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Relationship Between Notch Signaling Receptors and Lymphatic
Malformations
By: Epshita Das
Abstract
• The purpose of this project was to determine whether or not there was a relationship between the formation of lymphatic malformations, and a specific gene- Notch3. To determine if any relationship existed, tissues from a patient were stained with antibodies to show expression of proteins. After staining several sets of similar tissues data was collected showing levels of expression of certain proteins associated with LMs in those regions, which contained either a great population of LMECs or a great population of LMPCs. By analyzing and comparing the levels of expression of a specific antibody in areas with different cell types, the conclusion that was reached was that Notch3 is expressed in the LMPCs of lymphangiomatosis, and not LMECs.
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 rise of lymphatic malformations.
• Notch signaling also plays a key role in regulation of cell fate decisions and angiogenesis, and in mice, has been shown to regulate arterial and venous specifications of major vessels, such as the dorsal aorta [3,4,5]. In addition, Notch signaling pathway is used by endothelial cells in order to organize cellular behaviors during the sprouting stage [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 the growth and branching of blood and lymph vessels [7,8,9].
Background (continued)
• In terms of phenotypic characteristics, there are two distinct types of lymphatic malformation - microcystic and macrocystic. The main difference between the two types is the size and shape of the malformations.
• Lymphangiomatosis is a condition in which the body simultaneously has both microcystic and macrocystic lymphatic malformations, causing it to be categorized as 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 Notch1 being overexpressed in the LMEC population and Notch3 in the LMPCs [11].
• In addition, it has been noted that isolated lymphatic malformation endothelial cells tend to express high levels of Hey1, which suggests high Notch signal activation .
• 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 population they tend to be overly expressed in.
Research Problem
• What is the direct relationship between lymphangiomatosis and Notch pathway activities?
Hypothesis
• If there is Notch expression in the cells of a lymphatic malformation, then there is likely to be a direct connection between the formation of the LM and the protein receptor.
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.
Materials and Methods
• Immunohistochemistry is a process by which a set of cells are incubated with antibodies in order to “stain” them.
• For frozen tissue, the slides were “prefixed” in acetone for some time, and then washed in PBS (phosphate buffer saline).
• 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,” meaning each set was stained with two antibodies.
• 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, antigen retrieval induced in order to uncross linked proteins.
• The steps following the initial steps are the same standard ones as the steps followed for staining frozen tissue..
Figure 1: Expression of lymphatic endothelial cell and the stem cell markers normal on lymphatic malformation tissues. The tissues stained were acquired
form a sample of Normal foreskin, a microcystic LM, and a mixed macrocystic and microcystic LM. The tissues were stained using the antibodies
Podoplanin and CD133 to show expression of lymphatic endothelial cells and stem cells, respectively. DAPI was used to visualize the nuclei. Podoplanin
was highly expressed in the lymphatic vessels of normal tissue, while its expression was reduced or absent in LM tissues. CD133 expression was low in
the lymphatic vessels in normal tissue. In contrast, high CD133 staining was observed in the tissues of the lymphatic malformation tissues.
Results
Figure 2: Expression of lymphatic
endothelial cell marker and
lymphatic vessel marker on
lymphangiomatosis tissue. The
tissue that was stained was a paraffin
section from a lymphangiomatosis
patient, isolated from the jugular vein
region in the neck, the mesentery (gut
area), the lungs, and the thoracic duct
in the center of the body. The tissues
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
tissues collected from all four regions
(jugular/lymphatic, mesentery vessel,
lung, and thoracic duct), there was
high Podoplanin expression in the
vessels and surrounding tissue.
LYVE1 expression was much more
muted, and overlapped with that of
Podoplanin. The mesentery vessel,
out of all four regions, showed the
least expression of both markers,
although some degree of expression
is evident.
Figure 3: Expression of endothelial
cell 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 region in the
neck, the mesentery (gut area), the
lungs, and the thoracic duct in the
center of the body. The tissues were
stained using the antibodies
Podoplanin and Notch3 to show
expression of lymphatic endothelial
cells and Notch3 cell markers,
respectively, and DAPI was used to
visualize the nuclei. In the tissues
collected from the four
aforementioned regions, 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
to the other regions.
