In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.

AimsThe antidiabetic drug metformin is currently used prior and during pregnancy for polycystic ovary syndrome, as well as during gestational diabetes mellitus. We investigated the effects of prenatal metformin exposure on the metabolic phenotype of the offspring during adulthood in mice.MethodsMetformin (300 mg/kg) or vehicle was administered orally to dams on regular diet from the embryonic day E0.5 to E17.5. Gene expression profiles in liver and brain were analysed from 4-day old offspring by microarray. Body weight development and several metabolic parameters of offspring were monitored both during regular diet (RD-phase) and high fat diet (HFD-phase). At the end of the study, two doses of metformin or vehicle were given acutely to mice at the age of 20 weeks, and Insig-1 and GLUT4 mRNA expressions in liver and fat tissue were analysed using qRT-PCR.ResultsMetformin exposed fetuses were lighter at E18.5. There was no effect of metformin on the maternal body weight development or food intake. Metformin exposed offspring gained more body weight and mesenteric fat during the HFD-phase. The male offspring also had impaired glucose tolerance and elevated fasting glucose during the HFD-phase. Moreover, the expression of GLUT4 mRNA was down-regulated in epididymal fat in male offspring prenatally exposed to metformin. Based on the microarray and subsequent qRT-PCR analyses, the expression of Insig-1 was changed in the liver of neonatal mice exposed to metformin prenatally. Furthermore, metformin up-regulated the expression of Insig-1 later in development. Gene set enrichment analysis based on preliminary microarray data identified several differentially enriched pathways both in control and metformin exposed mice.ConclusionsThe present study shows that prenatal metformin exposure causes long-term programming effects on the metabolic phenotype during high fat diet in mice. This should be taken into consideration when using metformin as a therapeutic agent during pregnancy.

Macrophages are required for normal embryogenesis, tissue homeostasis and immunity against microorganisms and tumours. Adult tissue-resident macrophages largely originate from long-lived, self-renewing embryonic precursors and not from haematopoietic stem-cell activity in the bone marrow. Although fate-mapping studies have uncovered a great amount of detail on the origin and kinetics of fetal macrophage development in the yolk sac and liver, the molecules that govern the tissue-specific migration of these cells remain completely unknown. Here we show that an endothelium-specific molecule, plasmalemma vesicle-associated protein (PLVAP), regulates the seeding of fetal monocyte-derived macrophages to tissues in mice. We found that PLVAP-deficient mice have completely normal levels of both yolk-sac- and bone-marrow-derived macrophages, but that fetal liver monocyte-derived macrophage populations were practically missing from tissues. Adult PLVAP-deficient mice show major alterations in macrophage-dependent iron recycling and mammary branching morphogenesis. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis, interacts with chemoattractants and adhesion molecules and regulates the egress of fetal liver monocytes to the systemic vasculature. Thus, PLVAP selectively controls the exit of macrophage precursors from the fetal liver and, to our knowledge, is the first molecule identified in any organ as regulating the migratory events during embryonic macrophage ontogeny.

Macrophages serve multiple functions including immune regulation, morphogenesis, tissue homeostasis and healing reactions. The current paradigm holds that mammary gland macrophages first arise postnatally during the prepubertal period from the bone marrow-derived monocytes. Here we delineate the origins of tissue-resident mammary gland macrophages using high-dimension phenotypic analyses, cell-fate mapping experiments, gene-deficient mice lacking selective macrophage subtypes, and antibody-based depletion strategies. We show that tissue-resident macrophages are found in mammary glands already before birth, and that the yolk sac-derived and fetal liver-derived macrophages outnumber the adult-derived macrophages in the mammary gland also in the adulthood. In addition, fetal-derived mammary gland macrophages have a characteristic phenotype, display preferential periductal and perivascular localization, and are highly active in scavenging. These findings identify fetal-derived macrophages as the predominant leukocyte type in the adult mammary gland stroma, and reveal previously unknown complexity of macrophage biology in the breast.

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.