Surface Glycocalyx Research Articles

The comparative effects of high fat diet or disturbed blood flow on glycocalyx integrity and vascular inflammation

Background and aimsEndothelial surface glycocalyx shedding plays a role in endothelial dysfunction and increases vessel wall permeability, which can lead to inflammation and atherogenesis. We sought to elucidate whether a high fat diet (HFD) or disturbed blood flow conditions, both of which are atherogenic risk factors, would contribute more detrimentally to pre-atherosclerotic loss of endothelial glycocalyx integrity and vascular inflammation.MethodsSix to seven week-old C57BL/6-background apolipoprotein-E-knockout (ApoE-KO) male mice were either fed a chow diet, fed a modified Western HFD, and/or subjected to a partial left carotid artery (LCA) ligation procedure to induce disturbed blood flow patterns in the LCA. Mice were sacrificed after 1 week of experimental conditions. Both LCA and right carotid artery (RCA) vessels were dissected and preserved to compare glycocalyx coverage and thickness as well as macrophage accumulation in carotid arterial walls amongst and between cohorts.ResultsGlycocalyx coverage of the endothelium was significantly reduced in the LCAs of HFD fed mice when compared to the control. More significant reduction in glycocalyx coverage occurred in the LCAs of mice exposed to disturbed flow by partial LCA ligation when compared to the control. No differences were found in glycocalyx coverage of RCAs from all cohorts. Regarding inflammation, no difference in macrophage accumulation in carotid arterial walls was observed when comparing the LCAs and RCAs of control and HFD fed mice. However, macrophage infiltration in vessel walls showed a 20-fold increase in the LCAs exposed to disturbed flow following ligation, when compared to control LCAs, while no such statistical difference was observed between the RCAs of the group.ConclusionsIn our mouse model, endothelial glycocalyx integrity was compromised more by disturbed blood flow patterns than by exposure of the carotid vessel to HFD conditions. The pathophysiological implications include endothelial dysfunction, which correlates to macrophage infiltration in vessel walls and promotes atherogenesis.

Aldosterone induces albuminuria via matrix metalloproteinase–dependent damage of the endothelial glycocalyx

Aldosterone contributes to end-organ damage in heart failure and chronic kidney disease. Mineralocorticoid-receptor inhibitors limit activation of the receptor by aldosterone and slow disease progression, but side effects, including hyperkalemia, limit their clinical use. Damage to the endothelial glycocalyx (a luminal biopolymer layer) has been implicated in the pathogenesis of endothelial dysfunction and albuminuria, but to date no one has investigated whether the glomerular endothelial glycocalyx is affected by aldosterone. Invitro, human glomerular endothelial cells exposed to 0.1 nM aldosterone and 145 mMol NaCl exhibited reduced cell surface glycocalyx components (heparan sulfate and syndecan-4) and disrupted shear sensing consistent with damage of the glycocalyx. Invivo, administration of 0.6 μg/g/d of aldosterone (subcutaneous minipump) and 1% NaCl drinking water increased glomerular matrix metalloproteinase 2 activity, reduced syndecan 4 expression, and caused albuminuria. Intravital multiphoton imaging confirmed that aldosterone caused damage of the glomerular endothelial glycocalyx and increased the glomerular sieving coefficient for albumin. Targeting matrix metalloproteinases 2 and 9 with a specific gelatinase inhibitor preserved the glycocalyx, blocked the rise in glomerular sieving coefficient, and prevented albuminuria. Together these data suggest that preservation of the glomerular endothelial glycocalyx may represent a novel strategy for limiting the pathological effects of aldosterone.

Dynasore protects the ocular surface against damaging oxidative stress.

“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated in this case, and two of the inhibitors did not block dye uptake. The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Abstract 111: Endothelial Sodium Channel Activation Promotes Angiotensin II Induced Arterial Stiffness and Endothelial Dysfunction

