bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute and chronic cholinergic activity in the inflammatory and lipid regulation of epicardial stromal cells from patients with and without atrial fibrillation
Authors: Couselo Seijas M , Fu X , Lopez-Cano J.N , Rozados-Luis A , Fernandez A.L , Seoane L.M , Duran-Munoz D , Gonzalez-Melchor L , Rodriguez-Manero M , Gonzalez-Juanatey J.R , Eiras S
Published: November 2020

Aims

Acetylcholine (ACh) released modulation by botulinum toxin injection into epicardial fat diminish atrial fibrillation (AF) recurrence. These results suggest an interaction between autonomic imbalance and epicardial fat as risk factors of AF. Our aim was to study the inflammatory and lipid profile of epicardial stroma from AF patients and their regulation by high cholinergic activity.

Methods and results

We performed in vitro assays with primary cultures from paired subcutaneous and epicardial stromal cells from 33 patients. We analysed ACh effect on gene expression, intracellular calcium mobilization and neutrophil migration. Plasma protein regulation by parasympathetic denervation was performed in vagotomised rats. Acute ACh treatment up-regulated MCP1 levels on epicardial stromal cells and suggested a neutrophil infiltration enhancement. Patients with AF had a greater FABP4 gene expression (1.54±0.01 vs 1.47±0.01, p=0.005). Its plasma levels were pronouncedly declined on vagotomised rats (2.02±0.21 ng/mL vs 0.65±0.23 ng/mL, p<0.001). Additionally, chronic ACh treatment improved lipid accumulation within epicardial stromal cells (60.50% [22.82–85.13] vs 13.85% [6.17–23.16], p<0.001).

Conclusions

Acute ACh activity up-regulates MCP1 and calcium mobilization on epicardial stromal cells. Longer ACh treatment enhanced lipid accumulation. In this line, epicardial stroma from patients with permanent AF contains higher FABP4 expression levels. Thus, modulate cholinergic activity might reduce FABP4 since vagus nerve denervation is associated with a sharply decrease in FABP4 plasma levels.

FABP4 in human AF and vagotomised rats

Funding Acknowledgement

Type of funding source: Public Institution(s). Main funding source(s): Carlos III Health Institute; Health Research Institute of Santiago de Compostela
Pharmacological inhibition of acetylcholine-regulated potassium current (IK,ACh) and associated A1- and M2-pathways prevent atrial arrhythmogenic changes in a rat model for obstructive sleep apnea
Authors: Linz B , Hauge Thostrup A , Saljic A , Rombouts K , Wirth K , Hohl M , Linz D , Jespersen T
Published: November 2020

Background

In obstructive sleep apnea (OSA), intermittent hypoxemia and intrathoracic pressure fluctuations may increase vagal tone and adenosine release, potentially resulting in an increased acetylcholine-regulated potassium current (IK,ACh). Here we elucidated acute atrial electrophysiological effects of obstructive respiratory events simulated by intermittent negative upper airway pressure (INAP) and the role of atrial IKACh activation by A1-receptor and M2-receptor activation.

Methods

In sedated spontaneously breathing rats (2% isoflurane), either IK,ACh-inhibitor (XAF-1407: 1mg/kg), M2-receptor inhibitor (atropine; 1μg/kg), A1-receptor inhibitor (Rolofylline; 1μg/kg) or a buffer-based vehicle was perfused (Control). INAP was applied non-invasively by a negative pressure device 14 times throughout 70 minutes. Simulated apneas were maintained for one minute with a four minute resting period. Atrial effective refractory period (AERP) and atrial activation time were acquired by a programmed atrial pacing protocol before, during and after applied INAP throughout the study.

Results

Independent of IK,Ach-inhibition, single INAP applications prolonged transiently atrial activation times (Control: INAP vs. pre-INAP p=0.034; XAF-1407: INAP vs. pre-INAP p=0.039). In control-rats, seventy minutes of repetitive INAP decreased AERP by 15.45±0.06% (vs. baseline p=0.0015), which was reversible upon 1 hour of recovery. AERP shortening correlated with the cumulative pressure applied per body weight (Pearson r=−0.773; p=0.025). Whilst only XAF-1407 and atropine increased baseline AERP, all drug interventions, XAF-1407, atropine and Rolofylline could prevent INAP-associated AERP shortening (end INAP-protocol vs. respective baseline XAF-1407 p=0.994; atropine p=0.984; Rolofylline p=0.951). Drops in oxygen saturation and applied INAP were comparable in all groups.

Conclusion

Short-term simulated OSA is associated with progressive AERP shortening, which was determined by the cumulative negative airway pressure applied. This potentially represents an important insight in future OSA treatment. Pharmacological IK,ACh inhibition prevented INAP-associated AERP-shortening suggesting an involvement in acute atrial arrhythmogenesis in OSA. However, if IK,Ach and its potential upstream activating A1- and M2-pathway pose a pharmacological treatment target for OSA-patients with AF, requires further investigation.

Funding Acknowledgement

Type of funding source: None
New ex vivo calcification model for intact murine aortic valves
Authors: Kruithof B , Van De Pol V , Los T , Lodder K , De Ruiter M.C , Goumans M.J , Schalij M.J , Ajmone Marsan N
Published: November 2020

Introduction

Calcific aortic valve disease (CAVD) is a common, progressive disease of the aortic valves for which no medical treatment exists and surgery is the only therapeutic solution. The discovery of novel pharmacological treatment for CAVD has been hampered due to the lack of suitable test-systems.

