Influx Of Cells Research Articles

Inhibition of mitochondrial phosphate carrier prevents high phosphate-induced superoxide generation and vascular calcification

Vascular calcification is a serious complication of hyperphosphatemia that causes cardiovascular morbidity and mortality. Previous studies have reported that plasmalemmal phosphate (Pi) transporters, such as PiT-1/2, mediate depolarization, Ca2+ influx, oxidative stress, and calcific changes in vascular smooth muscle cells (VSMCs). However, the pathogenic mechanism of mitochondrial Pi uptake in vascular calcification associated with hyperphosphatemia has not been elucidated. We demonstrated that the phosphate carrier (PiC) is the dominant mitochondrial Pi transporter responsible for high Pi-induced superoxide generation, osteogenic gene upregulation, and calcific changes in primary VSMCs isolated from rat aortas. Notably, acute incubation with high Pi markedly increased the protein abundance of PiC via ERK1/2- and mTOR-dependent translational upregulation. Genetic suppression of PiC prevented Pi-induced ERK1/2 activation, superoxide production, osteogenic differentiation, and vascular calcification of VSMCs in vitro and aortic rings ex vivo. Pharmacological inhibition of mitochondrial Pi transport using butyl malonate (BMA) or mersalyl abolished all pathologic changes involved in high Pi-induced vascular calcification. BMA or mersalyl also effectively prevented osteogenic gene upregulation and calcification of aortas from 5/6 subtotal nephrectomized mice fed a high-Pi diet. Our results suggest that mitochondrial Pi uptake via PiC is a critical molecular mechanism mediating mitochondrial superoxide generation and pathogenic calcific changes, which could be a novel therapeutic target for treating vascular calcification associated with hyperphosphatemia.

Abstract P6-01-05: Platelet-derived growth factor-CC expression in primary triple-negative breast cancer is associated with the basal-like molecular subtype and increased immune infiltrate

Abstract Around 10-15% of all breast cancers are categorized clinically as triple-negative breast cancer (TNBC). TNBC is defined by lack of protein expression or over expression of treatment targets such as the Estrogen Receptor α (ER) and Human Epidermal Growth Factor Receptor-2 (HER2), and the prognostic factor PR (Progesterone Receptor). The negative definition of TNBC results in a heterogeneous mix of tumors with variable molecular characteristics and prognosis. Defining TNBC molecular subtypes in detail is of importance to improve prognostication and find new treatment options. Expression of Platelet-derived growth factor-CC (PDGF-CC) has previously been correlated with the TNBC subtype, and paracrine PDGF-CC signaling has been reported to be of importance for maintaining TNBC tumor cell phenotype. We aimed to characterize PDGF-CC expression within the TNBC patient population by combining studies of PDGF-CC in tissue microarrays (TMAs) with matching RNAseq data and clinical follow-up; all variables originating from the SCAN-B (Sweden Cancerome Analysis Network – Breast) clinical study (ClinicalTrials.gov: NCT02306096). TMAs constructed of primary TNBC patient samples were stained for PDGF-CC using the Dako PT Autostainer system. Tumor cell-specific expression of PDGF-CC intensity was scored as either absent (N=11), weak (N=86), intermediate (N=81) or strong (N=70), and the scores were used to create corresponding TNBC PDGF-CC subgroups. We then explored associations of these subgroups with clinicopathological variables and time-to-event outcomes. Intermediate and strong PDGF-CC scores were associated with Nottingham Histological Grade 3 (p=0.001), increased proliferation (p< 0.001) and younger patient age at diagnosis (p=0.002). RNAseq data corresponding to tumors included in the TMAs was then retrieved, and differentially expressed genes were identified and used to perform Gene Set Enrichment Analysis (GSEA) comparing the TMA-derived PDGF-CC subgroups. Immune-related signatures were found to be enriched in the strong PDGF-CC subgroup vs. intermediate. Interestingly, strong PDGF-CC intensity was associated with a decreased risk of recurrence in the chemotherapy treated patient group (HR 0.28, 95% CI 0.10-0.80, p=0.017). Finally, patient samples were assigned a PAM50 subtype and a TNBC molecular subtype by the TNBCtype algorithm. Ninety-four percent of tumors in the strong PDGF-CC subgroup were classified as basal-like, whereas the corresponding number in the weak and intermediate PDGF-CC subgroups were 51% and 84%, respectively. The TNBC molecular subtype termed ‘Immunomodulatory’ was more frequently represented in the strong PDGF-CC subgroup compared to weak and intermediate (33% vs. 13% and 16%, respectively). In conclusion, strong PDGF-CC protein expression identified basal-like TNBCs, with an increase in immune cell infiltrate shown by RNAseq analysis. Whether or not PDGF-CC has a direct effect on influx of immune cells into tumors remains to be investigated. Analyses are currently ongoing to better understand the improved outcome associated with strong PDGF-CC intensity and on the contrary, the worse outcome associated with weak and intermediate PDGF-CC intensity, and if paracrine PDGF-CC signaling may explain the discrepancy observed. Citation Format: Sophie Lehn, Gyula Pekar, Paulina Bolivar Balbas, Johan Staaf, Christina Möller, Kristina Lövgren, Anna Ehinger, Ana Bosch Campos, Åke Borg, Kristian Pietras. Platelet-derived growth factor-CC expression in primary triple-negative breast cancer is associated with the basal-like molecular subtype and increased immune infiltrate [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-05.

Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids.

There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation.

Imiquimod induces skin inflammation in humanized BRGSF mice with limited human immune cell activity.

Human immune system (HIS) mouse models can be valuable when cross-reactivity of drug candidates to mouse systems is missing. However, no HIS mouse models of psoriasis have been established. In this study, it was investigated if imiquimod (IMQ) induced psoriasis-like skin inflammation was driven by human immune cells in human FMS-related tyrosine kinase 3 ligand (hFlt3L) boosted (BRGSF-HIS mice). BRGSF-HIS mice were boosted with hFlt3L prior to two or three topical applications of IMQ. Despite clinical skin inflammation, increased epidermal thickness and influx of human immune cells, a human derived response was not pronounced in IMQ treated mice. However, the number of murine neutrophils and murine cytokines and chemokines were increased in the skin and systemically after IMQ application. In conclusion, IMQ did induce skin inflammation in hFlt3L boosted BRGSF-HIS mice, although, a limited human immune response suggest that the main driving cellular mechanisms were of murine origin.

An optimized IgG-based B7-H3xCD3 bispecific antibody for treatment of gastrointestinal cancers

T cell-based immunotherapy has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions remain rare, particularly in gastrointestinal cancers like colorectal cancer (CRC). B7-H3 is overexpressed in multiple cancer entities including CRC on both tumor cells and tumor vasculature, the latter facilitating influx of effector cells into the tumor site upon therapeutic targeting. We generated a panel of Tcell-recruiting B7-H3xCD3 bispecific antibodies (bsAbs) and show that targeting a membrane-proximal B7-H3 epitope allows for a 100-fold reduction of CD3 affinity. Invitro, our lead compound CC-3 showed superior tumor cell killing, Tcell activation, proliferation, and memory formation, whereas undesired cytokine release was reduced. Invivo, CC-3 mediated potent antitumor activity in three independent models using immunocompromised mice adoptively transferred with human effector cells with regard to prevention of lung metastasis and flank tumor growth as well as elimination of large established tumors. Thus, fine-tuning of both target and CD3 affinities as well as binding epitopes allowed for the generation of a B7-H3xCD3 bsAbs with promising therapeutic activity. CC-3 is presently undergoing good manufacturing practice (GMP) production to enable evaluation in a clinical "first-in-human" study in CRC.

The significance of mosquito saliva in arbovirus transmission and pathogenesis in the vertebrate host

Due to changes in climate, numerous mosquito species are continuously extending their geographical distributions, posing potential new public health threats as arbovirus infections emerge in these new areas. During probing and feeding on the vertebrate host, a mosquito can inject both arbovirus and saliva into the skin of the host. The presence of mosquito saliva in the host skin during arbovirus transmission contributes to high viral titers in the skin, enhanced viremia, and rapid dissemination of the virus to target organs. This enhanced phenotype effectuated by the presence of mosquito saliva in the skin can be partly ascribed to a polarization of the local immune balance towards a Th2 response, an increased permeability of the dermal endothelium, and the influx of virus-susceptible immune cells to the bite site. However, the complete identification and characterization of immunomodulatory salivary proteins from different mosquito species and the mechanisms by which these salivary proteins exert their effects synergistically or antagonistically remains to be further explored. Moreover, the effect of new virus-vector combinations on the outcome of arbovirus infection in a new host is limited. Here, we review the immunomodulatory effects of mosquito saliva in the skin and the proposed mechanisms by which mosquito saliva enhances arbovirus pathogenesis in the vertebrate host, and discuss potential differences between Aedes and Culex mosquito species, the main vectors for medically important arboviruses. Gaining more insight into the effect of mosquito saliva in the vector-virus-host triad aids in predicting the potential transmission risk and disease severity of emerging vector-borne diseases.

