Waon therapy mobilizes CD34+ cells and improves peripheral arterial disease

      Summary

      Background

      We previously reported that Waon therapy upregulates endothelial nitric oxide synthase protein, and augments ischemia-induced angiogenesis in mice with hindlimb ischemia, and it improves limb ischemia in patients with peripheral arterial disease (PAD). The aim of this study was to investigate the underlying mechanism of Waon therapy for the treatment of patients with PAD, and to determine whether Waon therapy can mobilize blood-derived progenitor cells.

      Methods

      21 consecutive PAD patients received standard medications, and were randomly divided into control (n = 10) and Waon therapy groups (n = 11). The Waon therapy group received Waon therapy daily for 6 weeks. The control group continued conventional therapy for 6 weeks. Leg pain was scored using a visual analogue scale. The ankle-brachial pressure index (ABPI) and the 6-min walking distance were measured at baseline and 6 weeks after therapy. Frequency of circulating CD34+ progenitor cell numbers was measured by quantitative real-time polymerase chain reaction, and the serum nitrate and nitrite levels were also measured at baseline and 6 weeks after therapy.

      Results

      The leg pain score, ABPI and the 6-min walking distance improved significantly after 6 weeks in the Waon therapy group, but not in the control group. Frequency of circulating CD34+ cells increased after 6 weeks of Waon therapy [2.0 ± 1.2 (×10−4) at baseline to 3.9 ± 1.9 (×10−4), p = 0.015], while it remained unchanged in the control group [1.8 ± 1.8 (×10−4) at baseline to 1.2 ± 0.9 (×10−4)]. Serum nitrate and nitrite levels increased significantly after Waon therapy (29.6 ± 17.6 to 36.0 ± 17.7 μmol/ml, p < 0.05), but not in the control group (34.4 ± 9.4 to 38.3 ± 8.8 μmol/ml).

      Conclusion

      Waon therapy mobilized circulating endothelial progenitor cells and improved limb ischemia in patients with PAD. Waon therapy is a highly promising therapy for patients with PAD.

