Influence of Rho Kinase Inhibitor Fasudil on Late Endothelial Progenitor Cells in Peripheral Blood of COPD Patients with Pulmonary Artery Hypertension

  • Pei Liu Intense Care Unit, Taihe Hospital of Hubei Medical University
  • Hongmei Zhang Department of Respiratoy Diseases, Taihe Hospital of Hubei Medical University
  • Yijun Tang Department of Respiratoy Diseases, Taihe Hospital of Hubei Medical University
  • Chunfeng Sheng Intense Care Unit, Taihe Hospital of Hubei Medical University
  • Jianxin Liu Department of Ultrasound Imaging, Taihe Hospital of Hubei Medical University Shiyan
  • Yanjun Zeng Beijing University of Technology
Keywords: Rho Kinase Inhibitor Fasudil, chronic obstructive pulmonary diseases, pulmonary artery hypertensio, endothelial progenitor cells, number and function of the late endothelial progenitor cells

Abstract

The objective of our work was to investigate the influence of Fasudil, a Rho inhibitor on the number and function of the late endothelial progenitor cells in peripheral blood of chronic obstructive pulmonary diseases (COPD) patients with pulmonary artery hypertension. Eighty COPD patients with pulmonary artery hypertension were selected and divided into two groups: the treatment group and the control group, which had 40 patients respectively. The control group received routine treatment, including oxygen uptake, anti-infection and phlegm dissolving. The treatment group received the Fasudil in addition to the routine treatment. The changes on the number and function of the late endothelial progenitor cells in peripheral blood of the patients before and after the treatment were compared between the two groups. The changes on the pulmonary artery pressure were also compared. The number of the late endothelial progenitor cells in peripheral blood of the treatment group increased and the function was enhanced. The pulmonary artery pressure was reduced. The difference before and after the treatment and with the control group was statistically significant (p<0.05). The changes on the number and function of the late endothelial progenitor cells in peripheral blood and the pulmonary artery pressure before and after the treatment of the control group were not statistically significant (p>0.05). The Rho-kinase inhibitor Fasudil increased the number and enhanced the function of the late endothelial progenitor cells in peripheral blood of COPD patients with pulmonary artery hypertension.

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Influence of Rho Kinase Inhibitor Fasudil on Late Endothelial Progenitor Cells in Peripheral Blood of COPD Patients with Pulmonary Artery Hypertension
Published
2014-05-20
How to Cite
1.
Liu P, Zhang H, Tang Y, Sheng C, Liu J, Zeng Y. Influence of Rho Kinase Inhibitor Fasudil on Late Endothelial Progenitor Cells in Peripheral Blood of COPD Patients with Pulmonary Artery Hypertension. Bosn J of Basic Med Sci [Internet]. 2014May20 [cited 2021Jul.30];14(1):40-4. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/2295
Section
Translational and Clinical Research

INTRODUCTION

Hypoxic pulmonary artery hypertension (PAH) is the core stage of the pathogenesis process of chronic obstructive pulmonary diseases (COPD). The decrease of the pulmonary artery pressure retards the pathogenesis and development of COPD significantly [1]. The search for the ideal method to decrease the pulmonary artery pressure is the key to prevent and cure COPD and cor pulmonate. Studies in recent years demonstrated that the damage and malfunction of endothelium play an important role in the pathogenesis and development of PAH [2]. The endothelial progenitor cells (EPC) participate not only in the formation of human embryonic vessels, but also in the neogenesis of blood vessels after birth and the repairment after endothelium damage [3]. Korean scientists, Hur et al. [4] reported in 2004 the presence of EPC of two kinds of different biological characteristics in adult peripheral blood and named these two kinds of peripheral blood EPC early EPC and late EPC. Following research considered the late EPC, coming from the bone marrow, as the real EPC, which directly takes part in the repairment of vascular endothelium and plays an important role in the neogenesis and repairment of blood vessels [5]. In recent years, the relationship between Rho/Rho-kinase signaling pathway and cardiopulmonary vascular diseases has drawn an increasing attention. This research investigated the influence of Fasudil, a Rho kinase inhibitor on the number and function of the late endothelial progenitor cells in peripheral blood of COPD patients with pulmonary artery hypertension by the intervention of Rho kinase inhibitor Fasudil on COPD patients with pulmonary artery hypertension.

