Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

OBJECTIVES: To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). METHODS: HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. RESULTS: The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. CONCLUSION: Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

Biological therapies in Crohn's disease: are they cost-effective? A critical appraisal of model-based analyses.

In refractory Crohn's disease, anti-TNF and anti-α 4 integrin agents are used for ameliorating disease activity but impose high costs to health-care systems. The authors systematically reviewed cost-effectiveness analyses based on decision models: most of the studies were judged to have a good quality, but a large portion assessed health and costs in a short time horizon, usually disregarding fistulizing disease and not considering safety. Infliximab induction followed by on-demand retreatment consistently proved to have a good cost per quality-adjusted life year, while maintenance treatment never satisfied commonly accepted cost-utility thresholds. Challenges in cost-effectiveness analysis include the lack of a standard model structure, a large variability in the costs of surgery and poor data on indirect costs. As clinical practice is moving to mucosal healing as a robust response marker, personalized schedules of anti-TNF therapies might prove cost-effective even in the perspective of the health-care system in the near future.

Efficacy and safety of natalizumab in multiple sclerosis: interim observational programme results.

BACKGROUND: Clinical trials established the efficacy and safety of natalizumab. Data are needed over longer periods of time and in the clinical practice setting. OBJECTIVE: To evaluate long-term safety of natalizumab and its impact on annualised relapse rate and Expanded Disability Status Scale (EDSS) progression in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: The Tysabri (natalizumab) Observational Program (TOP) is an open-label, multinational, 10-year prospective study in clinical practice settings. RESULTS: In this 5-year interim analysis, 4821 patients were enrolled. Follow-up for at least 4 years from natalizumab commencement in 468 patients and at least 2 years in 2496 patients revealed no new safety signals. There were 18 cases of progressive multifocal leucoencephalopathy reported, following 11-44 natalizumab infusions. Mean annualised relapse rate decreased from 1.99 in the 12 months prior to baseline to 0.31 on natalizumab therapy (p<0.0001), remaining low at 5 years. Lower annualised relapse rates were observed in patients who used natalizumab as first MS therapy, in patients with lower baseline EDSS scores, and in patients with lower prenatalizumab relapse rates. Mean EDSS scores remained unchanged up to 5 years. CONCLUSIONS: Interim TOP data confirm natalizumab's overall safety profile and the low relapse rate and stabilised disability levels in natalizumab-treated patients with RRMS in clinical practice. TRIAL REGISTRATION NUMBER: NCT00493298.

Oral treatment with a novel small molecule alpha 4 integrin antagonist, AJM300, prevents the development of experimental colitis in mice.

BACKGROUND AND AIMS: Inhibition of lymphocyte trafficking by treatment with an anti-α4 integrin antibody has been clinically validated as a therapeutic approach for inflammatory bowel disease (IBD), and the orally effective 'anti-α4 integrin therapy' may be more convenient in clinical practice. The aim of this study was to investigate the pharmacological profile and anti-inflammatory effect of a novel, orally active small molecule α4 integrin antagonist, AJM300. METHODS: The binding specificity/potency of HCA2969 (the active metabolite of AJM300) were investigated in vitro. The pharmacodynamics for α4 integrin antagonism of AJM300 was investigated in mice. The anti-inflammatory effect of AJM300 fed in a diet and the anti-α4 integrin monoclonal antibody was evaluated in a mouse colitis model induced by transfer of IL-10 deficient T cells. RESULTS: HCA2969 selectively inhibited the in vitro binding of α4 integrin (α4ß7/α4ß1) to the cell adhesion molecules. Oral treatment with AJM300 dose-dependently inhibited lymphocyte homing to Peyer's patches and increased the peripheral lymphocyte count in the same dose range. AJM300 dose-dependently prevented the development of experimental colitis in mice. A significant inhibition of colon weight increase was accompanied by inhibition of T-cell infiltration into the lamina propria of colon. The maximum efficacy of AJM300 (1% diet) was comparable to that achieved by the saturated α4 integrin blockade with antibody. CONCLUSIONS: Oral treatment with the selective small molecule α4 integrin antagonist (AJM300) prevented the development of colitis and its efficacy was comparable to that of the anti-α4 integrin antibody.

