Arginine methylation mediated by protein arginine methyltransferases (PRMTs) has been shown to be an important posttranslational mechanism involved in various biological processes. Herein, we sought to investigate whether PRMT5, a major type II enzyme, is involved in pathological angiogenesis and, if so, to elucidate the molecular mechanism involved. Our results show that PRMT5 expression is significantly upregulated in ischemic tissues and hypoxic endothelial cells (ECs). Endothelial-specific Prmt5-KO mice were generated to define the role of PRMT5 in hindlimb ischemia–induced angiogenesis. We found that these mice exhibited impaired recovery of blood perfusion and motor function of the lower limbs, an impairment that was accompanied by decreased vascular density and increased necrosis as compared with their WT littermates. Furthermore, both pharmacological and genetic inhibition of PRMT5 significantly attenuated EC proliferation, migration, tube formation, and aortic ring sprouting. Mechanistically, we showed that inhibition of PRMT5 markedly attenuated hypoxia-induced factor 1-α (HIF-1α) protein stability and vascular endothelial growth factor–induced (VEGF-induced) signaling pathways in ECs. Our results provide compelling evidence demonstrating a crucial role of PRMT5 in hypoxia-induced angiogenesis and suggest that inhibition of PRMT5 may provide novel therapeutic strategies for the treatment of abnormal angiogenesis-related diseases, such as cancer and diabetic retinopathy.
Qing Ye, Jian Zhang, Chen Zhang, Bing Yi, Kyosuke Kazama, Wennan Liu, Xiaobo Sun, Yan Liu, Jianxin Sun
The placenta is the primary organ for immune regulation, nutrient delivery, gas exchange, protection against environmental toxins, and physiologic perturbations during pregnancy. Placental inflammation and vascular dysfunction during pregnancy are associated with a growing list of prematurity-related complications. The goal of this study was to identify differences in gene expression profiles in fetal monocytes - cells that persist and differentiate postnatally - according to distinct placental histologic domains. Here, by using bulk RNA sequencing (RNA-Seq), we report that placental lesions are associated with gene expression changes in fetal monocyte subsets. Specifically, we found that fetal monocytes exposed to acute placental inflammation upregulate biological processes related to monocyte activation, monocyte chemotaxis, and platelet function while monocytes exposed to maternal vascular malperfusion lesions downregulate these processes. Additionally, we show that intermediate monocytes might be a source of mitogens, such as HBEGF, NRG1, and VEGFA, implicated in different outcomes related to prematurity. This is the first study to show that placental lesions are associated with unique changes in fetal monocytes and monocyte subsets. As fetal monocytes persist and differentiate into various phagocytic cells following birth, our study may provide insight into morbidity related to prematurity and ultimately potential therapeutic targets.
Abhineet M. Sharma, Robert Birkett, Erika T. Lin, Linda M. Ernst, William A. Grobman, Suchitra Swaminathan, Hiam Abdala-Valencia, Alexander V. Misharin, Elizabeth T. Bartom, Karen K. Mestan
Radiation causes a collapse of bone marrow cells and elimination of microvasculature. To understand how bone marrow recovers after radiation, we focused on mesenchymal lineage cells that provide a supportive microenvironment for hematopoiesis and angiogenesis in bone. We recently discovered a nonproliferative subpopulation of marrow adipogenic lineage precursors (MALPs) that express adipogenic markers with no lipid accumulation. Single-cell transcriptomic analysis revealed that MALPs acquire proliferation and myofibroblast features shortly after radiation. Using an adipocyte-specific Adipoq-Cre, we validated that MALPs rapidly and transiently expanded at day 3 after radiation, coinciding with marrow vessel dilation and diminished marrow cellularity. Concurrently, MALPs lost most of their cell processes, became more elongated, and highly expressed myofibroblast-related genes. Radiation activated mTOR signaling in MALPs that is essential for their myofibroblast conversion and subsequent bone marrow recovery at day 14. Ablation of MALPs blocked the recovery of bone marrow vasculature and cellularity, including hematopoietic stem and progenitors. Moreover, VEGFa deficiency in MALPs delayed bone marrow recovery after radiation. Taken together, our research demonstrates a critical role of MALPs in mediating bone marrow repair after radiation injury and sheds light on a cellular target for treating marrow suppression after radiotherapy.
