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662 SHOJI K et al. Circulation Journal ORIGINAL ARTICLE Circ J 2019; 83: 662 – 671 doi: 10.1253/circj.CJ-18-1044 Peripheral Vascular Disease

Impact of Therapeutic Angiogenesis Using Autologous Bone Marrow-Derived Mononuclear Cells Implantation in Critical Limb Ischemia With ― 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 disease (CD), particularly scleroderma (SSc), develop critical limb ischemia (CLI), which leads to limb amputation. However, conventional , 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 , PDE-5 inhibitors, and . 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

ritical limb ischemia (CLI) is caused by chronic CD, particularly with systemic sclerosis (SSc, scleroderma) arterial obstruction, mainly caused by arterioscle- have experienced Raynaud’s phenomenon or digital ulcers C rosis obliterans (ASO), thromboangiitis obliterans (DU) because of inadequate peripheral blood supply. (TAO), and (CD). Patients with CLI often Intractable ischemic ulcers negatively affect patients’ quality require limb amputation when conventional revascular- of life (QOL), and may lead to limb amputation.2 The ization methods such as bypass or endovascular European League against Rheumatism (EULAR) recom- treatment (EVT) fail or are not indicated.1 Patients with mends intravenous (IV) iloprost, PDE-5 inhibitors, and

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 , 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 , 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 , 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] ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Circulation Journal Vol.83, March 2019 BM-MNC Implantation for Scleroderma 663

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

bosentan as pharmacotherapies for DU in patients with Methods SSc.3 However, many patients with SSc are resistant to these medical treatments.4 Additionally, long-term outcomes Study Design after EVT or surgical bypass among CLI patients with CD This was a retrospective, observational non-controlled have been poor.5–7 Recently, treatments for DU in patients study. We investigated the long-term clinical outcomes of with SSc have been proposed in which accurate treatment all CLI patients who underwent BM-MNC implantation interventions are performed in the early stages of disease, under advanced medical treatment, and we reported the when lesions are reversible, to prevent progression. Thus, safety and efficacy of the procedure in 2018.16 In the present establishing new strategies to promote peripheral circulation study, we re-analyzed only the patients with CD, and we are needed for CLI patients with SSc. evaluated the clinical outcomes for each etiology of CD. It has been reported that one method among the cell The method of data collection for this study was similar therapies that promote peripheral circulation is therapeutic to that used in the long-term clinical outcomes survey,16 angiogenesis using bone marrow-derived mononuclear cells and data from some patients who underwent additional (BM-MNC).8–10 The first clinical pilot study, Therapeutic investigations extended the clinical outcomes survey until Angiogenesis by Cell Transplantation (TACT) study, April 2018. investigated this in CLI patients in 2002,11 and subsequently, We assessed the overall survival (OS), major amputation- several studies have reported the safety and efficacy of free (MAF) and amputation-free survival (AFS) rates. The BM-MNC implantation for CLI patients.12,13 Randomized AFS in this study included OS and major amputations. studies have reported that this cell could lead to The safety of the therapy was assessed in the context of significant improvements in limb ischemia, thus extending major adverse cardiovascular events (MACE: death, amputation-free intervals and survival rates.14,15 Moreover, non-fatal myocardial infarction, decompensated Kondo et al reported a very long-term clinical outcome failure, and stroke) and all-cause adverse events during survey of BM-MNC implantation in no-option CLI the 6-month follow-up after BM-MNC implantation. The patients, and indicated that BM-MNC implantation may participating hospitals’ ethics committees approved the be feasible and safe in CLI patients, particularly those with survey’s protocol. TAO and CD-associated (CDV).16 SSc is the most common cause of CLI among all patients with CD. Study Population Therefore, improving the salvage rate is desirable in CLI We included any CLI patients who underwent BM-MNC patients with SSc, which necessitates establishment of the implantation and met the following criteria: (1) Fontaine evidence for BM-MNC implantation compared with stage III–IV and Rutherford category 4–6 in upper or/and medical treatment and revascularization in no-option CLI lower limbs; (2) CLI caused by CD; (3) no clinical improve- patients with SSc. ment following conventional medical and surgical treat- In our previous study, the CDV group included SSc ments; and (4) provision of written informed consent. For because the of SSc may sometimes include a all patients, we confirmed that they were not candidates for mechanism similar to vasculitis.16,17 However, the histopa- surgical or non-surgical revascularization treatment of CLI thology of SSc vasculopathy reflects the pathogenesis, with according to the recommendations of vascular surgeons, myofibroblast proliferation and matrix deposition in the cardiologists and radiologists. All patients underwent a subendothelial layer leading to obliterative thickening of pre-assessment to determine whether they met any of the vessel walls. There is either no inflammatory infiltrate or exclusion criteria: untreated coronary disease or less compared with other CD-related vascular lesions.17 cerebrovascular disease; clinical or laboratory signs of Therefore, we present a subanalysis of the long-term chronic or acute ; a previous (past 5 years) outcomes of BM-MNC implantation in no-option CLI or current history of neoplasia; diabetes with untreated patients with CD, particularly those with SSc. retinopathy; age over 80 years; the possibility of pregnancy;

