Angiogenesis
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
Abstract
Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatic vessels in bone, gradually lose bone, and can have life-threatening complications such as chylothorax. The etiology of GSD is poorly understood and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a GSD patient and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.
Authors
Nassim Homayun Sepehr, Anna L. McCarter, Raphaël Helaers, Christine Galant, Laurence M. Boon, Pascal Brouillard, Miikka Vikkula, Michael T. Dellinger
Abstract
AbstractMillions of people are affected by hearing loss. When hearing loss is caused by noise or aging, it is often associated with breakdown of the barrier between the cochlea and its blood vessels. Pericytes populate many small vessels in the adult inner ear, however, their role in different forms of hearing loss is largely unknown. Using an inducible and conditional pericyte depletion mouse model, we show that loss of pericytes leads to marked changes in vascular structure, resulting in poor blood circulation and hearing loss. In vitro, using advanced tissue explants from pericyte fluorescence reporter models in combination with exogenous donor pericytes, we show pericytes, signaled by endothelial growth factor isoform A165 (VEGF-A165), vigorously drives new vessel growth in both adult and neonatal mouse inner ear tissue. In vivo, the delivery of an adeno-associated virus serotype 1 (AAV1)-mediated VEGF-A165 viral vector to pericyte depleted animals regenerated lost pericytes, improved blood supply, reduced loss of sensory hair cells, and attenuated hearing loss. These studies provide the first clear-cut evidence that pericytes are critical for adult hearing and can regenerate cochlear vasculature. The restoration of vascular function in the damaged inner ear with AAV1-mediated VEGF-A165 gene therapy is a new strategy for ameliorating vascular associated hearing disorders, including common forms of age-related hearing loss.
Authors
Jinhui Zhang, Zhiqiang Hou, Xiaohan Wang, Han Jiang, Lingling Neng, Yunpei Zhang, Qing Yu, George W. S. Burwood, Junha Song, Manfred Auer, Anders Fridberger, Michael Hoa, Xiaorui Shi
Abstract
EPAS1, encoding HIF-2α, mutations were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to our institution for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1-gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital two photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and Micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retina from corresponding developmental timepoints (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all of our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental timepoints. These findings add vascular malformation as a new clinical feature of EPAS1-gain-of-function syndrome.
Authors
Jared S. Rosenblum, Herui Wang, Pauline M. Dmitriev, Anthony J. Cappadona, Panagiotis Mastorakos, Chen Xu, Abhishek Jha, Nancy Edwards, Danielle R. Donahue, Jeeva Munasinghe, Matthew A. Nazari, Russell H. Knutsen, Bruce R. Rosenblum, James G. Smirniotopoulos, Alberto Pappo, Robert F. Spetzler, Alexander Vortmeyer, Mark R. Gilbert, Dorian B. McGavern, Emily Chew, Beth A. Kozel, John D. Heiss, Zhengping Zhuang, Karel Pacak
Abstract
Infantile hemangioma is a vascular tumor characterized by the rapid growth of disorganized blood vessels followed by slow spontaneous involution. The underlying molecular mechanisms that regulate hemangioma proliferation and involution still are not well elucidated. Our previous studies reported that NOGOB receptor (NGBR), a transmembrane protein, is required for the translocation of prenylated RAS from the cytosol to the plasma membrane and promotes RAS activation. Here, we show that NGBR is highly expressed in the proliferating phase of infantile hemangioma, but its expression decreases in the involuting phase, suggesting that NGBR may be involved in regulating the growth of proliferating hemangioma. Moreover, we demonstrated that NGBR knockdown in hemangioma stem cells (HemSCs) attenuates growth factors-stimulated RAS activation and diminishes the migration and proliferation of HemSCs, which is consistent with the effects of RAS knockdown in HemSCs. In vivo differentiation assay further showed that NGBR knockdown inhibits blood vessel formation and adipocyte differentiation of HemSCs in immunodeficient mice. Our data suggest that NGBR serves as a RAS modulator in controlling the growth and differentiation of HemSCs.
Authors
Wenquan Hu, Zhong Liu, Valerie Salato, Paula E. North, Joyce Bischoff, Suresh N. Kumar, Zhi Fang, Sujith Rajan, M. Mahmood Hussain, Qing R. Miao
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