bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor necrosis factor superfamily 14 (TNFSF14/ LIGHT) attenuates neovascularization by reducing circulating endothelial progenitor cells and function
Authors: Hsu C Y , Huang C Y , Lin S J , Lin F Y
Published: November 2023

Background

Tumor necrosis factor superfamily 14 (TNFSF14) is also known as LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) can bind to herpesvirus invasion mediator and lymphotoxin-β receptor to perform its biological activity. LIGHT signaling strengthens the synthesis of nitric oxide, reactive oxygen species, and cytokines and affects organ function. LIGHT also stimulates angiogenesis in tumors and induces synthesis of high endothelial venules, degrades extracellular matrix in thoracic aortic dissection, induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed this effect in the current study.

Methods

C57BL/6 mice underwent hind limb ischemia surgery for model construction. Doppler ultrasound, immunohistochemical staining, and western blotting were employed to analyze the effects of LIGHT on angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms.

Results

In the mice, LIGHT injection inhibited angiogenesis in ischemic limbs. In vitro, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the normal operation of intracellular Akt signaling pathway and endothelial nitrite oxide synthase (eNOS) function.

Conclusions

LIGHT inhibits angiogenesis after tissue ischemia. LIGHT inhibition of intracellular Akt-eNOS signaling axis and mitochondrial respiration in EPCs impair cell function. These events are the possible causes of impaired angiogenesis.LIGHT decreases the recovery of ischemiaSenescence and dysfunction of EPC
Impact of comorbidities on heart rate variability in patients with high cardiovascular risk
Authors: Gadaleta M , Mason F , Steinhubl S R , Quer G
Published: November 2023

Introduction

Heart rate variability (HRV) measures the variation in time intervals between successive heartbeats (RR intervals) and is considered a measure of a healthy regulatory system. It is influenced by a variety of factors, including age and comorbidities. We estimate temporal HRV metrics, such as the standard deviation of RR intervals (SDNN), and frequency metrics, such as the normalized high frequency component of the RR intervals (HFNU), to identify which were best for characterizing inter-individual differences by age, gender, and health status.

Purpose

Understand inter-individual differences in HRV and the influence of age, gender, and available comorbidities.

Methods

Data from at least one 2-week single-lead ECG were obtained from the 1,742 participants of the trial. Inclusion criteria for the study are defined as follows: age of 75 years or older, or a male older than age 55 years or female older than 65 years with 1 or more comorbidities that are risk factors for cardiovascular diseases. We adopted a linear regression method, where each HRV measure constitutes the outcome of a separate model, while the demographic characteristics (age and gender), the heart rate (HR), and the presence of comorbidities (yes/no for each comorbidity) are used as predictor variables. Age has been evaluated as a quadratic term to assess non-linearity of the association, and an age-gender interaction term has also been evaluated. Coefficients are expressed in terms of standard deviations, allowing for a direct comparison between metrics with different scales and variability. The HRV metrics are evaluated every 5 minutes, then the average value over a 1-day period is considered as input for the model.

Results

Temporal HRV metrics were lower among women compared with men and increased with age. They were higher in the presence of many disease states, including chronic heart failure (CHF), obesity (OBE), chronic obstructive pulmonary disease (COP), and sleep apnea (SA), but were lower among those with history of myocardial infarction (MI), chronic renal failure (CRF), and use of beta blocker medications (BB). There was no significant association of these metrics with hypertension or diabetes. For frequency metrics, analysis was limited to HFNU due to the high correlations among these measures. HFNU had strong positive associations with all co-morbidities except stroke, with particularly large coefficients for CHF, COP, and MI. HFNU values were also higher among subjects taking beta blockers. (Figure)

Conclusion

HRV can be an indicator of autonomic nervous system (ANS) dysfunction, which is often present in many diseases. By measuring HRV, clinicians can gain insights into the state of the ANS and potentially identify early warning signs of disease progression or complications.HRV and comorbidities
Immune system humanization in dyslipidemic Rag2-KO/IL2rg-KO/CD47-KO/LDL-R KO mice controls atherosclerosis
Authors: Bonacina F , Moregola A , Norata G
Published: November 2023

Background and aim

Given the key role of the immune response during atherosclerosis and the therapeutic interest of biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and to test therapeutic approaches for atherosclerosis is continuously increasing.

Here we describe the characteristics of an innovative immunodeficient mouse humanized with hCD34+ cells on an atheroprone background.

Methods

LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse recipient TKO-LDLR KO of human hematopoietic stem cells (hCD34+).

Results

TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet for 8 weeks, both male and females TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia (total cholesterol 870.9 and 890.1 mg/dL male and females respectively), steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next we tested the impact of immune system humanization on atherosclerosis. TKO-LDLR KO pups received a low-dose irradiation (150-200 cGy) and thereafter 1,5-2 x 10^5 hCD34+ were injected with in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. The humanization with hCD34+ cells affected atherosclerosis development as compared to non-humanized TKO-LDLR KO mice.

