The development of early human lymphatic vessels as characterized by lymphatic endothelial markers.

Lymphatic vessel development studies in mice and zebrafish models have demonstrated that lymphatic endothelial cells (LECs) predominantly differentiate from venous endothelial cells via the expression of the transcription factor Prox1. However, LECs can also be generated from undifferentiated mesoderm, suggesting potential diversity in their precursor cell origins depending on the organ or anatomical location. Despite these advances, recapitulating human lymphatic malformations in animal models has been difficult, and considering lymphatic vasculature function varies widely between species, analysis of development directly in humans is needed. Here, we examined early lymphatic development in humans by analyzing the histology of 31 embryos and three 9-week-old fetuses. We found that human embryonic cardinal veins, which converged to form initial lymph sacs, produce Prox1-expressing LECs. Furthermore, we describe the lymphatic vessel development in various organs and observe organ-specific differences. These characterizations of the early development of human lymphatic vessels should help to better understand the evolution and phylogenetic relationships of lymphatic systems, and their roles in human disease.

In vivo testing of novel nitric oxide-releasing nanoparticles for alleviating heart failure using the zebrafish embryo model.

Heart failure (HF) is a multifactorial, heterogeneous systemic disease that is considered one of the leading causes of death and morbidity worldwide. It is well-known that endothelial dysfunction (ED) plays an important role in cardiac disease etiology. A reduction in the bioavailability of nitric oxide (NO) in the bloodstream leads to vasoconstriction and ED. Many studies indicated diminishment of peripheral arteries vasodilation that is mediated by the endothelium in the of patients with chronic HF. With the advancement of nanomedicine, nanotechnology can provide adequate solutions for delivering exogenous NO with the aid of nanoparticles (NPs) to treat ED. The properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable both passive and active delivery of drugs. This prompted us to investigate the efficacy of our newly-developed hydrogel nanoparticles (NO-RPs) for the delivery and sustained release of NO gas to alleviate cardiac failure and inflammation in the heart failure zebrafish model. The hydrogel NO-RPs incorporate SPIONS and NO precursor. The sustainend release of NO in the NO-RPs (4200 s), overcomes the problem of the short half life of NO in vivo which is expected to ameliorate the reduced NO bioavailabilty, and its consequences in endothelial and cardiac dysfunction. Zebrafish embryos were used as the animal model in this study to determine the effect of SPIONs-loaded NO-RPs on the cardiovascular system. Cardiac failure was induced in 24hpf embryos by exposure to aristolochic acid (AA)(0.25, 0.5 µM) for 8 h, followed by the SPIONs-loaded NO-RPs (0.25, 0.5 mg/ml) for 48 h, experimental groups included: control group which is healthy non treated zebrafish embryos, AA injured zebrafish embryos (HF) model,and NO-RP treated HF zebrafish embryos. Survival rate was assessed at 72hpf. Cardiac function was also evaluated by analyzing cardiac parameters including heartbeat, major blood vessels primordial cardinal vein and dorsal aorta (PCV &DA) diameter, blood flow velocity in PCV & DA vessels, cardiac output, and PCV & DA shear stresses. All cardiac parameters were analyzed with the aid of MicroZebraLab blood flow analysis software from Viewpoint. In addition, we studied the molecular effects of the developed NO-RPs on the mRNA expression of selected pro-inflammatory markers: IL-6, and Cox-2. Our findings demonstrated that the NO-RPs improved the survival rate in the heart failure zebrafish model and reversed heart failure by enhancing blood flow perfusion in Zebrafish embryos, significantly. In addition, RT-PCR results showed that the NO-RPs significantly reduced the expression of pro-inflammatory markers (lL-6&COX-2) in the heart failure zebrafish model. Our study confirmed that the developed SPIONs-loaded NO-RPs are effective tool to alleviate cardiac failure and inflammation in the HF zebrafish model.

Effects and mechanisms of Porphyromonas gingivalis outer membrane vesicles induced cardiovascular injury.

