The research team
identified a new molecular pathway that could be exploited to directly target
pulmonary edema with minimal side effects, unlike currently available drugs
that exhibit many side effects. They have used genetically engineered
adeno-associated virus (AAV) vectors for recombinant gene delivery. Primary
human lung cells in a Lung Alveolus Chip have been used for the
experiments. The research has been
, which is a
constituent journal of the American Institute of Physics (AIP), Maryland, USA.
The study was led
by Dr. Donald E. Ingber, MD, PhD, who is the Founding Director of the Wyss
Institute for Biologically Inspired Engineering and also the Judah Folkman
Professor of Vascular Biology at Harvard Medical School and the Vascular
Biology Program at Boston Children’s Hospital (BCH), and Professor of
Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied
Sciences, Cambridge, Massachusetts, USA.
‘Gene therapy could be used for treating pulmonary edema. Delivery of specific genes into lung tissues stops the leakiness of the lung membranes, thereby preventing worsening of the symptoms of pulmonary edema.’
Dr. Ratnakar Potla,
MBBS, PhD, is a co-author of the paper. He was previously a Research Fellow in
the Vascular Biology Program at BCH and is currently at the Wyss Institute
for Biologically Inspired Engineering, Harvard University, Boston,
Prantil-Baun, PhD, is another co-author of the paper. She is a Senior Staff
Scientist at the Wyss Institute for Biologically Inspired Engineering, Harvard
University, Boston, Massachusetts, USA.
Pulmonary Edema Lacks Specific Targeted
Pulmonary edema is the leaking of fluid from
the blood capillaries surrounding the lungs into the air sacs or alveoli, where
the exchange of oxygen and carbon dioxide takes place during respiration.
Pulmonary edema develops in approximately 80 percent of heart failure patients.
In this condition, oxygen uptake by the lungs is severely hindered, resulting
in difficulty in performing everyday tasks and in severe cases, can be
are currently no drugs that directly treat the complications of pulmonary
edema. Those drugs that are available treat the condition indirectly by
lowering the blood
pressure and reducing the total fluid content of the body, in a bid
to reduce the damage that has been caused.
“Pulmonary edema is similar to a leak in a water pipe.
The most effective thing to do is to plug the leak, but current drugs on the
market work to shut off the water supply altogether and hope the leak fixes by
itself,” says Potla. “With our
method, we can actually target the leak, which is a much more efficient way to
address the problem.”
Mechanism of Pathogenesis of Pulmonary Edema
The mechanism of
pathogenesis of pulmonary edema was deciphered by studying a specific channel
protein called TRPV4 (Transient Receptor Potential Cation Channel Subfamily V
Member 4). This TRPV4 protein, encoded by the TRPV4
is a non-selective cation channel present in lung cell membranes. The TRPV4
protein is activated by mechanical stress that occurs when the lungs inflate
and deflate during breathing. TRPV4 is involved in the regulation of the flow
of substances, including cations such as calcium ions, between the blood
capillaries of the lungs and the alveoli. Activation of TRPV4 causes the lung
tissues to become ‘leaky’, resulting in accumulation of fluid in the alveoli,
thereby progressively worsening the pulmonary edema.
Problems of Current Drug Therapies for
for pulmonary edema are currently lacking. A drug that has been developed by
GlaxoSmithKline (GSK) inhibits TRPV4 by chemically blocking the channel. This
drug is currently undergoing Phase III clinical trials. However, inhibiting
TRPV4 is problematic, as this channel protein is involved in numerous other
physiological processes, including bone remodeling and maintaining the urinary
bladder tone. Therefore, inhibition of TRPV4 gives rise to many unwanted side
effects that limit its utility as a drug target.
How Did the Research Team Overcome the
The Wyss and BCH
researchers attempted to overcome the problems by shifting their attention from
TRPV4 to another membrane protein called CD98hc. This protein transmits
mechanical forces to the TRPV4 channel. The research team found that
overexpression of the high homology (HH) domain of the CD98hc protein blocked
the mechanical activation of TRPV4. Thus, the researchers opined that
delivering the HH domain of CD98hc to lung cells by means of gene therapy,
could prevent the leakage of fluid from the lung capillaries, thereby halting
the progression of pulmonary edema. Importantly, the researchers hypothesized that
this could act as a specific targeted therapy for treating pulmonary edema.
In order to confirm
their hypothesis, the researchers developed recombinant gene constructs using
AAV vectors derived from various serotypes of the virus. The specific DNA
(deoxyribonucleic acid) encoding the HH domain of the CD98hc protein was cloned
into these AAV vectors for gene delivery. The efficiency of gene transfer was
tested by transfecting human Pulmonary Alveolar Epithelial Cells (PAEpiC) and primary Human Pulmonary
Microvascular Endothelial Cells (HPMEC)
with the viral gene constructs. These cells were chosen for the gene
delivery experiments as they are situated at the interface between the alveoli
and the blood capillaries of the lungs and therefore play a crucial role in the
development of pulmonary edema.
Of the different AAV
vectors that were tested, the researchers found that AAV2.5T was the most
efficient in driving gene expression in both cell types – epithelial and
endothelial. Importantly, the cells in which the HH domain-encoding gene was
expressed were capable of completely inhibiting the mechanically-activated
calcium signaling pathway mediated through TRPV4. Moreover, a drug that
stimulated TRPV4 restored the activity of the calcium signaling pathway. This
confirmed that gene therapy was capable of specifically blocking TRPV4’s
mechanically-activated calcium signaling pathway, leaving its other
Lung Alveolus Chip Studies
Lung Alveolus Chip
is a clinically relevant human lung model developed by Wyss Institute
scientists. This chip was used to evaluate the efficiency of gene delivery and
the overexpression of the HH domain of CD98hc.
Lung Alveolus Chip consists of two narrow compartments separated by a permeable
membrane. One side of this membrane is lined with PAEpiC
and the other with HPMEC. The PAEpiC side is perfused with air, while the HPMEC
side is perfused with fluid. The motion of the lungs that occur during
breathing was simulated by alternate stretching and relaxing of the membrane
using a suction device.
It was observed
that when the air compartment of the chip was filled with fluid for four hours
to simulate pulmonary edema, the membrane partition between the air and fluid
compartments became compromised, and recovery was not possible even after
removal of the fluid.
following gene delivery into the Lung Alveolus Chip, fluid leakage into the air
compartment was significantly reduced, and the cells lining the membrane remained
intact, even under mechanical stress, mimicking the motion of the lungs during
“Remarkably, we saw a significant improvement in lung
function even though only about 30 percent of the lung epithelial cells and 10
percent of the vascular endothelial cells were successfully transfected with
the gene coding for the HH domain of CD98hc, indicating that this strategy is
extremely protective,” says Prantil-Baun.
are optimistic that the findings of the present study could pave the way
towards the development of mechanotherapeutic drugs for targeted gene therapy
for treating pulmonary edema.
“This is one the first examples of how
the mechanical signaling activity of a transmembrane molecule that is sensitive
to both physical forces and chemical cues can be selectively targeted with a
tailored mechanotherapeutic to provide a more specific therapeutic response. We
hope there will be many more to follow.”
The study was
supported by grants from the National Institutes of Health (NIH), Bethesda,
- AAV-mediated Gene Therapy Targeting TRPV4 Mechanotransduction for Inhibition of Pulmonary Vascular Leakage – (https://aip.scitation.org/doi/10.1063/1.5122967)
- A lifeline for leaky lung cells – (https://wyss.harvard.edu/news/a-lifeline-for-leaky-lung-cells/)