Conclusion

We have generated and characterized for the first time a humanized dyslipidemic TKO-LDLR KO mice. This mouse model presents human B and T cells and represent and important toll to investigate the impact of biologics targeted toward human targets in the context of atherosclerosis.
Growth differentiation factor 15 (GDF15) is elevated in human plaques and promotes endothelial-to-mesenchymal transition (EndMT)
Authors: Filipova V , Stuerzebecher P E , Kralisch S , Kokot K E , Kneuer J M , Erbe S , Toenjes A , Laufs U , Boeckel J N
Published: November 2023

Introduction and Purpose

Growth Differentiation Factor 15 (GDF15) is a member of the TGF-β superfamily and is upregulated under conditions of injury and stress such as hypoxia. GDF15 serum concentrations correlate with inflammation, cardiac fibrosis and unfavorable prognosis in cardiovascular diseases. The functional role of GDF15 in endothelial cells is largely unknown. Here, we investigate a possible role of GDF15 in the transdifferentiation of endothelial cells (EC) into mesenchymal cells (EndMT).

Methods and Results

GDF15 is expressed at mRNA and protein level in primary human ECs and is secreted as determined by qPCR, single cell sequencing (scRNA-seq) and enzyme-linked immunosorbent assay (mean ± SEM, 743.8±39.3 pg/ml, n=3). Analysis of vascular sections from Ldlr-/- mice fed a high-fat diet using spatial transcriptomics revealed increased GDF15 expression. ScRNA-seq analysis of human plaques and normal vessel sections from the same individuals revealed a 22-fold ± 5.48 (p<0.05) increase in GDF15 in ECs located in plaques compared to ECs in control vessel regions. Experiments in cultured human endothelial cells showed increased GDF15 mRNA expression qPCR (+1.7-fold control, p<0.05) and scRNA-seq analysis after EndMT induction.

Treatment with increasing levels of recombinant GDF15 (10, 50 and 150 ng/ml) induced EndMT as measured by expression of the EndMT marker calponin (+3.36, 7.99 and 9.53-fold, all p<0.05). Interestingly, EndMT induction decreased extracellular GDF15 levels (-54.6%, p<0.05), whereas intracellular levels in EC were increased (+1.9-fold control, p<0.05). The use of GDF15 neutralising antibodies (1 µg/ml) reduced soluble GDF15 levels by 97.7% as measured by ELISA (p<0.001). Treatment with neutralising antibodies against GDF15 reduced EndMT induction by 54% as measured by the EndMT marker calponin (p<0.05).

Conclusion

In conclusion, GDF15 is elevated in mouse and human plaques, and in EndMT. Treatment with GDF15 induces EndMT, whereas inhibition by neutralising antibodies reduces EndMT induction. These data are consistent with a functional role of GDF15 in endothelial dysfunction and atherogenesis.
Hypercoagulability impairs plaque stability in diabetes-induced atherosclerosis
Authors: Ambreen S , Fatima S , Elwakiel A , Rana R , Singh K
Published: November 2023

Introduction

Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accelerated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications are the major cause of mortality in diabetic patients. Efficient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Atherosclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation.

Methadology

Female ApoE-/- or TMPro/Pro ApoE-/- mice (age 6 to 8 weeks) were fed a normal chow diet and were made diabetic (DM) by injecting streptozotocin, reflecting type 1 DM. After 22 weeks of age, the body weight of the mice was measured, and the mice were sacrificed. Plasma and tissue samples were analysed for different parameters.

Results

Here, we demonstrate that atherosclerotic plaque stability is reduced in hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice), which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased fibrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently, more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice.

Conclusion

Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis.
Olfactory receptor 2 signaling drives experimental abdominal aortic aneurysm formation
Authors: Schelemei P , Picard F , Nemade H , Mehrkens D , Grimm S , Tinaz K , Wagner E , Orrechioni M , Wagenhaeuser M , Schelzig H , Roy J , Liljeqvist M , Mollenhauer M , Baldus S , Winkels H
Published: November 2023

Background

Abdominal aortic aneurysms (AAA) are defined as enlargement above 3cm or 50% of its original diameter. Therapeutic options are limited to surgical and endovascular treatment, while no pharmacological interventions are available.

Chronic inflammation of the vessel wall and in particularly macrophage infiltration has been associated with disease progression, leading to extensive tissue remodeling, yet the underlying mechanisms are incompletely understood.

A recent study has shown extra-nasal expression of the olfactory receptor 2 (OLFR2) on vascular macrophages, in which activation induced a pro-atherogenic response.

The role of macrophage OLFR2 signaling in AAA, however, is unknown.

Methods & Results

To determine whether the OLFR2 ortholog OR6A2 is expressed in human AAA, we performed in silico micro-array analysis of human AAA tissue. OR6A2 was shown to be specifically upregulated in AAA tissue with thrombus formation compared to healthy control tissue. We confirmed OR6A2 expression on macrophages by immunofluorescence.

To determine OLFR2 expression among vascular immune cells in mice, we induced AAA formation by porcine pancreas (PPE) infusion into the infrarenal aorta of C57BL6/J (WT) mice and performed spectral cytometry utilizing a 26-marker panel at baseline, day 7 and day 28. OLFR2 expression peaked at day 7 and decreased to baseline levels at day 28 particularly on pro-inflammatory monocytes, mixed resident / migratory-like macrophages and pro-inflammatory macrophages, while OLFR2 was not detectable on tissue resident-like macrophages.

To assess a functional role of OLFR2 in AAA formation, we induced AAA in OLFR2-deficient (KO) mice and WT controls by PPE. Ultrasound analysis of the abdominal aorta revealed protection from AAA formation in KO mice. Histological analysis showed significantly reduced macrophage while increased smooth muscle cell content in aortic sections of KO mice compared to WT controls. This was accompanied by higher collagen content in KO mice. Additionally, plasma levels of the pro-inflammatory cytokines TNF-alpha and Interferon-gamma were significantly reduced in KO mice 28 days post PPE.

