Developing a pro-angiogenic placenta derived amniochorionic scaffold with two exposed basement membranes as substrates for cultivating endothelial cells

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

Effect of eribulin on angiogenesis and endothelial adhesion molecules.

Tumor vasculature is an important component of the tumor microenvironment and deeply affect anticancer immune response. Eribulin is a non taxane inhibitor of the mitotic spindle. However, off-target effect interfering with the tumor vasculature have been reported. The mechanisms responsible of this effect is not clear. We designed an in vitro study to investigate the effect of eribulin on neo-angiogenesis and on the adhesion molecules ICAM-1 and VCAM-1, with or without TGF-beta. We also investigated the effects of paclitaxel and vinorelbine in the same experimental conditions. Eribulin was able to up-regulate the epithelial markers VE-cadherin and CD-31 in the HUVEC and tube formation in HUVEC cultured in Matrigel. The drug effectively arrested tube formation even in presence of TGF-beta. Eribulin counteracted the TGF-beta induced change in cell shape from the endothelial cobblestone-like morphology to an elongated spindle-shaped morphology that is characteristic of EndMT. We also observed that eribulin is able to upregulate ICAM-1 and to counteract its downregulation induced by TGF-beta. In this study, eribulin was able to inhibit the vasculature remodeling and the downregulation of ICAM-1 induced by TGF-beta. These effects might have important therapeutic consequence if the drug will be administered with immunotherapy.

Study of markers of systemic inflammation (matrix metalloproteinase-9, vascular endothelial adhesion molecules of type 1) in patients with ST -segment elevation myocardial infarction at the hospital and outpatient stages

Aim. To study the level of MMP-9 and VCAM-1 in patients with AMI with ST-segment elevation at the hospital stage and one year after the index event, depending on the development of complications and changes in BMI and waist size (WS). Material and methods. The study included 126 people with STEMI after PCI, as well as 27 people in the control group. The level of MMP-9 and VCAM-1 in peripheral blood was analyzed. In addition to the standard methods of examination and diagnosis, BMI and WS were measured to identify groups with visceral obesity. The assessment of the frequency and nature of complications after STEMI was performed. Results. The levels of biomarkers of vascular endothelial adhesion molecule type 1 and matrix metalloproteinase type 9 are increased in the acute period of STEMI, statistically significantly reduced, but remain elevated 12 months after the index event, with VCAM-1 by 3.5 times, and MMP by almost 2 times compared to the initial values. The level of MMP-9 is significantly higher in excess body mass index and waist size, and VCAM-1 has no association with visceral obesity. Vascular endothelial adhesion molecules of type 1 and matrix metalloproteinases of type 9 are increased in patients with a fatal outcome, as well as with an increase in the severity of OSN and CHF. The level of matrix metalloproteinase has a strong relationship with fatal outcome and repeated MI, and the vascular endothelial adhesion molecule type 1 has a stronger relationship with the severity of CHF. Conclusion. The article studied markers of systemic inflammation (matrix metalloproteinase type 9 and vascular endothelial adhesion molecules type 1) in patients with ST-segment elevation myocardial infarction, depending on the presence of obesity at the hospital and outpatient stages. Simultaneous determination of MMP-9 and VCAM-1 levels can be used to assess the intensity of the inflammatory process and the risk of adverse outcomes.

Manipulating Stress Receptor Signaling to Enhance Immunosuppression and Prolong Survival of Vascularized Composite Tissue

Vascularized composite tissue allotransplantation (VCA) can replace severely damaged body parts but the unavoidable toxicity of high doses of immunosuppressive drugs, such as tacrolimus, required results in significant morbidity. Here we tested whether we could suppress immune activity in a mouse model of VCA by mimicking the natural immune suppression generated by nervous system-induced signaling of adrenergic receptors (AR) by using a safe and well-studied β-AR agonist (terbutaline). Using wild-type and β2-AR-knockout (KO) mice, we found that increased β2-AR signaling results in delayed rejection in VCA recipients, even with subtherapeutic doses of tacrolimus, and this was associated with changes in immune contexture, expression of pro-inflammatory cytokines and chemokines, and function of endothelial adhesion molecules. We propose that β-AR agonists can be used safely to mimic the natural suppression of immune responses generated by adrenergic stress signaling and thereby reduce the dose needed of other more toxic immunosuppressive drugs.

Soluble Mediators Potentially Involved in Pruritus Associated to Cutaneous T-Cell Lymphomas and Mastocytosis: A Cross-Sectional Study

Pruritus is a major distressing symptom, common in inflammatory diseases, like Cutaneous T-Cell Lymphoma (CTCL) and mastocytosis. We aimed to study the involvement of some molecules, namely, cytokines, neuromediators, endothelial adhesion molecules and angiogenic factors, in the severity of pruritus associated to CTCL and mastocytosis. CTCL - Mycosis Fungoides (MF, n=17) and Sézary syndrome (SS, n=10) and mastocytosis patients (n=17) were evaluated. Interleukin (IL)-8, IL-31, Vascular Endothelial Growth Factor (VEGF), E-selectin, serotonin and C-reactive protein (CRP) levels, were assessed; tryptase was measured in mastocytosis. Pruritus severity was assessed, using a Visual Analogue Scale (VAS). Compared to controls (n=29), CTCL patients presented higher CRP and IL-31. SS patients had higher IL-31, E-selectin and CRP than MF patients and controls. Itch correlated with IL- 31 and E-selectin, when considering all CTCL patients; in SS, itch correlated with E-selectin. Advanced CTCL stages revealed higher IL-31, E-selectin and CRP than early stages, and controls; itch intensity correlated with IL-31 and E-selectin, in advanced stages. Mastocytosis showed higher serotonin and VEGF, compared to controls, and itch intensity correlated with tryptase. Data suggest that in mastocytosis, serotonin is an important biomarker and that tryptase levels reflect itch intensity; IL-31 and E-selectin appear to be more important mediators in CTCL and strongly correlated with itch severity. The different involvement of studied mediators, probably due to different immune responses, suggests that different mechanisms underlie these diseases and may lead to different itch mechanisms.

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.