Purpose

We aimed to establish an ex vivo calcification model for intact mouse aortic valves.

Methods

We used the ex vivo flow model for mouse hearts (Miniature Tissue Culture System) to induce calcification with culture media supplemented with 1) β-glycerophosphate, ascorbic acid, and dexamethasone (OSM) or 2) inorganic phosphates (PI) and compared this with in vitro calcification of mouse valvular interstitial cells (mVICs).

Results

Ex vivo cultured aortic valve leaflets calcified in the presence of PI, but not OSM. RUNX was upregulated in both OSM and PI conditions, whereas alkaline phosphatase (ALP) and cyclooxygenase-2 (COX2) were differentially expressed. Apoptosis was not observed, together indicating that osteogenic calcification took place. Interestingly, both OSM and PI were able to induce calcification in vitro, revealing in vitro-ex vivo differences. In addition to the calcification in the aortic valves, endochondral calcification was present in the sinus near the hinge of the aortic valve in both PI and OSM conditions as evidenced by the expression of collagen II, aggrecan and ALP.

Conclusions

Together these data show that we have established an ex vivo calcification model for mouse aortic valves. Osteogenic calcification is induced in the aortic leaflets, whereas endochondral calcification is induced in the aortic sinus, reflecting the calcification found in human CAVD. Using this model, we can now study the initiation and progression of aortic valve calcification.

Funding Acknowledgement

Type of funding source: Other. Main funding source(s): Dutch Heart Foundation, grant number 2013T093. Grants received from GE Healthcare, Lantheus medical imaging, St Jude Medical, Medtronic, Boston Scientific, Biotronik, and Edwards Lifesciences
Toll-like-receptor-3 function is critical for aortic valve stenosis development in mice
Authors: Niepmann S.T , Boucher A.S , Bulic M , Goody P.R , Jansen F , Treede H , Nickenig G , Zimmer S
Published: November 2020

Background

Aortic valve stenosis (AS) is the most common valve diseases in the western world. After having been considered a passive degenerative process, which develops as an inevitable consequence of age-related valvular degeneration, basic research of the last two decades has led to a paradigm shift. It is now believed that AS pathophysiology is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. In recent years, Toll-like-receptor-3 (TLR3) has emerged as a major regulator of vascular inflammation. TLR3 is a lysosomal pattern recognition receptor that recognizes single and double stranded RNA. Its activation leads to expression of pro-inflammatory cytokines via NFkb activation. The role of TLR3 in the development of AS has never been investigated.

Methods

Severe AS was induced in Wildtype-, ApoE- and TLR3/ApoE−/− mice. For this, a coronary springwire was used to induce an endothelial injury under echocardiographic guidance. Stenosis development was confirmed via ultrasound examinations. To inhibit TLR-3 activation, TLR3/RNA- Complex inhibitor C4a was injected every 48h after wire injury in WT mice. Valves were explanted and stained with hematoxylin/eosin (valve thickening) or anti-68 (macrophage infiltration). Valves from patients who received aortic valve replacement due to AS or aortic regurgitation (AR) were collected and mRNA levels of TLR3 and MyD88 were measured with use of quantitative-PCR.

Results

To evaluate weather TLR3 effects AS development in mice, we subjected TLR3/ApoE double- and ApoE knockout mice to our model of wire-induced AS. Surprisingly, TLR3 deficient mice failed to develop AS after wire injury. Peak velocity measurements showed no increase and histological analysis showed lower aortic valve area and macrophage infiltration compared to control mice. In order to pharmacological inhibit TLR3, WT mice were treated with C4a after wire injury. Compared to PBS control, C4a mice also did not develop AS upon wire injury. Trans-aortic valve peak velocity levels were significantly lower in C4a mice. Histological analysis underlined these results and showed thinner aortic valves and decreased macrophage infiltration in C4a mice comparted to control animals. To confirm our hypothesis, the expression of TLR3 and its downstream effector MyD88 were measured in human aortic valve specimens. qPCR analysis revealed decreased TLR3 and MyD88 expression in patients with AS compared to patients with AR.

Conclusion

In the presented study, we present first data that theTLR3 has a crucial role in the development of AS in mice. The exact downstream effects after TLR3 activation in AS need to be further investigated.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)
Presence of adipose tissue in aortic valves from aortic regurgitation patients: pathophysiological role and clinical implications
Authors: Sadaba Cipriain A , Navarro Echeverria A.M , Tiraplegui Garjon C.R , Garcia De La Pena Urtasun A , Arrieta Paniagua V , Matilla Cuenca L , Alvarez Asiain V , Fernandez-Celis A , Gainza Calleja A , Sadaba Sagredo R , Lopez-Andres N
Published: November 2020

Introduction

Adipose tissue is a common constituent of the heart and it is located, without great clinical relevance, frequently in the pericardium. The presence of adipose tissue in the aortic valve is rare, with unknown significance on valve structural properties and function. Aortic regurgitation (AR) is the third most prevalent valve disease, although it is uncommon to find it in isolation. Myxoid degeneration may be the cause or result of AR, although the pathophysiology remains poorly understood.

Purpose

To describe and characterize the presence of adipose tissue in the aortic valves from a cohort of AR patients.

Methods

116 patients undergoing aortic valve replacement due to severe AR were enrolled. We classified them in two groups according to the histological results showing presence or absence of adipose tissue in the aortic valves.