Leilei Zhong, Lutian Yao, Nicholas Holdreith, Wei Yu, Tao Gui, Zhen Miao, Yehuda Elkaim, Mingyao Li, Yanqing Gong, Maurizio Pacifici, Amit Maity, Theresa M. Busch, Kyu Sang Joeng, Keith Cengel, Patrick Seale, Wei Tong, Ling Qin
Hereditary hemorrhagic telangiectasia (HHT) patients have arteriovenous malformations (AVMs) with genetic mutations involving the activin-A receptor like type 1 (ACVRL1 or ALK1) and endoglin (ENG). Recent study showed that Neuropilin-1 (NRP-1) inhibits ALK1. We investigated the expression of NRP-1 in livers of patients with HHT and found that there was a significant reduction in NRP-1 in perivascular smooth muscle cells (SMCs). We used Nrp1SM22KO mice (Nrp1 was ablated in SMCs) and found hemorrhage, increased immune cell infiltration with decrease in SMCs and pericyte lining in lungs and liver in adult mice. Histologic examination revealed lung AVFs with enlarged liver vessels. Evaluation of the retina vessels at post-natal day 5 from Nrp1SM22KO mice demonstrated dilated capillaries with a reduction of pericytes. In inflow artery of surgical AVFs from the Nrp1SM22KO vs. WT mice, there was a significant decrease in Tgfb1, Eng and Alk1 expression, phosphorylated-SMAD (pSMAD)1/5/8, with an increase in apoptosis. TGF-β1 stimulated aortic SMCs from Nrp1SM22KO vs. WT mice have decreased pSMAD1/5/8 upon and increased apoptosis. Coimmunoprecipitation experiments revealed that NRP-1 interacts with ALK1 and ENG in SMCs. In sum, NRP-1 deletion in SMCs leads to reduced ALK1, ENG, pSMAD1/5/8 signaling, and cell death associated with AVM formation.
Sreenivasulu Kilari, Ying Wang, Avishek Singh, Rondell P. Graham, Vivek Iyer, Scott M. Thompson, Michael S. Torbenson, Debabrata Mukhopadhyay, Sanjay Misra
Arterial stiffness predicts cardiovascular disease and all-cause mortality but its treatment remains challenging. Mice treated with angiotensin-II (Ang-II) develop hypertension, arterial stiffness, vascular dysfunction, and a downregulation of Rho-related BTB domain-containing protein 1 (RhoBTB1) in the vasculature. RhoBTB1 is associated with blood pressure regulation but its function is poorly understood. We tested the hypothesis that restoring RhoBTB1 can attenuate arterial stiffness, hypertension, and vascular dysfunction in Ang-II-treated mice. Genetic complementation of RhoBTB1 in the vasculature was achieved utilizing mice expressing a tamoxifen-inducible, smooth muscle-specific RhoBTB1 transgene. RhoBTB1 restoration efficiently and rapidly alleviated arterial stiffness, but not hypertension or vascular dysfunction. Mechanistic studies revealed that RhoBTB1 had no substantial effect on several classical arterial stiffness contributors such as collagen deposition, elastin content, and vascular smooth muscle remodeling. Instead, Ang-II increased actin polymerization in the aorta which was reversed by RhoBTB1. Changes in the levels of two regulators of actin polymerization, Cofilin and Vasodilator Stimulated Phosphoprotein (VASP), in response to RhoBTB1 were consistent with an actin depolymerization mechanism. Our study reveals an important function of RhoBTB1 and demonstrates its vital role in antagonizing established arterial stiffness and further supports a functional and mechanistic separation between hypertension, vascular dysfunction, and arterial stiffness.
