bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial function after endogenous glutathione synthesis stimulation in old rats heart
Authors: Okhai I , Fedichkina R , Goshovska Y , Sagach V
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Aging is accompanied by alterations mitochondrial function, and decreasing mitochondrial membrane potential (Δψm) amid increasing ROS/RNS. Mitochondrial disfunction is one of the cause of age-relating cardiovascular deseases development. Previously, we showed that stimulation synthesis of antioxidant glutathione (using DL-propargylglycine (PAG) + L-cysteine) provided complete restoration of the heart and mitochondrial function, reduced ROS levels at ischemia-reperfusion model in adult rats.

The aim was to investigate whether PAG+L-cysteine stimulated glutathione synthesis in old rats, and cardiac Δψm, NOx (nitrite plus nitrate) and ROS production at endogenous stimulation glutathione synthesis.

Stimulation of glutathione synthesis was performed by combined pretreatment with 11.3 mg/kg PAG and 120 mg/kg L-cysteine injected intraperitoneally 40 and 30 min before sacrificing. Langendorff isolated rat hearts were underwent 20 min perfusion. Cardiac mitochondria were isolated and tested for respiration rate and Δψm with TPMP+-selective electrode. Proton-leak was titrated with malonate addition. Glutathione level was measured by Ellman's reagent. Generation rate of superoxide radical and hydrogen peroxide, malondialdehyde and NOx levels, iNOS and cNOS (inducible and constitutive nitric oxide synthase) activity was determined spectrophotometrically in heart tissue.

Pre-administration of PAG+L-cysteine to old rats increased the reduced glutathione level by 51.4%, P<0.05; significantly increased Δψm (P<0.01) and simultaneously decreased the rate of oxygen consumption by mitochondria (P <0.001); the proton-leak curve shifted to the right. It indicates improving the mitochondrial efficiency. At the same time, ROS generation was reduced by 48.2% and 21.7% for superoxide radical and hydrogen peroxide correspondingly, P<0.01. Malondialdehyde level dropped almost twice (P<0.01) too. Quantity NOx falled off by 24.5% with 52.5% decreased activity of iNOS and increased activity cNOS by 2.2 times, P<0.01. The reducing superoxide production and this condition of NO system may indicate of increase the coupling cNOS and ability heart to relax.

Thus, endogenous glutathione synthesis stimulation in conditions of aging caused increasing Δψm and improved respiratory chain efficacy. The redox balance and metabolism of nitric oxide were normalized in rat heart.
over-activated p21 protein activated kinase-3 (pak3) leads to myocardial lipotoxicity and cardiac dysfunction in diet-induced metabolic stress
Authors: Liu W , Ruiz-Velasco A , Raja R , Kaur N
Published: June 2022

Funding Acknowledgements

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

Background

In clinics, metabolic stress-induced cardiomyopathy is featured as diastolic/systolic dysfunction, at least partially resulting from impairment of metabolic flexibility in the myocardium, which leads to lipotoxicity and oxidative stress accompanied with cardiomyocytes death. P21-activated kinases (Paks) are a family of serine/threonine kinases, involved in cell survival, proliferation and cytoskeleton remodelling. In spite of the cardioprotective functions of Pak1 and Pak2 in the myocardium, the role of cardiac Pak3 is unexplored.

Purpose

The study is to investigate the function of cardiac Pak3 on myocardial steatosis in response to diet-induced metabolic stress, which will provide proof-of-concept evidence for the therapeutic potential for alleviating cardiac dysfunction by targeting lipotoxicity in the myocardium.

Methods

Pak3 overexpression in the myocardium was achieved by tail vein injection of adeno associated virus (AAV9) delivery of troponin T promoter-driven Pak3 on C57BL/6J mice. The mice were fed with high fat high sucrose diet, the metabolic profiles and cardiac function were measured at 4-week intervals. In addition, cardiac morphological and cellular changes were assessed, including cell size, fibrosis formation, cell death, lipid accumulation and lipid peroxidation. In addition, the transcript levels of the genes either participating lipid metabolism or regulating oxidative stress were also assessed by qPCR.

Results

First, we detected that Pak3 expression and activation was increased in the myocardium from human with obese or diabetes. Consistently, the augmented Pak3 was also detected in the failing heart from C57BL/6J mice, ob/ob and db/db mice. Next, in a time-course study, the mice with AAV9-delivery cardiac Pak3 overexpression were vulnerable to cardiac dysfunction upon diet-induced metabolic stress. Pak3 overexpression did not alter cardiomyocyte size; however, more fibrosis and cell death in the myocardium were detected due to Pak3 enhancement. More importantly, myocardial lipid accumulation and profound lipid peroxidation were determined by immunefluorescent staining of Perilipin 5 (PLIN5) and 4-Hydroxynonenal (4-NHE), respectively. Finally, the abnormal expression of the genes involved in lipid synthesis and fatty acid oxidation was detected in Pak3-overexpressed myocardium under metabolic stress, whereas the levels of anti-oxidative stress genes were decreased.