In the valve tissue molecular analysis were performed by RT-PCR, Western Blot and ELISA to analyze markers of adipocytes (leptin, adiponectin, resistin), inflammation (Rantes, interleukin-6, interleukin-1β), extracellular matrix remodeling (metalloproteinases-1, -2 and -9), proteoglycans (aggrecan, hyaluronan, lumican, syndecan-1, decorin) and fibrosis (collagens, fibronectin).

Results

Adipose tissue was found in 63% of the aortic valves analyzed. Baseline characteristics (age, hypertension, dyslipidemia, diabetes, smoking, left ventricular telediastolic diameter, left ventricular systolic function, ascending aorta) were similar in patients presenting valve adipose tissue as compared with patients without valve adipose tissue. Valves containing adipocytes exhibited a higher leptin content (p<0.001), fibronectin (p<0.01), decorin (p<0,0001), hyaluronan (p=0.03), aggrecan (p=0.04) and metalloproteinase 1 (p=0.03). Interestingly, the presence of adipocytes in the valve was positively correlated with valve thickness measured by echocardiogram (Pearson chi2 statistical significance = 26.3345 p<0.001).

Conclusion

To our knowledge, this is the first study that describes the presence of adipose cells in aortic valves from a cohort of AR patients. Aortic valves containing adipocytes were thicker and exhibited significant higher levels of proteoglycans, suggesting that adipocytes could contribute to the myxomatous degeneration process. Our results propose that the valve adipose tissue could play a role in the pathophysiology of AR.

Funding Acknowledgement

Type of funding source: Public hospital(s). Main funding source(s): Gobierno de Navarra
Clec4e signalling influences left-ventricular functional recovery in a murine model of myocardial ischemia-reperfusion injury
Authors: Veltman D , Gillijns H , Caluwe E , Wu M , Vanhaverbeke M , Gsell W , Himmelreich U , Sinnaeve P , Janssens S
Published: November 2020

Introduction

The acute inflammatory response contributes substantially to functional recovery and remodelling of the left ventricle after acute ischemic injury. Previously, we have shown that the C-Type Lectin Receptor CLEC4E plays a role in early leukocyte recruitment during the acute inflammatory response of ischemia-reperfusion injury (I/R). However, the role of CLEC4E signalling in functional recovery of the left ventricle after I/R remains unknown. Therefore, we studied the chronic inflammatory response and left-ventricular remodelling in murine gene deletion model of Clec4e, subjected to I/R.

Methods

In anesthetized C57Bl6/J wild-type (n=14) and Clec4e−/− (n=13) mice, we transiently occluded the left-descending artery for 60 min, followed by 4 weeks reperfusion (I/R). A blood sample was collected at 90 minutes reperfusion to measure high-sensitivity troponin I (TnI) levels, as a surrogate marker of cardiac damage. At 4 weeks, mice underwent MRI (7T) to investigate the effect of Clec4e-gene deletion on LV-remodelling.

Results

Plasma TnI-levels showed no statistical difference between both groups, indicating that the initial insult was comparable. In wild-type mice, plasma TnI-levels negatively correlated with ejection fraction (EF, R2=0.92 p<0.0001) at 4 weeks I/R, while Clec4e−/− mice showed preserved EF, irrespective of 90 minutes TnI-levels. MRI-analysis at 4 weeks after I/R showed significantly smaller end-diastolic and end-systolic volumes in Clec4e−/− mice, together with a trend towards a higher ejection fraction, suggesting better preserved structural and functional LV-remodelling (Fig.1).

Conclusion

The inflammatory leukocyte-associated Clec4e signalling pathway impairs functional recovery of the left ventricle after myocardial I/R injury. Inhibition of the Clec4e receptor may be a promising strategy in the treatment of ischemic injury.

Figure 1

Funding Acknowledgement

Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Scholarship Ir. Jozef en Mevr. Reinhilde De Swerts 2018-2022 by the Royal Academy of Medicine of Belgium
Nur77 deficiency exacerbates cardiac fibrosis after myocardial infarction through promoting endothelial to mesenchymal transition
Authors: Qin Q , Chen J.H , Jia J.B , Qian J.Y , Ge J.B
Published: November 2020

Background

Cardiac fibrosis is a reparative process after myocardial infarction (MI), which leads to cardiac remodeling and finally heart failure. Endothelial-to-mesenchymal transition (EndMT) is induced after MI and contributes to cardiac fibrosis after MI. Orphan nuclear receptor Nur77 is a key regulator of inflammation, angiogenesis, proliferation, and apoptosis in vascular endothelial cells. Here, we investigated the role of orphan nuclear receptor Nur77 in EndMT and cardiac fibrosis after MI.

Methods and results

Cardiac fibrosis was induced through MI by ligation of the left anterior descending coronary artery. Results suggested that Nur77 knockout aggravated cardiac dysfunction and cardiac fibrosis 30 days after MI. Moreover, Nur77 deficency resulted in enhanced EndMT as shown by increased expression of FSP-1, SM22α, Snail and decreased expression of PECAM-1 and eNOS compared with WT mice after MI. Then we found overexpression Nur77 in HCAECs significantly inhibited IL-1β and TGFβ2 induced EndMT, as shown by reduced transition to a fibroblast-like phenotype and preserved angiogenesis potential. Mechanistically, we demonstrated that Nur77 downregulated EndMT through inhibiting NF-κB-dependent pathway

Conclusion

Nur77 plays a role in cardiac fibrosis through inhibition of EndMT, and may be a promising target for therapy of cardiac fibrosis after MI.