      Keywords

      Introduction

      Peripheral arterial disease (PAD) is associated with decreased functional capacity and quality of life. Furthermore, patients with PAD have a higher rate of limb amputation and an increased risk of death compared with the general population. Patients with chronic critical limb ischemia have a 20% mortality rate per year with many patients being poor candidates for revascularization procedures [
      • Norgren L.
      • Hiatt W.R.
      • Dormandy J.A.
      • Nehler M.R.
      • Harris K.A.
      • Fowkes F.G.
      on behalf of the TASC II Working Group
      Inter-society consensus for the management of peripheral arterial disease (TASC II).
      ]. Therapeutic angiogenesis and vasculogenesis may provide a treatment option for those patients with critical limb ischemia who are not suited for conventional revascularization therapy [
      • Isner J.M.
      • Asahara T.
      Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization.
      ]. Some attempts at therapeutic angiogenesis have induced gene transfer using naked plasmid DNA encoding vascular endothelial growth factor (VEGF), which has demonstrated some promise in improving symptoms of critical limb ischemia [
      • Baumgartner I.
      • Pieczek A.
      • Manor O.
      • Blir R.
      • Keamey M.
      • Walsh K.
      • Isner I.M.
      Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.
      ], although not all studies have shown a benefit [
      • Yla-Herttuala S.
      • Rissanen T.T.
      • Vajanto I.
      • Hartikainen J.
      Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine.
      ,
      • Yla-Herttuala S.
      • Alitalo K.
      Gene transfer as a tool to induce therapeutic vascular growth.
      ]. In addition, research has examined endothelial progenitor cells, which originate from bone marrow and circulate in the peripheral blood, and are known to participate in postnatal neovascularization [
      • Asahara T.
      • Murohara T.
      • Sullivan A.
      • Silver M.
      • van der Zee R.
      • Li T.
      • Witzenbichler B.
      • Schantteman G.
      • Isner J.M.
      Isolation of putative progenitor endothelial cells for angiogenesis.
      ,
      • Asahara T.
      • Masuda H.
      • Takahashi T.
      • Kalka C.
      • Patore C.
      • Silver M.
      • Keame M.
      • Magner M.
      • Isner J.M.
      Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.
      ]. There is evidence that autologous implantation of bone marrow mononuclear cells could be effective for achieving therapeutic angiogenesis [
      • Tateishi-Yuyama E.
      • Matsubara H.
      • Murohara T.
      • Ikeda U.
      • Shintani S.
      • Masaaki H.
      • Amano K.
      • Kishimoto Y.
      • Yoshimoto K.
      • Akashi H.
      • Shimada K.
      • Iwasaka T.
      • Imaizumi T.
      Therapeutic Angiogenesis using Cell Transplantation (TACT) Study Investigators
      Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial.
      ,
      • Kajiguchi M.
      • Kondo T.
      • Izawa H.
      • Kobayashi M.
      • Yamamoto K.
      • Shintani S.
      • Numaguchi Y.
      • Naoe T.
      • Takamatsu J.
      • Komori K.
      • Murohara T.
      Safety and efficacy of autologous progenitor cell transplantation for therapeutic angiogenesis in patients with critical limb ischemia.
      ]. These therapeutic angiogenesis trials have shown promising early efficacy, but further trials are warranted.
      We have developed a form of thermal therapy, termed Waon therapy, which uses a dry sauna maintained at a temperature of 60 °C and differs from a traditional sauna. We previously reported that repeated Waon therapy improves hemodynamics and ameliorates symptoms and ventricular arrhythmias in patients with chronic heart failure (CHF) [
      • Tei C.
      • Horikiri Y.
      • Park J.C.
      • Jeong J.W.
      • Chang K.S.
      • Toyama Y.
      • Tanaka N.
      Acute hemodynamic improvement by thermal vasodilation in congestive heart failure.
      ,
      • Tei C.
      • Tanaka N.
      Thermal vasodilation as a treatment of congestive heart failure: a novel approach.
      ,
      • Kihara T.
      • Biro S.
      • Ikeda Y.
      • Fukudome T.
      • Shinsato T.
      • Masuda A.
      • Miyata M.
      • Hamasaki S.
      • Otsuji Y.
      • Minagoe S.
      • Akiba S.
      • Tei C.
      Effects of repeated sauna treatment on ventricular arrhythmias in patients with chronic heart failure.
      ]. Furthermore, we reported that repeated Waon therapy improves vascular endothelial dysfunction in patients with CHF, and in patients with coronary risk factors such as hypertension, hyperlipidemia, diabetes mellitus, and smoking [
      • Kihara T.
      • Biro S.
      • Imamura M.
      • Yoshifuku S.
      • Takasaki K.
      • Ikeda Y.
      • Minagoe S.
      • Toyama Y.
      • Tei C.
      Repeated thermal therapy treatment improves vascular endothelial and cardiac function in patients with chronic heart failure.
      ,
      • Imamura M.
      • Biro S.
      • Kihara T.
      • Yoshihuku S.
      • Takasaki K.
      • Otsuji Y.
      • Minagoe S.
      • Toyama Y.
      • Tei C.
      Repeated thermal therapy improves impaired vascular endothelial function in patients with coronary risk factors.
      ]. We also demonstrated that the molecular mechanism by which repeated Waon therapy improves vascular flow and endothelial function is through increased endothelial nitric oxide synthase (eNOS) expression [
      • Ikeda Y.
      • Biro S.
      • Kamogawa Y.
      • Yoshifuku S.
      • Eto H.
      • Orihara K.
      • Kihara T.
      • Tei C.
      Repeated thermal therapy upregulates arterial endothelial nitric oxide synthase expression in Syrian golden hamsters.
      ,
      • Ikeda Y.
      • Biro S.
      • Kamogawa Y.
      • Yoshifuku S.
      • Eto H.
      • Orihara K.
      • Kihara T.
      • Tei C.
      Repeated sauna therapy increases arterial endothelial nitric oxide synthase expression and nitric oxide production in cardiomyopathic hamsters.
      ].
      Nitric oxide (NO) is constitutively produced by eNOS, and is a mediator of angiogenesis. Waon therapy increases eNOS protein expression, blood flow, and capillary density in a mouse model of hindlimb ischemia [
      • Akasaki Y.
      • Miyata M.
      • Eto H.
      • Shirasawa T.
      • Hamada N.
      • Ikeda Y.
      • Biro S.
      • Otsuji Y.
      • Tei C.
      Repeated thermal therapy up-regulates endothelial nitric oxide synthase and augments angiogenesis in a mouse model of hindlimb ischemia.
      ]. Furthermore, Waon therapy does not increase blood flow and capillary density in eNOS-deficient mice, which demonstrates that eNOS is a critical regulator of angiogenesis during Waon therapy. Recently, we reported that Waon therapy is safe and improves leg pain, status, and blood flow in patients with PAD [
      • Tei C.
      • Shinsato T.
      • Miyata M.
      • Kihara T.
      • Hamasaki S.
      Waon therapy improves peripheral arterial disease.
      ,
      • Tei C.
      • Shinsato T.
      • Kihara T.
      • Miyata M.
      Successful thermal therapy for end-stage peripheral artery disease.
      ].
      This study was performed to confirm the beneficial effects of Waon therapy using the infrared-ray dry sauna, and to investigate the underlying mechanism of why Waon therapy may be beneficial in the treatment patients with PAD. In particular, we wanted to determine whether Waon therapy could mobilize blood-derived progenitor cells for local vascular regeneration.

