Robert W. Dettman

Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart.

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Emergence of Smooth Muscle Cell Endothelin B–Mediated Vasoconstriction in Lambs With Experimental Congenital Heart Disease and Increased Pulmonary Blood Flow

Background—Endothelin-1 (ET-1) has been implicated in the pathophysiology of pulmonary hypertension. In 1-month-old lambs with increased pulmonary blood flow, we have demonstrated early alterations in the ET-1 cascade. The objective of this study was to investigate the role of potential later alterations of the ET cascade in the pathophysiology of pulmonary hypertension secondary to increased pulmonary blood flow. Methods and Results—Eighteen fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 8 weeks after spontaneous delivery. Compared with age-matched control lambs, lung tissue ET-1 levels were increased in shunt lambs (317.2±113.8 versus 209.8±61.8 pg/g, P <0.05). In shunt lambs (n=9), exogenous ET-1 induced potent pulmonary vasoconstriction, which was blocked by the ETA receptor antagonist PD 156707 (n=3). This pulmonary vasoconstriction was mimicked by exogenous Ala1,3,11,15 ET-1 (4 Ala ET-1), the ETB receptor agonist, and was blocked by the ETB receptor antagonist BQ 788 (n=3). However, in control lambs (n=7), ET-1 and 4 Ala ET-1 did not change pulmonary vascular tone. In contrast to 4-week-old shunt lambs, immunohistochemistry revealed the emergence of ETB receptors on smooth muscle cells in the vasculature of 8-week-old shunt lambs. Conclusions—Over time, increased pulmonary blood flow and/or pressure results in the emergence of ETB-mediated vasoconstriction, which coincides with the emergence of ETB receptors on smooth muscle cells. These data suggest an important role for ETB receptors in the pathophysiology of pulmonary hypertension in this animal model of increased pulmonary blood flow.

Formation and remodeling of the coronary vascular bed in the embryonic avian heart

To study the formation of the coronary vessels in the developing avian heart, we stained developmentally staged quail hearts with the endothelial specific antibody QH‐1. QH‐1 reacted with individual cells in the proepicardial organ in Hamburger and Hamilton stage (HH) 17 embryos only after it had contacted the heart. In HH18–26 hearts, individual QH‐1+ cells accumulated over the surface of the atria and ventricles. The first endothelial vessels appeared in the dorsal atrioventricular groove in HH23 hearts. CD45+ hematopoietic precursors accumulated on the heart surface, demonstrating the close temporal relationship of hematopoiesis with vasculogenesis during heart development. However, CD45 expression preceded association of these cells with the vasculature, suggesting hematopoietic commitment precedes formation of blood islands in the coronary vasculature. Endothelial tubules first appeared on the dorsal and then the ventral aspects of the heart, coalescing into large sinusoids. These sinusoids remodeled into compact muscularized vessels by HH35. Smooth muscle cell markers were first expressed at HH27 and only in association with developing vasculature. We did not observe markers of smooth muscle differentiation in the proepicardium, but it remains uncertain whether cells in the proepicardium are committed to this cell fate. Our data support a strictly vasculogenic mechanism for the formation of the coronary vessels and blood islands. Developmental Dynamics 230:34–43, 2004.

Regulation of Hypoxia-induced Pulmonary Hypertension by Vascular Smooth Muscle Hypoxia-Inducible Factor-1α

RATIONALE Chronic hypoxia induces pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy. At present, little is known about mechanisms driving these responses. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of transcription in hypoxic cells, up-regulating genes involved in energy metabolism, proliferation, and extracellular matrix reorganization. Systemic loss of a single HIF-1α allele has been shown to attenuate hypoxic pulmonary hypertension, but the cells contributing to this response have not been identified. OBJECTIVES We sought to determine the contribution of HIF-1α in smooth muscle on pulmonary vascular and right heart responses to chronic hypoxia. METHODS We used mice with homozygous conditional deletion of HIF-1α combined with tamoxifen-inducible smooth muscle-specific Cre recombinase expression. Mice received either tamoxifen or vehicle followed by exposure to either normoxia or chronic hypoxia (10% O2) for 30 days before measurement of cardiopulmonary responses. MEASUREMENTS AND MAIN RESULTS Tamoxifen-induced smooth muscle-specific deletion of HIF-1α attenuated pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, right ventricular hypertrophy was unchanged despite attenuated pulmonary pressures. CONCLUSIONS These results indicate that HIF-1α in smooth muscle contributes to pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, loss of HIF-1 function in smooth muscle does not affect hypoxic cardiac remodeling, suggesting that the cardiac hypertrophy response is not directly coupled to the increase in pulmonary artery pressure.

Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study.

The transient and localized signaling events between invasive breast cancer cells and the underlying endothelial cells have remained poorly characterized. We report a novel approach integrating vascular engineering with three-dimensional time-lapse fluorescence resonance energy transfer (FRET) imaging to dissect how endothelial myosin light chain kinase (MLCK) is modulated during tumor intravasation. We show that tumor transendothelial migration occurs via both paracellular (i.e. through cell-cell junctions) and transcellular (i.e. through individual endothelial cells) routes. Endothelial MLCK is activated at the invasion site, leading to regional diphosphorylation of myosin-II regulatory light chain (RLC) and myosin contraction. Blocking endothelial RLC diphosphorylation blunts tumor transcellular, but not paracellular, invasion. Our results implicate an important role for endothelial myosin-II function in tumor intravasation.

Coronary endothelial proliferation and morphogenesis are regulated by a VEGF-mediated pathway

Though development of the coronary vasculature is a critical event during embryogenesis, the molecular mechanisms that regulate its formation are not well characterized. Two unique approaches were used to investigate interactions between cardiac myocytes and proepicardial (PE) cells, which are the coronary anlagen. One of these experimental approaches used a 3‐D collagen scaffold system on which specific cell‐cell and cell‐matrix interactions were studied. The other approach used a whole heart culture system that allowed for the analysis of epicardial to mesenchymal transformation (EMT). The VEGF signaling system has been implicated previously as an important regulator of coronary development. Our results demonstrated that a specific isoform of VEGF‐A, VEGF164, increased PE‐derived endothelial cell proliferation and also increased EMT. However, VEGF‐stimulated endothelial cells did not robustly coalesce into endothelial tubes as they did when cocultured with cardiac myocytes. Interestingly, blocking VEGF signaling via flk‐1 inhibition reduced endothelial tube formation despite the presence of cardiac myocytes. These results indicate that VEGF signaling is complex during coronary development and that combinatorial signaling by other VEGF‐A isoforms or other flk‐1‐binding VEGFs are likely to regulate endothelial tube formation. Developmental Dynamics 238:423–430, 2009.

A migratory role for EphrinB ligands in avian epicardial mesothelial cells

Little is known about the molecules that mediate the attachment of proepicardial cells to the heart. Ephrins are cell surface ligands for Eph tyrosine kinase receptors, molecules known to play a role in cell adhesion and migration. Here, we detected EphrinB ligands in proepicardial and epicardial mesothelial cells (EMCs) using reverse transcriptase‐polymerase chain reaction, immunoblotting, immunolocalization, and EphB‐Fc binding. Aggregated EphB‐Fc fragments clustered ephrinB1 ligands on living EMCs indicating that they are cell surface expressed. In vitro assays demonstrated that ephrinB ligands participate in EMC migration but not cell adhesion. Localization studies in hearts at Hamburger and Hamilton stage 30 and older revealed that ephrinB1 is expressed in the epicardium and subepicardial mesenchyme of the atrioventricular sulcus. EMCs treated with platelet‐derived growth factor‐BB expressed smooth muscle markers but not ephrinB1. Our study supports an early role for ephrinB ligands for migration of epicardial cells and a later role in perivascular fibroblasts of coronary vessels in the atrioventricular sulcus. Developmental Dynamics 239:598–609, 2010.

Myocardial Gene Expression Profiling to Predict and Identify Cardiac Allograft Acute Cellular Rejection: The GET-Study.

