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. 2013 Dec 3;22(2):378–389. doi: 10.1038/mt.2013.243

Repression of Choroidal Neovascularization Through Actin Cytoskeleton Pathways by MicroRNA-24

Qinbo Zhou 1, Chastain Anderson 1, Hongmei Zhang 2, Xinyu Li 2, Fiona Inglis 1, Ashwath Jayagopal 3, Shusheng Wang 1,4,*
PMCID: PMC3916039  PMID: 24297048

Abstract

Actin cytoskeleton is critical for cell motility and division, both of which are important for angiogenesis. MicroRNAs (miRNA/miR) are emerging as pivotal modulators of vascular development and disease. How miRNAs regulate actin cytoskeleton dynamics in endothelial cells (EC) and neovascularization is still unclear. Here, we report that miR-24 regulates actin dynamics in ECs through targeting multiple members downstream of Rho signaling, including Pak4, Limk2, and Diaph1 proteins. Overexpression of miR-24 in ECs blocks stress fiber and lamellipodia formation, represses EC migration, proliferation, and tube formation in vitro, as well as angiogenesis in an ex vivo aortic ring assay. Moreover, subretinal delivery of miR-24 mimics represses laser-induced choroidal neovascularization (CNV) in vivo. Mechanistically, knockdown of miR-24 target protein LIMK2 or PAK4 inhibits stress fiber formation and tube formation in vitro, mimicking miR-24 overexpression phenotype in angiogenesis, while overexpression of LIMK2 and PAK4 by adenoviruses partially rescued the tube formation defects in miR-24 overexpressing ECs. Taken together, these findings suggest that miR-24 represses angiogenesis by simultaneously regulating multiple components in the actin cytoskeleton pathways. Manipulation of actin cytoskeleton pathways by miR-24 may represent an attractive therapeutic solution for the treatment of wet age-related macular degeneration (AMD) and other vascular diseases.

Introduction

New blood vessel formation via angiogenesis is a critical process for both vertebrate embryonic development and vascular homeostasis in the adult. In response to an angiogenic signal, endothelial cells (EC) degrade and invade the extracellular matrix, migrate and proliferate to form a lumen, and finally stabilize the new vessel. Aberrant angiogenesis underlies the pathogenesis and progression of many diseases, including tumor growth, atherosclerosis, coronary artery disease, and age-related macular degeneration (AMD). AMD is a degenerative disease of the retina and the leading cause of blindness among the elderly in the industrialized world, affecting more than 30 million individuals globally.1 Wet (or neovascular) AMD, which accounts for the majority of acute vision loss in AMD, is characterized by choroidal neovascularization (CNV), a process involving abnormal growth of blood vessels from the choroid into the retina. Current therapeutic approaches for wet AMD are restricted to antiangiogenesis/permeability agents and photodynamic therapy. Several agents antagonizing vascular endothelial growth factor (VEGF) function, including Macugen, Lucentis, and Eylea, have been approved by FDA and are currently used clinically to treat CNV in wet AMD.2,3,4 Although anti-VEGF agents can markedly improve the clinical outcome of wet AMD, they have been unable to induce complete regression of the choroidal neovascular membranes, and only 30–40% of the individuals experienced vision improvement after treatment.5,6 This makes the development of novel and alternative therapeutics for wet AMD imperative.

Dynamic rearrangement of the cytoskeleton has been shown to be crucial for various steps of angiogenesis.7 Actin is a cytoskeleton element integral for cell division and motility, both of which are critical for angiogenesis. Actin rearrangement leads to formation of protrusive structures, including lamellipodia and filopodia, and generation of intracellular forces required for cell migration.8 The Rho family of small GTPases, including RhoA, Rac1, and Cdc42, are master regulators of the actin cytoskeleton and mediate critical steps in angiogenesis.9,10 It is generally believed that Cdc42 determines cell polarity and filopodia formation, Rac1 stimulates motility through lamellipodia, and RhoA stimulates contraction and force generation. Rho signaling has been shown to be essential for capillary formation in vitro and angiogenesis in vivo.11,12,13 Rho-associated kinases (Rock), mammalian homologs of Drosophila diaphanous (Diaph), and P21-activated kinase (Pak) family kinases are downstream mediators of Rho signaling. Rock and Pak proteins have been reported to phosphorylate substrates including LIM domain-containing kinases (Limk) and have been implicated in EC migration and angiogenesis. Both activated and dominant-negative forms of PAK inhibited EC migration, and a dominant-negative p65 PAK peptide was shown to inhibit angiogenesis.14,15 Diaph proteins promote actin nucleation and polymerization through binding to Profilin, which catalyzes the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) in the actin monomer. Limk1 and Limk2 are members of the PDZ/LIM family that function to slow down actin filament turnover through phosphorylation and inactivation of the actin-regulatory protein ADF/Cofilin. Cofilin acts to accelerate actin depolymerization and thus replenishes the monomer pool. While the function of Limk proteins in angiogenesis is not known, Limk1 and Limk2 have been shown to be important for tumor progression and metastasis.16 Based on these studies, pharmacological compounds that target actin cytoskeleton components may hold promise in combating pathological angiogenesis in disease settings.

