Gene therapy in coronary angioplasty and bypass

Details Gene therapy in coronary angioplasty and bypass Gene therapy in coronary angioplasty and bypass

1997-06-30

2002-01-01

Nguyen, Dave T. (Department: 1633)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C514S04400A, C435S320100, C435S455000

Reexamination Certificate

active

06335010

ABSTRACT:

BACKGROUND OF THE INVENTION
Vascular endothelial cells (ECs), located at the interface between the blood and the vessel wall, are exposed to the mechanical environment resulting from hemodynamic activities. Fluid shear stress is the hemodynamic force acting tangentially on the vascular ECs and it plays significant roles in atherogenesis and reperfusion injury. Many genes encoding for growth factors (e.g., platelet derived growth factor and transforming growth factor &bgr;-1), vasoconstrictors (e.g., endothelin-1), vasodilators (e.g., nitric oxide synthase), adhesion molecules (e.g., intercellular adhesion molecule-1, ICAM-1), and monocyte chemoattractants (e.g., monocyte chemotactic protein-1, MCP-I) in the ECs are modulated by fluid shearing (see7 for review). The induction of some, and perhaps the majority, of these inflammation-related genes is rapid and transient, and de novo protein synthesis is not required. These are the characteristics of the expression of immediate early (IE) genes induced by mitotic factors and agonists. The phorbol ester 12-0-tetradecanoyl-13-phorbol-acetate (TPA) responsive element, TRE, mediates the expression of many IE genes through its interaction with the transcription factor AP-1, a Jun/Fos heterodimer or a Jun/Jun homodimer (1). A divergent TRE in the 5′ promoter region of the MCP-1 gene has been found by the present inventors to be responsible for its mechanical inducibility (41). Consensus TRE with the sequence TGACTACA is sufficient for the shear-induced reporter activities in different types of cells. The applied fluid shearing probably exerts its actions on the cellular membrane to initiate biochemical signals which can then be transduced through the cytoplasm into the nucleus where the activation of AP-I/TRE occurs.
A major process through which extracellular stimuli can be transmitted into cells involves the membrane-associated p21
ras
and its downstream cytoplasmic kinase pathways, especially the members in the mitogen-activated protein kinases (MAPK) family. p21
ras
is a small GTPase molecule that plays a key role in the signal transduction pathways of cellular responses to stimuli by mitogins, cytokines, environmental stresses, and UV irradiation. p21
ras
cycles between an active GTP-bound state and an inactive GDP-bound state, thereby functioning as a molecular switch in response to extracellular stimuli in the control of normal and transformed cell growth. Activated p21
ras
triggers two protein kinases, Raf-1 and MEK (MAPK kinase) kinase (MEKK) which activate the downstream MAPKs, including c-Jun NH2-terminal kinases (JNK) and extracellular signal-regulated kinases ERK (11,32). Raf-1 activates ERK but not JNK, whereas MEKK mediates preferentially JNK over ERK (32,48). In different types of cells in response to UV irradiation, Ha-Ras expression, and osmotic shock, JNK kinase (JNKK) activates JNK by phosphorylating the Thr-Pro-Tyr phosphorylation sites, and the activated JNIK binds to c-Jun to specifically phosphorylate the -63 and -73 amino acids at the N-terminal (10, 29). In response to Ha-Ras expression, serum growth factor, or phorbol ester TPA stimulation, MEK activates ERK which in turn phosphorylates the transcription factor p62 ternary complex factor (p62TCF), leading to the activation of c-Fos (5, 18, 30, 37). In R.EF-52 fibroblasts, the activation of AP-I/TRE by these stimuli is mediated through ERK (16). It is not known where and how mechanical stimuli are transduced to biochemical signals. p21
ras
is a membrane-associated protein and its activation of the downstream Raf-1 and MEKK is through direct interactions on the membrane (27, 28, 36, 38, 43). Wang et al. (47) suggest that the integrins on the basal membrane constitute a mechano-receptor and that stress fibers are necessary to transmit the applied forces. Similarly, Davies et al. (8) suggest that focal adhesion complex at the abluminal endothelial membrane are mechanically responsive elements coupled to the cytoskeleton.
