3. Background
• Notch genes are required for proper lymphatic development in mice,
zebrafish, and several other animals. [1,2]. It’s hypothesized there’s a
direct relationship between level of expression of Notch genes and the
riseoflymphaticmalformations.
• Notch signaling also plays a key role in regulation of cell fate decisions
andangiogenesis,andinmice,hasbeenshowntoregulatearterialand
venousspecificationsofmajorvessels,suchasthedorsalaorta[3,4,5].In
addition,Notchsignalingpathwayisusedbyendothelialcellsinorderto
organizecellularbehaviorsduringthesproutingstage[1,6].
• Many comprehensive studies of the Notch signaling pathway have
revealed that there’s a direct relationship between the Hey1 and Hey2
genes and embryonic development, which later greatly affects physical
development after the prenatal stage, meaning it also directly affects
thegrowthandbranchingofbloodandlymphvessels[7,8,9].
4. Background (continued)
• In terms of phenotypic characteristics, there are two distinct types of lymphatic
malformation- microcystic and macrocystic. The main difference betweenthe two
typesisthesizeandshapeofthemalformations.
• Lymphangiomatosis is a condition in which the body simultaneously has both
microcysticandmacrocysticlymphaticmalformations,causingittobecategorizedas
a systemic LM. In most cases, lymphangiomatosis tends to involve several organ
systems.
• Lymphatic malformations are comprised of: lymphatic malformation endothelial
cells (LMECs) and lymphatic malformation progenitor cells (LMPC’s) [10]. It was
previously found that Notch1 and Notch3 were overexpressed in LM tissues, with
Notch1beingoverexpressedintheLMECpopulationandNotch3intheLMPCs[11].
• Inaddition,ithasbeennotedthatisolatedlymphaticmalformationendothelialcells
tendtoexpresshighlevelsofHey1,whichsuggestshighNotchsignalactivation.
• Because lymphangiomasotis is a collection of both types of LMs, it would be an
educated guess to hypothesize that lymphangiomatosis tissue would display an
overexpression of the two Notch genes (Notch1 and Notch3) in whatever cell
populationtheytendtobeoverlyexpressedin.
7. Significance
• Approximately 1 in 3500 births are affected by lymphatic
malformations, and this research would be greatly beneficial to and
alleviate the sufferings of a substantial portion of the population.
• A permanent treatment to this ailment would be of great value,
not only economic but also scientific.
• Normally, one surgery to remove or uproot a malformation can cost
anywhere from $10,000 to $45,000, which is very expensive and
often unaffordable for a middle class family.
• The scientific value of this research project is that, if the exact
relationship between Notch and malformations is discovered, then
that will allow scientists to develop drugs that could effectively
suppress the expression of those genes. Because this is also a
component of cancer research, research in this particular field
would lead to advancements in both fields.
8. Materials and Methods
• Immunohistochemistry is a process by which a set of cells are incubated with
antibodiesinorderto“stain”them.
• Forfrozentissue,theslideswere“prefixed”inacetoneforsometime,andthen
washedinPBS(phosphatebuffersaline).
• After incubation with blocking solution was complete, the antibodies were
administered to the tissue. Every set of tissue used in this project was “double-
stained,”meaningeachsetwasstainedwithtwoantibodies.
• After the staining process was completed, the slides were coverslipped and
observed under a fluorescence microscope, and the images taken were
documented.
• For paraffinized tissue, they were first washed in xylene and different
concentrations of ethanol in order to deparrafinize and rehydrate them. Next,
antigenretrievalinducedinordertouncrosslinkedproteins.
• The steps following the initial steps are the same standard ones as the steps
followedforstainingfrozentissue..
