Microrheological responses of red blood cells to hydrogen sulfide in normotensive and hypertensive individuals: Analysis of intracellular signaling pathways

Abstract

Background

It is now known regulatory effect of gaseous mediators (NO and H₂S) in many bodily functions. However, detailed data on the regulatory role of each of these gasotransmitters are still not sufficiently elucidated.

Objective

The aim of this study was to estimate RBC microrheological changes under the influence of H₂S donor in normotensive and hypertensive (AH) persons and the signaling pathways associated with these effects.

Methods

RBCs were incubated with: NaHS (H₂S donor), ODQ, methylene blue, L-NAME, and SNP (NO donor). RBC deformability (RBCD) and aggregation (RBCA) were evaluated and compared with the corresponding control cell suspension.

Results

There was a difference in RBCD and RBCA between healthy individuals and AH patients, 6 and 58%, respectively. NaHS increased RBCD in both groups of individuals and significantly reduced RBCA. These positive effects of NaHS were eliminated by inhibition NO signaling pathway. A greater microrheological effect was observed with the combined action of NO and NaHS.

Conclusion

NO and H₂S donors demonstrated cross-talk and caused a greater change in microrheological characteristics than their separate effects. The obtained data indicate that hydrogen sulfide, in microrheological responses to its exposure, may use the elements of the NO-associated signaling pathway.

Keywords

red blood cell deformability aggregation hydrogen sulphide signaling molecules

Introduction

There is evidence of a relationship between hemorheological parameters and blood pressure in arterial hypertension (AH).^1,2 Thus, it was shown that the microrheological characteristics of RBCs (deformability and aggregation) correlate with systolic (SBP) and diastolic blood pressure (DBP).^3,4 Since RBCs are the bulk of blood cells that provide tissue microperfusion and oxygenation, their microrheology is associated with the efficiency of microcirculation both in the norm,^5–10 and in pathology.^11,12 Red blood cells occupy an important strategic position in the microcirculation system: in response to mechanical stress or hypoxic stimuli, they produce signaling molecules such as nitric oxide (NO), nitrosothiols, ATP, for vascular smooth muscle cells, and for RBCs themselves.^13–15 As a result, these cells act as a sensor and regulator of arteriolar tone and their own microrheology.¹⁶ In vitro experiments have shown that RBCs, when incubated with NO and H₂S donors, positively change their microrheological characteristics: they increase deformability by 8%–10% and reduce aggregation by 20%–35%.^17,18

This exactly corresponds to the magnitude of negative changes in microrheological characteristics in AH patients.¹⁹ The combined action of two gasotransmitters (NO and H₂S), aimed at increasing and maintaining intracellular cGMP is important for the activation of protein kinase G, RBC microrheology, angiogenesis, and vasorelaxation.²⁰ Thus, it can be assumed that NO and H₂S are mutually necessary for the physiological control of vascular function and possibly RBC microrheological changes.¹⁴ Taking into account the above, the purpose of this study can be formulated as: to study the signaling pathways regulating the microrheological responses of red blood cells to hydrogen sulfide in arterial hypertension.

Methods

Patients and study design

This is a cross-sectional study on consecutive and unselected outpatients (n = 40, 22 women and 18 men, average age—53.0 ± 7.8 years) with arterial hypertension (АН), systolic blood pressure (SBP) > 140 mm Hg, and diastolic one (DBP) > 90 mm Hg. A group of healthy individuals was formed as a control (n = 36, 18 women and 18 men, average age—38.6 ± 5.5 years). The study was approved by the local Ethics Committee of the University, and an informed consent of all the subjects were obtained according to the recommendations of the Declaration of Helsinki.

RBC preparation

Whole blood samples (9 mL) from donors were drawn (via venipuncture), into EDTA vacuum tubes. RBCs were separated from plasma by centrifugation (15 min, 3000 rpm) and washed three times in isotonic NaCl solution. The RBCs were finally suspended in the Ringer solution (with addition of dextran 130%–10% HAES-steril, Fresenius Kabi, Germany), to stimulate RBC aggregation, at 40 ± 1% hematocrit (Hct) for its measurement and ∼1.0% Hct for deformability studies.

