RhoA Activation by Hypoxia in Pulmonary Arterial Smooth Muscle Cells Is Age and Site Specific

Abstract

Abstract-Hypoxia induces vasoconstriction of pulmonary arteries through contraction of smooth muscle cells (SMCs).The GTPase RhoA regulates smooth muscle contractility and actin cytoskeletal remodeling through the Rho-associated kinase (ROCK). We previously found that the postnatal fall in pulmonary vascular resistance was associated with actin cytoskeletal remodeling in porcine pulmonary arterial SMCs (PASMCs) in vivo. Here, we investigated the effects of acute and chronic hypoxia on the morphology and RhoA activity of PASMCs from fetal and neonatal piglets. Acute hypoxia enhanced actin stress fiber formation and RhoA activity in both inner and outer medial PASMCs from the fetus but only in the inner medial PASMCs from normal 3-day-old piglets. The increased stress fiber formation was dependent on Rho and ROCK. In outer medial PASMCs from 14-day-old animals, acute hypoxia decreased RhoA activity. Interestingly, outer medial PASMCs from animals exposed to chronic hypoxia had fewer stress fibers associated with a lower basal RhoA activity. Treatment of PASMCs from normal 3-day-old piglets with Rho or ROCK inhibitors for 24 hours induced a similar morphology. Rac activity was not altered by either acute or chronic hypoxia. These data show that acute hypoxia induces RhoA activation only in PASMCs from young animals, whereas chronic hypoxia selectively downregulates RhoA activity in outer medial PASMCs leading to an altered phenotype. Key Words: pulmonary Ⅲ smooth muscle cell Ⅲ hypoxia Ⅲ Rho Ⅲ Rac P ulmonary vascular resistance is high in utero and normally falls at birth when pulmonary arteries dilate and blood flow increases. Postnatal dilatation is characterized by elongation and thinning of medial pulmonary arterial smooth muscle cells (PASMCs) with associated actin cytoskeletal remodeling, transient disassembly of the actin cytoskeleton, and a reduction in total actin content. 1,2 Cytoskeletal change is most marked in inner medial SMCs. Failure to adapt normally to extrauterine life causes persistent pulmonary hypertension of the newborn (PPHN). The pulmonary arteries remain undilated and thick walled. 3,4 Hypoxia is a common cause of PPHN in human infants, and newborn piglets exposed to chronic hypoxia develop pulmonary hypertension and show pulmonary vascular abnormalities similar to those seen in sick infants. 5,6 Actin cytoskeleton remodeling is impaired in the PASMCs of chronically hypoxic newborn piglets, although the normal postnatal increase in proportion of monomer actin does take place. 2 These observations suggest that chronic hypoxia causes disregulation of actin filament turnover in the newborn.Rho-family GTPases are involved in actin remodeling: Rho regulates the formation of stress fibers and focal adhesions, 7,8 whereas Rac induces lamellipodia and membrane ruffles. 9 -11 Rho cycles between a GDP-bound inactive state and a GTP-bound active state, and when bound to GTP, activates its downstream targets, including the serine/threonine kinase Rho-associated kinase (ROCK). 12 The Rho-ROCK pathway c...

Role of Protein Kinase Cζ and Its Adaptor Protein p62 in Voltage-Gated Potassium Channel Modulation in Pulmonary Arteries

“…This response to U46619 in p62 Ϫ/Ϫ mice PA is similar to that observed in 2-week-old piglet PAs after inhibition of PKC (i.e., contraction without [Ca 2ϩ ] i signal) (Cogolludo et al, 2005). In these animals, there is an up-regulation of Rho kinase (Bailly et al, 2004), a key enzyme in Ca 2ϩ -sensitizing mechanisms. In addition, Rho kinase inhibitors were more effective inhibiting U46619 contractions in these piglets than in newborn piglets or adult rats (Cogolludo et al, 2005).…”

Relationship between Structural Remodeling and Reactivity in Pulmonary Resistance Arteries from Hypertensive Piglets

“…Phenotypic changes in response to hypoxia have been described previously in porcine, bovine, and rat conduit pulmonary arteries (18 -20). In SMC isolated from conduit arteries of neonatal piglets exposed to 11 d hypoxia, a stable change in SMC phenotype, manifested by a change in cell shape and regulation of the actin cytoskeleton, accompanied altered functional responses (21). Conduit vessels are composed of heterogenous SMC populations, but we found little structural variation between cells in the walls of resistance arteries, confirming previous studies (2) showing that the pulmonary resistance arteries of calves also have a homogeneous SMC population (22).…”

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

“…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. Numerous experimental evidences indicate that chronic hypoxia augments Rho kinase-induced vascular smooth muscle Ca 2ϩ sensitization, particularly in the pulmonary vasculature (13,27) McNamara et al (13) reported that pulmonary vascular RhoA is activated and Rho-kinase activity is increased in rat pups by exposure to hypoxia from birth for 14 d. Moreover, hypoxiainduced RhoA expression in the lung is age dependent and found to be greatest in the fetus (27). In the present study, we observed that the relaxation evoked by Y-27632 and hydroxyfasudil was similar in DAs from normoxic and hypoxic fetuses.…”