Patent Application: US-201515110108-A

Abstract:
the invention relates to the use of microrna 29 and precursors and mimics thereof for the modulation of tendon injury and the biomechanical properties of tendon . in particular , the invention derives from the finding that synthesis of type 1 collagen in tenocytes is less sensitive to mir - 29 than is synthesis of type 3 collagen , thus enabling the balance between the collagen subtypes to be modulated in favour of type 1 collagen , mitigating reduction in biomechanical properties during healing .

Description:
all procedures and protocols were approved by the ethics committee under acec no . 99 / 101 . fifteen supraspinatus tendon samples were collected from patients with rotator cuff tears undergoing shoulder surgery ( table 1 ). the mean age of the rotator cuff ruptured patients was 54 years ( range , 35 - 70 years )— the mean tear size was 2 . 5 cm . samples of the subscapularis tendon were also collected from the same patients . patients were only included if there was no clinically detectable evidence of subscapularis tendinopathy on a preoperative mri scan or macroscopic damage to the subscapularis tendon at the time of arthroscopy — by these criteria they represented a truly pre - clinical cohort . an independent control group was obtained comprising 10 samples of subscapularis tendon collected from patients undergoing arthroscopic surgery for shoulder stabilization without rotator cuff tears . the absence of rotator cuff tears was confirmed by arthroscopic examination . the mean age of the control group was 35 years ( range , 20 - 41 years ). arthroscopic repair of the rotator cuff was carried out using the standard three - portal technique as described previously described . the cross - sectional size of the rotator cuff tear was estimated and recorded as described previously 39 . the subscapularis tendon was harvested arthroscopically from the superior border of the tendon 1 cm lateral to the glenoid labrum . the supraspinatus tendon was harvested from within 1 . 5 cm of the edge of the tear prior to surgical repair . for immunohistochemical staining the tissue samples were immediately fixed in 10 % ( v / v ) formalin for 4 to 6 hours and then embedded in paraffin . sections were cut to 5 μm thickness using a leica - lm microtome ( leica microsystems , germany ) and placed onto superfrost ultra plus glass slides ( gerhard menzel , germany ). the paraffin was removed from the tissue sections with xylene , rehydrated in graded alcohol and used for histological and immunohistochemical staining per previously established methodologies 40 . human tendon derived cells were explanted from hamstring tendon tissue of 5 patients ( age 18 - 30 years ) undergoing hamstring tendon acl reconstruction . cultures were maintained at 37 ° c . in a humidified atmosphere of 5 % co 2 for 28 days . cells were subcultured and trypinized at subconfluency , cells from the 3 rd and 4 th passage were used in normoxic conditions . human sections were stained with haematoxylin and eosin and toluidine blue for determination of the degree of tendinopathy as assessed by a modified version of the bonar score 41 ( grade 4 = marked tendinopathy , grade 3 = advanced tendinopathy , 2 = moderate degeneration 1 = mild degeneration 0 = normal tendon ). this included the presence or absence of oedema and degeneration together with the degree of fibroblast cellularity and chondroid metaplasia . thereafter , sections were stained with antibodies directed against the following markers :— il - 33 ( alexis , mouse monoclonal ), st2 ( sigma aldrich , rabbit polyclonal ), il - 1racp ( prosci , rabbit polyclonal ) cd68 ( pan macrophages ), cd3 ( t cells ), cd4 ( t helper cells ), cd206 ( m 2 macrophages ), and mast cell tryptase ( mast cells ) ( vector labs ). endogenous peroxidase activity was quenched with 3 % ( v / v ) h 2 o 2 , and nonspecific antibody binding blocked with 2 . 5 % horse serum in tbst buffer for 30 minutes . antigen retrieval was performed in 0 . 01m citrate buffer for 20 minutes in a microwave . sections were incubated with primary antibody in 2 . 