Arterial stiffness is an independent risk factor for the development and progression of cardiovascular and kidney disease. Inappropriate activation of the renin-angiotensin-aldosterone system (RAAS) is seen in obesity and contributes significantly to development of arterial stiffness, vascular dysfunction and hypertension. We have recently observed that aldosterone increases the presence of epithelial sodium channels on endothelial cells (EnNaC), which results in stiffening of the endothelial cortical surface (glycocalyx) of the aorta. Accordingly, we posited that deletion of the critical alpha catalytic subunit of EnNaC would decrease Ang II induced arterial stiffening and vascular relaxation responses, in part, via decreases in oxidative stress. To investigate, we used mice with EC cell specific deletion of EnNaC, by serially crossing floxed” EnNaC mice with Tie 2-Cre transgene mice. EnNaC expression was markedly decreased in ECs from EnNaC KO mice while macrophage expression of EnNaC was unaffected. Ang II (500 ng/kg/min) was administered by infusion to 9 month old wild (WT) and EnNaC KO mice for three weeks with or without administration of the antioxidant tempol (2mM/day) in drinking water. Whole cell Na+ currents in ECs, as evaluated by patch clamp, were significantly increased by Ang II infusion, and this was markedly attenuated in EnNAC KO and tempol treated mice. Further, aortic endothelial stiffness as determined by atomic force microscopy was significantly increased in Ang II infused mice and this increase was attenuated in EnNaC KO mice. EnNaC deletion also attenuated impaired acetylcholine (eNOS)-mediated vasorelaxation. Thus, treatment with the antioxidant tempol reduced Ang II induced vascular stiffness and prevented impairment of NO mediated vascular relaxation. Moreover, tempol administration to Ang II infused EnNaC KO mice had similar effects in both Ang II-tempol and Ang II-EnNaC KO groups. Arterial stiffness was significantly decreased by EnNaC deletion or treatment with the antioxidant tempol in femoral vessels as well. These results are consistent with an important role for oxidative stress in Ang II promotion of EnNaC activation, endothelial stiffness and impaired eNOS mediated vascular relaxation.

Surface glycocalyx and glypican‐1 mediate tumor cell metastasis

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive renal carcinoma cells (SN12L1) with high metastatic potential have enhanced invasion rates compared to low metastatic (SN12C) cells in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) and hyaluronic acid (HA) in the glycocalyx play an important role in this flow mediated mechanotransduction and upregulation of invasive and metastatic potential. In our recent study, SN12L1 cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chambers with defined interstitial flow, we showed that flow‐enhanced invasion is suppressed in HS deficient cells. We also examined two prominent HSPGs on renal carcinoma cells – glypican‐1 and syndecan‐1 and one prominent HA receptor – CD44. We observed higher glypican‐1 levels in flow dependent SN12L1 cells when compared to SN12C cells. Caki‐1 (highly metastatic) cells did not display flow‐dependent invasion in vitro and did not display elevated glypican‐1 compared to low metastatic Caki‐2 cells. However we did observe significantly increased HS, HA, syndecan‐1 and CD44 in Caki‐1 compared to Caki‐2 cells suggesting an alternative mechanism for reported higher metastatic rates in these cells. All of our data are consistent with the hypothesis that glypican‐1 is the core protein responsible for flow sensing in metastatic cancer cells. This is also consistent with observations in endothelial cells. To assess the ability of tumor cells to metastasize in vivo, parental or HS knockdown SN12L1 cells expressing fluorescent reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from knockdown cells compared to control cells with intact HS. The reduction was even greater (98%) in lungs where most of the metastases from the control cells were observed. The ability of these knockdown cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx and glypican‐1 in tumor progression.Support or Funding InformationGrant: RO1CA204949 Targeting glycocalyx mediated mechanisms of tumor metastsisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy (STORM)

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs) and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of optical and electrical microscopy, the ultra-structure of ESG has not been revealed until recent development of super high resolution fluorescence optical microscope, STORM. We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC (bEnd3, mouse brain microvascular endothelial cells) monolayers. After confluent, the bEnd3 cells were immunolabeled with anti-HS, followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated HA binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM with a 100 x/1.49 oil immersed lens. Multiple Reporters of ATTO488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA. The field of 256 × 256 (40 × 40 μm2) of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame. The figure demonstrates the STORM images for the HA (top view, red, top row on the right) and HS (green, top row in the middle) and overlay (top row on the left) of the ESG focused at the bottom surface of the bEnd3 cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. The figure shows that HA is a long molecule weaving into a network, which is horizontal to the EC cell surface. In contrast, HS is a shorter molecule, which is perpendicular to the cell surface. The height of the HS is ~600 nm. HA and HS seem to overlap with each other at the EC surface. The revealed ultra-structure of ESG by STORM suggests that HS plays a major role in mechanosensing and HA plays a major role in forming the molecular sieve. Support or Funding Information NIH 1SC1CA153325-01 and NSF MRI CBET 1337746 STORM images showing distribution of ATTO 488 labeled anti-HS (green, middle) and Alexa Fluor 647 labeled anti-HA (red, right) and overlay (left) in the ESG of bEnd3 cell monolayer, focused on the bottom surface of the cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A positive feedback loop between IL-1β, LPS and NEU1 may promote atherosclerosis by enhancing a pro-inflammatory state in monocytes and macrophages

