hCALCRL mutation causes autosomal recessive nonimmune hydrops fetalis with lymphatic dysplasia.

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.

Acute small intestinal inflammation results in persistent lymphatic alterations.

Inflammatory bowel disease (IBD) has a complex pathophysiology with limited treatments. Structural and functional changes in the intestinal lymphatic system have been associated with the disease, with increased risk of IBD occurrence linked to a history of acute intestinal injury. To examine the potential role of the lymphatic system in inflammation recurrence, we evaluated morphological and functional changes in mouse mucosal and mesenteric lymphatic vessels, and within the mesenteric lymph nodes during acute ileitis caused by a 7-day treatment with dextran sodium sulfate (DSS). We monitored whether the changes persisted during a 14-day recovery period and determined their potential consequences on dendritic cell (DC) trafficking between the mucosa and lymphoid tissues. DSS administration was associated with marked lymphatic abnormalities and dysfunctions exemplified by lymphangiectasia and lymphangiogenesis in the ileal mucosa and mesentery, increased mesenteric lymphatic vessel leakage, and lymphadenopathy. Lymphangiogenesis and lymphadenopathy were still evident after recovery from intestinal inflammation and correlated with higher numbers of DCs in mucosal and lymphatic tissues. Specifically, a deficit in CD103+ DCs observed during acute DSS in the lamina propria was reversed and further enhanced during recovery. We concluded that an acute intestinal insult caused alterations of the mesenteric lymphatic system, including lymphangiogenesis, which persisted after resolution of inflammation. These morphological and functional changes could compromise DC function and movement, increasing susceptibility to further gastrointestinal disease. Elucidation of the changes in mesenteric and intestinal lymphatic function should offer key insights for new therapeutic strategies in gastrointestinal disorders such as IBD. NEW & NOTEWORTHY Lymphatic integrity plays a critical role in small intestinal homeostasis. Acute intestinal insult in a mouse model of acute ileitis causes morphological and functional changes in mesenteric and intestinal lymphatic vessels. While some of the changes significantly regressed during inflammation resolution, others persisted, including lymphangiogenesis and altered dendritic cell function and movement, potentially increasing susceptibility to the recurrence of gastrointestinal inflammation.

Loss of ADAMTS3 activity causes Hennekam lymphangiectasia-lymphedema syndrome 3.

Primary lymphedema is due to developmental and/or functional defects in the lymphatic system. It may affect any part of the body, with predominance for the lower extremities. Twenty-seven genes have already been linked to primary lymphedema, either isolated, or as part of a syndrome. The proteins that they encode are involved in VEGFR3 receptor signaling. They account for about one third of all primary lymphedema cases, underscoring the existence of additional genetic factors. We used whole-exome sequencing to investigate the underlying cause in a non-consanguineous family with two children affected by lymphedema, lymphangiectasia and distinct facial features. We discovered bi-allelic missense mutations in ADAMTS3. Both were predicted to be highly damaging. These amino acid substitutions affect well-conserved residues in the prodomain and in the peptidase domain of ADAMTS3. In vitro, the mutant proteins were abnormally processed and sequestered within cells, which abolished proteolytic activation of pro-VEGFC. VEGFC processing is also affected by CCBE1 mutations that cause the Hennekam lymphangiectasia-lymphedema syndrome syndrome type1. Our data identifies ADAMTS3 as a novel gene that can be mutated in individuals affected by the Hennekam syndrome. These patients have distinctive facial features similar to those with mutations in CCBE1. Our results corroborate the recent in vitro and murine data that suggest a close functional interaction between ADAMTS3 and CCBE1 in triggering VEGFR3 signaling, a cornerstone for the differentiation and function of lymphatic endothelial cells.

Dachsous1-Fat4 Signaling Controls Endothelial Cell Polarization During Lymphatic Valve Morphogenesis-Brief Report.

