Simultaneous Positron Emission Tomography and Molecular Magnetic Resonance Imaging of Cardiopulmonary Fibrosis in a Mouse Model of Left Ventricular Dysfunction.

BACKGROUND: Aging-associated left ventricular dysfunction promotes cardiopulmonary fibrogenic remodeling, Group 2 pulmonary hypertension (PH), and right ventricular failure. At the time of diagnosis, cardiac function has declined, and cardiopulmonary fibrosis has often developed. Here, we sought to develop a molecular positron emission tomography (PET)-magnetic resonance imaging (MRI) protocol to detect both cardiopulmonary fibrosis and fibrotic disease activity in a left ventricular dysfunction model. METHODS AND RESULTS: Left ventricular dysfunction was induced by transverse aortic constriction (TAC) in 6-month-old senescence-accelerated prone mice, a subset of mice that received sham surgery. Three weeks after surgery, mice underwent simultaneous PET-MRI at 4.7 T. Collagen-targeted PET and fibrogenesis magnetic resonance (MR) probes were intravenously administered. PET signal was computed as myocardium- or lung-to-muscle ratio. Percent signal intensity increase and Δ lung-to-muscle ratio were computed from the pre-/postinjection magnetic resonance images. Elevated allysine in the heart (P=0.02) and lungs (P=0.17) of TAC mice corresponded to an increase in myocardial magnetic resonance imaging percent signal intensity increase (P<0.0001) and Δlung-to-muscle ratio (P<0.0001). Hydroxyproline in the heart (P<0.0001) and lungs (P<0.01) were elevated in TAC mice, which corresponded to an increase in heart (myocardium-to-muscle ratio, P=0.02) and lung (lung-to-muscle ratio, P<0.001) PET measurements. Pressure-volume loop and echocardiography demonstrated adverse left ventricular remodeling, function, and increased right ventricular systolic pressure in TAC mice. CONCLUSIONS: Administration of collagen-targeted PET and allysine-targeted MR probes led to elevated PET-magnetic resonance imaging signals in the myocardium and lungs of TAC mice. The study demonstrates the potential to detect fibrosis and fibrogenesis in cardiopulmonary disease through a dual molecular PET-magnetic resonance imaging protocol.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Vascular smooth muscle ROCK1 contributes to hypoxia-induced pulmonary hypertension development in mice.

Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH.

Increased Transglutaminase 2 Expression and Activity in Rodent Models of Obesity/Metabolic Syndrome and Aging.

Diastolic dysfunction of the heart and decreased compliance of the vasculature and lungs (i.e., increased organ tissue stiffness) are known features of obesity and the metabolic syndrome. Similarly, cardiac diastolic dysfunction is associated with aging. Elevation of the enzyme transglutaminase 2 (TG2) leads to protein cross-linking and enhanced collagen synthesis and participates as a candidate pathway for development of tissue stiffness. With these observations in mind we hypothesized that TG2 may be elevated in tissues of a rat model of obesity/metabolic syndrome (the ZSF 1 rat) and a mouse model of aging, i.e., the senescent SAMP8 mouse. In the experiments reported here, TG2 expression and activity were found for the first time to be spontaneously elevated in organs from both the ZSF1 rat and the SAMP8 mouse. These observations are consistent with a hypothesis that a TG2-related pathway may participate in the known tissue stiffness associated with cardiac diastolic dysfunction in these two rodent models. The potential TG2 pathway needs better correlation with physiologic dysfunction and may eventually provide novel therapeutic insights to improve tissue compliance.

Glycolysis regulated transglutaminase 2 activation in cardiopulmonary fibrogenic remodeling.

