bluuebunny/crossref_metadata_2025 huggingface dataset:
S100A9 blockade ameliorates cardiac dysfunction and reduces myocardial immune infiltration in experimental autoimmune myocarditis
Authors: Jakobsson G , Mulholland M , Grentzmann A , Ljungcrantz I , Rattik S , Engelbertsen D , Schiopu A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Swedish Heart and Lung Foundation and Swedish Research Council

Background/Introduction

Autoimmune myocarditis is characterized by non-ischemic inflammatory heart injury, leading to myocardial damage and impaired cardiac function. S100A9 is an inflammatory alarmin present in large amounts in neutrophils. The role of S100A9 as a potential treatment target in myocarditis has not been fully explored.

Aim

We sought to investigate the impact of S100A9 blockade in experimental autoimmune myocarditis and assess the effects of treatment on cardiac function and immune infiltration.

Methods

BALB/C mice were immunized with αMHC peptide emulsified in Complete Freunds Adjuvant at day 0 and day 7 to induce the disease. The water-soluble small-molecule S100A9 blocker ABR-238901 was given continuously in drinking water starting on day 7. Echocardiography was performed weekly from day 21 to day 42 (n=11/group). For flow cytometry analysis, mice were sacrificed at day 21 (n=10/group).

Results

S100A9 blockade improved left ventricular ejection fraction [52.33% vs 44.87% on day 42 (p<0.001)]. Cardiac output was significantly improved on day 21 (12.69 vs 11.05 mL/min, p<0.05). We found a significant reduction of inflammatory cardiac infiltrates at day 21, characterized by reduced number of macrophages (p<0.05), neutrophils (p<0.01) and CD4+ T cells (p<0.05). The cardiac draining lymph nodes contained fewer dendritic cells (p<0.01), T cells (p<0.05), as well as reduced numbers of inflammatory CD4+ cells producing IL-17 (p<0.05).

Conclusion

Therapeutic S100A9 blockade inhibits inflammatory cardiac infiltration and improves cardiac function in experimental autoimmune myocarditis. Our findings highlight the important role of S100A9 in the pathogenesis of myocarditis and identify S100A9 blockade as a possible novel therapeutic avenue.
Collagen bundle orientation by Fourier analysis in myocardial infarction scarring
Authors: Marcos Garces V , Rios-Navarro C , Gomez-Torres F , Gavara J , De Dios E , Perez N , Diaz A , Minana G , Chorro FJ , Bodi V , Ruiz-Sauri A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” and Conselleria de Educación – Generalitat Valenciana.

Introduction

After acute myocardial infarction (AMI), the lack of oxygen and nutrients leads to cardiomyocyte necrosis and eventually to the formation of a collagen-based scar. Infarct scar characteristics, such as collagen bundle orientation, have a relevant influence on scar mechanics, the occurrence of cardiac arrhythmias, left ventricular dilation or aneurysm formation, wall stiffness, and the development of wall rupture or heart failure. However, the most adequate method for collagen bundle orientation (CBO) measurement in myocardial scar is not established.

Purpose

We aim to compare the measurement of collagen bundle orientation in infarct scar by Fourier analysis in three different histopathological techniques.

Methods

Juvenile swine (n=21) were subjected, by means of percutaneous balloon inflation, to a transient 90-min occlusion of mid left anterior descending artery followed by one month of reperfusion (chronic AMI group). Samples were obtained from the infarcted zone and stained with Masson’s trichrome, Picrosirius red and Haematoxylin-Eosin (H-E) standard protocols. Five microphotographs of the myocardial scar were taken at 200x magnification with light, polarised and confocal microscopy, respectively. A single observer measured CBO by means of Fast Fourier Transform analysis using a semi-automated protocol. Comparability between techniques was studied by the Intraclass Correlation Coefficient (ICC), the coefficient of variation (CV) and the Bland-Altman (B&A) plots and limits of agreement.

Results

Measurement of CBO in Masson’s trichrome tended to show higher (more "random-oriented") values than in Picrosirius and H-E+confocal techniques (ICC 0.79 and 0.7, p=0.001 and 0.005; B&A 0.29 to -0.02 and 0.43 to 0.01; CV 6.97% and 12.98%, respectively). However, measurement of CBO in Picrosirius and H-E+confocal techniques showed an "almost perfect" agreement (ICC 0.84, p<0.001; B&A 0.28 to -0.09; CV 17.33%). Selective staining and/or visualization of collagen in these latter techniques may underlie our findings, contrary to non-selective Masson’s trichrome.

Conclusion

Picrosirius red staining (visualized with polarised microscopy) and Haematoxylin-Eosin (visualized with confocal microscopy) are comparable in terms of collagen bundle orientation measurement by Fourier analysis in an animal model of chronic infarct scar. Masson’s trichrome (visualized with light microscopy) tends to show more "random-oriented" values, potentially due to non-specific staining and visualization of non-collagenous structures such as cells, and should not be recommended for this specific purpose.
Mice deficient in endothelial cell-selective adhesion molecule develop left ventricle diastolic dysfunction
Authors: Buncha V , Fopiano KA , Tian Y , Lang L , Bagi Z
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): American Heart Association

Background

Dysfunction of vascular endothelium plays a key role in the development of left ventricular diastolic dysfunction (LVDD) in patients with heart failure with preserved left ventricular ejection fraction (HFpEF). Recently, Endothelial Cell-Selective Adhesion Molecule (ESAM) was proposed as a novel biomarker in HFpEF.

