bluuebunny/crossref_metadata_2025 huggingface dataset:
Live imaging of nitric oxide release in vascular endothelial cells in response to mechanical stimuli on an organ chip
Authors: Takahashi K , Liu Y , Wang M , Liang Y , Naruse K
Published: October 2022

Background

Nitric oxide (NO), released from vascular endothelial cells in response to mechanical stimuli, regulates cardiac contractility and are also involved in the prevention of the development of cardiac hypertrophy.

Purpose

To establish an experimental system for live observation of NO release in response to mechanical stimuli on an organ chip.

Methods

Organ chips, which we used for the development of a heart-on-a-chip in the previous study [1], were used.

We seeded 300,000 human umbilical vein endothelial cells on a stretchable elastic membrane coated with Matrigel of a chip channel. Shear stress was applied to the cells by increasing flow rate of a peristaltic pump connected to the chip channel (Figure 1A). Pressure stimulus was applied by hydrostatic pressure. Stretch stimulus was applied by suction to the side ports of a chip using an electric syringe pump (Figure 1B). Cells were stained with 10 μM 4,5-diaminofluorescein diacetate for fluorescent live NO imaging.

Results

Monolayers of the endothelial cells formed intercellular junctions confirmed by CD31 staining (Figure 1C, yellow). Apparent permeability, which was measured by Texas red dye (MW 3000), was maintained at a low level of ∼3x10–6 cm/s until day 30, suggested the formation of robust intercellular junction.

When the endothelial cells were subjected to a pressure stimulus of 60 mmHg for 60 s, NO release was observed that lasted for >2 minutes (Figure 2A). A peak value of 1.46±1.08 (mean ± standard deviation) times the baseline was observed 271 s after the beginning of the pressure stimulus (n=251 cells). When the cells were subjected to a 1% stretch for 60 s, a peak value of 1.29±0.33 times the baseline was observed 105 s after the beginning of the stretch stimulus (Figure 2B). A shear stress of 0.01 dyn/cm2 hardly increased NO release (1.20±0.27 times the baseline, Figure 2C).

Conclusion

The system for live NO imaging in vascular endothelial cells in response to mechanical stimuli was established using organ-on-a-chip. The heart-on-a-chip with endothelial cells will be useful in elucidating the effects of mechanical stimulus such as hypertension on the contractile function and the remodeling of the heart.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science
Comparative prognostic utility of visual grade and heart-to-contralateral lung ratio in patients undergoing 99mTc-pyrophosphate radiotracer cardiac scintigraphy
Authors: Saad J M , Ahmed A I , Alahdab F , Han Y , Alfawara M , Kassi M , Al-Mallah M H
Published: October 2022

Introduction

Transthyretin (ATTR) cardiac amyloidosis (CA) is an underdiagnosed form of restrictive cardiomyopathy leading to a rapid progression into heart failure. 99mTc-labeled radiotracer cardiac scintigraphy is crucial for the evaluation of CA. Semiquantitative (Perugini visual grade) and quantitative (heart-to-contralateral lung ratio, H/CL) parameters are used to assessing CA via cardiac scintigraphy.

Purpose

We aimed to assess the comparative prognostic utility of the Perugini visual grade and heart to contralateral lung ratio.

Methods

The study population was identified based on an institutional registry of consecutive patients undergoing 99mTc-PYP radiotracer cardiac scintigraphy for suspected CA between January 2020-October 2021. The H/CL is calculated by the fraction of heart region of interest (ROI) mean uptake counts to the contralateral chest ROI. The visual scoring system compares uptake between bone (rib) and heart where 0 = absent cardiac uptake, 1 = uptake less than bone, 2 = uptake equal to bone, and 3 = is uptake greater than bone. Ejection fraction (EF) was obtained from echocardiographic studies done around the time of cardiac scintigraphy. MACE was defined as a composite of inclusive of all-cause death, myocardial infarction, admission for heart failure and late revascularization – percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) >90 days after imaging.

Results

A total of 330 patients (mean age 70.9±12.2 years, 62.2% male) were included in the analysis. Risk factors were prevalent (59.7% hypertension, Heart Failure 68.3%, 26.5% dyslipidemia, 26.2% diabetes) (Table 1). In total, 32.6% of the cohort had studies suggestive of ATTR CA. Median time between echocardiography and cardiac scintigraphy was 7 days (IQR 5–42 days). A higher frequency of MACE was reported in patients with a 2+ visual grade (30% vs 42%, p=0.03). A H/CL >1.5 was associated with borderline significance (43% vs 35% p=0.07).

Conclusion

Our analysis showed that the Perugini visual grading of transthyretin cardiac amyloidosis conferred the best prognostic utility contrary to heart to contralateral lung ratio in 99mTc-PYP radiotracer cardiac scintigraphy.

