Bischoff Lab

2016

Couto, Javier, Lan Huang, Matthew Vivero, Nolan Kamitaki, Reid Maclellan, John Mulliken, Joyce Bischoff, Matthew Warman, and Arin Greene. (2016) 2016. “Endothelial Cells from Capillary Malformations Are Enriched for Somatic GNAQ Mutations”. Plast Reconstr Surg 137 (1): 77e-82e. https://doi.org/10.1097/PRS.0000000000001868.

BACKGROUND: A somatic mutation in GNAQ (c.548G>A; p.R183Q), encoding Gαq, has been found in syndromic and sporadic capillary malformation tissue. However, the specific cell type containing the mutation is unknown. The purpose of this study was to determine which cells in capillary malformations have the GNAQ mutation. METHODS: Human capillary malformation tissue was obtained from 13 patients during a clinically indicated procedure. Droplet digital polymerase chain reaction, capable of detecting mutant allelic frequencies as low as 0.1 percent, was used to quantify the abundance of GNAQ mutant cells in capillary malformation tissue. Six specimens were fractionated by fluorescence-activated cell sorting into hematopoietic, endothelial, perivascular, and stromal cells. The frequency of GNAQ mutant cells in these populations was quantified by droplet digital polymerase chain reaction. RESULTS: Eight capillary malformations contained GNAQ p.R183Q mutant cells, two lesions had novel GNAQ mutations (p.R183L and p.R183G), and three capillary malformations did not have a detectable GNAQ p.R183 mutation. Mutant allelic frequencies ranged from 2 to 11 percent. Following fluorescence-activated cell sorting, the GNAQ mutation was found in the endothelial but not the platelet-derived growth factor receptor-β-positive cell population; mutant allelic frequencies were 3 to 43 percent. CONCLUSION: Endothelial cells in capillary malformations are enriched for GNAQ mutations and are likely responsible for the pathophysiology underlying capillary malformation.

Yu, Chen-Wei, Xiaoliang Liang, Samantha Lipsky, Cagatay Karaaslan, Harry Kozakewich, Gokhan Hotamisligil, Joyce Bischoff, and Sule Cataltepe. (2016) 2016. “Dual role of fatty acid-binding protein 5 on endothelial cell fate: a potential link between lipid metabolism and angiogenic responses”. Angiogenesis 19 (1): 95-106. https://doi.org/10.1007/s10456-015-9491-4.

Fatty acid-binding proteins (FABP) are small molecular mass intracellular lipid chaperones that are expressed in a tissue-specific manner with some overlaps. FABP4 and FABP5 share ~55 % amino acid sequence homology and demonstrate synergistic effects in regulation of metabolic and inflammatory responses in adipocytes and macrophages. Recent studies have shown that FABP4 and FABP5 are also co-expressed in a subset of endothelial cells (EC). FABP4, which has a primarily microvascular distribution, enhances angiogenic responses of ECs, including proliferation, migration, and survival. However, the vascular expression of FABP5 has not been well characterized, and the role of FABP5 in regulation of angiogenic responses in ECs has not been studied to date. Herein we report that while FABP4 and FABP5 are co-expressed in microvascular ECs in several tissues, FABP5 expression is also detected in ECs of larger blood vessels. In contrast to FABP4, EC-FABP5 levels are not induced by VEGF-A or bFGF. FABP5 deficiency leads to a profound impairment in EC proliferation and chemotactic migration. These effects are recapitulated in an ex vivo assay of angiogenesis, the aortic ring assay. Interestingly, in contrast to FABP4-deficient ECs, FABP5-deficient ECs are significantly more resistant to apoptotic cell death. The effect of FABP5 on EC proliferation and survival is mediated, only in part, by PPARδ-dependent pathways. Collectively, these findings demonstrate that EC-FABP5, similar to EC-FABP4, promotes angiogenic responses under certain conditions, but it can also exert opposing effects on EC survival as compared to EC-FABP4. Thus, the balance between FABP4 and FABP5 in ECs may be important in regulation of angiogenic versus quiescent phenotypes in blood vessels.

Dal-Bianco, Jacob, Elena Aikawa, Joyce Bischoff, Luis Guerrero, Jesper Hjortnaes, Jonathan Beaudoin, Catherine Szymanski, Philipp Bartko, Margo Seybolt, and Mark Handschumacher. 2016. “Myocardial infarction alters adaptation of the tethered mitral valve”. Journal of the American College of Cardiology 67 (3): 275-87.

BACKGROUND:

In patients with myocardial infarction (MI), leaflet tethering by displaced papillary muscles induces mitral regurgitation (MR), which doubles mortality. Mitral valves (MVs) are larger in such patients but fibrosis sets in counterproductively. The investigators previously reported that experimental tethering alone increases mitral valve area in association with endothelial-to-mesenchymal transition.

OBJECTIVES:

The aim of this study was to explore the clinically relevant situation of tethering and MI, testing the hypothesis that ischemic milieu modifies mitral valve adaptation.

METHODS:

Twenty-three adult sheep were examined. Under cardiopulmonary bypass, the papillary muscle tips in 6 sheep were retracted apically to replicate tethering, short of producing MR (tethered alone). Papillary muscle retraction was combined with apical MI created by coronary ligation in another 6 sheep (tethered plus MI), and left ventricular remodeling was limited by external constraint in 5 additional sheep (left ventricular constraint). Six sham-operated sheep were control subjects. Diastolic mitral valve surface area was quantified by 3-dimensional echocardiography at baseline and after 58 ± 5 days, followed by histopathology and flow cytometry of excised leaflets.

