Receptor Type Research Articles

High glucose elevates intracellular calcium level and induces ferroptosis in glomerular endothelial cells through the miR-223-3p/ITPR3 pathway

We investigated the link between ferroptosis and the miR-223-3p/inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) pathway in diabetic kidney disease (DKD). Blood samples from DKD patients and healthy controls were analysed for iron ions, calcium ions, and lipid peroxidation. High-glucose-induced glomerular endothelial cells were used to simulate DKD. MiR-223-3p overexpression or silencing was achieved using adenoviruses, affecting ferroptosis regulators (glutathione peroxidase 4 [GPX4], cystine/glutamate transporter (xCT), and long-chain acyl-CoA synthetase 4 [ACSL4]) and ITPR3. DKD patients showed elevated levels of iron ions, calcium ions, and lipid peroxidation. High glucose downregulated miR-223-3p, reducing xCT and GPX4 expression and increasing ACSL4 expression. MiR-223-3p was confirmed to target ITPR3 through luciferase reporter assay. MiR-223-3p overexpression reversed high-glucose-induced effects on ferroptosis markers and ITPR3 expression. In summary, high glucose levels decreased miR-223-3p expression, leading to increased calcium ion levels and ferroptosis, potentially through ITPR3 modulation. These findings provide insights into the mechanisms underlying DKD and its potential therapeutic targets.

Characterization of D1R and D2R neuronal subpopulations in the globus pallidus interna: Implications for Parkinson's disease pathogenesis.

Parkinson's disease (PD) ranks as the second most prevalent and rapidly growing neurodegenerative disorder. As a primary output nucleus within the basal ganglia (BG), the globus pallidus interna (GPi) is a key structure in BG information processing. It is also a key target for deep brain stimulation (DBS) to alleviate motor symptoms of PD. Previous studies have identifiedPD patients exhibiting abnormal neuronal activity in the GPi. On the other hand, various types of dopamine receptor (DR)-positive neurons have been identified within the GPi. However, the electrophysiological properties of specific DR-positive neurons within the GPi and their alterations in PD have not been addressed. In the present study, we used whole-cell patch-clamp recordings to identify two neuronal subpopulations within the GPi, dopamine D1 receptor (D1R)-positive, and dopamine D2 receptor (D2R)-positive neurons, which exhibited distinct electrophysiological properties. Additionally, significant alterations of electrophysiological properties of D2R-positive neurons within the GPi were observed in 6-hydroxydopamine (6-OHDA)-lesioned mice. These data suggest that the distinct electrophysiological properties of specific DR-positive neurons and their abnormal alteration in the GPi may be associated with PD's pathogenesis.

Calcium homeostasis imbalance mediates DEHP induced mitochondrial damage in cerebellum and the antagonistic effect of lycopene

Phthalates (PAEs), especially di (2-ethylhexyl) phthalate (DEHP), are generally considered to have adverse impact on nervous system. The residue of DEHP in the environment has gradually become a widely concerned environmental problem due to its widespread use in plastic items. Lycopene (LYC) as the readily available natural antioxidant is considered to have the potential to alleviate exogenous poisons-induced nerve damage. However, there is currently a lack of strategies to alleviate the neurotoxicity caused by DEHP, and it is also unknown whether LYC can alleviate the neurotoxicity caused by DEHP. The experiment demonstrated that LYC had the potential to mitigate DEHP-induced mitochondrial damage in cerebellum. DEHP induced the disorder of Ca2+ transport in cerebellum, thereby resulting in the imbalance of protein homeostasis. Such disruption in protein homeostasis further results in the overactivation of mitochondrial unfolded protein response (UPRmt) and mitochondrial injury. Mechanistically, LYC could alleviate the imbalance of calcium homeostasis and protein homeostasis induced by DEHP via regulating inositol 1, 4, 5-trisphosphate receptor type1 (IP3R1) and sarco/endoplasmic reticulum Ca (2+)-ATPase 2 (SERCA2), further alleviating mitochondrial damage in cerebellum. Subsequently, the present study suggested the mechanism of cerebellar injury induced by DEHP, and provided a novel approach to treating DEHP-induced neurotoxicity.

