Diabetes Volume 68, February 2019 349

Increased Expression of miR-483-3p Impairs the Vascular Response to Injury in Type 2 Diabetes

Kira Kuschnerus,1,2,3 Elisabeth T. Straessler,1,2 Maja F. Müller,4 Thomas F. Lüscher,4,5 Ulf Landmesser,1,2,4,5 and Nicolle Kränkel1,2,5

Diabetes 2019;68:349–360 | https://doi.org/10.2337/db18-0084

Aggravated endothelial injury and impaired endothelial (T2D), the excessive cardiovascular risk remains an unmet repair capacity contribute to the high cardiovascular risk clinical need (1). Patients with diabetes suffer from a more in patients with type 2 diabetes (T2D), but the underlying aggressive course of atherosclerosis and coronary artery mechanisms are still incompletely understood. Here we disease (CAD), and cardiovascular mortality is dispropor- describe the functional role of a mature form of miRNA tionally high in patients with diabetes (2,3). Among the (miR) 483-3p, which limits endothelial repair capacity in most relevant causes underlying this excessive risk profile patients with T2D. Expression of human (hsa)-miR-483-

is a severely impaired endothelial repair capacity (4). On PATHOPHYSIOLOGY 3p was higher in endothelial-supportive M2-type macro- the other hand, the necessity to replace damaged or de- phages (M2MFs) and in the aortic wall of patients with T2D than in control subjects without diabetes. Likewise, tached neighboring cells (e.g., in sections of nonlaminar fl the murine (mmu)-miR-483* was higher in T2D than in blood ow) or to populate nonendothelialized sections nondiabetic murine carotid samples. Overexpression of (e.g., after catheter intervention) arises more frequently miR-483-3p increased endothelial and macrophage ap- in patients with diabetes (5). optosis and impaired reendothelialization in vitro. The Functional impairment of the endothelial cells them- inhibition of hsa-miR-483-3p in human T2D M2MFs selves (6–10), as well as of “accessory” cells with paracrine transplanted to athymic nude mice (NMRI-Foxn1n/Foxn1n) activity (11–13), is involved in the reduced endothelial or systemic inhibition of mmu-miR-483* in B6.BKS(D)- repair capacity. Ex vivo cultured “early outgrowth cells” are db Lepr /J diabetic mice rescued diabetes-associated a heterogeneous myeloid cell population with regenerative impairment of reendothelialization in the murine carotid- potential and have been widely used to model and in- fi injury model. We identi ed the endothelial transcrip- vestigate alterations in patient-derived cells of the hema- fi tion factor vascular endothelial zinc nger 1 (VEZF1) topoietic lineage. Their exact composition varies between as a direct target of miR-483-3p. VEZF1 expression was isolation protocols, but most reports agree on a large reduced in aortae of diabetic mice and upregulated in component of M2-like macrophages (M2MFs), as well diabetic murine aortae upon systemic inhibition of mmu-483*. The miRNA miR-483-3p is a critical regulator as smaller components of lymphoid and progenitor cells F of endothelial integrity in patients with T2D and may (11,14,15). M2M s obtained from patients with high represent a therapeutic target to rescue endothelial re- cardiovascular risk, including T2D, exhibit altered secre- generation after injury in patients with T2D. tory activity, thus affecting the healing response of the adjacent endothelial cells (4,16). Mediators regulating Diabetes is among the most prevalent chronic diseases in the maintenance and restoration of endothelial integrity developed and developing countries. Despite substantial include peptide messengers; gaseous molecules, such as nitric progress in therapy for and prevention of type 2 diabetes oxide; radicals; and noncoding RNAs, such as miRNAs.

1Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, This article contains Supplementary Data online at http://diabetes Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0084/-/DC1. 2 DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, © 2018 by the American Diabetes Association. Readers may use this article as long Germany as the work is properly cited, the use is educational and not for profit, and the work 3 Deutsches Herzzentrum Berlin, Berlin, Germany is not altered. More information is available at http://www.diabetesjournals.org/ 4 University Hospital Zurich, Department of Cardiology, Zürich, Switzerland content/license. 5Center of Molecular Cardiology, University of Zurich, Zurich, Switzerland See accompanying article, p. 268. Corresponding author: Nicolle Kränkel, [email protected] Received 23 January 2018 and accepted 13 September 2018 350 miR-483-3p Counteracts Endothelial Regeneration Diabetes Volume 68, February 2019

