The expression and localization of RNase and RNase inhibitor in blood cells and vascular endothelial cells in homeostasis of the vascular system

<div><p>RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.</p></div

Localization of RNase 1 and VWF in EAhy926 cells and platelets.

<p>After fixation and blocking, cells were stained with antibodies against RNase 1 (green), VWF (red), and nuclei (blue). Scale bars = 10 μm.</p

RNase 1 and RI protein expression in supernatants of EAhy926 and blood cells.

<p>(A, B) Supernatants from the different cell types were prepared and analyzed by Western blot as described in Materials and Methods with antibodies against RNase 1 (poly) and RI (mono). Lane 1. positive control (A: 10 ng (0.56 pmol) of RNase 1, B: 30 ng (0.61 pmol) of RI), 2. EAhy926 cells, 3. platelets, 4. MNCs, 5. PMNs, 6. RBCs.</p

Localization of RNase 1 and VWF in EAhy926 cells and platelets by the pre-embedding method.

<p>EAhy926 cells (A, B) or platelets (C, D) immunostained with anti-RNase 1 (arrows, (A, C)) or anti-VWF antibodies (arrowheads, (B, D)). Scale bars = 500 nm.</p

Localization of RNase 1 and RI in EAhy926 cells and platelets.

<p>After fixation and blocking, cells were stained with antibodies against RNase 1 (green), RI (red), and nuclei (blue). Each image of platelets-2 and thrombin-activated platelets-2 is optical micrograph of platelets-1 and thrombin-activated platelets-1. Scale bars = 10 μm.</p

Localization of RNase 1 and VWF in EAhy926 cells and platelets by the post-embedding method.

<p>EAhy926 cells (A) or platelets (C) are TEM images. EAhy926 cells (B) or platelets (D) immunostained with anti-RNase 1 (arrows) and anti-VWF antibodies (arrowheads). Scale bars (A) 2 μm, (B) 100 nm, (C) 500 nm, (D) 100 nm.</p

RNase 1 and RI mRNA expression in EAhy926 and blood cells.

<p>Total cellular RNA was prepared from EAhy926 and blood cells, and RT-PCR was performed as described in Materials and Methods. Densitometric analysis of the gels were used to represent the mRNA ratios by using Scion Image PC (National Institutes of Health). Results were normalized to the expression levels (E) of GAPDH and expressed as the ratio E (target)/E (GAPDH) (target = RNase 1 or RI). Each value represents the mean ± SD (n = 6) compared with EAhy926 cells. * indicates a significant differences with p<0.05, whereas ** indicates a significant difference with p<0.01.</p

RNase activity in supernatant from EAhy926 and blood cells.

<p>RNase activity was determined in supernatant from EAhy926 cells, platelets, MNCs, PMNs, and RBCs. Each value represents the mean ± SD (n = 6). Significant differences are indicated as ○○ or ** for p<0.01. ○○ shows significant differences between EAhy926 cells and each cell type in the same time. ** shows differences between 0 min values and other min values of each cells. Whereas * indicates a significant difference with p<0.05.</p