Nur77 inhibited EndMT

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China
Circulating pro fibrotic protein promotes fibrosis in liver
Authors: Hsiao Y , Shimizu I , Yoshida Y , Ikegami R , Hayashi Y , Suda M , Katsuumi G , Wakasugi T , Nakao M , Minamino T
Published: November 2020

Background/Introduction

Non-alcoholic steatohepatitis (NASH), driven by the obesity epidemic, has become the most common form of liver disease. Inflamed visceral adipose tissue secretes pro-inflammatory adipokines that are causal for systemic metabolic disorders. Role of adipokines in NASH, especially those from brown adipose tissues (BATokine) remain unclear.

Purpose

To show the pathogenic role of BATokine in NASH.

Methods

To identify and characterize the pathological roles of pro-fibrotic BATokine, we generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice, and murine systemic or BAT specific knockout (KO) models. We also conducted functional in-vitro studies with differentiated brown adipocytes.

Results

Analyzing two sets of DNA micro array data with bioinformatics, we identified a secreted form pro-fibrotic protein (sPFP) expressed in dysfunctional brown adipose tissues (BAT) in mice. Testing our biobank samples, we found this protein increased in plasma of NASH patients. We generated a murine obese NASH model by imposing a high fat diet in C57BL6/NCr mice for 9–10 months since 4 weeks of age, and found that sPFP is produced predominantly by BAT. In this model, we also found that sPFP increased in plasma. We generated a murine systemic or BAT specific sPFP knockout (KO) models and found that liver fibrosis ameliorated in these models. We also suppressed circulating sPFP with a peptide vaccine targeting this molecule, and found that sPFP vaccination therapy inhibited liver fibrosis. Next, we generated sPFP gain of function (GOF) model by the administration of plasmid encoding sPFP into skeletal muscle. Liver fibrosis augmented in sPFP-GOF model, and these results suggested that sPFP has causal role for the progression of fibrotic response in liver. In vitro studies with differentiated brown adipocytes showed that metabolic stress increased c-Fos in nuclear, and this was causal for an increase in sPFP level.

Conclusions

Our results suggest that one of the BATokines, sPFP, contributes for the progression of fibrotic responses in obese-NASH model. Inhibition of sPFP may become a therapy for NASH or obesity related fibrotic disorders.

Funding Acknowledgement

Type of funding source: None
Smooth muscle cell-specific SOCS3 deficiency promote pericardial fibrosis and diastolic dysfunction in aging mice
Authors: Yanai T , Yasukawa H , Mawatari K , Sasaki T , Takahashi J , Nohara S , Shimozono K , Shibata T , Okabe K , Yamamoto M , Fukumoto Y
Published: November 2020

Background

Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible negative regulator of signal transducer and activator of transcription-3 (STAT3) signaling pathway. We have previously shown that cardiac-specific SOCS3 deficiency spontaneously develop cardiac dysfunction with advanced age. However, the role of SOCS3 in smooth muscle cells in cardiovascular pathophysiology remains elusive. In this study, we determined whether STAT3 and SOCS3 in smooth muscle cells would play a role in cardiovascular pathophysiology.

Methods and results

To target inactivation of the SOCS3 gene to smooth muscle cells, SOCS3-flox mice were bred with transgenic mice expressing Cre recombinase under control of the mouse SM22-α promoter (sm-SOCS3-KO mice). Left ventricular weight to body weight ratio was significantly increased in sm-SOCS3-KO mice compared with wild-type mice at 12 months of age (p<0.05). Echocardiographic analyses of smSOCS3-KO mice showed significantly increased left ventricular diastolic dysfunction compared with wild-type from 12 months of age (p<0.05). Sirius-red staining revealed that thickness of pericardium and cardiac interstitial fibrosis in sm-SOCS3-KO mice were markedly greater compared with wild-type mice at 12 months of age (p<0.05). Western blot analyses showed that phosphorylated STAT3 was significantly increased in sm-SOCS3-KO hearts compared with wild-type mice at 12 months of age (p<0.05), whereas no significant differences were observed at 2 months of age. To investigate the mechanism that gave rise to promoted cardiac fibrosis and diastolic dysfunction during aging in sm-SOCS3-KO, we conducted a real-time PCR array analysis for fibrosis. The expression of pro-fibrotic CTGF (connective tissue growth factor), PDGFb (platelet growth factor-b), and TGF (transforming growth factor) family genes including TGFb1, TGFb2, and TGFb3, were significantly higher in sm-SOCS3-KO hearts than those in wild-type at 6 months of age.

Conclusion

Thus, smooth muscle cell-specific SOCS3 deletion induces increased pericardial fibrosis, cardiac interstitial fibrosis, and increased diastolic dysfunction in aging mice, possibly through the augmentation of pro-fibrotic growth factors.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant JSPS KAKENHI
Mechanisms of endothelial cell activation by endocannabinoid 2-arachidonoylglycerol
Authors: Jehle J , Eich L , Avraamidou E , Tiyerili V , Bindila L , Lutz B , Nickenig G
Published: November 2020

Background

Endothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to restore vascular function and to prevent stent thrombosis. The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Earlier studies have demonstrated the relevance of this endocannabinoid in human pathophysiology during coronary artery disease and in murine experimental atherogenesis. However, evidence on the impact of 2-AG on endothelial cell function remains scarce.