Circulation Journal Vol.83, March 2019 664 SHOJI K et al.

Table 1. Baseline Characteristics of the Patients and the Lesions All SSc Non-SSc P value (n=69) (n=39) (n=30) 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 disease, n (%) 3 (4.3) 1 (2.6) 2 (6.7) 0.40 , 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 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 , (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.

or lack of informed consent. Our inclusion and exclusion unpaired Student’s t-tests for discrete and continuous criteria and assessment procedures were similar to those of variables, respectively. Visual analog scale (VAS) score the TACT study.11 The BM-MNC implantation was after BM-MNC implantation was compared with that at implemented with the approval of the ethics committee in baseline using a Wilcoxon signed-rank test in each group. each participating hospital for no-option CLI patients with A time-to-event analysis was performed by Kaplan-Meier CD, and we obtained written informed consent from all method to examine variables associated with OS, MAF, patients. and AFS. A log-rank test was conducted to examine the variables for associations with survival, major amputation, Statistical Analysis and AFS between groups. All statistical analyses were Continuous variables are presented as mean and standard performed using JMP® 14 (SAS Institute Inc., Cary, NC, deviation or median and interquartile range (IQR), as USA), with a value of P<0.05 considered to be statistically appropriate for the distribution of the data. Categorical significant. variables are presented as number (count). The SSc and non-SSc groups were compared by chi-squared and

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Table 2. All-Cause Adverse Events and MACE Within 6 Months After BM-MNC Implantation All SSc Non-SSc P value (n=69) (n=39) (n=30) 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.

Results polyangitis (EGPA) [n=1], Behçet’s disease [n=1], and vasculitis without definite diagnosis [n=7]) (Figure 1). A Study Flow Chart case in which a CD specialist strongly suspected a vasculitis BM-MNC implantation was performed in 69 patients with caused by a CD, but which did not result in a definitive CD in 10 hospitals. We divided these patients into a SSc diagnosis was defined as “vasculitis without definite group (SSc-related diseases, n=39) and a non-SSc group diagnosis”. (non-SSc-related diseases, n=30). The SSc group included some SSc-related diseases (SSc; [n=35] including CREST Patients’ Baseline and Procedural Characteristics syndrome (Calcinosis, Raynaud’s phenomenon, Esophageal The baseline characteristics of the patients are shown in dysmotility, Sclerodactyly, and Telangiectasia) [n=1], and Table 1. The average age was 54 years, women represented mixed connective tissue disease (MCTD) [n=3]). CREST 87% of the study population, and the rate of patients with syndrome is known as limited SSc, which is a subtype of Fontaine stage IV was 86%. The average age, percentage SSc.18 MCTD is characterized by overlapping features of of women, percentage of patients with Fontaine stage IV, SSc, systemic lupus erythematosus (SLE), and and body mass index (BMI) were not significantly different (PM)/ (DM).19 The vasculopathy in MCTD between the SSc and non-SSc groups. A history of smoking has been found to resemble that in SSc,20 so we classified in the SSc group was significantly more common than in CREST syndrome and MCTD into the SSc group. The the non-SSc group (P<0.01). In the SSc group, oral medi- non-SSc group included other CDs (SLE [n=5], rheumatoid cations including steroids or immunosuppressive agents arthritis (RA) [n=6], polyarteritis nodosa (PN) [n=7], were significantly less often administered than in the antiphospholipid syndrome (APS) [n=2], Sjögren’s syn- non-SSc group (P=0.01 and P=0.03, respectively), but oral drome (SjS) [n=1], eosinophilic granulomatosis with endothelin receptor antagonist medications was significantly

