Receptor-mediated Mechanism Research Articles

Human parietal epithelial cells as Trojan horses in albumin overload

Parietal Epithelial Cells (PECs) activation and proliferation are common to several distinct forms of glomerulopathies. Due to several stimuli, PECs can change to a progenitor (CD24+ and CD133/2+) or a pro-sclerotic (CD44+) phenotype. In addition, PECs, which are constantly exposed to filtered albumin, are known to be involved in albumin internalization, but how this mechanism occurs is unknown. We hypothesized that PECs can transport albumin via receptor-mediated endocytosis and that albumin overload may affect the state of PECs. Conditionally immortalized human PECs (hPECs) were incubated with different albumin concentrations at different times. Albumin internalization studies were performed. Protein expression was assessed using In-Cell Western and immunofluorescence. Cell morphology was analyzed by phase-contrast microscopy and F-actin staining. We demonstrate that hPECs internalize albumin via receptor-mediated mechanisms. Under albumin stimulation, megalin, cubilin, ClC-5, CD133/2, CD24, and CD44 were upregulated. The increase of pERK1/2, the upregulation of ROCK1, ROCK2, caspase -3, -6, and -7, and the morphological changes associated with loss of F-actin fibers indicated that inflammation, proliferation and apoptosis mechanisms had been activated. Our results demonstrate that long-term exposure to high doses of albumin induces up-regulation of molecules involved in the tubular protein uptake machinery and suggest that albumin overload is able to trigger a regenerative process as well as an activation state which might lead in vivo to glomerular crescent formation.

Distinct properties and activation of hexameric and heptameric Pannexin 1 channel concatemers.

Pannexin 1 (PANX1) is a member of a topologically related and stoichiometrically diverse family of large pore membrane ion channels that support the flux of signaling metabolites (e.g., ATP) and fluorescent dyes. High-resolution structural analyses have identified PANX1 as a heptamer despite early evidence suggesting that it might be a hexamer. To determine if PANX1 channel activity is supported in both hexameric and heptameric conformations, we examined properties of concatenated PANX1 constructs comprising either six or seven subunits with intact or truncated C-termini (the latter to mimic caspase-cleavage activation). In whole-cell recordings from PANX1-deleted cells, the C-tail-truncated hexameric and heptameric concatemers generated outwardly rectifying PANX1-like currents only after severing the intersubunit linkers. Surprisingly, α1D adrenoceptor stimulation activated constructs with intact or truncated C-tails, even without linker cleavage. In inside-out patches from PANX1-deleted cells, linker cleavage activated C-tail truncated channels derived from either hexameric or heptameric concatemers. The heptamers presented peak unitary conductance and mean open time that was similar to channels assembled from the expression of unlinked single PANX1 subunits and greater than from the hexamers. In addition, the linker-cleaved heptameric concatemers supported greater PANX1-dependent ATP release and TO-PRO-3 uptake than the corresponding hexamers. These data indicate that functional PANX1 channels can be obtained in either hexameric or heptameric conformations and suggest that the distinct unitary properties of heptameric channels are more conducive to large molecule permeation by PANX1; they also suggest that there are distinct structural requirements for C-tail cleavage and receptor-mediated PANX1 activation mechanisms.

HDL-Free Cholesterol Influx into Macrophages and Transfer to LDL Correlate with HDL-Free Cholesterol Content

High-density lipoprotein (HDL)-free cholesterol (FC) transfers to other lipoproteins and cells, the former by a spontaneous mechanism and the latter by both spontaneous and receptor-mediated mechanisms. Macrophages are an important cell type in all stages of atherosclerotic cardiovascular disease (ASCVD), and the magnitude of FC efflux from macrophages to HDL, a metric of HDL function, inversely associates with several metrics of ASCVD. Very high plasma HDL concentrations are associated with increased all cause and ASCVD mortality, suggesting that the reverse process, FC influx from HDL into macrophages, is atherogenic. We hypothesize that HDL-FC is a metric of dysfunctional HDL, and when combined with HDL particle number (HDL-P), is an ASCVD risk factor. The magnitude of FC influx from HDL to macrophages is expected to be a function of HDL-P and HDL-FC content. Here we show that plasma HDL-FC content varies 2-fold among normolipidemic human subjects and linearly correlates with low-density lipoprotein (LDL)-FC content. The influx of HDL-FC into macrophages and transfer to LDL increase linearly with HDL-FC. As expected, influx of HDL-FC into macrophages and transfer to LDL are positively correlated. These data support the hypothesis that high HDL FC content is a marker for dysfunctional HDL, resulting in greater influx into macrophages and greater HDL-FC transfer to LDL. HDL-FC transfer to LDL is a valid surrogate for influx into macrophages. This study of HDL composition and function of normolipidemic subjects provides the basis for further investigation and establishment of HDL-FC content as an ASCVD risk factor.