Bulk transcriptome analysis corroborated these findings and indicated increased pathways associated with vascular smooth muscle cell contractility while decreased leukocyte activation in KO aortas.

Conclusion

We show that OLFR2 deficiency in mice attenuates abdominal aortic aneurysm development, aortic remodeling and pro-inflammatory response. OLFR2 might thus present a novel target for drug-based AAA therapies.
Asymptomatic Brugada patients: long-term prognosis from a large prospective study
Authors: Giustetto C , Gaita F , Cerrato N , Martino A M , Millesimo M , Saglietto A , Bonacchi G , Barbonaglia L , Bianchi F , De Ferrari G M , Calo' L
Published: November 2023

Background

One of the greatest challenges concerning the Brugada syndrome is how to stratify and manage asymptomatic patients, who represent the majority of patients with Brugada ECG pattern (BrECG).

Purpose

Aim of our study was to evaluate the long-term prognosis of a large population of asymptomatic Brugada patients.

Methods

One thousand one hundred and forty-nine asymptomatic Brugada patients were collected. Patients were treated as follows: in the 539 subjects with spontaneous type 1 BrECG documented on basal ECG or at 12-lead 24-hour Holter monitoring (12L-Holter) an electrophysiological study (EPS) was proposed and ICD implantation was proposed on the basis of the EPS results; in the 610 subjects with drug-induced-only type 1, clinical follow-up with multiple ECGs and 12L-Holter was suggested to search for spontaneous type 1 BrECG.

Results

Over a median follow-up of 6 (4-9) years there were 17 arrhythmic events (1.5%). Sixteen events occurred in the 539 patients with spontaneous type 1 (3%; 0.4% person-years) and only 1 in the 610 patients with drug-induced type 1 BrECG (0.16%; 0.03% person-years, p 0.001). Patients with spontaneous type 1 had a 15-time higher risk of arrhythmic events than patients with drug- induced type 1 BrECG. Patients with spontaneous type 1 BrECG and positive EPS had a 3-time higher risk of arrhythmic events than patients with negative EPS (6.8% vs 1.6%, p 0.035). An ICD was implanted in 163 patients: 8 (5%) received an appropriate intervention, while 23% experienced device-related complications, including 1 death.

Conclusions

Asymptomatic patients with spontaneous type 1 BrECG have an event rate of 3% at a median follow-up of 6 years. In this group, EPS represents a useful risk stratification procedure, as a positive EPS identifies patients with a 3-time higher event rate as compared to subjects with negative EPS. A negative EPS identifies a population at low although not zero-risk. Asymptomatic patients with drug-induced only Brugada ECG pattern are a group at very low arrhythmic risk. ICD implantation is burdened by a high incidence of complications, therefore alternative therapeutic strategies are needed.
Role of S100A4 in the crosstalk between smooth muscle and inflammatory cells in atherosclerosis
Authors: Kapitanova K , Cardoso Dos Santos L M , Klingelhofer J , Azar P , Bochaton-Piallat M L
Published: November 2023

Background

During atherosclerotic plaque formation, smooth muscle cells (SMCs) accumulate in the intima and undergo a transition from a contractile to a synthetic phenotype. We previously showed that S100A4 is a marker of the synthetic SMCs in vitro and intimal SMCs in pig, human and mouse. In vitro, S100A4-treated SMCs acquire a pro-inflammatory phenotype. The systemic neutralization of extracellular S100A4 in ApoE-/- mice induces atherosclerotic plaque stabilization.

Purpose

To characterize the role of S100A4-expressing SMCs in atherosclerosis.

Methods and results

We established a coculture model between S100A4-stimulated human aortic SMCs and human blood-derived monocytes. Flow cytometry analysis of monocytes exposed to S100A4-treated SMCs for 3 days showed an upregulation of markers typical of inflammatory M1 (HLA-DR) and anti-inflammatory M2 (CD206) macrophages (fig. 1). Macrophages also upregulated mRNA expression of NPC Intracellular cholesterol transporter 1 and 2 by qPCR, suggesting modifications in their cholesterol metabolism. In vivo, in an SMC-lineage-tracing ApoE-/-mouse model, we identified S100A4-expressing SMCs at the onset of atherosclerosis in aortic media of 11-week-old mice fed a high cholesterol diet (HCD) for 3 weeks. Medial SMCs were characterized by means of single-cell RNA sequencing. Interestingly, 10 out of 11 identified clusters expressed S100A4, demonstrating broad and early involvement of S100A4 in shaping medial SMC identity. We assumed that blood-derived monocytes might be a driving force of early S100A4 activation in SMCs. To mitigate the impact of monocytes, 8-week-old ApoE-/- mice were fed an HCD for 9 weeks and injected with either clodronate-encapsulated or empty liposomes during the first 3 weeks. No differences in S100A4 expression in the media were observed by qPCR. Unexpectedly, ApoE-/- mice treated with clodronate demonstrated larger atherosclerotic lesions compared to control animals as measured by OilRedO staining (fig. 2). Studies are in progress to define the plaque composition.

