A platform method for charge heterogeneity characterization of fusion proteins by icIEF.

The charge heterogeneity of fusion proteins can vary dramatically compared with more traditional biopharmaceuticals like monoclonal antibodies, making the characterization of fusion proteins a challenge. A single platform method suitable for the analysis of multiple fusion proteins would reduce method development and streamline production workflows. Here, we develop a platform method to characterize the charge heterogeneity of a variety of fusion protein therapeutics using imaged capillary isoelectric focusing (icIEF). We describe the development of the platform method, and analyze 9 fusion protein therapeutics. The results are reproducible in peak group area percentage and apparent pI determination. We compare the platform icIEF method to traditional slab gel IEF, which is still used in many laboratories for the analysis of fusion proteins. The peak patterns obtained from the icIEF method is comparable to the band patterns of the gel IEF. The platform method can also be used as the starting point if further optimization is needed even when high resolution is required. The platform method described in this study can be applied as an identity and purity assay for fusion proteins in the biopharmaceutical industry.

Different effects of corticotropin-releasing factor and urocortin 2 on apoptosis of prostate cancer cells in vitro.

Urocortin (Ucn), a corticotropin-releasing factor (CRF)-related neuropeptide binding both CRF type 1 receptor (CRFR1) and CRFR2, has recently been found in prostate cancer. However, no report has yet been known to elucidate the roles of Ucn in prostate cancer via the two receptors. In this study, the expression of both CRFR1 and CRFR2 in the mouse prostate cancer cell line RM-1 were detected and cellular apoptosis was monitored in the presence of CRF or Ucn2, the CRFR1- and CRFR2-selective agonist respectively. CRF promoted apoptosis while Ucn2 exerted the opposite effect. CRF reduced Bcl-2 expression, induced Bax expression, and hyperpolarized the mitochondrial membrane potential to activate caspase-9. On the contrary, Ucn2 increased Bcl-2 expression and decreased Bax expression, in which phosphorylation of Akt and cyclic AMP response element-binding (CREB) was involved. Pretreatment with phosphatidylinositide 3-kinase/Akt inhibitor (LY-294002) prior to Ucn2 led to downregulation of CREB phosphorylation and hence reduced Bcl-2 expression. These effects of CRF and Ucn2 were abolished by antalarmin (Anta) and antisauvagine-30, the CRFR1- and CRFR2-selective antagonist respectively. In LNCaP cell line, similar effects on cell apoptosis by CRF and Ucn2 were observed. In summary, our results demonstrated CRFR1 and CRFR2 expression in prostate cancer and indicated the opposite apoptotic roles of the two different CRFRs. These data may contribute to uncovering the pathophysiological function of endogenous Ucn in prostate tumorigenesis and progression.

Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells.

BACKGROUND AND PURPOSE: Our previous study showed that urocortin (Ucn1) exacerbates the hypercoagulable state and vasculitis in a rat model of sodium laurate-induced thromboangiitis obliterans. Furthermore, the inflammatory molecules COX-2 and ICAM-1 may participate in this effect. In the present study, the effects of Ucn1 on COX-2 and ICAM-1 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) were investigated and the mechanisms involved explored. EXPERIMENTAL APPROACH: RAECs were isolated from adult male Wistar rats, and identified at the first passage. Experiments were performed on cells, from primary culture, at passages 5-8. The expression of COX-2 and ICAM-1 at both mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blot analysis. Levels of PGE(2) and soluble ICAM-1 (sICAM-1) in culture medium were measured by enzyme-linked immunosorbent assay. Furthermore, the phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-kappaB was analysed by Western blot; nuclear translocation of NF-kappaB was observed by immunofluorescence. KEY RESULTS: Ucn1 augmented LPS-induced expression of COX-2 and ICAM-1 in RAECs in a time- and concentration-dependent manner. Ucn1 increased PGE(2) and sICAM-1 levels. These effects were abolished by the CRF(2) receptor antagonist, antisauvagine-30, but not by the CRF(1) receptor antagonist, NBI-27914. Moreover, Ucn2 activated p38MAPK and augmented NF-kappaB nuclear translocation and phosphorylation, whereas ERK1/2, JNK and Akt pathways were not involved in this process. CONCLUSIONS AND IMPLICATIONS: These findings suggest that Ucn1 exerts pro-inflammatory effects by augmenting LPS-induced expression of COX-2 and ICAM-1 in RAECs via CRF(2) receptors and the activation of p38MAPK and NF-kappaB.

Urocortin promotes the development of vasculitis in a rat model of thromboangiitis obliterans via corticotrophin-releasing factor type 1 receptors.

