Coniferaldehyde Reverses PGK1–3-Phosphoglycerate–L-Serine Metabolic Reprogramming in Hindlimb Ischemia

Background/Objectives: Ischemic skeletal muscle undergoes metabolic remodeling that may influence vascular repair. We investigated whether coniferaldehyde (CA) regulates a phosphoglycerate kinase 1 (PGK1)–3-phosphoglycerate (3PG)–L-serine axis in mouse hindlimb ischemia (HLI). Methods: Gastrocnemius tissue from Sham, HLI, and HLI+CA mice (n = 6/group) underwent bulk RNA sequencing and untargeted metabolomics. Public single-cell data and hypoxic human skeletal muscle cells (HSkMCs) were used to examine PGK1. AlphaFold3 modeling, molecular dynamics simulation, and a cellular thermal shift assay (CETSA) were used to evaluate a potential CA–PGK1 interaction. HSkMC-conditioned media were applied to human umbilical vein endothelial cells (HUVECs), with 3PG supplementation where indicated. Results: Amino-acid biosynthesis and glycine/serine/threonine metabolism were enriched in HLI muscle. 3PG increased in HLI versus Sham (p = 0.0170) and decreased in HLI+CA versus HLI (p = 0.0358); L-serine followed the same pattern (p = 0.0274 and p = 0.0428, respectively). Pgk1 expression also increased in HLI versus Sham (p = 0.00348) and decreased in HLI+CA versus HLI (p = 0.00249). Hypoxia increased HSkMC PGK1 together with extracellular 3PG and serine, whereas CA attenuated all three. Modeling predicted a stable CA–PGK1 complex, and CETSA supported a potential CA–PGK1 interaction in cell lysates. Conditioned medium from CA-treated hypoxic HSkMCs improved HUVEC viability, redox, tube-formation, and wound-closure readouts, reduced reactive oxygen species, and increased glutathione peroxidase 4 (GPX4) and solute carrier, family 7 member 11 (SLC7A11); 3PG supplementation reversed these effects. Conclusions: CA modulates a PGK1-linked 3PG/L-serine metabolic axis, while 3PG exposure altered endothelial phenotypes in the conditioned-medium model.