What is the clinical evidence from this study?

Study design: Other. Population: Diabetes mellitus and cardiac fibrosis (n=178). Intervention: RAGE knockout / AGE/RAGE signaling inhibition vs. Wild-type / functional RAGE. Primary outcome: Cardiac fibroblast migration (migrated cells/% area) (p=0.0002).

February 25, 2020Open Access

The Impact of Diabetic Conditions and AGE/RAGE Signaling on Cardiac Fibroblast Migration

Key Result

Inhibiting the AGE/RAGE signaling pathway via genetic ablation significantly increased cardiac fibroblast migration in diabetic mice compared to diabetic controls (7.04 vs 3.19 migrated cells/% area).

Structured PICO

Does modifying the AGE/RAGE signaling pathway alter cell migration in diabetic and non-diabetic cardiac fibroblasts?

Population

178 adult male mice at 16 weeks of age, including diabetic, RAGE knockout, and Rap1a knockout models, used to isolate cardiac fibroblasts to study migration.

Intervention

Modification of AGE/RAGE signaling cascade using genetic knockouts (RAGE RKO, Rap1a KO) and pharmacological agents including ERK1/2 inhibitor (U0126, 5 µm), PKC-ζ inhibitor (pseudosubstrate PKC-ζ, 1 µg/mL), and exogenous AGEs (glycated albumin 0.5 mg/mL).

Comparator

Untreated fibroblasts, wild-type fibroblasts, or non-diabetic fibroblasts.

Outcome

Cardiac fibroblast migration measured by scratch assay (number of migrated cells normalized to calculated area of the scratch) at 24 hours.surrogate

Inhibiting the AGE/RAGE signaling pathway in cardiac fibroblasts increases cell migration, suggesting this pathway plays a role in the fibrotic response and left ventricular stiffening seen in diabetic cardiomyopathy.

Main Result

Absolute Event Rate: 7.04% vs 3.19%

p-value: p=0.0002

Limitations

Scratch assays may mimic an injury to confound experimental conditions, requiring future studies using Boyden chamber assays.
Further experiments are needed to determine if the observed in vitro responses exist in vivo.
Changes in migration protein markers need to be examined in future studies.

Abstract

Diabetic individuals have an increased risk for developing cardiovascular disease due to stiffening of the left ventricle (LV), which is thought to occur, in part, by increased AGE/RAGE signaling inducing fibroblast differentiation. Advanced glycated end-products (AGEs) accumulate within the body over time, and under hyperglycemic conditions, the formation and accumulation of AGEs is accelerated. AGEs exert their effect by binding to their receptor (RAGE) and can induce myofibroblast differentiation, leading to increased cell migration. Previous studies have focused on fibroblast migration during wound healing, in which diabetics have impaired fibroblast migration compared to healthy individuals. However, the impact of diabetic conditions as well as AGE/RAGE signaling has not been extensively studied in cardiac fibroblasts. Therefore, the goal of this study was to determine how the AGE/RAGE signaling pathway impacts cell migration in non-diabetic and diabetic cardiac fibroblasts. Cardiac fibroblasts were isolated from non-diabetic and diabetic mice with and without functional RAGE and used to perform a migration assay. Cardiac fibroblasts were plated on plastic, non-diabetic, or diabetic collagen, and when confluency was reached, a line of migration was generated by scratching the plate and followed by treatment with pharmacological agents that modify AGE/RAGE signaling. Modification of the AGE/RAGE signaling cascade was done with ERK1/2 and PKC-ζ inhibitors as well as treatment with exogenous AGEs. Diabetic fibroblasts displayed an increase in migration compared to non-diabetic fibroblasts whereas inhibiting the AGE/RAGE signaling pathway resulted in a significant increase in migration. The results indicate that the AGE/RAGE signaling cascade causes a decrease in cardiac fibroblast migration and altering the pathway will produce alterations in cardiac fibroblast migration.

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