Glutamate ionotropic receptor NMDA type subunit 1: A novel potential protein target of dapagliflozin against renal interstitial fibrosis

Renal interstitial fibrosis (RIF) is the final pathway for chronic kidney diseases (CKD) to end-stage renal disease, with no ideal therapy at present. Previous studies indicated that sodium glucose co-transporter-2 inhibitor (SGLT2i) dapagliflozin had the effect of anti-RIF, but the mechanism remains elusive and the renal protective effect could not be fully explained by singly targeting SGLT2. In this study, we aimed to explore the mechanism of dapagliflozin against RIF and identify novel potential targets. Firstly, dapagliflozin treatment improved pro-fibrotic indicators in unilateral ureteral obstruction mice and transforming growth factor beta 1 induced human proximal tubular epithelial cells. Then, transcriptomics and bioinformatics analysis were performed, and results revealed that dapagliflozin against RIF by regulating inflammation and oxidative stress related signals. Subsequently, targets prediction and analysis demonstrated that glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) was a novel potential target of dapagliflozin, which was related to inflammation and oxidative stress related signals. Moreover, molecular dynamics simulation revealed that dapagliflozin could stably bind to GRIN1 protein and change its spatial conformation. Furthermore, human renal samples and Nephroseq data were used for GRIN1 expression evaluation, and the results showed that GRIN1 expression were increased in renal tissues of CKD and RIF patients than controls. Additionally, further studies demonstrated that dapagliflozin could reduce intracellular calcium influx in renal tubular cells, which depended on regulating GRIN1 protein but not gene. In conclusion, GRIN1 is probably a novel target of dapagliflozin against RIF.

Probing the mechanism of annexin mediated plasma membrane repair.

The integrity of the plasma membrane is essential for maintaining cell homeostasis and eucaryotic cells have developed robust mechanisms for rapidly sealing plasma membrane ruptures. The repair process involves recruitment of multiple proteins to the damage site as triggered by the influx of calcium ions from the cytosol. Several members of the Annexin family are involved in membrane repair although their functional role in membrane remodeling is not fully understood. Biophysical experiments and modeling in synergy with cell experiments offer tools which can provide additional insight into Annexin mediated repair mechanisms. We review our recent studies into membrane remodeling and protein structures induced by Annexin binding. Using a unique platform of planar, free-edged membrane patches, the edge region of a membrane hole can be experimentally mimicked under controlled conditions. Using this platform, Annexins induce spontaneous membrane curvature leading to distinct, curved morphologies. We discuss the theoretical modeling of curvature effects and the formation of membrane necks around holes. We also describe AFM results resolving the organization of Annexins on membranes with molecular detail. Finally, we describe quantification of the calcium influx in cells during subjected to laser rupture. Detailed analysis and modeling of calcium diffusion and pumping allows extraction of key parameters for repair such as the timescale of hole closure.

Thymosin β4 and the anti-fibrotic switch

Wound healing involves a rapid response to the injury by circulating cells, followed by inflammation with an influx of inflammatory cells that release various factors. Soon after, cellular proliferation begins to replace the damaged cells and extracellular matrix, and then tissue remodeling restores normal tissue function. Various factors can lead to pathological wound healing when excessive and irreversible connective tissue/extracellular matrix deposition occurs, resulting in fibrosis. The process is initiated when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. TGFβ is the most well-characterized macrophage derived pro-fibrotic mediator. Other soluble mediators of fibrosis include connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and interleukin 10 (IL-10). Thymosin β4 (Tβ4) has shown therapeutic benefit in preventing fibrosis/scarring in various animal models of fibrosis/scarring. The mechanism of action of Tβ4 appears related, in part, to a reduction in the inflammatory response, including a reduction in macrophage infiltration, decreased levels of TGFβ and IL-10, and reduced CTGF activation, resulting in both prevention of fibroblast conversion to myofibroblasts and production of normally aligned collagen fibers. The amino N-terminal end of Tβ4, SDKP (serine-aspartate-lysine-proline), appears to contain the majority of anti-fibrotic activity and has shown excellent efficacy in many animal models of fibrosis, including liver, lung, heart, and kidney fibrosis. Ac-SDKP not only prevents fibrosis but can reverse fibrosis. Unanswered questions and future directions will be presented with regard to therapeutic uses alone and in combination with already approved drugs for fibrosis.