Recent studies have implicated pervasive roles for miRNAs in numerous diseases, including cardiovascular disease and cancer.17,18 An increasing list of miRNAs, termed “angiomiR”, has been shown to regulate angiogenesis either cell autonomously or noncell autonomously.19 Two miR-23~27~24 clusters exist in the vertebrate genome: an intergenic miR-23a~27a~24-2 cluster and an intronic miR-23b~27b~24-1 cluster. We and others have demonstrated that miR-23 and miR-27 are required for proper angiogenesis in vitro and in vivo through targeting Sprouty2 and Sema6A.20,21 In the current study, we investigated the role of miR-24 in angiogenesis and found that miR-24 represses angiogenesis by simultaneously regulating multiple components in the actin cytoskeleton pathways. Multiple genes downstream of Rho signaling, including Pak4, Limk2, and Diaph1, are direct miR-24 targets. Silencing of LIMK2 or PAK4 inhibits angiogenesis, mimicking the phenotype of miR-24 overexpression in vitro, while overexpression of LIMK2 and PAK4 by adenoviruses partially rescued the tube formation defects in miR-24 overexpressing ECs. Moreover, subretinal delivery of miR-24 mimics represses laser-induced CNV in vivo. These findings reveal a novel mechanism of miR-24 in regulating actin cytoskeleton dynamics and angiogenesis and suggest miR-24 as a potential novel therapeutic agent for combating aberrant angiogenesis through regulating actin cytoskeleton pathways.

Results

miR-24 targets multiple proteins involved in the actin cytoskeleton pathway

To begin to explore the function and underlying mechanisms of miR-24, we utilized DIANA-mirPath software (version 1) (diana.cslab.ece.ntua.gr/pathways) to search predicted target genes and signal pathways regulated by miR-24. Pathways involved in axon guidance, focal adhesion, and actin cytoskeleton dynamics were ranked as among the top pathways regulated by miR-24. Multiple proteins involved in regulating actin dynamics downstream of Rho signaling, including DIAPH1, PAK4, and LIMK2, were predicted to be miR-24 targets. Among these, PAK4 and LIMK2 are also involved in axon guidance and focal adhesion. As shown in Figure 1a, the 3′-untranslated region (3′-UTR) of these genes contain highly conserved sequences complementary to the miR-24 seed region. To test whether miR-24 represses the 3′-UTR activity of the predicted target genes, the 3′-UTRs of the three genes were fused downstream of the coding region of luciferase in a reporter vector, respectively. Each reporter was then cotransfected with a miR-24 mimic, a random control mimic, or a nonrelated miR-146 mimic in COS-1 cells. miR-24 mimic cotransfection resulted in a dose-dependent decrease in luciferase activity of the DIAPH1, LIMK2, and PAK4 3′-UTR reporters compared to the random control (Figure 1b). miR-146, which is not predicted to target these three 3′-UTRs, failed to repress the luciferase activity of the 3′-UTR reporters, suggesting the specificity of repression by miR-24 in the target 3′-UTRs. To rule out that miR-24 nonspecifically represses the activity of nonrelated 3′-UTRs, we also tested the effect on the activity of an EZH2 3′-UTR fragment, which does not contain miR-24 targeting site. We found that miR-24 has no effect on EZH2 3′-UTR activity as revealed by luciferase assay. To further examine whether miR-24 regulates the expression of these target proteins in vitro, human umbilical vein ECs (HUVECs) were transfected with miR-24 mimics or locked-nucleic acid (LNA) modified miR-24 anti-miR, and tested for DIAPH1, PAK4, and LIMK2 expression by western blot analyses. Transfection of miR-24 mimic led to ~50-fold increase in miR-24 expression, while miR-24 anti-miR transfection resulted in ~90% miR-24 knockdown, indicating the efficiency of miR-24 overexpression/silencing (Supplementary Figure S1). Of note, the expression of miR-23~27~24 family members miR-23a/b and miR-27a/b was not affected by either miR-24 mimic or anti-miR. Overexpression of miR-24 markedly repressed DIAPH1, PAK4, and LIMK2 protein expression, while silencing of miR-24 by LNA-anti-miR led to an increase in DIAPH1, PAK4, and LIMK2 expression (Figure 1c). Identification of PAK4 as a target of miR-24 is consistent with recent reports.22,23 PAK4-LIMK2 signaling has been shown to phosphorylate and inactivate cytoskeletal regulatory protein Cofilin, thereby inhibiting the activity of Cofilin in actin depolymerization. To further dissect whether the downregulation of PAK4 and LIMK2 by miR-24 leads to a decrease in Cofilin phosphorylation, phospho-cofilin and total cofilin levels were examined by western blot analyses upon miR-24 overexpression/knockdown. Consistently, miR-24 overexpression reduced the level of Cofilin phosphorylation, while miR-24 silencing led to a mild increase in Cofilin phosphorylation level. The total Cofilin level remained unchanged by miR-24 overexpression/knockdown. The PAK4-LIMK2-Cofilin pathway and its regulated stress fiber dynamics are required to sustain mitogen-activated protein kinase ERK1/2 activity. Specifically, PAK4 has been shown to phosphorylate and cooperate with RAF to activate the ERK pathway.24 To further test whether ERK1/2 phosphorylation is regulated by miR-24, phospho-ERK1/2, and total ERK1/2 levels were measured. As expected, miR-24 overexpression decreased phospho-ERK1/2 levels stimulated by VEGF, while miR-24 silencing had the opposite effect (Figure 1d). The level of total ERK1/2 was not changed by miR-24 overexpression/knockdown. Taken together, miR-24 is sufficient and necessary to regulate the actin polymerization/depolymerization pathway by targeting key proteins involved in actin cytoskeleton dynamics in ECs.

Figure 1.

Figure 1

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Regulation of miR-24 target proteins by miR-24 mimic and anti-miR in endothelial cells (Ecs). (a) Table showing several miR-24 target genes involved in cytoskeleton actin dynamics predicted by DIANA-mirPath software. The sequences in the target genes complementary to the miRNA seed regions are shown in red. The number of species in which the target sites are conserved is shown. (b) Dose-dependent repression of DIAPH1, PAK4, and LIMK2 3′ UTRs by miR-24 as shown by luciferase assays. miR-146, which was not predicted to target these UTRs, was used as controls. EZH2 UTR was used as a nonrelated control for testing miR-24 specificity. *P < 0.05; **P < 0.01; ***P < 0.0001; NS, not significant. (c) Regulation of miR-24 target proteins DIAPH1, PAK4, and LIMK2, as well as the phosphorylation of their downstream protein Cofilin by miR-24 mimic or anti-miR (100 µmol/l) in HUVECs, as shown by western blot analyses. Total Cofilin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as controls. Quantification compared with control (set as 1) is shown above the panel. (d) Regulation of vascular endothelial growth factor-induced ERK1/2 phosphorylation by miR-24 in human umbilical vein ECs, as revealed by western blot. Total ERK1/2 was used as a control. GAPDH served as a loading control. Quantification compared with control (set as 1) is shown above the panel.

