Pattern of P450 expression at the human blood-brain barrier: roles of epileptic condition and laminar flow

Abstract

Purpose: P450 enzymes (CYPs) play a major role in hepatic drug metabolism. It is unclear whether these enzymes are functionally expressed by the diseased human blood-brain barrier (BBB) and are involved in local drug metabolism or response. We have evaluated the cerebrovascular CYP expression and function, hypothesizing possible implication in drug-resistant epilepsy.

Methods: CYP P450 transcript levels were assessed by cDNA microarray in primary endothelial cultures established from a cohort of brain resections (n = 12, drug-resistant epilepsy EPI-EC and aneurism domes ANE-EC). A human brain endothelial cell line (HBMEC) and non-brain endothelial cell line (HUVEC) were used as controls. The effect of exposure to shear stress on CYP expression was evaluated. Results were confirmed by Western blot and immunohistochemistry on brain specimens. Endothelial drug metabolism was assessed by high performance liquid chromatography (HPLC-UV).

Results: cDNA microarray showed the presence of CYP enzymes in isolated human primary brain endothelial cells. Using EPI-EC and HBMEC we found that CYP mRNA levels were significantly affected by exposure to shear stress. CYP3A4 protein was overexpressed in EPI-EC (290 ± 30%) compared to HBMEC and further upregulated by shear stress exposure. CYP3A4 was increased in the vascular compartment at regions of reactive gliosis in the drug-resistant epileptic brain. Metabolism of carbamazepine was significantly elevated in EPI-EC compared to HBMEC.

Discussion: These results support the hypothesis of local drug metabolism at the diseased BBB. The direct association between BBB CYP enzymes and the drug-resistant phenotype needs to be further investigated.

Figure 1

Levels of CYP mRNAs in primary human brain endothelial cells. (A–B) Primary endothelial cell cultures (EPI-EC and ANE-EC) were established from brain resections (Table S1). cDNA analysis shows the presence of CYP enzymes in primary brain endothelial cells. CYP mRNA levels were increased compared to the controls (HBMEC and HUVEC). Levels of drug transporter proteins were also assessed (C). The levels of GAPDH mRNA were used as an internal control. Results are expressed as mean ± standard error of the mean (SEM) (one-way ANOVA, *p < 0.05 HBMEC vs. EPI-EC, **p < 0.05 HBMEC vs. ANE-EC, ^$p < 0.05 EPI-EC vs. ANE-EC). See also Fig. S1 and Table S2.

Figure 2

CYP mRNAs are under the control of shear stress. (A) Schematic representation of the experimental design adopted. In the dynamic in vitro (DIV) system, media is pumped in the luminal side of polypropylene fibers, which determines shear forces. Control endothelial cells or EPI-EC (Table S1) were seeded either in the luminal side (exposed to shear stress) or in the abluminal space (no shear) of the DIV. (B–D) Exposure to shear stress significantly increased the transcript levels of the CYPs and drug transporters [(mRNA)_shear/(mRNA)_no-shear], suggesting a positive effect of shear stress on transcription. The comparison between mRNA levels in EPI-EC and HBMEC unveiled cell-specific differences to flow response (one-way ANOVA, *p < 0.05 HBMEC vs. EPI-EC). Results are expressed as mean ± SEM. See text for details. See also Fig. S1.

Figure 3

Pattern of CYP3A4 protein expression in HBMEC and EPI-EC exposed to shear stress. (A–B) Western blot analysis shows that the CYP3A4 expression is significantly increased (p < 0.05) in EPI-EC (n = 4 patients, Table S1) compared to HBMEC (*). CYP3A4 expression was significantly increased by shear stress in both cell types (**). Expression of CYP3A4 in EPI-EC was comparable to hepatocytes. (C–D) Exposures to carbamazepine (CBZ) induced the expression of CYP3A4 in endothelial cells and hepatocytes (*). However, CBZ exposure did not exert significant effects on shear-stress preexposed endothelial cells (HBMEC-shear). In HBMEC with no-shear exposure CYP expression was significantly different compared to HBMEC shear and hepatocytes (**). The intensity of the bands obtained by western blot was normalized by β-actin value. Results are expressed as mean ± SEM [two-way analysis of variance (ANOVA), p < 0.05].

Figure 4

CYP3A4 expression at the blood–brain barrier of patients with drug-resistant epilepsy. (A) Montage reconstruction illustrates CYP3A4 expression in the temporal cortex of epileptic human brain. Both penetrating and small caliber vessels were positive for CYP3A4 staining (green). (B) CYP3A4 expression was more pronounced in regions of reactive gliosis, delimited by the dotted line. (D–E) Note the perivascular GFAP staining delimiting the vascular bed. (F–G) Endothelial expression of CYP3A4 colocalized with vWF. (C) CYPA4 expression (green bars) is significantly increased (*p < 0.05) in brain areas associated with reactive gliosis (bar graph n = 4 patients, three slices each). One-way analysis of variance (ANOVA) was used. Results are expressed as mean ± standard error of the mean (SEM).

Figure 5

Metabolism of carbamazepine (CBZ) at the blood–brain barrier. (A) CBZ was significantly metabolized within 72 h by dynamically grown control endothelial cells. An example of high-performance liquid chromatography (HPLC) traces demonstrated the decrease of CBZ, whereas bar-graph indicates the absolute values (μg/ml) of CBZ at time zero and 72 h after (standard curve: y = −396.5 + 567 X, r² = 0.98). Panel (B) displays the metabolic potency of hepatocytes evaluated in the same time frame. (C) Human drug-resistant epileptic blood–brain barrier (BBB) was established using EPI-EC cocultured with astrocytes (DIV-BBB). Note the reduction of CBZ in the luminal side and the near-zero levels of CBZ in the abluminal side (dotted line), suggesting poor penetration across the drug-resistant epileptic BBB. The amount of CBZ penetrating the BBB was higher in the control endothelium (dotted line in A). The relative percentage of CBZ metabolized by hepatocytes, HBMEC shear, and no-shear and epileptic is summarized in (D) and in Table 1 (One-way ANOVA, *p < 0.05 EC-shear vs. Epi-BBB; **p < 0.05 hepatocytes vs. EC-shear; ^$p < 0.05 HBMEC vs. HBMEC shear; ^$$p < 0.05 HBMEC shear vs. EPI-EC.) Results are expressed as mean ± SEM.