Bingmei M. Fu

Permeability of Endothelial and Astrocyte Cocultures: In Vitro Blood–Brain Barrier Models for Drug Delivery Studies

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (Lp), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that Lp and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2–4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

Non-invasive measurement of solute permeability in cerebral microvessels of the rat.

To quantify the solute permeability of rat cerebral microvessels, we measured the apparent permeability (P) of pial microvessels to various sized solutes. The pial microcirculation was observed by a high numerical aperture objective lens through a section of the frontoparietal bone thinned with a micro-grinder (a revised method from Easton and Fraser, 1994, J Physiol. 475:147-157, 1994). Sodium fluorescein (MW 376) at concentration 0.1 mg/ml or FITC-dextrans (MW 4k, 10k, 20k, 40k, 70k) at concentration 1 mg/ml in 1% BSA mammalian Ringer, was introduced into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of approximately 3 ml/min. P was determined using a highly sensitive quantitative fluorescence imaging and analyzing method. The mean P to sodium fluorescein was 2.71 (+/-0.76 SD, n=11)x10(-6) cm/s. The mean P to FITC-dextrans were 0.92 (+/-0.46 SD, n=10)x10(-6) cm/s for Dextran-4k, 0.31 (+/-0.13 SD, n=7)x10(-6) cm/s for Dextran-10k, 0.24 (+/-0.10 SD, n=6)x10(-6) cm/s for Dextran-20k, 0.19 (+/-0.11 SD, n=10)x10(-6) cm/s for Dextran-40k, and 0.15 (+/-0.05 SD, n=11)x10(-6) cm/s for Dextran-70k. These values were 1/10 to 1/6 of those of rat mesenteric microvessels for similar sized solutes (Fu, B.M., Shen, S., 2004. Acute VEGF effect on solute permeability of mammalian microvessels in vivo. Microvasc. Res. 68, 51-62.).

Mechano-sensing and transduction by endothelial surface glycocalyx: composition, structure, and function

The endothelial cells (ECs) lining every blood vessel wall are constantly exposed to the mechanical forces generated by blood flow. The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system. To ensure proper EC mechano‐sensing and transduction, there are a variety of mechano‐sensors and transducers that have been identified on the EC surface, intra‐ and trans‐EC membrane and within the EC cytoskeleton. Among them, the most recent candidate is the endothelial surface glycocalyx (ESG), which is a matrix‐like thin layer covering the luminal surface of the EC. It consists of various proteoglycans, glycosaminoglycans, and plasma proteins, and is close to other prominent EC mechano‐sensors and transducers. The ESG thickness was found to be in the order of 0.1–1 µm by different visualization techniques and in different types of vessels. Detailed analysis on the electron microscopy (EM) images of the microvascular ESG revealed a quasi‐periodic substructure with the ESG fiber diameter of 10–12 and 20 nm spacing between adjacent fibers. Atomic force microscopy and optical tweezers were applied to investigate the mechanical properties of the ESG on the cultured EC monolayers and in solutions. Enzymatic degradation of specific ESG glycosaminoglycan components was used to directly elucidate the role of the ESG in EC mechano‐sensing and transduction by measuring the shear‐induced productions of nitric oxide and prostacyclin, two characteristic responses of the ECs to the flow. The unique location, composition, and structure of the ESG determine its role in EC mechano‐sensing and transduction. WIREs Syst Biol Med 2013, 5:381–390. doi: 10.1002/wsbm.1211

The Structural Stability of the Endothelial Glycocalyx after Enzymatic Removal of Glycosaminoglycans

Rationale It is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown. Objective To evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme. Methods and Results Using confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large. Conclusion All GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.

Quantification of the endothelial surface glycocalyx on rat and mouse blood vessels.

The glycocalyx on the surface of endothelium lining blood vessel walls modulates vascular barrier function, cell adhesion and also serves as a mechano-sensor for blood flow. Reduction of glycocalyx has been reported in many diseases including atherosclerosis, inflammation, myocardial edema, and diabetes. The surface glycocalyx layer (SGL) is composed of proteoglycans and glycosaminoglycans, of which heparan sulfate is one of the most abundant. To quantify the SGL thickness on the microvessels of rat mesentery and mouse cremaster muscle in situ, we applied a single vessel cannulation and perfusion technique to directly inject FITC-anti-heparan sulfate into a group of microvessels for immuno-labeling the SGL. We also used anti-heparan sulfate for immuno-labeling the SGL on rat and mouse aortas ex vivo. High resolution confocal microscopy revealed that the thickness of the SGL on rat mesenteric capillaries and post-capillary venules is 0.9±0.1 μm and 1.2±0.3 μm, respectively; while the thickness of the SGL on mouse cremaster muscle capillaries and post-capillary venules is 1.5±0.1 μm and 1.5±0.2 μm, respectively. Surprisingly, there was no detectable SGL in either rat mesenteric or mouse cremaster muscle arterioles. The SGL thickness is 2.5±0.1 μm and 2.1±0.2 μm respectively, on rat and mouse aorta. In addition, we observed that the SGL is continuously and evenly distributed on the aorta wall but not on the microvessel wall.