Hypothesis. ESAM plays a mechanistic role in the development of both microvascular dysfunction and LVDD.

Methods

Animals: The study was performed utilizing ESAM knockout and wild type (WT) mice. Echocardiography: Left ventricular (LV) systolic and diastolic function and LV wall thickness were assessed by small animal ultrasound imaging (Vevo3100). Blood pressure measurement: Performed with tail-cuff plethysmography method. Vascular reactivity: Pulmonary arteries (2nd and 3rd order) were isolated from ESAM KO and WT mice, vascular reactivity was assessed with wire myography method. Evaluation of myocardial vascular density: Paraformaldehyde fixed paraffin embedded heart section (40 µm thick) from WT and ESAM KO mice stained with Tomato Lectin (Lycopersicon esculentum) DyLight594 to label vasculature were used. Unbiased automated tracing of the microvasculature was performed using the VesseLucida360 software (MBF), followed by a morphometric analysis (VesseLucida Explorer).

Results

Transthoracic echocardiography analysis showed that ESAM knockout mice displayed LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and increased E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure was not different between ESAM knockout and WT mice. Heart weight, related to body weight is increased in ESAM-deficient mice, pointing towards cardiac hypertrophy in these animals. Meanwhile, the analysis of the cardiac tissue vascularization with unbiased automated tracing of the microvasculature, reveals the decreased total vascular length per volume of the tissue in ESAM knockout mice. Using wire myography of the pulmonary artery vasodilator function we found that the endothelium-dependent, acetylcholine-induced relaxation of these arteries was significantly reduced in ESAM knockout mice, whereas endothelium-independent, nitric oxide donor, sodium nitroprusside-induced relaxation was similar in the two groups.

Conclusion

ESAM deficiency in mice resembles a HFpEF phenotype, in that ESAM knockout mice develop endothelial dysfunction and LVDD.
Optogenetic manipulation of cardiac electrical dynamics using sub-threshold illumination: dissecting the role of cardiac alternans in terminating rapid rhythms
Authors: Biasci V , Santini L , Hussaini S , Ferrantini C , Coppini R , Loew LM , Luther S , Campione M , Poggesi C , Pavone FS , Cerbai E , Bub G , Sacconi L
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ions channel recovery and intracellular Ca2+-cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose a novel approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination) but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsins-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We use an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role that cardiac alternans plays in spontaneous termination of ventricular tachycardias (VTs). We find that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT.
Size matters: Functional differences of progenitor cell-derived small extracellular vesicle subpopulations in cardiac repair
Authors: Van De Wakker S , Bauza Martinez J , Rios Arceo C , Willms EF , De Jong OJ , Wu W , Vader P , Sluijter JPG
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC EVICARE, Van Herk Foundation

Introduction

The use of cardiac progenitor cell (CPC)-derived small Extracellular Vesicles (sEVs) has shown great potential to stimulate cardiac repair post myocardial infarction. sEVs are released by cells and play a role in intercellular communication through transfer of their bioactive content, like RNAs and proteins. Increasing evidence indicates that sEVs present a heterogeneous mixture of vesicle populations, depicting different functional aspects, which hampers clinical translation. Studying sEV heterogeneity could provide new insights into contributing therapeutic mechanisms underlying sEV-mediated cardiac repair.

Methods

CPC-derived sEVs were purified by flow through chromatography followed by size-exclusion chromatography (SEC) and asymmetric flow field flow fractionation (A4F) for fractionation of different sEV-subfractions. Based on the differential expression of common sEV markers, different subpopulations were determined. sEV subpopulations were further characterized using western blot, nanoparticle tracking analysis, bicinchoninic acid protein assay, transmission electron microscopy and mass spectrometry (MS). Functional differences were studied using different cellular assays to determine AKT phosphorylation, wound healing, angiogenesis and proteasome activity.

Results

SEC or A4F were used to separate distinct subpopulations of CPC-derived sEVs with a different appearance, size and function. MS analysis confirmed the differences in proteomic expression levels of classical sEV markers, as well as annexins, rab proteins, integrins, histones and proteasomal proteins. Furthermore, differences in cellular components linked to their cellular origin were found. Exposure of recipient endothelial cells to sEV subpopulations demonstrated clear functional differences. Additionally, differences in proteolytic activity of the sEV subpopulations were identified.

Conclusions

SEC and AF4 allow for isolation and in-depth study of the functional heterogeneity of sEVs. In our study, we observed the presence of different subpopulations based on size, with a different composition and biological function. Increased knowledge of sEV heterogeneity will contribute to a better understanding of the mechanisms of action of sEVs and will improve translation to the clinic and potentially an off-the-shelf approach to stimulate cardiac repair.
HDL adverse remodelling and dysfunction is a temporal feature that can be reversed
Authors: Schoch L , Sutelman P , Padro T , Badimon L , Vilahur G
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e Innovación (MCIN)/AEI/10.13039/501100011033 and Fondo Europeo de Desarrollo Regional (FFEDER)

Background

High-density lipoprotein (HDL) particles have been shown to undergo adverse remodelling and loss of function in the presence of comorbidities. It has been demonstrated that high low-density lipoprotein cholesterol (LDL-C) plasma levels induce compositional changes in HDL particles that render them dysfunctional. It is unknown, however, whether this HDL adverse remodelling is reversible.

