Molecular Regulation of Sprouting Angiogenesis.

The term angiogenesis arose in the 18th century. Several studies over the next 100 years laid the groundwork for initial studies performed by the Folkman laboratory, which were at first met with some opposition. Once overcome, the angiogenesis field has flourished due to studies on tumor angiogenesis and various developmental models that can be genetically manipulated, including mice and zebrafish. In addition, new discoveries have been aided by the ability to isolate primary endothelial cells, which has allowed dissection of various steps within angiogenesis. This review will summarize the molecular events that control angiogenesis downstream of biochemical factors such as growth factors, cytokines, chemokines, hypoxia-inducible factors (HIFs), and lipids. These and other stimuli have been linked to regulation of junctional molecules and cell surface receptors. In addition, the contribution of cytoskeletal elements and regulatory proteins has revealed an intricate role for mobilization of actin, microtubules, and intermediate filaments in response to cues that activate the endothelium. Activating stimuli also affect various focal adhesion proteins, scaffold proteins, intracellular kinases, and second messengers. Finally, metalloproteinases, which facilitate matrix degradation and the formation of new blood vessels, are discussed, along with our knowledge of crosstalk between the various subclasses of these molecules throughout the text. Compr Physiol 8:153-235, 2018.

Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity.

Successful design of tissue engineering scaffolds must include the ability to stimulate vascular development by incorporating angiogenic growth factors. Current approaches can allow diffusion of growth factors, incorporate active factors randomly, or can leave residual toxins. We addressed these problems by genetically fusing the gene encoding Vascular Endothelial Growth Factor (VEGF) with the Ultrabithorax (Ubx) gene to produce fusion proteins capable of self-assembly into materials. We demonstrate that VEGF-Ubx materials enhance human endothelial cell migration, prolong cell survival, and dose-dependently activate the VEGF signaling pathway. VEGF-Ubx fibers attract outgrowing sprouts in an aortic ring assay and induce vessel formation in a chicken embryo chorioallantoic membrane (CAM) assay. Collectively, these results demonstrate that the activity of VEGF remains intact in Ubx materials. This approach could provide an inexpensive and facile mechanism to stimulate and pattern angiogenesis.

Culture of Tumorigenic Cells on Protein Fibers Reveals Metastatic Cell Behaviors.

Tumorigenic cell behaviors can be suppressed or enhanced by their physicochemical environment. As a first step toward developing materials that allow tumorigenic behaviors to be observed and manipulated, we cultured related MCF10 breast cell lines on fibers composed of the Drosophila protein Ultrabithorax (Ubx). These cell lines, originally derived from fibrocystic breast tissue, represent a continuum of tumorigenic behavior. Immortal but nontumorigenic MCF10A cells, as well as semitumorigenic MCF10AT cells, attached and spread on Ubx fibers. MCF10CA-1a cells, the most highly transformed line, secreted high concentrations of matrix metalloproteinases when cultured on Ubx materials, resulting in differences in cell attachment and cytoskeletal structure, and enabling invasive behavior. Because the mechanical and functional properties of Ubx fibers can be genetically manipulated, these materials provide a valuable tool for cancer research, allowing creation of diverse microenvironments that allow assessment of invasive, metastatic behavior.

Identification of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax.

The recombinant protein Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor, self-assembles into biocompatible materials in vitro that are remarkably extensible and strong. Here, we demonstrate that the strength of Ubx materials is due to intermolecular dityrosine bonds. Ubx materials auto-fluoresce blue, a characteristic of dityrosine, and bind dityrosine-specific antibodies. Monitoring the fluorescence of reduced Ubx fibers upon oxygen exposure reveals biphasic bond formation kinetics. Two dityrosine bonds in Ubx were identified by site-directed mutagenesis followed by measurements of fiber fluorescent intensity. One bond is located between the N-terminus and the homeodomain (Y4/Y296 or Y12/Y293), and another bond is formed by Y167 and Y240. Fiber fluorescence closely correlates with fiber strength, demonstrating that these bonds are intermolecular. To our knowledge, this is the first identification of specific residues that participate in dityrosine bonds in protein-based materials. The percentage of Ubx molecules harboring both bonds can be decreased or increased by mutagenesis, providing an additional mechanism to control the mechanical properties of Ubx materials. Duplication of tyrosine-containing motifs in Ubx increases dityrosine content in Ubx fibers, suggesting these motifs could be inserted in other self-assembling proteins to strengthen the corresponding materials.

Materials composed of the Drosophila Hox protein Ultrabithorax are biocompatible and nonimmunogenic.

Although the in vivo function of the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) is to regulate transcription, in vitro Ubx hierarchically self-assembles to form nanoscale to macroscale materials. The morphology, mechanical properties, and functionality (via protein chimeras) of Ubx materials are all easily engineered. Ubx materials are also compatible with cells in culture. These properties make Ubx attractive as a potential tissue engineering scaffold, but to be used as such they must be biocompatible and nonimmunogenic. In this study, we assess whether Ubx materials are suitable for in vivo applications. When implanted into mice, Ubx fibers attracted few immune cells to the implant area. Sera from mice implanted with Ubx contain little to no antibodies capable of recognizing Ubx. Furthermore, Ubx fibers cultured with macrophages in vitro did not lyse or activate the macrophages, as measured by TNF-α and NO secretion. Finally, Ubx fibers do not cause hemolysis when incubated with human red blood cells. The minimal effects observed are comparable with those induced by biomaterials used successfully in vivo. We conclude Ubx materials are biocompatible and nonimmunogenic.

Regional changes in elastic fiber organization and transforming growth factor ß signaling in aortas from a mouse model of marfan syndrome.

In Marfan Syndrome (MFS), development of thoracic aortic aneurysms (TAAs) is characterized by degeneration of the medial layer of the aorta, including fragmentation and loss of elastic fibers, phenotypic changes in the smooth muscle cells, and an increase in the active form of transforming growth factor-ß (TGFß), which is thought to play a major role in development and progression of the aneurysm. We hypothesized that regional difference in elastic fiber fragmentation contributes to TGFß activation and hence the localization of aneurysm formation. The fibrillin-1-deficient mgR/mgR mouse model of MFS was used to investigate regional changes in elastin fiber fragmentation, TGFß activation and changes in gene expression as compared to wild-type littermates. Knockdown of Smad 2 and Smad 3 with shRNA was used to determine the role of the specific transcription factors in gene regulation in aortic smooth muscle cells. We show increased elastin fiber fragmentation in the regions associated with aneurysm formation and altered TGFß signaling in these regions. Differential effects of Smad 2 and Smad 3 were observed in cultured smooth muscle cells by shRNA-mediated knockdown of expression of these transcription factors. Differential signaling through Smad 2 and Smad 3 in regions of active vascular remodeling likely contribute to aneurysm formation in the mgR/mgR model of MFS. Increased elastin fiber fragmentation in these regions is associated with these changes as compared to other regions of the thoracic aorta and may contribute to the changes in TGFß signaling in these regions.