Conclusion

Altogether, the study has demonstrated that Pak3 over-activation makes the heart more susceptible to myocardial lipotoxicity, which accelerates cardiac steatosis and dysfunction in the fact of metabolic stress.
Temporal and spatial dynamics in the regulation of myocardial metabolism during the ischemia-reperfusion process
Authors: De Dios E , Rios Navarro C , Moya J , Gavara J , Perez-Sole N , Marcos-Garces V , Forteza MJ , Diaz A , Ruiz-Sauri A , Chorro FJ , 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).

Introduction

In the context of severe myocardial ischemia, cardiac metabolism shifts from beta oxidation to glycolisis. However, the temporal and spatial dynamics of the main regulators of myocardial metabolism during the ischemia-reperfusion process in the infarcted heart has not been fully characterized.

Methods

Myocardial infarction (MI) was induced in swine by means of 90 minutes occlusion of the mid left anterior descending coronary artery using angioplasty balloons. Tetrazolium staining and intracoronary infusion of thioflavin-S were used to define the infarcted, adjacent, and remote areas. mRNA and protein expression of PGC1a, PPARa, ERRa, GLUT1, and GLUT4 were quantified in controls and in MI groups submitted to 48 hours and 3 weeks of reperfusion.

Results

Compared to controls, a severe and generalized drop of PGC1a mRNA gene and protein levels occurred in the infarcted, adjacent and remote areas since ischemia onset until 48 hours reperfusion that persists at 1 month in the infarcted region. Similar dynamics occurred in the infarcted, adjacent, and remote areas in the case of PPARa gene expresion; PPARa protein significantly decreased only until 48 hours reperfusion in the infarcted area. ERRa gene and protein expression persistenly decreased only in the infarcted region since ischemia onset until 1 month. Incrases in GLUT1 (since ischemia onset) and GLUT4 (at 1 month) were detected.

Conclusions

Dynamics and generalized changes in metabolism regulation to a shift from beta oxidation to glycolisis occur in the infarcted heart since ischemia onset until late after reperfusion. Further research in this field can be helpful for a better understanding of pathophysiology of myocardial infarction and to explore new therapeutic options.
Adipsin a regulatory protein found in the extracellular matrix is modulated by hypercholesterolemia in myocardial infarction
Authors: Padro T , Garcia-Arguinzonis M , Vilahur G , Diaz-Riera E , Badimon L
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): Agencia Estatal de Investigación (AEI)- Spanish Institute of Health Carlos III, ISCIII;

Agencia Estatal de Investigación (AEI)- Spanish Ministry of Economy Competitiveness of Science

Background

Local inflammation and innate immunity are key features during cardiac remodelling post myocardial infarction (MI). Adipsin, a component of the inflammatory response, is the major activator of the C3-complement pathway and exerts elastolytic activity. Beyond liver, adipsin is mostly secreted from adipocytes, monocytes and macrophages. It is to notice that intramyocardial lipid accumulation alters the fibrotic reparative process of the scar, resulting in larger infarcts and cardiac dysfunction in a pig model. Adipsin could directly contribute to collagen turnover in the cardiac extracellular matrix (ECM) during formation of the post-MI scar.

Purpose

The role of adipsin in the setting of myocardial infarction remains unknown. Here we investigated adipsin changes (content and activity) in the cardiac ECM of MI pigs and determine whether hypercholesterolemia relates to the turnover of the ECM-collagen pattern in association with the adipsin content.

Methods

Normo- (NC) and diet-induced hyper- (HC) cholesterolemic animals (N=28) were randomly distributed to MI-induction or sham-operation (N=7 animals/group). MI was induced by 90min closed-chest coronary balloon occlusion. After revascularization, animals were either maintained for 2.5hours or 30days before being sacrificed.  Adipsin, C3- activation products (as readout of adipsin activity) and collagen fragmentation products were determined in cardiac ECM-extracts from the ischemic and non-ischemic regions in all pigs.

Results

Adipsin and C3-activation products (iC3b, C3dg) were consistently detected in the cardiac ECM of sham-operated animals. Detection levels of adipsin, iC3b and C3dg were >2fold higher in HC vs NC pigs (p<0.05) suggesting higher adipsin activity in hearts of animals with high cholesterol levels. NC and HC MI-pigs subjected to 2.5hours reperfusion showed an acute and sharp increase in cardiac adipsin detection in the ischemic region as compared to sham and non-ischemic areas (p<0.05).  At 30days post-MI, adipsin content in the scar was comparable to that of sham-operated animals remaining significantly higher in HC vs NC pigs. Yet, adipsin cardiac levels in the scar of HC animals were associated with a lower content of fibrillar collagen-1 (MW: 235kDa) and a higher proportion of proteolytic collagen-fragments (MW: 135-48 kDa) (p<0.05 vs NC pigs)

Conclusions

Adipsin is a component of the cardiac ECM. Hypercholesterolemia enhances myocardial adipsin content and activity and promotes proteolysis of collagen fibrils in the forming scar contributing to the cardiac remodeling during post-infarction.
Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of Notch signaling in a murine model
Authors: Madonna R , Pieragostino DP , Pieragostino DP , Cufaro MC , Cufaro MC , Del Boccio PD , Del Boccio PD , Pucci AP , Pucci AP , Mattii LM , Mattii LM , Doria VD , Doria VD , Cadeddu Dessalvi CD , Cadeddu Dessalvi CD , Zucchi RZ , Zucchi RZ , Mercuro GM , Mercuro GM , De Caterina RD , De Caterina RD
Published: June 2022

Funding Acknowledgements

Type of funding sources: None.

