Plasma Membrane Of Fibroblasts Research Articles

Immune checkpoints: new insights into the pathogenesis of thyroid eye disease.

Thyroid eye disease (TED) is a disfiguring autoimmune disease characterized by changes in the orbital tissues and is caused by abnormal thyroid function or thyroid-related antibodies. It is the ocular manifestation of Graves' disease. The expression of thyroid-stimulating hormone receptor (TSHR) and the insulin-like growth factor-1 receptor (IGF-1 R) on the cell membrane of orbital fibroblasts (OFs) is responsible for TED pathology. Excessive inflammation is caused when these receptors in the orbit are stimulated by autoantibodies. CD34+ fibrocytes, found in the peripheral blood and orbital tissues of patients with TED, express immune checkpoints (ICs) like MHC II, B7, and PD-L1, indicating their potential role in presenting antigens and regulating the immune response in TED pathogenesis. Immune checkpoint inhibitors (ICIs) have significantly transformed cancer treatment. However, it can also lead to the occurrence of TED in some instances, suggesting the abnormality of ICs in TED. This review will examine the overall pathogenic mechanism linked to the immune cells of TED and then discuss the latest research findings on the immunomodulatory role of ICs in the development and pathogenesis of TED. This will offer fresh perspectives on the study of pathogenesis and the identification of potential therapeutic targets.

ROCK1 deficiency preserves caveolar compartmentalization of signaling molecules and cell membrane integrity.

In this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb-shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin-1 and -3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β-adrenergic signaling molecules including β1-adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin-1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions.

Synthesis and Preclinical Evaluation of Novel 99mTc-Labeled FAPI-46 Derivatives with Significant Tumor Uptake and Improved Tumor-to-Nontarget Ratios.

Fibroblast activation protein (FAP), which is expressed on the cell membranes of fibroblasts in most solid tumors, has become an important target for tumor diagnosis and treatment. However, previously reported 99mTc-labeled FAPI-04 complexes have high blood uptake, limiting their use in the clinic. In this work, six 99mTc-labeled FAPI-46 derivatives with different linkers (different amino acids, peptides, or polyethylene glycol) were prepared and evaluated. They had good in vitro stability, hydrophilicity, and good specificity for FAP. The biodistribution and MicroSPECT images revealed that they all had high specific tumor uptake for FAP, and their blood uptake was significantly decreased. Among them, [99mTc]Tc-6-1 exhibited the highest target-to-nontarget ratios (tumor/blood: 6.06 ± 1.19; tumor/muscle: 10.26 ± 0.44) and good tumor uptake (16.15 ± 0.83%ID/g), which also had significantly high affinity for FAP, good in vivo stability, and safety. Therefore, [99mTc]Tc-6-1 holds great potential as a promising molecular tracer for FAP tumor imaging.

(113) A Novel 3D Cellular Model for Peyronie's Disease Treatment Screening: PD.SPHERE

