Session I: Cell Biology of Rho GTPases: Angiogenesis/ Vasculogenesis

Chairs: Peter Jones and Troy Stevens

Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho. Accumulating evidence has demonstrated that Rho/Rho-kinase pathway plays an important role in various cellular functions, not only in vascular smooth muscle cell (VSMC) contraction but also in actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions, all of which may be involved in the pathogenesis of cardiovascular disease. At molecular level, Rho-kinase upregulates various molecules that accelerate inflammation/oxidative stress, thrombus formation, and fibrosis, whereas it downregulates endothelial nitric oxide synthase. The expression of Rho-kinase itself is mediated by protein kinase C/NF-kappaB pathway with an inhibitory and stimulatory modulation by estrogen and nicotine, respectively. At cellular level, Rho-kinase mediates VSMC hypercontraction, stimulates VSMC proliferation and migration, and enhances inflammatory cell motility. In animal studies, Rho-kinase has been shown to be substantially involved in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, stroke and heart failure, and to enhance central sympathetic nerve activity. Finally, in clinical studies, fasudil, a Rho-kinase inhibitor, is effective for the treatment of a wide range of cardiovascular disease, including cerebral and coronary vasospasm, angina, hypertension, pulmonary hypertension, and heart failure, with a reasonable safety. Thus, Rho-kinase is an important therapeutic target in cardiovascular medicine. Shimokawa, H., Takeshita, A. Rho-Kinase is an important therapeutic target in cardiovascular medicine. Arterioscler Thromb Vasc Biol 25(9):1767-75. Epub 2005 Jul 7.

Rho, Rac and Cdc42, three members of the Rho family of small GTPases, each control a signal transduction pathway linking membrane receptors to the assembly and disassembly of the actin cytoskeleton and of associated integrin adhesion complexes. Rho regulates stress fibre and focal adhesion assembly, Rac regulates the formation of lamellipodia protrusions and membrane ruffles, and Cdc42 triggers filopodial extensions at the cell periphery. These observations have led to the suggestion that wherever filamentous actin is used to drive a cellular process, Rho GTPases are likely to play an important regulatory role. Rho GTPases have also been reported to control other cellular activities, such as the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) cascades, an NADPH oxidase enzyme complex, the transcription factors NF-kappaB (nuclear factor kappaB) and SRF (serum-response factor), and progression through G1 of the cell cycle. Thus Rho, Rac and Cdc42 can regulate the actin cytoskeleton and gene transcription to promote co-ordinated changes in cell behavior. We have been analyzing the biochemical contributions of Rho GTPases in cell movement and have found that Rac controls cell protrusion, while Cdc42 controls cell polarity. Hall, A. Rho GTPases and the control of cell behavior., Biochem Soc Trans 33 (Pt 5):891-5.

Cells receive extracellular stimuli in the form of soluble molecules (growth factors, cytokines and hormones) that bind to cell surface receptors, adhesive interactions (extracellular matrix and cell-cell adhesion) or mechanical stresses (tension, compression and fluid shear stress). These stimuli act upon guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) to control the activation state of the small GTPases Rho, Rac and Cdc42. Once activated, the GTPases bind to a spectrum of effectors to stimulate downstream signaling pathways. Schwartz, M. Rho signaling at a glance. Journal of Cell Science 117, 5457-5458 (2004).  

Recent data have revealed new mechanisms that underlie endothelial cell (EC) lumen formation during vascular morphogenic events in development, wound repair, and other disease states. It is apparent that EC interactions with extracellular matrices (ECMs) establish signaling cascades downstream of integrin ligation leading to activation of the Rho GTPases, Cdc42 and Rac1, which are required for lumen formation. In large part, this process is driven by intracellular vacuole formation and coalescence, which rapidly leads to the creation of fluid-filled matrix-free spaces that are then interconnected via EC-EC interactions to create multicellular tube structures. EC vacuoles markedly accumulate in a polarized fashion directly adjacent to the centrosome in a region that strongly accumulates Cdc42 protein as indicated by green fluorescent protein (GFP)-Cdc42 during the lumen formation process. Downstream of Cdc42-mediated signaling, key molecules that have been identified to be required for EC lumen formation include Pak2, Pak4, Par3, Par6, and the protein kinase C (PKC) isoforms zeta and epsilon. Together, these molecules coordinately regulate the critical EC lumen formation process in three-dimensional (3D) collagen matrices. These events also require cell surface proteolysis mediated through membrane type 1 matrix metalloproteinase (MT1-MMP), which is necessary to create vascular guidance tunnels within the 3D matrix environment. These tunnels represent physical spaces within the ECM that are necessary to regulate vascular morphogenic events, including the establishment of interconnected vascular tube networks as well as the recruitment of pericytes to initiate vascular tube maturation (via basement membrane matrix assembly) and stabilization. Current research continues to analyze how specific molecules integrate signaling information in concert to catalyze EC lumen formation, pericyte recruitment, and stabilization processes to control vascular morphogenesis in 3D extracellular matrices. Davis, G.E., Koh, W., Stratman, A.N. Mechanisms controlling human endothelial lumen formation and tube assembly in three-dimensional extracellular matrices.  Birth Defects Res C Embryo Today, 2007 Dec; 81(4):270-85.

