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microRNAs play key roles in modulating a variety of cellular processes by post-transcriptional regulation of their target genes. VEGF, VEGFR-2 and FGFR-1 were identified by bioinformatic approaches and subsequently validated as targets of miR-16 and miR-424 in endothelial cells (ECs).
Mimetics of these microRNAs reduced VEGF, VEGFR-2 and FGFR-1 expression, whereas specific antagonists enhanced their expression. Expression of mature miR-16 and miR-424 was up-regulated upon VEGF or bFGF treatment. This up-regulation was accompanied by a parallel increase in pri-miR-16-1 and pri-miR-16-2 but not in pri-miR-424 levels, indicating a VEGF/bFGF-dependent transcriptional and post-transcriptional regulation of miR-16 and miR-424, respectively. Reduced expression of VEGFR2 and FGFR1 by miR-16 or miR-424 overexpression regulated VEGF and bFGF signaling through these receptors, thereby affecting the activity of downstream components of the pathways. Functionally, miR-16 or miR-424 overexpression reduced proliferation, migration and cord formation of ECs in vitro and, lentiviral overexpression of miR-16 reduced the ability of ECs to form blood vessels in vivo.
We conclude that these miRNAs finely tune the expression of selected endothelial angiogenic mediators in response to these growth factors. Altogether, these findings suggest that miR-16 and miR-424 play important roles in regulating cell-intrinsic angiogenic activity of ECs.
Angiogenesis is a process by which new vessels are generated from pre-existing vasculature1. Endothelial cells (ECs) play a key role in this process which depends on the proliferation, migration and differentiation of these cells2. A fine balance between positive and negative regulators controls angiogenesis3. While there are many angiogenic inducers, vascular endothelial growth factor (VEGF, also termed VEGF-A) and basic fibroblast growth factors (bFGF, also termed FGF2) are probably the most critical and potent ones4–6. The pro-angiogenic effect of VEGF and bFGF is mediated through the VEGF receptor 2 (VEGFR2, also termed Flk-1 or KDR), which is selectively expressed in vascular ECs5, or the FGF receptor 1 (FGFR1)6, respectively. Activation of these receptors stimulates the angiogenic cascade in vitro and in vivo5–8. The regulation of these pro-angiogenic factors and the signal transduction pathways that regulate proliferation, migration and differentiation of ECs upon activation of their respective receptors has been well studied. However, comparatively less is known about the regulation of VEGFR2 and FGFR1 in ECs, particularly concerning their post-transcriptional regulation involving miRNAs.
miRNAs play central roles in a broad range of biological processes9, and in many cases have been shown to modulate intracellular signaling pathways in animal cells10–12. miRNAs are initially transcribed as large pri-miRNAs that are matured through sequential steps to give rise to a heteroduplex RNA (mature miRNA). They regulate gene expression by inhibiting translation or promoting mRNA degradation mostly through canonical base paring between the seed sequence of the miRNA and its complementary seed match sequence, present in the 3′ untranslated regions (UTR)13.
Reduction of miRNAs by siRNA-mediated knockdown of Dicer in human ECs14, 15 or by knockout or conditional inactivation of Dicer in murine endothelium has revealed the importance of endothelial miRNAs in angiogenesis16, 17. To date, several miRNAs have been shown to participate in the control of angiogenesis18–22. In addition, very recent data also demonstrate that growth factors or cytokines can differentially regulate miRNA expression in ECs. In this scenario, they provide a first line of response following cytokine stimulation, promoting the modulation of EC targets involved in expression programs that control angiogenic responses16, 23.
