Dont underestimate this one either. I think Cacaito will "see" this for what its worth.....vbg
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MODULATION OF PERICYTE PROLIFERATION
This application claims benefit under 35 USC 119 (e) of U. S. provisional application Ser. No. 60/250,542, filed December 1,2000.
FIELD OF THE INVENTION
The present invention relates generally to novel therapeutic uses associated with the modulation of perivascular cell proliferation, including the use of
BPI protein products, such as BPI-derived peptides, to enhance desirable pericyte proliferation. The present invention additionally relates to the use of inhibitors of BPI protein product-induced pericyte proliferation to inhibit deleterious pericyte proliferation.
BACKGROUND OF THE INVENTION
Microvascular perivascular cells ("pericytes") are defined by their location in vivo. The pericyte is a small ovoid shaped cell with many finger-like projections that parallel the capillary axis and partially surround an endothelial cell in a microvessel. Pericytes share a common basement membrane with the endothelial cell. They are elongated cells with the power of contraction that have been observed to have a variety of functional characteristics. Pericytes are widely distributed in the body and include mesangial cells (in the glomeruli of the kidney), Rouget cells, or mural cells (in the retina of the eye) [Hirschi & D'Amore, Cardiovasc Res 1996
Oct; 32 (4): 687-98.]. Some of the pericyte functional characteristics observed in vivo and in vitro are that they regulate endothelial cell proliferation and differentiation, contract in a manner that either exacerbates or reduces endothelial cell junctional inflammatory leakage, synthesize and secrete a wide variety of vasoactive autoregulating agonists, and synthesize and release structural constituents of the basement membrane and extracellular matrix. [Shepro et al, FASEB J 1993
Aug ; 7 (11) : 1031-8.] Pericytes have thus been implicated as playing a role in vasoconstriction as well as a role in capillary blood flow, in the formation of blood vessels, in the immune response (particularly in the central nervous system), and in the extrinsic coagulation pathway. In the kidney, the contractile properties of the mesangial cells and their synthesis of various factors and structural proteins help to regulate the function of the glomerulus. [Schlandoiff, 1987, FASEB J, 1: 272-81.]
Pericytes have been suggested to be derived from undifferentiated mesenchymal cells that are recruited by primordial endothelium and then differentiate into pericytes in microvessels or smooth muscle cells in large vessels. Pericytes are also pluripotential progenitor cells and have been shown to differentiate into a variety of different cell types, including osteoblasts, chondrocytes, adipocytes, phagocytes, fibroblasts, and smooth muscle cells. [Sims, 2000, Clin. Exp. Ped. Physiol., 27: 842846.] Pericytes behave in a manner similar to osteoblasts in vitro, by forming a mineralized extracellular matrix and expressing a number of genes that are also expressed by osteoblasts. These cells also form a well-defined matrix of bone, cartilage, and fibrous tissue in vivo. [Doherty and Canfield, Crit Rev Eukaryot Gene Expr 9 (1) : 1-17,1999; Hirschi et al., CardiovascRes Oct; 32 (4): 687-98, 1996.]
The pericyte has been implicated in a variety of pathologies including hypertension, atherosclerosis, complications of diabetes (both insulin-dependent and non-insulin-dependent), ovarian failure, multiple sclerosis, and tumor vascularization, as well as in normal aging.
Microvessels of spontaneously hypertensive rat brains have been shown to have a relatively higher number of pericytes and an increased ratio of pericytes to endothelial cells, numbers which increased following the onset of chronic hypertension in the rats. Pericyte contraction has been suggested to play a pivotal role in regulating the flow of blood within the brain microcirculation and perhaps in the etiology and inception of cerebrovascular disease. [Herman IM et al., Tissue Cell 1987; 19 (2): 197-206.]
Pericytes have been identified in the inner intima, the outer media, and in the vasa vasora of the adventitia of large, medium and small human arteries.
Recent studies have suggested that pericytes in the arteries may be responsible, at least in part, for mediating the vascular calcification commonly associated with atherosclerosis [Canfield et al., ZKardiol 2000 ; 89 Suppl 2: 20-7.] Myxomatous tissue is a characteristic component of human coronary artery lesions and is found more often in restenotic lesions. This tissue represents a bulky accumulation of stellate- shaped cells of unknown histogenesis that are embedded in a loose stroma and may be involved in an immune response. Stellate cells represented a heterogenous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Some workers have concluded that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells, possibly pericytes, which is activated to express some markers of antigen-presenting cells.
