroidism (PHP). 16-year old previously healthy boy was admitted to intensive care unit with convulsions lasting for three minutes and a transient loss of consciousness. Laboratory results revealed severe hypocalcemia (total S-Ca < 1.0 mmol/L; normal 2.2-2.6 mmol/L), hyperphosphatemia (S-P 2.8 mmol/L; normal 0.6-1.6 mmol/L). Serum creatinkinase (S-CK) activity was 32 μkat/L (normal 0.57-2.45 μkat/L). Other basic biochemical parameters including creatinine, troponin, alkaline phosphatase were within normal values. Calcemia was gradually corrected within two weeks by intravenously and orally administered calcium and by cholecalciferol. S-CK reached a maximum of 222 μkat/L on day 4 and dropped to 7.2 μkat/L on day 14. Boy had no myalgias, neither clinical signs of myopathy. Echocardiography was normal with normal myocardial contractility, without any signs of calcification. The serum level of parathyroid hormone (S-PTH) was high (12 pmol/L; normal 0.7-5.5 pmol/L), fully compatible with the diagnosis of PHP. Molecular analysis revealed pseudohypoparathyroidism type Ib (PHPIb).In conclusion, manifest tetany and even mild myopathy with very high S-CK can occur in hypocalcemic patients and usually resolves after normalization of hypocalcemia.

A novel restorative pulmonary valved conduit in a chronic sheep model: Mid-term hemodynamic function and histologic assessment

ObjectiveTo evaluate the safety and the short-term function of a novel pulmonary valved conduit (Xeltis Pulmonary Valved Conduit; XPV) up to 12 months in a sheep model. MethodsXPV and Hancock bioprosthetic valved conduits (H, used as control) were implanted in adult sheep in the pulmonary artery position. Animals were killed at 2 months (n = 6 XPV), 6 months (n = 6 XPV and n = 3 H), and 12 months (n = 6 XPV) and examined histologically. During follow-up, function of the device as well as diameter of both XPV and H were assessed by transthoracic echocardiography. ResultsOf 18 animals that received an XPV, 15 survived until they were killed; 3 animals that received H survived the planned observational interval. XPV showed mild neointimal thickening and degradation beginning at 2 months with an ongoing process until 12 months. Only 1 of the 18 animals with XPV had significant calcification at 6 months. Pathologic specimen did not show any significant narrowing of the conduit whereas neointimal thickness showed a peak at 6 months. Inflammatory process reached a maximum at 6 months and the degradation process at 12 months. Gel permeation chromatography analysis showed molecular weight loss beginning at 2 months with a peak at 12 months for the conduit with slower absorption for the leaflets. The wall of the H conduits showed more neointimal thickening, narrowing, and calcification compared with XPV, but the leaflets demonstrated minimal changes. ConclusionsBoth conduits demonstrated an acceptable safety and functionality. Significant calcification was rarely observed in the XPV, whereas the H developed more neointimal thickness with calcification of the porcine aortic root portion of the wall.

Successful percutaneous coronary intervention for an in-stent chronic total occlusion in a patient with dextrocardia: a case report

Percutaneous coronary interventions of chronic total occlusion represent one of the most challenging issues in interventional cardiology. A Caucasian patient with dextrocardia presented with an in-stent chronic total occluded right coronary artery, a constellation which has not been described previously in the literature. A 69-year-old man with pre-known situs inversus totalis and a long history of coronary artery disease with multiple interventions and stent-implantations presented to our department suffering from episodes of chest pain under exercise. A coronary angiogram showed a completely occluded right coronary artery in the area of a drug-eluting stent which had been implanted eight years before. We found collaterals from the left coronary artery system and signs of calcification, so the lesion was classified as chronic total occlusion and the recanalization using an antegrade wire escalation technique successfully performed. This is the first reported case of an in-stent chronic total occlusion in situs inversus with dextrocardia. Our experience in this case demonstrates the feasibility of recanalization of in-stent chronic total occlusions even in the rare setting of this congenital cardiac malposition combined with severe coronary artery disease.