NKG2D Ligand Expression Induced by Oxidative Stress Mitigates Cutaneous Ischemia-Reperfusion Injury.

Pressure ulcers represent a crucial clinical problem, especially in hospitalized patients. Ischemia-reperfusion (I-R) is an important cause of these lesions. Natural killer (NK), invariant NK T (iNKT), and dendritic epidermal T-cells, which express the natural killer group 2, member D (NKG2D) receptor, have been reported to have physiological roles in skin tissue repair and wound healing. However, a role for NKG2D-NKG2D ligand interactions in I-R-induced skin injury has not been determined. Using a murine pressure ulcer model, we demonstrated that I-R-induced ulcers in NKG2D-deficient mice were larger than those in wild-type or T-cell receptor δ knockout mice. Histopathological evaluation revealed that accumulation of macrophages and neutrophils at the peripheral deep dermis and subcutaneous tissue of the ulcers was enhanced in NKG2D-deficient mice. Rae-1 mRNA, which encodes an NKG2D ligand, was induced, and RAE-1 protein was detected immunohistochemically in fibroblasts and inflammatory cells in the dermis after reperfusion. RAE-1 expression was also increased in primary mouse fibroblasts treated with sodium arsenite. These results suggested that NKG2D ligand expression was induced by oxidative stress after I-R injury and support a putative role for this ligand in wound repair. Furthermore, the influx of NKG2D-positive cells at I-R sites may mitigate pressure ulcers via NKG2D-NKG2D ligand interactions.

Phase Ib of pembrolizumab (pem) in combination with stereotactic body radiotherapy (SBRT) for resectable liver oligometastatic MMR-proficient (pMMR) colorectal cancer (CRC): Final results.

161 Background: SBRT is a standard treatment option for oligometastatic CRC and is associated with an increase in immunogenic antigen release and influx of immune cells. We hypothesized that radiation would enhance the immunogenicity of pMMR CRC and potentiate the effectiveness of PD-1 blockade. This phase Ib study examined the safety and efficacy of the sequential combination of SBRT and Pem in patients (pts) undergoing resection of their disease. Additionally, the accumulation and proteolysis of versican (VCAN), an immunoregulatory tumor matrix proteoglycan was examined as a novel immunotherapy biomarker. Proteolysis of VCAN results in the release of an immunostimulatory fragment, versikine. Cancers with low VCAN and high versikine (VCAN proteolysis predominant (VPP)) are hypothesized to respond better to immunotherapies. Methods: Eligibility criteria included resectable liver-confined metastatic pMMR CRC. Prior surgery and systemic chemotherapy were allowed. Subjects received sequential SBRT and cycle 1 of Pem prior to operative management and adjuvant Pem. The primary objectives were to determine the safety/tolerability of this regimen and the recurrence free survival (RFS) at 1 year following operative management. Correlative studies examined tumor infiltrating CD8+ T lymphocytes (TILs), VCAN, and versikine using immunohistochemistry. Results: 15 pts (median age 61.5 [range 39-69], 26% female) were enrolled. All pts had prior FOLFOX. The number of liver lesions ranged from 1-6. SBRT median dose was 50 Gy (40-60 Gy) to 1-2 liver lesions. Grade 3/4 AEs included one case of biliary tract injury and biloma, and one case of G3 hypophosphatemia. No grade 3/4 immune-related AEs occurred. All pts completed a minimum follow-up of 1 year post resection (median follow-up 41 months [range 15-64]). In the intention to treat analysis, the 1 year RFS was 67% (historic control 50%), 40% of patients remained cancer free, and 67% of pts are still alive. 2 of 3 pts with BRAF V600E mutations have had early recurrences. 2 pts had VCAN high tumors and both recurred prior to 1 year. 6 pts had VPP cancers and 83% were recurrence free at 1 year and 66% are currently without evidence of cancer. Conclusions: The combination of SBRT with Pem was well tolerated with no signal of increased immunotherapy-related toxicity. This study met its primary endpoint and this regimen deserves further investigation in confirmatory studies. Additionally, VCAN accumulation and proteolysis are promising biomarkers in this setting. Clinical trial information: NCT02837263 .