Efficacy of biological therapies in inflammatory bowel disease: systematic review and meta-analysis.

OBJECTIVES: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory disorders of the gastrointestinal tract of unknown etiology. Evidence for treatment of the condition with biological therapies exists, but no systematic review and meta-analysis has examined this issue in its entirety. METHODS: MEDLINE, EMBASE, and the Cochrane central register of controlled trials were searched (through to December 2010). Trials recruiting adults with active or quiescent CD or UC and comparing biological therapies (anti-tumor necrosis factor-α (TNFα) antibodies or natalizumab) with placebo were eligible. Dichotomous symptom data were pooled to obtain relative risk (RR) of failure to achieve remission in active disease and RR of relapse of activity in quiescent disease once remission had occurred, with a 95% confidence interval (CI). RESULTS: The search strategy identified 3,061 citations, 27 of which were eligible. Anti-TNFα antibodies and natalizumab were both superior to placebo in inducing remission of luminal CD (RR of no remission=0.87; 95% CI 0.80-0.94 and RR=0.88; 95% CI 0.83-0.94, respectively). Anti-TNFα antibodies were also superior to placebo in preventing relapse of luminal CD (RR of relapse=0.71; 95% CI 0.65-0.76). Infliximab was superior to placebo in inducing remission of moderate to severely active UC (RR=0.72; 95% CI 0.57-0.91). CONCLUSIONS: Biological therapies were superior to placebo in inducing remission of active CD and UC, and in preventing relapse of quiescent CD.

Aberrant DNA methylation of integrin alpha4: a potential novel role for metastasis of cholangiocarcinoma.

PURPOSE: Although the altered expression of integrin alpha4 is known to be associated with transformation or metastasis in several human cancers, the information on cholangiocarcinoma (CC) is still poor. In this study, we investigated the promoter methylation status of integrin alpha4 gene in CC. METHODS: A total of 29 CC, 19 adjacent non-tumor-containing tissue and 15 normal liver specimens were used for identification of gene methylation status by methylation-specific polymerase chain reaction. RESULTS: The frequency of DNA methylation was 55.17% (16 of 29) in the CC specimens (P < 0.001). Also, transcripts of the integrin alpha4 gene were decreased in all CC tissues in which there was DNA methylation of the integrin alpha4 gene. In addition, the downregulated expression of integrin alpha4 in CC cells with hypermethylation of the integrin alpha4 gene was restored by treatment with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. Moreover, we found that DNA methylation of integrin alpha4 was detected in all CC tissues obtained from patients with LN metastasis (7/7). Furthermore, phosphorylation of paxillin, cell migration-related molecule, was regulated by silencing of integrin alpha4. CONCLUSIONS: Taken together, these results suggest that loss of the integrin alpha4 gene is caused by aberrant DNA methylation of the 5'-CpG island site of the gene, and methylation of the integrin alpha4 gene can be a useful marker of metastasis of CC.

Evidence for a regulatory role of alpha 4-integrins in the maturation of eosinophils generated from the bone marrow in the presence of dexamethasone.

