The pro-healing effects of heparan sulfate and growth factors are enhanced by the heparinase enzyme: New association for skin wound healing treatment.

Effective treatment strategies for skin wound repair are the focus of numerous studies. New pharmacological approaches appear necessary to guarantee a correct and healthy tissue regeneration. For these reasons, we purposed to investigate the effects of the combination between heparan sulfate and growth factors further adding the heparinase enzyme. Interestingly, for the first time, we have found that this whole association retains a marked pro-healing activity when topically administered to the wound. In detail, this combination significantly enhances the motility and activation of the main cell populations involved in tissue regeneration (keratinocytes, fibroblasts and endothelial cells), compared with single agents administered without heparinase. Notably, using an experimental C57BL/6 mouse model of skin wounding, we observed that the topical treatment of skin lesions with heparan sulfate + growth factors + heparinase promotes the highest closure of wounds compared to each substance mixed with the other ones in all the possible combinations. Eosin/hematoxylin staining of skin biopsies revealed that treatment with the whole combination allows the formation of a well-structured matrix with numerous new vessels. Confocal analyses for vimentin, FAP1α, CK10 and CD31 have highlighted the presence of activated fibroblasts, differentiated keratinocytes and endothelial cells at the closed region of wounds. Our results encourage defining this combined treatment as a new and appealing therapy expedient in skin wound healing, as it is able to activate cell components and promote a dynamic lesions closure.

The Protecting Activity of RIPACUT®: A New Therapeutic Approach Preserving Epithelial Health Based on the Combination of Iceland Lichen Extract, Silver Salt, and Sodium Hyaluronate.

Epithelial integrity and function must be maintained in a dynamic healthy equilibrium, keeping unaltered the oxidative and inflammatory conditions and the microbiome of the cutaneous layers. Beside the skin, other mucous membranes can be injured, such as the nasal and anal ones, because of the contact with the external environment. Here, we detected the effects of RIPACUT®, a combination of Iceland lichen extract, silver salt and sodium hyaluronate that individually act in diverse biological ways. The findings we obtained on keratinocytes, nasal and intestinal epithelial cells reveal that this combination showed a marked antioxidant activity, further assessed by the DPPH assay. Additionally, by analyzing the release of the IL-1ß, TNF-α and IL-6 cytokines, we proved the anti-inflammatory effect of RIPACUT®. In both cases, the main preserving action was due to Iceland lichen. We also observed a notable antimicrobial activity mediated by the silver compound. These data suggest that RIPACUT® could signify the basis for an attractive pharmacological approach to maintaining healthy epithelial conditions. Interestingly, this may be extended to the nasal and anal areas where it protects against oxidative, inflammatory and infectious insults. Thus, these outcomes encourage the creation of sprays or creams for which sodium hyaluronate can guarantee a surface film-forming effect.

Role of Intracellular and Extracellular Annexin A1 in MIA PaCa-2 Spheroids Formation and Drug Sensitivity.

Among solid tumors, pancreatic cancer (PC) remains a leading cause of death. In PC, the protein ANXA1 has been identified as an oncogenic factor acting in an autocrine/paracrine way, and also as a component of tumor-deriving extracellular vesicles. Here, we proposed the experimental protocol to obtain spheroids from the two cell lines, wild-type (WT) and Annexin A1 (ANXA1) knock-out (KO) MIA PaCa-2, this last previously obtained through CRISPR/Cas9 genome editing system. The use of three-dimensional (3D) models, like spheroids, can be useful to mimic tumor characteristics and for preclinical chemo-sensitivity studies. By using PC spheroids, we have assessed the activity of intracellular and extracellular ANXA1. Indeed, we have proved that the intracellular protein influences in vitro tumor development and growth by spheroids analysis, in addition to defining the modification about cell protein pattern in ANXA1 KO model compared to the WT one. Moreover, we have tested the response to FOLFIRINOX chemotherapy regimen whose cytostatic effect appeared notably increased in ANXA1 KO spheroids. Additionally, this study has highlighted that the extracellular ANXA1 action is strengthened through the EVs supporting spheroids growth and resistance to drug treatment, mainly affecting tumor progression. Thus, our data interestingly suggest the relevance of ANXA1 as a potential therapeutic PC marker.

The combination of mesoglycan and VEGF promotes skin wound repair by enhancing the activation of endothelial cells and fibroblasts and their cross-talk.

