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Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Hiroshi Itoh, M.D., Ph.D., Department of Internal Medicine, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: hiito{at}kuhp.kyoto-u.ac.jp.
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Introduction |
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Natriuretic peptides (NPs) consist of atrial NP (ANP), brain NP (BNP), and C-type NP (CNP) and elicit various biological effects by activating particulate GCs: GC-A is a receptor selective for ANP and BNP, and GC-B is a receptor selective for CNP (4, 6, 7, 8). One of the major mediators of cGMP signaling is cGMP-dependent protein kinase (cGK) (4). ANP and BNP are secreted mainly from the atrium and ventricle of the heart, respectively, and act as cardiac hormones (4, 6, 7). The clinical significance of NPs is already recognized in the diagnosis and treatment of congestive heart failure (CHF). Recombinant human ANP and BNP are used for treating CHF, with the main expectation of diuretic and natriuretic effects (9, 10).
Recently, NPs have been revealed to have various effects on cell survival, proliferation, and differentiation. We reported that ANP at a physiological concentration induces endothelial regeneration in the human coronary artery and umbilical vein through the activation of ERK and phosphatidylinositol 3-kinase/Akt pathways (11). We used genetically engineered mice that overexpress BNP and type I cGK (cGKI), or otherwise lack cGKI, and demonstrated that BNP can promote vascular regeneration and accelerate the restoration of blood flow after the removal of a hind-limb artery in mice through the activation of the GC-A/cGMP/cGKI pathway (12, 13, 14). Meanwhile, CNP, which is secreted from endothelial cells (ECs) and acts as an endothelium-derived relaxing peptide (15), also induces redifferentiation of vascular smooth muscle cells (SMCs) while accelerating reendothelialization and suppressing neointimal hyperplasia in vein grafting or balloon injuries in rabbits, which simulate atherosclerotic lesions in humans (16, 17). These observations indicate that GC-A/cGMP and GC-B/cGMP signaling cascades have potential to promote vascular regeneration in PAD and to inhibit the progression of atherosclerotic lesions. On the other hand, we have reported previously that endothelial CNP expression is progressively reduced in accordance with the severity of human coronary atherosclerosis (18), which indicates that not only NO/soluble GC/cGMP signaling but also CNP/GC-B/cGMP signaling might be impaired in PAD. Therefore, the restoration of intracellular cGMP levels by the activation of GC-A, the third signaling pathway using cGMP as the second messenger in vascular SMCs and ECs, could improve PAD.
In this context, we hypothesized that an administration of ANP or BNP could, at least partly, compensate for impaired angiogenesis due to diminished intracellular cGMP levels in PAD patients by an activation of GC-A. In Japan, carperitide, a recombinant human ANP, is already approved and widely used for the treatment of CHF. By contrast, nesiritide, a recombinant human BNP, has not been approved in Japan, and it cannot be applied to rodent models because amino acid sequences and molecular forms of BNP are quite different between humans and rodents. In the present study, we therefore examined the effect of carperitide on vascular regeneration in animal models with diabetes, and we further tried to determine safety and to investigate any possible therapeutic effects of carperitide in PAD patients.
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Materials and Methods |
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An animal model of limb ischemia was made by a ligation of one femoral artery. The blood flow in both legs was assessed with a laser Doppler perfusion image analyzer (Moor Instruments, Devon, UK), and the blood flow recovery was assessed by the ischemic limb to normal limb ratio of blood flow, as we described previously (14).
To assess the effect of carperitide, a recombinant human ANP (Daiichi Asubio Pharma Co., Ltd., Tokyo, Japan), on angiogenesis in ischemic limbs, the femoral artery ligation was carried out at 16 wk after the first injection of STZ or vehicle, and carperitide at a dose of 2.2 µg/kg·min or equal volume of water (vehicle) was administered continuously and ip via a microosmotic pump (Alzet model 1002D; Alzet Pharmaceuticals, Palo Alto, CA), which was implanted ip at 3 d after the femoral artery ligation. Pumps were renewed at d 14 after primary implantation. At 28 d from the femoral artery ligation, mice were euthanized by an overdose of pentobarbital injection, and the ischemic hind limb was isolated for the histological analysis.
All experimental procedures were performed according to Kyoto University standards for animal care.
Histological analysis
After fixation with 4% paraformaldehyde, ischemic lower legs were embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) and frozen at –80 C. Cryostat sections (4–8 µm thick) of the tissue were stained with a rat antimouse platelet EC adhesion molecule-1 (PECAM-1) antibody (item 553370; PharMingen, San Diego, CA). Four random fields on two different sections (3 mm apart) from each mouse were photographed with a digital camera (Olympus, Tokyo, Japan). By computer-assisted analysis using NIH IMAGE, capillary density was calculated as the mean number of capillaries stained with PECAM-1, as we described previously (14).