Purpose

In this preclinical animal study we have investigated whether dyslipidaemia-induced HDL particle remodelling and dysfunction is a permanent adverse change or a temporal trait which can be regulated by dietary intervention.

Methods

After 10 days of high-fat diet (HFD) and reaching LDL-C levels of 392.8±94.9 mg/dL, the HDL particles of 15 pigs were characterized. Thereafter, pigs were randomly distributed into two arms. While group 1 was maintained on the same HFD (n=9), group 2 was switched to a normocholesterolemic diet (NCD; n=8) up to day 40. Blood was drawn at days 0 (baseline), 1, 3, 5, and 10 (to characterize HDL dysfunction over the 10-day period of dyslipidaemia induction), and at days 25 and 40 (to assess the HDL particle evolution in the intervention period). We assessed: a) HDL function [cholesterol efflux capacity (CEC) and antioxidant potential]; b) HDL particle number and size by nuclear magnetic resonance; c) oxidative susceptibility of LDL particles; d) HDL protein content of APOA-I, APOM, and APOC-III; and e) biochemical parameters.

Results

Up to day 10 there was a time-dependent increase in LDL-C levels that progressively induced the impairment of HDL CEC and antioxidant activity, higher HDL particle numbers, increased formation of conjugated dienes, and lower HDL-APOM content (p<0.01 vs baseline). All these parameters exhibited a plateau at day 10 and remained unchanged up to day 40 in those animals that were kept on HFD. Interestingly, in these animals a strong negative correlation was observed between HDL CEC and HDL-C levels (p<0.0001), with larger HDL particles being detected at day 40 (p<0.05 vs. baseline). On the contrary, all these parameters returned to physiological levels (baseline) in those animals that switched to a NCD (p=ns vs baseline). No changes were detected in APOA-I- and APOC-III- HDL content among animal groups and over time.

Conclusion

In the present study we evidence for the first time that dyslipidaemia-induced HDL particle remodelling and dysfunction is not a permanent adverse change. The implementation of a healthy dietary intervention acutely reverses HDL adverse remodelling and dysfunction in a preclinical animal model.
Expression of MMP2, MMP9 and PGP 9.5 in human aorta in patients with coronary artery disease
Authors: Patel N , Chandrashekhar R
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Atherosclerosis has been recognised as inflammatory conditions which involves activation of MMPs of which increased MMP2 & MMP9 expression has been correlated with the development of human aortic plaques.The Sympathetic Nervous System exerts complex control over inflammation in atherosclerosis both: on systemic scale and at the regional level.Protein gene product9.5 (PGP9.5) is a cytoplasmic neuron & is a reliable marker for visualisation of the cardiovascular system innervation.There have been no studies in the existing literature on the expression of PGP 9.5 and MMP2, MMP9 on human aorta.Aim:To study the expression of MMP2, MMP9 and PGP9.5 in the human aorta & gain insights into atherosclerosis & neural modulation in the aorta.Materials &

Methods

This is a single centre, prospective cohort, pilot study with a sample size of 87 patients,done over 2 years at AIMS,Kochi.During CABG, punch biopsy of the aorta taken & processed.One paraffin embedded section used for routine H&E staining & others used for IHC.IHC detection kits use 3, 3’-DABChromogen as a substrate of HRP for visualisation of antigenic structures in the tissues.Positive & negative control tissue were run for all 3 Antibodies.Interpretation done as Diffuse/Focal & Intense/Moderate/ Weak classification based on intensity signals observed on IHC by an experienced Cardiac Pathologist.Results:7 Controls with well matched baseline characteristics: Controls-Aorta samples taken from patients who were undergoing Aortic Valve Surgery for Aortic Valve disease with normal coronaries documented on a coronary angiogram.65.9% of patients with Positive MMP2 & 28.6 % of patients in the Control group was found to have significant difference when compared to positive & negative outcomes amongst the 2 groups.89.3% of patients with positive MMP2 had significant co-relation with PGP9.5 levels expressed in the same specimen analysed.MMP9 also had a p value of 0.002 suggesting a significant co-relation between MMP9 & PGP9.5.Statistically significant Co-relation seen between the Age, CRP & the levels of MMP 9 expressed in the Aorta observed.MMP2 & MMP9 expression have a definite & significant co-expression with PGP9.5 expression in human aorta,suggesting a novel marker of neuromodulation in atherosclerotic process in human aorta.

Expression of MMP2, MMP9 and PGP 9.5 in human aorta in patients with coronary artery disease
Targeting BACE1 to restore functional angiogenesis in type 2 diabetes
Authors: Clavane E , Cubbon RM , Caolo V , Meakin PJ
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

British Microcirculation and Vascular Biology Society

Background

β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease notorious for its contribution to amyloid plaque formation in the pathophysiology of Alzheimer’s disease (AD) [1]. Further research has suggested a role for BACE1 in vascular homeostasis [2,3] and has shown that it proteolytically cleaves various angiogenic signalling factors including VEGF receptor 1 (VEGFR1) [2], NOTCH ligands [4] and the insulin receptor [5]. Similar to AD, BACE1 activity is elevated in models of type 2 diabetes [5], suggesting a potential role for its contribution to aberrant vessel growth characteristic of diabetes-related complications.