      Methods

       Patients

      The present study included patients with PAD with intermittent claudication for a minimum of 4 weeks without evidence of improvement despite conventional therapies. Inclusion criteria were (1) a resting ankle-brachial pressure index (ABPI) <0.75 in the affected limb on 2 consecutive examinations performed at least 1 week apart, (2) lower limb artery lesions were detected with computed tomographic angiography (CTA), magnetic resonance angiography (MRA) or color duplex ultrasound. 21 consecutive PAD patients were enrolled, and were randomly divided into control (n = 10) and Waon therapy groups (n = 11). They were allowed to continue taking antiplatelet drugs, provided that these therapies had been used for a minimum of 6 months before the enrollment. In the Waon therapy group, they had Waon therapy daily for 6 weeks. In the control group, they had only conventional PAD therapy for 6 weeks. All patients gave their written informed consent. This study design was unanimously approved by the Ethics Committee of Kagoshima University.

       Waon therapy

      Waon therapy, a form of thermal therapy that uses a far infrared-ray dry sauna at 60 °C and differs from the traditional sauna in that it has no hydration pressure, was performed as previously reported [
      • Tei C.
      • Shinsato T.
      • Miyata M.
      • Kihara T.
      • Hamasaki S.
      Waon therapy improves peripheral arterial disease.
      ]. The patients were placed in a 60 °C dry sauna system for 15 min; after leaving the sauna, they underwent bed rest with a blanket to keep them warm for an additional 30 min. All patients were weighed before and after the therapy, and oral hydration with water was used to compensate for weight lost due to perspiration.
      Waon therapy was performed once a day, 5 days a week, for a total of 6 weeks in the Waon therapy group. To rule out an acute effect of Waon therapy, all measurements were performed before the first treatment and on the next day after the last treatment in the Waon therapy group.

       Measurements

      Leg pain was scored using a visual analogue scale, with a marked 10-cm line extending from “no pain: 0” to “severest pain: 10”. Resting ABPI was calculated by the quotient of the absolute ankle pressure to the right brachial pressure using an oscillometric method by Form/ABPI (Colin, Komaki, Japan) as reported previously [
      • Kubo T.
      • Miyata M.
      • Minagoe S.
      • Setoyama S.
      • Maruyama I.
      • Tei C.
      A simple oscillometric technique for determining new indices of arterial distensibility.
      ]. Six-minute (6-min) walking distance was measured to evaluate exercise capacity. The serum nitrate (NO3), nitrite (NO2), and VEGF levels were measured. The pain score, ABPI, and 6-min walking distance were measured at baseline and after 6 weeks.

