The rapid advancement of nanomedicine, particularly underscored by the clinical success of lipid nanoparticle (LNP)-based vaccines, has intensified the need for simple, accurate, and widely accessible analytical techniques. A critical yet often overlooked parameter in nanoparticle development is the precise determination of the nanoparticle concentration. Conventional methods such as nanoparticle tracking analysis and electron microscopy are costly, low-throughput, and unavailable in most laboratories, creating a significant bottleneck in nanomedicine research and quality control. We investigated the potential of derived count rate (DCR), a scattered intensity parameter inherent to dynamic light scattering (DLS) when using a Zetasizer Nano ZS (Malvern Panalytical Ltd.)─an instrument available in virtually all nanomedicine laboratories─as a quantitative tool for LNP concentration analysis. A comprehensive DCR-concentration-size map was established using model LNP batches with varying concentrations. This data was used to derive a model correlating DCR, LNP size, and concentration. The model's predictive power was rigorously validated through a double-blind study involving independently formulated LNP batches with randomized compositions and unknown concentrations, done by formulators of varying expertise. The predictive model demonstrated an excellent fit. In the double-blind validation, the model predicted LNP concentrations with an error of only ∼4% compared to the theoretical values. Crucially, the method proved robust and user-independent, showing consistent high precision and accuracy across beginner, junior, and senior formulators. This study establishes DCR from standard DLS measurements as a simple, accurate, and highly accessible method for LNP quantification, bypassing the need for specialized equipment.

Endothelial cells (ECs) are key regulators in vascular homeostasis, and their physiological state depends on hemodynamic forces produced by the blood flow. Laminar shear stress (LSS) maintains endothelial integrity, whereas oscillatory shear stress (OSS) leads to endothelial dysfunction and subsequently to cardiovascular disease. Here, we delineated the proteomic signatures of human umbilical vein endothelial cells (HUVECs) exposed to a protective flow (LSS) or a disturbed flow (OSS) in comparison to static conditions, using trapped ion mobility spectrometry coupled with parallel accumulation-serial fragmentation. OSS induced limited changes in cell morphology and the proteomic profile, whereas LSS triggered major modifications in cell shape and a proteomic signature related to the cell surface and extracellular matrix. Membrane subproteomic profiling confirmed the identification of membrane effectors involved in flow-mediated responses and in silico integrating promoter analysis suggested Krüppel-like Factor 4 (KLF4) as an important transcriptional regulator of this effector cluster. Using a novel EC model, TeloHAECs, we demonstrated that KLF4 likely contributes to LSS-induced alignment and elongation by increasing the expression of CD34, HEG1, PI16, and ITGB4 and that KLF4 is involved in PI16 and ITGB4 expression. Collectively, these findings provide a robust discovery data set on EC proteomic profiles related to flow and a refined view of KLF4-associated regulatory signatures at the level of membrane protein-coding genes in response to LSS.

Endothelial cells (ECs) are key regulators in vascular homeostasis, and their physiological state depends on hemodynamic forces produced by the blood flow. Laminar shear stress (LSS) maintains endothelial integrity, whereas oscillatory shear stress (OSS) leads to endothelial dysfunction and subsequently to cardiovascular disease. Here, we delineated the proteomic signatures of human umbilical vein endothelial cells (HUVECs) exposed to a protective flow (LSS) or a disturbed flow (OSS) in comparison to static conditions, using trapped ion mobility spectrometry coupled with parallel accumulation-serial fragmentation. OSS induced limited changes in cell morphology and the proteomic profile, whereas LSS triggered major modifications in cell shape and a proteomic signature related to the cell surface and extracellular matrix. Membrane subproteomic profiling confirmed the identification of membrane effectors involved in flow-mediated responses and in silico integrating promoter analysis suggested Kruppel-like Factor 4 (KLF4) as an important transcriptional regulator of this effector cluster. Using a novel EC model, TeloHAECs, we demonstrated that KLF4 likely contributes to LSS-induced alignment and elongation by increasing the expression of CD34, HEG1, PI16, and ITGB4 and that KLF4 is involved in PI16 and ITGB4 expression. Collectively, these findings provide a robust discovery data set on EC proteomic profiles related to flow and a refined view of KLF4-associated regulatory signatures at the level of membrane protein-coding genes in response to LSS.