Therapeutic potential of agents targeting cannabinoid type 2 receptors in organ fibrosis.

The endocannabinoid system has garnered attention as a potential therapeutic target in a range of pathological disorders. Cannabinoid receptors type 2 (CB2) are a class of G protein-coupled receptors responsible for transmitting intracellular signals triggered by both endogenous and exogenous cannabinoids, including those derived from plants (phytocannabinoids) or manufactured synthetically (synthetic cannabinoids). Recent recognition of the role of CB2 receptors in fibrosis has fueled interest in therapeutic targeting of CB2 receptors in fibrosis. Fibrosis is characterized by the alteration of the typical cellular composition within the tissue parenchyma, resulting from exposure to diverse etiological factors. The pivotal function of CB2 agonists has been widely recognized in the regulation of inflammation, fibrogenesis, and various other biological pathologies. The modulation of CB2 receptors, whether by enhancing their expression or activating their function, has the potential to provide benefits in numerous conditions, particularly by avoiding any associated adverse effects on the central nervous system. The sufficient activation of CB2 receptors resulted in the complete suppression of gene expression related to transforming growth factor β1 and its subsequent fibrogenic response. Multiple reports have also indicated the diverse functions that CB2 agonists possess in mitigating chronic inflammation and subsequent fibrosis development in various types of tissues. While currently in the preclinical stage, the advancement of CB2 compounds has garnered significant attention within the realm of drug discovery. This review presents a comprehensive synthesis of various independent experimental studies elucidating the pivotal role of identified natural and synthetic CB2 agonists in the pathophysiology of organ fibrosis, specifically in the cardiac, hepatic, and renal systems.

Methyl-Beta-Cyclodextrin Restores Aberrant Bone Morphogenetic Protein 2-Signaling in Bone Marrow Stromal Cells Obtained from Aged C57BL/6 Mice

During aging, disruptions in various signaling pathways become more common. Some older patients will exhibit irregular bone morphogenetic protein (BMP) signaling, which can lead to osteoporosis (OP)—a debilitating bone disease resulting from an imbalance between osteoblasts and osteoclasts. In 2002, the Food and Drug Administration (FDA) approved recombinant human BMP-2 (rhBMP-2) for use in spinal fusion surgeries as it is required for bone formation. However, complications with rhBMP-2 arose and primary osteoblasts from OP patients often fail to respond to BMP-2. Although patient samples are available for study, previous medical histories can impact results. Consequently, the C57BL/6 mouse line serves as a valuable model for studying OP and aging. We find that BMP receptor type Ia (BMPRIa) is upregulated in the bone marrow stromal cells (BMSCs) of 15-month-old mice, consistent with prior data. Furthermore, conjugating BMP-2 with Quantum Dots (QDot®s) allows effective binding to BMPRIa, creating a fluorescent tag for BMP-2. Furthermore, after treating BMSCs with methyl-β-cyclodextrin (MβCD), a disruptor of cellular endocytosis, BMP signaling is restored in 15-month-old mice, as shown by von Kossa assays. MβCD has the potential to restore BMPRIa function, and the BMP signaling pathway offers a promising avenue for future OP therapies.

C-C motif chemokine receptor-2 blockade ameliorates pulmonary hypertension in rats and synergizes with a pulmonary vasodilator