BACKGROUND: The outer membrane vesicles (OMVs) derived from Porphyromonas gingivalis (P. gingivalis) have long been acknowledged for their crucial role in the initiation of periodontitis. However, the implications of P. gingivalis OMVs in the context of cardiovascular disease (CVD) remain incompletely understood. This study aimed to clarify both the impact and the underlying mechanisms through which P. gingivalis OMVs contribute to the propagation of distal cardiovascular inflammation and trauma. METHODS: In this study, various concentrations (0, 1.25, 2.5, and 4.5 µg/µL) of P. gingivalis OMVs were microinjected into the common cardinal vein of zebrafish larvae at 48 h post-fertilization (hpf) to assess changes in cardiovascular injury and inflammatory response. Zebrafish larvae from both the PBS and the 2.5 µg/µL injection cohorts were harvested at 30 h post-injection (hpi) for transcriptional analysis. Real-time quantitative PCR (RT-qPCR) was employed to evaluate relative gene expression. RESULTS: These findings demonstrated that P. gingivalis OMVs induced pericardial enlargement in zebrafish larvae, caused vascular damage, increased neutrophil counts, and activated inflammatory pathways. Transcriptomic analysis further revealed the involvement of the immune response and the extracellular matrix (ECM)-receptor interaction signaling pathway in this process. CONCLUSION: This study illuminated potential mechanisms through which P. gingivalis OMVs contribute to CVD. It accentuated their involvement in distal cardiovascular inflammation and emphasizes the need for further research to comprehensively grasp the connection between periodontitis and CVD.

Live imaging of Fibronectin 1a-mNeonGreen and Fibronectin 1b-mCherry knock-in alleles during early zebrafish development.

Within the developing embryo, cells assemble and remodel their surrounding extracellular matrix during morphogenesis. Fibronectin is an extracellular matrix glycoprotein and is a ligand for several members of the Integrin adhesion receptor family. Here, we compare the expression pattern and loss of function phenotypes of the two zebrafish fibronectin paralogs fn1a and fn1b. We engineered two fluorescently tagged knock-in alleles to facilitate live in vivo imaging of the Fibronectin matrix. Genetic complementation experiments indicate that the knock-in alleles are fully functional. Fn1a-mNeonGreen and Fn1b-mCherry are co-localized in ECM fibers on the surface of the paraxial mesoderm and myotendinous junction. In 5-days old zebrafish larvae, Fn1a-mNeonGreen predominantly localizes to the branchial arches, heart ventricle, olfactory placode and within the otic capsule while Fn1b-mCherry is deposited at the pericardium, proximal convoluted tubule, posterior hindgut and at the ventral mesoderm/cardinal vein. We examined Fn1a-mNeonGreen and Fn1b-mCherry in maternal zygotic integrin α5 mutants and integrin ß1a; ß1b double mutants and find distinct requirements for these Integrins in assembling the two Fibronectins into ECM fibers in different tissues. Rescue experiments via mRNA injection indicate that the two fibronectins are not fully inter-changeable. Lastly, we examined cross-regulation between the two Fibronectins and find fn1a is necessary for normal Fn1b fibrillogenesis in the presomitic mesoderm, but fn1b is dispensable for the normal pattern of Fn1a deposition.

Fetal liver hematopoietic stem cell niches associate with portal vessels.

Whereas the cellular basis of the hematopoietic stem cell (HSC) niche in the bone marrow has been characterized, the nature of the fetal liver niche is not yet elucidated. We show that Nestin(+)NG2(+) pericytes associate with portal vessels, forming a niche promoting HSC expansion. Nestin(+)NG2(+) cells and HSCs scale during development with the fractal branching patterns of portal vessels, tributaries of the umbilical vein. After closure of the umbilical inlet at birth, portal vessels undergo a transition from Neuropilin-1(+)Ephrin-B2(+) artery to EphB4(+) vein phenotype, associated with a loss of periportal Nestin(+)NG2(+) cells and emigration of HSCs away from portal vessels. These data support a model in which HSCs are titrated against a periportal vascular niche with a fractal-like organization enabled by placental circulation.

Developmental anatomy of the liver from computerized three-dimensional reconstructions of four human embryos (from Carnegie stage 14 to 23).

