Abstract
Purpose :
Blood vessels and nerves travel together to supply most tissues in the body. Their direct interaction has not been elucidated in the cornea, a normally avascular and densely innervated tissue. In this study, we established trigeminal ganglion (TG) neurons-vascular endothelial cells (VEC) co-culture system to directly study the neuro-modulation of angiogenesis in dry eye disease (DED), and to investigate the role of substance P (SP), a neuropeptide secreted by TG neurons, in promoting angiogenesis after ocular surface inflammation in vitro and in vivo.
Methods :
DED was induced in C57BL/6 mice using a controlled environment chamber for 7 days. TG neurons were isolated from naïve and DED mice. The expression of SP in TG tissue and neurons were quantified using real-time PCR and ELISA. VEC were co-cultured with TG neurons using a transwell, or cultured in conditioned media (CM) collected from TG culture. VEC proliferation, migration and tube formation were assessed. SP and its receptor NK1R signaling was modulated with exogenous SP, Tac1 (SP precursor) siRNA, or NK1R antagonist Spantide I. Corneal neovascularization was induced by intra-stromal suture placement and vascularized area was photographed and quantified. Corneal hem- and lymph-angiogenesis was assessed using immunostaining with CD31 and LYVE1 antibodies, respectively.
Results :
Compared with those isolated from naïve mice, TG neurons and CM derived from DED mice significantly promoted VEC proliferation (27% and 15% increase), and tube formation with more than 3- and 2-fold increases in number of junctions and length of tubes formed, respectively (p<0.05). SP mRNA and protein expression were significantly higher in TG tissue and cultured TG neurons of DED mice than those of naïve mice (all p<0.05). SP potently promoted VEC proliferation (+50%, p<0.001) and tube formation (p=0.001 and 0.003, respectively). Blockade of SP/NK1R signaling with spantide I or Tac1 siRNA in TG neurons derived from DED mice significantly reduced VEC proliferation and tube formation. Compared with control group, Spantide I treatment significantly decreased vascularized area in the cornea in vivo (85.4%±8.2 in control vs. 66.4%±16.0 in Spantide Ι, p=0.03).
Conclusions :
Our results suggest that in response to acute ocular surface inflammation, sensory TG neurons increase SP expression and secretion, which in turn promotes corneal angiogenesis.
This is a 2020 ARVO Annual Meeting abstract.