Funding Acknowledgement

Type of funding sources: None.
Effect of coagulation factors FXIa, FXIIa, F alpha-XIIa, F beta-XIIa and FXIIIa on platelet function
Authors: Wildeis L , Ziese J , Helten C , Benkhoff M , Mourikis P , Zako S , Petzold T , Levkau B , Zeus T , Kelm M , Polzin A
Published: October 2022

Background

FXa has recently been shown to active platelets via protease activated receptor. Direct inhibition of FXa leads to reduced platelet reactivity and arterial thrombosis. It is not known, if other coagulation factors besides FXa and FIIa directly activate platelets. In this study, we hypothesized that FXIa, FXIIa, Fα-XIIa, Fβ-XIIa and FXIIIa directly active platelets.

Purpose

Direct inhibitors of above mentioned factors are currently in clinical trials. Hence, investigation of coagulation factors' non-canonical effects beyond activation of the coagulation cascade are of great importance.

Methods

Platelet reactivity was measured using light transmission aggregometry (LTA) and flow cytometry. Ex-vivo stimulation of washed platelets was conducted using adenosine diphosphate (ADP) and thrombin receptor activator peptide (TRAP).

Results

FXIa, FXIIa, Fα-XIIa, Fβ-XIIa and FXIIIa have no direct effect on platelet function in terms of aggregation (Maximum of aggregation: FXIa: 2.799±1,823%, FXIIa: 1,174±1,080%, Fα-XIIa: 1,663±1,680%, Fβ-XIIa: 1,060±1,356%, Fα-XIIa + Fβ-XIIa 1,790±2,327% and FXIIIa: 1,345±1,060%). Also, incubation of platelets does not cause expression of P-selectin (FXIa: 4,346±4,650%, FXIIa: 7,072±6,098%, Fα-XIIa: 9,124±6,075%, Fβ-XIIa: 4,556±4,693%, Fα-XIIa + Fβ-XIIa 8,070±4,342% and FXIIIa: 5,166±5,512%).

Conclusion

FXIa, FXIIa, Fα-XIIa, Fβ-XIIa and FXIIIa have no direct effect on platelet function in terms of aggregation and P-selectin expression. Thus, existing pharmacological inhibitors of these factors are to be classified as purely anticoagulant and do not exert non-canonical effects on platelet activation.

Funding Acknowledgement

Type of funding sources: None.
Long COVID-19 syndrome: association of cardiopulmonary impairment with a persistent platelet activation
Authors: Camera M , Brambilla M , Canzano P , Becchetti A , Conti M , Agostoni P G , Pengo M , Tortorici E , Mancini M E , Andreini D , Bonomi A , Parati G F
Published: October 2022

Background

A considerable proportion of patients do not fully recover from COVID-19 infection and report symptoms that persist beyond the initial phase of infection: this condition is defined long-COVID-19 syndrome (LCS). LCS can involve lungs as well as several extrapulmonary organs, including the cardiovascular system. The risk and 1-year burden of cardiovascular diseases (CVD) is increased in COVID-19 survivors, even in subjects at low risk of CVD. Recently, we documented that acute COVID-19 infection induces altered platelet activation state characterized by a prothrombotic phenotype and by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens. No data are yet available on the contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients.

Purpose

To study platelet activation status, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients enrolled at 6 months after resolution of the acute phase (6mo-FU), compared to acute COVID-19 infection patients.

Methods

6mo-FU COVID-19 patients (n=24) with established LCS were enrolled at Centro Cardiologico Monzino. Residual pulmonary impairment was assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows-CT scan evaluation. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were assessed by flow cytometry; pTGC by calibrated automated thrombogram. 46 patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison.

Results

Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-fold (1.5% [1.2–2.9] vs 2.4% [1.6–5.7]) and 2-fold (217/μl [137–275] vs 435/μl [275–633]) lower at 6mo-FU compared to acute phase, being comparable to HS. pTGC behaved similarly. At 6mo-FU, the MV profile, in terms of total number and cell origin, returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression (6.9% [3–13.5] vs 11.7% [5.2–18.9]) and PLA formation (35.5% [27.4–46.8] vs 67.7% [45.7–85.3]) was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed (r=−0.423; p=0.02).

Conclusion

These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health (Ricerca Corrente 2020 MPP COVID4)
Investigation of the effect of the angiotensin II type 2 receptor (AT2R) agonist C21 on plasma NT-proBNP, a diagnostic biomarker, in subjects hospitalised with COVID-19
Authors: Batta R , Tornling G , Rosendahl E , Bengtsson T , Raud J
Published: October 2022

The prohormone N-terminal pro-B-type natriuretic peptide (NT-proBNP) is released from stretched cardiac myocytes and is a diagnostic biomarker for heart failure and cardiac dysfunction as well as pulmonary embolism and pneumonia that are frequent complications to severe Coronavirus Disease 2019 (COVID-19). NT-proBNP is frequently elevated in COVID-19. In a recent publication, it was demonstrated that NT-proBNP was strongly associated with mortality in patients with COVID-19, and further investigation of its usefulness as a prognostic tool to predict disease outcomes in COVID-19 was suggested (1).

In the recently completed phase 2 trial (angiotensin II type 2 receptor agonist COVID-19 trial [ATTRACT]; NCT04452435) in subjects hospitalised with COVID-19, it was investigated whether treatment with the AT2R agonist C21 for 7 days affected the release of the plasma biomarker NT-proBNP.

