Proteomic studies in Collagen Induced Arthritis (CIA) rats

V{tÑàxÜ@H Proteomic studies in Collagen Induced Arthritis (CIA) rats

5.1. Introduction Over the last few decades, both naturally occurring and genetically engineered animal models of human diseases have been employed for evaluating new therapeutic options and for elucidating pathological pathways. The biological analysis of animal models that mimic human diseases has led to a better understanding of numerous pathological mechanisms and is extremely helpful in the initial testing of new therapeutic strategies (Glasser and Nogee, 2006; Hansen and Khanna, 2004; Monnet and Chachques, 2005; Patel and Goldstein, 2004). Although the usage of human cell cultures can give highly relevant data with respect to therapeutic dosage and toxic side effects (Findikli et al., 2006) but whole animal studies still forms a central part of pharmacological and biomedical evaluations (Contag, 2002). In pharmacological studies, cell culture models cannot be employed to mimic the complexity of adsorption, distribution, first pass hepatic elimination, modification and excretion of a drug. Also organ selective toxicity can only be studied at the whole body level (Boyd et al., 1983). Once a suitable animal model of a disease has been established, proteomic screening can be used to identify new candidate proteins with an altered expression level. While genomic data only predict the effects of changes in gene expression relating to the actual concentration of proteins or the extent of PTMs, proteomics can accurately provide the status of protein abundance, isoform expression patterns and peptide modification. Once interesting candidate proteins have been found through animal model proteomics, an informed decision can be made on the optimum design of subsequent clinical studies. Thus, the initial discovery of new biomarkers by proteomic studies in 81

animal model may have the potential for improving diagnostic procedures in the short term and will be helpful in the establishment of novel therapeutic targets in the long term. Collagen induced arthritis (CIA), as described in details in chapter 1, is an experimental model of arthritis induced in susceptible strains of rats by immunization with heterologous collagen type II, a joint specific protein, in complete Freund s adjuvant (Trentham et al., 1977). CIA has been extensively studied to elucidate the pathological mechanisms relevant to human RA and to identify potential therapeutic targets. The development of CIA, as of RA, is thought to depend on T cells, and disease susceptibility is linked to the MHC region (Wooley et al., 1981). Following T cell activation an inflammatory cascade is triggered that involves T cells, macrophages/monocytes, B cells and activated synoviocytes. The different leukocytes and synovial cells produce a complex array of cytokines and other soluble mediators that are thought to be responsible for cartilage destruction and bone erosion (Yang et al., 1999). Very few reports are available where the proteomic profiling of various tissues of CIA mice have been done to study disease specific changes in the protein expression (Biswas et al., 2009; Lorenz et al., 2003). In this study, we have developed the rat CIA model using porcine collagen II and investigated the plasma proteome of CIA rat. WGA affinity column was used to enrich plasma glycoproteins followed by 2 DE of WGA bound as well as unbound plasma proteins to study the differential expression of proteins. The proteins were identified by MALDI TOF MS followed by antibody validation of the most differentially expressed protein. Further, the gene expression levels of the two important proteins were checked by RT PCR analysis. 82

Thus, this study was conducted with the objective to ascertain the establishment of disease model of RA at proteomic level which may be important for testing various therapeutic agents. 5.2 Results 5.2.1. Visual evaluation of arthritis in CIA Fig. 5.1. (A) Representative photographs of hind paws of (a) Control and (b) CIA rats. (B) Percent change in body weight of control and CIA rats with reference to day 12 (appearance of inflammation) till the day of euthanization (day 42 th ). Animal group immunized with collagen showed symptoms of disease progression like inflammation in joints within 12 16 days period (Fig. 5.1 A). The visual examination showed that there was significant loss of body weight in CIA rats compared with control rats (Fig. 5.1 B). Similarly, the severity of disease as measured by arthritic index (Fig. 5.2 A) and arthritis score (Fig. 5.2 B) was found to be significantly increased in CIA rats as compared to control rats. 83