Conclusion

Our findings shed light on the role of S100A4 in the crosstalk between SMCs and inflammatory cells in atherosclerosis and the fine-tuning of the phenotype of medial SMCs prior to their accumulation in the intima. We suggest that temporary monocyte depletion at the onset of the disease alters the mechanisms underlying plaque formation, worsening the outcome of the disease, while not affecting S100A4 expression in medial SMCs.
Myeloid cannabinoid receptor CB1 deficiency confers atheroprotection in a sex-specific manner
Authors: Wang Y , Li G , Chen B , Shakir G , Volz M , Van Der Vorst E , Maas S , Hristov M , Lacy M , Enard W , Lutz B , Weber C , Herzig S , Guillamat-Prats R , Steffens S
Published: November 2023

Aim

Cardiovascular diseases (CVDs) are the leading cause of death globally, claiming an estimated 17.9 million lives each year. A better understanding of the underlying chronic inflammatory disease mechanisms in atherosclerosis might help finding novel treatments and reduce CVD deaths. In particular, the cannabinoid receptor CB1 has been implicated in atherosclerosis, while its cell-specific effects in this disease are not well understood.

Methods

Myeloid Cnr1 (CB1 encoding gene) knockout mice were generated on apolipoprotein E deficiency (Apoe-/-) background by crossing CB1flox mice with transgenic mice carrying Cre under control of the lysozyme M (LysM) promoter for selective expression in myeloid cells. Age - and sex-matched groups were analyzed at baseline, 4 and 16 weeks Western diet (0,15% cholesterol). For in vitro experiments, murine bone marrow-derived macrophages (BMDM) were used.

Results

Male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores compared to controls, while only minor effects in females were observed. Male Cnr1 deficient mice had reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in vitro in BMDMs and blunted by female hormone estradiol treatment. Array-based kinase activity profiling of BMDMs stimulated with a synthetic CB1 agonist revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling via bulk RNA-sequencing of CB1 agonist-stimulated BMDMs further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, again only in male mice.

Conclusion

Impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The biological effects of macrophage CB1 signaling seem to be more pronounced in male mice. This highlights the need to consider the biological sex as an important variable in preclinical studies.
A pH-Responsive and MRI-Functional nanomedicine enabling targeted delivery of simvastatin in atherosclerosis
Authors: Wang J W , Lin Y Z , Liu J J , Chong S Y , Kah J C Y
Published: November 2023

Background

Simvastatin is commonly used for the treatment of atherosclerosis. However, it causes side effects in some patients and not always effective. Moreover, although its treatment effects are often attributed to its cholesterol-lowering activity, some in vitro studies suggest that simvastatin also exerts antioxidant and anti-inflammatory effects.

Purpose

To develop an atherosclerotic plaque microenvironment-responsive nanoparticular system to deliver and release Gadolinium (Gd3+) for magnetic resonance imaging (MRI) and simvastatin for treatment within the plaque.

Methods

A nanoparticle was synthesized with nanoscale coordination polymers (NCPs), a pH-responsive linker, Gd3+, and simvastatin (ST), i.e. a ST/NCP-PEG nanoparticle. The biological effects of ST/NCP-PEG nanoparticle were tested on RAW264.7 macrophages. Four weeks old ApoE-/- mice were fed on high fat diet for 4 weeks and then randomly divided into three groups for a 8-week treatment regime via weekly intravenous administration of: 1) NCP-PEG nanoparticle without ST (control); 2) ST/NCP-PEG nanoparticles; or 3) free ST.

Results

We have developed a pH-responsive ST/NCP-PEG nanoparticle carrying an MRI contrast agent Gd3+ and a drug molecule ST, with an 80 nm diameter in spherical shape. This nanoparticle collapsed into biodegradable components while releasing both Gd3+ and ST under the atherosclerotic plaque microenvironment with a pH 5.6. ST/NCP-PEG nanoparticles exerted strong anti-oxidant and anti-inflammatory effects in vitro, as determined by an intracellular ROS indicator DCF-DA and expression levels of cytokines (e.g. IL-6, TNF-α and MCP-1), respectively. Upon intravenous administration, ST/NCP-PEG nanoparticles, as demonstrated by IVIS imaging system, accumulated specifically in the plaques in a time-dependent manner, reaching the peak concentration at 24 hours post administration. The plaques were readily visualized by MR imaging (T1-weighted signal) due to the enrichment of Gd3+. After 8 weeks treatment, compared to the empty NCP-PEG nanoparticle, treatment with ST/NCP-PEG and free ST reduced plaque lesion size by 55% (p<0.001) and 29% (p<0.05), respectively. Moreover, ST/NCP-PEG nanoparticles showed a larger reduction in plaque lesions than free ST (p<0.05). Oil red O staining of both the whole aorta and the aortic roots generated similar results in terms of plaque lesion changes. Mechanistically, both ST/NCP-PEG and free ST reduced ROS production and increased the ratios of M2/M1 macrophages within the plaque, with ST/NCP-PEG nanoparticle treatment exerted larger therapeutic effects. Moreover, long-term treatment with ST/NCP-PEG nanoparticles did not show obvious toxicity in all major organs.

Conclusions

An atherosclerotic plaque microenvironment-responsive MR imaging functional nanoparticle for targeted delivery of ST to the plaque could diagnose atherosclerotic plaque and improve treatment efficacy of ST.
Additional effects of combination therapy of antihypertensive medication on aortic elastic properties beyond blood pressure lowering effect in spontaneously hypertensive rats
Authors: Kim S A , Koh A R , Hong D K , Suh S W
Published: November 2023

Background

Combination therapy in the treatment of hypertension provides better therapeutic efficacy, improved tolerability by lowering the dose and reduced potential for side effects than monotherapy. Especially, combinations of calcium channel blocker (CCB), angiotensin receptor blocker (ARB) or diuretics (thiazide) are generally effective and well tolerated regimen in antihypertensive treatment. However, we have focused more on the antihypertensive power of the combination therapy rather than the effect on the vessel itself.