Differential regulation of miRNas in T2D appears to Table 1—Characteristics of healthy subjects for studies with provide an important link between metabolic cues and M2MFs expression, mediating even transgenerational effects (17). Apparently healthy We have previously observed a downregulation of the control subjects (n = 24) bradykinin B2 in patients with CAD (11), for Age (years) 57.6 6 1.3 which a mature form of miRNA (miR), 483-3p, was pre- Sex (% male) 70.8 dicted to be a potential translational regulator. miR-483- Blood pressure, 3p has been reported to be modulated in wound healing systolic (mmHg) 120.4 6 2.0 and cancer progression (18–20). Mechanistic studies point Blood pressure, to a role of metabolic dysregulation in the pathological diastolic (mmHg) 79.0 6 2.0 upregulation of miR-483-3p in tumor cells (21). Recent Heart rate (bpm) 60.3 6 2.0 data from in vitro models suggest a role in the apoptosis of BMI (kg/m2) 24.6 6 0.6 “endothelial progenitor cells” and cardiomyocytes in di- abetes (22,23). Yet, the in vivo relevance of miR-483-3p Hypertension (%) 0.0 for the vascular response to injury, which is delayed in Obesity (%) 0.0 patients with T2D, was not demonstrated before. Dyslipidemia (%) 8.3 We have analyzed the expression and functional rele- Family history (%) 0.0 vance of miR-483-3p in human M2MFs, which are known Smoking (n) 20 current smokers to crucially modulate the vascular repair process, in HAECs, in 24 ex-smokers two murine models of carotid artery injury, and in aortic LVEF (%) 59.3 6 0.9 specimens from human patients with T2D. miR-483-3p Glucose (mmol/L) 5.0 6 0.1 levels were higher in M2MFs and in the aortic wall of HbA1c (%) 5.5 6 0.0 patients with CAD who had T2D than in patients with Total cholesterol (mmol/L) 5.2 6 0.1 CAD who did not have T2D. We identified the transcrip- 6 tion factor vascular endothelial zinc finger 1 (VEZF1) as HDL (mmol/L) 1.7 0.1 a novel target of miR-483-3p. In subsequent functional LDL (mmol/L) 3.1 6 0.1 studies, we could establish that the overexpression of Triglycerides (mmol/L) 0.9 6 0.1 miR-483-3p and consecutive reduction of VEZF1 signaling Creatinine (mmol/L) 85.3 6 2.6 increases endothelial cell apoptosis, impairs M2MF- NT-proBNP (ng/L) 61.7 6 9.3 mediated regenerative function, and ultimately decreases Leukocyte count (103/mL) 5.0 6 0.2 endothelial regenerative capacity in vitro and in vivo. Erythrocyte count (10/mL) 4.7 6 0.1 6 RESEARCH DESIGN AND METHODS Hb (g/dL) 126.4 9.0 Thrombocyte count (103/mL) 245.5 6 9.7 Human Subjects The study was reviewed and approved by the local ethics Hb, hemoglobin; LVEF, left ventricular ejection fraction; committee (Kantonale Ethikkommission Zurich) and com- NT-proBNP, N-terminal prohormone brain natriuretic peptide. plies with International Conference on Harmonisation good clinical practice guidelines. All study participants provided written informed consent before participation The 10- to 12-week-old male athymic nude mice (NMRI- n n or tissue banking. CAD was defined as stenosis of .50% in Foxn1 /Foxn1 , for M2MF transplantation) and B6.BKS db a coronary artery confirmed by coronary angiography. (D)-Lepr /J and C57BL/6 wild-type mice (for systemic “Apparently healthy” control subjects were normoten- transduction) were given 200 mg of metamizole as pre- sive and normocholesterolemic, and of comparable age anesthetic analgesia and subjected to electrical injury of as patients without known cardiovascular disease or long- a 4-mm section of the left common carotid artery under term medication use. Human aortic samples were obtained general anesthesia with isoflurane as described previously from patients with CAD, with or without T2D, who were (11). Postoperative analgesia consisted of oral metamizole in the drinking water for 3 postoperative days. Three hours undergoing elective cardiovascular surgery. Patient char- n n acteristics are summarized in Tables 1 and 2. after the surgery, NMRI-Foxn1 /Foxn1 mice received 3 3 105 M2MFsi.v.frompatientswithCADandT2Dor Animals healthy control subjects, who had previously been trans- All animal experiments were performed in accordance fected with power inhibitor of hsa (human)-miR-483-3p with the local and national guidelines and were approved (T2D), mimic of hsa-miR-483-3p (healthy), or a scrambled by the local Animal Experiments Committee in Zurich and oligonucleotide (both groups) (all from Ambion/Exiqon, db db Berlin. B6.BKS(D)-Lepr /J and C57BL/6 wild-type mice Vedbaek, Denmark), and B6.BKS(D)-Lepr /J and C57BL/6 were purchased from Janvier Labs (Le Genest-Saint-Isle, mice received anti-miRNA or scrambled control (5 mg/kg n n France). NMRI-Foxn1 /Foxn1 mice were purchased from body weight, formulated with jetPEI) (Polyplus). Three Charles River Laboratories (Cologne, Germany). days after the injury/treatment, mice were injected with diabetes.diabetesjournals.org Kuschnerus and Associates 351

