Red blood cell stabilization reduces the effect of cell density on recovery following cryopreservation.

The relationship between red blood cell hematocrit and hemolysis during cryopreservation has been examined. Cells were frozen with glycerol, thawed, and deglycerolized in a model system based on the protocols used in transfusion medicine. Analysis included determination of hemolysis following thaw (Thaw) and deglycerolization (Overall) and osmotic fragility of the final cell suspensions. Results demonstrate that thaw hemolysis decreased with increasing hematocrit at all glycerol levels tested. Overall hemolysis increased with increasing hematocrit at low (15% w/v) glycerol and decreased with increasing hematocrit at high (40% w/v) glycerol levels. These results were paralleled by changes in the fragility index. Furthermore, these results indicate a distinction between freeze/thaw lysis and damage which leads to lysis during postthaw processing. To examine this further, a biochemical stabilizing solution, having no cryoprotective effects itself, was added to suboptimal glycerol concentrations. This addition resulted in hemolysis levels and fragility indices comparable to those using high (40% w/v) glycerol levels. Thus, the damage observed with increasing hematocrit is not necessarily a function of the packing on the volume of the ice-free zone, but rather an expression of cell damage. Furthermore, this damage is, in part, biochemical in nature and may be protected against through specific cellular stabilization prior to cryopreservation.

Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells.

Both nitric oxide (NO) and carbon monoxide (CO) are vessel wall-derived messenger molecules that cause platelet inhibition and vasodilation by activating guanylyl cyclase in target cells. Since vascular smooth muscle cells (SMCs) are exposed to shear and tensile stresses, this study examined the effects of these hemodynamic forces on the enzymes that generate NO and CO in SMCs. Monolayers of cultured rat aortic SMCs were subjected to shear stress using a modified cone and plate viscometer, or cyclic elongational stretch using a compliant silastic culture membrane. Shear stress stimulated time-dependent increases in mRNA and protein for inducible heme oxygenase-1 (HO-1), the enzyme which forms CO as a byproduct of heme degradation. The threshold level of shear necessary to induce HO-1 expression was between 5 and 10 dynes/cm2. In contrast, shear stress did not stimulate inducible NO synthase (iNOS) expression. Cyclic stretch also induced the expression of HO-1 but not of iNOS mRNA. Exposure of vascular SMCs to shear stress stimulated the production and release of CO as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. In addition, ADP-stimulated aggregation was inhibited in platelets exposed to sheared SMCs but not in platelets exposed to untreated control SMCs. Treatment of sheared SMCs with the HO-1 inhibitor, tin protoporphyrin-IX, blocked the antiaggregatory effect of the cells, whereas the iNOS inhibitor, methyl--arginine, had no effect. These results indicate that hemodynamic forces induce HO-1 gene expression and CO production in vascular SMCs, and that SMC-derived CO inhibits platelet aggregation. Thus, CO is a novel endogenous vessel wall-derived messenger molecule that may be selectively induced by hemodynamic forces to inhibit platelet reactivity and preserve blood fluidity at sites of vascular injury.

Epinephrine and shear stress synergistically induce platelet aggregation via a mechanism that partially bypasses VWF-GP IB interactions.

Elevated shear stress levels in pathologically stenosed vessels induce platelet activation and aggregation, and may play a role in the pathogenesis of arterial disease. Increased plasma catecholamine concentrations have also been implicated in the onset of acute coronary ischemic syndromes. This study was designed to examine the synergistic interaction of shear stress and epinephrine in the activation of platelets. Platelets (in PRP) sheared at 60 dyn/cm2 showed little or no aggregation unless pretreated with epinephrine. Pretreatment with 250 nM epinephrine followed by shear at 60 dyn/cm2 induced > 60% platelet aggregation. The specific alpha 2-adrenergic receptor antagonist yohimbine inhibited the synergistic aggregation, as did the ADP scavenging system phosphocreatine/creatine phosphokinase, indicating a three-way synergism with ADP. Chemical or monoclonal antibody blockade of von Willebrand factor (vWF) interactions with either platelet glycoprotein (Gp) Ib or Gp IIb/IIIa completely inhibited platelet aggregation induced by activating levels of shear stress alone. However, the combination of epinephrine and shear stress induced platelet aggregation that was blocked by 10E5, a monoclonal antibody that inhibits vWF binding to Gp IIb/IIIa, but not by aurin tricarboxylic acid or the monoclonal antibody 6D1, both of which inhibit vWF binding to Gp Ib. Synergistic platelet aggregation in response to epinephrine and shear stress was observed in washed platelets, platelet-rich plasma and whole blood in vitro, and also ex vivo following exercise to elevate endogenous levels of catecholamines. These results indicate that epinephrine synergizes with shear stress to induce platelet aggregation. This synergistic response requires functional Gp IIb/IIIa complexes, but is at least partially independent of vWF-Gp Ib interactions.

Chronic exercise and cardiac vascularization.

Male albino rats were trained on a motorized treadmill for 8 weeks, using eight training regimens which varied in severity from 0.313 m . s-1, 3 days/week for 30 min each day, to 0.447 m . s-1, 5 days/week for 60 min each day. At the end of the 8 week training period, the animals were anesthetized and Pelikan ink infused retrogradely through the aorta, into the coronary arteries and capillaries of the heart. The hearts were sectioned, stained and examined for the number of capillaries per mm2 and the capillary/muscle fiber (C/F) ratios. Six of the eight exercise groups had capillary densities significantly lower than the density in the sedentary control group of 3,022 cap mm-2. Likewise, five of the exercise groups had significantly lower C/F ratios than the sedentary group ratio of 1.077. The difficulties inherent in identification of cardiac muscle fibers make the C/F ratio a weaker measurement for determining cardiac vascularity. The lower capillary density in the trained hearts could be produced by muscle fiber hypertrophy which would push the capillaries farther apart. It is concluded that exercise training does not stimulate the multiplication of cardiac capillaries. The beneficial effect of exercise on the heart is probably a result of enlargement of the coronary arteries or the collateral circulation.