Vascular biology
Abstract
Mutation of KRAS in endothelial cells (KRAS-EC) leads to intracerebral hemorrhage (ICH) in brain arteriovenous malformations (bAVM), resulting in severe disabilities or even death. However, it is unclear what causes this hemorrhagic conversion of bAVM. Here, using a locally established, clinically-relevant sporadic bAVM mouse model, created by overexpressing mutant KRAS (KRASG12V) in the brain EC, we demonstrate that KRAS-EC act as trigger for microglia (MG) activation and infiltration of macrophages (Mϕ). Using three-dimensional immunostaining approach with cleared human and mouse bAVM tissues, we demonstrate an abundance of MG/Mϕ around the bAVM nidus. The presence of MG/Mϕ are correlated to the blood-brain barrier leakage in bAVM area. Time-lapsed intravital imaging in Cx3cr1-gfp;Ccr2-rfp reporter mice demonstrate the dynamic activation of MG and infiltration of Mϕ toward mutant KRAS-modified dysplastic vessels. Importantly, a time course analysis showed that these activated/infiltrated MG/Mϕ are present around the bAVMs prior to hemorrhagic conversion, and controlled depletion of MG/Mϕ reduced ICH incidence in bAVM. Inhibition of MG/Mϕ with long-term minocycline treatment attenuated the incidence of ICHs around bAVMs. Our study indicates that MG/Mϕ are involved in destabilization of KRAS-induced bAVM, leading to hemorrhagic conversion/ICH. Thus, modulation of MG/Mϕ may reduce ICH risk in bAVM patients.
Authors
Hyejin Park, Jung-Eun Park, Bridger H. Freeman, Bosco Seong Kyu Yang, Shun-Ming Ting, Alexander K. Suh, Jude P.J. Savarraj, Shuning Huang, Jakob Körbelin, Huimahn Alex Choi, Sean P. Marrelli, Jaroslaw Aronowski, Peng Roc Chen, Eunhee Kim, Eun S. Park
Abstract
Although renal fibrosis is predominantly driven by the accumulated inflammatory cells that secrete pro-inflammatory factors within the kidney, the key mechanisms underlying macrophage clearance from the kidney are not well understood. The interaction of hyaluronan (HA) with lymphatic endothelial hyaluronan receptor 1 (LYVE1) constitutes a critical initial step in macrophage adhesion and removal by lymphatic vessels. This study investigates alterations in LYVE1 during kidney disease and elucidates its role in macrophage trafficking. Three renal fibrosis models demonstrated a reduction in full-length LYVE1 and an increase in the soluble LYVE1 fragment. Immunostaining of fibrotic kidneys showed significantly reduced expression of soluble LYVE1 compared with intracellular fragment (Cyto-LYVE1), demonstrating ectodomain shedding of LYVE1 in vivo and in vitro. Functionally, human lymphatic endothelial cells exposed to TGF-β1 exhibited significant decrease in macrophage adhesion and transendothelial migration compared to controls. Mechanistic analyses identified increased matrix metalloproteinase (MMP)9 in renal injury as a key upstream regulator of LYVE1 shedding. MMP9 inhibitors reduced LYVE1 shedding, enhanced macrophage adhesion and trafficking, and mitigated macrophage accumulation and disease progression. In conclusion, MMP9-induced LYVE1 shedding is linked to progressive kidney fibrosis and macrophage accumulation. LYVE1 shedding inhibitors offer potential as therapeutic agents for mitigating immune overload and kidney fibrosis.
