The Annexin a2 Promotes Development in Arthritis through Neovascularization by Amplification Hedgehog Pathway.

The neovascularization network of pannus formation plays a crucial role in the development of rheumatoid arthritis (RA). Annexin a2 (Axna2) is an important mediating agent that induces angiogenesis in vascular diseases. The correlation between Axna2 and pannus formation has not been studied. Here, we provided evidence that compared to osteoarthritis (OA) patients and healthy people, the expression of Axna2 and Axna2 receptor (Axna2R) were up-regulated in patients with RA. Joint swelling, inflammation and neovascularization were increased significantly in mice with collagen-induced arthritis (CIA) that were exogenously added Axna2. Cell experiments showed that Axna2 promoted HUVEC proliferation by binding Axna2R, and could activate Hedgehog (HH) signaling and up-regulate the expression of Ihh and Gli. Besides, expression of Ihh, Patched (Ptc), Smoothened (Smo) and Gli and matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic growth factor of HH signaling downstream, were down-regulated after inhibition of expression Axna2R on HUVEC. Together, our research definitely observed that over-expression of Axna2 could promote the development of CIA, especially during the process of pannus formation for the first time. Meanwhile, Axna2 depended on combining Axna2R to activate and enlarge HH signaling and the expression of its downstream VEGF, Ang-2 and MMP-2 to promote HUVEC proliferation, and eventually caused to angiogenesis. Therefore, the role of Axna2 is instructive for understanding the development of RA, suppress the effect of Axna2 might provide a new potential measure for treatment of RA

Sca-1 is a marker for cell plasticity in murine pancreatic epithelial cells and induced by IFN-β in vitro

BACKGROUND & AIMS: Sca-1 is a surface marker for murine hematopoietic stem cells (HSCs) and type-I interferon is a key regulator for Lin$^{-}$Sca-1$^{+}$ HSCs expansion through Ifnar/Stat-1/Sca-1-signaling. In this study we aimed to characterize the role and regulation of Sca-1$^{+}$ cells in pancreatic regeneration. METHODS: To characterize Sca-1 in vivo, immunohistochemistry and immunofluorescence staining of Sca-1 was conducted in normal pancreas, in cerulein-mediated acute pancreatitis, and in Kras-triggered cancerous lesions. Ifnar/Stat-1/Sca-1-signaling was studied in type-I IFN-treated epithelial explants of adult wildtype, Ifnar$^{-}$$^{/-}$, and Stat-1$^{-}$$^{/}$$^{-}$ mice. Sca-1 induction was analyzed by gene expression and FACS analysis. After isolation of pancreatic epithelial Lin$^{-}$Sca-1$^{+}$cells, pancreatosphere-formation and immunofluorescence-assays were carried out to investigate self-renewal and differentiation capabilities. RESULTS: Sca-1$^{+}$ cells were located in periacinar and periductal spaces and showed an enrichment during cerulein-induced acute pancreatitis (23.2/100 μm$^{2}$ ± 4.9 SEM) and in early inflammation-mediated carcinogenic lesions of the pancreas of Kras$^{G12D}$ mice (35.8/100 μm$^{2}$ ± SEM 1.9) compared to controls (3.6/100 μm$^{2}$ ± 1.3 SEM). Pancreatic Lin$^{-}$Sca-1$^{+}$ cells displayed a small population of 1.46% ± 0.12 SEM in FACS. In IFN-β treated pancreatic epithelial explants, Sca-1 expression was increased, and Lin$^{-}$Sca-1$^{+}$ cells were enriched in vitro (from 1.49% ± 0.36 SEM to 3.85% ± 0.78 SEM). Lin$^{-}$Sca-1$^{+}$ cells showed a 12 to 51-fold higher capacity for clonal self-renewal compared to Lin$^{-}$Sca-1$^{-}$ cells and generated cells express markers of the acinar and ductal compartment. CONCLUSIONS: Pancreatic Sca-1$^{+}$ cells enriched during parenchymal damage showed a significant capacity for cell renewal and in vitro plasticity, suggesting that corresponding to the type I interferon-dependent regulation of Lin$^{-}$Sca-1$^{+}$ hematopoietic stem cells, pancreatic Sca-1$^{+}$ cells also employ type-I-interferon for regulating progenitor-cell-homeostasis

The relationship between Axna2/Axna2R and proliferation of HUVEC.

<p><b>(A)</b> Axna2 concentration-dependently promoted HUVEC proliferation, and at concentration of 1.0μg/ml achieved the maximum effect. <b>(B)</b> HUVEC cells were incubated with an anti-Axna2 antibody (green) and an anti-Axna2R antibody (red). Axna2 was marked by GFP, and Axna2R was marked by RFP. The staining was visualized with confocal microscopy. <b>(C)</b> After blocking Axna2R with the anti-Axna2R antibody, the promoting proliferation of Axna2 were weakened partly. <b>(D)</b> Detected the effect of plasmid transfection of knocking-down expression of Axna2R by using RT-PCR technology. plasmid C was selected for the following experiment based on the results of this experiment.<b>(E)</b> After silencing of Axna2R by plasmid C, the effect of the proliferation of Axna2 weakened.<b>(A, C and D)</b> *P < 0.05, **P < 0.005, ***P < 0.0005, One-way ANOVA Tukey's multiple comparisons test. <b>(E)</b> *P < 0.05, Mann Whitney test. Experiments were repeated at least 3 times. The bars above showed the mean±SEM.</p

Function of Axna2/Axna2R axis in promoting neovascularization of RA.

