Therapeutic Effect of a Recombinant betaig-h3 Fragment-RGD Peptide for Chronic Inflammatory Arthritis

OBJECTIVE: betaig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that betaig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with alphavbeta 3 integrin. We designed a recombinant betaig-h3 protein consisting of a fas-1 domain and RGD motif and evaluated the therapeutic efficacy in RA. METHODS: Inhibitory effect of adhesion and migration of NIH3T3 cell line was evaluated in 96 well microtiter and transwell plates coated with betaig-h3. Clinical arthritis index was evaluated after treating CIA mice with MFK12. Immunohistochemical staining in synovial tissues were performed. Expression of transcripts and proteins of inflammatory mediators were analyzed by semi-quantitative RT-PCR and immunoblotting. RESULTS: Recombinant protein consisted of 4th fas-1 domain truncated for H1 and H2 sequences and RGD peptide (MFK12), had M.W. of 10.4kDa. betaig-h3 mediated adhesion and migration of NIH3T3 cell line were significantly inhibited in a dose-dependent manner. Arthritis severity and incidence were efficiently reduced when CIA mice were treated with MFK12 at 30 mg/kg/day compared with the control. Immunohistochemical staining of joint tissues in MFK12 treated mice exhibited reduced angiogenesis. In treated mice, expression of transcripts regarding inflammatory mediators was markedly suppressed and immunoblotting of ICAM-1 and RANKL from whole extract of hind paws also showed a significant reduction. CONCLUSION: This study shows that MFK12 is effective in treating RA, although further study is warranted to improve the therapeutic efficacy.

Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.

Identification of TGF-beta-induced Gene Product, betaig-h3 in Ischemic Acute Renal Failure / 대한신장학회지

PURPOSE: Acute renal failure remains a potentially devastating clinical problem. This study aimed to examine whether the expression of TGF-beta-induced gene product, betaig-h3, is altered in ischemia- reperfusion (I/R) injury and urinary excretion of betaig-h3 is changed in I/R injury. METHODS: I/R injury was performed by clamping both renal arteries. Daily urine output, serum creatinine and urinary TGF-beta and betaig-h3 were measured after I/R injury. Also, the renal expression of betaig-h3 by western blotting and immunohistochemistry were investigated. In the second step, urinary betaig-h3 was measured at 4, 10, 16, and 24 hours after I/R injury to investigate whether it could be used as an early and sensitive marker for detecting I/R injury. RESULTS: Urinary betaig-h3 was significantly elevated at 24 hours and maintained higher than the controls until 2 days after I/R injury. In contrast, western blotting did not reveal any changes of betaig-h3 expression. Immunohistochemistry showed that labeling of betaig-h3 was seen at the basement membranes of proximal tubule cells mainly located at the medullary ray (S3 segment) in both groups. Following I/R injury, the labeling was also seen in the basement membrane of injured or regenerated proximal tubular epithelial cells. Within 24 hours, urinary betaig-h3 was significantly increased at 4 hours after I/R injury. Importantly, the urinary appearance of betaig-h3 preceded that of N-acetyl-beta-D-glucosaminidase. CONCLUSION: These results suggest that endogenous renal betaig-h3 may serve to promote tissue regeneration in I/R injury and urinary betaig-h3 could be used as an early and sensitive marker demonstrating I/R injury.

betaig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through alphavbeta5 integrin

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.

Expression of TGF-beta Induced Protein, betaig-h3, in Chronic Wound and It's Effect on Wound Healing

betaig-h3 is highly induced in A549 cells(human lung adenocarcinoma) after growth arrest by transforming growth factor-beta1. Although some reports describe variable functions of betaig-h3, it's function is in part controversial. betaig-h3 is a TGF-beta inducible cell adhesion molecule and some authors contend that recombinant betaig-h3 supports attachment and spreading of dermal fibroblast. Thus, it is expected that betaig-h3 plays a good role in wound healing. In order to investigate the effect of betaig-h3 in wound healing, The expression of TGF-beta and betaig-h3 in normal skin tissues and chronic wounds were examined by immunohistochemistry stain. In addition, 5 types of recombinant betaig-h3 were made, and among them, betaig-h3 b and betaig-h3 e were used for the experiment. Sprague- Dawley white rats served as the experimental models. Four round full-thickness skin defects of 2cm diameter were made on the back of each rat. The experiment was divided into 4 groups according to the content of ointment; group A: Topical application of ointment base only, group B: Topical application of ointment containing fibronectin 100microgram/ml, group C: Topical application of ointment containing betaig-h3 b 100 microgram/ml, group D: Topical application of ointment containing betaig-h3 e 100microgram/ml. In immunohistochemical study on normal human skin, the expression of TGF-beta was located in the upper dermal part, stratum basale, stratum spinosum and stratum granulosum in epidermis. The expression of betaig-h3 in normal dermis was similar with that of TGF-beta, but increased staining was observed in only stratum basale of normal epidermis. In chronic wounds, TGF-beta and betaig-h3 were highly expressed beneath the bottom and margin of wound and there were some cells showing positive staining within the nucleus. In animal models applied ointment, the reduction of wound size was more marked in group B, C, D than in group A. More rapid reepithelialization, less fibroplasia, less infiltration of inflammatory cells, more capillary formation in early period and more rapid collagen formation were seen in betaig-h3 treated wounds. In conclusion, betaig-h3 plays an important role in wound healing, and it is expected that recombinant betaig-h3 will become potent material for the treatment of chronic wound.

Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin

Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

Urinary Transforming Growth Factor-beta Induced Gene-h3 (betaig-h3)as a Marker of Lupus Activity in SLE with Nephritis / 대한류마티스학회지

BACKGROUND: TGF-beta-induced gene-h3 (betaig-h3) is a novel gene induced by active TGF-beta and the association with other renal disease is reported. Lupus nephritis is characterized by excessive extracelluar matrix accumulation and the implication that TGF-beta is increased in lupus nephritis is known. We measured the urinary betaig-h3 in lupus nephritis and sought its association with the activity of lupus nephritis through renal biopsy. The objective of this study was to examine urinary betaig-h3 excretion in lupus nephritis and the association with activity of lupus nephritis. METHODS: Fifteen patients (median age 32.6 2.9 years, range 18~64) who developed lupus nephritis underwent renal biopsy. At the time of biopsy, they showed significant proteinuria. Total urinary betaig-h3 concentration was assayed by enzyme-linked immunoabsorbent assay and expressed as a ratio to urinary creatinine concentration. RESULTS: There were correlations between urinary betaig-h3 and the reduction of C3 (r= 0.566, p=0.028<0.05), the magnitude of proteinuria (r=0.531, p=0.042<0.05). The Activity Index, Chronicity Index in the renal biopsy, C4, anti-dsDNA Ab titer were not significantly correlated with urinary betaig-h3 excretion, but the patients with high Activity Index had the increased level of urinary betaig-h3. Five patients who had fibrinoid necrosis in renal biopsy showed higher level of urinary betaig-h3 than the others (107.78 43.02 vs. 50.21 10.12 ng/ ml, p=0.061) CONCLUSION: In this study, There is some correlation between urinary betaig-h3 and the activity of lupus nephritis. Urinary betaig-h3 may play a role in predicting the active lupus nephritis. A further study is needed in large population and in situ expression of betaig-h3.

Effect of Chitosan, betaig-h3 and Human Bone Morphogenic Protein-4 on Early Bony Consolidation in Distraction Osteogenesis of the Canine Mandible

Sixteen dogs were used to study the effect of bone morphogenic protein(BMP-4), betaig-h3 and chitosan during the early bony consolidation stage in the distracted zones of mandibles. The lateral surface of the mandibular body was exposed in the subperiosteal plane and vertical osteotomy was carried out on the mandibular body. An external distraction device was applied to the mandibular body about 1 cm apart from the osteotomy line. Mandibular distraction was started 5 days after the mandibular osteotomy at a rate of 2 mm per day for a total of 10 mm distraction for 5 days. The experimental group was divided into 4 groups: control group, BMP-4 group, betaig-h3 group and chitosan group depending on the injected material into the distracted area. Four dogs were allocated to each group. On the day of completion of distraction, 0.5 ml of BMP-4, 0.5 ml of betaig-h3, 0.5 ml of 5% chitosan solution was injected respectively into the distracted area of each group with the same amount of tripolyphosphate in dual syringe for solidification of the injected solution. In the control group, 1 ml of tripolyphosphate was injected into the distracted area. After injection of the study materials, the distraction device was left in place for 4 or 7 weeks to allow bony consolidation. Radiographs were taken weekly. Two dogs in each group, a total of eight dogs, were sacrified in 4 weeks, and another eight dogs in 7 weeks after completion of distraction. Bone specimens of the distracted mandibles were taken for histologic examination. The mineral density of the distracted bone was measured during the radiological procedures and analysed by the computer. In the radiographs of the distracted areas of the mandibles, the control group has shown a mostly radiolucent zone but the other groups have shown the radiodense zones with various width of central radiolucent zones. The central radiolucent zone became narrower in time and vertical thickness of the radiodense zone was about twice thicker in 7 weeks than that of 4 weeks after finishing bone distraction. BMP-4 group showed the thickest radiodense zone and the chitosan group shows the thinnest radiodense zone. The mineral density of bone was highest in the BMP-4 group and lowest in the control group. In the histological findings of the distracted areas of mandibles, the control group showed whole fibrous tissue but the other groups showed new woven bones with central narrow fibrous interzone. The degree of new bone formation was most remarkable in the BMP-4 group and was least remarkable in the chitosan group. In conclusion, there was an active formation of a new bone in the distracted area of the mandible by injection of BMP-4, betaig-h3 and chitosan. The new bone formation was most remarkable in the BMP-4 group followed by betaig-h3, chitosan and control group. These findings suggest that BMP-4 is clinically worth using for early bony consolidation in the distraction osteogenesis.