Authors
Jing Liu, Yuqing Liu, Wenqian Zhou, Saiya Zhu, Jianyong Zhong, Haichun Yang, Annet Kirabo, Valentina Kon, Chen Yu
Abstract
BACKGROUND. Idiopathic pulmonary arterial hypertension (IPAH) alters right ventricular size and function, curtailing life-expectancy. Patients may experience angina and myocardial ischemia. However, the mechanisms underlying these changes are poorly understood. METHODS. A cross-sectional, case-control design of coronary pathophysiology (in vivo and ex vivo) in IPAH. Patients with IPAH (Group-1.1) undergoing clinically indicated right heart catheterization were prospectively enrolled. Participants underwent functional testing during coronary angiography using a dual pressure/temperature-sensitive guidewire. Cardiovascular magnetic resonance measured left and right ventricular mass and function. Autopsy cardiac tissues from end-stage PAH (Group-1) and control individuals were analyzed for right ventricular pathophysiology. RESULTS. Eleven participants with IPAH and 15 control participants completed the protocol (IPAH: 45±15 years, 73% female; controls: 58.3±9.1 years, 73% female). 73% (n=8) of IPAH patients had an elevated index of microcirculatory resistance (IMR >25) and 55% (n=6) had reduced coronary flow reserve (CRF <2.0). The mean IMR was significantly higher in IPAH participants (39.2±27.0 vs. 15.3±5.0, p=0.002) whereas mean CFR was lower (2.8±2.1 vs. 4.0±1.4; p=0.077). Paired right coronary artery/ventricular measurements (n=6) revealed IMR positively correlated with right ventricular mass (r=0.91, p=0.12), and negatively with CFR (r=-0.82, p=0.046). Compared to controls (n=5), PAH participants (n=4) had reduced right ventricular capillary density (111±18 vs. 167±20, p=0.032), increased cardiomyocyte area (383±118μm2 vs. 231±61μm2, p=0.0390), and increased mural area in small pre-capillary arterioles (127±10μm2 vs. 107±20μm2, p=0.041). CONCLUSIONS. Coronary microvascular dysfunction is prevalent in IPAH and correlates with increased right ventricular mass. Histopathology revealed vascular rarefaction and remodeling of pre-capillary arterioles. The clinical significance merits prospective evaluation. Invasive coronary function testing was feasible and safe in IPAH, providing a platform to assess therapeutic impacts on cardiac microvascular function.
Authors
Erin Boland, Michael G. Freeman, David S. Corcoran, Thomas J. Ford, Barry Hennigan, Damien Collison, Aida Llucià-Valldeperas, Frances S. de Man, Kanarath P. Balachandran, Martin Johnson, Colin Church, Colin Berry
Abstract
The central nervous system (CNS) parenchyma has conventionally been believed to lack lymphatic vasculature, likely owing to a non-permissive microenvironment that hinders the formation and growth of lymphatic endothelial cells (LECs). Recent findings of ectopic expression of LEC markers including prospero homeobox 1 (PROX1), a master regulator of lymphatic differentiation, and the vascular permeability marker plasmalemma vesicle–associated protein (PLVAP) in certain glioblastomas (GBM) and brain arteriovenous malformations have prompted investigation into their roles in cerebrovascular malformations, tumor environments, and blood-brain barrier (BBB) abnormalities. To explore the relationship between ectopic LEC properties and BBB disruption, we used endothelial cell–specific Prox1 overexpression mutants. When induced during embryonic stages of BBB formation, endothelial Prox1 expression induces hybrid blood-lymphatic phenotypes in the developing CNS vasculature. This effect is not observed when Prox1 is overexpressed during postnatal BBB maturation. Ectopic Prox1 expression leads to significant vascular malformations and enhanced vascular leakage, resulting in BBB disruption when induced during both embryonic and postnatal stages. Mechanistically, PROX1 downregulates critical BBB-associated genes, including β-catenin and claudin-5, which are essential for BBB development and maintenance. These findings suggest that PROX1 compromises BBB integrity by negatively regulating BBB-associated gene expression and Wnt/β-catenin signaling.
Authors
Sara González-Hernández, Ryo Sato, Yuya Sato, Chang Liu, Wenling Li, Zulfeqhar A. Syed, Chengyu Liu, Sadhana Jackson, Yoshiaki Kubota, Yoh-suke Mukouyama
Abstract
Clonal haematopoiesis of indeterminate potential (CHIP) is the expansion of blood stem cells and progeny after somatic mutation. CHIP associates with increased cardiovascular disease (CVD) with inflammation from macrophages a proposed common effector. However, mouse CHIP studies are discordant for clonal expansion and inflammation. Similarly, directionality of association between CHIP and CVD remains debated. We investigated effects of three CHIP mutations on macrophage cytokines, clonal expansion and atherosclerosis in parallel. We find that Tet2 and Dnmt3a mutations increase cytokines and inflammasome activation in Tet2 but decrease in Dnmt3a. However, Jak2 mutant macrophages produced equivalent cytokine as wild-type. In mice, Tet2 mutants clonally expanded, but Dnmt3a and Jak2 mutants didn’t. Expansion was unaffected by systemic inflammation, while hyperlipidemia expanded Tet2-/- cells, but not mono-allelic mutants. Similarly, human Mendelian randomisation showed no effect of serum cytokines or CVD on CHIP risk. Experimental atherosclerosis was increased in females with Tet2 and males with Jak2, but unchanged with Dnmt3a mutations. Together, common CHIP mutations have disparate effects on macrophage cytokines and clonal expansion, and sex-dependent effects on atherogenesis, suggesting a common mechanism across CHIP is unlikely. Thus, CHIP mutations differ in pathophysiology and clinical sequalae across sexes and should be treated as different entities.
