Rho activation causes increased pericyte contractility, loss of endothelial contact-mediated growth arrest, and disruption of cell-matrix interactions. On the level of the microvascular bed, pathological activation of Rho may lead to microvascular hypertension, pathological angiogenesis, and increased capillary permeability.

The Rho GTPase mechanochemical signaling apparatus links transmembrane and intracellular signaling-associated Rho GTPase with functional alterations in the actin cytoskeleton. Rho kinase, a principal downstream effector of Rho GTPase, mediates linkage to the actin cytoskeleton via four principle pathways discussed in the text: myosin light chain phosphatase (MLCP), ADF/cofilin, ERM proteins, and p38 MAP kinase.

Bovine retinal pericytes were transduced with dominant-active (RhoDA) or dominant-negative (RhoDN) Rho GTPase-expressing adenovirus, tetracycline-repressible transactivator (Tet)-expressing adenovirus, or were mock-infected (Control). Pericytes were then re-plated onto plasma glow discharge-prepared, Type I collagen-coated silicon substrates containing 0–10 μM of the small molecule Rho kinase inhibitor Y-27632, and monitored by real-time phase contrast imaging. A: Representative images are provided in as labeled, where arrows indicate substrate-wrinkling, actively contractile pericytes; images are 400x. B: After 24 hours, force transduction was quantified as the percentage of cells capable of producing substrate-deforming contractile force sufficient to produce substrate wrinkling visible by phase-contrast microscopy.

Bovine retinal pericytes were either transfected with plasmids (A) or infected with adenoviruses (B) containing dominant-active Rho GTPase (RhoDA) or dominant-negative Rho GTPase (RhoDN). Mock-transfected (Wild type) as well as tetracycline transactivator-infected (Tet) and mock-infected (Control) pericytes were used as controls as labeled. Rho-altered pericytes were then co-cultured with bovine retinal endothelial cells for 24 hours. BrdU was incorporated into the co-culture medium for the last 4 hours. A: Altered Rho-transfected pericyte co-cultures were then fixed and stained for the pericyte marker cytoplasmic alpha-smooth muscle actin and proliferative endothelial cell nuclear BrdU incorporation (αSMA + BrdU: red), the Myc epitope tag as a marker of transfection (Myc: green), and nuclei by Hoechst (Hoechst: blue). Parallel phase image are provided. Images are 200x. Arrows: BrdU-positive, pericyte-contacting proliferating endothelial cell. Arrowheads: BrdU-negative, pericyte-contacting quiescent endothelial cell. B: Adenoviral Rho GTPase-altered pericytes as well as dominant-active Ras, Rac1, Cdc42, and GTP control-transfected pericytes, were co-cultured for 24 hours with bovine retinal endothelial cells as above. Co-cultures were then scored for nuclear BrdU-positive endothelial proliferation as a function of pericyte contact and GTPase status. Results are expressed as mean percentages ± standard error (n > 400 cells/condition, experiments in triplicate). *: p < 0.05 for differences between lone and pericyte-contacting endothelial cells with same GTPase status; †, ‡: p < 0.05 for differences between lone endothelial cell populations with different Rho GTPase status.