Inflammation plays an important role in atherosclerosis, a notion supported by the beneficial effects of the IL-1β inhibitor canakinumab in the CANTOS trial. Sialic acids (Sias), components of the surface glycocalyx, regulate intercellular and intermolecular interactions. We investigated the expression of the Sia cleaving enzyme neuraminidase-1 (NEU1) in atherosclerotic plaques and its potential role in inflammatory processes.In isolated mononuclear blood cells from patients with myocardial infarction, NEU1 expression was increased compared to healthy controls. High expression of NEU1 in macrophages located on the intima layer, in calcified regions and the adventitia of the plaque was observed in human carotid arteries' atherectomies.IL-1β and LPS induced NEU1 expression in THP-1 monocytic cells. Lentiviral NEU1-overexpression in THP-1-cells enhanced expression of CD80, TNF-α, IL-1β, number of multinuclear cells, phagocytosis and chemotaxis indicative for M1 monocyte/macrophage polarization. CRISPR/Cas9-mediated knock-out of NEU1 in THP-1-cells did not affect differentiation of monocytes to macrophages but attenuated LPS- and IL-1β -induced TNF-α and IL-1β expression. SiRNA-mediated knock-down of NEU1 in M1-macrophages differentiated from primary human CD14+ monocytes reduced the expression of TNF-α and IL-1β.Thus, in monocytes/macrophages, LPS, NEU1 and IL-1β act in a positive feedback loop as enhancers of inflammation and may therefore promote atherosclerosis and plaque instability.

The Role of Endothelial Surface Glycocalyx in Mechanosensing and Transduction.

The endothelial cells (ECs) forming the inner wall of every blood vessel 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. A variety of mechanosensors and transducers, locating on the EC surface, intra- and trans-EC membrane, and within the EC cytoskeleton, have thus been identified to ensure proper functions of ECs. 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 mechanosensors and transducers. This chapter summarizes the ESG composition, thickness, and structure observed by different labeling and visualization techniques and in different types of vessels. It also presents the literature in determining the ESG mechanical properties by atomic force microscopy and optical tweezers. The molecular mechanisms by which the ESG plays the role in EC mechanosensing and transduction are described as well as the ESG remodeling by shear stress, the actin cytoskeleton, the membrane rafts, the angiogenic factors, and the sphingosine-1-phosphate.

AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes

Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11–19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression.

183 Novel glycomimetics inhibit gly-ldl induced smooth muscle cell calcification via creb

Advanced glycated end-products (AGEs) are known drivers of cardiovascular complications such as vascular calcification, which is currently untreatable, and are increased in diabetic subjects in part due to oxidative stress...

Sphingosine-1-phosphate reduces adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel walls by protecting endothelial surface glycocalyx.

Sphingosine-1-phosphate (S1P) is a sphingolipid in plasma that plays a critical role in cardiovascular and immune systems. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. To test the hypothesis that S1P can reduce tumor cell adhesion to microvessel walls by protecting the ESG, we quantified the ESG and MDA-MB-231 tumor cell adhesion in the presence and absence of 1μM S1P, and in the presence of the matrix metalloproteinase (MMP) inhibitor in post-capillary venules of rat mesentery. We also measured the microvessel permeability to albumin as an indicator for the microvessel wall integrity. In the absence of S1P, ESG was ~10% of that in the presence of S1P, whereas adherent tumor cells and the permeability to albumin and were ~3.5-fold (after 30 min adhesion) and ~7.7-fold that in the presence of S1P, respectively. In the presence of the MMP inhibitor, the results are similar to those in the presence of S1P. Our results conform to the hypothesis that protecting ESG by S1P inhibits MDA-MB-231 tumor cell adhesion to the microvessel wall.

