Lymphatics in health and disease: a new thematic series in vascular cell

Vascular Cell is launching new series on lymphatics, a vascular system required for physiological fluid balance and immunity, and whose damage leads to edema

Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity

In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis. We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression. Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression

Lens differentiation is controlled by the balance between PDGF and FGF signaling

How multiple receptor tyrosine kinases coordinate cell fate determination is yet to be elucidated. We show here that the receptor for platelet-derived growth factor (PDGF) signaling recruits the p85 subunit of Phosphoinositide 3-kinase (PI3K) to regulate mammalian lens development. Activation of PI3K signaling not only prevents B-cell lymphoma 2 (BCL2)-Associated X (Bax)- and BCL2 Antagonist/Killer (Bak)-mediated apoptosis but also promotes Notch signaling to prevent premature cell differentiation. Reducing PI3K activity destabilizes the Notch intracellular domain, while the constitutive activation of Notch reverses the PI3K deficiency phenotype. In contrast, fibroblast growth factor receptors (FGFRs) recruit Fibroblast Growth Factor Receptor Substrate 2 (Frs2) and Rous sarcoma oncogene (Src) Homology Phosphatase 2 (Shp2) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, which induces the Notch ligand Jagged 1 (Jag1) and promotes cell differentiation. Inactivation of Shp2 restored the proper timing of differentiation in the p85 mutant lens, demonstrating the antagonistic interaction between FGF-induced MAPK and PDGF-induced PI3K signaling. By selective activation of PI3K and MAPK, PDGF and FGF cooperate with and oppose each other to balance progenitor cell maintenance and differentiation

Myeloid Wnt ligands are required for normal development of dermal lymphatic vasculature

Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction

Notch regulates the angiogenic response via induction of VEGFR-1

Notch is a critical regulator of angiogenesis and arterial specification. We show that ectopic expression of activated Notch1 induces endothelial morphogenesis in human umbilical vein endothelial cells (HUVEC) in a VEGFR-1-dependent manner. Notch1-mediated upregulation of VEGFR-1 in HUVEC increased their responsiveness to the VEGFR-1 specific ligand, Placental Growth Factor (PlGF). In mice and human endothelial cells, inhibition of Notch signaling resulted in decreased VEGFR-1 expression during VEGF-A-induced neovascularization. In summary, we show that Notch1 plays a role in endothelial cells by regulating VEGFR-1, a function that may be important for physiological and pathological angiogenesis

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.</p

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs

VEGFR-1 blockade disrupts peri-implantation decidual angiogenesis and macrophage recruitment

Background: Angiogenesis and macrophage recruitment to the uterus are key features of uterine decidualization; the progesterone-mediated uterine changes that allow for embryo implantation and initiation of pregnancy. In the current study, we characterized the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) in macrophages and endothelial cells of the peri-implantation uterus and determined if VEGFR-1 function is required for decidual angiogenesis, macrophage recruitment, and/or the establishment of pregnancy. Methods: Expression of VEGFR-1 in uterine endothelial cells and macrophages was determined with immunohistochemistry. To assess the effect of continuous VEGFR-1 blockade, adult female mice were given VEGFR-1 blocking antibody, MF-1, every 3 days for 18 days. After 6 doses, females were mated and a final dose of MF-1 was given on embryonic day 3.5. Endothelial cells and macrophages were quantified on embryonic day 7.5. Pregnancy was analyzed on embryonic days 7.5 and 10.5. Results: F4/80+ macrophages are observed throughout the stroma and are abundant adjacent to the endometrial lumen and glands prior to embryo implantation and scatter throughout the decidua post implantation. VEGFR-1 expression is restricted to the uterine endothelial cells. F4/80+ macrophages were often found adjacent to VEGFR-1+ endothelial cells in the primary decidual zone. Continuous VEGFR-1 blockade correlates with a significant reduction in decidual vascular and macrophage density, but does not affect embryo implantation or maintenance of pregnancy up to embryonic day 10.5. Conclusions: We found that VEGFR-1 functions in both decidual angiogenesis and macrophage recruitment to the implantation site during pregnancy. VEGFR-1 is expressed by endothelial cells, however blocking VEGFR-1 function in endothelial cells results in reduced macrophage recruitment to the uterus. VEGFR-1 blockade did not compromise the establishment and/or maintenance of pregnancy

Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4)

Background: In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary. Methods: Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (α-SMA) detection. Results: We localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis. Conclusions: Our experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated

Unique functions for Notch4 in murine embryonic lymphangiogenesis

Publisher Copyright: © 2021, The Author(s).In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.Peer reviewe