Abstract Aims We investigated whether the disruption of C-C motif chemokine receptor (CCR) 2 may attenuate the development of pulmonary arterial hypertension (PAH) in any rat models with the reversal of the associated pro-inflammatory state and vascular dysfunction, and synergize with a conventional pulmonary vasodilator. Methods and Results Using Ccr2(-/-) rats generated by CRISPR/Cas9, we investigated pulmonary hypertension (PH) in Ccr2(+/+) or Ccr2(-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx) and chronic hypoxia (CH). Ccr2(-/-) decreased the right ventricular systolic pressure, an index of right ventricular hypertrophy and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (interleukin-6, tumor necrosis factor-α, C-C motif chemokine receptor (CCL)-2, interleukin-1β, transforming growth factor-β) in rats 3weeks after MCT injection, but not in SuHx or CH models. Consistently, Ccr2(-/-) decreased indices of pulmonary vascular diseases (PVD) and perivascular macrophage infiltration, as well as reversed impaired bone morphogenetic protein receptor type 2 signaling, increased endothelial apoptosis and impaired nitric oxide signaling and decreased phosphodiesterase-5 (PDE5) expression in lungs in MCT-treated rats. Gene expression of receptors for prostaglandin I2 and endothelin was not changed by Ccr2(-/-) in MCT-treated rats. In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2(-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. The whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2(-/-) PASMCs, which are related to regulation of cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2(-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2(-/-) rats but not in Ccr2(+/+) rats. Tadalafil further improved survival in MCT-treated Ccr2(-/-) rats. Conclusion The present findings demonstrated that CCR2 disruption ameliorated PAH in MCT-treated rats, which was associated with the reversal of dysregulated inflammatory pathways and vascular dysfunction and synergized with tadalafil. These findings suggest that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

Endothelin-1 receptor blockade impairs invasion patterns in engineered 3D high-grade serous ovarian cancer tumouroids.

High-grade serous ovarian cancer (HG-SOC), accounting for 70-80% of ovarian cancer deaths, is characterized by a widespread and rapid metastatic nature, influenced by diverse cell types, cell-cell interactions, and acellular components of the tumour microenvironment (TME). Within this tumour type, autocrine and paracrine activation of the endothelin-1 receptors (ET-1R), expressed in tumour cells and stromal elements, drives metastatic progression. The lack of three-dimensional models that faithfully recapitulate the unique HG-SOC TME has been the bottleneck in performing drug screening for personalized medicine. Herein, we developed HG-SOC tumouroids by engineering a dense central artificial cancer mass (ACM) containing HG-SOC cells, nested within a compressed hydrogel recapitulating the stromal compartment comprising type I collagen, laminin, fibronectin, and stromal cells (fibroblasts and endothelial cells). ET-1-stimulated HG-SOC cells in the tumouroids showed an altered migration pattern and formed cellular aggregates, mimicking micrometastases that invaded the stroma. Compared with control cells, ET-1-stimulated tumouroids showed a higher number of invasive bodies, which were reduced by treatment with the dual ET-1 receptor (ET-1R) antagonist macitentan. In addition, ET-1 increased the size of the invading aggregates compared with control cells. This study establishes an experimental 3D multicellular model eligible for mechanical research, investigating the impact of matrix stiffness and TME interactions, which will aid drug screening to guide therapeutic decisions in HG-SOC patients.

Discovery of highly potent and ALK2/ALK1 selective kinase inhibitors using DNA-encoded chemistry technology

Activin receptor type 1 (ACVR1; ALK2) and activin receptor like type 1 (ACVRL1; ALK1) are transforming growth factor beta family receptors that integrate extracellular signals of bone morphogenic proteins (BMPs) and activins into Mothers Against Decapentaplegic homolog 1/5 (SMAD1/SMAD5) signaling complexes. Several activating mutations in ALK2 are implicated in fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine gliomas, and ependymomas. The ALK2 R206H mutation is also present in a subset of endometrial tumors, melanomas, non-small lung cancers, and colorectal cancers, and ALK2 expression is elevated in pancreatic cancer. Using DNA-encoded chemistry technology, we screened 3.94 billion unique compounds from our diverse DNA-encoded chemical libraries (DECLs) against the kinase domain of ALK2. Off-DNA synthesis of DECL hits and biochemical validation revealed nanomolar potent ALK2 inhibitors. Further structure-activity relationship studies yielded center for drug discovery (CDD)-2789, a potent [NanoBRET (NB) cell IC50: 0.54 μM] and metabolically stable analog with good pharmacological profile. Crystal structures of ALK2 bound with CDD-2281, CDD-2282, or CDD-2789 show that these inhibitors bind the active site through Van der Waals interactions and solvent-mediated hydrogen bonds. CDD-2789 exhibits high selectivity toward ALK2/ALK1 in KINOMEscan analysis and NB K192 assay. In cell-based studies, ALK2 inhibitors effectively attenuated activin A and BMP-induced Phosphorylated SMAD1/5 activation in fibroblasts from individuals with FOP in a dose-dependent manner. Thus, CDD-2789 is a valuable tool compound for further investigation of the biological functions of ALK2 and ALK1 and the therapeutic potential of specific inhibition of ALK2.

