Advanced glycation end products facilitate the proliferation and reduce early apoptosis of cardiac microvascular endothelial cells via PKCβ signaling pathway: Insight from diabetic cardiomyopathy

Abstract

Objective: To investigate the effects of advanced glycation end products (AGEs) on the proliferation and apoptosis of cardiac microvascular endothelial cells (CMECs) in rats and their underlying signaling pathway. Methods: CMECs were isolated from Sprague–Dawley rats. We first examined the effects of AGEs on the proliferation and apoptosis of CMECs and then tested whether protein kinase C (PKC) β blockers could counteract the effects of AGEs. The PKC agonists phorbol 12-myristate 13-acetate (PMA) and PKCβ blockers were also used to verify whether PKC could act independently on CMECs. The receptor for AGEs (RAGE)–small interfering RNA (siRNA) transfection was used to verify the effect of AGEs on PKC. Following the above steps, we explained whether AGEs regulated the CMEC proliferation and early apoptosis through the PKCβ signaling pathway. Proliferation of CMECs was detected using the Cell Counting Kit-8 (CCK-8) assay, and early apoptosis was determined using the Annexin V- Fluorescein Isothiocyanate (FITC)/propidium iodide (PI) double staining. Expression of proliferation and apoptosis-related proteins and PKC phosphorylation were determined by western blotting analysis. Cell cycle distributions were assayed using a BD FACSCalibur cell-sorting system. Results: AGEs facilitated the proliferation of CMECs, upregulated phosphorylated extracellular signal regulated kinase (p-ERK), and accelerated the entry of cells from G1 phase to the S+G2/M phase, which was consistent with the upregulated cyclin D1 by AGEs. AGEs inhibited early apoptosis of CMECs by increasing the expression of survivin and decreasing the expression of cleaved-caspase3. All these effects can be reversed by PKCβ1/2inhibitors. In addition, AGE upregulated the RAGE expression and phosphorylation of PKCβ1/2 in CMECs, while the inhibition of RAGE reversed the phosphorylation, as well as the effects of AGEs on proliferation and apoptosis in CMECs. Conclusion: The study indicated that AGEs facilitated the proliferation and reduced early apoptosis of CMECs via the PKCβ signaling pathway.

Keywords

• advanced glycation end products
• cardiac microvascular endothelial cells
• diabetic cardiomyopathy
• protein kinase c