Elevated expression of the C-type lectin CD93 in the glioblastoma vasculature regulates cytoskeletal rearrangements that enhance vessel function and reduce host survival.

Glioblastoma is an aggressive brain tumor characterized by an abnormal blood vasculature that is hyperpermeable. Here, we report a novel role for CD93 in regulating angiogenesis in this setting by modulating cell-cell and cell-matrix adhesion of endothelial cells. Tissue microarray analysis demonstrated that vascular expression of CD93 was correlated with poor survival in a clinical cohort of patients with high-grade astrocytic glioma. Similarly, intracranial growth in the GL261 mouse model of glioma was delayed significantly in CD93(-/-) hosts, resulting in improved survival compared with wild-type mice. This effect was associated with increased vascular permeability and decreased vascular perfusion of tumors, indicating reduced vessel functionality in the absence of CD93. RNAi-mediated attenuation of CD93 in endothelial cells diminished VEGF-induced tube formation in a three-dimensional collagen gel. CD93 was required for efficient endothelial cell migration and proper cell polarization in vitro. Further, in endothelial cells where CD93 was attenuated, decreased cell spreading led to a severe reduction in cell adhesion, a lack of proper cell contacts, a loss of VE-cadherin, and aberrant actin stress fiber formation. Our results identify CD93 as a key regulator of glioma angiogenesis and vascular function, acting via cytoskeletal rearrangements required for cell-cell and cell-matrix adhesion.

Prediction of in vivo rat biliary drug clearance from an in vitro hepatocyte efflux model.

Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

Functional ATP-binding cassette drug efflux transporters in isolated human and rat hepatocytes significantly affect assessment of drug disposition.

Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.

The impact of solute carrier (SLC) drug uptake transporter loss in human and rat cryopreserved hepatocytes on clearance predictions.

Cryopreserved hepatocytes are often used as a convenient tool in studies of hepatic drug metabolism and disposition. In this study, the expression and activity of drug transporters in human and rat fresh and cryopreserved hepatocytes was investigated. In human cryopreserved hepatocytes, Western blot analysis indicated that protein expression of the drug uptake transporters [human Na(+)-taurocholate cotransporting polypeptide (NTCP), human organic anion transporting polypeptides (OATPs), human organic anion transporters, and human organic cation transporters (OCTs)] was considerably reduced compared with liver tissue. In rat cryopreserved cells, the same trend was observed but to a lesser extent. Several rat transporters were reduced as a result of both isolation and cryopreservation procedures. Immunofluorescence showed that a large portion of remaining human OATP1B1 and OATP1B3 transporters were internalized in human cryopreserved hepatocytes. Measuring uptake activity using known substrates of OATPs, OCTs, and NTCP showed decreased activity in cryopreserved as compared with fresh hepatocytes in both species. The reduced uptake in cryopreserved hepatocytes limited the in vitro metabolism of several AstraZeneca compounds. A retrospective analysis of clearance predictions of AstraZeneca compounds suggested systematic lower clearance predicted using metabolic stability data from human cryopreserved hepatocytes compared with human liver microsomes. This observation is consistent with a loss of drug uptake transporters in cryopreserved hepatocytes. In contrast, the predicted metabolic clearance from fresh rat hepatocytes was consistently higher than those predicted from liver microsomes, consistent with retention of uptake transporters. The uptake transporters, which are decreased in cryopreserved hepatocytes, may be rate-limiting for the metabolism of the compounds and thus be one explanation for underpredictions of in vivo metabolic clearance from cryopreserved hepatocytes.