Circulation Journal Vol.83, March 2019 666 SHOJI K et al.

Table 3. Long-Term Clinical Outcomes After BM-MNC Implantation All SSc Non-SSc P value (n=69) (n=39) (n=30) 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) 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.

more prevalent than in the non-SSc group (P<0.01). On the SSc group (P<0.01). the other hand, concomitant medical treatments such as antiplatelet therapy and peripheral vasodilator agents were Safety of BM-MNC Implantation in Patients With CLI not significant different between groups. The SSc group Caused by CD had more patients presenting with ischemic symptoms in We evaluated all-cause adverse events and MACE with the upper limbs than did the non-SSc group (P<0.01), and reference to the safety of BM-MNC implantation (Table 2). the BM-MNC implantation procedure was performed All-cause adverse events occurred in 8.7% of all patients more often in the upper limbs in the SSc group compared within 6 months after BM-MNC implantation. MACE did with the non-SSc group. The C-reactive protein values in not occur within 6 months after implantation. The most the non-SSc group were significantly higher than those in frequent adverse events in all patients with CD were severe

Circulation Journal Vol.83, March 2019 BM-MNC Implantation for Scleroderma 667

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.

bleeding/anemia (4.3%), worsening of liver function (2.9%), major and minor) occurred in 20.3% of all patients with and worsening of retinopathy (2.9%). The incidence of CD, in 12.8% of the SSc group, and in 30.0% of the non- all-cause adverse events or MACE was not significantly SSc group, respectively. In the SSc group, major and minor different between groups. No patients died, and no severe amputations tended to be fewer than in the non-SSc group adverse events associated with BM-MNC implantation (Table 3). were noted within 6 months after this therapy. All major amputations required in both groups were performed only on the lower limbs. Minor amputations of Improvement in VAS Scores After BM-MNC Implantation the lower limbs occurred more than those of the upper We evaluated rest pain using a VAS, on which 0 mm meant limbs. According to these results, in both groups there pain-free or no pain, and 100 mm indicated the most severe were more major and minor amputations in patients with pain. We evaluated VAS scores before and after BM-MNC ischemic symptoms in the lower limbs than in those with implantation compared only with patients with rest pain symptoms in the upper limbs. regardless of Fontaine stage (Figure 2). In the SSc group, Figure 3 shows the Kaplan-Meier analysis of OS, MAF, the VAS scores significantly improved within 6 months and AFS rates following BM-MNC implantation in all after BM-MNC implantation (P<0.01). It became slightly patients with CD. The 1-year OS, MAF, and AFS rates worse in 1 patient, and was unchanged in 2 patients. In the were 98.3%, 95.2%, and 93.5%, respectively. The 5-year non-SSc group, also, the VAS scores significantly improved OS, MAF, and AFS rates were 95.2%, 91.1%, and 86.5%, within 6 months after BM-MNC implantation (P<0.01). respectively. The 10-year OS, MAF, and AFS rates were Almost all patients in the non-SSc group had an improve- 68.1%, 91.1%, and 61.8%, respectively (Figure 3A–C). ment in their VAS score, and about half of those became Figure 4 shows the Kaplan-Meier analysis of OS, MAF, pain-free (Figure 2). and AFS rates between the SSc and non-SSc groups following BM-MNC implantation. The OS, MAF, and Long-Term Clinical Outcomes Survey AFS rates in each period were not significantly different The median follow-up duration for all patients was 36.5 between groups (log-rank test: P=0.82, P=0.09, and P=0.34, months (range: 0.4–165 months; mean: 47.9 months). respectively) (Figure 4A–C). However, major amputations Table 3 shows the long-term clinical outcomes after BM- often occurred within 2 years after BM-MNC implantation MNC implantation. During the follow-up period, all-cause in the non-SSc group compared with the SSc group deaths occurred in 10.1% of all patients with CD, in 10.3% (Figure 4B). of the SSc group, and in 10.0% of the non-SSc group. The Next, we analyzed only CLI patients who underwent rate of all-cause death was not significantly different BM-MNC implantation in the lower limbs. Figure 5 shows between groups. On the other hand, amputations (including the Kaplan-Meier analysis of OS, MAF, and AFS rates