MATERIALS AND METHODS

Research Objects

A total of 80 COPD patients with pulmonary artery hypertension hospitalized from July 2010 to April 2012 were selected, in which male patients represented 40 cases and female, 40 cases. The age of the patients ranges from 50 to 75 years old. The average age was 63.5±11.6 years old, conforming to the diagnosis criteria. The patients with diabetes, liver and kidney disease, blood diseases, recent surgery, wounds, inflammation, tumor, cerebrovascular disorder and acute myocardial infarction that could interfere with the EPC were ruled out. The selected patients did not receive statins. The patients were divided randomly into two groups. The treatment group had 40 patients, in which 22 male and 18 female. The control group also had 40 patients, in which 24 male and 16 female. The composition of age and sex of the two groups were not statistically different. All the research objects had been informed of the detailed research process. This experiment had the consent of the patients and was granted by the local ethnics committee.

Research Methods

All the patients of the two groups received such routine treatment as oxygen uptake, anti-infection and phlegm dissolving. The treatment group received intravenous drip of 30 mg of Fasudil hydrochloride injecta added by 100 ml physiological saline. It was applied twice a day and 21 days was a period of treatment.

Measurement of pulmonary artery pressure. The 5500 type color Doppler ultrasound of HP was used. Round chamber view section of the ventricular apex was selected to observe the regurgitation of the tricuspid valve and transprosthetic gradient (ΔP). It was estimated that the systolic pressure of pulmonary artery was ΔP+10mmHg (right atrial pressure).

Separation, culture and identification of late EPC. The peripheral vein blood of the subjects was collected and the mononuclear cells were separated by density gradient centrifugal method. The obtained mononuclear cell suspension was inoculated to a 24 pore cell culture plate covered by human fibronectin. The inoculation density was 2×l06cells/ml. The plate was cultured in an incubator of CO2 at 37°C. The fluid was changed for the first time after 4 days. Then, it was changed every 2 days until the 21st day of culture. The cells were washed with PBS for further analysis. The adherent cells were collected to measure the positivity of cell phenotypes CD34 and KDR with a flow cytometry and the absorption of DiLDL combined with FITC-UEA-I (double positivity) by the laser scanning confocal microscope.

Late EPC count. The obtained late EPC were counted under 200 times inverted microscope and the average was obtained. The cellular morphology of early and late EPC was observed respectively.

Measurement of the proliferation ability of late EPC. The adherent cells in the primary culture were digested by 0.25% trypsinase and prepared to be single cell suspension with culture fluid. The cell suspension concentration was adjusted to 3*105/mL. It was inoculated to a 96 pore plate with the density of 104 for every pore. After 48h of culture, its reproduction ability was measured by MTT method.

Measurement of adherence ability

The adherent cells were digested by 0.25% trypsinase and suspended in 50011L culture fluid for counting. Then the equal number of EPC was spread on a culture plate covered by HFN and cultured for 30 min at 37°C. Then, the adherent cells were counted under 200 time microscope.

Measurement of migration ability

The adherent cells were collected and counted as above. 2511L culture fluid and vascular endothelial growth factor (VEGF, 50 μg/L) were added to the lower chamber of the improved Boyden chambers. 2*104 EPC suspended in 50 μL culture fluid was added to the upper chamber. When cultured for 24 h, the unmigrated cells at the surface of the filter membrane were removed and fixated by methane and then stained by Giemsa. 3 random fields of microscope (x 400) were selected to count the cells migrated to the bottom.

Statistical analysis

Statistical analysis was conducted by SPSS13. 0 statistical software. The data was demonstrated by x ±s. The comparison between mean values was conducted with analysis of variance and the comparison between rates was conducted with χ2 test. Using P<0.05 as the variance was considered as statistically significant.

RESULTS

Basic Information

Comparison of age, sex, blood glucose, blood pressure, total cholesterol and low density lipoprotein (LDL) between the two groups.

The variance of age, sex, blood glucose, blood pressure, total cholesterol and low density lipoprotein betwen the two groups was not statistically significant. (Table 1)

TABLE 1: Comparison of the basic clinical data between the two groups

Late EPC Identification

The late EPC cultured in isolation was measured for the positivity of cell phenotypes of CD34 and KDR by a flow cytometry (Figure 1) and the absorption of DiLDL combining with FITC-UEA-I (double positivity) (Figure 2).