Proposed receptor-mediated mechanisms of melatonin in nitroglycerin-induced migraine-like hyperalgesic conditions in rats

Melatonin has a therapeutic effect on migraine, but the mechanisms underlying its antimigraine effect have not been elucidated. This study therefore investigated for the first time the receptor-mediated mechanisms of action of melatonin in nitroglycerin (NTG)- induced migraine-like hyperalgesic conditions in rats. Melatonin, nonselective MT1/MT2 antagonist luzindole, selective MT2 antagonist DH97 or potent MT3 antagonist prazosin, alone or in various combinations, were administered to NTG-induced migraine rats and ex-vivo meningeal preparations. Basal and drug-treated pain behaviors were assessed with the von-Frey test. CGRP levels in the trigeminal ganglia, trigeminal nucleus caudalis (TNC) and ex-vivo superfusate medium, as well as c-fos level in the TNC, were measured by ELISA. Meningeal mast cells were stained with toluidine-blue and examined histologically for their activation and count. Melatonin mitigated mechanical hyperalgesia, and c-fos and CGRP expression in the TNC, CGRP expression in trigeminal ganglia, CGRP release from meningeal afferents, all of which were induced by NTG, and also suppressed NTG-stimulated meningeal mast cell activation. The effects of melatonin were abolished in the presence of luzindole and DH97, respectively. However, prazosin did not reverse the effects of melatonin except for mechanical hyperalgesia. Luzindole and DH97 in combinations with prazosin also canceled the effects of melatonin, respectively, other than CGRP expression in the TNC. Melatonin exerts its anti-hyperalgesic effects through modulation of trigeminal expression and meningeal release of CGRP, and meningeal mast cell activation in experimental migraine-like conditions. The effects of melatonin are mainly mediated by MT2 receptors, without excluding a possible role for MT1.

Smart Mesoporous Silica Nanoparticles Loading Curcumin Inhibit Liver Cancer.

Curcumin (CUR) as one of the natural edible pigments is approved by the World Health Organization due to its nontoxic and anticancer effect. However, the utility of CUR is restricted due to its low oral bioavailability. Nanoparticle drug delivery systems like mesoporous silica nanoparticles (MSNs) have been extensively used due to their high specific surface area, high loading rate, and ease of modification. This study developed lactobionic acid (LA)-modified carboxymethyl chitosan (CMCS)-coated MSNs to deliver CUR specifically targeting hepatocellular carcinoma. Among these nanoparticles, LA targets liver cancer cells. CMCS utilizes pH-responsive release of CUR. The LA-CMCS-MSN@CUR (MSN@CUR) were evaluated using several methods, including Fourier transform infrared spectroscopy, transmission electron microscopy, and zeta potential measurements. Liver cellular uptake of MSN@CUR depends on a specific LA receptor-mediated endocytosis mechanism. Additionally, MSN@CUR performed with a better antitumor effect than Cur in H22 orthotopic transplantation of liver cancer and H22 solid tumor mouse models. Treatment with MSN@CUR significantly reduced the protein of VEGF, p-PI3K, and AKT, increased the protein of caspases 3 and 8, ultimately inhibited tumor migration, and promoted apoptosis. This study provides a new path for delivery of natural active ingredients with excellent bioavailability in the antitumor field.

