Impact of Therapeutic Angiogenesis Using Autologous Bone

Circulation Journal Vol.83, March 2019

662 SHOJI K et al.Circulation JournalCirc J 2019; 83: 662 – 671doi: 10.1253/circj.CJ-18-1044

CD, particularly with systemic sclerosis (SSc, scleroderma) have experienced Raynaud’s phenomenon or digital ulcers (DU) because of inadequate peripheral blood supply. Intractable ischemic ulcers negatively affect patients’ quality of life (QOL), and may lead to limb amputation.2 The European League against Rheumatism (EULAR) recom-mends intravenous (IV) iloprost, PDE-5 inhibitors, and

C ritical limb ischemia (CLI) is caused by chronic arterial obstruction, mainly caused by arterioscle-rosis obliterans (ASO), thromboangiitis obliterans

(TAO), and collagen disease (CD). Patients with CLI often require limb amputation when conventional revascular-ization methods such as bypass surgery or endovascular treatment (EVT) fail or are not indicated.1 Patients with

Received September 19, 2018; revised manuscript received December 24, 2018; accepted January 8, 2019; J-STAGE Advance Publication released online February 7, 2019 Time for primary review: 44 days

Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto (K.S., K.Y., S.M.); Department of Stem Cell and Immune Regulation, Yokohama City University Graduate School of Medicine, Yokohama (R.Y., N.H., H.N.); Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya (K. Kondo, T.M.); Department of Cardiology, National Hospital Organization Kumamoto Medical Center, Kumamoto (K.F.); Department of Cardiovascular Medicine, Shinshu University School of Medicine, Matsumoto (K. Kuwahara); Department of Cardiovascular Regeneration and Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima (Y.H.); and Department of Internal Medicine, Division of Cardiovascular Medicine, Kurume University School of Medicine, Kurume (Y.F.), Japan

The Guest Editor for this article was Dr. Yoshihiko Saito.TACT Follow up Study Investigators are listed in the Appendix.Mailing address: Kenji Yanishi, MD, PhD, Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine,

465 Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail: [email protected] All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Impact of Therapeutic Angiogenesis Using Autologous Bone Marrow-Derived Mononuclear Cells Implantation

in Critical Limb Ischemia With Scleroderma― Subanalysis of the Long-Term Clinical Outcomes Survey ―

Keisuke Shoji, MD; Kenji Yanishi, MD, PhD; Ryusuke Yoshimi, MD, PhD; Naoki Hamada, MD; Kazuhisa Kondo, MD, PhD; Kazuteru Fujimoto, MD, PhD;

Hideaki Nakajima, MD, PhD; Koichiro Kuwahara, MD, PhD; Yukihito Higashi, MD, PhD; Yoshihiro Fukumoto, MD, PhD; Toyoaki Murohara, MD, PhD; Satoaki Matoba, MD, PhD; TACT Follow up Study Investigators

Background: Many patients with collagen disease (CD), particularly scleroderma (SSc), develop critical limb ischemia (CLI), which leads to limb amputation. However, conventional therapies, including revascularization via surgical bypass, showed poor outcomes in CLI patients with CD. Many CLI patients with SSc showed poor responses to combination therapies including intravenous iloprost, PDE-5 inhibitors, and bosentan. Therefore, new methods of improving the peripheral circulation for limb salvage are required. This study was a subanalysis of the long-term clinical outcomes after autologous bone marrow-derived mononuclear cells (BM-MNC) in CLI patients with SSc.

Methods and Results: We assessed no-option CLI patients with CD who underwent BM-MNC implantation at 10 institutes; 69 patients (39 with SSc-related diseases (SSc group) and 30 with other CDs (non-SSc group)), were included. The median follow-up duration was 36.5 months. The 10-year overall survival rate was 59.1% in the SSc group and 82.4% in the non-SSc group. The 10-year major amputation-free rates were 97.4% and 82.6%, respectively. The number of major or minor amputations in the SSc group trended to be less than that in the non-SSc group. Significant improvements in visual analog scale scores were observed in both groups.

Conclusions: The BM-MNC implantation may be feasible in no-option CLI patients with CD. In the SSc group, limb salvage rate tended to be higher than in the non-SSc group.

Key Words: Critical limb ischemia; Scleroderma; Therapeutic angiogenesis

ORIGINAL ARTICLEPeripheral Vascular Disease


663BM-MNC Implantation for Scleroderma

MethodsStudy DesignThis was a retrospective, observational non-controlled study. We investigated the long-term clinical outcomes of all CLI patients who underwent BM-MNC implantation under advanced medical treatment, and we reported the safety and efficacy of the procedure in 2018.16 In the present study, we re-analyzed only the patients with CD, and we evaluated the clinical outcomes for each etiology of CD. The method of data collection for this study was similar to that used in the long-term clinical outcomes survey,16 and data from some patients who underwent additional investigations extended the clinical outcomes survey until April 2018.

We assessed the overall survival (OS), major amputation-free (MAF) and amputation-free survival (AFS) rates. The AFS in this study included OS and major amputations. The safety of the therapy was assessed in the context of major adverse cardiovascular events (MACE: death, non-fatal myocardial infarction, decompensated heart failure, and stroke) and all-cause adverse events during the 6-month follow-up after BM-MNC implantation. The participating hospitals’ ethics committees approved the survey’s protocol.