Background

Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms in general, and sex-related mechanisms in particular.

Objectives

To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model.

Methods

24-months-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days.

Results

PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a downregulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and - to a lesser extent - by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1).

Conclusions

We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity.
Immune checkpoint inhibition with PD-1 inhibitor induces cardiac dysfunction without overt myocarditis in C57BL/6J mice
Authors: Gergely T , Kucsera D , Toth VE , Petrovich B , Agg B , Onodi ZS , Ruppert M , Radovits T , Merkely B , Ferdinandy P , Varga ZV
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 739593

“Semmelweis 250+ Kiválósági PhD Ösztöndíj” (EFOP-3.6.3-VEKOP-16-2017-00009)

Gedeon Richter Talentum Foundation’s scholarship

Background

Immune checkpoint inhibitors have revolutionized the treatment of several form of malignancies (including metastatic melanoma) by enhancing the cytotoxic effects of T cells against cancer cells. Cancer cells evade immune surveillence by increasing the expression of T cell inhibitory molecules, also known as immune checkpoints, such  as programmed cell death-1 (PD-1). Pharmacological inhibition of these molecules by immune checkpoint inhibitors (ICI) will enhance the antitumor activity of T cells. However, enhanced T cell activity may cause immune related adverse effects, including cardiotoxicity.

Aims

We aimed to investigate the effect of PD-1 inhibition on cardiac function and the underlying mechanisms in mice.

Methods

8-10 weeks old C57BL6/J mice were treated with isotype control or anti-PD-1 antibody for 2 or 4 weeks. Cardiac function and morphology was assessed by echocardiography and histology, while the transcriptomic changes were analyzed via RNA sequencing. Nitrosative stress in the heart was assessed by immunohistochemistry and qRT-PCR. Inflammatory gene expression alterations were determined by qRT-PCR in the heart and thymus.

Results

Small animal echocardiography revealed cardiac dysfunction even after 2 weeks of anti-PD-1 treatment, with distinct transcriptomic changes. Nitrosative stress was found to be elevated in the myocardium due to anti-PD-1 treatment, however, histological and qRT-PCR analysis did not reveal T cell infiltration into the myocardium and only mild inflammation was seen in the heart. In contrast, inflammatory gene expression was significantly enhanced in the thymus of anti-PD-1-treated animals, where interleukin-17 showed the most prominent increase.

Conclusions

These findings characterize cardiac dysfunction as a form of ICI-induced cardiotoxicity, which may be mediated by increased thymic inflammatory activation and cytokine production.
Investigation of transcriptional changes underlying calcification of aortic valve
Authors: Semenova D , Zabirnyk A , Lobov A , Vaage J , Malashicheva A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): The Russian Science Foundation

Introduction

Aortic valve stenosis due to calcification of valve cusps is the most common valve disease in the world today. The main feature of this condition is a progressive mineralization of valve tissue. The mechanisms underlying this process is still unknown, but in recent years it has become clear that pathological mineralization of heart and blood vessels has some similarities with the physiological process of bone formation. It has been suggested that interstitial cells (VICs) are the main functional units in the valve that undergo calcification. However, the early initiating mechanisms that trigger osteogenic transformation of cells remain unclear.

Purpose

The aim of the present study was to elucidate the most responsive time point of osteogenic differentiation induction and to identify the main osteogenic markers that mediate pathological calcification in human aortic valve.

Methods

VICs were obtained from patients with aortic valve calcification and from healthy aortic valves. The effectiveness of cell cultures osteogenic differentiation was estimated by Alizarin Red staining. Investigation of gene expression changes upon osteogenic differentiation was performed by qPCR and RNA sequencing.

Results

We found that 48 hours after the induction of osteogenic differentiation is the most relevant time point to identify the early regulators of osteogenic transformation of the cells. That is the time when the most intensive response from osteogenic markers takes place – BGLAP, OPG, OGN, RUNX2 – in comparison to 24, 72 and 96 hours of differentiation in both patient’s and healthy cells. We found out that induction of osteogenic differentiation on early stages initiates transcriptional program that serve to induce the next molecular events which recruit phenotype-specific osteogenes. We revealed that 558 and 232 genes which were up and down regulated during differentiation were the same for healthy and patient’s cells. However, there was a number of genes which was specific for either patient’s or healthy cells.