Purpose

Type 2 diabetes dramatically increases an individual's risk of developing microvascular complications and consequent lower limb amputations [6]. Therefore, identifying novel roles for BACE1 in angiogenic dysregulation will aid progression of future biomedical interventions in this field.

Methods

Retinal staining and the fibrin gel angiogenesis assay were used to identify a role for BACE1 in vessel growth in vivo and in vitro, respectively. Endothelium of the developing retinal vasculature in BACE1-/- and wild type (WT) mice was stained with IsolectinB4-Alexa488 and imaged using confocal microscopy. Sprout formation was further analysed using the fibrin gel angiogenesis assay with human umbilical vein endothelial cells (HUVECs) treated with or without a highly specific BACE1 inhibitor or transfected to over-express BACE1. Primary isolated pulmonary endothelial cells (PECs) were isolated from BACE1-/- and wild type control mice prior to Western blots, and real-time PCR.

Results

BACE1-/- retinas had increased branch points, vasculature area and quantity of filopodia compared to WT mice. Moreover, BACE1-/- PECs had reduced NOTCH1 signalling (26.73% ± 14.15, P=0.05) and soluble Jagged-1 protein (28.48% ± 14.61, P=<0.05). HUVECs treated with a BACE1 inhibitor had increased sprouting (18.70%± 5.92, P=<0.05) as well as increased phosphorylation of eNOS (83% ± 22, P=<0.05) and Akt (85.5% ± 9.24, P=NS) compared to untreated cells. Moreover, HUVECs transfected to over-express BACE1 had decreased sprouting (35.22% ± 7.34, P=<0.01) and increased NOTCH1 signalling (23.4% ± 2.42, P=0.01).

Conclusion

Our findings indicate a role of BACE1 in negatively regulating angiogenesis, possibly via NOTCH1 or Akt/eNOS/NO signalling. This provides a potential therapeutic purpose for BACE1 inhibitors, previously trialled to treat AD, in normalising BACE1 levels in individuals with type 2 diabetes and preventing associated microvascular complications.
Deleterious effect of serum from stemi patients on endothelial cell viability: implication on the resulting cardiac structure
Authors: Rios Navarro C , De Dios E , Gavara J , Perez-Sole N , Marcos-Garces V , Chorro FJ , Ruiz-Sauri A , Bodi V
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by “Instituto de Salud Carlos III” and “Fondos Europeos de Desarrollo Regional FEDER” (grant numbers PI20/00637 and CIBERCV16/11/00486) and Conselleria de Educación – Generalitat Valenciana (PROMETEO/2021/008).

Aim

To evaluate the potential deleterious effect of serum from STEMI patients on cardiovascular magnetic resonance (CMR)-derived edema, hemorrhage and microvascular obstruction as well as on the grade of permeability in vitro by promoting endothelial cell apoptosis and necrosis in vitro.

Methods

Human coronary artery endothelial cells (HCAEC) were incubated with serum isolated 24h post-reperfusion from 43 STEMI patients submitted to CMR and from 10 control subjects. Using a multidisciplinary approach on HCAEC, the effect of serum from STEMI patients on the activation of apoptosis and necrosis as well as on changing the permeability and structure of the endothelial monolayer was assessed. The association between the serum-induced apoptosis and necrosis in vitro on HCAEC and CMR-derived edema, hemorrhage, and microvascular obstruction as well as the grade of permeability in vitro was also dissected.

Results

Serum from STEMI patients activated apoptosis (p<0.01) and necrosis (p<0.05) in HCAEC, evaluated by flow cytometry, enzymatic, genetic, and morphological analysis, and also provoked an increased in the permeability of the endothelial monolayer in vitro (p<0.01), due to enlarged intercellular spaces (p<0.05). Those patients whose serum induced higher necrosis in vitro displayed extensive CMR-derived edema (p<0.01), hemorrhage (p<0.01), and microvascular obstruction (p<0.05) as well as more endothelial permeability in vitro (p<0.05).

Conclusions

By activating apoptosis and necrosis on endothelial cells, serum from STEMI patients participates in the appearance of edema, hemorrhage, and microvascular obstruction as well as in increasing endothelium permeability. The negative effect of serum on the endothelial barrier can contribute to the adverse cardiac structure post-STEMI.
Complement C5 plasma levels are associated to abdominal aortic aneurysm prevalence and progression
Authors: Martinez-Lopez D , Mendez-Barbero N , Roldan-Montero R , Cerro-Pardo I , Picatoste B , Ortega-Villanueva L , Rodriguez De Cordoba S , Blanco-Colio LM , Linhdolt JS , Martin-Ventura JL
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Complemento II-CM

La Caixa

Aim

Previous human and experimental studies have suggested a role of complement activation in abdominal aortic aneurysm (AAA).  We recently described an increase in complement C5 levels in plasma of subjects with subclinical atherosclerosis (1). Our aim was to investigate the potential association of C5 with AAA presence and progression.

