Hu, Wenquan, Zhong Liu, Salato, Valerie, North, Paula E, Bischoff, Joyce, Kumar, Suresh N, Fang, Zhi, Rajan, Sujith, Hussain, M Mahmood, and Miao, Qing R. 2021. NOGOB receptor-mediated RAS signaling pathway is a target for suppressing proliferating hemangioma. JCI Insight, 6, 3.
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 was highly expressed in the proliferating phase of infantile hemangioma, but its expression decreased in the involuting phase, suggesting that NGBR may have been involved in regulating the growth of proliferating hemangioma. Moreover, we demonstrate that NGBR knockdown in hemangioma stem cells (HemSCs) attenuated growth factor-stimulated RAS activation and diminished the migration and proliferation of HemSCs, which is consistent with the effects of RAS knockdown in HemSCs. In vivo differentiation assay further shows that NGBR knockdown inhibited blood vessel formation and adipocyte differentiation of HemSCs in immunodeficient mice. Our data suggest that NGBR served as a RAS modulator in controlling the growth and differentiation of HemSCs.
Lupieri, Adrien, Nagata, Yasufumi, Passos, Livia S A, Beker-Greene, Dakota, Kirkwood, Katherine A, Wylie-Sears, Jill, Alvandi, Zahra, Higashi, Hideyuki, Hung, Judy W, Singh, Sasha A, Bischoff, Joyce, Levine, Robert A, and Aikawa, Elena. 2021. Integration of Functional Imaging, Cytometry, and Unbiased Proteomics Reveals New Features of Endothelial-to-Mesenchymal Transition in Ischemic Mitral Valve Regurgitation in Human Patients. Front Cardiovasc Med, 8, Pp. 688396.
Background: Following myocardial infarction, mitral regurgitation (MR) is a common complication. Previous animal studies demonstrated the association of endothelial-to-mesenchymal transition (EndMT) with mitral valve (MV) remodeling. Nevertheless, little is known about how MV tissue responds to ischemic heart changes in humans. Methods: MVs were obtained by the Cardiothoracic Surgical Trials Network from 17 patients with ischemic mitral regurgitation (IMR). Echo-doppler imaging assessed MV function at time of resection. Cryosections of MVs were analyzed using a multi-faceted histology and immunofluorescence examination of cell populations. MVs were further analyzed using unbiased label-free proteomics. Echo-Doppler imaging, histo-cytometry measures and proteomic analysis were then integrated. Results: MVs from patients with greater MR exhibited proteomic changes associated with proteolysis-, inflammatory- and oxidative stress-related processes compared to MVs with less MR. Cryosections of MVs from patients with IMR displayed activated valvular interstitial cells (aVICs) and double positive CD31+ αSMA+ cells, a hallmark of EndMT. Univariable and multivariable association with echocardiography measures revealed a positive correlation of MR severity with both cellular and geometric changes (e.g., aVICs, EndMT, leaflet thickness, leaflet tenting). Finally, proteomic changes associated with EndMT showed gene-ontology enrichment in vesicle-, inflammatory- and oxidative stress-related processes. This discovery approach indicated new candidate proteins associated with EndMT regulation in IMR. Conclusion: We describe an atypical cellular composition and distinctive proteome of human MVs from patients with IMR, which highlighted new candidate proteins implicated in EndMT-related processes, associated with maladaptive MV fibrotic remodeling.
Alvandi, Zahra and Bischoff, Joyce. 2021. Endothelial-Mesenchymal Transition in Cardiovascular Disease. Arterioscler Thromb Vasc Biol, 41, 9, Pp. 2357-2369.
Endothelial-to-mesenchymal transition is a dynamic process in which endothelial cells suppress constituent endothelial properties and take on mesenchymal cell behaviors. To begin the process, endothelial cells loosen their cell-cell junctions, degrade the basement membrane, and migrate out into the perivascular surroundings. These initial endothelial behaviors reflect a transient modulation of cellular phenotype, that is, a phenotypic modulation, that is sometimes referred to as partial endothelial-to-mesenchymal transition. Loosening of endothelial junctions and migration are also seen in inflammatory and angiogenic settings such that endothelial cells initiating endothelial-to-mesenchymal transition have overlapping behaviors and gene expression with endothelial cells responding to inflammatory signals or sprouting to form new blood vessels. Reduced endothelial junctions increase permeability, which facilitates leukocyte trafficking, whereas endothelial migration precedes angiogenic sprouting and neovascularization; both endothelial barriers and quiescence are restored as inflammatory and angiogenic stimuli subside. Complete endothelial-to-mesenchymal transition proceeds beyond phenotypic modulation such that mesenchymal characteristics become prominent and endothelial functions diminish. In proadaptive, regenerative settings the new mesenchymal cells produce extracellular matrix and contribute to tissue integrity whereas in maladaptive, pathologic settings the new mesenchymal cells become fibrotic, overproducing matrix to cause tissue stiffness, which eventually impacts function. Here we will review what is known about how TGF (transforming growth factor) β influences this continuum from junctional loosening to cellular migration and its relevance to cardiovascular diseases.