Angiogenesis plays a crucial role in numerous normal physiological processes, such as, embryonic development, wound healing and the menstrual cycle; as well as, in various pathological conditions like ischemic vascular diseases, diabetic retinopathy, rheumatoid arthritis and the development of cancer24. Recent insights suggest that future cancer therapies are likely to consist of a combination of anti-angiogenic agents (e.g. VEGF inhibitors) and cytotoxic chemotherapies as a venue to inhibit tumor growth. Therefore, the use of miRNAs mimics to regulate both angiogenesis and tumor cell survival might be an important finding in the research and development of effective therapeutic agents25. In different cancer cells, miR-15a/16-1 and miR-15b/16-2 clusters have been shown to play very important roles in regulating cell proliferation and apoptosis by targeting genes involved in cell cycle progression26–28, as well as anti-apoptotic proteins29. Interestingly, miR-15b and miR-16 have been shown to control the expression of VEGF in a carcinoma cell line30. In addition, miR-15b and miR-16 have also been shown to be differentially expressed in endometriosis, in which angiogenesis may be involved in the growth of the endothelium31, 32. Moreover, miR-424 has been reported to be downregulated in senile hemangioma, which is a common vascular anomaly associated with abnormal angiogenesis 33. Altogether, these reports indicate the in vivo relevance of miR-15b, miR-16, and miR-424 in diseases associated with vascular defects. However, the direct effect of these miRNAs on ECs has not been demonstrated. miR-15a, -15b, -16, -195, -424, and -497 have different genomic locations but possess the same seed sequence, which implies that these miRNAs share most of their target genes13, and furthermore, may strongly influence the expression of their common targets if co-expressed. In the present work, we investigated the role miR-16 and miR-424 in the cell-intrinsic angiogenic activity of ECs and determined their effects on neovascularization in vivo.
Bioinformatic analysis, 3′UTR Luciferase Reporter Assays, Western Blot Analysis, Quantitative real-time PCR, Cell number assessment, Crystal violet staining method, Cord formation assay, Migration Experiments, Lentivirus and EC transduction, Flow Cytometry and Immunofluorescence analyses are described in the Supplemental Data.
Human umbilical vascular ECs (HUVECs) were purchased from the tissue culture core laboratory from the Vascular Biology and Therapeutics program (Yale University) and serially cultured on 0.1% gelatin-coated flasks in M199/20%FBS supplemented with L-glutamine, penicillin/streptomycin (Gibco), and endothelial cell growth supplement (BD Biosciences) with heparin from porcine intestines as described15, 34, 35. Bovine Aortic ECs (BAECs) and human Aortic ECs (HAECs) were purchased from Lonza and cultured in DMEN/10%FBS and EBM-2/20%FBS, respectively.
All animal experiments were approved by the Institutional Animal Care Committee of New York University Medical Center. Twelve-week old C.B-17-SCID beige mice were obtained from Jackson Laboratory.
HUVECs, BAECs or HAECs were transfected with 30 nM miRNA mimics (miR-16 and miR-424) or with 60 nM miRNA inhibitors (anti-miR-16 and anti-miR-424) (Dharmacon) utilizing Oligofectamine (Invitrogen) as previously described15, 16, 35. The dose of mimics and inhibitors was selected based on dose response experiments. All experimental control samples were treated with an equal concentration of a non-targeting control mimic sequence (CM) or inhibitor negative control sequence (CI), for use as controls for non-sequence-specific effects in miRNA experiments. Mock transfected control (transfection reagent) did not produce any significant effect on any of the parameters analyzed. The efficiency of transfection was greater than 95%, as assessed by transfection with fluorescently labeled miRIDIAN miRNA mimic (miRNAmimic-AlexaFluor555) (Dharmacon) for 12 hrs, and visualized by fluorescence microscopy 12 h after transfection. Verification of the degree of miRNA overexpression and inhibition was determined using qRT-PCR.
Human microvessels were generated and implanted in the subcutaneous position on the abdominal wall of C.B-17-SCID beige mice as previously described34, 36. Briefly, transduced HUVECs with scr-miR or miR-16 were harvested and counted. 3.5×105 cells were suspended in a rat tail type I collagen-human plasma fibronectin gel and approximately 1ml of the cell suspension was gently poured into a single well of a 6-well tissue culture plate. The protein gel was polymerized at 37°C and an equal volume of M199/20%FBS supplemented with ECGS was added to the well. 18 hours after gel polymerization, the gels were removed, bisected, and implanted in the subcutaneous position on the abdominal wall. Two weeks after implantation, half of the animals were euthanized and the grafts were harvested for analysis of the human microvasculature. Recovered gels and surrounding soft tissue were snap frozen in Tissue-Tek OCT (Sakura Finetek) and used to prepare 6-μm cryosections, which were subsequently stained with hematoxylin and eosin. Sections were also stained with anti-human PECAM (eBioscience) or TRITC-conjugated Ulex europeus agglutinin (Uea-1) (Sigma).
All data are expressed as means ± SEM. Statistical differences were measured by either Student’s t test or two-way ANOVA with Bonferroni correction for multiple comparisons when appropriate. A value of P ≤ 0.05 was considered statistically significant. Data analysis was performed using the Prism program (Statistical Graphics).