Purpose

The purpose of this study is to determine the efficacy of antihypertensive medications as ARB and CCB and its combination with or without thiazide on the aortic structural and functional properties beyond the effect of blood pressure (BP) lowering.

Methods

Fourteen-week-old male spontaneous hypertensive rats were randomly divided into 5 groups, and treated with 1) vehicle (V), 2) amlodipine besylate (A), 3) olmesartan (O), 4) combined amlodipine and olmesartan (AO) or 5) combined amlodipine, olmesartan and thiazide (AOD), respectively. After 8 weeks of treatment, hemodynamic parameters and structural and functional properties of aorta as intima-media thickness (IMT) and velocity vector imaging (VVI) parameters of aorta including fractional area change (FAC), circumferential strain (CS) and strain rate (CSR) were assessed in each group and collagen contents of aorta were quantified as well.

Results

During the study period, systolic and diastolic BP were serially decreased in all treatment groups. Especially, AOD group showed lowest systolic and diastolic BP levels and also showed smallest IMT and vessel area and highest elasticity (FAC and CS) among the other groups (p<0.01) at 8 weeks (Table 1). In histologic analysis, collagen contents of aorta in all treatment groups showed significant associations with BP, IMT and VVI parameters (FAC, CS, CSR) (p<0.05), respectively. Particularly, aorta of AO and AOD groups showed significantly lower levels of collagen contents than that of control group. (p< 0.05) (Figure 1)

Conclusion(s)

Beyond the BP lowering effect, combination of amlodipine and olmesartan with or without thiazide minimize structural changes and shows better protection for the progression of arteriosclerosis than individual monotherapy in treatment of hypertension.Table 1Figure 1
CRIP1: Role in the pathogenesis of hypertension by regulating interactions with cytoskeletal proteins
Authors: Duque Escobar J , Boateng B , Schweigert O , Riedel R , Wenzel P , Zeller T
Published: November 2023

Background

Hypertension is a complex polygenic disease and the underlying molecular mechanisms remain unknown. In an interdisciplinary approach, we identified cysteine-rich protein 1 (CRIP1) in human monocytes as being strongly associated with hypertension, suggesting a link between CRIP1 and the pathophysiology of hypertension via the immune system. The identification of CRIP1 protein interaction partners and associated signaling pathways would provide insight into the pathophysiological significance of CRIP1 under hypertensive conditions.

Objective

The overall aim of our project is to evaluate the functional role of CRIP1 in hypertension and cardiovascular disease (CVD). Here, using systems medicine approaches including experimental settings and human cohort data, we focused on the identification of novel CRIP1 protein interaction partners that may be associated with the pathogenesis of hypertension and cardiovascular disease.

Methods

Monocytic CRIP1 was immunoprecipitated and co-immunoprecipitated proteins in the eluate were analyzed by LC-MS/MS and Pathway and Process Enrichment Analysis and validated by immunoblot techniques. Silencing of CRIP1 was established in the monocyte-like cell line THP-1 using a vector-based shRNA system. The association of CRIP1 with genes encoding cytoskeletal proteins was accessed in a human cohort by transcriptome-based analysis of human monocytes in the Guttenberg-Gesundheitsstudie (GHS; n = 1527).

Results

First, a Yeast Two-Hybrid Assay was performed and two potential protein interaction partners were predicted for CRIP1: KTN1 and TPM2. To obtain comprehensive information on interacting proteins, a co-immunoprecipitation assay with a specific antibody against monocytic CRIP1 confirmed these as interaction partners and revealed further protein interactions with proteins that are crucial for monocyte migration and invasion behavior of monocytes by LC-MS/MS and Pathway and Process Enrichment Analysis, and immunoblot analysis. In an in vitro experiment with the human monocytic cell line THP-1, downregulation of CRIP1 by >70% significantly reduce the abundance of these proteins. In addition, in a transcriptome-based analysis of human monocytes, most of the genes encoding proteins of microtubule cytoskeleton organization (GO:0000226), actin filament and cytoskeleton organization (GO:0007015 and GO:0030036), Arp2/3 complex-mediated actin nucleation (GO:0034314), and cytoskeleton-dependent intracellular transport (GO:0030705) were associated with CRIP1 expression.

Conclusion

As CRIP1 expression in human monocytes has already been linked to hypertension, our results suggest that CRIP1 may play a role in the pathogenesis of this disease by regulating interactions with cytoskeletal proteins that lead to impaired monocyte migration and invasion behaviour. Further experiments are underway to translate our findings into clinical application.
Accelerated arterial thrombus formation in Hutchinson Gilford Progeria Syndrome
Authors: Puspitasari Y M , Han J , Karch C , Ministrini S , Liberale L , Akhmedov A , Montecucco F , Beer J H , Bongiovanni D , Luscher T F , Camici G G
Published: November 2023

Introduction

Arterial thrombosis is an age-associated cause of myocardial infarction and stroke, which are the leading causes of mortality and morbidity worldwide. Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the lamin A gene. It is characterized by progressive premature aging, in which caused a decreased life expectancy. Two most frequent causes of death of children with HGPS are myocardial infarction and ischemic stroke – cardiovascular events that are tightly linked to arterial thrombosis. Despite this fact, the effect of the specific lamin A gene mutation on arterial thrombus formation remains unknown.

