Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.
Rui Si, Jody Tori O. Cabrera, Atsumi Tsuji-Hosokawa, Rui Guo, Makiko Watanabe, Lei Gao, Yun Sok Lee, Jae-Su Moon, Brian T. Scott, Jian Wang, Anthony W. Ashton, Jaladanki N. Rao, Jian-Ying Wang, Jason X.-J. Yuan, Ayako Makino
Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP–treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype–associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.
J. William Tierney, Brian C. Evans, Joyce Cheung-Flynn, Bo Wang, Juan M. Colazo, Monica E. Polcz, Rebecca S. Cook, Colleen M. Brophy, Craig L. Duvall
We recently described a previously unknown trans-tentorial venous system (TTVS) connecting venous drainage throughout the brain in humans. Prior to this finding, it was believed that the embryologic tentorial plexus regresses, resulting in a largely avascular tentorium. Our finding contradicted this understanding and necessitated further investigation into the development of the newly described TTVS. Herein we sought to investigate mice as a model to study the development of this system. First, using vascular casting and ex vivo micro-computed tomography (micro-CT), we demonstrate that this TTVS is conserved in adult mice. Next, using high-resolution magnetic resonance imaging (MRI), we found the primitive tentorial venous plexus in murine embryo at day 14.5. We also found that, at this embryologic stage, the tentorial plexus drains the choroid plexus. Finally, using vascular casting and micro-CT, we found that the TTVS is the dominant venous drainage in the early postnatal period (P8). Herein, we demonstrate that the TTVS is conserved between mice and humans and present a longitudinal study of its development. In addition, our findings establish mice as a translational model for further study of this newly described system and its relationship to intracranial physiology.
Pashayar P. Lookian, Vikram Chandrashekhar, Anthony Cappadona, Jean-Paul Bryant, Vibhu Chandrashekhar, Jessa M. Tunacao, Danielle R. Donahue, Jeeva P. Munasinghe, James G. Smirniotopoulos, John D. Heiss, Zhengping Zhuang, Jared S. Rosenblum
Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2-angiopoietin axis. Primary human endothelial cells treated with plasma from patients with severe COVID-19 upregulated expression of thromboinflammatory genes, inhibited expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from COVID-19 patients demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity and highest levels were associated with worse survival. These data highlight the disruption of Tie2-angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.
Alec A. Schmaier, Gabriel M. Pajares Hurtado, Zachary J. Manickas-Hill, Kelsey D. Sack, Siyu M. Chen, Victoria Bhambhani, Juweria Quadir, Anjali K. Nath, Ai-ris Y. Collier, Debby Ngo, Dan H. Barouch, Nathan I. Shapiro, Robert E. Gerszten, Xu Yu, Kevin G. Peters, Robert Flaumenhaft, Samir M. Parikh
Autophagy has long been associated with longevity and it is well established that autophagy reverts and prevents vascular deterioration associated with aging and cardiovascular diseases. Currently, our understanding of how autophagy benefits the vasculature is centered on the premise that reduced autophagy leads to the accumulation of cellular debris resulting in inflammation and oxidative stress, which are then reversed by reconstitution or upregulation of autophagic activity. Evolutionarily, autophagy also functions to mobilize endogenous nutrients in response to starvation. Therefore, we hypothesized that the biosynthesis of the most physiologically abundant ketone body, β-hydroxybutyrate (βHB), would be autophagy dependent, and exert vasodilatory effects via its canonical receptor, Gpr109a. We have revealed for the first time that the biosynthesis of βHB can be impaired by preventing autophagy. Subsequently, βHB caused potent vasodilation via potassium-channels, but not Gpr109a. Finally, we observed that chronic consumption of a high salt diet negatively regulates both βHB biosynthesis and hepatic autophagy, and that reconstitution of βHB bioavailability prevents high salt diet-induced endothelial dysfunction. In summary, this work offers an alternative mechanism to the anti-inflammatory and anti-oxidative stress hypothesis of autophagy-dependent vasculoprotection. Furthermore, it reveals a direct mechanism, by which ketogenic interventions (e.g., intermittent fasting) improve vascular health.
Cameron G. McCarthy, Saroj Chakraborty, Gagandeep Singh, Beng San Yeoh, Zachary J. Schreckenberger, Avinash Singh, Blair Mell, Nicole R. Bearss, Tao Yang, Xi Cheng, Matam Vijay-Kumar, Camilla F. Wenceslau, Bina Joe
The mutant nuclear lamin protein (progerin) produced in Hutchinson-Gilford progeria syndrome (HGPS) results in loss of arterial smooth muscle cells (SMCs), but the mechanism has been unclear. We found that progerin induces repetitive nuclear membrane (NM) ruptures, DNA damage, and cell death in cultured SMCs. Reducing lamin B1 expression and exposing cells to mechanical stress — to mirror conditions in the aorta — triggered more frequent NM ruptures. Increasing lamin B1 protein levels had the opposite effect, reducing NM ruptures and improving cell survival. Remarkably, raising lamin B1 levels increased nuclear compliance in cells and was able to offset the increased nuclear stiffness caused by progerin. In mice, lamin B1 expression in aortic SMCs is normally very low, and in mice with a targeted HGPS mutation (LmnaG609G), levels of lamin B1 decrease further with age while progerin levels increase. Those observations suggest that NM ruptures might occur in aortic SMCs in vivo. Indeed, studies in LmnaG609G mice identified NM ruptures in aortic SMCs, along with ultrastructural abnormalities in the cell nucleus that preceded SMC loss. Our studies identify NM ruptures in SMCs as likely causes of vascular pathology in HGPS.
