Stop the Divide and Build Coronary Arteries.

Understanding how arteries develop is critical to design repair strategies for coronary artery disease and treatment of arteriovenous malformations. Luo et al. report in Nature that NOTCH drives coronary artery formation by suppressing MYC, a transcription factor involved in cell cycle progression and cell metabolism.

Control of arterial branching morphogenesis in embryogenesis: go with the flow.

Formation of a properly branched vascular system during embryogenesis is crucial for embryo survival. Here we review the regulation of the morphogenesis of the arterial and venous system during embryogenesis. We show that in addition to deterministic patterning mechanisms and plasticity of endothelial cells, arterial-venous differentiation and branching morphogenesis involves a prominent role for blood flow. Based on in vivo observations of developing arteries, we identified a novel morphological event crucial for the morphogenesis of the arterial tree, disconnection of small side branches. This disconnection of side branches occurs exactly at the point of bifurcation. The rate of disconnection of side branches depends on flow velocity and branching angle. The balance between disconnection and maintenance of arterial side branches determines the number of side branches connected to a large artery. Based on these observations, we postulate that the number of pre-existing collaterals connected to a large artery is a function of the disconnection process and can be regulated by hemodynamics. We furthermore show that embryonic arteries already adapt their lumen diameter to the amount of flow carried. Taken together, we suggest that hemodynamics plays a pivotal role in shaping the arterial system. We suggest that flow-evoked remodeling processes determine the number of preexisting collaterals during critical periods of embryo-fetal development. Insight into these basic principles of arterial growth and branching during embryogenesis may aid to understanding the observed variability in the capacity to establish a collateral circulation in patients with ischemic diseases and finding new strategies for therapeutic arteriogenesis.

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.

A model for gene therapy of human hereditary lymphedema.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.

Development of the avian lymphatic system.

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo.

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.

Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo.

The expression of angiopoietin1 and 2 (ang1 and 2) and their receptor tie-2 was studied in avian embryos using in situ hybridization. Ang1 was transcribed in the mesenchymal cells surrounding venous endothelium expressing tie-2. By contrast, ang2 was transcribed around arteries in which the endothelium down-regulated tie-2 mRNA. The aorta and large arteries of the heart outflow tract were never surrounded by ang2 positive cells and maintained tie-2 expression.

Swelling of the proximal nail fold caused by underlying warts.

We report on 2 cases of patients with warts underlying the proximal nail fold which presented as tender erythematous nodules. The remarkable feature in both cases was a significant oedema of the proximal nail fold, resembling a reaction to a foreign body. One patient responded well to mild keratolytic treatment. The second patient was treated surgically with crescentic excision of the distal portion of the proximal nail fold. It is important to recognise this tumour variant in order to avoid unnecessary or excessive intervention.

The heterogeneous clinical spectrum of genital herpes.

BACKGROUND AND OBJECTIVE: Most data of genital herpes have been collected in STD clinics in the USA where unrecognized forms accounted for 80% of HSV-2 infections. Our aim was to study the clinical features in an outpatient clinic of dermatology. METHODS: The charts of 170 patients, previously monitored prospectively for a HIV prevalence study, with culture-confirmed genital herpes or herpetic infection with HSV-2 at any other site presenting between 1995 and 1999 were analyzed. RESULTS: 111 (65%) men and 59 (35%) women were identified with a mean age of 44 years. Only 49% had a typical cluster of genital lesions. Eighty-six (51%) presented with either lesions at extragenital sites [mostly the buttocks 33/170 (19%), thigh 10/170 (6%), anal region 9/170 (5%) and fingers 8/170 (5%)] or showed morphologically atypical forms of isolated genital lesions [single ulcer 16/170 (9%), erosion 6/170 (4%), crust 3/170 (2%) and fissure, edema or erythema each 1/170 (1%)]. Women significantly presented more often with extragenital infections of HSV-2 [36/59 (61%)] than men [18/111 (16%)]. CONCLUSIONS: More than half of the patients with genital herpes of a mainly immunocompetent population presented with atypical manifestations. The underdiagnosis of genital herpes seems largely due to misinterpretation of atypical genital and extragenital lesions.

