DREAM is a transcriptional repressor complex that regulates cell proliferation and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we utilized an assembly defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis, but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys, but not the brain or bone marrow. Using laser capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting AApoAIV amyloidosis. AApoAIV is a recently described form whereby wildtype apoA-IV has been shown to predominate in amyloid plaques. We determined that DREAM directly regulates Apoa4 by chromatin immunoprecipitation and that the histone variant H2AZ is reduced from the Apoa4 gene body in DREAM’s absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of non-genetic amyloid subtypes.
Pirunthan Perampalam, Haider M. Hassan, Grace E. Lilly, Daniel T. Passos, Joseph Torchia, Patti K. Kiser, Andrea Bozovic, Vathany Kulasingam, Frederick A. Dick
Propranolol, a pleiotropic β-adrenergic blocker, was anecdotally reported to reduce cerebral cavernous malformations (CCM) in humans. However, propranolol has neither been rigorously evaluated in animal models nor was its mechanism of action in CCM defined. We report that propranolol or its S(-) enantiomer dramatically reduced embryonic venous cavernomas in ccm2 mosaic zebrafish, whereas R-(+)-propranolol, lacking β-antagonism, had no effect. Silencing of β1, but not β2, adrenergic receptor mimicked the beneficial effects of propranolol in a zebrafish CCM model as did a β1-selective antagonist, metoprolol. Thus, propranolol ameliorates cavernous malformations by β1 adrenergic antagonism in zebrafish. Oral propranolol significantly reduced lesion burden in two chronic murine models of the exceptionally aggressive Pdcd10/Ccm3 form of CCM. Propranolol or other β1-selective antagonists may be beneficial in CCM disease.
Wenqing Li, Robert Shenkar, Matthew R. Detter, Thomas Moore, Christian R. Benavides, Rhonda Lightle, Romuald Girard, Nicholas Hobson, Ying Cao, Yan Li, Erin Griffin, Carol Gallione, Joseph M. Zabramski, Mark H. Ginsberg, Douglas A. Marchuk, Issam A. Awad
The development of ascites correlates with advanced-stage disease and poor prognosis in ovarian cancer. Vascular permeability is the key pathophysiological change involved in ascites development. Previously, we provided the first evidence that perivascular M2-like macrophages protect the vascular barrier through direct contact with endothelial cells (ECs). Here, we investigated the molecular mechanism and its clinical significance in the ovarian cancer setting. We found that upon direct coculture with the endothelium, M2 macrophages tuned down their VLA4 and reduced the levels of VCAM1 in ECs. On the other hand, ectopically overexpressing VLA4 in macrophages or VCAM1 in ECs induced hyperpermeability. Mechanistically, downregulation of VLA4 or VCAM1 led to reduced levels of RAC1 and reactive oxygen species (ROS), which resulted in decreased phosphorylation of PYK2 (p-PYK2) and VE-cadherin (p-VE-cad), hence enhancing cell adhesion. Furthermore, targeting the VLA4/VCAM1 axis augmented vascular integrity and abrogated ascites formation in vivo. Lastly, VLA4 expression on the macrophages isolated from ascites dictated permeability ex vivo. Importantly, VLA4 antibody acted synergistically with bevacizumab to further enhance the vascular barrier. Taken together, we reveal here that M2 macrophages regulate the vascular barrier though the VCAM1/RAC1/ROS/p-PYK2/p-VE-cad cascade, which provides specific therapeutic targets for the treatment of malignant ascites.
Shibo Zhang, Bingfan Xie, Lijie Wang, Hua Yang, Haopei Zhang, Yuming Chen, Feng Wang, Changqing Liu, Huanhuan He
The triggering receptor expressed on myeloid cells-1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of Angiotensin (Ang) II-induced AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalizes with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2 and Mmp9 mRNA expression, and led to a decreased macrophage content, due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L up-regulation and promoted pro-inflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII Receptor Type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared to patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in human.
Marie Vandestienne, Yujiao Zhang, Icia Santos-Zas, Rida Al-Rifai, Jeremie Joffre, Andreas Giraud, Ludivine Laurans, Bruno Esposito, Florence Pinet, Patrick Bruneval, Juliette Raffort, Fabien Lareyre, Jose Vilar, Amir Boufenzer, Lea Guyonnet, Coralie L. Guerin, Eric Clauser, Jean-Sébastien Silvestre, Sylvie Lang, Laurie Soulat-Dufour, Alain Tedgui, Ziad Mallat, Soraya Taleb, Alexandre Boissonnas, Marc Derive, Giulia Chinetti, Hafid Ait-Oufella
ABSTRACTIndividuals harboring the loss-of-function (LOF) proprotein convertase subtilising/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein (LDL) cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of pro-PCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes where it would be expected to contribute to ER storage disease (ERSD); a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than to induce them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress-driven conditions of the liver. In summary, we have uncovered a co-chaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.