ATTRACT was a randomised, double-blind, placebo-controlled, phase 2 trial that investigated the safety and efficacy of C21 treatment (100 mg twice daily) for 7 days in hospitalised subjects with COVID-19, not requiring mechanical ventilation. The results of the trial demonstrated that treatment with C21 on top of standard of care (vast majority of patients received glucocorticoids) significantly reduced the proportion of subjects requiring supplemental oxygen at Day 14, indicating faster recovery with C21 treatment compared to placebo. Blood samples for exploratory analysis were taken before and after 7 days of treatment with C21 or placebo.

Plasma NT-proBNP was markedly elevated in both treatment groups before treatment, with average values of 357 and 438 pg/mL in the placebo and C21 groups, respectively, as compared to normal levels of approximately <100 pg/mL. After 7 days of treatment, the C21 group experienced a dramatic reduction in plasma NT-proBNP (by 259 pg/mL) as compared to the placebo group (63 pg/mL) (p=0.02).

The results show that short-term C21 treatment decreased the release of NT-proBNP in subjects hospitalised with COVID-19. Further investigations are needed to elucidate whether this is related to effects on COVID-19-induced pulmonary damage or direct protective effects on the heart. We are currently conducting a global phase 3 trial (VP-C21–008) further investigating the effect of C21 in subjects hospitalised with COVID-19 including determination of NT-proBNP.

Funding Acknowledgement

Type of funding sources: Private company. Main funding source(s): Vicore Pharma AB
Identification of a PRDM1-regulated T cell network to regulate T cell driving plaque inflammation in human and mouse atherosclerosis
Authors: Maas S L , Jin H , Lu C , Nagenborg J , Karel J M H , Cavill R , Sikkink C J J M , Nadeau S , Gijbels M , Mees B M E , Smirnov E , Sluimer J C , Martins G A , Van Der Vorst E P C , Biessen E A L
Published: October 2022

T cells have a prominent role in the pathogenesis of atherosclerosis, although their exact function remains elusive. Here, we pursued a network-driven approach to identify T cell-associated gene programs driving the transition from low- to high-risk human plaques.

In this study 43 human carotid arterial plaques were collected and stratified based on absence (low-risk) or presence (high-risk) of intraplaque haemorrhage (IPH). Lesion RNA was subjected to microarray gene expression analysis and analysed by Weighted Gene Co-expression Network Analysis (WGCNA). We identified a co-expressed gene cluster displaying a strong T cell signalling signature in high- versus low-risk plaque, which was tightly connected to subnetworks of angiogenesis and interferon-signalling. WGCNA-based Bayesian network inference, cell-type deconvolution and single-cell gene expression revealed that this T cell-associated gene program was likely linked to effector-memory cytotoxic CD8+ T cells, underpinning the central role of T cells in plaque destabilization. Gene regulatory analysis identified cytotoxic T cell-related transcription factors, like PRDM1, regulating this plaque T cell gene program. Moreover, we demonstrated in LDL receptor knockout mice with T cell-specific Prdm1 deficiency, that lack of Prdm1 in T cells resulted in larger, more advanced plaques.

In conclusion, our study reveals a PRDM1-regulated T cell footprint in high- versus low-risk human atherosclerotic lesions and murine atherosclerotic plaque development, thereby identifying this network as a potential target for intervention in adverse T cell responses.

Funding Acknowledgement

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): The European Research Area Network on Cardiovascular Diseases (ERA-CVD and Dutch Heart Foundation)
Role of smooth muscle-derived S100A4 in an atherosclerotic mouse model
Authors: Cardoso Dos Santos L M , Azar P , Lemeille S , Bochaton-Piallat M L
Published: October 2022

Background

S100A4, a small calcium binding protein, plays an important role in vascular smooth muscle cell (SMC) phenotypic switch but its implication in atherosclerotic plaque development and particularly in SMC phenotypic plasticity is not clear yet. By neutralizing S100A4 using a monoclonal antibody, we have previously shown a reduction in overall atherosclerotic burden. However, this strategy did not distinguish the different contribution between SMCs or other S100A4-expressing cells (e.g. macrophages).

Methods

Herein, we used a lineage tracing mouse model in which we induced a SMC specific deletion of S100A4 (SMC-S100A4Δ/Δ) in an ApoE−/− background. High cholesterol diet was maintained for 12 weeks, after which, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed and aortas processed for staining and single cell RNA sequencing (scRNA-seq).

Results

We showed that S100A4 deletion modulated plaque composition rather than plaque size. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt showed a reduction in macrophages and dendritic cells and an increase in total SMCs, likely participating in fibrous cap stabilization. SMC populations showed a decrease in macrophage-like inflammatory phenotype and an increase in fibroblast-like repair phenotype, as well as retention of specific SMC markers, namely Acta2 and Myh11. Gene expression analysis revealed a reduction in the expression of inflammation markers, fatty acid metabolism, and extracellular matrix-related genes, and an increase in classical SMC markers and maintenance of the contractile phenotype.

