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1.  Relationships Between Lymphangiogenesis and Angiogenesis During Inflammation in Rat Mesentery Microvascular Networks 
Lymphatic Research and Biology  2012;10(4):198-207.
Abstract
Background
Lymphatic and blood microvascular systems play a coordinated role in the regulation of interstitial fluid balance and immune cell trafficking during inflammation. The objective of this study was to characterize the temporal and spatial relationships between lymphatic and blood vessel growth in the adult rat mesentery following an inflammatory stimulus.
Methods and Results
Mesenteric tissues were harvested from unstimulated adult male Wistar rats and at 3, 10, and 30 days post compound 48/80 stimulation. Tissues were immunolabeled for PECAM, LYVE-1, Prox1, podoplanin, CD11b, and class III β-tubulin. Vascular area, capillary blind end density, and vascular length density were quantified for each vessel system per time point. Blood vascular area increased compared to unstimulated tissues by day 10 and remained increased at day 30. Following the peak in blood capillary sprouting at day 3, blood vascular area and density increased at day 10. The number of blind-ended lymphatic vessels and lymphatic density did not significantly increase until day 10, and lymphatic vascular area was not increased compared to the unstimulated level until day 30. Lymphangiogenesis correlated with the upregulation of class III β-tubulin expression by endothelial cells along lymphatic blind-ended vessels and increased lymphatic/blood endothelial cell connections. In local tissue regions containing both blood and lymphatic vessels, the presence of lymphatics attenuated blood capillary sprouting.
Conclusions
Our work suggests that lymphangiogenesis lags angiogenesis during inflammation and motivates the need for future investigations aimed at understanding lymphatic/blood endothelial cell interactions. The results also indicate that lymphatic endothelial cells undergo phenotypic changes during lymphangiogenesis.
doi:10.1089/lrb.2012.0014
PMCID: PMC3525890  PMID: 23240958
2.  Expression of lymphatic markers during avian and mouse cardiogenesis 
The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just prior to septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.
doi:10.1002/ar.21043
PMCID: PMC3607319  PMID: 19938109
lymphatics; heart; Prox-1; LYVE-1; VEGFR-3; epicardium
3.  VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis 
Cardiovascular research  2007;78(2):315-323.
Aims
Vascular endothelial growth factor-C (VEGF-C) has been shown to stimulate both angiogenesis and lymphangiogenesis in some but not all models where VEGF-C is over-expressed. Our aim was to investigate the interaction between lymphangiogenesis and angiogenesis in adult tissues regulated by VEGF-C and identify evidence of polarized growth of lymphatics driven by specialized cells at the tip of the growing sprout.
Methods and results
We used an adult model of lymphangiogenesis in the rat mesentery. The angiogenic effect of VEGF-C was markedly attenuated in the presence of a growing lymphatic network. Furthermore, we show that this growth of lymphatic vessels can occur both by recruitment of isolated lymphatic islands to a connected network and by filopodial sprouting. The latter is independent of polarized tip cell differentiation that can be generated all along lymphatic capillaries, independently of the proliferation status of the lymphatic endothelial cells.
Conclusion
These results both demonstrate a dependence of VEGF-C-mediated angiogenesis on lymphatic vascular networks and indicate that the mechanism of VEGF-C-mediated lymphangiogenesis is different from that of classical angiogenic mechanisms.
doi:10.1093/cvr/cvm094
PMCID: PMC2613351  PMID: 18065770
Angiogenesis; Lymphangiogenesis; VEGF-C; Sprouting
4.  Lymphangiogenesis and Lymphatic Remodeling Induced by Filarial Parasites: Implications for Pathogenesis 
PLoS Pathogens  2009;5(12):e1000688.
Even in the absence of an adaptive immune system in murine models, lymphatic dilatation and dysfunction occur in filarial infections, although severe irreversible lymphedema and elephantiasis appears to require an intact adaptive immune response in human infections. To address how filarial parasites and their antigens influence the lymphatics directly, human lymphatic endothelial cells were exposed to filarial antigens, live parasites, or infected patient serum. Live filarial parasites or filarial antigens induced both significant LEC proliferation and differentiation into tube-like structures in vitro. Moreover, serum from patently infected (microfilaria positive) patients and those with longstanding chronic lymphatic obstruction induced significantly increased LEC proliferation compared to sera from uninfected individuals. Differentiation of LEC into tube-like networks was found to be associated with significantly increased levels of matrix metalloproteases and inhibition of their TIMP inhibitors (Tissue inhibitors of matrix metalloproteases). Comparison of global gene expression induced by live parasites in LEC to parasite-unexposed LEC demonstrated that filarial parasites altered the expression of those genes involved in cellular organization and development as well as those associated with junction adherence pathways that in turn decreased trans-endothelial transport as assessed by FITC-Dextran. The data suggest that filarial parasites directly induce lymphangiogenesis and lymphatic differentiation and provide insight into the mechanisms underlying the pathology seen in lymphatic filariasis.