We investigated the possible role of miR-16 and miR-424 in cell-intrinsic angiogenic activity of ECs. A direct effect of these miRNAs as regulators of angiogenesis in ECs has not been studied so far. miR-15a, -15b, -16 (including 16-1 and 16-2 which have the same whole mature sequence), -195, -497, and -424 have different genomic locations but possess the same seed sequence nucleotides 2–8 at their 5′end- (Supplemental Figure IA and IB), which implies that these miRNAs share most of their target genes13, 26–29, 37. Therefore, to simplify, here thereafter we will refer to these targets as “miR-16 predicted targets”. Perfect sequence complementarity to nucleotides 2–8 at the 5′-end of the miRNA, called the “seed” sequence, is the strongest characteristic for targeting activity, and holds true for the vast majority of targets characterized to date38. Other characteristics, such as site location within the 3′UTR, flanking region, and conservation across multiple species greatly increases the probability of a predicted target site to be real13. By combining these characteristics, numerous computational approaches have been developed to predict miRNA targets13, 38. By using these bioinformatic tools we determined if miR-16 predicted targets were preferentially connected to any specific biological process (Supplemental Material and Methods, Bioinformatic analysis). We saw enrichment for genes implicated in the control of transcription as well as in important cellular functions such as proliferation, cycle progression and apoptosis, the last of which were in agreement with the predictions and validated target genes reported by others26, 27, 29. Interestingly, we also observed enrichment for genes regulating angiogenesis and its related functions (proliferation, cell migration, cell differentiation and morphogenesis, as well as relevant transcriptional regulators). Analysis of the predicted targets involved in angiogenesis revealed that the majority of them (81%) were positive regulators of angiogenesis (Supplemental Figure II). Among the predicted targets found, of note were VEGR2 (aka KDR) and FGFR1 due to their important role in the regulation of angiogenic functions of ECs 8. These two genes along with VEGF, which was previously reported to be a target for miR-1630, 39, were selected for further analysis.
First, we analyzed the expression of the selected miRNAs (i.e. miR-16 and miR-424) in ECs. As shown in Supplemental Figure IIIA, the relative levels of these miRNAs in human primary ECs (HUVECs and HAECs) were very similar to other human primary cells, such as vascular smooth muscle cells (VSMCs) and fibroblasts, but very highly expressed in HeLa cells (a highly proliferative tumor cell line). Since these miRNAs play very important roles in regulating cell and cell cycle progression26, the reduced levels of miR-16/424 would allow HeLa cells to maintain a highly proliferative state with respect to the primary cell lines analyzed, including ECs.
We then analyzed whether “miR-16-like miRNAs” participate in the regulation of VEGFR2, FGFR1 and VEGF. Specifically, we analyzed the effect of miR-16 and miR-424 on VEGFR2, FGFR1 and VEGF. miR-16 was chosen as a representative member of miR-15 family. miR-424, although not a member of miR-15 family, was chosen because it shares the seed sequence with all the members of the miR-15 family but has a more divergent 3′ end (Supplemental Figure IB). We identified a putative binding site in the 3′UTR of VEGFR2 (Figure 1A). The miR-16/-424 predicted site is a canonical 7mer-m8 site (Supplemental Figure IC). In the specific case of miR-424, this site is supported by an additional 3′ pairing optimally centered on miRNA nt 13–1813 (Supplemental Figure IC). The “seed” region of miR-16/-424 complementary to the sequence in the VEGFR2 3′UTR is conserved across species (not shown) and extends in humans from nt 44 to 50 (Figure 1A and Supplemental Figure IC). In the FGFR1 3′UTR we found three predicted sites for miR-16/-424, the first of which is a conserved canonical 7mer-m8 located in the center of the FGFR1 3′UTR (Figure 1A and Supplemental Figure IC). This site is supported by an additional 3′ pairing for miR-16 but not for miR-424 (Supplemental Figure IC). The second site is located at the end of the FGFR1 3′UTR (Figure 1A and Supplemental Figure IC) and is a 7mer-8m8 for both miR-16 and -424 (Supplemental Figure IC). The third site is a 3′ compensatory site, or an imperfect match to the seed13; however we did not consider this site in our studies. The site predicted in the 3′UTR of VEGFA was previously reported30, 39 (Figure 1A) and is a conserved 8mer with an additional 3′ pairing for miR-16 but not for miR-424 (Supplemental Figure IC).
