Hyperglycemia is associated with increased susceptibility to atherothrombotic stimuli. The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the protective capacity of the ...vessel wall. We therefore evaluated whether hyperglycemia affects the glycocalyx, thereby increasing vascular vulnerability. The systemic glycocalyx volume was estimated by comparing the distribution volume of a glycocalyx permeable tracer (dextran 40) with that of a glycocalyx impermeable tracer (labeled erythrocytes) in 10 healthy male subjects. Measurements were performed in random order on five occasions: two control measurements, two measurements during normoinsulinemic hyperglycemia with or without N-acetylcysteine (NAC) infusion, and one during mannitol infusion. Glycocalyx measurements were reproducible (1.7 +/- 0.2 vs. 1.7 +/- 0.3 l). Hyperglycemia reduced glycocalyx volume (to 0.8 +/- 0.2 l; P < 0.05), and NAC was able to prevent the reduction (1.4 +/- 0.2 l). Mannitol infusion had no effect on glycocalyx volume (1.6 +/- 0.1 l). Hyperglycemia resulted in endothelial dysfunction, increased plasma hyaluronan levels (from 70 +/- 6 to 112 +/- 16 ng/ml; P < 0.05) and coagulation activation (prothrombin activation fragment 1 + 2: from 0.4 +/- 0.1 to 1.1 +/- 0.2 nmol/l; d-dimer: from 0.27 +/- 0.1 to 0.55 +/- 0.2 g/l; P < 0.05). Taken together, these data indicate a potential role for glycocalyx perturbation in mediating vascular dysfunction during hyperglycemia.
Vascular endothelial cells are shielded from direct exposure to flowing blood by the endothelial glycocalyx, a highly hydrated mesh of glycoproteins, sulfated proteoglycans, and associated ...glycosaminoglycans (GAGs). Recent data indicate that the incorporation of the unsulfated GAG hyaluronan into the endothelial glycocalyx is essential to maintain its permeability barrier properties, and we hypothesized that fluid shear stress is an important stimulus for endothelial hyaluronan synthesis. To evaluate the effect of shear stress on glycocalyx synthesis and the shedding of its GAGs into the supernatant, cultured human umbilical vein endothelial cells (i.e., the stable cell line EC-RF24) were exposed to 10 dyn/cm2 nonpulsatile shear stress for 24 h, and the incorporation of 3Hglucosamine and Na235SO4 into GAGs was determined. Furthermore, the amount of hyaluronan in the glycocalyx and in the supernatant was determined by ELISA. Shear stress did not affect the incorporation of 35S but significantly increased the amount of glucosamine-containing GAGs incorporated in the endothelial glycocalyx 168 (SD 17)% of static levels, P < 0.01 and shedded into the supernatant 231 (SD 41)% of static levels, P < 0.01. Correspondingly with this finding, shear stress increased the amount of hyaluronan in the glycocalyx from 26 (SD 24) x 10(-4) to 46 (SD 29) x 10(-4) ng/cell, static vs. shear stress, P < 0.05 and in the supernatant from 28 (SD 11) x 10(-4) to 55 (SD 16) x 10(-4) ng x cell(-1) x h(-1), static vs. shear stress, P < 0.05. The increase in the amount of hyaluronan incorporated in the glycocalyx was confirmed by a threefold higher level of hyaluronan binding protein within the glycocalyx of shear stress-stimulated endothelial cells. In conclusion, fluid shear stress stimulates incorporation of hyaluronan in the glycocalyx, which may contribute to its vasculoprotective effects against proinflammatory and pro-atherosclerotic stimuli.
Scarless wound healing is a unique and intrinsic capacity of the fetal skin that is not fully understood. Further insight into the underlying mechanisms of fetal wound healing may lead to new ...therapeutic approaches promoting adult scarless wound healing. Differences between fetal and adult wound healing are found in the extracellular matrix, the inflammatory reaction and the levels of growth factors present in the wound. This review focuses specifically on transforming growth factor β (TGF‐β), as this growth factor is prominently involved in wound healing and fibroblast‐to‐myofibroblast differentiation.
Although fetal fibroblasts do respond to TGF‐β, they lack a proliferative and a contractile response and display short‐lived myofibroblast differentiation, autocrine response, and collagen up‐regulation in comparison with adult fibroblasts. Curiously, prolonged TGF‐β activation is associated with fibrosis, and therefore, this short‐lived response in fetal fibroblasts might contribute to scarless healing. This review gives an overview of the current knowledge on TGF‐β signaling and the intracellular TGF‐β signaling pathway in fetal fibroblasts. Furthermore, this review also describes the various components that regulate the cellular TGF‐β response and hypothesizes about the possible roles these components might play in the altered response of fetal fibroblasts to TGF‐β.
Background: Microalbuminuria is a clinical parameter of vascular dysfunction in patients with diabetes. The endothelial glycocalyx plays a role in regulation of vascular permeability. Therapeutic ...interventions using a mixture of glycosaminoglycans (GAGs) containing 80% heparin and 20% dermatan sulfate have been shown to improve vascular barrier function by reducing microalbuminuria
Hypothesis: Exogenous GAGs attenuate hyperglycemia‐induced increases in endothelial permeability for albumin by restoring barrier properties of the endothelial glycocalyx.
Materials and methods: Human umbilical vein endothelial cells (HUVECs) were cultured on semi‐permeable inserts and exposed to normo‐ (5mM) or hyperglycemia (25mM) for 4 days, last 24h in presence of the GAG mixture. Endothelial permeability was assessed by determining FITC‐labeled albumin transfer over the monolayer (3 hours). Additionally, the glycocalyx glucosamine sugar residues on the endothelial cells were visualized with LEA‐lectin staining
Results: Albumin permeability of endothelial cells under hyperglycemia was increased to 122 ±8% (p<0.01) compared to normoglycemia. GAG incubation restored permeability in hyperglycemia (122 ±8% vs 85 ±6%; p<0.05). Additionally, LEA‐lectin revealed a 21% decrease in glucosamine staining in hyperglycemic cells which was restored following GAG mix incubation
Conclusion: GAG supplementation reverses the increased trans‐endothelial albumin leakage under hyperglycemic conditions by restoring the barrier properties of the endothelial glycocalyx layer in vitro
Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear+plasma membrane, cytosolic and microsomal, and used in in vitro ...phosphorylation of purified 20 kDa bovine amelogenin in the presence of 2P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca2+ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca2+ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca2+, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly Ca2+ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of 14C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, 14C-mercaptoethanol, 0.64 moles uC-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by 14C-mercaptoethanol, providing 0.46 and 0.88 moles 14C-incorporated/mole respectively. Further studies of the 14C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.
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