Akiko Mammoto

Local alterations in the mechanical compliance of the basement membrane that alter the level of isometric tension in the cell have been postulated to influence tissue morphogenesis. To explore whether cell tension contributes to tissue pattern formation in vivo, we modulated cytoskeletal force generation in embryonic mouse lung (embryonic days 12-14) rudiments using inhibitors of Rho-associated kinase (ROCK), myosin light chain kinase, myosin ATPase, and microfilament integrity, or a Rho stimulator (cytotoxic necrotizing factor-1). Tension inhibition resulted in loss of normal differentials in basement membrane thickness, inhibition of new terminal bud formation, and disorganization of epithelial growth patterns as well as disruption of capillary blood vessels. In contrast, increasing cell tension through Rho activation, as confirmed by quantitation of myosin light chain phosphorylation and immunohistocytochemical analysis of actin organization, accelerated lung branching and increase capillary elongation. These data suggest that changes in cytoskeletal tension mediated by Rho signaling through ROCK may play an important role in the establishment of the spatial differentials in cell growth and extracellular matrix remodeling that drive embryonic lung development. Copyright 2004 Wiley-Liss, Inc. Moore, K.A., Polte, T., Huang, S., Shi, B., Alsberg, E., Sunday, M.E., Ingber, D.E. Control of basement membrane remodeling and epithelial branching morphogenesis in embryonic lung by Rho and cytoskeletal tension. Dev Dyn2005 Feb; 232(2):268-81.

12:15 PM       Featured Abstract: The Guanine Nucleotide Exchange Factor, SmgGDS, is a Novel  

                        Regulator of Myosin Organization and Cellular Contraction in Vascular Smooth                        

The guanine nucleotide exchange factor SmgGDS activates small GTPases in the Ras and Rho families, including RhoA.  Although RhoA regulates vascular smooth muscle cell (VSMC) contraction, the expression and function of SmgGDS in VSMC have not been previously investigated.  We found by Western blotting that SmgGDS is expressed by VSMC from different vascular beds, and by the A7r5 VSMC line.  A7r5 cells transfected with SmgGDS siRNA or RhoA siRNA exhibited significantly reduced [3H] thymidine incorporation, suggesting that SmgGDS promotes cell proliferation by regulating small GTPases.  A7r5 cells with reduced SmgGDS expression have enhanced spreading and no longer exhibit the elongated shape that is characteristic of the VSMC morphology.  In contrast, cells with reduced RhoA expression are elongated with only minor cell spreading, suggesting that SmgGDS regulates VSMC morphology by controlling multiple small GTPases in addition to RhoA.  Immunofluorescent staining with antibody against myosin heavy chains revealed loss of myosin filaments in cells with reduced SmgGDS expression, which is consistent with inactivation of the Rho signaling pathway.  To determine whether the absence of SmgGDS affected myosin dynamics by altering Rho kinase activity, we examined phosphorylation of the Rho kinase substrate, myosin phosphatase (MYPT1).  Transfection of SmgGDS siRNA did not affect MYPT1 phosphorylation at either Thr696 or Thr850, even though the Rho kinase inhibitor Y27632 (3 M) significantly inhibited MYPT1 phosphorylation at Thr850.  However, transfection of SmgGDS siRNA or RhoA siRNA impaired the contraction of A7r5 cells following depolarization of the cells with 90 mM K+.  Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.  Thill, R., Lorimer, E.L., Williams, C.L. The Guanine Nucleotide Exchange Factor, SmgGDS, is a Novel Regulator of Myosin Organization and Cellular Contraction in Vascular Smooth Muscle Cells J. Cell Biochem Aug 1;104(5):1760-70

Session II:  Cell Biology of Rho GTPases: Endothelial Function

Chairs: Sharon Rounds and Jahar Bhattacharya

2:00 PM         Rho GTPases in Endothelial Cell Barrier Function

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling. Copyright 2005 Wiley-Liss, Inc. Birukova, A.A., Birukov, K.G., Adyshev, D., Usatyuk, P., Natarajan V, Garcia JG, Verin AD. Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction. J Cell Physiol204(3):934-47.Sep; 2005

2:40 PM         Rho GTPases and Ca++ signaling in regulation of EC Barrier Function               