To validate the predicted miRNA/mRNA interactions, the VEGFR2, FGFR1 and VEGF 3′-UTR were subcloned in a luciferase reporter vector. The resultant constructs were co-transfected into COS cells along with miR-16 or miR-424 mimic oligonucleotides or a non-targeting control mimic (CM). Transfection with the control luciferase reporter without any 3′UTR (empty vector) did not affect luciferase activity (data not shown). Interestingly, the relative luciferase activity was significantly reduced (≈20%) when cells were co-transfected with VEGFR2 3′UTR and miR-16 or miR-424 but not with CM (Figure 1B). Both miR-16 or miR-424 markedly repressed FGFR1 (Figure 1C) 3′UTR activity (≈50%); this effect is likely due to the presence of 3 binding sites13. Our data also indicate that miR-16, as well as miR-424, significantly reduced (≈20%) VEGF 3′UTR activity (Figure 1D), in agreement with previous reports30, 39. In all cases, when the miRNAs were transfected together, there was no difference in the overall luciferase activity as compared to their individual effect (data not shown). This is likely due to the identical seed sequences displayed by these miRNAs. Confirming the initial results, mutation of the miR-16/-424 site abrogated the repression of VEGFR2, and VEGF 3′UTR activity, consistent with a direct interaction of miR-16/-424 with the studied sites (Figure 1B–D). As indicated in Figure 1A, we found three predicted sites for miR-16/-424 in the FGFR1 3′UTR. Two of them had perfect sequence complementarities to the seed sequence, which is the strongest characteristic for targeting activity13 and more likely to provide the strongest effects. Mutation of these sites (1 and 2) produced a partial recovery of luciferase activity, therefore indicating that the effect of site 3 was not eliminated.
We further examined the effects of miR-16/-424 on VEGFR2, FGFR1 and VEGF expression in ECs (Figure 2 and Supplemental Figure IV). We first analyzed miRNA levels after transfection with miRNA mimics or inhibitors in order to measure transfection efficiency in ECs. As shown in Supplemental Figure IIIB, overexpression with both miR-16 and miR-424 mimics in HUVECs efficiently increased the levels of these miRNAs, and more importantly overexpression of one of them did not affect the expression of the other. Furthermore, inhibition of endogenous miR-16 and miR-424 with specific inhibitors reduced their intracellular levels in a specific manner (Supplemental Figure IIIC). Finally, we analyzed the effect of miR-16/-424 on VEGFR2, FGFR1 and VEGF expression. HUVECs were transfected with miR-16 or miR-424 mimics and the effect on protein and mRNA levels were analyzed 36 hours post-transfection. As shown in Figure 2A-C, both miR-16 and miR-424 significantly decreased VEGFR2, FGFR1 and VEGF protein and mRNA levels. Importantly, inhibition of endogenous miR-16 or miR-424 (I-miR-16 or I-miR-424) increased the expression of VEGFR2, FGFR1 and VEGF at both the protein and mRNA level (Figure 2B–D). The targeting activity of these miRNAs on VEGFR2 and FGFR1 was also relevant for HAECs (data not shown), a human adult EC type from a different vascular bed than HUVECs. In addition, and consistent with the conservation of these sites across species, similar results were obtained using bovine aortic ECs (BAECs) (Supplemental Figure IV).
It has been well established that both the VEGFR2 and FGFR-1 signaling pathways play crucial roles in angiogenesis. Interestingly, accumulating evidence has now implicated endothelial miRNAs in this process. To examine the potential relationship between VEGFR2 and FGFR-1 signaling and miR-16 or miR-424, we asked whether VEGF or bFGF modulated the cell-intrinsic expression of miR-16/-424 in HUVECs. As shown in Figure 3A, both cytokines regulated the expression of the mature form of miR-16 and miR-424. To identify whether these cytokines regulated the expression of these miRNAs at the transcriptional level, we examined the expression of primary transcripts (pri-miRNA) containing the stem-loop of the miRNA of interest. As shown in Figure 3B, both VEGF and bFGF increased the expression of pri-miR-16-1 (detects stem loops of miR-15b and miR-16-1 transcribed from Chromosome 13) and modestly, pri-miR-16-2 (detects stem loops of miR-15b and miR-16-1 transcribed from Chromosome 3), suggesting a transcriptional regulation of these two clusters by these cytokines. In contrast, the expression of pri-miR-424 remained essentially unchanged and, therefore suggests that VEGF and bFGF likely modulates the processing of miR-424 from the pre-existing primary transcript (i.e., the increase in the mature form, Figure 3A) without affecting its transcriptional expression.
Regardless of whether the effect is at the transcriptional or post-transcriptional level, both VEGF and bFGF have similar effects (stimulation) on miR-16 and miR-424 mature expression. We then investigated whether VEGF directly regulated VEGFR2 or FGFR1 3′ UTRs via miR-16 in ECs. For these experiments, we used the luciferase reporter assay, described above, directly in HUVECs. As shown in Figure 3C, stimulation of HUVECs with VEGF, reduced both VEGFR2 and FGFR1 3′ UTR activity, whereas no effect was observed when cells were transfected with the empty vector control, indicating that some endogenous miRNAs involved in the regulation of VEGFR2 and FGFR1 were induced to regulate their expression under stimulated conditions. Interestingly, endogenous inhibition of miR-16 or miR-424 prior to VEGF stimulation stored VEGFR2 and FGFR1 3′UTR activity (Figure 3D), indicating that these effects are likely to be mediated by miR-16 and miR-424 upregulation in ECs.
Next, we tested whether the effects observed on VEGFR2, FGFR1 and VEGF expression were functional. To this end, we examined the effects of miR-16 and miR-424 on three angiogenic phenotypes of ECs, namely proliferation, migration and morphogenesis (cord formation). Previous results have shown that both miR-16 and miR-424 regulate cell proliferation in different cell types26–28, 33. In agreement with this data, we observed that miR-16 or miR-424 had a negative effect on cell proliferation. As seen in Figure 4A, overexpression of miR-16 or miR-424 reduced cell proliferation in HUVECs without a significant induction of apoptosis assessed by induction of caspase-3 cleavage or increase in subG0/G1 population by flow cytometry (Supplemental Figure VA and VB, respectively). Moreover, in stress conditions, such as prolonged serum starvation 40, both miR-16 and miR-424 further reduced the number of cells assessed by cristal violet staining (Figure 4B). Interestingly, 9 additional hours in the absence of VEGF diminished the cell number in both control (CM) and miRNA overexpressing cells. Treatment with exogenous VEGF produced partial rescue in control cells without affecting miRNA overexpressing cells (Figure 4B), suggesting an altered response to VEGF in cells overexpressing miR-16 or miR-424. Next, we examined the effects of these miRNAs on EC migration. Overexpression of miR-16 or miR-424 reduced basal migration as well as VEGF or bFGF induced migration in BAECs (Figure 4C). miR-16 and miR-424 overexpression in HUVECs also resulted in significant impairment of cord formation under basal conditions and following stimulation with VEGF or bFGF (Figure 4D). Converse effects on migration and cord formation were obtained when the endogenous levels of these miRNAs were inhibited (Supplemental Figure VI). Importantly, we also overexpressed either VEGFR2 or FGFR1 cDNA together with a miR-16 mimic in HUVECs and evaluated their effect on EC migration in response to VEGF or bFGF (Supplemental Figure VII). As shown in Supplemental Figure VIIA, transfection of HUVECs with VEGFR2 or FGFR1 cDNA (without the respective 3′UTR) for 24h was very efficient and in both cases high levels of VEGFR2 and FGFR1 were obtained. Interestingly, VEGFR2 or FGFR1 overexpression rescued the inhibitory effect of miR-16 in EC migration. Altogether, these data indicate that miR-16/-424 regulates cell-intrinsic angiogenic responses in vitro and are consistent with their targeting activity on VEGFR2, FGFR1 and VEGF in ECs.
To test the potential involvement of these miRNAs on VEGFR2 and FGFR1 signaling more directly, we examined the effect of the reduction of VEGFR2 and FGFR1 via miR-16 overexpression on Akt and ERK1/2 activation since these downstream effectors (namely the PI3K/Akt and the Ras/Raf/Erk pathways) are activated after VEGF or bFGF stimulation in ECs. As seen in Figure 5A–B, Akt and ERK1/2 phosphorylation were reduced in response to VEGF or bFGF stimulation (Figure 5A–B, respectively) in ECs transfected with miR-16. Similar results were obtained when cells were transfected with miR-424 (Supplemental Figure VIII). Interestingly, when cells were stimulated with Sphingosine-1-phosphate (S1P), which signals primarily through a family of five G-protein-coupled receptors (EDG receptors)41, Akt and ERK1/2 activation was not significantly affected by miR-16 overexpression (Figure 5C). Altogether, this data suggests that these miRNAs are likely to control proliferation, migration and differentiation of ECs by regulation of VEGF and bFGF signaling through VEGFR2 and FGFR1.
