Intrapulmonary airway smooth muscle is hyperreactive with a distinct proteome in asthma.

Constriction of airways during asthmatic exacerbation is the result of airway smooth muscle (ASM) contraction. Although it is generally accepted that ASM is hypercontractile in asthma, this has not been unambiguously demonstrated. Whether airway hyperresponsiveness (AHR) is the result of increased ASM mass alone or also increased contractile force generation per unit of muscle directly determines the potential avenues for treatment.To assess whether ASM is hypercontractile we performed a series of mechanics measurements on isolated ASM from intrapulmonary airways and trachealis from human lungs. We analysed the ASM and whole airway proteomes to verify if proteomic shifts contribute to changes in ASM properties.We report an increase in isolated ASM contractile stress and stiffness specific to asthmatic human intrapulmonary bronchi, the site of increased airway resistance in asthma. Other contractile parameters were not altered. Principal component analysis (PCA) of unbiased mass spectrometry data showed clear clustering of asthmatic subjects with respect to ASM specific proteins. The whole airway proteome showed upregulation of structural proteins. We did not find any evidence for a difference in the regulation of myosin activity in the asthmatic ASM.In conclusion, we showed that ASM is indeed hyperreactive at the level of intrapulmonary airways in asthma. We identified several proteins that are upregulated in asthma that could contribute to hyperreactivity. Our data also suggest enhanced force transmission associated with enrichment of structural proteins in the whole airway. These findings may lead to novel directions for treatment development in asthma.

Characterization of cystic fibrosis airway smooth muscle cell proliferative and contractile activities.

Cystic fibrosis (CF) is a genetic disease that causes multiple airway abnormalities. Two major respiratory consequences of CF are airway hyperresponsiveness (AHR) and airway remodeling. Airway smooth muscle (ASM) is hypothesized to be responsible for the airway dysfunction, since their thickening is involved in remodeling, and excessive contraction by the ASM may cause AHR. It is unclear whether the ASM is intrinsically altered to favor increased contractility or proliferation or if microenvironmental influences induce pathological behavior in vivo. In this study, we examined the contractile and proliferative properties of ASM cells isolated from healthy donor and CF transplant lungs. Assays of proliferation showed that CF ASM proliferates at a higher rate than healthy cells. Through calcium analysis, no differences in contractile activation in response to histamine were found. However, CF ASM cells lagged in their reuptake of calcium in the sarcoplasmic reticulum. The combination CFTR corrector and potentiator, VX-809/770, used to restore CFTR function in CF ASM, resulted in a reduction in proliferation and in a normalization of calcium reuptake kinetics. These results show that impaired CFTR function in ASM cells causes intrinsic changes in their proliferative and contractile properties.

Tolerogenic signaling of alveolar macrophages induces lung adaptation to oxidative injury.

BACKGROUND: Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. OBJECTIVE: We sought to explore the mechanisms of airway adaptation to oxidative injury. METHODS: For a model to study pulmonary adaptation to oxidative stress-induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. RESULTS: Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-ß and prostaglandin E2. CONCLUSION: Pulmonary adaptation during oxidative stress-induced lung injury is mediated by a novel subset of forkhead box P3-positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-ß and prostaglandin E2.

Effect of bronchial thermoplasty on structural changes and inflammatory mediators in the airways of subjects with severe asthma.

BACKGROUND: Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects' symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. METHODS: Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-ß1 (TGF-ß1). RESULTS: The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-ß1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. CONCLUSION: and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

Contractile Properties of Intrapulmonary Airway Smooth Muscle in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Many patients with CF have asthma-like symptoms and airway hyperresponsiveness, which are potentially associated with altered airway smooth muscle (ASM) contractility. Our goal in this study was to assess the contractility of the CF intrapulmonary ASM. ASM strips were dissected from human control and CF intrapulmonary airways, and assessed for methacholine-induced shortening velocity, maximal force, and stress. We also assessed isoproterenol responses in maximally methacholine-contracted ASM. ASM strips were then incubated for 16 hours with IL-13 and measurements were repeated. Myosin light chain kinase (MLCK) expression was assessed by Western blotting. Airways were immunostained for morphometry. ASM mass was increased in CF airways, which likely contributes to airway hyperresponsiveness. Although ASM contractile properties were not intrinsically different between patients with CF and control subjects, CF ASM responded differently in the presence of the inflammatory mediator IL-13, showing impairment in ß-adrenergic-induced relaxation. Indeed, the percentage of relaxation measured at maximal isoproterenol concentrations in the CF ASM was significantly lower after incubation with IL-13 (46.0% ± 6.7% relaxation) than without IL-13 (74.0% ± 7.7% relaxation, P = 0.018). It was also significantly lower than that observed in control ASM incubated with IL-13 (68.8% ± 4.9% relaxation, P = 0.048) and without IL-13 (82.4% ± 9.9%, P = 0.0035). CF ASM incubated with IL-13 also expressed greater levels of MLCK. Thus, our data suggest that the combination of an increase in ASM mass, increased MLCK expression, and inflammation-induced ß-adrenergic hyporesponsiveness may contribute to airway dysfunction in CF.