Sphingosine 1-phosphate (S1P) ligation of endothelial differentiation gene-1 receptor coupled to the heterotrimeric G protein, Gi, promotes endothelial barrier strengthening via Rac-dependent assembly of adherens junctions (AJs). However, the mechanism of Rac activation induced by S1P stimulation remains unclear. In live endothelial cells expressing GFP-Rac, we observed that S1P induced the translocation of Rac to intercellular junctions, resulting in junctional sealing. We investigated the role of intracellular Ca2+ in signaling Rac activation and the enhancement of endothelial barrier function. We observed that S1P activated the release of Ca2+ from endoplasmic reticulum stores, and subsequent Ca2+ entry via lanthanum-sensitive store-operated Ca2+ channels (SOC) after store depletion. Inhibition of Gi, phospholipase C, or inositol trisphosphate receptor prevented the S1P-activated increase in intracellular Ca2+ as well as Rac activation, AJ assembly, and enhancement of endothelial barrier. Chelation of intracellular Ca2+ with BAPTA blocked S1P-induced Rac activation, indicating the requirement for Ca2+ in the response. Inhibition of SOC by lanthanum or transient receptor potential channel 1 (TRPC1), a SOC constituent, by TRPC1 antibody, failed to prevent S1P-induced Rac translocation to junctions and AJ assembly. Thus, our results demonstrate that S1P promotes endothelial junctional integrity by activating the release of endoplasmic reticulum-Ca2+, which induces Rac activation and promotes AJ annealing. Mehta, D., Konstantoulaki, M., Ahmmed, G.U., Malik, A.B. Sphingosine 1-phosphate-induced mobilization of intracellular Ca2+ mediates rac activation and adherens junction assembly in endothelial cells. J Biol Chem Apr 29; 280(17):17320-8. Epub 2005 Feb 22.

Isoprenylcysteine-O-carboxyl methyltransferase (ICMT) catalyzes methylation of proteins containing a C-terminal CAAX motif. We have previously shown that chemical inhibition of ICMT caused endothelial cell apoptosis, an effect correlated with decreased Ras and RhoA carboxyl methylation and GTPase activities. In the current study, proteomic analysis of pulmonary artery endothelial cells (PAEC) exposed to the ICMT inhibitor, N-acetyl-geranylgeranyl-cysteine (AGGC), demonstrated a shift in the isoelectric points (pI) of the glucose-regulated protein (GRP) 94. Two-dimensional PAGE and immunoblot analysis further documented that ICMT inhibition caused multiple changes in the pI of GRP94. GRP94 is an endoplasmic reticulum molecular chaperone, a component of the unfolded protein response (UPR), and is involved in apoptosis. Immunofluorescence analyses revealed redistribution and aggregation of GRP94 after 3 h exposure to AGGC. A similar finding was noted with calnexin. In addition, GRP94 protein levels were significantly diminished upon 18 h AGGC exposure or ICMT suppression. The effects of ICMT inhibition on changes in GRP94 subcellular localization and protein content were blunted by overexpression of constitutively active RhoA or a caspase inhibitor. Furthermore, GRP94 depletion augmented endothelial cell apoptosis induced by ICMT inhibition. These results indicate that ICMT inhibition leads to GRP94 relocalization, aggregation, and degradation; effects were dependent upon the activities of RhoA and caspases. We speculate that changes in the pI, subcellular localization, and protein level of GRP94 cause endothelial cell apoptosis, possibly through UPR dysfunction. These studies suggest a novel link between RhoA GTPases and the UPR. Lu, Q., Harrington, E.O., Newton, J., Jankowich, M., Rounds, S. Inhibition of ICMT induces endothelial cell apoptosis through GRP94. Am J Respir Cell Mol Biol. 2007 Jul; 37(1):20-30.

3:40 PM         Discussion                   

We have previously shown that the hyperpermeability-inducer thrombin is a potent activator of RhoA. Activation of RhoA induces endothelial barrier dysfunction by a contractile mechanism, involving its downstream target Rho kinase, which on its turn inactivates the Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to regulation of endothelial barrier integrity. To visualize basal Rho kinase activity, a phosphospecific antibody against the myosin phosphatase targeting subunit (Thr695-MYPT1) was used as a surrogate marker for Rho kinase activity. Under basal conditions a peripheral band of Rho kinase activity was observed by 3D deconvolution fluorescence microscopy, that colocalized with cortical F-actin. This suggested that inactivation of MP in junctional areas by Rho kinase contributes to barrier integrity. However, direct interaction of MP with junctional proteins VE-Cadherin/beta-catenin was not detectable. Remarkably, inhibition of basal Rho kinase activity disrupted endothelial barrier integrity, opposing previously observed protection from the thrombin response. The peripheral Rho kinase activity disappeared upon thrombin stimulation and was targeted to contractile F-actin stress fibers. To further explore the spatiotemporal activity of RhoA signalling, we used a set of live imaging sensors for RhoA activity, based on Fluorescence Resonance Energy Transfer. Those data will be presented at the meeting. Rho kinase has opposing activities in regulation of endothelial barrier function: 1- an intrinsic activity at the cell margins that contributes to barrier integrity, and 2- an induced activity at F-actin stress fibers, resulting in barrier disruption via inhibition of MP. Supported by NHF grant 2003T032. van Nieuw Amerongen, G.P., Hodzic, J., Beckers, C.M.L., Zeeman, S., Musters, R.J.P.,  Engelse, M.E., Vonk Noordegraaf, A.,  van Hinsbergh, V.W.M. Dual Role of Rho Kinase in Regulation of Endothelial Barrier Function. 