Rebound HIV-1 in cerebrospinal fluid after antiviral therapy interruption is mainly clonally amplified R5 T cell-tropic virus.

HIV-1 persists as a latent reservoir in people receiving suppressive antiretroviral therapy (ART). When ART is interrupted (treatment interruption/TI), rebound virus re-initiates systemic infection in the lymphoid system. During TI, HIV-1 is also detected in cerebrospinal fluid (CSF), although the source of this rebound virus is unknown. To investigate whether there is a distinct HIV-1 reservoir in the central nervous system (CNS), we compared rebound virus after TI in the blood and CSF of 11 participants. Peak rebound CSF viral loads vary and we show that high viral loads and the appearance of clonally amplified viral lineages in the CSF are correlated with the transient influx of white blood cells. We found no evidence of rebound macrophage-tropic virus in the CSF, even in one individual who had macrophage-tropic HIV-1 in the CSF pre-therapy. We propose a model in which R5 T cell-tropic virus is released from infected T cells that enter the CNS from the blood (or are resident in the CNS during therapy), with clonal amplification of infected T cells and virus replication occurring in the CNS during TI.

Myricetin alleviated immunologic contact urticaria and mast cell degranulation via the PI3K/Akt/NF-κB pathway.

Immunologic contact urticaria (ICU) is characterized by the wheal and flare reaction from direct contact with a chemical or protein agent, which involves a type I hypersensitivity mediated by allergen-specific immunoglobulin E (sIgE). Myricetin (Myr), a bioactive flavonoid, exhibits antiinflammatory activities. Our results showed that treatment with Myr could alleviate ICU symptoms, including a decrease in the number of wheals and scratching, and inhibit ear swelling in the IgE/DNFB-induced mice. The serum level of IgE, histamine, interleukin (IL)-4, TNF-α, and MCP-1 were reduced in Myr-treated mice. Myr also attenuated mast cells (MCs) degranulation and H-PGDS, TSLP, IL-33, PI3K, Akt, and NF-κB mRNA levels in ICU model. The IgE-mediated anaphylaxis mouse models demonstrated anti-allergic effects of Myr. In vitro analysis showed that Myr reduced IgE-induced calcium (Ca2+ ) influx, suppressed degranulation, and chemokine release in LAD2 cells (human primary mast cells). Myr can significantly inhibited PLCγ1, Akt, NF-κB, and p38 phosphorylation. In conclusion, the study demonstrated that Myr alleviate ICU symptoms and inhibit mast cell activation via PI3K/Akt/NF-κB signal pathway.

Biomineralized Nanoscavenger Abrogates Proinflammatory Macrophage Polarization and Induces Neutrophil Clearance through Reverse Migration during Gouty Arthritis.

The deposition of monosodium urate (MSU) crystals induces the overexpression of reactive oxygen species (ROS) and proinflammatory cytokines in residential macrophages, further promoting the infiltration of inflammatory leukocytes in the joints of gouty arthritis. Herein, a peroxidase-mimicking nanoscavenger was developed by forming manganese dioxide over albumin nanoparticles loaded with an anti-inflammatory drug, indomethacin (BIM), to block the secretion of ROS and COX2-induced proinflammatory cytokines in the MSU-induced gouty arthritis model. In the MSU-induced arthritis mouse model, the BIM nanoparticles alleviated joint swelling, which is attributed to the abrogation of ROS and inflammatory cytokine secretions from proinflammatory macrophages that induces neutrophil infiltration and fluid building up in the inflammation site. Further, the BIM nanoparticle treatment reduced the influx of macrophages and neutrophils in the injured region by blocking migration and inducing reverse migration in the zebrafish larva tail amputation model as well as in MSU-induced peritonitis and air pouch mouse models. Overall, the current strategy of employing biomineralized nanoscavengers for arthritis demonstrates clinical significance in dual blocking of peroxides and COX2 to prevent influx of inflammatory cells into the sites of inflammation.

Adar1 deletion causes degeneration of the exocrine pancreas via Mavs-dependent interferon signaling.