FIGURE 1: The late EPC cultured in isolation was measured for the positivity of cell phenotypes of CD34 and KDR by a flow cytometry.
FIGURE 2: The absorption of DiLDL combining with FITC-UEA-I (double positivity)

Comparison of systolic pressure of pulmonary artery before and after treatment of two groups.

In fasudil treatment group, the pulmonary arterial pressures were significantly decreased, the right ventricular outflow tract diameter was reduced and the ejection fraction was significantly improved. They were significantly different from those before treatment and from those in the control group (P<0.05). (Table 2)

TABLE 2: Comparison of the pulmonary arterial systolic pressures before and after treatment between the two groups

Changes on number and function of late EPC in perpheral blood before and after treatment of the two groups.

In fasudil treatment group, the number of late EPC in peripheral blood was increased; the migration, adhesion and proliferation capacities were also increased, which were significantly different from those before treatment and from those in the control group (P<0.05). (Table 3)

TABLE 3: Comparison of the changes in the number and function of late EPC in the peripheral blood before and after treatment between the two groups

DISCUSSION

The reformation of pulmonary vessels is the main factor for the pathogenesis of COPD with pulmonary artery hypertension. It is generally believed that systematic endothelial damage is the starting phase of pulmonary artery hypertension. The damage and / or malfunction of pulmonary endothelial cells might cause endogenous unbalance between pulmonary vasodilator and vasoconstrictor. Thus, the relaxation vascular active substance decreases and the contracting vascular active substance increases [6]. In addition, the damaged endothelial cells secrete multiple cell factors, such as fibroblast growth factor, platelet derived growth factor, etc, to promote the hyperplasia and hypertrophy of vascular smooth muscle cells. In a pulmonary artery hypertension case, the damage of vascular endothelial cells is one of the factors in the pathogenic processes. If this process is inhibited, the development of pulmonary arter hypertension could be possibly inhibited. EPC is reproductive and could be differentiated to be vascular endothelial cells to accelerate the repairment of damaged vascular endothelium. Studies showed that endogenous erythrocyte stimulating factor was able to promote the EPC mobilization from chronic hypoxia mouse bone marrow to prevent the PAH process and reduce the hypertrophy of the right ventricle and the reconstruction of pulmonary vessels [8]. As EPC has an important role in the maintaining of the complete functions of endothelium, the treatment of PAH by EPC also draws the attention of researchers home and abroad. This research found that the pulmonary artery pressure of the patients in the Fasudil treatment group was reduced significantly. The number and function of the late endothelial progenitor cells in peripheral blood were also enhanced. It is conjectured that the following mechanisms are possible. In recent years, the relationship between Rho/Rho-kinase signaling pathway and cardiopulmonary vascular diseases has drawn an increasing attention. Fasudil is a selective inhibitor of Rho-kinase signalling pathway. It competes for the combination with the ATO combination site in the kinase area of Rho-kinase with ATP, so as to block the activity of Rhokinase. It could also inhibit multiple proteinases such as myosin light chain proteinase and protein kinase, leading to the inhibition of phosphorylation at the final stage of contraction of smooth muscle. Thus, the vessels are expanded [9]. Fagan et al. [10] found that the selective inhibitors -Y-27632 and Fasudil of Rho/Rho-kinase signaling pathway were able to lower the pulmonary artery pressure and pulmonary vascular resistance in mice with pulmonary artery hypertension and had important adjustment effects on the vascular structure and function [11]. Recent studies proved that Fasudil could inhibit the synthesis and secretion of ET-1 by endothelial cells and promote the synthesis and secretion of NO, to improve the balance between ET-1 and NO. Therefore, the endothelium mediated relaxation vascular effect was enhanced [12]. Therefore, Fasudil could enlarge the vessels by Rho/Rho-kinase signaling pathway to reduce the pulmonary artery pressure; furthermore, it could adjust the endothelial cells function so that the latter decreases the secretion of blood vessel contraction substance and increase relaxation substance. In this way, the pulmonary artery pressure is lowered. With the development of COPD, the peripheral airway obstruction, lung parenchyma damage and abnormality of pulmonary blood vessels reduce the pulmonary gas exchange capacity and causes hyoxemia, even hypercapnia. Long term of chronic hypoxia results in the pulmonary artery hypertension. The main mechanism is hypoxic pulmonary vasoconstriction (HPV), dysfunction of endothelium of pulmonary vessels and reconstruction of pulmonary vessels. The vascular endothelial cells have important role in adjusting human pulmonary circulation. The endothelial cells could release multiple vascular relaxation substances to adjust the vascular reactions. These substances are called endothelium derived relaxation factors (EDRF) [13]. No is one of them. In COPD patients with pulmonary artery hypertension. EDRF mediated vascular contraction is damaged, pulmonary vascular resistance (RPV) increased. These are the results of endothelial damage caused by shear stress of hemodynamics and hypoxia [14]. The endothelial progenitor cells (EPCs) are precursor cells that can be differentiated into vascular endothelial cells directly. EPC plays an important role in the neogenesis and maintaining of the completeness of the function of endothelium of blood vessels [15]. Yamada et al. [16] proved that stem cells were necessary for the repairment of pulmonary tissues with the lipopolysaccharide mediated mouse pneumonia model. Nagaya et al. [17] reported that in the monocrotaline mediated pulmonary artery hypertension nude mouse model, xenotransplantation of EPC reduced the pulmonary vascular resistance by 16%. Gene infected EPC could reduce the pulmonary vascular resistance by 35% and its survival rate was higher [17]. These studies confirmed that EPC could enhance the damaged pulmonary artery endothelial repairment and the reconstruction of artery, so as to reduce the pulmonary artery pressure. In this research, Fasudil reduced the pulmonary artery pressure of COPD patients with pulmonary artery hypertension. One of the mechanisms is to increase the number and function of the late EPC in peripheral blood of the patients to reduce the damage of pulmonary vascular endothelial cells and improve the reconstruction of pulmonary vascular structure, so as to obtain the treatment effects.