BACKGROUND & AIM: Some aspects of human embryogenesis and organogenesis remain unclear, especially concerning the development of the liver and its vasculature. The purpose of this study was to investigate, from a descriptive standpoint, the evolutionary morphogenesis of the human liver and its vasculature by computerized three-dimensional reconstructions of human embryos. MATERIAL & METHODS: Serial histological sections of four human embryos at successive stages of development belonging to three prestigious French historical collections were digitized and reconstructed in 3D using software commonly used in medical radiology. Manual segmentation of the hepatic anatomical regions of interest was performed section by section. RESULTS: In this study, human liver organogenesis was examined at Carnegie stages 14, 18, 21 and 23. Using a descriptive and an analytical method, we showed that these stages correspond to the implementation of the large hepatic vascular patterns (the portal system, the hepatic artery and the hepatic venous system) and the biliary system. CONCLUSION: To our knowledge, our work is the first descriptive morphological study using 3D computerized reconstructions from serial histological sections of the embryonic development of the human liver between Carnegie stages 14 and 23.

Gastric and ectopic varices.

Although often considered together, gastric and ectopic varices represent complications of a heterogeneous group of underlying diseases. Commonly, these are known to arise in patients with cirrhosis secondary to portal hypertension; however, they also arise in patients with noncirrhotic portal hypertension, most often secondary to venous thrombosis of the portal venous system. One of the key initial assessments is to define the underlying condition leading to the formation of these portal-collateral pathways to guide management. In the authors' experience, these patients can be grouped into distinct although sometimes overlapping conditions, which can provide a helpful conceptual basis of management.

Evolution of congenital malformations of the umbilical-portal-hepatic venous system.

OBJECTIVE: The objective of this study is to describe the evolution of 8 cases of congenital malformations of the umbilical-portal-hepatic venous system diagnosed before the first month of life. MATERIALS AND METHODS: All cases of congenital malformation of the portal and hepatic venous system diagnosed prenatally or during the first month of life in our institution were systematically reviewed since November 2000. Clinical features, imaging, and anatomical findings were reviewed, focusing primarily on clinical and radiologic evolution. RESULTS: Eight cases of congenital malformation of the umbilical-portal-hepatic venous system were studied. Fifty percent of these malformations were diagnosed prenatally. We report 4 portosystemic shunts. Three involuted spontaneously, and the fourth one required surgical treatment. We report a variation of the usual anatomy of portal and hepatic veins that remained asymptomatic, an aneurysmal dilatation of a vitelline vein causing portal vein thrombosis that needed prompt surgical treatment with good result, a complex portal and hepatic venous malformation treated operatively, and a persistent right umbilical vein that remained asymptomatic. CONCLUSION: Prenatal diagnosis of malformations of the umbilical-portal-hepatic venous network is uncommon. Little is known about the postnatal prognosis. Clinical, biologic, and radiologic follow-up by ultrasonography is essential to distinguish pathologic situations from normal anatomical variants.

Treatment of growth-restricted human fetuses with amino acids and glucose supplementation through a chronic fetal intravascular perinatal port system.

OBJECTIVE: Intrauterine growth restriction (IUGR) carries an increased risk of mortality and morbidity. The accepted procedure to treat IUGR fetuses is premature delivery, which may increase neonatal mortality and morbidity and retards neonatal brain development. MATERIAL AND METHODS: We report here on intravascular supplementation with amino acids and glucose of an IUGR human fetus at 33 weeks of gestation with oligohydramnios and placental insufficiency using the port system (Norfolk Medical Products, Skokie, Ill., USA). The catheter was implanted into the umbilical vein (UV) by cordocentesis, and was then connected to a subcutaneously implanted port system. The treatment course included daily infusions of amino acid solution and 10% glucose into the UV. RESULTS: Daily intravascular fetal nutrition significantly improved both fetal condition and fetal weight gain. No complications were seen. The patient was delivered by cesarean section in the 38th week of gestation. The female newborn weighed 2,130 g and was 47 cm long. Blood sampling from the UV after delivery showed no deviations of amino acids in comparison to standardized curves. In one-year follow-up the child's development and weight gain was like that of children without IUGR in the anamnesis. CONCLUSION: This is the first report of the successful use of a subcutaneously implanted intravascular perinatal port system in IUGR human fetuses for long-term administration of nutriments into the UV of a fetus.