Value of mitral annular plane systolic excursion in the assessment of contractile reserve in patients with ischemic cardiomyopathy before cardiac revascularization

BackgroundMitral annular plane systolic excursion (MAPSE) is an M-mode derived echocardiographic marker of left ventricular longitudinal function, the aim of this study is to evaluate the value of MAPSE in assessment of contractile reserve in patients with ischemic cardiomyopathy before cardiac revascularization. MethodsThe study included 50 patients with ischemic cardiomyopathy with ejection fraction (EF) ≤35%, the patients presented to echocardiography laboratory for dobutamine stress echocardiography (DSE) to assess viability and contractile reserve before revascularization, patients with primary valvular disease, and those with significant mitral annular calcifications were excluded from the study. A low dose DSE was done to all patients using standardized incremental infusions of 5, 10, and 20 μg/kg/min and the following parameters were measured at both baseline and peak dose, (EF, wall motion score index(WMSI) and MAPSE). Contractile reserve was measured as the difference between the low dose and baseline values of the EF and WMSI. ResultsThe study included 50 patients aged 55.08±7.15 years, 94% were males, the DSE protocol was complete in all patients without serious side effects. A total of eight hundred segments were analyzed, at baseline 65% were dysfunctional including 31.2% hypokinetic, 28.8% were akinetic, and 5% were dyskinetic. At low dose study 70% of the dysfunctional myocardium showed viability, EF increased significantly from 30.84±4.56 to 42.24±8.15%, p <0.001, the WMSI reduced significantly from 1.92±0.33 to 1.47±0.39, and MAPSE increased significantly from 1.02±0.23 to 1.30±0.30mm. MAPSE showed a significant positive correlation with EF at both baseline and low dose study (r=0.283, p=0.046 & r=0.348, p=0.013) respectively and a significant negative correlation with WMSI at both baseline and low dose study (r=−0.3, p=0.034 & r=−0.409, p=0.003), respectively. By ROC curve analysis we found that Δ MAPSE ≥2mm can predict contractile reserve at Δ EF >10% (AUC=0.6, sensitivity 67.86, specificity 59.09), and Δ MAPSE ≥1.8mm can predict contractile reserve at ΔWMSI ≤0.20 (AUC=0.61, sensitivity 65.5, specificity 75.6). ConclusionsMAPSE is a rapid simple quantitative echocardiographic method that can asses contractile reserve in patients with ischemic cardiomyopathy before cardiac revascularization.

Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia.

ObjectiveOverexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium.Approach and resultsA vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0μm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330μm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622μm2) and lipids (30754 vs. 77317μm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls.ConclusionsEndogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.

Heterotopic Ossification after Arthroscopic Elbow Contracture Release

The excessive use of a radiaofrequency device in arthroscopic elbow release will result in an increased incidence of heterotopic ossification. This study investigates the prevalence and risk factors of heterotopic ossification (HO) in arthroscopic elbow release surgery. There were 101 elbows with arthroscopic release performed on 98 patients over the 5-year period from November 2011 to December 2015. They were divided into 3 groups. Group 1, elbow arthritis, included 46 elbows in 43 patients. Group 2, posttraumatic extrinsic elbow stiffness, included 23 elbows in 23 patients, and 32 elbows in 32 patients with intraarticular fractures and severe intrinsic contractures were in Group 3. Arthroscopic elbow release was performed under general anesthesia. In the postoperative period, x-ray or CT was followed up regularly to determine if there was HO, which was diagnosed when significant new calcifications were identified. The functional recovery was evaluated by comparing the range of motion and Mayo Elbow Performance Score (MEPS) pre and postoperatively in each group. Other complications were also assessed postoperatively. The patients’ mean age was 38.6 (range, 12–66) years old, with 57 males and 41 females. Mean follow-up was 21 (range, 4–56) months. HO developed in 25/101 (25%) elbows and 4 severe cases underwent repeat surgery. Group 1 patients were primarily arthritis and 3/46 (6.5%) elbows had minor HO on x-ray. In Group 2, 1/23 (4.3%) elbows had minor HO. However, in Group 3, 21/32 (65.6%) elbows developed HO, including 4 severe cases, 4 moderate cases and 13 minor cases. The average flexion– extension arc was improved from 93° to 126° in Group 1, 66° to 121° in Group 2, 46° to 91° in Group 3 at the last follow up. MEPS increased from 71.4 to 91.3 in Group 1, 65.6 to 93.5 in Group 2 and 52.3 to 80.6 in Group 3. In these severe intrinsic contractures cases, we routinely applied the radiofrequency device to create space for visualization and release, which was rarely used in Group 1 and 2. The occurrence of HO after arthroscopic elbow release for intrinsic posttraumatic stiffness was unexpectedly high. The predominant risk factor for the increased incidence of heterotopic ossification in this series was excessive use of radiofrequency, and this complication apparently impaired the otherwise predictable outcome.

Steps in the development of a VATS lobectomy program.

