Microcirculation and blood rheology abnormalities in chronic heart failure.

BACKGROUND: Generalized restricted blood flow is hallmark of CHF of any etiology, but the extent of microcirculation restriction and the role of intrinsic blood properties in heart failure remains unknown. OBJECTIVE: The aim of this study was to estimate the microvascular blood flow and hemorheological properties in chronic heart failure to test the hypothesis that CHF patients have altered peripheral blood flow which contributes to the tissue perfusion disturbances. METHODS: Cutaneous microvascular blood flow was estimated by Laser Doppler and Optical Tissue Oximetry techniques. Whole blood and plasma viscosity were measured by capillary viscometer, red blood cell aggregation was evaluated by direct microscopic method, erythrocyte deformability was assessed as elongation index in flow microchamber. Hematocrit-to-viscosity ratio was used as index of oxygen transport efficacy. RESULTS: Depression of the regulatory mechanisms of microvascular blood flow as well as decreased tissue perfusion indicated the restricted blood flow in microcirculatory network in CHF. Increased blood and plasma viscosity, enhanced red blood cell aggregation and decreased erythrocyte deformability were registered in CHF. CONCLUSIONS: Complex impairment of peripheral blood flow in CHF including restricted microcirculation, attenuated regulatory mechanisms and impaired hemorheological properties caused the reduced oxygen utilization contributing to symptoms and advance of heart failure.

Interrelation of blood coagulation and hemorheology in cancer.

Cancer progression is associated with activation of blood coagulation. Blood coagulation process, platelet hemostasis and hemorheological properties were evaluated in patients with solid tumors (n = 27) before and after surgery and in healthy control (n = 20). The main features of blood coagulation process in cancer patients were elevated intensity and shortened period of contact phase of coagulation and inhibited fibrinolysis stage. Such prothrombotic state was fixed before surgery as well as in early postoperative period in spite of preventing thromboprophylactic treatment. Platelets depletion within the high level of spontaneous and ADP-induced platelet aggregation was fixed in cancer. The main cause of blood viscosity decrease in cancer patients was dramatic fall of Hct, because blood viscosity adjusted by Hct 40% was increased owing to the rise of plasma viscosity and substantially worsened RBC microrheological properties. The results of our study indicated close correlation between hemorheological and hemostasis parameters; these interrelations were more numerous and strong in cancer. In cancer patients the combination of a high aggregation activity of platelets, reduced number of erythrocytes (Hct), an increase of RBC aggregation and plasma viscosity caused impairment of blood oxygen transportation efficacy that provoke hypoxia in the microcirculation favoring thrombosis, settlement of tumor and metastasis.

Signaling pathways regulating red blood cell aggregation.

The exposure of red blood cells (RBC) to some hormones (epinephrine, insulin and glucagon) and agonists of α- and ß-adrenergic receptors (phenylephrine, clonidine and isoproterenol) may modify RBC aggregation (RBCA). Prostaglandin E1 (PGE1) significantly decreased RBCA, and PGE2 had a similar but lesser effect. Adenylyl cyclase (AC) stimulator forskolin added to RBC suspension, caused a decrease of RBCA. More marked lowering of RBCA occurred after RBC treatment by dB-cAMP. Phosphodiesterase (PDE) inhibitors markedly reduced RBCA. Ca2+ influx stimulated by A23187 was accompanied by an increase of RBCA. The blocking of Ca2+ entry into the RBC by verapamil or the chelation of Ca2+ by EGTA led to a significant RBCA decrease. Lesser changes of aggregation were found after RBC incubation with protein kinase C stimulator phorbol 12-myristate 13-acetate (PMA). A significant inhibitory effect of tyrosine protein kinase (TPK) activator cisplatin on RBCA was revealed, while selective TPK inhibitor, lavendustin, eliminated the above mentioned effect. Taken together, the data demonstrate that changes in RBCA are connected with activation of different intracellular signaling pathways. We suggest that alterations in RBCA are mainly associated with the crosstalk between the adenylyl cyclase-cAMP system and Ca2+ control mechanisms.

Role molecular signaling pathways in changes of red blood cell deformability.