Study PopulationWe included any CLI patients who underwent BM-MNC implantation and met the following criteria: (1) Fontaine stage III–IV and Rutherford category 4–6 in upper or/and lower limbs; (2) CLI caused by CD; (3) no clinical improve-ment following conventional medical and surgical treat-ments; and (4) provision of written informed consent. For all patients, we confirmed that they were not candidates for surgical or non-surgical revascularization treatment of CLI according to the recommendations of vascular surgeons, cardiologists and radiologists. All patients underwent a pre-assessment to determine whether they met any of the exclusion criteria: untreated coronary artery disease or cerebrovascular disease; clinical or laboratory signs of chronic or acute inflammation; a previous (past 5 years) or current history of neoplasia; diabetes with untreated retinopathy; age over 80 years; the possibility of pregnancy;

bosentan as pharmacotherapies for DU in patients with SSc.3 However, many patients with SSc are resistant to these medical treatments.4 Additionally, long-term outcomes after EVT or surgical bypass among CLI patients with CD have been poor.5–7 Recently, treatments for DU in patients with SSc have been proposed in which accurate treatment interventions are performed in the early stages of disease, when lesions are reversible, to prevent progression. Thus, establishing new strategies to promote peripheral circulation are needed for CLI patients with SSc.

It has been reported that one method among the cell therapies that promote peripheral circulation is therapeutic angiogenesis using bone marrow-derived mononuclear cells (BM-MNC).8–10 The first clinical pilot study, Therapeutic Angiogenesis by Cell Transplantation (TACT) study, investigated this in CLI patients in 2002,11 and subsequently, several studies have reported the safety and efficacy of BM-MNC implantation for CLI patients.12,13 Randomized studies have reported that this cell therapy could lead to significant improvements in limb ischemia, thus extending amputation-free intervals and survival rates.14,15 Moreover, Kondo et al reported a very long-term clinical outcome survey of BM-MNC implantation in no-option CLI patients, and indicated that BM-MNC implantation may be feasible and safe in CLI patients, particularly those with TAO and CD-associated vasculitis (CDV).16 SSc is the most common cause of CLI among all patients with CD. Therefore, improving the salvage rate is desirable in CLI patients with SSc, which necessitates establishment of the evidence for BM-MNC implantation compared with medical treatment and revascularization in no-option CLI patients with SSc.

In our previous study, the CDV group included SSc because the pathology of SSc may sometimes include a mechanism similar to vasculitis.16,17 However, the histopa-thology of SSc vasculopathy reflects the pathogenesis, with myofibroblast proliferation and matrix deposition in the subendothelial layer leading to obliterative thickening of vessel walls. There is either no inflammatory infiltrate or less compared with other CD-related vascular lesions.17 Therefore, we present a subanalysis of the long-term outcomes of BM-MNC implantation in no-option CLI patients with CD, particularly those with SSc.

Figure 1. Study flow chart. APS, antiphos-pholipid syndrome; CD, collagen disease; CREST, Calcinosis, Raynaud’s phenom-enon, Esophageal dysmotility, Sclero-dactyly, and Telangiectasia; CLI, critical limb ischemia; EGPA, eosinophilic granu-lomatosis with polyangitis; MCTD, mixed connective tissue disease; PN, polyar-teritis nodosa; RA, rheumatoid arthritis; SjS, Sjögren’s syndrome; SLE, systemic lupus erythematosus; SSc, scleroderma.


664 SHOJI K et al.

unpaired Student’s t-tests for discrete and continuous variables, respectively. Visual analog scale (VAS) score after BM-MNC implantation was compared with that at baseline using a Wilcoxon signed-rank test in each group. A time-to-event analysis was performed by Kaplan-Meier method to examine variables associated with OS, MAF, and AFS. A log-rank test was conducted to examine the variables for associations with survival, major amputation, and AFS between groups. All statistical analyses were performed using JMP® 14 (SAS Institute Inc., Cary, NC, USA), with a value of P<0.05 considered to be statistically significant.

or lack of informed consent. Our inclusion and exclusion criteria and assessment procedures were similar to those of the TACT study.11 The BM-MNC implantation was implemented with the approval of the ethics committee in each participating hospital for no-option CLI patients with CD, and we obtained written informed consent from all patients.

Statistical AnalysisContinuous variables are presented as mean and standard deviation or median and interquartile range (IQR), as appropriate for the distribution of the data. Categorical variables are presented as number (count). The SSc and non-SSc groups were compared by chi-squared and

Table 1. Baseline Characteristics of the Patients and the Lesions

All (n=69)

SSc (n=39)

Non-SSc (n=30) P value

Age, years 54.8±14.0 56.7±12.1 52.4±16.1 0.23

Female, n (%) 60 (87.0) 33 (84.6) 27 (90.0) 0.39

Weight, kg 52.0±11.8 50.5±12.3 54.1±10.9 0.22

BMI, kg/m2 21.3±4.0　　 20.8±4.1　　 21.9±3.8　　 0.25

Hypertension, n (%) 24 (34.8) 16 (41.0) 8 (26.7) 0.21

Hyperlipidemia, n (%) 10 (14.5) 4 (10.3) 6 (20.0) 0.48

Diabetes mellitus, n (%) 8 (11.6) 4 (10.3) 4 (13.3) 0.49

Chronic kidney disease, n (%) 3 (4.3) 1 (2.6) 2 (6.7) 0.40

Hemodialysis, n (%) 1 (1.4) 0 (0)　　　 1 (3.3) 0.44

Smoking, n (%) 19 (27.5) 16 (41.0) 3 (10.0) <0.01　Medical therapy, n (%)