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine (A) to inosine (I). A-to-I editing alters post-transcriptional RNA processing, making ADAR1 a crucial regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we conditionally deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth, likely due to malabsorption associated with exocrine pancreatic insufficiency. Analyses of pancreata revealed ductal cell expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway, rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis.

The store-operated Ca2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1β, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1β lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1β is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCβ activation. Our results indicate that Orai1α interacts with PKCβ2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.

Screening of rosmarinic acid from Salvia miltiorrhizae acting on the novel target TRPC1 based on the ‘homology modelling–virtual screening–molecular docking–affinity assay–activity evaluation’ method

Context Salvia miltiorrhizae Bunge (Lamiaceae) is a traditional Chinese medicine (TCM) for the treatment of ‘thoracic obstruction’. Transient receptor potential canonical channel 1 (TRPC1) is a important target for myocardial injury treatment. Objective This work screens the active component acting on TRPC1 from Salvia miltiorrhizae. Materials and methods TCM Systems Pharmacology Database and Analysis Platform (TCMSP) was used to retrieve Salvia miltiorrhiza compounds for preliminary screening by referring to Lipinski’s rule of five. Then, the compound group was comprehensively scored by AutoDock Vina based on TRPC1 protein. Surface plasmon resonance (SPR) was used to determine the affinity of the optimal compound to TRPC1 protein. Western blot assay was carried out to observe the effect of the optimal compound on TRPC1 protein expression in HL-1 cells, and Fura-2/AM detection was carried out to observe the effect of the optimal compound on calcium influx in HEK293 cells. Results Twenty compounds with relatively good characteristic parameters were determined from 202 compounds of Salvia miltiorrhiza. Rosmarinic acid (RosA) was obtained based on the molecular docking scoring function. RosA had a high binding affinity to TRPC1 protein (KD value = 1.27 µM). RosA (50 μM) could reduce the protein levels (417.1%) of TRPC1 after oxygen-glucose deprivation/reperfusion (OGD/R) in HL-1 cells and it could inhibit TRPC1-mediated Ca2+ influx injury (0.07 ΔRatio340/380) in HEK293 cells. Discussion and conclusions We obtained the potential active component RosA acting on TRPC1 from Salvia miltiorrhizae, and we speculate that RosA may be a promising clinical candidate for myocardial injury therapy.

Structure-guided peptide engineering of a positive allosteric modulator targeting the outer pore of TRPV1 for long-lasting analgesia

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a classic analgesic target, but antagonists of TRPV1 failed in clinical trials due to their side effects like hyperthermia. Here we rationally engineer a peptide s-RhTx as a positive allosteric modulator (PAM) of TRPV1. Patch-clamp recordings demonstrate s-RhTx selectively potentiated TRPV1 activation. s-RhTx also slows down capsaicin-induced desensitization of TRPV1 in the presence of calcium to cause more calcium influx in TRPV1-expressing cells. In addition, our thermodynamic mutant cycle analysis shows that E652 in TRPV1 outer pore specifically interacts with R12 and K22 in s-RhTx. Furthermore, we demonstrate in vivo that s-RhTx exhibits long-lasting analgesic effects in noxious heat hyperalgesia and CFA-induced chronic inflammatory pain by promoting the reversible degeneration of intra-epidermal nerve fiber (IENF) expressing TRPV1 channels in mice, while their body temperature remains unaffected. Our results suggest s-RhTx is an analgesic agent as a PAM of TRPV1.

Clinical Approach to Corneal Ulcers

A corneal ulcer is a break or defect in the surface epithelium of the cornea, accompanied by tissue decay in the nearby area, leading to stromal necrosis. On a pathological level, there are swelling and an influx of cells in the affected region. Corneal ulcers often arise due to bacterial, viral, fungal or parasitic infections. However, non-infectious causes, such as dry eye, inflammatory disorders and trauma, also have a role. Delayed treatment can lead to blindness. Accurate diagnosis and prompt management are essential. Initially, patient history provides clues. A history of contact lens wear is often linked to bacterial or Acanthamoeba infections. Scratches may herald fungal infections, especially in agricultural settings. Viral aetiologies often correlate with dendritic patterns. Next, the slit-lamp examination can highlight corneal defects and infiltrates. Fluorescein staining may enhance the visibility of epithelial defects. Diagnostic procedures may include corneal scraping for microscopy, culture or polymerase chain reaction (PCR). This aids in identifying causative agents and tailoring antimicrobial therapy. Empirical treatment, especially in severe or progressing ulcers, begins before culture results. Bacterial ulcers frequently receive broad-spectrum antibiotics initially. Fungal infections warrant antifungal therapy, often with natamycin. Acanthamoeba keratitis necessitates specific antiamoebic therapy. Refractory or complex cases might demand additional interventions, such as corneal cross-linking or therapeutic keratoplasty. Appropriate patient education, such as contact lens hygiene, can serve as a preventative measure. Overall, understanding the underpinnings and a systematic approach to diagnosis and management are vital in preserving vision and avoiding complications in corneal ulcer patients.

