Macrocybin, a Natural Mushroom Triglyceride, Reduces Tumor Growth In Vitro and In Vivo through Caveolin-Mediated Interference with the Actin Cytoskeleton.

Mushrooms have been used for millennia as cancer remedies. Our goal was to screen several mushroom species from the rainforests of Costa Rica, looking for new antitumor molecules. Mushroom extracts were screened using two human cell lines: A549 (lung adenocarcinoma) and NL20 (immortalized normal lung epithelium). Extracts able to kill tumor cells while preserving non-tumor cells were considered "anticancer". The mushroom with better properties was Macrocybe titans. Positive extracts were fractionated further and tested for biological activity on the cell lines. The chemical structure of the active compound was partially elucidated through nuclear magnetic resonance, mass spectrometry, and other ancillary techniques. Chemical analysis showed that the active molecule was a triglyceride containing oleic acid, palmitic acid, and a more complex fatty acid with two double bonds. The synthesis of all possible triglycerides and biological testing identified the natural compound, which was named Macrocybin. A xenograft study showed that Macrocybin significantly reduces A549 tumor growth. In addition, Macrocybin treatment resulted in the upregulation of Caveolin-1 expression and the disassembly of the actin cytoskeleton in tumor cells (but not in normal cells). In conclusion, we have shown that Macrocybin constitutes a new biologically active compound that may be taken into consideration for cancer treatment.

Glu-tubulin is a marker for Schwann cells and can distinguish between schwannomas and neurofibromas.

Schwann cells generate myelin sheaths around the axons of the peripheral nervous system, thus facilitating efficient nerve impulse propagation. Two main tumor types can arise from peripheral nerves, schwannomas and neurofibromas, which are sometimes difficult to distinguish and may require the use of diagnostic biomarkers. Here, we characterize a new marker for Schwann cells and its potential use as a diagnostic marker for schwannomas. Immunohistochemistry for Glu-tubulin, a posttranslational modification of α-tubulin, was performed in mouse and human tissues. This technique labels Schwann cells but not oligodendrocytes. All peripheral nerves were immunoreactive for this antibody, including large nerve trunks, thin myelinated nerves, as well as the myenteric and submucous plexus of the digestive tract. In the mouse brain, many neurons were immunoreactive for Glu-tubulin but oligodendrocytes were negative. During embryo development, immunoreactive nerves were already found at E10. In Schwann cells, the staining is restricted to the myelin sheaths and is not present in the perinuclear cytoplasm or the Ranvier nodes. Primary cultures of fibroblasts and Schwann cells were established from mouse sciatic nerves, and Western blot analysis showed that Glu-tubulin immunoreactivity was found in the Schwann cells but not in the fibroblasts. Clinical specimens of schwannomas (n = 20) and neurofibromas (n = 20) were stained with anti-Glu-tubulin antibodies. Schwannomas presented a strong staining in all tumor cells, whereas neurofibromas had a light speckled staining pattern, easily distinguishable from the one found in schwannomas. In conclusion, Glu-tubulin can be used as a marker of Schwann cells and can help in diagnosing peripheral nerve tumors.

Adrenomedullin and tumour microenvironment.

Adrenomedullin (AM) is a regulatory peptide whose involvement in tumour progression is becoming more relevant with recent studies. AM is produced and secreted by the tumour cells but also by numerous stromal cells including macrophages, mast cells, endothelial cells, and vascular smooth muscle cells. Most cancer patients present high levels of circulating AM and in some cases these higher levels correlate with a worst prognosis. In some cases it has been shown that the high AM levels return to normal following surgical removal of the tumour, thus indicating the tumour as the source of this excessive production of AM. Expression of this peptide is a good investment for the tumour cell since AM acts as an autocrine/paracrine growth factor, prevents apoptosis-mediated cell death, increases tumour cell motility and metastasis, induces angiogenesis, and blocks immunosurveillance by inhibiting the immune system. In addition, AM expression gets rapidly activated by hypoxia through a HIF-1α mediated mechanism, thus characterizing AM as a major survival factor for tumour cells. Accordingly, a number of studies have shown that inhibition of this peptide or its receptors results in a significant reduction in tumour progression. In conclusion, AM is a great target for drug development and new drugs interfering with this system are being developed.

