Abstract
Aim: Peripheral artery disease (PAD) severely impairs patient prognosis and quality of life (QOL). Although lipoprotein apheresis (LA) has been applied to patients with PAD and elevated serum atherogenic lipoproteins, we hypothesized that LA can be effective for treating PAD even in patients with controlled serum lipoproteins through pleiotropic anti-atherosclerotic effects beyond lipoprotein removal. This study aimed to evaluate the efficacy of LA in patients with treatment-resistant PAD and controlled serum lipoproteins focusing on QOL.
Methods: In a single-arm prospective study, 30 patients with refractory PAD who had controlled serum lipoproteins underwent sequential LA sessions using dextran sulfate adsorption columns, aiming to complete 10 sessions. The ankle-brachial pressure index (ABI) and vascular QOL (VascuQOL) score were evaluated as the primary outcomes. Secondary outcomes included reactive hyperemia index (RHI) and biological antioxidant potential (BAP) as an endothelial function test and serum antioxidative-capacity evaluation, respectively.
Results: ABI significantly increased after LA sessions (pre-treatment 0.60±0.09 vs. post-treatment 0.65±0.13, p=0.023). Total VascuQOL score (3.7±1.1 vs 4.6±1.1, p＜0.001) and RHI (1.70±0.74 vs 2.34±1.76, p=0.023) significantly improved after the LA sessions. BAP tended to increase after the LA sessions, and the change reached statistical significance 3 months after treatment.
Conclusion: ABI and QOL improved after a series of LA sessions in conventional treatment-resistant PAD patients with controlled serum lipoprotein levels. Increased antioxidative capacity and ameliorated endothelial function were observed after the LA treatment.
See editorial vol. 31: 1365-1366
Introduction
Peripheral artery disease (PAD) is an atherosclerotic disorder that is significantly associated with morbidity and mortality1). Moreover, PAD severely impairs patients’ quality of life (QOL)2) due to its symptoms such as claudication, chronic pain, and ulcers, which negatively impact daily activities and emotional and social well-being2, 3). Despite current progress in medication and revascularization therapies, some lesions are resistant to these therapies, resulting in refractory symptoms and occasional amputations4). Therefore, adequate alternative therapies are an unmet medical need.
Lipoprotein apheresis (LA) was developed to remove atherogenic lipoproteins from circulation5, 6). It is an established treatment for PAD that is complicated by severe hypercholesterolemia7, 8) or elevated lipoprotein (a)9, 10). Due to the strict target serum cholesterol level recommended for this population11, 12) and the current progress in lipid-lowering medications, most patients with PAD are without such conditions13, 14).
We previously reported in an observational study that ankle-brachial index (ABI) and walking distance were improved after sequential LA in PAD patients undergoing hemodialysis, which were possibly mediated by amelioration of endothelial dysfunction15, 16). Other studies also reported that the anti-PAD effects of LA include increased microcirculatory blood flow17), suppression of inflammation18, 19), adhesion molecules20, 21), and oxidative stress5, 22, 23). However, factors critical for the beneficial effects of LA as a key mechanism are unclear, except for lipoprotein removal.
This study was designed to evaluate the efficacy and safety of LA in conventional therapy-resistant PAD patients with controlled serum lipoproteins and was conducted as an interventional trial strictly limited to such patients. Regarding the efficacy evaluations, ABI and disease-specific QOL scores were evaluated as the primary outcomes. ABI was measured as an absolute objective indicator, which is critical in single-arm settings. In contrast, the QOL score was assessed as a patient-centered evaluation. Improving QOL is one of the primary goals of PAD treatment and is important3). Nevertheless, no reports have examined whether LA therapy can improve the QOL of patients with PAD. Therefore, we focused on assessing the effect of LA on QOL of patients with PAD. Additionally, we investigated the mechanisms underlying the therapeutic effects of LA.
Aim
This study aimed to evaluate the efficacy of LA in patients with treatment-resistant PAD and controlled serum lipoproteins focusing on QOL.
Methods
The LDL Apheresis-Mediated Endothelial Activation Therapy to Severe-Peripheral Artery Disease (LETS-PAD) study is a single-center, single-arm interventional study conducted as an Advanced Medical Care B program approved by the Ministry of Health, Labor, and Welfare (MHLW), Japan24). The study protocol was approved by the Yokohama City University Certified Institutional Review Board and MHLW and was registered with UMIN Clinical Trials Registry (UMIN000021684) and with the Japan Registry of Clinical Trials (jRCTs032180100).
