Publications by Year: 2017

2017

KT, Kang, Lin RZ, Kupperman D, Melero-Martin J, and Bischoff J. 2017. “Endothelial Colony Forming Cells and Mesenchymal Progenitor Cells Form Blood Vessels and Increase Blood Flow in Ischemic Muscle”. Sci Rep.
Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.
Huang, Lan, Javier Couto, Anna Pinto, Sanda Alexandrescu, Joseph Madsen, Arin Greene, Mustafa Sahin, and Joyce Bischoff. (2017) 2017. “Somatic GNAQ Mutation is Enriched in Brain Endothelial Cells in Sturge-Weber Syndrome”. Pediatr Neurol 67: 59-63. https://doi.org/10.1016/j.pediatrneurol.2016.10.010.
BACKGROUND: Sturge-Weber syndrome (SWS) is a rare congenital neurocutaneous disorder characterized by facial and extracraniofacial capillary malformations and capillary-venule malformations in the leptomeninges. A somatic mosaic mutation in GNAQ (c.548G>A; p.R183Q) was found in SWS brain and skin capillary malformations. Our laboratory showed endothelial cells in skin capillary malformations are enriched for the GNAQ mutation. The purpose of this study is to determine whether the GNAQ mutation is also enriched in endothelial cells in affected SWS brain. METHODS: Two human SWS brain specimens were fractionated by fluorescence-activated cell sorting into hematopoietic (CD45), endothelial (CD31, VE-Cadherin, and vascular endothelial growth factor receptor 2), and perivascular (platelet-derived growth factor receptor beta) cells and cells negative for all markers. The sorted cell populations were analyzed for GNAQ p.R183Q mutation by droplet digital polymerase chain reaction. SWS patient-derived brain endothelial cells were selected by anti-CD31-coated magnetic beads and cultured in endothelial growth medium in vitro. RESULTS: The GNAQ p.R183Q mutation was present in brain endothelial cells in two SWS specimens, with mutant allelic frequencies of 34.7% and 24.0%. Cells negative for all markers also harbored the GNAQ mutation. The mutant allelic frequencies in these unidentified cells were 9.2% and 8.4%. SWS patient-derived brain endothelial cells with mutant allelic frequencies of 14.7% and 21% survived and proliferated in vitro. CONCLUSIONS: Our study provides evidence that GNAQ p.R183Q mutation is enriched in endothelial cells in SWS brain lesions and thereby reveals endothelial cells as a source of aberrant Gαq signaling. This will help to understand the pathophysiology of SWS, to discover biomarkers for predicting cerebral involvement, and to develop therapeutic targets to prevent neurological impairments in SWS.
Couto, Javier, Ugur Ayturk, Dennis Konczyk, Jeremy Goss, August Huang, Steve Hann, Jennifer Reeve, et al. (2017) 2017. “A somatic GNA11 mutation is associated with extremity capillary malformation and overgrowth”. Angiogenesis 20 (3): 303-6. https://doi.org/10.1007/s10456-016-9538-1.
BACKGROUND: Capillary malformation is a cutaneous vascular anomaly that is present at birth, darkens over time, and can cause overgrowth of tissues beneath the stain. The lesion is caused by a somatic activating mutation in GNAQ. In a previous study, we were unable to identify a GNAQ mutation in patients with a capillary malformation involving an overgrown lower extremity. We hypothesized that mutations in GNA11 or GNA14, genes closely related to GNAQ, also may cause capillary malformations. METHODS: Human capillary malformation tissue obtained from 8 patients that had tested negative for GNAQ mutations were studied. Lesions involved an extremity (n = 7) or trunk (n = 1). Droplet digital PCR (ddPCR) was used to detect GNA11 or GNA14 mutant cells (p.Arg183) in the specimens. Single molecule molecular inversion probe sequencing (smMIP-seq) was performed to search for other mutations in GNA11. Mutations were validated by subcloning and sequencing amplimers. RESULTS: We found a somatic GNA11 missense mutation (c.547C > T; p.Arg183Cys) in 3 patients with a diffuse capillary malformation of an extremity. Mutant allelic frequencies ranged from 0.3 to 5.0%. GNA11 or GNA14 mutations were not found in 5 affected tissues or in unaffected tissues (white blood cell DNA). CONCULSIONS: GNA11 mutations are associated with extremity capillary malformations causing overgrowth. Pharmacotherapy that affects GNA11 signaling may prevent the progression of capillary malformations.