Steroid 36 (52.2) 15 (38.5) 21 (70.0) 0.01

Immunosuppressive agent 9 (13.0) 2 (5.1) 7 (23.3) 0.03

Endothelin receptor antagonists 8 (11.6) 8 (20.5) 0 (0)　　　 <0.01　 Aspirin 18 (26.1) 8 (20.5) 10 (33.3) 0.28

Ticlopidine 2 (2.9) 0 (0)　　　 2 (6.7) 0.19

Cilostazol 19 (27.5) 9 (23.1) 10 (33.3) 0.42

Peripheral vasodilator agents 64 (92.8) 37 (94.9) 27 (90.0) 0.65

Statins 11 (15.9) 4 (10.3) 7 (23.3) 0.19

Complications, n (%)

Ischemic heart disease 2 (2.9) 1 (2.6) 1 (3.3) 0.68

Cerebrovascular disease 1 (1.4) 1 (2.6) 0 (0)　　　 0.57

Aortic disease 1 (1.4) 0 (0)　　　 1 (3.3) 0.44

Malignancy 0 (0)　　　 0 (0)　　　 0 (0)　　　 –

Fontaine classification, n

III/IV 10/59 5/34 5/25 0.45

Target limb, n (%)

Only lower limbs 35 (50.7) 11 (28.2) 24 (80.0) <0.01　 Only upper limbs 21 (30.4) 19 (48.7) 2 (6.7) <0.01　 Both lower and upper limbs 13 (18.8) 9 (23.1) 4 (13.3) 0.31

Total no. of target limbs, n (1/2/3/4) 32/25/7/5 18/13/5/3 14/12/2/2 0.58

Biochemistry

White blood cell count, (/μL) 7,000 (5,250–9,150) 6,500 (5,200–8,400) 7,400 (4,425–9,175) 0.55

Hemoglobin, (g/mL) 11.7 (10.5–13.2)　　 11.2 (11.0–13.3)　　 11.2 (9.4–12.5)　　　　 0.14

C-reaction protein, (mg/dL) 0.38 (0.11–1.15)　　 0.24 (0.09–0.60)　　 0.83 (0.29–1.40)　　 <0.01　Total no. of BM-MNC, (×109) 21.7±11.7 21.1±10.1 22.4±13.5 0.91

No. of BM-MNC per weight, (×108) 4.23±2.28 4.20±2.00 4.28±2.61 0.72

No. of BM-MNC per limb, (×109) 15.2±11.4 14.0±9.2　　 16.8±13.6 0.65

No. of CD34+ cells, (×107) 5.4±5.9 4.6±2.7 6.7±9.0 0.96

Data are presented as n (%), mean (standard deviation) or median (interquartile range). The P-values indicate the differences between groups. BM-MNC, bone marrow-derived mononuclear cells; BMI, body mass index; SSc, scleroderma.



polyangitis (EGPA) [n=1], Behçet’s disease [n=1], and vasculitis without definite diagnosis [n=7]) (Figure 1). A case in which a CD specialist strongly suspected a vasculitis caused by a CD, but which did not result in a definitive diagnosis was defined as “vasculitis without definite diagnosis”.

Patients’ Baseline and Procedural CharacteristicsThe baseline characteristics of the patients are shown in Table 1. The average age was 54 years, women represented 87% of the study population, and the rate of patients with Fontaine stage IV was 86%. The average age, percentage of women, percentage of patients with Fontaine stage IV, and body mass index (BMI) were not significantly different between the SSc and non-SSc groups. A history of smoking in the SSc group was significantly more common than in the non-SSc group (P<0.01). In the SSc group, oral medi-cations including steroids or immunosuppressive agents were significantly less often administered than in the non-SSc group (P=0.01 and P=0.03, respectively), but oral endothelin receptor antagonist medications was significantly

ResultsStudy Flow ChartBM-MNC implantation was performed in 69 patients with CD in 10 hospitals. We divided these patients into a SSc group (SSc-related diseases, n=39) and a non-SSc group (non-SSc-related diseases, n=30). The SSc group included some SSc-related diseases (SSc; [n=35] including CREST syndrome (Calcinosis, Raynaud’s phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia) [n=1], and mixed connective tissue disease (MCTD) [n=3]). CREST syndrome is known as limited SSc, which is a subtype of SSc.18 MCTD is characterized by overlapping features of SSc, systemic lupus erythematosus (SLE), and polymyositis (PM)/dermatomyositis (DM).19 The vasculopathy in MCTD has been found to resemble that in SSc,20 so we classified CREST syndrome and MCTD into the SSc group. The non-SSc group included other CDs (SLE [n=5], rheumatoid arthritis (RA) [n=6], polyarteritis nodosa (PN) [n=7], antiphospholipid syndrome (APS) [n=2], Sjögren’s syn-drome (SjS) [n=1], eosinophilic granulomatosis with

Table 2. All-Cause Adverse Events and MACE Within 6 Months After BM-MNC Implantation

All (n=69)

SSc (n=39)


All-cause adverse events, n (%) 6 (8.7) 2 (5.1) 4 (13.3) 0.22

MACE, n (%) 0 (0) 0 (0) 0 (0) –

Other, n (%) 6 (8.7) 2 (5.1) 4 (13.3) 0.22

Severe bleeding/anemia 3 (4.3) 0 (0) 3 (10.0) 0.08

Pelvic visceral disability 0 (0) 0 (0) 0 (0) –

Pelvic or sampling site pain 0 (0) 0 (0) 0 (0) –

Emergence of new tumor 0 (0) 0 (0) 0 (0) –

Severe infection 0 (0) 0 (0) 0 (0) –

Worsening of liver function 2 (2.9) 1 (2.6) 1 (3.3) 0.68

Worsening of kidney function 0 (0) 0 (0) 0 (0) –

Worsening of retinopathy 2 (2.9) 1 (2.6) 1 (3.3) 0.68

Data are presented as n (%). The P-values indicate the differences between groups. MACE, major adverse cardiac event. Other abbreviations as in Table 1.