Conclusion

We report an intrinsic function of S100A4 in the establishment of SMC fate within the plaque and, surprisingly, a global impact on the atherosclerotic plaque microenvironnement and inflammation status.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation
Targeting RNA elongation in endothelial-to-mesenchymal transition
Authors: Hoba J , Kokot K E , Andritschke M , Boeckel J N , Laufs U
Published: October 2022

Background and purpose

EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans.

Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4.

Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT.

Methods and results

After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT.

Conclusion

SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)
Arterial thrombosis in Hutchinson-Gilford Progeria Syndrome
Authors: Puspitasari Y M , Ministrini S , Karch C , Akhmedov A , Luscher T F , Camici G G
Published: October 2022

Introduction

Arterial thrombosis is the most common age-associated event underlying major adverse cardiovascular (CV) events. The interplay between the vascular endothelium, platelets, and the coagulation cascade leads to thrombus formation, which results in the cessation of blood supply to the downstream tissues. Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition with striking features of premature aging. It is caused by defects in the nuclear A-type lamin gene, leading to intracellular accumulation of progerin. This genetic disorder is characterized by shortened lifespan, primarily due to an increased incidence of myocardial infarction and ischemic stroke. Declined vascular function and compliance have been reported in HGPS patients. Nevertheless, the effect of the specific A-type lamin gene mutation on coagulation and thrombus formation has not been investigated previously.

Methods

28- to 30-week-old male and female transgenic heterozygous LmnaG609G knock-in (HGPS) mice and corresponding wild-type (WT) littermate controls were exposed to photochemically-induced carotid artery endothelial injury to trigger arterial thrombosis. Vascular and circulating levels of tissue factor (TF), plasminogen activator inhibitor (PAI)-1, and von Willebrand factor (vWF) were measured using enzyme-linked immunosorbent assay (ELISA). TF activity assay was also performed on carotid artery homogenates of WT and HGPS animals.

Results

HGPS mice displayed accelerated thrombus formation compared to the WT animals as underlined by a shortened time to occlusion. Although this finding suggests an increased activation of the extrinsic coagulation cascade, no significant differences were found in TF expression and activity in carotid artery lysates. Circulating and vascular expression of the fibrinolytic factor PAI-1 was also found to be similar between WT and HGPS animals. Furthermore, no significant difference in plasma vWF between the two groups was observed.

Conclusions

Our results show an increased arterial thrombotic response in HGPS mice as compared to WT littermates. This novel observation could provide a mechanistic explanation for the increased incidence of acute cardiovascular events observed in HGPS patients. Further studies will be conducted to investigate the molecular mechanism underlying the observed effects, in particular, on the potential involvement of platelets.

Funding Acknowledgement

Type of funding sources: None.
Totum-070 prevents diet-induced hypercholesterolemia in western diet fed mice and inhibits cholesterol uptake in human enterocyte caco2 cell line
Authors: Langhi C , Otero Y F , Le Joubioux F , Guigas B , Peltier S , Sirvent P
Published: October 2022

Introduction

It is well known that hypercholesterolemia is an important modifiable risk factor in the development of cardiovascular diseases. Lowering blood cholesterol level reduces the incidence of atherosclerotic disease. Totum-070 (T070) is a clinical stage polyphenol-rich active substance composed by the association of 5 plant extracts.

Purpose

We assessed the hypothesis that administration of T070 prevents hypercholesterolemia in western diet fed mice and explored the potential mechanisms involved in vitro in human enterocytes.

Methods

C57BL/6 mice were fed either a normal diet (ND) or western diet (WD) for 6 weeks and received either vehicle or T070 (1mg/kg or 3mg/kg) daily by gavage (n=14 mice per group). Food intake, body weight and plasma lipid were monitored along study duration. Liver and feces lipid content were quantified at the end of the study. For in vitro studies, human Caco2 cells were differentiated in transwells for 21 days. To perform cholesterol uptake assay, [1,2-3H(N)]-cholesterol was incubated in apical medium for 1 hour in presence or not of T070 prior to quantification of radioactivity in the cells.

Results

In mice, supplementation with T070 had no effect on food intake during the study. At the end of the in vivo study, body weight in WD-fed mice was increased to 28.3±0.8 g compared to the ND group (24.5±0.4 g, p<0.001). Interestingly, a significant (p<0.01) 29% reduction in body weight gain was observed in the T070 3mg/kg group compared to the WD group at the end of the study. Compared to WD group, total-cholesterol in mice supplemented by T070 was reduced in a dose dependent manner (298±8 mg/dl WD vs 276±9 mg/dl T070 1mg/kg, p=0.19, and 265±8 mg/dl T070 3mg/kg, p<0.05). Furthermore, hepatic steatosis induced by the WD was reduced by 73% (p<0.001) in the T070 3mg/kg group. Hepatic and intestinal gene expression were drastically altered by WD feeding compared to ND, while supplementation with T070 tended to restore normal expression levels for many genes implicated in lipid and lipoprotein metabolism. A significant increase in fecal cholesterol excretion was observed in mice supplemented with T070 3 mg/g compared to the mice fed with WD alone (442.3±151.7 nmol/day WD + T070 3mg/g vs 75.4±5.9 nmol/day WD, p<0.05). Studies in human enterocytes demonstrated that 1 g/l T070 reduced cholesterol uptake by 35% (p<0.001), suggesting that T070 inhibits intestinal cholesterol absorption.