[Tjurmin et al., Arterioscler Thromb Vasc Biol 1999 Jan; 19 (1) : 83-97.]
In diabetes mellitus, pericytes may be involved in the development of angiopathy, retinopathy, polyneuropathy and nephropathy. Hyperglycemia may promote apoptosis and a loss of retinal capillary pericytes very early in the development of diabetic retinopathy [Ruggiero et al., 1997-Diabetes Metabolism 23: 30-42; Hirschi & D'Amore, Cardiovasc Res 1996 Oct; 32 (4): 687-98.]. It has been suggested that the sensitivity of retinal pericytes to degeneration in diabetes is due to their lesser ability to reproliferate (compared to, e. g., brain pericytes) in response to the metabolic injury of diabetes. [Wong et al. Diabetologia 1992 Sep; 35 (9): 818-27.]
There is also a difference in pericyte/endothelial cell ratio in the eye (one pericyte per endothelial cell) relative to other locations (neural 1: 2, peripheral 1: 20) [Speiser et al., 1968 Arch Ophthalmol 80 : 332-337; Orlidge and D'Amore, 1987, J Cell Biol.
105: 1455-1462; Sims et al., 1994 AnatHistol Embryol 23 : 232-238.]. Pericyte degeneration has also been observed to precede development of diabetic polyneuropathy and is associated with its severity. [Giannini et al., Ann Neurol 1995
Apr; 37 (4): 498-504.] Pericytes have been implicated in the thickening of the glomerular capillary basement membrane observed in diabetic retinopathy. [Keys et al., 2000, FASEB J, 14: 439-47.] During diabetes, mesangial cells show increased synthesis of various extracellular matrix (ECM) components. This increased synthesis of ECM is also accompanied by a decreased degradation of ECM. The major enzymes responsible for ECM degradation are a large group of enzymes collectively known as matrix metalloproteinases (MMPs). The mesangial cell and its pericellular matrix have a very active plasminogen cascade that can liberate plasmin locally to mediate matrix degradation both directly and indirectly, by activating the MMPs. Thus, it is possible that degeneration of mesangial cells mediates the decrease in ECM degradation seen in diabetic nephropathy [McLennan et al., Cell Mol Biol (Noisy-le-gra71t) 1999 Feb; 45 (1) : 123-35.]
Pericyte degeneration has also been observed in animal models of ovarian failure. Ovaries of adult female rats treated with testosterone propionate and anovulatory ovaries of middle-aged female rats both exhibited regression of vascular pericytes, T-cells and dendritic cells within the interstitial glands. It appears that the function of ovarian steroidogenic cells may be regulated by mesenchymal cells.
[Bukovskya et al, Steroids 2000 Apr; 65 (4): 190-205.]
Changes in pericyte population have also been observed during aging.
There appears to be regional variation in the age-associated changes in the brain microvasculature. In the frontal cortex and hippocampus, there appears to be an increase in basement membrane with increasing age, accompanied by increased pericyte mitochondrial size. In the frontal cortex, there is increased capillary lumen area but in the hippocampus there is decreased capillary lumen area in the hippocampus. [Hicks P, Neurobiol Aging 1983 Spring; 4 (1) : 69-75.] The brains of aging rats have been found to have increased astrocyte and pericyte populations in the parietal cortex. [Peinado MA et al., Microsc Res Tech 1998 Oct 1: 43 (1) : 34-42.] At the ultrastructural level different anomalies of the cerebral microvasculature are encountered. These aberrations can either be attributed to degeneration processes or to the perivascular deposition of, e. g., collagen fibrils and other proteinaceous debris.
[de Jong Neurobiol. Aging 1992 Jan-Feb ; 13 (1) : 73-81.]
Of interest is the disclosure in Hu et al., Br. J. Exp. Pathol. 1989 Apr; 70 (2): 113-24 that intermittent treatment of mice with heparin has been shown to reduce the right ventricular hypertrophy caused by hypoxia; administration of heparin reduced the proportion of arteries that became muscularized, particularly at the alveolar duct level where the pericyte is the precursor smooth muscle cell. See also
Khoury et al., Am. J. Physiol. Lung Cell Mol. Physiol., 279: L252-L261,2000, a report that heparin-like molecules inhibit pulmonary vascular pericyte proliferation in vitro.