5 % ( w / v ) horse serum / human serum / tbst at 4 ° c . overnight . after two washes , slides were incubated with vector immpress reagent kit as per manufactures instructions for 30 minutes . the slides were washed and incubated with vector immpact dab chromagen solution for 2 minutes , followed by extensive washing . finally the sections were counterstained with hematoxylin . positive ( human tonsil tissue ) and negative control specimens were included , in addition to the surgical specimens for each individual antibody staining technique . omission of primary antibody and use of negative control isotypes confirmed the specificity of staining . we applied a scoring system based on previous methods 42 to quantify the immunohistochemical staining . ten random high power fields (× 400 ) were evaluated by three independent assessors ( nlm , jhr , alc ). in each field the number of positive and negatively stained cells were counted and the percentage of positive cells calculated giving the following semi - quantitative grading ; grade 0 = no staining , grade 1 =& lt ; 10 % cells stained positive , 2 = 10 - 20 % cells stained positive , grade 3 =& gt ; 20 % cells positive . mouse sections were processed using the above protocol with antibodies directed against the following markers :— il - 33 ( r & amp ; d systems , mouse monoclonal ), st2 ( sigma aldrich , rabbit polyclonal ), f4 / 80 ( serotec , mouse monoclonal ) and anti - histamine ( sigma aldrich , rabbit polyclonal ). tenocytes were evaluated for immunocytochemical staining of collagen 1 and collagen 3 to assess tenocyte matrix production ( abcam ). total soluble collagen was measured from cell culture supernatants using the sircol assay kit ( biocolor ltd , carrickfergus , northern ireland ) according to the manufacturer &# 39 ; s protocol . 1 ml of sircol dye reagent was ded to 100 μl test sample and mixed for 30 min at room temperature . the collagen - dye complex was precipitated by centrifugation at 10 , 000 × g for 10 min ; and then washed twice with 500 μl of ethanol . the pellet was dissolved in 500 μl of alkali reagent . the absorbance was measured at 540 nm by microplate reader . the calibration curve was set up on the basis of collagen standard provided by the manufacturer . additionally the concentration of human and mouse collagen 1 and 3 was assessed using elisa with colour change measured at 450 nm by microplate reader along with standards supplier by the manufacturer ( luscnk life science inc ). phosphorylation status of mitogen - activated protein kinases ( mapks ), extracellular signal regulated kinases ( erk1 / 2 ), c - jun n - terminal kinases ( jnks ) and p38 isoforms were evaluated using the human phospho - mapk array ( r & amp ; d systems europe , uk ) as per the manufacturer &# 39 ; s instructions . the erk inhibitor ( fr180204 ) was purchased from calbiochem ( merck kgaa , germany ) and used at ic 50 = 10 μm , a concentration previously determined to offer optimal specific inhibition relative to off target effects which was used previously in our laboratory 43 . phosphorylation of nfkβ p65 was assessed using the instantone elisa in cell lysates from treated and untreated tencocytes . the absorbance was measured at 450 nm by microplate reader with positive and negative controls supplied by the manufacturer . the relative absorbance of stimulated versus unstimulated cells was used to assess the total or phosphorylated nfkβ p65 in each sample . the cells isolated from the normoxic and hypoxic experiments trizol prior to mrna extraction . qiagen mini columns ( qiagen ltd , crawley uk ) were used for the rna clean - up with an incorporated on column dnase step as per manufactures instructions . cdna was prepared from rna samples according to affinityscript ™ ( agilent technologies , ca , usa ) multiple temperature cdna synthesis kit as per manufactures instructions . real time pcr was performed using sybr green or taqman fastmix ( applied biosystems , ca , usa ) according to whether a probe was used with the primers . the cdna was diluted 1 in 5 using rnase - free water . each sample was analysed in triplicate . primers ( integrated dna technologies , belgium ) were as follows : gapdh , 5 ′- tcg aca gtc agc cgc atc ttc ttt - 3 ′ ( f ) and 5 ′- acc aaa tcc gtt gac tcc gac ctt - 3 ′ ( r ); il - 33 human gga aga aca cag caa gca aag cct ( f ) taa ggc cag agc gga gct tca taa ( r ); il - 33 murine gga aga aca cag caa gca aag cct ( f ) taa ggc cag agc gga gct tca taa ( r ); total st2 human aca act gga cag cac ctc ttg agt ( f ) acc tgc gtc ctc agt cat cac att ( r ); sst2 murine cca atg tcc ctt gta gtc gg ( f ) ctt gtt ctc ccc gca gtc ( r ), tcc cca tct cct cac ctc cct taa t ( probe ); st2l murine tct gct att ctg gat act gct ttc , tct gtg gag tac ttt gtt cac c ( r ) aga gac ctg tta cct ggg caa gat g ( probe ); human st2l aca aag tgc tct aca cga ctg ( f ) tgt tct gga ttg agg cca c ( r ); ccc cat ctg tac tgg att tgt agt tcc g ( probe ); human sst2 gag acc tgc cac gat tac ac ( f ) tgttaaaccctgagttcccac ( r ), ccc cac acc cct atc ctt tct cct ( probe ); col 3a human ttg gca gca acg aca cag aaa ctg ( f ) ttg agt gca ggg tca gca cta ctt ( r ) col 3a mouse gct ttg tgc aaa gtg gaa cct gg ( f ) caa ggt ggc tgc atc cca att cat ( r ); col 1a1 human cca tgc tgc cct ttc tgc tcc ttt ( f ) cac ttg ggt gtt tga gca ttg cct ( r ) col 1a1 