Abstract
Purpose:
Diabetic retinopathy continues to be the leading cause of adult-onset blindness, globally. Photocoagulation therapies destroy pathologic vessels through laser ablation, yet pose risks for retinal detachment, while chronic anti-VEGF treatment may impair neural retinal survival/function. Thus, it is imperative to divulge mechanisms driving pathologic angiogenesis, to discover innovative medicines capable of preventing/protecting retinal function by promoting microvascular stability. To these ends, we investigated the role of retinal pericyte (RP) contractility on retinal endothelial cell (REC) dynamics and angiogenic potential.
Methods:
As the myosin-phosphatase RhoA interacting protein, MRIP, has been shown to regulate the RhoA/ROCK mediated inactivation of the myosin phosphatase and muscle cell contractility, we employed siRNA to silence MRIP in RP. We combined 2D and 3D co-culture based approaches in combination with biophysical, biochemical and high-resolution imaging to assess the role of RhoGTP and cytoskeletal effectors in modulating RP-REC interactions and angiogenic activation in vitro.
Results:
MRIP silencing induces RP hypertrophy including stress fiber assembly with increased isoactin incorporation/expression. MRIP-silencing also induces a marked enhancement in force generation since there is a 2.2 fold increase in substrate deformation. When MRIP-silenced RP are co-cultured with REC there is a ~2.5 fold increase in endothelial cell cycle re-entry. Similarly, MRIP-silenced RP induce a 1.5 fold increase in angiogenic sprouting when cells are placed together in a 3D model of angiogenesis.
Conclusions:
Altering RP contractile phenotype via manipulation of the Rho GTP cytoskeletal effector pathway markedly promotes angiogenesis, in part, by stimulating endothelial cell-cycle re-entry and angiogenic sprouting. Thus, subtle changes in RP mechanochemistry could represent key, initiating events in re-activating REC proliferation, promoting diabetes-induced, pathologic angiogenesis. In turn, this paradigm-shifting hypothesis suggests a mechanical component to the ‘angiogenic switch’ and calls into question whether pericyte loss/dropout is required for ocular pathologic angiogenic induction. Furthermore, we envision that manipulating RP cytoskeletal effector function could represent an innovative cell-based therapy for preventing and/or protecting retinal function during diabetes.
Keywords: 493 cytoskeleton •
499 diabetic retinopathy