10. Figure 2: Expression of lymphatic
endothelial cell marker and
lymphatic vessel marker on
lymphangiomatosis tissue. The
tissuethatwasstainedwasaparaffin
section from a lymphangiomatosis
patient,isolatedfromthejugularvein
regionintheneck,themesentery(gut
area),thelungs,andthethoracicduct
inthecenterofthebody.Thetissues
were stained using the antibodies
Podoplanin and LYVE1 to show
expression of lymphatic endothelial
cells and lymphatic vessels,
respectively. In addition, DAPI was
used to visualize the nuclei. In the
tissuescollectedfromallfourregions
(jugular/lymphatic,mesenteryvessel,
lung, and thoracic duct), there was
high Podoplanin expression in the
vessels and surrounding tissue.
LYVE1expressionwasmuchmore
muted, and overlapped with that of
Podoplanin. The mesentery vessel,
out of all four regions, showed the
least expression of both markers,
althoughsomedegreeofexpression
isevident.
11. Figure3:Expressionofendothelial
cellmarkerandNotch3markeron
lymphangiomatosis tissue. The
tissuethatwasstainedwasaparaffin
embedded section from
lymphangiomatosis patient, isolated
from the jugular vein region in the
neck, the mesentery (gut area), the
lungs, and the thoracic duct in the
centerofthebody.Thetissueswere
stained using the antibodies
Podoplanin and Notch3 to show
expression of lymphatic endothelial
cells and Notch3 cell markers,
respectively,andDAPIwasusedto
visualize the nuclei. In the tissues
collected from the four
aforementionedregions,Podoplanin
expression is very high. Notch3
expression, however, was much
lower in the same tissues, and its
level of expression is debatable.
Although there is still obvious
expression of Podoplanin in the
tissues from the lung, it is
significantly lower when compared
totheotherregions.
12. Figure4:Expressionofstemcell
marker and Notch 3 marker on
lymphangiomatosis tissue. The
tissue that was stained was a
paraffin embedded section from
lymphangiomatosis patient,,
isolated from the jugular vein
regionintheneck,themesentery
(gut area), the lungs, and the
thoracicduct inthecenterofthe
body. The tissues were stained
with the antibodies CD133 and
Notch3, to show expression of
stem cell markers and Notch3
markersinLMPCsonthetissue,
respectively; DAPI was used to
visualize the nuclei. There is
CD133 expression in tissues
fromthelung,jugular/lymphatic,
and mesentery vessel regions.
There is also Notch3 expression
in the tissues collected from the
aforementioned regions. In
contrast, there appears to be no
expression of either antibody in
the tissue from the thoracic duct
region. The tissue from the
mesentery vessel appears to
showaco-expressionofCD133
andNotch3.
13. Figure5:Expressionofendothelialcell
marker and notch activity on
lymphangiomatosis tissue. The tissue
that was stained was a paraffin
embedded section from
lymphangiomatosis patient,, isolated
fromthejugularveinregionintheneck,
the mesentery (gut area), the lungs, and
thethoracicductinthecenterofthebody.
The tissues were stained using the
antibodies Podoplanin and Hey2, to
show expression of endothelial cell
markers and notch activity in the tissue,
respectively, which in turn show Notch
activity in lymphatic endothelial cells.
DAPI was also used to visualize the
nuclei. There is expression of both
Podoplanin and Hey2 in all tissues;
however, there’s a greater level of
expression of Podoplanin and Hey2 in
the thoracic duct tissue. In the tissues
collected from the lung and jugular
lymphaticregions,there’saslightlylower
expressionofbothantibodies,andinthe
mesentery vessel tissue, there’s scarcely
anyexpressionofeitherantibody.
14. Discussion – Analysis of Results
• Inthelymphaticvesselsofnormaltissue,highPodoplaninexpressionwasobserved,while
its expression was greatly reduced in lymphatic malformation tissues, which suggests
lymphatic malformation endothelial cells (LMECs) usually lining lymphatic vessels are
greatlyaffectedbythelymphaticmalformation,tothepointofdegeneration
• CD133expressionwasreversed:itsexpressionwaslowinthenormaltissue,andhighin
the LM tissues. CD133 is a stem cell marker, so the absence of stem cells in the normal
tissueandtheirpresenceinLMtissuesuggeststhatthepresenceoftheseCD133positive
lymphatic malformation progenitor cells (LMPCs) is abnormal in lymphatic
malformations.