Methods

Transgenic heterozygous LmnaG609G knock-in (HGPS) mice and the corresponding wild-type (WT) littermate controls underwent photochemically-induced carotid artery endothelial injury to trigger thrombosis. Coagulation and fibrinolytic factors were measured in these animals. Ex-vivo platelet activation assay was also performed.

Results

HGPS mice displayed accelerated thrombus formation as underlined by shortened time to occlusion compared to WT littermates (Figure 1A-B). The level of tissue factor and von Willebrand factors are comparable between groups. No significant differences were found on factors involved in the fibrinolytic system, which was confirmed by the comparable level of D-dimers in both groups. A higher level of thrombin-antithrombin complex was observed, accompanied by lower level of antithrombin. Interestingly, HGPS animals displayed enhanced platelet activation markers expression upon ADP, collagen, and thrombin stimulation compared to WT group, demonstrating a higher platelet reactivity in progeria animals (Figure 1C).

Conclusions

Our study demonstrated an increased arterial thrombotic response in HGPS mice as compared to WT littermates which is, at least in part, mediated by higher platelet reactivity. This novel observation may provide a mechanistic explanation for the increased incidence of acute cardiovascular events observed in children with HGPS and warrant further clinical investigations also in consideration of available antiplatelet therapies.
S1P receptor-1 signaling reduces arterial thrombosis by upregulation of endothelial thrombomodulin expression
Authors: Mourikis P H I L I P , Benkhoff M A R C E L , Barcik M A I K E , Knoop B E T U E L , Trojovsky K A J E T A , Ahlbrecht S A M A N T , Metry S A R A , Helten C A R O L I , Dannenberg L I S A , Hohlfeld T H O M A S , Zeus T O B I A S , Kelm M A L T E , Levkau B O D O , Polzin A M I N
Published: November 2023

Background

Sphingosine-1-phosphate (S1P) is a bioactive lipid known for its cardioprotective effects. The extent to which S1P also has effects on arterial thrombosis has not yet been fully elucidated. Another important factor in arterial thrombosis is thrombomodulin (TM), an important endothelial antithrombotic factor. To what extent S1P, TM, and, in addition, platelet adhesion as an initial part influences arterial thrombosis is unclear and the aim of this work.

Methods

The influence of S1P on human umbilical cord venous endothelial cells (HUVECs) was investigated in cell culture. To investigate effects of S1P and TM on platelet adhesion, flow chamber experiments were performed. In addition, murine arterial thrombus formation was analyzed by intravital microscopy.

Results

S1P increased TM expression on HUVECs. This effect was abolished by S1P receptor1 (S1PR1) inhibition with W146 (S1P: 1.081 ± 0.091 vs. S1P + W146: 0.951 ± 0.073, normalized to control, p<0.0001). Sphingosine kinase-2 deficient mice (SphK1-/-) lack the kinase primarily producing sphingosine. Accordingly, these animals have less S1P. Here, TM expression was significantly lower in the aorta compared to wild-type mice (WT: 207.2 ± 45.8 pg/g aorta vs. SphK1-/- 124.5 ± 40.2 pg/g aorta, p<0.0001). S1P treatment decreased platelet adhesion from whole blood to endothelial cells in the flow chamber (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). W146 reversed this effect by S1PR1 inhibition (S1P: 80.91 ± 22.85%, vs. S1P + W146: 118.60 ± 33.65%, p=0.0067). Platelet adhesion to endothelial cells was assessed in the presence of a TM-neutralizing antibody. The TM antibody abolished the anti-adhesive effects of S1P (S1P: 54.79 ± 29.59% vs. S1P+AB: 105.54 ± 35.43%, p=0.0174). TM inhibited intravital murine arterial thrombus formation (time to first occlusion: con: 9.38 ± 1.02 min vs. TM: 60 ± 0 min, p=0.0286). Furthermore, the duration of the first occlusion was significantly shorter and the embolic rate as a marker for thrombus stability was higher.

Conclusion

We demonstrated that S1P inhibits arterial thrombosis via regulation of endothelial TM. In particular, platelet adhesion to endothelial cells is reduced. This effect is mediated via S1PR1. The extent to which S1PR1 represents a potential antithrombotic target must be clarified in future studies.
Sirtuin-1 directly binds and deacetylates hepatic PCSK9 to inhibit low-density lipoprotein receptor degradation
Authors: Velagapudi S , Xavier Miranda M , Mohammed S , Kraler S , Robert J , Rohrer L , Lee H , Jang H D , Camici G , Von Eckardstein A , Kim H S , Akhmedov A , Luscher T
Published: November 2023

Background

Low-density lipoprotein cholesterol (LDL-C) is causally involved in atherosclerotic cardiovascular disease (ASCVD). We have previously shown that pharmacological activation of Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, reduces plasma LDL-C levels by decreasing PCSK9 and increasing hepatic LDLR expression, thus exerts atheroprotective effects in mice; however, the mechanism of SIRT1 action remains elusive.

