Clinical Hemorheology and Microcirculation

The rheological properties of plasma and blood cells are markedly influenced by the surrounding milieu: physicochemical factors, metabolism and hormones. Acid/base status, osmolality, lipid status and plasma protein pattern are well known to exert a major influence. The oxidative stress induced by increased free radicals production decreases red cell deformability. Among circulating substances, the divalent cations magnesium and zinc improve red cell deformability probably via calcium antagonistic effects. Some metabolites like lactate or ketone bodies decrease red cell deformability, although the former has apparently the opposite effect in highly trained individuals. Endothelium‐derived factors such as nitric oxide (NO) and several arachidonic acid derivatives modulate both RBC and white cell mechanics. Endothelium regulates also blood rheology via the release of PAI‐1 which governs plasma fibrinogen levels. However, endothelium is not the only organ involved in the regulation of blood rheology: the kidney (by releasing erythropoietin which is a major “viscoregulatory” factor), the endocrine pancreas (via the action of insulin and glucagon on red cells), the adrenal gland (norepinephrine) and the endocrine heart (atrial natriuretic peptide) are also likely to exert important effects. Recently, increasing evidence is accumulating for a role of two other endocrine tissues in the regulation of blood rheology: the adipose tissue (free fatty acids, PAI‐1, IL‐6, leptin) and the pituitary gland (growth hormone–somatomedin axis, including the somatomedin carrier protein IGFBP1). These organs provide a link between body composition and hemorheology, since GH and somatomedins are major regulators of the body content in fat and water while the endocrine activity of fat mass is apparently proportional to its size. These mechanisms explain to some extent why many situations, either physiological (diet, exercise) or pathological (diabetes, uremia) are associated with marked changes in blood rheology that may in turn modify micro and macrocirculatory hemodynamics and the distribution of O₂ and fuels to tissues.

A number of clinical studies have demonstrated significant positive correlation between the severity of arterial hypertension (AHT) and whole blood viscosity. Red blood cell aggregation has also been associated with AHT especially in the severe form of the disease. The main possible cause of increased red blood cell aggregation is fibrinogen which can be found in a significantly higher concentration in patients with AHT than in healthy controls. On the other hand, blood pressure reduction with angiotensin‐converting‐enzyme inhibitors, calcium‐channel‐blocking agents, beta or alpha‐receptor blocking drugs leads to a significant improvement of blood rheology. It can be presumed that abnormal hemorheology and AHT are not directly linked but they share the same inductive genetic and/or environmental factors like obesity, chronic mental stress, physical inactivity and cigarette smoking. Regarding this hypothesis, the appropriate question is not whether hemorheological factors are causes or results of AHT but what their common origins are. Further studies are needed to clarify this hypothetical link between hemorheology and AHT.

It is known that intensive physical activity causes a loss of water. Since physical capacity is highly dependent upon fluidity of blood, the rheological characteristics of which are largely determined by water content, the aim of our study was to estimate rheological properties of blood under dehydration. To model the state of dehydration, the water deprivation was performed on rats (n=49) for 3 and 10 days. We registered hematocrit, apparent plasma viscosity; apparent viscosity, rigidity index (Tk), structural parameter (k), and extent of aggregation (“M” and “M1” indices) were determined in both unfractionated and density separated RBC suspensions in autologous plasma at Ht=40. The Ht/viscosity ratio as index of oxygen supply to tissues was calculated. The results of our study indicated that water deprivation caused progressive reduction of blood and plasma fluidity. The increase of Ht (22%, p<0.001) and significant elevation of erythrocyte aggregation (205%, p<0.001) were observed. In spite of Ht increase, oxygen transport was decreased by 18% (p<0.05). RBC rigidity was increased under water deprivation (13.6%, p<0.05) as well as during aging. Significant correlation was found between indices of aggregation and Tk and k indices in the course of dehydration. Thus, our data indicated that dehydration caused impairment of blood and RBC rheological properties.

Rheological and enzymatic properties of red blood cells (RBC) were investigated in vitro after the treatment with vasoactive drug – buflomedil (bfl) and toxic substance – 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Studies with bfl included two different concentrations of the drug: 90 μg/ml of blood and 10 μg/ml of blood. The former concentration of the drug corresponds to the amount of bfl which is taken daily by a patient, the latter one is the highest peak of this drug in plasma. The dosage of dioxin was 32 μg/ml of blood and 32 ng/ml of blood. Only the smaller dosage of this compound appears in the environment but the higher one may occur in human organs because of its cumulation.

