TGFß1 exacerbates blood-brain barrier permeability in a mouse model of hepatic encephalopathy via upregulation of MMP9 and downregulation of claudin-5.

Recent studies have found that vasogenic brain edema is present during hepatic encephalopathy following acute liver failure and is dependent on increased matrix metalloproteinase 9 (MMP9) activity and downregulation of tight junction proteins. Furthermore, circulating transforming growth factor ß1 (TGFß1) is increased following liver damage and may promote endothelial cell permeability. This study aimed to assess whether increased circulating TGFß1 drives changes in tight junction protein expression and MMP9 activity following acute liver failure. Blood-brain barrier permeability was assessed in azoxymethane (AOM)-treated mice at 6, 12, and 18 h post-injection via Evan's blue extravasation. Monolayers of immortalized mouse brain endothelial cells (bEnd.3) were treated with recombinant TGFß1 (rTGFß1) and permeability to fluorescein isothiocyanate-dextran (FITC-dextran), MMP9 and claudin-5 expression was assessed. Antagonism of TGFß1 signaling was performed in vivo to determine its role in blood-brain barrier permeability. Blood-brain barrier permeability was increased in mice at 18 h following AOM injection. Treatment of bEnd.3 cells with rTGFß1 led to a dose-dependent increase of MMP9 expression as well as a suppression of claudin-5 expression. These effects of rTGFß1 on MMP9 and claudin-5 expression could be reversed following treatment with a SMAD3 inhibitor. AOM-treated mice injected with neutralizing antibodies against TGFß demonstrated significantly reduced blood-brain barrier permeability. Blood-brain barrier permeability is induced in AOM mice via a mechanism involving the TGFß1-driven SMAD3-dependent upregulation of MMP9 expression and decrease of claudin-5 expression. Therefore, treatment modalities aimed at reducing TGFß1 levels or SMAD3 activity may be beneficial in promoting blood-brain barrier integrity following liver failure.

Ecallantide for treatment of acute attacks of acquired C1 esterase inhibitor deficiency.

Acquired C1 inhibitor (C1-INH) deficiency exposes patients to angioedema recurrences (acquired angioedema [AAE]) mediated by bradykinin pathway activation. C1-INH replacement and specific inhibition of plasma kallikrein with ecallantide have been successful in the treatment of hereditary angioedema (HAE), a more common related disorder. C1-INH replacement has also been used in the treatment of AAE, but because of the underlying mechanism of rapid catabolism, some patients may not respond. As part of preclinical investigation of ecallantide, a potent bradykinin pathway inhibitor, we evaluated three AAE patients treated successfully with that agent. This study was designed to assess ecallantide for treatment of attacks in AAE. Three patients with AAE were treated a total of 12 times with various dosing regimens of ecallantide based on the protocols established for the studies using ecallantide in HAE (Evaluation of DX-88's Effects in Mitigating Angioedema trials). Response to therapy was also based on outcome measures determined by these protocols. Ecallantide effectively relieved symptoms in three patients with various manifestations of AAE over 12 acute episodes. Kallikrein inhibition with ecallantide appears effective in the treatment of AAE and may be an alternative for patients with resistance to C1-INH replacement therapy.

A computational model of fibroblast growth factor-2 binding to endothelial cells under fluid flow.

Fibroblast growth factor-2 (FGF2) is an angiogenic growth factor that binds to cell surface receptors (FGFR) and heparan sulfate proteoglycans (HSPG), as well as HSPG in the basement membrane. FGF2 plays a critical role in angiogenesis, yet clinical FGF2 trials demonstrated limited success perhaps due to inadequate understanding of FGF2 binding in physiological conditions. We developed a computational model of FGF2 binding to isolated (HSPG or FGFR) or combined (HSPG and FGFR) binding sites under physiological fluid flow and predicted the effects of FGF2 concentration, binding site density, fluid flow rate, and delivery mode (continuous vs. bolus) on FGF2 complex formation. The isolated binding site models showed increased binding with FGF2 and binding site density. However, in the triad model, increasing FGF2 concentration decreased triads (FGF2-HSPG-FGFR) and increased FGF2-HSPG complexes. Fluid flow decreased time to equilibrium and dissociation in isolated binding site models, yet flow effect in the triad model depended on binding site density. Similarly, FGF2 capture and complex stability in bolus delivery depended on bolus size, flow rate, association and dissociation rate constants, as well as binding site density. This model shows the integrated effects of FGF2 binding stoichiometry, fluid flow, and delivery mode, and enhances our understanding of FGF2 complex formation under physiological conditions.