mouse ttc tcc tgg caa aga cgg act caa ( f ) gga agc tga agt cat aac cgc ca ( r ) total rna was isolated by mirneasy kit ( qiagen ). miscript reverse transcription kit ( qiagen ) was used for cdna preparation . taqman mrna assays ( applied biosystems ) or miscript primer assay ( qiagen ) were used for semi - quantitative determination of the expression of human mir - 29a ( ms ( ms00001701 ) 29b ( ms00006566 ) and c ( ms00009303 ) and mouse 29a ( ms00003262 ), 29b ( ms00005936 ) and c ( ms00001379 ). the expressions of u6b small nuclear rna or beta - actin were used as endogenous controls . the absolute levels of long and short 3 ′ utr forms of type 1 and 3 transcripts were determined by q - pcr relative to standards . cdna was generated using affinityscript ( agilent ) with both random hexamer and oligo - dt primers . sybr green quantitative - pcr was performed using the following primers : samples were normalised to gapdh endogenous control . col1a2_s fw 5 ′ gcctgcccttccttgatatt 3 ′ col1a2_s rev 5 ′ tgaaacagactgggccaatg 3 ′ col1a2_l fw 5 ′ tcagatacttgaagaatgttgatgg 3 ′ col1a2_l rev 5 ′ caccacacgatacaactcaatac 3 ′ col1a1_s fw 5 ′ cttcacctacagcgtcact 3 ′ col1a1_s rev 5 ′ ttgtattcaatcactgtcttgcc 3 ′ col1a1_l fw 5 ′ ccacgacaaagcagaaacatc 3 ′ col1a1_l rev 5 ′ gcaacacagttacacaaggaac 3 ′ col3a1_s fw 5 ′ ctatgacattggtggtcctgat 3 ′ col3a1_s rev 5 ′ tgggatttcagatagagtttggt 3 ′ col3a1_l fw 5 ′ ccaccaaatacaattcaaatgc 3 ′ col3a1_l rev 5 ′ gatgggctaggattcaaaga 3 ′ to characterize human sequences , 3 ′ race was performed on cdna that had been generated from total rna isolated from human tenocytes using mirscript ii reverse transcriptase kit ( qiagen ). cdna ends were amplified by pcr using the following gene specific forward primers listed below along with the universal reverse primer from the kit . to characterise horse sequences , the 3 ′ utrs of col1a1 , col1a2 and col3a1 transcripts expressed in equine tenocytes were amplified using 3 ′ rapid extension of cdna ends ( 3 ′ race ). the amplified cdna fragments were sequenced and the polya signal identified according to the location of aataaa canonical polya signal located 10 and 30 nucleotides 5 ′ to the polya tail . the resulting pcr products were cloned into pcr2 . 1 topo ( invitrogen ) and sequenced . mirna transfection cells were transfected with synthetic mature mirna for mir 29 a & amp ; b or with negative control ( c . elegans mir - 67 mimic labelled with dy547 , thermo scientific inc ) at a final concentration of 20 nm with the use of dharmacon ® dharmafect ® 3 sirna transfection reagents ( thermo scientific inc ). at 48 hours after transfection cellular lysates were collected to analyse the expression of genes of interest . transfection efficiency was assessed by flow cytometry using the labelled dy547 mimic and confirmed by quantitative pcr of control - scrambled mimic and the respective mir29 family mimic . luciferase reporter assay for targeting collagen 1 & amp ; 3 and soluble st2 the human 2 mirna target site was generated by annealing the oligos : for col 1 & amp ; 3 and soluble st2 3 ′ utr &# 39 ; s which were cloned in both sense and anti - sense orientations downstream of the luciferase gene in pmir - report luciferase vector ( ambion ). these constructs were sequenced to confirm inserts and named pmir - col i / col iii / sst2 - mir29a / b / c and pmir ( a / s )- col i / col iii / sst2 - mir29a / b / c , and used for transfection of hek293 cells . hek293 cells were cultured in 96 - well plates and transfected with 0 . 1 μg of either pmir - col i / col iii / sst2 - mir29a / b / c , pmir ( a / s )- col i / col iii / sst2 - mir29a / b / c or pmir - report , together with 0 . 01 g of prl - tk vector ( promega ) containing renilla luciferase and 40 nm of mir - 155 or scrambled mirna ( thermo scientific dharmacon ®). transfections were done using effectene ( qiagen ) according manufacturer &# 39 ; s instructions . twenty - four hours after transfection , luciferase activity was measured using the dual - luciferase reporter assay ( promega ). the 3 ′ utr of human sst2 was amplified from genomic dna using the following primers sst2fw 5 ′ agtttaaactggcttgagaaggcacaccgt3 ′ and sst2rev 5 ′ agtcgacgggccaagaaaggctccctgg3 ′ which created pmei and sali sites respectively . these sites where used to clone the pcr amplified product into the same sites of pmirglo ( promega ). the seed regions of the two targetscan predicted mir29a mre sites : 29 - 1 and 29 - 2 were mutated using the quickchange site - directed mutagenesis kit ( agilent ). each vector along with mir29a or scrambled control mimic were transfected into hek293 cells using attactene ( qiagen ) according to manufactures instructions . after 24 hours luciferase activity was measured using