Figure 2. Improvement in visual analog scale (VAS) scores within 6 months of implantation of autologous bone marrow-derived mononuclear cells in (A) the scleroderma (SSc) and (B) non-SSc groups. Data are expressed as median and interquartile range. The P-values indicate the differences before and after BM-MNC implantation.


666 SHOJI K et al.

the SSc group (P<0.01).

Safety of BM-MNC Implantation in Patients With CLI Caused by CDWe evaluated all-cause adverse events and MACE with reference to the safety of BM-MNC implantation (Table 2). All-cause adverse events occurred in 8.7% of all patients within 6 months after BM-MNC implantation. MACE did not occur within 6 months after implantation. The most frequent adverse events in all patients with CD were severe

more prevalent than in the non-SSc group (P<0.01). On the other hand, concomitant medical treatments such as antiplatelet therapy and peripheral vasodilator agents were not significant different between groups. The SSc group had more patients presenting with ischemic symptoms in the upper limbs than did the non-SSc group (P<0.01), and the BM-MNC implantation procedure was performed more often in the upper limbs in the SSc group compared with the non-SSc group. The C-reactive protein values in the non-SSc group were significantly higher than those in

Table 3. Long-Term Clinical Outcomes After BM-MNC Implantation

All (n=69)

SSc (n=39)


All-cause death, n (%) 7 (10.1) 4 (10.3) 3 (10.0) 0.65

Cause of death, n (%)

Pneumonia 1 (1.4) 1 (2.6) 0 (0)　　　　　 Lung cancer 1 (1.4) 1 (2.6) 0 (0)　　　　　 Subarachnoid hemorrhage 1 (1.4) 0 (0)　　　 1 (3.3)　　 Unknown 4 (5.8) 2 (5.1) 2 (6.7)　　Amputation, n (%) 14 (20.3) 5 (12.8) 9 (30.0) 0.07

Major amputation, n (%) 5 (7.2) 1 (2.6) 4 (13.3) 0.11

Lower limb 5 (7.2) 1 (2.6) 4 (13.3)

Upper limb 0 0 0

Minor amputation, n (%) 10 (14.5) 4 (10.3) 6 (20.0) 0.21

Lower limb 9 (13.0) 3 (7.7) 6 (20.0)

Upper limb 1 (1.4) 1 (2.6) 0 (0)　　　　　

Data are presented as n (%). The P-values indicate the differences between both groups. Amputation includes major or minor amputations. Abbreviations as in Table 1.

Figure 3. Kaplan-Meier analysis of (A) overall survival (OS), (B) major amputation-free (MAF) survival, and (C) amputation-free survival (AFS) following implantation of autologous bone marrow-derived mononuclear cells in all patients with collagen diseases.



major and minor) occurred in 20.3% of all patients with CD, in 12.8% of the SSc group, and in 30.0% of the non-SSc group, respectively. In the SSc group, major and minor amputations tended to be fewer than in the non-SSc group (Table 3).

All major amputations required in both groups were performed only on the lower limbs. Minor amputations of the lower limbs occurred more than those of the upper limbs. According to these results, in both groups there were more major and minor amputations in patients with ischemic symptoms in the lower limbs than in those with symptoms in the upper limbs.

Figure 3 shows the Kaplan-Meier analysis of OS, MAF, and AFS rates following BM-MNC implantation in all patients with CD. The 1-year OS, MAF, and AFS rates were 98.3%, 95.2%, and 93.5%, respectively. The 5-year OS, MAF, and AFS rates were 95.2%, 91.1%, and 86.5%, respectively. The 10-year OS, MAF, and AFS rates were 68.1%, 91.1%, and 61.8%, respectively (Figure 3A–C). Figure 4 shows the Kaplan-Meier analysis of OS, MAF, and AFS rates between the SSc and non-SSc groups following BM-MNC implantation. The OS, MAF, and AFS rates in each period were not significantly different between groups (log-rank test: P=0.82, P=0.09, and P=0.34, respectively) (Figure 4A–C). However, major amputations often occurred within 2 years after BM-MNC implantation in the non-SSc group compared with the SSc group (Figure 4B).

Next, we analyzed only CLI patients who underwent BM-MNC implantation in the lower limbs. Figure 5 shows the Kaplan-Meier analysis of OS, MAF, and AFS rates

bleeding/anemia (4.3%), worsening of liver function (2.9%), and worsening of retinopathy (2.9%). The incidence of all-cause adverse events or MACE was not significantly different between groups. No patients died, and no severe adverse events associated with BM-MNC implantation were noted within 6 months after this therapy.