BACKGROUND: Although eosinophils co-express multiple integrin receptors, the contributions of integrins to eosinophil development have not been explored. We previously described extensive aggregation and cytological immaturity in eosinophils developing in bone-marrow (BM) cultures exposed to dexamethasone. Here we examined the relationship of alpha 4 integrins with these effects of dexamethasone. OBJECTIVES: We evaluated: (a) the effects of exposure to dexamethasone in BM culture on eosinophil expression of alpha 4 integrin receptors and ligands; (b) the contribution of alpha 4 integrins to eosinophil aggregation and maturation. METHODS: Cultures were established with IL-5 (alone or with dexamethasone) for up to 7 days, and eosinophil production, alpha 4 integrin receptor/ligand expression, aggregation and morphology were evaluated before and after targeting alpha 4 integrin-dependent adhesions. Because prostaglandin E2 (PGE2) modifies the effects of dexamethasone on eosinophilopoiesis, PGE2 effects on alpha 4 integrin expression and function were also evaluated. RESULTS: Dexamethasone increased the yield of eosinophils up to day 7. The frequency of eosinophils expressing alpha 4, beta1 and beta 7 integrin receptors at day 7 was also increased by dexamethasone. Eosinophils also expressed the alpha 4 beta 1 ligand, VCAM-1. Dexamethasone increased the expression of alpha 4 integrin and VCAM-1 in aggregates containing eosinophils as early as day 3. PGE2, added up to day 3, modified the effects of dexamethasone to suppress the expression of alpha 4 integrin, decrease aggregation and promote cytological maturation of eosinophils recovered at day 7. Dissociation of immature eosinophils from clusters present at day 3 by reagents targeting alpha 4 or beta1 integrins or VCAM-1 also induced cytological maturation. The concordant effects of targeting alpha 4 integrins with drugs and antibodies support a relationship between alpha 4-mediated aggregation and maturational arrest. CONCLUSIONS: These observations support a novel role for alpha 4 integrin receptors and ligands in eosinophilopoiesis. In addition, increased alpha 4 expression following glucocorticoid exposure may contribute to the retention and accumulation of eosinophils in haemopoietic tissue.

Effect of plasma exchange in accelerating natalizumab clearance and restoring leukocyte function.

BACKGROUND: Accelerating the clearance of therapeutic monoclonal antibodies (mAbs) from the body may be useful to address uncommon but serious complications from treatment, such as progressive multifocal leukoencephalopathy (PML). Treatment of PML requires immune reconstitution. Plasma exchange (PLEX) may accelerate mAb clearance, restoring the function of inhibited proteins and increasing the number or function of leukocytes entering the CNS. We evaluated the efficacy of PLEX in accelerating natalizumab (a therapy for multiple sclerosis [MS] and Crohn disease) clearance and alpha4-integrin desaturation. Restoration of leukocyte transmigratory capacity was evaluated using an in vitro blood-brain barrier (ivBBB). METHODS: Twelve patients with MS receiving natalizumab underwent three 1.5-volume PLEX sessions over 5 or 8 days. Natalizumab concentrations and alpha4-integrin saturation were assessed daily throughout PLEX and three times over the subsequent 2 weeks, comparing results with the same patients the previous month. Peripheral blood mononuclear cell (PBMC) migration (induced by the chemokine CCL2) across an ivBBB was assessed in a subset of six patients with and without PLEX. RESULTS: Serum natalizumab concentrations were reduced by a mean of 92% from baseline to 1 week after three PLEX sessions (p < 0.001). Although average alpha4-integrin saturation was not reduced after PLEX, it was reduced to less than 50% when natalizumab concentrations were below 1 mug/mL. PBMC transmigratory capacity increased 2.2-fold after PLEX (p < 0.006). CONCLUSIONS: Plasma exchange (PLEX) accelerated clearance of natalizumab, and at natalizumab concentrations below 1 mug/mL, desaturation of alpha4-integrin was observed. Also, CCL2-induced leukocyte transmigration across an in vitro blood-brain barrier was increased after PLEX. Therefore, PLEX may be effective in restoring immune effector function in natalizumab-treated patients.

A small molecule inhibitor of alpha4 integrin-dependent cell migration.

A small molecule inhibitor of alpha4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with gamma-parvin. This molecule should serve as a useful tool to study alpha4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune diseases.

Increased serum fractalkine in systemic sclerosis. Down-regulation by prostaglandin E1.