The pathophysiology of pulmonary hypertension (PH) and heart failure (HF) includes fibrogenic remodeling associated with the loss of pulmonary arterial (PA) and cardiac compliance. We and others have previously identified transglutaminase 2 (TG2) as a participant in adverse fibrogenic remodeling. However, little is known about the biologic mechanisms that regulate TG2 function. We examined physiological mouse models of experimental PH, HF, and type 1 diabetes that are associated with altered glucose metabolism/glycolysis and report here that TG2 expression and activity are elevated in pulmonary and cardiac tissues under all these conditions. We additionally used PA adventitial fibroblasts to test the hypothesis that TG2 is an intermediary between enhanced tissue glycolysis and fibrogenesis. Our in vitro results show that glycolytic enzymes and TG2 are upregulated in fibroblasts exposed to high glucose, which stimulates cellular glycolysis as measured by Seahorse analysis. We examined the relationship of TG2 to a terminal glycolytic enzyme, pyruvate kinase M2 (PKM2), and found that PKM2 regulates glucose-induced TG2 expression and activity as well as fibrogenesis. Our studies further show that TG2 inhibition blocks glucose-induced fibrogenesis and cell proliferation. Our findings support a novel role for glycolysis-mediated TG2 induction and tissue fibrosis associated with experimental PH, HF, and hyperglycemia.

Transglutaminase 2 in pulmonary and cardiac tissue remodeling in experimental pulmonary hypertension.

Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a cross-linking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein cross-linking, extracellular matrix collagen accumulation, and fibroblast-to-myofibroblast transformation. We report here that TG2 expression and activity as measured by serotonylated fibronectin and protein cross-linking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein cross-linking activity, and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced cross-linking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 short interfering RNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.

Plasma 12- and 15-hydroxyeicosanoids are predictors of survival in pulmonary arterial hypertension.

This study aimed to characterize alterations in select eicosanoids in experimental and human pulmonary arterial hypertension (PAH) and to assess their potential utility as predictors of outcome. Using liquid chromatography-mass spectrometry, we performed targeted lipidomic analyses of the lungs and right ventricles (RVs) of chronically hypoxic rats and plasma of consecutive PAH patients and healthy controls. In rat lungs, chronic hypoxia was associated with significantly decreased lung prostacyclin (PGI2)/thromboxane B2 (TXB2) ratio and elevated lung 8-hydroxyeicosanoid (HETE) acid concentrations. RV eicosanoids did not exhibit any changes with chronic hypoxia. PAH treatment-naïve patients had significantly increased plasma concentrations of TXB2 and 5-, 8-, 12-, and 15-HETE. The PGI2/TXB2 ratio was lower in PAH patients than in controls, especially in the treatment-naïve cohort (median: 2.1, 0.3, and 1.3 in controls, treatment-naïve, and treated patients, respectively, P = 0.001). Survival was significantly worse in PAH patients with 12-HETEhigh (≥57 pg/mL) and 15-HETEhigh (≥256 pg/mL) in unadjusted and adjusted analyses (hazard ratio [HR]: 2.8 [95% confidence interval (CI): 1.1-7.3], P = 0.04 and HR: 4.3 [95% CI: 1.6-11.8], P = 0.004, respectively; adjustment was performed with the REVEAL [Registry to Evaluate Early and Long-Term PAH Disease Management] risk score). We demonstrate significant alterations in eicosanoid pathways in experimental and human PAH. We found that 12- and 15-HETE were independent predictors of survival in human PAH, even after adjusting for the REVEAL score, suggesting their potential role as novel biomarkers.

Alpha-Catulin Co-Localizes With Vimentin Intermediate Filaments and Functions in Pulmonary Vascular Endothelial Cell Migration via ROCK.

The ubiquitous α-catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α-catulin has homology to the cytoskeletal linkers α-catenin and vinculin, it appears to be functionally divergent. Here we further investigated α-catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α-catulin has a unique cytoskeletal role. Examination of endogenous α-catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double-immunofluorescence analyses of VEC showed endogenous α-catulin co-localization with vimentin intermediate filaments. Similar to vimentin, α-catulin was found to distribute into detergent-soluble and -insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α-catulin network distribution and altered α-catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α-catulin knock-down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α-catulin depletion was shown to reduce ROCK-induced signaling. These findings indicate that α-catulin has a unique function in co-localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. J. Cell. Physiol. 231: 934-943, 2016. © 2015 Wiley Periodicals, Inc.