Abstract Introduction Peyronie's disease (PD) is a common issue that affects approximately 10% of males. Currently, researchers primarily study the pathogenesis and potential treatments for PD using monolayer fibroblast cells and a limited number of animal models. However, these models do not accurately represent the complex cellular structure and interactions involved in PD pathogenesis. We have successfully developed a three-dimensional (3D) cellular model, called PD.SPHERE, using a ground-breaking 'micro-gravity' cell culture method. This model is cultivated from extracted human PD tissues and serves as a valuable tool for in vitro treatment screening for PD. However, we have yet to use this model to evaluate the effectiveness of available and potential treatments. Objective Our objective is to determine the efficacy of the novel 3D cellular PD model, PD.SPHERE, as a treatment screening tool. Methods To achieve this, we cultured fibroblasts isolated from human PD tissues using the 'micro-gravity' approach. In this method, cells were seeded in a non-adhesive 96-well plate, placed on an orbital shaker, and incubated at 37°C with 5% CO2 and 95% relative humidity. We confirmed the proper morphology and cellular composition through microscopic assays. This 3D model was then used to assess the effects of collagenase clostridium histolyticum (CCH) and the actinidin enzyme, obtained from kiwi fruits. The PD.SPHERE was treated with actinidin at 10 mg/ml for 48 hours. We subsequently quantified collagen content using a soluble collagen quantification kit and observed viability, morphology, and changes in cellular compositions of the spheroids through immunofluorescent techniques and confocal microscopy. Results Preliminary results from our model have indicated significant changes in cellular morphology. Treatment with actinidin led to a significant decrease in spheroid size and density compared to the control group (P<0.01). The ratio of vimentin and actin filaments to nuclei staining was slightly but significantly reduced (P<0.05) in the spheroids treated with actinidin at 10 mg/ml. The cellular viability of the treated group showed a slight decrease, as revealed by Sytox Green staining. The assessment of collagen contents in the treated spheroids is ongoing. Conclusions Preliminary results suggest that our PD.SPHERE model can be effectively used to assess morphological and cellular component changes in PD tissues after treatment. Actinidin appears to compromise the plasma membrane of fibroblasts in PD.SPHERE. Our model holds promise for personalized treatment planning and potential for developing high-throughput treatment screening platforms. It also demonstrates consistency and reproducibility while closely mimicking the morphology of a PD plaque. Further research is required to collect signature gene expression data, physiological and immunological information, such as collagen production, extracellular matrix organization, and cellular expression of tight junctions, chemokines, and cytokines. These additional measures will help verify our model's pathophysiological, immunological, and treatment response aspects. Disclosure No.

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters.

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

A Simple Kit Formulation for Preparation and Exploratory Human Studies of a Novel 99mTc-Labeled Fibroblast Activation Protein Inhibitor Tracer for Imaging of the Fibroblast Activation Protein in Cancers.

Fibroblast activation protein (FAP) is a potential target for tumor diagnosis and treatment because it is selectively expressed on the cell membrane of cancer-associated fibroblasts in most solid tumor stroma. The aim of this study was to develop a 99mTc-labeled fibroblast activation protein inhibitor (FAPI) tracer, evaluate its imaging efficacy in nude mice, and further explore its biodistribution in healthy volunteers and uptake in tumor patients. An FAPI-derived ligand (DP-FAPI) containing d-proline was designed and synthesized as a linker, and a stable hydrophilic 99mTc-labeled complex ([99mTc]Tc-DP-FAPI) was obtained by kit formulation. In vitro cellular uptake and saturation binding assays were performed in FAP-transfected HT-1080 cells (FAP-HT-1080). The biodistribution was characterized, and micro-single-photon emission computed tomography (SPECT) imaging was performed in BALB/c nude mice bearing U87 MG tumors. Furthermore, a first-in-man application was performed in four healthy volunteers and three patients with gastrointestinal tumors. In vitro, the nanomolar Kd values of [99mTc]Tc-DP-FAPI indicated that it had significantly high target affinity for FAP. Biodistribution and micro-SPECT imaging studies showed that [99mTc]Tc-DP-FAPI exhibited high uptake and high tumor-to-nontargeted ratios. The calculated effective dose for [99mTc]Tc-DP-FAPI was approximately <5 mSv in four healthy volunteers. In three patients with gastrointestinal tumors, [99mTc]Tc-DP-FAPI quantitative SPECT/CT revealed high and reliable uptake. [99mTc]Tc-DP-FAPI exhibited high selectivity and affinity for FAP in vitro. The safety and effectiveness of [99mTc]Tc-DP-FAPI in primary tumor imaging have been confirmed by animal and clinical studies, revealing the potential clinical application value of this tracer.

Macrophage-evading and tumor-specific apoptosis inducing nanoparticles for targeted cancer therapy.