Conclusions

We presume that a great amount of the main molecular participants of osteogenic differentiation is shared between different types of cells which are prone to differentiation. However, we perform the results about specificity and difference between the mechanisms of osteogenic differentiation of patient’s and healthy cells.
Sex-specific cell types and molecular pathways drive fibro-calcific aortic valve stenosis
Authors: Myasoedova V , Massaiu I , Moschetta D , Chiesa M , Valerio V , Bozzi M , Parisi V , Poggio P
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Fondazione Gigi e Pupa Ferrari ONLUS

Background

Aortic stenosis (AS) is the most common valve disorder characterized by fibro-calcific remodeling of valve leaflets. Recent evidence indicated that there is a sex-related difference in AS development and progression. Fibrotic remodeling is peculiar of women's aortic valve, while men's aortic leaflets are more calcified than in women.

Purpose

To assess aortic valve fibrosis (AVF) in a severe AS cohort using non-invasive diagnostic tools and determine whether sex-specific pathological pathways and cell types are associated with severe AS.

Methods

We have included 28 men and 28 women matched for age with severe AS who underwent Doppler echocardiography and cardiac contrast-enhanced computed tomography (CT) before intervention. The calcium and fibrosis volumes were assessed and quantified using the ImageJ thresholding method, indexed calcium and fibrosis volume were calculated by dividing the volume by the aortic annular area. Differentially expressed genes and functional inferences between women and men's aortic valves were carried out on a publicly available microarray-based gene expression dataset (GSE102249). Cell types enrichment analysis in stenotic aortic valve tissues was used to reconstruct the sex-specific cellular composition of stenotic aortic valves.

Results

We confirmed that women had significantly lower aortic valve calcium content compared to men, while fibrotic tissue composition was significantly higher in women than men. We identified that the expression profile of human stenotic aortic valves is sex-dependent. Pro-fibrotic processes were prevalent in women, while pro-inflammatory ones, linked to the immune response system, were enhanced in men. Cell-type enrichment analysis showed that mesenchymal cells were over-represented in AS valves of women, whereas signatures for monocytes, macrophages, T and B cells were enriched men ones.

Conclusions

Our data provide the basis that the fibro-calcific process of the aortic valve is sex-specific, both at gene expression and cell type level. The quantification of aortic valve fibrosis by CT could make it possible to perform population-based studies and non-invasive assessment of novel therapies to reduce or halt sex-related calcific aortic valve stenosis (CAVS) progression, acting in an optimal window of opportunity early in the course of the disease.
Paracrine signal emanating from stressed cardiomyocytes aggravates inflammatory microenvironment in diabetic cardiomyopathy
Authors: Kaur N , Ruiz A , Raja R , Howell G , Mace K , Binder P , Smith C , Swanton E , Wang X , Chernoff J , Cartwright EJ , Liu W
Published: June 2022

Funding Acknowledgements

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

Background

Cardiovascular issues associated with diabetes, such as diabetic cardiomyopathy (DCM), can lead to heart failure. DCM is etiologically related to myocardial inflammation and can stem from a complex interplay of different cell types. Cardiomyocyte as an active mediator of the inflammatory response is an emerging concept with limited mechanistic understanding.

Purpose

We aimed to address the knowledge gap of cardiomyocyte endoplasmic reticulum (ER) dysfunction-mediated macrophage response and provide functional evidence for the therapeutic feasibility of managing inflammatory paracrine signals in response to diet-induced metabolic stress.

Methods

In vivo mouse model of high fat high sucrose diet-induced diabetes, cardiomyocyte-specific p21-activated kinase 2 (PAK2) knockout model, echocardiography, histology, 3D imaging, qPCR, co-culture of H9c2 culturing medium and bone-marrow derived macrophages, immunoblotting, macrophage isolation from myocardium, flow cytometry and AAV9-gene therapy.

Results

In a time-course study, diet-induced diabetic mice demonstrated an association between cardiac ER stress and sustained myocardial inflammation, with a maladaptive shift in myocardial ER stress response over time. Furthermore, as a cardiac ER dysfunction model, mice with cardiac-specific PAK2 deletion exhibited heightened myocardial inflammatory response in diabetes. Using human and mice diabetic heart samples, we show that ER stress-induced CCAAT/enhancer-binding protein homologous protein (CHOP) is a novel transcriptional regulator of high mobility group box-1 (HMGB1). Cardiac stress-induced active release of HMGB1 facilitated M1 macrophage polarization, and aggravated myocardial inflammatory signatures. Therapeutically, sequestering the extracellular HMGB1 using Glycyrrhizin conferred cardioprotection through its anti-inflammatory action. Also, as functional evidence, we showed that un-mitigated cardiac ER response due to PAK2 loss under diabetes may account as a barrier for leveraging the anti-inflammatory potential of Vildagliptin.

Conclusion

Collectively, we introduce an ER stress-mediated cardiomyocyte-macrophage link, altering the macrophage response in the myocardium, thereby providing insight into therapeutic prospects for diabetes-associated cardiac dysfunction.
Tenascin-C provokes cardiac fibrosis and endothelial impairment in Duchenne Muscular Dystrophy
Authors: Szabo P L , Inci M , Hilber K , Abraham D , Trojanek S , Costantino S , Paneni F , Podesser B K , Kiss A
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): FWF Austrian Science Fund

Cardiac fibrosis and dilated cardiomyopathy are major contributors to mortality in Duchenne muscular dystrophy (DMD) patients. There is a growing collection of evidence that Tenascin C (TN-C) plays a maladaptive role in cardiac remodelling and fibrosis.

