Chemical Agonists Research Articles

TFEB-Mediated Pro-inflammatory Response in Murine Macrophages Induced by Acute Alpha7 Nicotinic Receptor Activation.

Transcription factors TFEB and TFE3 are crucial for regulating autophagy, lysosomal biogenesis, and lipid metabolism, and have significant roles in macrophage function and innate immunity. The alpha7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated Ca 2+ channel known for its therapeutic potential in neurological and inflammatory disorders, has been implicated in modulating immune responses by modulating macrophage function. Stimulation of α7nAChR with chemical agonists has been claimed to activate TFEB in pancreatic acinar cells and neurons. However, the impact of α7nAChR activation on TFEB and TFE3 in macrophages remained unknown, posing an important question due to the potential implications for inflammation regulation. This study investigates the effects of acute α7nAChR activation on TFEB-mediated responses in murine macrophages using the specific agonist PNU-282987. We demonstrate that α7nAChR stimulation triggers TFEB nuclear translocation and lysosomal expansion. Surprisingly, PNU-282987 induces a broad pro-inflammatory gene signature without concomitant cytokine secretion, suggesting an uncoupling of gene expression from cytokine release. Mechanistically, TFEB activation requires the lysosomal Ca 2+ exporter MCOLN1 and the Ca 2+ -dependent phosphatase PPP3/calcineurin. Additionally, PNU-282987 elevates reactive oxygen species (ROS) levels, and ROS are involved in TFEB activation by PNU-282987. Notably, even with α7nAChR deletion, compensatory ROS-mediated TFEB activation persists, suggesting the involvement of additional nicotinic receptors. Our findings reveal a novel α7nAChR-TFEB signaling axis in macrophages, offer new insights into the cholinergic regulation of immune responses, establish a baseline for comparison with disease states, and identify potential therapeutic targets for modulating inflammation.

Functional profiling of resident cells during vascular aging

Introduction: Depending on surrounding cell types, substrate mechanics, and chemical agonists, vascular cell types – including endothelial (ECs) and smooth muscle cells (VSMC) — modulate their behavior to maintain homeostasis. Aging perturbs this homeostasis resulting in EC and SMC dysfunction, marked by impaired barrier integrity and pathological ECM remodeling. Endothelial-to-mesenchymal transition (EndMT) in ECs and “dedifferentiation” of VSMCs towards a synthetic phenotype are phenomenon under investigation as cellular drivers of the widespread dysregulation seen with aging. The overall goal of this project is to understand the effect of cross-talk between ECM and growth factor signaling on vascular cell plasticity during aging. Materials and Methods: Human aortic endothelial cells (HAECs) were seeded onto transwell inserts after seven days treatment with or without TGF-β. After growing to confluence on the insert, cells were subjected to a FITC-dextran transwell assay. A parallel group of cells was seeded onto fibronectin coated glass-bottom dishes and stained for the expression of focal adhesion proteins and cell-type markers. Mouse aortic smooth muscle cells (maSMCs) were isolated from C57Bl6 mice at 3 (young), 6 (early middle-aged; EMA), 9 (middle-age; MA), or greater than 18 months of age (old). Cells were seeded onto dye-quenched (DQ) collagen type I (Col-I) substrate before gain of fluorescence was measured in proximity to the cell as well as in the surrounding media. A parallel group of cells were allowed to grow in media containing FITC Col-I, and gain of fluorescence was measured in proximity to the cells. Each cell type (3M, 6M, 9M, and 18M) in both experiments (DQ and FITC) were further treated with MMP inhibitor, TG2 inhibitor and TGF-β1 to measure their impacts on Col-I turnover. Results: Barrier function was not impaired in ECs cultured with TGF-β1, relative to their untreated counterparts. Mouse aortic SMCs isolated from older mice had decreased rates of Col-I cleavage and were more susceptible to MMP inhibition than their younger counterparts. Conclusions: maSMCs from older mice have lower Col-I turnover than their younger counterparts. This is consistent with bulk tissue behavior, as it is well known that relative collagen levels increase in the elastic vasculature as we age. TGF-β1 treatment did not have an effect on permeability of 4kd FITC-dextran, it is feasible that there will be an impact on the larger 70kd fragments. Travis Brady is supported by the NASEM Ford Foundation Pre-doctoral Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanosensor channels of the lens and ciliary body: patch clamp studies

