Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-14
2002-06-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024310
Reexamination Certificate
active
06399311
ABSTRACT:
BACKGROUND OF THE INVENTION
Atherosclerosis, a disease of arteries that is responsible for most cardiovascular-related morbidity and mortality, develops in predictable regions of the arterial tree that correlate with complex patterns of blood flow. Although many atherosclerotic lesions are mild and cause little harm, those that progressively obstruct the passage of blood may reduce oxygen delivery to levels below the needs of the tissue (e.g., angina pain when coronary arteries are affected) or precipitate an acute ischemia (e.g., heart attack, stroke) when blood clots form on a destabilized lesion surface. Another catastrophic consequence of advanced lesions is the weakening of the artery wall, leading to pressure-induced ballooning (aneurysm) and potential rupture. It has long been recognized that hemodynamics determines the location of lesions. Local vessel geometry (e.g., arterial branching and curvatures), and constraint of vessel motion by surrounding tissues (e.g., coronary arteries) lead to flow instabilities and separations that correlate with sites of lesion development.
The one-cell thick layer at the interface between flowing blood and the artery wall is called the endothelium. Two decades of intense research have shown that the endothelium, rather than being a simple passive barrier, is instead both i) a multifunctional effector of systemic and vessel wall biology, and ii) an exquisitely sensitive responder to the local environment. The endothelium is directly exposed to the hemodynamic shear stresses associated with all of the different flow characteristics found in the circulation.
Endothelial cell responses to the hemodynamic environment are frequently heterogeneous. Prominent examples are the expression of VCAM-protein in vivo (Walpola et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:2-10) and in vitro (Ohtsuka et al., 1993, Biochem. Biophys. Res. Comm. 193:303-310), VCAM-1 mRNA expression in vivo (McKinsey et al., 1995, FASEB J. A343), ICAM-1 protein expression in vivo (Walpola et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:2-10) and in vitro (Nagel et al., 1994, J. Clin. Invest. 94:885-891), elevation of intracellular calcium ([Ca
2+
]
I
) measured in vitro (Geiger et al., 1992, Am. J. Physiol. 262:C1411-C1417; Shen et al., 1992, Am. J. Physiol. 262:C384-C390) and in vivo (Falcone et al., 1993, Am. J. Physiol. 264:H653-659), induction of synthesis and nuclear localization of c-fos in vitro (Ranjan et al., 1993, Biochem. Biophys. Res. Comm. 196:79-84), expression of major histocompatibility complex (MHC) antigens in vitro (Martin-Mondiere et al., 1989, ASAIO Trans. 35:288-290), inhibition of endothelial cell division in vitro (Ziegler et al., 1994, Arterioscler. Thromb. 14:636-643), and re-localization of the Golgi apparatus and microtubule organizing center (MTOC) in vitro (Coan et al., 1993, J. Cell Sci. 104:1145-1153). In each of these cases, high levels of response in one cell or a group of cells are accompanied by absent or diminished responses in adjacent cells of the same endothelial monolayer despite exposure to a substantially identical bulk flow field in vitro, or location in a predicted uniform hemodynamic environment in vivo.
In vitro, nominal flow characteristics are defined by the geometry of the experimental system (e.g., flow tube, parallel plate, cone and plate, etc.). The average wall shear stress and shear stress gradient values can be accurately estimated or directly measured (Dewey et al., 1981, J. Biomech. Eng. 103:177-188; Davies et al., 1986, Proc. Nat. Acad. Sci. USA 83:2114-2118; Olesen et al., 1988, Nature 331:168-170; DePaola et al., 1992, Arterioscler. Thromb. 12:1254-1257). Although the flow characteristics are more complex in vivo, average shear stress values can be estimated from vessel geometry and flow rates (Zarins et al., 1983, Circ. Res. 53:502-514). Such measurements demonstrate that although all of the cells in a given region of the monolayer are estimated to be subject to very similar shear stresses calculated from bulk flow characteristics, there are substantial cell-to-cell differences in acute and chronic responses to flow. If, as a significant number of experiments demonstrate, the responses are related to hemodynamic forces, it has not been determined what accounts for the heterogeneous responses.
In vitro flow chamber models of disturbed and undisturbed blood flow as described herein have recently been used to identify regionally defined differential expression of connexin43, and early response genes (DePaola et al., 1999, Proc. Natl. Acad. Sci. USA, 96:3154-3159; Nagel et al., Arterioscler. Thromb. Vasc. Biol., In press.) In regional differential gene expression studies during flow in vitro, endothelial cells are typically isolated by scraping the regions of interest. If enough cells are recovered, quantitative estimates of regional up- or down-regulation of gene expression (i.e., an average from all of the cells isolated from a particular location) can be made by northern blot analyses using specific nucleic acid probes for each gene of interest.
A useful alternative for analyzing the smaller numbers of cells typically present in defined hemodynamic regions is differential-display PCR (ddPCR; Liang et al., 1992, Science, 257:967-971), which uses reverse-transcription PCR (RT-PCR) to amplify all expressing genes in the cell population. This allows evaluation of differential expression of multiple genes when PCR products derived from cells in different hemodynamic regions are displayed together (e.g., as in Topper et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10417-10422). Although ddPCR can be imprecise for quantitation of expression, this method has been used to identify differentially-expressed genes (e.g., Topper et al., 1997, Proc. Natl. Acad. Sci. USA, 94:9314-9319; Topper et al., 1997, J. Clin. Invest., 99:2942-2949; Topper et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 9314-9319).
Although regional differential gene expression studies as described above are of value, the hemodynamic effects which modulate endothelial gene expression through spatial and temporal shear-stress relationships are ultimately defined locally at the surface of individual endothelial cells. Surface topographies, and consequently the magnitudes and gradients of shear-stresses, vary considerably from cell to cell (Barbee et al., 1995, Am. J. Physiol., 268:H1765-H1772). Differences in hemodynamic signaling and gene expression that have been observed from region to region and from cell to cell in endothelium (both in culture and in tissues) are likely to arise from microscopic topographic differences at the interface of the fluid and the cell surface. Examples of such heterogeneity include variable expression of endothelial vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 proteins from cell to cell in vivo (Walpola et al., 1995, Arterioscler. Thromb. Vasc. Biol., 15:2-10 ; Nakashima et al., 1998, Arterioscler. Thromb. Vasc. Biol., 18:842-851) and in vitro (Nagel et al., 1994, J. Clin. Invest., 94:885-91), the elevation of intracellular calcium measured in vitro (Geiger et al., 1992, Am. J. Physiol., 262:C1411-1417; Shen et al., 1992, Am. J. Physiol., 262:C384-C390) and in vivo (Falcone et al., 1993 Am. J. Physiol., 264:H653-H659), the induction of synthesis and nuclear localization of c-Fos in vitro (Ranjan et al., 1993, Biochem, Biophys. Res. Commun., 196:79-84), and the expression of major histocompatibility complex antigens in vitro (Martin-Mondiere et al., 1989, ASAIO Trans., 35:288-290). In all of these studies, highly variable responses were observed in adjacent cells of the same endothelial monolayer exposed to a nominally identical flow field.
Two inter-related mechanisms may explain cell-to-cell differences in acute and chronic responses to hydrodynamic forces. First, there may be differential expression or sensitivity of mechano-sensing or transduction systems in the endothelial cells, ranging from a complete absence to super-sensitivity. Second, the shear stresses and shear stress g
Davies Peter F.
Polacek Denise C.
Akin Gump Strauss Hauer & Feld L.L.P.
Ketter James
The Trustees of the University of Pennsylvania
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