— according to the protocol of Nielsen et al. (24). Images were Table 2 Characteristics of patients with CAD with and 3 without T2D for studies with M2MFs acquired using a 20 objective with a Zeiss AxioScan Patients without Patients with Microscope and analyzed with ImageJ. Human formalin- T2D with CAD + T2D fixed, paraffin-embedded aortic biopsy specimens were CAD (n = 19) (n = 11) probed with a human miR-483-3p–specific probe (Affymet- Age (years) 60.4 6 1.6 61.5 6 2.1 rix) using the ViewRNA eZ-L Detection Kit–1-Plex Sex (% male) 84.2 81.8 (Red; Affymetrix) at Sophistolab (Muttenz, Switzerland). a Blood pressure, Human CD31 and -smooth muscle actin were visual- systolic (mmHg) 126.0 6 3.0 145.8 6 3.5*** ized by immunofluorescence staining (both from Abcam) Blood pressure, in additional sections to identify smooth muscle/endothe- diastolic (mmHg) 76.9 6 2.3 81.4 6 4.2 lial layers. Images were taken on a Keyence BZ-9000 fl 3 Heart rate (bpm) 64.1 6 2.2 69.1 6 3.4 inverted uorescence phase contrast microscope at 20 3 fi BMI (kg/m2) 28.0 6 1.0 32.2 6 1.2* and 40 magni cation. The area of miR-483-3p stain- ing was assessed after defining a red color threshold in Hypertension (%) 84.2 100.0 ImageJ and normalizing the miR-483-3p–positive area to Obesity (%) 36.8 72.7 the vessel wall area (px2/px2) visible in each view field. Dyslipidemia (%) 84.2 90.9 Family history (%) 63.2 72.7 Cell Culture and Media Smoking (n) 26 current 23 current HAECs were purchased from CellSystems (Troisdorf, Ger- smokers smokers many) and Lonza (Walkersville, MD) and propagated in 2 2 7 ex-smokers 8 ex-smokers fully supplemented EGM-2 with 10% FCS (Lonza) accord- LVEF (%) 58.2 6 2.2 54.6 6 2.8 ing to the manufacturer recommendations. HAECs were Glucose (mmol/L) 5.4 6 0.2 9.2 6 0.8**** used for assays at passages 6–8. The starvation medium