Epithelia from the lens and the ciliary body possess mechanosensors, including Piezo1 and transient receptor potential (TRP) channels, that may partake in the regulation of lens and aqueous humor volume. Although these mechanosensor channels commonly allow calcium entry, the outcomes of elevated intracellular calcium vary with the type of channel activated. For instance, separate activation of TRPV4 and TRPV1 increases Na-K-ATPase and Na/K/2Cl cotransporter activities, respectively. To further understand the role of mechanosensitivity in lens and ciliary body, we are currently using patch clamp techniques to characterize the response of primary cultured cells to chemical agonists and antagonists, as well as direct mechanical stimulation. Previously, we found that in mouse lens epithelial cells (mLEC), TRPV4 activation by agonist GSK1016790A (GSK) is followed by disappearance of a fast, transient K+-like outward current (Ipeak), decrease of a steady state outward current (Iss), fast depolarization of resting membrane potential (RMP) values from near -50 mV to near 0 mV, increase of Cx43 hemichannel-like events, and increase of overall membrane conductance ( gm) at holding potential (HP) = -50mV. More recently, we found that in mLEC, shear stress (streaming of external solution from a pipette tip over the cell membrane) reduced Ipeak but did not cause large RMP changes. Porcine non-pigmented epithelial cells (pNPE) in normal conditions display no Ipeak, small Iss, small RMP near -20 mV and spontaneous Cx43 hemichannel-like event activity. Nevertheless, GSK caused further depolarization of pNPE cells, and while no gm changes were seen when GSK was applied while holding cells at -50 mV, gm did increase when GSK exposure occurred during repeated ±80 mV pulses. Preliminary experiments show that activation of TRPM3 by its agonist pregnenolone causes a minimal hyperpolarization in mLEC. In comparison, in a line of human LEC (HLE B3) expressing TRPM3 channels, and which appear generally depolarized, pregnenolone caused a proportionally larger shift toward more negative RMP values; more intriguing, GSK has no measurable effect on the gm of HLE B3 cells. Taken together, and apart from considerations of cell- and species-specific differences, the data suggest that mechanosensor channels have different and/or opposite effects on the regulation of membrane ion channel activity, and thus on cell function. R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Isolation and purification of high molecular weight adiponectin from human plasma fraction

Adiponectin, a crucial protein hormone originating from adipose tissue, regulates key metabolic processes, including lipid metabolism, mitochondrial activity, and insulin sensitivity. These pleiotropic roles of adiponectin, along with its inverse correlation with metabolic disorders such as obesity, type II diabetes, and atherosclerosis, establish this protein as a potential therapeutic target. However, due to this complexity, challenges have arisen in its production with a natural conformation in bacterial or mammalian expression systems, hindering clinical translation. Furthermore, while inducers for adiponectin secretion or chemical agonists targeting adiponectin receptors have shown promise in laboratory settings, clinical studies with these agents have not yet been conducted. This study proposes a method for isolating and purifying natural high molecular weight (HMW) adiponectin from discarded plasma fractions during the conventional pharmaceutical protein manufacturing process. The process involved Cohn-Oncley fractionation, initial chromatography using reduced cellufine formyl, and subsequent purification via DEAE Sepharose chromatography. Characterization involved gel electrophoresis and biological assays on a hepatocyte cell-line. The purification process effectively captured adiponectin from the I + III paste, demonstrating that this fraction contained a significant portion of total plasma adiponectin. The two-step chromatography led to highly purified HMW adiponectin, confirmed by native-PAGE showing a 780 kDa multimeric complex. Biological assessments demonstrated normal downstream signaling, with HMW adiponectin inducing AMPK phosphorylation. This study demonstrates the feasibility of obtaining purified HMW adiponectin by repurposing plasma fractionation processes. It offers a promising avenue for the HMW adiponectin production, tapping into HMW adiponectin’s therapeutic potential against metabolic disorders while optimizing plasma resource utilization in healthcare.

Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

Agonists of melatonin receptors strongly promote the functional recovery from the neuroparalysis induced by neurotoxic snakes.

Snake envenoming is a major, but neglected, tropical disease. Among venomous snakes, those inducing neurotoxicity such as kraits (Bungarus genus) cause a potentially lethal peripheral neuroparalysis with respiratory deficit in a large number of people each year. In order to prevent the development of a deadly respiratory paralysis, hospitalization with pulmonary ventilation and use of antivenoms are the primary therapies currently employed. However, hospitals are frequently out of reach for envenomated patients and there is a general consensus that additional, non-expensive treatments, deliverable even long after the snake bite, are needed. Traumatic or toxic degenerations of peripheral motor neurons cause a neuroparalysis that activates a pro-regenerative intercellular signaling program taking place at the neuromuscular junction (NMJ). We recently reported that the intercellular signaling axis melatonin-melatonin receptor 1 (MT1) plays a major role in the recovery of function of the NMJs after degeneration of motor axon terminals caused by massive Ca2+ influx. Here we show that the small chemical MT1 agonists: Ramelteon and Agomelatine, already licensed for the treatment of insomnia and depression, respectively, are strong promoters of the neuroregeneration after paralysis induced by krait venoms in mice, which is also Ca2+ mediated. The venom from a Bungarus species representative of the large class of neurotoxic snakes (including taipans, coral snakes, some Alpine vipers in addition to other kraits) was chosen. The functional recovery of the NMJ was demonstrated using electrophysiological, imaging and lung ventilation detection methods. According to the present results, we propose that Ramelteon and Agomelatine should be tested in human patients bitten by neurotoxic snakes acting presynaptically to promote their recovery of health. Noticeably, these drugs are commercially available, safe, non-expensive, have a long bench life and can be administered long after a snakebite even in places far away from health facilities.

Role of TRPA1 in Painful Cold Hypersensitivity.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that plays a pivotal role in pain generation after exposure to irritant chemicals and is involved in the sensation of a wide variety of pathological pain. TRPA1 was first reported to be sensitive to noxious cold, but its intrinsic cold sensitivity still remains under debate. To address this issue, we focused on cold hypersensitivity induced by oxaliplatin, a platinum-based chemotherapeutic drug, as a peculiar adverse symptom of acute peripheral neuropathy. We and other groups have shown that oxaliplatin enhances TRPA1 sensitivity to its chemical agonists and reactive oxygen species (ROS). Our in vitro and animal model studies revealed that oxaliplatin, or its metabolite oxalate, inhibits hydroxylation of a proline residue within the N-terminus of human TRPA1 (hTRPA1) via inhibition of prolyl hydroxylase domain-containing protein (PHD), which induces TRPA1 sensitization to ROS. Although hTRPA1 is insensitive to cold, PHD inhibition endows hTRPA1 with cold sensitivity through sensing the small amount of ROS produced after exposure to cold. Hence, we propose that PHD inhibition can unveil the cold sensitivity of hTRPA1 by converting ROS signaling into cold sensitivity. Furthermore, in this review, we summarize the role of TRPA1 in painful cold hypersensitivity during peripheral vascular impairment.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

A NIR-II photothermal nanoplatform integrating intracellular Ca2+ elevation via TRPV1 activation for enhanced antitumor Therapy: A powerful combination of in situ tumor vaccination and vascular disruption