Alveolar Macrophages in the Resolution of Inflammation, Tissue Repair, and Tolerance to Infection.

Pathogen persistence in the respiratory tract is an important preoccupation, and of particular relevance to infectious diseases such as tuberculosis. The equilibrium between elimination of pathogens and the magnitude of the host response is a sword of Damocles for susceptible patients. The alveolar macrophage is the first sentinel of the respiratory tree and constitutes the dominant immune cell in the steady state. This immune cell is a key player in the balance between defense against pathogens and tolerance toward innocuous stimuli. This review focuses on the role of alveolar macrophages in limiting lung tissue damage from potentially innocuous stimuli and from infections, processes that are relevant to appropriate tolerance of potential causes of lung disease. Notably, the different anti-inflammatory strategies employed by alveolar macrophages and lung tissue damage control are explored. These two properties, in addition to macrophage manipulation by pathogens, are discussed to explain how alveolar macrophages may drive pathogen persistence in the airways.

Maintenance of contractile function of isolated airway smooth muscle after cryopreservation.

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

Modulation of the intrinsic neuronal excitability by multifunctional liposomes tailored for the treatment of Alzheimer's disease.

PURPOSE: Nanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved. MATERIALS AND METHODS: We utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood-brain barrier and to be effective on mouse models of Alzheimer's disease (AD), both in the absence and in the presence of ß-amyloid peptide oligomers. RESULTS: Data show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86 MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3 pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with ß-amyloid peptide oligomers suggest a retention of liposome efficacy. CONCLUSION: These data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability.

Tissue specificity of mitochondrial adaptations in rats after 4 weeks of normobaric hypoxia.

PURPOSE: Exposure to hypoxia has been suggested to activate multiple adaptive pathways so that muscles are better able to maintain cellular energy homeostasis. However, there is limited research regarding the tissue specificity of this response. The aim of this study was to investigate the influence of tissue specificity on mitochondrial adaptations of rat skeletal and heart muscles after 4 weeks of normobaric hypoxia (FiO2: 0.10). METHODS: Twenty male Wistar rats were randomly assigned to either normobaric hypoxia or normoxia. Mitochondrial respiration was determined in permeabilised muscle fibres from left and right ventricles, soleus and extensorum digitorum longus (EDL). Citrate synthase activity and the relative abundance of proteins associated with mitochondrial biogenesis were also analysed. RESULTS: After hypoxia exposure, only the soleus and left ventricle (both predominantly oxidative) presented a greater maximal mass-specific respiration (+48 and +25%, p < 0.05) and mitochondrial-specific respiration (+75 and +28%, p < 0.05). Citrate synthase activity was higher in the EDL (0.63 ± 0.08 vs 0.41 ± 0.10 µmol min- 1 µg- 1) and lower in the soleus (0.65 ± 0.17 vs 0.87 ± 0.20 µmol min- 1 µg- 1) in hypoxia with respect to normoxia. There was a lower relative protein abundance of PGC-1α (-25%, p < 0.05) in the right ventricle and a higher relative protein abundance of PGC-1ß (+43%, p < 0.05) in the left ventricle of rats exposed to hypoxia, with few differences for protein abundance in the other muscles. CONCLUSION: Our results show a muscle-specific response to 4 weeks of normobaric hypoxia. Depending on fibre type, and the presence of ventricular hypertrophy, muscles respond differently to the same degree of environmental hypoxia.

'In Vitro', 'In Vivo' and 'In Silico' Investigation of the Anticancer Effectiveness of Oxygen-Loaded Chitosan-Shelled Nanodroplets as Potential Drug Vector.

PURPOSE: Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects 'per se', using a mathematical model validated on experimental data. METHODS: OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section. In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN. RESULTS: OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results. CONCLUSIONS: Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.

Epithelial Cells Induce a Cyclo-Oxygenase-1-Dependent Endogenous Reduction in Airway Smooth Muscle Contractile Phenotype.

Airway smooth muscle cells (ASMCs) are phenotypically regulated to exist in either a proliferative or a contractile state. However, the influence of other airway structural cell types on ASMC phenotype is largely unknown. Although epithelial cells are known to drive ASM proliferation, their effects on the contractile phenotype are uncertain. In the current study, we tested the hypothesis that epithelial cells reduce the contractile phenotype of ASMCs. To do so, we measured force production by traction microscopy, gene and protein expression, as well as calcium release by Fura-2 ratiometric imaging. ASMCs incubated with epithelial-derived medium produced less force after histamine stimulation. We observed reduced expression of myocardin, α-smooth muscle actin, and calponin within ASMCs after coculture with epithelial cells. Peak calcium release in response to histamine was diminished, and depended on the synthesis of cyclo-oxygenase-1 products by ASM and on prostaglandin E receptors 2 and 4. Together, these in vitro results demonstrate that epithelial cells have the capacity to coordinately reduce ASM contraction by functional antagonism and by reduction of the expression of certain contractile proteins.

