RESEARCH REPORT
CCN1 suppresses pulmonary vascular smooth muscle contraction in response to hypoxia Seon-jin Lee,1,2 Meng Zhang,1 Kebin Hu,3 Ling Lin,3 Duo Zhang,1 Yang Jin1 1
Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; 3Division of Nephrology, Department of Medicine, Pennsylvania State University Medical Center, Hershey, Pennsylvania 17033, USA
Abstract: Pulmonary vasoconstriction and increased vascular resistance are common features in pulmonary hypertension (PH). One of the contributing factors in the development of pulmonary vasoconstriction is increased pulmonary artery smooth muscle cell (PASMC) contraction. Here we report that CCN1, an extracellular matrix molecule, suppressed PASMC contraction in response to hypoxia. CCN1 (Cyr61), discovered in past decade, belongs to the Cyr61-CTGF-Nov (CCN) family. It carries a variety of cellular functions, including angiogenesis and cell adhesion, death, and proliferation. Hypoxia robustly upregulated the expression of CCN1 in the pulmonary vessels and lung parenchyma. Given that CCN1 is a secreted protein and functions in a paracine manner, we examined the potential effects of CCN1 on the adjacent smooth muscle cells. Interestingly, bioactive recombinant CCN1 significantly suppressed hypoxia-induced contraction in human PASMCs in vitro. Consistently, in the in vivo functional studies, administration of bioactive CCN1 protein significantly decreased right ventricular pressure in three different PH animal models. Mechanistically, protein kinase A–pathway inhibitors abolished the effects of CCN1 in suppressing PASMC contraction. Furthermore, CCN1-inhibited smooth muscle contraction was independent of the known vasodilators, such as nitric oxide. Taken together, our studies indicated a novel cellular function of CCN1, potentially regulating the pathogenesis of PH. Keywords: CCN1, pulmonary hypertension, hypoxia, pulmonary artery smooth muscle cell. Pulm Circ 2015;5(4):716-722. DOI: 10.1086/683812.
Vasodilators are common agents utilized to decrease pulmonary vascular resistance and to relieve pulmonary hypertension (PH),1-3 including relatively new agents such as inhaled nitric oxide (NO), endothelin 1 (ET-1), and prostaglandins.1-3 “Hypoxic pulmonary vasoconstriction” (HPV) refers to a physiological status in which pulmonary arteries constrict in the presence of hypoxia. This process redirects blood flow to the alveoli with the higher oxygen content and consequently improves the ventilation/perfusion ratio and arterial oxygenation.4-7 HPV can help to maintain the normal pulmonary vascular tone,4-7 and this phenomenon has been studied extensively. However, in the presence of severe pulmonary disease, prolonged HPV increases pulmonary vascular resistance and consequently results in irreversible pulmonary vascular remodeling.4-7 The etiology of HPV is multifactorial, but detailed understanding of the exact mechanisms is lacking. Pulmonary artery smooth muscle cells (PASMCs) are clearly one of the main components involved in HPV. Endotheliumreleased mediators, such as endothelins, prostaglandins, serotonin, leukotrienes, histamine, angiotensin II, and NO, have been shown to play crucial roles in regulating smooth muscle contractility and proliferation.8-10 After prolonged hypoxia, endothelial cells (ECs) release these
mediators into the circulation and cause sustained HPV and subsequent vascular remodeling. CCN1 (formally named Cyr61) is a recently discovered earlystress-response gene product with diverse cellular functions.11-15 It belongs to the Cyr61-CTGF-Nov (CCN) family and is a secreted protein that functions in a paracrine and/or autocrine manner.16-20 CCN1’s functions include but are not limited to cytoprotection, angiogenesis, cell adhesion, and migration.11-15 The role of CCN1 in pulmonary diseases, especially pulmonary vascular diseases and PH, has not yet been explored. Here we report that CCN1 potentially is crucial in regulating smooth muscle contractility in pulmonary vasculature. In addition to the well-known NO, CCN1 potentially functions as a mediator in regulating right ventricular pressure (RVP) in hypoxia-induced PH. Our studies may suggest a novel target for PH research. ME T H O D S
Chemicals and reagents Recombinant CCN1 was obtained from R&D systems (Minneapolis, MN). Wortmannin, SB203580, U0126, Y-27632, and γ-secretase
Address correspondence to Dr. Yang Jin, Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA. E-mail: [email protected]. Submitted January 10, 2015; Accepted June 25, 2015; Electronically published October 7, 2015. © 2015 by the Pulmonary Vascular Research Institute. All rights reserved. 2045-8932/2015/0504-0013. $15.00.
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inhibitor IX were purchased from Calbiochem (Gibbstown, NJ). Purecol was obtained from Advanced Biomatrix (San Diego, CA). All cell culture reagents were obtained from Invitrogen (Carlsbad, CA) unless otherwise specified. We purchased the antibody to Cyr61 and endothelial NO synthase from Santa Cruz Biotechnology (Dallas, TX); antibodies to phospho–myosin light chain (MLC) were from Cell Signaling (Danvers, MA); antibodies to α-smooth muscle actin (SMA) and β-actin were from Sigma-Aldrich (St. Louis, MO); and enhanced chemiluminescence reagent was purchased from Thermo Scientific (Grand Island, NY). All short interfering RNA reagents were purchased from Gene Tools (Philomath, OR). All other reagents were obtained from Sigma-Aldrich unless otherwise specified.
Animals and in vivo exposures All animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care. The Animal Research Committee of Brigham and Women’s Hospital approved all protocols. Wild-type C57BL/6 mice were placed in a Plexiglas chamber maintained at 10% O2 (hypoxia group) or in a chamber open to room air (normoxic group), with a 12L ∶ 12D light cycle. The mice were exposed to hypoxia or normoxia for 3 weeks, and then their lungs were harvested and analyzed for Cyr61 expression by Western immunoblot analysis. For functional studies, age-matched littermates were exposed to 10% O2 (hypoxia group) or room air (normoxic group) in the presence of recombinant CCN1 protein (1 μg/kg), su5416 (mouse: 2 mg/kg twice per week; rat: 5 mg/kg every week), or vehicle for 3 weeks before functional measurements (15 mice in each group for each time point).
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Western immunoblotting and immunohistochemistry Western immunoblot analyses were performed as previously described.16 For Western blotting, protein concentrations of cell lysates and frozen tissue homogenates were determined with a Coomassie plus protein assay (Thermo Fisher Scientific, Pittsburgh, PA) and were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using NuPage Novex Bis-Tris 4%–12% polyacrylamide gels (Invitrogen). Lungs were inflated, harvested, fixed in 2% paraformaldehyde, and embedded in paraffin. Sections were stained with antimouse α-SMA and rabbit anti-CCN1 antibody (1 ∶ 100; Santa Cruz) for 3 hours at 4°C. The cells were washed and incubated with antirat IgG (immunoglobulin G)–fluorescein isothiocyanate and antirabbit IgG–tetramethylrhodamine isothiocyanate for 1 hour. After staining, the cells were mounted with mounting medium and observed by laser scanning confocal microscopy.
