Podoplanin and CLEC-2 drive cerebrovascular patterning and integrity during development

Abstract

Key Points• Podoplanin and CLEC-2 critically drive the formation and integrity of developing cerebral blood vessels.• Loss of cerebrovascular integrity is influenced by the loss of aIIb-mediated platelet aggregation and platelet secretion.Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin fl/fl mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin-and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet a-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage. (Blood. 2015;125(24):3769-3777)

Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes

“…To this aim, Pdpn fl/fl mice 28 were crossed with Prox1-Cre-ERT2 mice, 29 which express Cre recombinase under control of the lymphatic specification gene Prox1 30 in response to tamoxifen administration, obtaining the Pdpn ΔLEC line ( Figure 1A). Postnatal deletion of the podoplanin gene was induced by daily tamoxifen administrations from postnatal day (P) 1 to P3 ( Figure 1A).…”

“…24 In addition, several cardiac defects, including hypoplasia of the myocardium, were described in podoplanin-deficient embryos, [25][26][27] and deletion of podoplanin at the 2-cell stage causes cerebral hemorrhaging in embryos. 28 The broad expression of podoplanin, together with the lethal phenotype observed in global podoplanin-deficient mice, suggests an important role for podoplanin in diverse cell types and in the ontogeny of different organs. It has, however, not been possible to date to dissect its direct function specifically in the lymphatic endothelium at late postnatal or adult stages.…”

“…To specifically investigate the function of podoplanin in the postnatal lymphatic vasculature, we developed an inducible, lymphatic-specific podoplanin knockout mouse by crossing Pdpn fl/fl mice 28 and Prox1-Cre-ERT2 mice 29 to obtain Pdpn ΔLEC mice. Postnatal deletion of podoplanin was compatible with life and did not affect the maintenance of normal LV morphology and function.…”

CLEC-2 and podoplanin, partners again

“…4 In the European Union, defibrotide was approved for the treatment of severe VOD/SOS following HCT in October 2013, based on several studies carried out over the last 15 years, including those of the current authors. 5,6 In the United States, there are no approved therapies for VOD/SOS; however, the phase 3 study results by Richardson et al 2 …”

“…Mouse embryos lacking either podoplanin or platelet CLEC-2 exhibit brain hemorrhages, indicating that pairing of these 2 molecules protects neurovascular integrity in the developing brain as well. 6 After birth, platelet CLEC-2 was found to maintain the separated blood-lymphatic system by interacting with podoplanin at the lymphovenous junction. 7 Furthermore, platelet CLEC-2 was discovered to be critical for the integrity of high endothelial venules by interacting with podoplanin on fibroblastic reticular cells in the lymph node during lymphocyte trafficking.…”