Myeloid Cell Derived IL1ß Contributes to Pulmonary Hypertension in HFpEF.

BACKGROUND: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are poorly understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. METHODS: Eight-week-old male and female C57BL/6J mice received either Nγ-nitro-L-arginine methyl ester and high-fat diet or control water and diet for 2, 5, and 12 weeks. The db/db mice were studied as a second model of HFpEF. Early pathways regulating PH were identified by bulk and single-cell RNA sequencing. Findings were confirmed by immunostain in lungs of mice or lung slides from clinically performed autopsies of patients with PH-HFpEF. ELISA was used to verify IL-1ß (interleukin-1 beta) in mouse lung, mouse plasma, and also human plasma from patients with PH-HFpEF obtained at the time of right heart catheterization. Clodronate liposomes and an anti-IL-1ß antibody were utilized to deplete macrophages and IL-1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF in mouse models. RESULTS: Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice developed PH, small vessel muscularization, and right heart dysfunction. Inflammation-related gene ontologies were overrepresented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling showed an increase in IL-1ß in mouse and human plasma. Finally, clodronate liposome treatment in mice prevented PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice, and IL-1ß depletion also attenuated PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice. CONCLUSIONS: We report a novel model for the study of PH and right heart remodeling in HFpEF, and we identify myeloid cell-derived IL-1ß as an important contributor to PH in HFpEF.

Myeloid Cell Derived IL1ß Contributes to Pulmonary Vascular Remodeling in Heart Failure with Preserved Ejection Fraction.

Background: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are not well understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH in HFpEF, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. Methods: Eight week old male and female C57/BL6J mice were given either L-NAME and high fat diet (HFD) or control water/diet for 2,5, and 12 weeks. Bulk RNA sequencing and single cell RNA sequencing was performed to identify early and cell-specific pathways that might regulate pulmonary vascular remodeling in PH-HFpEF. Finally, clodronate liposome and IL1ß antibody treatments were utilized to deplete macrophages or IL1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF. Results: Mice given L-NAME/HFD developed PH, small vessel muscularization, and right heart dysfunction after 2 weeks of treatment. Inflammation-related gene ontologies were over-represented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling of mouse lung and plasma showed an increase in IL1ß, which was confirmed in plasma from patients with HFpEF. Single cell sequencing of mouse lungs also showed an increase in M1-like, pro-inflammatory populations of Ccr2+ monocytes and macrophages, and transcript expression of IL1ß was primarily restricted to myeloid-type cells. Finally, clodronate liposome treatment prevented the development of PH in L-NAME/HFD treated mice, and IL1ß antibody treatment also attenuated PH in L-NAME/HFD treated mice. Conclusions: Our study demonstrated that a well-accepted model of HFpEF recapitulates features of pulmonary vascular remodeling commonly seen in patients with HFpEF, and we identified myeloid cell derived IL1ß as an important contributor to PH in HFpEF.

Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-ß (transforming growth factor-ß) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

KCNK3 Mutation Causes Altered Immune Function in Pulmonary Arterial Hypertension Patients and Mouse Models.

Loss of function KCNK3 mutation is one of the gene variants driving hereditary pulmonary arterial hypertension (PAH). KCNK3 is expressed in several cell and tissue types on both membrane and endoplasmic reticulum and potentially plays a role in multiple pathological process associated with PAH. However, the role of various stressors driving the susceptibility of KCNK3 mutation to PAH is unknown. Hence, we exposed kcnk3fl/fl animals to hypoxia, metabolic diet and low dose lipopolysaccharide (LPS) and performed molecular characterization of their tissue. We also used tissue samples from KCNK3 patients (skin fibroblast derived inducible pluripotent stem cells, blood, lungs, peripheral blood mononuclear cells) and performed microarray, immunohistochemistry (IHC) and mass cytometry time of flight (CyTOF) experiments. Although a hypoxic insult did not alter vascular tone in kcnk3fl/fl mice, RNASeq study of these lungs implied that inflammatory and metabolic factors were altered, and the follow-up diet study demonstrated a dysregulation of bone marrow cells in kcnk3fl/fl mice. Finally, a low dose LPS study clearly showed that inflammation could be a possible second hit driving PAH in kcnk3fl/fl mice. Multiplex, IHC and CyTOF immunophenotyping studies on human samples confirmed the mouse data and strongly indicated that cell mediated, and innate immune responses may drive PAH susceptibility in these patients. In conclusion, loss of function KCNK3 mutation alters various physiological processes from vascular tone to metabolic diet through inflammation. Our data suggests that altered circulating immune cells may drive PAH susceptibility in patients with KCNK3 mutation.