Cold-shock proteins accumulate in centrosomes and their expression and primary cilium morphology are regulated by hypothermia and shear stress.

Primary cilia act as cellular sensors for multiple extracellular stimuli and regulate many intracellular signaling pathways in response. Here we investigate whether the cold-shock proteins (CSPs), CIRP and RBM3, are present in the primary cilia and the physiological consequences of such a relationship. R28, an immortalized retinal precursor cell line, was stained with antibodies against CIRP, RBM3, and ciliary markers. Both CSPs were found in intimate contact with the basal body of the cilium during all stages of the cell cycle, including migrating with the centrosome during mitosis. In addition, the morphological and physiological manifestations of exposing the cells to hypothermia and shear stress were investigated. Exposure to moderately cold (32°C) temperatures, the hypothermia mimetic small molecule zr17-2, or to shear stress resulted in a significant reduction in the number and length of primary cilia. In addition, shear stress induced expression of CIRP and RBM3 in a complex pattern depending on the specific protein, flow intensity, and type of flow (laminar versus oscillatory). Flow-mediated CSP overexpression was detected by qRT-PCR and confirmed by Western blot, at least for CIRP. Furthermore, analysis of public RNA Seq databases on flow experiments confirmed an increase of CIRP and RBM3 expression following exposure to shear stress in renal cell lines. In conclusion, we found that CSPs are integral components of the centrosome and that they participate in cold and shear stress sensing.

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses.

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.

In vivo biocompatibility testing of nanoparticle-functionalized alginate-chitosan scaffolds for tissue engineering applications.

Background: There is a strong interest in designing new scaffolds for their potential application in tissue engineering and regenerative medicine. The incorporation of functionalization molecules can lead to the enhancement of scaffold properties, resulting in variations in scaffold compatibility. Therefore, the efficacy of the therapy could be compromised by the foreign body reaction triggered after implantation. Methods: In this study, the biocompatibilities of three scaffolds made from an alginate-chitosan combination and functionalized with gold nanoparticles (AuNp) and alginate-coated gold nanoparticles (AuNp + Alg) were evaluated in a subcutaneous implantation model in Wistar rats. Scaffolds and surrounding tissue were collected at 4-, 7- and 25-day postimplantation and processed for histological analysis and quantification of the expression of genes involved in angiogenesis, macrophage profile, and proinflammatory (IL-1ß and TNFα) and anti-inflammatory (IL-4 and IL-10) cytokines. Results: Histological analysis showed a characteristic foreign body response that resolved 25 days postimplantation. The intensity of the reaction assessed through capsule thickness was similar among groups. Functionalizing the device with AuNp and AuNp + Alg decreased the expression of markers associated with cell death by apoptosis and polymorphonuclear leukocyte recruitment, suggesting increased compatibility with the host tissue. Similarly, the formation of many foreign body giant cells was prevented. Finally, an increased detection of alpha smooth muscle actin was observed, showing the angiogenic properties of the elaborated scaffolds. Conclusion: Our results show that the proposed scaffolds have improved biocompatibility and exhibit promising potential as biomaterials for elaborating tissue engineering constructs.

Endoglin and Activin Receptor-like Kinase 1 (Alk1) Modify Adrenomedullin Expression in an Organ-Specific Manner in Mice.