Endothelial cells (EC) play a pivotal role in vascular homeostasis. By sensing shear stress generated by blood flow, EC endorse vasculoprotection through mechanotransduction signaling pathways. Various ion channels are involved in mechanosignaling, and here, we investigated the endothelial voltage-gated Na+ channels (NaV channels), since their mechanosensitivity has been previously demonstrated in cardiomyocytes. First, we showed that EC from aorta (TeloHAEC) behave as EC from umbilical vein (HUVEC) under laminar shear stress (LSS). For both EC models, cell alignment and elongation occurred with the activation of the KLF2/KLF4 atheroprotective signaling pathways. We found that LSS decreased the expression of SCN5A, encoding NaV1.5, while LSS increased that of SCN3B, encoding NaVβ3. We demonstrated that the KLF4 transcription factor is involved in SCN3B expression under both static and LSS conditions. Interestingly, SCN3B silencing impaired EC alignment induced by LSS. The characterization of NaVβ3 interactome by coimmunoprecipitation and proteomic analysis revealed that mTOR, implicated in autophagy, binds to NaVβ3. This result was evidenced by the colocalization between NaVβ3 and mTOR inside cells. Moreover, we showed that SCN3B silencing led to the decrease in LC3B expression and the number of LC3B positive autophagosomes. Furthermore, we showed that NaVβ3 is retained within the cell and colocalized with LAMP1 and LC3B. Finally, we found that resveratrol, a stimulating-autophagy and vasculoprotective molecule, induced KLF4 together with NaVβ3 expression. Altogether, our findings highlight a novel role of NaVβ3 in endothelial function and cell alignment as an actor in shear stress vasculoprotective intracellular pathway through autophagy modulation.

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na+ (NaV) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that NaV channels, expressed in arteries, contribute to vascular tone in mouse mesenteric arteries (MAs). Here, we aimed to better characterize the mechanisms of action of VTD using mouse cecocolic arteries (CAs), a model of resistance artery. Using wire myography, we found that VTD induced vasorelaxation in mouse CAs. This VTD-induced relaxation was insensitive to prazosin, an α1-adrenergic receptor antagonist, but abolished by atropine, a muscarinic receptor antagonist. Indeed, VTD–vasorelaxant effect was totally inhibited by the NaV channel blocker tetrodotoxin (0.3 µM), the NO synthase inhibitor L-NNA (20 µM), and low extracellular Na+ concentration (14.9 mM) and was partially blocked by the NCX1 antagonist SEA0400 (45.4% at 1 µM). Thus, we assumed that the VTD-induced vasorelaxation in CAs was due to acetylcholine release by parasympathetic neurons, which induced NO synthase activation mediated by the NCX1-Ca2+ entry mode in endothelial cells (ECs). We demonstrated NCX1 expression in ECs by RT-qPCR and immunohisto- and western immunolabelling. VTD did not induce an increase in intracellular Ca2+ ([Ca2+]i), while SEA0400 partially blocked acetylcholine-triggered [Ca2+]i elevations in Mile Sven 1 ECs. Altogether, these results illustrate that VTD activates NaV channels in parasympathetic neurons and then vasorelaxation in resistance arteries, which could explain arterial hypotension after VTD intoxication.

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

The endothelial cell (EC) is a central actor to maintain vascular tone and homeostasis. Endothelial dysfunction is responsible for the development of many cardiovascular diseases including atherosclerosis. The endothelial function is mainly stimulated by the shear stress generated by blood flow. The physical forces produced by flow are detected by many mechanosensors in EC, which trigger intracellular signaling pathways influencing endothelial physiology. Among these mechanosensors, several ion channels have been identified. Voltage-gated Na+ (Nav) channels have been shown to be mechanosensitive in cardiac and intestinal cells. These ion channels are also expressed in EC and then we aim to investigate their role in EC responses to shear stress. Confluent Human Umbilical Vein EC (HUVEC) and Human Aorta EC (teloHAEC) were exposed to laminar (LSS, 20 dynes/cm2) or oscillatory shear stress (OSS, 20 dynes/cm2, 0.5 Hz frequency) using the Ibidi® pump system. Cell orientation was quantified by local gradient orientation method with Fiji software. RTqPCR and western blot were used to characterize Nav channel subunits at both transcriptional and protein levels. Exposition of HUVEC and TeloHAEC to a LSS for 24 hours and 4 days induced cell orientation in the direction of flow and activated the atheroprotective signaling pathway (KLF2/KLF4, eNOS). Interestingly, shear stress deep changes in several Nav channels subunits expression level. Notably, SCN3B expression, encoding the Ig-CAM Navβ3 protein, was increased with a 2.1 fold increase at 24 hours, 3.9 at 4 days and 12.6 at 7 days in HUVEC and 2.9 at 4 days in teloHAEC. Moreover, trans-resveratrol-induced KLF2 expression led to increase SCN3B expression, suggesting that this atheroprotective transcription factor might be involved in SCN3B regulation. Indeed, OSS, corresponding to an atherogenic flow, did not induce HUVEC alignment in the direction of flow neither activation of atheroprotective signaling pathway nor a significant increase of SCN3B expression. Taken together, our results suggest that Navβ3 could be a putative novel actor in the mechanotransduction of flow and thus in vasculoprotection.