Participants
Details of the study protocol have been previously described24). Fig.1 presents the inclusion and exclusion criteria. Eligible individuals were patients aged between 20 and 79 years with conventional treatment-resistant PAD (a Fontaine classification ≥ IIb and ABI ＜0.7), despite the normal range of atherogenic lipoproteins (total cholesterol (TC) ≤ 220 mg/dL and LDL-C ≤ 140 mg/dL)24). In addition, eligible individuals were determined by a third-party committee to be refractory to conventional revascularization therapies. During the study period, changes in medications (such as antiplatelet therapy, anticoagulation, and lipid-lowering medications) were avoided, except when the physician in charge decided to reduce or discontinue the dosage (due to bleeding complications, etc). All patients provided written informed consent before enrollment, and the study was conducted in accordance with the Declaration of Helsinki.
Lipoprotein Apheresis
Ten LA sessions were conducted once or twice a week. Hollow polysulfone fibers (Sulflux FP-08, Kaneka, Tokyo, Japan) and dextran sulfate cellulose columns (Liposorber LA-15, Kaneka, Tokyo, Japan) were used as plasma separators and adsorbers, respectively. During each session 3000–4000 mL of plasma was processed. Nafamostat mesylate was used as the first-line anticoagulant. Vascular access for hemodialysis was used in patients undergoing hemodialysis, whereas catheters were inserted in the jugular veins in other cases.
Evaluations of Outcome
The primary outcomes were ABI and vascular QOL (VascuQOL) score, which is a PAD-specific QOL evaluation25). Changes in these parameters between baseline and 1 month (30±7 days) after the last LA session were assessed. A form of the VascuQOL questionnaire was administered and retrieved from each patient before examination by the attending physician. The VascuQOL questionnaire comprises 25 items allocated to either of the following five domains: symptoms, activities, pain, emotional, and social domains. Each item is rated on a 7-point scale, with 1 and 7 representing the worst and best conditions, respectively26). The total and domain scores were calculated as an arithmetic mean of all 25 items or items in that domain, respectively.
Secondary outcomes included pain-free and maximally tolerated walking distance, rest pain, ulcers, skin perfusion pressure, endothelial function, oxidative stress, serum lipids, serum C-reactive protein (CRP), and plasma fibrinogen level. These evaluations were performed at baseline and 1 (30±7 days) and 3 months (90±14 days) after the last LA session. Rest pain in lower extremities was measured using a visual analog scale. Patients were asked to draw a vertical line along a 100-mm horizontal line, which indicates the intensity of pain, with 0 mm and 100 mm indicating no pain and the worst pain, respectively. Ulcers were graded according to size, inflammation/infection, granulation, and necrosis by plastic surgeons using a scale derived from the DESIGN-R classification developed by the Japanese Association of Pressure Ulcers27, 28), along with photographic records. Skin perfusion pressure (SPP) was measured with a laser Doppler probe beneath a blood pressure cuff placed on the dorsum and sole of the feet using a SensiLase PAD3000 Doppler Waveform Analyzer (Kaneka Medix Corp., Osaka, Japan). Endothelial function was evaluated in the form of the reactive hyperemia index (RHI) using an EndoPAT 2000 (Itamar Medical Inc., Israel) according to the manufacturer’s instructions. Occlusion of the brachial arteries was performed in the non-vascular access arms. To evaluate the oxidative stress status, the derivatives of reactive oxidative metabolites (d-ROMs) and biological antioxidant potential (BAP) were measured using the FREE Carpe diem (Wismerll Co. Ltd., Tokyo, Japan) according to the manufacturer’s instructions. In the d-ROM and BAP assays, the oxidative capacity of serum hydroperoxides and the reducing power of serum antioxidant components were quantified using a color reaction. Results of d-ROMs is expressed in Carr unit (1 U.CARR is equal to 0.08 mg/dL of hydrogen peroxide). Finally, all adverse events from the initiation of LA sessions to 1 month after the last session were recorded for the safety evaluation.
Statistical Analysis
The target sample size was calculated using the paired t-test for the change in the ABI before and after the protocol treatment24). The effect size and the standard deviation (SD) were estimated to be 0.075 and 0.16, respectively, according to our previous data15). Using a one-tailed significance level of 0.05 and power of 0.8, the required sample size was calculated to be 29 24). Data are presented as the mean±standard deviation (SD) and as frequencies for continuous and categorical variables, respectively. The difference between baseline and 1 or 3 months after the last LA session was evaluated using a one-sided paired t-test or Wilcoxon signed-rank test, as appropriate. For the primary outcomes, a change in the VascuQOL score was analyzed only if a change in the ABI reached statistical significance to handle multiplicity issues. The minimal important difference (MID) and SD for the VascuQOL questionnaire were estimated to be 0.4 to 0.8 and 1.0 to 1.2, respectively, based on the previous studies24, 26, 29). We concluded that a targeted sample size of 29 would have sufficient power (＞80%) to detect changes in VascuQOL scores. Statistical analyses were performed using R software, version 4.0 or later (R, Foundation for Statistical Computing, Vienna, Austria). The statistical significance was set at one-sided 0.05 for the primary outcomes. For the secondary outcomes, statistical significance was set at p＜0.025 and ＜0.050 for one- and two-sided test, respectively.