5.2.2. Radiographic evaluation Typical radiographs of the hind limbs in each group were obtained on 42 nd day. Radiographic joint damage including bone erosion and loss of joint space was detected in X rays of hind limbs from CIA rats unlike those of control rats (Fig. 5.3 A) Fig. 5.2. (A) % mean arthritic index and (B) mean arthritic score of control and CIA rats till the day of euthanization (day 42). 5.2.3. Histopathological evaluation 84

Hematoxylin and eosin stained joint tissue slides were analyzed for cartilage destruction. Histological photomicrographs (Fig. 5.3 B) shows active synovitis and bone erosion in CIA (b) as compared to the control (a). Arthritic joints in CIA illustrate synovial hyperplasia, massive inflammatory cells infiltration (monomorphonuclear and polymorphonuclear cells), erosion of bone Fig. 5.3. (A) Representative radiographs of the hind limbs (showing the tibiotarsal and tibiofemoral joints) of control and CIA rats. Arrows indicate the bone damage in both tibiotarsal as well as tibiofemoral joints. (B) Representative histological figures (hematoxylin and eosin stained slides) of knee joints showing smooth and monolayer synovial linings and uniform synovial space of control rats. Hyperplastic synovial cells indicated by circle, erosion and disruption of synovial linings as indicated by arrows was observed in CIA rats. m=meniscus, f=fibula, js=joint space, t=tibila. 85

and cartilage. These histological analyses of hind limb joints were in agreement to the radiographic studies which demonstrate a significant joint destruction in CIA rats. 5.2.4. Proteomic analysis of plasma in CIA rats WGA affinity chromatography was used to enrich plasma glycoproteins and deplete high abundant proteins such as albumin. 2 DE profiling of WGA bound (Fig. 5.4) and unbound (Fig. 5.5) plasma proteins showed the presence of Fig. 5.4. A representative 2 DE image of WGA bound plasma of pooled control and CIA rats from each group. Three hundred microgram of protein was focused in ph 3 10 IPG strips followed by second dimensional separation in 12 % polyacrylamide gel. many protein spots, which were differentially present in the control and CIA rats. However, the protein spots, which were successfully identified by MS/MS analysis by MALDI TOF (Fig. 5.6), were given in Table 5.1. Most of the differentially expressed proteins were found to be positive acute phase proteins, such as orosomucoid (AGP), hemopexin, kininogen, and alpha 1 major acute 86

phase protein, which were found to be over expressed in the CIA rats. The changes in fold expression of each identified spot were given in Table 5.1. 5.2.5. Western blotting The expression level of spot no 16 identified as alpha 1 major acute phase protein or T kininogen 1, which was highly expressed in CIA rats, was further validated by 2 D Western blot analysis of plasma of rats. It is an Fig 5.5. A representative 2 DE image of flow through of WGA bound plasma of pooled control and CIA rat from each group. Three hundred microgram of protein was focused in ph 3 10 IPG strips followed by second dimensional separation in 12 % polyacrylamide gel. important inflammation related protein. The Western blot confirms the increased expression of T Kininogen 1 in CIA rats (Fig 5.7.) 5.2.6. Gene expression studies in CIA Estimation of m RNA levels of three genes in liver tissues of CIA and co 87

Fig. 5.6. Mascot search result showing the significance of the result and MS/MS sequence spectra of alpha 1 major acute phase protein. ntrol rats after euthanization on 42 th day, using semi quantitative RT PCR analysis (Fig. 5.8 A). The densitometric analysis revealed the ~ 3 fold increase, with respect to housekeeping gene GAPDH, in the level of kininogen mrna with statistical significance. Similarly, the level of clusterin mrna was found to be increased by ~1.2 fold in CIA albeit statistical significance (Fig. 5.8 B). 5.3. Discussion CIA is a widely used experimental model of polyarthritis that has many histopathological features in common with RA in human. It has been widely demonstrated that the disease progression in RA is reflected in the plasma concentration of acute phase proteins which are also denoted as disease biomarkers. It is considered that therapeutic agents that affect the acute phase 88