LncRNA MYOSLID contributes to PH via targeting BMPR2 signaling in pulmonary artery smooth muscle cell.

The pathogenesis and vascular remodeling during pulmonary hypertension (pH) have been associated with dysregulation of bone morphogenetic protein receptor type 2 (BMPR2) and transforming growth factor-β (TGF-β) signaling in pulmonary artery smooth muscle cells (PASMCs). Evidence suggests that the human-specific lncRNA MYOSLID is a transcriptional target of the TGF-β/SMAD pathway. In this study, we investigated the involvement of MYOSLID in the pathogenesis of PH. Lung tissues from PH patients and rat PH models were analyzed to assess clinical relevance. RNA-Seq was performed to identify target genes. Pulmonary artery smooth muscle cells (PASMCs) were used to evaluate function and underlying mechanisms. RNA-Seq analysis of PASMCs stimulated by TGF-β1 revealed significantly dysregulated lncRNAs. MYOSLID expression was markedly elevated in lung tissues from PH patients and in PASMCs stimulated with TGF-β1. Mechanistically, loss of MYOSLID inhibited the TGF-β pathway by reducing SMAD2/3 pHosphorylation and activated the BMPR2 pathway by enhancing SMAD1/5/9 phosphorylation and increasing ID genes expression in PASMCs. DAZAP2, a target gene of MYOSLID, functions as an inhibitor of BMPR2 signaling. Moreover, DAZAP2 expression was significantly elevated in lung tissues from PH patients and rat PH models. Functionally, knockdown of MYOSLID and DAZAP2 reduced proliferation, migration, and apoptosis resistance in PASMCs. The activation of the MYOSLID-DAZAP2-BMPR2 axis contributes to pulmonary vascular remodeling, and targeting MYOSLID and DAZAP2 may represent novel therapeutic strategies for PH treatment.

Cannabinoid Receptor type 2 agonist GP1a attenuates macrophage activation induced by M. bovis-BCG by inhibiting NF-ĸB signaling.

Tuberculosis (TB) is one of the leading causes of death worldwide and a major public health problem. Immune evasion mechanisms and antibiotic resistance highlight the need to better understand this disease and explore alternative treatment approaches. Mycobacterial infection modulates the macrophage response and metabolism to persist and proliferate inside the cell. Cannabinoid receptor type 2 (CB2) is expressed mainly in leukocytes and modulates the course of inflammatory diseases. Therefore, our study aimed to evaluate the effects of the CB2-selective agonist GP1a on irradiated M. bovis-BCG (iBCG)-induced J774A.1 macrophage activation. We observed increased expression of CB2 in macrophages after iBCG stimulation. The pretreatment with CB2-agonists, GP1a, JWH-133, and GW-833972A (10 µM), reduced iBCG-induced TNF-α and IL-6 release by these cells. Moreover, the CB2-antagonist AM630 (200nM) treatment confirmed the activity of GP1a on CB2 by scale down its effect on cytokine production. GP1a pretreatment (10 µM) also inhibited the iBCG-induced production of inflammatory mediators as prostaglandin (PG)E2 and nitric oxide (NO) by macrophages. Additionally, GP1a pretreatment also reduced the transcription of proinflammatory genes (inos, il1b, cox2) and genes related to lipid metabolism (dgat1, acat1, plin2, atgl, cd36). Indeed, lipid droplet accumulation was reduced by GP1a treatment which was partially blockade by AM630 pretreatment. Finally, GP1a pretreatment reduced the activation of the NF-κB signaling pathway. In conclusion, the activation of CB2 by GP1a modulated the macrophage response to iBCG by reducing inflammatory mediator levels and metabolic reprogramming.