Conclusion

Our results show the beneficial effect of supplementation with Totum-070 to prevent induction of hypercholesterolemia in a nutritional mouse model. Investigation of mechanisms of action in enterocytes demonstrate the role of Totum-070 to inhibit intestinal cholesterol absorption. These results highlight the interest of using Totum-070 for the management of hypercholesterolemia.

Funding Acknowledgement

Type of funding sources: None.
Ceramide-based lipid profiles and the prevalence of type 2 diabetes differ between patients with coronary artery disease and those with peripheral artery disease
Authors: Leiherer A , Muendlein A , Brandtner E , Saely C H , Vonbank A , Mader A , Sprenger L , Maechler M , Jylha A , Laaperi M , Laaksonen R , Maerz W , Fraunberger P , Kleber M , Drexel H
Published: October 2022

Introduction

Serum lipids and metabolic diseases, in particular type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), predict the atherosclerotic diseases coronary artery disease (CAD) and peripheral arterial disease (PAD).

Purpose

The purpose of this study was to investigate in how far a more detailed characterization including serum lipids improves discrimination of PAD from CAD.

Method

A cohort of 274 statin-naïve patients with either PAD (n=89) or stable CAD (n=185) were referred to metabolic screening and were characterized using nuclear magnetic resonance- and liquid chromatography-tandem mass spectrometry based advanced lipid and lipoprotein analysis. Results were validated in an independent cohort of 1239 patients with PAD or CAD.

Results

We found a significant difference in T2D prevalence and in the ceramide-based lipid profile between PAD and CAD patients. However, neither cholesterol-based markers (including LDL-C, HDL-C) and detailed lipoprotein profiles nor the LD status differed significantly between PAD and CAD patients (figure). The difference between ceramide-based lipid profiles of CAD and PAD remained significant also after adjusting for body composition, smoking, inflammatory parameters, and T2D.

Conclusion

We conclude that PAD and CAD differ in ceramide-based lipid profiles and T2D status, but not in other lipid characteristics or metabolic diseases.

Funding Acknowledgement

Type of funding sources: None.
Acute arterial haemodynamics activation of endothelial to mesenchymal transition in long saphenous veins. Impact on vein graft disease
Authors: Ladak S , McQueen L , Joeldavid L , Murphy G , Zakkar M
Published: October 2022

Introduction

The long saphenous vein (LSV) is frequently used in cardiac surgery; however, its use is complicated by late stenosis or occlusion due to the development of intimal hyperplasia (IH). TGF-β has been implicated in the process of IH however the impact of acute haemodynamic changes on the activation of TGF-β endothelial-to mesenchymal transition (EndMT) has not been assessed and it's the focus of this study.

Purpose

To assess the role of acute haemodynamics changes in veins implanted into arterial circulation on EndMT.

Methods

Surplus LSV were exposed to acute arterial haemodynamics using a perfusion bioreactor. Changes in EndMT markers at the RNA and protein level evaluated by quantitative real time PCR, RNAScope and immunofluorescence.

Results

The acute exposure of veins to arterial haemodynamics ex-vivo induced significant increase in inflammatory marker IL-8 expression and transcription factor TWIST1 in LSV (both p≤0.01, Figure 1A) endothelium. Furthermore, immunostaining demonstrated the activation of pSMAD (p≤0.01, Figure 1B) acutely in endothelium after 45 minutes of exposure to arterial haemodynamics. This was followed by significant increase of SMC related markers (Vimentin and α-SMA; Figure 1C, both p≤0.001) and the suppression of endothelial cell related marker CD31 after 4 hours of LSV exposure to acute arterial haemodynamics. RNAScope and IHC results showed localisation of TWIST1 RNA and protein in CD31+ (p≤0.001) and VECAD+ (p≤0.001) cells respectively following exposure to acute arterial haemodynamics (Figure 1D). Furthermore, TGF-β pathway phosphorylation array identified the activation of Smad1 (p≤0.05) but not TAK1 in LSV endothelial cells indicating that acute arterial haemodynamics activates the TGF-β–SMAD pathway specifically (Figure 1E).

Conclusion

The exposure of LSV to acute arterial haemodynamics is associated with the activation of TGF-β–SMAD pathway leading to EndMT changes in the endothelium of vein grafts ex-vivo. This contributes to our understanding of the changes that occur in veins after implantation into arterial circulation and that the acute changes in the endothelium and suggests that strategies to modulate TGF-β–SMAD can be utilised to modulate IH in vein grafts.

Funding Acknowledgement

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Van Geest Foundation Heart and Cardiovascular Diseases Research Fund
Endothelial retargeting of AAV9 in vivo
Authors: Bozoglu T , Lee S , Ziegler T , Kupatt C
Published: October 2022

Background

Adeno-associated virus (AAV) is favorable choice for gene transfer and gene editing applications in vivo, due to its low integration rate and low immunogenicity, as well as a variety of natural and engineered serotypes providing different tissue and cell type tropism. Endothelial cells, however, are remarkably resistant to transduction by AAVs.