Fig 5.7. Validation of expression of alpha 1 major acute phase protein (T kininogen 1) in CIA rat by 2 DE Western blotting. protein profile of plasma in RA not only impart relief from the disease symptoms but can also ensure intervention in the disease progression itself. Therefore, we investigated the changes in the expression of plasma proteins in CIA rats. Since most of the plasma proteins are glycoproteins in nature, we studied both WGA bound as well as unbound plasma by 2 DE for complete proteomic profiling. WGA bound plasma showed the complete depletion of albumin which may otherwise interfere in the 2 DE profiling. Most of the differentially expressed proteins like Clusterin, AGP, hemopexin, alpha 1 major acute phase protein (T kininogen 1), are the acute phase glycoproteins. Kininogens are important precursor molecules for vasodilator peptide called bradykinin via plasma kallikrein kinin system (KKS) which participates in the pathogenesis of various inflammatory reactions like involved in cellular injury, coagulation, complement activation, cytokine secretion, release of proteases. It has been demonstrated that therapy with a specific plasma kallikrein inhibitor modulated the experimental arthritis and systemic inflammation (Isordia Salas et al., 2005). 89

Fig. 5.8. (A) Representative gel image of semi quantitative RT PCR amplified mrna of three genes namely Clusterin, Kinninogen, and GAPDH in liver tissues of control and CIA rat groups. (B) Densities of the above mrnas were calculated with respect to housekeeping gene GAPDH resulting in relative density values (IDV). Values are expressed as means ± S.D. (n=5 rats per group). Only expression of kininogen was found to be statistically significant (p<0.05). Earlier studies have shown that the total plasma level of kininiogen is increased to many folds in adjuvant induced arthritis. Analysis of the kininogens demonstrated that increase in T kininogen 1 was the major reason for the rise in total kininogen level. High molecular weight and low molecular weight kininogens showed little or no change (Barlas et al., 1985). In our study, we have also found the ~6 fold increase in the level of T kininogen 1 while very slight decrease in the level of LMW kininogen was observed in plasma of CIA. Clusterin has been reported to function in many inflammatory and 90

immunological processes, particularly, autoimmunity (Falgarone and Chiocchia, 2009). The expression level of clusterin, both at genomics and proteomics level has been detected in synovial tissues in RA patients and healthy controls and has been found to be decreased in RA patients (Devauchelle et al., 2006). AGP, as described in details in chapter 3, is one of the major acute phase proteins. It is synthesized in large quantities during acute phase reactions and accumulates at the vessel wall surface via binding to E selectin providing an anchoring template for plasminogen activator inhibitor type 1 (PAI 1) on the cellular surface and stabilizes its inhibitory activity toward plasminogen activators (Boncela et al., 2001). Its expression is almost twofold increased in CIA plasma which is in agreement with plasma studies in RA. Hemopexin or Beta 1B glycoprotein is an another important acute phase protein which has been reported to be increased in inflammation (Marinkovic et al., 1989) and adjuvant induced arthritic experimental animals (Saso et al., 1992). It functions as a heme scavenging molecule protecting cells from oxidative damage by free heme molecules released by hemolysis and preserves the iron level in the body. Low hemopexin level shows the degradation of hemoglobin which indicates hemolysis. Thus, low hemopexin level is one of the diagnostic features of a hemolytic anemia. 5.4 Conclusion Thus, the proteomic analysis of plasma proteins in CIA showed that this model mimics the disease i.e. RA at the proteomic level. This study can be very useful where the effects of different compounds are tested on the animal model of various diseases. Besides many biochemical and histochemical assays used for this study, the effects of these compounds can be checked on the expression levels of these plasma proteins. A study by Sahu et al has shown the effect of a 91

well known synthetic compound suramin on the level of few plasma acute phase proteins in CIA which were brought to normalization after treatment with suramin (Sahu et al., 2011). 92