OBJECTIVES: To evaluate serum levels of fractalkine (FKN), a mediator of leukocyte transmigration, C-reactive protein (CRP) and expression of integrins CD11a and CD49d on peripheral blood lymphocytes in systemic sclerosis (SSc) and to investigate whether they are modulated by intravenous prostaglandin E1 (PGE1). METHODS: Serum levels of fractalkine and C-reactive protein and expression of CD11a and CD49d on peripheral blood lymphocytes were assessed in 50 SSc patients and in 18 healthy controls. In 25 SSc patients studied parameters were evaluated also after 3 consecutive daily PGE1 infusions (20 microg-40 microg-60 microg) and after 4 weeks. RESULTS: In SSc fractalkine basal level was significantly higher than in controls (9.04+/-1.79 ng/ml vs. 1.17+/-0.1 ng/ml; p<0.0001) and decreased significantly after PGE1 (5.16+/-1.27 ng/ml, p<0.05). After four weeks fractalkine level was still significantly lower than baseline 7.70+/-2.19 ng/ml (p<0.05). Basal percentage of CD11a (+) nor CD49d (+) lymphocytes in SSc (82.38+/-1.60%, 70.74+/-1.68%, respectively) did not differ from controls (85.73+/-2.04%, 75.62+/-2.48%; respectively, p>0.05). PGE1 treatment resulted in decrease of both CD11a (+) (67.72+/-3.34%, p<0.0001) and CD49d (+) lymphocytes (65.32+/-1.62%, p<0.0001). After 4 weeks the percentage of CD11a (+) and CD49d (+) lymphocytes remained significantly lower than at baseline (77.80+/-2.47% and 65.32+/-1.62%, respectively, both p<0.001). In SSc CRP basal level was significantly higher than in controls (4.70+/-2.01 mg/dl vs. 1.40+/-1.79 mg/dl, p<0.005) and reduced significantly after PGE1 (3.39+/-2.06 mg/dl, p<0.05). After 4 weeks, CRP level (4.38+/-2.19 ng/ml) was significantly lower than baseline (p<0.05). CONCLUSION: Fractalkine may play an important role in the pathogenesis of vascular dysfunction in systemic sclerosis. Prostaglandin E1 down-regulates serum fractalkine level, as well as CD11a and CD49d expression on peripheral blood lymphocytes, which suggests additional mechanisms in which this vasodilatatory agent exerts its efficacy in systemic sclerosis.

Inhibition of alpha(4) integrin mediated adhesion was involved in the reduction of B16-F10 melanoma cells lung colonization in C57BL/6 mice treated with gambogic acid.

Gambogic acid, the major active ingredient of gamboge, has been shown to exhibit anti-cancer activity both in vivo and in vitro. However, its potential activity in tumor metastasis remains unclear. In the present study, we found that gambogic acid strongly inhibited the adhesion of highly metastatic mouse melanoma B16-F10 cells in vitro. Gambogic acid also exhibited significant anti-metastasis activity on the development of in vivo artificial metastases (i.e. following tail vein i.v. injection of the B16-F10 melanoma tumor cells in C57BL/6 mice). Flow cytometric analysis and Western blot showed that gambogic acid inhibited the cell surface expression of alpha(4) integrin, while exhibited negligible effects on the expression of alpha(5) and beta(1) integrin. Thus we concluded for the first time that gambogic acid could inhibit the adhesion and migration of B16-F10 cells in vitro and in vivo, in which down-regulation of alpha(4) integrin expression was involved.

Blockade of alpha4 integrin signaling ameliorates the metabolic consequences of high-fat diet-induced obesity.