Shi Fang, Jing Wu, John J. Reho, Ko-Ting Lu, Daniel T. Brozoski, Gaurav Kumar, Alec M. Werthman, Sebastiao Donato Silva Jr, Patricia C. Muskus Veitia, Kelsey K. Wackman, Angela J Mathison, Bi Qing Teng, Chien-Wei Lin, Frederick W. Quelle, Curt D. Sigmund
The capacity of ADAMTS3 to cleave proVEGFC into active VEGFC able to bind its receptors and to stimulate lymphangiogenesis has been clearly established during the embryonic life. However such function of ADAMTS3 is unlikely to persist in adulthood because of its restricted expression pattern after birth. Since ADAMTS2 and ADAMTS14 are closely related to ADAMTS3 and are mainly expressed in connective tissues where the lymphatic network extends, we hypothesized that they could substitute ADAMTS3 during adulthood in mammals for proteolytic activation of proVEGFC. Here, we demonstrated that ADAMTS2 and ADAMTS14 are able to process proVEGFC and activate the downstream pathway as efficiently as ADAMTS3. In vivo, adult mice lacking Adamts2 develop skin lymphedema due to a reduction of the density and diameter of lymphatic vessels leading to a decrease of lymphatic functionality, while genetic ablation of Adamts14 has no impact. In a model of thermal cauterization of cornea, lymphangiogenesis was significantly reduced in Adamts2 and Adamts14 knockout mice, and further repressed in Adamts2/Adamts14 double knockout mice. In summary, we have demonstrated that ADAMTS2 and ADAMTS14 are as efficient as ADAMTS3 for proVEGFC activation and are involved in the homeostasis of the lymphatic vasculature in adulthood, both in physiological and pathological processes.
Laura Dupont, Loïc Joannes, Florent Morfoisse, Silvia Blacher, Christine Monseur, Christophe F. Deroanne, Agnès Noël, Alain CMA Colige
Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). Very low-density lipoprotein receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids (FA). Since FA uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We find that excess circulating lipids restrain retinal autophagy, which contributes to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived FA sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased Sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.
Emilie Heckel, Gael Cagnone, Tapan Agnihotri, Bertan Cakir, Ashim Das, Jin Sung Kim, Nicholas Kim, Geneviève Lavoie, Anu Situ, Sheetal Pundir, Ye Sun, Florian Wünnemann, Kerry A. Pierce, Courtney Dennis, Grant A. Mitchell, Sylvain Chemtob, Flavio A. Rezende, Gregor Andelfinger, Clary B. Clish, Philippe P. Roux, Przemyslaw Sapieha, Lois E.H. Smith, Jean-Sébastien Joyal
Kawasaki disease (KD) is the leading cause of non-congenital heart disease in children. Studies in mice and humans propound the NLRP3-IL-1β pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of Inositol Requiring Enzyme-1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1β secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis, and highlight IRE1 RNase activity as a potential new therapeutic target.
Stefanie Marek-Iannucci, Asli D. Yildirim, Syed M. Hamid, Asli B. Ozdemir, Angela C. Gomez, Begüm Kocatürk, Rebecca A. Porritt, Michael C. Fishbein, Takao Iwawaki, Magali Noval Rivas, Ebru Erbay, Moshe Arditi
Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as a low flow-sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation - the physical removal of cilia from the cell surface - is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress will manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer ciliary-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared to healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (Arl13b) on their surface post-adhesion to brain ECs. Brain ECs post interaction with sickle RBCs show high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreases cilia protein levels on RBCs and rescues ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.
Ankan Gupta, Karthikeyan Thirugnanam, Madhan Thamilarasan, Ashraf M. Mohieldin, Hadeel T. Zedan, Shubhangi Prabhudesai, Meghan R. Griffin, Andrew D. Spearman, Amy Pan, Sean P. Palecek, Huseyin C. Yalcin, Surya M. Nauli, Kevin R. Rarick, Rahima Zennadi, Ramani Ramchandran
Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II–induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II–induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.
Changshun He, Bo Jiang, Mo Wang, Pengwei Ren, Sae-Il Murtada, Alexander W. Caulk, Guangxin Li, Lingfeng Qin, Roland Assi, Constantinos J. Lovoulos, Martin A. Schwartz, Jay D. Humphrey, George Tellides
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