Regulation of actin cytoskeleton dynamics in ECs by miR-24 in vitro

DIAPH1 has been shown to promote actin polymerization through recruiting Profilin, while phosphorylation of Cofilin by PAK4/LIMK2 is known to block the function of Cofilin as an actin depolymerizing protein. To further examine whether downregulation of DIAPH1 and PAK4/LIMK2/Cofilin pathway by miR-24 leads to a defect in actin polymerization, the amount and distribution of filamentous F-actin and the number of stress fibresin ECs were visualized by labeling with phalloidin upon miR-24 overexpression/silencing. Overexpression of miR-24 in HUVECs resulted in a loss of normal stress fibers as revealed by phalloidin staining (Figure 2a) and F-actin quantification (Supplementary Figure S2a). The miR-24 overexpressed cells appeared smaller and rounder and contained a prominent band of polymerized actin around the cell periphery. The specificity of miR-24 mimic on stress fiber formation was shown by the rescue of the miR-24 mimic stress fiber defects by cotransfection miR-24 anti-miR (Supplementary Figure S2b). In contrast, silencing of miR-24 mildly but significantly increased the stress fiber amount under normal conditions. To examine whether the formation of stress fibers is affected by miR-24, miR-24 mimic or anti-miR transfected HUVECs were pretreated for 1 hour with Y-27632 (10 µmol/l), a Rock inhibitor, to collapse the actin stress fibers. Normally actin stress fibers reappear after the removal of the ROCK inhibitor Y-27632. However, in contrast to the control, in miR-24 transfected HUVECs, stress fibers failed to reappear at 20 and 40 minutes after the removal of Y-27632 (Figure 2b). Conversely, compared to the control, miR-24 silencing caused only a slight increase in stress fiber formation at 20 minutes after inhibitor removal (Supplementary Figure S2c). Taken together, these data indicate an important role for miR-24 in regulating actin fiber formation.

Figure 2.

Figure 2

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Regulation of endothelial cell (EC) shape and actin dynamics by miR-24. (a). Stress fiber and shape changes in ECs transfected with miR-24 mimic, anti-miR or controls, as revealed by phalloidin staining. Scale = 50 µm. (b) Stress fiber formation in miR-24 mimic transfected ECs after 1 hour Rho inhibitor Y-27632 treatment. Scale = 50 µm.

Regulation of angiogenesis by miR-24 in vitro and ex vivo

The actin cytoskeleton plays an essential role in maintaining the normal function of the cell by modulating cell shape, migration, and proliferation, which are processes critical for angiogenesis. To study the function of miR-24 in angiogenesis, an in vitro Matrigel tube formation assay was performed in HUVECs transfected with miR-24 mimic or anti-miR. Under normal conditions, when cultured on Matrigel for 6–8 hours, HUVECs form a primary vascular tubular network. Overexpression of miR-24 with miRNA mimics disrupted tube formation as quantified by drastically reduced number of branch points, while silencing of miR-24 with LNA-anti-miR mildly enhanced the formation of tubular structures (Figure 3a,b). To dissect the cellular mechanism whereby miR-24 regulates angiogenesis, a bromodeoxyuridine incorporation assay and a scratch wound assay were utilized to analyze EC proliferation and migration upon miR-24 overexpression. As shown in Figure 3c,d, compared to the control, miR-24 overexpression strongly repressed EC proliferation in EGM2 medium after overnight starvation, while silencing of miR-24 with LNA-anti-miR slightly increased EC proliferation. In a scratch wound cell migration assay, overexpression of miR-24 inhibited EC migration into the wound region in HUVECs compared to the nontransfection and mimic control cells, while LNA-miR-24 anti-miR showed a trend to increase EC migration (Figure 3e). The effect of miR-24 mimic on EC migration was also visualized for 6 hours by time-course live cell imaging (see representative pictures in Figure 3f). Compared to the massive lamellipodia formation and active migration in control cells, miR-24 overexpressed ECs had many fewer lamellipodia protrusions and remained stagnant.

Figure 3.

Figure 3

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Regulation of angiogenesis by miR-24 in vitro. (a) Representative pictures of in vitro endothelial cell (EC) tube formation after miR-24 mimic or anti-miR transfection and 8-hour culture in the Matrigel. (b) Quantification of branch points per field in (a). ***P < 0.001; *P < 0.05. (c) Quantification of EC proliferation in EGM2 medium after starvation indicated by bromodeoxyuridine incorporation after miR-24 mimic transfection. (d) Quantification of EC proliferation in EGM2 medium after starvation indicated by BrDU incorporation after miR-24 anti-miR transfection. (e) Quantification of scratch wound EC migration after miR-24 mimic or anti-miR transfection in ECs. ***P < 0.001; n.s., not significant. (f) Representative real-time pictures showing scratch wound EC migration after miR-24 mimic transfection. Red lines indicated the initial positions of cells. Time points were indicated.

To further investigate the role of miR-24 in sprouting angiogenesis, miR-24 mimic or anti-miR was transfected into aortic ring segments in EGM2 medium, and the sprouting of aortic ring cells was quantified after an ex vivo aortic ring assay. As shown in Supplementary Figure S3a,b, miR-24 overexpression significantly repressed the outgrowth of aortic ring cells at 6 days after culture, while silencing of miR-24 seemed not to affect aortic ring cell outgrowth. Taken together, our data suggest that miR-24 represses angiogenesis in vitro and ex vivo.