Thus, there exists a need for an agents that modulate pericyte proliferation. hi conditions where proliferation of pericytes is desirable, there is a need for agents that allow or enhance such proliferation to be enhanced. In conditions where proliferation of pericytes is deleterious, there is a need for agents that inhibit such proliferation.
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254 : 11000 (1979)] orE. coli affinity chromatography [Weiss, et al., Blood, 69 : 652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in U. S. Patent No. 5,198,541 and Figure 1 of Gray et al., J. Biol.
Chem., 264 : 9505 (1989), incorporated herein by reference. The Gray et al. nucleic acid and amino acid sequence are set out in SEQ ID NOS: 1 and 2 hereto. U. S. Patent
No. 5,198,541 discloses recombinant genes encoding and methods for expression of
BPI proteins, including BPI holoprotein and fragments of BPI. Recombinant human
BPI holoprotein has also been produced in which valine at position 151 is specified by GTG rather than GTC, residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of-3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein.
[Ooi et al., J Bio. Chem., 262 : 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp.
Med., 174 : 649 (1991).] An N-terminal BPI fragment of, approximately 23 kD, referred to as"rBPI23,"has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. [Gazzano-Santoro et al., Itifect. Iinmun. 60 : 4754-4761 (1992).] An N-terminal analog designated rBPI1 (also referred to as rBPI (1-193) aval32) has been described in U. S. Patent No. 5,420,019.
The bactericidal effect of BPI was originally reported to be highly specific to gram-negative species, e. g., in Elsbach and Weiss, Inflammation : Basic
Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd.
(1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. In susceptible gram-negative bacteria,
BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death.
[Elsbach and Weiss (1992), supra]. LPS has been referred to as"endotoxin"because of the potent inflammatory response that it stimulates, i. e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock.
BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
BPI protein products have a wide variety of beneficial activities. BPI protein products are bactericidal for gram-negative bacteria, as described in U. S.
Patent Nos. 5,198,541,5,641,874,5,948,408,5,980,897 and 5,523,288. International
Publication No. WO 94/20130 proposes methods for treating subjects suffering from an infection (e. g. gastrointestinal) with a species from the gram-negative bacterial genus Helicobacter with BPI protein products. BPI protein products also enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U. S. Patent Nos. 5,948,408,5,980,897 and 5,523,288 and International Publication
Nos. WO 89/01486 (PCT/US99/02700) and WO 95/08344 (PCT/US94/11255). BPI protein products are also bactericidal for gram-positive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U. S. Patent Nos. 5,578,572 and 5,783,561 and International Publication
No. WO 95/19180 (PCT/US95/00656). BPI protein products exhibit antifungal activity, and enhance the activity of other antifimgal agents, as described in U. S.
Patent No. 5,627,153 and International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for BPI-derived peptides in U. S. Patent
No. 5,858,974, which is in turn a continuation-in-part of U. S. Application Serial No.
08/504,841 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), as well as in U. S. Patent
Nos. 5,733,872,5,763,567,5,652,332,5,856,438 and corresponding International
Publication Nos. WO 94/20532 (PCT/US/94/02465) and WO 95/19372 (PCT/US94/10427). BPI protein products exhibit anti-protozoan activity, as described in U. S. Patent Nos. 5,646,114 and 6,013,629 and International Publication
No. WO 96/01647 (PCT/US95/08624). BPI protein products exhibit anti-chlamydial activity, as described in co-owned U. S. Patent No. 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPI protein products exhibit anti-mycobacterial activity, as described in co-owned, co-pending U. S. Application Serial No. 08/626, 646, which is in turn a continuation of U. S. Application Serial No. 08/285, 803, which is in turn a continuation-in-part of U. S. Application Serial No. 08/031,145 and corresponding
International Publication No. WO 94/20129 (PCT/US94/02463).
The effects of BPI protein products in humans with endotoxin in circulation, including effects on TNF, IL-6 and endotoxin are described in U. S. Patent
Nos. 5,643,875,5,753,620 and 5,952,302 and corresponding International Publication
No. WO 95/19784 (PCT/US95/01151).
BPI protein products are also useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in U. S. Patent Nos.
5,888,977 and 5,990,086 and International Publication No. W097/42966 (PCT/US97/08016), hemorrhage due to trauma in humans, (as described in U. S.