Right ventricular systolic pressure and heart weight measurements Measurements were obtained with a PC-driven PowerLab (ADInstruments, Colorado Springs, CO). The system was calibrated before each experiment. Each mouse was weighed and injected with sodium pentobarbital (60 mg/kg) to induce anesthesia with maintenance of spontaneous respiration. The abdomen was opened and the left and right ventricles visualized through an intact diaphragm. A 23-G needle was then inserted into the right ventricle, and RVP was recorded. The diaphragm was opened, and then a repeat measurement of RVP, as well as left ventricular pressure, was obtained. The right ventricular (RV) free wall was dissected and weighed separately from the left ventricle and septum (LV + S), which were weighed together.
Monocrotaline (MCT) We assigned adult male Sprague-Dawley rats (200–250 g in body weight; Charles River Laboratories, Wilmington, MA) to groups receiving either subcutaneous injection of 60 mg/kg body weight of MCT or saline alone. We performed hemodyamic measurements and gene expression analysis 3 weeks after injection (10 rats per group for each time point).
Vascular cell culture Human PASMCs (HPASMCs; Lonza, Walkersville, MD) at passages 7–10 were grown to ∼80% confluence in smooth muscle cell growth medium 2 (SmGM2) supplemented with SmGM2 SingleQuots and 5% fetal bovine serum (Lonza). Cells were cultured in humidified incubators containing 95% air and 5% CO2 at 37°C. Cells were grown in 100-mm dishes, detached with 0.05% trypsin, resuspended in complete growth medium, and seeded into 35-mm, 6-well, and 12-well plates for individual experiments. For hypoxic exposures, HPASMCs were placed in an airtight modular incubator chamber (Billups-Rothenberg, San Diego, CA), flushed continuously (10 minutes) with a premixed gas (1% O2, 5% CO2, 94% N2), and then incubated for the indicated intervals. Corresponding normoxic controls were maintained for equivalent times in humidified incubators in an atmosphere of 95% air and 5% CO2.
Collagen matrix contraction assay To assess contractility of HPASMCs on a collagen gel matrix, 2 × 105 cells were plated on type I collagen gel matrices in 24-well dishes in triplicate and exposed to hypoxia or normoxia for 24 hours. Collagen matrices were prepared with Vitrogen 100 collagen (Celltrix, Santa Clara, CA), 10× Dulbecco’s modified Eagle’s medium, and HEPES (pH 8.5). After hypoxic exposure, the matrix was released to initiate contraction, and the cells were then incubated in normoxia at 37°C. Gel size was defined as the sum of the two longest gel diameters, and gel contraction was expressed as a percentage of the original gel size. In certain experiments, cells were treated with CCN1 (100 ng/mL), protein kinase inhibitor (PKI; 1 μM), wortmannin (100 nM), SP600126 (20 μM), cholera toxin (5 μg/mL), U0126 (10 μM), Arg-Gly-Asp (RGD; 10 μM), γ-secretase inhibitor (5 μM), SB203586 (10 μM), Z-Leu-Leu-Leu-al (10 μM), Y-27632 (20 μg/mL), or vehicle (the same volume of dimethyl sulfoxide) during hypoxic exposure.
Statistics Data are presented as mean ± SD. Paired analysis was performed with the Student t test as appropriate. Parametric analyses were carried out by 1-way ANOVA with Tukey’s method. Statistical significance was accepted at P < 0.05.
Figure 1. A, Expression of CCN1 in pulmonary vasculature, in the presence and absence of hypoxia. Rats were exposed to normoxia or hypoxia (10% oxygen). After 3 weeks, pulmonary vascular sections were stained with CCN1 using immunohistochemistry (IHC). Red arrow indicates CCN1. EC: endothelial cell; L: lumen; SMC: smooth muscle cell. B, Stain of α-smooth muscle actin (α-SMA) and CCN1 in mouse lung distal vessels. Mice were exposed to room air or hypoxia (10% oxygen). After 3 weeks, lung sections were fixed and stained with anti-α-SMA or anti-CCN1. Confocal microscopy was used to determine colocalization. Green: α-SMA; red: CCN1; yellow: merge. C, Stain of PECAM (platelet endothelial cell adhesion molecule) and CCN1 in mouse lung distal vessels. Mice were exposed to room air or hypoxia (10% oxygen). After 3 weeks, lung sections were fixed and stained with anti-PECAM or anti-CCN1. Confocal microscopy was used to determine colocalization (arrowheads). Green: PECAM; red: CCN1; yellow: merge. Each figure represents 2 independent experiments. *, # P < 0.05. D–G, Expression of CCN1 in pulmonary vascular cells and lung tissue, in the presence and absence of hypoxia, analyzed with Western blot analysis. D, Expression of CCN1 in human pulmonary endothelial cells (HPAECs). E, Expression of CCN1 in human pulmonary artery smooth muscle cells (HPASMCs). F, Expression of CCN1 in mouse lung tissue. G, Expression of CCN1 in rat lung tissue. Each panel represents 2 independent experiments. *P < 0.05.
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RESU LTS
Hypoxia upregulated CCN1 expression in pulmonary vasculature We evaluated the expression of CCN1 in pulmonary vasculature in the presence and absence of hypoxia, a common trigger for PH. CCN1 was upregulated robustly in ECs and smooth muscle cells (SMCs; Fig. 1A). Given that CCN1 is a secreted protein and functions in a paracrine/autocrine manner, SMCs in the medium layer of pulmonary vasculature certainly are one of the potential targets of lung-derived CCN1. We next stained the CCN1, along with the SMC marker protein α-SMA and the EC marker PECAM (platelet endothelial cell adhesion molecule). As shown in Figures 1B and 1C, hypoxia indeed induced markedly increased CCN1 in pulmonary vasculature. We confirmed this result with Western blotting analysis. CCN1 was significantly induced both in human pulmonary artery ECs (Fig. 1D) and in HPASMCs (Fig. 1E) after hypoxia. In addition, consistent results were obtained in mouse (Fig. 1F) and rat (Fig. 1G) lung tissue homogenates.
