Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL–induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.
Shu-lin Liu, Anamika Bajpai, Elizabeth A. Hawthorne, Yongho Bae, Paola Castagnino, James Monslow, Ellen Puré, Kara L. Spiller, Richard K. Assoian
Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210– and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients’ cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210– and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.
Anika Wehrle, Tomasz M. Witkos, Judith C. Schneider, Anselm Hoppmann, Sidney Behringer, Anna Köttgen, Mariet Elting, Jürgen Spranger, Martin Lowe, Ekkehart Lausch
Mechanical injury to the brain triggers multiple biochemical events whose specific contributions to the pathogenesis define clinical manifestations and the overall outcome. Among many factors, mitochondrial injury has recently attracted much attention due to the importance of the organelle for bioenergetics as well as intra- and extracellular signaling and cell death. Assuming the essentiality of a mitochondria-unique phospholipid, cardiolipin (CL), for the structural and functional organization of mitochondria, here we applied global (phospho) lipidomics and redox lipidomics to reveal and identify CL modifications during controlled cortical impact (CCI). We revealed 2 major pathways activated in the CCI-injured brain as time-specific responses: early accumulation of oxidized CL (CLox) products was followed by hydrolytic reactions yielding monolyso-CLs (mCLs) and free fatty acids. To quantitatively assess possible specific roles of peroxidation and hydrolysis of mitochondrial CL, we performed comparative studies of CL modifications using an animal model of Barth syndrome where deficiency of CL reacylation (Tafazzin [Taz] deficiency) was associated exclusively with the accumulation of mCLs (but not CLox). By comparing the in vitro and in vivo results with genetic manipulation of major CL-, CLox-, and mCL-metabolizing enzymes, calcium-independent phospholipase A2γ and Taz, we concluded that the 2 processes — CL oxidation and CL hydrolysis — act as mutually synergistically enhancing components of the pathogenic mechanism of mitochondrial injury in traumatic brain injury. This emphasizes the need for combined therapeutic approaches preventing the formation of both CLox and mCL.
Honglu Chao, Tamil S. Anthonymuthu, Elizabeth M. Kenny, Andrew A. Amoscato, Laura K. Cole, Grant M. Hatch, Jing Ji, Valerian E. Kagan, Hülya Bayır
Elevated blood pressure (BP) and renal dysfunction are complex traits representing major global health problems. Single nucleotide polymorphisms identified by genome-wide association studies have identified the Alström syndrome 1 (ALMS1) gene locus to render susceptibility for renal dysfunction, hypertension, and chronic kidney disease (CKD). Mutations in the ALMS1 gene in humans causes Alström syndrome, characterized by progressive metabolic alterations including hypertension and CKD. Despite compelling genetic evidence, the underlying biological mechanism by which mutations in the ALMS1 gene lead to the above-mentioned pathophysiology is not understood. We modeled this effect in a KO rat model and showed that ALMS1 genetic deletion leads to hypertension. We demonstrate that the link between ALMS1 and hypertension involves the activation of the renal Na+/K+/2Cl– cotransporter NKCC2, mediated by regulation of its endocytosis. Our findings establish a link between the genetic susceptibility to hypertension, CKD, and the expression of ALMS1 through its role in a salt-reabsorbing tubular segment of the kidney. These data point to ALMS1 as a potentially novel gene involved in BP and renal function regulation.
Ankita Bachhawat Jaykumar, Paulo S. Caceres, Keyona N. King-Medina, Tang-Dong Liao, Indrani Datta, Dipak Maskey, Jürgen K. Naggert, Mariela Mendez, William H. Beierwaltes, Pablo A. Ortiz
Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.
Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr
Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.
