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 6

Hhip targets Hedgehog Signaling Pathway to mediate angiogenic changes in mECs.

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Hhip targets Hedgehog Signaling Pathway to mediate angiogenic changes i...
(A) Relative mRNA expression levels of Hhip in KO SMCs with siRNA-mediated (si-mediated) knockdown of Hhip or nonspecific control (si-NC) (n = 6). (B) representative images of the WB of HHIP protein from WT or KO SMC with si-NC and si-Hhip transfection. (C) the quantification of HHIP protein expression between Carmn WT or KO SMCs. (D) the HHIP concentration measured by ELISA in supernatants harvested from the indicated groups of SMCs. (E) quantification of BrdU incorporation in mECs incubated with supernatants collected from the indicated groups of SMCs. (F) the representative WB images of specific AKT and eNOS proteins in mECs incubated with supernatants collected from KO SMCs transfected with si-NC or si-Hhip. (G and H) the quantification of relative expression of p-AKT or p-eNOS of mECs incubated with KO SMC si-NC or si-Hhip supernatants. (I) representative WB images of the indicated protein expression of the Hedgehog signaling pathway in Carmn WT or KO SMCs. (J) the quantification of relative expression of proteins in I. (K) representative images of spheroids cocultured with supernatants collected from the indicated 4 groups of SMCs. Scale bar: 100 μm. (L) quantification of branch length and number of sprouts in K. For all panels, error bars represent SEM. P value was determined by unpaired 2-tailed Student’s t test (A, C, G, and H) or 1-way ANOVA with Bonferroni post test (D, E, J, and L).