CCN1 suppressed hypoxia-induced smooth muscle contraction Initially, we found that bioactive CCN1 recombinant protein robustly suppressed hypoxia-induced smooth muscle contraction in vitro (Fig. 2A). CCN1 (100 ng/mL) was added into cell culture media as described in “Methods.” After 12 hours of hypoxia (5% oxygen), CCN1 significantly blocked HPASMC contraction. Pretreatment of the cells with protein kinase A (PKA) pathway inhibitor PKI markedly reversed the effects of CCN1 on HPASMC relaxation (Fig. 2A). Furthermore, pretreatment of the cells with wortmannin (the specific inhibitor of phosphoinositide 3-kinase [PI3K]), SP600126 (Jun N-terminal kinase [JNK] inhibitor), and RGD peptide (Arg-Gly-Asp, an integrin-binding protein) partially reversed the effects of CCN1 (Fig. 2A). The percentage of the original gel size summarized from 3 independent assays is shown in Figure 2B. In addition, we tested the effects of a variety of inhibitors for multiple pathways, including cholera toxin (G-protein signal pathway), Z-LeuLeu-Leu-al (NF-κB pathway), NMA (NO inhibitor), PD98059 (MEK1 pathway), γ-secretase (notch-mediated pathway), U0126 (MEK1/2 inhibitor), SB203580 (p38 pathway), and Y-27632 (Rho-associated
Figure 2. CCN1 prevented hypoxia-induced human pulmonary artery smooth muscle cell (HPASMC) contraction via multiple pathways. First, 2 × 105 HPASMC cells were plated on type I collagen gel matrices in 24-well dishes in triplicate and exposed to hypoxia or normoxia for 24 hours. After hypoxic exposure, the matrix was released to initiate contraction, and the cells were then incubated in normoxia at 37°C. Gel size is defined as the sum of the two longest
gel diameters, and gel contraction is expressed as a percentage of the original gel size. In certain experiments, cells were treated with CCN1 (100 ng/mL), protein kinase inhibitor (PKI; 1 μM), wortmannin (Wort; 100 nM), SP600126 (SP; 20 μM), Arg-Gly-Asp (RGD; 10 μM), or vehicle (CTL; the same amount of dimethyl sulfoxide) during hypoxic exposure. These inhibitors were used to block certain signaling pathways. A, Left (dashed frame): cells exposed to room air (normoxia); right: cells exposed to hypoxia. PBS: phosphate-buffered saline. B, Percentage of original gel size. The data shown in Figure 1A are presented here as a bar graph. C, Evaluation of multiple signaling pathways on hypoxia-induced HPASMC contraction in the presence of CCN1; see “CCN1 suppressed the hypoxia-induced smooth muscle contraction” for details. mTOR: mammalian target of rapamycin; Tx: toxin. D, Percentage of original gel size. Each figure represents 3 independent * experiments. P < 0.05.
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coil kinase [ROCK] inhibitor). None of these inhibitors showed significant effects of blocking CCN1-associated HPASMC relaxation (Fig. 2C, 2D).
CCN1 decreased RVP in animal models of PH We used 4 animal models of PH to examine the effects of CCN1 on RVP: (1) hypoxia-induced PH model in mice, (2) hypoxia-induced PH model in rats, (3) MCT-induced PH model in rats, and (4) sugen 5416 (vascular endothelial growth factor receptor inhibitor su5416) followed by hypoxia in mice. As shown in Figure 3, all animals were exposed to room air, hypoxia (10% oxygen), or MCT for 3 weeks as described in “Methods.” In the su5416-hypoxia model, we treated mice with su5416, along with hypoxia, as described above. After 3 weeks, we administrated recombinant CCN1 protein (1 μg/kg) 12 hours before measurement. We found that CCN1 significantly decreased RVP in all 4 of these models (Fig. 3). As expected, the ratio of right ventricular weight to body weight (RVW/BW, mg/g) and the ratio of right ventricular weight to left ventricular weight plus septa (Fulton index; RV/(LV + S)) were not affected (Figs. S1– S4, available online).
DISCUSSION Although the pathogenesis remains not completely clear, vasoconstriction and subsequent pulmonary vascular remodeling have been noted in all types of PH regardless of its causes.17-21 Medium/ smooth muscle contractions are crucial in the vasoconstriction involved in PH.17-21 In recent years, accumulating evidence has suggested an essential role of extracellular matrix (ECM) proteins in the development of pulmonary vascular remodeling and vasoconstriction.17-21 However, it is still not completely understood which specific ECM proteins are playing the crucial roles, despite the discovery of NO, endothelin, and prostacyclin (PGI). Our studies have revealed that CCN1 potentially is a novel and effective inhibitor of hypoxia-induced PASMC contraction. CCN1 has many unique features that initiated our interest. First, CCN1 is a secreted protein that functions in a paracrine/autocrine manner.11-15 Thus, lung tissue/cell-derived CCN1 may exert essential regulation on the adjacent SMCs. This secreted molecule can potentially function as a communicator or messenger among epithelial cells, ECs, SMCs, fibroblasts, and even inflammatory cells. Second, CCN1 binds a variety of integrins and exerts differential biological functions based on the binding integrins, including β1-containing integrin(s). The β1-containing integrins are essential in regulating smooth muscle contractility.22 Our data, from the collagen contraction assays, showed that CCN1 prevented hypoxia-induced HPASMC contraction (Fig. 1). The mechanisms by which CCN1 mediated the HPASMC relaxation could be mutifactorial. Nevertheless, we showed that integrin, PI3K, JNK, and PKA are all involved in the regulation of HPASMC relaxation caused by CCN1 (Fig. 1). All these pathways potentially manipulate MLC phosphorylation and subsequently control SMC contraction and relaxation. Previous reports have demonstrated
Figure 3. Effects of CCN1 on right ventricular pressure (RVP), from animal models of pulmonary hypertension (PH). A, C57BL/6 mice were exposed to hypoxia (10%). After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via intraperitoneal (ip) injection. RVP was determined with right heart catheterization (n = 10, *P < 0.05). B, Sprague-Dawley rats were exposed to hypoxia (10%). After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via ip injection (n = 6, *P < 0.05). C, C57BL/6 mice were exposed to hypoxia (10%) and su5416 (2 mg/kg, twice per week) or vehicle (phosphate-buffered saline [PBS]). After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via ip injection. RVP was determined with right heart catheterization (n = 15, *P < 0.05). D, Sprague-Dawley rats were given either subcutaneous injection of monocrotaline (MCT; 60 mg/ kg) or saline alone, in a single dose. After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via ip injection. RVP was determined with right heart catheterization (n = 6, *P < 0.05).