Authors
Paul R. Carter, Lauren Kitt, Amanda Rodgers, Nichola Figg, Ang Zhou, Chengrui Zhu, Ziyang Wang, Peter Libby, Stephen Burgess, George S. Vassiliou, Murray CH. Clarke
Abstract
BACKGROUND Platelets are increasingly recognized as active participants in immune signaling and systemic inflammation. Upon activation, platelets form monocyte platelet aggregates (MPA) representing the crossroads of thrombosis and inflammation. We hypothesized that platelet transcriptomics could capture this thromboinflammatory axis and identify individuals at elevated cardiovascular risk.METHODS: MPA levels, defined as CD14+CD61+ cells, were measured using flow cytometry at 2 time points, 4 weeks apart, in healthy individuals Platelets were isolated and sequenced. Individuals were categorized as MPAhi or MPAlo based on consistently high or low MPA levels across time points.RESULTS Among 149 participants (median age 52 years, 57% female, 50% non-White), MPAhi individuals exhibited increased expression of platelet activation markers P-selectin (P < 0.001), PAC-1 (P = 0.021), and CD40L (P < 0.001) and enriched immune signaling pathways. Informed by MPA levels and derived from the platelet transcriptome, we developed a 42-gene thromboinflammation platelet signature (TIPS), which correlated with MPA levels in multiple cohorts and was reproducible over time. TIPS was elevated in patients with COVID-19 (P = 0.0002) and myocardial infarction (Padj = 0.008), and as in predicted future cardiovascular events in patients who underwent lower extremity revascularization after a median follow-up of 18 months (adjusted for age, sex, race, and ethnicity [adjHR] 1.55, P = 0.006). Notably, TIPS was modifiable by ticagrelor (P = 0.002) but not aspirin.CONCLUSION These findings establish MPA as a biomarker of thromboinflammation and introduce TIPS, a platelet RNA signature, that captures thromboinflammation and provides a promising tool for cardiovascular risk stratification and a potential therapeutic target.TRIAL REGISTRATION NCT04369664FUNDING NIH R35HL144993, NIH R01HL139909, and AHA 16SFRN2873002 to JSB, DFG Walter-Benjamin-Programme 537070747 to AB.
Authors
Antonia Beitzen-Heineke, Matthew A. Muller, Yuhe Xia, Elliot Luttrell-Williams, Florencia Schlamp, Deepak Voora, Kelly V. Ruggles, Michael S. Garshick, Tessa J. Barrett, Jeffrey S. Berger
Abstract
Pathogenic variants in kinesin KIF11 underlie microcephaly-lymphedema-chorioretinopathy (MLC) syndrome. Although well known for regulating spindle dynamics ensuring successful cell division, the association of KIF11 (encoding EG5) with development of the lymphatic system, and how KIF11 pathogenic variants lead to lymphatic dysfunction and lymphedema remain unknown. Using patient-derived lymphoblastoid cells, we demonstrated that MLC patients carrying pathogenic stop-gain variants in KIF11 have reduced mRNA and protein levels. Lymphoscintigraphy showed reduced tracer absorption, and intestinal lymphangiectasia was detected in one patient, pointing to impairment of lymphatic function caused by KIF11 haploinsufficiency. We revealed that KIF11 is expressed in early human and mouse development with the lymphatic markers VEGFR3, Podoplanin and PROX1. In zebrafish, scRNA-seq identified kif11 specifically expressed in endothelial precursors. In human lymphatic endothelial cells (LECs), EG5 inhibition with Ispinesib, reduced VEGFC-driven AKT phosphorylation, migration and spheroid sprouting. KIF11 knockdown reduced PROX1 and VEGFR3 expression, providing for the first time a link between KIF11 and drivers of lymphangiogenesis and lymphatic identity.