HbA1c (%) 5.6 6 0.1 7.3 6 0.3**** used was EBM-2 (Lonza) supplemented with 100 IU/mL m Total cholesterol penicillin, 100 g/mL streptomycin (both Invitrogen, Carls- (mmol/L) 4.5 6 0.3 4.2 6 0.3 bad, CA), and 0.5% FCS. Peripheral blood mononuclear cells HDL (mmol/L) 1.3 6 0.1 1.0 6 0.1* were isolated from patients, healthy control subjects, or F LDL (mmol/L) 2.5 6 0.3 2.3 6 0.3 buffy coats, and were used for the generation of M2M s by adhesion culture, as described previously (11). Triglycerides (mmol/L) 1.6 6 0.2 2.3 6 0.3 Transfection Creatinine (mmol/L) 80.2 6 3.2 85.8 6 6.6 Transfections with mimic or power inhibitor of miR-483- NT-proBNP (ng/L) 96.7 6 36.4 419.9 6 183.5 3p (Ambion and Exiqon, respectively) and silencing RNA for VEZF1 (Ambion) and scrambled oligonucleotide Leukocyte count (103/mL) 7.3 6 0.6 8.4 6 0.8 (Ambion) were performed by electroporation using the Neon Transfection System and Kit (Thermo Fisher Scien- Erythrocyte fi count (103/mL) 4.8 6 0.1 4.7 6 0.1 ti c). Single pulses of 1,400 V for 20 ms (for HAECs) or 1,900 V for 15 ms (for M2MFs) were used. Hb (g/dL) 142.9 6 3.5 141.9 6 31.4 Thrombocyte Quantitative RT-PCR count (103/mL) 247.3 6 20.8 254.7 6 25.3 Total RNA was isolated (miRNeasy; Qiagen) and retro- Table shows differences between patients without T2D with transcribed for quantification of either miRNA (miRCURY CAD and patients with T2D with CAD. CAD + T2D, patients with T2D with CAD; Hb, hemoglobin; LVEF, left ventricular LNA Universal cDNA Synthesis Kit II; Exiqon) or mRNA ejection fraction; NT-proBNP, N-terminal prohormone brain (High Capacity cDNA Reverse Transcription Kit; Applied natriuretic peptide. *P , 0.05; ***P , 0.001; ****P , 0.0001. Biosystems). Quantitative PCR was performed in a Viia7 real-time thermal cycler (for miRNA: miRCURY LNA Uni- versal RT miRNA PCR Kit and corresponding primer sets [Exiqon]; for mRNA: SYBR Select Master Mix [Applied Evans blue under anesthesia, and vessels were harvested Biosystems]; and oligonucleotide primers were synthe- for quantification of persisting endothelial injury (11) and sized at TIB MOLBIOL [Berlin]). analysis of miR-483* expression. Endothelial Cell Gap Closure Assay In Situ Hybridization The capacity of a confluent cell layer of transfected endo- Murine cryopreserved carotid arteries were probed with thelial cells to close a scratched gap within 16 h was miRCURY LNA miRNA In Situ Hybridization Optimiza- assessed as described previously (11). For assessing the tion Kit and probes (sequence: 59-AAGACGGGAGGAGAG effect of miR-483-3p overexpression on M2MF paracrine GAGTGA-39; Exiqon) at Bioneer (Hørsholm, Denmark) effects, untransfected HAECs were used as described and 352 miR-483-3p Counteracts Endothelial Regeneration Diabetes Volume 68, February 2019 transfected M2MFs were added to the freshly scratched secretome analysis. A P , 0.05 was considered significant. HAECs. Box plots show medians with interquartile ranges and ranges. Apoptosis Rate HAECs and M2MFs were cultured and transfected as RESULTS described above. Prior to assessment of the apoptosis Expression of hsa-miR-483-3p Is Increased in M2MFs rate, cells were kept in starvation medium containing of Patients With T2D and Impairs In Vitro a a 10 ng/mL tumor necrosis factor- (TNF- ) for 16 h. Reendothelialization Mitochondrial membrane potential was assessed by JC-1 Expression of hsa-miR-483-3p was higher in M2MFs B B (Supplementary Figs. 4 and 5 ). Binding of annexin V obtained from human patients with T2D compared with and uptake of propidium iodide (both from BioLegend) control subjects without diabetes, both with concomitant fl C were assessed by ow cytometry (Supplementary Figs. 4 CAD (Fig. 1A). hsa-miR-483-3p expression was not differ- C fl and 5 ). Overall cell viability was assessed by ow cytometry ent between subjects without diabetes with CAD and sub- (Zombie Fixable Viability Kit; Life Technologies). jects without diabetes and without CAD. M2MFsobtained from patients with diabetes and prediabetes lose their VEZF1 Quantification capacity to support vascular healing (4). Increased expres- F Protein amount of VEZF1 in HAECs and M2M s was sion levels of miR-483-3p in this cell type might potentially fl assessed by Western blot and immuno uorescence stain- contribute to their dysfunction. Supporting this hypothesis, m ing in 25 g of lysate and adherent cells, respectively, using transfection with a mimic of miR-483-3p (Fig. 1B and anti-VEZF1 (Sigma-Aldrich) and anti-GAPDH antibodies Supplementary Fig. 1) reduced the capacity of transplanted (Western blot; Cell Signaling Technology), or Hoechst M2MFs from healthy human individuals to support the fl 33342 (immuno uorescence staining). ImageJ was used reendothelialization of an injury of defined length inflicted fl for densitometric analysis of protein bands and for uo- upon the common carotid artery in the recipient mice, rescence intensity analysis of VEZF1 in adherent cells. whereas transfection with a power inhibitor of miR-483- 3p partially rescued the capacity of T2D M2MFstosupport Luciferase Assay in vivo reendothelialization (Fig. 1B–D). In situ hybridiza- Cells were cotransfected with mimic of miR-483-3p or tion analysis of human aortic sections verified increased 9 scrambled control oligonucleotide and empty vector, 3 miR-483-3p expression in the vascular wall of human T2D 9 untranslated region (UTR) positive control, or VEZF1 3 patients compared with patients without diabetes with UTR of the GoClone luciferase constructs (Switchgear comparable underlying pathology (Fig. 2). Genomics, La Hulpe, Belgium) according to the manufac- turer protocol, and were plated in 96-well white-bottom Endothelial Expression of mmu-miR-483* Is plates. After 24 h, LightSwitch assay solution was added. Modulated by T2D in Mice and Impairs In Vivo Luminescence was read in a Tecan Infinite F200 Pro (Tecan Reendothelialization GmbH, Grödig, Austria). Results were plotted as log2 Semiquantitative analysis by in situ hybridization indi- ratios. cated elevated levels of mmu-miR-483* in the endothelial and smooth muscle layers of T2D obese (B6.BKS(D)- db Secretome Analysis Lepr /J) mouse aortae (Fig. 3A and B). Intravenous M2MFs were obtained and transfected as described above. injection of an LNA-based inhibitor of mmu-miR 483* Twenty-four hours after transfection, cells were washed decreased miR-483* expression in the vascular wall of with sterile PBS and starvation medium was added diabetic mice (Fig. 3A and B). An induced injury of the (100 mL per 1 3 105 cells). After additional an 24 h, common carotid artery is reendothelialized faster in wild- medium was carefully collected and centrifuged to pellet type mice of the BL/6 background compared with T2D db cell debris, and the supernatant was used for analysis. obese mice of the same background (B6.BKS(D)-Lepr /J) Unconditioned medium was used as a control, and values (Fig. 3C). Systemic injection of inhibitor of the mmu-483* lower than in unconditioned medium were considered to rescued carotid reendothelialization capacity in diabetic be zero. Analytes were quantified using proximity exten- obese mice (Fig. 3A and C). No effect on cardiac capil- sion assay by Olink Proteomics (Uppsala, Sweden). larization or arteriole number was observed in the treated mice (Supplementary Fig. 2). Statistical Analysis Depending on the matching of samples and the normality Cell Type–Specific Upregulation of hsa-miR-483-3p in of distribution (D’Agostino-Pearson test), paired or un- HAECs and M2MFs paired t test, Wilcoxon signed rank test, or Mann-Whitney Exposure of HAECs and M2MFs to stimuli mimicking U test was used to compare two groups. Three or more aspects of increased inflammatory state (TNF-a)andmet- groups of unmatched samples were compared by Kruskal- abolic dysregulation (hyperinsulinemia, hyperglycemia, and Wallis test or one-way ANOVA, depending on normality. lipotoxicity), all of which are considered to mediate specific Two-way ANOVA followed by Sidak post-test was used for mechanisms of vascular dysfunction in T2D, resulted in diabetes.diabetesjournals.org Kuschnerus and Associates 353