Session III:  Rho GTPases in Pulmonary Hypertension: Vascular Tone

Chairs: Mark Gillespie and Karen Fagan

Ca2+ sensitivity of smooth muscle and nonmuscle myosin II reflects the ratio of activities of myosin light-chain kinase (MLCK) to myosin light-chain phosphatase (MLCP) and is a major, regulated determinant of numerous cellular processes. We conclude that the majority of phenotypes attributed to the monomeric G protein RhoA and mediated by its effector, Rho-kinase (ROK), reflect Ca2+ sensitization: inhibition of myosin II dephosphorylation in the presence of basal (Ca2+ dependent or independent) or increased MLCK activity. We outline the pathway from receptors through trimeric G proteins (Galphaq, Galpha12, Galpha13) to activation, by guanine nucleotide exchange factors (GEFs), from GDP. RhoA. GDI to GTP. RhoA and hence to ROK through a mechanism involving association of GEF, RhoA, and ROK in multimolecular complexes at the lipid cell membrane. Specific domains of GEFs interact with trimeric G proteins, and some GEFs are activated by Tyr kinases whose inhibition can inhibit Rho signaling. Inhibition of MLCP, directly by ROK or by phosphorylation of the phosphatase inhibitor CPI-17, increases phosphorylation of the myosin II regulatory light chain and thus the activity of smooth muscle and nonmuscle actomyosin ATPase and motility. We summarize relevant effects of p21-activated kinase, LIM-kinase, and focal adhesion kinase. Mechanisms of Ca2+ desensitization are outlined with emphasis on the antagonism between cGMP-activated kinase and the RhoA/ROK pathway. We suggest that the RhoA/ROK pathway is constitutively active in a number of organs under physiological conditions; its aberrations play major roles in several disease states, particularly impacting on Ca2+ sensitization of smooth muscle in hypertension and possibly asthma and on cancer neoangiogenesis and cancer progression. It is a potentially important therapeutic target and a subject for translational research. Somlyo A.P., Somlyo A.V. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.  Physiol. Rev. 83: 1325-1358, 2003 

We have examined the effects of Y-27632, a specific inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM - 3 microM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10-600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV. Robertson TP, Dipp M, Ward JP, Aaronson PI, Evans AM. Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat. Br J Pharmacol Sep; 131(1):5-9 2000.

 9:40 AM         Discussion

There is current controversy regarding whether vasoconstriction plays a significant role in the elevated pressure of severe, advanced stages of pulmonary hypertension. Results of acute vasodilator testing using conventional vasodilators in such patients suggest there is only a minor contribution of vasoconstriction. However, there is a possibility that these results may underestimate the contribution of vasoconstriction because the most effective vasodilators have not yet been tested. This issue has not been addressed even experimentally, due mainly to a lack of appropriate animal models. A few animal models that mimic the pathology of human severe pulmonary hypertension more closely (i.e., development of occlusive neointimal lesions in small pulmonary arteries/arterioles) have been introduced, including rat models of left lung pneumonectomy plus monocrotaline injection and vascular endothelial growth factor inhibition plus exposure to chronic hypoxia. We have observed that Rho kinase inhibitors, a novel class of potent vasodilators, reduce the high pulmonary artery pressure of these models acutely and markedly, suggesting that vasoconstriction can significantly be involved in pulmonary hypertension with severely remodeled (occluded) pulmonary vessels. This chapter describes methods used for evaluation of the involvement of Rho kinase-mediated vasoconstriction in rat models of pulmonary hypertension. Oka M, Homma N, McMurtry IF. Rho kinase-mediated vasoconstriction in rat models of pulmonary hypertension. Methods Enzymol439:191-204   2008.

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca(2+)](i)). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca(2+) desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca(2+)-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca(2+)](i). We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca(2+) sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca(2+) desensitization through inhibition of RhoA/ROK. Jernigan NL, Walker BR, Resta TC. Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling. Am J Physiol Lung Cell Mol Physiol 287(6):L1220-9. Epub 2004 Aug 13

11:30 AM       Discussion

 Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15-500 microg) and HA-1077 (500 microg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 +/- 0.18 to 0.16 +/- 0.02 mmHg x ml (-1) x min (-1) (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 +/- 0.03 to 0.19 +/- 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15-30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 +/- 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension. Parker TA, Roe G, Grover TR, Abman SH. Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung. Am J Physiol Lung Cell Mol Physiol 2006 Nov; 291(5):L976-82. Epub June  30.