Registration of RBC microrheological responses to gasotransmitter donor and other compounds

To study the red blood cell sensitivity to gasotransmitters (GT) in in vitro experiments, their suspension was divided into 6 aliquots and the cells were incubated at 37°C for 30 min with each of the following compounds:

(1) H₂S donor, sodium hydrosulfide (NaHS, 100 μM);

(2) 1H-[1, 2, 4]- oxadiazole [4, 3-a]quinoxalin-l-one (ODQ, 1.0 μM), a soluble guanylate cyclase (sGC) inhibitor;

(3) methylene blue, as a cGMP inhibitor (MB, 50 μM);

(4) inhibitor of NO synthase activity, N-Nitroarginine methyl ester (L-NAME, 200 μM);

(5) nitric oxide (NO) donor, sodium nitroprusside (SNP, 100 μM);

(6) only in Ringer’s solution (without any active compounds)—control samples.

RBC suspension was incubated the someway (duration and temperature) but in a drug-free Ringer’s solution was used as a control sample. All compounds were purchased from Sigma-Aldrich (USA). They were dissolved in distilled water or DMSO (Dimethyl sulfoxide). Preliminary experiments with incubation of RBCs in isotonic Ringer’s solution containing DMSO did not reveal significant differences in RBCD and RBCA compared to control samples (without DMSO). The effectiveness of the influence of GT donors and other compounds was assessed by the magnitude of the microrheological responses of red blood cells to them (changes in deformability and aggregation). All measurements of RBC microrheological properties were carried out within 3 min after incubation without additional washing and centrifugation to avoid redundant strong mechanical stress on RBC membrane.

Hemorheological measurements

Red blood cell aggregation was assessed by the Myrenne aggregometer which provides an index of RBC aggregation facilitated by low shear. The suspension was subjected to a short period of high shear to disrupt pre-existing aggregates, following which the shear was abruptly reduced to 3 s^-1 and light transmission through the suspension that was integrated for 10 seconds; the resulting index, termed “M1” by the manufacturer and “RBCA” herein, increased with enhanced RBC aggregation. A parallel plate flow microchannel was used to estimate red blood cell deformability (for details see.²¹: In brief, the cells were attached to bottom part of the chamber with “one point” and then they were deformed by shear flow, under constant shear stress (0.36 N/m²). The length (L) and width (W) of each of about hundred cells were measured and red blood cell elongation index of individual red blood cells was calculated as an index of red blood cell deformability (RBCD) according to: RBCD = L/W, where L is RBC length and W is RBC width.

Statistics and data presentation

Statistical processing included obtaining the mean (M) and standard deviation (SD). The sampling distribution was tested using the Shapiro-Wilk test. Nonparametric statistics of the program Statistica 10.0 (StatSoft Inc., USA) was used. When conducting paired comparisons of indicators within groups during repeated measurements, the Wilcoxon test was used. Differences at p < 0.05 and p < 0.01 were taken as statistically significant. The data correlation hypothesis was tested using Pearson’s correlation coefficients.

Results and discussion

Under different pathological conditions, negative changes in the microrheological red blood cell (RBC) characteristics are observed.³ Thus, in arterial hypertension (AH), we found a 6% (p < 0.01) decrease in RBCD from 2.07 ± 0.06 units in healthy individuals to 1.94 ± 0.04 in AH patients (Figure 1(a)). As for RBC aggregation, it was 53% (Figure 1(b), p < 0.01) higher in individuals with AH (8.60 ± 1.01 units in healthy individuals and 13.14 ± 1.01 units in hypertensive patients).

Figure 1.

Changes in red blood cell deformability (RBCD) and aggregation (RBCA) in patients with arterial hypertension (b) relative to healthy individuals (a). Note: Data are expressed as median±interquartile intervals.