Huang, Lan, Bichsel, Colette, Norris, Alexis, Thorpe, Jeremy, Pevsner, Jonathan, Alexandrescu, Sanda, Pinto, Anna, Zurakowski, David, Kleiman, Robin J, Sahin, Mustafa, Greene, Arin K, and Bischoff, Joyce. 2021. Endothelialp.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels. Arterioscler Thromb Vasc Biol, Pp. ATVBAHA121316651.
OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by qPCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.
Seebauer, Caroline T, Graus, Matthew S, Huang, Lan, McCann, Alex J, Wylie-Sears, Jill, Fontaine, Frank R, Karnezis, Tara, Zurakowski, David, Staffa, Steven J, Meunier, Frédéric A, Mulliken, John B, Bischoff, Joyce, and Francois, Mathias. 2021. Non-β-blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma. J Clin Invest.
Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of β-blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSC) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial differentiation. As our previous work implicated the transcription factor SRY(Sex Determining Region Y)-Box Transcription Factor-18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with a similar efficacy as the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical and quantitative molecular imaging assays we show that R(+) propranolol directly interferes with SOX18 target gene trans-activation, disrupts SOX18-chromatin binding dynamics and reduced SOX18 dimer formation. We suggest the R(+) enantiomers of widely used β-blockers could be repurposed to increase efficiency of current IH treatment and lower adverse associated side effects.


Huang, Lan and Bischoff, Joyce. 2020. Isolation of Stem Cells, Endothelial Cells and Pericytes from Human Infantile Hemangioma. Bio Protoc, 10, 2, Pp. e3487.
Infantile hemangioma (IH) is a vascular tumor noted for its excessive blood vessel formation during infancy, glucose-transporter-1 (GLUT1)-positive staining of the blood vessels, and its slow spontaneous involution over several years in early childhood. For most children, IH poses no serious threat because it will eventually involute, but a subset can destroy facial structures and impair vision, breathing and feeding. To unravel the molecular mechanism(s) driving IH-specific vascular overgrowth, which to date remains elusive, investigators have studied IH histopathology, the cellular constituents and mRNA expression. Hemangioma endothelial cells (HemEC) were first isolated from surgically removed IH specimens in 1982 by Mulliken and colleagues ( Mulliken et al., 1982 ). Hemangioma stem cells (HemSC) were isolated in 2008, hemangioma pericytes in 2013 and GLUT1-positive HemEC in 2015. Indeed, as we describe here, it is possible to isolate HemSC, GLUT1-positive HemEC, GLUT1-negative HemEC and HemPericytes from a single proliferating IH tissue specimen. This is accomplished by sequential selection using antibodies against specific cell surface markers: anti-CD133 to select HemSC, anti-GLUT1 and anti-CD31 to select HemECs and anti-PDGFRβ to select HemPericytes. IH-derived cells proliferate well in culture and can be used for in vitro and in vivo vasculogenesis and angiogenesis assays.
Dong, Yunzhou, Lee, Yang, Cui, Kui, He, Ming, Wang, Beibei, Bhattacharjee, Sudarshan, Bo Zhu, Yago, Tadayuki, Kun Zhang, Deng, Lin, Ouyang, Kunfu, Wen, Aiyun, Cowan, Douglas B, Song, Kai, Lili Yu, Brophy, Megan L, Liu, Xiaolei, Wylie-Sears, Jill, Hao Wu, Wong, Scott, Cui, Guanglin, Kawashima, Yusuke, Matsumoto, Hiroyuki, Kodera, Yoshio, Wojcikiewicz, Richard J H, Srivastava, Sanjay, Bischoff, Joyce, Wang, Da-Zhi, Ley, Klaus, and Hong Chen. 2020. Epsin-mediated degradation of IP3R1 fuels atherosclerosis. Nat Commun, 11, 1, Pp. 3984.
The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.
Marsit, Ons, Clavel, Marie-Annick, Côté-Laroche, Claudia, Hadjadj, Sandra, Bouchard, Marc-André, Handschumacher, Mark D, Clisson, Marine, Drolet, Marie-Claude, Boulanger, Marie-Chloé, Kim, Dae-Hee, Guerrero, J Luis, Bartko, Philipp Emanuel, Couet, Jacques, Arsenault, Marie, Mathieu, Patrick, Pibarot, Philippe, Aikawa, Elena, Bischoff, Joyce, Levine, Robert A, and Beaudoin, Jonathan. 2020. Attenuated Mitral Leaflet Enlargement Contributes to Functional Mitral Regurgitation After Myocardial Infarction. J Am Coll Cardiol, 75, 4, Pp. 395-405.