Patent Nos. 5,756,464 and 5,945,399, U. S. Application Serial No. 08/862,785 and corresponding International Publication No. WO 97/44056 (PCT/US97/08941), burn injury (as described in U. S. Patent No. 5,494,896 and corresponding International
Publication No. WO 96/30037 (PCT/US96/02349)) ischemia/reperfusion injury (as described in U. S. Patent No. 5,578,568), and depressed RES/liver resection (as described in co-owned, co-pending U. S. Application Serial No. 08/582,230 which is in turn a continuation of U. S. Application Serial No. 08/318,357, which is in turn a continuation-in-part of U. S. Application Serial No. 08/132,510, and corresponding
International Publication No. WO 95/10297 (PCT/US94/11404).
BPI protein products also neutralize the anticoagulant activity of exogenous heparin, as described in U. S. Patent No. 5,348,942, neutralize heparin in vitro as described in U. S. Patent No. 5,854,214, and are useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis, for inhibiting endothelial cell proliferation, and for inhibiting angiogenesis and for treating angiogenesis-associated disorders including malignant tumors, ocular retinopathy and endometriosis, as described in U. S. Patent Nos. 5,639,727,5,807,818 and 5,837,678 and International Publication No. WO 94/20128 (PCT/US94/02401).
BPI protein products are also useful in antithrombotic methods, as described in U. S. Patent Nos. 5,741,779 and 5,935,930 and corresponding
International Publication No. WO 97/42967 (PCT/US7/08017).
SUMMARY OF THE INVENTION
The present invention provides novel therapeutic methods of modulating proliferation of pericytes, including mural cells of the retina and mesangial cells of the kidney. In conditions where pericyte proliferation is desirable, the invention provides methods of using a therapeutically effective amount of a BPI protein product to enhance pericyte proliferation. Such conditions include complications of diabetes (both insulin-dependent and non-insulin-dependent), other diseases associated with the presence of autoantibodies to pericytes, age-related macular degeneration (ARMD), ovarian failure, multiple sclerosis, Alzheimer's disease, traumatic brain injury or other conditions involving perturbation of the bloodbrain-barrier, partial seizures and placental development in pregnancy. In particular, sequelae of diabetes mellitus include diabetic retinopathy, diabetic polyneuropathy, diabetic nephropathy, skeletal muscle degeneration after pericyte degeneration, or other organ complications.
In conditions where pericyte proliferation is deleterious, the invention provides methods of inhibiting pericyte proliferation using therapeutically effective amounts of inhibitors capable of inhibiting the type of proliferation induced by BPI protein product, e. g., antibodies or other agents capable of binding to BPI protein product or otherwise inhibiting interaction with its receptor or ligand, or antagonists of the pericyte receptor that recognizes BPI protein products. Such conditions include hypertension, vascular disease, atherosclerosis, including formation of vascular calcifications and atherosclerotic plaques, restenosis, acute respiratory distress syndrome (ARDS), endometriosis or adenomyosis, and normal aging. In particular, sequelae of hypertension, atherosclerosis and other vascular diseases include cerebrovascular ischemia or stroke, coronary artery disease and myocardial ischemia or infarction, peripheral vascular disease, Raynaud's syndrome, early occlusion of peripheral arteries or vascular remodeling associated with pulmonary hypertension.
Uses of BPI protein products according to the invention are specifically contemplated in mammals, particularly humans, for prophylactic or therapeutic treatment of disease states or conditions mediated or exacerbated by pericyte proliferation or degeneration.
Studies suggest that undifferentiated mesenchymal cells or fibroblasts transfonn into capillary pericytes which in turn transform into vascular smooth muscle and other related cells such as adipocytes, osteoblasts and phagocytes [Rhodin & Fujita 1989 Submicrosc Cytol Pathol 21 : 1-34; Doherty & Canfield 1999 Crit Rev
Euk Gene Exp 9 (1) : 1-17]. Thus, further provided are methods for enhancing the production or formation of osteoblasts, chondrocytes, adipocytes, phagocytes, fibroblasts, and smooth muscle cells from pericytes and thus repairing or replacing damaged tissue, e. g. in wounds. In addition to enhancing proliferation of such cells per se, BPI protein products, including BPI-derived peptides, may be expected to enhance differentiation of pericytes into certain cell types, or to enhance proliferation of the finally differentiated cell types. Such methods are expected to be useful in a variety of conditions including enhancing wound healing, treatment of bone fractures or bone degenerative disorders.