Video-assisted thoracoscopic surgery (VATS) lobectomy has been employed in recent decades for the treatment of non-small cell lung cancer (NSCLC). Although trials have shown this procedure to be safe and feasible a VATS approach for lobectomy has not been widely used yet. Surgeons can go beyond this limit by following a specific operative plan focused on learning the minimally invasive technique in centers of excellence and then including: a pre-operative phase based on the radioclinical assessments and an operating phase designed to develop a methodical approach to VATS technique. At the beginning it's recommended to follow a learning curve with careful selection of patients keeping in mind that intraoperative complications may occur even later, especially in presence of clinical nodal disease or significant calcification on preoperative CT scan, which represents the main reason for conversion from VATS to thoracotomy.

The degree of hair graying as an independent risk marker for coronary artery disease, a CT coronary angiography study.

BackgroundCardiovascular disease is a leading cause of death worldwide. Aging is an unavoidable coronary risk factor and is associated with dermatological signs that could be a marker for increased coronary risk. We tested the hypothesis that hair graying as a visible marker of aging is associated with risk of coronary artery disease (CAD) independent of chronological age.MethodsThis cross-sectional study included 545 males who underwent a computed tomography coronary angiography (CTCA) for suspicious of CAD, patients were divided into subgroups according to the percentage of gray/white hairs (Hair Whitening Score, HWS: 1–5) and to the absence or presence of CAD.ResultsCAD was prevalent in 80% of our studied population, 255 (46.8%) had 3 vessels disease with mean age of 53.2 ± 10.7 yrs. Hypertension, diabetes and dyslipidemia were more prevalent in CAD group (P = 0.001, P = 0.001, and P = 0.003, respectively). Patients with CAD had statistically significant higher HWS (32.1% vs 60.1%, p < 0.001) and significant coronary artery calcification (<0.001). Multivariate regression analysis showed that age (odds ratio (OR): 2.40, 95% confidence interval (CI): [1.31–4.39], p = 0.004), HWS (OR: 1.31, 95% CI: [1.09–1.57], p = 0.004), hypertension (OR: 1.63, 95% CI: [1.03–2.58], p = 0.036), and dyslipidemia (OR: 1.61, 95% CI: [1.02–2.54], p = 0.038) were independent predictors of the presence of atherosclerotic CAD, and only age (p < 0.001) was significantly associated with HWS.ConclusionsHigher HWS was associated with increased coronary artery calcification and risk of CAD independent of chronological age and other established cardiovascular risk factors.

A Biomimetic Heparinized Composite Silk-Based Vascular Scaffold with sustained Antithrombogenicity

Autologous grafts, as the gold standard for vascular bypass procedures, associated with several problems that limit their usability, so tissue engineered vessels have been the subject of an increasing number of works. Nevertheless, gathering all of the desired characteristics of vascular scaffolds in the same construct has been a big challenge for scientists. Herein, a composite silk-based vascular scaffold (CSVS) was proposed to consider all the mechanical, structural and biological requirements of a small-diameter vascular scaffold. The scaffold’s lumen composed of braided silk fiber-reinforced silk fibroin (SF) sponge covalently heparinized (H-CSVS) using Hydroxy-Iron Complexes (HICs) as linkers. The highly porous SF external layer with pores above 60 μm was obtained by lyophilization. Silk fibers were fully embedded in scaffold’s wall with no delamination. The H-CSVS exhibited much higher burst pressure and suture retention strength than native vessels while comparable elastic modulus and compliance. H-CSVSs presented milder hemolysis in vitro and significant calcification resistance in subcutaneous implantation compared to non-heparinized ones. The in vitro antithrombogenic activity was sustained for over 12 weeks. The cytocompatibility was approved using endothelial cells (ECs) and vascular smooth muscle cells (SMCs) in vitro. Therefore, H-CSVS demonstrates a promising candidate for engineering of small-diameter vessels.

Routine CT angiography to detect severe coronary artery disease prior to transcatheter aortic valve replacement.

Patients undergoing TAVR undergo routine CT angiography (CTA) to assess aorto-iliac pathology and annular dimensions. While coronary CTA may exclude severe CAD in younger patients, its efficacy in defining CAD severity prior to TAVR may be limited. We retrospectively studied 50 consecutive patients undergoing both invasive coronary angiography (ICA) and routine pre-TAVR CTA. Severe CAD was defined as ≥50% stenosis by quantitative coronary angiography and compared to a blinded CTA visual estimation of ≥50% stenosis. The analysis was confined to four segments: left main and three proximal to mid major coronaries to maximize myocardial territory at risk. Coronary assessment was performed using standard reconstructed ECG phases from pre-TAVR chest CTA on a Philips 256 iCT scanner. Nearly ¾ of patients were ≥75 years old, 57% were female, half were diabetic and 45% had prior PCI. By ICA, 49% had significant coronary calcification. The incidence of severe proximal to mid vessel CAD by ICA was 39%. Similarly, a third of patients required PCI prior to TAVR. CTA was unable to exclude severe proximal to mid vessel CAD in 88% of patients in all four segments: non-diagnostic CTA readings were mainly due to calcification (60%) or motion artifact (28%). Non-diagnostic coronary CTA readings ranged from 25 to 72% according to segment analyzed: only the left main segment had diagnostic quality CTA in the majority of patients (p < 0.01). PCI is performed frequently prior to TAVR based upon invasive coronary angiographic assessment. Routine chest CTA algorithms do not provide adequate diagnostic information to exclude severe CAD, primarily due to severe coronary calcification in the TAVR population.

Relation between coronary artery calcium score and serum tenascin-C level in patients without known coronary artery disease.

Presence of coronary artery disease (CAD) also in subjects without traditional risk factors leads to a search for new risk factors. Tenascin-C (TNC), an extracellular matrix glycoprotein is normally found in very low concentrations in tissues and serum of adults, whereas its expression is enhanced in case of pathological conditions accompanied by inflammation. This study aimed to investigate the relation between coronary artery calcium score (CACS)and serum TNC level. Ninety patients (age range, 18-75 years) presenting with chest pain but without known CAD were divided according to their CACSs as control (CACS = 0, n = 30), low CACS (CACS between 0 and 400, n = 30), and high CACS (CACS ≥ 400, n = 30) groups. The patients were questioned about risk factors and underwent laboratory analyses for biochemical parameters including TNC. The mean TNC level was significantly higher in the high CACS group as compared to both the low CACS group and the control group [4.7 (0.03-30.7), 28 (0.7-212) and 84 (2-456) ng/mL, respectively; P < 0.01]. A positive correlation was determined between serum TNC level and CACS (r = 0.641, P < 0.001). In the ROC curve analysis, when the cut-off value for TNC was accepted as 8.09 ng/mL, the sensitivity and specificity in detecting patients with CACS > 100 (moderate or significant calcification) were 72% and 82%, respectively. A significant relationship was found between CACS and serum TNC level. Thus, measurement of TNC level can be used in determining elevated CACS and thereby the risk for CAD.

The Consumption of Plasminogen Following Severe Burn and Its Implications in Muscle Calcification

INTRODUCTIONBurn and blast injuries present with serious risk for complications including fibrosis and calcification of the soft tissues. Plasmin, the active form of plasminogen, is the key serine protease responsible for the degradation of fibrin (fibrinolysis). Our lab has previously shown that plasmin is essential for the prevention of muscle fibrosis and calcification following injury. We hypothesized that 1) plasminogen is consumed in response to severe burn, and 2) this transient plasminogen deficiency impairs proper muscle regeneration.METHODSTo test our first hypothesis in humans, we collected blood from 10 control subjects at one time‐point and 20 burn patients with burns ranging from 25–75% total body surface area (TBSA) at 0, 3, 7, and 14 days post‐admission. Plasma plasminogen levels were assessed by human plasminogen ELISA. Methods including human subjects were pre‐approved by Vanderbilt's IRB.Because our lab has demonstrated that plasmin activity prevents muscle calcification, we used the formation of dystrophic calcification and as an output measure for impaired muscle regeneration. To assess muscle regeneration following severe burn in mice, we induced bilateral muscle injuries in the mice by injecting cardiotoxin (CTX) into the calf muscles and then we administered a 30% TBSA burn to the dorsum by scalding in 100°C water. Control groups received either the burn alone or the CTX injection alone. Another group of mice received the same injury and burn concurrent with a subcutaneous injection of either an antisense oligonucleotide (ASO) against plasmin's inhibitor, α2‐antiplasmin, or a control ASO, beginning 2 weeks prior to injury. Following the burn injury, weekly radiographs of the lower extremities were taken up to 28 days post‐injury (DPI) to assess dystrophic calcification. Following sacrifice at 28 DPI, ex vivo micro‐computed tomography (μCT) was performed on the lower extremities to further assess muscle calcification.To assess plasminogen consumption following severe burn in mice, we collected blood from animals with either CTX‐induced injury, injury with burn (as described above) or no injury following sacrifice at 3 DPI. Plasma plasminogen levels were assessed by mouse plasminogen ELISA. Vanderbilt IACUC pre‐approved all animal experiments.RESULTSBurn patients exhibited a significant plasminogen deficit compared to control subjects at 0 and 3 days post‐admission, followed by a return to control plasminogen levels between 7 and 14 days (Figure 1).Consistent with the human subjects, mice exhibited a significant deficit in circulating plasminogen following burn when compared to both uninjured mice and mice given CTX injection alone.In the animal models assessing muscle regeneration, following the CTX‐induced injury alone, mice did not form significant dystrophic calcification in the injured muscles, but the mice that received the CTX injection and the burn developed dystrophic calcification in the injured muscle at 7 DPI. This phenotype in the burn mice was rescued by a systemic increase in plasmin activity through weekly administration of α2AP ASO (Figure 2).DISCUSSIONWhile many response mechanisms following severe burn have been implicated in impaired healing, the loss of plasminogen and the consequences of this have not been well characterized. Conclusively, this study suggests that severe burn increases the risk of the formation of calcification in injured muscle due to consumption of plasminogen.Support or Funding InformationNIH T32 AR059039‐06A1 Rheumatology Training Grant, Vanderbilt Department of Orthopaedics and Rehabilitation, Caitlin Lovejoy Fund