Hereditary hemorrhagic telangiectasia (HHT) is a rare disease characterized by vascular malformations and profuse bleeding. The disease is caused by mutations in the components of the BMP-9 receptor: endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1) genes. Recently, we reported that HHT patients expressed higher serum levels of adrenomedullin (AM) than healthy volunteers; thus, we studied the expression of AM (by enzyme immunoassay, qRT-PCR, immunohistochemistry, and Western blotting) in mice deficient in either one of the receptor components to investigate whether these defects may be the cause of that elevated AM in patients. We found that AM expression is not affected by these mutations in a consistent pattern. On the contrary, in some organs (blood, lungs, stomach, pancreas, heart, kidneys, ovaries, brain cortex, hippocampus, foot skin, and microvessels), there were no significant changes, whereas in others we found either a reduced expression (fat, skin, and adrenals) or an enhanced production of AM (cerebellum and colon). These results contradict our initial hypothesis that the increased AM expression found in HHT patients may be due directly to the mutations, but open intriguing questions about the potential phenotypic manifestations of Eng and Acvrl1 mutants that have not yet been studied and that may offer, in the future, a new focus for research on HHT.

Agaricus Mushroom-Enriched Diets Modulate the Microbiota-Gut-Brain Axis and Reduce Brain Oxidative Stress in Mice.

Neurodegenerative diseases pose a major problem for developed countries, and stress has been identified as one of the main risk factors in the development of these disorders. Here, we have examined the protective properties against brain oxidative stress of two diets supplemented with 5% (w/w) of Agaricus bisporus (white button mushroom) or Agaricus bisporus brunnescens (Portobello mushroom) in mice. These diets did not modify the weight gain of the animals when compared to those fed with a regular diet, even after feeding on them for 15 weeks. The long-term modification of the microbiota after 12 weeks on the diets was investigated. At the phylum level, there was a large increase of Verrucomicrobia and a reduction of Cyanobacteria associated with the mushroom diets. No changes were observed in the Firmicutes/Bacteroidetes ratio, whose stability is a marker for a healthy diet. At the family level, three groups presented significant variations. These included Akkermansiaceae and Tannerellaceae, which significantly increased with both diets; and Prevotellaceae, which significantly decreased with both diets. These bacteria participate in the generation of microbiota-derived short-chain fatty acids (SCFAs) and provide a link between the microbiota and the brain. Mice subjected to restraint stress showed an upregulation of Il-6, Nox-2, and Hmox-1 expression; a reduction in the enzymatic activities of catalase and superoxide dismutase; and an increase in lipid peroxidation in their brains. All these parameters were significantly prevented by feeding for 3 weeks on the Agaricus-supplemented diets. In summary, the supplementation of a healthy diet with Agaricus mushrooms may significantly contribute to prevent neurodegenerative diseases in the general population.

Mild hypothermia and vitrification increase the mRNA expression of cold-inducible proteins in bovine oocytes and cumulus cells.

The cold-inducible RNA-binding protein (CIRBP) assists cells in adapting to new environmental conditions stabilizing specific mRNAs and promoting their translation. CIRBP participates in anti-apoptotic and anti-senescence processes, and its expression is induced by mild hypothermia, which may be advantageous to oocytes during vitrification. Several newly discovered small molecules, like zr17-2, mimic the effects of cold temperatures by increasing the expression of CIRBP at normothermia. This study aimed to evaluate the mRNA changes of a group of cold-inducible protein-encoding and apoptotic genes in response to exogenous supplementation of zr17-2 (experiment 1) or CIRBP (experiment 2) in vitro matured bovine oocytes and their cumulus cells. In experiment 1, cumulus-oocyte complexes (COCs) were randomly exposed to three concentrations of zr17-2 (1, 10, and 100 µM) during 24 h of in vitro maturation (IVM) under normothermia (38.5 °C) or mild hypothermia (34 °C) culture conditions. In experiment 2, COCs were randomly exposed to three concentrations of CIRBP (2, 4, and 6 µg/mL) or subjected to mild hypothermia (34 °C), followed by oocyte vitrification/warming after 20 h of IVM. The quantification of the selected gene transcript expression was performed separately in oocytes and cumulus cells by quantitative real-time PCR. We show that oocytes and cumulus cells exhibited similar mRNA expression responses to mild hypothermia and vitrification. However, minor differences were observed when COCs were exposed to exogenous supplementation with zr17-2 and CIRBP. In conclusion, the alterations observed in the mRNA expression in these experimental conditions may impact the quality of the cumulus-oocyte complexes in terms of vitrification and sublethal hypothermia challenges. In this sense, our results should complement other oocyte quality assessments for its application in future assisted reproductive techniques in the bovine species, including improving oocyte cryotolerance to vitrification.

