S-08-3: ROCK2 SPECIFIC INHIBITION DECREASES TH17/TREG BALANCE AND REDUCES HYPERTENSION AND HYPERTENSION-ASSOCIATED END-ORGAN DAMAGE

Abstract

Hypertension is characterized by immune activation and inflammatory end-organ damage, yet there are no therapies for hypertension that specifically target the immune system. We previously demonstrated a critical role for pro-inflammatory CD4+ T cells and their cytokines, interleukin 17A (IL-17A) and IL-21, in the full development of hypertension and hypertension-associated renal and vascular damage. Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been shown to function as a molecular switch upregulating Th17 and inhibiting anti-inflammatory regulatory T cell (Treg) differentiation in autoimmune diseases. We found that ROCK2 expression is upregulated in splenic and renal CD4+ T cells in a DOCA (deoxycorticosterone) salt model of hypertension. We hypothesized that hypertensive stimuli increase T cell ROCK2 activity, leading to increased Th17/Treg ratios, and that selective ROCK2 inhibition is a potential novel therapeutic target for hypertension-associated end-organ damage. We showed in vitro that KD025, an orally bioavailable ROCK2 inhibitor, inhibits Th17 proliferation and IL-17A/IL-21 production. We then tested ROCK2 inhibition in vivo where DOCA salt-induced hypertension (uninephrectomy + 100 mg DOCA pellet + 1% NaCl drinking water) was initiated in all mice allowing for hypertension to develop for 10 days, followed by treatment with KD025 (50 mg/kg daily, n = 11) or vehicle (n = 8) starting on day 11 through the remainder of the protocol. KD025 treatment significantly attenuated cardiac hypertrophy (8.7 ± 0.3 vs 7.6 ± 0.2 mg/grams body weight, p = 0.005) and left ventricular (LV) fibrosis (4.9 ± 1.7 vs 2.4 ± 1.3% LV area, p = 0.008). Flow cytometric analysis revealed that KD025 decreased F4/80+ infiltrating cardiac macrophages (24,000 ± 578 vs 18,918 ± 1,430 cells, p = 0.011) and decreased the Th17/Treg ratio (0.251 ± 0.041 vs 0.117 ± 0.009, p = 0.003). Furthermore, KD025 attenuated renal infiltration of CD3+ T cells (59.0 ± 5.6 vs 43.5 ± 3.8 cells/mg tissue, p = 0.019). Additional studies using the Angiotensin II (Ang II, 490ng/kg/min) model of hypertension demonstrated that KD025 treatment starting on Day 14 of Ang II infusion significantly reduced blood pressures from Day 14 (154 ± 5.5mmHg) to Day 28 of the study (141.9 ± 5.9mmHg, p = 0.043, n = 5) which was not observed in vehicle controls (Day 14 157.0 ± 5.4mmHg, Day 28 154.9 ± 3.7mmHg, n = 4). These data indicate that the ROCK2-specific inhibitor KD025 may be a novel therapeutic for the treatment of hypertension and the associated end-organ damage.