Table 5.1. List of differentially expressed 2 DE protein spots in WGA bound and unbound plasma fractions of CIA as identified by MALDI TOF MS. Spot no Protein name *Accession number M.W./P.I Fold change Precursor ions Sequence of peptide matched 1 Immunoglobulin J chain precursor isoform gi 62660301 18,229/4.74 1.85 1721.8190 IIPSPEDPNEDIVER 100 2 Immunoglobulin J chain precursor isoform gi 62660301 18,229/4.74 1.89 1721.8634 IIPSPEDPNEDIVER 92 3 Complement C1q subcomponent, B chain gi 9506433 26,801/9.13 2.22 1432.6762, VITNVNDNYEPR, 72 precursor 1438.7827 TVNSALRPNQAIR 4 Complement C1q subcomponent subunit A gi 1168714 26,198/9.16 3.19 1256.7490, DQPRPAFSAIR, 153 1576.8938 QNPPTYGNVVVFDK 5 Preprohaptoglobin gi 204657 38,563/6.10 1.50 1372.6358 SCAVAEYGVYVR 54 6 Clusterin gi 461756 24,930/4.91 1.48 1800.9403 LTQQYNELLHSLQSK 42 7 Orosomucoid 1 precursor (AGP) gi 16757980 23,731/5.64 2.14 1884.8402 DVGMDESEIVFVDWTK 77 8 Ig gamma 2A chain C region gi 121052 50,268/7.52 79.0 1828.9760 VTCVVVDISQNDPEVR 43 9 Coagulation factor X gi 455396 18,322/8.80 1.50 1358.6198 DTYDFDIAMLR 67 10 Corticosteroid binding globulin gi 57527135 44,813/4.80 2.60 1544.8136, GIWELPFSPENTR, 181 1824.9833 GLAPTNVDFAFNLYQR 11 Alpha 1 major acute phase protein gi 205308 48,324/5.94 6.36 1815.8030 YNAELESGNQFLLYR 112 12 Leukemia inhibitory factor receptor alpha gi 4379217 123,808./5.56 2.72 1270.6306, WDDIPVEELR 180 chain 2341.1323 IASMEIPNDDITVEQAVGLGNR 13 Alpha 1 macroglobulin gi 21955142 168,422/6.46 82.17 1269.6417, AISYLISGYQR 112 1349.7096 ALLAYAFALAGNR 14 Beta 2 glycoprotein (Apolipoprotein H) gi 1351954 39,743/8.58 14.50 1529.7823, VCPFAGILENGVVR 106 2365.0318 TFYDPGEQIVYSCKPGYVSR 15 Hemopexin gi 1881768 51,351/7.58 1.52 2472.0557 LFQEEFPGIPYPPDAAVECHR 75 16 Fibrinogen beta chain gi 32527707 54,896/6.86 10.73 1902.063 LYIDETVNDNIPLNLR 101 17 Complement component 3 gi 158138561 187,825/6.06 5.91 1403.730, TIYTPGSTVFYR, 240 2454.3896 EPGQDLVVLSLPITPEFIPSFR 18 Transferrin precursor gi 61556986 78,090/7.14 2.81 1656.9152,2 KPVDQYEDCYLAR, 274 586.5103 LPEGTTYEEYLGAEYLQAVGNIR 19 Albumin precursor gi 124028612 70,670/6.09 3.19 1465.8009, LGEYGFQNAVLVR, 194 1609.8114 DVFLGTFLYEYSR 20 Transferrin gi 1854476 78,512/7.14 13.17 1392.7804, 1539.7327 LYLGHSYVTAIR, DQYELLCLDNTR 87 Ion score

21 Alpha 1B glycoprotein gi 84028169 57,127/5.68 5.55 1231.6548 VLNIQGFSPTR 65 22 Complement component 3 gi 158138561 187,825/6.06 4.85 1683.8276, VYSYYNLEESCTR, 220 2113.1965 ILLQGTPVAQMAEDAVDGER 23 Leukemia inhibitory factor receptor gi 7305235 122,394/5.56 1.50 2342.1396 IASMEIPNDDITVEQAVGLGNR 133 24 Murinoglobulin 1 precursor gi 12831225 166,590/5.68 3.72 1819.9276, 2721.4155 ALGCLEASWETIEQGR, SEGYLYTPQASSAEVEMSAYVVLAR 278 *NCBI accession number average M.W and p.i values as available in the NCBI database. fold expression values were calculated in CIA with respect to control and difference values 1.5 fold were considered. indicates up regulation and indicates down regulation of proteins, measured as ratio of spot densities of CIA/control.