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Thrombospondin-1 induced vascular smooth muscle cell migration and proliferation are functionally dependent on microRNA-21 Jeffrey J. Stein, MD,a,b Chinenye Iwuchukwu, MD, MPH,a,b Kristopher G. Maier, PhD,a,b and Vivian Gahtan, MD,a,b Syracuse, NY

Objectives. Thrombospondin-1 (TSP-1) is a matricellular glycoprotein released from platelets at sites of arterial injury and is important in neointima development after balloon angioplasty. MicroRNAs are small noncoding RNAs that function by binding target gene mRNA and inhibiting protein translation. MicroRNA-21 (miR-21) is up-regulated after angioplasty, and inhibition of miR-21 leads to decreased intimal hyperplasia. In this study, we examined the effects of miR-21 inhibition on vascular smooth muscle cell (VSMC) processes. Methods. VSMCs were exposed to TSP-1 and miR-21 inhibitor for 20 minutes. TSP-1 induced migration was assessed with a modified Boyden microchemotaxis chamber and proliferation with calceinAM fluorescence. Phosphorylated extracellular signaling kinase (ERK) 1/2 expression was determined by Western Blot and densitometry. Quantitative real-time polymerase chain reaction for TSP-1, hyaluronic acid synthase 2 (HAS2), and transforming growth factor beta 2 (TGFb2) was performed. Statistical analysis was performed with analysis of variance (P < .05). Results. Inhibition of miR-21 blocked TSP-1 induced VSMC migration, proliferation, and ERK 1/2 phosphorylation (P < .05) and had no effect on TSP-1 stimulated expression of genes for TSP-1, HAS2, or TGFb2 (P > .05). Conclusion. Acute inhibition of miR-21 led to a decrease in VSMC migration and proliferation caused by TSP-1. The decrease in TSP-1’s activation of ERK 1/2 after acute miR-21 inhibition indicates an active role for miR-21 in TSP-1’s cell signaling cascade. No effect on TSP-1-induced expression of the pro-stenotic genes thbs1, tgfb2, or has2, occurred after acute miR-21 inhibition. These data indicate that miR-21 directly modulates cell function and signaling pathways in ways other than inhibition of protein translation. (Surgery 2013;j:j-j.) From the Department of Surgery,a SUNY Upstate Medical University; and Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse,b Syracuse, NY

PERIPHERAL ARTERY DISEASE is a growing problem that is treated by endovascular intervention with balloon angioplasty or stenting to improve arterial flow to the extremities. This procedure, however, leads to a local vascular injury that can result in

Supported by Veteran’s Affairs Merit Award # 1I01BX00124301A2 and a Bridge Grant from SUNY Upstate Medical University. K.G.M and V.G contributed equally to this work. Accepted for publication August 12, 2013. Reprint requests: Vivian Gahtan, MD, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2013.08.003

the development of intimal hyperplasia (IH), resulting in restenosis and reconstructive failure. MicroRNAs are small, noncoding RNAs that recently have been discovered to regulate genes at the posttranscriptional level by binding the 39-untranslated regions of mRNA.1-3 Wang et al4 reported that miR-21 was the most up-regulated microRNA in human atherosclerotic arteries compared with normal arteries. In a rat carotid artery injury model, blocking miR-21 inhibited neointima formation after angioplasty and proliferation of cultured vascular smooth muscle cells (VSMCs).5 In addition, inhibition of miR-21 lead to a reduction in platelet-derived growth factor-BB induced VSMC migration and proliferation,4 a growth factor important in IH. We have shown that in the presence of hyperglycemia, thrombospondin-1 (TSP-1) stimulates the expression of miR-21 by SURGERY 1

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Fig 1. Diagram depicting how TSP-1 may regulate miR21 and ERK 1/2 ineraction to stimulate VSMC migration and proliferation.

