MiR-33 regulation of stretch-induced intimal hyperplasia in vein grafts

This editorial refers to ‘MicroRNA-33 protects against neointimal hyperplasia induced by arterial mechanical stretch in the grafted vein’ by K. Huang et al., pp. 488–497.

Vein graft bypass surgery has become the most commonly performed revascularization technique in patients with coronary artery disease, the leading cause of mortality and morbidity worldwide. Vein grafts adapt to the new arterial environment, and the structural vascular remodelling and intimal thickening in the vein graft wall is the main cause of restenosis after vascular reconstruction.¹ Intimal accumulation of smooth muscle cells (SMCs) contributes to the thickening and narrowing of the vessel lumen through pro-inflammatory cytokine-induced cell migration and local cell proliferation.¹ Proliferation of SMCs is a crucial event in the pathogenesis of intimal hyperplasia, which is thought to be an important determinant of successful vein graft adaptation. Although the disease process has been described, the underlying mechanisms are still unclear. Work over the last decade has uncovered prominent roles for non-coding RNAs in several cardiovascular disorders including the failure of vein graft bypass.^2,3 MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules involved in the regulation of gene expression at the post-transcriptional level.⁴ Huang and co-workers⁵ identified microRNA-33 (miR-33) as a major regulator of SMC proliferation and neointimal hyperplasia in vein grafts. Huang and colleagues found that miR-33 expression was markedly attenuated in grafted veins. The authors observed an inverse correlation between miR-33 levels and increased intimal thickening and SMC proliferation. To determine whether miR-33 directly controls SMC proliferation, the authors performed a series of elegant studies, including BrdU incorporation and CCK-8 assays. They found that miR-33 overexpression markedly inhibited SMC proliferation. By contrast, antagonism miR-33 enhances SMC proliferation. These findings are consistent with previous reports establishing miR-33 as an important regulator of cell proliferation and cell cycle progression.⁶

To dissect the molecular mechanisms by which miR-33 controls SMCs proliferation, Huang et al.⁵ analysed miR-33-predicted targets mRNAs using a number of computational algorithms. They identified bone morphogenetic protein 3 (BMP3) as a novel miR-33 target-gene. BMP3 is a member of the transforming growth factor beta (TGF-β) superfamily and promotes mesenchymal stem cell proliferation though the TGF-β/Activin signalling pathway.⁷ The authors found significantly upregulated BMP3 expression in grafted veins, while miR-33 showed an opposite regulation. They also utilized gain- and loss- of function approaches to demonstrate that exogenous BMP3 accelerated venous SMC proliferation, whereas knock-down of BMP3 exhibited the opposite effect. Most importantly, exogenous BMP3 abolished the inhibitory effects of miR-33 on SMC proliferation. Further studies showed that the phosphorylation of SMAD2 and SMAD5, two molecules downstream of BMP3, were regulated by miR-33 in a BMP3-dependent manner. Together, these observations support the hypothesis that miR-33 protects SMCs proliferation and neointimal hyperplasia by repressing BMP3.

The authors also analysed the function of miR-33 in venous SMC proliferation in response to mechanical cyclic stretch, the predominant mechanical force influencing SMCs structural organization, function, and gene expression. Consistent with the in vivo vein graft model, cyclic stretch decreased the expression of miR-33 accompanied by elevated BMP3 expression and increased phosphorylation of SMAD2 and SMAD5 in vitro. By treating the SMCs with miR-33 mimics or BMP3 specific siRNA, the authors further validated the important role of miR-33 and BMP3 on venous SMC proliferation in response to cyclic stretch. Notably, injection of agomiR-33 attenuated neointimal formation and repressed cell proliferation in grafted veins by regulating BMP3 expression and phosphorylation of SMAD2 and SMAD5. As expected, BMP3 overexpression using lentivirus negated the effects of agomiR-33 on intimal thickening occurring in the vein grafts, suggesting the effects of miR-33 on venous SMC proliferation and neointimal hyperplasia are dependent on BMP3 expression.

It is worth noting that each microRNA can regulate multiple target mRNAs and each target mRNA can also be regulated by multiple microRNAs.⁴ Previously, miR-33 has been demonstrated to play an important role in the regulation of cell proliferation and cell cycle progression by targeting cyclin-dependent kinase 6 (CDK6), cyclin D1, and p53, by which control hepatocyte proliferation, replicative senescence of mouse embryonic fibroblasts and haematopoietic stem cell self-renewal.^6–9 CDK6 is a D-cyclin-activated kinase involved in driving the cell cycle through interactions with cyclins D1, D2, and D3 in G1 phase of the cell cycle, while p53 induces G1 arrest in the cell cycle by regulating p21 expression. Whether these target genes are involved in miR-33-dependent regulation of mechanical stretch-induced proliferation of venous SMCs needs to be addressed in the future. On the other hand, the proliferation of SMCs can be regulated by many miRNAs including microRNA-143/145, miRNA-21 and microRNA-181,^2,3,10 some of which are also predicted to target BMP3. Thus, it would be interesting to investigate the role of these miRNAs in vein graft-induced neointimal hyperplasia through the regulation of BMP3.

Although the work of Huang et al.⁵ demonstrates that miR-33 influences vein graft-induced neointimal hyperplasia, a number of important questions still need to be addressed, including the molecular mechanism and mechanosensors that control miR-33 expression in response to arterial stretch of SMCs and vascular injury. Previous work has established an essential role of miR-33 in regulating lipid metabolism. miR-33 is encoded within the intron of SREBP2 gene and regulates cholesterol homeostasis in concert with its host gene.^11–13 Most of these reports have demonstrated that miR-33 is cotranscribed with SREBP2, thus, it would be interesting to determine how SREBP2 expression is regulated in vein grafts and the importance of SREBP2-regulated cholesterol metabolism during neointimal hyperplasia. Indeed, statins and ezetimibe, drugs that lower circulating cholesterol and increase SREBP2 expression and processing, reduced expansive remodelling and intimal hyperplasia.^14,15 Finally, another aspect that requires further investigation is the study of miR-33 in human vein graft adaptation. This will be critical because the human genome encodes two different miR-33 isoforms, miR-33a and miR-33b, encoded within SREBP2 and SREBP1 genes respectively.¹³ Thus, additional studies using human samples or alternative animal models such as non-human primates that express miR-33a and miR-33b will be necessary to translate these findings to the intimal hyperplasia observed in human vein grafts.

In summary, this interesting study revealed a novel mechanism by which miR-33 plays a prominent role in arterial mechanical stretch-induced venous SMC proliferation and neointimal hyperplasia (Figure1) and suggests that targeting miR-33 might provide a novel therapeutic strategy to prevent vein graft failure and neointimal hyperplasia.

Figure 1.

A proposed model of how miR-33 regulates mechanical stretch-induced SMC proliferation and neointimal hyperplasia of vein grafts. (A) Placement of vein grafts to the new arterial environment induces mechanical cyclic stretch of SMC, which is sensed by mechanosensors. The mechanosignal inhibits the expression of miR-33 and upregulates the novel miR-33 target gene, BMP3. Upregulation of BMP3 results in increased venous SMC proliferation and neointima hyperplasia by phosphorylating the downstream molecules, SMAD2 and SMAD5. (B) Addition of agomiR-33 attenuates neointimal formation and represses cell proliferation in grafted veins by regulating BMP3 expression and phosphorylation of SMAD2 and SMAD5.

Conflict of interest: none declared.

Funding

This work was supported by grants from the National Institutes of Health (R35HL135820) the American Heart Association (16EIA27550005), and the Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research MIRVAD.