Author Summary
The nematode parasites Brugia malayi and Wuchereria bancrofti are the major organisms responsible for lymphatic filariasis. Lymphatic filariasis is characterized by the dysfunction of the lymphatics that can lead to severe (and often) irreversible lymphedema and elephantiasis. Current advances in distinguishing blood vascular from lymphatic endothelial cells have allowed the direct study of the interaction between live filarial parasites and their lymphatic niche. In the quest towards understanding parasite-lymphatic endothelium interactions, we observed that the filarial antigens have a specific but differential stimulatory capacity towards the lymphatics and cause them to differentiate into tube-like vascular networks in vitro that resemble the formation of collateral lymphatics in vivo. This was a lymphatic-specific phenomenon, as the filarial parasites or antigen did not exhibit similar effects on the human umbilical vein endothelial cells. The differentiation of the lymphatic endothelial monolayers into vascular networks was not dependent on typical markers of lymphangiogenesis but rather involves the matrix metalloproteases and their inhibitors that suggest lymphatic matrix remodeling rather than rendering of the lymphatics hyper-permeable as has been postulated previously.
doi:10.1371/journal.ppat.1000688
PMCID: PMC2781552  PMID: 20011114
5.  NFATc1 regulates lymphatic endothelial development 
Mechanisms of development  2009;126(5-6):350-365.
NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin-NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin-NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Aβ regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.
doi:10.1016/j.mod.2009.02.003
PMCID: PMC2680470  PMID: 19233265
NFATc1; lymphatic; lymphangiogenesis; calcineurin
6.  Immunohistochemical Examination on the Distribution of Cells Expressed Lymphatic Endothelial Marker Podoplanin and LYVE-1 in the Mouse Tongue Tissue 
The clinical study for lingual disease requires the detailed investigation of the lingual lymphatic network and lymphatic marker-positive cells. Recently, it has been reported that several tissue cells and leukocytes express lymphatic markers, LYVE-1 and podoplanin. This study was aimed to clarify the lingual distribution of cells expressing LYVE-1 and podoplanin. In the mouse tongue, podoplanin is expressed in nerve sheaths, lingual gland myoepithelial cells, and lymphatic vessels. LYVE-1 is expressed in the macrophage marker Mac-1-positive cells as well as lymphatic vessels, while factor-VIII was detected in only blood endothelial cells. α-SMA was detected in vascular smooth muscle and myoepithelial cells. Therefore, identification of lymphatic vessels in lingual glands, the combination of LYVE-1 and factor-VIII, or LYVE-1 and Mac-1 is useful because myoepithelial cells express podoplanin and α-SMA. The immunostaining of factor-VIII on lymphatic vessels was masked by the immunostaining to LYVE-1 or podoplanin because lymphatic vessels express factor-VIII to a far lesser extent than blood vessels. Therefore, except for the salivary glands, the combination of podoplanin and α-SMA, or factor-VIII is useful to identify lymphatic vessels and blood vessels with smooth muscle, or blood capillaries.
doi:10.1267/ahc.10008
PMCID: PMC2875857  PMID: 20514293
podoplanin; LYVE-1; lymphatic vessels; macrophages
7.  Digging deeper into lymphatic vessel formation in vitro and in vivo 
BMC Cell Biology  2011;12:29.
Background
Abnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures.
Results
In both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation.