Expression of a Human Caveolin-1 Mutation in Mice Drives Inflammatory and Metabolic Defect-Associated Pulmonary Arterial Hypertension.

Background: In 2012, mutations in Cav1 were found to be the driving mutation in several cases of heritable pulmonary arterial hypertension (PAH). These mutations replaced the last 21 amino acids of Cav1 with a novel 22-amino-acid sequence. Because previously only Cav1 knockouts had been studied in the context of PAH, examining the in vivo effects of this novel mutation holds promise for new understanding of the role of Cav1 in disease etiology. Methods: The new 22 amino acids created by the human mutation were knocked into the native mouse Cav1 locus. The mice underwent hemodynamic, energy balance, and inflammatory measurements, both at baseline and after being stressed with either a metabolic or an inflammatory challenge [low-dose lipopolysaccharide (LPS)]. To metabolically challenge the mice, they were injected with streptozotocin (STZ) and fed a high-fat diet for 12 weeks. Results: Very little mutant protein was found in vivo (roughly 2% of wild-type by mass spectrometry), probably because of degradation after failure to traffic from the endoplasmic reticulum. The homozygous mutants developed a mild, low-penetrance PAH similar to that described previously in knockouts, and neither baseline nor metabolic nor inflammatory stress resulted in pressures above normal in heterozygous animals. The homozygous mutants had increased lean mass and worsened oral glucose tolerance, as previously described in knockouts. Novel findings include the preservation of Cav2 and accessory proteins in the liver and the kidney, while they are lost with homozygous Cav1 mutation in the lungs. We also found that the homozygous mutants had a significantly lower tolerance to voluntary spontaneous exercise than the wild-type mice, with the heterozygous mice at an intermediate level. The mutants also had higher circulating monocytes, with both heterozygous and homozygous animals having higher pulmonary MCP1 and MCP5 proteins. The heterozygous animals also lost weight at an LPS challenge level at which the wild-type mice continued to gain weight. Conclusions: The Cav1 mutation identified in human patients in 2012 is molecularly similar to a knockout of Cav1. It results in not only metabolic deficiencies and mild pulmonary hypertension, as expected, but also an inflammatory phenotype and reduced spontaneous exercise.

The BDNF rs6265 Polymorphism is a Modifier of Cardiomyocyte Contractility and Dilated Cardiomyopathy.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

Antagonism of the Thromboxane-Prostanoid Receptor as a Potential Therapy for Cardiomyopathy of Muscular Dystrophy.

Background Muscular dystrophy (MD) causes a progressive cardiomyopathy characterized by diffuse fibrosis, arrhythmia, heart failure, and early death. Activation of the thromboxane-prostanoid receptor (TPr) increases calcium transients in cardiomyocytes and is proarrhythmic and profibrotic. We hypothesized that TPr activation contributes to the cardiac phenotype of MD, and that TPr antagonism would improve cardiac fibrosis and function in preclinical models of MD. Methods and Results Three different mouse models of MD (mdx/utrn double knockout, second generation mdx/mTR double knockout, and delta-sarcoglycan knockout) were given normal drinking water or water containing 25 mg/kg per day of the TPr antagonist ifetroban, beginning at weaning. After 6 months (10 weeks for mdx/utrn double knockout), mice were evaluated for cardiac and skeletal muscle function before euthanization. There was a 100% survival rate of ifetroban-treated mice to the predetermined end point, compared with 60%, 43%, and 90% for mdx/utrn double knockout, mdx/mTR double knockout, and delta-sarcoglycan knockout mice, respectively. TPr antagonism improved cardiac output in mdx/utrn double knockout and mdx/mTR mice, and normalized fractional shortening, ejection fraction, and other parameters in delta-sarcoglycan knockout mice. Cardiac fibrosis in delta-sarcoglycan knockout was reduced with TPr antagonism, which also normalized cardiac expression of claudin-5 and neuronal nitric oxide synthase proteins and multiple signature genes of Duchenne MD. Conclusions TPr antagonism reduced cardiomyopathy and spontaneous death in mouse models of Duchenne and limb-girdle MD. Based on these studies, ifetroban and other TPr antagonists could be novel therapeutics for treatment of the cardiac phenotype in patients with MD.

A potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a deadly disease with no cure. Alternate conversion of angiotensin II (AngII) to angiotensin-(1-7) (Ang-(1-7)) by angiotensin-converting enzyme 2 (ACE2) resulting in Mas receptor (Mas1) activation improves rodent models of PAH. Effects of recombinant human (rh) ACE2 in human PAH are unknown. Our objective was to determine the effects of rhACE2 in PAH.We defined the molecular effects of Mas1 activation using porcine pulmonary arteries, measured AngII/Ang-(1-7) levels in human PAH and conducted a phase IIa, open-label pilot study of a single infusion of rhACE2 (GSK2586881, 0.2 or 0.4 mg·kg-1 intravenously).Superoxide dismutase 2 (SOD2) and inflammatory gene expression were identified as markers of Mas1 activation. After confirming reduced plasma ACE2 activity in human PAH, five patients were enrolled in the trial. GSK2586881 was well tolerated with significant improvement in cardiac output and pulmonary vascular resistance. GSK2586881 infusion was associated with reduced plasma markers of inflammation within 2-4 h and increased SOD2 plasma protein at 2 weeks.PAH is characterised by reduced ACE2 activity. Augmentation of ACE2 in a pilot study was well tolerated, associated with improved pulmonary haemodynamics and reduced markers of oxidant and inflammatory mediators. Targeting this pathway may be beneficial in human PAH.

rhACE2 Therapy Modifies Bleomycin-Induced Pulmonary Hypertension via Rescue of Vascular Remodeling.

Background: Pulmonary hypertension (PH) is a progressive cardiovascular disease, characterized by endothelial and smooth muscle dysfunction and vascular remodeling, followed by right heart failure. Group III PH develops secondarily to chronic lung disease such as idiopathic pulmonary fibrosis (IPF), and both hastens and predicts mortality despite of all known pharmacological interventions. Thus, there is urgent need for development of newer treatment strategies. Objective: Angiotensin converting enzyme 2 (ACE2), a member of the renin angiotensin family, is therapeutically beneficial in animal models of pulmonary vascular diseases and is currently in human clinical trials for primary PH. Although previous studies suggest that administration of ACE2 prevents PH secondary to bleomycin-induced murine IPF, it is unknown whether ACE2 can reverse or treat existing disease. Therefore, in the present study, we tested the efficacy of ACE2 in arresting the progression of group 3 PH. Methods: To establish pulmonary fibrosis, we administered 0.018 U/g bleomycin 2x/week for 4 weeks in adult FVB/N mice, and sacrificed 5 weeks following the first injection. ACE2 or vehicle was administered via osmotic pump for the final 2 weeks, beginning 3 weeks after bleomycin. Echocardiography and hemodynamic assessment was performed prior to sacrifice and tissue collection. Results: Administration of bleomycin significantly increased lung collagen expression, pulmonary vascular remodeling, and pulmonary arterial pressure, and led to mild right ventricular hypertrophy. Acute treatment with ACE2 significantly attenuated vascular remodeling and increased pulmonary SOD2 expression without measurable effects on pulmonary fibrosis. This was associated with nonsignificant positive effects on pulmonary arterial pressure and cardiac function. Conclusion: Collectively, our findings enumerate that ACE2 treatment improved pulmonary vascular muscularization following bleomycin exposure, concomitant with increased SOD2 expression. Although it may not alter the pulmonary disease course of IPF, ACE2 could be an effective therapeutic strategy for the treatment of group 3 PH.

Isolation and characterization of endothelial-to-mesenchymal transition cells in pulmonary arterial hypertension.

Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose polarity and cell-to cell contacts, and undergo a dramatic remodeling of the cytoskeleton. It has been implicated in initiation and progression of pulmonary arterial hypertension (PAH). However, the characteristics of cells which have undergone EndMT cells in vivo have not been reported and so remain unclear. To study this, sugen5416 and hypoxia (SuHx)-induced PAH was established in Cdh5-Cre/Gt(ROSA)26Sortm4(ACTB-tdTomato,EGFP)Luo/J double transgenic mice, in which GFP was stably expressed in pan-endothelial cells. After 3 wk of SuHx, flow cytometry and immunohistochemistry demonstrated CD144-negative and GFP-positive cells (complete EndMT cells) possessed higher proliferative and migratory activity compared with other mesenchymal cells. While CD144-positive and α-smooth muscle actin (α-SMA)-positive cells (partial EndMT cells) continued to express endothelial progenitor cell markers, complete EndMT cells were Sca-1-rich mesenchymal cells with high proliferative and migratory ability. When transferred in fibronectin-coated chamber slides containing smooth muscle media, α-SMA robustly expressed in these cells compared with cEndMT cells that were grown in maintenance media. Demonstrating additional paracrine effects, conditioned medium from isolated complete EndMT cells induced enhanced mesenchymal proliferation and migration and increased angiogenesis compared with conditioned medium from resident mesenchymal cells. Overall, these findings show that EndMT cells could contribute to the pathogenesis of PAH both directly, by transformation into smooth muscle-like cells with higher proliferative and migratory potency, and indirectly, through paracrine effects on vascular intimal and medial proliferation.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/ß-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/ß-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor ß (TGF-ß) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-ß signaling.

Serotonin 2B Receptor Antagonism Prevents Heritable Pulmonary Arterial Hypertension.

Serotonergic anorexigens are the primary pharmacologic risk factor associated with pulmonary arterial hypertension (PAH), and the resulting PAH is clinically indistinguishable from the heritable form of disease, associated with BMPR2 mutations. Both BMPR2 mutation and agonists to the serotonin receptor HTR2B have been shown to cause activation of SRC tyrosine kinase; conversely, antagonists to HTR2B inhibit SRC trafficking and downstream function. To test the hypothesis that a HTR2B antagonist can prevent BMRP2 mutation induced PAH by restricting aberrant SRC trafficking and downstream activity, we exposed BMPR2 mutant mice, which spontaneously develop PAH, to a HTR2B antagonist, SB204741, to block the SRC activation caused by BMPR2 mutation. SB204741 prevented the development of PAH in BMPR2 mutant mice, reduced recruitment of inflammatory cells to their lungs, and reduced muscularization of their blood vessels. By atomic force microscopy, we determined that BMPR2 mutant mice normally had a doubling of vessel stiffness, which was substantially normalized by HTR2B inhibition. SB204741 reduced SRC phosphorylation and downstream activity in BMPR2 mutant mice. Gene expression arrays indicate that the primary changes were in cytoskeletal and muscle contractility genes. These results were confirmed by gel contraction assays showing that HTR2B inhibition nearly normalizes the 400% increase in gel contraction normally seen in BMPR2 mutant smooth muscle cells. Heritable PAH results from increased SRC activation, cellular contraction, and vascular resistance, but antagonism of HTR2B prevents SRC phosphorylation, downstream activity, and PAH in BMPR2 mutant mice.

Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling.

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-ß1 (TGF-ß1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-ß1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-ß1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Levuglandin forms adducts with histone h4 in a cyclooxygenase-2-dependent manner, altering its interaction with DNA.

Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has long been linked with the development of cancer, although little is known about any epigenetic effects of COX-2. A product of COX-2 activation, levuglandin (LG) quickly forms covalent bonds with nearby primary amines, such as those in lysine, which leads to LG-protein adducts. Here, we demonstrate that COX-2 activity causes LG-histone adducts in cultured cells and liver tissue, detectable through LC-MS, with the highest incidence in histone H4. Adduction is blocked by a γ-ketoaldehyde scavenger, which has no effect on COX-2 activity as measured by PGE2 production. Formation of the LG-histone adduct is associated with an increased histone solubility in NaCl, indicating destabilization of the nucleosome structure; this is also reversed with scavenger treatment. These data demonstrate that COX-2 activity can cause histone adduction and loosening of the nucleosome complex, which could lead to altered transcription and contribute to carcinogenesis.

