Interleukin-17 and -22 synergy linking inflammation and EMT-dependent fibrosis in Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory, autoimmune and systemic disorder commonly associated with dry eyes and a dry mouth. Recently, the hypothetical link between epithelial-mesenchymal transition (EMT)-dependent salivary gland (SG) fibrosis and chronic inflammatory conditions has been suggested. In this study, we present data demonstrating a negative correlation of the epithelial marker E-cadherin expression and a positive correlation of mesenchymal vimentin and collagen type I expression with increasing degrees of tissue inflammation in pSS SG specimens. In addition, as it is not clear whether dysregulated cytokines in pSS, interleukin (IL)-17 and IL-22 may also contribute to the EMT-dependent fibrosis process, the effect of IL-17 and IL-22 treatment on EMT-dependent SG fibrosis was evaluated in primary human salivary gland epithelial cells (SGEC) isolated from healthy subjects. Here we present data demonstrating that IL-17 and IL-22 can induce SGEC to undergo a morphological and phenotypical transition to a mesenchymal phenotype. In support of this, vimentin and collagen type I were up-regulated while a decreased expression of E-cadherin occurs after interleukin treatment, and co-operation between IL-17 and Il-22 was required to induce the EMT.

Sjögren's syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB.

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren's syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

Osteopontin is expressed and functional in human eosinophils.

BACKGROUND: Eosinophils are critically involved in allergic inflammation and tissue remodeling. Osteopontin (OPN) is a glycoprotein molecule which exhibits pro-fibrogenic and pro-angiogenic properties and has recently also been implicated in allergic diseases. In this study, we investigated the expression and function of OPN in human eosinophils. METHODS: Osteopontin mRNA (RT-PCR) and protein (immunofluorescence) expression in peripheral blood eosinophils from atopic human subjects were evaluated. Soluble OPN release was determined in resting and activated eosinophils. The contribution of OPN to eosinophil-induced angiogenesis was determined using the chick embryo chorio- allantoic membrane (CAM) assay and OPN-induced eosinophil chemotaxis was determined (ChemoTx System microplate wells). Finally, OPN expression in bronchoalveolar lavage (BAL) fluids from mild asthmatic and normal control subjects was determined. RESULTS: Osteopontin is expressed in human eosinophils and is increased following GM-CSF and IL-5 activation. Eosinophil-derived OPN contributes to eosinophil-induced angiogenesis. Recombinant OPN promotes eosinophil chemotaxis in vitro and this effect is mediated by alpha(4)beta(1) integrin binding. Soluble OPN is increased in the bronchoalveolar lavage fluid from mild asthmatic subjects and correlates with eosinophil counts. CONCLUSIONS: We therefore conclude that OPN is likely to contribute to the process of angiogenesis observed in the airways in asthma.

Mast cells and basophils: a potential link in promoting angiogenesis during allergic inflammation.

Mast cells and basophils are granulated metachromatic cells which possess complex and partially overlapping roles in acquired and innate immunity, including both effector and regulatory activities. Mast cells and basophils cooperate in exacerbating and/or modulating inflammation as well as in mediating subsequent tissue repair. Mast cells release a series of potent proangiogenic molecules during inflammation that stimulate vessel sprouting and new vessel formation. Recent data suggest that basophils may also play a role in inflammation-related angiogenesis, principally but not exclusively through the expression of several forms of vascular endothelial growth factors and their receptors. This review focuses on the potential cooperative link between mast cells and basophils in promoting angiogenesis during allergic inflammation. We discuss the multifaceted roles of mast cells and basophils in inflammatory mechanisms of allergic diseases and whether these cells can be both source and target of proangiogenic mediators.

VEGF concentration from plasma-activated platelets rich correlates with microvascular density and grading in canine mast cell tumour spontaneous model.

Canine cutaneous mast cell tumour (CMCT) is a common cutaneous tumour in dog, with a higher incidence than in human. CMCT is classified in three subgroups, well and intermediately differentiated (G1 and G2), corresponding to a benign disease, and poorly differentiated (G3), corresponding to a malignant disease, which metastasize to lymph nodes, liver, spleen and bone marrow. In this study, we have evaluated serum (S), platelet-poor plasma (P-PP), plasma-activated platelet rich (P-APR) and cytosol vascular endothelial growth factor (VEGF) concentrations, microvascular density (MVD) and mast cell density (MCD) in a series of 86 CMCTs and we have correlated these parameters with each other, by means of ELISA detection of VEGF and immunohistochemistry. Results show that VEGF level from cytosol P-APR and MVD were significantly higher in G3 CMCTs as compared to G1 or G2 subgroups. Moreover, a significantly strong correlation among VEGF levels from P-PAR and cytosol, MVD and MCD was found in G3 subgroup. Because VEGF levels from P-APR well correlated with MVD and malignancy grade in CMCT, we suggest that VEGF might be secreted from MCs and it may be a suitable surrogate inter-species angiogenetic markers of tumour progression in CMCT. Finally, CMCT seems to be a useful model to study the role of MCs in tumour angiogenesis and inhibition of MCs degranulation or activation might be a new anti-angiogenic strategy worthy to further investigations.