Virus-mimicking nanodrug active crossing of the blood-brain barrier via transcytosis against central nervous system leukemia

The poor central nervous system leukemia (CNSL) clinical efficacy of conventional doses of chemotherapy is mainly attributed to the limited permeability of chemotherapy agents caused by the blood-brain barrier (BBB). Effectively enhancing the accumulation of drugs across the BBB in the central nervous system is one of the key challenges in improving patient compliance and clinical efficacy of CNSL. Here, we find that the VP1 protein, the functional module of the John Cunningham (JC) virus, can safely penetrate the BBB through a sialic acid receptor-mediated transcytosis mechanism. Based on this, we develop a JC virus-mimicking nanodrug delivery platform based on VP1 protein-conjugated self-assembled nanoparticles (MFHV), which can active target and cross the BBB via a receptor-mediated transcytosis for safe and effective low-dose chemotherapy against CNSL after systemic administration. The results demonstrate that such a platform can penetrate the BBB through the dual mechanism of clathrin-mediated endocytosis and micropinocytosis pathway. When further synergistic with ferroptosis and histamine metabolism, the long-term survivors of low-dose MTX are significantly enhanced by 83.3 % and 56.7 % in two CNSL mice models. Collectively, this study takes a new perspective on natural living materials and molecule targeting of the BBB to present a promising strategy for low-dose chemotherapy against CNSL with safety and efficacy, which might provide a clinically translatable option for the prevention and treatment of CNSL.

Pyroptosis-associated genes and tumor immune response in endometrial cancer

The occurrence and progression of tumors are linked to the process of pyroptosis. However, the precise involvement of pyroptosis-associated genes (PRGs) in endometrial cancer (EC) remains uncertain. 29 PRGs were identified as being either up-regulated or down-regulated in EC. PRGs subgroup analysis demonstrated distinct survival outcomes and diverse responses to chemotherapy and immune checkpoint blockade therapy. A higher expression of GPX4 and NOD2, coupled with lower levels of CASP6, PRKACA, and NLRP2, were found to be significantly associated with higher overall survival (OS) rates (p < 0.05). Conversely, lower expression of NOD2 was linked to lower progression-free survival (p = 0.021) and advanced tumor stage(p = 0.0024). NOD2, NLRP2, and TNM stages were identified as independent prognostic factors (p < 0.001). The LASSO prognostic model exhibited a notable decrease in OS among EC patients in the high-risk score group (ROC-AUC10-years: 0.799, p = 0.00644). Furthermore, NOD2 displayed a positive correlation with the infiltration of immune cells and the expression of immune checkpoints (p < 0.001). GPX4 and CASP6 are significantly associated with TMB and MSI (RTMB = 0.39; RMSI = 0.23). Additionally, a substantial upregulation of NOD2 was confirmed in both EC cells and tissue, indicating a positive relationship between advanced TNM stage (p < 0.0001) and infiltration of M1 phenotype macrophages. Nonetheless, its impact on patient OS did not reach statistical significance (p = 0.141). Our findings have contributed to the advancement of a prognostic model for EC patients. NOD2 receptor-mediated pyroptosis mechanism potentially regulates tumor immunity and promotes the transformation of macrophages from the M2 phenotype to the M1 phenotype, which significantly impacts the progression of EC.

Potential Advantages of Idarubicin-Loaded Trastuzumab-Coated Liposomes for Combating Head and Neck Squamous Cancer Cells.

