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, N^G-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.

Although anti‐angiogenic therapies (AATs) have some effects against multiple malignancies, they are limited by subsequent tumor vasculogenesis and progression. To investigate the mechanisms by which tumor vasculogenesis and progression following AATs, we transfected microRNA (miR)‐9 into human umbilical vein endothelial cells (HUVECs) to mimic the tumor‐associated endothelial cells in hepatocellular carcinoma and simulated the AATs in vitro and in vivo. We found that administration of the angiogenesis inhibitor vandetanib completely abolished miR‐9‐induced angiogenesis and promoted autophagy in HUVECs, but induced the release of vascular endothelial growth factor (VEGF)‐enriched exosomes. These VEGF‐enriched exosomes significantly promoted the formation of endothelial vessels and vasculogenic mimicry in hepatocellular carcinoma and its progression in mice. Anti‐autophagic therapy is proposed to improve the efficacy of AATs. However, similar effects by AATs were observed with the application of anti‐autophagy by 3‐methyladenine. Our results revealed that tumor vasculogenesis and progression after AATs and anti‐autophagic therapies were due to the cross‐talk between endothelial and tumor cells via VEGF‐enriched exosomes.

Zn²⁺ is an essential element for cell survival/growth, and its deficiency is linked to many disorders. Extracellular Zn²⁺ concentration changes participate in modulating fundamental cellular processes such as proliferation, secretion, ion transport, and cell signal transduction in a mechanism that is not well understood. Here, we hypothesize that the Zn²⁺-sensing receptor ZnR/G protein-coupled receptor 39 (GPR39), found in tissues where dynamic Zn²⁺ homeostasis takes place, enables extracellular Zn²⁺ to trigger intracellular signaling pathways regulating key cell functions in vascular cells. Thus, we investigated how extracellular Zn²⁺ regulates cell viability, proliferation, motility, angiogenesis, vascular tone, and inflammation through ZnR/GPR39 in endothelial cells. Knockdown of GPR39 through siRNA largely abolished Zn²⁺-triggered cellular activity changes, Ca²⁺ responses, as well as the downstream activation of Gαq-PLC pathways. Extracellular Zn²⁺ promoted vascular cell survival/growth through activation of cAMP and Akt as well as overexpressing of platelet-derived growth factor-α receptor and vascular endothelial growth factor A. It also enhanced cell adhesion and mobility, endothelial tubule formation, and cytoskeletal reorganization. Such effects from extracellular Zn²⁺ were not observed in GPR39^−/− endothelial cells. Zn²⁺ also regulated inflammation-related key molecules such as heme oxygenase-1, selectin L, IL-10, and platelet endothelial cell adhesion molecule 1, as well as vascular tone-related prostaglandin I2 synthase and nitric oxide synthase-3. In sum, extracellular Zn²⁺ regulates endothelial cell activity in a ZnR/GPR39-dependent manner and through the downstream G_αq-PLC pathways. Thus, ZnR/GPR39 may be a therapeutic target for regulating endothelial activity.

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (D_eff) in rat brain tissue 100–250 μm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ∼3 ml/min. P and D_eff were determined from the rate of tissue solute accumulation and the radial concentration gradient around individual microvessels in the brain tissue. The mean apparent permeability P values for sodium fluorescein (molecular weight (MW) 376 Da), dextran-4k, -20k, -40k, -70k, and IgG (MW ∼160 kDa) were 14.6, 6.2, 1.8, 1.4, 1.3, and 0.54 × 10⁻⁷cm/s, respectively. These P values were not significantly different from those of rat pial microvessels for the same-sized solutes (Yuan et al., 2009, “Non-Invasive Measurement of Solute Permeability in Cerebral Microvessels of the Rat,” Microvasc. Res., 77(2), pp. 166–73), except for the small solute sodium fluorescein, suggesting that pial microvessels can be a good model for studying BBB transport of relatively large solutes. The mean D_eff values were 33.2, 4.4, 1.3, 0.89, 0.59, and 0.47 × 10⁻⁷ cm²/s, respectively, for sodium fluorescein, dextran-4k, -20k, -40k, -70k, and IgG. The corresponding mean ratio of D_eff to the free diffusion coefficient D_free, D_eff/D_free, were 0.46, 0.19, 0.12, 0.12, 0.11, and 0.11 for these solutes. While there is a significant difference in D_eff/D_free between small (e.g., sodium fluorescein) and larger solutes, there is no significant difference in D_eff/D_free between solutes with molecular weights from 20,000 to 160,000 Da, suggesting that the relative resistance of the brain tissue to macromolecular solutes is similar over a wide size range. The quantitative transport parameters measured from this study can be used to develop better strategies for brain drug delivery.