Results
Study Population and Conduct of LA Sessions
Fig.1 presents the study’s flowchart. Of 72 screened patients, 32 met the inclusion criteria and were enrolled in the study. However, post-treatment evaluations were not conducted in two patients: one underwent revascularization due to acute limb ischemia, and another was admitted to the high-care unit considering that the general condition deteriorated due to severe ischemic enteritis. The remaining 30 patients underwent post-treatment evaluation and comprised the full analysis set (Fig.1). Furthermore, 26 patients completed 10 LA sessions, three patients discontinued after five sessions due to hemorrhagic complications, and one withdrew consent after 4 sessions because of insufficient improvement in symptoms. Five patients were treated once a week with sequential LA sessions, whereas four were treated twice a week. The remaining patients underwent LA sessions either once or twice a week, on an irregular basis. The average duration of LA sessions was 53±20 days. During the study period, some patients changed their medications. Lipid-lowering drugs were discontinued in one patient due to hemoptysis and difficulty taking the medications. Additionally, antiplatelet medications were discontinued in three patients, while anticoagulants were discontinued in two patients, all due to bleeding complications.
Baseline Characteristics
The baseline characteristics of the patients are shown in Table 1. The mean age was 72.2±5.8 years and 76.7% of patients were male. Further, 53.3% of patients had critical limb ischemia (Fontaine classification III or IV), 70.0% had diabetes mellitus, 70.0% were on maintenance hemodialysis, and 96.7% of patients had concomitant cardiovascular disease. Overall, 62.1% of patients had a history of revascularization therapy on the target limb and the average duration from the last revascularization was almost 2 years. Serum LDL-C level was 76.9±28.2 mg/dL, with 86.7% of the patients receiving lipid-lowering agents. Most patients were on antiplatelet or anticoagulant therapies.
Table 1.Baseline characteristics of the study population
|
N = 30 |
| Age, years |
72.2 (±5.8) |
| Male, n (%)
|
23 (76.7) |
| Body mass index |
21.4 (±2.9) |
| Fontaine classification, n (%)
|
|
| Stage IIb: moderate-severe claudication |
14 (46.7) |
| Stage III: rest pain |
5 (16.7) |
| Stage IV: ulceration or gangrene |
11 (36.7) |
| Ankle-Brachial Index |
0.60 (±0.09) |
| Comorbidities, n (%)
|
|
| Hypertension |
26 (86.7) |
| Diabetes mellitus |
21 (70.0) |
| Chronic kidney disease |
29 (96.7) |
| Dialysis-dependent CKD |
21 (70.0) |
| Non-dialysis-dependent CKD |
8 (26.7) |
| Cardiovascular disease |
29 (96.7) |
| Coronary artery disease |
25 (83.3) |
| Congestive heart failure |
9 (30.0) |
| Stroke |
18 (60.0) |
| Smoking status, n (%)
|
|
| Current smoker |
3 (10.0) |
| Former smoker |
24 (80.0) |
| Never smoked |
3 (10.0) |
| Medication, n (%)
|
|
| Lipid-lowering therapy |
26 (86.7) |
| Statin |
21 (70.0) |
| Others |
5 (16.7) |
| Antihypertensive agent |
21 (70.0) |
| Antiplatelet agent |
29 (96.7) |
| Anticoagulant agent |
6 (20.0) |
| Peripheral artery disease history |
|
| Previous revascularization on the target limb, n (%)
|
18 (62.1) |
| Time from the last revascularization on the target limb, months |
24.8 (±30.8) |
| Previous limb amputation, n (%)
|
3 (10.0) |
| Laboratory findings |
|
| Total cholesterol, mg/dL |
145.3 (±36.3) |
| LDL cholesterol, mg/dL |
76.9 (±28.2) |
| HDL cholesterol, mg/dL |
47.4 (±14.9) |
| Triglycerides, mg/dL |
107.6 (±49.3) |
| C-reactive protein, mg/dL |
1.0 (±1.8) |
| Fibrinogen, mg/dL |
378.8 (±94.1) |
The parameters are shown as means (±SD) or percentages.
Abbreviations: CKD, chronic kidney disease; LDL, low-density lipoprotein; HDL, high-density lipoprotein; SD, standard deviation
Fig.2 shows the results of the primary outcomes. ABI significantly improved 1 month after the last LA session compared with that at the baseline (0.60±0.09 vs. 0.65±0.13, p=0.023). The total VascuQOL score also significantly improved 1 month after the last apheresis session compared with that at the baseline (3.7±1.1 vs. 4.6±1.1, p＜0.001). Regarding the domain scores of VascuQOL, all domain scores except the social domain score significantly improved 1 month after the last LA session (Table 2).