Hypocretin modulation of behavioral coping strategies for social stress.

Best known for promoting wakefulness and arousal, the neuropeptide hypocretin (Hcrt) also plays an important role in mediating stress responses, including social stress. However, central and systemic manipulation of the Hcrt system has produced diverse behavioral outcomes in animal models. In this review, we first focus on studies where similar manipulations of the Hcrt system led to divergent coping behaviors. We hypothesize that Hcrt differentially facilitates active and passive coping behaviors in response to social stress by acting in different brain regions and on different cell types. We then focus on region and cell type-specific effects of Hcrt in the ventral pallidum, lateral habenula, ventral tegmental area, nucleus accumbens, amygdala, and bed nucleus of the stria terminalis. Overall, the evidence suggests that rather than enhancing or inhibiting behavioral responses to social stress, Hcrt may signal the heightened arousal associated with stressful contexts. The resulting behavioral effects depend on which circuits Hcrt release occurs in and which receptor types are activated. Further study is needed to determine how and why circuit specific activation of Hcrt neurons occurs.

Possible Mechanism for Perception of Auditory-Verbal Hallucinations in Schizophrenia and Approaches to Their Weakening

We put forward a hypothesis that the processing and perception of auditory-verbal hallucinations in schizophrenia — internally generated speech, occurs in the same neural circuits as speech from external sources. These topographically organized cortico—basal ganglia—thalamocortical neural circuits include auditory, language, and frontal neocortical areas. It follows from our proposed mechanism for sound processing, that the increased action on dopamine D2 receptors on striatal neurons, which is considered the cause of hallucinations, should lead to a determined reorganization of activity in these neural circuits. As a result of this reorganization, the inhibition of neurons in the thalamic nuclei, including the internal geniculate body, should decrease synergistically through the direct and indirect pathways in the basal ganglia, and the excitation of connected with them cortical neurons, in the activity of which speech is represented, should increase. From this mechanism it follows that in order to weaken the perception of auditory-verbal hallucinations, it is necessary to increase the inhibition of thalamic neurons. Taking into account the known data on the distribution of receptors of different types on neurons in the striatum, thalamus and neocortex, as well as the previously formulated unified modification rules for the effectiveness of synaptic transmission in different structures, we proposed that agonists of adenosine A1 and muscarinic M4 receptors located on striatonigral spiny cells, giving rise to a direct disinhibitory pathway through the basal ganglia as well as antagonists of delta-opioid and cannabinoid CB1 receptors, located on striatopallidal spiny cells, giving rise to an indirect inhibitory pathway through the basal ganglia may be useful to weaken the perception of hallucinations. In addition, activation of A1 receptors can directly weaken the activity of neurons in the thalamus and neocortex due to the induction of long-term depression in the efficiency of their excitation. Inactivation of cannabinoid CB1 receptors on the projection GABAergic cells of the reticular thalamic nucleus may enhance their inhibitory effect on neurons of different thalamic nuclei. Since the proposed substances only indirectly affect the dopaminergic system, their use should not cause such pronounced side effects as D2 receptor antagonists, antipsychotics that are widely used to suppress auditory-verbal hallucinations.

Angiotensin II Type-1 Receptor Antibody in Solid Organ Transplantation – Is It Time to Test?

Angiotensin II type-1 receptor antibody (AT1R-Ab) has been mooted as a potential effector of both acute and chronic antibody mediated rejection (AMR). A growing body of literature on the topic is now coming under scrutiny in the context of the evolving Banff AMR diagnostic classification system and refinement of recommendations for histocompatibility testing by the Sensitization in Transplantation Assessment of Risk (STAR) workgroup. This mini-review discusses the latest understanding of pathophysiological mechanisms, clinical evidence for the pathogenicity of AT1R-Ab, and methods of laboratory testing.

Association of dietary manganese intake and the IL1R1 rs3917225 polymorphism with thyroid cancer risk: a prospective cohort study in Korea.