• Characterization of lymphangiomatosis tissue revealed that lymphatic
endothelial cell markers Podoplanin and LYVE1 were expressed in several cell
typeswithdifferentlymorphology,besidesthetypicallymphaticvessels,causing
the resulting vessels to appear grossly deformed. This staining pattern was
consistent with that for LMPCs observed in the LM tissues, suggesting LMPCs
arepresentinlargequantitiesinlymphangiomatosistissue.
• Notch3 was not co-expressed with high Podoplanin levels in the
lymphangiomatosistissuesamples,indicatingit’snothighlyexpressedinLMECs.
15. Discussion – Analysis of Results (continued)
• Notch3 was not co-expressed with high Podoplanin levels in the
lymphangiomatosistissuesamples,indicatingit’snothighlyexpressedinLMECs.
• AsshowninFig.4,Notch3wasalsoexpressedintheCD133positivecellsinthe
samples while it was greatly muted or absent in the tissue samples from the
same area (when the jugular/lymphatic and thoracic duct regions of Fig. 3 are
comparedtothesameregionsinFig.4),indicativeofitssuspectedexpressionin
the LMPC population. This data suggests that Notch3 may function in the
lymphaticmalformationprogenitorcellpopulation.
• Hey2 expression was observed in the same cells that expressed high levels of
CD133andNotch3,andbecauseHey2isadownstreameffectorofNotchsignal
activationandwasabsentincellsshowinghighlevelsofPodoplanin(whichare,
therefore,lymphaticmalformationendothelialcells),itsuggeststhattheNotch3
gene has direct correlations with the formation of a lymphatic malformation
consistingprimarilyofLMECs.
• The absence of Notch3 and Hey2 in the Podoplanin positive LMECs suggests
that these proteins and Notch signaling do not function simultaneously in the
cellsfoundinlymphangiomatosistissue.
18. References
• 1.ShawberC,Kitajewski,J.Notchfunctioninthevasculature:insightsfromzebrafish,mouseandman.
BioEssays.2004;26.3:225-234
• 2.Schweisguth,F.RegulationofNotchSignalingActivity.CurrentBiology.2004;14:129-138
• 3. Funahashi Y, Shawber C, Kitajweski J. Notch genes: orchestrating endothelial differentiation.
EndothelialBiomedicine.41:368-374
• 4.NiessenK,ZhangG,RidgwayJRetal.TheNotch1-Dll4signalingpathwayregulatesmousepostnatal
lymphaticdevelopment.Blood.2011;118(7):1989-1997
• 5. Zheng W, Tammela T, Yamamoto M et al. Notch restricts lymphatic vessel sprouting induced by
vascularendothelialgrowthfactor.Blood.2011;118(4):1154-1162
• 6. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in
development.Science.1999;284(5415):770-776
• 7.XueY,LindsellCE,NortonCRetal.EmbryoniclethalityandvasculardefectsinmicelackingtheNotch
ligandJagged1.HumMolGenet.1999;8(5);723-730
• 8.TienAC,RajanA,BellenHJ.ANotchUpdated.J.CellBiol.2009;184:621-629
• 9.OliverG,SrinivasanRS.Endothelialcellplasticity:howtobecomeandremainalymphaticendothelial
cell.Development.2010;137:363-372
• 10. Wu JK, Adepoju O, De Silva D et al. A switch in Notch gene expression parallels stem cell to
endothelialtransitionininfantilehemangioma.SpringerScience+BusinessMediaBV.2010;
• 11. Johnson NC, Dillard ME, Baluk P et al. Lymphatic endothelial cell identity is reversible and its
maintenancerequiresProx1activity.GenesDev.2008;22:3282-3291.
19. Acknowledgements
• I’d like to thank my mentor Carrie Shawber for
allowing my to work with her in the
Kitajewski/Shawber lab in the Columbia
University Medical Center, and for providing
me with all the necessary protocols and
materials, along with guidance and
mentorship.