Hemangioblast commitment in the avian allantois: cellular and molecular aspects.

We recently identified the allantois as a site producing hemopoietic and endothelial cells capable of colonizing the bone marrow of an engrafted host. Here, we report a detailed investigation of some early cytological and molecular processes occurring in the allantoic bud, which are probably involved in the production of angioblasts and hemopoietic cells. We show that the allantois undergoes a program characterized by the prominent expression of several "hemangioblastic" genes in the mesoderm accompanied by other gene patterns in the associated endoderm. VEGF-R2, at least from stage HH17 onward, is expressed and is shortly followed by transcription factors GATA-2, SCL/tal-1, and GATA-1. Blood island-like structures differentiate that contain both CD45(+) cells and cells accumulating hemoglobin; these structures look exactly like blood islands in the yolk sac. This hemopoietic process takes place before the establishment of a vascular network connecting the allantois to the embryo. As far as the endoderm is concerned, GATA-3 mRNA is found in the region where allantois will differentiate before the posterior instestinal portal becomes anatomically distinct. Shortly before the bud grows out, GATA-2 was expressed in the endoderm and, at the same time, the hemangioblastic program became initiated in the mesoderm. GATA-3 is detected at least until E8 and GATA-2 until E3 the latest stage examined for this factor. Using in vitro cultures, we show that allantoic buds, dissected out before the establishment of circulation between the bud and the rest of the embryo, produced erythrocytes of the definitive lineage. Moreover, using heterospecific grafts between chick and quail embryos, we demonstrate that the allantoic vascular network develops from intrinsic progenitors. Taken together, these results extend our earlier findings about the commitment of mesoderm to the endothelial and hemopoietic lineages in the allantois. The detection of a prominent GATA-3 expression restricted to the endoderm of the preallantoic region and allantoic bud, followed by that of GATA-2, is an interesting and novel information, in the context of organ formation and endoderm specification in the emergence of hemopoietic cells.

Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.

We have cloned a novel quail cDNA with strong homology to the pim family of proto-oncogenes. The deduced amino acid (aa) sequence of the cDNA, named qpim, is more closely related to Xenopus Pim and to the recently identified rat Pim-3 than to human or rodent Pim-1 or Pim-2. The protein encoded by the qpim cDNA can autophosphorylate itself and share substrates with murine Pim-1, suggesting functional redundancy to other Pim family serine/threonine kinases. We have compared the expression of qpim in avian embryos to mouse pim-1, -2 and -3 by in situ hybridization. qpim shows a highly dynamic expression pattern, particularly at early developmental stages. Surprisingly, its expression pattern is not identical to any of the murine pim genes, which show complementary and/or partially overlapping expression sites both in- and outside of the hematopoietic system. Altogether, our results suggest novel functions for Pim family kinases during embryonic development, in particular in epithelia and in the central nervous system.

[Receptors and development of endothelial and hematopoietic cells]. / Récepteurs et développement des cellules endothéliales et hématopoïétiques.

During vertebrate embryonic development, the endothelial and hemopoietic systems are the first system to be specified. In this review, we will summarize recent findings about the molecular mechanisms responsible for the successive steps of the development of these systems: the differentiation of mesodermal cells to endothelial and hemopoietic cells, their proliferation and their interactions to form the vascular system.

[Secondary prevention of malignant melanoma]. / Sekundäre Prävention des malignen Melanoms.

Secondary prevention of melanoma covers early recognition of malignant melanoma and precursors. The aims of secondary prevention are: decrease of tumor thickness at the time of first detection and first therapy. decrease of melanoma mortality. Screening of high-risk-persons for melanoma is a fair strategy for Switzerland: easy to perform and also efficient.

