**Background:** Metabolic diseases, particularly diabetes, are major risk factors for cardiovascular disease (CVD). Endothelial dysfunction, driven by high glucose levels, is an early event in CVD pathogenesis. Mitochondrial dysfunction, including excessive reactive oxygen species (ROS) production and altered mitochondrial dynamics (imbalance between fission and fusion), contributes to endothelial damage. PDGF-C, a growth factor binding to PDGFRα and PDGFRαβ receptors, was previously shown by this group to reduce mitochondrial superoxide production in high-glucose-treated human aortic endothelial cells (HAECs) via upregulation of SOD2. This study extends those findings by investigating PDGF-C's effects on mitochondrial morphology and bioenergetics under acute high glucose stress.
**Methods:** HAECs (passages 4-7) were cultured in EGM-2 medium containing 5.5 mmol/L glucose. After 12-hour serum deprivation, cells were exposed to 35 mmol/L d-glucose (HG) for 6-9 hours. PDGF-C (50 ng/mL) was added for the final hour of glucose exposure. Mitochondrial network integrity was assessed by confocal microscopy using MitoTracker Green FM and Hoechst staining, with analysis of branch count, junction count, and mitochondrial area using Fiji/ImageJ. Expression of fusion proteins (OPA1, MFN1, MFN2) and fission proteins (DRP1, FIS1, and DRP1 phosphorylated at Ser616) was measured by western blot. Bioenergetic analysis was performed using a Seahorse XFe24 analyzer with Mito Stress Test and Energy Phenotype Test protocols, measuring oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Parameters assessed included basal respiration, maximal respiration, spare respiratory capacity, ATP production, proton leak, non-mitochondrial oxygen consumption, and OCR/ECAR ratios.
**Key Results:** Confocal microscopy revealed that HG induced a fragmented mitochondrial morphology with a 64% reduction in branch count (p < 0.001), 63% reduction in junction count (p < 0.001), and 71% reduction in mitochondrial area (p < 0.0001) compared to normal glucose (NG). PDGF-C treatment significantly increased branch count (p < 0.05) and junction count (p < 0.05) in HG-exposed cells, with a tendency to increase total mitochondrial area. Western blot analysis showed HG did not significantly alter MFN1 or MFN2 expression, but reduced OPA1 expression at 6 and 7 hours (p < 0.05), which was restored by PDGF-C (p = 0.0486). HG did not change FIS1 or total DRP1 expression, but significantly increased DRP1 phosphorylation at Ser616 at 6 hours (p < 0.01) and 7 hours (p < 0.001). PDGF-C reduced this phosphorylation to basal levels (p < 0.001). Bioenergetic analysis showed HG significantly reduced basal respiration (p < 0.01), maximal respiration (p < 0.01), spare respiratory capacity (p < 0.01), non-mitochondrial oxygen consumption (p < 0.05), and ATP production (p < 0.05). PDGF-C significantly increased non-mitochondrial oxygen consumption in HG conditions (p < 0.05). Energy phenotype testing showed HG reduced baseline OCR (p < 0.05), baseline OCR/ECAR ratio (p < 0.01), stressed OCR (p < 0.01), and stressed OCR/ECAR ratio (p < 0.0001). PDGF-C increased stressed OCR (p < 0.05) and stressed ECAR (p < 0.05) in HG conditions. In normal glucose conditions, PDGF-C significantly reduced metabolic potential (% baseline OCR; p < 0.05) and increased metabolic potential (% baseline ECAR; p < 0.05), suggesting a shift toward glycolytic metabolism.
**Clinical Implications:** This study demonstrates that acute high glucose exposure induces mitochondrial fragmentation and bioenergetic dysfunction in macrovascular endothelial cells, and identifies PDGF-C as a potential modulator of these effects. PDGF-C appears to protect mitochondrial integrity by restoring OPA1 expression and reducing DRP1 Ser616 phosphorylation, thereby promoting fusion over fission. However, PDGF-C only partially restored bioenergetic parameters (primarily non-mitochondrial oxygen consumption), suggesting its effects are more pronounced on mitochondrial morphology than on functional recovery in this acute model. The findings highlight the importance of mitochondrial dynamics in glucose-induced endothelial dysfunction and suggest PDGF-C signaling as a potential therapeutic target for preserving endothelial function in diabetes. The authors note that these results represent an initial approach and that future studies should assess endothelial function parameters (e.g., angiogenic capacity, nitric oxide production) and evaluate individual mitochondrial complex behavior.