Extended circulation of anticancer nanodrugs in blood stream is essential for their clinical applications. However, administered nanoparticles are rapidly sequestered and cleared by cells of the mononuclear phagocyte system (MPS). In this study, we developed a biomimetic nanosystem that is able to efficiently escape MPS and target tumor tissues. The fabricated nanoparticles (TM-CQ/NPs) were coated with fibroblast cell membrane expressing tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). Coating with this functionalized membrane reduced the endocytosis of nanoparticles by macrophages, but increased the nanoparticle uptake in tumor cells. Importantly, this membrane coating specifically induced tumor cell apoptosis via the interaction of TRAIL and its cognate death receptors. Meanwhile, the encapsulated chloroquine (CQ) further suppressed the uptake of nanoparticles by macrophages, and synergized with TRAIL to induce tumor cell apoptosis. The vigorous antitumor efficacy in two mice tumor models confirmed our nanosystem was an effective approach to address the MPS challenge for cancer therapy. Together, our TM-CQ/NPs nanosystem provides a feasible approach to precisely target tumor tissues and improve anticancer efficacy.

From Flat to Bulb ‐ Novel Insights in Caveolae Membrane Curvature

Caveolae are 60‐100 nm sized bulb‐shaped plasma membrane invaginations found in various cell types including adipocytes, endothelial cells and myocytes. Their cellular functions comprise endocytosis and signaling processes, cellular lipid uptake, as well as sensing and regulation of membrane tension. Thereby, it was shown previously that caveolae can change their membrane shape from invaginated bulbs to flat domains and vice versa. However it is currently not clear which proteins are responsible for the membrane curvature changes. Here, we report the first detailed structural investigation of caveolae membrane shapes in vivo. To do so, Platinum replica EM images of the plasma membrane in mouse embryonic fibroblasts and endothelial cells were correlated to super‐resolution (STED) fluorescence intensities of proteins associated with caveolae domains. This correlative imaging approach (CLEM) allowed us to precisely localize caveolae related proteins to the underlying membrane curvature. The major structural caveolae proteins such as Caveolins and Cavins were found in all caveolae membrane domains independent of their curvature. Surprisingly, EHD2, a caveolar neck protein, was not only detected at bulb‐shaped invaginations but also at flat caveolae. Contrarily, Pacsin/Syndapin2 and EHBP1 were predominantly surrounding flat caveolae domains indicating that both proteins promote the neck formation. Because dynamin was previously associated with caveolae endocytosis and mobility we analyzed its spatial distribution to caveolae. However, our detailed CLEM analysis failed to determine dynamin to caveolae membrane domains in several cell types. Additionally, dynamin lacking cells did not show severe morphological changes of caveolae suggesting that dynamin is not involved in caveolae membrane curvature changes. Taken together, this study gives novel insights in caveolae structure and their formation from flat to invaginated membrane domains.

Abstract 515: Inhibiting Dpp4 In Senescent Vascular Smooth Muscle Cells Suppresses A Coagulation Program

In older persons, senescent vascular smooth muscle cells (VSMCs) express increased levels of senescence-associated secretory phenotype (SASP) factors such as cytokines, chemokines, adhesion molecules, matrix-remodeling enzymes, and angiogenic factors. The SASP contributes to destabilizing the plaque cap, suggesting that VSMC senescence promotes disease development. As a result of their prevalence in many diseases, major efforts are underway to develop therapies directed at senescent cells (senotherapies). Dipeptidyl Peptidase 4 (DPP4), a serine protease, was elevated on the plasma membrane of senescent fibroblasts. While the effect of DPP4 on VSMCs is not well understood, DPP4 inhibitors such as Vildagliptin are clinically used to treat diabetes, and in animal models, gliptins reduced atherosclerosis and inflammation independently of DPP4’s canonical role in glucose metabolism. We hypothesized that DPP4 inhibitors may reduce the progression and burden of atherosclerosis by preventing DPP4 enzymatic function on senescent VSMCs. Towards this end, we discovered that DPP4 protein levels and enzymatic activity increased in senescent human VSMCs (hVSMCs) senescence. We also found elevated DPP4 in human atherosclerotic plaques, and high levels of DPP4 colocalized with the senescence marker p16 in murine atherosclerosis. Strikingly, silencing DPP4 in senescent hVSMCs increased cell death and caspase activity. Proteomic analysis of conditioned media from senescent hVSMCs treated with DPP4 inhibitors revealed a reduction in numerous complement and coagulation factors, indicating that DPP4 partially governs hemostasis and the proteolytic events that promote thrombotic signaling. We propose that DPP4 inhibition suppresses the pro-coagulation/complement phenotype of senescent hVSMCs to disrupt their function and improve vascular disease progression.