dual - glo luciferase assay ( promega ) with luciferase activity being normalized to renilla . normalized luciferase activity was expressed as a percentage of scrambled control for the same constructs . a 25 - plex human cytokine assay evaluated the in vitro quantitative determination of 25 separate human cytokines using luminex technology . supernatants ( n = 3 ) in preparation for the surgical procedure , mice were anesthetised with a mixture of isofluorane ( 3 %) and oxygen ( 1 %) and both hind limbs were shaved . during the surgical procedure , anaesthesia was delivered via a nose cone with the level of isofluorane reduced to 1 % with the oxygen . following a skin incision , two cuts parallel to the tendon were made in the retinaculum on each side , a set of flat faced scissors were then placed underneath the patellar tendon . with the scissor blades serving as a support , a 0 . 75 mm diameter biopsy punch ( world precision instruments ) was used to create a full thickness partial transection in the right patellar tendon . the left patellar tendon underwent a sham procedure , which consisted of only placing the plastic backing underneath the tendon without creating and injury . the skin wounds were closed with skin staples and the mice were sacrificed at 1 day , 3 days and 7 and 21 days post - surgery . mice were sacrificed by co 2 inhalation and immediately weighted . mice from two groups balb / c control ( ctl ) and st2 −/− balb / c were used . each group contained 16 mice ( n = 8 st2 −/− balb / c and 8 balb / c ) per time point . these experiments were repeated on 4 separate occasions . to test if il - 33 induced tendon matrix dysregulation a cytokine injection model was established . il - 33 was tested in a previously reported model initially described for the application of il - 23 or il - 22 44 - 45 . st2 −/− mice ( n = 4 / group / treatment / experiment ) were injected i . p . daily with il - 33 ( 0 . 2 μg per mouse diluted in 100 μl pbs ) on days - 3 , - 2 , - 1 and the day of injury . 24 hours following the final injection mice were culled as per protocol . control mice similarly received an equal volume of pbs . we also tested neutralising antibodies to il - 33 ( 0 . 5 μg / ml r & amp ; d systems ) by injecting i . p immediately post injury in wt and st2 −/− mice with igg controls again with 4 / group / treatment / experiment . for the biomechanical analysis , the patellar tendons of mice from each group were injured and eight mice sacrificed at one of three time points for mechanical testing as described previously by lin et al 10 . briefly , the patellar tendons were dissected and cleaned , leaving only the patella , patellar tendon and tibia as one unit . tendon width and thickness were then quantified and cross sectional area was calculated as the product of the two . the tibia was the embedded in isopon p38 ( high build cellulose filler ) in a custom designed fixture and secured in place in a metal clamp . the patella was held in place by vice grips used with the bose electroforce ® 3200 test instrument . each tendon specimen underwent the following protocol immersed in a 370 c saline bath — reloaded to 0 . 02n , preconditioned for 10 cycles from 0 . 02 to 0 . 04 at a rate of 0 . 1 %/ s ( 0 . 003 mm / s ), and held for 10 s . immediately following , a stress relaxation experiment was performed by elongating the tendon to a strain of 5 % ( 0 . 015 mm ) at a rate of 25 % ( 0 . 75 mm / s ), followed by a relaxation for 600 s . finally a ramp to failure was applied at a rate of 0 . 1 %/ s ( 0 . 003 mm / s ). from these tests , maximum stress was determined and modulus was calculated using linear regression from the near linear region of the stress strain curve . a transfection complex was prepared containing 150 ng / ml mir - 29a mimic , 9 μg / ml polyethylenimine ( pei ) and 5 % glucose . 50 μl of this complex was injected into mouse patellar tendon immediately after surgery . animals were sacrificed after 1 and 3 days and col1a1 and col3a1 mrna and protein levels were measured . fluorescently labelled mir - 29a mimic was used to assess the in vivo distribution of mir - 29a mimic in the tendon by immunofluorescence , using counterstains for phalloidin ( to show cytoskeletal structure ) and nuclei ( dapi ). all results are displayed as mean +/− standard error mean ( sem ) and all statistical analysis was done either by students t test , anova test or mann whitney test , as indicated in figure legends , using the graph pad prism 5 software . a p value of & lt ; 0 . 