Our aims were to 1) assess the vascular dysfunction and cardiac fibrosis and its link to TN-C in a mouse model of DMD and 2) explore the effect of knocking out TN-C in dystrophic mice.

Male wt, mdx and mdx TN-C KO age-matched mice were used. Cardiac fibrosis was assessed on tissue sections. Wire myography was used to test the vascular reactivity and endothelial cells (ECs) were isolated from mouse lung tissues to characterize the oxidative stress and inflammatory marker expression. To study the signalling pathways contributing to cardiac fibrosis, human cardiac fibroblasts (hCFs) were treated with TN-C or TGF-β and gene expression and epigenetic regulation of p65 were assessed.

Cardiac fibrosis was markedly increased in mdx mice which was accompanied with elevated TN-C level in cardiac tissue and plasma compared to wt animals. In addition, endothelial cells isolated from mdx mice also showed a marked upregulation of oxidative stress and inflammatory markers and in line with that vascular endothelial function was impaired in mdx mice. Interestingly, mdx- TN-C KO mice showed preserved vascular function as well as reduced cardiac fibrosis compared to age-matched mdx mice. hCFs treated with TN-C or TGF-β showed increased collagen and α-SMA expressions which could be reduced by TN-C siRNA. In addition, both TN-C and TGF-β promote p65/NF-κB promoter demethylation and subsequently stimulate pro-inflammatory and pro-fibrotic signalling, which could be reversed by applying p38 MAPK inhibitor in hCFs.

TN-C promotes oxidative stress and inflammation in ECs and fibroblasts, contributing to severe endothelial dysfunction and cardiac fibrosis. In addition, activation of NF-κB p65 signalling pathway may play a role in TN-C induced fibrosis. Thus, TN-C may be a critical mediator and potential target for therapy in DMD.
Dysregulation of the endomembrane homeostasis triggers vascular smooth muscle cell phenotype switching
Authors: Hense N , Mir B , Marx N , Goettsch C
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Background

Cardiovascular diseases are a leading cause of mortality globally, with cardiovascular calcification as a prominent predictor and contributor. Notably, microcalcifications might cause atherosclerotic plaque rupture, leading to myocardial infarction. Calcified plaque remodeling accompanies phenotypic modulation of vascular smooth muscle cells (SMCs). Previously, we showed that extracellular vesicles (EVs) released from calcifying SMCs are pivotal for microcalcification formation. EVs originate from the endolysosomal system. The lipid kinase FYVE-type zinc finger containing phosphoinositide kinase (PIKfyve) plays a role in the endolysosomal maturation. Here, we investigated how modulation of the endolysosomal system intersects with SMC phenotype regulation.

Methods and Results

In calcified arteries and SMCs, we observed increased PIKfyve expression. Inhibition of PIKfyve with the small molecule Apilimod inhibited osteogenic differentiation of SMCs in an in vitro calcification model. Apilimod reduced tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA (-79%, p<0.01), protein (-96%, p<0.01) and activity (-92%, p<0.01) levels. Additionally, matrix mineralization and collagen network formation are inhibited. SMC-secreted EVs exhibited a lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Transcriptome analyses revealed that the anti-calcifying effect is accompanied by a decreased expression of contractile SMC genes. Validation by RT-PCR supported that SMC-specific markers (ACTA2, SM22) and the key SMC transcription factor MYOCD were downregulated, while the expression of macrophage-like SMC markers (LGALS3, CD68) was induced. On the other hand, Apilimod increased the expression of adipogenic transcription factors (CEBPA, PPARG) and genes of the cholesterol and fatty acid metabolism pathways (SREBF2, HMGCR, FABP3, CD36) and induced the nuclear translocation of PPARγ, the main regulator of adipogenic differentiation. Fluorescence-based assays revealed increased fatty acid and disrupted low-density lipoprotein (LDL) uptake in Apilimod-treated SMCs. In Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks and treated with Apilimod for 5 weeks, we found differentially distributed plaque lipid depositions detected by Oil Red O staining.

Conclusion

Disrupting the endolysosomal maturation by PIKfyve inhibition ameliorates SMC calcification and induces a phenotypic adipogenic-like, pro-inflammatory adaption. Our data elucidates the importance of SMC phenotype monitoring during therapeutic interference. The exact lineage characteristics of these SMCs remain to be further investigated.
High pulsatile load ex vivo decreases arterial stiffness by altering the viscous, but not elastic, mechanical properties of the arterial wall
Authors: Neutel C , Wesley C , Puylaert P , De Meyer GRY , Martinet W , Guns PJ
Published: June 2022

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Research Foundation - Flanders (Brussels, BE):

==> [DUTCH]: "Fonds voor Wetenschappelijk Onderzoek (FWO) - Vlaanderen"

Introduction

The pulsatile character of blood flow in the central arteries results in a local transmission of energy to the arterial wall, acting on both the viscous and elastic components of the arterial wall. The amplitude of the "pulsatile load" is known to modulate the mechanosensitive properties of blood vessels.