Methods

Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO. The residual endothelial gap at five days was visualized by Evan's blue staining in either group. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. Activation of HCAEC adhesion molecules was characterized by flow cytometry.

Results

Elevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery, resulting in a residual denudation at 5 days of 2291±286 μm vs. 1505±223 μm (n=18–19; p<0.05). In vitro, 2-AG significantly reduced viability of HCAEC at 24 hours (0.31±0.10 vs. 1.00±0.08; n=3; p<0.01). Finally, 2-AG promoted HCAEC activation resulting in a significant increase in THP-1 monocyte adhesion to HCAEC following pre-treatment of HCAEC with 2-AG (0.17±0.03 THP-1 cells per HCAEC vs. 0.07±0.01 THP-1 cells per HCAEC; n=3; p<0.05). Adhesion molecules E-selectin, ICAM-1 and VCAM-1, that are known to be regulated by 2-AG in the venous endothelium, remained unchanged in arterial endothelial cells. Besides, HCAEC migration, ROS-production, expression of NADPH oxidases and secretion of inflammatory cytokines were unaffected by 2-AG.

Conclusion

Elevated 2-AG levels hamper endothelial repair and impair HCAEC proliferation while facilitating adhesion of monocytes. Intriguingly, the underlying mechanisms in the arterial vascular bed appear distinct from venous endothelium. Given that 2-AG is elevated during coronary artery disease in humans, 2-AG might impair reendothelialization after angioplasty and thus impact on clinical outcomes.

Funding Acknowledgement

Type of funding source: None
The role of B cells and IgG in aortic dissection
Authors: Nishida N , Furusho A , Aoki H , Ohno-Urabe S , Nishihara M , Hirakata S , Hayashi M , Ito S , Majima R , Hashimoto Y , Nakao E , Fukumoto Y
Published: November 2020

Background

Aortic dissection (AD) is one of the destructive and fatal aortic diseases, for which molecular pathogenesis is largely unknown. Recent studies have highlighted the importance of inflammatory response in AD. We and others reported that B cells and immunoglobulins participate in pathogenesis of abdominal aortic aneurysm, another form of aortic destructive disease, by promoting inflammatory response. It is not known whether and how B cells participate in AD pathogenesis.

Methods and results

Immunohistochemical staining of human AD tissue revealed that B cells were clustered together with T cells, macrophages and neutrophils at the entry site of AD with medial disruption. B cell cluster was also observed at the site of medial disruption in mouse model of AD that was induced by continuous infusion of beta-aminopropionitrile and angiotensin II (BAPN+AngII). In muMT mouse, which is deficient for B cells and immunoglobulins due to genetic deletion of immunoglobulin heavy chain, BAPN+AngII induced significantly less severe AD compared to that in wild type. Depositions of IgG and fibrinogen, one of the endogenous antigen for natural IgG, were observed after BAPN+AngII infusion before and after AD development in wild type mice. Deposition of fibrinogen was also observed in mMT mice after BAPN+AngII infusion. The rate of aortic rupture and sudden death was approximately 42% in wild type mice, while that in muMT mouse was 12% (P<0.05). Administration of mouse normal polyclonal IgG to muMT mice resulted in dramatic increase in aortic rupture and sudden death, starting at day 7 of BAPN+AngII infusion, and reaching 69% of rupture rate, indicating the critical role of IgG in AD.

Conclusion

These findings demonstrated B cells and IgG are critically involved in the destructive inflammation of AD pathogenesis. Further, the deposition of fibrinogen, one of the targets of natural IgG, precedes the development of AD. Our findings may provide the conceptual foundation of the diagnostic strategy for on-going tissue destruction and for the therapeutic opportunities to intervene the progressive tissue destruction in AD.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Japan Society for the Promotion of Science
Nox2+ myeloid cells drive vascular inflammation and endothelial dysfunction in heart failure after myocardial infarction via angiotensin II receptor type 1
Authors: Molitor M , Rudi W.S , Garlapati V.S , Finger S , Wild J , Knopp T , Karbach S , Ruf W , Muenzel T , Wenzel P
Published: November 2020

Background

Ischemic heart failure (HF) ensuing myocardial infarction (MI) leads to impaired left ventricular function, reduced cardiac output and counterregulatory activation of angiotensin II (AngII) levels. Furthermore, it is characterized by the initiation of a systemic inflammatory response.

Objective

We aimed to elucidate the impact of myelomonocytic cells and their activation by angiotensin II on vascular endothelial function in a mouse model of HF after MI.

Results

HF was induced in male C57BL/6J mice by permanent ligation of the left anterior descending coronary artery. Compared to sham, HF mice had significantly impaired endothelial function accompanied by enhanced mobilization of Sca-1+c-Kit+ hematopoietic stem cells and Sca-1-c-Kit+ common myeloid and granulocyte-macrophage progenitors in the bone marrow as well as increased vascular infiltration of CD11b+Ly6G-Ly6Chigh monocytes and accumulation of CD11b+ F4/80+ macrophages, assessed by flow cytometry. Using mice with Cre-inducible expression of diphtheria toxin receptor in myeloid cells, we selectively depleted lysozyme M+ myelomonocytic cells for 10 d starting 28d after MI. While the cardiac phenotype remained unaltered until 38d post MI, myeloid cell depletion attenuated vascular accumulation of Nox2+CD45+ cells, endothelial dysfunction, oxidative stress and vascular expression of adhesion molecules and angiotensin II receptor type 1 (AT1R). Pharmacological blockade of this receptor for 4 weeks did not significantly alter cardiac function, but mimicked the effects of myeloid cell depletion: Telmisartan (20 mg/kg/d, fed to C57BL/6J mice) diminished bone marrow myelopoesis and myeloid ROS production, attenuated endothelial leukocyte rolling and vascular accumulation of CD11b+Ly6G-Ly6Chigh monocytes and macrophages, resulting in improved vascular function with less abundance of Nox2+CD45+ cells.