Veratridine (VTD), a selective activator of voltage-gated Na + (Nav) channels, triggers contractile responses in mesenteric arteries (MA), by increasing intracellular Ca2+ mediated by Na + /Ca 2+ exchanger (NCX) in smooth muscle cells (SMC). However, VTD induces endothelium-dependent relaxation of retinal and cremaster arteries. These opposite VTD-responses gives rise to the question as to whether Nav channels play a role in vascular tone. We aim to characterize the contribution of Nav channels to vascular tone in MA. Since Nav channels are expressed in SMC, endothelial cells (EC) and nerve terminal endings of arteries, our aim is also to identify those which are responsible of VTD-evoked vasomotor responses. The VTD-induced contractile responses of first order mesenteric arteries (FOMA) and cecolic arteries (CA) of mice were recorded by wire myography. EC were used to characterize Ca 2+ responses using FURA-2 probe. Western blot and absolute RT-qPCR were used to characterize Nav channels and NCX expression. Nav1.2 channels are expressed in both SMC and EC of MA. VTD elicits contraction of FOMA, which is abolished by prazosin. Surprisingly, VTD induces relaxation of CA. This vasorelaxation is resistant to prazosin and supressed by atropine. VTD-induced relaxation is inhibited by eNO-synthase inhibitor (L-NNA) and NCX antagonists. Morever, NCX1 is the main NCX isoform expressed in EC. Finally, acetylcholine-induced Ca 2+ reponses are inhibited by NCX antagonists in endothelial cells. VTD induces opposite contractile responses in isolated mesenteric arteries. In fact, VTD activates Nav channels of sympathetic neurons in FOMA, and parasympathetic neurons in CA, leading to noradrenaline and acetylcholine release, respectively. Thus, the vascular tone of both arteries are differently regulated by the autonomic nervous system. Finally, acetylcholine triggers Ca 2+ -activation of eNO-synthase mediated by NCX1 in CA.

Voltage-gated sodium (Na V ) channels are the hallmarks of excitable cells and involved in the cardiac excitation-contraction coupling. They are made of a pore-forming α subunit encoding by 9 genes (SCN1A–5A and 8A–11A) associated with β auxiliary subunits (SCN1B–4B genes). Interestingly, Na V channels are also expressed in non-excitable cells such as endothelial cells (EC) but their role is still not yet totally understood. The aim of our study is to explore the role of Na V channels: – in proliferation and migration of EC, key process of vascular remodeling; – as new vascular mechanosensor since Na V channels have been shown to be sensitive to mechanical forces in cardiomyocytes and EC are constitutively exposed to physical forces created by blood flow, especially to shear stress. Proliferation and migration of Human Umbilical Vein Endothelial Cells (HUVEC) were studied using the live-cell imaging system IncuCyte S3 and shear stress using the Ibidi pump system. Blocking Na V activity using the selective inhibitor tetrodotoxin inhibited significantly proliferation and migration of HUVEC. Exposition of HUVEC to a laminar shear stress (20 dynes/cm 2 ) for 4 days induced a cell orientation in the flow direction and activated the atheroprotective signaling pathway (KLF2, eNOS, CYP1B1). Moreover, shear stress led to a profound transcriptional remodeling of different subunits of Na V channels on HUVEC with a down-regulation for the cardiac SCN5A by 5-fold and up-regulation for SCN8A, SCN9A, SCN1B and SCN3B. Since SCN3B is strongly upregulated by shear stress (9-fold), overexpression and silencing of this protein are currently under process in order to better understand the involvement of this transmembrane protein belong to Ig-CAM family in EC functions and its regulation. Our data highlight the contribution of Na V channels in EC functions and suggest a new role of SCN3B as a potential vascular mechanotransductor.

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action, whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα ( Esr1 -/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H 2 O 2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.