During the development of the pulmonary vasculature in the fetus, many structural and functional changes occur to prepare the lung for the transition to air breathing. The development of the pulmonary circulation is genetically controlled by an array of mitogenic factors in a temporo-spatial order. With advancing gestation, pulmonary vessels acquire increased vasoreactivity. The fetal pulmonary vasculature is exposed to a low oxygen tension environment that promotes high intrinsic myogenic tone and high vasocontractility. At birth, a dramatic reduction in pulmonary arterial pressure and resistance occurs with an increase in oxygen tension and blood flow. The striking hemodynamic differences in the pulmonary circulation of the fetus and newborn are regulated by various factors and vasoactive agents. Among them, nitric oxide, endothelin-1, and prostaglandin I(2) are mainly derived from endothelial cells and exert their effects via cGMP, cAMP, and Rho kinase signaling pathways. Alterations in these signaling pathways may lead to vascular remodeling, high vasocontractility, and persistent pulmonary hypertension of the newborn.

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. cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries. Am J Physiol Heart Circ Physiol 307: H328 -H336, 2014. First published June 6, 2014; doi:10.1152/ajpheart.00132.2014cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3=,5=-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (PO2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5=-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr 853 , which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK. soluble guanylyl cyclase; inosine 3=,5=-cyclic monophosphate; hypoxic vasoconstriction; endothelium A PREVIOUS STUDY reported that acute hypoxia caused a rapid further increase in tension of contracted canine saphenous veins (31). Subsequent findings demonstrated that such a phenomenon, termed hypoxic augmentation of vasoconstriction (5, 15), occurs in a number of blood vessel types (5,7,8,15,21,23,25,26) contracted with norepinephrine (7, 8), phenylephrine (23), PGF 2␣ (15,25), and the TP receptor agonist U-46619 (21), indicating that it is not unique for a specific vasoconstrictor. It occurs in isolated coronary arteries with but not without endothelium (5,15,25,26). The phenomenon is not affected by bosentan, a blocker of endothelin receptors (5), but is prevented by inhibitors of nitric oxide (NO) synthase (5,15,25). Hypoxic augmentation also occurs in arteries without endothelium treated with an exogenous NO donor (5) However, hypoxic augmentation is not accompanied by changes in the intracellular leve...

Pulmonary veins have been seen primarily as conduit vessels; however, over the past two decades, a large amount of evidence has accumulated to indicate that pulmonary veins can exhibit substantial vasoactivity. In this review, the role of veins in regulation of the pulmonary circulation, particularly during the perinatal period and under certain pathophysiological conditions, is discussed. In the fetus, pulmonary veins contribute a significant fraction to total pulmonary vascular resistance. At birth, the veins as well as the arteries relax in response to endothelium-derived nitric oxide and dilator prostaglandins, thereby assisting in the fall in pulmonary vascular resistance. These effects are oxygen dependent and modulated by cGMP-dependent protein kinase. Under chronic hypoxic conditions, pulmonary veins undergo remodeling and demonstrate substantial constriction and hypertrophy. In a number of species, including the human, pulmonary veins are also the primary sites of action of certain vasoconstrictors such as endothelin and thromboxane. In various pathological conditions, there is an increased synthesis of these vasoactive agents that may lead to pulmonary venous constriction, increased microvascular pressures for fluid filtration, and formation of pulmonary edema. In conclusion, the significant role of veins in regulation of the pulmonary circulation needs to be appreciated to better prevent, diagnose, and treat lung disease.

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.