Table 2.Changes in VascuQOL Domain Scores
| Domain scores |
Baseline |
1 month after the treatment |
3 months afterthe treatment |
P-value a 1 month after the treatment
|
P-value b 3 months after the treatment
|
| Symptoms |
4.3 (±1.3) |
5.1 (±1.3) |
5.2 (±1.2) |
＜0.01 |
＜0.01 |
| Activities |
3.2 (±1.1) |
4.1 (±1.3) |
4.3 (±1.3) |
＜0.01 |
＜0.01 |
| Pain |
4.0 (±1.6) |
5.0 (±1.3) |
5.0 (±1.3) |
＜0.001 |
＜0.01 |
| Emotional |
3.6 (±1.3) |
4.7 (±1.3) |
5.0 (±1.0) |
＜0.001 |
＜0.0001 |
| Social |
4.3 (±1.6) |
4.9 (±1.3) |
5.5 (±1.1) |
0.084 |
＜0.01 |
a vs. baseline (1 month after treatment) (one-sided paired t-test)
b vs. baseline (3 months after treatment) (one-sided paired t-test).
The parameters are presented as mean (±SD). NS indicates no statistically significant difference.
Abbreviation: VascuQOL, vascular quality of life; SD, standard deviation
The secondary outcomes are summarized in Table 3. Significant improvements in ABI and VascuQOL were maintained for 3 months after sequential treatment.
Table 3.Short- and long-term effects of lipoprotein apheresis on clinical and laboratory parameters
|
Baseline |
1 month after the treatment |
3 months after the treatment |
P-value a 1 month after the treatment
|
P-value b 3 months after the treatment
|
| Clinical symptoms |
|
|
|
|
|
| Walking distance, m |
|
|
|
|
|
| Pain-free |
69.7 (±84.4) |
111.9 (±111.5) |
133.4 (±147.4) |
0.040 |
＜0.01 |
| Maximum tolerated |
250.6 (±199.0) |
265.8 (±141.3) |
333.2 (±249.0) |
0.38 |
0.047 |
| Rest pain (VAS), mm |
34.9 (±29.4) |
17.7 (±29.6) |
24.2 (±30.2) |
0.049 |
0.061 |
| VascuQOL (total score) |
3.7 (±1.1) |
4.6 (±1.1) |
4.8 (±1.0) |
＜0.001 |
＜0.001 |
| Physiological examinations |
|
|
|
|
|
| ABI |
0.60 (±0.09) |
0.65 (±0.13) |
0.69 (±0.20) |
0.023 |
0.011 |
| SPP, mmHg |
|
|
|
|
|
| Dorsal |
40.5 (±17.7) |
41.9 (±21.5) |
47.9 (±20.8) |
0.34 |
0.12 |
| Plantar |
40.7 (±17.6) |
37.7 (±17.3) |
41.7 (±16.5) |
0.80 |
0.59 |
| Endothelial function (RHI) |
1.70 (±0.74) |
2.34 (±1.76) |
1.88 (±0.76) |
0.023 |
0.33 |
| Laboratory findings |
|
|
|
|
|
| Serum lipids, mg/dL |
|
|
|
|
|
| LDL-cholesterol |
76.9 (±28.2) |
78.8 (±29.8) |
81.4 (±29.7) |
0.67 |
0.71 |
| HDL-cholesterol |
47.4 (±14.9) |
52.0 (±19.0) |
54.0 (±17.4) |
0.020 |
0.013 |
| Total cholesterol |
145.3 (±36.3) |
152 (±40.3) |
158.3 (±38.6) |
0.86 |
0.96 |
| Triglyceride |
107.6 (±49.3) |
111.9 (±64.4) |
117.6 (±71.9) |
0.72 |
0.76 |
| C-reactive protein, mg/dL |
1.0 (±1.8) |
1.8 (±2.6) |
0.9 (±1.4) |
0.94 |
0.34 |
| Fibrinogen, mg/dL |
378.8 (±94.1) |
389.2 (±86.4) |
371.3 (±66.3) |
0.78 |
0.26 |
| Oxidative stress |
|
|
|
|
|
| d-ROMs, U.CARR |
346.2 (±66.2) |
340.4 (±70.0) |
329.8 (±57.0) |
0.42 |
0.046 |
| BAP, μmol/L |
2449.9 (±340.9) |
2575.3 (±307.3) |
2590.2 (±332.5) |
0.039 |
0.016 |
a vs. baseline (1 month after treatment)
b vs. baseline (3 months after treatment)
Parameters are shown as the mean (±SD). NS indicates no statistically significant difference.
