Iron transport kinetics through blood-brain barrier endothelial cells

Abstract

Background: Transferrin and its receptors play an important role during the uptake and transcytosis of iron through blood-brain barrier (BBB) endothelial cells (ECs) to maintain iron homeostasis in BBB endothelium and brain. Any disruptions in the cell environment may change the distribution of transferrin receptors on the cell surface, which eventually alter the homeostasis and initiate neurodegenerative disorders. In this paper, we developed a comprehensive mathematical model that considers the necessary kinetics for holo-transferrin internalization and acidification, apo-transferrin recycling, and exocytosis of free iron and transferrin-bound iron through basolateral side of BBB ECs.

Methods: Ordinary differential equations are formulated based on the first order reaction kinetics to model the iron transport considering their interactions with transferrin and transferrin receptors. Unknown kinetics rate constants are determined from experimental data by applying a non-linear optimization technique.

Results: Using the estimated kinetic rate constants, the presented model can effectively reproduce the experimental data of iron transports through BBB ECs for many in-vitro studies. Model results also suggest that the BBB ECs can regulate the extent of the two possible iron transport pathways (free and transferrin-bound iron) by controlling the receptor expression, internalization of holo-transferrin-receptor complexes and acidification of holo-transferrin inside the cell endosomes.

Conclusion: The comprehensive mathematical model described here can predict the iron transport through BBB ECs considering various possible routes from blood side to brain side. The model can also predict the transferrin and iron transport behavior in iron-enriched and iron-depleted cells, which has not been addressed in previous work.

Keywords: Apo-transferrin; Blood-brain barrier; Holo-transferrin; Transferrin receptors.

Fig. 1

Iron transport through transferrin receptor-mediated pathway across BBB endothelium.

Fig. 2

The overall modeling scheme depicting the essential pathways and parameters for the receptor-mediated transcytosis of iron across the BBB endothelial cells.

Fig. 3

Calibration curve for a) binding rate constant of holo-transferrin and transferrin receptors, k₁, and b) internalization rate constant of holo-transferrin-receptor complex, k₂. Numerical solutions for different value of k₁ and k₂ are shown as color lines and experimental results from Raub and Newton [19] are shown as green circles. The experiment was performed by culturing the brain microvessel endothelial cells with 10.0 nM of holo-transferrin in the apical side. Similar condition is considered in the numerical simulation to find out the optimized rate constants.

Fig. 4

Accumulation of transferrin inside the brain microvessel endothelial cells for different initial concentration of holo-transferrin at the apical side at 60 mins. Experimental result of Raub and Newton [19] are used for comparison of our model results. The experimental values are converted from ng/dish to ng/cm² by dividing the area of dish. For numerical results, the accumulations are converted from ng/ml to ng/cm² by considering volume to surface area ratio as 390. Here term ‘volume’ refers the volume occupied by a single cell and term ‘surface area’ refers the apical or basolateral surface area of a single cell. It has been reported that volume and surface area of an endothelial cell varies from 1,000 to 3,000 μm³ and from 35 to 350 μm² [68] respectively. In this study, we have considered the cell volume as 3,000 μm³ and the surface area of a cell is 120 μm².

Fig. 5

Holo-transferrin transported to brain side (basolateral side) as a function of time for an initial holo-transferrin concentration of 1,400 ng/ml (17.5 nM) in the blood side (apical side). Experimental result from Descamps et al. [21] is shown as green circles, where they cultured their brain capillary endothelial cells with 17.5 nM of holo-transferrin in the apical side.

Fig. 6

Temporal distribution of transferrin recycles to apical side, exocytoses to basolateral side and remains inside the endothelial cell. Here all concentrations are normalized with initially endocytosed holo-transferrin amount after first 1 hr of incubation. The pulse-chase experiments of Descamps et al. [21] reported that 10% of the transferrin is recycled to the apical side, while 75% of the transferrin is exocytosed to the basolateral side at 30 mins. The concept of apical and basolateral side in a cell culture is shown in inset.

Fig. 7

Effect of ligand (holo-transferrin) concentration variation on (a) holo-transferrin internalization through the apical side, (b) number of surface receptors (free) per cell on apical surface, (c) recycled apo-transferrin concentration in the apical side and, (d) amount of holo-transferrin transported to the basolateral side. Related rate constants and initial concentrations of presented in Table 2 and Table 3.

Fig. 8

Comparison of transport processes among normal, iron-enriched and iron-depleted cells. Concentration of a) apo-transferrin recycled back to blood side, b) holo-transferrin transported to brain side, c) (free) iron accumulated in LIP and d) free iron transported to brain side. In all cases, the cell is incubated with 17.5 nM of holo-transferrin in apical side for 2 hrs.

Fig. 9

Bar chart showing transferrin bound (holo-transferrin) and free iron transport in the brain side. All simulation conditions are same as in Fig. 8.