Conclusions
In this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.
doi:10.1186/1471-2121-12-29
PMCID: PMC3141733  PMID: 21702933
8.  Characterization of lymphangiogenesis in a model of adult skin regeneration 
To date, adult lymphangiogenesis is not well understood. In this study we describe the evolution of lymphatic capillaries in regenerating skin and correlate lymphatic migration and organization with the expression of matrix metalloproteinases (MMPs), immune cells, the growth factors VEGF-A and VEGF-C, and the heparan sulfate proteogylcan perlecan, a key component of basement membrane. We show that while lymphatic endothelial cells (LECs) migrate and organize unidirectionally, in the direction of interstitial fluid flow, they do not sprout into the region but rather migrate as single cells that later join together into vessels. Furthermore, in a modified “shunted flow” version of the model, infiltrated LECs fail to organize into functional vessels, indicating that interstitial fluid flow is necessary for lymphatic organization. Perlecan expression on new lymphatic vessels was only observed after vessel organization was complete and also appeared first in the distal region, consistent with the directionality of lymphatic migration and organization. VEGF-C expression peaked at the initiation of lymphangiogenesis but was reduced to lower levels throughout organization and maturation. In mice lacking MMP-9, lymphatics regenerated normally, suggesting that MMP-9 is not required for lymphangiogenesis, at least in mouse skin. This study thus characterizes the process of adult lymphangiogenesis and differentiates it from sprouting blood angiogenesis, verifies its dependence on interstitial fluid flow for vessel organization, and correlates its temporal evolution with those of relevant environmental factors.
doi:10.1152/ajpheart.00038.2006
PMCID: PMC2751590  PMID: 16648194
lymphatic; vasculogenesis; interstitial fluid flow; matrix metalloproteinase-9; perlecan
9.  CD34+VEGFR-3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells 
Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34+VEGFR-3+ EPCs were isolated from mononuclear cells of human cord blood by fluorescence-activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF-C for 2 weeks, CD34+VEGFR-3+ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′-nucleotidase, LYVE-1 and Prox-1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary-like structures in three-dimensional collagen gel and Matrigel. VEGF-C enhanced VEGFR-3 mRNA expression. After interfering with VEGFR-3 siRNA, the effects of VEGF-C were diminished. These results demonstrate that there is a population of CD34+VEGFR-3+ EPCs with lymphatic potential in human cord blood. VEGF-C/VEGFR-3 signalling pathway mediates differentiation of CD34+VEGFR-3+ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood-derived CD34+VEGFR-3+ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.
doi:10.1111/jcmm.12233
PMCID: PMC3955149  PMID: 24450475
Endothelial progenitor cells; VEGFR-3; VEGF-C; Differentiation; Lymphangiogenesis
10.  Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature 
Background
In vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo.
Results
Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.
Conclusion
We present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.
doi:10.1186/1471-213X-10-72
PMCID: PMC2909156  PMID: 20584329
11.  Vascular Endothelial Growth Factor Receptor-2 Promotes the Development of the Lymphatic Vasculature 
PLoS ONE  2013;8(9):e74686.
Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed by lymphatic endothelial cells and has been shown to stimulate lymphangiogenesis in adult mice. However, the role VEGFR2 serves in the development of the lymphatic vascular system has not been defined. Here we use the Cre-lox system to show that the proper development of the lymphatic vasculature requires VEGFR2 expression by lymphatic endothelium. We show that Lyve-1wt/Cre;Vegfr2flox/flox mice possess significantly fewer dermal lymphatic vessels than Vegfr2flox/flox mice. Although Lyve-1wt/Cre;Vegfr2flox/flox mice exhibit lymphatic hypoplasia, the lymphatic network is functional and contains all of the key features of a normal lymphatic network (initial lymphatic vessels and valved collecting vessels surrounded by smooth muscle cells (SMCs)). We also show that Lyve-1Cre mice display robust Cre activity in macrophages and in blood vessels in the yolk sac, liver and lung. This activity dramatically impairs the development of blood vessels in these tissues in Lyve-1wt/Cre;Vegfr2flox/flox embryos, most of which die after embryonic day14.5. Lastly, we show that inactivation of Vegfr2 in the myeloid lineage does not affect the development of the lymphatic vasculature. Therefore, the abnormal lymphatic phenotype of Lyve-1wt/Cre;Vegfr2flox/flox mice is due to the deletion of Vegfr2 in the lymphatic vasculature not macrophages. Together, this work demonstrates that VEGFR2 directly promotes the expansion of the lymphatic network and further defines the molecular mechanisms controlling the development of the lymphatic vascular system.
doi:10.1371/journal.pone.0074686
PMCID: PMC3759473  PMID: 24023956
12.  Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer 
Oncoimmunology  2014;3:e29080.
Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133+CD34+ progenitors into podoplanin+ cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin+ cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34+ cord blood progenitors into hemangiogenic and lymphangiogenic CD11b+ myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b+ cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer.
doi:10.4161/onci.29080
PMCID: PMC4121340  PMID: 25101222
lymphatic endothelial cells; angiogenesis; lymphangiogenesis; podoplanin; vasculogenesis; bone-marrow derived cells; breast cancer
13.  Podoplanin-expressing Cells Derived from Bone Marrow Play a Crucial Role in Postnatal Lymphatic Neovascularization 
Circulation  2010;122(14):1413-1425.
Background
Emerging evidence has suggested a contribution of bone marrow (BM) cells to lymphatic vessel formation; however, the exact phenotype of the cells with lymphatic endothelial progenitor cell (LEPC) function has yet to be identified. Here we investigate the identity of BM-derived LEPCs and their role in lymphatic neovascularization.
Methods and Results
Culture of BM-mononuclear cells (MNCs) in the presence of VEGFA, VEGFC and EGF resulted in expression of lymphatic endothelial cell (LEC) markers. Among these cells, podoplanin+ cells were isolated by magnetic-labeled cell separation system (MACS) and characterized by FACS and immunocytochemistry. These podoplanin+ cells highly express markers for LECs, hematopoietic lineages, and stem/progenitor cells, and upon further cultivation, generate LECs. We further confirmed that podoplanin+ cells exist in small numbers in BM and peripheral blood (PB) of normal mice, but are significantly (15 fold) augmented upon lymphangiogenic stimuli such as tumor implantation. Next, to evaluate the potential of podoplanin+ cells for the formation of new lymphatic vessels in vivo, we injected culture-isolated or freshly isolated BM-derived podoplanin+ cells into wound and tumor models. Immunohistochemistry demonstrated that the injected cells were incorporated into the lymphatic vasculature, displayed LEC phenotypes, and increased lymphatic vascular density in tissues, suggesting lymphvasculogenesis. Podoplanin+ cells also expressed high levels of lymphangiogenic cytokines and increased proliferation of LECs during co-culture, suggesting a lymphangiogenic or paracrine role.
Conclusions
Our results provide compelling evidence that BM-derived podoplanin+ cells, a previously unrecognized cell type, function as LEPCs and participate in postnatal lymphatic neovascularization through both lymphvasculogenesis and lymphangiogenesis.
doi:10.1161/CIRCULATIONAHA.110.941468
PMCID: PMC2989430  PMID: 20855662
Bone Marrow; Podoplanin; Lymphangiogenesis; Lymphvasculogenesis
14.  Vascular islands during microvascular regression and regrowth in adult networks 
Objective: Angiogenesis is the growth of new vessels from pre-existing vessels and commonly associated with two modes: capillary sprouting and capillary splitting. Previous work by our laboratory suggests vascular island incorporation might be another endothelial cell dynamic involved in microvascular remodeling. Vascular islands are defined as endothelial cell segments disconnected from nearby networks, but their origin remains unclear. The objective of this study was to determine whether vascular islands associated with microvascular regression are involved in network regrowth.
Methods: Mesenteric tissues were harvested from adult male Wistar rats according to the experimental groups: unstimulated, post stimulation (10 and 70 days), and 70 days post stimulation + restimulation (3 and 10 days). Stimulation was induced by mast cell degranulation via intraperitoneal injections of compound 48/80. Tissues were immunolabeled for PECAM (endothelial cells), neuron-glial antigen 2 (NG2) (pericytes), collagen IV (basement membrane), and BrdU (proliferation).
Results: Percent vascular area per tissue area and length density increased by day 10 post stimulation compared to the unstimulated group. At day 70, vascular area and length density were then decreased, indicating vascular regression compared to the day 10 levels. The number of vascular islands at day 10 post stimulation was dramatically reduced compared to the unstimulated group. During regression at day 70, the number of islands increased. The disconnected endothelial cells were commonly bridged to surrounding networks by collagen IV labeling. NG2-positive pericytes were observed both along the islands and the collagen IV tracks. At 3 days post restimulation, vascular islands contained BrdU-positive cells. By day 10 post restimulation, when vascular area and length density were again increased, and the number of vascular islands was dramatically reduced.
Conclusion: The results suggest that vascular islands originating during microvascular regression are capable of undergoing proliferation and incorporation into nearby networks during network regrowth.
doi:10.3389/fphys.2013.00108
PMCID: PMC3655287  PMID: 23720632
angiogenesis; microcirculation; mesentery; proliferation; endothelial cell; disconnected segment; vascular island
15.  Blood flow reprograms lymphatic vessels to blood vessels 
The Journal of Clinical Investigation  2012;122(6):2006-2017.