In a final series of experiments, we evaluated the effect of miR-16 on the ability of human ECs to form capillary-like structures in vivo using a previously described model for forming tubes within a three-dimensional gel with cultured HUVECs 34, 42. We used lentiviral vectors to manipulate the levels of miRNAs in vivo as reported previously43. HUVECs were efficiently transduced with a miR-16 lentiviral vector or scrambled miRNA (scr-miR) (Figure 6A) and then suspended in collagen-fibronectin protein gels34, 36, 42. These gels were then implanted into the abdominal wall of immunocompetent mice. Consistent with previous reports, HUVEC-derived cords formed in vitro survive and evolve into tubes that inosculated with the host microcirculation34, 42. Grafts were explanted for evaluation at 14 and 21 days. Interestingly, 7 days after implantation, HUVECs transduced with either scr-miR or miR-16 that were kept in culture in parallel, maintained GFP expression (Figure 6B). Moreover, miR-16 transduced HUVECs presented increased levels of miR-16 (Figure 6C) and, as expected, a concomitant decrease in the expression of VEGFR2, FGFR1 and VEGF (Figure 6D). Gross visualization of constructs harvested 14 days after implantation appeared to be blood-perfused by mouse circulation, however those containing miR-16 transduced HUVECs seemmed to be, in general, less efficiently perfused and cellularized and slightly smaller than scr-miR implants (Figure 6). Moreover, miR-16 implants contained significantly fewer capillary structures (Figure 6F), as assessed by both human PECAM or UEA-1 lectin staining (reacts with the blood group ABH expressed on human EC) for detection of human EC-lined vessels within engrafted protein gels, respectively. In both cases, the majority of the structures were wholly composed of human ECs, as anti-mouse PECAM antibodies reacted with fewer than 1% of the vascular profiles within the constructs (data not shown), confirming that the vessel-like structures detected were not formed by mouse neovascularization of the gel and consistent with previous reports of this model42. After 21 days of implantation, PECAM-positive structures were present throughout the collagen-fibronectin gel of scr-miR implants, however they were largely absent in the gels containing miR16-HUVECs (Supplemental Figure IX). Altogether, these data indicate that miR-16 participates in the regulation of neovascularization in vivo by controlling the cell-intrinsic angiogenic activity of ECs.
A growing body of evidence indicates that miRNAs actively participate in the control of angiogenesis12, 18–22, 33. In the present study, we have investigated the function of miR-16 and miR-424, as representative members of a group of miRNAs that share the same seed sequence, in different aspects of EC biology pertinent to angiogenesis. Seed sequences of miRNAs are arranged between the second and eighth nucleotide in the 5′ end and are the most critical determinants of miRNA targeting activity13. miR-15a, -15b, -16 (1 and 2), -195, -424, and -497 have different genomic locations but possess the same seed sequence, implying that these miRNAs share most of their target genes13. In agreement with previous experimental data26–29, 37, our analysis showed that indeed, these miRNAs share most of their targets. Therefore, the slight differences observed in target prediction by using other bioinformatic algorithms were likely due to differences at the level of the pairing to the miRNA 3′ end and/or the degree of binding site conservation across species13. Functional annotation of the predicted targets for miR-15a, -15b, -16, -195, -424, and -497 suggests that these miRNAs control a complex network of genes involved in cell cycle, proliferation, apoptosis, and survival26, 27, 29. More appealing to us was the identification of target genes connected to angiogenesis and their related functions (proliferation, cell migration, cell differentiation, morphogenesis, as well as cell signaling and relevant transcriptional regulators)2, suggesting that miR-16 and its related miRNAs may participate in the control of angiogenesis in multiple ways. Given their important role in the regulation of angiogenic functions in ECs2, 5–7, of special interest was the identification of VEGR2, FGFR1 and VEGF as target genes for miR-16.