Expression of Retinal G Protein-Coupled Receptor, a Member of the Opsin Family, in Human Skin Cells and Its Mediation of the Cellular Functions of Keratinocytes.

Background: Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported. Objectives: We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes. Methods: The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology. Results: The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis. Conclusions: This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 μM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5–10 μM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

BAG3 induces α-SMA expression in human fibroblasts and its over-expression correlates with poorer survival in fibrotic cancer patients.

Hypoxia and angiogenesis in solid tumors are often strictly linked to the development of fibrotic tissues, a detrimental event that compromises the antitumor immunity. As a consequence, tumor aggressiveness and poor patient prognosis relate to higher incidence of tissue fibrosis and stromal stiffness. The molecular pathways through which normal fibroblasts are converted in cancer‐associated fibroblasts (CAFs) have a central role in the onset of fibrosis in tumor stroma, thus emerging as a strategic target of novel therapeutic approaches for cancer disease. Several studies addressed the role of BAG3 in sustaining growth and survival of cancer cell and also shed light on the different mechanisms in which the intracellular protein is involved. More recently, new pieces of evidence revealed a pivotal role of extracellular BAG3 in pro‐tumor cell signaling in the tumor microenvironment, as well as its involvement in the development of fibrosis in tumor tissues. Here we report further data showing the presence of the BAG3 receptor (Interferon‐induced transmembrane protein [IFITM]‐2) on the plasma membrane of normal dermal fibroblasts and the activity of BAG3 as a factor able to induce the expression of α‐smooth muscle actin and the phosphorylation of AKT and focal adhesion kinase, that sustain CAF functions in tumor microenvironment. Furthermore, in agreement with these findings, bag3 gene expression has been analyzed by high throughput RNA sequencing databases from patients‐derived xenografts. A strong correlation between bag3 gene expression and patients' survival was found in several types of fibrotic tumors. The results obtained provide encouraging data that identify BAG3 as a promising therapeutic target to counteract fibrosis in tumors.

Signaling and function of cardiac autonomic nervous system receptors: Insights from the GPCR signalling universe.

The two branches of the autonomic nervous system (ANS), adrenergic and cholinergic, exert a multitude of effects on the human myocardium thanks to the activation of distinct G protein-coupled receptors (GPCRs) expressed on the plasma membranes of cardiac myocytes, cardiac fibroblasts, and coronary vascular endothelial cells. Norepinephrine (NE)/epinephrine (Epi) and acetylcholine (ACh) are released from cardiac ANS terminals and mediate the biological actions of the ANS on the heart via stimulation of cardiac adrenergic or muscarinic receptors, respectively. In addition, several other neurotransmitters/hormones act as facilitators of ANS neurotransmission in the heart, taking part in the so-called nonadrenergic noncholinergic (NANC) part of the ANS's control of cardiac function. These NANC mediators also use several different cell membrane-residing GPCRs to exert their effects in the myocardium. Cardiac ANS dysfunction and an imbalance between the activities of its two branches underlie a variety of cardiovascular diseases, from heart failure and hypertension to coronary artery disease, myocardial ischemia, and arrhythmias. In this review, we present the main well-established signaling modalities used by cardiac autonomic GPCRs, including receptors for salient NANC mediators, and we also highlight the latest developments pertaining to cardiac cell type-specific signal transduction, resulting in cell type-specific cardiac effects of each of these autonomic receptors.

