Bionegineered In Vitro Lymphatic Model May Lead to New Therapies
The lymphatic system plays important roles in maintaining health, such as fluid drainage to remove toxins and prevent tissue swelling, fat transport to facilitate metabolism, and immune cell transport to promote immune defense. As a result, health conditions including cancer, infections, cardiovascular disease, and neurodegenerative diseases have been associated with a malfunctioning lymphatic system. Thus, regenerating the lymphatic system can help afflicted patients recover from such diseases.
However, there is a lack of a suitable in vitro model to study how lymphangiogenesis, or formation of the lymphatic network, is regulated. Adequate understanding of the molecular process of lymphangiogenesis is necessary in order to develop effective therapeutic strategies for restoring a dysfunctional lymphatic system. Current in vitro models fail to mimic lymphangiogenesis in the human body. So, Dr. Hanjaya-Putra and researchers at the University of Notre Dame engineered a two-dimensional biomimetic lymphatic tubule system using hyaluronic acid-based hydrogels (HA-hydrogels), which act as support structures, to help stimulate lymphatic endothelial cells (LECs) to form their networks. The advantages of these hydrogels are that they are non-immunogenic, meaning they do not cause an undesired immune response by the body, and they are convenient to chemically modify.
First, the researchers tested to confirm that LECs cultured on HA-hydrogels do not lose their phenotypes, which are their expression of several distinct markers: LYVE-1, PDPN, CD44, and PROX-1. These markers are key to lymphangiogenesis, and so if any of them were negatively affected by the hydrogels, the hydrogels wouldn’t be a suitable in vitro model because they would fail to replicate how lymphangiogenesis actually occurs in the body. For instance, LYVE-1 uniquely binds to the hydrogels, and PROX-1 has a role in regulating the development of the lymphatic system. Fortunately, the researchers discovered that the LECs maintained expression of these four markers, and furthermore, LYVE-1 and PROX-1 expression increased while PDPN and CD44 expression remained the same. In fact, LYVE-1 and PROX-1 expression increased even more so on the softer matrix created by the hydrogels.
However, there is a lack of a suitable in vitro model to study how lymphangiogenesis, or formation of the lymphatic network, is regulated. Adequate understanding of the molecular process of lymphangiogenesis is necessary in order to develop effective therapeutic strategies for restoring a dysfunctional lymphatic system. Current in vitro models fail to mimic lymphangiogenesis in the human body. So, Dr. Hanjaya-Putra and researchers at the University of Notre Dame engineered a two-dimensional biomimetic lymphatic tubule system using hyaluronic acid-based hydrogels (HA-hydrogels), which act as support structures, to help stimulate lymphatic endothelial cells (LECs) to form their networks. The advantages of these hydrogels are that they are non-immunogenic, meaning they do not cause an undesired immune response by the body, and they are convenient to chemically modify.
First, the researchers tested to confirm that LECs cultured on HA-hydrogels do not lose their phenotypes, which are their expression of several distinct markers: LYVE-1, PDPN, CD44, and PROX-1. These markers are key to lymphangiogenesis, and so if any of them were negatively affected by the hydrogels, the hydrogels wouldn’t be a suitable in vitro model because they would fail to replicate how lymphangiogenesis actually occurs in the body. For instance, LYVE-1 uniquely binds to the hydrogels, and PROX-1 has a role in regulating the development of the lymphatic system. Fortunately, the researchers discovered that the LECs maintained expression of these four markers, and furthermore, LYVE-1 and PROX-1 expression increased while PDPN and CD44 expression remained the same. In fact, LYVE-1 and PROX-1 expression increased even more so on the softer matrix created by the hydrogels.
Dysfunctions of the lymphatic system can contribute to chronic conditions such as cancer. Therefore, it is important to research lymphangiogenesis in order to restore the lymphatic system’s function in patients.
Image Source: marijana1
Next, they tested how changing the concentrations of VEGF-C and varying the matrix stiffness of the hydrogels affected lymphangiogenesis. VEGF-C is a molecule known to induce lymphangiogenesis. They found that high concentrations of VEGF-C greatly increased lymphangiogenesis in soft hydrogels. Soft hydrogels also expressed more VEGFR-3, a receptor that binds to VEGF-C, and thereby could further amplify lymphangiogenesis. Additionally, increased VEGFR-3 and VEGF-C binding augments expression of matrix metalloproteinases (MMPs), molecules important in LEC migration and thus essential in lymphangiogenesis.
To understand more of the mechanisms behind why changes in stiffness affect lymphangiogenesis, Hanjaya-Putra and his team examined two proteins called YAP and TAZ, which are known to act as mechanosensors. Low matrix stiffness resulted in cellular destruction of YAP and TAZ, which in turn increased expression of PROX-1 and its targets, VEGFR-3 and MMP-14. These relationships between markers and proteins explains why low matrix stiffness led to increased lymphangiogenesis.
Overall, the data demonstrates HA-hydrogels’ ability to support LECs and lymphangiogenesis in a viable in vitro model. Furthermore, lymphangiogenesis was also able to be modulated by precisely controlling VEGF-C concentrations and matrix stiffness. Thus, this novel model could be instrumental in future research and applications that aim to revitalize malfunctioning lymphatic systems.
To understand more of the mechanisms behind why changes in stiffness affect lymphangiogenesis, Hanjaya-Putra and his team examined two proteins called YAP and TAZ, which are known to act as mechanosensors. Low matrix stiffness resulted in cellular destruction of YAP and TAZ, which in turn increased expression of PROX-1 and its targets, VEGFR-3 and MMP-14. These relationships between markers and proteins explains why low matrix stiffness led to increased lymphangiogenesis.
Overall, the data demonstrates HA-hydrogels’ ability to support LECs and lymphangiogenesis in a viable in vitro model. Furthermore, lymphangiogenesis was also able to be modulated by precisely controlling VEGF-C concentrations and matrix stiffness. Thus, this novel model could be instrumental in future research and applications that aim to revitalize malfunctioning lymphatic systems.
Featured Image Source: National Cancer Institute
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