Aims Serial invasive endomyocardial biopsies (EMB) remain the gold standard for acute cellular rejection (ACR) diagnosis. However histological grading has several limitations. We aimed to explore the value of myocardial Gene Expression Profiling (GEP) for diagnosing and identifying predictive biomarkers of ACR. Methods A case-control study nested within a retrospective heart transplant patients cohort included 126 patients with median (IQR) age 50 (41–57) years and 111 (88%) males. Among 1157 EMB performed, 467 were eligible (i.e, corresponding to either ISHLT grade 0 or ≥3A), among which 36 were selected for GEP according to the grading: 0 (CISHLT, n = 13); rejection ≥3A (RISHLT, n = 13); 0 one month before ACR (BRISHLT, n = 10). Results We found 294 genes differentially expressed between CISHLT and RISHLT, mainly involved in immune activation, and inflammation. Hierarchical clustering showed a clear segregation of CISHLT and RISHLT groups and heterogeneity of GEP within RISHLT. All EMB presented immune activation, but some RISHLT EMB were strongly subject to inflammation, whereas others, closer to CISHLT, were characterized by structural modifications with lower inflammation level. We identified 15 probes significantly different between BRISHLT and CISHLT, including the gene of the muscular protein TTN. This result suggests that structural alterations precede inflammation in ACR. Linear Discriminant Analysis based on these 15 probes was able to identify the histological status of every 36 samples. Conclusion Myocardial GEP is a helpful method to accurately diagnose ACR, and predicts rejection one month before its histological occurrence. These results should be considered in cardiac allograft recipients’ care.

The differential expression of MC1R regulators in dorsal and ventral quail plumages during embryogenesis: Implications for plumage pattern formation

Melanin pigmentation patterns are ubiquitous in animals and function in crypsis, physical protection, thermoregulation and signalling. In vertebrates, pigmentation patterns formed over large body regions as well as within appendages (hair/feathers) may be due to the differential distribution of pigment producing cells (melanocytes) and/or regulation of the melanin synthesis pathway. We took advantage of the pigmentation patterns of Japanese quail embryos (pale ventrum and patterned feathers dorsally) to explore the role of genes and their transcripts in regulating the function of the melanocortin-1-receptor (MC1R) via 1. activation: pro-opiomelanocortin (POMC), endoproteases prohormone convertase 1 (PC1) and 2 (PC2), and 2. inhibition—agouti signaling and agouti-related protein (ASIP and AGRP, respectively). Melanocytes are present in all feather follicles at both 8 and 12 days post-fertilisation (E8/E12), so differential deposition of melanocytes is not responsible for pigmentation patterns in embryonic quail. POMC transcripts expressed were a subset of those found in chicken and POMC expression within feather follicles was strong. PC1 was not expressed in feather follicles. PC2 was strongly expressed in all feather follicles at E12. ASIP transcript expression was variable and we report four novel ASIP transcripts. ASIP is strongly expressed in ventral feather follicles, but not dorsally. AGRP expression within feather follicles was weak. These results demonstrate that the pale-bellied quail phenotype probably involves inhibition of MC1R, as found previously. However, quail may require MC1R activation for eumelanogenesis in dorsal feathers which may have important implications for an understanding of colour pattern formation in vertebrates.

The Murine Bladder Supports a Population of Stromal Sca-1+/CD34+/lin- Mesenchymal Stem Cells.

Bladder fibrosis is an undesired end point of injury of obstruction and often renders the smooth muscle layer noncompliant. In many cases, the long-term effect of bladder fibrosis is renal failure. Despite our understanding of the progression of this disease, little is known about the cellular mechanisms that lead to a remodeled bladder wall. Resident stem (progenitor) cells have been identified in various organs such as the brain, heart and lung. These cells function normally during organ homeostasis, but become dysregulated after organ injury. Here, we aimed to characterize a mesenchymal progenitor cell population as a first step in understanding its role in bladder fibrosis. Using fluorescence activated cell sorting (FACS), we identified a Sca-1+/ CD34+/ lin- (PECAM-: CD45-: Ter119-) population in the adult murine bladder. These cells were localized to the stromal layer of the adult bladder and appeared by postnatal day 1. Cultured Sca-1+/ CD34+/ lin- bladder cells self-renewed, formed colonies and spontaneously differentiated into cells expressing smooth muscle genes. These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium. Both acute and partial obstruction of the bladder reduced expression of CD34 and changed localization of Sca-1 to the urothelium. Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population. Our data indicate a resident, mesenchymal stem cell population in the bladder that is altered by bladder obstruction. These findings provide new information about the cellular changes in the bladder that may be associated with bladder fibrosis.