05 was considered statistically significant . we first investigated il - 33 expression in human tendinopathy using our previously developed model 22 . il - 33 , soluble and membrane bound st2 transcripts were significantly upregulated in early tendinopathy compared to control or torn tendon biopsies ( fig1 a - c ). early tendinopathy tissues exhibited significantly greater staining for il - 33 and st2 compared to torn tendon or control biopsies ( fig1 d ). staining was prominent in endothelial cells and particularly fibroblast - like cells , namely tenocytes that are considered pivotal to the regulation of early tendinopathy ( data not shown ). in parallel , in vitro cultured tenocytes expressed nuclear il - 33 that was up regulated at both mrna and protein levels following stimulation by tnf and il - 1β ( fig1 e and data not shown ). in contrast st2 was constitutively expressed in both resting and unstimulated tenocytes ( data not shown ). matrix dysregulation towards collagen 3 expression is a key early phenotypic change in tendinopathy thereby hastening repair ; collagen 3 is however biomechanically inferior . il - 33 induced dose and time dependent upregulation of total collagen protein ( data not shown ), accounted for by increased expression of type 1 but particularly type 3 collagen mrna and protein ( fig1 f , g ). following array analysis ( data not shown ) and consistent with reported il - 33 downstream signalling 12 , 16 , this was abrogated by erk inhibition ( data not shown ). rhil - 33 also significantly elevated production of il - 6 , il - 8 and mcp - 1 ( data not shown ), which was regulated by nf - kb inhibition suggesting that il - 33 operates in tenocytes via its canonical il - 1r signalling pathway ( data not shown ). in contrast we found no effect on production of other cytokines in keeping with previously reported il - 33 induced cytokine production profiles in fibroblasts 20 - 23 . we extended these observations to a well - established in vivo model of tendon injury . il - 33 mrna was elevated on days 1 and 3 post tendon injury in wt mice ( fig2 a ). this was significantly reduced in injured st2 −/− mice suggesting autocrine regulation . soluble st2 was significantly up regulated at all time points post injury in wt mice compared to uninjured controls ( fig2 b ) whereas membrane st2 mrna was elevated only by day 3 post injury ( data not shown ). no significant changes in il - 33 or st2 transcript or protein expression were found in wt mice at days 7 or 21 post - injury , or for il - 33 expression in st2 −/− mice , suggesting that the impact of il - 33 expression is manifest early , in keeping with ‘ alarmin ’ type activity in tendon injury / repair . analysis of collagen synthesis revealed significantly greater expression of collagen 3 at all time points post injury in wt mice compared to uninjured controls or injured st2 −/− mice ( fig2 e , f & amp ; data not shown ). collagen 1 was initially down regulated ( days 1 , 3 ) at mrna levels ( fig2 c ) in wt injured mice but reverted towards pre - injury levels by days 7 and 21 ( data not shown ) with a similar trend in collagen 1 protein expression ( fig2 d ). in contrast , st2 −/− injured mice showed prolonged reduction of collagen 1 synthesis ( days 1 , 3 & amp ; 7 ) returning to baseline only by day 21 ( data not shown ). importantly injury of wt mice tendons resulted in a significant decrease in biomechanical strength at day 1 post injury compared to st2 −/− ( fig2 g ) that recovered by days 7 and 21 ( data not shown ). these data suggest altered collagen matrix synthesis in st2 −/− mice implicating il - 33 / st2 as an early modulator of collagen changes in tendon injury that has biomechanical significance . to confirm this possibility we sought to directly modify il - 33 effector biology in vivo . administration of rhil - 33 did not affect collagen 1 synthesis ( fig3 a , b ) but did significantly increase collagen 3 synthesis particularly in injured tendons ( fig3 d , e and data not shown ). moreover , rhil - 33 administration significantly reduced ultimate tendon strength at all time points post injection in wt mice ( fig3 e and data not shown ) suggesting that such changes were of functional impact . il - 33 administration did not affect collagen matrix synthesis or ultimate tendon strength of the healing tendon in st2 −/− mice confirming that il - 33 acted via an st2 - dependent pathway ( data not shown ). we next directly targeted il - 33 in vivo . neutralising antibodies to il - 33 attenuated the collagen 1 to 3 switch at days 1 and 3 post injury in wt injured mice ( fig3 f - i ) resulting in a significant increase in biomechanical strength at day 1 post injury wt mice tendons ( fig3 j ). this effect was not seen at later time points ( data not shown ). in control experiments we observed no effect on st2 −/− mice ( data not shown ) further confirming the contribution of endogenous il - 33 to injury - induced