Conclusion

Endothelial dysfunction in HF ensuing MI is mediated by inflammatory Nox2+ myeloid cells infiltrating the vessel wall that can be targeted by AT1R blockade.

Funding Acknowledgement

Type of funding source: None
PHACTR1, a pro-atherosclerotic mechanosensitive PPARgamma corepressor in endothelial cells
Authors: Jiang D , Liu H , Zhu G , Li X , Fan L , Yu Z , Wang S , Rhen J , Yin Y , Gu Y , Xu X , Fisher E , Ge J , Xu Y , Pang J
Published: November 2020

Background

Numerous genome-wide association studies revealed that SNPs at phosphatase and actin regulator 1 (PHACTR1) locus are strongly correlated with coronary artery disease (CAD). However, the mechanism linking these variants to CAD remains uncertain.

Purpose

We studied the biological functions and molecular mechanisms of PHACTR1 in atherosclerosis.

Methods and results

Analysis of GTEx database showed that CAD-related SNPs in PHACTR1 are cis-eQTLs for PHACTR1 in arteries. Therefore, we generated Phactr1 knockout mice and crossed them with apolipoprotein E-deficient (ApoE−/−) mice to induce atherosclerosis by high-fat/high-cholesterol (HF-HC) diet. Phactr1 deficiency significantly inhibited atherosclerosis with decreased inflammatory cell infiltration. Western blot showed that PHACTR1 was restricted to endothelial cells (ECs) in mice. Mechanistically, RNAseq of aortic ECs revealed that the major molecular function of PHACTR1 was transcriptional regulation. PPARγ/RXRα was the top transcription factor, and PPARγ target gene expression substantially increased in Phactr1−/− mice. Moreover, we generated endothelial cell specific Phactr1−/−, ApoE−/− mice and found decreased atherosclerotic plaque area in aortic sinus. In vitro, PHACTR1 associated with PPARγ and inhibited PPARγ transcriptional activity. The inhibitory effect of PHACTR1 on PPARγ required its shuttling from cytosol to nucleus triggered by disturbed flow, a well-established pro-atherosclerotic stimulus.

Conclusion

Our results identified PHACTR1 as a mechanosensitive corepressor of PPARγ in ECs to promote atherosclerosis. Endothelial PHACTR1 is a potential therapeutic target for atherosclerosis treatment.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China (NSFC), China Postdoctoral Science Foundation (CPSF)
Beneficial effect of colchicine in an animal model of atherosclerosis
Authors: Cecconi A , Vilchez-Tschischke J.P , Mateo J , Sanchez-Gonzalez J , Espana S , Fernandez-Jimenez R , Lopez-Melgar B , Fernandez Friera L , Lopez-Martin G.J , Fuster V , Ruiz-Cabello J , Ibanez B
Published: November 2020

Background

Atherosclerosis is a chronic inflammatory disease and colchicine demonstrated clinical benefits in the treatment of stable coronary artery disease.

Purpose

Our aim was to assess the anti-inflammatory effects of colchicine on atherosclerotic vascular disease.

Methods

Atherosclerosis was induced in the abdominal aorta of 20 rabbits with high-cholesterol diet and balloon endothelial denudation. The study protocol lasted 36 weeks. After 18 weeks rabbits were randomized to receive either colchicine or placebo. Two animals died in each group. At randomization and at the end of the study, all animals underwent MRI and positron 18F-FDG PET/CT. In this post-hoc subgroup analyses, animals of each group in the first quartile of cholesterol levels were excluded. For MRI plaque volume values were expressed as Normalized Wall Index (NWI) and for 18F-FDG PET/CT values were expressed as mean maximum standard uptake (meanSUVmax). Statistical comparisons were made by using the Mann-Whitney U test for unpaired data and Wilcoxon signed-rank test for paired data.

Results

Results are summarized in Table 1. Animals with higher levels of cholesterol (6 per group) showed significant differences in favor to colchicine group, both as NWI at the end of the protocol 0.47 (IQR 0.05) in the colchicine group versus 0.52 (IQR 0.03) in the placebo; p=0.01] and as relative increase in meanSUVmax [−4.0% (IQR 30.1) in the colchicine group versus 35.1 (IQR 59.0) in the placebo; p 0.041] (Figure 1).

Conclusions

Colchicine may stabilize atherosclerotic plaque by reducing inflammatory activity and plaque burden.

Figure 1

Funding Acknowledgement

Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was supported by a grant from the Sociedad Española de Cardiología and a grant from the Carlos III Institute of Health of Spain and Fondo Europeo de Desarrollo Regional (FEDER, “Una manera de hacer Europa”)
Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of vascular smooth muscle cells to osteochondrocytic-like phenotype
Authors: Miyawaki D , Yamada H , Kubota H , Sugimoto T , Saburi M , Wakana N , Matoba S
Published: November 2020

Background and objective

Maternal high-fat diet (HFD) has been shown to modulate vascular function and remodeling in adult offspring. Atherosclerotic vascular calcification is closely associated with the onset of cardiovascular event. We therefore investigated the impact of maternal HFD on calcification of atherogenic plaques.