Dietary Mn intake may have a beneficial effect in reducing cancer risk; however, its association with thyroid cancer (TC) risk remains inadequately understood. Additionally, Mn was associated with inflammation markers. Thus, we examined whether dietary Mn intake emerges a protective role against TC and whether this preventative effect has an interaction with IL1 receptor type 1 (IL1R1) rs3917225. The prospective study was designed at National Cancer Center in Korea between October 2007 and December 2020 including 17754 participants. We identified TC cases by following participants until December 2020. Mn intake was collected using a semiquantitative food frequency questionnaire (SQFFQ). Genotyping was performed to determine IL1R1 rs3917225. The hazard ratios (HR) and 95 % confidence interval (CI) were calculated with a Cox proportional hazards model. We ascertained 108 incident TC cases throughout follow-up duration. Dietary Mn intake was found to be inversely associated with TC risk (HR (95 % CI)=0·64 (0·44, 0·95)). However, IL1R1 rs3917225 seemed to modify this association; the protective effect was limited to G-allele carriers (HR = 0·30 (0·11, 0·86), P interaction=0·028). A higher dietary Mn was suggested to be a protective factor against TC. Additionally, we drew a potential biological interaction between Mn intake and IL1R1 rs3917225 with a greater effect among individuals with a minor allele. This implies that when considering the cancer-preventive role of Mn, it is important to account for the influence of inflammatory gene participation.

Development of a hamster model of spontaneous hypertriglyceridemia in diabetes.

Hypertriglyceridemia (HTG) often accompanies diabetes and is considered a risk factor for diabetic vascular complications. However, inducing diabetic HTG typically requires high-fat diets in certain animal models. Leveraging our newly developed LDL receptor knockout hamster model, which exhibits features akin to human lipid metabolism, we sought to determine whether these animals would develop HTG without dietary manipulations in diabetes. Diabetes was induced via intraperitoneal injection of STZ in wild type and heterozygous LDL receptor deficient hamsters. Blood glucose, triglyceride, and cholesterol were measured over 60 days. Plasma TG clearance was determined via olive oil gavage. The effect of insulin on diabetic HTG was assessed on Day 60 post-diabetes induction. Blood glucose increased over threefold, while plasma insulin decreased to 30% of controls after STZ injection in both wild type and heterozygous hamsters by Day 7, remaining stable for 60 days. Plasma TG in wild-type hamsters remained unchanged at Day 7 post-STZ injection but increased slightly thereafter. Conversely, heterozygous hamsters exhibited severe HTG by Day 7 until the end of the study. Olive oil gavage revealed much slower plasma triglyceride clearance in heterozygous hamsters compared to WT animals, despite significantly reduced lipoprotein lipase activity in post-heparin plasma in both animals. Hyperglycemia and HTG in heterozygous hamsters were reversed to pre-diabetic levels following intraperitoneal insulin administration. In conclusion, severe HTG in diabetic heterozygous LDL receptor deficient hamsters developed spontaneously and was insulin-dependent. Thus, this hamster model holds promise for effectively studying the complications associated with human diabetic HTG.

Sphingosine-1-phosphate (S1P) receptor type 1 signaling in macrophages reduces atherosclerosis in LDL receptor-deficient mice.

Sphingosine 1-phosphate (S1P) is a lysosphingolipid with anti-atherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet-fed LDL receptor-deficient mice with high or low overexpression levels of S1P receptor type 1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, IRF8, and LXR and were skewed towards a M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5, increased ATP-binding cassette transporter A1- and G1-dependent cholesterol efflux, increased expression of MerTK and efferocytosis, and reduced apoptosis due to elevated Bcl6 and MafB. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to down-regulation of the cAMP-dependent protein kinase A and activation of the signaling cascade encompassing protein kinases Akt and mTOR complex 1 (mTORC1) as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (Lamtor-1). Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages towards an anti-atherogenic functional phenotype and countervails the development of atherosclerosis in mice.