Authors
Kazim Ogmen, Sara E. Dobbins, Rose Yinghan Behncke, Ines Martinez-Corral, Ryan C.S. Brown, Michelle Meier, Sascha Ulferts, Nils Rouven Hansmeier, Ege Sackey, Ahlam Alqahtani, Christina Karapouliou, Dionysios Grigoriadis, Juan C. Del Rey Jimenez, Michael Oberlin, Denise Williams, Arzu Ekici, Kadri Karaer, Steve Jeffery, Peter Mortimer, Kristiana Gordon, Kazuhide S. Okuda, Benjamin M. Hogan, Taija Mäkinen, René Hägerling, Sahar Mansour, Silvia Martin-Almedina, Pia Ostergaard
Abstract
Authors
Filip Hanak, Jessica L. Swanson, Krzysztof Felczak, Prakashkumar Dobariya, Ursula C.H. Girdwood, Kenneth E. Bernstein, Swati S. More, Patrick E. Rothwell
Abstract
Pulmonary arterial hypertension (PAH) is a rare and incurable disease characterized by progressive narrowing of pulmonary arteries (PA), resulting in right ventricular (RV) hypertrophy, RV failure, and eventually death. Orai1 inhibition has emerged as promising therapeutic approach to mitigate PAH. In this study, we investigated the efficacy of a clinically applicable selective Orai1 inhibitor, CM5480, and its effects when combined with standard PAH therapies in a preclinical PAH model. In male and female monocrotaline PAH-rats, CM5480 monotherapy improved hemodynamics, PA, and RV remodeling, as confirmed by RV catheterization, echocardiography, histology, and unbiased RNA-Seq. Standard PAH therapies, ambrisentan or sildenafil, achieved modest improvements in experimental PAH. In contrast, combination therapies with CM5480 yielded significantly greater benefits in reducing PA remodeling and improving cardiac function compared with monotherapies. Furthermore, in vitro experiments showed that Orai1 knockdown reduced pulmonary endothelial cell dysfunction in PAH and that the Orai1 pathway is independent of standard PAH-targeted pathways in PA smooth muscle cells (PASMCs). Finally, we found enhanced Orai1 expression/function in PASMCs and pulmonary vein SMCs from patients with pulmonary veno-occlusive disease. These findings suggest that Orai1 inhibition represents a potentially novel and complementary therapeutic strategy for PAH by acting at pulmonary vascular and RV levels.
Authors
Anaïs Saint-Martin Willer, Grégoire Ruffenach, Bastien Masson, Kristelle El Jekmek, Angèle Boët, Rui Adão, Mathieu Gourmelon, Antoine Beauvais, Jessica Sabourin, Mary Dutheil, Maria-Rosa Ghigna, Laurent Tesson, Séverine Ménoret, Ignacio Anegon, Fabrice Bauer, Vincent de Montpréville, Sudarshan Hebbar, Carmen Brás-Silva, Kenneth Stauderman, Marc Humbert, Olaf Mercier, David Montani, Véronique Capuano, Fabrice Antigny
Abstract
Angiopoietin-like 3 (ANGPTL3) is a major regulator of lipoprotein metabolism. ANGPTL3 deficiency results in lower levels of triglycerides, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C), and may protect from cardiovascular disease. ANGPTL3 oligomerizes with ANGPTL8 to inhibit lipoprotein lipase (LPL), the enzyme responsible for plasma triglyceride hydrolysis. Independent of ANGPTL8, oligomers of ANGPTL3 can inhibit endothelial lipase (EL), which regulates circulating HDL-C and LDL-C levels through the hydrolysis of lipoprotein phospholipids. The N-terminal region of ANGPTL3 is necessary for both oligomerization and lipase inhibition. However, our understanding of the specific residues that contribute to these functions is incomplete. In this study, we performed mutagenesis of the N-terminal region to identify residues important for EL inhibition and oligomerization. We also assessed the presence of different ANGPTL3 species in human plasma. We identified a motif important for lipase inhibition, and protein structure prediction suggested that this region interacted directly with EL. We also found that recombinant ANGPTL3 formed a homotrimer and was unable to inhibit EL activity when trimerization was disrupted. Surprisingly, we observed that human plasma contained more monomeric ANGPTL3 than trimeric ANGPTL3. An important implication of these findings is that previous correlations between circulating ANGPTL3 and circulating triglyceride-rich lipoproteins need to be revisited.
Authors
Sydney G. Walker, Yan Q. Chen, Kelli L. Sylvers-Davie, Alex Dou, Eugene Y. Zhen, Yuewei Qian, Yi Wen, Mariam E. Ehsani, Sydney A. Smith, Rakshya Thapa, Maxwell J. Mercer, Lucy Langmack, Bharat Raj Bhattarai, Michael Ploug, Robert J. Konrad, Brandon S.J. Davies
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