This study was designed to investigate the dependency of the red blood cell deformability upon activation of extra- and intracellular signaling pathways. Exposures of red blood cells (RBCs) to catecholamines and to insulin led to positive change in the RBC deformability. When forskolin, a stimulator of adenylyl cyclase (AC), was added to RBC suspension, the RBC deformability was increased. Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. The inhibitors of phosphodiesterase (PDE) activity increased red cell deformability. These results revealed a considerable role of the AC-cAMP signaling system in the regulation of red blood cell deformability. The rise of the red blood cell Ca(2+) influx, stimulated by mechanical loading or A23187 was accompanied by a marked lowering of RBC deformability. At the same time blocking of Ca(2+) entry into RBC by verapamil or Ca(2+) chelating by EGTA led to significant deformability rise. The comparison of the effect of the different protein kinases on the red blood cell deformability showed that it was altered more considerable under PKA activation by forskolin or dB-cAMP than by other protein kinases. There was a lesser but quite statistically significant effect of tyrosine protein kinase (TPK) on RBC microrheology. Whereas the microrheological effect of PKC was not so considerable. The problem of the short-term regulation of red blood cell microrheology is examined. The latter includes: the modes of activation of extra- and intracellular molecular signaling pathways, ligand - receptor interaction, second messengers, membrane protein phosphorylation. On the whole the total data clearly show that the red cell deformability changes are connected with activation of different extra - and intracellular signaling pathways. It seems reasonable to suppose that red blood cell deformability changes were mainly associated with activation of the AC-cAMP-PKA pathway, and with decrease of Ca(2+) entry into cells.

Role Ca(2+) in mechanisms of the red blood cells microrheological changes.

To assess the physiological role of intracellular Ca(2+) in the changes of microrheological red blood cell (RBC) properties (RBC deformability and aggregation), we employed several types of chemicals that can increase and decrease of the intracellular Ca(2+) concentration. The rise of Ca(2+) influx, stimulated by mechanical loading, A23187, thrombin, prostaglandin F(2α) was accompanied by a moderate red cell deformability lowering and an increase of their aggregation. In contrast, Ca(2+) entry blocking into the red cells by verapamil led to a significant RBC aggregation decrease and deformability rise. Similar microrheological changes were observed in the red blood cells treated with phosphodiesterase inhibitors IBMX, vinpocetine, rolipram, pentoxifylline. When forskolin (10 µM), an AC stimulator was added to RBC suspension, the RBC deformability was increased (p <0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. Red cell aggregation was significantly decreased under these conditions (p<0.01). On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of both intracellular signaling pathways: Ca(2+) regulatory mechanism and Gs-protein/adenylyl-cyclase-cAMP system. And the final red cell microrheological regulatory effect is connected with the crosstalk between these systems.

Microcirculation and blood rheology in patients with cerebrovascular disorders.

We estimated hemorheological parameters of vein blood samples and cutaneous microvascular blood flow in patients with acute ischemic stroke and in controls. The worsened blood rheological properties were registered in patients with stroke: the enhanced whole blood viscosity was due to the substantial increase of plasma viscosity and the impairment of microrheological blood properties: elevated erythrocyte aggregability and decreased deformability compared to the healthy group. The decrease of oxygen consumption fixed by rheological methods and by laser Doppler flowmetry led us to conclude that the tissue hypoxia took place in patients with stroke. The regulatory mechanisms aimed to maintain blood supply to tissue were activated under cerebral infarction and the impact of unfavorably changed rheological blood properties was markedly enhanced. Revealed close interrelations between rheological and microcirculation parameters testified the important role of hemorheological factors in maintenance of microvascular blood flow and oxygen delivery to tissue.

Macro- and microrheological parameters of blood in patients with cerebral and peripheral atherosclerosis: the molecular change mechanisms after pentoxifylline treatment.