       Analysis of CD34 mRNA for detection of circulating CD34+ cells in the periphery

      To detect the shift of circulating CD34+ progenitor cells in the periphery with higher sensitivity, quantitative real-time polymerase chain reaction (PCR) for CD34 messenger RNA (mRNA) was performed with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. Patients’ peripheral blood mononuclear cells were harvested at baseline and after 6 weeks. Peripheral blood mononuclear cells (PBMC) (approximately 106/tube) were stored at −80 °C and later used for RNA extraction, cDNA synthesis, and quantitative real-time PCR.
      Total RNA was extracted from patients’ PBMC using the guanidine isothiocyanate method (RNeasy Mini Kit, Qiagen, Hilden, Germany), and was then transcribed to cDNA (Transcriptor First Strand cDNA Synthesis Kit, Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Real-time quantitative PCR analysis was performed using a LightCycler system and LC FastStart DNA Master Hybridization Probes (Roche Diagnostics), and 1 μl of cDNA in a final reaction volume of 20 μl. Primers for human CD34 (GenBank accession no. M81104) were designed as follows: (forward) 5′-CTCAGTGTCTACTGCTGGTCT-3′ and (reverse) 5′-GGAATAGCTCTGGTGGCTT-3′. The taqman probe sequence was 5′-FAM-CAACTTATGAAAAAGCACCAATCTGACCTG-TAMRA-3′. Thermal cycler parameters included 10 min at 95 °C, 40 cycles of 95 °C for 10 s, and 62 °C for 20 s. The size of the amplicon of the CD34 gene was 143 bp. The expression level of the GAPDH (GenBank accession no. M33197) gene was measured as an internal reference with a standard curve for determining the integrity of template RNA for all specimens. All primers were purchased from Nihon Gene Research Laboratories (Sendai, Japan). The frequency of CD34+ cells in the periphery was determined as the ratio of the mRNA level of these genes as follows: absolute copy number of CD34 gene in 1 μl of cDNA/absolute copy number of GAPDH gene in 1 μl of cDNA.

       Statistical analysis

      All data are expressed as mean values ± SD. Data at baseline and after 6 weeks were compared using the paired t-test. Differences in patients’ characteristics were evaluated using the chi-squared test and the unpaired t-test. A value of p < 0.05 was considered statistically significant.

      Results

       Clinical characteristics

      The patients’ clinical characteristics are summarized in Table 1. There were no significant differences in age, body mass index, ABPI, hyperlipidemia, diabetes mellitus, hypertension, and smoking between the control and the Waon therapy groups. In addition, there were no significant differences in the use of medications for PAD, such as antiplatelets, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or statins between the two groups. There was a significant difference in gender between the two groups.
      Table 1Characteristics of patients.
      Control (n = 10)Waon therapy (n = 11)p-Value
      Gender (male/female)10/07/4<0.05
      Age (years)75 ± 575 ± 6N.S.
      BMI24.0 ± 1.323.0 ± 1.9N.S.
      ABPI0.50 ± 0.210.55 ± 0.22N.S.
      Hyperlipidemia, n (%)5 (50%)7 (63%)N.S.
      Diabetes mellitus, n (%)6 (60%)8 (72%)N.S.
      Hypertension, n (%)9 (90%)8 (72%)N.S.
      Smoking, n (%)8 (80%)8 (72%)N.S.
      Medications
       Antiplatelets, n (%)10 (100%)11 (100%)N.S.
       ACEI or ARB, n (%)4 (40%)5 (45%)N.S.
       Statin, n (%)5 (50%)6 (54%)N.S.
       PGI2 analogue, n (%)3 (33%)3 (27%)N.S.
      All patients enrolled in the trial completed the study without any adverse events. During the trial, no patients developed significant laboratory test abnormalities, including those for blood cell counts, electrolyte concentrations, chemistry and lipid profiles, tumor markers, and inflammatory markers. No changes in diabetic retinopathy were observed on fundoscopic examination, and no latent neoplasms developed. Transient leg pain during Waon therapy was seen, but it disappeared after a few sessions of Waon therapy.