Abbreviations: VAS, visual analog scale; ABI, ankle brachial pressure index; SPP, skin perfusion pressure; RHI, reactive hyperemia index; d-ROMs, derivatives of reactive oxidative metabolites; BAP, biological antioxidant potential; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VascuQOL, vascular quality of life; SD, standards deviation; U.CARR Carr unit.
Regarding PAD symptoms, pain-free walking distance tended to extend 1 month after the sequential treatment compared with that at the baseline (pre-treatment) and it was significantly longer 3 months after the end of the treatment compared with that at the baseline. The maximum tolerated walking distance did not significantly increase 1 month after the sequential treatment compared with that at the baseline but tended toward extension 3 months after the sequential treatment. Rest pain temporarily tended to mitigate 1 month after the treatment compared with that at the baseline; however, no difference was found 3 months after the treatment (Table 3).
Additionally, ulcer size significantly improved after treatment (Table 4). Of the 10 patients who had ulcers before the start of the treatment, complete epithelization was achieved 1 month after the treatment in 6 patients and 3 months after the treatment in one additional patient.
Table 4.Change in the distribution of each category score of the DESIGN-R classification
| category scores |
Baseline |
1 month after the treatment |
3 months after the treatment |
P-value a 1 month after the treatment
|
P-value b 3 months after the treatment
|
| Size |
|
|
|
＜0.01 |
＜0.01 |
| s0 |
0 (0) |
6 (60) |
7 (87.5) |
|
|
| s3 |
7 (70) |
2 (20) |
0 (0) |
|
|
| s6 |
2 (20) |
0 (0) |
1 (12.5) |
|
|
| s8 |
1 (10) |
2 (20) |
0 (0) |
|
|
| s9 |
0 (0) |
0 (0) |
0 (0) |
|
|
| s12 |
0 (0) |
0 (0) |
0 (0) |
|
|
| S15 |
0 (0) |
0 (0) |
0 (0) |
|
|
| Inflammation/infection |
|
|
|
1.0 |
- |
| i0 |
9 (90) |
9 (90) |
8 (100) |
|
|
| i1 |
1 (10) |
0 (0) |
0 (0) |
|
|
| I3 |
0 (0) |
1 (10) |
0 (0) |
|
|
| I9 |
0 (0) |
0 (0) |
0 (0) |
|
|
| Granulation tissue |
|
|
|
0.063 |
0.12 |
| g0 |
4 (40) |
7 (70) |
7 (87.5) |
|
|
| g1 |
0 (0) |
0 (0) |
0 (0) |
|
|
| g3 |
0 (0) |
0 (0) |
0 (0) |
|
|
| G4 |
0 (0) |
0 (0) |
0 (0) |
|
|
| G5 |
0 (0) |
1 (10) |
0 (0) |
|
|
| G6 |
6 (60) |
2 (20) |
1 (12.5) |
|
|
| Necrotic tissue |
|
|
|
0.25 |
0.13 |
| n0 |
3 (30) |
6 (60) |
7 (87.5) |
|
|
| N3 |
3 (30) |
0 (0) |
0 (0) |
|
|
| N6 |
4 (40) |
4 (40) |
1 (12.5) |
|
|
a vs. baseline (1 month after treatment) (Wilcoxon signed-rank test). b vs. baseline (3 months after treatment) (Wilcoxon signed-rank test). The parameters are presented as percentages (%). NS indicates no statistically significant difference.
SPP, which is an indicator of microcirculation at the skin tissue level, did not differ significantly from the baseline either at 1 or 3 months after treatment. However, endothelial function, measured as the RHI, significantly improved 1 month after the treatment compared with the baseline (1.70±0.74 vs.2.34±1.76, p=0.023). Among laboratory data, serum high-density lipoprotein-cholesterol (HDL-C) significantly increased 1 month after the LA sessions compared with the baseline and remained elevated three months after treatment. Serum TC, LDL-C, triglyceride, CRP, and plasma fibrinogen levels did not significantly change from baseline (Table 3). Oxidative stress status was ameliorated after treatment, with the oxidative capacity (d-ROMs) tending to decline and the antioxidative capacity (BAP) significantly increasing 3 months after treatment (Table 3, Fig.3).
Evaluations of Safety
Severe adverse events, defined as Common Terminology Criteria for Adverse Events version 4.0 Grade 3 or more, included vascular catheter-related bloodstream infection and hematoma in three and two patients, respectively, and acute coronary syndrome in one patient. Non-severe adverse events included fever, hypotension, nausea, appetite loss, abdominal pain, hematoma, catheter insertion site hemorrhage, dermatitis, pruritus, coagulation of the extracorporeal circuit, anemia, neutrophilia, and hypofibrinogenemia.