Paul H. Kim, Natalie Y. Chen, Patrick J. Heizer, Yiping Tu, Thomas A. Weston, Jared L.-C. Fong, Navjot Kaur Gill, Amy C. Rowat, Stephen G. Young, Loren G. Fong
The prevalence of hypertension is increasing globally, while strategies for prevention and treatment of hypertension remain limited. FG-4592 (Roxadustat) is a novel, orally active small-molecule HIF stabilizer, and is being used clinically to treat CKD anemia. In the present study, we evaluate the effects of FG-4592 on hypertension. In an Ang II hypertension model, FG-4592 abolished hypertensive responses, prevented vascular thickening, cardiac hypertrophy, and kidney injury, downregulated AGTR1 expression, and enhanced AGTR2, eNOS, and HIF1α protein levels in the aortas of mice. Additionally, the levels of thiobarbituric acid reactive substances (TBARs) in blood and urine were diminished by FG-4592 treatment. In vascular smooth muscle cells, FG-4592 treatment reduced AGTR1 and increased AGTR2 levels, while preventing Ang II-induced oxidative stress. In vascular endothelial cells, FG-4592 upregulated total and phosphorylated eNOS. Moreover, FG-4592 treatment was hypotensive in L-NAME-induced hypertension. In summary, FG-4592 treatment remarkably ameliorated hypertension and organ injury, possibly through stabilizing HIF1α and subsequently targeting eNOS, AGTR1, AGTR2, and oxidative stress. Therefore, in addition to its role in treating CKD anemia, FG-4592 could be explored as a treatment for hypertension associated with high RAS activity or eNOS defects.
Jing Yu, Shuqin Wang, Wei Shi, Wei Zhou, Yujia Niu, Songming Huang, Yue Zhang, Aihua Zhang, Zhanjun Jia
Neutrophils provide a critical line of defense in immune responses to various pathogens, but also inflict self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients suffering from COVID-19. Here, we utilize state-of-the art mass spectrometry-based proteomics, transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic interleukin-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophil-driven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces SARS-CoV-2 spike protein-induced, hACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8-axis as promising therapeutic target in severe SARS-CoV-2 infection.
Rainer Kaiser, Alexander Leunig, Kami Pekayvaz, Oliver Popp, Markus Joppich, Vivien Polewka, Raphael Escaig, Afra Anjum, Marie-Louise Hoffknecht, Christoph Gold, Sophia Brambs, Anouk Engel, Sven Stockhausen, Viktoria Knottenberg, Anna Titova, Mohamed Haji, Clemens Scherer, Maximilian Muenchhoff, Johannes C. Hellmuth, Kathrin Saar, Benjamin Schubert, Anne Hilgendorff, Christian Schulz, Stefan Kääb, Ralf Zimmer, Norbert Hübner, Steffen Massberg, Philipp Mertins, Leo Nicolai, Konstantin Stark
Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Murine and human data suggest that the NLRP3-IL-1β pathway is the main driver of KD pathophysiology. NLRP3 can be activated during defective autophagy/mitophagy. We used the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, to examine the role of autophagy/mitophagy on cardiovascular lesion development. LCWE-injected mice had impaired autophagy/mitophagy and increased levels of ROS in cardiovascular lesions, together with increased systemic 8-OHdG release. Enhanced autophagic flux significantly reduced cardiovascular lesions in LCWE-injected mice, whereas autophagy blockade increased inflammation. Vascular smooth muscle cell specific deletion of Atg16l1 and global Parkin-/- significantly increased disease formation, supporting the importance of autophagy/mitophagy in this model. Ogg1-/- mice had significantly increased lesions with increased NLRP3 activity, whereas treatment with MitoQ, reduced vascular tissue inflammation, ROS production and systemic 8-OHdG release. Treatment with MN58b or Metformin (increasing AMPK and reducing ROS), resulted in decreased disease formation. Our results demonstrate that impaired autophagy/mitophagy and ROS-dependent damage exacerbate the development of murine KD vasculitis. This pathway can be efficiently targeted to reduce disease severity. These findings enhance our understanding of KD pathogenesis and identify novel therapeutic avenues for KD treatment.
Stefanie Marek-Iannucci, A. Beyza Ozdemir, Debbie Moreira, Angela C. Gomez, Malcolm Lane, Rebecca A Porritt, Youngho Lee, Kenichi Shimada, Masanori Abe, Aleksandr Stotland, David Zemmour, Sarah Parker, Elsa Sanchez-Lopez, Jennifer Van Eyk, Roberta A. Gottlieb, Michael Fishbein, Michael Karin, Timothy R Crother, Magali Noval Rivas, Moshe Arditi
Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbate disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in COVID-19 patients and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, two signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in COVID-19 patients. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation leading to production of pathological HA fragments which are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.
Kimberly A. Queisser, Rebecca A. Mellema, Elizabeth A. Middleton, Irina Portier, Bhanu Kanth Manne, Frederik Denorme, Ellen J. Beswick, Matthew T. Rondina, Robert A. Campbell, Aaron C. Petrey
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