Head and neck squamous cell carcinoma (HNSCC) with a high mortality rate is among the most common types of cancer in the world. Human epidermal growth factor receptor 2 (HER2) is expressed higher than normal level in the most HNSCC tumors, making them resistant to chemotherapy and radiotherapy. Therefore, HER2 has been introduced as a suitable target for anticancer drugs. The aim of this study is to examine the efficacy of a treatment protocol involving targeted delivery of idarubicin encapsulated in trastuzumab-decorated liposomes to HNSCC cells. In the current experimental study, efficacies of idarubicin, prepared liposomal idarubicin, and constructed immunoliposomal idarubicin (trastuzumab-decorated) were investigated in killing HN5 cells, a HER2- overexpressing HNSCC-originating cell line. Liposomal content of idarubicin and trastuzumab were qualified by UVVisible spectroscopy and preparations were characterized for shape and size by atomic force microscopy (AFM) and dynamic light scattering (DLS). To clarify role of the missing parts of the available crystal structure (PDB ID: 1n8z) within trastuzumab-HER2 interactions, we used a 40 ns molecular dynamic simulation approach. Based on the obtained results, liposomal idarubicin showed higher toxicity of the encapsulated drug on HN5 cells compared to the traditional free drug formulations. The immunoliposomal form of idarubicin was more effective than the liposomal formulation, in killing of HN5 cells. In addition, simulation of interactions between trastuzumab and HER2 revealed that the missing parts (in the crystal structure) of HER2 have critical interaction with trastuzumab, through salt-bridges and hydrogen bonds. It seems that the prepared immunoliposomes could attach more efficiently to HER2 overexpressing cells, which consequently leads to increasing cellular uptake of idarubicin through a receptor-mediated endocytosis mechanism. Moreover, simulation of the interaction between HER2 and trastuzumab suggested considerable possibilities for increasing trastuzumab affinity to HER2.

Role of Vasoactive Hormone-Induced Signal Transduction in Cardiac Hypertrophy and Heart Failure.

Heart failure is the common concluding pathway for a majority of cardiovascular diseases and is associated with cardiac dysfunction. Since heart failure is invariably preceded by adaptive or maladaptive cardiac hypertrophy, several biochemical mechanisms have been proposed to explain the development of cardiac hypertrophy and progression to heart failure. One of these includes the activation of different neuroendocrine systems for elevating the circulating levels of different vasoactive hormones such as catecholamines, angiotensin II, vasopressin, serotonin and endothelins. All these hormones are released in the circulation and stimulate different signal transduction systems by acting on their respective receptors on the cell membrane to promote protein synthesis in cardiomyocytes and induce cardiac hypertrophy. The elevated levels of these vasoactive hormones induce hemodynamic overload, increase ventricular wall tension, increase protein synthesis and the occurrence of cardiac remodeling. In addition, there occurs an increase in proinflammatory cytokines and collagen synthesis for the induction of myocardial fibrosis and the transition of adaptive to maladaptive hypertrophy. The prolonged exposure of the hypertrophied heart to these vasoactive hormones has been reported to result in the oxidation of catecholamines and serotonin via monoamine oxidase as well as the activation of NADPH oxidase via angiotensin II and endothelins to promote oxidative stress. The development of oxidative stress produces subcellular defects, Ca2+-handling abnormalities, mitochondrial Ca2+-overload and cardiac dysfunction by activating different proteases and depressing cardiac gene expression, in addition to destabilizing the extracellular matrix upon activating some metalloproteinases. These observations support the view that elevated levels of various vasoactive hormones, by producing hemodynamic overload and activating their respective receptor-mediated signal transduction mechanisms, induce cardiac hypertrophy. Furthermore, the occurrence of oxidative stress due to the prolonged exposure of the hypertrophied heart to these hormones plays a critical role in the progression of heart failure.

Precision targeting in hepatocellular carcinoma: Exploring ligand-receptor mediated nanotherapy.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and poses a major challenge to global health due to its high morbidity and mortality. Conventional chemotherapy is usually targeted to patients with intermediate to advanced stages, but it is often ineffective and suffers from problems such as multidrug resistance, rapid drug clearance, nonspecific targeting, high side effects, and low drug accumulation in tumor cells. In response to these limitations, recent advances in nanoparticle-mediated targeted drug delivery technologies have emerged as breakthrough approaches for the treatment of HCC. This review focuses on recent advances in nanoparticle-based targeted drug delivery systems, with special attention to various receptors overexpressed on HCC cells. These receptors are key to enhancing the specificity and efficacy of nanoparticle delivery and represent a new paradigm for actively targeting and combating HCC. We comprehensively summarize the current understanding of these receptors, their role in nanoparticle targeting, and the impact of such targeted therapies on HCC. By gaining a deeper understanding of the receptor-mediated mechanisms of these innovative therapies, more effective and precise treatment of HCC can be achieved.