Paul F. Lebeau, Hanny Wassef, Jae Hyun Byun, Khrystyna Platko, Brandon Ason, Simon Jackson, Joshua Dobroff, Susan Shetterly, William G. Richards, Ali A. Al-Hashimi, Kevin D. Won, Majambu Mbikay, Annik Prat, An Tang, Guillaume Paré, Renata Pasqualini, Nabil G. Seidah, Wadih Arap, Michel Chretien, Richard C. Austin
Tertiary lymphoid organs are aggregates of immune and stromal cells including high endothelial venules and lymphatic vessels that resemble secondary lymphoid organs and can be induced at nonlymphoid sites during inflammation. The function of lymphatic vessels within tertiary lymphoid organs remains poorly understood. During lung transplant tolerance, Foxp3+ cells accumulate in tertiary lymphoid organs that are induced within the pulmonary grafts and are critical for the local downregulation of alloimmune responses. Here, we showed that tolerant lung allografts could induce and maintain tolerance of heterotopic donor-matched hearts through pathways that were dependent on the continued presence of the transplanted lung. Using lung retransplantation, we showed that Foxp3+ cells egressed from tolerant lung allografts via lymphatics and were recruited into donor-matched heart allografts. Indeed, survival of the heart allografts was dependent on lymphatic drainage from the tolerant lung allograft to the periphery. Thus, our work indicates that cellular trafficking from tertiary lymphoid organs regulates immune responses in the periphery. We propose that these findings have important implications for a variety of disease processes that are associated with the induction of tertiary lymphoid organs.
Wenjun Li, Jason M. Gauthier, Alice Y. Tong, Yuriko Terada, Ryuji Higashikubo, Christian C. Frye, Margaret S. Harrison, Kohei Hashimoto, Amit I. Bery, Jon H. Ritter, Ruben G. Nava, Varun Puri, Brian W. Wong, Kory J. Lavine, Ankit Bharat, Alexander S. Krupnick, Andrew E. Gelman, Daniel Kreisel
Pathologic lymphatic remodeling in lymphedema evolves during periods of tissue inflammation and hypoxia through poorly defined processes. In human and mouse lymphedema, there is a significant increase of hypoxia inducible factor (HIF)-1α, but a reduction of HIF-2α protein expression in lymphatic endothelial cells (LECs). We questioned whether dysregulated expression of these transcription factors contributes to disease pathogenesis and found that LEC-specific deletion of Hif-2α exacerbated lymphedema pathology. Even without lymphatic vascular injury, the loss of LEC-specific Hif-2α caused anatomic pathology and a functional decline in fetal and adult mice. These findings suggest that HIF-2α is an important mediator of lymphatic health. HIF-2α promoted protective phosphorylated TIE2 (p-TIE2) signaling in LECs, a process also replicated by upregulating TIE2 signaling through adenovirus-mediated angiopoietin-1 (Angpt1) gene therapy. Our study suggests that HIF-2α normally promotes healthy lymphatic homeostasis and raises the exciting possibility that restoring HIF-2α pathways in lymphedema could mitigate long-term pathology and disability.
Xinguo Jiang, Wen Tian, Eric J. Granucci, Allen B. Tu, Dongeon Kim, Petra Dahms, Shravani Pasupneti, Gongyong Peng, Yesl Kim, Amber H. Lim, F. Hernan Espinoza, Matthew Cribb, J. Brandon Dixon, Stanley G. Rockson, Gregg L. Semenza, Mark R. Nicolls
Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF ("TFshort") in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant negative mechanism. F3+/- mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation.
Sol Schulman, Emale El-Darzi, Mary HC Florido, Max Friesen, Glenn Merrill-Skoloff, Marisa A. Brake, Calvin R. Schuster, Lin Lin, Randal J. Westrick, Chad A. Cowan, Robert Flaumenhaft, NIHR BioResource, Willem H. Ouwehand, Kathelijne Peerlinck, Kathleen Freson, Ernest Turro, Bruce Furie
Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II–induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II–induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II–induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A2 analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.
Wakako Kawarazaki, Risuke Mizuno, Mitsuhiro Nishimoto, Nobuhiro Ayuzawa, Daigoro Hirohama, Kohei Ueda, Fumiko Kawakami-Mori, Shigeyoshi Oba, Takeshi Marumo, Toshiro Fujita
Diabetic patients develop endothelial dysfunction shortly after diabetes onset that progresses to vascular disease underlying the majority of diabetes associated comorbidities. Increased lipid peroxidation, mitochondrial calcium overload and mitochondrial dysfunction are characteristics of dysfunctional endothelial cells in diabetic patients. We here identified that targeting the lipid peroxidation product 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) induced activation of the intracellularly located cation channel transient receptor potential vanilloid 1 (TRPV1) in endothelial cells is a means to causally control early stage vascular disease in type I diabetic mice. Mice with an inducible, endothelial specific 12/15 lipoxygenase (12/15Lo) knock out were similarly protected from type I diabetes induced endothelial dysfunction and impaired vascular regeneration following arterial injury as TRPV1 knock out mice. Both 12(S)-HETE in concentrations found in diabetic patients and TRPV1 agonists triggered mitochondrial calcium influx and mitochondrial dysfunction in endothelial cells and 12(S)-HETE effects were absent in endothelial cells from TRPV1 knock out mice. As a therapeutic consequence, we found that a peptide targeting 12(S)-HETE induced TRPV1 interaction at the TRPV1 TRP box ameliorated diabetes-induced endothelial dysfunction and augmented vascular regeneration in diabetic mice. Our findings suggest that pharmacological targeting of increased endothelial lipid peroxidation can attenuate diabetes induced comorbidities related to vascular disease.
Mandy Otto, Clarissa Bucher, Wantao Liu, Melanie Müller, Tobias Schmidt, Marina Kardell, Marvin Noel Driessen, Jan Rossaint, Eric R. Gross, Nana-Maria Wagner