in the laboratory of Marlene Rabinovitch, M.D.
Development of a mouse model for pulmonary hypertension
Reversing pulmonary vascular disease
Serotonin transport and the regulation of Mts1-induced pulmonary vascular disease
BMP type II receptor and the pathobiology of pulmonary hypertension
The role of LC3 in cell motility and growth
LC3 expression during mouse development
Development of a mouse model for pulmonary hypertension
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Our lab is developing a powerful animal model of severe pulmonary hypertension (PH) using a mouse that constitutively overexpresses the Mts-1 gene, first described in cancer cells.
The Goal of this study is to
understand the global changes in gene expression that accompany the
onset and progression of PPH in Mts-1 mice, employing
microarray analysis.
Compared with age matched isogenic mice, some Mts-1 expressing mice
develop obliterative pulmonary arterial lesions, right ventricular
hypertrophy
and have elevated right ventricular systolic pressures.
Pioneered at Stanford, microarray technology allows one to simultaneously
quantify the expression levels of thousands of genes in a single experiment.
We have used microarrays to compare levels of gene expression found
in the lungs of normal and Mts-1 expressing mice.
Future directions include using laser capture microscopy combined with amplification protocols and gene array technology to assess gene expression in small numbers of cells collected from pulmonary arterial lesions.
The image shows the hybridization pattern
found on a DNA microarray after it was hybridized with fluorescently
labeled cDNAs prepared from
a reference RNA (red), and RNA from Mts-1 mouse lungs (green). Each spot
represents a single gene and genes that are expressed equally in both
samples (Mts-1 lung and reference) appear yellow in the image. Genes
expressed more highly in the reference sample produce a red spot while
those more prevalent in the RNAs of Mts-1 mouse lungs produce a green
spot. Image analysis software is then used to capture pixel number and
intensity of each spot, and these values are then used to quantify gene
expression levels using a variety of bioinformatic algorithms.
REVERSING PULMONARY VASCULAR DISEASE
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A mouse that over-expresses the calcium-binding
protein Mts-1 develops severe obliterative pulmonary vascular disease
observed in patients with this fatal condition.
The goal of this study is to develop ways to reverse these obliterative
changes that occur as a result of smooth muscle cell proliferation and
migration by inducing programmed cell death.
Panel A in the picture shows an occluded vessel in an Mts-1 mouse, and Panel B is a normal vessel, full of red blood cells, in a normal mouse.
SEROTONIN TRANSPORT REGULATES MTS1-INDUCED PULMONARY VASCULAR DISEASE
Recent advances in genetic screening have identified a number of potentially important mutations with regard to the development of pulmonary hypertension.
We are investigating whether two of these mutations, malfunction of the bone morphogenetic protein receptor 2 (BMPR2), and the serotonin transporter (SERT) are connected to a newly described metastases associated protein Mts1.
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Figure 1 is a densitometry on western
immunoblotting for Mts1 activity in bovine PASMC lysates at 1, 6, and
24h after stimulation.
We have shown that stimulation of pulmonary artery smooth muscle cells
(PASMC) with serotonin increases the production and subsequent secretion
of Mts1 (Figure 1). The effect appears to be dependent on the SERT since
it is blocked with ipronazid. We are now studying the mechanism of this
activation.
Figure 2 is
a densitometry on western immunoblotting for Mts1 activity in bovine
PASMC lysates at 1, 6, and 24h after stimulation.
We have shown that stimulation of pulmonary artery smooth muscle cells (PASMC)
with serotonin increases the production and subsequent secretion of Mts1 (Figure
1). The effect appears to be dependent on the SERT since it is blocked with ipronazid.
We are now studying the mechanism of this activation.
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BMP TYPE II RECEPTOR (BMPR-II) AND THE PATHOBIOLOGY OF PULMONARY HYPERTENSION
Primary pulmonary hypertension (PPH) is a rare disease characterized by vascular smooth muscle cell (VSMC) proliferation and obliteration of small pulmonary arteries leading to severe pulmonary hypertension and development of right ventricular failure. A germline mutation in the bone morphogenetic protein type II receptor (BMPR-II) gene has been identified in familial and sporadic PPH but its relationship to pathophysiology is still unknown. BMPs are growth factors of the TGF-? family that with no mutation in their receptor will inhibit vascular smooth muscle cell proliferation. Knocking out BMPR-II in transgenic mice is lethal at an early embryonic stage. We will therefore selectively and conditionally knock out BMPR-II in transgenic mice. The goals of this project are to study the effect of conditional K/O of BMPR-II and the resultant alteration in its functional activity on the proliferation and migration of vascular smooth muscle cells. This will enable us to unravel the molecular mechanisms behind the alteration in BMPR-II function and the pathogenesis and development of PPH. The picture shows the strategy used to conditionally Knock-Out (K/O) BMPR-II in mice. We use the Cre/LoxP system that allows homologous recombination between two loxP sites excising any unwanted gene. By crossing a transgenic mouse expressing Cre under the regulation of an arterial SMC promoter (SM22?-tTA+/Cre+) with a transgenic mouse with floxed BMPR-II (the functional domain of the receptor is flanked by two LoxP sites), we will create a transgenic mouse with conditionally deleted BMPR-II in vascular smooth muscle cells that can be temporally controlled by administering doxycycline.
THE ROLE OF STROMAL DERIVED FACTOR (SDF-1) AND STEM CELL MOBILIZATION AND DIFFERENTIATION IN PULMONARY HYPERTENSION
The chemokine SDF-1 and its receptor CXCR-4 play a critical role
in stem cell mobilization as well as vasculogenesis and angiogenic
remodeling.
The picture is a section of myocardium following transplant of CD44 stem cells from ROSA mice where the ßgal positive stem cells stain red, cells with MEF-2 (cardiac myocyte marker) stain green, and double positive differentiated stem cells are yellow. In keeping with these observations, an improvement in cardiac function (reduced end systolic and diastolic dimensions) was present in mice with elastase inhibitors with higher SDF-1 levels.
THE ROLE OF LC3 IN CELL MOTILITY AND GROWTH LC3, a microtubule associated protein, regulates messenger RNA (mRNA) transcription through interaction between an arginine-rich (RRR) domain in LC3 and a sequence (UUAUUUAU) in the mRNA from a variety of genes, including fibronectin and apolipoprotein D, implicated in vascular cell migration and in preventing cancerous transformation. The goals of this study are to establish the mechanisms regulating interaction between LC3 and fibronectin mRNA, as well as other mRNAs which may influence tumor growth and invasion. Panel a in the picture shows dividing fibrosarcoma (cancer) cells stained for tubulin. Panel b shows the same cells following transfection with LC3. The cells are elongated and grow significantly more slowly, i.e., are less tumorigenic. We are now studying how LC3 regulates translation of fibronectin mRNA and other genes in cancer and vascular cells.
LC-3 EXPRESSION DURING MOUSE DEVELOPMENT
Regulation of gene expression
occurs at many levels but perhaps one of the least understood
regulatory steps is post-transcriptional control of mRNA translation.
The image shows an embryonic day 12 mouse embryo which has been hybridized with an RNA probe to detect the expression of LC-3. Distinct tissues expressing LC-3 mRNA can be seen. |
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