This study was designed to evaluate hemorheological changes in patients with cerebrovascular disease (CVD) and peripheral arterial disease (PAD) after 4 weeks of pentoxifylline therapy as well as to study red blood cell microrheological variables after the cell incubation with pentoxifylline and some phosphodiesterase (PDE) activity inhibitors. The patients with CVD (n = 50) and PAD (n = 33) were treated with pentoxifylline (400 mg, thrice a day) for 4 weeks. Before and after drug therapy the hemorheological measurements including plasma and whole blood viscosity, red blood cell aggregation (RBCA) and deformability (RBCD) were completed. In vitro study RBCs were incubated with: 1) Vinpocetine--inhibitor PDE-1, 10 µM; 2) Rolipram--PDE-4, 10 µM; 3) Isobutyl-methylxanthine (IBMX)--nonselective PDE inhibitor, 100 µM and with pentoxifylline, 10 µM The cell incubation was performed at 37 °C for 15 min. There were the positive changes of hemorheological profile after 4 weeks of the pentoxifylline therapy both in CVD and PAD patients. The marked RBCD changes were observed after the in vitro cell pentoxifylline treatment as well. Perhaps it is connected with the inhibition of the phosphodiesterase activity in RBCs. An application of drugs and chemicals that can inhibit the PDE activity resulted in RBCD rise and RBCA decrease. The experiments with the use of selective PDE inhibitors have revealed the similar red cell deformability changes. Vinpocetine increased RBCD significantly (p < 0.05). PDE-4 inhibitor--Rolipram stimulated RBCD by 15% (p < 0.05). Some more effective was IBMX. After cell incubation with it a significant rise of the deformability (by 27%; p < 0.05) was found. All drugs, having PDE activity decreased RBCA, but the most pronounced effect had Vinpocetine (50%; p < 0.05). Thus, administered pentoxifylline, daily (1200 mg), during four weeks improves hemorheological profile and especially its microrheological part as well as the blood transport capacity in subjects with cerebral and peripheral vascular disorders. It is most probably red cell microrheological control mechanisms may be associated with the phosphodiesterase activity alterations.

Crosstalk between adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism under red blood cell microrheological changes.

There are evidences that red blood cell (RBC) deformation and aggregation change under their incubation with catecholamines and it is connected with activation of intracellular signaling pathways. The present study was designed to explore the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism of RBCs together with their microrheological changes. The washed RBCs were resuspended in PBS. In each of the three research sessions RBC suspensions were divided into two aliquots: 1) control (without drug) and 2) with an appropriate drug. After cell incubation RBC deformability (RBCD) and aggregation (RBCA) were estimated. RBC incubation with catecholamines resulted in RBCD changes by 18-30%. RBCs incubation with forskolin facilitated an increase of RBCD by 17% (p < 0.05). A significant deformability rise under dB-AMP incubation was found by 27% (p < 0.01). Ca2+ cell influx, stimulated by A23187, was accompanied by an increase of RBCA; whereas red cell deformability was changed only slightly. On the other hand, Ca2+ entry blocking into the cells by verapamil has led to significant RBCA decrease and RBCD rise. The obtained results make us believe that RBCD change was closely associated with Ca2+ control mechanisms. An effect of Ca2+ concentration increase on RBC microrheology was removed, if it was preliminary added to incubation medium EGTA as Ca2+ chelator. It was found that all four PDE inhibitors: IBMX, vinpocetine, rolipram, pentoxifylline decreased RBCA significantly and, quite the contrary, they increased red cell deformability. Our data have shown that Ca2+ entry increase was accompanied by red cell aggregation rise, while adenylyl cyclase-cAMP system stimulation led to red cell deformability increase and its aggregation lowered. The crosstalk between two intracellular signaling systems is probably connected with phosphodiesterase activity.

Extra- and intracellular signaling pathways under red blood cell aggregation and deformability changes.

Exposure of red blood cells (RBCs) to catecholamines (epinephrine, phenylephrine, an agonist of alpha1-adrenergic receptors, clonidine, an agonist of alpha2-adrenergic receptors and isoproterenol, an agonist of beta-adrenergic receptors) led to change in the RBC microrheological properties. When forskolin (10 microM), an AC stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 17% (p<0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 27%; p<0.01). Red blood cell aggregation (RBCA) was significantly decreased under these conditions (p<0.01). All drugs having PDE activity increased red cell deformability similarly. Some more changes of deformability was found after RBC incubation with pentoxifylline--25% (p<0.05) and IBMX incubation was accompanied only by 15% rise of RBC deformability. The drugs with PDE inhibitory activity reduced red cell aggregation. The most significant RBCA reduction effect was found under cell incubation with pentoxifylline and inhibitor PDE1-vinpocetine. On the whole RBCA reduction averaged 36% (p<0.05) under RBCs incubation with PDE inhibitors. The rise of Ca2+ influx, stimulated by A23187, was accompanied by an increase of RBCA, whereas red cell deformability was changed insignificantly. At the same time Ca2+ entry blocking into the red cells by verapamil or its chelating in medium by EGTA led to significant RBCA decrease and deformability rise (p<0.05).On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways. It seems reasonable to suppose that RBCA decrease was mainly associated with an activation of the adenylyl-cyclase-cAMP system, while the red cell deformability was closely associated with Ca2+ control mechanisms.