For further analysis of STAT3 involvement, we studied miR-21 that contains 2 STAT3 binding sites in its promoter. In separate experiments, exposure to Ang II resulted in 1.9-fold stimulation of miR-21 measured by real-time PCR (P < 0.05; Figs. 4A, B). As shown in Figures 4B and C compared with the cultures transfected with scrambled sequence, cells transfected with miR-21 inhibitor lost responsiveness to Ang II and did not exhibit any increase in tube formation.
In the present study, we have shown that HCAECs react to Ang II in moderate concentrations by upregulating several genes implicated in blood vessel development and angiogenesis. Moreover, we have shown that proangiogenic effects of Ang II are, in part, linked to STAT3 and miR-21.
Our microarray data offer interesting clues regarding possible mechanisms implicating STAT3 as a key player in Ang II–mediated proangiogenic signaling. In a number of reports, Ang II has been shown to activate STAT3 signaling cascade in various cell types and utilizing both JAK2-dependent and JAK2-independent pathways.45,46 It is also interesting that STAT3 consensus sequences are found in miR-21 promoter as well. Mir-21 seems to be central to angiogenic action of Ang II as its blockade using miR-21 inhibitor made endothelial cells unable to form tubular networks on Matrigel in response to Ang II exposure.
The actions of miR21 are attributed to the central role of its primary target PTEN (phosphatase and tensin homolog deleted from chromosome 10) in Akt signaling. PTEN antagonizes PI3K by cleaving its major product, lipid PtdIns(3,4,5)P3, and thus preventing activation of downstream Akt signaling cascade. In recent studies,52 pharmacological inhibition of PI3Kγ inhibition in ApoE or LDLR KO significantly reduced the formation of atherosclerotic lesions.
Judging from the pattern emerging from the PCR array analysis, Ang II triggers multilayered stimulation of angiogenesis and proliferation-related miRNAs while suppressing a number of miRNAs responsible for cell arrest and apoptosis. In our data set, expression of many members of Let-7 family (let-7a, c, d, e, f, and g) was stimulated by Ang II (see Table 2, Supplemental Digital Content 1, http://links.lww.com/JCVP/A153). Let-7g was shown to be necessary—via its effect on TIMP1/MMP activities—for the tube-forming ability of endothelial cells.22 Members of 17–92 cluster are regulated by MYC and have been directly implicated in various aspects of tumor angiogenesis due (presumably) to inhibition of their target genes thrombospondin-1 and connective tissue growth factor.53 However, recent studies showed that overexpression of miR-19a and several other cluster members had negative effect in 3-dimensional model of angiogenesis.54 Several miRNAs, including miR-19, miR-21, miR-23b-3p, and miR-24-3p, converge on the stimulation of prosurvival Akt pathway through inhibition of PTEN.17,19,20,48
In agreement with experimentally validated proangiogenic action of Ang II, many antiangionenic miRNAs were downregulated. On the other hand, a number of inhibiting miRNAs were overexpressed, probably reflecting the initiation of self-balancing program involving critical aspects of Ang II proangiogenic signaling. Several upregulated and downregulated miRNAs (miR-125, miR-128, miR-15a-5p, miR-16-5p and miR-20b-5p) target VEGF. It has also been shown that increase in VEGF production triggers the synthesis of miR-15555 (found to be upregulated in our study) that targets AT1R.15
It should also be noted that many miRNAs that were found to be differentially expressed in Ang II–treated cultures are directly or indirectly implicated in STAT3 signaling and that observed shifts in their expression were consistent with STAT3 stimulation (see Figure 2, Supplemental Digital Content 2). For example, a number of miRNAs, including miR-21 and members of 17–92 cluster, are directly stimulated by STAT3.56,57 In line with the expectations, miR-21, miR-18a, miR-19a, and miR-20 were significantly upregulated in our data set (see Table 2, Supplemental Digital Content 1, http://links.lww.com/JCVP/A153).
In summary, our studies show that one of the dominant themes of Ang II transcriptional signature in endothelial cells is stimulation of angiogenesis. Strong suppression of Ang II–mediated tube formation in response to inhibition of STAT3 and miR-21 suggests that the activation of STAT3–miR-21 signaling axis is one of the prerequisites of proangiogenic effect of Ang II.
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