Figure 1—Upregulation of miR-483-3p in T2D M2MFs and effect on endothelial regeneration. A: Increased miR-483-3p levels were observed in M2MFs isolated from human (H) patients with T2D in comparison with subjects without diabetes, with concomitant cardio- vascular disease (n =7pergroup).B–D: Transfection of human M2MFs with mimic of miR-483-3p (mi483) reduced their capacity to support reendothelialization in a mouse model of induced vascular injury (n = 9/10 per group). the upregulation of hsa-miR-483-3p expression in a cell was observed for palmitic acid, mimicking lipotoxicity (Fig. type–specific manner. In endothelial cells, hyperinsulinemia 4A). For M2MFs, TNF-a was the main stimulus, whereas strongly upregulated miR-483-3p, and a trend (P =0.051) palmitic acid induced a downregulation of miR-483-3p (Fig.

Figure 2—miR-483-3p is upregulated in endothelial and vascular smooth muscle cells of the aortic wall of human T2D patients. miR-483-3p expression was stronger in aortic sections of human patients with T2D (C, E, and G) than in those of patients without diabetes with similar vascular pathology (D, F, and G), as assessed by in situ hybridization in vascular wall of human aorta samples (n = 5 [CAD]; n = 7 [CAD + T2D]). Representative images of vascular morphology (A, B, E, and F) and miR-483-3p signal (C, D, E, and F), as well as the red and green fluorescence channels (C and D) are shown. miR-483-3p is visible in pink in panels E and F, and in red fluorescence in panels C and D. Arrows indicate examples of miR-483-3p. Overlay of images obtained in the red and green channels results in yellow/orange appearance of lamina/ vascular smooth muscle cell layer (autofluorescence). aSMA, a-smooth muscle actin. 354 miR-483-3p Counteracts Endothelial Regeneration Diabetes Volume 68, February 2019

Figure 3—mmu-483* is upregulated in endothelial and vascular smooth muscle cells of murine carotid arteries in T2D and affects endothelial regeneration. A and B: miR-483-3p expression was stronger in carotid arteries of db/db mice as assessed by in situ hybridization (n =8 per group). A and C: Reendothelialization of a deendothelialized area of the arteria carotis communis progressed slower in diabetic db/db mice than in wild-type animals (n =7–10 per group). C: Systemic treatment with an inhibitor of mmu-miR-483* (anti483) rescued reendothelialization response in the diabetic animals (n = 9 per group).

4B). Hyperglycemia did not result in a significant upregu- endothelial cell layer was less efficient in the presence of lation of miR-483-3p in our models. M2MFs from healthy human donors transfected with a mimic of miR-483-3p compared with scrambled control- miR-483-3p Modulates Cellular Functions Relevant to transfected M2MFs from the same donors (Fig. 5B). The Reendothelialization closure of a scratch injury within an endothelial monolayer HAECs transfected with a mimic of miR-483-3p were severely relies on several cellular functions, including migration, pro- impaired in their capacity to effect reendothelialization (Fig. liferation, and cell death. Using the fluorescent label CFSE 5A). In separate experiments, closure of a scratch in the (carboxyfluorescein succinimidyl ester), the proliferation of

Figure 4—Factors inducing miR-483-3p upregulation in endothelial cells and M2MFs. HAECs and M2MFs were exposed in vitro to stimuli mimicking inflammation, hyperinsulinemia, hyperglycemia (HG), and lipotoxicity. Upregulation of miR-483-3p was observed upon challenge with high insulin and with palmitic acid (PalmAc) in endothelial cells (A)(n = 8 per condition), whereas in M2MFs(B)(n = 8 per condition), miR-483-3p was upregulated upon treatment with TNF-a. StM, starvation medium (control). diabetes.diabetesjournals.org Kuschnerus and Associates 355

Figure 5—MiR-483-3p impairs reendothelialization and cell survival. Transfection with mimic of miR-483-3p reduced the capacity of HAECs to reendothelialize a decellularized area under limiting conditions in vitro (A)(n = 7 per group) or the capacity of M2MF to support reendothelialization by HAECs (B)(n =6–9 per group). An increase of intracellular miR-483-3p levels led to an increased induction of apoptosis-associated processes (loss of mitochondrial membrane potential [C and D], externalization of phosphatidylserine [E and F], disruption of cellular membrane integrity [G and H] in HAECs [A, C, E, and G][n = 10] and in M2MFs[B, D, F, and H][n =8–10]). I: The proresolving factors TIMD4 and LYVE1 were released to a greater degree by M2MFs transfected with a power inhibitor of miR-483-3p (anti483) and to a lesser degree after transfection with a mimic of miR-483-3p (mi483) (n = 4, matched biological replicates). I: The factors IL-7R, TNC, MET, PRSS2, and FCN2 (ficolin-2), which might have a role in the proinflammatory activation of M2MFs, are released in higher amounts after transfection with a mimic of miR-483-3p (mi483) and in lower amounts after transfection with a power inhibitor of miR-483-3p (anti483). HAEC, human aortic endothelial cells.