Rheological properties of erythrocytes were examined using a laser diffractometer Rheodyne SSD (Myrenne). The deformability of RBC was expressed as an elongation index IE which was counted from the equation: EI=(L−W)/(L+W) where L is the length of cell and W is the width of cell. As far as the impact of bfl on RBC rheology is concerned studies were conducted in two different ways: (1) RBC were incubated with bfl directly, (2) RBC before incubation with bfl were treated with diamide to cause their rigidity. The action of bfl seems to be not efficient enough as data are not statistically significant in those two cases. Enzymatic properties of RBC were investigated using the methods of Beutler [7]. The activity of three enzymes was measured (acetylcholinesterase – Ache, dehydrogenase glucoso‐6‐phosphate – G‐6‐PD and gluthatione reductase – GR) for both bfl and TCDD‐treated RBC. For TCDD‐treated RBC additionally malonyldialdehyde (MDA) level was assessed.

Experiments in rat mesenterium were carried out under conditions when both pressure gradient in the chosen microvessels and their diameters were preserved constant. All details of the hemorheological events were directly visualized and documented by usage of appropriate microscopic video techniques. Intensified RBC aggregation locally produced in individual capillaries, immediately disturbs the normal blood flow structure inside their lumina and deranges the rheological properties of blood flow in the microvessels, which slows down till a full stop. The RBC aggregates gradually grow up due to addition of new cells, which become compressed and appear homogeneous. This usually interferes with restoration of blood flow in capillaries. Further the RBC aggregates can move slowly towards veins, while the flow accelerates immediately as soon as the aggregates reach the larger venules.

The objective of our research was to reveal the effects of different shear stresses on the expression of proto‐oncogenes c‐fos and c‐myc in cultured human umbilical vein endothelial cells (HUVEC). A parallel plate flow chamber was used to control the value and duration of shear stress (SS), and the expression of c‐fos and c‐myc protein was measured by immunocytochemistry methods and image analysis software. Some important conclusions were drawn. In the stationary state, c‐fos protein levels were very low. The SS's of 4 dyn/cm² and 10 dyn/cm² induced rapid increases of c‐fos protein levels, especially the SS of 10 dyn/cm². The levels peaked at 1.0 h. Then, c‐fos protein levels began to decrease, after 2.5 h, they declined to almost basal levels. In the stationary state, the c‐myc protein levels were also very low slowly increasing after the onset of shear stress. The effects of 4 dyn/cm² and 10 dyn/cm² SS on c‐myc protein expression levels had no difference and apparently were less than the effect on c‐fos protein levels. The c‐myc protein levels peaked at 1.5 h. Subsequently, they fell to basal levels at 2.5 h. Increased expression of these proto‐oncogenes mediated by shear stress may have important effects on the regulation of critical cell activities, such as proliferation and differentiation.

This study was designed to examine changes of hemorheological parameters in essential arterial hypertension subjects following antihypertensive drug therapy. Eighty two female subjects were enrolled, and sub‐divided into two groups based upon their high shear whole blood viscosity being lower (L) or higher (H) than normal controls. Equal numbers of L and H subjects were then treated for four weeks with one of four agents: angiotensin‐converting enzyme inhibitor (ACE‐inhibitor, Spirapril – 6 mg/day); calcium antagonist (Isradipin – 5 mg/day); beta‐1‐blocker (Talinolol – 100 mg/day); diuretic (Indapamide – 1.5 mg/day). Both prior to and following drug treatment for six weeks, hemorheological measurements included plasma viscosity; high and low shear whole blood viscosity, hematocrit, fibrinogen and RBC aggregation. Treatment with each of the four drugs significantly (p<0.05) reduced blood pressure in both the L and H groups. However, the hemorheological effects of antihypertensive drug therapy differed markedly between groups: plasma and whole blood viscosity were significantly elevated in the L groups whereas these parameters were significantly decreased in the H groups. Fibrinogen levels and RBC aggregation decreased in both groups, whereas hematocrit was unaffected. These results thus suggest that the rheologic effects of antihypertensive drug therapy depend strongly on the initial, pre‐treatment status of the subject, and that for some subjects, such therapy can result in adverse hemorheological alterations.