Anti-angiogenic activity of carebastine: a plausible mechanism affecting airway remodelling.

Ebastine is a well-known selective second-generation histamine H(1) receptor antagonist, which is used for seasonal and perennial allergic rhinitis and chronic urticaria. Angiogenesis plays a crucial role in the development of airway inflammation and remodelling in allergic rhinitis and asthmatic patients, in whom, indeed, the mucosa displays increased vascularity and overexpression of vascular endothelial growth factor (VEGF). The aim of the present study was to evaluate the anti-angiogenic properties of carebastine, the active metabolite of ebastine. The effects of carebastine on human umbilical vein endothelial cell (EC) (HUVEC) and human pulmonary artery EC (HPAEC) proliferation, migration and capillary-like tube formation were investigated in vitro, and in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Moreover, the effect of carebastine on phosphorylation of the cell VEGF receptor fetal liver kinase-1, or VEGF receptor 2 (VEGFR-2), and Akt kinase (Akt) was evaluated by Western blotting. Carebastine inhibited VEGF-induced HUVEC and HPAEC proliferation, migration and angiogenesis in a dose-dependent manner in vitro. Cell proliferation was inhibited by 42 and 64% in HUVECs and 62 and 75% in HPAECs upon exposure for 48 and 72 h, respectively, to 20 microM carebastine (p < or = 0.03), and even more with 30 microM carebastine. Cell migration was inhibited by 37 and 70% in HUVECs (p < or = 0.03) and 60 and 78% in HPAECs (p < or = 0.01) in the presence of 10 and 30 microM carebastine, respectively. Carebastine (20 microM) caused a significant reduction (70-86%; p<0.01) in topological parameters of the capillary network produced in vitro by both EC lines on a basement membrane extract. Carebastine (30 and 50 microM) inhibited the VEGF-induced angiogenesis in the CAM assay in vivo two- and three-fold, respectively (p<0.001). Finally, both EC lines, on exposure to 10 and 20 microM carebastine, showed a four- to six-fold reduction (p < or = 0.01) in both VEGF- and H1 receptor-induced VEGFR-2 and Akt phosphorylation. Overall, these data provide the first evidence regarding the anti-angiogenic activity of ebastine, and suggest its potential use as an anti-angiogenic molecule, besides its antihistaminic activity for the treatment of allergic diseases in which angiogenesis takes place.

Angiogenesis in asthma.

Asthma is a chronic inflammatory disease of the airways characterized by infiltration and activation of inflammatory cells and by structural changes, including subepithelial fibrosis, smooth muscle cells hypertrophy/hyperplasia, epithelial cell metaplasia and angiogenesis. These structural changes are thought to correlate with asthma severity and to account for the development of progressive lung function deterioration. The mechanism underlying airway angiogenesis in asthma and its precise clinical relevance have not yet been completely elucidated. This review provides recent data showing the contribution of allergic inflammation in increased airway vascularity and potential therapeutical approaches in asthma treatment by acting on bronchial microvascular changes.

The role of eosinophil major basic protein in angiogenesis.

BACKGROUND: Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. METHODS: Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. RESULTS: Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly-L-arginine does not induce any angiogenic effects. CONCLUSIONS: These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.

Transferrin promotes endothelial cell migration and invasion: implication in cartilage neovascularization.

During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50-70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin. The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium. Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.

Enhancement of fibroblast proliferation, collagen biosynthesis and production of growth factors as a result of combining sodium hyaluronate and aminoacids.

Fibroblasts play a key role in tissue healing by producing the majority of extracellular matrix components, favouring granulation tissue formation, and stimulating re-epithelialization. Hyaluronan is a component of ECM and its anti-inflammatory effects and properties in enhancing wound closure are well known. In this study, we examined the effects of Aminogam gel, a new pharmacological preparation suggested to improve wound healing, composed of hyaluronic acid, proline, lysine, glycine and leucine, on human fibroblasts. Results show that fibroblasts treated with hyaluronic acid plus aminoacid solution increased their proliferative activity, collagen I and III, and fibronectin synthesis. Moreover, HA plus aminoacid solution increased the expression of transforming growth factor beta, connective tissue growth factor, interleukin-6 and -8, assayed by RT-PCR. These results suggested that Aminogam gel, involved in several stages of wound healing, as fibroblast proliferation, granulation tissue formation, ECM component deposition, and production of cytokines, may be a useful device to favour and accelerate wound closure.

