Ion Channel and Transporter Involvement in Chemotherapy-Induced Peripheral Neurotoxicity.

The peripheral nervous system can encounter alterations due to exposure to some of the most commonly used anticancer drugs (platinum drugs, taxanes, vinca alkaloids, proteasome inhibitors, thalidomide), the so-called chemotherapy-induced peripheral neurotoxicity (CIPN). CIPN can be long-lasting or even permanent, and it is detrimental for the quality of life of cancer survivors, being associated with persistent disturbances such as sensory loss and neuropathic pain at limb extremities due to a mostly sensory axonal polyneuropathy/neuronopathy. In the state of the art, there is no efficacious preventive/curative treatment for this condition. Among the reasons for this unmet clinical and scientific need, there is an uncomplete knowledge of the pathogenetic mechanisms. Ion channels and transporters are pivotal elements in both the central and peripheral nervous system, and there is a growing body of literature suggesting that they might play a role in CIPN development. In this review, we first describe the biophysical properties of these targets and then report existing data for the involvement of ion channels and transporters in CIPN, thus paving the way for new approaches/druggable targets to cure and/or prevent CIPN.

Group 1 metabotropic glutamate receptors trigger glutamate-induced intracellular Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity causes an increase in local cerebral blood flow (CBF) to ensure local supply of oxygen and nutrients to the activated areas. The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-D-aspartate receptors to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca2+ signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca2+ signals followed by NO release by activating muscarinic 5 receptors in hCMEC/D3 cells, a widely employed model of human brain microvascular endothelial cells. Herein, we sought to assess whether also glutamate elicits metabotropic Ca2+ signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) that was blocked by α-methyl-4-carboxyphenylglycine and phenocopied by trans-1-amino-1,3-cyclopentanedicarboxylic acid, which, respectively, block and activate group 1 metabotropic glutamate receptors (mGluRs). Accordingly, hCMEC/D3 expressed both mGluR1 and mGluR5 and the Ca2+ response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca2+ response to glutamate was initiated by endogenous Ca2+ release from the endoplasmic reticulum and endolysosomal Ca2+ store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca2+ entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca2+]i. These data demonstrate for the first time that glutamate may induce metabotropic Ca2+ signals in human brain microvascular endothelial cells. The Ca2+ response to glutamate is likely to support NVC during neuronal activity, thereby reinforcing the emerging role of brain microvascular endothelial cells in the regulation of CBF.

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation.

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 µM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3 -dependent Ca2+ release in mouse brain endothelial cells.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 µM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

In Vivo Comparative Study on Acute and Sub-acute Biological Effects Induced by Ultrafine Particles of Different Anthropogenic Sources in BALB/c Mice.

Exposure to ultrafine particles (UFPs) leads to adverse effects on health caused by an unbalanced ratio between UFPs deposition and clearance efficacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute effects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50 µg UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant (HO-1, iNOS, Cyp1b1, Hsp70) protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of different responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison.

Artificial apolipoprotein corona enables nanoparticle brain targeting.

Many potential therapeutic compounds for brain diseases fail to reach their molecular targets due to the impermeability of the blood-brain barrier, limiting their clinical development. Nanotechnology-based approaches might improve compounds pharmacokinetics by enhancing binding to the cerebrovascular endothelium and translocation into the brain. Adsorption of apolipoprotein E4 onto polysorbate 80-stabilized nanoparticles to produce a protein corona allows the specific targeting of cerebrovascular endothelium. This strategy increased nanoparticle translocation into brain parenchyma, and improved brain nanoparticle accumulation 3-fold compared to undecorated particles (119.8 vs 40.5 picomoles). Apolipoprotein decorated nanoparticles have high clinical translational potential and may improve the development of nanotechnology-based medicine for a variety of neurological diseases.

The hunt for brain Aß oligomers by peripherally circulating multi-functional nanoparticles: Potential therapeutic approach for Alzheimer disease.