cells transfected with scrambled oligonucleotide or a mimic and subjected to starvation, and apoptosis-associated of miR-483-3p in fully supplemented growth medium was characteristics were analyzed. The disruption of mito- traced. No significant differences were observed for CFSE chondrial membrane potential was examined by JC-1 fluorescence between scrambled and mimic-transfected staining, phosphatidylserine exposure on the cell surface HAECs after 48 h (Supplementary Fig. 3). HAECs and by annexin V binding in the absence of propidium iodide M2MFs were transfected with a mimic of miR-483-3p uptake, and the disruption of cell membrane integrity by 356 miR-483-3p Counteracts Endothelial Regeneration Diabetes Volume 68, February 2019 propidium iodide uptake (Supplementary Figs. 4 and 5). response to injury in M2MFs and HAECs, including the In HAECs and M2MFs, miR-483-3p overexpression led secreted growth factors IGF-I, platelet-derived growth to an increase in those markers of compromised cell factor-b, and kit ligand; as well as apoptosis-related viability (Fig. 5C–H). By proximity extension assay, we factors FAIM (Fas apoptosis inhibitory molecule), BIRC6 analyzed the release of 92 with cardiometabolic (baculoviral IAP repeat-containing protein 6), RNF 32 iso- relevance by M2MFs transfected with either mimic or form 2, GUCY1 (guanylate cyclases, soluble), PIP5KA power inhibitor of hsa-miR-483-3p or scrambled oligo- (phosphatidylinositol 4-phosphate-5 kinase A), SAMD9 nucleotide (Supplementary Fig. 6). We identified the (SAM domain–containing protein 9), CFLAR (CASP8 GAS6 gene to be reduced, and ICAM1, CES1, QPCT, and FADD-like apoptosis regulator), NOL3 (nucleolar pro- IL-7R, and MET to be increased in the supernatant tein 3), and ANGPTL7 (angiopoietin-related protein 7); of M2MFs transfected with a mimic of hsa-miR-483-3p and the receptors TrkC (tropomyosin receptor kinase C), (Supplementary Fig. 6A). When assessing the opposite B2R (bradykinin receptor B2), and TGFb-R (transform- regulation of protein release by miR-483-3p overexpres- ing growth factor-b receptor). Of those, only VEZF1, sion versus inhibition, a reduction in the release of lym- a previously associated with endo- phatic vessel endothelial hyaluronan receptor 1 (LYVE1) thelial dysfunction in diabetes (25), showed a significant and T-cell Ig and mucin domain containing 4 (TIMD4) with repression in mimic-transfected HAECs and M2MFs miR-483-3p overexpression and their increased release compared with scrambled-transfected controls (Fig. 6A– with miR-483-3p was observed (Fig. 5I and Supplemen- D and Supplementary Figs. 7–9). miR-483-3p is predicted tary Fig. 6B). Vice versa, the release of interleukin (IL)-7R to bind to a sequence in the 39 UTR of the VEZF1 transcript (IL-7R), tenascin-C (TNC), the hepatocyte growth factor conserved among mammals (Supplementary Fig. 10). receptorMET,theserineproteasePRSS2,andficolin-2 We could verify direct targeting of the VEZF1 39 UTR by was enhanced with miR-483-3p overexpression and re- miR-483-3p by luciferase reporter assay (Fig. 6E). VEZF1 duced with miR-483-3p inhibition (Fig. 5I and Supple- expression was reduced in aortic homogenates of human mentary Fig. 6B). CAD patients with T2D compared with nondiabetic CAD patients (Supplementary Fig. 11A). Finally, systemic VEZF1 Is a Target Protein of miR-483-3p injection of power inhibitor of mmu-miR-483* rescued In silico analyses have indicated a number of potential vascular VEZF1 expression, which was barely detectable targets of miR-483-3p, which might influence the vascular in aortae of db/db mice (Fig. 6F and Supplementary Fig.

Figure 6—VEZF1 is a target protein of miR-483-3p. VEZF1 is reduced in HAECs (A)(n = 4) and M2MFs(B)(n = 21) after transfection with miR- 483-3p mimic (mi483) at mRNA (A and B) and protein (C and D)(n = 6 per group) levels. Luciferase reporter assay confirmed miR-483-3p to directly interact with the VEZF1 promotor (E)(n = 8). In diabetic db/db mice, systemic inhibition of mmu-miR-483* (anti483) rescued VEZF1 expression (F)(n =15–16). Silencing of VEZF1 in HAECs (siVEZF1) mimicked the effect of miR-483-3p overexpression, impairing HAEC survival (G)(n =17–18) and reendothelialization capacity in vitro (H)(n =19–20). HAEC, human aortic endothelial cells. diabetes.diabetesjournals.org Kuschnerus and Associates 357