Session IV:  Rho GTPases in Pulmonary Hypertension: Vascular Remodeling

Chairs: Brian Fouty and Norbert Voelkel

2:00 PM         RhoA / Rho Kinase Pathway Regulates Smooth Muscle Cell Differentiation                     

                        Julian Solway

RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics. Liu HW, Halayko AJ, Fernandes DJ, Harmon GS, McCauley JA, Kocieniewski P, McConville J, Fu Y, Forsythe SM, Kogut P, Bellam S, Dowell M, Churchill J, Lesso H, Kassiri K, Mitchell RW, Hershenson MB, Camoretti-Mercado B, Solway J. The RhoA/Rho kinase pathway regulates nuclear localization of serum response factor. Am J Respir Cell Mol Biol 29(1):39-47. Epub 2003 Jan 10.

2:20  PM       Discussion   

2:40 PM        Substrate and Shape - Dependent Control of Pulmonary Vascular Remodeling:

Tenascin-C (TN-C) is an extracellular matrix (ECM) protein expressed within remodeling systemic and pulmonary arteries (PAs), where it supports vascular smooth muscle cell (SMC) proliferation. Previously, we showed that A10 SMCs cultivated on native type I collagen possess a spindle-shaped morphology and do not express TN-C, whereas those on denatured collagen possess a well-defined F-actin stress fiber network, a spread morphology, and they do express TN-C. To determine whether changes in cytoskeletal architecture control TN-C, SMCs on denatured collagen were treated with cytochalasin D, which decreased SMC spreading and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling effectors required for TN-C transcription. Next, to determine whether cell shape, dictated by the F-actin cytoskeleton, regulates TN-C, different geometries of SMCs (ranging from spread to round) were engineered on denatured collagen: as SMCs progressively rounded, ERK1/2 activity and TN-C transcription declined. Because RhoA and Rho kinase (ROCK) regulate cell morphology by controlling cytoskeletal architecture, we reasoned that these factors might also regulate TN-C. Indeed, SMCs on denatured collagen possessed higher levels of RhoA activity than those on native collagen, and blocking RhoA or ROCK activities attenuated SMC spreading, ERK1/2 activity, and TN-C expression in SMCs on denatured collagen. Thus, ROCK controls the configuration of the F-actin cytoskeleton and SMC shape in a manner that is permissive for ERK1/2-dependent production of TN-C. Finally, we showed that inhibition of ROCK activity suppresses SMC TN-C expression and disease progression in hypertensive rat PAs. Thus, in addition to its role in regulating vasoconstriction, ROCK also controls matrix production. Chapados R, Abe K, Ihida-Stansbury K, McKean D, Gates AT, Kern M, Merklinger S, Elliott J, Plant A, Shimokawa H, Jones PL. ROCK controls matrix synthesis in vascular smooth muscle cells: coupling vasoconstriction to vascular remodeling. Circ Res 2006 Oct 13;99(8):837-44. Epub 2006 Sep 2.

Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by endothelial actin cytoskeletal and adherens junction remodeling, but the mechanisms involved are uncertain. We therefore measured the activities of the Rho GTPases Rac1, RhoA, and Cdc42 during hypoxia/reoxygenation and correlated them with changes in endothelial permeability, remodeling of the actin cytoskeleton and adherens junctions, and production of ROS. Dominant negative forms of Rho GTPases were introduced into cells by adenoviral gene transfer and transfection, and inhibitors of NADPH oxidase, PI3 kinase, and Rho kinase were used to characterize the signaling pathways involved. In some experiments constitutively activated forms of RhoA and Rac1 were also used. We show for the first time that hypoxia/reoxygenation-induced changes in endothelial permeability result from coordinated actions of the Rho GTPases Rac1 and RhoA. Rac1 and RhoA rapidly respond to changes in oxygen tension, and their activity depends on NADPH oxidase- and PI3 kinase-dependent production of ROS. Rac1 acts upstream of RhoA, and its transient inhibition by acute hypoxia leads to activation of RhoA followed by stress fiber formation, dispersion of adherens junctions, and increased endothelial permeability. Reoxygenation strongly activates Rac1 and restores cortical localization of F-actin and VE-cadherin. This effect is a result of Rac1-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA, and lysophosphatidic acid. Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodeling, junctional integrity, or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function. Wojciak-Stothard B, Tsang LY, Haworth SG. Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol. 2005 Apr;288(4):L749-60. Epub 2004 Dec 10.