The treatment of triple-negative breast cancer (TNBC) remains a challenging issue in clinical settings due to the absence of effective therapeutic targets. Recent studies have revealed that transient receptor potential vanilloid type 1 (TRPV1), a non-selective Ca2+ channel responsive to noxious thermal stimuli, is frequently overexpressed in both malignant breast cancers and vascular cells, expected to be a promising therapeutic target. Herein, we developed a second near-infrared (NIR-II) photothermal nanoplatform named CT@hCuS to enhance immunotherapy and exacerbate tumor starvation, constructed from hollow CuS nanoparticles (hCuS) with tannic acid (TA) surface-coating and efficient Ca2+ loading via stable TA/Ca2+ complexation. The CT@hCuS platform releases Ca2+ in response to the mildly acidic pH of the tumor microenvironment. When exposed to intermittent 1064 nm laser irradiation, CT@hCuS delivers precise and controllable photothermy while avoiding potential adverse events of chemical TRPV1 agonists, activating TRPV1 and initiating an intracellular influx of Ca2+, which could not only destroy tumor vessels but also induce the immunogenic death of tumor cells. The resulting tumor antigens can be captured by CT@hCuS through binding with TA, leading to in situ tumor vaccination. Moreover, the nanoplatform via local administration contributes to durable drug release. Both in vitro and in vivo experiments demonstrated that CT@hCuS, combined with intermittent NIR-II laser irradiation, significantly enhanced the synergistic effects of intracellular Ca2+ elevation for tumor vaccination and tumor vascular disruption, which not only selectively block tumor nutrient sources but also transforms the immunosuppressive tumor environment into an immunoreactive phenotype for inhibiting TNBC growth and metastasis. This work presents a powerful NIR-II photothermal nanoplatform and proposes a unique synergistic strategy of immunotherapy and vascular disruption therapy for clinical TNBC treatment.

Prediction and assessment of xenoestrogens mixture effects using the in vitro ERα-CALUX assay.

Introduction: Many natural or synthetic compounds used in foods, dietary supplements, and food contact materials (FCMs) are suspected endocrine disruptors (EDs). Currently, scientific evidence to predict the impacts on biological systems of ED mixtures is lacking. In this study, three classes of substances were considered: i) phytoestrogens, ii) plant protection products (PPP) and iii) substances related to FCMs. Fourteen compounds were selected based on their potential endocrine activity and their presence in food and FCMs. Methods: These compounds were evaluated using an in vitro gene expression assay, the ERα-CALUX, to characterize their responses on the estrogen receptor alpha. Cells were exposed to fixed ratio mixtures and non-equipotent mixtures of full and partial agonists. The concentration-response curves measured for the three classes of compounds were characterized by variable geometric parameters in terms of maximum response (efficacy), sensitivity (slope) and potency (median effective concentration EC50). To account for these variations, a generic response addition (GRA) model was derived from mass action kinetics. Results: Although GRA does not allow us to clearly separate the concentration addition (CA) and independent action (IA) models, it was possible to determine in a statistically robust way whether the combined action of the chemicals in the mixture acted by interaction (synergy and antagonism) or by additive behavior. This distinction is crucial for assessing the risks associated with exposure to xenoestrogens. A benchmark dose approach was used to compare the response of phytoestrogen blends in the presence and absence of the hormone estradiol (E2). At the same time, 12 mixtures of 2-5 constituents including phytoestrogens, phthalates and PPPs in proportions close to those found in food products were tested. In 95% of cases, the response pattern observed showed a joint and independent effect of the chemicals on ER. Discussion: Overall, these results validate a risk assessment approach based on an additive effects model modulated by intrinsic toxicity factors. Here, the CA and IA approaches cannot be distinguished solely based on the shape of the concentration response curves. However, the optimized GRA model is more robust than CA when the efficacy, potency, and sensitivity of individual chemical agonists show large variations.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

TRPM8 Contributes to Early Temperature-Induced Platelet Activation

Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.

The conducting state of TRPA1 modulates channel lateral mobility

We have studied Danio rerio (Zebrafish) TRPA1 channel using a method that combines single channel electrophysiological and optical recordings to evaluate lateral mobility and channel gating simultaneously in single channels. TRPA1 channel activation by two distinct chemical ligands: allyl isothiocyanate (AITC) and TRPswitch B, results in substantial reduction of channel lateral mobility at the plasma membrane. Incubation with the cholesterol sequestering agent methyl-β-cyclodextrin (MβCD), prevents the reduction on lateral mobility induced by the two chemical agonists. This results strongly suggest that the open conformation of TRPA1 modulates channel lateral mobility probably by facilitating the insertion of the channel into cholesterol-enriched domains at the plasma membrane.