Repression of laser-induced CNV by miR-24 mimic in mice

Our previous results have shown that miR-23~27~24 members were upregulated in the retina/choroid in a laser-induced CNV mouse model. To test the role of miR-24 in pathological angiogenesis induced by laser injury, cholesterol-conjugated miR-24 mimic was injected subretinally into mice at 0, 4, and 9 days after laser injury, and CNV was quantified at 2 weeks post injury after ICAM-2 staining and confocal flat mount imaging. Cholesterol conjugation of the miRNA mimic eliminates the requirement of transfection reagents for in vivo miRNA mimic delivery. First, we tested the efficiency of miRNA mimic delivery. To do so, carboxyfluorescein (FAM)-labeled miR-24 mimic was injected subretinally into mice at 7 days after laser injury, and the distribution of labeled mimics was visualized by ICAM-2 costaining and flat mount imaging 4 days later. As shown in Supplementary Figure S4, miR-24 mimic was successfully delivered into the injured region of the retina and partially overlapped with the vasculature. By real-time reverse transcription polymerase chain reaction, 1 µl of miR-24 mimic (200 ng/µl) injection led to an ~20-fold increase in miR-24 expression in the posterior eye cup (Figure 4a). These indicate efficient delivery of miRNA mimics into the choroid by subretinal injection in vivo. We next examined the effect of miR-24 mimic on laser-induced CNV in mice. Quantification of the CNV area revealed that miR-24 overexpression led to about 60% decrease in the neovascularization of the choroid (3031 ± 496 µm2, n = 33 in the mimic control, versus 1243 ± 317 µm2, n = 36 in miR-24 mimic injected samples (Figure 4b,c). Compared to the saline control, the control mimic led to a small but insignificant decrease in CNV (P = 0.31). Decreased neovascularization of the choroid by miR-24 mimic was also confirmed by ICAM-2 staining of frozen sections (Figure 4d). These results indicate that miR-24 overexpression is sufficient to repress CNV in vivo.

Figure 4.

Figure 4

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Repression of laser-induced CNV by miR-24 in vivo. (a) Real-time polymerase chain reaction showing upregulation of miR-24 by miR-24 mimic in the posterior eyes. ***P < 0.0001. (b) Representative images showing repression of laser-induced CNV by miR-24 mimic. Scale bar = 40 µm. (c) Quantification of CNV area (µm2) in (b). Saline and control mimic were used as controls. **P < 0.01. (d) Representative images of ICAM-2 staining from frozen sections of laser lesions. Scale bar = 50 µm. miRNA mimic treatments were indicated, and the lesion areas were labeled by dashed lines.

Mimicking of miR-24 overexpression angiogenic phenotype by PAK4 or LIMK2 silencing in vitro

Given the in vitro and in vivo evidence that miR-24 represses angiogenesis, we set to further dissect the mechanism by which miR-24 regulates cytoskeleton dynamics and angiogenesis. miR-24 target gene DIAPH1, LIMK2, or PAK4 was silenced with siRNAs in HUVECs, and actin cytoskeleton structure and distribution was visualized with phalloidin staining. As shown by western blots in Figure 5a, siRNAs against DIAPH1, LIMK2, and PAK4 were efficient in silencing their respective genes when transfected at either 50 or 100 nmol/l concentration in HUVECs. We also designed siRNA to silence LIMK1 and found that LIMK2 siRNA did not affect the expression of its family member LIMK1, and vice versa, suggesting the specificity of the siRNAs. Functionally, silencing of LIMK2 or PAK4 also decreased the level of phosphorylated Cofilin, consistent with them being upstream of ADF/Cofilin/F-actin pathway (Supplementary Figure S5). Phalloidin staining revealed that, compared to control siRNA, silencing of LIMK2 or PAK4, and to a less extent DIAPH1, disrupted stress fibers in HUVECs (Figure 5b). In the case of LIMK2 silencing, cells appeared smaller and rounder, and actin aggregates were observed near to the nucleus.

Figure 5.

Figure 5

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Mimicking of the miR-24 overexpression phenotype by silencing of miR-24 target genes in vitro. (a) Western blots showing efficient silencing of DIAPH1, LIMK2, PAK4, and LIMK1 in human umbilical vein endothelial cells (ECs) with specific siRNAs at 50 and 100 nmol/l. (b) Representative phalloidin staining showing actin fiber structure and its cellular distribution after DIAPH1, LIMK2, or PAK4 knockdown in ECs. (c) Representative images of in vitro matrigel assays after DIAPH1, LIMK2, or PAK4 knockdown in ECs. (d) Quantification of EC migration in response to wound scratch after DIAPH1, LIMK2, or PAK4 knockdown. (e) Quantification of EC proliferation after LIMK2 or PAK4 knockdown.

To further study whether silencing of miRNA target genes mimics the phenotype of miR-24 overexpression in angiogenesis, an in vitro Matrigel tube formation assay was performed after siRNA silencing of the miR-24 target genes in HUVECs. Consistent with the essential role of LIMK2 and PAK4 in actin skeleton dynamics, knockdown of LIMK2 or PAK4 blocked EC tube formation in the Matrigel (Figure 5c). However, knockdown of DIAPH1 only had a mild effect in EC tube formation. Quantification of angiogenic tube formation also showed a dramatic decrease in branch points in the Matrigel after LIMK2 or PAK4 silencing and a slight decrease after DIAPH1 or LIMK1 knockdown (Supplementary Figure S6). We further performed experiments to determine whether EC proliferation and migration is repressed by LIMK2 or PAK4 knockdown. Consistently, LIMK2 or PAK4 silencing led to a significant decrease in cell migration after scratch wound and cell proliferation by bromodeoxyuridine incorporation assay (Figure 5d,e). These results indicate that LIMK2 and PAK4 silencing recapitulates the phenotype of miR-24 overexpression in actin cytoskeleton dynamics and angiogenesis in vitro, suggesting LIMK2 and PAK4 as important miR-24 target genes mediating its function in angiogenesis.

