High glucose conditioned neonatal astrocytes results in impaired mitogenic activity in cerebral microvessel endothelial cells in co-culture

Abstract

Angiogenesis is a highly complex and coordinated process in the brain. Under normal conditions, it is a vital process in growth and development, but under adverse conditions such as diabetes mellitus, it can lead to severe pathology. Astrocytes are a key constituent of the neurovascular unit and contribute to cerebral function, not only bridging the gap between metabolic supplies from blood vessels to neurons, but also regulating angiogenesis. Astrocytes affect angiogenesis by secreting angiogenic factors such as vascular endothelial growth factor (VEGF) into its microenvironment and regulating mitogenic activity in cerebral microvessel endothelial cells (CMEC). We hypothesized that astrocytes conditioned in high glucose media would produce and secrete decreased VEGF which would lead to impaired proliferation, migration, and tube formation of CMEC in vitro. Using neonatal rat astrocytes, we used normal glucose (NG, 5.5mM) vs. high glucose (HG, 25mM) feeding media and measured VEGF message and protein levels as well as secreted VEGF. We co-cultured conditioned astrocytes with isolated rat CMEC and measured mitogenic activity of endothelial cells using BrdU assay, scratch recovery assay, and tube formation assay. HG astrocytes produced and secreted decreased VEGF protein and resulted in impaired mitogenic activity when co-cultured with CMEC as demonstrated by decreased BrdU uptake, decreased scratch recovery, and slower tube formation. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross-talk between astrocytes and CMEC which could be one explanation for cerebral microangiopathy seen in diabetic conditions.

Keywords: Cell biology; Neuroscience.

Fig. 1

Astrocytes cultured in high glucose media had significantly decreased VEGF mRNA levels compared to normal glucose media (Difference between the means -88.42 ± 2.91%, n = 12, p < 0.0001).

Fig. 2

Astrocytes cultured in high glucose media had significantly decreased VEGF protein level compared to normal glucose media (n = 4, p < 0.01).

Fig. 3

Astrocytes cultured in high glucose media secreted significantly decreased levels of VEGF into media compared to normal glucose media (n = 6, *p < 0.001).

Fig. 4

Astrocytes conditioned in high glucose media and co-cultured with cerebral microvessel endothelial cells resulted in decreased mitogenic activity. Astrocytes conditioned in high glucose media and co-cultured with cerebral microvessel endothelial cells resulted in decreased BrdU levels (Fig. 4A: n = 7, *p < 0.05), decreased scratch recovery assay results (Fig. 4B: Time accounts for 66.13% of the total variance between the two curves at time point 5 hours, where F = 56.01, p value < 0.0001), and slower tube formation compared to astrocytes conditioned to normal glucose media (Fig. 4C: n = 5).