Purpose

The aim of this study is to retarget wild type AAV9 vectors using a combination of polyamidoamine dendrimers (PAMAM) and peptides with endothelial affinity.

Methods

An endothelial-affine peptide sequence was obtained via bio-panning of a phage display library on endothelial culture cells. Second-generation PAMAM dendrimers were linked to the peptide via PEG linker. AAV9 vectors were coated with these modified dendrimers (G2CNN) immediately before in vivo applications.

Results

mTmG-mice or mTmG-pigs were utilized as reporter organisms, where red-to green fluorescence change upon Cre expression displays cellular resolution of transduction. Cre encoding AAV coated with G2CNN application systemically (mice) or locally (pigs) transduced over 30% of CD31+ cells in skeletal muscle and heart.

Functional relevance of endothelial retargeting was assessed by systemic injections of G2CNN coated AAV9 encoding for three transgenes:

1) An artificial adhesion molecule (S1FG). In wild type mice, S1FG expression in endothelial cells increased leukocyte adhesion in cremaster muscle vasculature (day 10 post injection).

2) Anti-inflammatory Annexin A1 (Anxa1). In ApoE−/− mice on high-fat diet, Anxa1 expression reduced long term leukocyte recruitment in carotid artery (day 28 post injection).

3) sgRNA targeting the vasodilatory enzyme eNOS (endothelial nitric oxide synthase). In conditional Cas9 transgenic mice, editing of eNOS via AAV delivered Cre and sgRNA caused increase of blood pressure (day 56 post injection).

Conclusion

Endothelial retargeted AAV9 efficiently transduced endothelial cells of skeletal muscle and heart in vivo. Accordingly, in vascular and atherosclerosis models, these modified vectors may enable gene transfer.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Bundesministerium für Bildung und ForschungDeutsche Forschungsgemeinschaft
Prognostic value of PET derived coronary flow reserve: should we correct for resting perfusion product?
Authors: Ahmed A I , Saad J M , Alahdab F , Han Y , Alfawara M , Nabi F , Mahmarian J , Al-Mallah M H
Published: October 2022

Introduction

Positron emission tomography (PET) myocardial perfusion imaging (MPI) derived coronary flow reserve (CFR) is an important risk-stratifying tool to tailor management of patients with coronary artery disease. Resting myocardial blood flow (MBF) is positively correlated to the product of resting heart rate and systolic blood product (resting pressure product – RPP), and can in turn lead to lower CFR in the setting of normal stress MBF.

Purpose

We aimed to assess the comparative incremental prognostic value of uncorrected to RPP-corrected CFR in predicting incident events.

Methods

Consecutive patients who had clinically indicated PET MPI were included. Patients were followed from the date of imaging to incident major adverse cardiovascular event (MACE: inclusive of all-cause death, myocardial infarction, admission for heart failure and late revascularization – percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) >90 days after imaging).

Results

The study population consisted of 2,893 patients with clinically indicated PET MPI (mean age 67 (±12), 53% male, 41% Caucasian). Cardiovascular risk factors were prevalent (88% hypertensive, 80% dyslipidemic, 47% diabetics, 18% ever smoking). After a mean follow-up of 8±7 months, 250 patients (8.6%, 10.7 per 1000 person-year) experienced MACE (90 D/38 MI/51 PCI/11 CABG/113 HF). Both uncorrected and corrected CFR were independently associated with incident MACE (HR 0.38, 95% CI 0.29–0.51, p<0.001 and 0.61, 95% CI 0.50–0.76, p<0.001 respectively). A comparison of model incremental prognostic value showed the model with uncorrected CFR had a statistically higher Harrell's C (0.76 vs 0.77, p=0.23).

Conclusion

Our findings showed that the prognostic value of CFR was preserved even in the setting of high resting blood pressure or heart rate. This suggests that correction for RPP should be selective.

Funding Acknowledgement

Type of funding sources: None.
Problem of implicit assumptions about left ventricular geometry in the calculation of myocardial mass by echocardiography – a validation study with cardiac magnetic resonance
Authors: Winkler A , Sveric K , Doering C , Mierke J , Svitil J , Heidrich F M , Linke A , Ulbrich S
Published: October 2022

Background/Introduction

Cardiac magnetic resonance imaging (CMR) is regarded as the reference method in assessing left ventricular (LV) myocardial mass. However, 2-dimensional echocardiography (Echo) is still used due to availability and practicability.

Purpose

We sought to assess measurements of LV myocardial mass from Echo exams and validate the results with CMR on a large cohort of patients.

Methods

We analyzed 357 patients who underwent clinically indicated CMR examinations and performed a standard Echo at the same day. In Echo, the commonly used Devereux formula (CUBE) was assessed for the calculation of LV mass: 0.8 × {1.04 × [([LV end-diastolic diameter + diastolic septum-thickness + posterior wall-thickness]3 − LV end-diastolic diameter3)]} + 0.6. Mass was indexed on body surface area (g/m2). Correlation (r), regression (R2) and Bland-Altman analysis were performed in order to assess bias between the two methods. Significance was defined as a 2-tailed P value <0.05.