There is now considerable evidence supporting a mitogenic action of serotonin (5-HT) on vascular smooth muscle cells (SMC) that might participate in pulmonary hypertension (PH). Our previous studies have demonstrated that 5-HT-induced proliferation depends on the generation of reactive oxygen species and activation of extracellular signal-regulated kinase (ERK) 1/ERK2. Activation of Rho kinase (ROCK) in SMC also may be important in PH. We undertook the present study to assess the role of Rho A/ROCK and its possible relation to ERK1/ERK2 in 5-HT-induced pulmonary artery SMC proliferation. We found that this stimulation of SMC proliferation requires Rho A/ROCK as inhibition with Y27632, a ROCK inhibitor, or dominant negative (DN) mutant Rho A blocks 5-HT-induced proliferation, cyclin D1 expression, phosphorylation of Elk, and the DNA binding of transcription factors, Egr-1 and GATA-4. 5-HT activated ROCK, and the activation was blocked by GR 55562 and GR127935, 5-HT 1B/1D receptor antagonists, but not by serotonin transport (SERT) inhibitors. Activation of Rho kinase by 5-HT was independent of activation of ERK1/ERK2, and 5-HT activated ERK1/ERK2 independently of ROCK. Treatment of SMC with Y27632 and expression of DNRho A in cells blocked translocation of ERK1/ERK2 to the cellular nucleus. Depolymerization of actin with cytochalasin D (CD) and latrunculin B (latB) failed to block the translocation of ERK, suggesting that the actin cytoskeleton does not participate in the translocation. The studies show for the first time to our knowledge combinational action of SERT and a 5-HT receptor in SMC growth and Rho A/ROCK participation in 5-HT receptor 1B/1D-mediated mitogenesis of vascular SMCs through an effect on cytoplasmic to nuclear translocation of ERK1/ERK2. Liu Y, Suzuki YJ, Day RM, Fanburg, BL. Rho kinase-induced nuclear translocation of ERK1/ERK2 in smooth muscle cell mitogenesis caused by serotonin. Circ Res 2004 Sep 17; 95(6):579-86. Epub 2004 Aug 5.

Session V: Rho GTPases and Inflammation

Chairs: Rubin Tuder and Asrar Malik

8:15 AM         Inflammation in Pulmonary Hypertension

Inflammatory mechanisms are believed to play a role in pulmonary arterial hypertension (PAH) [1]. Indeed, severe PAH may occur in a subset of patients displaying systemic inflammatory conditions, and treatments with corticosteroids and/or immunosuppressants sometimes dramatically improve PAH complicating the POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, and monoclonal immunoglobulin and skin changes)  [2], Castleman’s disease  [3] , systemic lupus erythematosus  [4, 5] , mixed connective tissue disease  [4, 5]  and primary Sjögren’s syndrome  [6] . Autoimmunity and inflammation have also been demonstrated to contribute to idiopathic PAH pathogenesis [7–11]. Idiopathic PAH patients are characterized by circulating autoantibodies (antinuclear, anti-endothelial and anti-fibroblast anti- bodies) and elevated serum concentrations of proinflammatory cytokines, e.g. interleukin (IL)-1 and IL-6 [7–11]. Of note, pathogenic autoantibodies targeting endothelial cells might be capable of inducing vascular endothelial apoptosis which may in turn promote PAH development [10]. Lung pathology of patients displaying severe PAH in the context of connective tissue diseases further indicates that inflammation and remodeling are key contributors to pulmonary vascular disease complicating inflammatory diseases [12]. In addition, idiopathic PAH frequently harbors inflammatory infiltrates, e.g. macrophages, lymphocytes and dendritic cells, in plexiform lesions with local expression of chemokines CCL2 (MCP-1), CCL5 (RANTES) and CX3CL1 (fractalkine) [12–16]. Experimental data from monocrotaline-induced pulmonary hypertension in rats further support a direct link between pulmonary vascular inflammation and remodeling [1]. Perros F, Cohen-Kaminsky S, Humbert M. Understanding the role of CD4+CD25 (high) (so-called regulatory) T cells in idiopathic pulmonary arterial hypertension. Respiration. 2008; 75(3):253-6. Epub 2008 Feb 15

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-β-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-β-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-β-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil. Fessler, M.B., Arndt, P.G., Frasch, S.C., Lieber, J.G., Johnson, C.A., Murphy, R.C., Nick, J.A., Bratton, D.L., Malcolm, K.C.,  Worthen, G.S. Lipid Rafts Regulate Lipopolysaccharide-induced Activation of Cdc42 and Inflammatory Functions of the Human Neutrophil.  Journal of Biological Chemistry, 279, 39989-39998.