Kim, Dae-Hee, Ran Heo, Mark Handschumacher, Sahmin Lee, Yun-Sil Choi, Kyu-Ri Kim, Yewon Shin, et al. 2017. “Mitral Valve Adaptation to Isolated Annular Dilation: Insights Into the Mechanism of Atrial Functional Mitral Regurgitation”. JACC Cardiovasc Imaging. https://doi.org/10.1016/j.jcmg.2017.09.013.
OBJECTIVES: This study hypothesized that compensatory mitral leaflet area (MLA) adaptation occurs in patients with persistent atrial fibrillation (AF) without left ventricular (LV) dysfunction but has limitations that augment mitral regurgitation (MR). The study also explored whether asymmetrical annular dilation is matched by relative leaflet enlargement. BACKGROUND: Functional MR occurs in patients with AF and isolated annular dilation, but the relationship of MLA adaptation with annular area (AA) is unknown. METHODS: Three-dimensional echocardiographic images were acquired from 86 patients with quantified MR: 53 with nonvalvular persistent AF (23 MR+ with moderate or greater MR, 30 MR-) without LV dysfunction or dilation and 33 normal controls. Comprehensive 3-dimensional analysis included total diastolic MLA, adaptation ratios of MLA to annular area and MLA to leaflet closure area, and annular and tenting geometry. RESULTS: Total MLA was 22% larger in patients with AF than in controls, thus paralleling the increased AA. However, as AA increased, adaptive indices (MLA/AA ratio and ratio of MLA to closure area) plateaued, becoming lowest in MR+ patients (ratio of MLA to closure area = 1.63 ± 0.17 controls, 1.60 ± 0.11 MR-, 1.32 ± 0.10 MR+; p < 0.001). MR increased as the ratio of MLA to closure area decreased (R = 0.68; p < 0.001). The posterior-to-anterior MLA ratio remained constant, whereas the posterior-to-anterior mitral annulus perimeter increased (1.21 ± 0.16 controls, 1.32 ± 0.20 MR-, 1.46 ± 0.19 MR+; p < 0.001). Multivariate MR determinants were annular area, total MLA to closure area, and posterior-to-anterior perimeter ratios. CONCLUSIONS: MLA adaptively increases in AF with isolated annular dilation and normal LV function. This compensatory enlargement becomes insufficient with greater annular dilation, and the leaflets fail to match asymmetrical annular remodeling, thereby increasing MR. These findings can potentially help optimize therapeutic options and motivate basic studies of adaptive growth processes.
Beaudoin, Jonathan, Jacob Dal-Bianco, Elena Aikawa, Joyce Bischoff, Luis Guerrero, Suzanne Sullivan, Philipp Emanuel Bartko, et al. (2017) 2017. “Mitral Leaflet Changes Following Myocardial Infarction: Clinical Evidence for Maladaptive Valvular Remodeling”. Circ Cardiovasc Imaging 10 (11). https://doi.org/10.1161/CIRCIMAGING.117.006512.