PIEZO1 is downregulated in glenohumeral chondrocytes in early cuff tear arthropathy following a massive rotator cuff tear in a mouse model.

Introduction: A massive rotator cuff tear (RCT) leads to glenohumeral joint destabilization and characteristic degenerative changes, termed cuff tear arthropathy (CTA). Understanding the response of articular cartilage to a massive RCT will elucidate opportunities to promote homeostasis following restoration of joint biomechanics with rotator cuff repair. Mechanically activated calcium-permeating channels, in part, modulate the response of distal femoral chondrocytes in the knee against injurious loading and inflammation. The objective of this study was to investigate PIEZO1-mediated mechanotransduction of glenohumeral articular chondrocytes in the altered biomechanical environment following RCT to ultimately identify potential therapeutic targets to attenuate cartilage degeneration after rotator cuff repair. Methods: First, we quantified mechanical susceptibility of chondrocytes in mouse humeral head cartilage ex vivo with treatments of specific chemical agonists targeting PIEZO1 and TRPV4 channels. Second, using a massive RCT mouse model, chondrocytes were assessed for mechano-vulnerability, PIEZO1 expression, and calcium signaling activity 14-week post-injury, an early stage of CTA. Results: In native humeral head chondrocytes, chemical activation of PIEZO1 (Yoda1) significantly increased chondrocyte mechanical susceptibility against impact loads, while TRPV4 activation (GSK101) significantly decreased impact-induced chondrocyte death. A massive RCT caused morphologic and histologic changes to the glenohumeral joint with decreased sphericity and characteristic bone bruising of the posterior superior quadrant of the humeral head. At early CTA, chondrocytes in RCT limbs exhibit a significantly decreased functional expression of PIEZO1 compared with uninjured or sham controls. Discussion: In contrast to the hypothesis, PIEZO1 expression and activity is not increased, but rather downregulated, after massive RCT at the early stage of cuff tear arthropathy. These results may be secondary to the decreased axial loading after glenohumeral joint decoupling in RCT limbs.

Abstract 28 A Novel Chemically Defined Culturing System to Support Hematopoietic Stem Cell Expansion

Abstract Introduction Hematopoietic stem cells (HSCs) are known to be multipotent and can reconstitute the entire blood and immune system following HSC transplantation (HSCT). However, the current HSCTs have many unresolved issues including difficulties in finding donors for bone marrow transplantation, preparing a sufficient HSC dose in cord blood transplantation, and the risk of graft-versus-host disease (GvHD) for all allogeneic HSCTs. In recent years, there have been substantial efforts to research and understand the biology of HSCs to develop cell therapy products that meet these challenges and provide an alternative to traditional HSCTs. Several culturing methods have been developed to stimulate HSC expansion ex vivo, but the use of biological materials, such as albumin, has not been ideal for pharmaceutical manufacturing, and it has been difficult inhibiting differentiation of HSCs into lineage-committed cells while culturing in a cytokine-filled medium. We have developed a novel albumin-free culturing system that supports proliferation of long-term HSCs ex vivo by replacing cytokines and albumin with chemical agonists and a caprolactam-based polymer. Objectives The objective of this study is to characterize our expanded cell population as part of an analysis to determine whether the population reconstitutes bone marrow and is therefore suitable for development of a cell therapy product. Methods Here we report the in vitro and in vivo characterization of proliferated HSCs using our novel culturing system compared with a nicotinamide system which is known to support HSC proliferation ex vivo. A colony forming assay, cell surface marker analysis using a flow cytometer, and in vivo repopulating analysis using the NOD/Shi-scid IL-2Rγnull (NOG) mouse were conducted. Results Surface marker analysis of the proliferated population shows more than 70% remaining as undifferentiated CD34+. Of which, a higher number of CD201+CD90+CD45RA- exists compared with the nicotinamide-cultured population. Colony forming unit assays indicate that our proliferated cell population contains a higher number of multipotent progenitors. The repopulating cell assay also shows that the proliferated cells have bone marrow reconstitution capability in bone marrow-ablated NOG mice. Discussion We believe that our chemically defined culturing system can selectively proliferate HSCs while restraining their differentiation and will contribute to the development of novel HSC therapy products.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Molecular determinants of TRPM8 function: key clues for a cool modulation.

Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.

Potential of omega-3 and conjugated fatty acids to control microglia inflammatory imbalance elicited by obesogenic nutrients

High-fat diet-induced obesity detrimentally affects brain function by inducing chronic low-grade inflammation. This neuroinflammation is, at least in part, likely to be mediated by microglia, which are the main immune cell population in the brain. Microglia express a wide range of lipid-sensitive receptors and their activity can be modulated by fatty acids that cross the blood-brain barrier. Here, by combining live cell imaging and FRET technology we assessed how different fatty acids modulate microglia activity. We demonstrate that the combined action of fructose and palmitic acid induce Ikβα degradation and nuclear translocation of the p65 subunit nuclear factor kB (NF-κB) in HCM3 human microglia. Such obesogenic nutrients also lead to reactive oxygen species production and LynSrc activation (critical regulators of microglia inflammation). Importantly, short-time exposure to omega-3 (EPA and DHA), CLA and CLNA are sufficient to abolish NF-κB pathway activation, suggesting a potential neuroprotective role. Omega-3 and CLA also show an antioxidant potential by inhibiting reactive oxygen species production, and the activation of LynSrc in microglia. Furthermore, using chemical agonists (TUG-891) and antagonists (AH7614) of GPR120/FFA4, we demonstrated that omega-3, CLA and CLNA inhibition of the NF-κB pathway is mediated by this receptor, while omega-3 and CLA antioxidant potential occurs through different signaling mechanisms.

Effect of ECM and mechanical strain on vascular cell plasticity

Introduction: Depending on surrounding cell types, substrate mechanics, and chemical agonists, vascular smooth muscle cells (VSMCs) alternate between a contractile and synthetic phenotype. While endothelial cells (ECs) do not modulate their phenotype in this way, their behavior is highly dependent on location within the body. These variations are part of healthy vascular function, but improper phenotype has been implicated in cardiovascular disease. Atherosclerotic lesions, for example, have been found to be high in dedifferentiated, synthetic VSMCs as well as endothelial cells that have undergone endothelial to mesenchymal transition (EndMT). The overall goal of this project is to understand the effect of cross-talk between ECM and mechanical strain on vascular cell plasticity during aging. Materials and Methods: Mouse aortic smooth muscle cells (maSMCs) were isolated from C57Bl6 mice at less than 3 months (young), 6 to 10 months of age (middle-aged; MA), or greater than 15 months of age (old). Cells were then seeded onto dye-quenched (DQ) collagen type I (Col-I) substrate and measured for gain of fluorescent signal over 3 days. Human aortic endothelial cells (HAECs) were seeded onto glass cover slips or fibronectin coated PDMS chambers. Cells were further treated with or without TGF-β2 with daily media changes over 3 days. IF images were captured with a Leica Confocal microscope and quantification was performed using ImageJ. Results: ECs cultured on glass or treated with TGF-β2 show increased expression of SM22α and decreased expression of VECAD. Physiological strain (10%) did not cause changes in expression relative to static (0%). Mouse aortic SMCs isolated from older mice had significantly decreased rates of Col-I cleavage. Conclusions: maSMCs from older mice have lower Col-I turnover than their younger counterparts. This is consistent with bulk tissue behavior, as it is well known that relative collagen levels increase in the elastic vasculature as we age. HAECs on stiff substrates are more likely to show signs of EndMT – consistent with other studies. The addition of agonists such as TGF-β2 can reproduce this behavior in cells cultured on soft substrates. NASEM Ford Foundation Pre-Doctoral Fellowship and NHBLI grant R01HL148112 01 (L.S.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1