RBC deformability significantly negatively correlated with whole blood viscosity (WBV). In the group of healthy individuals, the negative correlation coefficients between RBCD and WBV were—0.45 (p < 0.05), and in hypertensive patients—0.52 (p < 0.05); therefore, deformability could significantly affect blood fluidity, as a parameter inverse to its viscosity and its oxygen transport potential.^8,22 Since AH is accompanied by decreased RBCD (p < 0.01) and increased RBCA (p < 0.01), the question arises about the possibility of correcting these pathological changes. It is known that endogenously synthesized gasotransmitters (GT) in the cells of the circulatory system, for example, nitric oxide (NO), have a positive effect on the RBC microrheology.^10,23,24 Much less is known about the microrheological responses of RBCs to hydrogen sulfide.¹⁷

Changes in RBCD and RBCA in response to the H₂S donor, sodium hydrosulfide were studied in healthy individuals and in AH patients (Figure 2(a) and (b)). It was found that RBCD increased statistically significantly in both groups by 8 and 6%, respectively (Figure 2(a)). At the same time, the RBCD increase in the group of healthy individuals was 0.190 ± 0.040 units, and in patients with AH—0.103 ± 0.001 units only (p = 0.009; Figure 2(b)).

Figure 2.

Changes in RBC deformability under the influence of hydrogen sulfide donor (NaHS) in healthy individuals and hypertensive patients (a) and the difference in RBCD increase in the groups of healthy individuals and hypertensive patients (b).

It was found that NaHS reduced RBCA by 38% (p = 0.002) in the healthy group and somewhat less, by 30% in the AH group (p = 0.0015; Figure 3(a)). Since RBC aggregation was significantly higher in AH patients than in healthy individuals (Figure 1(b)), the reduction in this microrheological characteristic was more noticeable (Figure 3(a)). However, the difference in RBCA decrease under the influence of NaHS between the two groups was not statistically significant (Figure 3(b)).

Figure 3.

Changes in RBC aggregation under the influence of hydrogen sulfide donor (NaHS) in healthy individuals and hypertensive patients (a) and the difference in the magnitude of RBCA increment in healthy individuals and AH patients (b).

ATP-dependent potassium channels are mainly considered as a molecular cellular target for hydrogen sulfide.^25–28 However, we have previously shown that blocking K_ATP with glibenclamide did not eliminate the positive microrheological effects of the H₂S donor, sodium hydrosulfide.²⁹ Therefore, red blood cell membrane K_ATP channels probably aren’t a molecular cellular target for hydrogen sulfide. It can be assumed that mature RBCs have retained only relics of cation channels of this type.

On the other hand, there is abundant evidence of cross-talk between H₂S and NO^30,31 and also data on the use of the NO-associated signaling pathway by hydrogen sulfide.^32,33 There is evidence that H₂S can activate soluble guanylate cyclase (s-GC) and cGMP as an element of the signaling pathway.^34,35 When s-GC was inhibited by preincubation RBCs with ODQ, the increase in RBCD and decrease in RBCA induced by NaHS were almost completely eliminated (Figure 4(a) and (b)).

Figure 4.

Changes in RBCD (a) and RBCA (a) under the influence of NaHS before and after inhibition of soluble guanylate cyclase by ODQ. Note: RBCD—red blood cell deformability; RBCA—red blood cell aggregation; NaHS—sodium hydrosulfide; ODQ – 1H-[1,2,4]-oxadiazolo [4,3-a][quinoxalin-l-one.

The second element of the NO-associated signaling cascade is guanosine-5′-monophosphate (cGMP). H₂S is known to increase the intracellular concentration of cGMP in a NO-dependent manner³⁰ and also activates protein kinase G (PKG) and its effectors, such as RBC cytoskeletal spectrins, which is associated with an increase in RBCD.³⁶ We showed that while NaHS significantly increased RBCD by 7% (p < 0.01) and decreased RBCA by 30% (p < 0.01), prior inhibition of cGMP with methylene blue completely eliminated these RBC microrheological responses (Figure 5(a)).

Figure 5.

Changes in RBCD (a) and RBCA (b) under the influence of NaHS before and after inhibition of guanosine-5′-monophosphate (cGMP) by methylene blue. Note: RBCD—red blood cell deformability; RBCA—red blood cell aggregation; NaHS—sodium hydrosulfide; MB—methylene blue.