Prenatal diagnosis of congenital agenesis of the fetal portal venous system.

OBJECTIVE: To describe the prenatal diagnosis and review our experience of fetal congenital agenesis of the portal venous system (CAPVS) and to review the current literature on this poorly documented vascular malformation. METHODS: This was a retrospective survey covering the 12-year period between 1996 and 2008. The database of a single, large, ultrasonographic tertiary academic referral center in Israel was analyzed and cases with a prenatal diagnosis of CAPVS were identified. All fetuses underwent detailed biometric and structural ultrasound examinations and a precise anatomical description of the fetal umbilical, portal and hepatic venous system was noted, as well as the presence of aberrant vessels, shunt location and the presence or absence of the DV. Results of fetal echocardiography, karyotyping and toxoplasma, rubella, cytomegalovirus and herpes evaluations were determined. Medical records were evaluated. Diagnosis was confirmed by pathology, postmortem venography or neonatal ultrasound or venography. Liveborns were examined by a certified neonatologist and long-term follow-up from pediatric gastroenterology units was determined. RESULTS: Nine cases with CAPVS were studied. In all cases an aberrant umbilical-portal vein was the primary indication for detailed portal system evaluation. Five fetuses demonstrated total CAPVS (Type I) and four showed partial agenesis of the portal vein (Type II). Among the five Type I fetuses, there was a shunt from the umbilical vein to the inferior vena cava in three (60%), to the right atrium in one and to the coronary sinus in one. In this group, in only one case could we delineate a common confluence between the splenic vein and the superior mesenteric vein shunting to the inferior vena cava. In four cases termination of pregnancy was performed due to additional findings: one case with hydrothorax, ascites and mitral atresia, one with cleft lip/palate and one with trisomy 21. One case had no additional anomalies, but the parents elected to terminate the pregnancy. All four of the Type II fetuses had a portosystemic shunt: in two cases to the right atrium, in one to the iliac vein and in one to the right hepatic vein. In three, the shunt resolved spontaneously. In only one case was abnormal liver function present over a follow-up period of 2-10 years. CONCLUSION: CAPVS can be detected prenatally. An abnormal course of the umbilical vein necessitates prompt sonographic evaluation of the umbilical-portal venous system and meticulous investigation for additional anomalies. Complete CAPVS may be associated with remote clinical consequences of which the parents should be informed. Partial CAPVS has a favorable prognosis.

p75 Neurotrophin receptor is a marker for precursors of stellate cells and portal fibroblasts in mouse fetal liver.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are 2 distinct mesenchymal cells in adult liver. HSCs in sinusoids accumulate lipids and express p75 neurotrophin receptor (p75NTR). HSCs and PFs play pivotal roles in liver regeneration and fibrosis. However, the roles of mesenchymal cells in fetal liver remain poorly understood. In this study, we aimed to characterize mesenchymal cells in mouse fetal liver. METHODS: We prepared an anti-p75NTR monoclonal antibody applicable for flow cytometry and immunohistochemistry. p75NTR(+) cells isolated from fetal liver by flow cytometry were characterized by reverse-transcription polymerase chain reaction, immunohistochemistry, and cell cultivation. Lipid-containing cells were visualized by Oil-red O staining. RESULTS: p75NTR(+) cells in fetal liver were clearly distinct from endothelial cells and showed characteristics of mesenchymal cells. At embryonic day (E) 10.5, p75NTR(+) cells were present at the periphery of the liver bud in close contact with endothelial cells, and spread over the liver at E11.5. With the formation of the liver architecture, they began to localize to 2 distinct areas, parenchymal and portal areas, and lipid-containing p75NTR(+) cells increased accordingly. p75NTR(+) cells around portal veins were adjacent to cholangiocytes and expressed Jagged1, a crucial factor for the commitment of hepatoblasts to cholangiocytes. By cultivation, p75NTR(+) cells showed features of adult HSCs with markedly increased expression of glial fibrillary acidic protein and alpha-smooth muscle actin. CONCLUSIONS: p75NTR(+) mesenchymal cells in fetal liver include progenitors for HSCs and PFs, and the anti-p75NTR monoclonal antibody is useful for their isolation.

Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development.