Purpose

We found diminished levels of circulating SIRT1 in atherosclerotic mice compared to wild-type healthy controls. We hypothesized that this decrease in SIRT1 levels may promote dysregulated lipid metabolism and ASCVD in humans. Herein, we aimed to restore the reduced systemic Sirtuin-1 plasma levels in ASCVD by injecting recombinant murine Sirtuin-1 (rmSIRT1) in atherosclerotic mice.

Methods

Twelve-week-old apolipoprotein E-deficient (ApoE-/-) male mice fed a high-cholesterol diet (1.25% w/w) were randomized to receive rmSIRT1 (n=6; 0.3mg/kg BW i.p.) or vehicle treatment (n=6) every third day over 4-weeks. Mice were euthanized and organs were harvested to study lipid metabolism and ASCVD progression. Human hepatocytes (Huh7) cells were used to perform radioactive assays to study cellular uptake of radioiodinated LDL, surface plasmon resonance to study binding and site-directed mutagenesis to dissect the underlying molecular mechanism exerted by circulating SIRT1.

Results

Boosting circulating SIRT1 levels increased hepatic LDLR expression, reduced plasma LDL-C levels and progression of plaques in ApoE-/- mice (Figure 1). rmSIRT1 treatment did not change hepatic expression of total PCSK9 but increased its deacetylated status. Mechanistically, rmSIRT1 directly bound to hepatic PCSK9 and deacetylated PCSK9 at 3 sites: Lys243, Lys421 and Lys506 as shown by Surface Plasmon Resonance and mass spectrometry, respectively. In vitro mutagenesis to triple deacetylation mimetic (3KR) in hepatocytes (Huh7 cells) reduced SIRT1-induced PCSK9 activity, as evidenced by increased cellular binding and association of 125I-LDL through LDLR in Huh7cells. Finally, plasma levels of SIRT1 and PCSK9 were assessed at baseline in patients with acute coronary syndromes (ACS) where plasma SIRT1 showed an inverse correlation with PCSK9 and conferred a reduced risk of major adverse cardiovascular events.

Conclusion

SIRT1 directly binds PCSK9 and decreases its activity by deacetylation, thereby enhancing LDL-C clearance by hepatic LDLR upregulation. Increased circulating SIRT1 exerts atheroprotective effects and is associated with improved prognosis in patients with ACS.Figure 1
Canagliflozin enhances endogenous eliminatiton of senescent cells through downregulation of PD-L1
Authors: Katsuumi G , Suda M , Shimizu I , Yoshida Y , Furuuchi R , Hsiao C L , Hsiao Y T , Minamino T
Published: November 2023

Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been developed as a new class of anti-diabetic drug, and shown cardio- and renal-protective effects in patients with diabetes, heart failure, and chronic kidney diseases. Also, there's evidence that canagliflozin, one of the SGLT2 inhibitors has expanded lifespan in aged mice, but their detailed protective machinery is still to be clarified. Here we demonstrate that canagliflozin has a effect to eliminate senescent cells from pathological tissues, so-called "senolysis" in some murine disease models and alleviates their pathology.

We treated canagliflozin mixed in food at 0.03% (w/w) to high-fat-diet(HFD)-fed obese mice and found that canagliflozin reduced senescent cells in visceral adipose tissue within 1 week. Also, we found that senolytic effect of canagliflozin was provided through enhancing endogenous senolytic function by NK or T cells. This machinery was mediated by enhanced AMPK signaling, which leads to the downregulation of PD-L1 expression on senescent cells. Furthermore, we treated canagliflozin to ApoE-knockout atherosclerotic mice with western diet feeding, progeroid mice and chronological aged mice. Canagliflozin reduced their senescent cell burden, resulting in alleviation of atherosclerosis, extended lifespan of progeroid, and altered physical function due to chronological aging.

Our results would be promising evidence that SGLT2 inhibitors can be used as not only cardio- or renal-protective drugs but also anti-aging drugs.
The COVID-19 mRNA vaccine Comirnaty induces anaphylactic shock in an anti-PEG hyperimmune large animal model
Authors: Barta B A , Radovits T , Dobos A B , Kozma G T , Meszaros T , Berenyi P , Facsko R , Merkely B , Szebeni J
Published: November 2023

Background

The polyethylene-glycol (PEG)-containing COVID-19 mRNA vaccines can cause hypersensitivity reactions (HSRs), or rarely, life-threatening anaphylaxis in a small fraction of patients. The role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet been proven in an animal model.

Purpose

The aim of this study was to provide such evidence using an anti-PEG hyperimmune porcine model wherein the Ab levels are increased by PEGylated liposomes called Doxebo. We also aimed to find evidence for a role of complement activation and thromboxane A2 release in blood.

Methods

Experimental pigs (n=6) were immunized with 0.1 mg/kg BW Doxebo i.v., and the rise of anti-PEG Abs were measured with ELISA. 2-3 weeks later the animals were injected with 1/3 human dose of Pfizer’s COVID-19 mRNA vaccine Comirnaty i.v., and the hemodynamic, hematological and blood immune mediator changes were measured to assess the severity of hypersensitivity reaction (HSR). The endpoints were PAP, SAP, HR, EtCO2, WBC, granulocyte, lymphocyte and platelet counts, C3a and plasma TXB2.