Ras can activate both ERK and JNK pathways (27, 32). The signaling in response to growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF) is mediated through both the Ras/ERK and Ras/JNK pathways in PCI2, MRCS, and HeLa cells (32, 33). In contrast, inflammation—related cytokines (e.g., TNF and IL-1), environment stresses (e.g., osmoic pressure), and UV irradiation selectively activate the Ras/JNK, but not the ERK, pathway (17, 19, 26, 32, 42, 44). Mechanical shearing is a form of force borne by vascular ECs and many other cell types, such as the osteoblasts, under physiological conditions. The application of such physiological forces on static cells cultured in the flow chambers provides a sudden change of hemodynamic environment. This in vitro system mimics the pathophysiological changes during reperfusion after flow stoppage. Fluid shearing of vascular EC caused the activation of JNK by more than 10-fold and the activation of ERK by a much lesser magnitude (1.8 fold) and shorter duration (FIG.
3
). Morooka et al. demonstrated that reperfusion of ischemic kidney induced a rapid activation of JNK (35). Bogoyevitch et al. reported that reperfusion of rat heart induced a 10- to 50-fold activation of JNK, but not ERK (2).
SUMMARY OF THE INVENTION
While several laboratories have shown that fluid shearing induces a variety of transient responses in the endothelial cytoplasm and nucleus, there is little, if any, knowledge on how ECs transduce the mechanical stimuli into biochemical signals which ultimately activate the downstream gene expression. The present inventors have found that fluid shearing, a physiological form of hemodynamic forces, activates p21
ras
in ECs in a rapid and transient manner, and that it is followed by the activation of the MEKK-JNK pathway, leading to the induction of the AP-1/TRE mediated gene expression in the nucleus. In contrast, the ERK pathway is weakly activated by fluid shearing and is not essential for the shear-induced activation of AP-I/TRE. These results indicate that hemodynamic forces share the same signaling pathways as a variety of stimuli, including osmotic pressure, chemical stress, and UV irradiation, in activating the promoter regions of IE genes.
Percutaneous transluminal coronary angioplasty (PTCA) causes restenosis mainly from proliferation of vascular smooth muscle cells (VSMC) gene expression and proliferation. Recognizing this, an effective gene therapy method for the reduction of the high incidence of restenosis after angioplasty is provided in the present invention.
The present invention demonstrates that p21
ras
plays critical roles in the responses of vascular ECs to fluid shearing. First, the guanine nucleotide exchange on Ras, i.e., the conversion of Ras-GDP to Ras-GTP, was promoted by fluid shearing. Second, the dominant negative mutant of p21
ras
RasN17, inhibited the shear-induced signal transduction pathway including JNK and its downstream c-Jun transcriptional activity. Third, RasNI7 also abrogated the reporter activities of 4×TRE-Luc and MCP1-Luc-540, two chimeric constructs whose induction by fluid shearing is mediated by AP-1/TRE (41). The present inventors establish that the mechanical-biochemical transmitting process occurs at least in part on the cellular membrane. The present inventors propose that the signals initiated by fluid shearing originate from the abluminal side of ECs. In contrast, the burst production of nitric oxide is dependent upon the activation of G proteins (25) which are located at the luminal surface.
Besides the activation of ERK, the inventors also demonstrate that the blockage of the p21
ras
-RAF-ERK pathway by Raf.301, K7 I R, or K52R did not affect the TRE-mediated reporter activities in response to fluid shearing (FIG.
6
). In contrast, the blockade of the p21
ras
-MEKK-JNK pathway by either MEKK(K-M) or JNK(K-R) significantly attenuated the shear induced reporter activities (FIG.
5
). MEKK(K.M) and JN.K(K-R), which were mutated at the ATP binding sites with the conserved Lys replaced by either a Met or an Arg,

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