Tissue transglutaminase promotes serotonin-induced AKT signaling and mitogenesis in pulmonary vascular smooth muscle cells.

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50 µM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling.

Role of hypoxia-induced transglutaminase 2 in pulmonary artery smooth muscle cell proliferation.

We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [(3)H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH.

Evaluation and management of heart rhythm disturbances due to cardiac sarcoidosis.

Cardiac sarcoidosis (CS) affects less than 5% of patients with pulmonary or systemic sarcoidosis, but when present is often associated with a spectrum of clinically significant conduction abnormalities and arrhythmias. The cardinal manifestations of CS include conduction disturbances, arrhythmias, or congestive heart failure. Less commonly, there is concealed subclinical disease. The electrophysiologic evaluation for CS includes a history and physical exam, ECG, and echocardiogram for all sarcoidosis patients, along with MRI, PET/nuclear scans, and EPS for certain subsets of patients. Despite variable data to support their efficacy, glucocorticoids should still be considered in the treatment plan of CS. Antiarrhythmics in isolation are often ineffective in controlling ventricular arrhythmias. Cardiac pacemakers have provided important therapy for patients with conduction defects and implantable cardioverter defibrillator (ICD) therapy provides the strongest insurance to prevent fatal arrhythmias from CS. A recent consensus statement provides guidance for clinicians on the diagnosis and management of arrhythmias associated with CS including indications for ICDs. The use of pacemakers, ICD implantation and early implementation of corticosteroid therapy have led to an improvement in the overall prognosis and clinical outcomes of CS.

Differences between basal lung levels of select eicosanoids in rat and mouse.

Metabolites of arachidonic acid play an important role in mediating inflammation, cell proliferation, and oxidative stress that contribute to many pulmonary diseases. We hypothesized that the substantial differences between rats and mice in their responses to experimental pulmonary hypertensive stimuli would be associated with parallel differences in their basal eicosanoid profile. Rat and mouse lung extracts were subjected to liquid chromatography tandem mass spectrometry that was optimized for simultaneous separation and rapid quantification of the major hydroxyeicosatetraenoic acids (HETEs) and prostaglandins (PGs). Basal levels (pg/µg protein) of arachidonic acid metabolites differed significantly between rat and mouse lungs. Median values of the following major eicosanoids were significantly higher in mouse than in rat lungs: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGE2, and PGI2, as well as isoprostane-E2 and -F2α. In addition, the PGI2/TXB2 ratio was increased in mouse relative to rat lungs. On the basis of the important roles that these compounds play in determining pulmonary vascular tone, the differences in select eicosanoid profiles, especially the PGI2/TXB2 ratio, between rat and mouse lungs may underlie the interspecies differences in susceptibility to the development of pulmonary hypertension.

Mineralocorticoid receptor antagonism attenuates experimental pulmonary hypertension.

Mineralocorticoid receptor (MR) activation stimulates systemic vascular and left ventricular remodeling. We hypothesized that MR contributes to pulmonary vascular and right ventricular (RV) remodeling of pulmonary hypertension (PH). We evaluated the efficacy of MR antagonism by spironolactone in two experimental PH models; mouse chronic hypoxia-induced PH (prevention model) and rat monocrotaline-induced PH (prevention and treatment models). Last, the biological function of the MR was analyzed in cultured distal pulmonary artery smooth muscle cells (PASMCs). In hypoxic PH mice, spironolactone attenuated the increase in RV systolic pressure, pulmonary arterial muscularization, and RV fibrosis. In rat monocrotaline-induced PH (prevention arm), spironolactone attenuated pulmonary vascular resistance and pulmonary vascular remodeling. In the established disease (treatment arm), spironolactone decreased RV systolic pressure and pulmonary vascular resistance with no significant effect on histological measures of pulmonary vascular remodeling, or RV fibrosis. Spironolactone decreased RV cardiomyocyte size modestly with no significant effect on RV mass, systemic blood pressure, cardiac output, or body weight, suggesting a predominantly local pulmonary vascular effect. In distal PASMCs, MR was expressed and localized diffusely. Treatment with the MR agonist aldosterone, hypoxia, or platelet-derived growth factor promoted MR translocation to the nucleus, activated MR transcriptional function, and stimulated PASMC proliferation, while spironolactone blocked these effects. In summary, MR is active in distal PASMCs, and its antagonism prevents PASMC proliferation and attenuates experimental PH. These data suggest that MR is involved in the pathogenesis of PH via effects on PASMCs and that MR antagonism may represent a novel therapeutic target for this disease.