Potent "clicked" MMP2 inhibitors: synthesis, molecular modeling and biological exploration.

A new series of MMP2 inhibitors is described, following a fragment-based drug design approach. One fragment containing an azide group and a well known hydroxamate Zinc Binding Group in a α-sulfone, α-tetrahydropyrane scaffold, has been synthesized. Water-LOGSY, STD and competition-STD experiments indicate that this fragment binds to the active site of the enzyme. A click chemistry reaction was used to connect the azide to lipophilic alkynes selected to interact selectively with the S1' subunit of MMP2, as shown by docking and molecular dynamic experiments of the designed compounds. The most potent compounds 18 and 19 displayed an IC(50) of 1.4 and 0.3 nM against MMP2 respectively, and showed negligible activity towards MMP1 and MMP7, two metalloproteinases which have a shallow S1' subsite. Compound 18 also showed a promising selectivity profile against some antitarget metalloproteinases, such as MMP8, and considerably less activity against MMP14 (IC(50) = 65 nM), and MMP9 (IC(50) = 98 nM), other MMPs characterized by having a deep S1' pocket and, therefore, more similar to MMP2.

Natural Food Supplements Reduce Oxidative Stress in Primary Neurons and in the Mouse Brain, Suggesting Applications in the Prevention of Neurodegenerative Diseases.

Neurodegenerative diseases pose a major health problem for developed countries, and stress has been identified as one of the main risk factors in the development of these disorders. Here, we have examined the protective properties against oxidative stress of several bioactive natural food supplements. We found that MecobalActive®, Olews®, and red and white grape seed polyphenol extracts may have a neuroprotective effect in vitro, both in the SH-SY 5Y cell line and in hippocampal neuron cultures, mainly by reducing reactive oxygen species levels and decreasing caspase-3 activity. In vivo, we demonstrated that oral administration of the supplements reduces the expression of genes involved in inflammation and oxidation mechanisms, whereas it increments the expression of genes related to protection against oxidative stress. Furthermore, we found that preventive treatment with these natural extracts increases the activity of antioxidant enzymes and prevents lipid peroxidation in the brain of stressed mice. Thus, our results indicate that some natural bioactive supplements may have important protective properties against oxidative stress processes occurring in the brain.

A Grape Juice Supplemented with Natural Grape Extracts Is Well Accepted by Consumers and Reduces Brain Oxidative Stress.

Neurodegenerative diseases pose a major health problem for developed countries. Stress, which induces oxidation in the brain, has been identified as the main risk factor for these disorders. We have developed an antioxidant-enriched drink and have examined its protective properties against acute oxidative stress. We found that addition of red grape polyphenols and MecobalActive® to grape juice did not provoke changes in juice organoleptic characteristics, and that the pasteurization process did not greatly affect the levels of flavonoids and vitamin B12. Out of all combinations, grape juice with red grape polyphenols was selected by expert judges (28.6% selected it as their first choice). In vivo, oral administration of grape juice supplemented with red grape polyphenols exerted an antioxidant effect in the brain of stressed mice reducing two-fold the expression of genes involved in inflammation and oxidation mechanisms and increasing three-fold the expression of genes related to protection against oxidative stress. In addition, we found that this drink augmented antioxidant enzyme activity (17.8 vs. 8.2 nmol/mg), and prevented lipid peroxidation in the brain (49.7 vs. 96.5 nmol/mg). Therefore, we propose supporting the use of this drink by the general population as a new and global strategy for the prevention of neurodegeneration.