VEGF and VEGFRs expression in oral squamous cell carcinoma.

Oral cancer is an important cause of worldwide morbidity and mortality, with substantial economic, physiological, and psychosocial impacts due to its treatment modality and a great risk for recurrences and second primary oral squamous cell carcinomas (OSCC) development. Therefore, it is very important to understand the underlying cell biology of such tumors. It is now a well-accepted fact that angiogenesis is essential for the growth and metastasis of solid tumors, including head and neck squamous cell carcinoma. The main factor responsible for angiogenesis is VEGF and its receptors. It has been demonstrated that VEGFRs are also present on tumor cells themselves and other cells from the tumor microenvironment, in addition to tumoral endothelial cells (ECs). Therefore between these cells take place numerous and different interactions mediated via paracrine/autocrine pathways that promote angiogenesis, uncontrolled tumor proliferation and metastasation. In consequence, estimation of VEGF expression and its receptors became a reliable prognostic tool in OSCCS, predicting the poor disease-free survival, poor overall survival, and metastatic disease. Understanding the distribution and role of VEGF and its receptors in the progression of OSCC will be essential to the development and design of new therapeutic strategies.

Treatment of finger degloving injury with acellular dermal matrices: Functional and aesthetic results.

INTRODUCTION: Finger degloving injuries (FDIs) represent a challenge in hand surgery. When replantation is not possible, several techniques including loco-regional flaps, pocket abdominal flaps and free flaps have been described as methods to provide skin cover and avoid finger shortening. The aim of this study is to present our experience with acellular dermal matrices (ADMs) in the treatment of FDI. MATERIALS AND METHODS: We retrospectively reviewed the charts of 18 patients who presented with FDI and were treated with ADM between December 2015 and July 2017. Surgical outcomes including complications were analysed, and patient-centred assessments were performed at 12 months of follow-up. RESULTS: The follow-up period ranged from 10 to 20 months. All patients showed good integration and vascularisation of the ADM. All the fingers covered with ADM were firm and soft, with a slim and satisfactory appearance at a mean follow-up of 12 months. No limitations in tendon sliding were observed at dynamic sonography one year after surgery. CONCLUSION: ADMs could be regarded as a viable option when dealing with FDIs, if replantation is not possible and finger length is to be preserved. On the basis of these results, the surgical treatment of FDI with ADM is a viable option that produces good functional outcomes and cosmetic appearance.

Clodronate inhibits angiogenesis in vitro and in vivo.

The effects of amino-bisphosphonate clodronate on endothelial cell functions involved in angiogenesis, namely proliferation and morphogenesis on matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo. This was performed by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, clodronate inhibited the endothelial cell proliferation in a dose-dependent fashion, peaking at 30 microM. At the same concentration, clodronate inhibited the fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on matrigel. In vivo, when tested with the CAM assay, clodronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that antiangiogenesis by clodronate can be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.

Angiogenesis in vasculitides.

Vasculitides, including Wegener's granulomatosis, Takayasu's arteritis, giant cell arteritis, Kawasaki disease, Behçet disease, thromboangiitis obliterans and erythema elevatum diutinum, are inflammatory diseases of blood vessel wall characterized by myointimal proliferation, fibrosis and thrombus formation leading to stenosis or occlusion of the vascular lumen, and finally to tissue ischemia. In these diseases the hypoxic environment subsequent to stenosis or occlusion of the vascular lumen is a potent signal for the generation of new blood vessels. Angiogenesis may be a compensatory response to ischemia and to the increased metabolic activity and may be also a further inflammatory stimulus because endothelial cells of newly-formed vessels express adhesion molecules and produce colony-stimulating factors and chemokines for leukocytes.

The history of the angiogenic switch concept.

Spontaneously arising tumor cells are not usually angiogenic at first. The phenotypic switch to angiogenesis is usually accomplished by a substet that induces new capillaries that then converge toward the tumor. The switch clearly involves more than simple upregulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators. Tumor growth is although to require disruption of this balance and hence this switch must turned on for cancer progression. Progenitor endothelial cells, the crosstalk between angiogenic factors and their receptors and the interaction between vasculogenesis and lymphangiogenesis are all factors that may contribute to the switch. Its promotion is also the outcome of genetic instability resulting in the emergence of tumor cell lines. This review describes the history of the angiogenic switch illustrated in the literature and with particular reference to the three transgenic mouse models, namely RIP1-TAG2, keratin-14 (K14) (human papilloma virus) HPV16 and papilloma virus, used for stage-specific assessment of the effects of antiangiogenic and antitumorigenic agents.