Improvement in VAS Scores After BM-MNC ImplantationWe evaluated rest pain using a VAS, on which 0 mm meant pain-free or no pain, and 100 mm indicated the most severe pain. We evaluated VAS scores before and after BM-MNC implantation compared only with patients with rest pain regardless of Fontaine stage (Figure 2). In the SSc group, the VAS scores significantly improved within 6 months after BM-MNC implantation (P<0.01). It became slightly worse in 1 patient, and was unchanged in 2 patients. In the non-SSc group, also, the VAS scores significantly improved within 6 months after BM-MNC implantation (P<0.01). Almost all patients in the non-SSc group had an improve-ment in their VAS score, and about half of those became pain-free (Figure 2).

Long-Term Clinical Outcomes SurveyThe median follow-up duration for all patients was 36.5 months (range: 0.4–165 months; mean: 47.9 months). Table 3 shows the long-term clinical outcomes after BM-MNC implantation. During the follow-up period, all-cause deaths occurred in 10.1% of all patients with CD, in 10.3% of the SSc group, and in 10.0% of the non-SSc group. The rate of all-cause death was not significantly different between groups. On the other hand, amputations (including

Figure 4. Kaplan-Meier analysis of (A) OS, (B) MAF survival, and (C) AFS following implantation of autologous bone marrow-derived mononuclear cells in the SSc and non-SSc groups. The log-rank test P-values indicate the differences in time-to-event between groups. Abbreviations as in Figures 1,3.


668 SHOJI K et al.

with SSc specifically can develop ischemic ulcers on their fingertips or toes because of these factors. In this study, more than half of the CLI patients with CD had SSc. According to data from the University of Pittsburgh, 58% of patients with SSc experience a DU during their disease duration. In addition, 32% of all patients with SSc have had persistent DUs (persistent or recurrent ulcers for at least 6 months), and of these, 30% of cases were severe (complicated by gangrene, or requiring digital sympathec-tomy or amputation).22

In managing DUs in SSc, vasodilator treatment efficacies are reported. EULAR recommends IV iloprost, PDE-5 inhibitors, and bosentan as pharmacotherapies for DU in SSc.3 IV iloprost and PDE-5 inhibitors significantly reduced the number of DUs and improved healing compared with placebo in some RCTs.23,24 Bosentan significantly reduced new DUs in the RAPIDS-1 and RAPIDS-2 studies.25,26 However, in Japan, only bosentan is recommended in the Dermatological Association guidelines, and vasodilator treatments are not recommended because of a lack of expert consensus as to their efficacy in patients with SSc. Combination therapy of iloprost and bosentan for DUs in SSc patients has promoted healing of ischemic ulcers with poor curative tendency when treated with only iloprost. However, ulcers occurring with severe cases of fibrosis, or in cases of lower limb ulcers, have shown poor response to combination therapy, and 24.6% of patients are non-responders.4 Patients with CLI in SSc may not continue therapy because of intolerability. CLI in SSc among patients treated with pharmacotherapy alone could progress to a need for limb amputation because of drug resistance or

between the SSc and non-SSc groups following BM-MNC implantation in the lower limbs. The OS, MAF, and AFS rates in each period were not significantly different between groups (log-rank test: P=0.94, P=0.39 and P=0.47, respec-tively) (Figure 5A–C).

According to these results, BM-MNC implantation provided a high salvage rate for no-option CLI patients with CD, especially those with SSc. Furthermore, in the SSc group, the salvage rate was high regardless of the target limb (upper or lower). In addition, all patients who underwent BM-MNC implantation in the upper limbs had good outcomes with a very low rate of amputations, either major or minor.

DiscussionThis study showed the very long-term clinical outcomes after BM-MNC implantation in CLI patients with CD in Japan. According to these results, we suggest that BM-MNC implantation may be a safe and feasible therapy for all no-option CLI patients with CD.

Peripheral circulation failure caused by arteriosclerosis is the main pathology in CLI patients with ASO, but in those with CD it is not the only cause. In SSc vasculopathy, intimal hyperplasia, abnormal vascular contraction, and intravascular micro-thrombus from autoimmune abnor-malities are the main causes of inadequate blood supply that leads to the development of CLI.21 Moreover, in other vasculitides, the presence of inflammation cells infiltrates and destruction of the vascular wall also lead to vascular stenosis or obstruction. Among CDs, many patients

Figure 5. Kaplan-Meier analysis of (A) OS, (B) MAF survival, and (C) AFS following implantation of autologous bone marrow-derived mononuclear cells in the lower limbs in the SSc and non-SSc groups. The log-rank test P-values indicate the differences in time-to-event between groups. Abbreviations as in Figures 1,3.



reduced from those recorded in the Pittsburgh Scleroderma Databank.33 In that report, the 9-year cumulative survival was 38% in patients with severe organ involvement and 72% in patients with mild organ involvement. Moreover, the presence of DUs in SSc predicts cardiovascular events and reduced survival rates.34,35 In our study, we did not evaluate in detail whether patients with each type of CD had severe complications within the follow-up period. However, our 10-year OS rate in the SSc group was 59.1%, which is similar to that in a past report. Moreover, deaths, MACE, or severe adverse events associated with BM-MNC implantation were not noted during the 6 months after this therapy. So, we suggest that BM-MNC implantation may be safe for treating CLI patients with CD. The lifetime prognosis for SSc has steadily improved, but this will likely increase the number of patients with SSc who will experience ischemic pain or ulcers.36

BM-MNC implantation showed good long-term salvage rates compared with conventional treatments, including revascularization, in no-option CLI patients with CD. In addition, the effects of revascularization after BM-NMC implantation might be maintained over the long term. Cell therapy contributed to improved patient QOL by relieving pain. In CLI patients, clinical outcomes after EVT or surgical bypass are poor and vasodilator treatments are insufficient, especially in Japan. Therefore, in CLI patients with CD, early BM-MNC implantation or combination treatment with conventional therapies including revascu-larization may contribute to improve QOL and limb salvage over the long term. A RCT is needed to further evaluate the efficacy of BM-MNC implantation in CLI patients with CD. In addition, if it is possible, we would like to measure expression levels of some growth factors after BM-MNC implantation.