BACKGROUND: Ischemic mitral regurgitation (MR) is classically ascribed to functional restriction of normal leaflets, but recent studies have suggested post-myocardial infarction (MI) mitral valve (MV) leaflet fibrosis and thickening, challenging valve normality. Progression of leaflet thickness post-MI has not been studied. We hypothesized that excessive MV remodeling post-MI contributes to MR. Our objectives are to characterize MV changes after MI and relate them to MR. METHODS AND RESULTS: Three groups of 40 patients with serial echocardiograms over a mean of 23.4 months were identified from an echocardiography database: patients first studied early (6±12 days) and late (12±7 years) after an inferior MI and normal controls. MV thickness was correlated with MR. We studied the mechanisms for MV changes in a sheep model (6 apical MI versus 6 controls) followed for 8 weeks, with MV cellular and histopathologic analyses. Early post-MI, leaflet thickness was found to be similar to controls (2.6±0.5 vs 2.5±0.4 mm; =0.23) but significantly increased over time (2.5±0.4 to 2.9±0.4 mm; <0.01). In this group, patients tolerating maximal doses of renin-angiotensin blocking agents had less thickening (25% of patients; <0.01). The late-MI group had increased thickness (3.2±0.5 vs 2.5±0.4 mm; <0.01) without progression. At follow-up, 48% of post-MI patients had more than mild MR. Increased thickness was independently associated with MR. Experimentally, 8 weeks post-MI, MVs were 2-fold thicker than controls, with increased collagen, profibrotic transforming growth factor-β, and endothelial-to-mesenchymal transformation, confirmed by flow cytometry. CONCLUSIONS: MV thickness increases post-MI and correlates with MR, suggesting an organic component to ischemic MR. MV fibrotic remodeling can indicate directions for future therapy.
Bartko, Philipp, Jacob Dal-Bianco, Luis Guerrero, Jonathan Beaudoin, Catherine Szymanski, Dae-Hee Kim, Margo Seybolt, et al. 2017. “Effect of Losartan on Mitral Valve Changes After Myocardial Infarction”. J Am Coll Cardiol 70 (10): 1232-44. https://doi.org/10.1016/j.jacc.2017.07.734.
BACKGROUND: After myocardial infarction (MI), mitral valve (MV) tethering stimulates adaptive leaflet growth, but counterproductive leaflet thickening and fibrosis augment mitral regurgitation (MR), doubling heart failure and mortality. MV fibrosis post-MI is associated with excessive endothelial-to-mesenchymal transition (EMT), driven by transforming growth factor (TGF)-β overexpression. In vitro, losartan-mediated TGF-β inhibition reduces EMT of MV endothelial cells. OBJECTIVES: This study tested the hypothesis that profibrotic MV changes post-MI are therapeutically accessible, specifically by losartan-mediated TGF-β inhibition. METHODS: The study assessed 17 sheep, including 6 sham-operated control animals and 11 with apical MI and papillary muscle retraction short of producing MR; 6 of the 11 were treated with daily losartan, and 5 were untreated, with flexible epicardial mesh comparably limiting left ventricular (LV) remodeling. LV volumes, tethering, and MV area were quantified by using three-dimensional echocardiography at baseline and at 60 ± 6 days, and excised leaflets were analyzed by histopathology and flow cytometry. RESULTS: Post-MI LV dilation and tethering were comparable in the losartan-treated and untreated LV constraint sheep. Telemetered sensors (n = 6) showed no significant losartan-induced changes in arterial pressure. Losartan strongly reduced leaflet thickness (0.9 ± 0.2 mm vs. 1.6 ± 0.2 mm; p < 0.05; 0.4 ± 0.1 mm sham animals), TGF-β, and downstream phosphorylated extracellular-signal-regulated kinase and EMT (27.2 ± 12.0% vs. 51.6 ± 11.7% α-smooth muscle actin-positive endothelial cells, p < 0.05; 7.2 ± 3.5% sham animals), cellular proliferation, collagen deposition, endothelial cell activation (vascular cell adhesion molecule-1 expression), neovascularization, and cells positive for cluster of differentiation (CD) 45, a hematopoietic marker associated with post-MI valve fibrosis. Leaflet area increased comparably (17%) in constrained and losartan-treated sheep. CONCLUSIONS: Profibrotic changes of tethered MV leaflets post-MI can be modulated by losartan without eliminating adaptive growth. Understanding the cellular and molecular mechanisms could provide new opportunities to reduce ischemic MR.