Exploring the Relationship between Primary Endpoints, Endothelial Function, and Oxidative Stress
Next, we examined whether the increase in ABI following LA treatment was associated with improved endothelial function. However, there was no significant correlation between the changes in ABI and RHI at both 1 month and 3 months after the sequential treatment (Supplementary Fig.1A, 1B). Furthermore, we examined the relationship between oxidative stress status and improvements in ABI and VascuQOL. There was no correlation between changes in ABI or VascuQOL and changes in dROMs or BAP after 1 or 3 months of treatment (Supplementary Fig.1C-1J). In addition, there was no significant correlation between the changes in HDL-C and d-ROMs or BAP (Supplementary Fig.2).
Finally, we compared baseline clinical and laboratory parameters by dividing the patients into responders and non-responders based on their response to the LA treatment (Supplementary Table 1). Responders were individuals who demonstrated an increase in ABI one month following the sequential treatment; conversely, non-responders were individuals whose ABI decreased. Among the participants, there were 17 responders and 13 non-responders. The responders tended to have a lower incidence of current smoking (0% vs. 23.1%, respectively), a higher rate of previous revascularization, and a higher baseline ABI compared to the non-responders (Supplementary Table 1).
Supplementary Table 1.Baseline characteristics of the study population according to the response to lipoprotein apheresis
|
Respondera n = 17
|
Non-respondera n = 13
|
P value
|
| Age, years |
70.9 (±6.2) |
73.8 (±4.9) |
0.14 |
| Male, n (%)
|
14 (82) |
9 (69.2) |
0.67 |
| Body mass index |
21.8 (±3.0) |
20.8 (±2.9) |
0.23 |
| Fontaine classification, n (%)
|
|
|
0.30 |
| Stage IIb: moderate-severe claudication |
6 (35.3) |
8 (61.5) |
|
| Stage III: rest pain |
4 (23.5) |
1 (7.7) |
|
| Stage IV: ulceration or gangrene |
7 (41.2) |
4 (30.8) |
|
| Comorbidities, n (%)
|
|
|
|
| Hypertension |
15 (88.2) |
11 (84.6) |
1.00 |
| Diabetes mellitus |
11 (64.7) |
10 (76.9) |
0.69 |
| Chronic kidney disease |
|
|
|
| Dialysis-dependent CKD |
12 (70.5) |
9 (69.2) |
1.00 |
| Non-dialysis-dependent CKD |
5 (29.4) |
4 (30.8) |
1.00 |
| Cardiovascular disease |
17 (100.0) |
12 (92.3) |
0.43 |
| Coronary artery disease |
16 (94.1) |
9 (69.2) |
0.14 |
| Congestive heart failure |
7 (41.2) |
2 (15.4) |
0.23 |
| Stroke |
11 (64.7) |
7 (53.8) |
0.71 |
| Smoking status, n (%)
|
|
|
0.061 |
| Current smoker |
0 (0) |
3 (23.1) |
|
| Former smoker |
16 (94.1) |
8 (61.5) |
|
| Never smoked |
1 (5.9) |
2 (15.4) |
|
| Medication, n (%)
|
|
|
|
| Lipid-lowering therapy |
|
|
|
| Statin |
13 (76.5) |
8 (61.5) |
0.39 |
| Others |
3 (17.6) |
2 (15.4) |
|
| Antihypertensive agent |
12 (70.5) |
9 (69.2) |
1.00 |
| Antiplatelet agent |
16 (94.1) |
13 (100.0) |
1.00 |
| Anticoagulant agent |
3 (17.6) |
3 (23.1) |
1.00 |
| Peripheral artery disease history |
|
|
|
| Previous revascularization on the target limb, n (%)
|
13 (76.5) |
5 (45.5) |
0.061 |
| Time from the last revascularization on the target limb, months |
26.3±35.5 |
22.3±19.0 |
0.88 |
| Previous limb amputation, n (%)
|
2 (11.8) |
1 (7.7) |
1.00 |
| Clinical symptoms |
|
|
|
| Walking distance, m |
|
|
|
| Pain-free |
64.6 (±103.0) |
77.4 (±46.3) |
0.15 |
| Maximum tolerated |
234.2 (±211.4) |
275.9 (±184.9) |
0.41 |
| Rest pain (VAS), mm |
31.4 (±29.4) |
44.8 (±31.3) |
0.48 |
| VascuQOL (total score) |
90.5 (±29.4) |
95.3 (±24.0) |
0.60 |
| Physiological examinations |
|
|
|
| ABI |
0.59 (±0.09) |
0.61 (±0.09) |
0.080 |
| SPP, mmHg |
|
|
|
| Dorsal |
38.4 (±14.2) |
44.8 (±21.9) |
0.55 |
| Plantar |
43.2 (±14.4) |
39.6 (±20.4) |
0.51 |
| Endothelial function (RHI) |
1.82 (±0.87) |
1.45 (±0.25) |
0.27 |
| Laboratory findings |
|
|
|
| Total cholesterol, mg/dL |
139.0 (±29.2) |
153.5 (±43.7) |
0.66 |
| LDL cholesterol, mg/dL |
73.5 (±22.3) |
81.2 (±35.0) |
0.67 |
| HDL cholesterol, mg/dL |
43.9 (±8.3) |
51.8 (±20.2) |
0.36 |
| Triglycerides, mg/dL |
111.2 (±49.4) |
102.8 (±50.8) |
0.45 |
| Lipoprotein(a), mg/dL |
13.8 (±7.3) |
20.5 (±13.7) |
0.39 |
| C-reactive protein, mg/dL |
0.9 (±1.4) |
1.2 (±2.2) |
0.70 |
| Fibrinogen, mg/dL |
372.4 (±71.5) |
387.3 (±120.2) |
0.73 |
| Oxidative stress |
|
|
|
| d-ROMs, U.CARR |
341.2 (±64.7) |
352.7 (±70.2) |
0.80 |
| BAP, μmol/L |
2556.5 (±320.7) |
2387.3 (±447.5) |
0.32 |
The parameters are shown as means (±SD) or percentages.