Purpose

To evaluate the behavior of both the viscous and elastic components of the mouse aorta under altered pulsatile loading. Moreover, we aimed to assess regional differences in (arterial) tissue stiffness, as well as the contribution of vascular smooth muscle cell (VSMC) contractility under altered pulsatile conditions.

Methods

Aortic segments from C57Bl6 (n=9) mice were mounted in a Rodent Oscillatory Set-up for Arterial Compliance (ROTSAC), and subjected to high frequency (10 Hz) cyclic stretch at alternating loads (i.e. 20 to 100 mmHg with mean pressures ranging from 80 to 200 mmHg). Diastolic and systolic diameter, compliance, and the Peterson elastic modulus (Ep), as a measure of aortic stiffness, were determined. Viscous modulus (EV) was extracted from pressure-diameter tracings by eliminating loop hysteresis. Afterwards, the elastic modulus (EE) was calculated as the slope of the resulting pressure-diameter tracing:

(P_elastic)= (P_total) - (EV * (dD/dt))

To assess regional differences in response to changes in pulsatile load, the most proximal aortic region (i.e., thoracic ascending aorta) was compared to a more distal aortic region (i.e., abdominal infrarenal aorta).

Results

Increasing pulse pressure from 40 to 100 mmHg decreased arterial stiffness (298 ± 9 vs. 270 ± 9 mmHg; p < 0.0001). Analyzing viscoelasticity revealed that increasing pulsatile load from 40 to 100 mmHg decreased EV (0.24 ± 0.02 vs. 0.21 ± 0.02 mmHg.s/mm; p < 0.01) but not EE. 50 mM KCl-induced VSMC contraction increased vessel stiffness (298 ± 9 mmHg vs. 395 ± 12 mmHg; p < 0.0001). This effect was pronounced at low pulse pressures (20 and 40 mmHg) while at higher pulse pressures (60, 80 and 100 mmHg) contraction-induced stiffness did not occur. Interestingly, the distal region of the aorta was stiffer than the proximal region (367 ± 60 vs. 289 ± 36 mmHg; p < 0.01) and had a higher EV (0.77 ± 0.2 vs. 0.21 ± 0.05 mmHg.s/mm; p < 0.0001). Increasing pulsatile load from 40 to 100 mmHg decreased the EV from distal aortic tissue more than the EV of tissue from the proximal region (-0.26 ± 0.02 vs. -0.06 ± 0.007 mmHg.s/mm).

Conclusion

High pulsatile load decreased arterial stiffness by decreasing the viscous mechanical properties. This effect was more pronounced in the distal region of the aorta.
Deciphering the role of Store Operated Ca2+ entry in post-ischemic angiogenesis
Authors: Galeano-Otero I , Del Toro R , Mayoral-Gonzalez I , Ordonez-Fernandez A , Smani T
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación

Background

Post-ischemic angiogenesis is essential to improve tissue repair and heart recovery after myocardial infarction (MI). This process is tightly controlled by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which in turn promotes signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recently, we demonstrated that store-operated calcium entry (SOCE) plays a key role in angiogenesis. However, the function of SOCE in post-ischemic angiogenesis remains unknown. The aim of this study is to explore the role of SOCE in this crucial repairing process.

Materials and methods

In vitro angiogenesis was studied using Human Umbilical Vein Endothelial Cells (HUVEC) for tube formation assay over Matrigel supplemented with serum from rat model of ischemia/reperfusion (I/R) and from ST elevation MI (STEMI) patients who underwent primary percutaneous coronary intervention (pPCI). We evaluated serum VEGF-A levels using ELISA. Genes and protein expression was determined by RT-qPCR and western blot, respectively. HUVEC migration was assessed by wound healing assay. Moreover, changes in [Ca2+]i were measured using fura-2 AM.

Results

We found that STEMI patients had higher levels of circulating VEGF-A, as compared to controls. The incubation of HUVEC seeded on Matrigel with the serum of these patients promoted vessels-like formation since it enhanced the number of meshes and junctions. Likewise, serum from I/R rats also boosted HUVEC angiogenesis over Matrigel and promoted faster gap closure in wound healing assay, as compared to cells incubated with sham rats’ serum. Furthermore, serum from both rats and patients modulated the expression Hes1 and Hey1, well-known mediators of Notch1 signaling pathway. Moreover, we found that knockdown of Orai1, the pore-forming subunit of the store-operated calcium channel (SOCC), using siRNA impairs VEGF-mediated [Ca2+]i increase, as well as HUVEC tube formation, proliferation, and migration.