Abstract A018: Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells

Abstract Introduction: Enzalutamide is indicated for treatment of castrate-resistant prostate cancer (CRPC), but therapeutic effect is transient, with improvement in survival of approximately 4 months. The mechanisms by which prostate cancer (PCa) cells survive Enzalutamide therapy are poorly understood, but androgen receptor (AR) and non-AR mediated mechanisms have been reported from transcriptome studies. We performed unbiased high resolution proteomic analyses to identify proteins and pathways that are involved in survival and progression in Enzalutamide treatment failure. Materials &amp; Methods: Enzalutamide-resistant MR49F and MR42D cell lines were maintained in standard medium (DMEM with 5% FBS) with Enzalutamide 10µM. Parental LNCaP cells were grown in standard medium. For perturbation, MR49F and MR42D cells were then changed to standard medium with either Enzalutamide 10µM (ENZA) or DMSO (control) for 7 days in. Medium was changed every 48 hours. Protein lysates from 4 biological replicates were labeled using a Tandem Mass Tag (TMT) assay and subjected to mass spectrometry (MS). Protein abundance was measured and significant differences in expression were analysed using a 2-way t-test with a permutation based false detection rate (FDR) of 0.05 and 250 randomisations in the Perseus software suite. MR49F and MR42D were compared to parental LNCaP, and MR49F and MR42D were compared on and off Enzalutamide treatment pressure. Functional biological pathways involved were identified using the DAVID and WebGestalt online pathway analysis tools. Results: MS identified 5364 proteins. Of these, 247 (180 up, 67 down) were differentially expressed by at least 50% in both MR49F and MR42D cell lines relative to parental LNCaP cells. When MR49F and MR42D cell lines were grown with or without Enzalutamide pressure, 5 proteins (4 up, 1 down) were regulated by at least 50% in both cell lines (ANLN, NIN, UQCRB, HIST1H3A and S100P). Pathway analyses with the differentially expressed and regulated proteins identified perturbations in biological pathways with a high representation of pathways related to metabolism, including amino acid and fatty acid metabolism, ascorbate and propanoate metabolism, drug transport and metabolism, and cell adhesion molecules. Conclusions: This unbiased proteomic analysis of Enzalutamide-resistant PCa cells identified proteins and pathways that may be essential for PCa cell survival and progression during Enzalutamide therapy failure. Enzalutamide therapy failure appears to involve some non-AR mediated mechanisms which may be targeted for therapeutic benefit. A better understanding of the mechanisms of Enzalutamide resistance could lead to development of novel therapies for post-Enzalutamide disease progression. Citation Format: Chidi N. Molokwu, Anders Kristensen, Chris Sutton. Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A018.

Microbially catalyzed conjugation of GABA and tyramine to bile acids.

Bile acids (BAs) are cholesterol-derived molecules that aid in digestion and nutrient absorption, regulate host metabolic processes, and influence physiology of the gut microbiota. Both the host and its microbiome contribute to enzymatic modifications that shape the chemical diversity of BAs in the gut. Several bacterial species have been reported to conjugate standard amino acids to BAs, but it was not known if bacteria conjugate BAs to other amine classes. Here, we show that Bacteroides fragilis strain P207, isolated from a bacterial bloom in the J-pouch of a patient with ulcerative colitis pouchitis, conjugates standard amino acids and the neuroactive amines γ-aminobutyric acid (GABA) and tyramine to deoxycholic acid. We extended this analysis to other human gut isolates and identified species that are competent to conjugate GABA and tyramine to primary and secondary BAs, and further identified diverse BA-GABA and BA-tyramine amides in human stool. A longitudinal metabolomic analysis of J-pouch contents of the patient from whom B. fragilis P207 was isolated revealed highly reduced levels of secondary bile acids and a shifting BA amide profile before, during, and after onset of pouchitis, including temporal changes in several BA-GABA amides. Treatment of pouchitis with ciprofloxacin was associated with a marked reduction of nearly all BA amides in the J-pouch. Our study expands the known repertoire of conjugated bile acids produced by bacteria to include BA conjugates to GABA and tyramine and demonstrates that these molecules are present in the human gut. IMPORTANCE BAs are modified in multiple ways by host enzymes and the microbiota to produce a chemically diverse set of molecules that assist in the digestive process and impact many physiological functions. This study reports the discovery of bacterial species that conjugate the neuroactive amines, GABA and tyramine, to primary and secondary BAs. We further present evidence that BA-GABA and BA-tyramine conjugates are present in the human gut, and document a shifting BA-GABA profile in a human pouchitis patient before, during, and after inflammation and antibiotic treatment. GABA and tyramine are common metabolic products of the gut microbiota and potent neuroactive molecules. GABA- and tyramine-conjugated BAs may influence receptor-mediated regulatory mechanisms of humans and their gut microbes, and absorption of these molecules and their entry into enterohepatic circulation may impact host physiology at distal tissue sites. This study defines new conjugated bile acids in the human gut.