Exemplary BPI protein products include recombinantly-produced Nterminal analogs or fragments of BPI, especially those having a molecular weight of approximately between 20 to 25 kD such as rBPkl, BPI23, rBPI (10-193) C132A (also designated rBPI (10-1 93) alal32), dimerie fonns of these N-terminal polypeptides (e. g., rBPI2 dimer), or BPI-derived peptides. Exemplary BPI-derived peptides include peptides derived from BPI domain II, such as XMP. 679 ([SEQ ID NO: 3]), the structure and activity of which are described in co-owned U. S. Serial No. 09/602,811 filed June 23,2000, which is a continuation-in-part of U. S. Serial No. 09/344,219 filed June 25,1999, each incorporated herein by reference.
It is contemplated that the administration of a BPI protein product may be accompanied by the concurrent administration of other therapeutic agents, such as growth factors that enhance proliferation of pericytes or inhibitors of such growth factors, depending on the condition.
The invention also provides methods of screening for other BPI protein products that enhance pericyte proliferation. Such methods would comprise steps of, e. g., detecting or measuring growth or proliferation of pericytes in the presence and optionally the absence of a BPI protein product. Optionally the screening methods involve a further step of testing selected candidate compounds in animal models of pericyte proliferation wherein the proliferation results in desirable effects. The screening methods may also involve a further step of testing selected candidate compounds for ability to inhibit endothelial cell proliferation or angiogenesis (vasculogeriesis). BPI protein products, including BPI-derived peptides, can be screened for proliferation-promoting activity using these methods. In addition, the rational design of molecules that function like pericyte proliferation-enhancing BPI protein products is contemplated. For example, peptides or other organic molecules may be synthesized that mimic the structure and function of BPI protein products with the desired pericyte proliferation-enhancing activity.
The invention also provides methods of screening for a candidate enhancer of pericyte proliferation including (a) measuring proliferation of pericytes in the presence and absence of a test compound, (b) measuring proliferation in the presence of the test compound and a BPI protein product (preferably at a concentration effective to enhance pericyte proliferation), and identifying the test compound as a candidate enhancer of pericyte proliferation when pericyte proliferation is increased in step (a) but not further increased in step (b), or when the increase in pericyte proliferation measured in step (a) is about the same as or less than the increase in pericyte proliferation measured in step (b).
The invention further provides methods of screening organic or inorganic compounds for the ability to inhibit proliferation induced by BPI protein products. Suitable standards for use in such screening assays include any BPI protein product that enhances pericyte proliferation, e. g., rBPI21 or XMP. 679. Such methods would comprise steps of, e. g., contacting pericytes with a BPI protein product and a candidate compound, and detecting or measuring growth or proliferation of the cells in the presence and absence of the test compound. A test compound is identified as a candidate inhibitor of BPI-induced proliferation when proliferation of the pericytes induced by the BPI protein product is reduced in the presence of the test compound.
Optionally, as a control, the growth or proliferation of the pericytes is also measured in the presence and absence of the test compound alone (in the absence of BPI protein product). The screening methods may involve a further step of testing selected candidate compounds in animal models of pericyte proliferation wherein the proliferation results in deleterious effects. These methods according to the invention can be used for high throughput screening of libraries of molecules, such as inorganic or organic compounds (including bacterial, fungal, mammalian, insect or plant products, peptides, peptidomimetics and/or organomimetics).
Further provided are compounds identified by the screening methods described herein as well as methods of treatment using these compounds, to modulate (i. e., allow or enhance or inhibit) pericyte proliferation.
Yet another aspect of the invention provides methods for enhancing epithelial cell proliferation, particularly in the retina, by administering an effective amount of a BPI protein product, and methods for screening for compounds, either
BPI protein products, BPI protein product mimetics, or small molecules, that allow or enhance epithelial cell proliferation, particularly in the retina (e. g. retinitis pigmentosa or choroidal neovascularisation seen in age-related macular degeneration [Campochiaro et al., Molecular Vision, 1999,5: 34 (1999)]), in wounds or bone fractures. Corresponding use of BPI protein products in preparation of a medicament for the treatment of diseases involving epithelial cell degeneration or diseases that would benefit from epithelial cell (particularly retinal epithelial cell) proliferation is also contemplated. In addition, this aspect of the invention includes corresponding methods of screening for other BPI protein products for the ability to enhance epithelial cell proliferation, particularly retinal epithelial cell proliferation, and methods of screening organic or inorganic compounds for the ability to inhibit epithelial cell proliferation induced by BPI protein products. |