Abstract 4136605: Effect Of A Bone Morphogenetic Protein Receptor Type 2 Mutation On The Cardiomyocyte Response To Pressure Overload

Introduction: A mutation in the Bone Morphogenetic Protein Receptor Type 2 ( BMPR2 ) gene is in 70% of hereditary pulmonary arterial hypertension (hPAH) patients the causative mutation of the disease. Previous work demonstrated that right ventricular function is more impaired in hPAH patients with a BMPR2 mutation. However, the underlying mechanism remains elusive. Moreover, natriuretic peptides, such as N-terminal Brain Natriuretic Peptides (NTproBNP) and Mid Regional proAtrial Natriuretic Peptides (MRproANP), are secreted from cardiomyocytes upon stretch as adaptation to increased pressure overload. We hypothesize that the BMPR2 mutation impairs the response to increased pressure overload. For this purpose, we make use of ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of hPAH patients carrying a BMPR2 mutation and their isogenic controls in which the mutation was corrected. Aim: To study the effect of a BMPR2 mutation on the response to pressure overload in vitro . Methods: iPSCs from 2 hPAH patients and its isogenic controls were used. iPSCs were differentiated into ventricular iPSC-CMs and stretched 10% equiaxially for 24h at 1Hz on the Flexcell FX-6000 system. Natriuretic peptides gene expressions were quantified via RT-PCR. NTproBNP and MRproANP release were measured on cell supernatants throughout specific immunoassays. Normality of the data was checked, and statistics were performed accordingly. Results: No differences were observed on natriuretic peptides gene expression on static and stretched iPSC-CMs. There is a clear correlation between NTproBNP and MRproANP release (R 2 =0.83, p<0.0001, Figure 1A). Interestingly, hPAH iPSC-CMs showed higher baseline values for NTproBNP release compared to isogenic iPSC-CMs (p<0.01, Figure 1B). Furthermore, stretching resulted in increased NTproBNP and MRproANP secretion in both hPAH and isogenic iPSC-CMs (Figure 1B-C). Conclusions: Secretion of NTproBNP and MRproANP was closely correlated in iPSC-CMs. iPSC-CMs of hPAH patients with a BMPR2 mutation showed higher baseline values of NTproBNP. Increased secretion of NTproBNP and MRproANP upon mechanical stimulation was similar between hPAH and isogenic iPSC-CMs. Experiments in atrial iPSC-CMs are currently ongoing to test potential differences in natriuretic peptide secretion between ventricular and atrial cardiomyocytes as the right atrium is also under stretch in PAH.

Abstract 4137693: CC-Chemokine Receptor 2 Inhibition Prevents Monocyte/Macrophages Recruitment in Abdominal Aortic Aneurysms

Intro: Abdominal aortic aneurysm (AAA) is a prevalent condition in the elderly, with a significant risk of mortality if it ruptures. Despite this, there is currently no effective medical therapy to prevent AAA growth and rupture. The monocyte chemoattractant protein (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis appears to play a role in AAA development. We demonstrated the potential of CCR2 as a theranostic marker for predicting AAA rupture, along with the effectiveness of a CCR2 inhibitor (RS-504393) in mitigating rupture. We further hypothesized that CCR2 inhibition can impact monocytes/macrophages trafficking in aortic tissue. Mehtods: Sprague-Dawley rats (N=54) underwent intraluminal aortic exposure to porcine pancreatic elastase (PPE) to induce aneurysmal degeneration. To stimulate AAA rupture, rats also recieved β-aminopropionitrile (BAPN). After 3 days of PPE exposure, a group of rats with AAA were administered a CCR2 inhibitor (CCR2i) either daily until the end of the study (CCR2i Full), or discontinued at day 5 (CCR2i Short), while the remaining rats proceeded without intervention (Fig1A). Ultrasound (12MHz) was performed at 7&14 days to assess diameter, while another cohort of animals were euthanized at 6 days, prior to rupture, for single-cell RNA sequencing analysis. The relative content of inflammatory cells within the aortic tissue was analyzed. Results: Both CCR2i Short and CCR2i Full treated, exhibited a significant reduction in AAA diameter at day 7 and 14 compared to controls (p<0.05; Fig1B&C). Notably, rats treated with CCR2i Full did not experience any AAA ruptures, while controls had a 56% risk of rupture. In contrast, CCR2i Short delayed the occurrence of ruptures without reducing the rupture rate (p<0.01; Fig1D). Single-cell RNA seq analysis of aortic tissue revealed that CCR2 inhibition led to a decrease in the number of recruited monocytes/macrophages. Cell recruitment was also relative to the duration of CCR2i treatment (Fig1E-G). Conclusions: Our study findings highlight the impact of CCR2-targeting in reducing AAA growth and the risk of rupture. Interestingly, length of CCR2i was a crucial variable for AAA rupture prevention. Furthermore, treatment length impacted the relative recruitment of monocytes/macrophages to AAA tissue, highlighting the role these cells in AAA disease pathology. Future analysis of CCR2+ inflammatory cell subgroups may help elucidate how CCR2 mechanistically modulates AAA progression and rupture risk.