tendinopathy . il - 33 promotes differential regulation of collagen 1 / 3 via mir - 29 in tenocytes having established that il - 33 drives differential regulation of collagen 1 and 3 in tenocytes we postulated a mechanistic role for the mirna network in this process . previous studies have shown that the mir - 29 family directly targets numerous extracellular matrix genes , including type 1 and 3 collagens 24 - 25 and is implicated in regulation of innate and adaptive immunity 26 . computational algorithms predict that mir - 29 may also target sst2 . we found that all members of the mir - 29 family were expressed in human tendon biopsies and explanted tenocytes ( fig4 a ) with mir - 29a showing the most altered expression . in tenocyte culture il - 33 significantly reduced the expression of mir - 29a at 6 , 12 and 24 hours ( fig4 b ) acting via nfκb dependent signalling whereas we observed inconsistent effects on mir - 29b and c ( data not shown ). since il - 33 mediated collagen 3 matrix changes could be regulated by mir - 29a we analysed the functional effects of mir - 29a manipulation on collagen matrix synthesis in vitro . firstly , using luciferase assays , we confirmed that mir - 29a directly targets col 1a1 and 3a1 as previously demonstrated 27 ( fig7 b ). we also observed a previously unrecognised interaction with col 1a2 subunit transcript ( fig7 ). to test whether mir - 29a indeed regulates the levels of candidate target mrnas in disease relevant cells , we transfected tenocytes with mir - 29a mimic and antagomir . mir - 29a manipulation selectively regulated collagen 3 but not collagen 1 mrna and protein expression in primary tenocytes ( fig4 c , d ). moreover , mir - 29a over expression significantly abrogated il - 33 induced collagen 3 mrna and protein synthesis ( fig4 e ). additionally mir - 29a inhibition resulted in a significant increase in col 3a1 expression indicating that mir - 29a is not only actively regulating these transcripts in human tenocytes but whose loss is an important factor in the increase of type 3 collagen production observed in tendinopathy . in contrast col 1a1 transcript levels were unchanged ( fig4 i ). given that mir - 29a was capable of repressing col 1a1 and 1a2 with equal or greater efficiency than collagen 3 in luciferase reporter assays , this was unlikely to be the result of mir - 29a having greater affinity for its mres in type 3 transcripts ( fig4 f ). one well - documented mechanistic explanation for transcripts to modulate their sensitivity to mirna regulation is through the utilisation of alternative polyadenylation signals ( fig4 g ). to test this , we compared levels of full - length ( mir - 29a containing ) transcripts to total levels by q - pcr ( fig4 h ) showing that in tenocytes , less than 5 % of col 1a1 and 1a2 transcripts make use of the distal polyadenylation signal whereas the majority of col 3a1 transcripts do . this was confirmed by 3 ′ rapid amplification of cdna ends ( race ) ( fig7 e ) confirming that both col 1a1 and 1a2 , but not col 3a1 , make use of previously unrecognized polyadenylation signals ( fig4 g ). the resulting truncated 3 ′ utr lack mir29a mres . ( it will be appreciated that the sequences shown in fig7 e are cdna sequences ; the corresponding mrna sequences would of course contain u rather than t .) these data suggest that in tenocytes , mir - 29a specifically regulates col 3a1 , while both col 1a1 and col 1a2 are rendered insensitive to mir - 29a inhibition due to the utilisation of alternative polyadenylation signals . this utilisation of alternative polyadenylation signals was not influenced by the presence of il - 33 ( data not shown ). loss of mir - 29a upon il - 33 signalling results in depression of collagen 3 likely contributing to the increase of this collagen observed in injured tendons . the 3 ′ race results from human tenocytes revealed two col 3a1 utrs , the shorter of which [ designated col3a1 ( short 3 ′ utr ) in fig7 e ] contains one mir - 29a mre , while the longer one contains two . both are regulated by mir - 29a as shown in fig7 d . characterisation of the 3 ′ utrs of col1a1 , co1a2 and col3a1 transcripts expressed in equine tenocytes showed that they utilise the same conserved polya signals used in the orthologous collagen transcripts expressed in human tenocytes . in col1a1 and co1a2 , use of these proximal polya signals results in transcripts with 3 ′ utrs that are between 100 and 350 nucleotides in length and which do not contain mir - 29 binding sites and therefore insensitive to regulation by this mirna . in contrast both col3a1 3 ′ utrs contain mir - 29 binding sites rendering them sensitive to regulation by mir - 29 . computational analysis revealed that soluble st2 can