Study LimitationsThe first limitation of this study was its non-randomized, single-arm, open-labeled, and non-controlled retrospective study design. The second limitation was the possibility that patients with insufficient pharmacotherapy (IV iloprost, PDE 5 inhibitor, or bosentan) were included. In 2015, bosentan was approved for patients with DUs in SSc in Japan. In this study, bosentan was administered to only 8 patients (20.5% of patients with SSc) before performing BM-MNC implantation because we began case registrations in 2004. Those 8 patients’ ischemic ulcers were not healed and BM-MNC implantation was necessary to avoid amputation. Therefore, it is possible that pharmacotherapy was not tried in all patients with SSc, and those patients may have had good outcomes or tolerated the therapy. We demonstrated that BM-MNC implantation was effective in the long term among patients with SSc cases in whom drugs were ineffective or not tolerable. It is possible that even better outcomes might be achieved by combining BM-MNC implantation and pharmacotherapy, which requires further investigation in the future. The third limitation was that there are few reports showing long-term clinical outcomes such as limb salvage after conventional treatments in CLI patients with all types of CD. So it was difficult to compare sufficiently the clinical outcomes of this study and historical data between the SSc and non-SSc groups. Therefore, in the SSc group, we could not neces-sarily conclude that BM-MNC implantation was more significantly effective in terms of limb salvage than in the non-SSc group at this time. The fourth limitation was that

medication intolerance.In patients with SSc and DUs, 11% of patients in the

Pittsburgh database that underwent amputation or experi-enced gangrene were prospectively followed for an average of 10 years.22 In the RAPIDS-2 trial conducted in patients with active DUs, the amputation incidence was 11% (1–2% per patient-year follow-up).27 According to a recent study, the outcome of SSc-related ischemic lower limb ulcers was poor and the amputation rate was 28.6%.28 Moreover, digital amputations in SSc patients with skin ulcers receiving conventional therapy were necessary in 11.5% (mean follow-up 5.8±4.6 SD years). In addition, the lower limb amputation rate was 26.8%, and the upper limb amputation rate was 7.8%.29 Compared with those previous studies, the lower and upper limb amputation rates in the present study tended to be equal to or less than those reported with conventional treatments despite our targeting SSc cases with more severe patient background (no-option CLI). Considering that no-option CLI was the subject of our study, we suggest that BM-MNC implantation might be an acceptable therapy for CLI patients with SSc. On the other hand, in patients with angiitis, it was reported that the 3-year limb savage rate was 67.2%, and the 3-year bypass patency was poor.5 In our study, the 5-year limb salvage rate was 82.6%, and the VAS scores significantly improved within 6 months. So, we also suggest that BM-MNC implantation might became a feasible therapy in CLI patients with other CDs. However, further reports showing long-term outcomes after conventional treatments are desirable because there are few reports about the clinical outcomes for CLI patients with CD.

In CLI patients resistant to medical therapy, the first-line treatment for limb salvage is revascularization because the major amputation rate is 25–40% with conservative therapy alone.1,30 Surgical bypass using autologous great saphenous vein grafts is the most recommended revascularization method because of high graft patency and long-term limb salvage.31 Therefore, surgical bypass might be the best revascularization method for CLI patients with CD. However, the limb salvage rate and patency of surgical bypass in CLI patients with CD were significantly lower than in those with ASO. In that report, the 3-year primary and secondary patency rates of surgical bypass in patients with angiitis were 38.9% and 61.5%, respectively.5 In another report, 12 of 13 CLI patients with CD underwent EVT or surgical bypass. Of the 5 patients who had EVT, 4 failed, 2 immediately and 2 subsequently. Seven patients had a major amputation at 6 months. At 2 years, 11 of 13 patients (84.6%) had major amputations.7 In a report on CLI with SSc, 5 of 6 patients had graft occlusions within 18 months of surgical bypass, and 2 required major amputations.6 With vascular stenosis or occlusion caused by CD, peripheral arteries targeted for bypass are often absent and long-term results are poor, even after surgical bypass, possibly because of micro-artery occlusions in the skin or subcutaneous tissue via immunological mechanisms. Interventions, including EVT and surgical bypass, among CLI patients with CD show poor outcomes compared with the general vascular population. Accordingly, many challenges remain with revascularization for CLI patients with CD.

For SSc, the median survival is approximately 11 years in the USA.32 A recent study reported survival rates among patients with SSc and severe organ involvement (skin, lung, gastrointestinal, heart or kidney) that were markedly


670 SHOJI K et al.

Shintani S, Masaki H, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: A pilot study and a randomised controlled trial. Lancet 2002; 360: 427 – 435.

12. Saigawa T, Kato K, Ozawa T, Toba K, Makiyama Y, Aizawa Y, et al. Clinical application of bone marrow implantation in patients with arteriosclerosis obliterans, and the association between efficacy and the number of implanted bone marrow cells. Circ J 2004; 68: 1189 – 1193.

13. Matoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, Ito M, et al. Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia. Am Heart J 2008; 156: 1010 – 1018.

14. Idei N, Soga J, Hata T, Fujii Y, Fujimura N, Mikami S, et al. Autologous bone-marrow mononuclear cell implantation reduces long-term major amputation risk in patients with critical limb ischemia: A comparison of atherosclerotic peripheral arterial disease and Buerger disease. Circ Cardiovasc Interv 2011; 4: 15 – 25.

15. Pignon B, Sevestre MA, Kanagaratnam L, Pernod G, Stephan D, Nguyen P, et al. Autologous bone marrow mononuclear cell implantation and its impact on the outcome of patients with critical limb ischemia: Results of a randomized, double-blind, placebo-controlled trial. Circ J 2017; 81: 1713 – 1720.

16. Kondo K, Yanishi K, Hayashida R, Shintani S, Shibata R, Matoba S, et al. Long-term clinical outcomes survey of bone marrow-derived cell therapy in critical limb ischemia in Japan. Circ J 2018; 82: 1168 – 1178.

17. Kao L, Weyand C. Vasculitis in systemic sclerosis. Int J Rheumatol 2010; 2010: 3859938.

18. Adigun R, Bhimji SS. Systemic sclerosis (CREST syndrome). StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing, 2018 – 2017 Apr 19. https://www.ncbi.nlm.nih.gov/books/NBK430875 (accessed August 30, 2018).

19. Sharp GC, Irvin WS, Tan EM, Gould RG, Holman HR. Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 1972; 52: 148 – 159.

20. Grader-Beck T, Wigley FM. Raynaud’s phenomenon in mixed connective tissue disease. Rheum Dis Clin North Am 2005; 31: 465 – 481.

21. Schiopu E, Impens AJ, Phillips K. Digital ischemia in scleroderma spectrum of diseases. Int J Rheumatol, doi:10.1155/2010/923743.

22. Ingraham KM, Steen VD. Morbidity of digital tip ulcerations in scleroderma (abstract). Arthritis Rheum 2006; 54: P578.

23. Wigley FM, Wise RA, Seibold JR, McCloskey DA, Kujala G, Medsger TA, et al. Intravenous iloprost infusion in patients with Raynaud phenomenon secondary to systemic sclerosis: A multicenter, placebo-controlled, double-blind study. Ann Intern Med 1994; 120: 199 – 206.

24. Tingey T, Shu J, Smuczek J, Pope J. Meta-analysis of healing and prevention of digital ulcers in systemic sclerosis. Arthritis Care Res (Hoboken) 2013; 65: 1460 – 1471.

25. Korn JH, Mayes M, Matucci Cerinic M, Rainisio M, Pope J, Hachulla E, et al. Digital ulcers in systemic sclerosis: Prevention by treatment with bosentan, an oral endothelin receptor antagonist. Arthritis Rheum 2004; 50: 3985 – 3993.

26. Matucci-Cerinic M, Denton CP, Furst DE, Mayes MD, Hsu VM, Carpentier P, et al. Bosentan treatment of digital ulcers related to systemic sclerosis: Results from the RAPIDS-2 randomised, double-blind, placebo-controlled trial. Ann Rheum Dis 2011; 70: 32 – 38.

27. Steen V, Denton CP, Pope JE, Matucci-Cerinic M. Digital ulcers: Overt vascular disease in systemic sclerosis. Rheumatology 2009; 48: 19 – 24.

28. Bohelay G, Blaise S, Levy P, Claeys A, Baudot N, Senet P, et al. Lower-limb ulcers in systemic sclerosis: A multicentre retrospective case–control study. Acta Derm Venereol 2018; 98: 677 – 682.

29. Giuggioli D, Manfredi A, Lumetti F, Colaci M, Ferri C. Scleroderma skin ulcers definition, classification and treatment strategies our experience and review of the literature. Autoimmun Rev 2018; 17: 155 – 164.

30. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): Executive summary. A collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine

we did not evaluate some parameters showing peripheral blood flow assessment such as skin perfusion pressure (SPP) or transcutaneous oxygen pressure (TcpO2). So, we could not discuss any improvement in peripheral blood flow. The last limitation was that we could evaluate MACE only up to 6 months after BM-MNC implantation. In addition, it was difficult to evaluate accurately the long-term MACE at the time of the outcome survey. In the next trial, we would like to accurately evaluate efficacy parameters such as SPP, TcpO2, and MACE for a longer period.

ConclusionsWe conclude that therapeutic angiogenesis using autologous BM-MNC implantation might be a feasible treatment option in no-option CLI patients with CD. In CLI patients with SSc, BM-MNC implantation tended to salvage isch-emic limbs more than for those with other CDs.

AcknowledgmentsThe authors appreciate the help and support of all members of the committee for this project and members of the institutes that partici-pated in the TACT Follow-up Study (Appendix).

Financial DisclosureThe authors received no specific funding for this work.

Conflicts of InterestThe authors declare no conflicts of interest associated with this manuscript.

References 1. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA,

Fowkes FG, et al. Inter-society consensus for the management of peripheral arterial disease (TASC II). J Vasc Surg 2007; 45(Suppl S): S5 – S67.