Interestingly, miR-15b and miR-16 have been shown to control the expression of VEGF in a carcinoma cell line30 and in a human breast cancer cell line39. In agreement with these previous reports, our studies also indicate that miR-16, as well as miR-424, significantly reduced VEGF 3′UTR activity and therefore targeted VEGF. However, a key difference between these experiments and those previously reported30, 39 is that in those reports they tested the fragments of the 3′UTR containing the target sequence whereas here we tested the sequence in the context of the entire 3′UTR. There is increasing evidence that contextual features of the 3′UTR, such as secondary structures or local AU-rich regions, among others, can govern miRNA/mRNA interactions13. This approach, together with the mutation of the predicted binding site (also in the context of the complete 3′ UTR), shows more unequivocally that this interaction is, indeed, functional. Because VEGFR2 and FGFR1 play key roles in angiogenesis5, 6 and little is known about their posttranscriptional regulation by miRNAs, we additionally validated VEGFR2 and FGFR1 as targets for miR-16 and miR-424. Moreover, we provide evidence that these interactions are relevant in an EC context, since both gain and loss of function experiments revealed that these miRNAs regulate the expression of VEGFR2, FGFR1 and VEGF. Therefore, it appears likely that miR-16 related miRNAs participate in the regulation of angiogenesis in the context of ECs, at least in part by the modulation of these receptors together with the regulation of endothelial VEGF.
VEGF has been recognized as a paracrine factor in both developmental and pathological settings44. However, a critical role of endogenous VEGF expression in EC functions related to viability and survival has been demonstrated40. Interestingly, our data show that miR-16/-424 overexpression reduced EC growth in both basal and stressed conditions and that exogenous VEGF could not rescue compromised survival of ECs over-expressing miR-16. A similar phenotype was observed in VEGFECKO cells40, however, in our case these effects may also be explained by the miR-16-mediated reduction of VEGFR2 protein levels in ECs, which compromises its activation and thus, the survival-promoting activity of exogenous VEGF through the activation PI3-kinase-Akt pathway45. Additionally, and in agreement with the regulation of FGFR1 by miR-16, we found that miR-16/-424 also diminished bFGF signaling through FGFR1. Moreover, migration and cord formation was significantly reduced in miR-16/-424 overexpressing cells in response to exogenous VEGF of bFGF. Altogether, our data indicate that these miRNAs affect the activity of downstream components of the pathways that regulate proliferation, migration and cord formation of ECs in vitro by regulating the expression of key endothelial angiogenic proteins (i.e. VEGFR2, FGFR1).
Previous reports have shown that in different cell types miR-15a/16-1 and miR-15b/16-2 clusters, in addition to miR-195 and miR-424, play very important roles in regulating cell proliferation and apoptosis by targeting cell cycle progression proteins26–29, 33, 46. Consistent with these findings, it has recently been shown, that the downregulation of miR-424 in senile hemangioma contributes to abnormal angiogenesis33. Specifically the authors showed that miR-424 negatively regulates the proliferative activity of human endothelial microvascular cells (HDMECs) via MEK1 and cyclin E1. 26–29, 33, 46. Altogether these data, in addition to the new functions we describe here for miR-16 and/-424, indicate that these miRNAs participate in the regulation of the angiogenic functions of ECs. In contrast, a recent report shows that miR-424 expression is upregulated in HUVECs by hypoxia, thereby promoting angiogenesis by targeting cullin 2 and increasing hypoxia-inducible factor 1α levels47. However, under standard culture conditions (normoxia) the authors showed that overexpression of miR-424 stimulates proliferation, migration and cord formation47, which is contrary to both our findings as well as the data reported by Nakashima et al.33. In our experimental conditions, miR-424 overexpression is performed by using microRNA mimics, which are double-stranded RNA oligonucleotides that supplement miRNA activity by effectively mimicing the endogenous mature miRNA function. Unlike the endogenous miRNA duplex, however, the active strand of the miRNA mimic is preferentially incorporated into the RISC-like complex, while the passenger strand is excluded through chemical modification. In their transient expression studies, miR-424 was PCR amplified from human genomic DNA and cloned into the EcoRI-BamHI sites of the pGSU6 vector and then transfected into HUVECs. In this scenario, the passenger strand could potentially be selected; therefore the authors cannot rule out the effect of miR-424*. By using gain and loss of function approaches (using mimics or inhibitors for these miRNAs) we have shown that their targeting activity towards VEGFR2, FGFR1 and VEGF is indeed relevant in the context of ECs. Furthermore, we obtained the same results using a lentiviral approach to overexpress miR-16. In our in vivo model we introduced stably transduced HUVECs with miR-16 into a synthetic vascular bed to specifically address the effect of miR-16 in regulating the cell-intrinsic angiogenic activity of ECs and avoid the effect of overexpression of the microRNA in other cell types, which cannot be ruled out in the Gosh et al. in vivo studies. Our data indicate that miR-16 regulates capillary tube formation of human ECs in vivo. Although, we have not performed the experiments to test the effect of miR-424 in vivo, our in vitro data suggests that miR-424 has the same functions as miR-16 in ECs (i.e. targeting VEGFR2, FGGR1 and VEGF in ECs and negatively regulating proliferation, migration and cord formation) and therefore, similar results would be expected.