BACKGROUND AND PURPOSE: Urocortin is a locally expressed pro-inflammatory peptide. Here we have examined the effects of urocortin on sodium laurate-induced peripheral arterial vasculitis in rats, modelling the mechanisms of thromboangiitis obliterans (TAO). EXPERIMENTAL APPROACH: Peripheral vasculitis in rats was induced by sodium laurate and graded by gross appearance on the 12th day after injection. Histological changes in rat femoral arteries were assessed by histopathology and transmission electron microscopy. Blood cell counts, blood rheology, blood coagulation and plasma urocortin, thromboxane B(2), prostaglandin E(2) and soluble intercellular adhesion molecule-1 levels were measured. Expression of urocortin, corticotrophin-releasing factor (CRF(1/2)) receptors, cyclooxygenase (COX)-2 and intercellular adhesion molecule-1 (ICAM-1) at both mRNA and protein levels were determined by RT-PCR and Western blot. KEY RESULTS: Rats showed grossly visible signs and symptoms of TAO on the 12th day after sodium laurate injection. In these rats, blood was in a hypercoagulable state; plasma urocortin, prostaglandin E(2) and soluble intercellular adhesion molecule-1 levels were elevated; and the expression of urocortin, CRF(1) and CRF(1alpha)-receptors, COX-2 and ICAM-1 in rat femoral arteries were markedly increased. Exogenous urocortin, given for 12 days after sodium laurate, exacerbated the hypercoagulable state and augmented expression of CRF(1alpha)-receptors, COX-2 and ICAM-1. These effects were abolished by a CRF(1)-receptor antagonist, NBI-27914, or a non-selective CRF-receptor antagonist, astressin, but not by the CRF(2)-receptor antagonist, antisauvagine-30, given with exogenous urocortin. CONCLUSION AND IMPLICATIONS: Urocortin exacerbated the hypercoagulable state and vasculitis in a model of TAO induced by sodium laurate in rats, via CRF(1)-receptors. COX-2 and ICAM-1 might also have contributed to this exacerbation.

Urocortin inhibits mesenteric arterial remodeling in spontaneously hypertensive rats.

Urocortin (UCN), a newly isolated corticotrophin-releasing factor (CRF) related peptide, has been found to have potent cardiovascular protective effects. This study aimed to investigate the long-term effects of UCN on arterial remodeling and related functional alterations. UCN (7 microg/kg/d) was administered to spontaneously hypertensive rats (SHR) for 8 weeks. Systolic blood pressure (SBP) was measured weekly. Functional studies were performed on isolated mesenteric arterial segments. Also, by light microscope and electron microscope, the morphology of mesenteric arteries was examined. Our results showed that mean SBP in UCN-treated SHRs was about 40 mm Hg lower than that of the control SHR group, and was similar to that of the enalapril-treated group. In the mesenteric arterial segments pre-contracted with norepinephrine (0.001-10 microM), the maximal relaxation rate induced by acetylcholine (10 microM) in UCN-treated group (about 93.3%) was higher than that in SHR control group (about 40.0%) (n=6, P<0.01). Furthermore examination under light microscope showed that UCN (3.5 microg/kg/d) treatment significantly reduced media thickness, media/lumen ratio, resulting in larger lumen diameter while analysis of transmission electron microscopic findings revealed that chromatin, internal elastic lamina and densely packed mitochondria displayed a close-to-normal distribution after UCN treatment. These results suggested that long-term UCN treatment not only had hypotensive effects but may also inhibited development of vascular remodeling in mesenteric arteries in SHR.

COX-2 as a novel target of CRF family peptides' participating in inflammation.

In mammals, corticotropin-releasing factor (CRF) family peptides include CRF, Urocortin (Ucn) 1, Ucn2, and Ucn3. In contrast to their systemic indirect immunosuppressive effects on the hypothalamic-pituitary adrenal axis, CRF family peptides act as locally expressed autocrine or paracrine pro-inflammatory factors in a series of inflammatory diseases. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in metabolism of arachidonic acid, has been abundantly reported to take part in inflammatory diseases. Recently, reports indicate that CRF family peptides may play an important role in the regulation of COX-2 under inflammatory conditions. Moreover, CRF receptors are involved in this process. This review aims to highlight the current novel findings on regulation of COX-2 by CRF family peptides in inflammation. Furthermore, the relevant mechanisms are discussed.

Enhanced expression of vascular cell adhesion molecule-1 by corticotrophin-releasing hormone contributes to progression of atherosclerosis in LDL receptor-deficient mice.