Material and methods

Tissue and tissue-conditioned media from AAA (thrombus and wall) or healthy samples were obtained from surgical repair or brain-deceased organ donors, respectively. Blood samples from 490 AAA patients (maximal aortic diameter ≥30 mm) were obtained within a population-based ultrasound-screening trial in Danish men and from 176 age-matched screened negative controls. C5 protein levels were assessed by immunohistochemistry and ELISA.

Results

High C5 positive immunostaining was observed in AAA thrombus and media, while the staining in healthy arteries was faint. C5 levels were higher in tissue-conditioned media from AAA thrombus and AAA media as compared to healthy arteries (7.1±1 vs 2.9 ±0.5 vs 1.2±0.2ug/ml, p<0.0001 for both). C5 plasma levels in AAA patients were higher than in controls (116±8 vs 61±7 ug/ml, P<0.001). AAA patients at the upper tertile of C5 at baseline had a 25% higher risk of needing surgical repair during the follow-up (Hazard Ratio=1.25, 95% confidence interval, 1.045;1.511, P<0.05).

Conclusions

C5 is associated to AAA presence and progression suggesting its potential use as a prognostic marker. Future studies are needed to clarify the pathogenic role of C5 in AAA.
Identification of CD8+ T cell PRDM1 in high-risk human plaques and its regulatory role in murine lesion development
Authors: Maas SL , Jin H , Lu C , Nagenborg J , Manca M , Karel JMH , Cavill R , Waring O , Sikkink CJJM , Mees BME , Daemen MJAP , Smirnov E , Sluimer J , Van Der Vorst EPC , Biessen EAL
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fritz Thyssen Stiftung

T cells have a prominent role in the pathogenesis of atherosclerosis, although their function in atherosclerotic plaques is only partly understood. In this study, we utilize the advantages of high-throughput techniques and data analytic strategies to compare the inherent biological changes of T cells during plaque transition from a stable, non-haemorrhaged (low-risk) to a rupture-prone, haemorrhaged (high-risk) phenotype.

We classified 43 human carotid arterial lesions into high- and low-risk plaques based on the presence/absence of intraplaque hemorrhages. RNA from these lesions was isolated and microarray gene expression data was obtained and analyzed by Weighted Gene Co-expression Network Analysis. A strong T cell signalling signature was identified in high- versus low-risk plaques, influencing angiogenesis and interferon-related processes. Bayesian network inference, cell type deconvolution and single-cell RNA sequencing analysis revealed that the T cell-associated gene program was linked to effector-memory cytotoxic, CD8+ T cells. This gene program appeared driven by CD8+ T cell-related transcription factors, including RUNX3, IRF7 and most importantly PRDM1. To validate these findings, we demonstrated in a murine model that T cell PRDM1 plays a key role in plaque formation, as atherosclerotic mice with a T cell specific Prdm1 deficiency developed larger and more advanced atherosclerotic plaques compared to control mice.

In conclusion, our study unveils a clear PRDM1-regulated effector-memory cytotoxic CD8+ T cell footprint in plaque development and the shift from low- to high-risk plaques, thereby revealing CD8+ T cells and PRMD1 as potential targets for intervention in adverse T cell responses in human atherosclerotic lesions.
Oscillatory shear stress augments endothelial pannexin1 by inhibiting macro-autophagy
Authors: Molica F , Hautefort A , Idris T , Pelli G , Foglia B , Kwak BR
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Introduction

Atherosclerotic lesions preferentially develop in arterial regions exposed to disturbed blood flow, which are characterized by a dysfunctional proinflammatory endothelial cell (EC) phenotype. ECs release ATP in response to changes in wall shear stress (WSS), which subsequently regulates the inflammatory response. ATP can be released from cells in a controlled manner through Pannexin1 (Panx1) channels.

Objective

To study the expression of Panx1 in response to WSS and its role in the endothelium.

Methods

Human ECs (HUVECs or EA.hy926) were exposed to physiological high laminar shear stress (HLSS) and atheroprone oscillatory shear stress (OSS) for 48h using an orbital shaker. Panx1 transcript and protein levels were determined by qPCR and Western blot, respectively. mRNA was extracted for RNAseq. WSS-modifying casts were placed for 1 week around the carotid artery of mice and Panx1 expression was analyzed en face. Carotid WSS-modifying casts were also placed in Tie2CreTgPanx1fl/flApoE-/- and Panx1fl/flApoE-/- mice followed by 9 weeks high fat diet. CD68+ cells were quantified in OSS-induced atherosclerotic lesions.

Results

We found that Panx1 expression was increased in carotid regions exposed to OSS as compared to HLSS regions. These results were confirmed in vitro in HUVECs. In silico analysis of the promotor of Panx1 revealed binding sites for the WSS-sensitive transcription factors NF-kB and CREB. NF-kB and CREB mRNA levels were similar under OSS and HLSS, however NF-kB activation (phospho-NF-kB) was detected in ECs under OSS. Surprisingly, Panx1 mRNA level was not affected under these conditions, suggesting that the increased Panx1 protein observed under OSS may be due to decreased Panx1 degradation rather than to increased synthesis of the protein. Unbiased analysis of differential gene expression in ECs exposed to HLSS or OSS revealed 320 up-regulated and 353 down-regulated genes under OSS. Down-regulated genes included proteins involved in macro-autophagy. Inhibition of macro-autophagy in ECs by exposure to chloroquine for 6h increased the expression of glycosylated Panx1, suggestive for more Panx1 channels at the plasma membrane under these conditions. Finally, we observed that atherosclerotic lesions in OSS regions of Tie2CreTgPanx1fl/flApoE-/- mice contained more CD68+ cells than Panx1fl/flApoE-/- controls.

Conclusion

Endothelial Panx1 expression is upregulated in response to OSS. Absence of OSS effects on NF-kB, CREB and Panx1 mRNA revealed that the upregulation of Panx1 protein is not due to transcriptional effects of OSS. Expression of genes involved in the macro-autophagic process were down-regulated under OSS, and chemical inhibition of macro-autophagy augmented the plasma membranous form of Panx1. OSS-induced decrease in autophagic flux may enhance ATP release through Panx1 channels, thereby counterbalancing leukocyte recruitment in atherosclerosis.
Comparison of lung functions of normal and hypertensive individuals
Authors: Oza Patel F , Vyas HARITA , Tanna KRIMA
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Introduction

Hypertension is a medical condition in which the blood pressure is chronically elevated. It normally refers to arterial hypertension. In past years, the diastolic value was emphasized in assessing HTN. However elevation SBP is also associated with increased incidence of coronary and cardiovascular diseases (e.g. stroke). Therefore both SBP and DBP value are important to note.

Pulmonary function test measures the function of lung capacity and lung and chest wall mechanics to determine whether or not the patients have a lung problem. Lung functions tests are commonly referred as —PFTs||. When a patient is referred for PFTs, it means that a battery of tests may be carried out including: simple screening spirometry, static lung volume measurement, diffusing capacity of carbon monoxide, airway resistance, respiratory muscle strength and arterial blood gases. Forced vital capacity (FVC) is the maximum volume of air which can be exhales or inspired during forced maneuver. Forced expired volume in one second (FEV1) is the volume of air expired in the first second of maximal expiration after a full inspiration. The total volume of air that is exhaled during forced expiration is called FVC. It is the FEV1 expressed as percentage of FVC and give the clinically useful index of airflow limitation. The greatest flow rate achieved during forceful expiration are called Peak Expiratory Flow Rate (PEFR) and usually taken as an indicator of larger airway resistance.

Aim

To compare the lung functions of normotensive and hypertensive individuals.

Methodology

Study Design: Cross-sectional Study

Study Duration: Total study duration was 6 months.

Subjects: 30 subjects

Sampling Design: Purposive sampling

Procedure- Ethic's approval and written informed consent of the subjects was taken prior to study. Procedure and purpose of the study was explained to the participants. In this study male and female between age group of 35 to 65 years who were Non-smokers and willing to participate were included. Subjects who were Smokers or who had URTI, any cardio Respiratory Illness or Any major Neuromuscular or musculoskeletal disorders were excluded. Participants were divided into two groups: one who was having HTN and other who were normotensive.

Outcome measures:

FEV1 % of predicted

FVC% of predicted

PEFR% of predicted

Results

Statistical analysis was done by SPSS version 16.Mann-Whitney U test was applied to see difference of lung function in hypertensive and normotensive individuals. Pulmonary function test parameters in hypertensives were statistically significantly lesser than normal controls (p<0.05).

Conclusion

Lung functions of hypertensive individuals shows significant reduction as compared to the normotensive individuals. Thus, it can be concluded that persistent high blood pressure is responsible for altered pulmonary functions.
Necroptosis, unlike pyroptosis mediates right ventricular damage in pulmonary arterial hypertension
Authors: Jarabicova I , Horvath C , Velasova E , Bies Pivackova L , Veteskova J , Klimas J , Krenek P , Adameova A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic

The Slovak Research and Development Agency

Introduction

Necroptosis has been found to underlie myocardial damage in various pathological states, however its role as well as its potential interconnection to another cell damaging processes under conditions of pulmonary arterial hypertension (PAH) remains insufficiently understood.

Purpose

In view of these facts, we aimed to investigate the signalling of both necroptosis and pyroptosis in the lung and right ventricular (RV) tissue affected by PAH and we also examined the circulating levels of receptor-interacting protein kinase 3 (RIP3).

Methods

Male Wistar rats were administrated either vehicle (Control group) or monocrotaline (MCT) (60 mg/kg, s. c.) to induce PAH. The MCT-treated animals were sacrificed 4 weeks after the treatment (MCT group) or prematurely, based on rapid health deterioration, thereby indicating the advanced stage of PAH (ptMCT group). Pulse oximetry, gravimetry and evaluation of myocardial damage markers were used to assess the development of PAH. The molecular analyses (ELISA, RT-qPCR and immunoblotting) were used to examine the potential underlying mechanisms of tissue damage due to cell death.

Results

In the RVs, the expression of both pThr231/Ser232-RIP3 and pSer345-mixed linkage kinase domain-like protein was elevated equally in both PAH stages, strongly indicating promotion of necroptosis. Contrary in the lungs affected by PAH, the upregulation of pThr231/Ser232-RIP3 likely proceeded to pyroptotic rather than necroptotic cell death activation, as evidenced by caspase-1 and N-terminal gasdermin D increase. Moreover, we found that the plasma RIP3 levels increased with the severity of PAH stage and positively correlated with RV hypertrophy, but not with cardiac hemodynamic stress markers.

Conclusions

In summary, we showed for the first time that PAH-induced damage of RV and lung tissue might be mediated by different necrosis-like cell death forms with a crucial role pThr231/Ser232-RIP3. Furthermore, we suggested that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac damage due to PAH.
Evaluation of the quality of screening for hypertension
Authors: Bekbergenova ZH , Derbissalina G , Umbetzhanova A , Batarbekova SH , Masharipova A , Mauletbayeva G , Akhmetova D , Kelimberdiyeva E
Published: June 2022

Funding Acknowledgements

Type of funding sources: None. Main funding source(s): -

Background/Introduction

Hypertension is one of the most common causes of myocardial infarction and stroke in the Republic of Kazakhstan (RK). In RK, screening for hypertension is performed with 40-years of age.

Purpose

To assess the quality of the screening for hypertension.

Methods

A survey of 354 patients with hypertension identified at screening was carried out.

Results

The average age of patients is 54 years [Q1-Q3: 50-54], of which 36.2% are men, 63.8% are women. In 66.7% of patients, heredity is burdened. According to the analysis of medical records, 117 (33.1%) patients smoke, while polling the same patients, 175 (49.4%) people smoke, which is 16.3% higher. According to the analysis, 38 (10.7%) patients drink, when we interviewed the same patients, 62 (17.5%) people drink, which is 6.8% higher. According to the analysis, 219 (61.9%) patients perform daily physical activity, according to our data, 111 (31.4%) people, which is 30.5% lower.

Middle systolic blood pressure is 140 [Q1-Q3: 130-140] mm Hg. Most of the respondents have varying degrees of obesity. The average body mass index is 31.86 [Q1-Q3: 29.4-31.86]. The average waist is 95 [Q1-Q3: 92-95]. Mean values of total cholesterol. 5.8 [Q1-Q3: 5.6-5.8]. During the electrocardiographic study, changes were identified in 69 patients (32 women, 37 men).

Conclusion (s)

During the initial questionnaire survey and clinical examination of patients with arterial hypertension at screening in 2015 and comparison with retrospective data, it became clear that such risk factors as tobacco smoking, alcohol consumption and daily physical activity were unreliable. Unfortunately, our work also has limitations and may be subject to subjective error, for example, we were unable to establish contact with the patient and a trusting relationship with the patient, or memory errors.
Diurnal variation in thrombotic and fibrinolytic status in healthy volunteers
Authors: Gue Y , Memtsas V , Kanji R , Gorog D
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): East and North Hertfordshire NHS Trust

Background

Previous studies assessing markers of fibrinolysis in healthy volunteers and patients with coronary artery disease (CAD) indicate circadian variation, primarily attributable to changes in plasminogen activator inhibitor-1 (PAI-1) concentrations, the main inhibitor of fibrinolysis, peaking between midnight and 06:00 hrs, with a nadir at 01:00 hrs. Whether the elevated PAI-1 translates into a global impairment of fibrinolysis, is not known.

Purpose

It was our aim to assess diurnal variation in global fibrinolytic activity in healthy subjects.

Methods

A single centre, prospective, observational study was conducted in healthy volunteers. Blood samples were obtained at two different time points on the same day, with a minimum inter-sample interval of 8 hours and a maximum of 12 hours. Venous blood was assessed to determine global thrombotic and fibrinolytic status in native whole blood status using the automated, point-of-care Global Thrombosis Test (GTT). This utilises non-anticoagulated blood to assess the time taken to form an occlusive thrombus under high shear (occlusion time) and the time taken for spontaneous restart of flow as a measure of endogenous fibrinolysis (lysis time).

Results

A total of 25 healthy volunteers (36% male), aged 34±12 y were included. All were non-smokers with a BMI of 25 (22.3 – 27.5) kg/m². The median interval between samples was 8:05 [hh:mm] (IQR 8:30 – 9:10). Occlusion time was similar in the morning and evening samples (median 451s [389 – 590] vs. 524s [428 – 597], p=0.207). Lysis time was also similar between morning and evening (median 1792s [1622 -2097] vs. 1726s [IQR 1517 – 1891], p=0.265).

Conclusion

Compared to the known circadian variation of thrombotic and fibrinolytic activity attributable to PAI-1, global thrombotic and fibrinolytic status do not appear to exhibit diurnal variation. Further studies are needed to assess how this correlates to t-PA and PAI-1 levels and the status in patients with cardiovascular disease.
Association postprandial hypertriglyceridemia, levels of leptin and carotid intima-media thickness in patients with coronary artery disease combined with nonalcoholic liver steatosis
Authors: Grechanyk M , Kuryata O
Published: June 2022

Funding Acknowledgements

Type of funding sources: None. Main funding source(s): -

Objective

To estimate the frequency of postprandial hypertriglyceridemia (PPG), levels of leptin, carotid intima-media thickness in patients with coronary artery disease (CHD) combined with nonalcoholic liver steatosis (NALS).

Methods

We studied 61 patients with CHD in combination with NALS mean age 57,8±2,04 (group A) and 20 patients with CHD without NALS mean age 56,6±4,6 (group B). Patients with diabetes were excluded. Studied PPG, levels of leptin and carotid intima-media thickness.