Serotonylated fibronectin is elevated in pulmonary hypertension.

Serotonin (5-HT) and fibronectin (FN) have been associated with pulmonary hypertension (PH). We previously reported that FN is posttranslationally modified by tissue transglutaminase (TGase) to form serotonylated FN (s-FN) in pulmonary artery smooth muscle cells and that serotonylation stimulates their proliferation and migration, hallmarks of PH. We hypothesized that s-FN and its binding to TGase are elevated in human and experimental PH. To assess this hypothesis, FN isolation and electrophoretic, immunoblotting, and densitometric techniques were used. Mean ratio of serum s-FN to total FN level (s-FN/FN) was elevated in 19 consecutive pulmonary arterial hypertension (PAH) patients compared with 25 controls (0.3 ± 0.18 vs. 0.05 ± 0.07, P < 0.001). s-FN/FN also was increased in lungs of mice and rats with hypoxia-induced PH and in rats with monocrotaline-induced PH. In mice, the increase was detected at 1 wk of hypoxia, preceding the development of PH. Hypoxic rats had elevated serum s-FN/FN. Enhanced binding of TGase to its substrate FN occurred in serum from patients with PAH (mean 0.50 ± 0.51 vs. 0.063 ± 0.11, P = 0.002) and s-FN/FN and TGase-bound FN were highly correlated (R(2) = 0.77). TGase-bound FN also was increased in experimental PH. We conclude that increased serotonylation of FN occurs in human and experimental PH and may provide a biomarker for the disease.

Serotonin transporter interacts with the PDGFß receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle cells.

The serotonin transporter (SERT) and the platelet-derived growth factor receptor (PDGFR) have been implicated in both clinical and experimental pulmonary hypertension (PH) and the facilitation of pulmonary artery smooth muscle cell (PASMC) growth. To gain a better understanding of the possible relationship of these two cell surface molecules we have explored interactions between SERT and PDGFR. We have previously demonstrated that SERT transactivates PDGFRß in serotonin-stimulated PASMC proliferation. We now provide evidence for a role for SERT in PDGF-BB signaling and PASMC proliferation by using pharmacological inhibitors, genetic ablation, and construct overexpression of SERT. The results show that four tested SERT blockers dose dependently inhibit PDGF-stimulated human and bovine PASMC proliferation with comparable efficacy to that of PDGFR inhibitors, whereas 5-HT1B or 5-HT2A receptor inhibitors had no effect. Combinations of the SERT and PDGFR inhibitors led to synergistic/additive inhibition. Similarly, PDGF-induced PASMC proliferation was attenuated by small interfering RNA downregulation of SERT. Inhibition of SERT in PASMCs attenuated PDGF-induced phosphorylation of PDGFRß, Akt, and p38 but not Erk. Overexpression of SERT in HEK293 cells led to enhanced Akt phosphorylation by PDGF, which was blunted by a SERT PDZ motif mutant, indicating the mechanistic need for the PDZ motif of SERT in PDGF signaling. Furthermore, coimmunoprecipitation experiments showed that SERT and PDGFRß become physically associated upon PDGF stimulation. In total, the data show for the first time an important interactive relationship between SERT and the PDGFRß in the production of PASMC proliferation triggered by PDGF that may be important in PH.

Role of protein transamidation in serotonin-induced proliferation and migration of pulmonary artery smooth muscle cells.