Endogenous cannabinoid signaling is required for voluntary exercise-induced enhancement of progenitor cell proliferation in the hippocampus.

Voluntary exercise and endogenous cannabinoid activity have independently been shown to regulate hippocampal plasticity. The aim of the current study was to determine whether the endocannabinoid system is regulated by voluntary exercise and if these changes contribute to exercise-induced enhancement of cell proliferation. In Experiment 1, 8 days of free access to a running wheel increased the agonist binding site density of the cannabinoid CB(1) receptor; CB(1) receptor-mediated GTPgammaS binding; and the tissue content of the endocannabinoid anandamide in the hippocampus but not in the prefrontal cortex. In Experiment 2, the CB(1) receptor antagonist AM251 (1 mg kg(-1)) was administered daily to animals given free access to a running wheel for 8 days, after which cell proliferation in the hippocampus was examined through immunohistochemical analysis of the cell cycle protein Ki-67. Voluntary exercise increased proliferation of progenitor cells, as evidenced by the increase in the number of Ki-67 positive cells in the granule cell layer of the dentate gyrus (DG) in the hippocampus. However, this effect was abrogated by concurrent treatment with AM251, indicating that the increase in endocannabinoid signaling in the hippocampus is required for the exercise-induced increase in cell proliferation. These data demonstrate that the endocannabinoid system in the hippocampus is sensitive to environmental change and suggest that it is a mediator of experience-induced plasticity.

Characterization of covalent adducts of nucleosides and DNA formed by reaction with levuglandin.

Enhanced expression of cyclooxygenase-2 (COX-2) is associated with development of several cancers. The product of COX-2, prostaglandin H(2) (PGH(2)), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form the highly reactive levuglandin E(2) (LGE(2)) or D(2) (LGD(2)). Incubation with LGE(2) causes DNA-protein cross-linking in cultured cells, suggesting that levuglandins can directly react with DNA. We report the identification by liquid chromatography-tandem mass spectrometry of a stable levuglandin-deoxycytidine (LG-dC) adduct that forms upon reaction of levuglandin with DNA. We found that LGE(2) reacted with deoxycytidine, deoxyadenosine, or deoxyguanosine in vitro to form covalent adducts with a dihydroxypyrrolidine structure, as deduced from selective ion fragmentation. For LG-deoxycytidine adducts, the initial dihydroxypyrrolidine structure converted to a pyrrole structure over time. Reaction of LG with DNA yielded a stable LG-dC adduct with a pyrrole structure. These results describe the first structure of levuglandinyl-DNA adducts and provide the tools with which to evaluate the potential for LG-DNA adduct formation in vivo.

Circulating endocannabinoids and N-acyl ethanolamines are differentially regulated in major depression and following exposure to social stress.

Central endocannabinoid signaling is known to be responsive to stressful stimuli; however, there is no research to date characterizing the effects of stress on peripheral endocannabinoid content. The current study examined serum content of the endocannabinoid ligands N-arachidonylethanolamide (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), and the non-cannabinoid N-acyl ethanolamine (NAE) molecules palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) under basal conditions, immediately following the Trier Social Stress Test (TSST), and 30 min thereafter, in 15 medication-free women diagnosed with major depression, and 15 healthy matched controls. Basal serum concentrations of AEA and 2-AG, but not PEA or OEA, were significantly reduced in women with major depression relative to matched controls, indicating a deficit in peripheral endocannabinoid activity. Immediately following the TSST, serum 2-AG concentrations were increased compared to baseline; serum AEA concentration was unchanged at this time point. Serum concentrations of PEA and OEA were significantly lower than baseline 30 min following the cessation of the TSST. The magnitude of these responses did not differ between depressed and control subjects. These are the first data to demonstrate that the peripheral endocannabinoid/NAE system is responsive to exposure to stress.