UNLABELLED: Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis. CONCLUSION: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.

The human fetal venous system: normal embryologic, anatomic, and physiologic characteristics and developmental abnormalities.

OBJECTIVE: The introduction of high-resolution ultrasonography combined with color-coded Doppler imaging offered a breakthrough in the evaluation of the human fetal venous system, considerably enhancing our understanding of fetal venous circulation in normal physiologic conditions, as well as providing us the ability to study circulatory changes in abnormal circumstances. The purpose of this study was to describe the normal anatomic development and complex of anomalies of the human fetal venous system and to review recently published series of these anomalies. METHODS: Normal embryologic and anatomic development is described. An English language literature search of recent MEDLINE listings was performed to glean data from recently published series reporting prenatal diagnosis of the various anomalies and their associated malformations. RESULTS: Anomalies of the human fetal venous system occur sporadically, often associated with cardiac or other malformations. The pathophysiologic mechanisms leading to abnormal in utero development of the human venous system remain largely undetermined. On the basis of the type of vein involved, embryologic precursor, and etiologic correlation (primary or secondary), classification into 4 major groups is described. CONCLUSIONS: Prenatal evaluation of fetuses found to have anomalies of the venous system should include a careful search for cardiac anomalies, including pulmonary venous drainage, and a detailed anatomic survey of the umbilical, portal, hepatic, and ductal systems to determine aberrant communication and, if possible, to discover clues to systemic diseases or thromboembolic phenomena.

The correlation between portal myofibroblasts and development of intrahepatic bile ducts and arterial branches in human liver.

BACKGROUND/AIMS: The development of the intrahepatic bile ducts most likely requires interactions between epithelial and mesenchymal cells. In view of the epithelial-mesenchymal interactions between portal myofibroblasts (pMFs) and biliary epithelial cells in adult diseases of the bile ducts, we investigated the presence and function of pMFs during the development of intrahepatic bile ducts, as well as the development of intrahepatic branches of the hepatic artery. METHODS: We performed haematoxylin-eosin-stainings and immunohistochemistry for alpha-smooth-muscle actin, cytokeratin 19 and vimentin on serial sections of 45 fetal and postnatal liver biopsies. RESULTS: The mesenchyme of portal tracts in the ductal plate stage devoid of a hepatic artery branch, contained numerous and diffusely scattered pMFs. Portal tracts with a hepatic artery branch were always larger than those without and showed a decreasing number of pMFs. In the remodeling stage, all portal tracts contained a hepatic artery branch, and pMFs were restricted to the periductal mesenchyme. These periductal pMFs disappeared after full incorporation of the bile duct. CONCLUSION: Our findings strongly suggest interactions between pMFs and epithelial cells of the developing bile ducts. The development of the intrahepatic arterial branches always precedes the incorporation of the tubular segments of the ductal plate.

Assessment of fetal cardiovascular function: ultrasound study of the fetal circulatory compartments.

Recent advances in high-resolution ultrasonography combined with color-coded Doppler analysis have enabled a new insight to be gained into fetal physiology and allowed the investigation of small, previously inaccessible fetal vessels under normal and abnormal conditions. In addition, the findings in some cases contradict the accepted fetal pathophysiology derived from animal studies. The aim of the present study is to describe new reports assessing the major compartments of the fetal cardiovascular system (heart, lung, splanchnic and fetal venous circulations) and to discuss the developments in these fields.

Congenital anomalies of the portal venous system--CT appearances with embryological considerations.

The portal vein is formed by the union of the splenic and superior mesenteric veins behind the neck of the pancreas. This system is derived from the vitelline veins, a component of the extraembryonic venous system. In this paper we report three cases, each of which illustrates a congenital variant of the portal venous system, describe their computed tomography appearances and discuss the embryological processes accounting for these anomalies.

[Ectasia of the umbilical recess during the antenatal period. 3 case reports]. / Ectasie du récessus ombilical en période anténatale. A propos de 3 observations.

Having found three cases of ectasia of the umbilical recess in the antenatal period we decided to review the embryology and the ultrasound of the anatomy of the portal system. We consider that this is a normal anatomic variant of the portal system because the lesion is asymptomatic and regresses spontaneously.