VSMCs,6 which is important because chronic hyperglycemia such as that seen in diabetes contributes to vascular disease.6 TSP-1, a 420-kDa matricellular glycoprotein constituent of platelet alpha granules, is released at sites of local vascular injury and is known to contribute to the development of IH.7 The functional importance of TSP-1 in IH was previously shown by Moura et al,7 who demonstrated that TSP-1 knockout mice had attenuated IH development after arterial injury. Furthermore, TSP-1 can stimulate VSMC migration and proliferation, both of which contribute to IH formation.8-10 TSP-1 induced VSMC migration is dependent on activation of the extracellular-signal regulated kinase 1/2 (ERK 1/2) pathway (Fig 1).8 We have shown in a previous microarray analysis that TSP-1 (thbs1), transforming growth factor 2 (tgfb2), and hyaluronan synthase 2 (has2) genes were up-regulated by TSP-1 at 6 hours.6 The purpose of this study was to examine the effects of miR-21 inhibition on TSP-1 induced VSMC migration, proliferation, and cellular signaling. Our hypothesis was that miR-21 is necessary for TSP-1 to stimulate VSMC migration, proliferation, and prostenotic gene expression (thbs1, tgfb2, has2), and that these processes will correlate with ERK 1/2 phosphorylation. METHODS Materials. TSP-1 was purchased from Athens Biochemical (Athens, GA). miR-21 inhibitor was purchased from Thermo Scientific Dharmacon (Lafayette, CO). Human aortic VSMCs, serumfree media (SFM, Dulbecco’s Modified Eagle Medium), and basal medium (BM) were purchased from Cell Applications (San Diego, CA). Western

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blotting materials were purchased from Bio-Rad (Hercules, CA). Phosphorylated ERK, b-tubulin, and goat anti-rabbit antibodies were purchased from Cell Signaling (Danvers, MA). The cDNA reverse transcriptase kit and polymerase chain reaction (PCR) probes for thbs1, tgfb2, and has2 were purchased from Applied Biosystems (Austin, TX). The cyclophilin A PCR probe was used as the control (ThermoFisher-Dharmacon, Lafayette, CO). SYBR Green master mix for quantitative realtime (qRT)-PCR was purchased (Bio-Rad). Cell treatment. For all experiments, VSMCs were made quiescent by culturing in SFM or BM for 48 hours. Then, VSMCs were pretreated for 20 minutes with 25 nM miR-21 inhibitor. 20 mg/mL of TSP-1 was used in all experiments. Migration assay. Migration of VSMCs (50,000 cells per well) pretreated with miR-21 inhibitor towards TSP-1 was examined with a modified Boyden microchemotaxis chamber as previously described.11 The migration assay was run for 4 hours in triplicate and repeated 3 times. Results were recorded as cells migrated per 5 high power fields and then converted to percent positive control. Proliferation assay. Quiescent cells (2,500 cells/ well) were cultured in SFM with 0.2% fetal bovine serum in a 384-well plate. SFM-treated and miR-21 inhibitor pretreated VSMCs were exposed to TSP1 for 48 hours. They were then exposed to 20 mM Calcien-AM (Sigma-Aldrich, St. Louis, MO) for 20 minutes at 378C. Results were read using a BioTek Flourescent plate reader (Winooski, VT) at 485 nm excitation and 528-nm emission and converted to percent negative control. Each experiment was done in triplicate and repeated 3 times. ERK activation Western blot. SFM treated and miR-21 inhibitor pretreated VSMCs were exposed to TSP-1 for 20 minutes. The cells were lysed using RIPA buffer (Sigma-Aldrich, St. Louis, MO), spun at 14,500g for 10 minutes, and the supernatant was collected. The supernatant was electrophoresed on 18.5% Tris-HCl gel, and the protein was then transferred to nitrocellulose blotting paper. The blots were probed with a phosphorylated ERK 1/2 antibody, which was then developed on X-ray film. Densitometry was normalized to background and b-tubulin, and results recorded as percent negative control. qRT-PCR. BM-treated and miR-21 inhibitor pretreated VSMCs were exposed to TSP-1 for 6 hours or 24 hours (n = 6). Total RNA was extracted with the use of a QIAGEN miRNeasy kit (Valencia, CA). Quality analysis of the RNA was performed with an Agilent 2100 Bioanalyzer (Santa Clara, CA). cDNA was made from RNA and then probed for expression of thbs1, tgfb2, and has2 on a Light

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Fig 2. TSP-1 increases VSMC migration. miR-21 inhibitor pretreatment returns TSP-1 stimulated VSMC migration to levels comparable with SFM.

Fig 3. VSMC proliferation is stimulated by exposure to TSP-1. Pretreatment with a miR-21 inhibitor before exposure to TSP-1 results in VSMC proliferation levels comparable with SFM.

Cycler 1800 qRT-PCR machine (Roche Applied Science, Indianapolis, IN). Results are displayed as relative quantity (RQ) of expressed gene. Each experiment was done in triplicate and repeated 3 times. Statistics. All results are expressed as ± SEM mean. All statistical comparisons are made to the positive control, TSP-1. Significance was assessed by the use of ANOVA. RESULTS miR-21 inhibition reduced TSP-1 induced VSMC migration (Fig 2). TSP-1 stimulated VSMC migration greater than SFM alone (100% ± 9.6 vs 43.7% ± 7.4, P < .001). When pretreated with the miR-21 inhibitor, TSP-1 induced VSMC migration returned to levels consistent with the negative control (44.7% ± 4.9 vs 43.7% ± 7.4, P = .8931). miR-21 inhibition prevents TSP-1 induced VSMC proliferation (Fig 3). VSMC proliferation

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was stimulated by TSP-1 compared with SFM alone (100% ± 3.7 vs 78.8% ± 2.9, P = .0026). Pretreatment with miR-21 inhibitor decreased TSP-1 induced VSMC proliferation to levels consistent with SFM alone (77.2% ± 6.3 vs 78.8% ± 2.9, P = .6861). ERK 1/2 activation by TSP-1 is reduced by miR-21 inhibition (Fig 4, A and B). TSP-1 increased ERK activation (phosphorylated ERK) in VSMCs compared with negative control for both ERK 1 (100% ± 0 vs 78.6% ± 6.3, P = .0125) and ERK 2 (100% ± 0 vs 76.2% ± 2.3, P = .0284). Pretreatment with miR-21 inhibitor decreased TSP-1 activation of ERK 1 (77% ± 6.2 vs 100% ± 0, P = .0093) and ERK 2 (71.1% ± 9.8 vs 100% ± 0, P = .0135). miR-21 inhibition has no effect on TSP1 induced thbs1 expression. Treatment of VSMCs for 6 hours with TSP-1 increased the expression of thbs1 compared with BM (average RQ 1.8 vs 1, P = .0003). miR-21 inhibition had no effect on thbs1 expression by TSP-1 (average RQ 1.6 vs 1.8, P = .2925; Fig 5, A). VSMCs treated for 24 hours with TSP-1 did not alter the expression of thbs1 compared with BM (average RQ 1.2 vs 1, P = .6848). Similarly, miR-21 inhibtion had no effect on thbs1 expression compared with TSP-1 (average RQ 0.62 vs 1.2 P = .1649; Fig 5, B). miR-21 inhibition has no effect on TSP1 induced tgfb2 expression. Treatment of VSMCs for 6 hours with TSP-1 increased the expression of tgfb2 compared with BM (average RQ 1.4 vs 1.0, P = .0300; Fig 6, A). Inhibition of miR-21 had no effect on the 6-hour expression level compared to TSP-1 (average RQ 1.6 vs 1.4, P = .2871). Treatment of VSMCs for 24 hours with TSP-1 decreased the expression of tgfb2 compared with BM (average RQ 0.43 vs 1.0, P = .0161) and was not altered by miR-21 inhibition compared with TSP-1 (average RQ 0.26 vs 0.43, P = 1361; Fig 6, B). miR-21 inhibition has no effect on TSP1 induced has2 expression. Treatment of VSMCs for 6 hours with TSP-1 increased the expression of has2 compared with BM (average RQ 2.5 vs 1.0, P = .0024; Fig 7, A). Inhibition of miR-21 had no effect on the 6-hour expression level compared with TSP1 (average RQ 2.1 vs 2.5, P = .5413). Treatment of VSMCs for 24 hours with TSP-1 had no significant effect on the expression of has2 compared with BM (average RQ 0.5 vs 1.0, P = .4822) and was not altered by miR-21 inhibtion compared with TSP-1 (average RQ 0.46 vs 0.50, P = .5933; Fig 7, B). DISCUSSION TSP-1 is known to stimulate VSMC functions important for the development of IH, including migration, proliferation, and certain cellular

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Fig 4. (A) A representative Western blot of ERK 1/2 phosphorylation in VSMCs exposed to SFM, TSP-1, or a miR-21 inhibitor followed by TSP-1. b-tubulin served as the loading control. (B) Combined densitometry showing that exposure to TSP-1 results in an increase in activation of ERK 1 and 2. Pretreatment with a miR-21 inhibitor decreased TSP-1 activation of ERK 1 and 2 to levels comparable with SFM.