Neuropilin-1 directs PDGFRα-entry into lung fibroblasts and signaling from very early endosomes.

Platelet-derived growth factor receptor-α (PDGFRα) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRα resides intracellularly as well as on the plasma membrane of murine lung fibroblasts (LF). We have examined how neuropilin-1 (Nrp1), a surface receptor without kinase activity, regulates the intracellular trafficking of PDGFRα in LF obtained from mice, some bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay, we observed that PDGFRα and Nrp1 colocalized in both early antigen-1 (EEA1) containing sorting endosomes and with adaptor protein containing a pleckstrin homology domain and a phosphotyrosine-binding domain-1 (APPL1) in very early endosomes (VEE). These findings were confirmed using live-cell imaging, which demonstrated that recently internalized PDGFRα was observed in Rab5-containing vesicles residing within 100 nm of the plasma membrane. Nrp1 deletion reduced the phosphorylation of Akt (protein kinase B), the major downstream target of PDGFRα, and limited accumulation of inositol-3 phosphates in APPL1-containing endosomes after exposure to PDGFA. PDGFRα co-immunoprecipitated with APPL1, indicating that PDGFRα enters VEE. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGFA. Our findings demonstrate that Nrp1 enhances the entry of PDGFRα into APPL1 containing VEE and that APPL1 enhances PDGFRα signaling. Therefore, Nrp1 promotes endosomal signaling by PDGFRα offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.

Doxyl Nitroxide Spin Probes Can Modify Toxicity of Doxorubicin towards Fibroblast Cells.

The biological properties of doxyl stearate nitroxides (DSs): 5-DS, Met-12-DS, and 16-DS, commonly used as spin probes, have not been explored in much detail so far. Furthermore, the influence of DSs on the cellular changes induced by the anticancer drug doxorubicin (DOX) has not yet been investigated. Therefore, we examined the cytotoxicity of DSs and their ability to induce cell death and to influence on fluidity and lipid peroxidation (LPO) in the plasma membrane of immortalised B14 fibroblasts, used as a model neoplastic cells, susceptible to DOX-induced changes. The influence of DSs on DOX toxicity was also investigated and compared with that of a natural reference antioxidant α-Tocopherol. By employing the trypan blue exclusion test and double fluorescent staining, we found a significant level of cytotoxicity for DSs and showed that their ability to induce apoptosis and modify plasma membrane fluidity (measured fluorimetrically) is more potent than for α-Tocopherol. The most cytotoxic nitroxide was 5-DS. The electron paramagnetic resonance (EPR) measurements revealed that 5-DS was reduced in B14 cells at the fastest and Met-12-DS at the slowest rate. In the presence of DOX, DSs were reduced slower than alone. The investigated compounds, administered with DOX, enhanced DOX-induced cell death and demonstrated concentration-dependent biphasic influence on membrane fluidity. A-Tocopherol showed weaker effects than DSs, regardless the mode of its application—alone or with DOX. High concentrations of α-Tocopherol and DSs decreased DOX-induced LPO. Substantial cytotoxicity of the DSs suggests that they should be used more carefully in the investigations performed on sensitive cells. Enhancement of DOX toxicity by DSs showed their potential to act as chemosensitizers of cancer cells to anthracycline chemotherapy.

Воздействие наноразмерных анионов кремний-молибденовой кислоты на плазматическую мембрану фибробластов эмбриона человека