11B). Silencing of VEZF1 in HAECs increased cell death suggest potential further mechanisms of miR-483-3p tran- (Fig. 6G) and reduced in vitro reendothelialization ca- scriptional regulation by insulin-responsive/sensitive ele- pacity (Fig. 6H). ments and/or by inflammatory transcription factors, such as nuclear factor-kB. Together with the cell type–specific DISCUSSION differences in target genes and functional effects of miR- 483-3p, our data therefore suggest that also transcrip- A dysfunctional vascular response to injury has been de- tional regulation of miR-483-3p depends on additional scribed for patients and animal models of T2D and is factors, which differ between cell types and pathological associated with higher cardiovascular morbidity and mor- settings (33). tality in patients with diabetes (26). Among several candidates, we have identified VEZF1 as In this translational study, we have observed that miR- a target of miR-483-3p. This transcription factor is essen- 483-3p , which is extremely low abundant in nondiabetic tial for embryonic angiogenesis (34). Our own present and conditions, is upregulated in individuals with T2D, both previous findings indicate a role of VEZF1 not only in em- murine and human. Consequently, we have investigated bryonic vascular development, but also in the maintenance of fi the signi cance of this upregulation in the vasculature and the adult endothelium and especially in diabetes-associated F in M2M s. Our study thereby addresses two aspects endothelial dysfunction (25). VEZF1 has been identified as — relevant to the maintenance of vascular integrity the a coactivator of IL-3 expression (35). However, we could not regenerative capacity of the endothelial layer, and the detect changes in IL-3 protein levels in supernatant of modulation of endothelial survival and function by alter- M2MFs after the inhibition of miR-483-3p or after the F fi natively activated M2M s. Our ndings support the silencing of VEZF1 (Supplementary Fig. 12). Aitsebaomo hypothesis that elevated levels of miR-483-3p impair et al. (36) have described mechanistically distinct effects of endothelial cell survival under stress, thereby limiting VEZF1 on the transcriptional regulation of endothelin-1 and fi vascular repair capacity upon injury. We have identi ed IL-3, depending on differential effects of the transcrip- – miR-483-3p mediated downregulation of the vascular tional activator Tax. Further potential actions of VEZF1 transcription factor VEZF1 as a key mechanism for the include the repression of the antiangiogenic factor CITED2 fi loss of endothelial regeneration ef ciency in T2D. Vice (37), which we could not verify in our setting, and the versa, the inhibition of miR-483-3p restored endothelial upregulation of the proangiogenic factor strathmin (38). repair capacity in vitro and in vivo, either in the endo- By extension, those data illustrate that the presence of thelium via systemic delivery of power inhibitor of miR- additional cofactors and the baseline expression levels of 483-3p or by transplanting ex vivo transfected human target genes modulate the target spectrum of VEZF1 in a cell M2MFs into a murine model of vascular injury. type–specific manner. miR-483-3p has been studied mainly in the field of In addition to modulating target gene transcription, cancer and only recently for cardiovascular and metabolic VEZF1 has been shown to affect DNA methylation and diseases (18,19,23,27,28). Although miR-483-3p is upreg- elongation during transcription, via regulating the expres- ulated in certain tumors as well as in diabetes, its role can sion of the full-length versus the short isoform of DNA change from a factor supporting tumor cell proliferation methyltransferase Dnmt3b1 (39). In a subsequent study and survival in cancer to inducing or permitting apopto- (40), the same group identifiedVEZF1tobindtoCpG sis in a cardiovascular setting (22,23,27,29,30). islands, thereby protectingthemfromdenovoDNA Studies from the field of cancer biology have shown that methylation. These findings are in line with observations beyond transcriptional regulation together with its host of aberrant DNA methylation being involved in the path- gene, IGF2, miR-483-3p transcription can be regulated ogenesis of type 1 diabetes and T2D (41), which is assumed to independently of IGF2 transcription via b-catenin/Wnt play a role in phenomena like the higher frequency of T2D in signaling (31). b-Catenin/Wnt signaling is also enhanced children born to mothers suffering from malnutrition during via high glucose in diabetes, thus providing a link for the pregnancy (42). Interestingly, miR-483-3p expression in upregulation of miR-483-3p in both T2D and tumors, adipose tissue of low–birth weight adult humans and in albeit with different downstream effects on cell prolifer- prediabetic adult rats that had been exposed to suboptimal ation and survival. Increased glucose levels have also been nutrition in early life was elevated, thus suggesting an described to enhance miR-483-3p transcriptional activa- epigenetic priming by nutrition in early life (17). One might tion via the O-linked N-acetylglucosamine transferase, therefore speculate about the existence of an epigenetic which stabilizes the transcriptional complex at the miR- feedback mechanism that aggravates both the causes and 483 promoter (32). Those findings point to a crucial role of effects of miR-483-3p overexpression in pre-T2D. glucose levels for the transcription of miR-483-3p. Al- As is typical for miRNAs, miR-483-3p also has multiple though our data tentatively suggest increased expression targets, with greater or lesser relevance in certain cell types of miR-483-3p in HAECs and in M2MFs after exposure to and pathological/physiological situations. Of a number of hyperglycemia, this effect was not significant, in contrast in silico–predicted and previously described potential tar- to hyperinsulinemia in HAECs and the inflammatory gets, only the transcription factor VEZF1 was verified by stimulus TNF-a