Methods and results

Eight-week-old female apo-E−/− mice (C57BL/6) were fed an HFD or a normal diet (ND) one week prior to mating, and the diet was continued throughout gestation and lactation. Offspring of both groups were fed a high-cholesterol diet (HCD) from 8 weeks of age. Ex vivo osteogenic activity of aortic root and aortic arch was analyzed using in vivo imaging system (IVIS) with OsteoSense 680. Sixteen-week-old male offspring of HFD-fed dams (O-HFD) showed a 1.4-fold increase in fluorescent intensity compared with those of ND-fed dams (O-ND) (p<0.05). Likewise, female O-HFD showed a significantly increased osteogenic activity in aortic arch (154%, p<0.05). Percentages of plaque area and oil red O-positive area were comparable between O-ND and O-HFD of both genders, suggesting that augmented osteogenic activity in O-HFD is not dependent on the plaque size. To investigate the underlying mechanism of augmented calcified plaque formation in O-HFD, vascular smooth muscle cells (VSMCs) of thoracic aorta form 8-week-old male offspring were primarily cultured and VSMCs calcification was induced by treatment with calcification media supplemented with phosphate (2.6 mM). Alizarin-red-positive area upon 10 days stimulation showed a 3.4-fold increase in VSMCs from O-HFD compared with that from O-ND (p<0.01). Consistently, western blotting analysis revealed that expression level of osteocalcin was significantly higher in O-HFD than O-ND, suggesting that osteochondrocytic transformation of VSMCs is augmented in O-HFD.

Conclusion

Our findings demonstrate that maternal HFD accelerates the development of atherogenic calcification independent of plaque size. In vitro transformation to osteochondrocytic-like cells is enhanced in VSMCs from offspring of HFD-fed dams. Inhibition of VSMCs skewing toward osteochondrocytic-like cells could be a potential therapeutic target for preventing the development of atherosclerotic vascular calcification.

Funding Acknowledgement

Type of funding source: None
Molecular structure analysis of ascending aorta aneurysm upon atherosclerosis
Authors: Mamarelis I , Mamareli V , Kyriakidou M , Tanis O , Mamareli C , Kotoulas S , Subramaniyan Parimalam S , Packirisamy M , Badilescu S , Anastassopoulou J , Kotoulas C , Theophanides T
Published: November 2020

Background

The atherosclerotic ascending aorta could represent a potential source of emboli or could be an indicator of atherosclerosis in general with high mortality. The mechanism of aneurysm formation and atherosclerosis of the ascending aorta at the molecular level has not yet been clarified. To approach the mechanism of ascending aortic lesions and mineralization at a molecular level, we used the non-destructive FT-IR, Raman spectroscopy, SEM and Hypermicroscope.

Methods

Six ascending aorta biopsies were obtained from patients who underwent aortic valve replacement (AVR) cardiac surgery. CytoViva (einst inc) hyperspectral microscope was used to obtain the images of ascending aorta. The samples were dissolved in hexane on a microscope glass plate. The FT-IR and Raman spectra were recorded with Nicolet 6700 thermoshintific and micro-Raman Reinshaw (785nm, 145 mwatt), respectively. The architecture of ascending aorta biopsies was obtained by using scanning electron microscope (SEM of Fei Co) without any coating.

Results

FT-IR and Raman spectra showed changes arising from the increasing of lipophilic environment and aggregate formation (Fig. 1). The band at 1744 cm–1 is attributed to aldehyde CHO mode due to oxidation of lipids. The shifts of the bands of the amide I and amide II bands to lower are associated with protein damage, in agreement with SEM data. The bands at about 1170–1000 cm–1 resulted from the C-O-C of advanced glycation products as result of connecting tissues fragmentations and polymerization. The spectroscopic data were analogous with the lesions observed with SEM and hypermicroscopic images.

Conclusions

The present innovate molecular structure analysis showed that upon ascending aorta aneurysm development an excess of lipophilic aggregate formation and protein lesions, changing the elasticity of the aorta's wall. The released Ca2+ interacted mostly with carbonate-terminal of cellular protein chains accelerated the ascending aorta calcifications.

Figure 1. FT-IR and Raman spectra

Funding Acknowledgement

Type of funding source: None
A novel three-dimensional visualization system revealed an essential adaptive angiogenic response during the early phase of pulmonary hypertension
Authors: Fujiwara T , Takeda N , Hatano M , Nishimura S , Komuro I
Published: November 2020

Background

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance and right heart failure with progressive narrowing or occlusion of the pulmonary artery. However, the assessment of vascular remodeling is mostly limited to averaged increases in wall thickening, and even the role of vascular endothelial growth factor (VEGF), remains incompletely understood; Although abundantly expressed VEGF is expected to elicit angio-obliteration and the knockout of hypoxia inducible factor (HIF) prevents PH in mice, VEGF inhibitor Sugen exacerbates hypoxia (Hx)-induced PH model, which is referred to as VEGF paradox.

Purpose

To analyze three-dimensional (3D) spatiotemporal changes of pulmonary microstructure and function, which reflect the disease activity and lead to resolve the paradox.