RESULTS:

Tethered plus MI leaflets were markedly thicker than tethered-alone valves and sham control subjects. Leaflet area also increased significantly. Endothelial-to-mesenchymal transition, detected as α-smooth muscle actin-positive endothelial cells, significantly exceeded that in tethered-alone and control valves. Transforming growth factor-β, matrix metalloproteinase expression, and cellular proliferation were markedly increased. Uniquely, tethering plus MI showed endothelial activation with vascular adhesion molecule expression, neovascularization, and cells positive for CD45, considered a hematopoietic cell marker. Tethered plus MI findings were comparable with external ventricular constraint.

CONCLUSIONS:

MI altered leaflet adaptation, including a profibrotic increase in valvular cell activation, CD45-positive cells, and matrix turnover. Understanding cellular and molecular mechanisms underlying leaflet adaptation and fibrosis could yield new therapeutic opportunities for reducing ischemic MR.

Rossi, Elisa, David Smadja, Elisa Boscolo, Carmen Langa, Miguel Arevalo, Miguel Pericacho, Luis Gamella-Pozuelo, et al. (2016) 2016. “Endoglin regulates mural cell adhesion in the circulatory system”. Cell Mol Life Sci 73 (8): 1715-39. https://doi.org/10.1007/s00018-015-2099-4.

The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for β1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or β1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5β1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng (+/-) mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia.

Ye, Xi, Yassir Abou-Rayyah, Joyce Bischoff, Alison Ritchie, Neil Sebire, Patrick Watts, Amanda Churchill, and David Bates. 2016. “Altered ratios of pro- and anti-angiogenic VEGF-A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma”. J Pathol 239 (2): 139-51. https://doi.org/10.1002/path.4715.

Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF-A), which consists of both the pro- and anti-angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF-A variants in IH progression and its spontaneous involution is unknown. Using patient-derived cells and surgical specimens, we showed that the relative level of VEGF-A165 b was increased in the involuting phase of IH and the relative change in VEGF-A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF-A165 b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF-A165 b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta-like ligand 4 (DLL4) expression. These results indicate that VEGF-A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte-derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF-A isoform-dependent manner, and in perivascular cells in a VEGF-independent manner. The contribution of VEGF-A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Ayturk, Ugur, Javier Couto, Steven Hann, John Mulliken, Kaitlin Williams, August Yue Huang, Steven Fishman, et al. 2016. “Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital Hemangioma”. Am J Hum Genet 98 (6): 1271. https://doi.org/10.1016/j.ajhg.2016.05.010.

Congenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Gln209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Gln209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffinembedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Gln209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors’ different postnatal behaviors

Bischoff, Joyce, Guillem Casanovas, Jill Wylie-Sears, Dae-Hee Kim, Philipp Bartko, Luis Guerrero, Jacob Dal-Bianco, et al. 2016. “CD45 Expression in Mitral Valve Endothelial Cells After Myocardial Infarction”. Circ Res 119 (11): 1215-25. https://doi.org/10.1161/CIRCRESAHA.116.309598.

RATIONALE: Ischemic mitral regurgitation, a complication after myocardial infarction (MI), induces adaptive mitral valve (MV) responses that may be initially beneficial but eventually lead to leaflet fibrosis and MV dysfunction. We sought to examine the MV endothelial response and its potential contribution to ischemic mitral regurgitation. OBJECTIVE: Endothelial, interstitial, and hematopoietic cells in MVs from post-MI sheep were quantified. MV endothelial CD45, found post MI, was analyzed in vitro. METHODS AND RESULTS: Ovine MVs, harvested 6 months after inferior MI, showed CD45, a protein tyrosine phosphatase, colocalized with von Willebrand factor, an endothelial marker. Flow cytometry of MV cells revealed significant increases in CD45 endothelial cells (VE-cadherin/CD45/α-smooth muscle actin [SMA] and VE-cadherin/CD45/αSMA- cells) and possible fibrocytes (VE-cadherin/CD45/αSMA) in inferior MI compared with sham-operated and normal sheep. CD45 cells correlated with MV fibrosis and mitral regurgitation severity. VE-cadherin/CD45/αSMA cells suggested that CD45 may be linked to endothelial-to-mesenchymal transition (EndMT). MV endothelial cells treated with transforming growth factor-β1 to induce EndMT expressed CD45 and fibrosis markers collagen 1 and 3 and transforming growth factor-β1 to 3, not observed in transforming growth factor-β1-treated arterial endothelial cells. A CD45 protein tyrosine phosphatase inhibitor blocked induction of EndMT and fibrosis markers and inhibited EndMT-associated migration of MV endothelial cells. CONCLUSIONS: MV endothelial cells express CD45, both in vivo post MI and in vitro in response to transforming growth factor-β1. A CD45 phosphatase inhibitor blocked hallmarks of EndMT in MV endothelial cells. These results point to a novel, functional requirement for CD45 phosphatase activity in EndMT. The contribution of CD45 endothelial cells to MV adaptation and fibrosis post MI warrants investigation.

Bai, Shoumei, Patrick Ingram, Yu-Chih Chen, Ning Deng, Alex Pearson, Yashar Niknafs, Patrick O’Hayer, et al. 2016. “EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells”. Cancer Res 76 (21): 6396-6409. https://doi.org/10.1158/0008-5472.CAN-16-0225.

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.