Rescue of actin cytoskeletal and angiogenic defects in miR-24 overexpressing ECs by its downstream targets

Our results showed that miR-24 decreases the level of phosphorylated Cofilin through PAK4/LIMK2 pathways, which indirectly increases the pool of nonphosphorylated Cofilin and therefore actin depolymerization. We reasoned that knockdown of Cofilin in ECs may rescue the stress fiber defects in miR-24 overexpressing cells. Cofilin-1 was knocked down with specific siRNAs and efficient Cofilin silencing was achieved in HUVECs as revealed by western blot analysis (Figure 6a). Indeed, knockdown of Cofilin in ECs at least partially rescued the disappearance of the stress fibers and small cell size in miR-24 overexpressing ECs (Figure 6b), indicating that PAK4/LIMK2/Cofilin pathway mediates miR-24 function in regulating actin cytoskeleton. We further tested whether Cofilin knockdown can rescue tube formation defects in miR-24 overexpressing cells. As shown in Figure 6c, Cofilin-1 knockdown itself did not significantly affect vascular tube formation in ECs. However, the tube formation defects upon miR-24 overexpression were at least partially rescued by Cofilin knockdown, indicating the importance of Cofilin pathway in regulating miR-24 function in angiogenesis.

Figure 6.

Figure 6

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Rescue of tube formation phenotype in Matrigel assay in miR-24 overexpressing endothelial cells (ECs) by adenoviral overexpression of its target genes or Cofilin knockdown. (a) Western blots showing efficient silencing of Cofilin in human umbilical vein ECs with specific siRNAs at 50 and 100 nmol/l. (b) Rescue of stress fiber formation in ECs transfected with miR-24 mimic by knockdown of Cofilin by siRNA, as revealed by phalloidin staining. Scale = 50 µm. (c) Quantification of matrigel tube formation in ECs transfected with miR-24 mimic and/orCofilin-1 siRNA. (d) Western blots showing efficient overexpression of LIMK2 and PAK4 in human umbilical vein ECs by adenoviruses. (e) Significant rescue of matrigel tube formation in ECs transfected with miR-24 mimic by overexpression of LIMK2 and PAK4. Scale = 200 µm. (f) Quantification of branching points in (e). ***P < 0.001.

To directly test the importance of miR-24 target genes LIMK2 and PAK4 in mediating miR-24 function in angiogenesis, we have generated adenoviruses overexpressing LIMK2 and PAK4 coding cDNAs. Overexpression of LIMK2, PAK4 by adenoviruses in ECs was confirmed by western blot analyses (Figure 6d). In support of a major role for these target genes in mediating miR-24 angiogenic function, overexpression of LIMK2 and PAK4 by viruses significantly rescued the tube formation defects in miR-24 overexpressing ECs (Figure 6e,f).

Discussion

Our results reveal an important role for miR-24 in regulating actin cytoskeleton dynamics and angiogenesis in ECs. We found that multiple proteins downstream of Rho signaling, including LIMK2, PAK4, and DIAPH1, are direct miR-24 targets. Either overexpression of miR-24 or knockdown of its target genes LIMK2 or PAK4, results in a block in actin fiber formation, as well as repression in angiogenesis in vitro. Subretinal delivery of miR-24 mimic represses laser-induced CNV in vivo. These findings support a novel model for miR-24 in repressing angiogenesis by simultaneously regulating multiple components in the actin cytoskeleton pathways (Figure 7). In this model, miR-24 targets on PAK4 and LIMK2 downstream of Rho signaling, therefore repressing Cofilin phosphorylation and promoting actin-depolymerization. In addition, miR-24 also targets on DIAPH1 downstream of Rho signaling, therefore repressing the actin polymerization through Profilin. By targeting multiple components downstream of Rho signaling, miR-24 exerts an inhibitory effect on actin skeleton dynamics, and represses EC migration, proliferation, and angiogenesis.

Figure 7.

Figure 7

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A model for miR-24 in angiogenesis. In response to extracellular stimuli, Rho signaling activates DIAPH1, PAK4, and LIMK2, which function to regulate actin polymerization/ depolymerization. miR-24 targets on PAK4 and LIMK2 downstream of Rho signaling, therefore repressing Cofilin phosphorylation and promoting actin-depolymerization. In addition, miR-24 also targets on DIAPH1 downstream of Rho signaling, therefore repressing the actin polymerization through Profilin. By targeting key proteins involved in actin cytoskeleton dynamics, miR-24 represses angiogenesis and choroidal neovascularization in the retina.