Results

LV myocardial mass was measured in 345 patients with CMR and Echo. The median age was 61 years [19–79], 44% were female and CMR was performed due to coronary artery diseases (45%), suspected or florid myocarditis (31%) or further diagnosis of non-ischemic heart failure (24%). LV ejection fraction (EF) ranged between 13 and 71%. We found a significant relation of measurements for myocardial mass between Echo and CMR (R2=0.6, p<0.001) (Figure 1, left). However, there was a significant proportional bias (R2=0.47, p<0.001) depending in the amount of LV mass measured (Figure 1, right). As expected, the measurement bias correlated not only with the amount of mean wall-thickness (septum & posterior wall) from Echo, but also with LV end-diastolic diameter (r=−0.55 and −0.41, p<0.001 for all). Interestingly, the amount of LV EF correlated reversely with the proportional bias (r=0.34, p<0.001).

Conclusion

The results provided by our study, highlight the problem of implicit assumptions of LV geometry in the calculation of LV myocardial mass by Echo (CUBE) compared to CMR. Furthermore, the strongly biased differences between these two measurement methods are not negligible and warrant further investigation.

Funding Acknowledgement

Type of funding sources: None.
Feasibility and role of echocontrast evaluation of patients with LVAD
Authors: Dellino C , Pergola V , Baroni G , Semeraro L , Mastro F , Arura P , Cecchetto A , Previtero M , Tarzia V , Mele D , Gerosa G , Iliceto S
Published: October 2022

Background

Advanced heart failure is a clinical syndrome characterized by persistent or progressive symptoms of heart failure despite optimal medical therapy. Left ventricular assist device (LVAD) brings survival benefits and improvement in quality of life, compared with conventional medical treatments. Development of Right ventricle failure in patients with LVAD has a direct effect on mortality and hospitalization.

Purpose

Evaluation of clinical safety and feasibility of echocontrast in patients implanted with 3 different types of LVAD; improvement in the visualization of heart structures; intra and inter-operator agreement of RV assesement with and without contrast.

Methods

43 patients were implanted with LVAD, 7 patients (16%) with Jarvik 2000, 31 (72%) with HeartMAte 3, 5 (12%) with (HeartWAre HVAD). Nine patients (21%) had contraindication or refused contrast. In 3 (7%) patients was technically challenging to obtain apical images at all levels. Two (5%) patients lost their follow-up. Our final population was of 29 (67%) patients (mean age 65±7 y; 100% Male). We assessed the reproducibility of these measurements between two different expert blind operators.

Results

Total 329 (64%) of 516 RV wall segments were available for qualitative analysis without contrast vs 451 (87%) with contrast (p<0.001) with a significant improvement of the evaluability of regional contractility (especially due to the better evaluation of medial and apical segments of lateral and anterior walls) and FAC (41% vs 90%, p<0.001). Evaluation of TAPSE, TR and sPAP was similar with and without contrast (p=NS). All the RV parameters showed little inter-operator variability when measured with contrast. TAPSE, FAC, and RWMA showed an excellent reproducibility (ICC >0.86) while it was good for 2D-baseline derived parameters (ICC = 0.74) showing improvement of inter operator reproducibility in the evaluation of regional contractility in the contrast echocardiography modality.

Conclusion

EC is safe with all the types of LVAD examined. Accurate and reproducible visualization of RV is imperative for reliability of information, a routine use of EC could play a pivotal role in interpreting RV features. EC improves RV morphologic and functional judgment allowing greater accuracy and precision in the assessment of both global and regional RV functions. This finding may have important clinical improvement, especially in the future for analysis focused in RV prognostic role in LVAD patients.

Funding Acknowledgement

Type of funding sources: None.
Accuracy of diagnostic score by echocardiography for the detection of cardiac amyloidosis in patients with proven extracardiac amyloidosis
Authors: Bauer F , Fauvel C H , Raitiere O , Si Belkacem N , Colleville B , Mulder P
Published: October 2022

Background

In 2021, a position statement of the ESC Working Group on Myocardial and Pericardial Diseases suggested to use an echocardiographic score (i.e., ECHO score) to confirm cardiac amyloidosis (CA) thus to avoid heart biopsy in patients with left ventricular (LV) hypertrophy >12 mm and proven extracardiac amyloidosis.

Purpose

To test the accuracy of ECHO-score in a broad spectrum of patients with proven cardiac amyloidosis and anatomical LV hypertrophy measured by echocardiography.

Methods

One hundred and twenty patients with proven CA (both from ATTR and AL forms) underwent comprehensive echocardiography including parietal wall thickness (+3 points), Doppler E/e' ratio (+1 point), TAPSE (+2 points), LV global longitudinal strain (1 point) and systolic longitudinal strain apex to base ratio (+3 points) and compared to 13 controls. A score ≥8 points in the presence of LV wall >12 mm which predicts CA, was investigated in patients with LV hypertrophy from 12.1 to 15.0 mm, from 15.1 to 20 mm and over 20 mm. Cardiovascular magnetic resonance (CMR) imaging was available in 63 patients for extracellular volume measurement >0.40% and diffuse late gadolinium enhancement assessment, both present to confirmed amyloidosis.

