Coronary vessel development is dependent on the type III transforming growth factor beta receptor

Abstract-Transforming growth factor (TGF)␤ receptor III (TGF␤R3), or ␤-glycan, binds all 3 TGF␤ ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the type I and type II receptors (TGF␤R1, TGF␤R2). TGF␤R3 facilitates signaling via TGF␤R1/TGF␤R2 but also has been suggested to play a unique and nonredundant role in TGF␤ signaling. Targeted deletion of Tgfbr3 revealed a requirement for Tgfbr3 during development of the coronary vessels. Coronary vasculogenesis is significantly impaired in null mice, with few vessels evident and numerous, persistent blood islands found throughout the epicardium. Tgfbr3-null mice die at embryonic day 14.5, the time when functional coronary vasculature is required for embryo viability. However, in null mice nascent coronary vessels attach to the aorta, form 2 coronary ostia, and initiate smooth muscle recruitment by embryonic day 14. Analysis of earlier developmental stages revealed defects in the epicardium. At embryonic day 13.5, these defects include an irregular and hypercellular epicardium with abundant subepicardial mesenchyme and a thin compact zone myocardium. Tgfbr3-null mice also displayed other defects in coronary development, including dysmorphic and distended vessels along the atrioventricular groove and subepicardial hemorrhage. In null mice, vessels throughout the yolk sac and embryo form and recruit smooth muscle in a pattern indistinguishable from heterozygous or wild-type littermates. These data demonstrate a requirement for Tgfbr3 during coronary vessel development that is essential for embryonic viability. (Circ Res. 2007;101:000-000.) Key Words: coronary vessels Ⅲ transforming growth factor ␤ receptor Ⅲ mice, null C oronary artery disease is responsible for 54% of all cardiovascular disease in the United States. 1 Coronary vessels have a unique derivation from mesothelial cells that form a transitory structure termed the proepicardium. Proepicardial cells are transferred to the heart, form the epicardium, and give rise to endothelial cells, smooth muscle cells, and cardiac fibroblasts (reviewed elsewhere 2,3 ). Endothelial cells derived from the epicardium form a vascular plexus by the process of vasculogenesis. This vascular network attaches to the aorta and recruits epicardially derived mesenchyme to become vascular smooth muscle. The identification of the molecular and cellular processes that regulate coronary vessel development may provide insight into coronary vessel disease and reveal novel therapeutic opportunities. The transforming growth factor (TGF)␤ family of growth factors regulates cell growth and differentiation in the cardiovascular system during both development and disease. 4 -6 Three ligands, TGF␤1, TGF␤2, and TGF␤3, 7-9 bind 4 cell surface proteins. These include two transmembrane serine/ threonine kinase receptors, the type I TGF␤ receptor (TGF␤R1) and the type II TGF␤ receptor (TGF␤R2). 10 -12 Several type I receptors, termed activin receptor-like kinases (ALKs), have been described. TGF␤R2 has a constitutively active cytoplasmic kinase domain and an extracellular domain that binds TGF␤1 and TGF␤3 with high affinity. Materials and Methods Generation of Null Mice A targeting vector was made to delete exon 3 that encodes the N terminus, including a portion of the extracellular ligand binding domain. Construction and validation of the targeting vector is described in the online data supplement at http://circres.ahajournals.org (see supplemental Histology, Whole-Mount Immunohistochemistry, and ␤-Galactosidase Staining Detailed methodology is described in the online data supplement. Results Deletion of Tgfbr3 Results in Embryonic Lethality Exon 3 was targeted as depicted in the online data supplement (supplemental Tgfbr3-Null Mice Display Defects in Coronary Vasculogenesis Because embryonic death coincides temporally with the known dependency of embryo viability on the formation of the coronary circulation, we examined null embryos for the presence of coronary vessels. Whole-heart immunostaining for the vascular endothelial cell marker platelet endothelial cell adhesion molecule (PECAM) (also known as CD31) at E14.0 revealed dramatically decreased immunoreactivity in nulls Percentage of Observed Defects in Embryos Tgfbr3-Null Mice Have Abnormal Epicardium and Dysmorphic Coronary Vessels The epicardium is derived from the proepicardium and contains coronary vessel precursor cells. At E13.5, the epicardium forms a tightly apposed monolayer on the surface on the atria and ventricles. In the region of the atrioventricular groove, the epicardium is separated from the myocardium by a layer of epicardial-derived mesenchyme ( Tgfbr3-Null Mice Initiate Recruitment of Smooth Muscle to Extracardiac and Coronary Vessels TGF␤ has multiple roles during blood vessel formation, including vascular smooth muscle cell recruitment. After right and left coronary arteries attach to the systemic circulation at the aorta, smooth muscle recruitment commences at the coronary ostia and progresses distally. As noted in Discussion Disruption of Tgfbr3 revealed a requirement for coronary vessel development and embryonic viability. Null embryos die at E14.5 with defects in coronary vessel development, whereas vasculature outside of the coronary circulation appears normal. Although greatly reduced in size, nascent coronary vessels attach to the aorta and initiate smooth muscle recruitment. Coronary vessel defects are coincident with epicardial abnormalities that include increased space between the epicardium and myocardium, abundant subepi- cardial mesenchyme, and persistent blood islands. In addition to coronary vessel anomalies, null embryos have OFT abnormalities and myocardial thinning. Presumably, the greatly reduced coronary vasculature in null embryos is not sufficient to adequately perfuse the heart resulting in embryonic death. Despite the abundance of data implicating TGF␤ signaling in vasculogenesis and angiogenesis, these processes appear to occur normally outside of the coronary vessels in nulls. The localization of defects to the coronary vessels may be explained by the unique derivation of these vessels (reviewed elsewhere 2,3 ). The proepicardium, adjacent to the liver rudiment, is transferred to the heart and gives rise to the epicardium as well as coronary endothelial cells, smooth muscle cells, and cardiac fibroblasts. Although targeted gene deletion in mice has uncovered roles for several molecules in coronary vessel development, none has a phenotype similar to Tgfbr3 nulls. Deletion of vascular cell adhesion molecule-1 40 or the counter receptor ␣4 integrin Defects in coronary vasculogenesis in Tgfbr3 nulls do not appear to result from defects in angioblast or endothelial differentiation because PECAM-positive cells do appear. A failure in coronary vasculogenesis could result from an insufficient population of angioblasts or endothelial cells, a possibility we cannot exclude because the numbers of these cells was not quantitated. At E14.5, we saw fewer vessels on the heart and a complete absence of large vessels formed by remodeling of the primary vascular plexus. This observation suggests a defect in angioblast or endothelial cell assembly or remodeling. TGF␤R3 contains a cytoplasmic PDZ domain that has been shown to bind glycoinositolphosphorylceramide or synectin