2. Reidy ME, Steen V, Nicholas JJ. Lower extremity amputation in scleroderma. Arch Phys Med Rehabil 1992; 73: 811 – 813.

3. Kowal-Bielecka O, Fransen J, Avouac J, Becker M, Kulak A, Allanore Y, et al. Update of EULAR recommendations for the treatment of systemic sclerosis. Ann Rheum Dis 2017; 76: 1327 – 1339.

4. De Cata A, Inglese M, Molinaro F, De Cosmo S, Rubino R, Bernal M, et al. Digital ulcers in scleroderma patients: A retro-spective observational study. Int J Immunopathol Pharmacol 2016; 29: 180 – 187.

5. Miyahara T, Suhara M, Nemoto Y, Shirasu T, Haga M, Mochizuki Y, et al. Long-term results of treatment for critical limb ischemia. Ann Vasc Dis 2015; 8: 192 – 197.

6. Deguchi J, Shigematsu K, Ota S, Kimura H, Fukuyama M, Miyata T. Surgical result of critical limb ischemia due to tibial arterial occlusion in patients with systemic scleroderma. J Vasc Surg 2009; 49: 918 – 923.

7. Harries RL, Ahmed M, Whitaker C, Majeed MU, Williams DT. The influence of connective tissue disease in the management of lower limb ischemia. Ann Vasc Surg 2014; 28: 1139 – 1142.

8. Fujita Y, Kinoshita M, Furukawa Y, Nagano T, Hashimoto H, Hirami Y, et al. Phase II clinical trial of CD34+ cell therapy to explore endpoint selection and timing in patients with critical limb ischemia. Circ J 2014; 78: 490 – 501.

9. Liotta F, Annunziato F, Castellani S, Boddi M, Alterini B, Maggi E, et al. Therapeutic efficacy of autologous non-mobilized enriched circulating endothelial progenitors in patients with critical limb ischemia: The SCELTA trial. Circ J 2018; 82: 1688 – 1698.

10. Horie T, Yamazaki S, Hanada S, Kobayashi S, Tsukamoto T, Fukushima M, et al. Outcome from a randomized controlled clinical trial: Improvement of peripheral arterial disease by granulocyte colony-stimulating factor-mobilized autologous peripheral-blood-mononuclear cell transplantation (IMPACT). Circ J 2018; 82: 2165 – 2174.

11. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U,



sclerosis, 1972–2002. Ann Rheum Dis 2007; 66: 940 – 944.

AppendixStudy InvestigatorsDepartment of Cardiovascular Medicine, Kyoto Prefectural University of Medicine; Keisuke Shoji, Kenji Yanishi, and Satoaki Matoba, Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine; Naoki Hamada, Ryusuke Yoshimi, and Hideaki Nakajima, Department of Cardiology, Nagoya University Graduate School of Medicine; Ryo Hayashida, Kazuhisa Kondo, Satoshi Shintani, Rei Shibata, and Toyoaki Murohara, Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Nagoya; Kenta Murotani, Masahiko Ando, Masaaki Mizuno, and Tadami Fujiwara, Department of Cardiology, the National Hospital Organization Kumamoto Medical Center; Kazuteru Fujimoto, Department of Cardiovascular Medicine, Shinshu University School of Medicine; Tamon Kato and Koichiro Kuwahara, Department of Cardiovascular Regeneration and Medicine, Research Institute for Radiation Biology and Medicine, Department of Cardiovascular Regeneration and Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University; Masato Kajikawa and Yukihito Higashi, Department of Internal Medicine, Division of Cardiovascular Medicine, Kurume University School of Medicine; Masanori Ootsuka, Kenichiro Sasaki, Yoshihiro Fukumoto, Department of Cardiology, Yokohama City University Hospital; Tomoaki Ishigami, First Department of Internal Medicine, Nara Medical University; Yoshihiko Saito, and Metabolism, Endocrinology, Department of Premier Preventive Medicine, Osaka City University Graduate School of Medicine; Shinya Fukumoto.

and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease) endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. J Am Coll Cardiol 2006; 47: 1239 – 1312.

31. Aboyans V, Ricco JB, Bartelink MEL, Björck M, Brodmann M, Cohnert T, et al. 2017 ESC Guidelines on the Diagnosis and Treatment of Peripheral Arterial Diseases, in collaboration with the European Society for Vascular Surgery (ESVS). Eur Heart J 2018; 39: 763 – 816.

32. Mayes MD, Lacey JV Jr, Beebe-Dimmer J, Gillespie BW, Cooper B, Laing TJ, et al. Prevalence, incidence, survival, and disease characteristics of systemic sclerosis in a large US population. Arthritis Rheum 2003; 48: 2246 – 2255.

33. Steen VD, Medsger TA Jr. Severe organ involvement in systemic sclerosis with diffuse scleroderma. Arthritis Rheum 2000; 43: 2437 – 2444.

34. Mihai C, Landewé R, van der Heijde D, Walker UA, Constantin PI, Gherghe AM, et al. Digital ulcers predict a worse disease course in patients with systemic sclerosis. Ann Rheum Dis 2016; 75: 681 – 686.

35. Tolosa-Vilella C, Morera-Morales ML, Simeón-Aznar CP, Marí-Alfonso B, Colunga-Arguelles D, Callejas Rubio JL, et al. Digital ulcers and cutaneous subsets of systemic sclerosis: Clinical, immunological, nailfold capillaroscopy, and survival differences in the Spanish RESCLE Registry. Semin Arthritis Rheum 2016; 46: 200 – 208.

36. Steen VD, Medsger TA. Changes in causes of death in systemic

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