Circulation Journal Vol.83, March 2019 668 SHOJI K et al.

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.

between the SSc and non-SSc groups following BM-MNC with SSc specifically can develop ischemic ulcers on their implantation in the lower limbs. The OS, MAF, and AFS fingertips or toes because of these factors. In this study, rates in each period were not significantly different between more than half of the CLI patients with CD had SSc. groups (log-rank test: P=0.94, P=0.39 and P=0.47, respec- According to data from the University of Pittsburgh, 58% tively) (Figure 5A–C). of patients with SSc experience a DU during their disease According to these results, BM-MNC implantation duration. In addition, 32% of all patients with SSc have provided a high salvage rate for no-option CLI patients had persistent DUs (persistent or recurrent ulcers for at with CD, especially those with SSc. Furthermore, in the least 6 months), and of these, 30% of cases were severe SSc group, the salvage rate was high regardless of the (complicated by gangrene, or requiring digital sympathec- target limb (upper or lower). In addition, all patients who tomy or amputation).22 underwent BM-MNC implantation in the upper limbs had In managing DUs in SSc, vasodilator treatment efficacies good outcomes with a very low rate of amputations, either are reported. EULAR recommends IV iloprost, PDE-5 major or minor. inhibitors, and bosentan as pharmacotherapies for DU in SSc.3 IV iloprost and PDE-5 inhibitors significantly reduced Discussion the number of DUs and improved healing compared with placebo in some RCTs.23,24 Bosentan significantly reduced This study showed the very long-term clinical outcomes new DUs in the RAPIDS-1 and RAPIDS-2 studies.25,26 after BM-MNC implantation in CLI patients with CD in However, in Japan, only bosentan is recommended in the Japan. According to these results, we suggest that BM-MNC Dermatological Association guidelines, and vasodilator implantation may be a safe and feasible therapy for all treatments are not recommended because of a lack of no-option CLI patients with CD. expert consensus as to their efficacy in patients with SSc. Peripheral circulation failure caused by arteriosclerosis Combination therapy of iloprost and bosentan for DUs in is the main pathology in CLI patients with ASO, but in SSc patients has promoted healing of ischemic ulcers with those with CD it is not the only cause. In SSc vasculopathy, poor curative tendency when treated with only iloprost. intimal hyperplasia, abnormal vascular contraction, and However, ulcers occurring with severe cases of , intravascular micro-thrombus from autoimmune abnor- or in cases of lower limb ulcers, have shown poor response malities are the main causes of inadequate blood supply to combination therapy, and 24.6% of patients are non- that leads to the development of CLI.21 Moreover, in other responders.4 Patients with CLI in SSc may not continue vasculitides, the presence of inflammation cells infiltrates therapy because of intolerability. CLI in SSc among patients and destruction of the vascular wall also lead to vascular treated with pharmacotherapy alone could progress to a stenosis or obstruction. Among CDs, many patients need for limb amputation because of drug resistance or