Conclusion

Our data show for the first that serum from both STEMI patients and I/R rats promote angiogenesis, involving Orai1 and Notch1 signaling pathways.
Predictive value of HOMAIR indices for severity of left ventricular diastolic dysfunction and NT-proBNP activity
Authors: Sapojnic N , Caraush AP , Moscalu V , Sasu DB , Bitca AI , Durnea AS , Caraush MP
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Academy of Sciences of Moldova

Hypertension (HT) and insulin resistance (IR) often coexist and condition LV diastolic function's alteration. Diastolic dysfunction (DD) identifies hypertensives with high cardiovascular risk independent of LV mass and BP level. NT-pro BNP level serve as a diagnostic and prognostic tools in patient with DD. Several studies suggest that DD severity could be interdependent with some biomarkers (HOMAIR, NT-proBNP).

Aim

To determine if HOMA IR indices could play a predictive role in appreciating severity of LV diastolic dysfunction/ NT-proBNP activity.

Methods

101 hypertensive patients (mean age 50.11±0.79 yrs; 48.51% of men, SBP/DBP: 201.31±7.41/106.25±5.54 mmHg, BMI- 29.38±0.22 kg/m2 ,  HOMA IR - 2.67±0.81) with DD and IR (HOMAIR > 2.5) were enrolled for the study. Ambulatory blood pressure monitoring (ABPM), transthoracic echocardiography (TE), HOMAIR and NT-pro BNP were performed at baseline and after 6, 12- months period of treatment. DD patterns were appreciated according to ASE/EACVI 2016 guideline. NT-proBNP cut-off value was considered > 125 pg/ml (ESC). Correlation analysis was performed using Pearson’s test. The correlation coefficient was considered weak at <0.3, medium-0.3-0.7 and strong > 0.7-1.0. Statistical significance was appreciated at a P-value <0.05 and highly significant at a P-value <0.001.

Results

The baseline characteristics of bunch are shown in Fig 1. Diastolic dysfunction, HOMA-IR and NT-proBNP levels demonstrated a statistically significant correlation (p< 0.001). A more advanced patterns of diastolic dysfunction demonstrated a particularly strong relationship with higher HOMA-IR and NT-proBNP levels ( Tab. 1).

Conclusion

In hypertensives with increase in insulin resistance, progression of the left ventricle disorder could develop. The interrelation is in such manner, that the higher is HOMAIR level, the more advanced patterns of diastolic dysfunction and NT-pro BNP activity occure.
APAC treatment limits collar-induced carotid atherosclerotic plaque development in apoE-/- mice
Authors: Bot I , Delfos L , Hemme E , Kovanen PT , Jouppila A , Lassila R
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation

Introduction and aim: Mimics of mast cell-derived heparin proteoglycans (HEP-PG) can be tailored to molecules carrying both antiplatelet and anticoagulant properties. These dual antiplatelet and anticoagulant (APAC) constructs can also shield adhesion molecules such as P-selectin and VCAM-1 expressed by endothelial cells upon atherosclerosis development. We hypothesize that via this way, APAC prevents macrophage accumulation and lesion development. In this study, we therefore determined the efficacy of APAC in inhibiting atherosclerosis.

Methods

Male western-type diet fed apoE-/- mice were equipped with perivascular carotid artery collars to induce atherosclerosis. In this collar model, mRNA expression of adhesion molecules such as ICAM-1, VCAM-1, P-Selectin but also of Platelet Factor 4 (PF4) are significantly upregulated upon lesion development (all P<0.05 at 2 weeks after collar placement compared with control arteries). From lesion initiation, mice were treated with 0.2 mg/kg APAC or vehicle control (i.v, 3x per week, n=12-14 per group) for 2.5 weeks. At 5 weeks after collar placement, mice were sacrificed.

Results

APAC treatment did not affect body weight or plasma total cholesterol levels of the mice during the experiment. Interestingly, carotid artery plaque size was reduced by over 50% upon APAC treatment (APAC: 50±10*10E3 versus controls: 102±13*10E3 square µm; P<0.01). This observation was aligned with reduced plaque macrophage area (APAC: 20±5*10E3 versus controls: 33±5*10E3 square µm) and collagen content (APAC: 13±4*10E3 versus controls: 28±6*10E3 square µm; P<0.05).

Conclusion

We here show that APAC effectively inhibits atherosclerotic lesion development when administered during lesion initiation and may have potential as therapeutic agent to prevent atherosclerosis.
White matter penetrating arteriole dysfunction correlates with MRI-defined white matter integrity in patients with Alzheimer's disease
Authors: Bagi ZSOLT , Kroenke C , Keene CD , Sherman LS , Back SA
Published: June 2022

Funding Acknowledgements

Type of funding sources: Public Institution(s). Main funding source(s): National Institute of Health

Objectives

Patients with Alzheimer’s Disease (AD) display cerebral white matter (WM) hyperintensities in association with microvascular brain injury (mVBI) and cerebral microinfarcts. We determined the extent to which vasomotor dysfunction of WM penetrating arterioles is associated with mVBI in autopsy brains from patients with low or high AD neuropathology, and determined if these changes correlate with quantitative indices of MRI-defined WM integrity.