Skin wound healing requires accurate therapeutic topical managements to accelerate tissue regeneration. Here, for the first time, we found that the association mesoglycan/VEGF has a strong pro-healing activity. In detail, this combination induces angiogenesis in human endothelial cells promoting in turn fibroblasts recruitment. These ones acquire a notable ability to invade the matrigel coating and to secrete an active form of metalloproteinase 2 in presence of endothelial cells treated with mesoglycan/VEGF. Next, by creating intrascapular lesions on the back of C57Bl6 mice, we observed that the topical treatments with the mesoglycan/VEGF promotes the closure of wounds more than the single substances beside the control represented by a saline solution. As revealed by eosin/hematoxylin staining of mice skin biopsies, treatment with the combination mesoglycan/VEGF allows the formation of a well-structured matrix with a significant number of new vessels. Immunofluorescence analyses have revealed the presence of endothelial cells at the closed region of wounds, as evaluated by CD31, VE-cadherin and fibronectin staining and of activated fibroblasts assessed by vimentin, col1A and FAP1α. These results encourage defining the association mesoglycan/VEGF to activate endothelial and fibroblast cell components in skin wound healing promoting the creation of new vessels and the deposition of granulation tissue.

The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs.

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.

ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis.

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.

The Procoagulant Activity of Emoxilane®: A New Appealing Therapeutic Use in Epistaxis of the Combination of Sodium Hyaluronate, Silver Salt, α-tocopherol and D-panthenol.

Epistaxis is one of the most frequent hemorrhages resulting from local or systemic factors. Its management without hospitalization has prompted an interest in locally applied hemostatic agents. Generally, the therapy approaches involve sprays or creams acting as a physical barrier, even used as tampons or gauze. In this study, we have investigated the activity of Emoxilane®, a combination of sodium hyaluronate, silver salt, α-tocopherol acetate and D-panthenol, which is known to be able to separately act in a different biological manner. Our in vitro results, obtained on endothelial and nasal epithelial cells, have shown that the association of these molecules presented a notable antioxidant activity mainly due to the α-tocopherol and D-panthenol and a significant antimicrobial role thanks to the silver compound. Moreover, remarkable hemostatic activity was found by evaluating plasmin inhibition attributable to the sodium hyaluronate. Interestingly, on human plasma, we have confirmed that Emoxilane® strongly induced the increase of thrombin levels. These data suggest that the use of this association could represent an appealing pharmacological approach to actively induce hemostasis during epistaxis. Our future perspective will aim to the creation of a formulation for an easy topical application in the nose which is able to contrast the bleeding.

The promising pro-healing role of the association of mesoglycan and lactoferrin on skin lesions.

Skin wound repair represents an important topic for the therapeutic challenges. Many molecules are commonly used as active principles of topical devices to induce the correct tissue regeneration. Among these molecules, mesoglycan, a mixture of glycosaminoglycans, and the lactoferrin have recently aroused interest. Here, for the first time, we used mesoglycan/lactoferrin to treat the cell populations mainly involved in wound healing. We showed that human keratinocytes, fibroblasts and endothelial cells migrate and invade more rapidly when treated with the association. Moreover, we found that mesoglycan/lactoferrin, are able to trigger the differentiation process of keratinocytes, the switch of the fibroblasts into myofibroblasts, the acquisition of a mesenchymal phenotype for the endothelial cells which, in this way, start to form the capillary-like structures. Additionally, we proved that the well known antimicrobial behavior of lactoferrin encourages the inhibition of S. aureus and P. aeruginosa biofilm formation by the whole association, providing an appealing feature for this formulation. Finally, by the in vivo analysis, we showed that the mesoglycan/lactoferrin favors the closure of skin wounds performed on the mice back. Beside the decrease of the lesion diameters, by a confocal analysis of mice biopsies we found that the use of the association strongly promote cell activation underlying the correct tissue regeneration. These results encourage to further investigation aiming the development of a new topical patch that includes this association.

Mesoglycan connects Syndecan-4 and VEGFR2 through Annexin A1 and formyl peptide receptors to promote angiogenesis in vitro.

Mesoglycan is a mixture of glycosaminoglycans (GAG) with fibrinolytic effects and the potential to enhance skin wound repair. Here, we have used endothelial cells isolated from wild-type (WT) and Syndecan-4 null (Sdc4-/-) C57BL/6 mice to demonstrate that mesoglycan promotes cell motility and in vitro angiogenesis acting on the co-receptor Syndecan-4 (SDC4). This latter is known to participate in the formation and release of extracellular vesicles (EVs). We characterized EVs released by HUVECs and assessed their effect on angiogenesis. Particularly, we focused on Annexin A1 (ANXA1) containing EVs, since they may contribute to tube formation via interactions with Formyl peptide receptors (FPRs). In our model, the bond ANXA1-FPRs stimulates the release of vascular endothelial growth factor (VEGF-A) that interacts with vascular endothelial receptor-2 (VEGFR2) and activates the pathway enhancing cell motility in an autocrine manner, as shown by wound healing/invasion assays, and the induction of endothelial to mesenchymal transition (EndMT). Thus, we have shown for the first time that mesoglycan exerts its pro-angiogenic effects in the healing process triggering the activation of the three interconnected molecular axis: mesoglycan-SDC4, EVs-ANXA1-FPRs, and VEGF-A-VEGFR2.

Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2.

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there is no information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.

Annexin A1 Released in Extracellular Vesicles by Pancreatic Cancer Cells Activates Components of the Tumor Microenvironment, through Interaction with the Formyl-Peptide Receptors.