that activation of MLC via phosphorylation by MLC kinase results in smooth muscle contraction. On the other side, MLC phosphatase dephosphorylates MLC and leads to relaxation.23-26 Two phases of contraction occur in smooth muscle. The initial phase is regulated
Pulmonary Circulation
by inositol 1,4,5-trisphosphate (Ins (1,4,5)P3)-dependent Ca2+release and Ca2+/calmodulin-dependent activation of MLC kinase.23-26 The late-phase contraction of vascular smooth muscle is mediated by phosphorylation of Ser19 on the regulatory myosin light chain MLC20.26-28 This contraction is RhoA dependent and occurs via inhibition of MLC phosphatase. Membrane-anchored RhoA activates Rho kinase and phospholipase D (PLD).26-28 Rho kinase and PLD both inhibit MLC phosphatase, either directly or indirectly via the PKC pathway.26-28 Interestingly, PKA induces relaxation via inactivation of the membrane-bound RhoA. Inactivation of membrane-bound RhoA fails to act on its membrane-bound substrates, Rho kinase and PLD.26-28 Since Rho kinase and PLD inhibit MLC phosphatase and result in sustained phosphorylation of MLC20 and muscle contraction, PKA favors muscle relaxation via inducing inactivated membranebound RhoA and, subsequently, inactive Rho kinase and PLD. Our data shown in Figure 2 indicate that the initial and late phases of SMC contraction were modulated by CCN1, via PI3K- and PKAmediated pathways, respectively. However, the detailed mechanisms require further investigation and will be included in our future studies. Endogenous vasodilators derived from pulmonary vascular endothelium, particularly NO and PGI, play important effects in maintaining low pressure and low resistance in pulmonary vasculature.17-21,29,30 This low-pressured and compliant pulmonary vasculature is essential to maintaining RV function and provides a large surface area for gas exchange.17-21 Thus, current PH therapy involves in treating vasoconstriction with NO and PGI analogs. Interestingly, NO and PGI both increase cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophasphate (cAMP). These secondary messengers, cGMPs and cAMPs, induce the relaxation of arterial smooth muscle via regulation of the intracellular Ca2+ concentrations, particularly the smooth muscle Ca2+ sensitivity of the contractile elements.29,30 In vivo, we showed that hypoxia upregulated the expression of CCN1, particularly in intima/ECs. Presumably, this EC-derived CCN1 will act on SMCs to prevent hypoxia-induced contraction. However, one question that arises is, why can administration of exogenous CCN1 still decrease RVP dramatically in animal models of PH, despite the robustly upregulated CCN1 in ECs? Multiple potential explanations should be explored in future studies. Hypoxia may have significantly downregulated CCN1 receptors, particularly the β1-containing integrin(s) in SMCs/medium. As discussed above, β1-containing integrin(s) play crucial roles in regulating smooth muscle contractility.20 Therefore, matrix CCN1, even if it reaches the medium layer of artery, may not function efficiently because of a lack of functional receptors. Near-future directions should include exploring the expression of β1-containing integrin(s) in SMCs/ medium. Next and potentially more important, CCN1 alternative splicing has been widely reported in the latest literature. One of the alternatively spliced CCN1 forms results in truncation of two important functional domains.31,32 Therefore, we will vigorously explore the alternatively spliced CCN1 protein in the setting of hypoxiaassociated pulmonary vascular remodeling. CCN1 appears to function as an independent vasodilator to prevent hypoxia-induced smooth muscle contraction, via a distinct path-
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way other than NO-, PGI-, or ET-1-meditated signals. It is a conservative gene product, with 93% homology between humans and rodents. Therefore, our studies have potentially uncovered a novel, yet important vasodilator for treatment of PH. Whether this molecule has synergistic effects with existing vasodilators remains unknown. Its effects on systemic vasculature also require further investigation. In addition, the mechanisms by which CCN1 decreases RVP in MCT-induced PH models may differ from those in hypoxiainduced PH models. In summary, our studies indicate a novel function of CCN1 in regulating SMC contractility in the presence of hypoxia and show that CCN1 decreased RVP in animal models of PH. This work potentially provides novel insights and targets for PH research.
ACKNOW LEDGMENTS We thank Drs. Augustine M. K. Choi, Lester F. Lau, and Mark A. Perrella for their scientific advice and support. Source of Support: Nil. Conflict of Interest: None declared. REFER E NCES 1. Palevsky HI, Schloo BL, Pietra GG, Weber KT, Janicki JS, Rubin E, Fishman AP. Primary pulmonary hypertension: vascular structure, morphometry, and responsiveness to vasodilator agents. Circulation 1989;80 (5):1207–1221. 2. Tsai BM, Wang M, Turrentine MW, Mahomed Y, Brown JW, Meldrum DR. Hypoxic pulmonary vasoconstriction in cardiothoracic surgery: basic mechanisms to potential therapies. Ann Thorac Surg 2004; 78(1):360–368. 3. Lowson S. Inhaled alternatives to nitric oxide. Anesthesiology 2002;96 (6):1504–1513. 4. Blaise G, Langleben D, Hubert B. Pulmonary arterial hypertension: pathophysiology and anesthetic approach. Anesthesiology 2003;99(6): 1415–1432. 5. Abe K, Toba M, Alzoubi A, Ito M, Fagan KA, Cool CD, Voelkel NF, McMurtry IF, Oka M. Formation of plexiform lesions in experimental severe pulmonary arterial hypertension. Circulation 2010;121(25):2747– 2754. 6. Stenmark KR, Meyrick B, Galiè N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol 2009;297(6):L1013–L1032. 7. Ciuclan L, Bonneau O, Hussey M, Duggan N, Holmes AM, Good R, Stringer R, et al. A novel murine model of severe pulmonary arterial hypertension. Am J Respir Crit Care Med 2011;184(10):1171–1182. 8. Pappert D, Busch T, Gerlach H, Lewandowski K, Radermacher P, Rossaint R. Aerosolized prostacyclin versus inhaled nitric oxide in children with severe acute respiratory distress syndrome. Anesthesiology 1995;82(6):1507–1511. 9. Langer F, Wilhelm W, Tscholl D, Schramm R, Lausberg H, Wendler O, Schäfers H-J. Intraoperative inhalation of the long-acting prostacyclin analog iloprost for pulmonary hypertension. J Thorac Cardiovasc Surg 2003;126(3):874–875. 10. Vincent JL, Carlier E, Pinsky MR, Goldstein J, Naeije R, Lejeune P, Brimioulle S, Leclerc JL, Kahn RJ, Primo G. Prostaglandin E1 infusion for right ventricular failure after cardiac transplantation. J Thorac Cardiovasc Surg 1992;103(1):33–39.
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11. Babic AM, Kireeva ML, Kolesnikova TV, Lau LF. CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc Natl Acad Sci USA 1998;95(11):6355– 6360. 12. Perbal B. CCN proteins: multifunctional signalling regulators. Lancet 2004;363(9402):62–64. 13. O’Brien TP, Yang GP, Sanders L, Lau LF. Expression of cyr61, a growth factor-inducible immediate-early gene. Mol Cell Biol 1990;10(7):3569– 3577. 14. Grzeszkiewicz TM, Lindner V, Chen N, Lam SC, Lau LF. The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin α6ß1 and cell surface heparan sulfate proteoglycans. Endocrinology 2002;143 (4):1441–1450. 15. Lau LF. CCN1/Cyr61: the very model of a modern matricellular protein. Cell Mol Life Sci 2011;68(19):3149–3163. 16. Jin Y, Kim HP, Cao J, Zhang M, Ifedigbo E, Choi AMK. Caveolin-1 regulates the secretion and cytoprotection of Cyr61 in hyperoxic cell death. FASEB J 2009;23(2):341–350. 17. Pietra GG, Capron F, Stewart S, Leone O, Humbert M, Robbins IM, Reid LM, Tuder RM. Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 2004;43(1):25–32. 18. Humbert M. Update in pulmonary hypertension 2008. Am J Respir Crit Care Med 2009;179(8):650–656. 19. Mooi WJ, Grünberg K. Histopathology of pulmonary hypertensive diseases. Curr Diagn Pathol 2006;12(6):429–440. 20. Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, McMurtry IF, et al. Cellular and molecular basis of pulmonary arterial hypertension J Am Coll Cardiol 2009;54(1 suppl.):S20–S31. 21. Meyrick BO, Perkett EA. The sequence of cellular and hemodynamic changes of chronic pulmonary hypertension induced by hypoxia and other stimuli. Am Rev Respir Dis 1989;140(5):1486–1489. 22. Dahm LM, Bowers CW. Vitronectin regulates smooth muscle contractility via αv and β1 integrin. J Cell Sci 1998;111(9):1175–1183.