Patients
Participants were a series of 13 Japanese patients including 11 males and two females, aged 38–92 yr, who had already been diagnosed with PAD and hospitalized in our department from June 2003 to August 2005 (Table 1
). Patients classified as Fontaine’s classes II–IV or with characteristic symptoms of PAD were included. Diseases accompanying PAD were defined as follows: type 2 diabetes mellitus, following the diagnostic criteria of Japan Diabetes Society; hypertension, blood pressure is equal to or greater than 140/90 mm Hg; end-stage renal disease, chronic renal failure on indispensable renal replacement therapy; ischemic heart disease, history of angina pectoris or myocardial ischemia with or without present medication; CHF, past diagnosis of CHF with or without present medication; hyperlipidemia, low-density lipoprotein-cholesterol is equal to or greater than 140 mg/dl, or triglyceride is equal to or greater than 150 mg/dl; obesity, body mass index is greater than 25 kg/m2. Exclusion criteria were contraindications for carperitide: possibility of immediate surgery, suffering from malignancy, febrility, an inability to declare subjective symptoms, pregnancy, or other unfavorable statuses. The study was conducted in accordance with the guidelines in the Declaration of Helsinki. The study protocol was approved by the Ethics Committee Graduate School and Faculty of Medicine, Kyoto University. Patients were fully informed of the aim of the study, and their written informed consent was obtained.
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Pain was assessed when present with a numerical rating scale from 0–10; grade 0 indicated no pain and grade 10 the strongest pain the patient could imagine. The ankle-brachial pressure index (ABI) was assessed by an automated measurement device (BP-203RPEII; Colin Medical Technology Corp., Aichi, Japan). An exercise tolerance test was carried out weekly for patients with intermittent claudication. Pain-free walking distance on a flat ground was assessed. A stair-climb test was performed when walking on flat ground did not induce claudication. The test assessed how many floors a patient could climb without pain on the stair of our internal medicine ward building. Blood sampling was performed immediately before the beginning of carperitide administration and weekly during the administration for routine blood examination. It was also performed to determine the plasma levels of ANP, cGMP, and vascular endothelial growth factor (VEGF).
Analysis of blood samples
The blood samples from mice were withdrawn in an ice-cold tube containing 0.5 M Na2EDTA final concentration and mixed well. Aprotinin was added at 500 U/ml when a sample was used for human ANP measurement. The plasma was immediately isolated by a centrifugation and stored at –20 C until further processing. Plasma concentrations of cGMP, VEGF, and human ANP were analyzed by SRL, Inc. (Tokyo, Japan).
Statistical analysis
Results are presented as mean ± SEM unless otherwise indicated. The statistical significance of differences in means was evaluated by ANOVA supplemented with Fisher’s least-significant difference in comparisons among three or more groups in animal experiments and by paired t tests between before and after the carperitide administration in the human study. A P value < 0.05 was considered significant.
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Results |
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At 4 wk after the induction of diabetes, blood flow recovery of the STZ-diabetic group was similar to that of nondiabetic controls (Fig. 1A). But after a long-term hyperglycemic state of 16 or 26 wk, recovery was suppressed in the STZ-diabetic group by 26 or 32%, respectively, when compared with the control mice (Fig. 1, B and C).
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Blood glucose levels at femoral artery ligation were 116 ± 4 mg/dl in the vehicle-treated nondiabetic group, 122 ± 3 mg/dl in the carperitide-treated nondiabetic group, 343 ± 42 mg/dl in the vehicle-treated STZ-diabetic group, and 366 ± 42 mg/dl in the carperitide-treated STZ-diabetic group. In nondiabetic mice, the carperitide administration significantly accelerated blood flow recovery compared with the vehicle-treated group. The ischemic/normal limb blood flow ratio measured at 21 d after the surgery was 0.58 ± 0.03 in the vehicle-treated nondiabetic group (n = 13) and was significantly augmented in the carperitide-treated nondiabetic group (0.74 ± 0.06, n = 7; P < 0.05). The accelerating effect of carperitide on blood flow recovery was also seen in STZ-diabetic mice. The ischemic/normal limb blood flow ratio at 21 days after surgery was 0.52 ± 0.05 in the carperitide-treated STZ-diabetic group (n = 8) and significantly higher than that in the vehicle-treated STZ-diabetic group (0.37 ± 0.06, n = 7; P < 0.05) (Fig. 2B). The time course of blood flow recovery in each group was shown in Fig. 2A.
