The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential
modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA
knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network
formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA
plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in
actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation.
Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage,
and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines
revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the
transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in
xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that
ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations
in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic
targeting of ROCK signaling may have profound impacts for targeting angiogenesis.
Keywords: Angiogenesis, cytoskeleton, endothelial cell, rho kinase, ROCK, vascular endothelial growth factor
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