Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-11-06
2001-06-19
Clark, Deborah J. R. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S009000, C800S013000, C800S014000, C800S018000, C424S009200, C424S569000, C435S001100, C435S320100, C600S036000
Reexamination Certificate
active
06248933
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to vascular graft stenosis. About 400,000 patients undergo aorta-coronary artery bypass graft surgery annually in the United States. Unfortunately, the grafts often require subsequent intervention because of the development of hemodynamically significant lesions on the lumenal side of the intima, which is the innermost layer of the artery. The occlusion in the artery resulting from the lesion is known as a stenosis. Within two years of the surgery, up to 30% of vein grafts develop stenosis. Ten years after surgery, stenosis has occurred in approximately 50% of vein grafts.
The factors involved in causing graft stenosis are largely unknown, and there is a lack of an adequate animal model for characterizing graft stenosis.
SUMMARY OF THE INVENTION
The invention features a rodent model, e.g., a mouse model, of vascular graft stenosis in which the stenosis develops rapidly and closely mimics the development of vascular graft stenosis in humans.
The invention provides a surgically modified mouse in which a blood vessel, e.g, an artery or vein, has been transplanted into, i.e., spliced into, an endogenous artery. Preferably, a segment of a histocompatible, e.g., autogenous, vessel is used. Graft stenosis is detected within 30 days, preferably within 20 days, more preferably within 10 days, and most preferably within 7 days of transplantation. The transplanted vessel is preferably 10% occluded, more preferably 25% occluded, even more preferably 50% occluded, and most preferably 75-100% occluded.
By “autogenous” is meant that the donor vessel is obtained from the recipient animal.
The donor blood vessel can be any vein or artery, e.g., the jugular or saphenous, vein. The endogenous artery (i.e., the tissue serving as the recipient of the grafted tissue) can be any suitable artery, e.g., the carotid, femoral or aortic artery. Preferably, the transplanted artery is the carotid artery.
The mouse of the invention may be made using mouse strains harboring one or more mutations, e.g., gene deletions, e.g., in genes encoding platelet derived growth factor (PDGF) or plasminogen; OP/OP homozygous mice (a spontaneous mutant strain in which macrophage colony stimulation factor (MCSF) is absent with consequent marked reduction in macrophage production); Beige (Bg) mice (a spontaneous mutant strain in which natural killer cell function is deficient); or mice lacking the CC chemokine receptor 1 gene, the cystathione beta-synthase (CBS) gene, or the apoE gene.
The invention also provides an in vivo screening assay to determine whether a compound reduces vein graft stenosis. The method involves the steps of providing a first and second mouse, each of which contains a histocompatible autogenously transplanted vein; contacting the first mouse with a candidate compound and the second mouse with a vehicle alone; and comparing the degree of vein graft stenosis in the grafts of each mouse within 20 days after transplantation. Alternatively, the second mouse may simply be maintained in the absence of the candidate compound. The candidate compound is preferably a compound which inhibits vascular smooth muscle cell proliferation or inflammation, e.g., macrophage activity, either directly or indirectly. Evidence of vein graft stenosis can be detected in the vein grafts as early as 7 days after transplantation. The vein grafts may be removed from the mice at any point during the assay and preserved, e.g., frozen for subsequent analysis. The candidate compound or vehicle may be administered locally to each mouse so as to contact the vein graft directly; alternatively, the compound or vehicle may be delivered systemically so the vein graft is contacted with compound via the circulatory system. A lesser degree of stenosis in the graft of the first mouse compared to the graft of the second mouse is an indication that the candidate compound reduces vein graft stenosis. In the absence of contact with the candidate compound, vein graft stenosis in the second mouse is at least 100% greater, more preferably at least 150% greater, and most preferably 300% greater, than that of the first mouse.
Also included in the invention is an in vivo screening assay to determine whether a gene encodes a gene product that increases or decreases vein graft stenosis. The method includes the steps of providing a first mouse and second mouse: the first mouse has a mutation in a gene to be tested and the second mouse has at least one functional copy of the gene. The first mouse further includes a vein autogenously transplanted into an artery of the first mouse, and the second mouse further includes a second vein autogenously transplanted into an artery of the second mouse. The degree of vein graft stenosis in the transplanted veins in the first mouse is compared to that of the second mouse. An increase or decrease in vein graft stenosis in the first mouse compared to said second mouse indicates that the gene product increases or decreases, respectively, vein graft stenosis.
Compounds that inhibit the expression or function of genes or gene products that increase vein graft stenosis are administered to patients to prevent or decrease the development of stenosis associated with vascular injury, e.g., vein graft stenosis.
Degree of vein graft stenosis may be determined by physically measuring neointimal thickening, or by characterizing and enumerating cells involved in the rejection process, e.g., smooth muscle cells, leukocytes, macrophages, and subpopulations of lymphocytes (CD4
+
and CD8
+
cells). The intima is defined as the region between the lumen and the internal elastic lamina of the artery; the media is defined as the region between the internal and external elastic laminae; and the adventitia is defined as the region outside the external elastic lamina of the artery. Functional activity, e.g., production of cytokines, of the infiltrating cells can also be measured to determine the degree of graft stenosis. Replicating cells which contribute to the formation of the graft stenosis may be detected by expression of proliferating cell nuclear antigen (PCNA).
The invention also includes a method of making the surgically modified mouse. The method can be carried out as follows: a mouse is provided; a blood vessel, preferably a vein, which is not part of a solid organ, or which has been removed from a solid organ (“the transplant vessel”) is surgically removed from the mouse, and the vessel is surgically attached (spliced) to an endogenous artery of either the first mouse or a second mouse, such that the functional integrity of the blood vessel is restored, i.e., pulsation of the vessel and blood flow through the artery is evident.
The invention also includes a method of inhibiting vein graft stenosis in a recipient mammal by administering to the recipient a compound which inhibits vascular smooth muscle cell proliferation, or by administering to the recipient a compound which inhibits macrophage activity. Alternatively, the method may be carried out by contacting a donor blood vessel or organ either in vivo or ex vivo with a compound which inhibits macrophage activity, and if the contacting step was carried out ex vivo, then transplanting the organ into the recipient mammal. Preferably, the compound inhibits expression or activity of macrophage colony stimulating factor or galactose/N-acetylgalactosamine macrophage lectin. The compound may also inhibit expression of the oligosaccharide, galactose/N-acetylgalactosamine (which binds to galactose/N-acetylgalactosamine macrophage lectin), in the donor blood vessel or organ. Antibodies which bind to either the macrophage lectin or its oligosaccharide ligand may be used to block binding of lectin to its oligosaccharide ligand, thereby inhibiting macrophage activity at the graft site. The compound may inhibit macrophage colonization of the donor organ or blood vessel, for example, by inhibiting expression or activity of a macrophage or monocyte chemoattractant protein. An antibody which binds to a macrophage or monocyte chemoattracta
Haber Carol
Haber Edgar
Shi Chengwei
Sibinga Nicholas E. S.
Beattie Ingrid A.
Clark Deborah J. R.
Haber Carol
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
President and Fellows of Harvard College
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