Donald R. Bell

Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis

The size of hyaluronan in solid tissue was measured using a combination of agarose gel electrophoresis and a radiometric assay. Radiolabeled hyaluronan binding proteins, used in the radiometric assay, were also used to detect hyaluronan after transfer to a nylon membrane following gel electrophoresis. Lane intensity on the autoradiograph was linearly related to the amount applied to the gel between 10 and 100ng. The intensity was independent of the hyaluronan molecular weight for standards with molecular weights equal to or greater than 790,000. The radiometric assay was used to measure hyaluronan irrespective of size, while gel electrophoresis was used to measure hyaluronan with molecular weights greater than 0.79x10(6) or 4x10(6). Deferoxamine was used to inhibit depolymerization during the digestion of tissue samples with protease. The molecular weight pattern was similar for skin, skeletal muscle, heart, lung, small intestine, and large intestine despite large differences in hyaluronan content. For all tissues, 58% of the hyaluronan had a molecular weight greater than 4million. All tissues showed an absence of hyaluronan with a molecular weight below 790,000. The procedure can be used to study changes in hyaluronan size in tissue during inflammation and other pathological states.

Permeability of rabbit skin and muscle microvasculature after saline infusion

The protein permeability characteristics of skin and skeletal muscle microvasculature were examined in rabbits following saline infusion equivalent to 9.5% of the body weight. Prenodal lymph was collected from these two tissues. In muscle the lymph flow increased from 0.94 ± 0.12 to 2:91 ± 0.36 microliters (μl)/min over baseline, and the lymph-to-plasma total protein ratio (RT) decreased from 0.561 ± 0.033 to 0.364 ± 0.016. In skin the lymph flow increased from 1.56 ± 0.25 to 4.01 ± 0.64 microliters (μl)/min, and the RT decreased from 0.340 ± 0.014 to 0.230 ± 0.016. In both tissues, the lymph-to-plasma ratios for albumin were greater than those for immunoglobulin G in controls and showed the same relationship after infusion. Thus, membrane selectivity and protein sieving did not change with saline expansion of the plasma volume. No change in microvasculature permeability occurred in these tissues, which contain 70% of the interstitial volume, following a large intravenous infusion of crystalloid.

Platelet Phospholipids Decrease Vascular Endothelial Permeability via a Novel Signaling Pathway Independent of cAMP/Protein Kinase A

Maintenance of the vascular endothelium as a semipermeable membrane to the passage of water and protein affords protection against the development of tissue edema. Platelets contribute to this maintenance of the vascular endothelial barrier. A low platelet count in the blood causes purpuric hemorrhages in the skin1 and has been associated with an increase in vascular endothelial permeability to protein in various organ beds.2,3 Repletion with platelet-rich plasma reverses these changes.1–3 Platelet conditioned medium (PCM), which contains releasate from platelets, replicates the permeability-decreasing activity of whole platelets.4-7 Two important questions remain concerning platelets and endothelial permeability, that is, the identity of the platelet factor(s) and the cellular mechanism. Much effort has been focused on identifying the active platelet factor(s). Proposed mediators that have been eliminated from contention are serotonin, norepinephrine, cyclooxygenase metabolites, adenosine, adenine nucleotides, and a >100-kD protein. A recent publication by Alexander et al.4 suggested that the active platelet factor was lysophosphatidic acid (LPA). We used gel filtration chromatography to show that phospholipids attached to albumin are responsible for the decreased vascular endothelial permeability induced by PCM. Permeability was assessed across endothelial cell monolayers derived from bovine pulmonary artery using electric cell-substrate impedance sensing (ECIS), which measures real-time changes in endothelial electrical resistance. Activity was present in a Sephacryl S-200 fraction associated with the calibrated albumin standard. Subsequent studies demonstrated that activity could be found in the albumin immunoprecipitate of PCM (FIG. 1) and in the fraction of PCM bound to a Blue Sepharose column, implying that the active factor associates with albumin. To confirm that the activity of the albumin fraction that we obtained by immunoprecipitation was a phospholipid, the albumin immunoprecipitate was extracted with methanol. Activity was present in the methanol extract and removed from the extracted albumin immunoprecipitate (FIG. 1). Since LPA was proposed as the active

Transvascular albumin and IgG flux in skin and skeletal muscle following plasmapheresis

The extravascular uptake for labeled albumin and IgG and the extravascular masses for endogenous albumin and IgG were measured in skin and skeletal muscle from anesthetized rabbits following 24 hr of intermittent plasmapheresis. An amount of protein equivalent to the total intravascular protein mass was removed. There was a significant reduction in the extravascular mass for albumin in both tissues and for IgG in skin. The shift of albumin out of the extravascular space of skin and skeletal muscle could account for 95% of the vascular replacement of albumin. The extravascular uptake for the labeled proteins was measured as the 1-hr extravascular distribution space at plasma concentration divided by time and expressed as a plasma clearance. The plasma volume in the tissue samples was estimated from the 3-min distribution space for labeled transferrin. Following plasmapheresis the rate of extravascular uptake for both labeled proteins was greater than that for control or sham-operated animals, suggesting an increase in transvascular protein permeability. The transvascular fluxes for native albumin and IgG were not increased due to the decrease in the plasma concentrations. The results were consistent with the major mechanism for a shift of plasma proteins being due to a decrease in plasma protein concentration and a subsequent increase in lymph flow instead of a decrease in transvascular protein permeability.