Sphingosine‐1‐phosphate reduces adhesion of malignant mammary tumor cells MDA‐MB‐231 to microvessel walls by protecting endothelial surface glycocalyx

To test the hypothesis that the plasma phospholipid S1P can reduce tumor cell adhesion to microvessel walls by protecting the endothelial surface glycocalyx (ESG), we quantified the ESG by fluorescence immunostaining of heparan sulfate (HS), the most abundant glycosaminoglycans in the ESG, the microvessel permeability to albumin, and MDA‐MB‐231 tumor cell adhesion in the presence and absence of S1P in post‐capillary venules of rat mesentery. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously and kept warm on a heating pad. A midline incision (~2 inch) was made in the abdominal wall and the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. A post‐capillary venule of 35–50μm diameter was cannulated by a glass micropipette; after 20 min perfusing with 1% fatty acid free bovine serum albumin Ringer solution with and without 1 μM S1P, the intensity of FITC‐anti‐HS, permeability to albumin and tumor cell adhesion rates were quantified, respectively, in 3 groups of experiments. In the absence of S1P, FITC‐anti‐HS labeled ESG was ~10% of that in the presence of S1P, whereas the permeability to albumin and adherent tumor cells were ~7‐fold and ~3.5‐fold (after 30 min adhesion) those in the presence of S1P, respectively. Our results conform to the hypothesis that protecting ESG by S1P inhibits tumor cell adhesion to the microvessel wall.Support or Funding InformationSupported by NIH SC1CA153325.

Changes in surface glycosylation and glycocalyx shedding in Trichobilharzia regenti (Schistosomatidae) during the transformation of cercaria to schistosomulum.

The invasive larvae (cercariae) of schistosomes penetrate the skin of their definitive hosts. During the invasion, they undergo dramatic ultrastructural and physiological transitions. These changes result in the development of the subsequent stage, schistosomulum, which migrates through host tissues in close contact with host’s immune system. One of the striking changes in the transforming cercariae is the shedding of their thick tegumental glycocalyx, which represents an immunoattractive structure; therefore its removal helps cercariae to avoid immune attack. A set of commercial fluorescently labeled lectin probes, their saccharide inhibitors and monoclonal antibodies against the trisaccharide Lewis-X antigen (LeX, CD15) were used to characterize changes in the surface saccharide composition of the neuropathogenic avian schistosome Trichobilharzia regenti during the transformation of cercariae to schistosomula, both in vitro and in vivo. The effect of various lectins on glycocalyx shedding was evaluated microscopically. The involvement of peptidases and their inhibitors on the shedding of glycocalyx was investigated using T. regenti recombinant cathepsin B2 and a set of peptidase inhibitors. The surface glycocalyx of T. regenti cercariae was rich in fucose and mannose/glucose residues. After the transformation of cercariae in vitro or in vivo within their specific duck host, reduction and vanishing of these epitopes was observed, and galactose/N-acetylgalactosamine emerged. The presence of LeX was not observed on the cercariae, but the antigen was gradually expressed from the anterior part of the body in the developing schistosomula. Some lectins which bind to the cercarial surface also induced secretion from the acetabular penetration glands. Seven lectins induced the shedding of glycocalyx by cercariae, among which five bound strongly to cercarial surface; the effect could be blocked by saccharide inhibitors. Mannose-binding protein, part of the lectin pathway of the complement system, also bound to cercariae and schistosomula, but had little effect on glycocalyx shedding. Our study did not confirm the involvement of proteolysis in glycocalyx shedding.

Endothelial antigen assembly leads to thrombotic complications in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.

Vascular smooth muscle cell glycocalyx mediates shear stress-induced contractile responses via a Rho kinase (ROCK)-myosin light chain phosphatase (MLCP) pathway

The vascular smooth muscle cells (VSMCs) are exposed to interstitial flow induced shear stress that may be sensed by the surface glycocalyx, a surface layer composed primarily of proteoglycans and glycoproteins, to mediate cell contraction during the myogenic response. We, therefore, attempted to elucidate the signal pathway of the glycocalyx mechanotransduction in shear stress regulated SMC contraction. Human umbilical vein SMCs (HUVSMCs) deprived of serum for 3–4 days were exposed to a step increase (0 to 20 dyn/cm2) in shear stress in a parallel plate flow chamber, and reduction in the cell area was quantified as contraction. The expressions of Rho kinase (ROCK) and its downstream signal molecules, the myosin-binding subunit of myosin phosphatase (MYPT) and the myosin light chain 2 (MLC2), were evaluated. Results showed that the exposure of HUVSMCs to shear stress for 30 min induced cell contraction significantly, which was accompanied by ROCK1 up-regulation, re-distribution, as well as MYPT1 and MLC activation. However, these shear induced phenomenon could be completely abolished by heparinase III or Y-27632 pre-treatment. These results indicate shear stress induced VSMC contraction was mediated by cell surface glycocalyx via a ROCK-MLC phosphatase (MLCP) pathway, providing evidence of the glycocalyx mechanotransduction in myogenic response.