PS-BPB01-6: PROTECTIVE NOX4 EFFECT IN ENDOTHELIAL CELLS INVOLVES ACTIVATION OF TRANSIENT RECEPTOR POTENTIAL MELASTATIN 2 (TRPM2) CATION CHANNEL

Objective: In endothelial cells, Nox4 is the most prevalent isoform and, in contrast to other Noxes, which generate superoxide O2, Nox4 produces mainly H2O2. Low H2O2 levels have been described to lower blood pressure, which suggests that a certain level of reactive oxygen species (ROS) can have protective effects on the cardiovascular system. We demonstrated that TRPM2 is a target of H2O2 in vascular cells. TRPM2 is a redox-sensitive Ca2+ channel and once activated leads to Ca2 influx. Endothelial dysfunction involves a reduction in eNOS activation by Ca2+ and is potentiated in the absence of Nox4. As TRPM2 channel is a H2O2 sensor and regulates Ca2+ entry, we questioned whether protective Nox4 effect in endothelial cells involves the H2O2-TRPM2-Ca2+ axis, with consequent regulation of NO signalling. Design and Methods: WT and TTRhRen hypertensive mice were crossed with Nox4 KO mice. Vascular function was measured by wire myograph in resistance arteries. ROS and H2O2 generation were assessed by lucigenin and amplex red, and Ca2+ influx by fluorescence microscopy in rat aortic endothelial cells (RAEC). eNOS activation was assessed by immunoblotting. Results: Blood pressure was significantly increased in TTRhRen (130.3 ± 7.0 mmHg) and TTRhRen/Nox4 KO mice (141.3 ± 18 mmHg) versus control mice (98.1 ± 8.0 mmHg). TTRhRen mice had reduced endothelium-dependent relaxation (Emax: WT 83.5 ± 4.03 vs TTRhRen 59.1 ± 3.5); an effect worsened by Nox4 KO (37.9 ± 5.4), p < 0.05. The TRPM2 activator, ADPR, improved vascular relaxation in TTRhRen/Nox4 KO mice (75.9 ± 7.7); an effect recapitulated by H2O2 (74.2 ± 15.4), p < 0.05. Ang II-induced ROS and H2O2 generation in RAEC, effect reversed by Nox4 siRNA and followed by increased Ca2+ influx (AUC - Ca2+: 20440.3333 ± 1870.9), important in eNOS activation. Ang II-induced Ca2+ influx in endothelial cells was reduced by TRPM2 siRNA, TRPM2 inhibitors (AUC - Ca2+: 8-br-cADPR 15232.2 ± 1052.1; Olaparib 14952.6 ± 843.2 and 2-APB 13270.8333 ± 277.4, p < 0.05) and by Nox4 siRNA and Nox4 inhibitor GKT137831 (AUC - Ca2+: GKT 15067.5 ± 255.6). Ang II-induced eNOS activation (Ser1177) (%: 72.3 ± 24.7) and NO release were blocked by Nox4 and TRPM2 sirNA, GKT137831, PEG-catalase and the TRPM2 inhibitor 8-br-cADPR (p < 0.05). Conclusions: We defined a novel molecular pathway in endothelial cells, where Nox4-induced H2O2 production activates PARP/TRPM2 signalling followed by Ca2+ influx, eNOS activation and NO release. The absence of Nox4 impairs Ca2+ homeostasis leading to endothelial dysfunction, an effect exacerbated in hypertension. The protective role of Nox4 corroborates the idea that the use of antioxidants based on the sole evidence that decreased oxidative stress is cardioprotective, it is not effective.