OBJECTIVE: The purpose of this study was to investigate the role of Fat4 and Dachsous1 signaling in the lymphatic vasculature. APPROACH AND RESULTS: Phenotypic analysis of the lymphatic vasculature was performed in mice lacking functional Fat4 or Dachsous1. The overall architecture of lymphatic vasculature is unaltered, yet both genes are specifically required for lymphatic valve morphogenesis. Valve endothelial cells (Prox1high [prospero homeobox protein 1] cells) are disoriented and failed to form proper valve leaflets. Using Lifeact-GFP (green fluorescent protein) mice, we revealed that valve endothelial cells display prominent actin polymerization. Finally, we showed the polarized recruitment of Dachsous1 to membrane protrusions and cellular junctions of valve endothelial cells in vivo and in vitro. CONCLUSIONS: Our data demonstrate that Fat4 and Dachsous1 are critical regulators of valve morphogenesis. This study highlights that valve defects may contribute to lymphedema in Hennekam syndrome caused by Fat4 mutations.

Sjögren's syndrome associated with protein losing gastroenteropathy manifested by intestinal lymphangiectasia successfully treated with prednisolone and hydroxychloroquine.

Protein-losing gastroenteropathy (PLGE), a rare manifestation of primary Sjögren's syndrome (SS), is characterized by profound edema and severe hypoalbuminemia secondary to excessive serum protein loss from the gastrointestinal tract and is clinically indistinguishable from nephrotic syndrome. We report a case of a 30-year-old Taiwanese woman with PLGE-associated SS. In addition to a positive Schirmer's test, she had eye-dryness, thirst, and high levels of anti-SSA antibodies, fulfilling SS criteria. PLGE diagnosis was highly appropriate given the clinical profile of hypoalbuminemia, hypercholesterolemia, pleural effusion, and ascites, with absent cardiac, hepatic, or renal disease. We were unable to perform technetium-99 m-labeled human serum albumin scintigraphy ((99m)Tc-HAS). However, the patient's edema and albumin level improved dramatically in response to a 3-month regime of oral prednisolone followed by oral hydroxychloroquine.

Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.

RATIONALE: Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. OBJECTIVE: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. METHODS AND RESULTS: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. CONCLUSIONS: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.

Intestinal lymphangiectasis and lipidosis in rats following subchronic exposure to indole-3-carbinol via oral gavage.

To investigate the toxicity and carcinogenic potential of indole-3-carbinol (I3C), the National Toxicology Program has conducted 13-week subchronic studies in Fisher 344 rats and B6C3F1 mice, and chronic 2-year bioassays in Sprague-Dawley rats and B6C3F1 mice. While the chronic study results are not yet available, subchronic study results and short-term special evaluations of interim sacrifices in the 2-year rat bioassay are presented. F344 rats were orally gavaged ≤300 mg I3C/kg body weight 5 days a week for 13 weeks. Rats treated with ≥150 mg/kg demonstrated a dose-related dilation of lymphatics (lymphangiectasis) of the duodenum, jejunum, and mesenteric lymph nodes. Material within dilated lacteals stained positively for Oil Red O and Sudan Black, consistent with lipid. Electron microscopic evaluation confirmed extracellular lipid accumulation within the villar lamina propria, lacteals, and within villar macrophages. Analyses of hepatic and pulmonary CYP1A enzymes demonstrated dose-dependent I3C induction of CYP1A1 and 1A2. B6C3F1 mice orally gavaged ≤250 mg I3C/kg body weight did not demonstrate histopathological changes; however, hepatic CYP induction was similar to that in rats. The histopathologic changes of intestinal lymphangiectasis and lipidosis in this study share similarities with intestinal lymphangiectasia as observed in humans and dogs. However, the resultant clinical spectrum of protein-losing enteropathy was not present.

Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

BACKGROUND AND AIM: Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. METHODS: Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. RESULTS: In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. CONCLUSIONS: There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

Fat absorption in patients with functional intestinal lymphangiectasia and lymphangiectic cysts.

Nine patients with endoscopically identified dilated lacteals of the duodenum were studied for evidence of pathologic intestinal lymphangiectasia. Three of the nine patients also had lymphangietic cysts in association with dilated lacteals. Duodenal biopsies, laboratory data, and imaging studies were performed in each patient. In addition, a 14C triolein fat absorption study was performed to assess subclinical malabsorption. Biopsies revealed dilated lymphatic channels in all patients, but laboratory studies failed to suggest intestinal losses of protein or fat, and radiographic abdominal imaging failed to define any of the causes of secondary lymphangiectasia. Eight of the nine patients had adequate fat absorption as measured by the 14C triolein breath test. Our data suggest that patients with incidentally discovered dilated lacteals and no clinical evidence of malabsorption may have a functional intestinal lymphangiectasia. Follow-up endoscopy probably is not warranted in this population.