Key Words: smooth muscle cells Ⅲ phenotype Ⅲ COMP Ⅲ integrin Ⅲ neointima U nlike cardiac or skeletal muscle cells, vascular smooth muscle cells (VSMCs) have a unique property of plasticity and can undergo reversible changes in phenotype. 1,2 Normally, they are mainly restricted to the media of adult blood vessels, express a repertoire of contractile proteins such as smooth muscle (SM) myosin heavy chain, SM ␣-actin, SM-22␣ and calponin, and have low rate of replication. However, on various environmental cues, VSMCs can undergo transition from a quiescent, contractile/differentiated phenotype to a synthetic/dedifferentiated phenotype, with a high rate of migration/proliferation and a concomitant reduction in expression of VSMC marker proteins. 1,3 Phenotypic switching of VSMCs plays an essential role in the development of cardiovascular diseases such as atherosclerosis, postangioplastic restenosis and hypertension. Studies have demonstrated the contribution to cell dedifferentiation of growth factors, mitogenic cytokines, reactive oxygen species, stretch or injury. 2,4,5 Nevertheless, how normal VSMCs maintain the differentiated state is much less understood and has been largely ignored. Understanding the mechanisms that conserve a differentiated phenotype is critical to interfere with the development of cardiovascular diseases. MethodsAnimal care and use of carotid-artery injury model in male SpragueDawley rats were in accordance with institutional guidelines. VSMCs were isolated from the thoracic aortic arteries of rats by collagenase digestion. Small interfering (si)RNA against COMP, ␣ 7 integrin, and ␣ 8 integrin were transfected in vitro by use of Oligofectamine (Invitrogen). The adenovirus for COMP was constructed and a single exposure of 5ϫ10 8 plaque forming units was luminally delivered to balloon-injured carotid segments for in vivo studies. The promoter constructs of SM ␣-actin-luc and SM22␣-luc was transfected into VSMCs and the relative promoter activities were detected by use of a luciferase assay kit. For cell adhesion assay, nontreated 48-well plates were coated with purified COMP, fibronectin, or polymerized collagen I and incubated with neutralizing anti-␣ 7 integrin monoclonal antibody or mouse normal IgG. The contractility of VSMCs and vessel tension measurement was examined as described previously.An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Results COMP Level Is Decreased in Balloon-Injured Rat Carotid ArteriesUsing a rat balloon-injury model, we first examined the expression of COMP in carotid arteries after injury. COMP protein level was markedly lower in injured vessels than in sham-operated vessels 4 to 14 days after injury and was paralleled by an increase of COMP degradation fragment (Figure 1). Combined with our previous observation that degradation of COMP promoted VSMC migration and neointima formation, 6 COMP may negatively regulate VSMC activation in response to injury. Association of COMP and VSMC Markers In VitroVSMC...

In perinatal lungs, veins contribute substantially to total pulmonary vascular resistance. The present study was designed to determine the role of endothelium-derived nitric oxide (EDNO) in modulating the responsiveness of pulmonary veins and arteries in newborn lambs. Fourth-order pulmonary arterial and venous rings of newborn lambs were suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2/5% CO2, 37 degrees C), and their isometric force was measured. Nitro-L-arginine had no effect on the resting tension of pulmonary arteries but caused an endothelium-dependent contraction of pulmonary veins. During contraction to endothelin-1 or U46619, acetylcholine, bradykinin, and calcium ionophore A23187 induced larger endothelium-dependent relaxation in pulmonary veins than in arteries. The endothelium-dependent relaxation of pulmonary vessels was abolished by nitro-L-arginine. In vessels without endothelium, nitric oxide induced significantly greater relaxation in pulmonary veins than in arteries. All vessels relaxed similarly to 8-bromo-cGMP. Radioimmunoassay showed that the basal level of intracellular cGMP of pulmonary veins with endothelium was higher than that of arteries with endothelium. Acetylcholine, bradykinin, and calcium ionophore A23187 induced a significantly larger endothelium-dependent increase in the intracellular content of cGMP in pulmonary veins than in arteries. In vessels without endothelium, nitric oxide induced a larger increase in cGMP content in pulmonary veins than in arteries. The present study suggests that EDNO may play a larger role in modulation of pulmonary venous than arterial reactivity, which may be mainly due to a difference in the activity of soluble guanylate cyclase in vascular smooth muscle.