Sialyl LewisX glycomimetics bearing an extended anionic chain targeting E- and P- selectin binding sites

Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties.

Cardioprotective effects of Moku-boi-to and its impact on AngII-induced cardiomyocyte hypertrophy.

Cardiomyocyte hypertrophy, induced by elevated levels of angiotensin II (AngII), plays a crucial role in cardiovascular diseases. Current therapeutic approaches aim to regress cardiac hypertrophy but have limited efficacy. Widely used Japanese Kampo medicines are highly safe and potential therapeutic agents. This study aims to explore the impact and mechanisms by which Moku-boi-to (MBT), a Japanese Kampo medicine, exerts its potential cardioprotective benefits against AngII-induced cardiomyocyte hypertrophy, bridging the knowledge gap and contributing to the development of novel therapeutic strategies. By evaluating the effects of six Japanese Kampo medicines with known cardiovascular efficiency on AngII-induced cardiomyocyte hypertrophy and cell death, we identified MBT as a promising candidate. MBT exhibited preventive effects against AngII-induced cardiomyocyte hypertrophy, cell death and demonstrated improvements in intracellular Ca2+ signaling regulation, ROS production, and mitochondrial function. Unexpectedly, experiments combining MBT with the AT1 receptor antagonist losartan suggested that MBT may target the AT1 receptor. In an isoproterenol-induced heart failure mouse model, MBT treatment demonstrated significant effects on cardiac function and hypertrophy. These findings highlight the cardioprotective potential of MBT through AT1 receptor-mediated mechanisms, offering valuable insights into its efficacy in alleviating AngII-induced dysfunction in cardiomyocytes. The study suggests that MBT holds promise as a safe and effective prophylactic agent for cardiac hypertrophy, providing a deeper understanding of its mechanisms for cardioprotection against AngII-induced dysfunction.

Protective effect of MitoTEMPO against cardiac dysfunction caused by ischemia–reperfusion: MCAO stroke model study

Purpose Neurological impairments are the leading cause of post-stroke mortality, while stroke-related cardiovascular diseases rank second in significance. This study investigates the potential protective effects of MitoTEMPO (2,2,6,6-tetramethyl-4-[[2-(triphenylphosphonio) acetyl] amino]-1-piperidinyloxy, monochloride, monohydrate), a mitochondria-specific antioxidant, against cardiac and neurological complications following stroke. The objective is to assess whether MitoTEMPO can be utilized as a protective agent for individuals with a high risk of stroke. Materials and methods Seventeen-week-old male Wistar Albino rats were randomly assigned to three groups: SHAM, ischemia–reperfusion and MitoTEMPO + ischemia–reperfusion (MitoTEMPO injection 0.7 mg/kg/day for 14 days). The SHAM group underwent a sham operation, while the ischemia–reperfusion group underwent 1-h middle cerebral artery occlusion followed by three days of reperfusion. Afterwards, noninvasive thoracic electrical bioimpedance and electrocardiography measurements were taken, and sample collection was performed for histological and biochemical examinations. Results Our thoracic electrical bioimpedance and electrocardiography findings demonstrated that MitoTEMPO exhibited a protective effect on most parameters affected by ischemia–reperfusion compared to the SHAM group. Furthermore, our biochemical and histological data revealed a significant protective effect of MitoTEMPO against oxidative damage. Conclusions The findings suggest that both ischemia–reperfusion-induced cardiovascular abnormalities and the protective effect of MitoTEMPO may involve G-protein coupled receptor-mediated signaling mechanisms. This study was conducted with limitations including a single gender, a uniform age group, a specific stroke model limited to middle cerebral artery, and pre-scheduled only one ischemia–reperfusion period. In future studies, addressing these limitations may enable the implementation of preventive measures for individuals at high risk of stroke.