Pathological and pathophysiological study on intestinal lymphatic system in fat absorption.

Pathological and pathophysiological study on the intestinal lymphatics was carried out clinically and experimentally. Jejunal biopsy study revealed marked dilatation of intestinal lymphatics in liver cirrhosis with ascites and Behçet's disease as well as protein-losing enteropathy. Lymphangiographic study showed also abnormal findings in above-mentioned diseases. In cases of McKee dogs in which mechanism of blockade of lymphatic flow was already clarified, McKee dogs showed marked malabsorptive of fat. When lymphatic flow was blocked mechanically at the thoracic duct, fat accumulation was seen in the absorptive cells as well as in dilated intestinal lymphatics. In administration of colchicine to rats, fat accumulation was seen in the matrix of absorptive cells as well as in the endoplasmic reticulums and Golgi apparatus. However, amorphous substances instead of chylomicrons were seen in dilated intestinal lymphatics. These studies suggested that a tract of fat transportation from intestinal cells to the thoracic duct should be considered as a functional unit in fat absorption.

Endogenous faecal calcium in chronic malabsorption syndromes and in intestinal lymphangiectasia.

Endogenous faecal calcium was measured by an isotopic technique in five patients with protein-losing enteropathy due to intestinal lymphangiectasia and in ten patients with chronic malabsorption due to severe Crohn's disease (one patient) or extensive small-bowel resection (nine patients). In most patients absorption of dietary calcium and calcium balance were also determined. Endogenous faecal calcium and digestive juice calcium were highly increased in 3 patients with intestinal lymphangiectasia and normal or subnormal in the remaining 12 patients. Absorption of dietary calcium was normal in patients with intestinal lymphangiectasia but extremely low in most patients with chronic malabsorption syndromes. It is concluded that a net loss of calcium in stools in patients with intestinal lymphangiectasia is due to increased endogenous faecal calcium. In contrast, a net loss of calcium in stools in patients with extensive small-bowel resection is due to decreased absorption of dietary calcium with normal or almost normal endogenous faecal calcium.

Protein-losing enteropathy secondary to intestinal lymphangiectasia in a dog.

Protein-losing enteropathy secondary to intestinal lymphangiectasia was diagnosed in a 1 1/2 -year-old female Doberman Pinscher. Poor growth rate, intermittent diarrhea, ascites, edema, hypoproteinemia, grossly dilated intestinal lymphatics, and impaired fat absorption characterized the disease. There was histopathologic evidence of dilatation of lacteals and excessive chromium (51Cr)-labeled human albumin in the feces following its intravenous administration. Sixteen weeks of a special diet (high in carbohydrates and medium-chain triglycerides; low in long-chain triglycerides) led to remission of clinical signs. Serum albumin increased over its initial value of 1.09 to 1.7 g/dl, while serum globulin increased from 1.71 to 1.9 g/dl. Following 9 months of dietary therapy, serum albumin increased to 2.1 g/dl and serum globulin increased to 2.6 g/dl.

Limitations of the usefulness of the d-xylose absorption test.

The results of the conventional 5-hour d-xylose absorption test were surveyed in 38 subjects with disease of the jejunal mucosa, giardiasis or bacterial overgrowth, or no small-bowel disorder. The test was in error in 20-40% of cases, depending on the disease category, and the error for the entire group was 30%. We conclude that the test yields little guidance for diagnosis or therapy of clinical problems and is superfluous when a jejunal biopsy can be obtained.

The relative importance of the factors involved in the absorption of vitamin E in children.

The vitamin E status and ease of repletion in groups of children with coeliac disease, intestinal lymphangiectasia, and abetalipoproteinaemia was studied and compared with earlier studies in cystic fibrosis and obstructive jaundice. Each group represents an experimental model in which one of the transport steps involved in the absorption of vitamin E is defective or absent and thus the relative importance of these factors could be determined. Chylomicron formation and an adequate intraluminal concentration of bile salts were found to be the most important factors for the efficient absorption of the vitamin. The results in the five groups of patients have therapeutic implications if it is considered desirable to correct vitamin E deficiency states.