A model for interpreting the tracer labeling of interendothelial clefts

We extended the model describing the low molecular weight electron dense tracer wake in the interendothelial cleft and surrounding tissue to describe the time-dependent transport of intermediate size solutes of 1.0–3.5 nm radius by convection and diffusion in an interendothelial cleft containing a fiber matrix. This model provides a quantitative basis on which to reinterpret electron microscopic studies of the distribution of tracers such as horseradish peroxidase (HRP; molecular weight=40,000; Stokes radius =3.0 nm) along the interendothelial cell cleft from the lumen to the tissue for example, we show that, in contrast to our results with low molecular weight tracers, the wake of large molecular weight tracers on the abluminal side of the junctional strand is not likely to be detected, because the concentration of the tracer is predicted to be very low in most experiments. Thus the lack of a tracer such as HRP on the abluminal side of the junctional strand and in the tissue is not as strong evidence against the presence of a cleft pathway as suggested previously. The model does provide the basis for the design of experiments to locate both the principal molecular sieve and breaks in the junctional strand from the standing gradient on the luminal side of the junctional strand. An important experimental variable is the pressure in the vessel lumen which can be varied between 0 and 30 cm H2O to change the contributions of diffusive and convective transport to transcapillary exchange through the interendothelial cleft. This approach will also allow the testing of models for transcapillary pathways for large molecules by measuring the distribution of fluorescent tracers across the microvessel wall and in the tissue surrounding the microvessel using confocal microscopy.

Mechanical mechanisms of thrombosis in intact bent microvessels of rat mesentery

The hypothesis that thrombus can be induced by localized shear stresses/rates, such as in the bent/stretched microvessels, was tested both experimentally and computationally. Our newly designed in vivo experiments were performed on the microvessels (post-capillary venules, 20-50 microm diameter) of rat mesentery. These microvessels were bent/stretched with no/minimum injuries. In less than 60 min after the microvessels were bent/stretched, thrombi were formed in 19 out of 61 bent locations (31.1%). Interestingly, thrombi were found to be initiated at the inner wall of the curvature in these bent/stretched vessels. To investigate the mechanical mechanisms of thrombus induction, we performed a 3-D computational simulation using commercial software, FLUENT. To simulate the bending and stretching, we considered the vessels with different curvatures (0 degrees , 90 degrees and 180 degrees ) as well as different shaped cross-sections (circular and elliptic). Computational results demonstrated that the highest shear stress/rate and shear stress/rate gradient are located at the inner wall of the curved circular-shaped vessels. They are located at the two apexes of the wall with shorter axis for the 0 degrees (straight) elliptic-shaped vessel and towards the inner side when the vessels are bent. The differences of the shear stresses/rates and of the shear stress/rate gradients between the inner and outer walls become larger in more bent and elliptic-shaped microvessels. Comparison of our experimental and numerical simulation results suggests that the higher shear stress/rate and the higher shear stress/rate gradient at the inner wall are responsible for initiating the thrombosis in bent post-capillary venules.

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-Mediated Secretion of VEGF

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium.

Endothelial Surface Glycocalyx Can Regulate Flow-Induced Nitric Oxide Production in Microvessels In Vivo

Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250–300g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.

Modulation of the blood–brain barrier permeability by plasma glycoprotein orosomucoid

Previous studies have shown that the glycoprotein orosomucoid modulates permeability of peripheral microvessels to charged molecules by contributing to the net charge on the microvessel wall. To investigate whether or not orosomucoid also modulates the permeability of the blood-brain barrier (BBB) by a similar mechanism, we measured the permeability (P) of rat pial microvessels to similar-sized molecules with different charges: alpha-lactalbumin (-10, Stokes radius 2.08 nm) and ribonuclease (+4, Stokes radius 2.01 nm). Tests were performed under control conditions with a Ringer-BSA (bovine serum albumin) perfusate and with 0.1mg/ml orosomucoid in Ringer-BSA perfusate. The pial circulation was observed through a section of frontoparietal bones thinned with a micro-grinder, and P was determined using a quantitative fluorescence video microscopy. In the absence of orosomucoid, the permeability of pial microvessels to positively charged ribonuclease was 4-fold that to negatively charged alpha-lactalbumin. In contrast, in the presence of orosomucoid, permeability to ribonuclease was 12-fold that to alpha-lactalbumin. On the basis of these experimental data, our theoretical model predicted that the charge density of the endothelial glycocalyx layer at the luminal surface of the BBB increased 2.8-fold in the presence of 0.1 mg/ml orosomucoid, while the charge density of the BBB basement membrane increased 1.8-fold, compared to their control values. Our results indicate that orosomucoid can modulate the permeability of the BBB to charged molecules by adding negative charge to the matrix components of the BBB.