[Endothelial cell precursors in the avian embryo]. / Les précurseurs des cellules endothéliales chez l'embryon d'oiseau.

Whereas the origin and migration of endothelial cells (ECs) have been studied primarily in the avian embryo, the molecular mechanisms governing these events in birds and mammals were unraveled following the identification of specific growth factors and of their receptors. In particular, analytic and experimental studies of vascular endothelial growth factor (VEGF) and its receptors have provided significant insights into the developmental biology of the vascular system. VEGFR2 is the earliest marker expressed by EC precursors in chickens and mice. Based on the localization of VEGFR2-positive cells in the avian embryo and on findings from clonal culture experiments, two types of EC precursors can be differentiated as early as the gastrulation stage, namely posterior mesoderm hemangioblasts capable of differentiating into both ECs and hematopoietic cells and anterior angioblasts capable of yielding only ECs.

Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny.

We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be determined by detecting the common leukocyte antigen CD45. VEGF-R2 and CD45 are expressed in complementary fashion, namely the hemopoietic cluster-bearing floor of the aorta is CD45(+)/VEGF-R2(-), while the rest of the aortic endothelium is CD45(-)/VEGF-R2(+). To determine if the hemopoietic clusters are derived from endothelial cells, we tagged the E2 endothelial tree from the inside with low-density lipoproteins (LDL) coupled to DiI. 24 hours later, hemopoietic clusters were labelled by LDL. Since no CD45(+) cells were inserted among endothelial cells at the time of vascular labelling, hemopoietic clusters must be concluded to derive from precursors with an endothelial phenotype.

Lectitis purulenta et granulomatosa (granulomatous purulent nail bed inflammation).

A granulomatous purulent nail bed inflammation of the great toenails in a 69-year-old female patient is described. The successful treatment consisted of intra- and perilesional corticosteroide injections in addition to systemic antibiotic treatment.

Paracrine and autocrine regulation of vascular endothelial growth factor during tissue differentiation in the quail.

The expression of vascular endothelial growth factor (VEGF) has been described to coincide both temporally and spatially with angiogenesis suggesting a role as a paracrine stimulator of endothelial cells. We have used digoxigenin labelled RNA probes to VEGF and the VEGF receptor-2 (Quek1) to investigate the relationship between VEGF expression and vascular events in quail embryos from day 1 to 13 of incubation. Furthermore, the effect of exogenously applied VEGF was studied in day 4 quail embryos using polyclonal anti-VEGF antibodies. Expression of VEGF mRNA was observed in day 1 and 2 embryos in regions of active angiogenesis and hemangiopoiesis. VEGF mRNA expression was found at high levels in the ventral aspect of the neural tube and Quek1 mRNA expression in the accompanying endothelial cells of day 3 embryos, suggesting a function in brain angiogenesis. However, in the neural tube, thyroid gland and cartilaginous skeleton VEGF mRNA was expressed at least 1 day before the ingrowth of vessels, suggesting that additional mechanisms are involved in control of angiogenesis. This is supported by the observation that application of VEGF165 into the midbrain induced dilatation of perineural vessels, while the intraneural vessels remained almost unaffected. Expression of VEGF mRNA was also observed at high levels in podocytes during all stages, indicative of its importance in glomerular development and function. The results of the present study indicate that as angiogenesis occurred in other tissues and organs (day 13 metanephros, dorsal third of the day 7 neural tube, skeletal muscle, and many mesodermal compartments), there was concurrent paracrine expression of VEGF mRNA and Quek1 mRNA. One noteable exception was the hepatocytes of the developing liver which appeared to remain VEGF-negative throughout the study. However, a small number of endothelial cells within liver sinuses, and additionally within the kidney and the elastic arteries, expressed VEGF mRNA. These results suggest that VEGF may also act as an autocrine mediator of angiogenesis, possibly as a result of localised tissue hypoxia.