be targeted by mir - 29a suggesting a feasible regulatory role in il - 33 effector functions . a luciferase reporter gene was generated that contains the 3 ′ utr of human sst2 predicted to possess two potential mir - 29abc binding sites . cotransfection of sst2 - luciferase reporter plasmid with mir - 29 mimics resulted in significant reduction in luciferase activity relative to scrambled control ( fig7 b ) furthermore luciferase activity was fully restored when the seed regions of both mir - 29 mres in sst2 were mutated , demonstrating conclusively that sst2 is a direct target of mir - 29a ( fig5 a ). to investigate whether mir - 29a does indeed regulate the levels of the candidate target mrna in tenocytes we again transfected mir - 29a mimic and antagomir into human tenocytes . soluble st2 message was significantly ( p & lt ; 0 . 01 ) altered by transfection with mir - 29a mimic / antagomir by approximately 5 fold ( fig5 b ) with a corresponding significant change in soluble st2 protein confirming mir29a as a target for soluble st2 ( fig5 c ). il - 33 / sst2 regulates mir - 29 expression in in vivo models of tendon healing finally , we investigated mir - 29a expression in our in vivo tendinopathy model . tendon injury in wt mice resulted in a 22 fold decrease in mir29a on day 1 which reverted to a 6 fold decrease ( versus baseline ) by day 3 ( fig5 d & amp ; data not shown ) with no significant difference by day 7 . this effect was significantly abrogated in st2 −/− mice ( data not shown ). in addition , administration of exogenous rh - il - 33 reduced mir - 29a expression in uninjured tendons at all - time points compared to pbs injected controls ( data not shown ). this effect was most profound in injured wt mice , with the addition of rhil - 33 mediating a further 10 fold reduction in mir - 29a ( fig5 e ). addition of rhil - 33 in st2 −/− mice had no significant effect on mir - 29a expression in injured or uninjured tendons again suggesting that mir - 29a down regulation is in part directly mediated by il - 33 / st2 dependent signalling . the addition of neutralising antibody to il - 33 significantly reduced the effect of injury on mir - 29a gene expression at days 1 and 3 post injury ( fig5 f ). in vivo administration of mir29a mimic in patellar tendon injury model mir - 29a mimic was delivered to tenocytes in wt mouse patellar tendons via direct injection of a mir - 29a / pei complex . immunofluorescence staining for the mimic ( red ), counterstained with phalloidin ( green , for cytoskeletal structure ) and dapi ( to show nuclei ) was used to visualise the localisation of mimic around tenocytes at 24 h post injection of mir - 29a mimic ( not shown ). as shown in fig8 , collagen 3 mrna and protein levels were significantly reduced in tendons injected with mir - 29a mimic compared to controls . in contrast collagen 1 levels were unchanged . micrornas have emerged as powerful regulators of diverse cellular processes with important roles in disease and tissue remodeling . these studies utilising tendinopathy as a model system reveal for the first time the ability of a single microrna ( mir - 29 ) to cross regulate inflammatory cytokine effector function and extracellular matrix regulation in the complex early biological processes leading to tissue repair . we herein provide new evidence for a role of il - 33 in the initial steps that lead to the important clinical entity of tendinopathy . il - 33 has recently become increasingly associated with musculoskeletal pathologies 16 . our data show il - 33 to be present in human tendon biopsies at the early stage of disease while end stage biopsies have significantly less il - 33 expression at the message and protein level promoting the concept of il - 33 as an early tissue mediator in tendon injury and subsequent tissue remodelling . upon cell injury endogenous danger signals , so called damage associated molecular patterns , are released by necrotic cells including heat shock proteins 28 , hmgb 29 , uric acid 30 and il - 1 family members 31 - 32 including il - 33 33 - 34 . these danger signals are subsequently recognised by various immune cells that initiate inflammatory and repair responses . our data implicate il - 33 as an alarmin in early tendinpoathy , and importantly , our biomechanical data suggest such expression has a pathogenically relevant role . the addition of rhil - 33 significantly reduced the load to failure of wt mice by approximately 30 % at early time points , likely as a consequence of the concomitant collagen 3 matrix changes which result in mechanically inferior tendon 35 . thus one plausible mechanism for the events mediating early tendon repair that is biomechanically inferior , may be that upon repeated micro