Platelet Release from Infused CD34+-Derived Human Megakaryocytes: Lessons from a Natural-Occurring Platelet Bioreactor

The release of biologically intact platelets from in vitro-grown megakaryocytes remains one of the challenges in an attempt to replace donor-derived platelets with platelets prepared from in vitro-grown megakaryocytes. Thrombopoiesis, the process by which megakaryocytes release platelets, is thought to occur as megakaryocytes exit from the medullar space and transit into the blood stream. Two-photon fluorescence microscopy studies by several groups have documented megakaryocytes transitioning into the blood stream and extending mostly a single protrusion that appears to release large cytoplasmic fragments downstream. These studies have been widely interpreted to support platelet release in the bone marrow environment. A counter proposal made by Dr. William Howell over 75 years ago is that either whole megakaryocytes or large cytoplasmic fragments circulate to the lungs and release platelets in the lungs. Studies by our group infusing in vitro-grown megakaryocytes (both human and mouse) have shown that infused megakaryocytes do indeed become entrapped in the lungs and release platelets. These in vivo-released platelets have a much longer half-life than in vitro-prepared platelets from static cultures and have the same size distribution and nearly the same functionality as infused donor-derived platelets. Using confocal intravital microscopy, we now directly visualized the lungs of recipient NOD-SCID interferon 2 receptor γ-deficient mice during the infusion of calcein-loaded CD34+-derived megakaryocytes. Infused human megakaryocytes were immediately arrested in the pulmonary bed in vessels ~50 µm in diameter. Each cell then extended several distinct protrusions winding down presumed pulmonary capillaries that are presumably wrapped around alveoli. Some of these protrusions reached 200-300 µm in length and assumed the appearance of beads on a string. Consistent with our recently published data, this thrombopoiesis process appears to take 30 minutes to be near-complete, similar to the timeframe we showed for detecting newly released human platelet-like particles after megakaryocyte infusion. At 30 minutes, remaining bodies of the megakaryocytes are still present and we presume these are mostly the remaining nuclei. To better define whether the pulmonary bed is unique for releasing platelets, we also infused megakaryocytes intra-arteriole rather than intravenously to have the megakaryocytes encounter other organ capillary beds before the lung. These studies showed poor platelet release compared to parallel studies in mice receiving the megakaryocytes intravenously. Many of the intra-arterial infused megakaryocytes were entrapped in the spleen and very few were notable in other organs, including the lungs, liver and kidney. Our studies showed that megakaryocytes can shed platelets in the lungs where they may take advantage of the unique three-dimensional organization of the pulmonary vascular bed, flow conditions, vascular surface receptors and glycocalyx as well as a sudden shift from hypoxic to normoxic conditions. Whether these features of the pulmonary vascular bed can be simulated ex vivo and whether this will enhance true platelet release from megakaryocytes in vitro needs to be examined. DisclosuresNo relevant conflicts of interest to declare.

Differential effect of rebamipide on transmembrane mucin biosynthesis in stratified ocular surface epithelial cells

Mucins are a group of highly glycosylated glycoproteins responsible for the protection of wet-surfaced epithelia. Recent data indicate that transmembrane mucins differ in their contribution to the protective function of the ocular surface, with MUC16 being the most effective barrier on the apical surface glycocalyx. Here, we investigated the role of the mucoprotective drug rebamipide in the regulation of transmembrane mucin biosynthesis using stratified cultures of human corneal and conjunctival epithelial cells. We find that the addition of rebamipide to corneal, but not conjunctival, epithelial cells increased MUC16 protein biosynthesis. Rebamipide did not affect the levels of MUC1, 4 and 20 compared to control. In these experiments, rebamipide had no effect on the expression levels of Notch intracellular domains, suggesting that the rebamipide-induced increase in MUC16 biosynthesis in differentiated corneal cultures is not regulated by Notch signaling. Overall these findings indicate that rebamipide induces the differential upregulation of MUC16 in stratified cultures of human corneal epithelial cells, which may have implications to the proper restoration of barrier function in ocular surface disease.