Circulating Levels of Calcitonin Gene-Related Peptide Are Lower in COVID-19 Patients.

BACKGROUND: To better understand the biology of COVID-19, we have explored the behavior of calcitonin gene-related peptide (CGRP), an angiogenic, vasodilating, and immune modulating peptide, in severe acute respiratory syndrome coronavirus 2 positive patients. METHODS: Levels of CGRP in the serum of 57 COVID-19 patients (24 asymptomatic, 23 hospitalized in the general ward, and 10 admitted to the intensive care unit) and healthy donors (n = 24) were measured by enzyme-linked immunosorbent assay (ELISA). In addition, to better understand the physiological consequences of the observed variations, we investigated by immunofluorescence the distribution of receptor activity modifying protein 1 (RAMP1), one of the components of the CGRP receptor, in autopsy lung specimens. RESULTS: CGRP levels were greatly decreased in COVID-19 patients (P < 0.001) when compared to controls, and there were no significant differences due to disease severity, sex, age, or comorbidities. We found that COVID-19 patients treated with proton pump inhibitors had lower levels of CGRP than other patients not taking this treatment (P = 0.001). RAMP1 immunoreactivity was found in smooth muscle cells of large blood vessels and the bronchial tree and in the airways´ epithelium. In COVID-19 samples, RAMP1 was also found in proliferating type II pneumocytes, a common finding in these patients. CONCLUSIONS: The lower levels of CGRP should negatively impact the respiratory physiology of COVID-19 patients due to vasoconstriction, improper angiogenesis, less epithelial repair, and faulty immune response. Therefore, restoring CGRP levels in these patients may represent a novel therapeutic approach for COVID-19.

Bioorthogonal Self-Immolative Linker Based on Grob Fragmentation.

A self-immolative bioorthogonal conditionally cleavable linker based on Grob fragmentation is described. It is derived from 1,3-aminocyclohexanols and allows the release of sulfonate-containing compounds in aqueous media. Modulation of the amine pKa promotes fragmentation even at slightly acidic pH, a common feature of several tumor environments. The Grob fragmentation can also occur under physiological conditions in living cells, highlighting the potential bioorthogonal applicability of this reaction.

Hypothermic Shock Applied After Perinatal Asphyxia Prevents Retinal Damage in Rats.

Perinatal asphyxia (PA) can cause retinopathy and different degrees of visual loss, including total blindness. In a rat model of PA, we have previously shown a protective effect of hypothermia on the retina when applied simultaneously with the hypoxic insult. In the present work, we evaluated the possible protective effect of hypothermia on the retina of PA rats when applied immediately after delivery. Four experimental groups were studied: Rats born naturally as controls (CTL), animals that were exposed to PA for 20 min at 37°C (PA), animals exposed to PA for 20 min at 15°C (HYP), and animals that were exposed to PA for 20 min at 37°C and, immediately after birth, kept for 15 min at 8°C (HYP-PA). To evaluate the integrity of the visual pathway, animals were subjected to electroretinography at 45 days of age. Molecular (real time PCR) and histological (immunohistochemistry, immunofluorescence, TUNEL assay) techniques were applied to the eyes of all experimental groups collected at 6, 12, 24, and 48 h, and 6 days after birth. PA resulted in a significant reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram. All animals treated with hypothermia had a significant correction of the a-wave and OP, but the b-wave was fully corrected in the HYP group but only partially in the HYP-PA group. The number of TUNEL-positive cells increased sharply in the ganglion cell layer of the PA animals and this increase was significantly prevented by both hypothermia treatments. Expression of the cold-shock proteins, cold-inducible RNA binding protein (CIRP) and RNA binding motif protein 3 (RBM3), was undetectable in retinas of the CTL and PA groups, but they were highly expressed in ganglion neurons and cells of the inner nuclear layer of the HYP and HYP-PA groups. In conclusion, our results suggest that a post-partum hypothermic shock could represent a useful and affordable method to prevent asphyxia-related vision disabling sequelae.

Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer.

To investigate the contribution of adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we studied the effects of four small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. For each SM, four experimental groups of male mice were used: (i) Control group; (ii) SM group; (iii) DSS group (injected with azoxymethane [AOM] and drank dextran sulfate sodium [DSS]); and (iv) DSS + SM group (treated with AOM, DSS, and the SM). None of the mice in groups i and ii developed tumors, whereas all mice in groups iii and iv developed colon neoplasias. No significant differences were found among mice treated with PAMP modulators (87877 and 106221). Mice that received the AM negative modulator, 16311, had worse colitis symptoms than their control counterparts, whereas mice injected with the AM positive modulator, 145425, had a lower number of tumors than their controls. SM 145425 regulated the expression of proliferation marker Lgr5 and had an impact on microbiota, preventing the DSS-elicited increase of the Bacteroides/Prevotella ratio. These results suggest that treatment with AM or with positive modulator SMs may represent a novel strategy for colon cancer.

Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays.

BACKGROUND: Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB) macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia. RESULTS: RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4%) from 12 farms, including 9 farms (14 samples) located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species. CONCLUSION: This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial tests like RLB that clearly identify the parasite and can detect subclinical infections are essential to establish good control measures.

Adrenomedullin regulates club cell recovery following lung epithelial injury.

The equilibrium between lung epithelium damage and recovery in the context of chronic injury is at the basis of numerous lung diseases, including lung cancer and COPD. Understanding the contribution of growth factors and other molecular intermediates to this crosstalk may help in devising new therapeutic approaches. To better understand the contribution of adrenomedullin (AM) to lung homeostasis, we built club cell-specific conditional knockout (KO) mice for AM and subjected them to naphthalene injury. Untreated KO mice had lower levels of club cell 10 KDa protein (CC10) immunoreactivity than their wild type (WT) littermates in both terminal and regular bronchioles. Naphthalene injury resulted in a rapid necrosis of club cells followed by a progressive recovery of the epithelium. Club cells proliferated at higher rates in the KO mice and at 21 days post-injury the club cell coverage of the main bronchioles was higher and more homogeneous than in the WT animals. In conclusion, the paracrine/autocrine influence of AM in club cells subtly modulates their proliferation and spreading kinetics during lung epithelium recovery.

Bioactive bilayered dressing for compromised epidermal tissue regeneration with sequential activity of complementary agents.

The article deals with the design, preparation, and evaluation of a new bilayered dressing for application in the healing of compromised wounds. The system is based on the sequential release of two complementary bioactive components to enhance the activation of the regeneration of dermal tissue. The internal layer is a highly hydrophilic and biodegradable film of gelatin and hyaluronic acid (HG), crosslinked with the natural compound genipin, which reacts with the amine groups of gelatin. This film is loaded with the proangiogenic, anti-inflammatory, and antibacterial peptide, proadrenomedullin N-terminal 20 peptide (PAMP), that is released slowly in the wound site. The external layer, more stable and less hydrophilic, is constituted by a biodegradable polyurethane derived from poly(caprolactone) and pluronic L61. This layer is loaded with resorbable nanoparticles of bemiparin (a fractionated low molecular weight heparin), which promotes the activation of growth factors, FGF and VEGF, and provides a good biomechanical stability and controlled permeability of the bilayered dressing. Experiments carried out in mice demonstrate the excellent angiogenic effect of the HG film in the dermal tissue. Application of the bilayered dressing in the wound healing rabbit ear model shows an improved cicatrization of the wound in both ischemic and non-ischemic defects, favoring epithelialization and reducing noticeably the contraction and the inflammation.