Actin cytoskeleton is crucial for cell motility and cytokinesis, and therefore involved in numerous developmental and disease processes, including angiogenesis.7 A few miRNAs have been implicated in actin cytoskeleton dynamics. miR-124 contributes to the control of neurite outgrowth by regulating Rho GTPase Cdc42.25 miR-206 also targets Cdc42 and represses cancer cell invasion and migration.26 miR-143/145 controls smooth muscle cell plasticity through regulation of multiple genes involved in actin dynamics.27 miR-200c represses cancer cell invasion by targeting WAVE3, a protein involved in actin cytoskeleton remodeling.28 miR-34a regulates RhoA/Rac1 crosstalk and negatively modulates actin cytoskeleton reorganization in chondrocytes.29 Our results provide evidence that miR-24 regulates actin cytoskeleton dynamics in ECs by targeting multiple proteins downstream of Rho signaling, namely LIMK2, PAK4, and DIAPH1. Overexpression of miR-24 blocks stress fiber and lamellipodia formation, therefore repressing EC migration and proliferation, while knockdown of miR-24 shows a trend to enhance stress fiber formation. Identification of miR-24 as a key regulator of cytoskeleton dynamics is consistent with a very recent report that miR-24 controls epidermal differentiation through regulating actin adhesion and cell migration.23 Consistent with the critical role of actin cytoskeleton in angiogenesis, we found that, through targeting actin cytoskeleton pathways, miR-24 represses angiogenesis both in vitro and in vivo. Silencing of miR-24 target genes LIMK2 or PAK4 recapitulates miR-24 overexpression phenotype in actin dynamics and angiogenesis, while overexpression of LIMK2 and PAK4 by adenoviruses partially rescued the tube formation defects in miR-24 overexpressing ECs, indicating these two miR-24 target genes play an important role in mediating miR-24 function in actin cytoskeleton remodeling and angiogenic response in ECs. Identification of PAK4 as a bona fide miR-24 target is consistent with recent reports.22,23 Interestingly, these reports and ours support an important role of PAK4 in mediating miR-24 function in angiogenesis. While Thomas and colleagues attribute mechanism of PAK4 function to repression of EC apoptosis, our studies indicate a new mechanism for PAK4 in angiogenesis, which is to regulate EC migration and proliferation via modulation of actin cytoskeleton remodeling. Consistent with the results of Thomas and colleagues, we also have results that miR-24 modestly promotes EC cell death by Anexin V (Supplementary Figure S7). Conflicting results regarding miR-24 in apoptosis have been reported.22,30,31,32 By real-time polymerase chain reaction of the published and predicted miR-24 target genes related to apoptosis, we found that proapoptotic genes BIM (BCL2L11) and IL-1R1, and anti-apoptotic genes PAK4 and BCL2 are downregulated by miR-24 (Supplementary Figure S8). The conflicting results regarding miR-24 in apoptosis likely result from cellular context and the relative expression level of its target genes. Among these genes, PAK4 likely has multiple functions in ECs and is required for EC proliferation, migration, and survival. Consistent with LIMK proteins as downstream targets for PAK and ROCK pathways, our results show that, LIMK2 plays a critical role in regulating actin cytoskeleton remodeling and angiogenesis in ECs. By fine-tuning multiple genes in the actin cytoskeleton pathway network, miR-24 exerts strong regulation of actin dynamics and angiogenesis in ECs. Although we believe that LIMK2 and PAK4 play important roles in mediating miR-24 function in angiogenesis, the role of other miR-24 target proteins cannot be ruled out. For example, besides LIMK2 and PAK4, other known miR-24 targets, such as E2F2 and cMyc, may contribute to miR-24 function in EC proliferation.33

An increasing list of miRNAs, including miR-126, miR-23/27, miR-132, and miR-17~92 cluster, has been shown to regulate angiogenesis.19 We and others have demonstrated that miR-23 and miR-27 are required for proper angiogenesis in vitro and in vivo through regulating VEGF receptor 2 (VEGFR2) phosphorylation and mitogen-activated protein kinase pathways.20,21 In this report, we extend our study to investigate the miR-23~27~24 family member miR-24 in angiogenesis. Our results reveal that miR-24 represses angiogenesis through a novel mechanism of regulating actin cytoskeleton dynamics. Overexpression of miR-24 in HUVECs represses EC migration and proliferation in intro, tube formation in Matrigel and sprouting angiogenesis in the aortic ring assays. Similar effect of miR-24 on tube formation was also observed in primary choroidal EC, suggesting a general mechanism of miR-24 in EC behavior (Supplementary Figure S9). Of significance, pharmacological delivery of miR-24 mimics represses laser-injury induced CNV in mice. All these in vitro and in vivo data unequivocally point to a repressive role for miR-24 in angiogenesis. This is consistent with a recent report that blocking miR-24 function improves cardiac function and survival after myocardial infarction through enhancing vascularity in the heart.22 Compared to their in vivo results, the effect of miR-24 silencing in angiogenesis in vitro is relatively moderate in our hand. This is likely due to the different systems used in our studies. Other miRNAs or compensatory mechanisms might compromise the loss of miR-24 function in vitro, while the effect of in vivo miR-24 silencing might be attributed to its effect in multiple cell types. Our results that miR-23~27~24 members, miR-24 and miR-23/27, have opposite function in angiogenesis are intriguing. Based on this, we speculate that miR-23~27~24 clusters function as a rheostat to keep angiogenesis under check under extreme conditions. Proper miR-23~27~24 concentration is likely ideal for maximal angiogenesis. It is interesting that, in disease settings, miR-23/27/24 have been shown to be differentially regulated.34