Human vascular malformations cause disease as a result of changes in blood flow and vascular hemodynamic forces. Although the genetic mutations that underlie the formation of many human vascular malformations are known, the extent to which abnormal blood flow can subsequently influence the vascular genetic program and natural history is not. Loss of the SH2 domain–containing leukocyte protein of 76 kDa (SLP76) resulted in a vascular malformation that directed blood flow through mesenteric lymphatic vessels after birth in mice. Mesenteric vessels in the position of the congenital lymphatic in mature Slp76-null mice lacked lymphatic identity and expressed a marker of blood vessel identity. Genetic lineage tracing demonstrated that this change in vessel identity was the result of lymphatic endothelial cell reprogramming rather than replacement by blood endothelial cells. Exposure of lymphatic vessels to blood in the absence of significant flow did not alter vessel identity in vivo, but lymphatic endothelial cells exposed to similar levels of shear stress ex vivo rapidly lost expression of PROX1, a lymphatic fate–specifying transcription factor. These findings reveal that blood flow can convert lymphatic vessels to blood vessels, demonstrating that hemodynamic forces may reprogram endothelial and vessel identity in cardiovascular diseases associated with abnormal flow.
doi:10.1172/JCI57513
PMCID: PMC3366395  PMID: 22622036
16.  Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity 
The Journal of Clinical Investigation  2014;124(9):3960-3974.
Schlemm’s canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin α9 and exhibited continuous vascular endothelial–cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.
doi:10.1172/JCI75392
PMCID: PMC4153702  PMID: 25061877
17.  Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer 
Background
It has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression.
Methods
We initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3) and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells.
Results
Lymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium), although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating endothelial cells, and the cross sectional area of vessel profiles were significantly increased in response to VEGF-D in comparison to control tumor cells. In contrast, no significant changes were noted in myometrial blood vessels. In addition, examples of invading cells or tumor emboli were observed in mice receiving VEGF-D expressing 293EBNA cells.
Conclusions
These results illustrate that VEGF-D over-expression has differential effects on the uterine vasculature. These effects may facilitate VEGF-D's ability to promote endometrial cancer metastasis and disease progression.
doi:10.1186/1477-7827-8-84
PMCID: PMC2909246  PMID: 20615255
18.  Hypoxia and Extracellular Matrix Proteins Influence Angiogenesis and Lymphangiogenesis in Mouse Embryoid Bodies 
Regulatory mechanisms for angiogenesis are relatively well established compared to lymphangiogenesis. Few studies have shown that a combination of vascular endothelial growth factor VEGF-A/C with hypoxia or collagen matrix promotes lymphatic structures along with blood vessel development in mouse embryoid bodies (EB). In this study we tested the hypothesis that while hypoxia combined with prolonged VEGF-A/C treatment would induce early lymphangiogenesis in addition to angiogenesis in mouse EBs, under similar conditions specific extracellular matrix (ECM) proteins would promote lymphatic vessel-like structures over angiogenesis. EBs were subjected to four conditions and were maintained under normoxia and hypoxia (21% and 2.6% O2, respectively) with or without VEGF-A/C. Microarray analyses of normoxic and hypoxic EBs, and immunofluorescence data showed very low expression of early lymphatic endothelial cell (LEC) markers, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and prospero-related homeobox 1 (Prox1) at early time points. Double immunofluorescence using MECA-32 and Prox1/LYVE1 demonstrated that combined hypoxia and VEGF-A/C treatment promoted formation of blood vessel-like structures, whereas only Prox1+/LYVE1+ LECs were detected in EBs at E22.5. Furthermore, EBs were grown on laminin or collagen-I coated plates and were subjected to the four treatments as described above. Results revealed that LECs in EBs at E36.5 attached better to collagen-I, resulting in an organized network of lymphatic vessel-like structures as compared to EBs grown on laminin. However, blood vessel-like structures were less favored under these same conditions. Collectively, our data demonstrate that hypoxia combined with growth factors promotes angiogenesis, whereas combination of these conditions with specific ECM proteins favors lymphangiogenesis processes in mouse EBs.
doi:10.3389/fphys.2011.00103
PMCID: PMC3243103  PMID: 22194726
angiogenesis; lymphangiogenesis; embryoid body; hypoxia; VEGF
19.  Detection of lymphangiogenesis in non-small cell lung cancer and its prognostic value 
Background
Our aim was to detect lymphatic endothelial marker podoplanin, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR)-3 and study the prognostic relevance of lymphangiogenesis in non-small cell lung cancer (NSCLC).