Results

The level of anti-PEG IgM rose 5-10-thousand-fold in all pigs immunized with Doxebo by day 10. Each of the 6 animals developed an anaphylactic shock to injection of 1/3 human dose of Comirnaty that required cardiopulmonary resuscitation in 4 cases. The reaction, starting within 1 min involved maximal pulmonary hypertension (PAP 13±2 before vs. 37±6mmHg after Comirnaty, median±MAD, P<0.05) and severe systemic hypotension (SAP 63±8 before vs. 42±11mmHg after Comirnaty, median±MAD, P<0.05), tachycardia, granulo- and thrombocytopenia, and skin flushing or rash. These hemodynamic changes were paralleled by C3a and TXB2 levels in blood.

Conclusion

We have established the first relevant large animal model of anaphylactic reaction induced by an mRNA vaccine. Consistent with previous studies, our current data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty, which involves complement activation, and, hence, it represents CARPA (complement activation-related pseudo-allergy).
Inclisiran reduces expression of Toll-like receptor-4 on circulating monocytes
Authors: Bernhard J , Engelberger K , Galli L , Haider P , Ungersbaeck K , Hengstenberg C , Speidl W S , Krychtiuk K A
Published: November 2023

Background

Toll-like receptor 4 (TLR4) is a key regulator of both innate and adaptive immune responses and its functions affects the initiation and progression of atherosclerosis. In addition, it has recently been shown that TLR4 regulates foam cell formation by catabolism of aggregated LDL and that TLR4 is induced by proprotein convertase subtilisin/kexin type 9 (PCSK9) in vitro. The aim of this study was to examine the effects of siRNA-induced knockdown of PCSK9 by inclisiran on the expression of TLR4 in vivo.

Methods

Patients on stable lipid lowering therapy for at least 4 weeks were treated with a single dose of inclisiran (284 mg s. c.) and blood samples were taken directly before treatment and after 1 and 3 months. Monocyte expression of TLR4 was analyzed by flow-cytometry.

Results

We included 22 consecutive patients, 54.5% of patients were male and median age was 63 (IQR 60-75) years. 15 patients were treated with high dose statin and 7 patients did not tolerate statin treatment. At baseline TLR4 expression tended to be lower in statin-treated patients (MFI: 2136 IQR 1427-4011 vs. 3115 IQR 2187 vs. 5851; p=0.06). TLR4 expression after one month was comparable to baseline. However, 3 months after after inclisiran treatment, TLR4 expression on monocytes significantly decreased as compared to baseline expression (MFI baseline: 2396 IQR 1802-4408; MFI 3 months: 1575 IQR 1263-2233; p=0.006). This effect was more pronounced in statin-naïve as compared to statin-treated patients (TLR4 MFI 3 months vs baseline no statin: -58.8 IQR -41.9 to -81.2 %; statin: -27.1 IQR +2.2 to -42.1 %).

Conclusions

siRNA-induced knockdown of PCSK9 by inclisiran was associated with significant decreased expression of TLR4 on circulating monocytes. This effect was more pronounced in patients with statin intolerance.
Semaglutide effects on human epicardial and subcutaneous fat released exosomes, aortic endothelial cells and neutrophils
Authors: Sanchez-Lopez D , Lestegas-Soto A , Garcia-Vega D , Vinuela J E , Reija-Lopez L , Martinez-Cereijo J M , Fernandez-Lopez A L , Gonzalez-Juanatey J R , Eiras S
Published: November 2023

Background

Obesity has increased in recent years with consequences on diabetes and other comorbidities. Thus, 1 out of 3 diabetic patients have cardiovascular disease (CVD) and obese patients have a 95% risk of heart failure (HF) and, consequently, of death. The proinflammatory profile of epicardial fat (EAT) participates in this pathological mechanism. New drugs based on GLP-1 receptor agonists (GLP-1-RA) have demonstrated benefits on CVD. Semaglutide is a new GLP-1-RA that can reduce body weight and atherogenesis process.

Purpose

To study the semaglutide effects on human epicardial fat, aortic endothelial cells and neutrophils as main involved-cardiovascular cells on atherogenesis.

Materials and Methods

Epicardial (EAT) and subcutaneous fat (SAT) biopsies were collected from patients undergoing open-heart surgery. All the patients signed an informed consent. Differential fat-released exosomes, isolated by Exo-Spin Exosome Purification Kit, regarding semaglutide or/and insulin treatment were characterized by proteomics Triple TOF (n=3). Modulation of human neutrophils (dHL60 cells) phenotype, obtained after promyelocytic cell differentiation, and adhesion into human aortic endothelial cells (HAEC) by Semaglutide were analyzed by flow cytometry and functional analysis, respectively. Semaglutide effects on angiogenesis were performed on HAEC according to the manufacturer’s protocol. GLP-1 receptor expression levels were confirmed by real time-PCR.

Results

Fat biopsies, neutrophils and HAEC expressed GLP-1 receptor, measured by qPCR. We did not observe differential exosomes-cargo after semaglutide treatment on EAT. However, the released-exosomes from SAT with ANXA5, FABP4, GSN, SERPINF1 and PRDX1 were modulated by Semaglutide treatment. In addition, the CD11b molecule expression on neutrophils (p=0.0482) and their adhesion (p=0.0148) into HAEC treated with high glucose (22.2 mM) were modulated by Semaglutide treatment. Finally, GLP-1-RA improves angiogenesis, measured by loop formation after 20 hours.

Conclusion

Semaglutide modulates the released exosomes by SAT, the neutrophils adhesion into endothelial cells and enhances the angiogenesis process. These results suggest a new anti-inflammatory and anti-atherogenic role of this molecule in the human cardiovascular cell system.Figure 1Figure 2