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 µM forskolin plus 1 µM IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of 32P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of %7e23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction. Rho guanosine 5'-triphosphate; protein kinase A inhibitor; guanine nucleotide dissociation inhibitor; endothelial resistance. Qiao, J. Huang, F., Lum, H. PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol. Vol. 284, Issue 6, L972-L980, June 2003. 

11:10 AM       Circulation Progenitor Cells in Pulmonary Hypertension             

Functional phenotypes of each of the vascular cell types, from the hilum of the lung to the most peripheral vessels in the alveolar wall. The magnitude and the specific profile of the changes depend on the species, sex, and the developmental stage at which the exposure to hypoxia occurred. Further, hypoxia-induced changes are site specific, such that the remodeling process in the large vessels differs from that in the smallest vessels. The cellular and molecular mechanisms vary and depend on the cellular composition of vessels at particular sites along the longitudinal axis of the pulmonary vasculature, as well as on local environmental factors. Each of the resident vascular cell types (i.e., endothelial, smooth muscle, adventitial fibroblast) undergo site- and time-dependent alterations in proliferation, matrix protein production, expression of growth factors, cytokines, and receptors, and each resident cell type plays a specific role in the overall remodeling response. In addition, hypoxic exposure induces an inflammatory response within the vessel wall, and the recruited circulating progenitor cells contribute significantly to the structural remodeling and persistent vasoconstriction of the pulmonary circulation. The possibility exists that the lung or lung vessels also contain resident progenitor cells that participate in the remodeling process. Thus the hypoxia-induced remodeling of the pulmonary circulation is a highly complex process where numerous interactive events must be taken into account as we search for newer, more effective therapeutic interventions. This review provides perspectives on each of the aforementioned areas. Stenmark, K.R., Fagan, K. A., Frid, M.G. Hypoxia-Induced Pulmonary Vascular Remodeling   (Circulation Research. 2006;99:675.)

11:50 AM        Featured Abstract: A Crucial Role of Rho/ Rho Kinase Pathway in Construction of 

                         Human and Rabbit Ductus Arteriosus

Background – At birth, functional closure of the ductus arteriosus (DA) is initiated by inhibition of oxygen-sensitive, voltage-gated K+ channels in DA smooth muscle cells. The resulting membrane depolarization and activation of L-type calcium channels initiates vasoconstriction. We hypothesized that maintenance of vasoconstriction, in both human and rabbit DA, requires activation of the Rho-kinase pathway of calcium sensitization. Methods and Results–DA constriction to O2 was assessed in freshly isolated rings from human infants or rabbit pups. Two different Rho-kinase inhibitors (Y-27632 and fasudil) rapidly lower basal tone in hypoxic human DAs and reverse O2 induced constriction.  O2 constriction in term rabbit DA persists in the absence of extracellular calcium, albeit at a significantly reduced level, consistent with calcium sensitization. Rho-kinase inhibitors also cause dose-dependent relaxation of oxygen-constricted rabbit DA and attenuate constriction to endothelin, phenylephrine and KCl. Rho-kinase inhibitor-induced relaxation is greater in DA than aorta. The mechanism by which O2 activates Rho-kinase in the DA by a redox-dependent mechanism, which is mimicked by H2O2. Exposure to O2 for 1-hour increases expression of Rho/Rho-kinase proteins in Human DA smooth muscle cells. Conclusions – In the ductus, activation of the Rho-kinase pathway is crucial to maintaining basal tone and to sustaining oxygen constriction. Oxygen activates Rho-kinase both functionally (by an acute redox mechanism) and by enhancing expression of Rho/ROCK-1. Rho-kinase inhibition has the potential to maintain DA patency as a bridge to surgery in patients with DA-dependent congenital heart disease. Kajimoto, H., Hashimoto, K., Bonnet, S. N., Haromy A., Moudgil, R., Rebeyka, I., Archer, S.L. A Crucial Role of Rho/Rho-Kinase Pathway in Constriction of Human and Rabbit Ductus Arteriosus.  