Abstract 4115947: Empagliflozin Inhibits the Mobilization of CCR2+ Monocyte-Derived Macrophages by Reducing CCL2 Expression in Fibroblasts and Slows the Progression of Heart Failure After Pressure Overload.

Background: The positive effects of sodium glucose co-transporter-2 (SGLT2) inhibitors on reducing cardiovascular mortality and hospitalizations due to heart failure have been well established. Although numerous theories propose explanations for the cardioprotective actions of SGLT2 inhibitors, these hypotheses primarily rely on initial findings and require further validation. Studies have shown that immune cells, particularly macrophages marked by C-C chemokine receptor type 2 (CCR2), contribute to the progression of heart failure by infiltrating the myocardium and inducing detrimental structural changes following myocardial infarction or pressure overload. Purpose: This study aims to investigate whether SGLT2 inhibitors can mitigate heart damage by inhibiting the migration of CCR2+ monocyte-derived macrophages. Methods: In this experiment, male B6 mice were subjected to transverse aortic constriction (TAC) to induce heart failure. Two weeks post-TAC, with heart function confirmed as equal across all groups by echocardiography, one group began receiving daily oral doses of empagliflozin, while another served as the control. At four weeks post-TAC, cardiac function was reassessed. The animals were then euthanized, and their hearts were analyzed using flow cytometry, staining, and qPCR to evaluate heart failure and the involvement of immune cells. Additionally, isolated cardiac fibroblasts were subjected to cyclic stretching to mimic post-TAC mechanical stress. Results: Treatment with empagliflozin resulted in improved cardiac function and reduced congestion in the heart and lungs, indicating alleviation of heart failure. The treatment notably decreased myocardial fibrosis, evident from Picrosirius-red staining and reduced expression of fibrosis-related mRNA. Importantly, empagliflozin specifically reduced the number of CCR2-positive macrophages in the heart, without affecting other types of immune cells. In cardiac fibroblasts that underwent cyclic stretching, empagliflozin pre-treatment diminished the expression of C-C motif chemokine ligand 2 (CCL2), the ligand for CCR2. Conclusion: Empagliflozin’s ability to suppress CCL2 expression in fibroblasts and curtail the recruitment of CCR2+ macrophages may represent a potential strategy to decelerate heart failure progression. These findings advance our understanding of the cardioprotective mechanisms of SGLT2 inhibitors and propose novel therapeutic avenues for heart failure management.

Angiotensin type-1 receptor autoantibodies promote alpha-synuclein aggregation in dopaminergic neurons

Angiotensin, through its type-1 receptor (AT1), is a major inducer of inflammation and oxidative stress, contributing to several diseases. Autoimmune processes have been involved in neurodegeneration, including Parkinson’s disease (PD). AT1 autoantibodies (AT1-AA) enhance neurodegeneration and PD, which was related to increased neuronal oxidative stress and neuroinflammation. However, the effect of AT1-AA on α-synuclein aggregation, a major factor in PD progression, has not been studied. In cultures of dopaminergic neurons, we observed that AT1-AA promote aggregation of α-synuclein, as AT1-AA upregulated major mechanisms involved in the α-synuclein aggregation process such as NADPH-oxidase activation and intracellular calcium raising. The results further support the role of AT1 receptors in dopaminergic neuron degeneration, and several recent clinical studies observing the neuroprotective effects of AT1 receptor blockers.