Methods and results

We developed a novel 3D visualization system of microstructural networks in whole mouse organ with single-cell resolution, using combined tissue clearing technique called CUBIC and multiphoton excitation microscope. The system enabled the simultaneous 3D evaluation of microvascular structure, invaded macrophages and fibrosis with effective penetration of several mm (whole organ). Three-dimensional observations of PH mice models including Hx, Sugen/Hx, and human-like Alk1+/− hereditary PH models, revealed that not only inward (negative) microvessel remodeling with stenosis, but also marked elongation of microvascular ECs, was evident except Sugen/Hx model at the early phase, which had not been detected by 2D histological sections. Comparable transcriptome analysis revealed that PGC1α, which regulates HIF-independent VEGF expression and angiogenesis, plays an important role in the characteristic response for mitochondrial and microvascular maintenance. PGC1α was up-regulated in the early phage in Hx and Alk1+/− PH models with microvascular angiogenetic change, whereas Sugen/Hx-model did not increase PGC1α expression and did not show microvascular remodeling. Furthermore pulmonary ECs-specific PGC1α-deficient mice exacerbated Hx-PH model with decreased VEGF expression and microvessel density, and administration of Baicalin, a flavonoid enhancing PGC1α expression, ameliorated Hx-PH model with increased VEGF expression.

Conclusions

The 3D visualization system disclosed an unexpected change of angiogenic microvascular structure in the early phage of PH, which is regulated by EC PGC1α. Microvascular angiogenesis which is induced by up-regulation in PGC1α -VEGF pathway is a crucial factor for compensation of PH in the early phase, which provides a potential novel therapeutic target for PH.

Figure 1

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSJP
Treating ApoE−/− mice with Toll-like receptor 7 ligand lead to expansion of marginal zone B cells, reduced plasma cholesterol and decreased atherosclerosis
Authors: Paulsson-Berne G , Karadimou G , Gistera A , Gallina A , Centa M , Salagianni M , Andreakos E , Hansson G.K , Malin S , Olofsson P
Published: November 2020

Background

The interplay between innate and adaptive immunity is involved in the cause of the life-threatening events in atherosclerosis, such as myocardial infarction and stroke. We have previously shown that higher levels of Toll-like receptor 7 (TLR7) expression in human atherosclerotic lesions are correlated to better patient outcome, emphasizing the role of the innate pattern recognizing receptors in the pathogenesis of atherosclerosis.

Purpose

Firstly, to investigate if activation of the innate immune receptor TLR7 in vivo in experimental atherosclerosis is a potential way to reduce disease and secondly to identify pathways upon TLR7 ligation treatment.

Methods

Apolipoprotein E (ApoE−/−) deficient mice with established disease were injected intraperitoneal with a TLR7 ligand. Local effects were evaluated by characterization of atherosclerotic lesions. Systemic effects of the treatment were investigated by immune composition analysis in the spleen and by plasma measurements

Results

In vivo treatment of ApoE−/−mice with TLR7 ligand arrested atherosclerotic lesion development in the aortic root. Moreover, in the lesions of the treated mice we detected decrease in necrotic area and decrease in accumulation of apoptotic cells. In the spleen we detected an expansion of marginal zone B cells accompanied with an increase in IgM antibodies against oxidized low-density lipoprotein (oxLDL). The treated mice had reduced plasma cholesterol levels.

Conclusions

In vivo administration of TLR7 ligand ameliorated the atherosclerotic burden in old ApoE−/− mice. Our findings indicate that TLR7 ligation is involved in control of inflammatory responses in atherosclerosis, suggesting that TLR7 is a potential therapeutic target. Further analysis is needed in order to elucidate the underlying mechanisms by which TLR7 exerts its protective role.

Funding Acknowledgement

Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Swedish Heart-Lung Foundation
IgG1 antibodies against phosphorylcholine are associated with protection in SLE and atherosclerosis: potential underlying mechanisms
Authors: Frostegard J , Steen J , Fiskesund R , Frostegard A.G , Rahman M , Lundstrom S , Thiagarajan D
Published: November 2020

Background

The risk of cardiovascular disease (CVD) and atherosclerosis is very high in SLE. This is a clinical problem, and could also shed light on immunity and atherosclerosis in general. IgM antibodies against phosphorylcholine (anti-PC) may be protective in atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE). We here study IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE.

Methods

We determined anti-PC by ELISA in 116 SLE-patients and 110 age- and sex-matched controls. For functional studies, we used three in-house generated, fully human monoclonal IgG1 anti-PC (A01, D05, E01). Apoptosis was induced in Jurkat T-cells and pre-incubated with A01, D05, E01 or isotype control IgG1 and effects on efferocytosis by human macrophages studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach.

Results

IgG1 but not IgG2 anti-PC levels were higher among SLE patients (p=0.02). IgG1 anti-PC was negatively associated with SLICC and SLEDAI (OR: 2,978 CI: 0.876–10.098, OR: 5.108 CI 1.3 20.067 respectively) and negatively associated with CVD, atherosclerotic plaques and echolucent (potentially vulnerable plaques) but the association for the two former was not significant after controlling for confounders. D05 had maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC associated neo-epitopes. Peptide analysis showed difference in the CDR3 region of the three anti-PC IgG1 clones which are crucial for recognition of PC on apoptotic cell surface and other neo-epitopes.

Conclusion

Anti-PC IgG1 is negatively associated with disease activity, and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells.

Funding Acknowledgement

Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swedish Heart and Lung Foundation, Reumatikerfonden