Методом времяпролетной масс-спектрометрии вторичных ионов (ToF-SIMS) исследованы изменения состава плазматических мембран (ПМ) фибробластов эмбриона человека под воздействием наноразмерных анионов кремний-молибденовой кислоты (КМК). Измерены и интерпретированы зависимости масс-спектров основных липидов ПМ от концентрации КМК, коррелирующие с их послойными распределениями и со сродством холестерина к фосфолипидам. Обнаружен новый для биохимии клетки эффект – значительное снижение относительного содержания в ПМ холестерина и сфингомиелина под воздействием многозарядных анионов гетерополикислоты (ГПК). Для водных растворов КМК при концентрации С ~ 10мкМ/л и периода воздействия 48 ч обнаружено, что количество холестерина в ПМ снижается в 2–2.5 раза, при этом количество сфингомиелина уменьшается на 20–25%. Предложен новый механизм начального воздействия ГПК на плазматические мембраны, сводящийся к селективному травлению многозарядными анионами. Согласно предлагаемому механизму на первом этапе взаимодействия аниона полиоксометаллата с клеткой осуществляется экстракция из ПМ холестерина и сфингомиелина – основных регуляторов проницаемости и микровязкости ПМ. Как следствие возросшей проницаемости ПМ в клетках возможно ускорение жизненно важных трансмембранных и латеральных процессов.

Cell‐specific heterogeneity of Cx43‐positive gap junctions in the mammalian kidney

ObjectiveConnexin 43 (Cx43) is a gap junction‐forming protein which mediates the intercellular passage of molecules &lt; 1000 Da. In the kidney, Cx43 was reported in glomerular vessels and mesangium. Own results prompted us to reevaluate the distribution of Cx43 and its phosphorylated form, phospho‐S368‐Cx43 (pCx43), within the different zones of the rat kidney and in cultured fibroblasts in more detail.MethodsCx43 and pCx43 were localized by immunofluorescence in rat kidney sections and cultured human fibroblasts. High‐resolution immunostaining of Cx43 was achieved by ultrastructural immunogold labelling on electron microscopy sections. Double‐labelings with anti‐aquaporin 1 (AQP1), aquaporin 2 (AQP2), zonula occludens‐1 (ZO‐1), and cyclooxygenase‐2 (COX‐2) were conducted to clarify Cx43 distribution in the rat renal medulla. Cultured human fibroblasts served to study intercellular transfer of fluorescent lucifer yellow.ResultsCx43 was distributed in renal arterial and arteriolar endothelia, lymphatic vessel walls, peritubular capillaries, and glomerular compartments. In the interstitium, Cx43 was present as linear or punctate signals of the cell membrane of fibroblasts, in colocalization with pCx43. Cx43‐positive medullary fibroblasts co‐expressed COX‐2. No colocalization was detected between Cx43 and AQP1 or AQP2. Ultrastructural analysis showed Cx43 immunoreactivity at membrane contact zones between fibroblasts and in isolated cell processes of inner medullary interstitium. Cultured fibroblasts showed extensive, punctate Cx43 and pCx43 signals between adjacent cells. Signal was further distributed in non‐junctional areas of plasma membrane, suggesting Cx43 hemichannel functions. Lucifer yellow spread rapidly (10 min) between fibroblasts suggesting intercellular exchange via gap junctions, which was blocked by the gap junctional inhibitor, 18β‐glycyrrhetinic acid.ConclusionsCx43 in rat kidney shows prominent glomerular, vascular, and interstitial distribution, while epithelia, with exception of the podocytes, were negative. Focusing on interstitial fibroblasts, Cx43‐mediated information transfer was demonstrated. A constitutive nature of Cx43 phosphorylation was established in these cells. Our data suggest a cell‐specific heterogeneity of Cx43‐positive gap junctions mediating information exchange among renal cell types.