a Responders were defined as those who exhibited an increase in ABI one month after the sequential treatment, while non-responders were those who showed a decrease in ABI. Abbreviations: ABI, ankle brachial pressure index; BAP, biological antioxidant potential; CKD, chronic kidney disease; d-ROMs, derivatives of reactive oxidative metabolites; HDL, high-density lipoprotein; LDL, low-density lipoprotein; RHI, reactive hyperemia index; SD, standard deviation, SPP, skin perfusion pressure; U.CARR, Carr unit; VAS, visual analog scale; VascuQOL, vascular quality of life
Discussion
In this prospective study targeting 30 patients with PAD and controlled serum lipoproteins, ABI and VascuQOL scores significantly improved after a series of LA sessions. These improvements were sustained for 3 months after treatment. Antioxidative capacity and endothelial function also significantly improved after the LA sessions.
Although LA indication is limited to patients with elevated atherogenic lipoproteins in most countries30), the results of the present study demonstrated the effectiveness of LA in patients with PAD without such conditions. Notably, the mean serum LDL-C level of our patients was 76.9±28.2 mg/dL. According to the Japan Atherosclerosis Society Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases12), the target control levels for LDL-C are less than 100 mg/dL for primary prevention in high-risk patients and less than 70 mg/dL for secondary prevention in patients with a history of cerebral infarction or coronary artery disease. Additionally, in this study, baseline CRP levels were 1.0±1.8 mg/dL. Since chronic inflammation contributes to arteriosclerosis and CRP levels are associated with a high risk of cardiovascular diseases31, 32), these elevated CRP levels might have reflected chronic inflammation in patients with severe arteriosclerosis.
ABI significantly increased after LA sessions, which is consistent with previous reports8, 15, 21). Although several studies on patients with dyslipoproteinemia have shown that continuous LA treatment results in a plaque reduction in the coronary33, 34) and peripheral arteries10), at least 1–2 years are required for such an effect. Therefore, the increase in ABI observed in this study may not reflect a reduction in the stenotic lesions. The amelioration of blood flow in the lower extremities due to reduced vascular tone and suppressed coagulation, which were induced by the improved endothelial function, have reportedly been associated with increased ABI10, 35, 36). However, there was no correlation between improved endothelial function and ABI in the present study. This may be due to the small sample size, or ABI may be improved by other mechanisms.
LA sessions also significantly improved the patients’ QOL. The average changes in the total VascuQOL score from baseline to 1 and 3 months after treatment were greater than the prespecified MID24), indicating the clinical significance of these changes. Among the domain scores, the pain and emotional domain score particularly improved between baseline and 1 month after the LA sessions. The increase in the pain domain score was consistent with the observed trend toward improvement in rest pain and pain-free walking distance. Therefore, mitigating this pain may have resulted in relief of discomfort, which was reflected in the emotional domain score. The social domain score largely increased from 1 to 3 months after treatment, with a slight increase in other domain scores. These observations suggest that a series of LA sessions not only improve each PAD symptom but also beneficially affect comprehensive QOL, including emotional and social aspects. To the best of our knowledge, this is the first study to assess the impact of LA on QOL in patients with PAD.