Maria Papadaki, Ronald J. Holewinski, Samantha Beck Previs, Thomas G. Martin, Marisa J. Stachowski, Amy Li, Cheavar A. Blair, Christine S. Moravec, Jennifer E. Van Eyk, Kenneth S. Campbell, David M. Warshaw, Jonathan A. Kirk
Molecular mechanisms underlying the cancer stroma in metastasis need further exploration. Here, we discovered that cancer-associated fibroblasts (CAFs) produced high levels of IL-33 that acted on tumor-associated macrophages (TAMs), causing them to undergo the M1 to M2 transition. Genomic profiling of metastasis-related genes in the IL-33–stimulated TAMs showed a >200-fold increase of MMP9. Signaling analysis demonstrated the IL-33-ST2-NF-κB-MMP9-laminin pathway that governed tumor stroma–mediated metastasis. In mouse and human fibroblast-rich pancreatic cancers, genetic deletion of IL-33, ST2, or MMP9 markedly blocked metastasis. Pharmacological inhibition of NF-κB and MMP9 also blocked cancer metastasis. Deletion of IL-33, ST2, or MMP9 restored laminin, a key basement membrane component associated with tumor microvessels. Together, our data provide mechanistic insights on the IL-33-NF-κB-MMP9-laminin axis that mediates the CAF-TAM–committed cancer metastasis. Thus, targeting the CAF-TAM-vessel axis provides an outstanding therapeutic opportunity for cancer treatment.
Patrik Andersson, Yunlong Yang, Kayoko Hosaka, Yin Zhang, Carina Fischer, Harald Braun, Shuzhen Liu, Guohua Yu, Shihai Liu, Rudi Beyaert, Mayland Chang, Qi Li, Yihai Cao
Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient–derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvβ3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvβ3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvβ3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvβ3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.
Vincent F. Fiore, Simon S. Wong, Coleen Tran, Chunting Tan, Wenwei Xu, Todd Sulchek, Eric S. White, James S. Hagood, Thomas H. Barker
Cardiac Nav1.5 and Kir2.1–2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems — rat ventricular cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) — demonstrated that endoplasmic reticulum (ER) trafficking–defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking–defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking–defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.
Marta Pérez-Hernández, Marcos Matamoros, Silvia Alfayate, Paloma Nieto-Marín, Raquel G. Utrilla, David Tinaquero, Raquel de Andrés, Teresa Crespo, Daniela Ponce-Balbuena, B. Cicero Willis, Eric N. Jiménez-Vazquez, Guadalupe Guerrero-Serna, Andre M. da Rocha, Katherine Campbell, Todd J. Herron, F. Javier Díez-Guerra, Juan Tamargo, José Jalife, Ricardo Caballero, Eva Delpón
AEP is an age-dependent lysosomal asparaginyl endopeptidase that cleaves numerous substrates including tau and α-synuclein and mediates their pathological roles in neurodegenerative diseases. However, the molecular mechanism regulating this critical protease remains incompletely understood. Here, we show that Akt phosphorylates AEP on residue T322 upon brain-derived neurotrophic factor (BDNF) treatment and triggers its lysosomal translocation and inactivation. When BDNF levels are reduced in neurodegenerative diseases, AEP T322 phosphorylation is attenuated. Consequently, AEP is activated and translocates into the cytoplasm, where it cleaves both tau and α-synuclein. Remarkably, the unphosphorylated T322A mutant increases tau or α-synuclein cleavage by AEP and augments cell death, whereas phosphorylation mimetic T322E mutant represses these effects. Interestingly, viral injection of T322E into Tau P301S mice antagonizes tau N368 cleavage and tau pathologies, rescuing synaptic dysfunction and cognitive deficits. By contrast, viral administration of T322A into young α-SNCA mice elicits α-synuclein N103 cleavage and promotes dopaminergic neuronal loss, facilitating motor defects. Therefore, our findings support the notion that BDNF contributes to the pathogenesis of neurodegenerative diseases by suppressing AEP via Akt phosphorylation.
Zhi-Hao Wang, Wanqiang Wu, Seong Su Kang, Xia Liu, Zhiping Wu, Junmin Peng, Shan Ping Yu, Fredric P. Manfredsson, Ivette M. Sandoval, Xuebo Liu, Jian-Zhi Wang, Keqiang Ye
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