Results

Revealed an increase in the level of triglycerides after fat load test in group A by 57% (from 2.27 [1.74; 2.77] mmol/l to 3.56 [2.60; 4.89] mmol/l), in the group B by 203% (s 1.24 [1.05; 1.86] mmol/l to 3.76 [1.64; 5.51] mmol/l). In patients in a group A identified the highest leptin levels 20,9 [12,3; 24,6] ng/ml than  in a group B- 13,2 [7,3; 16,9] ng/ml, (p <0,02). Carotid wall thickness was lower in a group A (0,14±0,02 cm)  than in a group B (0,16±0,01 cm, p =0,01). Frequency of exposure of atherosclerotic carotid plaques in a group A – 82%, was higher than in a group B – 64% (p <0,05). In group A unstable plaques found significantly more often (31%, p = 0,01), than in the control group B (8%). There was correlation between PPG and unstable plaques (r=0,75, p=0,03) in a subgroup with obesity grade 2.

Conclusion

The incidence of unstable atherosclerotic plaques of carotid arteries in the group with CAD and NALS was higher (34%) unlike patients with CAD without NALS (5%). It has been shown for the first time that under the conditions of the combined course of CAD with liver steatosis, an increase in body weight is associated with a reduced reaction of postprandial TG levels with an increase in leptin levels. According to the results of ROC analysis of significant predictors of liver steatosis in the group of patients with CAD were levels of leptin (AUC for leptin – 0.69 (0.56-0.82)).
Virus specific CD8+ T-cells accumulate, but do not recognize antigen, in the atherosclerotic lesion
Authors: De Jong M , Depuydt MAC , Lozano Vigario F , Van Veelen PA , Kuiper J , Slutter BA
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERA CVD

Dutch Heart Foundation

Viral infections have been associated with the progression of atherosclerosis. CD8+ T-cells directed against common viruses, such as influenza and Epstein-Barr virus, have been detected inside of human atherosclerotic lesions. These virus specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis, however, their mechanism of action in the atherosclerotic lesion remains unclear. This study aimed to characterize the behavior of virus specific CD8+ T-cells in the atherosclerotic lesion.

The presence of virus specific CD8+ T-cells in atherosclerotic lesions was assessed by performing T-cell receptor (TCR)-β sequencing on human endarterectomy samples and patient matched blood samples (N=10). These TCRs were subsequently compared to known virus specific TCR sequences. Virus specific CD8+ T-cells seemed to accumulate in the atherosclerotic lesion (mean=3.5%), compared to patient matched blood samples (mean=2.0%) (p=0.053). Moreover, these virus specific CD8+ T-cells produced significantly more IFN-γ (p=0.0009) and Granzyme B (p=0.0038) in response to external stimuli, suggesting these T-cells may play a pro-inflammatory role in the pathogenesis of atherosclerosis.

To investigate if virus specific CD8+ T-cells can be stimulated in situ, the immunopeptidome of 51 pooled human plaques was determined and matched with various viral peptidomes. Only one peptide presented in the atherosclerotic lesions matched a viral peptidome, rendering almost all virus specific CD8+ T-cells in the lesion antigen non-specific.

In conclusion, virus specific CD8+ T-cells are enriched in atherosclerotic lesions and have an activated phenotype, compared to other plaque residing CD8+ T-cells. The absence of virus specific antigen presentation in the atherosclerotic lesion suggests that if these CD8+ T-cells contribute to inflammation, they do so in an antigen independent manner.
Granzyme B+ CD4+ T cells associate with an unstable plaque phenotype in advanced human atherosclerosis
Authors: Depuydt MAC , Smit V , Lozano Vigario F , Bernabe Kleijn MNA , De Vries MR , Quax PHA , Wezel A , Smeets HJ , Kuiper J , Foks AC , Bot I , Slutter B
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): The Dutch Heart Foundation

Cytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with the pathophysiology of atherosclerosis has not been established.

Single-cell RNA sequencing was performed on human carotid atherosclerotic plaques of 18 patients to identify specific T cell populations (2). Next, human femoral and carotid atherosclerotic plaques (n=95) and matched blood samples (n=49) were analyzed by flow cytometry for the presence of CD4+GZMB+ T cells. Plaque morphology was assessed by Movat’s Pentachrome staining.

A distinct cytotoxic GZMB+ PRF1+ CD28- CD4+ T cell cluster was identified using single-cell RNA sequencing. Furthermore, flow cytometry analysis showed that the percentage of GZMB+CD4+ T cells was significantly elevated in plaque compared to blood (Blood: 12.17±2.0 vs. Plaque: 17.40±1.0; P=0.0002). Moreover, a significant positive correlation was observed between the percentage of GZMB+CD4+ T cells in blood versus plaque (P=0.031). In line with the proinflammatory character of these cells, we found a positive association of GZMB+CD4+ T cells (P=0.036) with necrotic core size, whereas no correlation was found with this subtype in the circulation.

In this study we have shown an enrichment of cytotoxic CD4+ T cells in atherosclerotic lesions, which positively correlate with necrotic core size. Future studies are aimed at elucidating the role of these cells in advanced atherosclerosis.