Materials
82 paraffin-embedded tissues and 40 fresh frozen tissues from patients with NSCLC were studied. Tumor samples were immunostained for the lymphatic endothelial markers. Lymphangiogenesis was assessed by immunohistochemical double stains for Podoplanin and Ki-67. The prognostic relevance of lymphangiogenesis-related clinicopathological parameters in NSCLC was evaluated.
Results
We found that the number of podoplanin positive vessels was correlated positively with the number of LYVE-1 positive vessels. Most of VEGFR-3 positive, few of LYVE-1 positive and none of podoplanin positive vessels were blood vessels. Peritumoral lymphatic vessel density (ptLVD), pathologic stage, lymph node status, lymphatic vessel invasion (LVI), vascular endothelial growth factor-C (VEGF-C) expression and Ki-67 index of the endothelium cells of the micro lymphatic vessels (Ki67%) were associated significantly with a higher risk of tumor progress. ptLVD, pathologic stage, lymph-node metastasis and Ki67% were independent prognostic parameters for overall survival.
Conclusion
Podoplanin positive ptLVD might play important roles in the lymphangiogenesis and progression of NSCLC. Patients with high podoplanin+ ptLVD have a poor prognosis.
doi:10.1186/1756-9966-28-21
PMCID: PMC2647904  PMID: 19216806
20.  Initial Afferent Lymphatic Vessels Controlling Outbound Leukocyte Traffic from Skin to Lymph Nodes 
Tissue drains fluid and macromolecules through lymphatic vessels (LVs), which are lined by a specialized endothelium that expresses peculiar differentiation proteins, not found in blood vessels (i.e., LYVE-1, Podoplanin, PROX-1, and VEGFR-3). Lymphatic capillaries are characteristically devoid of a continuous basal membrane and are anchored to the ECM by elastic fibers that act as pulling ropes which open the vessel to avoid edema if tissue volume increases, as it occurs upon inflammation. LVs are also crucial for the transit of T lymphocytes and antigen presenting cells from tissue to draining lymph nodes (LN). Importantly, cell traffic control across lymphatic endothelium is differently regulated under resting and inflammatory conditions. Under steady-state non-inflammatory conditions, leukocytes enter into the lymphatic capillaries through basal membrane gaps (portals). This entrance is integrin-independent and seems to be mainly guided by CCL21 chemokine gradients acting on leukocytes expressing CCR7. In contrast, inflammatory processes in lymphatic capillaries involve a plethora of cytokines, chemokines, leukocyte integrins, and other adhesion molecules. Importantly, under inflammation a role for integrins and their ligands becomes apparent and, as a consequence, the number of leukocytes entering the lymphatic capillaries multiplies several-fold. Enhancing transmigration of dendritic cells en route to LN is conceivably useful for vaccination and cancer immunotherapy, whereas interference with such key mechanisms may ameliorate autoimmunity or excessive inflammation. Recent findings illustrate how, transient cell-to-cell interactions between lymphatic endothelial cells and leukocytes contribute to shape the subsequent behavior of leukocytes and condition the LV for subsequent trans-migratory events.
doi:10.3389/fimmu.2013.00433
PMCID: PMC3856852  PMID: 24368908
dendritic cell; T cell; lymphatic vessel; migration; inflammation
21.  POTENTIAL THERAPEUTIC STRATEGIES FOR LYMPHATIC METASTASIS 
Microvascular research  2007;74(2-3):145-158.
Physiologically, the lymphatic system regulates fluid volume in the interstitium and provides a conduit for immune cells to travel to lymph nodes, but pathologically, the lymphatic system serves as a primary escape route for cancer cells. Lymphatic capillaries have a thin discontinuous basement membrane, lack pericyte coverage, and often contain endothelial cell gaps that can be invaded by immune cells (or tumor cells). In addition, tumor cells and stromal cells in the tumor microenvironment secrete factors that stimulate lymphangiogenesis, the growth of lymphatic endothelial cells and the sprouting of lymphatic capillaries. As a result, many tumors are surrounded by large, hyperplastic, peri-tumoral lymphatic vessels and less frequently are invaded by intra-tumoral lymphatic vessels. Carcinoma cells commonly metastasize through these lymphatic vessels to regional lymph nodes. The presence of metastatic cells in the sentinel lymph node is a prognostic indicator for many types of cancer, and the degree of dissemination determines the therapeutic course of action. Lymphangiogenesis is currently at the frontier of metastasis research. Recent strides in this field have uncovered numerous signaling pathways specific for lymphatic endothelial cells and vascular endothelial cells. This review will provide an overview of tumor lymphangiogenesis and current strategies aimed at inhibiting lymphatic metastasis. Novel therapeutic approaches that target the tumor cells as well as the vascular and lymphatic endothelial compartments are discussed.
