Modification of collagenous materials and medical treatment, diagnosis and monitoring of fibrotic conditions

Abstract

The present invention relates to the gene PLOD2 which codes for telopeptide lysyl hydroxylase (TLH). This enzyme converts telopeptidyl Lys into telopeptidyl Hyl, that can subsequently be converted into hydroxyallysine cross-links. Collagen with hydroxyallysine cross-links shows a higher resistance to degradation by proteinases than collagen with cross-links derived from allysine. In one aspect, the invention provides methods and compositions to prepare collagenous materials with varying biodegradation rates by varying the ratio of hydroxyallysine cross-links over allysine cross-links. In another aspect, the invention provides methods and compositions to lower the ratio of hydroxyallysine cross-links over allysine cross-links in fibrotic processes, in order to obtain a collagenous network that is more easy to degrade. Furthermore, the invention provides methods to diagnose and/or monitor fibrotic processes by measuring mRNA levels of PLOD2, by measuring protein levels of the translated mRNA, and/or by measuring enzymatic activity levels of TLH. The invention also provides the description of a high through-put system facilitating the screening of antagonists of telopeptide lysyl hydroxylase.

1 . A method for obtaining a collagenous matrix which comprises cross-linked collagen molecules, wherein the resistance of said collagenous matrix against proteolytic degradation is controlled by controlling the ratio of hydroxyallysine cross-links to allysine cross-links in the collagenous matrix.  
     
     
         2 . The method of  claim 1  wherein the ratio of hydroxyallysine cross-links to allysine cross-links in the collagenous matrix is controlled by controlling the lysyl hydroxylation level of the collagen telopeptides.  
     
     
         3 . The method of  claim 2  wherein the lysyl hydroxylation level of the collagen telopeptides is controlled by controlling the level of telopeptide lysyl hydroxylase activity.  
     
     
         4 . The method of  claim 3  wherein the level of telopeptide lysyl hydroxylase activity is controlled by controlling the expression of a PLOD2 gene or by controlling the telopeptide lysyl hydroxylase activity of a PLOD2 expression product.  
     
     
         5 . The method of  claim 1  comprising obtaining collagen from tissues which show an increased ratio of hydroxyallysine cross-links to allysine cross-links and using said collagen to prepare a collagenous matrix with an increased resistance against proteolytic degradation.  
     
     
         6 . The method of  claim 1  comprising obtaining collagen from tissues which predominantly show allysine cross-links and using said collagen for preparing a collagenous matrix, wherein a collagenous matrix with an increased resistance against proteolytic degradation is obtained by admixing collagen obtained from tissues which show an increased ratio of hydroxyallysine cross-links to allysine cross-links.  
     
     
         7 . The method of  claim 1  comprising culturing cells producing collagen with telopeptides having an increased ratio of hydroxylysine to lysine residues and using the collagen produced for preparing a collagenous matrix having an increased resistance against proteolytic degradation due to an increased ratio of hydroxyallysine cross-links to allysine cross-links.  
     
     
         8 . The method of  claim 7  wherein said collagen-producing cells comprise a recombinant, constitutively expressed PLOD2 gene.  
     
     
         9 . The method of  claim 7  wherein said collagen-producing cells are cultured in the presence of a composition which stimulates the expression of a PLOD2 gene.  
     
     
         10 . The method of  claim 1  comprising obtaining collagen from tissues which show a decreased ratio of hydroxyallysine cross-links to allysine cross-links and using said collagen to prepare a collagenous matrix with a decreased resistance against proteolytic degradation.  
     
     
         11 . The method of  claim 1  comprising obtaining collagen from tissues which predominantly show hydroxyallysine cross-links and using said collagen for preparing a collagenous matrix, wherein a collagenous matrix with a decreased resistance against proteolytic degradation is obtained by admixing collagen obtained from tissues which show a decreased ratio of hydroxyallysine cross-links to allysine cross-links.  
     
     
         12 . The method of  claim 1  comprising culturing cells producing collagen with telopeptides having a decreased ratio of hydroxylysine to lysine residues and using the collagen produced for preparing a collagenous matrix having a decreased resistance against proteolytic degradation due to a decreased ratio of hydroxyallysine cross-links to allysine cross-links.  
     
     
         13 . The method of  claim 12  wherein said collagen-producing cells are cultured in the presence of a composition which inhibits the activity or production of PLOD2-encoded telopeptide lysyl hydroxylase.  
     