in M2MFs. Our data would therefore our analyses in macrovascular endothelial cells and M2- 358 miR-483-3p Counteracts Endothelial Regeneration Diabetes Volume 68, February 2019 type macrophages. Other targets, such as IGF-I (22), were including microvesicle shedding and release of apoptotic not confirmed by our analyses (Supplementary Fig. 7 and bodies during apoptosis. Supplementary Table 4), potentially because of cell type– Our in situ hybridization analyses have also revealed an specific differences in basal target and in expression and regulation of miR-483-3p in the vascular the presence of transcriptional coregulators. smooth muscle layer of human aortic biopsy specimens. In order to better understand the paracrine mecha- The overall vascular effects of miR-483-3p therefore likely nisms affecting endothelial regeneration upon miR-483-3p exceed the mechanisms described here for impairment of modulation in M2MFs, we have used a proximity exten- endothelial regeneration and might potentially also in- sion assay to analyze a panel of 92 proteins with cardio- clude the regulation of vascular tone. metabolic relevance. Upon miR-483-3p overexpression, Therefore, our observations, together with reports by reduced amounts of the hyaluronan receptor LYVE1, as other working groups (18,22,23), indicate a potential for well as the phosphatidylserine receptor TIMD4 were re- the therapeutic downregulation of miR-483-3p in patients leased, which have both been reported to support pro- with T2D. This particular patient population might benefit resolving macrophage functions, such as efferocytosis from improving their response to vascular injury, as is for (43,44). Vice versa, both membrane receptors were re- example evoked by percutaneous catheter intervention, leased in greater amount upon miR-483-3p inhibition. a frequent clinical intervention in patients with T2D be- This could, in principle, be explained by increased shedding cause of their higher risk for coronary artery disease (2). of microvesicles from the cell surface or disintegration of Recent advances in RNA therapeutics have raised hopes the cell into apoptotic bodies, as our preparation removed that these approaches might be translated into a realistic larger cell debris. Because of the opposite direction of treatment option in the future (51), especially as the en- change in apoptosis rate in our setting, microvesicle release dothelium is an organ readily addressable with current might be more plausible, although further studies are transfection approaches. needed to verify this hypothesis. In addition, the IL-7R, TNC, the hepatocyte growth factor receptor MET, the serine protease PRSS2, and ficolin-2 were released in Acknowledgments. The authors thank Sabine Knüppel (Charité– higher amounts with elevated miR-483-3p levels and re- Universitätsmedizin Berlin) for excellent technical support. The authors also leased in lower amounts with reduced miR-483-3p levels. thank iPATH.Berlin, Immunopathology for Experimental Models, core unit of For those factors, we assume that their increased release the Charité–Universitätsmedizin Berlin, for support with the histopathological from miR-483-3p–overexpressing M2MFs and reciprocal analysis. regulation by a power inhibitor of miR-483-3p reflect Funding. The project was supported by the Novartis Foundation (grant 11A26), a response to increased activation and/or death and the Bank Vontobel Foundation, the German Center for Cardiovascular Research, partner site Berlin (grant FKZ 81Z2100202), and the Zurich Heart House– the resulting release of proinflammatory cytokines and Foundation for Cardiovascular Research. K.K. was supported by the Deutsche damage-associated molecular patterns, which then activate Herzstiftung (Kaltenbach Stipendium) and the EMDO Foundation. T.F.L. is sup- neighboring cells. In support of this hypothesis, IL-7R ported by the Swiss National Science Foundation (SNSF) (grant 310030_166576). and MET have been shown to be upregulated by lipopo- U.L. is a professor at the Berlin Institute of Health. N.K. was supported by the SNSF lysaccharides, IL-1b,andTNF-a (45–47). TNC was (ambizione fellowship PZ00P3_126621). reported to be upregulated under pathological conditions Duality of Interest. No potential conflicts of interest relevant to this article caused by inflammation and infection (48). Little is known were reported. about the role of the serine protease PRSS2 in macrophage Author Contributions. K.K. designed the experiments, obtained patient function, but it has been observed to be upregulated in material, performed cell culture and animal experiments and data analysis, wrote Mycobacterium bovis–infected, bovine monocyte–derived the article, and reviewed and approved the article. E.T.S. and M.F.M. obtained macrophages (49). The opsonin ficolin-2 activates the patient material, performed cell culture and animal experiments, and reviewed and approved the article. T.F.L. and U.L. contributed to and discussed research lectin complement pathway and has recently been identi- fi fl strategy and data interpretation and reviewed and approved the article. N.K. ed to stimulate macrophage proin ammatory M1 polar- conceived and supervised the project, designed the experiments, performed data ization through toll-like receptor four activation (50). analysis, and wrote the article. K.K. and N.K. are the guarantors of this work and, Those initial data would tentatively support a role of as such, had full access to all the data in the study and take responsibility for the PRSS2 and ficolin-2 in (secondary) macrophage activation integrity of the data and the accuracy of the data analysis. upon miR-483-3p upregulation, as speculated for IL7-R, TNC, and MET. Of note, we do not have information on References the molecular mechanisms causing the altered secretion pro- 1. Sarwar N, Gao P, Seshasai SR, et al.; Emerging Risk Factors Collabo- file upon miR-483-3p manipulation. The proteins identified ration. 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