23. Morgado M, Cairrão E, Santos-Silva AJ, Verde I. Cyclic nucleotidedependent relaxation pathways in vascular smooth muscle. Cell Mol Life Sci 2012;69(2):247–266. 24. Pfitzer G. Regulation of myosin phosphorylation in smooth muscle. J Appl Physiol 2001;91(1):497–503. 25. Hartshorne DJ, Ito M, Erdödi F. Myosin light chain phosphatase: subunit composition, interactions and regulation. J Muscle Res Cell Motil 1998;19(4):325–341. 26. Kamm KE, Stull JT. Dedicated myosin light chain kinase with diverse cellular functions. J Biol Chem 2001;276(7):4527–4530. 27. Azam MA, Yoshioka K, Ohkura S, Takuwa N, Sugimoto N, Sato K, Takuwa Y. Ca2+-independent, inhibitory effects of cyclic adenosine 5′monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2α, Rho, and myosin phosphatase in vascular smooth muscle. J Pharmacol Exp Ther 2007;320(2):907–916. 28. Murthy KS, Zhou H, Grider JR, Makhlouf GM. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA. Am J Physiol Gastrointest Liver Physiol 2003;284(6):G1006–G1016. 29. Body SC, Hartigan PM, Shernan SK, Formanek V, Hurford WE. Nitric oxide: delivery, measurement, and clinical applications. J Cardiothorac Vasc Anesth 1995;9(6):748–763. 30. Zwissler B, Welte M, Messmer K. Effects of inhaled prostacyclin as compared with inhaled nitric oxide on right ventricular performance in hypoxic pulmonary vasoconstriction. J Cardiothorac Vasc Anesth 1995; 9(3):283–289. 31. Hirschfeld M, zur Hausen A, Bettendorf H, Jäger M, Stickeler E. Alternative splicing of Cyr61 is regulated by hypoxia and significantly changed in breast cancer. Cancer Res 2009;69(5):2082–2090. doi:10.1158 /0008-5472.CAN-08-1997. 32. Grzeszkiewicz TM, Kirschling DJ, Chen N, Lau LF. CYR61 stimulates human skin fibroblast migration through integrin αvβ5 and enhances mitogenesis through integrin αvβ3, independent of its carboxyl-terminal domain. J Biol Chem 2001;276(24):21943–21950.
CCN1 suppresses pulmonary vascular smooth muscle contraction in response to hypoxia Seon-jin Lee,1,2 Meng Zhang,1 Kebin Hu,3 Ling Lin,3 Duo Zhang,1 Yang Jin1 1
Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; 3Division of Nephrology, Department of Medicine, Pennsylvania State University Medical Center, Hershey, Pennsylvania 17033, USA
Abstract: Pulmonary vasoconstriction and increased vascular resistance are common features in pulmonary hypertension (PH). One of the contributing factors in the development of pulmonary vasoconstriction is increased pulmonary artery smooth muscle cell (PASMC) contraction. Here we report that CCN1, an extracellular matrix molecule, suppressed PASMC contraction in response to hypoxia. CCN1 (Cyr61), discovered in past decade, belongs to the Cyr61-CTGF-Nov (CCN) family. It carries a variety of cellular functions, including angiogenesis and cell adhesion, death, and proliferation. Hypoxia robustly upregulated the expression of CCN1 in the pulmonary vessels and lung parenchyma. Given that CCN1 is a secreted protein and functions in a paracine manner, we examined the potential effects of CCN1 on the adjacent smooth muscle cells. Interestingly, bioactive recombinant CCN1 significantly suppressed hypoxia-induced contraction in human PASMCs in vitro. Consistently, in the in vivo functional studies, administration of bioactive CCN1 protein significantly decreased right ventricular pressure in three different PH animal models. Mechanistically, protein kinase A–pathway inhibitors abolished the effects of CCN1 in suppressing PASMC contraction. Furthermore, CCN1-inhibited smooth muscle contraction was independent of the known vasodilators, such as nitric oxide. Taken together, our studies indicated a novel cellular function of CCN1, potentially regulating the pathogenesis of PH. Keywords: CCN1, pulmonary hypertension, hypoxia, pulmonary artery smooth muscle cell. Pulm Circ 2015;5(4):716-722. DOI: 10.1086/683812.
Vasodilators are common agents utilized to decrease pulmonary vascular resistance and to relieve pulmonary hypertension (PH),1-3 including relatively new agents such as inhaled nitric oxide (NO), endothelin 1 (ET-1), and prostaglandins.1-3 “Hypoxic pulmonary vasoconstriction” (HPV) refers to a physiological status in which pulmonary arteries constrict in the presence of hypoxia. This process redirects blood flow to the alveoli with the higher oxygen content and consequently improves the ventilation/perfusion ratio and arterial oxygenation.4-7 HPV can help to maintain the normal pulmonary vascular tone,4-7 and this phenomenon has been studied extensively. However, in the presence of severe pulmonary disease, prolonged HPV increases pulmonary vascular resistance and consequently results in irreversible pulmonary vascular remodeling.4-7 The etiology of HPV is multifactorial, but detailed understanding of the exact mechanisms is lacking. Pulmonary artery smooth muscle cells (PASMCs) are clearly one of the main components involved in HPV. Endotheliumreleased mediators, such as endothelins, prostaglandins, serotonin, leukotrienes, histamine, angiotensin II, and NO, have been shown to play crucial roles in regulating smooth muscle contractility and proliferation.8-10 After prolonged hypoxia, endothelial cells (ECs) release these
mediators into the circulation and cause sustained HPV and subsequent vascular remodeling. CCN1 (formally named Cyr61) is a recently discovered earlystress-response gene product with diverse cellular functions.11-15 It belongs to the Cyr61-CTGF-Nov (CCN) family and is a secreted protein that functions in a paracrine and/or autocrine manner.16-20 CCN1’s functions include but are not limited to cytoprotection, angiogenesis, cell adhesion, and migration.11-15 The role of CCN1 in pulmonary diseases, especially pulmonary vascular diseases and PH, has not yet been explored. Here we report that CCN1 potentially is crucial in regulating smooth muscle contractility in pulmonary vasculature. In addition to the well-known NO, CCN1 potentially functions as a mediator in regulating right ventricular pressure (RVP) in hypoxia-induced PH. Our studies may suggest a novel target for PH research. ME T H O D S
Chemicals and reagents Recombinant CCN1 was obtained from R&D systems (Minneapolis, MN). Wortmannin, SB203580, U0126, Y-27632, and γ-secretase
Address correspondence to Dr. Yang Jin, Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA. E-mail: [email protected]. Submitted January 10, 2015; Accepted June 25, 2015; Electronically published October 7, 2015. © 2015 by the Pulmonary Vascular Research Institute. All rights reserved. 2045-8932/2015/0504-0013. $15.00.
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inhibitor IX were purchased from Calbiochem (Gibbstown, NJ). Purecol was obtained from Advanced Biomatrix (San Diego, CA). All cell culture reagents were obtained from Invitrogen (Carlsbad, CA) unless otherwise specified. We purchased the antibody to Cyr61 and endothelial NO synthase from Santa Cruz Biotechnology (Dallas, TX); antibodies to phospho–myosin light chain (MLC) were from Cell Signaling (Danvers, MA); antibodies to α-smooth muscle actin (SMA) and β-actin were from Sigma-Aldrich (St. Louis, MO); and enhanced chemiluminescence reagent was purchased from Thermo Scientific (Grand Island, NY). All short interfering RNA reagents were purchased from Gene Tools (Philomath, OR). All other reagents were obtained from Sigma-Aldrich unless otherwise specified.