Accelerated wound healing of oral soft tissues and angiogenic effect induced by a pool of aminoacids combined to sodium hyaluronate (AMINOGAM).

In this study we investigated the property of a new medical substance, in the form of a gel compound containing four aminoacids (glycine, leucine, proline, lysine) and sodium hyaluronate (AMINOGAM), to accelerate the wound healing process of the soft oral tissues and to promote angiogenesis in vivo in the vascular proliferation in chick embryo chorioallantoic membrane (CAM) assay. Furthermore, we investigated the capacity of AMINOGAM to induce the expression of an angiogenic cytokine, namely vascular endothelial growth factor (VEGF) in human fibroblasts in vitro. Results showed that AMINOGAM promoted wound healing in post-surgical wounds (after teeth extraction, oral laser surgery with secondary healing without direct suture of the surgical wound, and after dental implant insertion). Stimulated angiogenesis in vivo in the CAM assay and the response was similar to that obtained with vascular endothelial growth factor, a well-known angiogenic cytokine, tested in the same assay, and confirmed by clinical outcomes, which showed reduction of the healing time of oral soft tissues after three different kinds of surgery and also the absence of post-operative infections.

Podoplanin expression in advanced-stage gastric carcinoma and prognostic value of lymphatic microvessel density.

In gastric cancer, lymph node metastasis is a major prognostic factor. Tumor lymphangiogenesis promotes metastasis in experimental models, but in human tumors data about the presence and clinical significance of lymphatic vessels in the tumor area are controversial. We investigated 70 patients with advanced-stage gastric carcinoma and the pathological examination showed 40 cases with intestinal subtype and 30 cases with diffuse subtype. Forty three from 70 cases had regional lymph node metastasis. Additional slides were stained with an antibody against podoplanin, and lymphatic microvessel density (LMVD) was evaluated in the tumoral and peritumoral areas. Lymphatic vessels were identified in tumor area in all cases and LMVD was higher in the peritumoral than in the tumor area. Podoplanin-positive vessels in tumor area were usually small, with narrow lumen. A significant correlation was found between LMVD and stage of the tumor (p<0.002) and lymph node metastasis (p<0.031), but not with the pathological subtype and grade of the tumor. We found tumor cells in the lumen of lymphatic vessels in 11 cases, whereas tumor cells expressing podoplanin were found in 4 cases of less differentiated diffuse subtype gastric carcinoma. In conclusion, our results suggest that LMVD predicts tumor stage and lymph node metastasis, and podoplanin-positive tumor cells select a subgroup of tumors with high potential of invasion and metastasis.

The chick embryo chorioallantoic membrane in the study of tumor angiogenesis.

The chick embryo chorioallantoic membrane (CAM) is commonly used as an experimental in vivo assay to study both angiogenesis and antiangiogenesis in response to tissues, cells or soluble factors. This article summarizes literature data about the use of the CAM in the study of tumor angiogenesis and particularly our experimental data concerning the study of angiogenesis in multiple myeloma and in neuroblastoma.

Hydrogels for biomedical applications from glycol chitosan and PEG diglycidyl ether exhibit pro-angiogenic and antibacterial activity.

We aimed at producing a hydrogel from a chitosan (CS) derivative soluble in physiological conditions to avoid any purification step thus allowing to use the materials also as an in-situ forming material. So, we crosslinked glycol chitosan (GCS) with poly(ethylene glycol) diglycidyl ether (PEGDE) in water at 37 °C. The scaffolds, referred as GCS-PEG, were specifically designed to be used as wound dressing materials as such (after crosslinking) or as in-situ forming materials. Different amounts of PEGDE were tested. The obtained scaffolds showed macroscopic pores and a tailorable swelling in water by controlling the crosslinking degree. Moreover, GCS-PEG scaffolds displayed a significant antimicrobial activity against Staphylococcus aureus. In-vivo study using the chick embryo choriallantoic membrane resulted in a highly pronounced pro-angiogenic activity suggesting important tissue regeneration properties. Moreover, the employed materials are commercially available, no organic solvents are required and the scaling up is quite predictable.

Importance of the bone marrow microenvironment in inducing the angiogenic response in multiple myeloma.

Tumor microenvironment is essential for tumor cell proliferation, angiogenesis, invasion and metastasis through its provision of survival signals, secretion of growth and pro-angiogenic factors, and direct adhesion molecule interactions. This review examines its importance in the induction of an angiogenic response in multiple myeloma (MM). The encouraging results of preclinical and clinical trials in which MM has been treated by targeting the tumor microenvironment are also discussed.