We previously showed the ability of liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide (mApoE-PA-LIP) to reduce brain Aß in transgenic Alzheimer mice. Herein we investigated the efficacy of mApoE-PA-LIP to withdraw Aß peptide in different aggregation forms from the brain, using a transwell cellular model of the blood-brain barrier and APP/PS1 mice. The spontaneous efflux of Aß oligomers (Aßo), but not of Aß fibrils, from the 'brain' side of the transwell was strongly enhanced (5-fold) in presence of mApoE-PA-LIP in the 'blood' compartment. This effect is due to a withdrawal of Aßo exerted by peripheral mApoE-PA-LIP by sink effect, because, when present in the brain side, they did not act as Aßo carrier and limit the oligomer efflux. In vivo peripheral administration of mApoE-PA-LIP significantly increased the plasma Aß level, suggesting that Aß-binding particles exploiting the sink effect can be used as a therapeutic strategy for Alzheimer disease. From the Clinical Editor: Alzheimer disease (AD) at present is an incurable disease, which is thought to be caused by an accumulation of amyloid-ß (Aß) peptides in the brain. Many strategies in combating this disease have been focused on either the prevention or dissolving these peptides. In this article, the authors showed the ability of liposomes bi-functionalized with phosphatidic acid and with an ApoE- derived peptide to withdraw amyloid peptides from the brain. The data would help the future design of more novel treatment for Alzheimer disease.

Multifunctional liposomes reduce brain ß-amyloid burden and ameliorate memory impairment in Alzheimer's disease mouse models.

Alzheimer's disease is characterized by the accumulation and deposition of plaques of ß-amyloid (Aß) peptide in the brain. Given its pivotal role, new therapies targeting Aß are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aß assemblies and evaluated their efficiency in reducing the Aß burden in Alzheimer's disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood-brain barrier targeting and with phosphatidic acid for Aß binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aß assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aß1-42 (-33%), assessed by ELISA, and the number and total area of plaques (-34%) detected histologically. Also, brain Aß oligomers were reduced (-70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [(11)C]Pittsburgh compound B (PIB). The reduction of brain Aß was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aß aggregates and promote peptide removal across the blood-brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimer's disease.

Corrigendum: Precision medicine: a new era for inner ear diseases.

[This corrects the article DOI: 10.3389/fphar.2024.1328460.].

Precision medicine: a new era for inner ear diseases.

The inner ear is the organ responsible for hearing and balance. Inner ear dysfunction can be the result of infection, trauma, ototoxic drugs, genetic mutation or predisposition. Often, like for Ménière disease, the cause is unknown. Due to the complex access to the inner ear as a fluid-filled cavity within the temporal bone of the skull, effective diagnosis of inner ear pathologies and targeted drug delivery pose significant challenges. Samples of inner ear fluids can only be collected during surgery because the available procedures damage the tiny and fragile structures of the inner ear. Concerning drug administration, the final dose, kinetics, and targets cannot be controlled. Overcoming these limitations is crucial for successful inner ear precision medicine. Recently, notable advancements in microneedle technologies offer the potential for safe sampling of inner ear fluids and local treatment. Ultrasharp microneedles can reach the inner ear fluids with minimal damage to the organ, collect µl amounts of perilymph, and deliver therapeutic agents in loco. This review highlights the potential of ultrasharp microneedles, combined with nano vectors and gene therapy, to effectively treat inner ear diseases of different etiology on an individual basis. Though further research is necessary to translate these innovative approaches into clinical practice, these technologies may represent a true breakthrough in the clinical approach to inner ear diseases, ushering in a new era of personalized medicine.

Small-angle X-ray scattering unveils the internal structure of lipid nanoparticles.

Lipid nanoparticles own a remarkable potential in nanomedicine, only partially disclosed. While the clinical use of liposomes and cationic lipid-nucleic acid complexes is well-established, liquid lipid nanoparticles (nanoemulsions), solid lipid nanoparticles, and nanostructured lipid carriers have even greater possibilities. However, they face obstacles in being used in clinics due to a lack of understanding about the molecular mechanisms controlling their drug loading and release, interactions with the biological environment (such as the protein corona), and shelf-life stability. To create effective drug delivery carriers and successfully translate bench research to clinical settings, it is crucial to have a thorough understanding of the internal structure of lipid nanoparticles. Through synchrotron small-angle X-ray scattering experiments, we determined the spatial distribution and internal structure of the nanoparticles' lipid, surfactant, and the bound water in them. The nanoparticles themselves have a barrel-like shape that consists of coplanar lipid platelets (specifically cetyl palmitate) that are covered by loosely spaced polysorbate 80 surfactant molecules, whose polar heads retain a large amount of bound water. To reduce the interface cost of bound water with unbound water without stacking, the platelets collapse onto each other. This internal structure challenges the classical core-shell model typically used to describe solid lipid nanoparticles and could play a significant role in drug loading and release, biological fluid interaction, and nanoparticle stability, making our findings valuable for the rational design of lipid-based nanoparticles.