Circulation Journal Vol.83, March 2019 BM-MNC Implantation for Scleroderma 669 medication intolerance. reduced from those recorded in the Pittsburgh Scleroderma In patients with SSc and DUs, 11% of patients in the Databank.33 In that report, the 9-year cumulative survival Pittsburgh database that underwent amputation or experi- was 38% in patients with severe involvement and enced gangrene were prospectively followed for an average 72% in patients with mild organ involvement. Moreover, of 10 years.22 In the RAPIDS-2 trial conducted in patients the presence of DUs in SSc predicts cardiovascular events with active DUs, the amputation incidence was 11% (1–2% and reduced survival rates.34,35 In our study, we did not per patient-year follow-up).27 According to a recent study, evaluate in detail whether patients with each type of CD the outcome of SSc-related ischemic lower limb ulcers was had severe complications within the follow-up period. poor and the amputation rate was 28.6%.28 Moreover, However, our 10-year OS rate in the SSc group was 59.1%, digital amputations in SSc patients with skin ulcers receiving which is similar to that in a past report. Moreover, deaths, conventional therapy were necessary in 11.5% (mean MACE, or severe adverse events associated with BM-MNC follow-up 5.8±4.6 SD years). In addition, the lower limb implantation were not noted during the 6 months after this amputation rate was 26.8%, and the upper limb amputation therapy. So, we suggest that BM-MNC implantation may rate was 7.8%.29 Compared with those previous studies, the be safe for treating CLI patients with CD. The lifetime lower and upper limb amputation rates in the present prognosis for SSc has steadily improved, but this will likely study tended to be equal to or less than those reported with increase the number of patients with SSc who will experience conventional treatments despite our targeting SSc cases ischemic pain or ulcers.36 with more severe patient background (no-option CLI). BM-MNC implantation showed good long-term salvage Considering that no-option CLI was the subject of our rates compared with conventional treatments, including study, we suggest that BM-MNC implantation might be revascularization, in no-option CLI patients with CD. In an acceptable therapy for CLI patients with SSc. On the addition, the effects of revascularization after BM-NMC other hand, in patients with angiitis, it was reported that implantation might be maintained over the long term. Cell the 3-year limb savage rate was 67.2%, and the 3-year therapy contributed to improved patient QOL by relieving bypass patency was poor.5 In our study, the 5-year limb pain. In CLI patients, clinical outcomes after EVT or salvage rate was 82.6%, and the VAS scores significantly surgical bypass are poor and vasodilator treatments are improved within 6 months. So, we also suggest that BM- insufficient, especially in Japan. Therefore, in CLI patients MNC implantation might became a feasible therapy in with CD, early BM-MNC implantation or combination CLI patients with other CDs. However, further reports treatment with conventional therapies including revascu- showing long-term outcomes after conventional treatments larization may contribute to improve QOL and limb salvage are desirable because there are few reports about the clinical over the long term. A RCT is needed to further evaluate outcomes for CLI patients with CD. the efficacy of BM-MNC implantation in CLI patients In CLI patients resistant to medical therapy, the first-line with CD. In addition, if it is possible, we would like to treatment for limb salvage is revascularization because the measure expression levels of some growth factors after major amputation rate is 25–40% with conservative therapy BM-MNC implantation. alone.1,30 Surgical bypass using autologous great saphenous vein grafts is the most recommended revascularization Study Limitations method because of high graft patency and long-term limb The first limitation of this study was its non-randomized, salvage.31 Therefore, surgical bypass might be the best single-arm, open-labeled, and non-controlled retrospective revascularization method for CLI patients with CD. study design. The second limitation was the possibility that However, the limb salvage rate and patency of surgical patients with insufficient pharmacotherapy (IV iloprost, bypass in CLI patients with CD were significantly lower PDE 5 inhibitor, or bosentan) were included. In 2015, than in those with ASO. In that report, the 3-year primary bosentan was approved for patients with DUs in SSc in and secondary patency rates of surgical bypass in patients Japan. In this study, bosentan was administered to only 8 with angiitis were 38.9% and 61.5%, respectively.5 In patients (20.5% of patients with SSc) before performing another report, 12 of 13 CLI patients with CD underwent BM-MNC implantation because we began case registrations EVT or surgical bypass. Of the 5 patients who had EVT, 4 in 2004. Those 8 patients’ ischemic ulcers were not healed failed, 2 immediately and 2 subsequently. Seven patients and BM-MNC implantation was necessary to avoid had a major amputation at 6 months. At 2 years, 11 of 13 amputation. Therefore, it is possible that pharmacotherapy patients (84.6%) had major amputations.7 In a report on was not tried in all patients with SSc, and those patients CLI with SSc, 5 of 6 patients had graft occlusions within may have had good outcomes or tolerated the therapy. We 18 months of surgical bypass, and 2 required major demonstrated that BM-MNC implantation was effective in amputations.6 With vascular stenosis or occlusion caused the long term among patients with SSc cases in whom by CD, peripheral targeted for bypass are often drugs were ineffective or not tolerable. It is possible that absent and long-term results are poor, even after surgical even better outcomes might be achieved by combining bypass, possibly because of micro-artery occlusions in the BM-MNC implantation and pharmacotherapy, which skin or subcutaneous tissue via immunological mechanisms. requires further investigation in the future. The third Interventions, including EVT and surgical bypass, among limitation was that there are few reports showing long-term CLI patients with CD show poor outcomes compared clinical outcomes such as limb salvage after conventional with the general vascular population. Accordingly, many treatments in CLI patients with all types of CD. So it was challenges remain with revascularization for CLI patients difficult to compare sufficiently the clinical outcomes of with CD. this study and historical data between the SSc and non-SSc For SSc, the median survival is approximately 11 years groups. Therefore, in the SSc group, we could not neces- in the USA.32 A recent study reported survival rates among sarily conclude that BM-MNC implantation was more patients with SSc and severe organ involvement (skin, significantly effective in terms of limb salvage than in the lung, gastrointestinal, heart or kidney) that were markedly non-SSc group at this time. The fourth limitation was that

