Deleterious Effects of Intra-arterial Administration of Particulate Steroids on Microvascular Perfusion in a Mouse Model.

Purpose To determine the in vivo effects of several particulate steroids on microvascular perfusion by using intravital microscopy in a mice model and to investigate the in vitro interactions between these particulate steroids and red blood cells (RBCs). Materials and Methods The study was conducted in agreement with the guidelines of the National Committee of Ethic Reflection on Animal Experimentation. By using intravital microscopy of mouse cremaster muscle, the in vivo effects of several particulate steroids on microvascular perfusion were assessed. Four to five mice were allocated to each of the following treatment groups: saline solution, dexamethasone sodium phosphate, a nonparticulate steroid, and the particulate steroids cortivazol, methylprednisolone, triamcinolone, and prednisolone. By using in vitro blood microcinematography and electron microscopy, the interactions between these steroids and human RBCs were studied. All results were analyzed by using nonparametric tests. Results With prednisolone, methylprednisolone, or triamcinolone, blood flow was rapidly and completely stopped in all the arterioles and venules (median RBC velocity in first-order arterioles, 5 minutes after administration was zero for these three groups) compared with a limited effect in mice treated with saline, dexamethasone, and cortivazol (20.3, 21.3, and 27.5 mm/sec, respectively; P < .003). This effect was associated with a large decrease in the functional capillary density (4.21, 0, and 0 capillaries per millimeter for methylprednisolone, triamcinolone, or prednisolone, respectively, vs 21.0, 21.4, and 19.1 capillaries per millimeter in mice treated with saline, dexamethasone, and cortivazol, respectively; P < .003). This was because of the rapid formation of RBC aggregates. However, no change in microvascular perfusion was associated with administration of cortivazol or dexamethasone. In vitro experiments confirmed the formation of RBC aggregates associated with the transformation of RBCs into spiculated RBCs with the same steroids. Conclusion Several particulate steroids have an immediate and massive effect on microvascular perfusion because of formation of RBC aggregates associated with the transformation of RBCs into spiculated RBCs. (©) RSNA, 2016 Online supplemental material is available for this article.

Vascular endothelial growth factor receptor-1 (VEGFR-1) expression in human corneal fibroblast decreased with age.

PURPOSE: Mechanisms by which fibroblast networks between stromal lamellae are laid in the corneal stroma are far from clear. We have investigated the role of vascular endothelial growth factor receptors (VEGFRs) by in vitro studies in the human corneal network formation obtained from donors whose ages ranged from 19 to 89 years. METHODS: Corneal fibroblasts were prepared from cornea donations. The functional properties of these cells to form networks were analyzed using a semi solid matrix (substratum) of Matrigel. The presence of VEGF receptor-1 (VEGFR-1) and the functionality in these fibroblasts were investigated using immunofluorescence, molecular analysis (gene microarray, reverse transcription polymerase chain reaction [RT-PCR] and VEGFR siRNA transfections), and cell culture. RESULTS: Corneal fibroblasts from 61 donors were classified into two groups according to whether they formed (82%) a reticulum on Matrigel or not (18%). By RT-PCR and immunofluorescence analysis, we showed that corneal fibroblasts expressed VEGFR-1 (mRNA and protein). Further, cell culture analysis revealed that only the network (reticulum) forming corneal fibroblast expressed VEGFR-1 in contrast to non network-forming fibroblasts. Use of inhibitors such as VEGFR-1 siRNA transfection or neutralizing antibody (Avastin) indicated that VEGFR-1 was essential to the formation of the corneal network in vitro. CONCLUSIONS: The cell reticulum formation seemed to be directly related to the expression of VEGFR-1 in the corneal fibroblast, and this expression decreased with age. The decrease in VEGFR-1 expression is probably related to the diminution of autocrine functions, which may alter the overall tissular homeostasis. This may culminate in the gradual development of poor vision, which is observed in certain pathologies and in aging individuals.

Plasma endothelial protein C receptor influences innate immune response in ovarian cancer by decreasing the population of natural killer and TH17 helper cells.

In spite of the growing importance of endothelial protein C receptor/active protein C (EPCR/aPC) in tumor biology, their impact on immunological homeostasis remains largely unexplored. The objective of this study was to assess whether soluble plasma endothelial protein C receptor (sEPCR), which is a regulator of circulating aPC, is involved in innate immune response in cancer patients. In the Ovcar-3 ovarian cancer line, the role of aPC in secretion of cytokines was analyzed. In parallel, in 33 patients, with a diagnosis of ovarian epithelial cancer, sEPCR was quantified, blood immune cell phenotypes were determined by flow cytometry and plasma cytokines were evaluated using a protein array. Spearman's rank correlation coefficients (r) and coefficient significance was determined by a statistical hypothesis test (α=0.05). Our results show that i) aPC induced the secretion of several cytokines in Ovcar-3 cells; ii) 61% of patients exhibited a concentration of plasma sEPCR well above the baseline (normal plasma level, 100 ± 28 ng/ml); iii) comparing immune cell phenotypes in patients having a normal level of sEPCR with those having a high level of sEPCR, it was found that sEPCR levels were correlated with high intensity of cells expressing CD45ra, CD3, CD8, CD25 and low intensity of cells expressing CD56 (NK cells), CD294 (TH2 cells), IL-2, IL-10, IL-17a (TH17 cells), IL-21 (TH21 cells) and CD29 markers (r ≥ 0.60); and iv) high levels of sEPCR correlate with high levels of plasma bioactive proteins such as insulin-like growth factor-2 (IGFII), IL-13rα, macrophage inflammatory protein (MIP1α) and matrix metalloproteinase-7 (MMP-7) that have already been proposed as biomarkers for ovarian cancer and particularly those with poor prognosis. In conclusion, sEPCR produced by ovarian cancer cells, by modulating circulating aPC, influences the secretory behavior of tumor cells (cytokines and interleukins). Consequently, sEPCR in turn acts on the innate immune response by decreasing effector cells such as natural killer and T helper cells (TH2, TH17 and TH21).

Endothelial protein C receptor expressed by ovarian cancer cells as a possible biomarker of cancer onset.

Coagulation disorders often accompany cancer onset and evolution, which, if not properly managed, could have grave consequences. Endothelial protein C is an important regulator of homeostasis and acts through its high affinity binding to its transmembrane receptor (EPCR). Soluble (sEPCR) which results from the proteolytic cleavage of the membrane bound form can trap activated endothelial protein C and deprive it of its anti-coagulant function. In this study, the expression of EPCR and its soluble form (sEPCR) released into plasma as a result of proteolytic cleavage were investigated in ovarian, breast, lung and colorectal cancer biopsies, as well as in ascitic cell clusters and peritoneal fluid from ovarian cancer samples. In parallel, breast, ovarian, lung and colorectal cancer cell lines were investigated for the expression of EPCR. The integrity of the EPCR gene sequence as well gene haplotypes were ascertained in the established cancer cell lines in order to understand their eventual regulatory functions. The results from the present study indicate that in cancer patients, the levels of sEPCR are significantly higher than the normal range compared to healthy volunteers. The increase in the levels of sEPCR parallels the increase in CA125, showing a close correlation. Therefore, the detection of sEPCR in cancer and during the post-treatment period could be taken into account as an additional marker that could re-inforce the one obtained using CA125 alone as a marker of cancer cell mass.