Animals and in vivo exposures All animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care. The Animal Research Committee of Brigham and Women’s Hospital approved all protocols. Wild-type C57BL/6 mice were placed in a Plexiglas chamber maintained at 10% O2 (hypoxia group) or in a chamber open to room air (normoxic group), with a 12L ∶ 12D light cycle. The mice were exposed to hypoxia or normoxia for 3 weeks, and then their lungs were harvested and analyzed for Cyr61 expression by Western immunoblot analysis. For functional studies, age-matched littermates were exposed to 10% O2 (hypoxia group) or room air (normoxic group) in the presence of recombinant CCN1 protein (1 μg/kg), su5416 (mouse: 2 mg/kg twice per week; rat: 5 mg/kg every week), or vehicle for 3 weeks before functional measurements (15 mice in each group for each time point).
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Western immunoblotting and immunohistochemistry Western immunoblot analyses were performed as previously described.16 For Western blotting, protein concentrations of cell lysates and frozen tissue homogenates were determined with a Coomassie plus protein assay (Thermo Fisher Scientific, Pittsburgh, PA) and were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using NuPage Novex Bis-Tris 4%–12% polyacrylamide gels (Invitrogen). Lungs were inflated, harvested, fixed in 2% paraformaldehyde, and embedded in paraffin. Sections were stained with antimouse α-SMA and rabbit anti-CCN1 antibody (1 ∶ 100; Santa Cruz) for 3 hours at 4°C. The cells were washed and incubated with antirat IgG (immunoglobulin G)–fluorescein isothiocyanate and antirabbit IgG–tetramethylrhodamine isothiocyanate for 1 hour. After staining, the cells were mounted with mounting medium and observed by laser scanning confocal microscopy.
Right ventricular systolic pressure and heart weight measurements Measurements were obtained with a PC-driven PowerLab (ADInstruments, Colorado Springs, CO). The system was calibrated before each experiment. Each mouse was weighed and injected with sodium pentobarbital (60 mg/kg) to induce anesthesia with maintenance of spontaneous respiration. The abdomen was opened and the left and right ventricles visualized through an intact diaphragm. A 23-G needle was then inserted into the right ventricle, and RVP was recorded. The diaphragm was opened, and then a repeat measurement of RVP, as well as left ventricular pressure, was obtained. The right ventricular (RV) free wall was dissected and weighed separately from the left ventricle and septum (LV + S), which were weighed together.
Monocrotaline (MCT) We assigned adult male Sprague-Dawley rats (200–250 g in body weight; Charles River Laboratories, Wilmington, MA) to groups receiving either subcutaneous injection of 60 mg/kg body weight of MCT or saline alone. We performed hemodyamic measurements and gene expression analysis 3 weeks after injection (10 rats per group for each time point).
Vascular cell culture Human PASMCs (HPASMCs; Lonza, Walkersville, MD) at passages 7–10 were grown to ∼80% confluence in smooth muscle cell growth medium 2 (SmGM2) supplemented with SmGM2 SingleQuots and 5% fetal bovine serum (Lonza). Cells were cultured in humidified incubators containing 95% air and 5% CO2 at 37°C. Cells were grown in 100-mm dishes, detached with 0.05% trypsin, resuspended in complete growth medium, and seeded into 35-mm, 6-well, and 12-well plates for individual experiments. For hypoxic exposures, HPASMCs were placed in an airtight modular incubator chamber (Billups-Rothenberg, San Diego, CA), flushed continuously (10 minutes) with a premixed gas (1% O2, 5% CO2, 94% N2), and then incubated for the indicated intervals. Corresponding normoxic controls were maintained for equivalent times in humidified incubators in an atmosphere of 95% air and 5% CO2.
Collagen matrix contraction assay To assess contractility of HPASMCs on a collagen gel matrix, 2 × 105 cells were plated on type I collagen gel matrices in 24-well dishes in triplicate and exposed to hypoxia or normoxia for 24 hours. Collagen matrices were prepared with Vitrogen 100 collagen (Celltrix, Santa Clara, CA), 10× Dulbecco’s modified Eagle’s medium, and HEPES (pH 8.5). After hypoxic exposure, the matrix was released to initiate contraction, and the cells were then incubated in normoxia at 37°C. Gel size was defined as the sum of the two longest gel diameters, and gel contraction was expressed as a percentage of the original gel size. In certain experiments, cells were treated with CCN1 (100 ng/mL), protein kinase inhibitor (PKI; 1 μM), wortmannin (100 nM), SP600126 (20 μM), cholera toxin (5 μg/mL), U0126 (10 μM), Arg-Gly-Asp (RGD; 10 μM), γ-secretase inhibitor (5 μM), SB203586 (10 μM), Z-Leu-Leu-Leu-al (10 μM), Y-27632 (20 μg/mL), or vehicle (the same volume of dimethyl sulfoxide) during hypoxic exposure.
Statistics Data are presented as mean ± SD. Paired analysis was performed with the Student t test as appropriate. Parametric analyses were carried out by 1-way ANOVA with Tukey’s method. Statistical significance was accepted at P < 0.05.
Figure 1. A, Expression of CCN1 in pulmonary vasculature, in the presence and absence of hypoxia. Rats were exposed to normoxia or hypoxia (10% oxygen). After 3 weeks, pulmonary vascular sections were stained with CCN1 using immunohistochemistry (IHC). Red arrow indicates CCN1. EC: endothelial cell; L: lumen; SMC: smooth muscle cell. B, Stain of α-smooth muscle actin (α-SMA) and CCN1 in mouse lung distal vessels. Mice were exposed to room air or hypoxia (10% oxygen). After 3 weeks, lung sections were fixed and stained with anti-α-SMA or anti-CCN1. Confocal microscopy was used to determine colocalization. Green: α-SMA; red: CCN1; yellow: merge. C, Stain of PECAM (platelet endothelial cell adhesion molecule) and CCN1 in mouse lung distal vessels. Mice were exposed to room air or hypoxia (10% oxygen). After 3 weeks, lung sections were fixed and stained with anti-PECAM or anti-CCN1. Confocal microscopy was used to determine colocalization (arrowheads). Green: PECAM; red: CCN1; yellow: merge. Each figure represents 2 independent experiments. *, # P < 0.05. D–G, Expression of CCN1 in pulmonary vascular cells and lung tissue, in the presence and absence of hypoxia, analyzed with Western blot analysis. D, Expression of CCN1 in human pulmonary endothelial cells (HPAECs). E, Expression of CCN1 in human pulmonary artery smooth muscle cells (HPASMCs). F, Expression of CCN1 in mouse lung tissue. G, Expression of CCN1 in rat lung tissue. Each panel represents 2 independent experiments. *P < 0.05.