Mature human RBCs contain NO synthase (NOS).^13,14,37,38 It was shown that its non-selective inhibitor, L-NAME, caused a significant concentration-dependent decrease in basal endogenous H₂S levels. On the other hand, sodium nitroprusside, as NO donor, was found to stimulate an increase in H₂S levels in cells.³¹

In our experiments, NaHS caused an increase in RBCD by 8% (p < 0.01) and a significant decrease in RBCA by 21% (p < 0.05). Testing the effect of L-NAME on RBC microrheology showed that inhibition of eNOS in RBCs eliminated the increase in RBCD and decrease in RBCA under the influence of cell incubation with NaHS. This was indicated by a reliable difference in the RBCD and RBCA values obtained after incubation of cells with NaHS and after exposure to “L-NAME + NaHS” (Figure 6(a) and (b)).

Figure 6.

Changes in RBCD (a) and RBCA (b) under the influence of NaHS before and after inhibition NO-synthase (NOS) by N-nitroarginine methyl ester (L-NAME). Note: RBCD—red blood cell deformability; RBCA—red blood cell aggregation; NaHS—sodium hydrosulfide; L-N—L-NAME.

In this case under the influence of separately applied GT donors, the increase in RBCD for SNP was 8%, for NaHS – 9%, and when they used together (SNP + NaHS), RBCD was increased by 12% (p < 0.01). This was significantly greater than the increases in this microrheological characteristic for SHP and NaHS separately (Figure 7(a)). The same pattern of change was revealed when analyzing the effect of GT donors on RBC aggregation. A 38% reduction in RBCA was recorded in response to the separate action of SHP and NaHS and a significantly greater decrease in this microrheological characteristic with the simultaneous use of two GTs (by 42%, p < 0.01, Fig. 7b).

Figure 7.

Changes in RBCD (a) and RBCA (b) under the influence of SNP and NaHS separately and when used together (SNP + NaHS). Note: Data are expressed as median±interquartile intervals.

Thus, more pronounced red blood cell microrheological responses to the influence of two gasotransmitter donors at once may be evidence of their cross-talk under these conditions.

Conclusion

Red blood cell deformability was significantly reduced by 6% (p < 0.01) in AH patients. This relatively small negative change in red blood cell microrheology should nevertheless significantly affect tissue perfusion. Red blood cells perform up to 2000 capillary transits daily, adapting their shape to the geometry of exchange capillaries.³⁹ Therefore red blood cell deformability is critical for optimal microrheology, microperfusion and gas exchange functionality during capillary flow.^8,40 Whole blood fluidity was found to be significantly correlated with RBCD. In the AH group, this was indicated by a significant negative correlation between WBV and RBCD (r = −0.52, p < 0.01). As for RBC aggregation, the difference between the groups of healthy individuals and AH patients was significantly greater and was equal to 53% (0.01). However, no reliable correlations were found between blood viscosity and RBCA (0.16 in the control group and 0.22 in the AH group).

In vitro experiments showed that the H₂S donor increased RBCD by 6%–9% (p < 0.01). In the hypertensive group, this would completely restore RBC deformability to the level of healthy individuals. Thus, measures to restore the metabolism and bioavailability of GT, including H₂S, can improve the RBC microrheological characteristics and tissue microperfusion. The obtained data on the probable use of the H₂S NO-mediated signaling pathway suggest cross-talking and cooperative regulatory effects not only in the management of arteriolar tone,^32,41–43 but also in the red blood cell microrheological responses.

Taken together, the results of the study showed that the H₂S increased RBCD in both groups, while restoring the reduced RBC deformability of hypertensive individuals to the level of healthy ones and significantly reducing RBCA. The two donors showed cross-talk and caused a greater change in microrheological characteristics than their individual effects. Data were obtained indicating the possible use of the NO-mediated signaling pathway by H₂S in human RBCs in regulatory changes in their microrheology. Thus, although there is sufficient information indicating the physiological regulation of H₂S and NO, much work is needed to investigate the cross-talk between H₂S and NO. A better understanding of the interactions will lead to the development of new therapeutic strategies for cardiovascular diseases.

Footnotes

Author contributions

A. Muravyov, A. Priezzhev, supervised the experimental study design, interpretation and wrote the article;

I. Tikhomirova included the patients and conducted the experiments;

A. Lugovtsov conducted the experiments and interpretation performed the data analysis;

E. Volkova conducted the experiments and statistical processing of the obtained data;

P. Mikhailov conducted data analysis, worked with literature data, and produced figures for the article.

All authors read and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Russian Science Foundation (Grant No. 25-15-00172).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Appendix

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