Pulmonary hypertension is characterized by elevated pulmonary artery pressure and pulmonary artery smooth muscle cell (SMC) proliferation and migration. Clinical and experimental evidence suggests that serotonin (5-HT) is important in these responses. We previously demonstrated the participation of the 5-HT transporter and intracellular 5-HT (5-HTi) in the pulmonary vascular SMC-proliferative response to 5-HT. However, the mechanism underlying the intracellular actions of 5-HT is unknown. We speculated that 5-HTi activates SMC growth by post-translational transamidation of proteins via transglutaminase (TGase) activity, a process referred to as serotonylation. To test this hypothesis, serotonylation of pulmonary artery SMC proteins, and their role in 5-HT-induced proliferative and migratory responses, were assessed. 5-HT caused dose- and time-dependent increase in serotonylation of multiple proteins in both bovine and rat pulmonary artery SMCs. Inhibition of TGase with dansylcadaverin blocked this activity, as well as SMC-proliferative and migratory responses to 5-HT. Serotonylation of proteins also was blocked by 5-HT transporter inhibitors, and was enhanced by inhibition of monoamine oxidase, an enzyme known to degrade 5-HTi, indicating that 5-HTi levels regulate serotonylation. Immunoprecipitation assays and HPLC-mass spectral peptide sequencing revealed that a major protein serotonylated by TGase was fibronectin (FN). 5-HT-stimulated SMC serotonylation and proliferation were blocked by FN small interfering (si) RNA. These findings, together with previous observations that FN expression in the lung strongly correlates with the progression of pulmonary hypertension in both experimental animals and humans, suggest an important role of FN serotonylation in the pathogenesis of this disease.

The Lbc Rho guanine nucleotide exchange factor α-catulin axis functions in serotonin-induced vascular smooth muscle cell mitogenesis and RhoA/ROCK activation.

Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knockdown by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent serum response factor-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knockdown reduced 5-HT-induced RhoA/ROCK activation, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA, and the α-catulin scaffold. Furthermore, α-catulin knockdown attenuated 5-HT-induced PASMC thymidine uptake. 5-HT-induced PASMC mitogenesis was reduced by dominant-negative G(q) protein, suggesting cooperation with Lbc/α-catulin. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/α-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.

JNK regulates serotonin-mediated proliferation and migration of pulmonary artery smooth muscle cells.

JNK is a member of the MAPK family and has essential roles in inflammation and cell differentiation and apoptosis. In recent years, there have been accumulating data indicating a novel role for JNK in cell growth and migration. In this report, we demonstrate that JNK activity is necessary for serotonin (5-HT)-induced proliferation and migration of bovine pulmonary artery smooth muscle cells (PASMCs). Stimulation with 5-HT was found to lead to activation of JNK with a maximal activation at 10 min. Inhibition of JNK with its specific inhibitor, SP-600125, or its dominant-negative form, DN-JNK, significantly reduced 5-HT-stimulated [(3)H]thymidine incorporation and cyclin D1 expression. A similar inhibitory effect on SMC migration produced by 5-HT, as detected by a wound healing assay, was observed with inhibition of JNK. Furthermore, inhibition of 5-HT receptors (1B) and (2A), but not inhibition of the 5-HT transporter, blocked 5-HT-induced JNK activation. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 and wortmannin had little or no effect on 5-HT-induced JNK phosphorylation, but JNK inhibitor SP-600125 and DN-JNK blocked 5-HT-stimulated phosphorylation of Akt and its downstream effectors, p70S6K1 and S6, indicating that Akt is a downstream effector of JNK. Activation of Akt by 5-HT was blocked only minimally, if at all, by inhibitors of ERK and p38 MAPK, indicating a uniqueness of JNK MAPK in this activation of Akt. Coimmunoprecipitation showed binding of Akt to JNK, further supporting the interaction of JNK and Akt. Thus JNK is a critical molecule in 5-HT-induced PASMC proliferation and migration and may act at an important point for cross talk of the MAPK and PI3K pathways. Its activation by 5-HT is initiated through 5-HT (1B) and (2A) receptors, and its stimulation of SMC proliferation and migration occurs through the Akt pathway.