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RESU LTS
Hypoxia upregulated CCN1 expression in pulmonary vasculature We evaluated the expression of CCN1 in pulmonary vasculature in the presence and absence of hypoxia, a common trigger for PH. CCN1 was upregulated robustly in ECs and smooth muscle cells (SMCs; Fig. 1A). Given that CCN1 is a secreted protein and functions in a paracrine/autocrine manner, SMCs in the medium layer of pulmonary vasculature certainly are one of the potential targets of lung-derived CCN1. We next stained the CCN1, along with the SMC marker protein α-SMA and the EC marker PECAM (platelet endothelial cell adhesion molecule). As shown in Figures 1B and 1C, hypoxia indeed induced markedly increased CCN1 in pulmonary vasculature. We confirmed this result with Western blotting analysis. CCN1 was significantly induced both in human pulmonary artery ECs (Fig. 1D) and in HPASMCs (Fig. 1E) after hypoxia. In addition, consistent results were obtained in mouse (Fig. 1F) and rat (Fig. 1G) lung tissue homogenates.
CCN1 suppressed hypoxia-induced smooth muscle contraction Initially, we found that bioactive CCN1 recombinant protein robustly suppressed hypoxia-induced smooth muscle contraction in vitro (Fig. 2A). CCN1 (100 ng/mL) was added into cell culture media as described in “Methods.” After 12 hours of hypoxia (5% oxygen), CCN1 significantly blocked HPASMC contraction. Pretreatment of the cells with protein kinase A (PKA) pathway inhibitor PKI markedly reversed the effects of CCN1 on HPASMC relaxation (Fig. 2A). Furthermore, pretreatment of the cells with wortmannin (the specific inhibitor of phosphoinositide 3-kinase [PI3K]), SP600126 (Jun N-terminal kinase [JNK] inhibitor), and RGD peptide (Arg-Gly-Asp, an integrin-binding protein) partially reversed the effects of CCN1 (Fig. 2A). The percentage of the original gel size summarized from 3 independent assays is shown in Figure 2B. In addition, we tested the effects of a variety of inhibitors for multiple pathways, including cholera toxin (G-protein signal pathway), Z-LeuLeu-Leu-al (NF-κB pathway), NMA (NO inhibitor), PD98059 (MEK1 pathway), γ-secretase (notch-mediated pathway), U0126 (MEK1/2 inhibitor), SB203580 (p38 pathway), and Y-27632 (Rho-associated
Figure 2. CCN1 prevented hypoxia-induced human pulmonary artery smooth muscle cell (HPASMC) contraction via multiple pathways. First, 2 × 105 HPASMC cells were plated on type I collagen gel matrices in 24-well dishes in triplicate and exposed to hypoxia or normoxia for 24 hours. After hypoxic exposure, the matrix was released to initiate contraction, and the cells were then incubated in normoxia at 37°C. Gel size is defined as the sum of the two longest
gel diameters, and gel contraction is expressed as a percentage of the original gel size. In certain experiments, cells were treated with CCN1 (100 ng/mL), protein kinase inhibitor (PKI; 1 μM), wortmannin (Wort; 100 nM), SP600126 (SP; 20 μM), Arg-Gly-Asp (RGD; 10 μM), or vehicle (CTL; the same amount of dimethyl sulfoxide) during hypoxic exposure. These inhibitors were used to block certain signaling pathways. A, Left (dashed frame): cells exposed to room air (normoxia); right: cells exposed to hypoxia. PBS: phosphate-buffered saline. B, Percentage of original gel size. The data shown in Figure 1A are presented here as a bar graph. C, Evaluation of multiple signaling pathways on hypoxia-induced HPASMC contraction in the presence of CCN1; see “CCN1 suppressed the hypoxia-induced smooth muscle contraction” for details. mTOR: mammalian target of rapamycin; Tx: toxin. D, Percentage of original gel size. Each figure represents 3 independent * experiments. P < 0.05.
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coil kinase [ROCK] inhibitor). None of these inhibitors showed significant effects of blocking CCN1-associated HPASMC relaxation (Fig. 2C, 2D).
CCN1 decreased RVP in animal models of PH We used 4 animal models of PH to examine the effects of CCN1 on RVP: (1) hypoxia-induced PH model in mice, (2) hypoxia-induced PH model in rats, (3) MCT-induced PH model in rats, and (4) sugen 5416 (vascular endothelial growth factor receptor inhibitor su5416) followed by hypoxia in mice. As shown in Figure 3, all animals were exposed to room air, hypoxia (10% oxygen), or MCT for 3 weeks as described in “Methods.” In the su5416-hypoxia model, we treated mice with su5416, along with hypoxia, as described above. After 3 weeks, we administrated recombinant CCN1 protein (1 μg/kg) 12 hours before measurement. We found that CCN1 significantly decreased RVP in all 4 of these models (Fig. 3). As expected, the ratio of right ventricular weight to body weight (RVW/BW, mg/g) and the ratio of right ventricular weight to left ventricular weight plus septa (Fulton index; RV/(LV + S)) were not affected (Figs. S1– S4, available online).
DISCUSSION Although the pathogenesis remains not completely clear, vasoconstriction and subsequent pulmonary vascular remodeling have been noted in all types of PH regardless of its causes.17-21 Medium/ smooth muscle contractions are crucial in the vasoconstriction involved in PH.17-21 In recent years, accumulating evidence has suggested an essential role of extracellular matrix (ECM) proteins in the development of pulmonary vascular remodeling and vasoconstriction.17-21 However, it is still not completely understood which specific ECM proteins are playing the crucial roles, despite the discovery of NO, endothelin, and prostacyclin (PGI). Our studies have revealed that CCN1 potentially is a novel and effective inhibitor of hypoxia-induced PASMC contraction. CCN1 has many unique features that initiated our interest. First, CCN1 is a secreted protein that functions in a paracrine/autocrine manner.11-15 Thus, lung tissue/cell-derived CCN1 may exert essential regulation on the adjacent SMCs. This secreted molecule can potentially function as a communicator or messenger among epithelial cells, ECs, SMCs, fibroblasts, and even inflammatory cells. Second, CCN1 binds a variety of integrins and exerts differential biological functions based on the binding integrins, including β1-containing integrin(s). The β1-containing integrins are essential in regulating smooth muscle contractility.22 Our data, from the collagen contraction assays, showed that CCN1 prevented hypoxia-induced HPASMC contraction (Fig. 1). The mechanisms by which CCN1 mediated the HPASMC relaxation could be mutifactorial. Nevertheless, we showed that integrin, PI3K, JNK, and PKA are all involved in the regulation of HPASMC relaxation caused by CCN1 (Fig. 1). All these pathways potentially manipulate MLC phosphorylation and subsequently control SMC contraction and relaxation. Previous reports have demonstrated
Figure 3. Effects of CCN1 on right ventricular pressure (RVP), from animal models of pulmonary hypertension (PH). A, C57BL/6 mice were exposed to hypoxia (10%). After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via intraperitoneal (ip) injection. RVP was determined with right heart catheterization (n = 10, *P < 0.05). B, Sprague-Dawley rats were exposed to hypoxia (10%). After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via ip injection (n = 6, *P < 0.05). C, C57BL/6 mice were exposed to hypoxia (10%) and su5416 (2 mg/kg, twice per week) or vehicle (phosphate-buffered saline [PBS]). After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via ip injection. RVP was determined with right heart catheterization (n = 15, *P < 0.05). D, Sprague-Dawley rats were given either subcutaneous injection of monocrotaline (MCT; 60 mg/ kg) or saline alone, in a single dose. After 3 weeks, 12 hours before measurement, CCN1 recombinant protein (1 μg/kg) was administrated via ip injection. RVP was determined with right heart catheterization (n = 6, *P < 0.05).
