Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-11
2002-07-16
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200, C514S002600
Reexamination Certificate
active
06420119
ABSTRACT:
BACKGROUND
1. Field of the Invention
This invention relates to novel methods for diagnosing, preventing or delaying the onset of, and treating urinary incontinence. The methods involve detecting and modulating the proteolysis of collagen in pelvic supporting tissue. More particularly, the methods of this invention relate to the differential expression of the matrix metalloproteinases (MMPs) known to degrade extracellular matrix collagen and the tissue inhibitors of metalloproteinases (TIMPs) which specifically inhibit MMP proteolytic activity. The invention also relates to a novel method of screening for specific modulators and inhibitors of pelvic collagen degradation.
2. State of the Art
Urinary incontinence and pelvic floor dysfunction are a major public health problem in the United States. A decade ago the National Institutes of Health estimated that at least 10 million American adults suffered from urinary incontinence (1). In addition, two-thirds of the burden of urinary incontinence is borne by women with prevalence rates ranging from 14% to 41% depending on the study population definition. Currently, American women can expect a 11.1% lifetime risk of undergoing surgery for incontinence or pelvic organ prolapse by the age of 80 and of those who do undergo surgery, close to 30% will require a repeat operation for recurrent urinary dysfunction (2), thus defining a growing area of disability for women for which no medical prophylaxis or therapy currently exists.
1. Collagen Content and Expression.
Collagen is composed of three polypeptide chains assembled in a triple helix, which is then supercoiled (3). Collagen forms the matrix of connective tissue and is made through very complex post-translational processing which ultimately requires crosslinking of the fascial collagen chains by pyridinoline in order to mature into a high tensile strength support structure (4). Over a dozen types of collagen have been identified, with types I and III being the main structural components of epithelial tissue (5), vaginal epithelium, and endopelvic fascia (6). Type I collagen is most abundant in the skin, tendons, ligaments, bones, and cornea where it comprises over 80% of the total collagen. Type II collagen is primarily present in cartilage, and type III collagen has the same distribution as does type I, but the ratio between the two varies. Type III collagen appears to be more abundant in the dermis during early fetal development and is thought to contribute to the elastic properties of the tissue (5).
Mechanical stability of the genito-urinary tract depends on intact, functional collagen fibers, which support the bladder neck, urethra, and pelvic organs. There are several reports in the literature that suggest that women with stress urinary incontinence, and perhaps those with prolapse as well (7), have lower levels of total collagen in vesico-vaginal fascia (8), abdominal skin, and round ligament (9) than control, continent women. Collagenous tissue from the vaginal wall is representative of tissue from the uterosacral and round ligaments (7) as well as tissue supporting the urethra, bladder, and bladder base (6). The content of type III collagen in perineal skin, uterosacral ligaments, and round ligaments of women with stress urinary incontinence was shown to be significantly reduced compared to that of women without SUI (6). In addition, there was a positive correlation between urethral pressure measurements and collagen content in abdominal skin as measured by hydroxyproline (10). Consistent with these in vivo observations, an in vitro study of fibroblasts cultured from skin biopsies of women with stress incontinence showed a 30% decrease in collagen accumulation compared to cultures from continent women (11).
Other studies have compared mRNA levels for specific collagen types (12), the pyridinoline crosslink content of collagen (13,14), collagen fiber size by transmission electron microscopy (12), ratios of type I/type III collagens (14), levels of type I collagen (15). However, such studies have yielded conflicting and/or inconclusive results.
2. Collagen Metabolism
Collagen is a metabolically active structure with constant turnover mediated through proteolysis by a group of enzymes termed matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of matrix metalloproteinase (TIMPs).
Matrix metalloproteinases (MMPs) are a family of structurally related proteins which degrade extracellular matrix and basement membrane components (16). They are produced as proenzymes and activated through proteolytic cleavage by other MMPs or plasmin. Interstitial collagens (types I, II, and III), the most abundant connective tissue proteins, are cleaved by the interstitial collagenases (MMP-1, MMP-8, and MMP-13) which specifically cleave native triple helical collagen at a single peptide bond in each a chain, yielding two fragments (17). This single site for cleavage (Gly
775
-Leu/Ile
776
) produces both three-quarter (TC
A
) and one-quarter (TC
B
) length collagen fragments (18) generating two neoepitopes: (COL2-3/4C
short
) on the carboxy-terminal end and COL2-1/4N1 on the amino-terminus. Specific antibodies to these carboxy- and amino-terminal neoepitopes have been utilized to document increased collagenase activity in cartilage from patients with osteoarthritis compared to nonarthritic cartilage (19) and excessive collagenase activity in human atheromatous plaques (20).
These one-quarter and three-quarter length collagen fragments are very susceptible to rapid gelatinase (MMP-2 and MMP-9) degradation to amino acids (21). Importantly, MMP-2 may also act on intact collagen degrading it to quarter length fragments similarly to MMP-1 (17). Thus, the interstitial collagenases and the gelatinases together are capable of completely degrading intact collagen fibers. As such, MMP-2 and MMP-9 may be critically important to loss of strength of fibrous collagen and, ultimately, with sufficient degradation, to loss of tissue strength.
In the extracellular matrix, the activity of MMPs is tightly regulated by a family of natural inhibitors, the tissue inhibitors of metalloproteinase (TIMPs). TIMP-1, TIMP-2, TIMP-3, and TIMP-4 have been described (22). TIMP-1 is a glycoprotein of molecular weight 28.5 kDa which binds stoichiometrically with MMP-9. TIMP-2 is a 21 kDa protein which binds to MMP-2. TIMP-3, a novel member of the TIMP family, has inhibitory activity against MMP-9 as well as stromelysin-1 and MMP-1. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix and it appears to be the most effective inhibitor of MMP-9 (23). The most recently discovered TIMP-4 appears primarily in cardiac tissue and may function to maintain extracellular matrix hemostasis (22). A single study addressed MMP levels and has shown significant increases in MMP-2 and MMP-9 in pelvic tissue from premenopausal women with prolapse, but not incontinence, when compared with normal women (7), implying increased metabolic turnover, and decreased mechanical strength in the tissue. There is no information about TIMP expression in pelvic tissue in incontinent women.
The MMP-2/TIMP-2 ratio has been found to correlate with the invasiveness of breast carcinomas, suggesting the potential utility of this ratio as an early prognostic tool to determine more reliably the aggressiveness of the breast cancer (24).
3. Modulators of MMP and/or TIMP Expression.
(a) GnRH Agonist: Trophoblast invasion is regulated in part by MMP-9 which can be inhibited by both TIMP-1 and TIMP-3 (25). We have shown that TIMP-1 and TIMP-3 mRNA expression in cultured human endometrial stromal cells and protein secretion into the medium were significantly decreased by GnRH agonist when compared to control cells. Moreover, the expression of TIMP-1 appeared to be affected to a much greater extent than that of TIMP-3 (26).
(b) Cytokines and Growth Factors: The cytokine IL-1&bgr; has also been shown to stimulate a two-fold increase in procollagenase production in pregnant Guinea pig cervix (27). Our labora
Chen Bertha
Polan Mary Lake
Friedman Susan J.
Horlick Kenneth R.
Speckman Ann W.
The Board of Trustees of the Leland Stanford Jr. University
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