Pancreatic cancer (PC) is one of the most aggressive cancers in the world. Several extracellular factors are involved in its development and metastasis to distant organs. In PC, the protein Annexin A1 (ANXA1) appears to be overexpressed and may be identified as an oncogenic factor, also because it is a component in tumor-deriving extracellular vesicles (EVs). Indeed, these microvesicles are known to nourish the tumor microenvironment. Once we evaluated the autocrine role of ANXA1-containing EVs on PC MIA PaCa-2 cells and their pro-angiogenic action, we investigated the ANXA1 paracrine effect on stromal cells like fibroblasts and endothelial ones. Concerning the analysis of fibroblasts, cell migration/invasion, cytoskeleton remodeling, and the different expression of specific protein markers, all features of the cell switching into myofibroblasts, were assessed after administration of wild type more than ANXA1 Knock-Out EVs. Interestingly, we demonstrated a mechanism by which the ANXA1-EVs complex can stimulate the activation of formyl peptide receptors (FPRs), triggering mesenchymal switches and cell motility on both fibroblasts and endothelial cells. Therefore, we highlighted the importance of ANXA1/EVs-FPR axes in PC progression as a vehicle of intercommunication tumor cells-stroma, suggesting a specific potential prognostic/diagnostic role of ANXA1, whether in soluble form or even if EVs are captured in PC.

Heparan sulfate binds the extracellular Annexin A1 and blocks its effects on pancreatic cancer cells.

In pancreatic cancer (PC) progression the protein Annexin A1 (ANXA1) has been described as oncogenic factor. Thus, the need to inhibit its action, mainly the extracellular form, has become an appealing cue for the anti-cancer research. Heparan sulfate (HS) is a glycosaminoglycan of the extracellular matrix known to bind several molecules, as growth factors and cytokines, generating a kind of reservoir in the extracellular environment. Here, we started our study by showing the physical calcium-dependent interaction between HS and ANXA1 as both full-length protein and N-terminal portion, Ac2-26 by biophysical techniques. HS is able to inhibit the migration/invasion process of human PC MIA PaCa-2 cells and partially revert their mesenchymal phenotype as reported through the expression of specific protein markers and the growth in colonies and in 3D-spheroids. Furthermore, both on MIA PaCa-2 and PANC-1 cells, HS blocks the effects of Ac2-26, which enhances the aggressive behavior of PC cells if added alone. These effects appear evident also on endothelial cells whose activation is promoted by Ac2-26 but not in presence of HS. Thus, the interference of the interaction ANXA1-HS on angiogenesis strongly emerges. Moreover, once sequestered by HS, ANXA1 is not more able to bind the formil-peptide receptors (FPRs) preventing the increase of calcium mobilization, peculiar for cell motility. These findings introduce a new important tale in the knowledge about the inhibition of the ANXA1 action in PC development. Further information will be useful to highlight the interaction of HS with the protein, focusing on the characterization of the glycosaminoglycan and on in vivo assays.

Manipulation of the Immune System for Cancer Defeat: A Focus on the T Cell Inhibitory Checkpoint Molecules.

The immune system actively counteracts the tumorigenesis process; a breakout of the immune system function, or its ability to recognize transformed cells, can favor cancer development. Cancer becomes able to escape from immune system control by using multiple mechanisms, which are only in part known at a cellular and molecular level. Among these mechanisms, in the last decade, the role played by the so-called "inhibitory immune checkpoints" is emerging as pivotal in preventing the tumor attack by the immune system. Physiologically, the inhibitory immune checkpoints work to maintain the self-tolerance and attenuate the tissue injury caused by pathogenic infections. Cancer cell exploits such immune-inhibitory molecules to contrast the immune intervention and induce tumor tolerance. Molecular agents that target these checkpoints represent the new frontier for cancer treatment. Despite the heterogeneity and multiplicity of molecular alterations among the tumors, the immune checkpoint targeted therapy has been shown to be helpful in selected and even histologically different types of cancer, and are currently being adopted against an increasing variety of tumors. The most frequently used is the moAb-based immunotherapy that targets the Programmed Cell Death 1 protein (PD-1), the PD-1 Ligand (PD-L1) or the cytotoxic T lymphocyte antigen-4 (CTLA4). However, new therapeutic approaches are currently in development, along with the discovery of new immune checkpoints exploited by the cancer cell. This article aims to review the inhibitory checkpoints, which are known up to now, along with the mechanisms of cancer immunoediting. An outline of the immune checkpoint targeting approaches, also including combined immunotherapies and the existing trials, is also provided. Notwithstanding the great efforts devoted by researchers in the field of biomarkers of response, to date, no validated FDA-approved immunological biomarkers exist for cancer patients. We highlight relevant studies on predictive biomarkers and attempt to discuss the challenges in this field, due to the complex and largely unknown dynamic mechanisms that drive the tumor immune tolerance.