PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts
Mogher Khamaisi, … , Amy Wagers, George L. King
Mogher Khamaisi, … , Amy Wagers, George L. King
Published January 25, 2016
Citation Information: J Clin Invest. 2016;126(3):837-853. https://doi.org/10.1172/JCI82788.
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Research Article Endocrinology

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts

Abstract

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

Authors

Mogher Khamaisi, Sayaka Katagiri, Hillary Keenan, Kyoungmin Park, Yasutaka Maeda, Qian Li, Weier Qi, Thomas Thomou, Danielle Eschuk, Ana Tellechea, Aris Veves, Chenyu Huang, Dennis Paul Orgill, Amy Wagers, George L. King

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Figure 1

Effect of glucose, insulin, and hypoxia on VEGF expression.

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Effect of glucose, insulin, and hypoxia on VEGF expression. Basal VEGF p...
Basal VEGF protein (A) and mRNA (B) levels (cells were incubated with DMEM medium only) after incubation with 100 nM insulin and after incubation for 16 hours under 5% O2 hypoxic conditions. VEGF protein levels secreted into the medium were measured using an ELISA kit, which determined mainly VEGF165. qPCR using the human VEGF primers detailed in Supplemental Figure 9 was performed to determine VEGF mRNA expression levels. Data represent the mean ± SD for 7 control subjects and 26 Medalists, each in triplicate. (C) VEGF protein expression after incubation of control and Medalist fibroblasts in 5.6 mM or 25 mM glucose for 24, 48, and 72 hours. Osmolality in 5.6 nM conditions was corrected using mannitol. A Student’s t test or χ2 test was used for 2-way comparisons based on the distribution and number of observations of the variable.