A smooth muscle cell lncRNA controls angiogenesis in chronic limb-threatening ischemia through miR-143-3p/HHIP signaling
Ming Zhai, Anurag Jamaiyar, Jun Qian, Winona W. Wu, Emre Bektik, Vinay Randhawa, Camila Vaz, Arvind K. Pandey, Akm Khyrul Wara, Madhur Sachan, Yi Hu, Jéssica L. Garcia, Claire E. Alford, Terence E. Ryan, Wenhui Peng, Mark W. Feinberg
Ming Zhai, Anurag Jamaiyar, Jun Qian, Winona W. Wu, Emre Bektik, Vinay Randhawa, Camila Vaz, Arvind K. Pandey, Akm Khyrul Wara, Madhur Sachan, Yi Hu, Jéssica L. Garcia, Claire E. Alford, Terence E. Ryan, Wenhui Peng, Mark W. Feinberg
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Research Article Angiogenesis Vascular biology

A smooth muscle cell lncRNA controls angiogenesis in chronic limb-threatening ischemia through miR-143-3p/HHIP signaling

Abstract

Peripheral artery disease (PAD) often advances to chronic limb-threatening ischemia (CLTI), resulting in severe complications such as limb amputation. Despite the potential of therapeutic angiogenesis, the mechanisms of cell-cell communication and transcriptional changes driving PAD are not fully understood. Profiling long noncoding RNAs (lncRNAs) from gastrocnemius muscles of participants with or without CLTI revealed that a vascular smooth muscle cell–enriched (SMC-enriched) lncRNA, CARMN, was reduced with CLTI. This study explored how a SMC lncRNA-miRNA signaling axis regulates angiogenesis in limb ischemia. CARMN-KO mice exhibited reduced capillary density and impaired blood flow recovery and tissue necrosis following limb ischemia. We found that CARMN-KO SMC supernatants inhibited endothelial cell (EC) proliferation, spheroid sprouting, and network formation. RNA-seq identified downregulation of the Hedgehog signaling pathway in CARMN-KO models and revealed that CARMN regulates this pathway through its downstream miRNA, miR-143-3p, which targets Hedgehog-interacting protein (HHIP), an antagonist of Hedgehog signaling. Delivery of HHIP-specific siRNA or miR-143-3p mimics rescued EC angiogenic defects and improved blood flow recovery in both CARMN-KO and WT mice. These findings underscore the critical role of CARMN in modulating angiogenesis through the miR-143-3p-HHIP-Hedgehog signaling axis, providing insights into SMC-EC interactions and potential therapeutic strategies for CLTI.

Authors

Ming Zhai, Anurag Jamaiyar, Jun Qian, Winona W. Wu, Emre Bektik, Vinay Randhawa, Camila Vaz, Arvind K. Pandey, Akm Khyrul Wara, Madhur Sachan, Yi Hu, Jéssica L. Garcia, Claire E. Alford, Terence E. Ryan, Wenhui Peng, Mark W. Feinberg

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Figure 8

Carmn can inhibit the expression of Hhip through miR-143-3p signaling.

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Carmn can inhibit the expression of Hhip through miR-143-3p signaling. ...
(A) Schematic of binding sites and sequence complentarity for miR143 and miR145 in the Hhip 3′ UTR. (B) the relative expression of miR-143-3p, miR-143-5p, miR-145-3p, and miR-145-5p between Carmn WT and KO SMCs in vivo and in vitro. (C) the relative expression of miR-143-3p, miR-143-5p, miR-145-3p, and miR-145-5p in WT SMC transfected with nonspecific control inhibitor (NSi), miR143 inhibitor, and miR145 inhibitor. (D) the relative expression of Hhip mRNA among WT SMCs transfected with nonspecific control (NSi), miR143 inhibitor, and miR145 inhibitor. (E) the protein expression of HHIP between WT SMCs transfected with nonspecific control (NSi), miR143 inhibitor, and miR145 inhibitor. (F) the quantification of relative expression of HHIP among the 3 groups in E. (G) (Left) Schematic of binding sites between miR-143-3p and Hhip 3′ UTR. (Right) Relative luciferase units (RLU) of WT Hhip 3′ UTR and mutated (MUT) Hhip 3′ UTR luciferase reporter assay with NS mimic or miR-143-3p mimic (n = 6). (H) the BrdU assay of mECs incubated with supernatants collected from WT SMCs transfected with nonspecific control, miR143 inhibitor, and miR145 inhibitor. (I) the representative images of mEC spheroids cocultured with supernatants collected from WT SMCs transfected with nonspecific control (NSi), miR143 inhibitor, and miR145 inhibitor. Scale bar: 100 μm. (J) the quantification of spheroids branch length and the number of branches of spheroids in I. (K and L) the relative expression of miR-143-3p and miR-145-3p between groups of KO SMCs transfected with nonspecific control (NSm), miR-143-3p mimic, or miR-143-5p mimic. (M) the relative expression of Hhip in KO SMCs transfected with nonspecific control (NSm), miR-143-3p mimic, or miR-143-5p mimic. (N) The WB representative images and related quantification of HHIP in KO SMC transfected with nonspecific control, miR-143-3p mimic or miR-143-5p mimic. (O) BrdU incorporation assay of mECs incubated with supernatants collected from KO SMCs transfected with nonspecific control, miR-143-3p mimic, or miR-143-5p mimic. (P) representative images of mEC spheroid cocultured with supernatants collected from KO SMCs transfected with nonspecific control, miR-143-3p mimic, or miR-143-5p mimic. Scale bar: 100 μm. (Q) quantification of spheroid branch length and the number of branches of spheroid in P. For all panels, error bars represent SEM. P value was determined by unpaired 2-tailed Student’s t test (B) or 1-way ANOVA with Bonferroni post test (C, D, F–H, J–O, and Q).