<p>In the pathogenesis of RA, the over-expression of Axna2 on vascular endothelial cells could bind Axna2R and activate the HH signaling, causing increasd expression of angiogenic factor, and ultimately promote neovascularization.</p

Axna2 enhanced the inflammatory changes and promoted angiogenesis in mice with CIA.

<p><b>(A)</b> The differential expression of the CⅡ-specific antibodies between the early initial immunization (day 0) and the peak of arthritis (day 40) in the serum of the CIA mice. <b>(B)</b> H&E staining of joint tissue showed a remarkable increase in synovial inflammation as well as cartilage and bone destruction in the Axna2/CⅡ^+/+ mice compared to that in the other three groups(original magnification×40,×100 with an electron microscope). <b>(C and D)</b> The IHC <b>(C)</b> and IF <b>(D)</b> (original magnification×200) analysis of the CD31 by using fluorescence microscopy. The number of neovascularization was larger significantly in Axna2/CⅡ^+/+ mice than the other three groups. <b>(E)</b> Three vies of the original magnification×200 were selected for the quantitative analysis of the expression of CD31. The highest expression of CD31 was found in the Axna2/CⅡ^+/+ group. <i>P</i><0.05 represented a significant statistical difference. <b>(A)</b> ***<i>P</i> < 0.0001, Two-way ANOVA, experiments were repeated at least 3 times. <b>(E)</b> **<i>P</i> < 0.005, ***<i>P</i> < 0.0005, One-way analysis of variance. <b>(A and E)</b> the bars in showed the mean±SEM.</p

The expression of Axna2 and Axna2R were up-regulated in the synovial tissues of RA compared to OA and healthy people.

<p><b>(A)</b> GEO DataSets showed that the expression of Axna2 mRNA in the synovial tissues of the normal controls (n = 5) and the patients with OA (n = 5) and RA (n = 5). <b>(B)</b> GEO DataSets indicated the expression of Axna2R mRNA in the synovial tissues of patients with OA (n = 5) and RA (n = 7). <b>(C)</b> The expression of Axna2 in the serum of patients with RA (n = 5) and pancreatitis(n = 7) were detected by ELISA. <b>(A)</b> *P < 0.05, **P < 0.005, ***P < 0.0005, One-way ANOVA Tukey's multiple comparisons test. <b>(B and C)</b> **P < 0.005, Mann Whitney test. The bars above showed the mean±SEM.</p

Exogenously added Axna2 facilitated the progression of arthritis in mice with CIA.

<p><b>(A)</b> There was no swollen paws in Control and Axna2/CⅡ^+/- mice, the number of swollen paws in Axna2/CⅡ^+/+ mice was larger than that in Axna2/CⅡ^-/+ mice. <b>(B)</b> The number of swollen paws of the mice represented the incidence of arthritis. Incidence of CIA in Axna2/CⅡ^+/+ mice was higher than that in Axna2/CⅡ^-/+ mice. <b>(C and D)</b> Plantar thickness <b>(C)</b> and arthritis score <b>(D)</b> were clinical indicators of the arthritis changes in the four groups, the largest change happened in Axna2/CⅡ^+/+ mice and statistically significant. <b>(E)</b> Physical photograph displayed the change of paw swelling in the four group mice. Radiography and Micro-CT of the hind paws showed the joint bone destruction of CIA mice. P value represented Axna2/CⅡ^+/+ group compared to Axna2/CⅡ^-/+group. <b>(B and D)</b> *P < 0.05, **P < 0.005, ***P <0.0005, Mann Whitney test. <b>(C)</b>**P < 0.005, One-way analysis of variance. The bars in <b>(B-D)</b> showed the mean±SEM.</p

Plasmid Sequences for Gene silencing of Annexin a2 receptor.

<p>Plasmid Sequences for Gene silencing of Annexin a2 receptor.</p

The Axna2/Axna2R axis affected HH signaling.

<p><b>(A and B)</b> The expression of Ihh and Gli increased after being treated with 1.0μg/ml Axna2 for 24 hours by using RT-PCR and western blotting. <b>(C)</b> Knockdown of Axna2R on the surface of HUVEC could down-regulate the expression of Ihh, Ptc, Smo and Gli. <b>(D)</b> Knockdown of Axna2R on the surface of HUVEC decreased the expression of HH signaling downstream MMP-2, VEGF and Ang-2. <b>(E)</b> After inhibition of HH signaling by CYC, the effect of Axna2 in promoting endothelial cell proliferation was significantly decreased. The bars showed the mean±SEM. <b>(A, C and D)</b> *P < 0.05, **P < 0.005,*** P < 0.0005, Mann Whitney test. <b>(E)</b> *P < 0.05, **P < 0.005,*** P < 0.0005, One-way ANOVA Tukey's multiple comparisons test. Experiments were repeated at least 3times.</p

Development of a Novel PET Tracer [¹⁸F]AlF-NOTA-C6 Targeting MMP2 for Tumor Imaging

<div><p>Background and Objective</p><p>The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [¹⁸F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH₂ (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many <i>in vivo</i> tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (¹⁸F) is the optimal PET radioisotope, and the creation of a [¹⁸F]AlF-peptide complex is a simple procedure.</p><p>Methods</p><p>C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [¹⁸F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [¹⁸F]AlF-NOTA-C6 were tested <i>in vitro</i> and <i>in vivo</i>.</p><p>Results</p><p>The non-decay corrected yield of [¹⁸F]AlF-NOTA-C6 was 46.2–64.2%, and the radiochemical purity exceeded 95%. [¹⁸F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [¹⁸F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.</p><p>Conclusions</p><p>[¹⁸F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.</p></div