Brain vascular calcification is a prevalent age-related condition often accompanying neurodegenerative and neuroinflammatory diseases. The pathogenesis of large vessel calcifications in peripheral tissue is well-studied, but microvascular calcification in the brain remains poorly understood. Here, we report that elevated platelet-derived growth factor BB (PDGF-BB) from bone preosteoclasts contribute to cerebrovascular calcification in male mice. Aged male mice exhibited higher serum PDGF-BB levels and a significantly higher incidence of brain calcification compared to young mice, mainly in the thalamus. Transgenic mice with preosteoclast-specific Pdgfb overexpression exhibited elevation of serum PDGF-BB levels and recapitulated age-associated thalamic calcification. Conversely, mice with preosteoclast-specific Pdgfb deletion displayed diminished age-associated thalamic calcification. In an ex vivo cerebral microvascular culture system, PDGF-BB dose-dependently promoted vascular calcification. Analysis of osteogenic gene array and single-cell RNA sequencing revealed that PDGF-BB upregulates multiple osteogenic differentiation genes and the phosphate transporter Slc20a1 in cerebral microvessels. Mechanistically, PDGF-BB stimulated the phosphorylation of its receptor PDGFRβ (pPDGFRβ) and ERK (p-ERK), leading to the activation of RUNX2. This activation, in turn, induced the transcription of the osteoblast differentiation genes in pericytes and upregulated Slc20a1 in astrocytes. Thus, bone-derived PDGF-BB induces brain vascular calcification by activating the pPDGFRβ/p-ERK/RUNX2 signaling cascade in cerebrovascular cells.
Jiekang Wang, Ching-Lien Fang, Kathleen Noller, Zhiliang Wei, Guanqiao Liu, Ke Shen, Kangping Song, Xu Cao, Mei Wan
The endothelium plays a critical role in the host response to infection, and has been a focus of investigation in sepsis. While it is appreciated that intravascular thrombus formation, severe inflammation, and loss of endothelial integrity impair tissue oxygenation during sepsis, the precise molecular mechanisms that lead to endothelial injury remain poorly understood. We demonstrate herein that endothelial ADAM10 is essential for the pathogenesis of Staphylococcus aureus sepsis, contributing to a-toxin (Hla)-mediated microvascular thrombus formation and lethality. As ADAM10 is essential for endothelial development and homeostasis, we examined whether other major human sepsis pathogens also rely on ADAM10-dependent pathways in pathogenesis. Mice harboring an endothelial-specific knockout of ADAM10 are protected against lethal Pseudomonas aeruginosa and Streptococcus pneumoniae sepsis, yet remain fully susceptible to Group B Streptococci and Candida albicans sepsis. These studies illustrate a previously unknown role for ADAM10 in sepsis-associated endothelial injury, and suggest that understanding pathogen-specific divergent host pathways in sepsis may enable more precise targeting of disease.
Danielle N. Alfano, Mark J. Miller, Juliane Bubeck Wardenburg
Endothelial dysfunction is a critical and initiating factor of the vascular complications of diabetes. Inflammation plays an important role in endothelial dysfunction regulated by epigenetic modifications. N6-methyladenosine (m6A) is one of the most prevalent epigenetic modifications in eukaryotic cells. In this research, we identified an m6A demethylase, fat mass and obesity-associated protein (FTO), as an essential epitranscriptomic regulator in diabetes-induced vascular endothelial dysfunction. We showed that enhanced FTO reduced the global level of m6A in hyperglycemia. FTO knockdown in endothelial cells (ECs) resulted in less inflammation and compromised ability of migration and tube formation. Compared with EC Ftofl/fl diabetic mice, EC-specific Fto-deficient (EC FtoΔ/Δ) diabetic mice displayed less retinal vascular leakage and acellular capillary formation. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) combined with RNA-Seq indicated that Tnip1 served as a downstream target of FTO. Luciferase activity assays and RNA pull-down demonstrated that FTO repressed TNIP1 mRNA expression by erasing its m6A methylation. In addition, TNIP1 depletion activated NF-κB and other inflammatory factors, which aggravated retinal vascular leakage and acellular capillary formation, while sustained expression of Tnip1 by intravitreal injection of adeno-associated virus alleviated endothelial impairments. These findings suggest that the FTO-TNIP1-NF-κB network provides potential targets to treat diabetic vascular complications.
Chuandi Zhou, Xinping She, Chufeng Gu, Yanan Hu, Mingming Ma, Qinghua Qiu, Tao Sun, Xun Xu, Haibing Chen, Zhi Zheng
Endothelial phospholipase Cγ (PLCγ) is essential for vascular development, however, its role in healthy, mature or pathological vessels is unexplored. PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2+/Y1173F mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B-cells, CD4+ and regulatory T-cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS, and suppressed vascular leakage. Thus, we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier resulting in loss of anti-tumor immunity.
