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 1

Carmn expression is reduced with limb ischemia and deficiency of Carmn-impaired perfusion recovery after hindlimb ischemia in mice.

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Carmn expression is reduced with limb ischemia and deficiency of Carmn-...
(A) (top) Overlap between differentially expressed lncRNAs screened from hypoxia-stimulated SMCs and differentially expressed lncRNAs screened from human gastrocnemius muscles collected from patients with PAD, healthy adults (HA), or patients with CLTI (GSE120642). (bottom) Heatmap of differentially expressed lncRNAs in primary SMCs between normoxia and hypoxia conditions (P = 0.00012). (B) The expression of Carmn in primary SMCs exposed to hypoxia or in gastrocnemius muscles of patients with or without CLTI (P = 1.44 × 10–8). (C) the relative expression of Carmn in endothelial cells (ECs) and non-ECs. (D) the representative image of Carmn colocalized within the nucleus of α-SMA+ SMCs within the gastrocnemius muscle harvested from Carmn WT mice. (E) Representative Laser Doppler Imaging (LDI) images of hindlimbs immediately after FAL and at different time points. (F) Quantification of blood flow (surgical / contralateral limb) by LDI images, normalized to the nonsurgery limb between 2 groups, (n = 6). (G, Necrosis score of ischemic foot 2 weeks after FAL. (H) the CD31 immunofluorescence staining in WT and Carmn-KO mice. Scale bar: 20 μm. (I) the quantification of CD31+ capillary density, α-SMA+ arteriole density, and diameter of α-SMA+ arterioles. (J) the representative images of H&E staining of gastrocnemius slides from WT or Carmn-KO mice. Scale bar: 100 μm. (K) representative images of Masson trichrome staining of gastrocnemius harvested from Carmn WT or Carmn-KO mice. Scale bar: 100 μm. (L) the quantification of the fibrosis areas between groups. For all panels, error bars represent SEM. P value was determined by unpaired 2-tailed Student’s t test (B, C, I, and L) or 1-way ANOVA with Bonferroni post test (F).