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Microrna-Regulated Pathways of Flow-Stimulated Angiogenesis And Henn et al. J Transl Med (2019) 17:22 https://doi.org/10.1186/s12967-019-1767-9 Journal of Translational Medicine RESEARCH Open Access MicroRNA‑regulated pathways of fow‑stimulated angiogenesis and vascular remodeling in vivo Dominic Henn1† , Masood Abu‑Halima2†, Dominik Wermke3, Florian Falkner1, Benjamin Thomas1, Christoph Köpple1, Nicole Ludwig2, Matthias Schulte1, Marc A. Brockmann4, Yoo‑Jin Kim5, Justin M. Sacks6, Ulrich Kneser1, Andreas Keller3, Eckart Meese2‡ and Volker J. Schmidt1*‡ Abstract Background: Vascular shear stress promotes endothelial cell sprouting in vitro. The impact of hemodynamic forces on microRNA (miRNA) and gene expression within growing vascular networks in vivo, however, remain poorly investi‑ gated. Arteriovenous (AV) shunts are an established model for induction of neoangiogenesis in vivo and can serve as a tool for analysis of hemodynamic efects on miRNA and gene expression profles over time. Methods: AV shunts were microsurgically created in rats and explanted on postoperative days 5, 10 and 15. Neoan‑ giogenesis was confrmed by histologic analysis and micro-computed tomography. MiRNA and gene expression pro‑ fles were determined in tissue specimens from AV shunts by microarray analysis and quantitative real-time polymer‑ ase chain reaction and compared with sham-operated veins by bioinformatics analysis. Changes in protein expression within AV shunt endothelial cells were determined by immunohistochemistry. Results: Samples from AV shunts exhibited a strong overexpression of proangiogenic cytokines, oxygenation- associated genes (HIF1A, HMOX1), and angiopoetic growth factors. Signifcant inverse correlations of the expressions of miR-223-3p, miR-130b-3p, miR-19b-3p, miR-449a-5p, and miR-511-3p which were up-regulated in AV shunts, and miR-27b-3p, miR-10b-5p, let-7b-5p, and let-7c-5p, which were down-regulated in AV shunts, with their predicted interacting targets C–X–C chemokine receptor 2 (CXCR2), interleukin-1 alpha (IL1A), ephrin receptor kinase 2 (EPHA2), synaptojanin-2 binding protein (SYNJ2BP), forkhead box C1 (FOXC1) were present. CXCL2 and IL1A overexpression in AV shunt endothelium was confrmed at the protein level by immunohistochemistry. Conclusions: Our data indicate that fow-stimulated angiogenesis is determined by an upregulation of cytokines, oxygenation associated genes and miRNA-dependent regulation of FOXC1, EPHA2 and SYNJ2BP. Keywords: AV shunt, Shear stress, Microarray, Chemokines *Correspondence: Volker.schmidt@bgu‑ludwigshafen.de †Dominic Henn and Masood Abu-Halima contributed equally to this work ‡Eckart Meese and Volker J. Schmidt contributed equally to this work 1 Department of Hand, Plastic and Reconstructive Surgery, University of Heidelberg, BG Trauma Center Ludwigshafen, Ludwig‑Guttmann Str. 13, 67071 Ludwigshafen, Germany Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henn et al. J Transl Med (2019) 17:22 Page 2 of 19 Background strategy for the suppression of neoangiogenesis in oncol- Vascular remodeling and angiogenesis play important ogy patients [17]. roles in the pathophysiology of cardiovascular diseases. Arteriovenous (AV) shunts are an established model Te development of new blood vessels is governed for in vivo induction of neoangiogenesis through elevated by an interplay of biochemical and mechanical stim- blood fow [18, 19]. Here, an AV shunt is microsurgically uli. Te pulsatile blood fow generated by the cardiac created by interposing a vein graft between the saphen- cycle exposes endothelial cells (ECs) to two mechani- ous artery and vein on the hind limbs of a rat [20], leading cal forces, namely circumferential stretch acting per- to an increase in blood fow by 4.5-fold within the vascu- pendicularly, and shear stress acting tangentially to the lar construct [21]. Elevated shear stress on the vascular vascular wall [1]. Elevated shear stress has been shown wall due to increased blood fow triggers rapid sprouting to promote EC migration and regeneration, as well as of new blood vessels from the AV shunt leading to the diferentiation of embryonic stem cells into ECs [2, 3]. development of a microvascular network within 15 days In vitro studies have shown that mechanical forces act- [21]. Terefore, AV shunts are an ideal tool for in vivo ing on ECs and vascular smooth muscle cells (VSMCs) analyses of the efects of elevated vascular shear stress are translated into biochemical signals by mechanosen- on miRNA and gene expression profles over time, which sory proteins [4, 5]. Tese promote intracellular path- regulate fow-stimulated angiogenesis and remodeling. ways, which lead to altered gene expression profles [6, We determined the expression profles of 758 miRNAs 7] with up-regulated proangiogenic factors like vascu- and 30,584 messenger RNAs (mRNAs) by microarray lar endothelial growth factor (VEGF) [3]. analysis in venous tissue samples from rat AV shunts after Angiogenesis, however, is a delicately regulated pro- exposure to elevated blood fow for 5, 10 and 15 days cess, which goes far beyond EC sprouting, and requires (n = 7 per group) (Fig. 1). Expression profles were com- a concerted action of ECs, VSMCs, as well as signals pared to sham-operated veins (end-to-end anastomosis, from the extracellular matrix (ECM) and cells like mac- n = 8) in order to eliminate efects of the surgical pro- rophages and fbroblasts chemotactically attracted cedure and mechanical infuence caused by the opera- to areas of neoangiogenesis [8, 9]. In vitro studies on tion itself on miRNA and gene expression. To identify ECs have shown that elevated shear stress induces the miRNA–mRNA interactions that are relevant for the expression of microRNAs (miRNAs) that afect vascular regulation of angiogenic processes, a gene ontology (GO) remodeling and angiogenesis [10, 11]. Current knowl- enrichment analysis was performed with GeneTrail2 [22, edge of diferential signaling in angiogenesis largely 23]. We determined miRNA/mRNA pairs with inverse −5 stems from in vitro studies by means of EC sprouting correlations (r < 0.5) and a P-value < 5 × 10 , which assays or co-culture systems [12]. Still, these models are were associated with GO terms for positive, respec- limited in representing in vivo physiologic conditions. tively negative regulation of angiogenic processes (A Te development of new therapeutic strategies targeting list of employed GO terms is shown in Additional fle 1: angiogenesis requires more realistic model systems and a Table S1). We used TargetScan release 7.1 for miRNA/ more detailed understanding of the complex in vivo envi- mRNA target prediction analysis since it has proven to ronment determining angiogenic processes. Terefore, be the most robust prediction tool for identifcation of studies integrating the molecular signals from diferent miRNA/mRNA target interactions [24, 25]. In mammals, cellular players and miRNAs which govern angiogenesis cumulative weighted context++ scores of the binding are needed. sites are calculated and used for prediction of efcacy So far, evidence is lacking on how elevated shear stress of targeting according to Agarwal et al. [24]. Te con- in vivo infuences miRNA expression profles within the text++ model has shown to be more predictive than any vascular wall, and to what extent altered miRNA signa- previously published model, being as predictive as most tures infuence the expression levels of pro- and anti- in vivo crosslinking approaches [24]. Neoangiogenesis angiogenic genes. Tis knowledge may soon gain clinical originating from AV shunts was assessed by morphologi- value as RNA-based therapeutics are an emerging feld cal analysis of histologic cross-sections and micro-com- in cardiovascular pharmacology, and studies in small puted tomography (micro-CT). Te expression changes and large animal models of cardiovascular diseases have of strongly deregulated mRNAs were confrmed at the yielded promising results [13–15]. MiRNA-mediated protein level by immunohistochemical analysis. silencing of anti-angiogenic genes by means of synthetic RNA mimics appears to be a promising approach for the Methods promotion of local neoangiogenesis in ischemic myo- Microsurgical AV shunt creation cardium [16]. Conversely, miRNA-mediated down-reg- Te experiments were performed in accordance with the ulation of proangiogenic genes constitutes a treatment German Animal Welfare Act and approved by the local Henn et al. J Transl Med (2019) 17:22 Page 3 of 19 Fig. 1 a Experimental setup and micro CT analysis. An arteriovenous shunt is microsurgically created on a rat’s hind limb by anastomosing a saphenous vein graft (green) from the contralateral leg between saphenous artery (red) and vein (blue). b The AV shunt is placed around four pins (P) for stabilization within a Tefon chamber (C). Two layers of acellular dermal substitute (ADS) are placed below and above the vascular construct (upper layer not shown). A saphenous artery, V saphenous vein, VG vein graft. c Analysis of an explanted AV shunt on postoperative day 15 by micro‑computed tomography revealed a dense microvascular sprouting from the shunt vessels governmental authorities [Landesuntersuchungsamt dissected after midventral cutaneous incision on both Rheinland-Pfalz (G15-7-047)] on 47 female Sprague– hind limbs. A 20 mm long saphenous vein graft was har- Dawley rats (Charles River Laboratories, Sulzfeld, Ger- vested from the left leg and anastomosed between the many) weighing 280–320 g at ages 11–15 months. Te right saphenous artery and vein in an end-to-end fash- animals had access to food and water and were kept at a ion, thereby creating an AV shunt (Fig.
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