Elin Sjöberg, Marit Melssen, Mark Richards, Yindi Ding, Catarina Chanoca, Dongying Chen, Emmanuel Nwadozi, Sagnik Pal, Dominic T. Love, Takeshi Ninchoji, Masabumi Shibuya, Michael Simons, Anna Dimberg, Lena Claesson-Welsh
Human cancers induce a chaotic, dysfunctional vasculature that promotes tumor growth and dampens most current therapies, but the underlying mechanism has been unclear. Here we show that SPEN (split end), a transcription repressor, coordinates ribosome RNA (rRNA) synthesis in endothelial cells (ECs) and is required for physiological and tumor angiogenesis. SPEN deficiency attenuated EC proliferation and blunted retinal angiogenesis, which was attributed to p53 activation. Furthermore, SPEN knockdown activated p53 by upregulating the noncoding promoter RNA (pRNA), which represses rRNA transcription and triggers p53-mediated nucleolar stress. In human cancer biopsies, low endothelial SPEN level correlated with extended overall survival. Consistently in mice, endothelial SPEN deficiency compromised rRNA expression and repressed tumor growth and metastasis by normalizing tumor vessels, which was abrogated by p53 haploinsufficiency. rRNA gene transcription is driven by RNA polymerase I (RNPI). We found that CX-5461, an RNPI inhibitor, recapitulated the effect of Spen ablation on tumor vessel normalization, and combining CX-5461 with cisplatin substantially improved the efficacy on treating tumors in mice. Together, these results demonstrate that SPEN is required for angiogenesis by repressing pRNA to enable rRNA gene transcription and ribosomal biogenesis, and that RNPI represents a target for tumor vessel normalization therapy of cancer.
Zi-Yan Yang, Xian-Chun Yan, Jia-Yu-Lin Zhang, Liang Liang, Chun-Chen Gao, Pei-Ran Zhang, Yuan Liu, Jia-Xing Sun, Bai Ruan, Juan-Li Duan, Ruo-Nan Wang, Xing-Xing Feng, Bo Che, Tian Xiao, Hua Han
Vascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with Bone Morphogenic Protein 9 (BMP9) and BMP10. Conversely, mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in vascular development and homeostasis, and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between fluid shear stress and SMAD signaling in vascular stability and its failure in HHT. We have now found that loss of Smad4 increases ECs’ sensitivity to flow by lowering the FSS set point with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling leading to cell cycle progression - mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors, CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.
Kuheli Banerjee, Yanzhu Lin, Johannes Gahn, Julio Cordero, Purnima Gupta, Islam Mohamed, Mariona Graupera, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
BACKGROUND. Cellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than High Density Lipoprotein-Cholesterol (HDL-C) but is not suitable as a routine clinical assay. METHODS. We developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD); Study I: NIH severe-CAD (n=50) and non-CAD (n=50) subjects, frequency matched for gender, BMI, Type 2-diabetes mellitus and smoking; Study II: Japanese CAD (n=70) and non-CAD (n=154) subjects. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 cases to 340 controls individually matched for age, sex, smoking and HDL-C levels. RESULTS. Receiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. AUC in Study I: HDL-SPE, 0.68; apoA-I, 0.62; HDL-C, 0.63; CEC, 0.52. AUC in Study II: HDL-SPE, 0.83; apoA-I, 0.64; HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with odds ratios ˂ 0.2 per SD increment in the PREVEND study (p<0.001). CONCLUSION. HDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery. TRIAL REGISTRATION. ClinicalTrials.gov: NCT01621594; Jichi Medical University study protocols C17-R007, 122, 142 and 158; University Medical Center Groningen, Netherlands study approval number: MEC96/01/022. FUNDING. This work was supported by the NIH, NHLBI Intramural Research Program.
Masaki Sato, Edward B. Neufeld, Martin P. Playford, Yu Lei, Alexander V. Sorokin, Angel M. Aponte, Lita A. Freeman, Scott M. Gordon, Amit K. Dey, Kianoush Jeiran, Masato Hamasaki, Maureen L. Sampson, Robert D. Shamburek, Jingrong Tang, Marcus Y. Chen, Kazuhiko Kotani, Josephine L.C. Anderson, Robin P.F. Dullaart, Nehal N. Mehta, Uwe J.F. Tietge, Alan T. Remaley
Nirmal S. Sharma, Kapil Patel, Ezgi Sari, Shruti Shankar, Maria G. Gastanadui, Diego Moncada-Giraldo, Yixel M. Soto-Vázquez, Delores A. Stacks, Louise Hecker, Kevin G. Dsouza, Mudassir Banday, Edward O'Neill, Paul Benson, Gregory A. Payne, Camilla Margaroli, Amit Gaggar
Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4–expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.
Pearl Quijada, Shuin Park, Peng Zhao, Kamal S.S. Kolluri, David Wong, Kevin D. Shih, Kai Fang, Arash Pezhouman, Lingjun Wang, Ali Daraei, Matthew D. Tran, Elle M. Rathbun, Kimberly N. Burgos Villar, Maria L. Garcia-Hernandez, Thanh T.D. Pham, Charles J. Lowenstein, M. Luisa Iruela-Arispe, S. Thomas Carmichael, Eric M. Small, Reza Ardehali
Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage, and secrete factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse senohemostasis, and improve vascular disease.
Allison B. Herman, Dimitrios Tsitsipatis, Carlos Anerillas, Krystyna Mazan-Mamczarz, Angelica E. Carr, Jordan M. Gregg, Mingyi Wang, Jing Zhang, Marc Michel, Charnae' Henry-Smith, Sophia C. Harris, Rachel Munk, Jennifer L Martindale, Yulan Piao, Jinshui Fan, Julie A. Mattison, Supriyo De, Kotb Abdelmohsen, Robert W. Maul, Toshiko Tanaka, Ann Z. Moore, Megan E. DeMouth, Simone Sidoli, Luigi Ferrucci, Yie Liu, Rafael de Cabo, Edward G. Lakatta, Myriam Gorospe