       Changes in leg pain, walking distance, and ABPI

      Therapeutic benefit was demonstrated by regression of leg pain in all patients treated with Waon therapy. The pain score evaluated using a visual analogue scale was significantly decreased in the Waon therapy group after 6 weeks (8.1 ± 0.7 to 2.8 ± 1.1, p < 0.05), while it remained unchanged in the control group (8.4 ± 0.8 to 8.2 ± 1.1, Fig. 1A ).
      Figure thumbnail gr1
      Figure 1Changes in leg pain, walking distance, and ankle-brachial pressure index (ABPI). (A) Pain score evaluated using a visual analogue scale (10: severe pain; 0: no pain). (B) 6-min walking distance. (C) ABPI open bars: at baseline; solid bars: after 6 weeks.
      Exercise performance improved significantly in the Waon therapy group (Fig. 1B). After 6 weeks of Waon therapy, the 6-min walking distance increased from 149 ± 104 m at baseline to 230 ± 154 m at 6 weeks after Waon therapy (p < 0.01), while it did not change in the control group (136 ± 76 to 132 ± 72 m, Fig. 1B).
      The mean ABPI increased significantly from 0.55 ± 0.20 at baseline to 0.60 ± 0.22 after 6 weeks in the Waon therapy group (p < 0.01, Fig. 1C), but it did not change in the control group (0.50 ± 0.21 to 0.49 ± 0.20).

       Detection of circulating CD34+ cells by quantitative real-time PCR

      Frequency of circulating CD34 positive cell numbers was measured at baseline and after 6 weeks by quantitative real-time PCR using the mRNA CD34/GAPDH ratio (Fig. 2). In the PAD group, the mRNA CD34/GAPDH ratio increased 2.0-fold, from 2.0 ± 1.2 (×10−4) at baseline to 3.9 ± 1.9 (×10−4) after 6 weeks of Waon therapy (p = 0.015), while the mRNA CD34/GAPDH ratio in the control group was 1.8 ± 1.8 (×10−4) at baseline and 1.2 ± 0.9 (×10−4) after 6 weeks (no significant difference). Repeated Waon therapy appears to mobilize progenitor cells among PAD patients, and this might be helpful for the repair or regeneration of damaged vessels.
      Figure thumbnail gr2
      Figure 2Changes in frequency of mobilized circulatory CD34+ cells. Open bars: at baseline; solid bars: after 6 weeks. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

       NO production and VEGF levels

      Serum nitrate (NO3), nitrite (NO2), and VEGF levels were measured in both groups. In the Waon therapy group, the serum nitrate and nitrite levels increased significantly after 6 weeks of Waon therapy (29.6 ± 17.6 to 36.0 ± 17.7 μmol/ml, p < 0.05), while it remained unchanged in the control group (34.4 ± 9.4 to 38.3 ± 8.8 μmol/ml, Fig. 3).
      Figure thumbnail gr3
      Figure 3Changes in serum nitrate and nitrite levels. Open bars: at baseline; solid bars: after 6 weeks.
      There were no significant differences in VEGF levels at baseline and after 6 weeks in both groups (control group: 39.2 ± 21.7 to 42.5 ± 21.6 pg/ml; Waon therapy group: 70.2 ± 46.3 to 40.2 ± 40.5 pg/ml, Fig. 4). VEGF level at baseline of Waon therapy group was significantly higher than that of control group (p = 0.041).
      Figure thumbnail gr4
      Figure 4Changes in serum vascular endothelial growth factor (VEGF) levels. Open bars: at base line; solid bars: after 6 weeks.