Figure 4: Expression of stem cell
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
region in the neck, the mesentery
(gut area), the lungs, and the
thoracic duct in the center of the
body. The tissues were stained
with the antibodies CD133 and
Notch3, to show expression of
stem cell markers and Notch3
markers in LMPCs on the tissue,
respectively; DAPI was used to
visualize the nuclei. There is
CD133 expression in tissues
from the lung, 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
show a co-expression of CD133
and Notch3.
Figure 5: Expression of endothelial cell
marker and notch activity on
lymphangiomatosis tissue. The tissue
that was stained was a paraffin
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 center of the body.
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
lymphatic regions, there’s a slightly lower
expression of both antibodies, and in the
mesentery vessel tissue, there’s scarcely
any expression of either antibody.
Discussion – Analysis of Results • In the lymphatic vessels of normal tissue, high Podoplanin expression was observed, while
its expression was greatly reduced in lymphatic malformation tissues, which suggests lymphatic malformation endothelial cells (LMECs) usually lining lymphatic vessels are greatly affected by the lymphatic malformation, to the point of degeneration
• CD133 expression was reversed: its expression was low in the normal tissue, and high in the LM tissues. CD133 is a stem cell marker, so the absence of stem cells in the normal tissue and their presence in LM tissue suggests that the presence of these CD133 positive 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 types with differently morphology, besides the typical lymphatic vessels, causing the resulting vessels to appear grossly deformed. This staining pattern was consistent with that for LMPCs observed in the LM tissues, suggesting LMPCs are present in large quantities in lymphangiomatosis tissue.
• Notch3 was not co-expressed with high Podoplanin levels in the lymphangiomatosis tissue samples, indicating it’s not highly expressed in LMECs.
Discussion – Analysis of Results (continued)
• Notch3 was not co-expressed with high Podoplanin levels in the lymphangiomatosis tissue samples, indicating it’s not highly expressed in LMECs.
• As shown in Fig. 4, Notch3 was also expressed in the CD133 positive cells in the 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 compared to the same regions in Fig. 4), indicative of its suspected expression in the LMPC population. This data suggests that Notch3 may function in the lymphatic malformation progenitor cell population.
• Hey2 expression was observed in the same cells that expressed high levels of CD133 and Notch3, and because Hey2 is a downstream effector of Notch signal activation and was absent in cells showing high levels of Podoplanin (which are, therefore, lymphatic malformation endothelial cells), it suggests that the Notch3 gene has direct correlations with the formation of a lymphatic malformation consisting primarily of LMECs.
• The absence of Notch3 and Hey2 in the Podoplanin positive LMECs suggests that these proteins and Notch signaling do not function simultaneously in the cells found in lymphangiomatosis tissue.
Discussion – Main Conclusion
• This study demonstrated that Notch3 is expressed in the progenitor cell population in lymphangiomatosis tissues, and that the expression of the Notch target gene, Hey2, was unregulated, and also consistent with that of Notch3 actively signaling.
Future Research
• Because these data were collected from only one patient, the results are pretty inconclusive. Several future studies using tissue samples from many other lymphangiomatosis patients would lead to more verifiable results.
• Only one specific Notch gene was studied in this project, whereas the results could be accounted for or influenced by other genes. Future research can focus on the extent to which other Notch genes impact LM formation.
References • 1. Shawber C, Kitajewski, J. Notch function in the vasculature: insights from zebrafish, mouse and man.
BioEssays. 2004; 26.3: 225-234
• 2 . Schweisguth, F. Regulation of Notch Signaling Activity. Current Biology. 2004; 14: 129-138
• 3. Funahashi Y, Shawber C, Kitajweski J. Notch genes: orchestrating endothelial differentiation. Endothelial Biomedicine. 41:368-374
• 4. Niessen K, Zhang G, Ridgway JR et al. The Notch1-Dll4 signaling pathway regulates mouse postnatal lymphatic development. Blood. 2011; 118(7): 1989-1997
• 5. Zheng W, Tammela T, Yamamoto M et al. Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor. 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. Xue Y, Lindsell CE, Norton CR et al. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet. 1999; 8(5); 723-730
• 8. Tien AC, Rajan A, Bellen HJ. A Notch Updated. J. Cell Biol. 2009; 184: 621-629
• 9. Oliver G, Srinivasan RS. Endothelial cell plasticity: how to become and remain a lymphatic endothelial 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 endothelial transition in infantile hemangioma. Springer Science+Business Media BV. 2010;
• 11. Johnson NC, Dillard ME, Baluk P et al. Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity. Genes Dev. 2008; 22: 3282-3291.
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.