The folate-linked chitosan-coated Kaempferol/HSA nano-transporters (FCKH-NTs) as the selective apoptotic inducer in human MCF-7 breast cancer cell line

Background Kaempferol, the natural bioactive flavonoid, has been utilized as an efficient anti-breast cancer compound. In the current study, the Kaempferol’s cellular uptake and its aqueous solubility were improved by using human serum albumin (HSA) as the Kaempferol adjuvant and encapsulating it with the folate-linked chitosan polymer to evaluate the apoptotic, activity of the novel-formulated Kaempferol in human MCF-7 breast cancer cells. Methods The folate-linked chitosan-coated Kaempferol/HSA nano-transporters (FCKH-NTs) were synthesized and characterized using FTIR, FESEM, DLS, and Zeta potential analysis. The nano-transporters’ selective cytotoxicity was studied by applying an MTT assay on the cancerous MCF-7 cells compared with normal HFF cell lines. Cell death type determination was determined by analyzing the expression of apoptotic (BAX and Cas-8) and anti-apoptotic genes (BCL2 and NF-κB). The FCKH-NTs apoptotic activity was verified by studying the flow cytometry and AO/PI staining results. Result The 126-nm FCKH-NTs (PDI = 0.282) selectively induced apoptotic death in human MCF-7 breast cancer cells by up-regulating the BAX, Nf- κB, and Cas-8 gene expression. The apoptotic activity of FCKH-NTs was verified by detecting the SubG1-arrested cancer cells and increased apoptotic bodies in AO/PI staining images. Conclusion The FCKH-NTs exhibited a selective-cytotoxic impact on human MCF-7 breast cancer cells compared with normal HFF cells, which can be due to the folate receptor-mediated endocytosis mechanism of the nano-transporters. Therefore, the FCKH-NTs have the potential to be used as a selective anti-breast cancer compound.

Patient-specific models link neurotransmitter receptor mechanisms with motor and visuospatial axes of Parkinson’s disease

Parkinson’s disease involves multiple neurotransmitter systems beyond the classical dopaminergic circuit, but their influence on structural and functional alterations is not well understood. Here, we use patient-specific causal brain modeling to identify latent neurotransmitter receptor-mediated mechanisms contributing to Parkinson’s disease progression. Combining the spatial distribution of 15 receptors from post-mortem autoradiography with 6 neuroimaging-derived pathological factors, we detect a diverse set of receptors influencing gray matter atrophy, functional activity dysregulation, microstructural degeneration, and dendrite and dopaminergic transporter loss. Inter-individual variability in receptor mechanisms correlates with symptom severity along two distinct axes, representing motor and psychomotor symptoms with large GABAergic and glutamatergic contributions, and cholinergically-dominant visuospatial, psychiatric and memory dysfunction. Our work demonstrates that receptor architecture helps explain multi-factorial brain re-organization, and suggests that distinct, co-existing receptor-mediated processes underlie Parkinson’s disease.

Sex-dependent neuronal effects of α-synuclein reveal that GABAergic transmission is neuroprotective of sleep-controlling neurons