CONCLUSION

Fasudil as a Rho-kinase inhibitor can increase the number and function of late EPC in the peripheral blood of COPD patients combined with pulmonary hypertension.

DECLARATION OF INTEREST

The authors declare no conflict of interest.

REFERENCES

  1. , , , , , (). Tissue Doppler evaluation of tricuspid annulus for estimation of pul-monary artery pressure in patients with COPD. Lung.
  2. , , , , , (). Smoking is associated with depletion of circulating endothelial progenitor cells and elevated pulmonary artery systolic pressure in patients with coronary artery disease. Am J Cardiol.
  3. , , , , , (). Differential activity of bone marrow hematopoietic stem cell subpopulations for EPC development and ischemic neovascu-larization. J Mol Cell Cardiol.
  4. , , , , , (). Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol.
  5. , , , , , (). Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases. Circulation.
  6. , , (). Vascular endothelial growth factor as a non-invasive marker of pulmonary vascular remodeling in patients with bronchitis-type of COPD. Respir Res.
  7. , , , , , (). VEGF increases the proliferative capacity and eNOS/NO levels of endothelial progenitor cells through the calcineurin/NFAT signalling pathway. Cell Biol Int.
  8. , , , , , (). Autologous transplantation of bone marrow-derived endothelial progenitor cells attenuates monocrotaline-induced pulmonary arterial hypertension in rats. Crit Care Med.
  9. , , , (). Fasudil, a Rho-kinase inhibitor, protects against excessive endurance exercise training-induced cardiac hypertrophy, apoptosis and fibrosis in rats. Eur J Appl Physiol.
  10. , , , , , (). Attenuation of acute hypoxic pulmonary vasoconstriction and hy-poxic pulmonary hypertension in mice by inhibition of Rho-kinase. Am J Physiol Lung Cell Mol Physiol.
  11. , , , , (). Rho kinase-me-diated vasoconstriction in pulmonary hypertension. Adv Exp Med Biol.
  12. , , , , , (). The Rho kinase inhibitor fasudil augments the number of functional endothelial progenitor cells in ex vivo cultures. Int J Mol Med.
  13. , , (). Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells. 1985. Br J Pharmacol. discussion 491-493
  14. , , , , , (). Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2. Circ Res.
  15. , , , , , (). Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction. J Am Coll Cardiol.
  16. , , , , , (). Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury. J Immunol.
  17. , , , , , (). Effects of adrenomedullin inhalation on hemodynamics and exercise capacity in patients with idiopathic pulmonary arterial hypertension. Circulation.