Methods

We analyzed 28 consecutive cases of prefrontal rapid autopsies in a population-based cohort with low or high AD neuropathological changes, and with or without cerebral microinfarcts (mVBI). WM penetrating and pial surface arteriolar responses to the endothelium-dependent agonist, bradykinin were assessed ex vivo with videomicroscopy. Expression of vascular endothelial nitric oxide synthase (eNOS) and NAD(P)H-oxidase (Nox1,2 and 4 isoforms) was measured with quantitative PCR. Diffusion tensor imaging was used to measure mean apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in post-mortem prefrontal WM.

Results

Patients with high AD neuropathology and mVBI exhibited a significantly reduced dilation in response to bradykinin selectively in WM arterioles, when compared to low or high AD cases without mVBI or to pial surface arterioles. Expression of eNOS was reduced, whereas Nox-1 expression was increased in WM arterioles in AD and mVBI cases. Moreover, we found that in cases with low AD pathology the magnitude of bradykinin-induced WM arteriole dilation was correlated with higher FA and lower ADC. In contrast, dilation to bradykinin was associated with lower FA and higher ADC in cases with high AD neuropathology.

Interpretation: Selectively impaired vasodilation to the endothelium-dependent agonist, bradykinin occurs in WM penetrating arterioles in AD patients with WM microinfarcts, which is likely due to reduced nitric oxide and an increase in Nox1-derived reactive oxygen species production. Notably, the bradykinin response of WM arterioles strongly correlated with MRI-defined WM integrity suggesting that impaired bradykinin-mediated vasodilation in WM penetrating arterioles contributes to disrupted white matter microstructural integrity as defined by MRI.
Analysis of miRNA profile in circulating extracellular vesicles of patients with pulmonary arterial hypertension
Authors: Khandagale A , Corcoran P , Nikpour M , Siegbahn A , Christersson C
Published: June 2022

Funding Acknowledgements

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

Introduction

Pulmonary arterial hypertension (PAH) is characterized by a progressive elevation of pulmonary pressure leading to right ventricular dysfunction and is associated with a poor prognosis. Patients with PAH have increased numbers of circulating extracellular vesicles (EVs), which are small membrane vesicles (<1 µm) released from different cells. EVs contain surface antigens, proteins and genetic material, and serve as vectors of intercellular communication. MicroRNAs (miRs) are small RNAs that act as post-transcriptional regulators inhibiting gene expression. Alteration of miR expression is associated with stage, progression and severity of different diseases making them suitable as potential biomarker. The aim of the study was to evaluate the miRNA profile contained in EVs derived from plasma of both control healthy subjects and patients with PAH.

Methods

Circulating EVs, purified from platelet free plasma, from 9 healthy controls (HC) and 10 PAH patients were analyzed. After total RNA isolation, fixed volumes of total RNA were biotinylated to generate fragmented cRNA. Samples were then subjected to Microarray analysis using GeneChip miRNA 4.0 Array. Data was further analysed using various bioinformatics tools.

Results

Total of 762 miRs were identified while comparing PAH vs HC groups, of which 22 were found to be down-regulated and 3 were up-regulated in PAH group (Table 1). Gene ontology enrichment analysis revealed various cell signaling pathways where-in identified miRs are involved.

Conclusions

PAH patients pack and transport a differentiated expression profile of miRs, suggesting that EVs might serve as signaling vectors. Further analyses of the functionality of EVs' miRs is currently undergoing in our lab to understand its role during PAH progression and/or inhibition.
Influence of factors on systolic blood pressure in patients with hypertension identified during screening
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). Blood pressure (BP) is an easily measured indicator of hypertension.

Purpose

To assess the effect of various factors on systolic blood pressure (SBP) in patients with hypertension identified at screening.

Methods

A survey of 354 patients with hypertension identified at screening was carried out and the influence of various factors on SBP was assessed.

Results

The average age of patients is 54 years [Q1-Q3: 50-54], of which 36.2% are men, 63.8% are women. 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].

With an increase in age by 1 full years, an increase in SBP by 0.148 mm.Hg. should be expected. In accordance with the coefficient of determination R2 in the resulting model, 0.56% of the factors influencing the value of SBP were taken into account.

With an increase in body mass index by 1, a decrease in SBP by 0.059 mm Hg should be expected. In accordance with the coefficient of determination R2 in the resulting model, 0.04% of the factors influencing the SBP were taken into account.

With an increase in the waist by 1 cm, an increase in SBP by 0.059 mm Hg should be expected. In accordance with the coefficient of determination R2 in the resulting model, 0.09% of the factors influencing the SBP were taken into account.

With an increase in total cholesterol by 1 mmol/l. an increase in SBP by 24.466 mm Hg should be expected. In accordance with the coefficient of determination R2, the resulting model took into account 34.27% of the factors affecting the SBP. The effect of total cholesterol on SBP was statistically significant (p = 0.001) (methods used: Kruskal-Wallis test).

Conclusion (s)

Thus, the effect of total cholesterol on SBP is statistically significant (p = 0.001). With an increase in total cholesterol by 1 mmol/l. an increase in SBP by 24.466 mm Hg should be expected.