OBJECTIVE: Many prevalent diseases of advanced societies, such as obesity-induced type 2 diabetes, are linked to indolent mononuclear cell-dependent inflammation. We previously proposed that blockade of alpha4 integrin signaling can inhibit inflammation while limiting mechanism-based toxicities of loss of alpha4 function. Thus, we hypothesized that mice bearing an alpha4(Y991A) mutation, which blocks signaling, would be protected from development of high-fat diet-induced insulin resistance. RESEARCH DESIGN AND METHODS: Six- to eight-week-old wild-type and alpha4(Y991A) C57Bl/6 male mice were placed on either a high-fat diet that derived 60% calories from lipids or a chow diet. Metabolic testing was performed after 16-22 weeks of diet. RESULTS: Alpha4(Y991A) mice were protected from development of high-fat diet-induced insulin resistance. This protection was conferred on wild-type mice by alpha4(Y991A) bone marrow transplantation. In the reverse experiment, wild-type bone marrow renders high-fat diet-fed alpha4(Y991A) acceptor animals insulin resistant. Furthermore, fat-fed alpha4(Y991A) mice showed a dramatic reduction of monocyte/macrophages in adipose tissue. This reduction was due to reduced monocyte/macrophage migration rather than reduced monocyte chemoattractant protein-1 production. CONCLUSIONS: Alpha4 integrins contribute to the development of HFD-induced insulin resistance by mediating the trafficking of monocytes into adipose tissue; hence, blockade of alpha4 integrin signaling can prevent the development of obesity-induced insulin resistance.

Natalizumab: targeting alpha4-integrins in multiple sclerosis.

In 1992, it was shown that monoclonal antibodies blocking alpha(4)-integrins prevent the development of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (MS). As alpha(4)beta(1)-integrin was demonstrated to mediate the attachment of immune-competent cells to inflamed brain endothelium in experimental autoimmune encephalomyelitis, the therapeutic effect was attributed to the inhibition of immune cell extravasation and inflammation in the central nervous system. This novel therapeutic approach was rapidly and successfully translated into the clinic. The humanized anti-alpha(4)-integrin antibody natalizumab demonstrated an unequivocal therapeutic effect in preventing relapses and slowing down the pace of neurological deterioration in patients with relapsing-remitting MS in phase II and phase III clinical trials. The occurrence of 3 cases of progressive multifocal leukoencephalopathy in patients treated with natalizumab led to the voluntary withdrawal of the drug from the market. After a thorough safety evaluation of all patients receiving this drug in past and ongoing studies for MS and Crohn's disease, natalizumab again obtained approval in the US and the European Community. A treatment targeting leukocyte trafficking in MS has now re-entered the clinic. Further thorough evaluation is necessary for a better understanding of the risk-benefit balance of this new treatment option for relapsing MS. In this review, we discuss the basic mechanism of action, key clinical results of clinical trials and the emerging indication of natalizumab in MS.

Connexin 43 gap junction proteins are up-regulated in remyelinating spinal cord.

Alterations in the expression of gap junction proteins have previously been observed in several diseases affecting the central nervous system; however, the status of connexin 43 (Cx43) has not yet been reported in spinal cord remyelination. We studied Cx43 expression in demyelination and remyelination by using a chronic guinea pig model of experimental allergic encephalomyelitis (EAE). Hartley guinea pigs were immunized with homogenized whole CNS and complete Freund's adjuvant. Animals became chronically ill by day 40 postimmunization, and animals with paralysis were entered into the study. Animals were treated on days 40-60 postimmunization with either saline or drugs that promote remyelination: an adenosine amine congener (100 mug/kg), an anti-alpha4-integrin blocker (CT301; ELN 69299; 30 mg/kg), or a combination of both drugs. Remyelination was induced in all drug-treated groups. Cx43 expression was virtually absent in demyelinated lesions of saline-treated controls compared with healthy tissue and normal appearing white matter (P < 0.001), whereas Cx43 was considerably increased (300-500%) in remyelinating lesions of all treatment groups (P < 0.001), most notably in CT301-treated animals. These changes in Cx43 expression indicate that Cx43 may beimportant for recovery from neuroinflammation.