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.

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.

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. Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. It afflicts >700,000 individuals annually in the United States alone, has mortality rates of 30%, and is the 11th leading cause of death. One of the key molecular causes of Gram-negative septicemia is endotoxin that consists of lipopolysaccharides (LPSs) bound with high affinity to LPS-binding glycoprotein. Complex LPS-binding glycoprotein is recognized by cognate receptor Toll-like receptor 4 and co-receptor CD14 on monocytes/macrophages and endothelial cells. 1 Considering the systemic nature of septicemia, vascular endothelium represents the first line of exposure to bacterial endotoxins. 2 It responds to endotoxins with a complex system of danger signals, which are chronologically sequenced and spatially propagated. 3 Functionally, these waves of danger signaling tend to secure proper organismal responses, both proinflammatory and anti-inflammatory.Among the earliest responses of activated endothelial cells to endotoxin are exocytosis of Weibel-Palade bodies (WPBs) and secretory lysosomes. 4 WPBs are rod-shaped members of lysosome-related organelles (0.2 mm by 2 to 3

Sphagnum moss has been used as the major substrate for cultivating Phalaenopsis spp. in China, Japan, and Taiwan. With a lengthened duration of cultivation, the pH of the moss gradually declines. It is not understood what causes this decline in substrate pH. Using the vegetatively propagated Phal. Sogo Yukidian ‘V3’, this study investigated if substrate, fertilization, light, and plant roots could be the cause of pH decline in the substrate. The results showed that, although increasing fertilizer concentration resulted in a low initial pH (pH measured by the pour-through technique at first fertilization), fertilization itself was not the primary cause of the long-term pH decline. Regardless of whether the sphagnum moss was fertilized, the pH of the substrate without plants increased as time progressed, whereas the pH of the substrate in which living Phalaenopsis plants were growing declined with time. Although the magnitude and course of pH decline were different in various substrates, the pH of sphagnum moss, artificial textile fiber, and pine bark substrates in which living plants were growing declined with time. Whether the substrate was exposed to light (clear pots) or not (opaque pots) had no effect on substrate pH, indicating that algae were not a factor in pH decline. Therefore, the roots of Phalaenopsis may be the major contributor to substrate pH decline during production.

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In the past years, there have been many new developments in biological imaging, ranging from synchrotron X-ray crystallography for protein structure to 3D maps of entire human body [1]. Confocal microscopy, one of the most rapidly growing areas, is commonly used to construct the 3D images of biological samples where the area of interest is stained with fluorescent dyes conjugated to specific targeting antibodies. However, the physical resolution limit of light microscopy (~0.2µm) does not permit visualization of features that are on the nanoscale. Transmission electron microscopy, TEM, is another popular technique where serial sectioning has permitted 3D reconstruction of biological elements. However, this technique is very difficult and requires extremely accurate positioning of the sections, which are each floated from the air/water interface. Lately, focused ion beam (FIB), has been introduced as a viable alternative, which can produce rapid and precise milling (sectioning) of the sample. In combination with scanning electron microscopy (SEM), it becomes a straightforward and powerful tool for 3D imaging of biological samples. Yet, despite its accuracy and high resolution, the EM techniques are not able to uniquely identify the proteins, which comprise the observed structures, or functional domains and receptors which determine the structure of the adhered cells. Hence immunohistochemical staining with fluorescently labeled antibodies has recently been used in combination with electron microsopy, otherwise known as, correlative light and electron microscopy (CLEM). It is a convenient technique which can be used to obtain comprehensive 3D images of tissues elucidating structure, composition, and cell lineage.An elegant application of CLEM regards the influence of nanoparticles on tissue composition and development. Nanoparticles have been applied to drug delivery systems, targeted organ imaging, as well as cancer therapy [2]. Numerous evidences have shown that toxicological influence is not only on cell viability but on protein expression, extracellular matrix production, and cell mechanics. It has also been shown that substrate mechanics/ or topography can have influence on the cellular uptake of nanoparticles [3]. However, nanoparticle uptake as well as cell/substrate interactions can have very large variabilities in samples. Previous reports of these factors have been ensemble averages, making it very difficult to obtain the correlation between nanoparticle uptake in a specific cell and the consequent effect on a specific response. Furthermore, nanoparticles can only be imaged using electron microscopy, following sectioning and embedding, which requires removal of the cells from their substrates, impeding the ability to observe the response from cells to substrate mechanics. Hence, we developed a new approach, which is different from coventional CLEM, permits us to investigate the correlation between targeting protein expression of the cell, particle distributions, and particle uptake at the single cell lev...

To test the hypothesis that endothelial surface glycocalyx (ESG) plays a role in mechanosensing and transduction of the microvessel wall, we used fluorescence microscopy to measure the NO levels in the post‐capillary venules of rat mesentery under low and high flow conditions and with/out enzyme treatment for removing heparan sulfate (HS) of the ESG. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously, the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. After perfusion for 1 hr with 1% BSA Ringer for the control or with 50 mU/mL heparanase III for the treatment, an individual post‐capillary venule (35–50 μm) was loaded for 45 min with 5 μM 4, 5‐Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO was measured for ~10min under a low flow (< 300 μm/s) as the baseline and for ~60min under a high flow (1500–2000 μm/s mean velocity). In 1 min after switching to the high low, NO increased by 1.14 ± 0.05‐fold (n=3) under control and 1.15 ± 0.04‐fold (n=3) under enzyme treatment. NO continuously increased after 1 min under high flow, reached a plateau of 1.42 ± 0.02‐fold in ~40 min under control and of 1.22 ± 0.03 in ~15min under treatment. The results suggest that ESG participate in EC mechanosensing and transduction through its HS and other components. Supported by NIH R01HL094889–01.