Anti-VEGF agents are the current mainstay in treating wet AMD. Only 30–40% of the individuals experienced vision improvement after treatment, and about 15% patients do not respond to the treatment.6 The multifactorial feature of the disease calls for combinational or multifunctional drugs for efficient AMD treatment. miRNAs have been shown to simultaneously target mRNAs in multiple signaling pathways or multiple components in a given signal pathway, making them an attractive option for AMD. A group of miRNAs have been shown to be substantially decreased in a laser-induced CNV model.35 Overexpression of miR-31, miR-150, or miR-21 represses neovascularization in a laser-induced CNV model.35,36 We recently showed that the expression of miR-23~27~24 cluster members is upregulated in the eye in a laser-induced CNV model.20 Moreover, silencing of miR-23 and miR-27 repressed laser-induced CNV by targeting MAPK and VEGFR2 signaling. In this report, we found that miR-24 overexpression is sufficient to repress laser-induced CNV in mice through targeting multiple components involved in regulating actin cytoskeleton dynamics. Since miR-23/27 and miR-24 function in different pathways, it would be interesting to see whether miR-24 mimic and miR-23/27 LNA-anti-miR have a combinational effect in repressing CNV. miR-24 has been shown to inhibit cell proliferation and regulate apoptosis,22,30,33,37,38,39,40 as well as skeletal muscle differentiation41 and erythropoiesis.42 CNV can lead to hemorrhage and scar formation beneath the central retina, which accounts for most cases of legal blindness from AMD. Currently, there is no drug available for treating fibrosis in wet AMD. miR-24 has been recently shown to repress cardiac fibrosis and scar formation through a furin-TGF-β pathway after myocardial infarction.43 In this context, it would be appealing to develop miR-24 or target actin cytoskeletal pathway as potential drug targets for both CNV and fibrosis in wet AMD. Besides CNV, miR-24 was also shown to regulate vascularity after myocardial infarction.22 Moreover, the potential of miR-24 as therapeutic target for other angiogenesis-associated vascular diseases including tumor growth, and metastasis should not be overlooked. In all, our data support an important role of miR-24 in regulating actin cytoskeleton dynamics and angiogenesis, which will have implication in a variety of vascular disorders.

Materials and Methods

Animals. Animal studies were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and were approved by the Institutional Animal Care and Use Committees at the University of Texas Southwestern Medical Center and Tulane University. C57BL/6 male mice (6–8 weeks of age) were used for the studies in laser-induced CNV and aortic ring assays.

Laser-induced CNV and retinal vasculature staining. Laser-induced CNV was induced in 6- to 8-week-old male C57BL/6J mice as described previously.20 Briefly, the pupils of anesthetized animals were dilated with 1% tropicamide (Alcon Laboratories, Forth Worth, TX). Three 532-nm diode laser spots (140 mW, 100 msec, and 100 µm; OcuLight GL Photocoagulator, Iridex, Mountain View, CA) were applied to each fundus of adult mice using a coverslip as a contact lens. Formation of a bubble at the time of laser application indicates rupture of Bruch membrane and successful laser injury. Animals were injected subretinally with 1 µl of 200 ng/µl solution of miR-24 mimic or scramble control, or PBS, after laser photocoagulation. Two more injections were performed at days 5 and 9 after injury. For visualizing the distribution of miRNA mimic, FAM-labeled miRNA was delivered at 7 days after laser injury, and the injected eyes were collected for flat mount imaging at 4 days later. At 14 days after laser injury, the treated eyes were fixed in 4% paraformaldehyde and subjected to flat mount ICAM-2 staining or staining of frozen sections. For visualization of the retinal vasculature, retinas were dissected from pups or adult mice and stained with Alexa-594 conjugated isolectin B4 (Life Technologies, Grand Island, NY) or ICAM-2. Images of CNV were captured using a Leica SP2 multiphoton Laser Scanning confocal microscope, and CNV volume were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

miRNA mimics, LNA-anti-miRs, and siRNAs. LNA-anti-miRs for miR-24 or scramble controls for in vitro were synthesized from Exiqon (Woburn, MA). miR-24 or control pre-miR precursor for in vitro study were synthesized from Life Technologies, while miR-24 or control mimic for in vivo studies were synthesized from Shanghai Gene Pharma (Shanghai, China). Sequence for control LNA-antimiR is: 5′-GTGTAACACGTCTATACGCCCA-3′. Sequence for miR-24 LNA-antimiR is: 5′-CUGUUCCUGCUGAACUGAGCCA-3′. Sequences for control mimic are: [sense] 5′-p-fUsfUfCfUfCfCGAAfCGfUGfUfCAfCsGfUsTsT-3′ and [antisense] 5′-Chol-sAsfCGfUGAfCAfCGfUfUfCGGAGAAsTsT-3′ (f: 2′-deoxy-2′-fluro nucleotides, Chol: cholesterol, p: phosphate group, s:phosphorothioate linkages). Sequences for miR-24 mimic are: [sense] 5′-p-fUsGGfCfUfCAGfUfUfCAGfCAGGAAfCsAsG-3′ and [antisense] 5′-Chol-sGsfUfUfCfCfUGfCfUGAAfCfUGAGfCfCAsfUsfU-3′. siRNAs for LIMK1, LIMK2, PAK4, DIAPH1, and COFILIN were purchased from Sigma (St Louis, MO). The sequences are as follows: human LIMK1siRNA: 5′-CUCUGAGUCCCUCCGCGUA[dT][dT]-3′ human LIMK2siRNA: 5′-GCUUCUUUGGGCAGGCUAU[dT][dT]-3′ human PAK4siRNA: 5′-GGCUGAAGCUGUCAGACUU[dT][dT]-3′ human DIAPH1siRNA: 5′-CAUGUGAGGAGUUACGUAA[dT][dT]-3′ human COFILINsiRNA: 5′-CCUCUAUGAUGCAACCUAU[dT][dT]-3′.

Plasmid construction and reporter assay. 3′-UTRs of miR-24 target genes were polymerase chain reaction amplified from human placenta race-ready cDNAs and cloned into pmiR-REPORT vector (Life Technologies). The sequences for UTR cloning are: LIMK2, 5′-atcggccggcAGGAGGCAAGTGGGCGCAGC-3′ and 5′-atcggtttaaacGGCCCAGTTCAGGCCCACCA-3′ PAK4, 5′-atcggagctcCTGGGGGTAGATGAG ACCCTACT-3′ and 5′-atcgaagcttCTGGTTCTTCAGGCAGTGGTT-3′ DIAPH1, 5′-atcgactagtGTGACCGCGGCAGCTCCTCA-3′ and 5′-atcggtttaaacTGGGCCAAGCCCAAGAGTGCC-3′ EZH2, 5′-atcggagctcTTCCCCTTCTCTCTGAAACAGC-3′ and 5′-atcgaagctt CAACAAGTTCAAGTATTCTTTATTCAA-3′. Reporter assays to test UTR activities were performed as described.20