BackgroundSleep disorders (SDs) are a symptom of the prodromal phase of neurodegenerative disorders that are mechanistically linked to the protein α-synuclein (α-syn) including Parkinson’s disease (PD). SDs during the prodromal phase could result from neurodegeneration induced in state-controlling neurons by accumulation of α-syn predominant early in the disease, and consistent with this, we reported the monomeric form of α-syn (monomeric α-syn; α-synM) caused cell death in the laterodorsal tegmental nucleus (LDT), which controls arousal as well as the sleep and wakefulness state. However, we only examined the male LDT, and since sex is considered a risk factor for the development of α-syn-related diseases including prodromal SDs, the possibility exists of sex-based differences in α-synM effects. Accordingly, we examined the hypothesis that α-synM exerts differential effects on membrane excitability, intracellular calcium, and cell viability in the LDT of females compared to males.MethodsPatch clamp electrophysiology, bulk load calcium imaging, and cell death histochemistry were used in LDT brain slices to monitor responses to α-synM and effects of GABA receptor acting agents.ResultsConsistent with our hypothesis, we found differing effects of α-synM on female LDT neurons when compared to male. In females, α-synM induced a decrease in membrane excitability and heightened reductions in intracellular calcium, which were reliant on functional inhibitory acid transmission, as well as decreased the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) with a concurrent reduction in action potential firing rate. Cell viability studies showed higher α-synM-mediated neurodegeneration in males compared to females that depended on inhibitory amino acid transmission. Further, presence of GABA receptor agonists was associated with reduced cell death in males.ConclusionsWhen taken together, we conclude that α-synM induces a sex-dependent effect on LDT neurons involving a GABA receptor-mediated mechanism that is neuroprotective. Understanding the potential sex differences in neurodegenerative processes, especially those occurring early in the disease, could enable implementation of sex-based strategies to identify prodromal PD cases, and promote efforts to illuminate new directions for tailored treatment and management of PD.

D2 dopamine receptor-mediated mechanisms of dopaminergic system modulation in in vivo and in vitro experimental models of migraine.

The dopaminergic system is implicated in the pathophysiology of migraine. However, the underlying mechanisms remain unclear. We explored the effects and mechanisms of dopaminergic system modulation in the in vivo and in vitro rat models of migraine. Dopaminergic agonist apomorphine, D2 receptor antagonists metoclopramide and haloperidol and 5-HT3 receptor antagonist ondansetron alone and together were tested in nitroglycerin-induced migraine model, in vivo. Likewise, the combinations of drugs were also tested on basal calcitonin gene-related peptide (CGRP) release in vitro hemiskull preparations. Mechanical allodynia was tested by von Frey filaments. CGRP concentrations in trigeminovascular structures and in vitro superfusates and c-Fos levels in the brainstem were determined by enzyme-linked immunosorbent assay. Meningeal mast cells were evaluated with toluidine blue staining. Apomorphine further enhanced nitroglycerin-induced mechanical allodynia, brainstem c-fos expression, trigeminal ganglion and brainstem CGRP concentrations and meningeal mast cell degranulation, in vivo. Haloperidol completely antagonised all apomorphine-induced effects and also alleviated changes induced by nitroglycerin without apomorphine. Metoclopramide and ondansetron partially attenuated apomorphine- or nitroglycerin-induced effects. A combination of haloperidol and ondansetron decreased basal CGRP release, in vitro, whereas the other administrations were ineffective. Apomorphine-mediated dopaminergic activation exacerbated nitroglycerin-stimulated nociceptive reactions by further enhancing c-fos expression, CGRP release and mast cell degranulation in strategical structures associated with migraine pain. Metoclopramide partially attenuated the effects of apomorphine, most likely because it is also a 5-HT3 receptor antagonist. Haloperidol with pure D2 receptor antagonism feature appears to be more effective than metoclopramide in reducing migraine-related parameters in dopaminergic activation- and/or NTG-induced migraine-like conditions.

GDF15 increases insulin action in the liver and adipose tissue via a β-adrenergic receptor-mediated mechanism

Growth differentiation factor 15 (GDF15) induces weight loss and increases insulin action in obese rodents. Whether and how GDF15 improves insulin action without weight loss is unknown. Obese rats were treated with GDF15 and displayed increased insulin tolerance 5h later. Lean and obese female and male mice were treated with GDF15 on days 1, 3, and 5 without weight loss and displayed increased insulin sensitivity during a euglycemic hyperinsulinemic clamp on day 6 due to enhanced suppression of endogenous glucose production and increased glucose uptake in WAT and BAT. GDF15 also reduced glucagon levels during clamp independently of the GFRAL receptor. The insulin-sensitizing effect of GDF15 was completely abrogated in GFRAL KO mice and also by treatment with the β-adrenergic antagonist propranolol and in β1,β2-adrenergic receptor KO mice. GDF15 activation of the GFRAL receptor increases β-adrenergic signaling, in turn, improving insulin action in the liver and white and brown adipose tissue.