injury il - 33 is up regulated with its subsequent release through mechanical stress / necrosis , which in turn drives the matrix degeneration and proinflammatory cytokine production propelling the tendon toward a pathological state such as that seen in early tendinopathy biopsies . interestingly the addition of neutralising antibodies to injured mice did reverse the collagen 3 phenotype but this was only able to temporarily improve tendon strength on day 1 post injury . whilst this may negate blocking il - 33 in longer term sports injuries the repetitive microtrauma associated with pathological tendon changes may conversely allow neutralising il - 33 to act as a check rein to further unwanted matrix dysregulation . emerging studies highlight mirnas as key regulators of leukocyte function and the cytokine network while orchestrating proliferation and differentiation of stromal lineages that determine extracellular matrix composition 36 . the novel finding of a role for mir - 29a in the regulation of il - 33 ‘ alarmin ’ mediated effects provides mechanistic insight into mirna cross - regulatory networks involving inflammation and matrix regulation in tissue repair . our data provide convincing evidence for a functional role for mir - 29 as a posttranscriptional regulator of collagen in murine and human tendon injury . the regulation of collagens by the mir - 29 family has been highlighted in several prior studies 37 27 , 38 . our results now suggest that mir - 29 acts as a critical repressor to regulate collagen expression in tendon healing . moreover its reduced expression in human biopsies suggests that its functional diminution permissively permits development of tendinopathy . despite tendon pathology being characterised by increased collagen 3 deposition resulting in biomechanical inferiority and degeneration the molecular premise for this collagen ‘ switch ’ has hitherto been unknown . we describe for the first time that il - 33 induced deficiency in mir - 29a results in an over - production of collagen 3 whilst simultaneously setting in motion , via sst2 inhibition of il - 33 , the ultimate resolution of this early repair process . contrary to expectations in human tenocytes , mir - 29 was only capable of influencing the expression of col 3a1 and not type 1 collagens . subsequent characterisation of the 3 ′ utr of type 1 and 3 collagens revealed a previously unreported pattern of alternative polyadenylation in both type 1 subunits , resulting in transcripts lacking mir29a binding sites rendering them insensitive to repression by this mirna . this was not the case for type 3 collagen transcripts , which retain both mir - 29a binding sites . in human tenocytes , collagen 3 is actively repressed by mir - 29a , as demonstrated by the ability of mir - 29a inhibitors to significant increase collagen 3 levels while supplementing tenocytes with mir - 29a in the presence of il - 33 was sufficient to inhibit the increase in collagen 3 production . importantly in our model system mir - 29a additionally targeted the il - 33 decoy receptor sst2 . thus il - 33 driven loss of mir - 29a expression results in the simultaneous repression of collagen 3 and sst2 , with a subsequent auto - regulatory inhibition of il - 33 promoting the resolution of the immediate alarmin response . based on this work we propose il - 33 as an influential alarmin in the unmet clinical area of early tendon injury and tendinopathy , which may be important in the balance between reparation and degeneration . a novel role for mir - 29 as a posttranscriptional regulator of matrix / inflammatory genes in tendon healing and tendinopathy has been uncovered . one of the great promises of exploiting mirnas for therapeutic purposes has been the potential of a single microrna to regulate functionally convergent target genes . our discovery of a single microrna dependent regulatory pathway in early tissue healing , highlights mir - 29 replacement therapy as a promising therapeutic option for tendinopathy with implications for many other human pathologies in which matrix dysregulation is implicated . while the invention has been described in conjunction with the exemplary embodiments described above , many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure . accordingly , the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting . various changes to the described embodiments may be made without departing from the spirit and scope of the invention . all documents cited herein are expressly incorporated by reference . 1 . eming , s . a ., krieg , t . & amp ; davidson , j . m . inflammation in wound repair : molecular and cellular mechanisms . j invest dermatol 127 , 514 - 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