BACKGROUND: Mitral leaflet enlargement has been identified as an adaptive mechanism to prevent mitral regurgitation in dilated left ventricles (LVs) caused by chronic aortic regurgitation (AR). This enlargement is deficient in patients with functional mitral regurgitation, which remains frequent in the population with ischemic cardiomyopathy. Maladaptive fibrotic changes have been identified in post-myocardial infarction (MI) mitral valves. It is unknown if these changes can interfere with valve growth and whether they are present in other valves. OBJECTIVES: This study sought to test the hypothesis that MI impairs leaflet growth, seen in AR, and induces fibrotic changes in mitral and tricuspid valves. METHODS: Sheep models of AR, AR + MI, and controls were followed for 90 days. Cardiac magnetic resonance, echocardiography, and computed tomography were performed at baseline and 90 days to assess LV volume, LV function, mitral regurgitation and mitral leaflet size. Histopathology and molecular analyses were performed in excised valves. RESULTS: Both experimental groups developed similar LV dilatation and dysfunction. At 90 days, mitral valve leaflet size was smaller in the AR + MI group (12.8 ± 1.3 cm2 vs. 15.1 ± 1.6 cm2, p = 0.03). Mitral regurgitant fraction was 4% ± 7% in the AR group versus 19% ± 10% in the AR + MI group (p = 0.02). AR + MI leaflets were thicker compared with AR and control valves. Increased expression of extracellular matrix remodeling genes was found in both the mitral and tricuspid leaflets in the AR + MI group. CONCLUSIONS: In these animal models of AR, the presence of MI was associated with impaired adaptive valve growth and more functional mitral regurgitation, despite similar LV size and function. More pronounced extracellular remodeling was observed in mitral and tricuspid leaflets, suggesting systemic valvular remodeling after MI.
Goss, Jeremy A, Konczyk, Dennis J, Smits, Patrick, Sudduth, Christopher L, Bischoff, Joyce, Liang, Marilyn G, and Greene, Arin K. 2020. Diffuse capillary malformation with overgrowth contains somatic PIK3CA variants. Clin Genet, 97, 5, Pp. 736-740.
Diffuse capillary malformation with overgrowth (DCMO) is a clinical diagnosis describing patients with multiple, extensive capillary malformations (CMs) associated with overgrowth and foot anomalies. The purpose of the study was to identify somatic variants in DCMO. Skin containing CM and overgrown subcutaneous adipose tissue was collected from patients with DCMO. Exons from 447 cancer-related genes were sequenced using OncoPanel. Variant-specific droplet digital PCR (ddPCR) independently confirmed the variants and determined variant allele frequencies (VAF). One subject contained a somatic PIK3CA p.G106V variant. A second patient had a PIK3CA p.D350G variant. VAF was 27% to 29% in skin and 16% to 28% in subcutaneous adipose. Variants were enriched in endothelial cells (VAF 50%-51%) compared to nonendothelial cells (1%-8%). DCMO is associated with somatic PIK3CA variants and should be considered on the PIK3CA-related overgrowth spectrum (PROS). Variants are present in both skin and subcutaneous adipose and are enriched in endothelial cells.


Overman, Jeroen, Fontaine, Frank, Wylie-Sears, Jill, Moustaqil, Mehdi, Huang, Lan, Meurer, Marie, Chiang, Ivy Kim, Lesieur, Emmanuelle, Patel, Jatin, Zuegg, Johannes, Pasquier, Eddy, Sierecki, Emma, Gambin, Yann, Hamdan, Mohamed, Khosrotehrani, Kiarash, Andelfinger, Gregor, Bischoff, Joyce, and Francois, Mathias. 2019. R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma. Elife, 8.
Propranolol is an approved non-selective β-adrenergic blocker that is first line therapy for infantile hemangioma. Despite the clinical benefit of propranolol therapy in hemangioma, the mechanistic understanding of what drives this outcome is limited. Here, we report successful treatment of pericardial edema with propranolol in a patient with Hypotrichosis-Lymphedema-Telangiectasia and Renal (HLTRS) syndrome, caused by a mutation in . Using a mouse pre-clinical model of HLTRS, we show that propranolol treatment rescues its corneal neo-vascularisation phenotype. Dissection of the molecular mechanism identified the R(+)-propranolol enantiomer as a small molecule inhibitor of the SOX18 transcription factor, independent of any anti-adrenergic effect. Lastly, in a patient-derived in vitro model of infantile hemangioma and pre-clinical model of HLTRS we demonstrate the therapeutic potential of the R(+) enantiomer. Our work emphasizes the importance of SOX18 etiological role in vascular neoplasms, and suggests R(+)-propranolol repurposing to numerous indications ranging from vascular diseases to metastatic cancer.