Assessment of dynamic mechanical properties of the respiratory system during high-frequency oscillatory ventilation*.

OBJECTIVES: 1) To investigate the possibility of estimating respiratory system impedance (Zrs, forced oscillation technique) by using high-amplitude pressure oscillations delivered during high-frequency oscillatory ventilation; 2) to characterize the relationship between Zrs and continuous distending pressure during an increasing/decreasing continuous distending pressure trial; 3) to evaluate how the optimal continuous distending pressure identified by Zrs relates to the point of maximal curvature of the deflation limb of the quasi-static pressure-volume curve. DESIGN: Prospective laboratory animal investigation. SETTING: Experimental medicine laboratory. SUBJECTS: Eight New Zealand rabbits. INTERVENTIONS: The rabbits were ventilated with high-frequency oscillatory ventilation. Zrs was measured while continuous distending pressure was increased and decreased between 2 and 26 cm H2O in 1-minute steps of 4 cm H2O. At each step, a low-amplitude (6 cm H2O) sinusoidal signal was alternated with a high-amplitude (18 cm H2O) asymmetric high-frequency oscillatory ventilation square pressure waveform. Pressure-volume curves were determined at the end of the continuous distending pressure trial. All measurements were repeated after bronchoalveolar lavage. MEASUREMENTS AND MAIN RESULTS: Zrs was estimated from flow and pressure measured at the inlet of the tracheal tube and expressed as resistance (Rrs) and reactance (Xrs). Linear correlation between the values, measured by applying the small-amplitude sinusoidal signal and the ventilator waveform, was good for Xrs (r = 0.95 ± 0.04) but not for Rrs (r = 0.60 ± 0.34). Following lavage, the Xrs-continuous distending pressure curves presented a maximum on the deflation limb, identifying an optimal continuous distending pressure that was, on average, 1.1 ± 1.7 cm H2O below the point of maximal curvature of the deflation limb of the pressure-volume curves. CONCLUSIONS: Xrs can be accurately measured during high-frequency oscillatory ventilation without interrupting ventilation and/or connecting additional devices. An optimal continuous distending pressure close to the point of maximal curvature of the deflation limb of quasi-static pressure-volume curve can be identified by measuring Zrs during a decreasing continuous distending pressure trial. Zrs might constitute a useful bedside tool for monitoring lung mechanics and improving the continuous distending pressure optimization during high-frequency oscillatory ventilation.

Aß42 production in brain capillary endothelial cells after oxygen and glucose deprivation.

Although the diverse triggers of AD are still under debate, the hypothesis of the contribution of cerebrovascular deficiencies has emerged in recent years. Cerebrovascular dysfunction may precede cognitive decline and onset of neurodegeneration. Indeed, the toxic Aß(42) aggregates constituting senile plaques, one of AD hallmarks, is often detected as amorphous material or fine fibrils in the brain capillary of AD patients. Aß(42) causing cerebral microangiopathy might originate either from the circulating blood, the vessel wall itself or the brain parenchyma. In the present investigation we show, for the first time, that in rat brain capillary endothelial cells (RBE4), in vitro oxygen glucose deprivation treatment elicits 250% of Aß(42) peptide production increase through a mechanism that involves the hypoxia inducible factor-1-mediated ß-secretase (BACE1) up-regulation. Furthermore, we observed a time dependent increase of amyloid protein precursor (AßPP) gene and protein expression, confirming previous reports which established the existence of AßPP in the cerebrovascular domain. Our experimental evidences point out that ischemic events may directly contribute in brain capillary endothelial cells to the enhancement of the amyloidogenic metabolism, leading to intracellular deposition of Aß(42). This events may contribute to the impairment of Aß brain clearance and AD related blood brain barrier dysfunctions.

Neuromodulatory functions exerted by oxytocin on different populations of hippocampal neurons in rodents.

Oxytocin (OT) is a neuropeptide widely known for its peripheral hormonal effects (i.e., parturition and lactation) and central neuromodulatory functions, related especially to social behavior and social, spatial, and episodic memory. The hippocampus is a key structure for these functions, it is innervated by oxytocinergic fibers, and contains OT receptors (OTRs). The hippocampal OTR distribution is not homogeneous among its subregions and types of neuronal cells, reflecting the specificity of oxytocin's modulatory action. In this review, we describe the most recent discoveries in OT/OTR signaling in the hippocampus, focusing primarily on the electrophysiological oxytocinergic modulation of the OTR-expressing hippocampal neurons. We then look at the effect this modulation has on the balance of excitation/inhibition and synaptic plasticity in each hippocampal subregion. Additionally, we review OTR downstream signaling, which underlies the OT effects observed in different types of hippocampal neuron. Overall, this review comprehensively summarizes the advancements in unraveling the neuromodulatory functions exerted by OT on specific hippocampal networks.