doi:10.1016/j.mvr.2007.08.006
PMCID: PMC2525453  PMID: 17950368
22.  Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages 
Journal of Clinical Investigation  2005;115(9):2363-2372.
In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow–derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.
doi:10.1172/JCI23874
PMCID: PMC1193872  PMID: 16138190
23.  Structure of Microvascular Networks in Genetic Hypertension 
Methods in Enzymology  2008;444:271-284.
Microvascular rarefaction, defined by a loss of terminal arterioles, small venules and/or capillaries, is a common characteristic of the hypertension syndrome. While rarefaction has been associated with vessel specific free radical production, deficient leukocyte adhesion, and cellular apoptosis, the relationships of rarefaction with structural alterations at the network and cellular level remain largely unexplored. The objective of this study was to examine the architecture and perivascular cell phenotypes along microvascular networks in hypertensive versus normotensive controls in the context of imbalanced angiogenesis. Mesenteric tissues from age-matched adult male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were harvested and immnolabeled for PECAM and neuron-glia antigen 2 (NG2). Evaluation of intact rat mesenteric microvascular networks rats suggests that network alterations associated with hypertension are more complex than just a loss of vessels. Typical SHR versus WKY networks demonstrate a reduced branching architecture marked by more proximal arteriole/venous anastomoses and an absence of NG2 labeling along arterioles. Although less frequent, larger SHR microvascular networks display regions of dramatically increased vascular density. SHR and WKY lymphatic networks demonstrate increased vessel diameters and vascular density compared to networks in normotensive Wistar rats (the strain from which both the SHR and WKY originated). These observations provide a rationale for investigating the presence of local angiogenic factors and response of microvascular networks to therapies aimed at reversing rarefaction in genetic hypertension.
doi:10.1016/S0076-6879(08)02812-7
PMCID: PMC3325543  PMID: 19007669
24.  Recent Developments in Morphology of Lymphatic Vessels and Lymph Nodes 
Annals of Vascular Diseases  2012;5(2):145-150.
This paper reviews the morphology of lymphatics and lymphangiogenesis in vivo, microenvironments that promote lymphangiogenesis, and the structure and function of lymph nodes. Lymphatic capillaries consist of a single layer of lymphatic endothelial cells (LECs) and have valves, while collecting lymphatics are endowed with smooth muscle cells (SMCs) and valves besides a single layer of LECs. In the embryonic rat diaphragm, LECs first migrate presumably according to interstitial fluid flow and later join to form lymphatic vessels. SMCs of the collecting lymphatics are apparently differentiated from mesenchymal cells. LECs cultured on Cell Culture Inserts under a low oxygen condition proliferate very well and form a lymphatic network. LECs cultured on a collagen fiber network with a natural three-dimensional (3D) architecture under low oxygen rapidly form a 3D lymphatic network. The lymph node initiates an immune response as a critical crossroads for the encounter between antigen-presenting cells, antigens from lymph, and lymphocytes recruited into nodes from the blood. The node consists of spaces lined with LECs and parenchyma. High endothelial venules in the node strongly express Aquaporin-1, suggesting their involvement in the net absorption of water from lymph coming through afferent lymphatics. SMCs in node capsules seem to be involved in squeezing out lymphocytes and lymph. (*English Translation of J Jpn Col Angiol 2008; 48: 107-112.)
doi:10.3400/avd.ra.11.00099
PMCID: PMC3595868  PMID: 23555502
lymphatic vessel; lymphangiogenesis; lymph node; metastasis; lymphedema
25.  Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas 
BMC Cancer  2007;7:105.
Background
Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels.
Methods
Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis.
Results
LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-α1 and -α9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs.
Conclusion
LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.
doi:10.1186/1471-2407-7-105
PMCID: PMC1925108  PMID: 17584927

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