Min Zeng

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.).

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.

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.

Adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel wall increases microvascular permeability via degradation of endothelial surface glycocalyx

To investigate the effect of tumor cell adhesion on microvascular permeability (P) in intact microvessels, we measured the adhesion rate of human mammary carcinoma MDA-MB-231, the hydraulic conductivity (L(p)), the P, and reflection coefficient (σ) to albumin of the microvessels at the initial tumor cell adhesion and after ∼45 min cell perfusion in the postcapillary venules of rat mesentery in vivo. Rats (Sprague-Dawley, 250-300 g) were anesthetized with pentobarbital sodium given subcutaneously. A midline incision was made in the abdominal wall, and the mesentery was gently taken out and arranged on the surface of a glass coverslip for the measurement. An individual postcapillary venule was perfused with cells at a rate of ∼1 mm/s, which is the mean blood flow velocity in this type of microvessels. At the initial tumor cell adhesion, which was defined as one adherent cell in ∼100- to 145-μm vessel segment, L(p) was 1.5-fold and P was 2.3-fold of their controls, and σ decreased from 0.92 to 0.64; after ∼45-min perfusion, the adhesion increased to ∼5 adherent cells in ∼100- to 145-μm vessel segment, while L(p) increased to 2.8-fold, P to 5.7-fold of their controls, and σ decreased from 0.92 to 0.42. Combining these measured data with the predictions from a mathematical model for the interendothelial transport suggests that tumor cell adhesion to the microvessel wall degrades the endothelial surface glycocalyx (ESG) layer. This suggestion was confirmed by immunostaining of heparan sulfate of the ESG on the microvessel wall. Preserving of the ESG by a plasma glycoprotein orosomucoid decreased the P to albumin and reduced the tumor cell adhesion.

Vascular Endothelial Growth Factor Enhances Cancer Cell Adhesion to Microvascular Endothelium in vivo

To investigate whether vascular endothelial growth factor (VEGF) enhances cancer cell adhesion to normal microvessels, we used in vivo video microscopy to measure adhesion rates of MDA‐MB‐435s human breast cancer cells and ErbB2‐transformed mouse mammary carcinomas in the postcapillary venules of rat mesentery. An individual postcapillary venule in the mesentery was injected via a glass micropipette with cancer cells either in a perfusate of mammalian Ringer solution containing 1% bovine serum albumin as a control, or with the addition of 1 nm VEGF for test measurements. Cell adhesion was measured as either the number of adherent cells or the fluorescence intensity of adherent cells in a vessel segment for ∼60 min. Our results showed that during both control and VEGF treatments, the number of adherent cells increased almost linearly with time over 60 min. The VEGF treatment increased the adhesion rates of human tumour cells and mouse carcinomas 1.9‐fold and 1.8‐fold, respectively, over those in control conditions. We also measured cancer cell adhesion after pretreatment of cells with an antibody blocking VEGF or an antibody blocking α6 integrin, and pretreatment of the microvessel with VEGF receptor (KDR/Flk‐1) inhibitor, SU1498, or anti‐integrin extracellular matrix ligand antibody, anti‐laminin‐5. All antibodies and inhibitor significantly reduced adhesion, with anti‐VEGF and SU1498 reducing it the most. Our results indicate that VEGF enhances cancer cell adhesion to the normal microvessel wall, and further suggest that VEGF and its receptor, KDR/Flk‐1, as well as integrins of tumour cells and their ligands at the endothelium, contribute to mammary cancer cell adhesion to vascular endothelium in vivo.

Sphingosine-1-phosphate Maintains Normal Vascular Permeability by Preserving Endothelial Surface Glycocalyx in Intact Microvessels

S1P was found to protect the ESG by inhibiting MMP activity‐dependent shedding of ESG in cultured endothelial cell studies. We aimed to further test that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels.

Exocytosis of Endothelial Lysosome-Related Organelles Hair-Triggers a Patchy Loss of Glycocalyx at the Onset of Sepsis.

Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality.

Quantification of transient increase of the blood–brain barrier permeability to macromolecules by optimized focused ultrasound combined with microbubbles

Radioimmunotherapy using a radiolabeled monoclonal antibody that targets tumor cells has been shown to be efficient for the treatment of many malignant cancers, with reduced side effects. However, the blood–brain barrier (BBB) inhibits the transport of intravenous antibodies to tumors in the brain. Recent studies have demonstrated that focused ultrasound (FUS) combined with microbubbles (MBs) is a promising method to transiently disrupt the BBB for the drug delivery to the central nervous system. To find the optimal FUS and MBs that can induce reversible increase in the BBB permeability, we employed minimally invasive multiphoton microscopy to quantify the BBB permeability to dextran-155 kDa with similar molecular weight to an antibody by applying different doses of FUS in the presence of MBs with an optimal size and concentration. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a micro-grinder. About 5 minutes after applying the FUS on the thinned skull in the presence of MBs for 1 minute, TRITC (tetramethylrhodamine isothiocyanate)-dextran-155 kDa in 1% bovine serum albumin in mammalian Ringer’s solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump. Simultaneously, the temporal images were collected from the brain parenchyma ~100–200 μm below the pia mater. Permeability was determined from the rate of tissue solute accumulation around individual microvessels. After several trials, we found the optimal dose of FUS. At the optimal dose, permeability increased by ~14-fold after 5 minutes post-FUS, and permeability returned to the control level after 25 minutes. FUS without MBs or MBs injected without FUS did not change the permeability. Our method provides an accurate in vivo assessment for the transient BBB permeability change under the treatment of FUS. The optimal FUS dose found for the reversible BBB permeability increase without BBB disruption is reliable and can be applied to future clinical trials.