that activation of MLC via phosphorylation by MLC kinase results in smooth muscle contraction. On the other side, MLC phosphatase dephosphorylates MLC and leads to relaxation.23-26 Two phases of contraction occur in smooth muscle. The initial phase is regulated
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by inositol 1,4,5-trisphosphate (Ins (1,4,5)P3)-dependent Ca2+release and Ca2+/calmodulin-dependent activation of MLC kinase.23-26 The late-phase contraction of vascular smooth muscle is mediated by phosphorylation of Ser19 on the regulatory myosin light chain MLC20.26-28 This contraction is RhoA dependent and occurs via inhibition of MLC phosphatase. Membrane-anchored RhoA activates Rho kinase and phospholipase D (PLD).26-28 Rho kinase and PLD both inhibit MLC phosphatase, either directly or indirectly via the PKC pathway.26-28 Interestingly, PKA induces relaxation via inactivation of the membrane-bound RhoA. Inactivation of membrane-bound RhoA fails to act on its membrane-bound substrates, Rho kinase and PLD.26-28 Since Rho kinase and PLD inhibit MLC phosphatase and result in sustained phosphorylation of MLC20 and muscle contraction, PKA favors muscle relaxation via inducing inactivated membranebound RhoA and, subsequently, inactive Rho kinase and PLD. Our data shown in Figure 2 indicate that the initial and late phases of SMC contraction were modulated by CCN1, via PI3K- and PKAmediated pathways, respectively. However, the detailed mechanisms require further investigation and will be included in our future studies. Endogenous vasodilators derived from pulmonary vascular endothelium, particularly NO and PGI, play important effects in maintaining low pressure and low resistance in pulmonary vasculature.17-21,29,30 This low-pressured and compliant pulmonary vasculature is essential to maintaining RV function and provides a large surface area for gas exchange.17-21 Thus, current PH therapy involves in treating vasoconstriction with NO and PGI analogs. Interestingly, NO and PGI both increase cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophasphate (cAMP). These secondary messengers, cGMPs and cAMPs, induce the relaxation of arterial smooth muscle via regulation of the intracellular Ca2+ concentrations, particularly the smooth muscle Ca2+ sensitivity of the contractile elements.29,30 In vivo, we showed that hypoxia upregulated the expression of CCN1, particularly in intima/ECs. Presumably, this EC-derived CCN1 will act on SMCs to prevent hypoxia-induced contraction. However, one question that arises is, why can administration of exogenous CCN1 still decrease RVP dramatically in animal models of PH, despite the robustly upregulated CCN1 in ECs? Multiple potential explanations should be explored in future studies. Hypoxia may have significantly downregulated CCN1 receptors, particularly the β1-containing integrin(s) in SMCs/medium. As discussed above, β1-containing integrin(s) play crucial roles in regulating smooth muscle contractility.20 Therefore, matrix CCN1, even if it reaches the medium layer of artery, may not function efficiently because of a lack of functional receptors. Near-future directions should include exploring the expression of β1-containing integrin(s) in SMCs/ medium. Next and potentially more important, CCN1 alternative splicing has been widely reported in the latest literature. One of the alternatively spliced CCN1 forms results in truncation of two important functional domains.31,32 Therefore, we will vigorously explore the alternatively spliced CCN1 protein in the setting of hypoxiaassociated pulmonary vascular remodeling. CCN1 appears to function as an independent vasodilator to prevent hypoxia-induced smooth muscle contraction, via a distinct path-
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way other than NO-, PGI-, or ET-1-meditated signals. It is a conservative gene product, with 93% homology between humans and rodents. Therefore, our studies have potentially uncovered a novel, yet important vasodilator for treatment of PH. Whether this molecule has synergistic effects with existing vasodilators remains unknown. Its effects on systemic vasculature also require further investigation. In addition, the mechanisms by which CCN1 decreases RVP in MCT-induced PH models may differ from those in hypoxiainduced PH models. In summary, our studies indicate a novel function of CCN1 in regulating SMC contractility in the presence of hypoxia and show that CCN1 decreased RVP in animal models of PH. This work potentially provides novel insights and targets for PH research.
ACKNOW LEDGMENTS We thank Drs. Augustine M. K. Choi, Lester F. Lau, and Mark A. Perrella for their scientific advice and support. Source of Support: Nil. Conflict of Interest: None declared. REFER E NCES 1. Palevsky HI, Schloo BL, Pietra GG, Weber KT, Janicki JS, Rubin E, Fishman AP. Primary pulmonary hypertension: vascular structure, morphometry, and responsiveness to vasodilator agents. Circulation 1989;80 (5):1207–1221. 2. Tsai BM, Wang M, Turrentine MW, Mahomed Y, Brown JW, Meldrum DR. Hypoxic pulmonary vasoconstriction in cardiothoracic surgery: basic mechanisms to potential therapies. Ann Thorac Surg 2004; 78(1):360–368. 3. Lowson S. Inhaled alternatives to nitric oxide. Anesthesiology 2002;96 (6):1504–1513. 4. Blaise G, Langleben D, Hubert B. Pulmonary arterial hypertension: pathophysiology and anesthetic approach. Anesthesiology 2003;99(6): 1415–1432. 5. Abe K, Toba M, Alzoubi A, Ito M, Fagan KA, Cool CD, Voelkel NF, McMurtry IF, Oka M. Formation of plexiform lesions in experimental severe pulmonary arterial hypertension. Circulation 2010;121(25):2747– 2754. 6. Stenmark KR, Meyrick B, Galiè N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol 2009;297(6):L1013–L1032. 7. Ciuclan L, Bonneau O, Hussey M, Duggan N, Holmes AM, Good R, Stringer R, et al. A novel murine model of severe pulmonary arterial hypertension. Am J Respir Crit Care Med 2011;184(10):1171–1182. 8. Pappert D, Busch T, Gerlach H, Lewandowski K, Radermacher P, Rossaint R. Aerosolized prostacyclin versus inhaled nitric oxide in children with severe acute respiratory distress syndrome. Anesthesiology 1995;82(6):1507–1511. 9. Langer F, Wilhelm W, Tscholl D, Schramm R, Lausberg H, Wendler O, Schäfers H-J. Intraoperative inhalation of the long-acting prostacyclin analog iloprost for pulmonary hypertension. J Thorac Cardiovasc Surg 2003;126(3):874–875. 10. Vincent JL, Carlier E, Pinsky MR, Goldstein J, Naeije R, Lejeune P, Brimioulle S, Leclerc JL, Kahn RJ, Primo G. Prostaglandin E1 infusion for right ventricular failure after cardiac transplantation. J Thorac Cardiovasc Surg 1992;103(1):33–39.
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