Oxygen Activates the Rho/Rho-Kinase Pathway and Induces RhoB and ROCK-1 Expression in Human and Rabbit Ductus Arteriosus by Increasing Mitochondria-Derived Reactive Oxygen Species

Abstract

Background-Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O 2 -sensitive K ϩ channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction. Methods and Results-The effect of increased PO 2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (nϭ15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O 2 constriction. Heterogeneity exists in the sensitivity of constrictors (PO 2 ϭendothelinϭphenylephrineϾKCl) and of fetal vessels (DAϭpulmonary arteryϾaorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O 2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca 2ϩ -ATPase. In term DA, an increase in PO 2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a PO 2 -dependent, rotenone-sensitive increase in mitochondrion-derived reactive O 2 species. O 2 effects on Rho-kinase are mimicked by exogenous H 2 O 2 . In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O 2 constriction and lack of PO 2 -dependent upregulation of Rho-kinase expression. Conclusions-O 2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.

Developmental Absence of the O2 Sensitivity of L-Type Calcium Channels in Preterm Ductus Arteriosus Smooth Muscle Cells Impairs O2 Constriction Contributing to Patent Ductus Arteriosus

“…In both term and preterm DAs approximately two thirds of O 2 constriction depends on calcium influx via the Ca L and thus is inhibited by a Ca L blocker, consistent with previous reports (9,16,17). The remaining one third of constriction appears to reflect calcium sensitization (i.e.…”

“…Patency is favored in preterm DA by several mechanisms, including (relative to term DA) increased production of vasodilator prostanoids (6), reduced production of endothelin (6), decreased expression and function of O 2 -sensitive Kv (6,11) and more recently decreased rho-kinase activity (16,17). The current investigation identifies a new, developmentally regulated mechanism of O 2 sensitivity that is absent in preterm DA and that contributes to O 2 -induced constriction in term DAs.…”

Morphological and Functional Alterations of the Ductus Arteriosus in a Chicken Model of Hypoxia-Induced Fetal Growth Retardation

“…Recently, Rho kinase activation has been implicated in DA constriction in several mammalian species (25,26). Our observation of the Rho-kinase inhibitors Y-27632 and hydroxyfasudil concentration dependently decreasing tone of KClprecontracted chicken DA suggests that this pathway also plays a role in the regulation of DA contraction in this species.…”

“…Activation of the RhoA/Rho-kinase pathway can induce Ca 2ϩ sensitization, a phenomenon in which sustained vasoconstriction occurs, independent of ongoing increases in cytosolic Ca 2ϩ (25,26). Recently, Rho kinase activation has been implicated in DA constriction in several mammalian species (25,26).…”

Progerin Expression during Normal Closure of the Human Ductus Arteriosus: A Case of Premature Ageing?

“…This process is associated with the initiation of apoptosis in these progerin-expressing cells [12], being characteristic for degeneration in the DA media [13]. The cause for progerin increase in the closing DA needs further investigation, but it is known that alternative splicing is affected by oxidative stress [14] which is activated during normal DA closure [15]. Accumulation of reactive oxygen species has also been observed during vascular ageing [10] in which an increase of progerin has been described in the smooth muscle cells of the coronary vessels of elderly persons as well as a gradual increase of progerin-expressing cells in other vessels with upclimbing age [7,10].…”