Abstract-Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascularresistance caused by narrowing and loss of pulmonary microvasculature, which in its late stages becomes refractory to traditional therapies. We hypothesized that bone marrow-derived endothelial progenitor cells (EPCs), which normally function to repair and regenerate blood vessels, would restore pulmonary hemodynamics and increase microvascular perfusion in the rat monocrotaline (MCT) model of PAH. Mononuclear cells were isolated from the bone marrow of syngeneic Fisher-344 rats by Ficoll gradient centrifugation and cultured for 7 to 10 days in endothelial growth medium. Fluorescently labeled endothelial-like progenitor cells (ELPCs) engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCT-injured lung. The administration of ELPCs 3 days after MCT nearly completely prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone (31.5Ϯ0.95 versus 48Ϯ3 mm Hg, respectively; PϽ0.001), whereas injection of skin fibroblasts had no protective effect (50.9Ϯ5.4 mm Hg). Delayed administration of progenitor cells 3 weeks after MCT prevented the further progression of PAH 2 weeks later (ie, 5 weeks after MCT), whereas only animals receiving ELPCs transduced with human endothelial NO-synthase (eNOS) exhibited significant reversal of established disease at day 35 (31Ϯ2 mm Hg, PϽ0.005) compared with day 21 (50Ϯ3 mm Hg). Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT, whereas arteriolar-capillary continuity and microvascular architecture was preserved with the administration of syngeneic ELPCs. Moreover, the delivery of ELPCs to rats with established PAH resulted in marked improvement in survival, which was greatest in the group receiving eNOS-transduced cells. We conclude that bone marrow-derived ELPCs can engraft and repair the MCT-damaged lung, restoring microvasculature structure and function. Therefore, the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with PAH. ( Key Words: progenitor cells Ⅲ pulmonary hypertension Ⅲ endothelium Ⅲ endothelial nitric oxide synthase P ulmonary arterial hypertension (PAH) is a devastating disease that in its most severe form, idiopathic PAH, leads to progressive debilitation and death, often within 2 to 3 years after its initial diagnosis. 1 Despite significant advances in the therapy of PAH during the last decade, the prognosis remains poor. Although the genetic basis for some patients with familial PAH has been elucidated, 2-4 how these mutations are causally linked to the development of PAH remains unclear. 5,6 Evidence from experimental models as well as lung specimens from patients with PAH underlines the importance of microvascular occlusion in the pathogenesis of this disease, 7 especially in its advanced stages; however, the precise mechanisms...

Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1 -/-mice were absent in Cav1 -/-Nos3 -/-mice. Further, treatment of Cav1 -/-mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1 -/-lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1 -/-lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality.

Background-Endothelium-derived nitric oxide (NO) is produced by an oxidative reaction catalyzed by endothelial NO synthase (eNOS). NO plays a crucial role in controlling cell growth and apoptosis, as well as having well-characterized vasodilator and antithrombotic actions. More recently, endothelium-derived NO was shown to be involved in postdevelopmental vascular remodeling and angiogenesis, as well as in the formation of limb vasculature during embryogenesis. Therefore, we investigated the role of endothelium-derived NO during cardiovascular development using mice deficient in eNOS. Methods and Results-We examined the hearts of 12 mature eNOS-deficient and 26 mature wild-type mice. Five of the mature eNOS-deficient mice had a bicuspid aortic valve; none of the 26 wild-type animals exhibited identifiable valvular or cardiac abnormalities. Immunohistochemical analysis revealed prominent eNOS expression localized to the endothelium lining the valve cusps of the aorta in mature wild-type mice; expression was localized to the myocardium and endothelial cell monolayer lining the valve leaflets in the developing embryo. Conclusions-These results show a strong association between eNOS deficiency and the presence of a bicuspid aortic valve; they provide the first molecular insight into one of the most common types of congenital cardiac abnormality. Key Words: nitric oxide Ⅲ endothelium Ⅲ mice, knockout Ⅲ heart defects, congenital Ⅲ valves N itric oxide (NO) is a free radical gas that is synthesized from L-arginine in a complex oxidative reaction catalyzed by 3 distinct isoforms of NO synthase: endothelial (eNOS), inducible (iNOS), and neuronal (nNOS). 1 The neuronal isoform is highly expressed in neuronal cells and skeletal muscle, whereas the endothelial isoform is predominantly expressed in endothelial cells and produces small amounts of NO in response to intimal shear stress or agonists such as bradykinin. 2,3 The inducible isoform is expressed in many cell types, including macrophages and smooth muscle cells, primarily in response to inflammatory cytokines. Once expressed, this isoform can produce large amounts of NO in a continuous manner. 1,2 Endothelium-derived NO is crucial in the regulation of cell growth and apoptosis; it also has a well-characterized role as a vasodilator and antithrombotic agent. 1 More recently, NO derived from the endothelium was shown to be involved in postdevelopmental vascular remodeling and angiogenesis, as well as in the formation of limb vasculature during embryogenesis. 4 -6 The valve leaflets of the heart originate from mesenchymal outgrowths known as cardiac cushions. Cushion formation is localized to the atrioventricular canal and ventricular outflow tract regions of the primary heart tube. These formations arise from regional thickenings of the cardiac jelly, the extracellular matrix that resides between the myocardium and endocardium of the primitive heart tube. This event involves the transformation of a subset of endothelial cells of the endocardium into mesenchyme. The mo...

IntroductionIdiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown cause characterised by progressive fibrotic formation in lung tissue. We hypothesise that disrupted metabolic pathways in IPF contribute to disease pathogenesis.MethodsMetabolomics of human IPF was performed using mass spectroscopy (IPF lung=8; donor lung=8). Gene expression of key metabolic enzymes was measured using microarrays. Of the 108 metabolites whose levels were found altered, 48 were significantly increased, whereas 60 were significantly decreased in IPF samples compared with normal controls.ResultsSpecific metabolic pathways mediating the IPF remodelling were found with a downregulated sphingolipid metabolic pathway but an upregulated arginine pathway in IPF. In addition, disrupted glycolysis, mitochondrial beta-oxidation and tricarboxylic acid cycle, altered bile acid, haem and glutamate/aspartate metabolism were found in IPF samples compared with control.ConclusionsOur results show alterations in metabolic pathways for energy consumption during lung structural remodelling, which may contribute to IPF pathogenesis. We believe that this is the first report of simultaneously and systemically measuring changes of metabolites involving nine metabolic pathways in human severe IPF lungs. The measurement of the metabolites may serve in the future diagnosis and prognosis of IPF.

Glycolysis is critical for cancer stem cell reprogramming; however, the underlying regulatory mechanisms remain elusive. Here, we show that pyruvate dehydrogenase kinase 1 (PDK1) is enriched in breast cancer stem cells (BCSCs), whereas depletion of PDK1 remarkably diminishes ALDH+ subpopulations, decreases stemness-related transcriptional factor expression, and inhibits sphere-formation ability and tumor growth. Conversely, high levels of PDK1 enhance BCSC properties and are correlated with poor overall survival. In mouse xenograft tumor, PDK1 is accumulated in hypoxic regions and activates glycolysis to promote stem-like traits. Moreover, through screening hypoxia-related long non-coding RNAs (lncRNAs) in PDK1-positive tissue, we find that lncRNA H19 is responsible for glycolysis and BCSC maintenance. Furthermore, H19 knockdown decreases PDK1 expression in hypoxia, and ablation of PDK1 counteracts H19-mediated glycolysis and self-renewal ability in vitro and in vivo. Accordingly, H19 and PDK1 expression exhibits strong correlations in primary breast carcinomas. H19 acting as a competitive endogenous RNA sequesters miRNA let-7 to release Hypoxia-inducible factor 1α, leading to an increase in PDK1 expression. Lastly, aspirin markedly attenuates glycolysis and cancer stem-like characteristics by suppressing both H19 and PDK1. Thus, these novel findings demonstrate that the glycolysis gatekeeper PDK1 has a critical role in BCSC reprogramming and provides a potential therapeutic strategy for breast malignancy.

Background-Pulmonary arterial hypertension is characterized by increased pulmonary vascular resistance secondary to a decrease in the caliber and number of pulmonary vascular channels. We hypothesized that the targeted overexpression of an angiogenic factor within the lung would potentially minimize the development and progression of pulmonary arterial hypertension by preventing the loss of existing vessels or by inducing the development of new blood vessels within the lung. Methods and Results-We used a cell-based method of gene transfer to the pulmonary microvasculature by delivering syngeneic smooth muscle cells overexpressing vascular endothelial growth factor (VEGF)-A to inbred Fisher 344 rats in which pulmonary hypertension was induced with the pulmonary endothelial toxin monocrotaline. Four weeks after simultaneous endothelial injury and cell-based gene transfer, right ventricular (RV) hypertension and RV and vascular hypertrophy were significantly decreased in the VEGF-treated animals. Four weeks after gene transfer, the plasmid VEGF transcript was still detectable in the pulmonary tissue of animals injected with VEGF-transfected cells, demonstrating survival of the transfected cells and persistent transgene expression. In addition, delay of cell-based gene transfer until after the development of pulmonary hypertension also resulted in a significant decrease in the progression of RV hypertension and hypertrophy. Conclusions-These results indicate that cell-based VEGF gene transfer is an effective method of preventing the development and progression of pulmonary hypertension in the monocrotaline model and suggest a potential therapeutic role for angiogenic factors in the therapy of this devastating disease.

Although multiple gene and protein expression have been extensively profiled in human pulmonary arterial hypertension (PAH), the mechanism for the development and progression of pulmonary hypertension remains elusive. Analysis of the global metabolomic heterogeneity within the pulmonary vascular system leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways in the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to increased ATP synthesis for the vascular remodeling process in severe pulmonary hypertension. These identified metabolites may serve as potential biomarkers for the diagnosis of PAH. By profiling metabolomic alterations of the PAH lung, we reveal new pathogenic mechanisms of PAH, opening an avenue of exploration for therapeutics that target metabolic pathway alterations in the progression of PAH.