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

ABSTRACT: Peripheral artery disease (PAD), affecting over 200 million people globally, often advances to chronic limb-threatening ischemia (CLTI), resulting in severe complications such as limb amputation and heightened mortality. Despite the potential of therapeutic angiogenesis, the mechanisms of cell-cell communication and transcriptional changes driving PAD are not fully understood. Profiling long non-coding RNAs (lncRNAs) from human gastrocnemius muscles of human subjects with or without CLTI revealed that a vascular smooth muscle cell (SMC)-enriched lncRNA CARMN was reduced with CLTI. This study explored the role of how SMC-driven regulation of angiogenesis in limb ischemia is conferred via a unique lncRNA-miRNA coupled signaling pathway.To investigate this interesting project, we perform three RNA-seq project. The first one was performed between hypoxia and normoxia VSMCs to mimic the HLI disease in patients. The second one was performed between CARMN KO SMC and CARMN WT SMC to find the potential downstream signaling pathway. The third one was about the RNA-seq of the gastro muscle obtained from CARMN KO and CARMN WT mice after HLI surgery.

ORGANISM(S): Mus musculus

PROVIDER: GSE290218 | GEO | 2025/03/01

REPOSITORIES: GEO

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Project description:Background: Lower-limb peripheral artery disease (PAD) is one of the major complications of diabetes mellitus. PAD is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the PAD population. Praliciguat is an orally available soluble guanylate cyclase (sGC) stimulator that has been reported both pre-clinically and in early-stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in PAD. Objective: We evaluated the effect of praliciguat on hind limb ischemia (HLI) recovery in a mouse model of type 2 diabetes. Methods: HLI was induced in leptin receptor-deficient (Leprdb/db) mice by ligation and excision of the left femoral artery. Praliciguat (10 mg/kg/day) was administered in the diet starting 3 days before surgery. Results: 28 days after surgery, ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (i.e., weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased intercellular adhesion molecule 1 (ICAM1) expression, and prevented the accumulation of oxidative pro-angiogenic and pro-inflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression in cultured myoblasts and that conditioned medium form praliciguat-treated myoblast decreased ICAM-1 mRNA expression in endothelial cells. These results suggest that praliciguat therapy may decrease ICAM-1 expression in endothelial cells by downregulating Cxcl12 in myocytes. Conclusion: Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM-1 expression.

Project description:Background: Endothelial cells (ECs) use glycolysis to produce energy. In pre-clinical models of peripheral arterial disease (PAD), the further activation of EC glycolysis was ineffective and/or deleterious in promoting hypoxia-dependent angiogenesis while pentose phosphate pathway (PPP) activation was effective. Hexosamine biosynthesis pathway (HBP), PPP, and glycolysis are closely linked. Glucosamine directly activates HBP.Methods:Hind-limb ischemia (HLI)ineNOS-/-and Balb/c mice was used. Glucosamine (600 ug/g/day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging (LDPI), angiogenesis was studied by CD31immunostaining.In-vitro: human umbilical vein ECs (HUVECs) and mouse microvascular EC with glucosamine, L-glucose or vascular endothelial growth factor (VEGF165a), were tested underhypoxiaand serum starvation (HSS). Cell counting kit-8 (CCK-8), tube formation, intracellular reactive oxygen species (ROS), Electric Cell-substrate Impedance Sensing (ECIS) and FITC dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time qPCR, and western blot. Human muscle biopsies from patients with PAD were assessed for EC O-GlcNAcylation before and after supervised exercise vs. standard medical care. Results: Day-3 post-HLI,glucosamine vs. control-treated eNOS-/-had less necrosis.Beginning Day-7 post-HLI, glucosamine vs. control-treated Balb/chad higherblood flowthat persisted to Day-21whereischemic musclesshowed greaterCD31 staining/muscle fiber. In-vitro,glucosamine vs. L-glucoseshowedimproved EC survival and tube formation. RNA-sequencing of glucosamine vs. L-glucose showed increased amino acid metabolism. That resulted in increased oxidative phosphorylation and serine biosynthesis pathway (SBP) without an increase in glycolysis or PPP genes. This was associated with better barrier function and less ROS compared to activating glycolysis by VEGF165a. These effects were mediated by activating transcription factor 4 (ATF4); a driver of exercise-induced angiogenesis. Finally, in muscle biopsies from humans with PAD,EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise vs. standard medical care. Conclusion: In-cells, mice, and humans activation of HBP by glucosamine in PAD inducesan “exercise-like” angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder.

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

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