Adenovirus generation and infection. Generation and infection of adenoviruses were performed as described.44 Briefly, human LIMK2, PAK4, and DIAPH1 cDNAs without 3-UTRs were cloned into the pShuttle-CMV vector after polymerase chain reaction and digestion with KpnI/XhoI, respectively. These vectors were then recombined into adenovirus vectors and be used to generate adenovirus. Adeno-LacZ virus was used as a control for the experiments. Normally, viruses with multiplicity of infection (MOI) of 50–100 were used to infect EC. Sequences for cloning human LIMK2, PAK4, and DIAPH1 into pShuttle vectors are: 5′-ATCGggtaccCGGGCTCCCGGGACCATGT-3′ and 5′-ATCGctcgagTGGGCCAGGGCTAGGGAGGT-3′ for LIMK2; 5′-ATCGggtaccatgtttgggaagaggaagaagc-3′ and 5′-ATCGctcgagctcatctggtgcggttctg-3′ for PAK4; 5′-ATCGggtaccgctggctaaagagcgactgg-3′ and 5′-ATCGctcgagtcacaggacccacattagcttg-3′ for DIAPH1.

Cell culture and aortic ring assay. HUVEC (ATCC, Manassas, VA) cells were grown in EC growth medium (Lonza, Allendale, NJ). Human choroidal EC were isolated as described.45 More than 85% purity of the cells was achieved as assessed by fluorescence-activated cell sorting analysis after isolectin B4 staining. For VEGF treatment, HUVECs were starved with EC basal medium-2 with 0.1% fetal bovine serum for overnight and then treated with VEGF for the indicated periods of time. miRNA pre-miR/mimic or LNA-anti-miR transfection in cell culture and aortic ring culture were performed as described.46 For visualizing actin fiber in cells, ECs were fixed with 4% paraformaldehyde and stained with 50 mg/ml fluorescein isothiocyanate-labeled phalloidin (Sigma) at room temperature for 1 hour. For quantification of F-actins, images were obtained from randomly selected areas, and about 50 cells per each group selected randomly were used to quantify F-actin amount using the Image J. For ROCK inhibitor treatment, HUVECs were pretreated with Y-27632 (10 µmol/l). The inhibitor was washed away at 1 hour after, and the cells were subjected to phalloidin staining at indicated time points.

RNA and western blot analysis. Total RNA was isolated from mouse tissues or cell lines using TRIzol reagent (Life Technologies). Northern blots to detect miRNA level were performed using Starfire probes as described.46 miRNA real-time reverse transcription polymerase chain reaction was performed using qScript cDNA synthesis and microRNA quantification system (Quanta Biosciences, Gaithersburg, MD). For western blot analysis, protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted using standard procedures. Antibodies used were as follows: mDIA1 (Thermo, Franklin, MA), LIMK1 (Cell signaling, Danvers, MA), LIMK2 (Cell signaling), PAK4 (Cell signaling), Cofilin (Sigma), phosphor-Cofilin (Sigma), ERK1/2 (Cell signaling), Phospho-ERK1/2 (Cell signaling), and glyceraldehyde 3-phosphate dehydrogenase (Millipore, Billerica, MA) as loading control.

In vitro matrigel assay, cell proliferation, scratch-wound assay, and cell death assay. In vitro matrigel assay, cell proliferation, and scratch-wound assay were performed as described.20 Cell death was performed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using the in situ cell death detection kit and an Annexin V cell death kit (Invitrogen) according to the manufacturer's protocol.

Living cell time-lapse imaging and analysis. For live cell imaging, HUVECs were transfected with control and miR-24 mimic in 35 mm glass bottom dish (MatTek, Ashland, MA). Scratch wound was generated using 200 µl pipette tip 3 days later. Cells were then cultured in a stage-top incubator built on a Zeiss Axio observer microscope, which maintains 37 °C temperature and 5% CO2 level. Images were acquired every 5 minutes for 6 hours using 20X objective on the microscope. Time-lapse images were converted to audio/video interleave (AVI) movies using image J (National Institutes of Health).

Statistics. Each experiment was repeated at least three times. Student's t-tests were used to determine statistical significance between groups. P values < 0.05 were considered to be statistically significant.

SUPPLEMENTARY MATERIAL Figure S1. The expression of miR-23~27~24 family members after miR-24 mimic or anti-miR transfection in HUVECs by real-time PCR. Figure S2. Regulation of EC shape and actin dynamics by miR-24. Figure S3. Regulation of angiogenesis by miR-24 ex vivo. Figure S4. Flat mount imaging of FAM labeled miRNA mimic after suretinal injection. Figure S5. Repression of Cofilin phosphorylation after LIMK2 and PAK4 knockdown in HUVECs as shown by western blot analyses. Figure S6. Quantification of Matrigel tube formation after DIAPH1, LIMK2, PAK4 knockdown in ECs. Figure S7. Statistics of Annexin V staining after miR-24 mimic or antimiR transfection in ECs. Figure S8. Real-time RT-PCR analyses of miR-24 target gene related to apoptosis. Figure S9. Regulation of EC Tube formation by miR-24 in matrigel.

Acknowledgments

We are grateful to Eric Olson for his encouragement of the project. S.W. was supported by a Startup fund from Tulane University, President's Research Council New Investigator Award from UT Southwestern Medical Center, NIH Grant EY021862, a career development award from the Research to Prevent Blindness foundation, and a BrightFocus Foundation Award in age-related macular degeneration. Q.Z. was supported by an American Heart Association Southeast Affiliate Postdoctoral fellowship. The authors declare no fanancial conflict of interest related to the submitted manuscript.

Supplementary Material

Supplementary Information
Click here for additional data file. (24MB, pdf)

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