Fig 5. (A) TSP-1 increases expression of its own gene, thbs1 in VSMCs compared with BM at 6 hours. Pretreatment with a miR-21 inhibitor had no effect on TSP-1 induced thbs1 expression. (B) TSP-1 does not increase the expression of thbs1 in VSMCs compared with BM at 24 hours. Pretreatment with a miR-21 inhibitor had no effect on TSP-1 induced thbs1 expression level at 24 hours.

signaling. Recently recognized are microRNAs, which affect protein translation, are important in the development of vascular diseases, and are

being studied as therapeutic targets.12 miR-21 is one such microRNA, and its role in the development of IH is well established.4,13-18 The current

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Fig 6. (A) TSP-1 increases expression of the prostenotic gene tgfb2 at 6 hours. Pretreatment with miR-21 inhibitor did not affect TSP-1 induced tgfb2 expression at 6 hours. (B) TSP-1 decreases the expression of tgfb2 in VSMCs compared with BM at 24 hours. Pretreatment with a miR-21 inhibitor had no effect on TSP-1-induced tgfb2 expression level at 24 hours.

Fig 7. (A) TSP-1 stimulates expression of the prostenotic gene has2 at 6 hours. Pretreatment with miR-21 inhibitor had no affect on TSP-1 induced has2 expression at 6 hours. (B) TSP-1 decreases the expression of has2 in VSMCs compared with BM at 24 hours. Pretreatment with a miR-21 inhibitor had no effect on TSP-1-induced has2 expression level at 24 hours.

study demonstrated that miR-21 inhibition blocked TSP-1 induced VSMC migration and proliferation. Because our laboratory has previously shown that TSP-1 induces phosphorylation of ERK1/2 in VSMCs and TSP-1 induced VSMC migration is dependent on ERK 1/2,19 we investigated the effect of miR-21 inhibition on TSP-1 stimulated phosphorylation of ERK 1/2. The results show that TSP-1 induced ERK 1/2 phosphorylation requires active miR-21. Finally, miR-21 activity was not required for TSP-1 to upregulate the expression of the prostenotic genes thbs1, tgfb2, or has2 after 6 hours (an early acutephase time point). Thus, that inhibition of miR21 inhibits TSP-1-induced ERK 1/2 activation but not prostenotic gene expression would indicate that the expression of these genes is independent of ERK 1/2 signaling. We further examined the effect of TSP-1 on prostenotic gene expression in VSMCs at 24 hours. We found that at 24 hours, TSP-1 did not enhance

the expression of thbs1, tgfb2, or has2 and that inhibition of miR-21 had no effect. Indeed, in the TSP1 treated samples the expression of tgfb2, and has2 was reduced compared with the negative control. It is likely that by 24 hours the RNA has been translated into protein which causes a negative feedback loop to halt transcription to maintain homeostasis. These findings are in line with the concept that TSP-1 acts primarily as an acute phase reactant.7 Interestingly, microRNAs were previously thought to only work at a translational level by binding mRNA in the 39-untranslated region. However, using a 20-minute pretreatment with miR-21 inhibitor, we were able to prevent TSP1 induced VSMC migration, proliferation, and signal transduction for ERK1/2. We believe that such a short treatment should not result in an effect on protein translation, implying that microRNAs may be able to affect cellular functions using a different mechanism yet to be determined.

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Indeed, our findings suggest that miR-21 also may affect cellular functions on a shorter time scale and perhaps may modulate cellular functions through pathways other than inhibition of protein translation.

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Thrombospondin-1-induced vascular smooth muscle cell migration and proliferation are functionally dependent on microRNA-21.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein released from platelets at sites of arterial injury and is important in neointima development...
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