Angiogeneses
Abstract
Loss of function mutations in CCM genes and gain of function mutation in the MAP3K3 gene encoding MEKK3 cause cerebral cavernous malformation (CCM). Deficiency of CCM proteins leads to the activation of MEKK3-KLF2/4 signaling, but it is not clear how this occurs. Here we demonstrate that deletion of the CCM3 interacting kinases STK24/25 in endothelial cells cause defects in vascular patterning during development as well as CCM lesion formation during postnatal life. While permanent deletion of STK24/25 in endothelial cells caused developmental defects of the vascular system, inducible postnatal deletion of STK24/25 impaired angiogenesis in the retina and brain. More importantly, deletion of STK24/25 in neonatal mice led to the development of severe CCM lesions. At the molecular level, a hybrid protein consisting of the STK kinase domain and the MEKK3 interacting domain of CCM2 rescued the vascular phenotype caused by the loss of ccm gene function in zebrafish. Our study suggests that CCM2/3 proteins act as adapters to allow recruitment of STK24/25 to limit the constitutive MEKK3 activity that contributes to vessel stability. Loss of STK24/25 causes MEKK3 activation leading to CCM lesion formation.
Authors
Xi Yang, Shi-Ting Wu, Rui Gao, Rui Wang, Yixuan Wang, Zhenkun Dong, Lu Wang, Chunxiao Qi, Xiaohong Wang, M. Lienhard Schmitz, Renjing Liu, Zhiming Han, Lu Wang, Xiangjian Zheng
Abstract
Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and characterize the transcriptional profiles and metabolic pathways of pro-angiogenic microglia in a mouse model of oxygen-induced proliferative retinopathy (OIR). Using transcriptional single-cell sorting, we comprehensively map all microglia populations in retinas of room air (RA) and OIR mice. We unveil several unique types of PR-associated microglia (PRAM) and identify markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hyper-metabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes and pro-angiogenic insulin-like growth factor 1. Immunohistochemical staining shows these PRAMs were spatially located within or around neovascular (NV) tufts. These unique microglia-types have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis-related diseases.
Authors
Zhiping Liu, Huidong Shi, Jiean Xu, Qiuhua Yang, Qian Ma, Xiaoxiao Mao, Zhimin Xu, Yaqi Zhou, Qingen Da, Yongfeng Cai, David J.R. Fulton, Zheng Dong, Akrit Sodhi, Ruth B. Caldwell, Yuqing Huo
Abstract
The (Pro)renin receptor ((P)RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell (EC)-specific Atp6ap2 knockout mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2 deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the extracellular-signal-regulated kinase (ERK) pathway. Furthermore, Atp6ap2 deficient retinas exhibited reduced revascularization in an oxygen induced retinopathy model. Collectively, our results demonstrated a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.
Authors
Nehal R. Patel, Rajan K C, Avery E. Blanks, Yisu Li, Minolfa C. Prieto, Stryder M. Meadows
Abstract
Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation as well as immune and vascular signaling by protease activated receptors (PARs). Here, we show that endothelial EPCRPAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCRPAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR−PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.
Authors
Magdalena L. Bochenek, Rajinikanth Gogiraju, Stefanie Großmann, Janina Krug, Jennifer Orth, Sabine Reyda, George S. Georgiadis, Henri Spronk, Stavros Konstantinides, Thomas Münzel, John H. Griffin, Philipp S. Wild, Christine Espinola-Klein, Wolfram Ruf, Katrin Schäfer
Abstract
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.
Authors
Qing Ye, Jian Zhang, Chen Zhang, Bing Yi, Kyosuke Kazama, Wennan Liu, Xiaobo Sun, Yan Liu, Jianxin Sun
Abstract
Malignant pleural effusion (MPE) is an incurable common manifestation of many malignancies. Its formation is orchestrated by complex interactions among tumor cells, inflammatory cells, and the vasculature. Tumor-associated macrophages present the dominant inflammatory population of MPE, and M2 macrophage numbers account for dismal prognosis. M2 polarization is known to be triggered by CSF1/CSF1 receptor (CSF1R) signaling. We hypothesized that CSF1R+ M2 macrophages favor MPE formation and could be therapeutically targeted to limit MPE. We generated mice with CSF1R-deficient macrophages and induced lung and colon adenocarcinoma–associated MPE. We also examined the therapeutic potential of a clinically relevant CSF1R inhibitor (BLZ945) in lung and colon adenocarcinoma–induced experimental MPE. We showed that CSF1R+ macrophages promoted pleural fluid accumulation by enhancing vascular permeability, destabilizing tumor vessels, and favoring immune suppression. We also showed that CSF1R inhibition limited MPE in vivo by reducing vascular permeability and neoangiogenesis and impeding tumor progression. This was because apart from macrophages, CSF1R signals in cancer-associated fibroblasts leading to macrophage inflammatory protein 2 secretion triggered the manifestation of suppressive and angiogenic properties in macrophages upon CXCR2 paracrine activation. Pharmacological targeting of the CSF1/CSF1R axis can therefore be a vital strategy for limiting MPE.
Authors
Chrysavgi N. Kosti, Photene C. Vaitsi, Apostolos G. Pappas, Marianthi P. Iliopoulou, Katherina K. Psarra, Sophia F. Magkouta, Ioannis T. Kalomenidis
Abstract
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.
Authors
Laura Dupont, Loïc Joannes, Florent Morfoisse, Silvia Blacher, Christine Monseur, Christophe F. Deroanne, Agnès Noël, Alain CMA Colige
Abstract
Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss of function mutations in RASA1 or EPHB4 genes that encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4) respectively. However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell (EC)-specific disruption of Ephb4 in mice results in accumulation of collagen IV in the EC endoplasmic reticulum leading to EC apoptotic death and defective developmental, neonatal and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice can be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4 mutant mice that express a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity show normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications with regards possible means of treatment of this disease.
Authors
Di Chen, Elizabeth D. Hughes, Thomas L. Saunders, Jiangping Wu, Magda N. Hernández Vásquez, Taija Makinen, Philip D. King
Abstract
SNHG12, a long non-coding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease (PAD) has not been elucidated. Hindlimb ischemia studies using femoral artery ligation in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain and loss of function studies demonstrated that SNHG12 regulated angiogenesis – SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including insulin growth factor 2 mRNA binding protein 3 (IGF2BP3/IMP3). RNA-seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IGF2BP3/IMP3. Knockdown of SNHG12 in mice undergoing femoral artery ligation using injected gapmeRs decreased angiogenesis, an effect that was more pronounced in a model of insulin resistant db/db mice. RNA-seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12-knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.
Authors
David A. Gross, Henry S. Cheng, Rulin Zhuang, Michael G. McCoy, Daniel Pérez-Cremades, Zachary Salyers, A.K.M. Khyrul Wara, Stefan Haemmig, Terence E. Ryan, Mark W. Feinberg
Abstract
Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex ‘organization’ of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter, in mice, to study expression of its ligand, ephrinB2, and analysed developmental phenotypes following conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells, and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization, and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.
Authors
Oliver Lyons, James Walker, Christopher Seet, Mohammed Ikram, Adam Kuchta, Andrew Arnold, Magda Hernández-Vásquez, Maike Frye, Gema Vizcay-Barrena, Roland A. Fleck, Ashish S. Patel, Soundrie Padayachee, Peter Mortimer, Steve Jeffery, Siren Berland, Sahar Mansour, Pia Ostergaard, Taija Makinen, Bijan Modarai, Prakash Saha, Alberto Smith
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