Session VI: Abstract/Poster Review

Chairs (Facilitators): Bruce Pitt and Mary Townsley

Session VII: Medical Implications of Rho GTPases

Chairs: Ken Wier and John Weil

Pulmonary vascular injury by toxins can induce neointimal formation, pulmonary arterial hypertension (PAH), right ventricular failure, and death. We showed previously that simvastatin attenuates smooth muscle neointimal proliferation and pulmonary hypertension in pneumonectomized rats injected with the alkaloid toxin monocrotaline. The present study was undertaken to investigate the efficacy of simvastatin and its mechanism of reversing established neointimal vascular occlusion and pulmonary hypertension. METHODS AND RESULTS: Pneumonectomized rats injected with monocrotaline at 4 weeks demonstrated severe PAH at 11 weeks (mean pulmonary artery pressure [mPAP]=42 versus 17 mm Hg in normal rats) and death by 15 weeks. When rats with severe PAH received simvastatin (2 mg x kg (-1) x d (-1) by gavage) from week 11, there was 100% survival and reversal of PAH after 2 weeks (mPAP=36 mm Hg) and 6 weeks (mPAP=24 mm Hg) of therapy. Simvastatin treatment reduced right ventricular hypertrophy and reduced proliferation and increased apoptosis of pathological smooth muscle cells in the neointima and medial walls of pulmonary arteries. Longitudinal transcriptional profiling revealed that simvastatin downregulated the inflammatory genes fos, jun, and tumor necrosis factor-alpha and upregulated the cell cycle inhibitor p27Kip1, endothelial nitric oxide synthase, and bone morphogenetic protein receptor type 1a. CONCLUSIONS: Simvastatin reverses pulmonary arterial neointimal formation and PAH after toxic injury. Nishimura, T., Vaszar, L.T., Faul, J.L., Zhao, G., Berry, G.J., Shi, L., Qiu, D., Benson, G., Pearl, R.G., Kao, P.N. Simvastatin rescues rats from fatal pulmonary hypertension by inducing apoptosis of neointimal smooth muscle cells. Circulation 2003 Sep 30; 108(13):1640-5. Epub 2003 Sep 8.

The statins, a class of HMG-CoA reductase inhibitors, directly affect multiple vascular processes via inhibition of geranylgeranylation, a covalent modification essential for Rho GTPase interaction with cell membrane-bound activators. We explored simvastatin effects on endothelial cell actomyosin contraction, gap formation, and barrier dysfunction produced by the edemagenic agent, thrombin. Human pulmonary artery endothelial cells exposed to prolonged simvastatin treatment (5 microM, 16 h) demonstrated significant reductions in thrombin-induced (1 U/ml) barrier dysfunction ( approximately 70% inhibition) with accelerated barrier recovery, as measured by transendothelial resistance. Furthermore, simvastatin attenuated basal and thrombin-stimulated (1 U/ml, 5 min) myosin light chain diphosphorylation and stress fiber formation while dramatically increasing peripheral immunostaining of actin and cortactin, an actin-binding protein, in conjunction with increased Rac GTPase activity. As both simvastatin-induced Rac activation and barrier protection were delayed (maximal after 16 h), we assessed the role of gene expression and protein translation in the simvastatin response. Simultaneous treatment with cycloheximide (10 microg/ml, 16 h) abolished simvastatin-mediated barrier protection. Robust alterations were noted in the expression of cytoskeletal proteins (caldesmon, integrin beta4), thrombin regulatory elements (PAR-1, thrombomodulin), and signaling genes (guanine nucleotide exchange factors) in response to simvastatin by microarray analysis. These novel observations have broad clinical implications in numerous vascular pathobiologies characterized by alterations in vascular integrity including inflammation, angiogenesis, and acute lung injury. Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. Jacobson JR, Dudek SM, Birukov KG, Ye SQ, Grigoryev DN, Girgis RE, Garcia JG. Am J Respir Cell Mol Biol 30(5):662-70. Epub 2003 Nov 20.

9:40  AM      Discussion 

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca2+ sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50–300 µm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca2+ concentration ([Ca2+]i). Basal VSM [Ca2+] i was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 µM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 µM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5–45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50–200 µm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 µM) but not by GF109203X. VSM [Ca2+]i was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200–300 µm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca2+ sensitization. Broughton, B.R.S., Walker, B.R., Resta, T.C. Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 294: L797-L806, 2008.  

10:20 AM     Discussion                 

The pulmonary circulation differs from the systemic in several important aspects, the most important being that pulmonary arteries constrict to moderate physiological (20-60 mmHg PO2) hypoxia, whereas systemic arteries vasodilate. This phenomenon is called hypoxic pulmonary vasoconstriction (HPV), and is responsible for maintaining the ventilation-perfusion ratio during localized alveolar hypoxia. In disease, however, global hypoxia results in a detrimental increase in total pulmonary vascular resistance, and increased load on the right heart. Despite many years of study, the precise mechanisms underlying HPV remain unresolved. However, as we argue below, there is now overwhelming evidence that hypoxia can stimulate several pathways leading to a rise in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMC). This rise in [Ca2+]i is consistently found to be relatively small, and HPV seems also to require rho kinase-mediated Ca2+ sensitization. There is good evidence that HPV also has an as yet unexplained endothelium dependency. In this brief review, we highlight selected recent findings and ongoing controversies which continue to animate the study of this remarkable and unique response of the pulmonary vasculature to hypoxia. Aaronson PI, Robertson TP, Knock GA, Becker S, Lewis TH, Snetkov V, Ward JP. Hypoxic pulmonary vasoconstriction: mechanisms and controversies. J Physiol 2006 Jan 1;570(Pt 1):53-8. Epub 2005 Oct 27.