Circulation Journal Vol.83, March 2019 670 SHOJI K et al. we did not evaluate some parameters showing peripheral Shintani S, Masaki H, et al. Therapeutic angiogenesis for blood flow assessment such as skin perfusion pressure patients with limb ischaemia by autologous transplantation of bone-marrow cells: A pilot study and a randomised controlled (SPP) or transcutaneous oxygen pressure (TcpO2). So, we trial. Lancet 2002; 360: 427 – 435. could not discuss any improvement in peripheral blood 12. Saigawa T, Kato K, Ozawa T, Toba K, Makiyama Y, Aizawa flow. The last limitation was that we could evaluate MACE Y, et al. Clinical application of bone marrow implantation in only up to 6 months after BM-MNC implantation. In patients with arteriosclerosis obliterans, and the association addition, it was difficult to evaluate accurately the long-term between efficacy and the number of implanted bone marrow cells. Circ J 2004; 68: 1189 – 1193. MACE at the time of the outcome survey. In the next trial, 13. Matoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, we would like to accurately evaluate efficacy parameters Ito M, et al. Long-term clinical outcome after intramuscular such as SPP, TcpO2, and MACE for a longer period. 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. Conclusions 14. Idei N, Soga J, Hata T, Fujii Y, Fujimura N, Mikami S, et al. Autologous bone-marrow mononuclear cell implantation We conclude that therapeutic angiogenesis using autologous reduces long-term major amputation risk in patients with critical BM-MNC implantation might be a feasible treatment limb ischemia: A comparison of atherosclerotic peripheral arterial disease and Buerger disease. Circ Cardiovasc Interv 2011; 4: option in no-option CLI patients with CD. In CLI patients 15 – 25. with SSc, BM-MNC implantation tended to salvage isch- 15. Pignon B, Sevestre MA, Kanagaratnam L, Pernod G, Stephan emic limbs more than for those with other CDs. 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, Acknowledgments placebo-controlled trial. Circ J 2017; 81: 1713 – 1720. The authors appreciate the help and support of all members of the 16. Kondo K, Yanishi K, Hayashida R, Shintani S, Shibata R, committee for this project and members of the institutes that partici- Matoba S, et al. Long-term clinical outcomes survey of bone pated in the TACT Follow-up Study (Appendix). 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 Financial Disclosure 2010; 2010: 3859938. The authors received no specific funding for this work. 18. Adigun R, Bhimji SS. Systemic sclerosis (CREST syndrome). 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