Several lines of evidence indicate that the regulation of miRNA levels by different stimuli may serve as points of crosstalk between signaling pathways, thereby contributing to the regulation of the specific stimulus-induced responses10. Recent insights indicate that signal transduction pathways are prime candidates for miRNA-mediated regulation in animal cells, and therefore are ideal targets for specific fine tuned cell responses10. miR-16/-424 regulation of VEGFR2, FGFR1 and VEGF, as we present in this report, could be considered an example of the multi-gene regulatory capacity of miRNAs to remodel the signaling landscape in an effective and timely manner10. In fact, our data indicate that angiogenic growth factors such as, VEGF and bFGF, stimulate the expression of mature miR-16 and miR-424 in HUVECs to fine tune the levels of angiogenic mediators in ECs (i.e. VEGFR2, FGFR1 and VEGF) thereby participating in the maintenance of EC steady state conditions and conferring signal robustness10. miRNA processing is faster than protein translation, allowing miRNAs to affect gene expression with shorter delay than transcriptional repressors, conferring exquisite temporal and quantitative precision over cell signaling. Given their essential roles in multiple processes9, miRNA expression needs to be tightly regulated10. Thus the identification of the elements implicated in their regulation is essential to dissect the role of miRNAs in signaling networks10. miRNA abundance can be controlled at the level of transcription of the primary transcript (pri-miRNA), during the maturation steps, or through turnover of the mature miRNA. Our data underlie the complexity of the regulation of miRNAs, whereas miR-16 seems to be regulated by VEGF/bFGF at the level of transcription; increased levels of miR-424 after VEGF/bFGF stimulation are likely due to a positive regulation of miRNA maturation/processing of from the pre-existing primary transcript (i.e., the increase in the mature) without affecting its expression (i.e., no change in pri-miR-424). These data highlight the different mechanisms by which miRNAs can be regulated. The importance of understanding how these stimuli might affect miRNA levels in ECs needs further investigation.
miRNAs have tremendous therapeutic potential for the treatment of vascular diseases associated with aberrant pathological angiogenesis19. In addition to their roles as potent regulators of cell proliferation in different cancer cell lines26–28, 46, of interest are the miRNAs encoded by the miR-17-92 cluster. These miRNAs are known to act as oncogenes but also have essential functions in tumor formation and normal development of the heart, lungs, and immune system. Although other cell types, such as VSMCs and circulating progenitors, play an important role in neovascularization, in the context of ECs they have been shown to provide cell-intrinsic antiangiogenic activity 22. The miR-15 family members may also be implicated in the non-cell-autonomous30, 48, as well as in the cell-autonomous, regulation of angiogenesis, as we show here. Therefore, the identification of miRNAs regulating both angiogenesis and tumor cell survival might be a meaningful approach for cancer therapy. In fact, miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts49, and mir-16 and 15a have been recently reported to act as tumor suppressors both in tumor and stromal cells by targeting FGFR148. Thus, we hypothesize that the use of miRNA mimics of miR-16 may be an attractive anti-angiogenesis strategy that could target tumor cell survival and proliferation while disrupting cell-intrinsic angiogenic activity of ECs. Interestingly, there is now direct evidence that synthetic miRNA mimics can be systemically delivered and support the promise of miRNAs as a future targeted therapy for cancer 25.
Although we have begun to appreciate the importance of miRNAs in the regulation of angiogenic signaling in ECs, much work remains to be done to determine the miRNAs involved and the target pathways affected19.
We thank Dr. B. R. Shepherd for helpful comments and advice for the in vivo study, Dr. M.V. Guijarro for assistance with immunofluorescent microscopy, and Leigh Goedeke for the editing work on this manuscript.
Source of funding
This work was supported by SDG-AHA 0835481N to Y.S and 0835585D to C. F-H and by the National Institutes of Health (1P30HL101270-01) and R01HL105945 from the National Heart, Lung, And Blood Institute to Y.S and R01HL107953 and R01HL16063 to C.F-H.