LAKE NONA, Fla.—To date, the primary therapeutic focus when it comes to encouraging angiogenesis, or the development of new blood vessels, is vascular endothelial growth factor (VEGF). But a signaling pathway discovered by researchers at the Sanford Burnham Prebys Medical Discovery Institute (SBP) as being pivotal for angiogenesis could offer a better option. This work was detailed in a paper titled “R-Ras-Akt axis induces endothelial lumenogenesis and regulates the patency of regenerating vasculature,” which appeared in Nature Communications.
VEGF does encourage vascularization, but as the team—led by Dr. Masanobu Komatsu, associate professor at SBP’s Lake Nona campus—showed with 3D culture and living tissue, the resulting vessels are unstable and non-functional. Specifically, “Functional vessels need to have a lumen, a pipe-like opening that allows oxygenated blood and nutrients to travel through the body, and VEGF alone cannot fully support the formation of such a vessel structure,” Komatsu said in a press release. As the authors note in their paper, “The formation of endothelial lumen is fundamental to angiogenesis and essential to the oxygenation of hypoxic tissues.”
The other needed parts of the process are Akt, a protein kinase, and the protein R-Ras. Akt is activated by R-Ras, a Ras homolog, which then “stabilizes the microtubule cytoskeleton in endothelial cells leading to endothelial lumenogenesis,” per the paper. In contrast, “The activation of Akt by the potent angiogenic factor VEGF-A does not strongly stabilize microtubules or sufficiently promote lumen formation, hence demonstrating a distinct role for the R-Ras-Akt axis.”
The team began their work in vitro with a fibrin gel 3D culture of endothelial cells. Endothelial cells were “coated onto microbeads and embedded in fibrin gel with pericytes seeded on the top of the gel as feeder cells,” the authors wrote. Silencing R-Ras with short hairpin RNA proved to disrupt lumenogenesis in the endothelial cells, and many of the cells did not undergo morphogenesis.
“Generating new blood vessels is similar to the way trees grow; sprouts develop from existing vessels and then branch out further and further to restore vascularity,” reported Dr. Fangfei Li, a postdoctoral associate in Komatsu’s lab and lead author of the paper. “This study shows that there are distinct steps and signals that control the process.
“First, VEGF activates Akt to induce endothelial cells to sprout. Then, R-Ras activates Akt to induce lumen formation. The second step involving Akt activation by R-Ras stabilizes the microtubule cytoskeleton in endothelial cells, creating a steady architecture that promotes lumen formation.”
When the team moved from samples to rodents, their in-vivo work in mice demonstrated that R-Ras activating Akt is a pivotal part of the process of lumen development in “reparative angiogenesis.” Without R-Ras, the authors note, an abundance of vessels develop in ischemic tissue that lack lumens, which means they are useless for circulation and do not aid recovery.
Komatsu tells DDNews that “In our study, we found that if you upregulate R-Ras expression, increase the production of R-Ras protein in these blood vessels, they become mature. So you can repair the function of blood vessels by upregulating R-Ras, and R-Ras uses the Akt pathway for promoting vessel maturation.”
“We propose that VEGF and R-Ras activation of Akt signaling are complementary to each other, both are necessary to generate fully functional blood vessels to repair ischemic tissue,” Komatsu explained. “Our next step is to work toward promoting the combined signaling of Akt in clinical studies, prompting R-Ras activation through either gene therapy or pharmacologically in parallel with VEGF therapy.”
The difficulty lies in the fact that R-Ras is difficult to target therapeutically, he says.
“Akt is one of the downstream R-Ras signaling pathways that we looked at,” Komatsu explains. “We also have found several other downstream pathway mechanisms, but what we still don't know much about is the upstream of R-Ras. We don't know how R-Ras expression is regulated by blood vessels or the endothelial cells in blood vessels. The new question is, how can you upregulate R-Ras? Now we are looking into the upstream mechanisms, regulators of the R-Ras gene. And once we find those regulators, hopefully some of those regulator pathways are easy to target. R-Ras is not an ideal target, but maybe the upstream of R-Ras can provide a new target that can be druggable.”
This is not the first time Komatsu has explored R-Ras and its role in angiogenesis. In a 2012 Cancer Cell article, “Small GTPase R-Ras Regulates Integrity and Functionality of Tumor Blood Vessels,” Komatsu and colleagues found that R-Ras helps to normalize blood vessels in tumors to prevent metastasis.
Rampant angiogenesis is a hallmark of cancer. As tumor tissue is usually hypoxic, or a low-oxygen environment, more blood vessels are needed to bring oxygen to the cancerous tissue. Komatsu says that previous efforts at fighting cancer have focused on trying to starve tumors by disrupting angiogenesis, but they weren't successful. “Even though in the clinic they still use anti-angiogenic drugs in combination with chemotherapy, we now know that anti-angiogenic therapy does not really prolong patient survival—maybe only six to eight months. It's not a cure, it's not very effective.”
“Tumor tissue produces a lot of blood vessels, but these blood vessels are dysfunctional,” he explains. “Tumor angiogenesis is induced by growth factors; they induce the sprouting of blood vessels, but they do not have the chance to mature. They are very chaotic and immature and leaky, and this leakiness allows the cancer cells to penetrate into the bloodstream. Once the cancer cells penetrate into the bloodstream, it facilitates distant metastasis. Blood vessels are made by endothelial cells, those are the cells that line the lumen of blood vessels. It's kind of like tile in your bathroom; the cells attach to each other to make a seal. But this seal is broken in blood vessels in tumor tissue, so cancer cells can get into the bloodstream. What we found was if you upregulate R-Ras in these blood vessels, you can repair the leakiness. So in other words, you can inhibit metastasis.”
This recent work in Nature Communications was funded in part by the National Cancer Institute, the National Science Foundation Grant, the American Heart Association, the Bankhead-Coley Cancer Research Program and the Florida Breast Cancer Foundation.