Mechanotransduction of the endothelial glycocalyx mediates nitric oxide production through activation of TRP channels.

The endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the vascular endothelium, serving critical functions in the mechanotransduction of blood flow-induced forces. One of the most important protective functions of the ESG is to mediate the production of nitric oxide (NO) in response to blood flow. However, the detailed mechanism underlying ESG's mechanotransduction of the production of NO has not been completely identified. Herein, using the cultured rat brain microvascular endothelial cells (bEnd.3) as a model system, we have implemented a combined atomic force and fluorescence microscopy approach to show that the ESG senses and transduces vertical mechanical stretch to produce NO. This rapid NO production is dependent on the presence of both heparan sulfate (HS) and hyaluronic acid (HA) in ESG, as the removal of HS and/or HA leads to a significant decrease in NO production. Moreover, the production of NO is dependent on the intake of Ca2+ via endothelial transient receptor potential (TRP) channels. Together, our results demonstrate the molecular mechanism of rapid production of NO in response to vertical mechanical stretch.

Dual Regulation of L-Selectin-Mediated Leukocyte Adhesion by Endothelial Surface Glycocalyx.

Endothelial surface glycocalyx (ESG) is a carbohydrate-rich, gel-like layer found on vascular endothelium, serving critical functions in mechanotransduction of blood flows, maintenance of the endothelial permeability, and the control of leukocyte adhesion and inflammation. This study aimed to clarify the role of ESG in the adhesion between leukocytes and Human Umbilical Vein Endothelial Cells (HUVECs) under resting or inflammatory conditions. Using an atomic force microscopy-based single-cell adhesion assay, we directly quantified the detachment force and work perpendicular to the cell membrane. Detachment force and work were measured for every separation event of a leukocyte from a HUVEC with ESG, or with the major ESG glycosaminoglycan components, heparan sulfate (HS) and hyaluronic acid (HA) removed. For the resting HUVECs, when HS and/or HA were removed, the detachment force and work increased dramatically. For the HUVECs activated by inflammatory cytokine tumor necrosis factor alpha, we observed increases in the detachment force and work compared to the resting HUVECs, and removal of HS and/or HA resulted in significant decreases in the detachment force and work. The results demonstrate that the ESG layer serves a dual function: (1) on resting endothelium, it prevents leukocyte adhesion, and (2) under inflammatory conditions, it participates in endothelial-leukocyte interactions with molecules such as selectins.

Endothelial glycocalyx, apoptosis and inflammation in an atherosclerotic mouse model

Background and aimsPrevious experiments suggest that both increased endothelial cell apoptosis and endothelial surface glycocalyx shedding could play a role in the endothelial dysfunction and inflammation of athero-prone regions of the vasculature. We sought to elucidate the possibly synergistic mechanisms by which endothelial cell apoptosis and glycocalyx shedding promote atherogenesis. Methods4- to 6-week old male C57Bl/6 apolipoprotein E knockout (ApoE−/−) mice were fed a Western diet for 10 weeks and developed plaques in their brachiocephalic arteries. ResultsGlycocalyx coverage and thickness were significantly reduced over the plaque region compared to the non-plaque region (coverage plaque: 71 ± 23%, non-plaque: 97 ± 3%, p = 0.02; thickness plaque: 0.85 ± 0.15 μm, non-plaque: 1.2 ± 0.21 μm, p = 0.006). Values in the non-plaque region were not different from those found in wild type mice fed a normal diet (coverage WT: 92 ± 3%, p = 0.7 vs. non-plaque ApoE−/−, thickness WT: 1.1 ± 0.06 μm, p = 0.2 vs. non-plaque ApoE−/−). Endothelial cell apoptosis was significantly increased in ApoE−/− mice compared to wild type mice (ApoE−/−:64.3 ± 33.0, WT: 1.1 ± 0.5 TUNEL-pos/cm, p = 2 × 10−7). The number of apoptotic endothelial cells per unit length was 2 times higher in the plaque region than in the non-plaque region of the same vessel (p = 3 × 10−5). Increased expression of matrix metalloproteinase 9 co-localized with glycocalyx shedding and plaque buildup. ConclusionsOur results suggest that, in concert with endothelial apoptosis that increases lipid permeability, glycocalyx shedding initiated by inflammation facilitates monocyte adhesion and macrophage infiltration that promote lipid retention and the development of atherosclerotic plaques.