Results

ATTR and AL amyloidosis accounted for 77% and 23%, respectively. Age averaged 76±7yo and 72% of patients were male. LV ejection fraction averaged 61±5%. The ECHO score averaged 6.4±2.5 in patients with LV hypertrophy from 12.1 to 15.0 mm (n=29), 7.9±2.6 in patients with LV hypertrophy from 15.1 to 20.0 mm (n=59) and 9.8±1.7 when LV hypertrophy was >20 mm (n=32). Figure 1 depicts the sensitivity of ECHO score alone and in addition to CMR as a function of LV hypertrophy range. In patients with LV hypertrophy >20 mm, ECHO score alone has a sensitivity near 100% and CMR is useless to confirm cardiac amyloidosis. Between 15.1 to 20.0 mm of hypertrophy, both ECHO and CMR are necessary to reach a sensitivity of 100%. Unfortunately, below 15 mm of LV hypertrophy, neither ECHO nor CMR altogether predict cardiac amyloidosis with good accuracy, meaning heart biopsy is still useful.

Conclusion

Upon the degree of LV hypertrophy in a patient with extrcardiac amyloidosis, ECHO score alone or in combination with MRI are efficient only if myocardial parietal thickness is above 15 mm.

Funding Acknowledgement

Type of funding sources: None.
Prognostic implications of biventricular and left radial strain in heart failure
Authors: Matshela M R
Published: October 2022

Background

Chronic heart failure (CHF) is associated with the high ventricular filling pressures with subsequent negative impact on the left (LV) and right ventricular (RV), and left atrium (LA) mechanics, and pulmonary vasculature. All these could lead to poor long-term outcome.

Purpose

Assess the prognostic implications of LA, LV and RV mechanical dysfunction, and pulmonary hypertension, in CHF based on advanced echocardiographic technology.

Methods

A total of 2110 CHF patients were retrospectively reviewed and enrolled for the analyses. The 2D-Speckle tracking echocardiographic analysis was used to measure the peak LV, RV and LA longitudinal strain. The long-term outcomes were evaluated for all-cause CVS outcomes and recurrent hospitalization.

Results

For the 941eligible patients [mean age: 61±4; 62% male sex], 244 reached the combined endpoint. Population was divided into three groups based on the mean pulmonary artery systolic pressure (mPASP): Group 1, 25<mPASP≤40 mmHg; Group 2, 40mPASP 55 mmHg. Mean follow-up was 2.5±0.6 years. The higher mPASP was demonstrated in those with higher left atrial volume ndex (LAVI), NYHA ≥II class, pulmonary diseases, presence of at least moderate mitral and/or tricuspid regurgitation (TR) grades, significantly lower LVEF, lower strain values (LA, LV, RV), and tricuspid annular plane systolic excursion (TAPSE), p<0.001). High mPASP, lower LVEF and RV dysfunction were independent predictors of survival after multiple adjustments using different models.

Conclusions

Multiple factors are associated with poor outcomes in CHF; particularly the presence of RV dysfunction, severe PH and severely impaired, which would guide further manage of CHF and to consider other treatment options including device therapy or even transplantation. All these factors could strongly influence the risk stratification of patients with CHF

Funding Acknowledgement

Type of funding sources: None.
Impact of atrial fibrillation on outcome of percutaneous mitral valve repair (PMVR) procedure
Authors: Mohrez Y , Strohmaier J , Paiva T C , Forkmann M , Butz S , Acil T , Schnupp S , Mahnkopf C , Busch S
Published: October 2022

Background

Limited evidence exists regarding the impact of atrial fibrillation (AF) on outcomes after percutaneous mitral valve repair (PMVR). The aim of the present study was to compare 1-year clinical outcomes following PMVR in patients with and without AF.

Methods

426 patients with PMVR for severe MR were included in the COburg MItraclip (COMI) registry between 2016 and 2021. Patients were divided into an AF group (group A, n=284) and a non-AF group (group B, n=142). Clinical profiles, PMVR procedure characteristics and the clinical follow-up were compared between the two groups.

Results

Patients with AF were older (79.3±6.3 [AF] vs. 77.8±8 years [non-AF], p=0.03), had higher N-terminal B-type natriuretic peptide levels (NT-pro BNP: 5675±5544.2 vs 8516±11184.5, p=0.004), more often a coronary artery disease (62% vs 38%, p=0.03) and had a lower stroke risk (CHA2DS2-VASc score: 4.6±1.2 vs 4.9±1.3, p=0.01). There were no significant differences between groups with regard to the gender, logistic Euroscore or prevalence of chronic heart failure, hypertension, diabetes mellitus or severe mitral regurgitation. Acute procedural success (post-procedural 2. MR, 95.1% vs. 94.4%; p=0.76) and procedure-related major complications were similar.

There were no significant differences in 1-year MACCE (death, MI, stroke, bleeding), health-related quality of life or clinical improvement.

Conclusion

In patients with mitral regurgitation who underwent PMVR, AF is common and appears not to have a relevant negative impact on the clinical outcome at 1 year compared to patients with sinus rhythm.

Funding Acknowledgement

Type of funding sources: None.