Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown. Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH. Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B 2/2 ), or early growth response-1 (Egr-1 2/2 ), were evaluated for vascular morphology and hemodynamics. Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B 2/2 or Egr-1 2/2 , but not Beclin 1 1/2 , mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxiainducible factor-1a stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia. Conclusions:The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B 2/2 and Egr-1 2/2 mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.Keywords: autophagy; hypoxia; hypertension, pulmonary Pulmonary hypertension (PH) is functionally characterized by elevations in mean pulmonary pressure and the development of secondary right ventricular failure. Category I PH, also known as pulmonary arterial hypertension (PAH), includes idiopathic (IPAH), familial, and acquired forms resulting from collagen vascular disease, drugs, HIV infection, portal hypertension, and pulmonary artery shunts (1-5). Categories II-V of PH include those forms secondary to left-heart disease; lung disease or hypoxia; chronic thrombotic or embolic disease; or other diseases rarely associated with PH, such as sarcoid or mediastinitis (1, 2). The molecular and cellular basis for the vascular changes associated with PH remains unclear (6). Impaired production of nitric oxide can result in vasoconstriction and smooth muscle cell proliferation, suggesting a role for endothelial dysfunction in PH (7). In addition, variations in the production, expression, or activity of vascular mediators (i.e., endothelin-1, prostacyclins, voltage-gated K 1 -channels), signaling molecules, and growth factors (e.g., transforming growth factor-b, platelet-derived growth factor [PDGF]) have been implicated in hypoxic remodelin...
Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-β, induces β-catenin (β-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA–Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both β-C– and Dvl-mediated RhoA–Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2–mediated angiogenesis.
Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. ). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.Regulation of protein degradation by the ubiquitin-proteasome pathway enables cells rapidly to reduce levels of defined proteins that control diverse processes, such as gene expression, cell signaling, immune responses, and stress adaptation. Therefore, proteasome-mediated degradation has to display a high degree of specificity, carried out by complex cascades of enzymes, toward its numerous substrates (6). Recently, a variety of inhibitors of the 26S proteasome have been identified. For example, the peptide aldehyde MG132 and the natural products lactacystin and its derivative clasto-lactacystin -lactone all selectively inhibit degradation of proteins by the ubiquitin-proteasome pathway (15).When the ubiquitin-proteasome network is down-regulated, certain heat shock proteins, such as Hsp70, and other molecular chaperones are induced (5,13,14,21,22,45). Expression of heat shock genes is regulated at the transcriptional level by activation of the heat shock transcription factors (HSFs) (25,42). Unlike in yeast and Drosophila, the existence of multiple HSFs in eukaryotes suggests that the various HSFs might have specialized functions in response to distinct physiological and environmental stimuli. Among the three known mammalian HSFs (HSF1, HSF2, and HSF4), HSF1 mediates the stress response induced by heat, heavy metals, and oxidants, while HSF2 has been suggested to have a role as a developmental regulator (1,2,23,31,32,34,35,37,38), and the role of HSF4 is still unknown (27). Recently, the phenotype of homozygous HSF1-deficient (hsf1 Ϫ/Ϫ ) mice was reported to include defects of the chorioallantoic placenta and increased prenatal lethality, growth retardation, fe...
Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and z15% of patients with IPAH, their low penetrance (z20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown.Objectives: To determine whether WES can help identify novel gene modifiers in patients with IPAH.Methods: Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR. Measurements and Main Results:A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival.Conclusions: WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individual's risk of developing IPAH.
Vascular smooth muscle cell motility relies on interdependent activation of canonical and noncanonical Wnt signal transduction pathways; fibronectin, produced in response to BMP-2–mediated activation of β-catenin, promotes motility by activating an integrin-linked kinase via α4-integrin.
Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells. The role of GATA4 in Leydig cell development, however, remains controversial, because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses. We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are down-regulated or up-regulated after small interfering RNA (siRNA)-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1. These same genes were dysregulated when primary cultures of Gata4(flox/flox) adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Among the down-regulated genes were enzymes of the androgen biosynthetic pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a). Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed alteration of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, Gpi1, Pfkp, and Pgam1), lower intracellular levels of ATP, and increased extracellular levels of glucose. Our findings suggest that GATA4 plays a pivotal role in Leydig cell function and provide novel insights into metabolic regulation in this cell type.
Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.