Progress Report for American Heart Pre-Doctoral Fellowship
Justin L. Grobe
May 2004
(ROUGH DRAFT)
The primary goals of my original proposal were aimed at developing a regulatable genetic
therapy for hypertension, which could be delivered to an animal after it had developed
hypertension. In my proposal, I suggested altering the expression levels of angiotensinogen
and/or angiotensin converting enzyme 2 (ACE2). Decreasing expression of angiotensinogen, or
increasing expression of ACE2, it was proposed, should alleviate the hypertension.
Successfully cloning the entire tetracycline-sensitive promoter system and a regulatable
transgene (antisense to angiotensinogen) into our lenti-vector viral delivery system has been
accomplished, but use of this system for experimental purposes has shown little promise.
First, cloning of the tetracycline-sensitive system into two separate viral vectors was
accomplished. This two-vector method was used in vitro with some success, but in vivo
expression was very low. Our lab group hypothesized that in vivo, the probability of two viral
vectors infecting the same cell successfully was low, and subsequently the entire tetracycline
sensitive promoter system and transgene were cloned into a single viral vector. The expression
levels of transgene in vitro and in vivo in this new single-vector system, though, are too high
basally (an inherent “leak” in the regulation), and expression levels after induction are not
significantly increased over basal expression levels.
One major criticism of my original American Heart Association application was a lack of
physiological hypothesis testing. Between our recognition of this criticism, and the lack of
significant success in the development of the proposed tetracycline regulatable system, my
mentor and I decided to change the focus of my work while still in the infant stages of my predoctoral work. We decided to shift my efforts toward understanding the physiological regulation
(and importance in hypertension) of the newly discovered ACE2 gene, rather than focusing on
physiological effects of manipulating expression of such a gene (as was proposed originally).
To these ends, the sequence for the endogenous promoter for the ACE2 gene was obtained from
the NCBI website. This sequence was examined using the transcription factor binding site
analysis software package __________. It was noted that many potential transcription factor
binding sequences were present within the first two kilobases of the ACE2 transcription start
site.
Of particular interest, multiple estrogen receptor, progesterone receptor, and androgen receptor
potential binding sequences were discovered in this promoter region for the ACE2 gene (see
table 1). This discovery, along with recent publication of papers suggesting cardiac endothelial
cell expression of ACE2, prompted us to examine the effects of sex steroids on endogenous
ACE2 expression.
To examine expression of the ACE2 gene in response to sex steroids, human coronary artery
endothelial cells were grown to confluence in six-well plates. Estrogen (17-beta-estradiol) was
then added to the media of a subset of the wells, and allowed to incubate overnight. Cells were
harvested the next day, and RNA was isolated using an RNaqueous-4-PCR kit from Ambion.
This RNA was used to perform real-time RT-PCR for ACE2. These experiments revealed
decreased expression of ACE2 with low-level estrogen application (figure 1a).
Further experiments were carried out in ovariectomized rats with and without estrogen
replacement. Animals were ovariectomized, and a subset of animals had 17-beta-estradiol
pellets (Innovative Research of America) implanted subcutaneously. After three weeks of
treatment, animals were sacrificed, and the ventricles and the kidney cortex were removed. RNA
we then isolated from these tissues using an RNeasy Fibrous Tissue mini kit from Qiagen. This
RNA was used for real-time RT-PCR analysis of ACE2 gene expression. Again, these
experiments revealed decreased ACE2 expression with low-dose estrogen application (figures 1b
and 1c). Taken together, these in vitro and in vivo experiments suggested that i) low-dose
estrogen affects ACE2 gene transcription, ii) this effect appears to happen both in ventricles and
kidney, and iii) these effects are seen in coronary artery endothelial cells.
Expression of the ACE2 gene has been shown to affect blood pressure regulation by multiple
independent research groups. One physiological state in which sex steroids are significantly
altered (and therefore in which ACE2 gene expression may be significantly altered) is in
pregnancy. Many changes happen in blood pressure regulation during pregnancy, and thus a
new hypothesis was formed; sex steroid fluctuations during pregnancy may alter expression of
the ACE2 gene, and thus mediate the cardiovascular alterations during pregnancy.
To examine this new hypothesis, new in vitro and in vivo experiments were undertaken. First, a
more definitive dose-response relationship needed to be established in vitro, and secondly, the
effects of other sex steroids on ACE2 expression needed to be examined. Human coronary
artery endothelial cells and human coronary artery smooth muscle cells were both grown to
confluence in six-well plates. These cells were then exposed to different doses of estrogen or
progesterone, and incubated overnight. Cells were then harvested, and RNA and protein were
isolated. RNA is currently being used for real-time RT-PCR analysis, and protein is being used
for an ACE2 activity assay.
In addition, animal experiments evaluating blood pressure responses to angiotensin II in steroidmanipulated female (figure 2a) and male (figure 2b) rats have been performed. Blood was
collected from these animals to examine the circulating levels of angiotensin 1-7, which is the
major metabolic product of ACE2 enzymatic activity. Tissues (atria, ventricles, kidney cortex
and medulla, lung, and uterus) were collected for RNA and protein extraction, to be analyzed by
real-time RT-PCR and ACE2 activity analysis. Results from all of these real-time RT-PCR and
ACE2 activity assays (in vitro and in vivo experiments) should be obtained by the end of the
summer.
Further experiments are planned to examine the effects of ACE2 manipulation on vascular
reactivity, and also to examine effects of other doses of estrogens, progesterones, and possibly
androgens on ACE2 transcription.
During the course of my training thus far, I have learned many new techniques, including various
cloning methods, one- and two-step real-time RT-PCR, multiple surgical techniques, and many
tissue handling and processing methods. These skills, combined with the novel and (likely)
publishable results of these experiments, should prepare me for a good post-doctoral experience
and career in the physiological sciences.
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Table 1: Sex steroid binding sequences in ACE2 promoter region
Figure 1: ACE2 mRNA expresssion in (a) human coronary artery endothelial cells, and in rat (b)
ventricles and (c) kidney in response to 17-beta-estradiol replacement
Figure 2: Ang II pressor responses in (a) female and (b) male steroid-manipulated animals.