The finding, reported in the October 1 issue of Genes & Development, is a notable advance in an effort that has met with setbacks. Researchers have tried to generate the production of healthy blood vessels by inducing over-expression of the growth factor VEGF. But studies in mice have shown that while over-expression of VEGF induces the growth of blood vessels, the capillaries are leaky, the tissues are inflamed and swollen, and the blood vessels have an abnormal “corkscrew-like” shape.
In the current study, researchers genetically engineered mice to overexpress the HIF-1α gene in skin cells. In response, the number of capillaries in the mice’s skin increased by nearly 70 percent. More importantly, the blood vessels did not leak, cause swelling or inflammation.
“The vessels looked like normal capillaries,” says senior author Jeffrey M. Arbeit, MD, UCSF associate professor of surgery, and a member of the UCSF Comprehensive Cancer Center. “This finding, together with the fact that the vessels didn't leak, is extremely exciting.” The increase in healthy blood vessels was evident in the mice’s significantly pinker ears, paws and tails.
Notably, in the current study the overexpression of the HIF-1α gene caused a 13-fold increase in the expression of the VEGF gene. The fact that HIF-1α had an effect on VEGF expression is not surprising in itself, as HIF-1α is a sub unit of the HIF-1 transcription factor, which regulates the expression of numerous genes, including VEGF. However, the finding does prompt the question of why the blood vessels were robust, given that previous studies involving elevated expression of VEFG led to the development of weak, leaky vessels.
“We know that VEGF plays a crucial role in blood vessel growth. We need to determine how overexpression of HIF-1α harnesses VEGF in a way that could be beneficial therapeutically,” says lead author David Elson, BA, UCSF staff research associate in the Arbeit lab.
The potential clinical implications of the finding are significant. The HIF-1α gene is already being explored as a stimulant to promote blood vessel growth in oxygen-deprived, or ischemic, tissue such as that associated with diabetic peripheral vascular disease, which can cause chronic leg ulcers that often precipitate amputation. It is being investigated as therapy to increase blood flow into cardiac tissue deprived of oxygen due to clogged arteries, and as therapy to treat recalcitrant wounds resulting from lack of blood flow to the legs caused by atherosclerosis alone or in association with diabetes. It could also be used to promote the grafting of artificial skin into tissues of the body, either in burn or diabetic patients. Once a graft had fused with the skin, the gene could be “turned off.”
On the flip side, HIF-1α could prove a potent target for cancer therapy. Malignant tumors must recruit blood vessels to fuel their growth. Scientists have known that HIF-1α is over-expressed by malignant tumors, and NIH investigators currently are exploring its potential as a therapeutic target. However, the gene’s specific role in cancer development has not been known. The discovery that overexpression of the gene generates the growth of robust blood vessels will assist ongoing therapeutic studies.
There are various possible explanations for why the new blood vessels in the UCSF study were robust despite the elevated expression of VEFG, says Elson. In the current study, over-expression of HIF-1α caused the induction of the naturally occurring VEGF gene. In previous studies, scientists engineered the expression of various splicings, or isoforms, of the VEGF gene. It may be, says Elson, that the spectrum of alternatively spliced isoforms created by the naturally occurring VEGF does not cause leakage. Alternatively, he says, the HIF-1 transcription factor may increase expression of an as-yet-unidentified target that modulates vascular permeability independent of VEGF function.
The current finding leads scientists a step closer to teasing out the specific role of HIF-1α. The HIF-1 transcription factor regulates the activity of numerous genes, some of which promote blood vessel growth, or angiogenesis, in response to oxygen deprivation. And scientists have known that the HIF-1α gene, a sub unit of the transcription factor, activates genes required for energy metabolism and tissue perfusion during periods of oxygen deprivation and is likewise necessary for embryonic development. They have also known that the gene is over-expressed during myocardial infarction (when blood flow is blocked from reaching the heart) in wound healing (which requires oxygen for tissue repair) and in malignant tumors. But its specific role in these conditions has not been known. Once expressed, HIF-1α is swiftly degraded at the protein level in healthy adult cells.
In their study, the researchers created mice genetically engineered to maintain expression of the gene in an attempt to tease out its impact. They did so by inserting normal HIF-1α into skin cells or inserting copies of the HIF-1α gene lacking the portion of the gene that normally degrades HIF-1α. This region is known as the oxygen-dependent degradation domain (ODD).
While researchers still must determine how HIF-1α prompts the development of healthy blood vessels in spite of over-expression of VEGF, the study reinforces the importance of the sub-unit. The finding also suggests the importance of focusing on the overall influence of the HIF-1 transcription factor, says Arbeit.
HIF-1, like the hormone estrogen, is a master regulatory transcription factor, meaning that it controls the expression of a vast number of genes representing various functions. HIF-1 is known to regulate 30 genes, but it may regulate as many as 100 genes, says Arbeit. The more scientists learn about the role of the genes in such molecular pathways the more opportunity they have for learning to manipulate them to treat disease.
A surgeon by training, Arbeit has seen the impact of both unwanted blood vessel growth, as in cancer, and oxygen-starved tissue, as in recalcitrant wounds.
“I don’t ascribe to the hope for a magic bullet for treating disease,” he says, “but targeting one molecule sitting at the head of an interlocking genetic network is a powerful therapeutic concept.”
Other co-authors of the study were Gavin Thurston, PhD, formerly UCSF adjunct assistant professor of anatomy and now at Regeneron Pharmaceuticals, Inc; David G. Ginzinger, PhD, director of the UCSF Genome Analysis Core in the UCSF Comprehensive Cancer Center; Donald M. McDonald, MD, PhD, UCSF professor of anatomy and a member of the UCSF Cardiovascular Research Institute; L. Eric Huang, PhD, of the Laboratory of Human Carcinogenesis, National Cancer Institute; and Randall S. Johnson, PhD, associate professor of biology, UC San Diego.
The study was funded by the National Institutes of Health.
Journal
Genes & Development