     
         14 . The method of  claim 13  wherein said composition does not affect the level of lysyl oxidase.  
     
     
         15 . The method of  claim 1  comprising treating a fibrotic condition in a mammal by administering to said mammal an effective amount of a compound or composition which reduces the lysyl hydroxylation level of collagen telopeptides and thereby results in a collagenous matrix having a decreased ratio of hydroxyallysine cross-links to allysine crosslinks.  
     
     
         16 . The method of  claim 15  comprising administration of an effective amount of a compound or composition that inhibits the activity or production of telopeptide lysyl hydroxylase encoded by a PLOD2 gene but not the activity or production of lysyl oxidase.  
     
     
         17 . The method of  claim 16  wherein said compound or composition comprises a compound that binds to the subsite or subsites of the active site of said telopeptide lysyl hydroxylase destined for binding of the co-substrate 2-oxoglutarate, thereby replacing the 2-oxoglutarate.  
     
     
         18 . The method of  claim 16  wherein said compound or composition comprises a compound that chelates the Fe 2+  bound in the active site of said telopeptide lysyl hydroxylase, thereby inhibiting the oxygen atom acceptor function of said Fe 2+ and/or inhibiting of said Fe 2+  to 2-oxoglutarate.  
     
     
         19 . The method of  claim 16  wherein said compound or composition comprises an anthracycline or a coumalic acid analogue that syncatalytically inactivates said telopeptide lysyl hydroxylase.  
     
     
         20 . The method of  claim 16  wherein said compound or composition comprises a peptide or peptidomimetic containing a non-physiological lysine derivative in a hydroxylatable position which syncatalytically nactivates said telopeptide lysyl hydroxylas  
     
     
         21 . The method of  claim 16  wherein said compound or composition comprises a hydroxylatable peptide or peptidomimetic that competes with the natural substrate for the active site of said telopeptide lysyl hydroxylase.  
     
     
         22 . The method of  claim 16  wherein said compound or composition comprises a Pon-hydroxylatable peptide or peptidomimetic that competes with the natural substrate for the active site of said telopeptide lysyl hydroxylase.  
     
     
         23 . The method of  claim 16  wherein said compound or composition comprises a compound that is a non-reducing ascorbate that binds to the active site of said telopeptide lysyl hydroxylase, thereby replacing ascorbate.  
     
     
         24 . The method of  claim 16  wherein said compound or composition comprises a peptide or peptidomimetic with a non-hydroxylatable sequence capable of enhancing the uncoupled reaction of said telopeptide lysyl hydroxylase, resulting in an increased level of the inactive self-oxidized form of said telopeptide lysyl hydroxylase.  
     
     
         25 . The method of  claim 16  wherein said compound or composition comprises a compound found by means of screening compound libraries (combinatorial chemistry).  
     
     
         26 . The method of  claim 16  wherein said compound or composition comprises an antibody or an aptamer that selectively inhibits the activity of telopeptide lysyl hydroxylase.  
     
     
         27 . The method of  claim 26  wherein said antibody or aptamer reacts with or near the active site of said telopeptide lysyl hydroxylase.  
     
     
         28 . The method of  claim 26  wherein said antibody or aptamer reacts with the telopeptides, thereby inhibiting the reaction of said enzyme by means of steric hindrance.  
     
     
         29 . The method of  claim 16  wherein said compound or composition contains a compound that selectively inhibits the transcription of a telopeptide lysyl hydroxylase gene.  
     
     
         30 . The method of  claim 29  wherein said compound is minoxidil or a minoxidil analogue.  
     
     
         31 . The method of  claim 16  wherein said compound or composition contains an antisense RNA in order to inhibit the translation of mRNA derived from a telopeptide lysyl hydroxylase gene.  
     
     
         32 . The method of  claim 16  wherein said compound or composition contains a recombinant gene encoding a mutated telopeptide lysyl hydroxylase that shows no activity towards telopeptides but that is competitive to endogenous telopeptide lysyl hydroxylase with respect to its natural substrate (collagen telopeptides).  
     
     
         33 . The method of  claim 16  wherein said compound or composition is a hydroxylatable peptide or a sequence encoding such a peptide that is competitive to collagen telopeptides with respect to the reaction that is normally catalyzed by telopeptide lysyl hydroxylase.  
     
     
         34 . The method of  claim 15  wherein said mammal is a human being.  
     
     
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         42 . (canceled)