Peripherally produced corticotrophin-releasing hormone (CRH) is a strong proinflammatory factor involved in many inflammatory diseases. However, to date, there is no evidence about the action of CRH on atherosclerosis, a chronic disease characterized by inflammatory reactions. In this study we observed the effect of CRH on atherosclerosis in low-density lipoprotein receptor-deficient (LDLr-/-) mice. Twelve-week-old, male LDLr-/- mice were subcutaneously injected with CRH (10microg/kg) or vehicle once a day for 8 weeks. The results indicated aortic atherosclerotic lesions were larger (P<0.01) in CRH-treated mice than those in untreated mice. CRH significantly up-regulated the expression of both protein and mRNA for vascular cell adhesion molecule-1 (VCAM-1), together with a markedly increased activation of nuclear factor kappa B (NF-kappaB) in aortas. In addition, the blood lipid levels were not influenced by CRH subcutaneous injection. The significant proatherogenic effect of CRH in LDLr-/- mice was largely attenuated by selective CRH receptor 1 (CRHR1) antagonist NBI27914 but not by specific CRH receptor 2 (CRHR2) antagonist antisauvagine-30 (anti-Svg-30). Meanwhile, both the enhanced expression of VCAM-1 and increased activation of NF-kappaB induced by CRH in aortas of LDLr-/- mice were also largely suppressed by NBI27914, whereas these inhibitory effects were not observed in anti-Svg-30 group. Taken together, these findings indicated that CRH may accelerate atherosclerosis progression in LDLr-/- mice via CRHR1. The enhanced VCAM-1 expression which probably resulted from increased activation of NF-kappaB induced by CRH, may be one of the important molecular mechanisms by which CRH accelerates atherosclerosis. This study provides a new insight into the effect of CRH on atherosclerosis and suggests a potential target for the prevention and treatment of atherosclerosis.

Genistein inhibits the development of atherosclerosis via inhibiting NF-kappaB and VCAM-1 expression in LDLR knockout mice.

Diet can be an important factor that influences risks for cardiovascular disease. Genistein (4',5,7-trihydroxyisoflavone), rich in soy, is one candidate that may benefit the cardiovascular system. Here, we explored the effect of genistein in atherosclerosis (AS) development in an in vivo mouse model. Low-density lipoprotein receptor (LDLR) knockout mice were allocated to control, model, and genistein groups. Our results showed that genistein significantly reduced the formation and development of atherosclerotic plaques ((4.68 +/- 1.18) x106 versus (6.65 +/- 1.51) x106 microm2, p < 0.05). In the genistein group, compared with the model group, total antioxidant capacity (TAC) level was 85.5 +/- 15.6 versus 203.4 +/- 32.6 mmol/L (p < 0.01); malondialdehyde (MDA) level was 3.79 +/- 0.28 versus 3.06 +/- 0.31 mmol/L (p < 0.01), and superoxide dismutase (SOD) activity was 86.1 +/- 6.1 versus 139.1 +/- 25.1 U/mL (p < 0.01). Therefore, genistein was able to enhance serum antioxidative ability in our mouse model. Genistein had no influence, however, on serum cholesterol and lipid profiles. Genistein also markedly downregulated the expression of nuclear factor (NF)-kappaB and vascular cell adhesion molecule (VCAM)-1 in aortas of mice (p < 0.05). These observations suggest that genistein may inhibit AS in LDLR-/- mice via enhancing serum antioxidation and downregulating NF-kappaB and VCAM-1 expression in the aorta.

Urocortin's inhibition of tumor growth and angiogenesis in hepatocellular carcinoma via corticotrophin-releasing factor receptor 2.

Urocortin (UCN) functions via corticotrophin-releasing factor receptors (CRFRs), CRFR1 & 2. CRFR2 is reported to be a tonic suppressor of vascularization, implying its role in tumor angiogenesis. Here, it was found that UCN inhibited the growth of hepatocellular carcinoma (HCC) and reduced tumor microvessel density in nude mice. Hepatoma cells didn't express CRFRs whereas vessels expressed CRFRs, mainly CRFR2. In vitro three-dimensional culture assay showed UCN inhibited angiogenesis, this effect was abolished by CRFR2 antagonist, anti-sauvagine-30, demonstrating involvement of CRFR2. Furthermore, UCN inhibited the proliferation and promoted the apoptosis of endothelial cells and down-regulated VEGF expression in vivo via CRFR2.

Enhanced intracellular calcium induced by urocortin is involved in degranulation of rat lung mast cells.

Corticotropin-releasing factor (CRF), which activates the hypothalamic-pituitary- adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN (0.1, 1 and 10 microM) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1 (CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2 (CRF-R2) antagonist antisauvagine-30 (anti-Svg-30). The results also showed that UCN caused a rapid peak increase in [Ca(2+)](i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase in [Ca(2+)](i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN on [Ca(2+)](i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value of [Ca(2+)](i) (P < 0.01). Taken together, our present study suggested that UCN induced the increase of [Ca(2+)](i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation.