Effect of Diabetes‐Associated Metabolic Factors on Placental Growth Factor in Skeletal Muscle Cells
Authors: Rouf Farzana , Lloyd Pamela G 
Published: November 2023

Peripheral artery disease (PAD), a chronic circulatory problem, is characterized by narrowing of the vasculature due to plaque deposition, limiting blood and nutrient supply to the limbs. Around 18 million people in the US have been diagnosed with PAD, and the number is predicted to reach 21 million by 2020. Diabetics have increased prevalence of PAD and suffer greater complications. Currently, ~29 million people in the US have diabetes and ~40% of them suffer from PAD. Since diabetes impairs wound healing, stimulation of peripheral blood vessel growth is a more desirable therapy than surgical intervention in these patients. Arteriogenesis is a compensatory mechanism to occlusion, in which pre‐existing collateral arteries remodel to increase their flow capacity, reducing ischemic tissue damage. Placental growth factor (PLGF), a member of the VEGF family, is a key arteriogenic growth factor. Results from previous in vivo studies in our lab demonstrated that metabolic dysfunction induced by feeding a Western diet for 6 mo decreases PLGF expression in skeletal muscle, potentially contributing to impaired collateral growth in diabetes. Therefore, the goal of this study was to identify which specific features of metabolic dysfunction (hypercholesterolemia, hyperglycemia, hyperinsulinemia, and/or oxidative stress) have the greatest effect on PLGF expression. To achieve this goal, mouse and human skeletal muscle cells (SKMC) were treated with low density lipoprotein (LDL, 0.2 mg/mL), oxidized LDL (ox‐LDL, 0.2 mg/mL), glucose (5–20 mM), advanced glycation end products (AGE, both glucose and glyceraldehyde derived, 1 mg/mL), insulin (100–500 mU/L), and hydrogen peroxide (H2O2, 0.1–0.3 mM) for 24 h. Since PLGF is a secreted protein, media samples were collected after 4, 8, and 24 h of treatment and probed for PLGF protein expression (ELISA). PLGF expression was normalized to total protein (BCA assay) and data analysis was conducted using one‐way ANOVA followed by the Student‐Newman‐Keuls post hoc test. PLGF expression appeared to be more sensitive to metabolic dysfunction in mouse SKMC than human SKMC. Ox‐LDL (formed during hypercholesterolemia), AGE (a reactive adduct formed during long‐term hyperglycemia), and 0.3 mM H2O2 (generated during oxidative stress) all decreased PLGF by ~30–40% in mouse SKMC, while LDL reduced PLGF by ~10% (P<0.05 for all). Surprisingly, hyperglycemia (15–20 mM glucose) induced a ~40–200% increase (P<0.05) in PLGF expression in mouse SKMC; whereas, hyperinsulinemia had no effect. In human SKMC, PLGF expression was significantly reduced only in the presence of hyperglycemic conditions (by ~30% in the presence of AGE, and by ~15% in the presence of glucose; P<0.05). A time‐dependent effect was observed, as the reduction in PLGF was most prominent after 24 h. These results suggest that long‐term hyperglycemia may be the major contributor to impaired PLGF expression and arteriogenesis in diabetic skeletal muscle. However, other metabolic parameters may also play a role. Our results have important clinical implications, as modulation of PLGF expression in diabetic patients is a potential therapeutic approach to treat diabetic PAD.Support or Funding InformationNIH R01 HL‐084494 (PL)