Predicting protein decomposition: the case of aspartic-acid racemization kinetics

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L- isomer has been widely used in archaeology and geochemistry as a tool for dating. The method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathe- matical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn+Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemi- zation kinetics in proteins at high temperatures (95-140 °C). The model fails to predict racemization kinetics in dentine collagen at 37 °C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly in£uences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx re£ects the proportion of non- helical to helical collagen, overlain by the e¡ects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement

Reflections on the 'History and Historians' of the black woman's role in the community of slaves: enslaved women and intimate partner sexual violence

Taking as points of inspiration Peter Parish’s 1989 book, Slavery: History and Historians, and Angela Davis’s seminal 1971 article, “Reflections on the black woman’s role in the community of slaves,” this probes both historiographically and methodologically some of the challenges faced by historians writing about the lives of enslaved women through a case study of intimate partner violence among enslaved people in the antebellum South. Because rape and sexual assault have been defined in the past as non-consensual sexual acts supported by surviving legal evidence (generally testimony from court trials), it is hard for historians to research rape and sexual violence under slavery (especially marital rape) as there was no legal standing for the rape of enslaved women or the rape of any woman within marriage. This article suggests enslaved women recognized that black men could both be perpetrators of sexual violence and simultaneously be victims of the system of slavery. It also argues women stoically tolerated being forced into intimate relationships, sometimes even staying with “husbands” imposed upon them after emancipation

Images of the month: Cavernous sinus venous thrombosis secondary to Streptococcus milleri maxillary sinusitis: An unusual cause of diplopia and headache.

Cavernous sinus venous thrombosis is an uncommon condition associated with high mortality rates if not recognised early. Symptoms include headache, visual loss, ophthalmoplegia, altered consciousness, proptosis and periorbital oedema. High-quality imaging is critical in early diagnosis and successful management. Primary infection (such as sinusitis) and possible complications (including meningitis) should be considered as potential aetiologies of cavernous sinus venous thrombosis, especially in those with a preceding history of localised infection. We present a case of a 50-year-old man with a bilateral cavernous sinus venous thrombosis with associated meningitis caused by Streptococcus milleri, secondary to maxillary sinusitis and otomastoiditis. He was successfully treated with antimicrobial treatment, surgical drainage and anticoagulation