Insulin-like growth factor-1 enhances the expression of functional TSH receptor in orbital fibroblasts from thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), an autoimmune disease, occurs in approximately 50 % of patients with Graves' hyperthyroidism. Thyroid-stimulating hormone receptor (TSHR) that is expressed in orbital fibroblasts is the autoimmune target in the development of TAO. In addition to thyroid-stimulating immunoglobulin (TSI), insulin-like growth factor (IGF)-1 is also involved in the development of TAO. IGF-1 has been reported to potentiate the effects of thyroid-stimulating hormone (TSH) and TSI on TSHR signaling. In the current study, we investigated the effects of IGF-1 on the cell surface expression of the functional TSHR and its possible mechanism of action in human orbital fibroblasts. Our results show that orbital fibroblasts from the TAO patients expressed higher levels of IGF-1 receptor (IGF-R), compared to control subjects. Treatment with IGF-1 enhanced the expression of surface TSHR in orbital fibroblasts from TAO patients, but not from control subjects. In addition, treatment with IGF-1 increased the level of TSHR at both the protein and mRNA levels. Furthermore, pre-treatment with IGF-1 potentiated TSH-induced cAMP production, compared to cells that were treated with only TSH. Our results also show that pre-treatment with cycloheximide, an inhibitor of mRNA translation, partially, but not completely, inhibited the expression of TSHR on the cell surfaces of orbital fibroblasts from TAO patients. These collective results show that IGF-1enhances the cell surface expression of functional TSHR, not only by increasing TSHR expression, but also by inducing TSHR translocation to the plasma membrane in orbital fibroblasts from TAO.

Liposomal delivery of functional transmembrane ion channels into the cell membranes of target cells; a potential approach for the treatment of channelopathies

Defects in transmembrane ion channels underlie many disorders, commonly known as channelopathies. Current therapies are mostly symptomatic and do not treat the underlying cause. Here, we demonstrate the delivery of functional ion channels in protein form into the membrane of target cells using fusogenic proteoliposomes. The glycine receptor (GlyR) was adopted as a model channel. HEK293 cells were transfected with GlyR and GlyR-rich cell membrane fragments (CMF) were incorporated into fusogenic liposomes. Proteoliposomes were generated using 1,2-dioleoylphosphoethanolamine (DOPE) as the fusogenic lipid, lecithin, 1,2-distearoylphosphoethanolamine (DSPE), and cholesterol (Chol). Three formulations were prepared Non-fuse (2.5:0.5 Lecithin: Chol), Fuse1 (1.25:0.25:0.25:0.25) and Fuse2 (1.25:0.5:0.5:0.25 Lecithin: DOPE: DSPE: Chol). Proteoliposomes were assessed for their ability to (1) incorporate GlyR rich CMF (2) fuse with L929 fibroblast cell membrane and (3) deliver functional GlyR to these cells. All formulations were capable of integrating CMF, with Fuse2 showing highest CMF incorporation (1.2 and 1.4 folds relative to Non-fuse and Fuse1 respectively). All liposomes showed ability to fuse with the fibroblast cell membrane, with Fuse2 showing highest fusion. Patch-clamp analysis demonstrated successful delivery of functional GlyR into the fibroblast cell membrane. Thus, proof of principle was established for the use of liposomes to deliver functional ion channels to living cells.

The Effect of Nanosized Silicon Molybdate Anions on the Plasma Membrane of Human Fetal Fibroblasts

We used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to study changes in the composition of the plasma membranes of human fetal fibroblasts under the action of nanosized anions of silicon molybdic acid. The dependences of the mass spectra of the main lipids of the plasma membranes on the silicon molybdate concentration were measured and interpreted; the dependences correlate with the layer-by-layer distributions and with the affinity of cholesterol for phospholipids. A new effect for cell biochemistry was discovered, that is, a significant decrease in the relative concentrations of cholesterol and sphingomyelin in plasma membranes under the effect of multiply charged heteropoly anions (HPAs). In aqueous silicon molybdate solutions with a concentration of c ≈ 10 µM/L and an exposure time of 48 h, the amount of cholesterol in plasma membranes decreased by 2–2.5 times, while the amount of sphingomyelin decreased by 20–25%. A new mechanism is proposed for the initial effect of HPA on plasma membranes, which consists of selective etching by multiply charged anions. According to the proposed mechanism, cholesterol and sphingomyelin, the main regulators of permeability and microviscosity of plasma membranes, are extracted from the plasma membrane at the first stage of the interaction of the polyoxometallate anion with the cell. As a consequence of the increased permeability of the plasma membranes in cells, acceleration of vital transmembrane and lateral processes may occur.