Protein could help rejuvenate oxygen-starved cardiac tissue, heal wounds

By Jennifer O'Brien

A UCSF-led team is reporting striking results in mice that indicate that a
molecule known as HIF-1 could prove an effective target for inducing the growth
of blood vessels in oxygen-starved tissues. The strategy is sought for treating
cardiac and peripheral vascular disease, diabetes-damaged tissues and
intractable wounds.

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-1 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-1 gene caused a
13-fold increase in the expression of the VEGF gene. The fact that HIF-1 had an
effect on VEGF expression is not surprising in itself, as HIF-1 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-1 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-1
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-1 could prove a potent target for cancer therapy.
Malignant tumors must recruit blood vessels to fuel their growth. Scientists
have known that HIF-1 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-1 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-1. 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-1 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-1 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-1 into skin cells or inserting copies of the HIF-1 gene
lacking the portion of the gene that normally degrades HIF-1. This region is
known as the oxygen-dependent degradation domain (ODD).

While researchers still must determine how HIF-1 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

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

The study was funded by the National Institutes of Health.