Scaffold-free high throughput generation of quiescent valvular microtissues

AimsThe major challenge of working with valvular interstitial cells in vitro is the preservation or recovery of their native quiescent state. In this study, a biomimetic approach is used which aims to engineer small volume, high quality valve microtissues, having a potential in regenerative medicine and as a relevant 3D in vitro model to provide insights into valve (patho)biology. Methods and resultsTo form micro-aggregates, porcine valvular interstitial cells were seeded in agarose micro-wells and cultured in medium supplemented with 250μM Ascorbic Acid 2-phosphate for 22days. Histology showed viable aggregates with normal nuclei and without any signs of calcification. Aggregates stained strongly for GAG and collagen I and reticular fibers were present. ECM formation was quantified and showed a significant increase of GAG, elastin and Col I during aggregate culture. Cultivation of VIC in aggregates also promoted mRNA expression of Col I/III/V, elastin, hyaluronan, biglycan, decorin, versican MMP-1/2/3/9 and TIMP-2 compared to monolayer cultured VIC. Phenotype analysis of aggregates showed a significant decrease in α-SMA expression, and an increase in FSP-1 expression at any time point. Furthermore, VIC aggregates did not show a significant difference in OCN, Egr-1, Sox-9 or Runx2 expression. ConclusionIn this study high quality valvular interstitial cell aggregates were generated that are able to produce their own ECM, resembling the native valve composition. The applied and completely cell driven 3D approach overcomes the problems of VIC activation in 2D, by downregulating α-SMA expression and stimulating a homeostatic quiescent VIC state.

In vitro comparative assessment of decellularized bovine pericardial patches and commercial bioprosthetic heart valves

Notwithstanding their wide exploitation, biological prosthetic heart valves are characterized by limited durability (10–15 years). The treatment of biological tissues with chemical crosslinking agents such as glutaraldehyde accounts for the enhanced risk of structural deterioration associated with the early failure of bioprosthetic valves. To overcome the shortcomings of the currently available solutions, adoption of decellularized biological tissues of animal origin has emerged as a promising approach. The present study aims to assess in vitro cardiovascular scaffolds composed of bovine pericardium decellularized with the novel TRITDOC (TRIton-X100 and TauroDeOxyCholic acid) procedure. The effects of the treatment have been assessed by means of histological, biomolecular, cellular, biochemical and biomechanical analyses. The TRITDOC procedure grants the complete decellularization of bovine pericardial scaffolds while preserving the extracellular matrix architecture and the biomechanical properties. With a dedicated ELISA test, the TRITDOC procedure has been proven to ensure the complete removal of the alphaGal antigen, responsible for hyperacute rejection and for long-term deterioration of xenogenic biomaterials. Static seeding of the acellular pericardial patches with human adipose-derived stem cells resulted in an evenly repopulated scaffold without signs of calcification. The in vitro cyto-/immuno-compatibility response of the TRITDOC-bovine pericardium was compared with glutaraldehyde-treated xenogenic pericardium collected from two bioprosthetic devices currently used in clinical practice: PERIMOUNT MAGNA and TRIFECTATM. TRITDOC-bovine pericardium exhibited lower complement activation, lower cytotoxicity and a lower tendency to secrete pro-inflammatory cytokines compared to the tested commercial bioprostheses. Therefore, TRITDOC-decellularized pericardium could be considered as possible candidate material for the production of prosthetic heart valves.