GABAA and GABAB Receptors Mediate GABA-Induced Intracellular Ca2+ Signals in Human Brain Microvascular Endothelial Cells.

Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABAA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.

Extracellular transglutaminase-2, nude or associated with astrocytic extracellular vesicles, modulates neuronal calcium homeostasis.

We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+ concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]i rise, which was reversible upon TG2 removal and was dependent on Ca2+ influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarization and activation of inward currents, due to the Na+/Ca2+-exchanger (NCX) operating in the reverse mode and indirect activation of L-type VOCCs, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+ influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]i through release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation.

Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model.

A promising strategy to enhance blood-brain barrier penetration by drugs is the functionalization of nanocarriers with uptake-facilitating ligands. We studied the cellular uptake, by cultured RBE4 brain capillary endothelial cells, of nanoliposomes (NLs) covalently coupled with monomer or tandem dimer of apolipoprotein E (ApoE)-derived peptides (residues 141-150), at various densities. NLs without functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy and quantified by fluorescence-activated cell sorting. Functionalization with peptides mediated an efficient NLs uptake that increased with peptide density; NLs carrying monomeric peptide performed the best. Moreover, we studied the ability of ApoE-NLs to enhance the transport of a drug payload through a RBE4 cell monolayer. The permeability of a tritiated curcumin derivative was enhanced after its entrapment into ApoE-NLs, in particular those functionalized with the dimer (+83% with respect to free drug, P < 0.01). Thus, these NLs appear particularly suitable for implementing further strategies for drug brain targeting.

Pulmonary Interstitial Matrix and Lung Fluid Balance From Normal to the Acutely Injured Lung.

This review analyses the mechanisms by which lung fluid balance is strictly controlled in the air-blood barrier (ABB). Relatively large trans-endothelial and trans-epithelial Starling pressure gradients result in a minimal flow across the ABB thanks to low microvascular permeability aided by the macromolecular structure of the interstitial matrix. These edema safety factors are lost when the integrity of the interstitial matrix is damaged. The result is that small Starling pressure gradients, acting on a progressively expanding alveolar barrier with high permeability, generate a high transvascular flow that causes alveolar flooding in minutes. We modeled the trans-endothelial and trans-epithelial Starling pressure gradients under control conditions, as well as under increasing alveolar pressure (Palv) conditions of up to 25 cmH2O. We referred to the wet-to-dry weight (W/D) ratio, a specific index of lung water balance, to be correlated with the functional state of the interstitial structure. W/D averages ∼5 in control and might increase by up to ∼9 in severe edema, corresponding to ∼70% loss in the integrity of the native matrix. Factors buffering edemagenic conditions include: (i) an interstitial capacity for fluid accumulation located in the thick portion of ABB, (ii) the increase in interstitial pressure due to water binding by hyaluronan (the "safety factor" opposing the filtration gradient), and (iii) increased lymphatic flow. Inflammatory factors causing lung tissue damage include those of bacterial/viral and those of sterile nature. Production of reactive oxygen species (ROS) during hypoxia or hyperoxia, or excessive parenchymal stress/strain [lung overdistension caused by patient self-induced lung injury (P-SILI)] can all cause excessive inflammation. We discuss the heterogeneity of intrapulmonary distribution of W/D ratios. A W/D ∼6.5 has been identified as being critical for the transition to severe edema formation. Increasing Palv for W/D > 6.5, both trans-endothelial and trans-epithelial gradients favor filtration leading to alveolar flooding. Neither CT scan nor ultrasound can identify this initial level of lung fluid balance perturbation. A suggestion is put forward to identify a non-invasive tool to detect the earliest stages of perturbation of lung fluid balance before the condition becomes life-threatening.

Multifunctional Liposomes Modulate Purinergic Receptor-Induced Calcium Wave in Cerebral Microvascular Endothelial Cells and Astrocytes: New Insights for Alzheimer's disease.

In light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca2+) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ response to ATP is mainly due to endogenous Ca2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aß-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer's disease (AD).