Directional preference of airway smooth muscle mass increase in human asthmatic airways.

Airway smooth muscle (ASM) orientation and morphology determine the ability of the muscle to constrict the airway. In asthma, ASM mass is increased, but it is unknown whether ASM orientation and morphology are altered as well or whether the remodeling at the source of the mass increase is ongoing. We dissected human airway trees from asthmatic and control lungs. Stained, intact airway sections were imaged in axial projection to show ASM bundle orientation, whereas cross-sectional histological slides were used to assess ASM area, bundle thickness, and ASM bundle-to-basement membrane distance. We also used these slides to assess cell size, proliferation, and apoptosis. We showed that ASM mass increase in cartilaginous airways is primarily the result of an increase of ASM bundle thickness (as measured radially in an airway cross section) and coincides with an increased distance of the ASM bundles to the airway perimeter. ASM orientation was unchanged in all airways. Apoptosis markers and cell size did not show differences between asthmatics and controls. Our findings show that ASM mass increase likely contributes to the airway-constricting capacity of the muscle. Both the increased bundle thickness and increased thickness of the airway wall inwards of the ASM bundles could further enhance this capacity. Turnover of ASM appears to be the same in airways and biopsies, but the lack of correlation between different markers of proliferation casts doubt on the specificity of markers generally used to assess proliferation.

Fenotipos del asma, ¿son importantes? / Asthma Phenotypes; Do They Matter?

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Inflammation and airway hyperresponsiveness after chlorine exposure are prolonged by Nrf2 deficiency in mice.

RATIONALE: Chlorine gas (Cl2) is a potent oxidant and trigger of irritant induced asthma. We explored NF-E2-related factor 2 (Nrf2)-dependent mechanisms in the asthmatic response to Cl2, using Nrf2-deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. METHODS: Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48h after a 5-min nose-only exposure to 100ppm Cl2 of Nrf2-deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. RESULTS: Cl2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2-deficient mice and wild-type mice at 24h after Cl2 exposure, were significantly greater at 48h post exposure in Nrf2-deficient mice. Lung GSH and mRNA for Nrf2-dependent phase II enzymes (NQO-1 and GPX2) were significantly lower in Nrf2-deficient than wild-type mice after Cl2 exposure. BSO reduced GSH levels and promoted Cl2-induced airway inflammation in wild-type mice, but not in Nrf2-deficient mice, whereas SFN suppressed Cl2-induced airway inflammation in wild-type but not in Nrf2-deficient mice. AHR was not affected by either BSO or SFN at 48h post Cl2 exposure. CONCLUSIONS: Nrf2-dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR.

Acetylation mediates Cx43 reduction caused by electrical stimulation.

Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43.

Dextran-shelled oxygen-loaded nanodroplets reestablish a normoxia-like pro-angiogenic phenotype and behavior in hypoxic human dermal microvascular endothelium.

In chronic wounds, hypoxia seriously undermines tissue repair processes by altering the balances between pro-angiogenic proteolytic enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) released from surrounding cells. Recently, we have shown that in human monocytes hypoxia reduces MMP-9 and increases TIMP-1 without affecting TIMP-2 secretion, whereas in human keratinocytes it reduces MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. Provided that the phenotype of the cellular environment is better understood, chronic wounds might be targeted by new oxygenating compounds such as chitosan- or dextran-shelled and 2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets (OLNs). Here, we investigated the effects of hypoxia and dextran-shelled OLNs on the pro-angiogenic phenotype and behavior of human dermal microvascular endothelium (HMEC-1 cell line), another cell population playing key roles during wound healing. Normoxic HMEC-1 constitutively released MMP-2, TIMP-1 and TIMP-2 proteins, but not MMP-9. Hypoxia enhanced MMP-2 and reduced TIMP-1 secretion, without affecting TIMP-2 levels, and compromised cell ability to migrate and invade the extracellular matrix. When taken up by HMEC-1, nontoxic OLNs abrogated the effects of hypoxia, restoring normoxic MMP/TIMP levels and promoting cell migration, matrix invasion, and formation of microvessels. These effects were specifically dependent on time-sustained oxygen diffusion from OLN core, since they were not achieved by oxygen-free nanodroplets or oxygen-saturated solution. Collectively, these data provide new information on the effects of hypoxia on dermal endothelium and support the hypothesis that OLNs might be used as effective adjuvant tools to promote chronic wound healing processes.

Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing.

BACKGROUND: In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. OBJECTIVE: To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. METHODS: HaCaT cells were treated for 24h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. RESULTS: Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. CONCLUSION: Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds.

Oxygen-Loaded Nanodroplets Effectively Abrogate Hypoxia Dysregulating Effects on Secretion of MMP-9 and TIMP-1 by Human Monocytes.

Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP-) based oxygen-loaded nanodroplets (OLNs). Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation.

2H,3H-decafluoropentane-based nanodroplets: new perspectives for oxygen delivery to hypoxic cutaneous tissues.

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues.