Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis.

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Macroscopic and microscopic fluorescence spectroscopy of colorectal benign and malignant lesions - diagnostically important features.

Fluorescence spectroscopy is a sensitive, fast and non-invasive tool for a diagnostics of cancerous gastrointestinal lesions. It could be applied for in situ detection of tumours during primary endoscopic observations or as add-on measurement modality during microscopic observations of tissue histology slides for their initial or retrospective diagnosis. Therefore, we are looking for diagnostically important features of normal and cancerous tissue areas in a broad spectral range for gastrointestinal tissues ex vivo using two steady-state macroscopic fluorescent spectroscopic modalities and by confocal fluorescent microscopic detection. Results obtained from autofluorescence spectroscopy of benign and malignant lower part gastrointestinal tract (GIT) lesions from freshly excised tissues during surgical removal of the lesions in 18 patients (22 lesions), were compared with the spectral measurements obtained during confocal fluorescent microscopy observations of unstained tissue slides using 405 nm excitation. Excitation-emission matrices (EEMs) were used for ex vivo measurements with applied excitation in 280-440 nm spectral region and emission observed between 300 and 700 nm. Synchronous fluorescence spectroscopy (SFS) approach was also applied to improve the spectral resolution of the observed complex emission spectra. Specific fluorescent features observed, related to presence of structural proteins, co-enzymes and endogenous porphyrins in the tissues investigated, allow discriminating normal mucosa from benign polyps and malignant carcinoma lesions with diagnostic accuracy up to 94.4%.

Proanthocyanidin may improve the shear bond strength at the composites/dentine interface.

TThis study aimed at assessing whether proanthocyanidin, a collagen cross-linker, affects the adhesion strength of resin composites on the dentine surface. Freshly extracted, caries-free, human molars (N=55) were embedded in transparent resin and bisected. The halves were then assigned to either a treated or a non-treated group, where the treatment consisted of a 10 min incubation in a 6.5% proanthocyanidin solution in PBS. A resin composite cylinder was polymerized perpendicularly to the dentinal surfaces and shear tests were made, using an Instron-like machine. The fracture surfaces were characterized by optical (Picro-Sirius Red stain) and electron microscopy (FESEM EDX analysis). Mean bond strength values were 10.73 MPa (SD 3.70) for the treated group and 8.69 MPa (SD 3.20) for the non-treated group (p less than 0.05 Student’s t-test). No constant fracture patterns could be found within the two groups. Proanthocyanidin treatment may improve the adhesion properties of the dentine-bonding interface.

Cytokine, Chemokine, and Growth Factor Profile Characterization of Undifferentiated and Osteoinduced Human Adipose-Derived Stem Cells.

Bone is the second most manipulated tissue after blood. Adipose-derived stem cells (ASCs) may become a convenient source of MSC for bone regenerative protocols. Surprisingly, little is known about the most significant biomolecules these cells produce and release after being osteoinduced. Therefore, the present study aimed at dosing 13 candidates chosen among the most representative cytokines, chemokines, and growth factors within the conditioned media of osteodifferentiated and undifferentiated ASCs. Two acknowledged osteoblastic cell models, that is, MG-63 and SaOs-2 cells, were compared. Notably, IL-6, IL-8, MCP-1, and VEGF were highly produced and detectable in ASCs. In addition, while IL-6 and IL-8 seemed to be significantly induced by the osteogenic medium, no such effect was seen for MCP-1 and VEGF. Overall SaOS-2 had a poor expression profile, which may be consistent with the more differentiated phenotype of SaOs-2 compared to ASCs and MG-63. Instead, in maintaining medium, MG-63 displayed a very rich production of IL-12, MCP-1, IP-10, and VEGF, which were significantly reduced in osteogenic conditions, with the only exception of MCP-1. The high expression of MCP-1 and VEGF, even after the osteogenic commitment, may support the usage of ASCs in bone regenerative protocols by recruiting both osteoblasts and osteoclasts of the host.

Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 µM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1-10 µM result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium.

Cytokine, chemokine, and growth factor profile of platelet-rich plasma.

During wound healing, biologically active molecules are released from platelets. The rationale of using platelet-rich plasma (PRP) relies on the concentration of bioactive molecules and subsequent delivery to healing sites. These bioactive molecules have been seldom simultaneously quantified within the same PRP preparation. In the present study, the flexible Bio-Plex system was employed to assess the concentration of a large range of cytokines, chemokines, and growth factors in 16 healthy volunteers so as to determine whether significant baseline differences may be found. Besides IL-1b, IL-1ra, IL-4, IL-6, IL-8, IL-12, IL-13, IL-17, INF-γ, TNF-α, MCP-1, MIP-1a, RANTES, bFGF, PDGF, and VEGF that were already quantified elsewhere, the authors reported also on the presence of IL-2, IL-5, IL-7, IL-9, IL-10, IL-15 G-CSF, GM-CSF, Eotaxin, CXCL10 chemokine (IP-10), and MIP 1b. Among the most interesting results, it is convenient to mention the high concentrations of the HIV-suppressive and inflammatory cytokine RANTES and a statistically significant difference between males and females in the content of PDGF-BB. These data are consistent with previous reports pointing out that gender, diet, and test system affect the results of platelet function in healthy subjects, but seem contradictory when compared to other quantification assays in serum and plasma. The inconsistencies affecting the experimental results found in literature, along with the variability found in the content of bioactive molecules, urge further research, hopefully in form of randomized controlled clinical trials, in order to find definitive evidence of the efficacy of PRP treatment in various pathologic and regenerative conditions.

Plasma of Argon Affects the Earliest Biological Response of Different Implant Surfaces: An In Vitro Comparative Study.

The aim of this in vitro study was to evaluate the early cell response and protein adsorption elicited by the argon plasma treatment of different commercially available titanium surfaces via a chair-side device. Sterile disks made of grade 4 titanium (n= 450, 4-mm diameter) with 3 surface topographies (machined, plasma sprayed, and zirconia blasted and acid etched) were allocated to receive 4 testing treatments (2% and 10% protein adsorption and cell adhesion with MC3T3-E1 and MG-63). Furthermore, the specimens were divided to undergo 1) argon plasma treatment (10 W, 1 bar for 12 min) in a plasma reactor, 2) ultraviolet (UV) light treatment for 2 h (positive control group), or 3) no treatment (control group). Pretreatment surface analyses based on a scanning electron microscope and profilometer images were also performed. Profilometric analysis demonstrated that the evaluated specimens perfectly suit the standard parameters. The use of argon plasma was capable of affecting the quantity of proteins adsorbed on the different surfaces, notwithstanding their roughness or topographic features at a low fetal bovine serum concentration (2%). UV light treatment for 2 h attained similar results. Moreover, both the plasma of argon and the UV light demonstrated a significant increase in the number of osteoblasts adherent at 10 min in all tested surfaces. Within its limitations, this in vitro study highlights the potential biological benefits of treating implant surfaces with plasma of argon or UV, irrespective of the roughness of the titanium surface. However, in vivo experiments are needed to confirm these preliminary data and settle the rationale of a treatment that might be clinically relevant in case of bone-reparative deficiencies.

Overcoming physical constraints in bone engineering: 'the importance of being vascularized'.

Bone plays several physiological functions and is the second most commonly transplanted tissue after blood. Since the treatment of large bone defects is still unsatisfactory, researchers have endeavoured to obtain scaffolds able to release growth and differentiation factors for mesenchymal stem cells, osteoblasts and endothelial cells in order to obtain faster mineralization and prompt a reliable vascularization. Nowadays, the application of osteoblastic cultures spans from cell physiology and pharmacology to cytocompatibility measurement and osteogenic potential evaluation of novel biomaterials. To overcome the simple traditional monocultures in vitro, co-cultures of osteogenic and vasculogenic precursors were introduced with very interesting results. Increasingly complex culture systems have been developed, where cells are seeded on proper scaffolds and stimulated so as to mimic the physiological conditions more accurately. These bioreactors aim at enabling bone regeneration by incorporating different cells types into bio-inspired materials within a surveilled habitat. This review is focused on the most recent developments in the organomimetic cultures of osteoblasts and vascular endothelial cells for bone tissue engineering.

Interaction of air pollution and hyperbaric oxygen on virus replication.

A study was undertaken to weigh the effects of air pollution and oxygen at high pressure on the susceptibility of mice to Coxsackievirus B1 infection. Animals exposed to air pollutants or oxygen at high pressure were found to contain higher amounts of recoverable virus than control animals. Animals exposed to both air pollutants and oxygen at high pressure were found to contain the greatest levels of recoverable virus. This same group of animals was also found to have hearts which were smaller than any other group. Animals maintained in an ambient atmosphere had higher levels of recoverable virus and smaller hearts than animals exposed to terpene vapors and hyperbaric oxygen. The results of this study suggest that terpene vapors may nullify the activity of oxygen at high pressure.

Anti-kidney cytotoxic antibodies in human renal allografts.

Periodic comparisons were made of sera from two groups of patients, ten who rejected their renal transplants within a year after transplantation and ten who successfully maintained their transplanted kidneys for five years or more. What appeared to be anti-kidney cytotoxic antibodies were found in much higher levels in the sera of those patients with the short-lived transplants, the difference in titer levels between the two groups being significant at the .0001 levels. This antibody showed no correlation with either the presence or the absence of lymphocytotoxic antibodies, nor did it appear to have any relationship to the HL-A antigens or the previous renal disease of the recipient patient. While it was cytotoxic to tissue cultures of cells obtained from random human kidneys, human kidney tumor cells (Wilms), and human embryonic kidneys, this antibody did not react with non-renal human tissues (lung, spleen, deltoid muscle, foreskin). It did not react with nonhuman (simian) kidney tissue culture cells. The findings suggest the appearance of an organ-specific, cytotoxic anti-kidney antibody in patients undergoing renal homograft rejection.