      Discussion

      We have developed thermal therapy using a 60 °C dry sauna and showed that it improves hemodynamic parameters, endothelial function, clinical symptoms, and prognosis in many CHF patients [
      • Tei C.
      • Horikiri Y.
      • Park J.C.
      • Jeong J.W.
      • Chang K.S.
      • Toyama Y.
      • Tanaka N.
      Acute hemodynamic improvement by thermal vasodilation in congestive heart failure.
      ,
      • Tei C.
      • Tanaka N.
      Thermal vasodilation as a treatment of congestive heart failure: a novel approach.
      ,
      • Kihara T.
      • Biro S.
      • Ikeda Y.
      • Fukudome T.
      • Shinsato T.
      • Masuda A.
      • Miyata M.
      • Hamasaki S.
      • Otsuji Y.
      • Minagoe S.
      • Akiba S.
      • Tei C.
      Effects of repeated sauna treatment on ventricular arrhythmias in patients with chronic heart failure.
      ,
      • Miyata M.
      • Kihara T.
      • Kubozono T.
      • Ikesa Y.
      • Shinsato T.
      • Izumi T.
      • Matsuzaki M.
      • Yamaguchi T.
      • Kasanuki H.
      • Daida H.
      Beneficial effects of Waon therapy on patients with chronic heart failure: results of a prospective multicenter study.
      ,
      • Basford J.R.
      • Oh J.K.
      • Allison T.G.
      • Sheffield C.G.
      • Manahan B.G.
      • Hodge D.O.
      • Tajik A.J.
      • Rodeheffer R.J.
      • Tei C.
      Safety, acceptance, and physiologic effects of sauna bathing in people with chronic heart failure: a pilot report.
      ,
      • Kihara T.
      • Miyata M.
      • Fukudome T.
      • Ikeda Y.
      • Shinsato T.
      • Kubozono T.
      • Fujita S.
      • Kuwahata S.
      • Torii H.
      • Lee S.
      • Toda H.
      • Tei C.
      Waon therapy improves the prognosis of patients with chronic heart failure.
      ]. Generally, thermal therapy at very high temperature was originally used to treat localized cancer. However, the therapy we developed to treat cardiovascular diseases is quite different in that it consists of systemic soothing warmth that comfortably refreshes the mind and body. Therefore, we have changed the name from thermal to Waon therapy, since “Waon” in Japanese means soothing warmth [
      • Tei C.
      Waon therapy: soothing warmth therapy.
      ].
      In the present study, it was confirmed that Waon therapy is safe and potentially effective for patients with severe PAD, as evidenced by substantial decreases in the pain score and increases in ABPI and walking distance.
      The goal in this study was to examine the underlying mechanism of Waon therapy for the treatment of PAD, and to see whether Waon therapy could mobilize blood-derived progenitor cells for local vascular regeneration. Recently, flow cytometry has been used to detect CD34+ progenitor cells in the periphery; however, the frequency of CD34+ cells in the periphery under normal, steady state conditions is less than 0.01%, which is just at the limit of the detectable range of flow cytometers. It is widely known that granulocyte-colony stimulating factor (G-CSF) can mobilize a large number of CD34+ hematopoietic progenitor cells from the bone marrow, which can be easily detected by flow cytometry. Nonetheless, we speculated that progenitor mobilization by our Waon therapy might be much weaker than that with G-CSF administration. To detect the change in circulating CD34+ cells with higher sensitivity, we developed a quantitative real-time PCR for CD34 mRNA using the GAPDH gene as an internal control, and found that circulating CD34+ cells were mobilized by repeated Waon therapy in patients with PAD.
      We previously reported that angiogenesis was induced via eNOS using Waon therapy in mice with hindlimb ischemia [
      • Akasaki Y.
      • Miyata M.
      • Eto H.
      • Shirasawa T.
      • Hamada N.
      • Ikeda Y.
      • Biro S.
      • Otsuji Y.
      • Tei C.
      Repeated thermal therapy up-regulates endothelial nitric oxide synthase and augments angiogenesis in a mouse model of hindlimb ischemia.
      ]. NO is a mediator of angiogenesis and plays a key role in angiogenesis induced by Waon therapy. Repeated Waon therapy increases cardiac output, shear stress of the vessel wall, and, ultimately, eNOS expression.
      PAD patients are characterized by reduced systemic NO bioactivity. The impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells [
      • Aicher A.
      • Heeschen C.
      • Mildner-Rihm C.
      • Urbich C.
      • Ihling C.
      • Technau-Ihling K.
      • Zeiher A.M.
      • Dimmeler S.
      Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells.
      ]. In the present study, Waon therapy increased NO and circulating endothelial progenitor cells (EPC), which suggests that this therapy may mobilize EPC through an eNOS-dependent mechanism.
      Some strategies to regulate the mobilization of progenitor cells through the eNOS pathway have been reported [
      • Aicher A.
      • Heeschen C.
      • Mildner-Rihm C.
      • Urbich C.
      • Ihling C.
      • Technau-Ihling K.
      • Zeiher A.M.
      • Dimmeler S.
      Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells.
      ]. Statin therapy has improved EPC mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction [
      • Landmesser U.
      • Engberding N.
      • Bahlmann F.H.
      • Schaefer A.
      • Wiencke A.
      • Heineke A.
      • Spiekermann S.
      • Hilfiker-Kleiner D.
      • Templin C.
      • Kotlarz D.
      • Mueller M.
      • Fuchs M.
      • Hornig B.
      • Haller H.
      • Drexler H.
      Statin-induced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction requires endothelial nitric oxide synthase.
      ]. Estrogen therapy has augmented EPC incorporation into sites of myocardial neovascularization after myocardial infarction [
      • Iwakura A.
      • Luedemann C.
      • Shastry S.
      • Hamada H.
      • Kawamoto A.
      • Kishore R.
      • Zhu Y.
      • Qin G.
      • Silver M.
      • Thorne T.
      • Eaton L.
      • Masuda H.
      • Asahara T.
      • Losordo D.W.
      Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury.
      ] and physical training also has increased EPCs and enhanced angiogenesis [
      • Laufs U.
      • Werner N.
      • Link A.
      • Endres M.
      • Wassmann S.
      • Jürgens K.
      • Miche E.
      • Böhm M.
      • Nickenig G.
      Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis.
      ].
      NO is an angiogenic mediator acting downstream of VEGF; impaired angiogenesis in eNOS-deficient mice was not improved by administration of VEGF [
      • Murohara T.
      • Asahara T.
      • Silver M.
      • Bauters C.
      • Masuda H.
      • Kalka C.
      • Kearney M.
      • Chen D.
      • Symes J.F.
      • Fishman M.C.
      • Huang P.L.
      • Isner J.M.
      Nitric oxide modulates angiogenesis in response to tissue ischemia.
      ], and transgenic mice overexpressing eNOS did not show a greater increase in VEGF expression in hindlimb ischemia [
      • Amano K.
      • Matsubara H.
      • Iba O.
      • Okigaki M.
      • Fujiyama S.
      • Imada T.
      • Kojima H.
      • Nozawa Y.
      • Kawashima S.
      • Yokoyama M.
      • Iwasaka T.
      Enhancement of ischemia-induced angiogenesis by eNOS overexpression.
      ]. Although Waon therapy did not increase expression of VEGF in the ischemic hindlimb, this therapy augmented angiogenesis by increasing eNOS expression in the course of ischemia-induced VEGF expression [
      • Akasaki Y.
      • Miyata M.
      • Eto H.
      • Shirasawa T.
      • Hamada N.
      • Ikeda Y.
      • Biro S.
      • Otsuji Y.
      • Tei C.
      Repeated thermal therapy up-regulates endothelial nitric oxide synthase and augments angiogenesis in a mouse model of hindlimb ischemia.
      ]. Therefore, we think CD34+ cells were recruited in PAD patients who had the ischemia-induced VEGF. Furthermore, in this study, the concentration of VEGF which was upregulated by ischemia decreased in the course of improvement of limb ischemia after Waon therapy.

      Study limitations

      There are several limitations in this study. VEGF levels at baseline of the two groups were different. This difference may have an influence on results, although Waon therapy did not increase the VEGF levels. Further large study is required to resolve this grouping problem.
      There was no significant correlation between CD34+ cells, NO, and VEGF in the present study. There may be a time lag for a rise of these factors. Further study that measures these factors in several points will clarify the correlation of these factors.

      Conclusions

      Waon therapy mobilized circulating endothelial progenitor cells and improved limb ischemia in PAD patients. Waon therapy is a highly promising therapy for PAD patients.

      Acknowledgments

      The authors gratefully acknowledge Mrs. Eriko Masuyama, Mrs. Mitsuko Nakayama, Mrs. Michiko Takeshita, and Miss Aya Nagaki for their kind assistance. This study was supported by Grants-in-Aid from the Japan Society for the Promotion of Science ( #16300184 ).

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