The effect of LA, represented by a significant increase in ABI and VascuQOL, was sustained for 3 months after the last session. Oxidative stress amelioration may have played a role in this long-term effect; the antioxidative capacity (BAP) significantly increased 3 months after the LA sessions, with the oxidative capacity (d-ROMs) tending to decrease concomitantly. Oxidative stress, which is induced by an imbalance between reactive oxygen species (ROS) production and antioxidative clearance36), is an established pathogenesis and promoter of PAD37, 38). Although many reports have demonstrated the reduction of ROS and its metabolites after LA22, 23, 36), few studies have investigated the effect of LA on antioxidative capacity. One study reported that LA partly corrected the impaired antioxidative function of HDL-C particles in familial hypercholesterolemia39). In the present study, HDL-C levels significantly increased after LA sessions. However, this increase did not correspond with elevated antioxidative capacity. The activity of antioxidative enzymes and qualitative assessment of HDL, such as morphology and composition of HDL, were not conducted in this study. Therefore, the mechanism underlying this increased antioxidative capacity should be further studied.
Other proposed LA mechanisms on PAD include improving endothelial function and microcirculation. Regarding endothelial function, the RHI, which evaluates endothelial-dependent vasodilative function in a completely automated procedure40), was significantly improved 1 month after the LA sessions. This finding is consistent with a previous report that demonstrated a significant increase in flow-mediated dilation (FMD)41). Although RHI and FMD are both established modalities for evaluating an endothelial function, the latter requires skill, and the results depend on the examiners42). Several studies have shown that excessive ROS induces endothelial dysfunction through inflammatory signals and impaired nitric monoxide bioavailability43, 44).
The beneficial effects of LA on microcirculation have been repeatedly documented45). The underlying mechanisms include improved rheology via fibrinogen adsorption46), reduction of erythrocyte aggregation47), and activated production of bradykinin48) and nitric monoxide49). Here, we investigated SPP to evaluate microcirculation. No significant difference in SPP was observed between baseline and 1 month after treatment. Although several reports have shown increased SPP by LA50, 51), evaluations were conducted immediately after LA sessions in these reports. Our results suggest that the effects of LA on microcirculation are not sustained after discontinuing the procedure. Collectively, in this study, the sustained ameliorative effects on PAD were observed after the LA treatment, concomitant with increased antioxidant capacity and improved endothelial function, rather than improved microcirculation.
This study had some limitations. First, this is a single-arm intervention design. Therefore, attributing the observed effects solely to the LA was challenging. Improved management of atherosclerotic risk factors during participation might have partially affected the results, although medication alterations were avoided to the utmost extent. Placebo effect, concerning subjective symptoms, cannot be excluded. Second, no correlation was found between the primary endpoint of improvement in ABI or VascQOL and endothelial function or oxidative stress. Consequently, the mechanism underlying the improvement in ABI following the LA treatment remains unclear. Third, because patients with ABI ≥ 0.7 were excluded, our findings may not apply to patients with normal or increased ABI due to severe arterial calcification or pedal lesions. Fourth, the study population was dominantly complicated by CKD, which makes it difficult to generalize our results to patients without CKD. Fifth, the follow-up period in this study was relatively short. Sixth, the safety of LA in patients with strictly lowered LDL-C levels should be assessed separately; in our patients, the baseline LDL-C level was relatively higher than the most recent recommendations52) for the highest risk. Despite these limitations, the results of the present study were worthwhile because it was very hard to target patients with PAD who were refractory to conventional revascularization therapies.
Conclusions
ABI and QOL improved after a series of LA sessions in patients with conventional treatment-resistant PAD despite the normal range of atherogenic lipoproteins. These effects were sustained for 3 months after treatment discontinuation. Increased antioxidant capacity and improved endothelial dysfunction were observed after the LA treatment.
Acknowledgements
The authors thank Takayasu Ohtake, Yasuyuki Mochida, and Kazuo Takahashi for their contributions to the Data and Safety Monitoring Committee and Tatsuo Hashimoto and Naohiro Komura for their oversight of protocol compliance. The authors also thank the staff of the YCU Center for Novel and Exploratory Clinical Trials for their support in study management. Finally, the authors are grateful for the collaboration between the patients and the dedicated staff of Yokohama City University Hospital.
Conflicts of Interest and Source of Funding:
The plasma separators (Sulflux FP-08), dextran sulfate cellulose columns for adsorption (Liposorber LA-15) and extracorporeal circuit used in this study were provided by Kaneka Corporation, Tokyo, Japan, under the contract of the industry-university collaboration. This work was supported by grants from the Yokohama Foundation for Advancement of Medical Science; the Uehara Memorial Foundation; the Japan Society for the Promotion of Science; the Japan Kidney Association-Nippon Boehringer Ingelheim Joint Research Program; the Japanese Association of Dialysis Physicians; the Salt Science Research Foundation; the Strategic Research Project of Yokohama City University; the Moriya Scholarship Foundation the Japan Agency for Medical Research and Development (AMED); the Translational Research program, Strategic Promotion for Practical Application of Innovative Medical Technology (TR-SPRINT) from AMED; the Bayer Scholarship for Cardiovascular Research; the Japan Science and Technology Agency (Grant Number JPMJPF2303); and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.
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