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, C and D). The capillary density tended to be higher in the carperitide-treated nondiabetic group (1604 ± 108 counts/mm2, n = 6) than in the vehicle-treated nondiabetic group. Among STZ-diabetic mice, the carperitide administration significantly increased the capillary density to 1180 ± 95 counts/mm2 (n = 6; P < 0.05). In this study, 4-wk administration of carperitide to mice increased plasma human ANP levels from under the detection limit (10 pg/ml) to 156 ± 79 pg/ml (n = 5 each) and plasma cGMP levels from 8.9 ± 1.1 nM (n = 7) to 20.0 ± 2.9 nM (n = 6, P < 0.05). The carperitide administration altered blood pressure from 106 ± 3/73 ± 3 mm Hg to 94 ± 4/62 ± 4 mm Hg (n = 4 each; P < 0.05).
Human study
All patients had characteristic symptoms of PAD (Fontaine’s class: I, one; II, five; III, two; and IV, five) (Table 2). A patient who was Fontaine’s class I had a cold sensation in the lower extremities. The diagnosis was confirmed by ABI measurement, ultrasound velocity spectroscopy, or magnetic resonance angiography.
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). Among diabetic subjects, HbA1c levels were 7.7 ± 0.5%, and disease duration was 16.5 ± 2.1 yr. Seven patients suffered from end-stage renal diseases and were on hemodialysis. Eight patients had a past history of an ischemic heart disease, CHF, or both, and all of them were in stable condition with or without medication Plasma ANP levels were 315 ± 130 pg/ml, and ejection fractions measured by ultrasonic echocardiography were 49.9 ± 6.2%.
Plasma ANP levels were elevated from 224 ± 93 pg/ml at baseline to 400 ± 125 pg/ml during the administration (n = 12; P < 0.05; data were lacking in patient 5). Plasma cGMP levels were elevated from 14.4 ± 3.5 to 24.0 ± 4.5 nM (n = 9; P < 0.01). No significant differences were seen in plasma VEGF levels: 92.2 ± 25.4 pg/ml at the baseline and 65.2 ± 11.1 pg/ml in the course of administration (n = 8). The blood pressure of patients (excepting those on hemodialysis) fell from 143 ± 8/74 ± 2 mm Hg to 123 ± 7/69 ± 3 mm Hg (n = 5; P < 0.05). An excessive decrease in systolic blood pressure to less than 90 mm Hg was observed in a few patients on hemodialysis and could be quickly reversed by reducing the carperitide infusion rate. Medications except for injections were continued during carperitide injection without any changes. Details of medications especially for PAD are shown in Table 2. Alprostadil (prostaglandin E) had been iv administrated daily for a week to patients 2 and 3, and for a month to patients 6 and 11, and was stopped at least 3 d before the beginning of carperitide administration. Phosphodiesterase inhibitors other than cilostazaol were not used in patients enrolled in this study. Smoking status was not changed in five never-smokers (patients 1, 3, 5, 12, and 13) and seven former smokers (patients 2, 4, 7, 8, 9, 10, and 11) during this study. One patient (no. 6) was a current smoker (20 cigarettes/d) at the enrollment and stopped smoking 7 d before the administration.
The ABI of the affected limb (or worse side when both limbs affected) was significantly elevated from 0.61 ± 0.08 at the baseline to 0.72 ± 0.09 on the 14th day of administration (n = 12; P < 0.05) except for patient 5, for whom the administration was stopped within a week (Table 3 and Fig. 3b). Brachial systolic blood pressure values for ABI calculations before and on the 14th day of administration were 140 ± 10 and 132 ± 8 mm Hg, respectively (n = 12; P = 0.5). Ankle systolic blood pressure values at affected limb were 84 ± 13 mm Hg before administration and were increased to 94 ± 11 mm Hg on the 14th day of administration (n = 12; P = 0.4).
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). Rest pain disappeared in three of the six patients (patient 6, 4/0; patient 9, 4/0; and patient 13, 3/0, as before/after the administration of carperitide) and was reduced in another patient (no. 1, 3/1). In patient 4, although the pain once worsened in the early phase of administration (from 4 to 6), the injections were continued, and the pain was reduced to level 1 within a week. In another patient (no. 5), the carperitide infusion was stopped at d 7 because rest pain had worsened (4 to 6) (Fig. 3A). All patients who felt the rating score of rest pain reduced could stop to use pain relievers or hypnotics.
Exercise performance was carried out on all patients with intermittent claudication except for those who could not walk as a result of rest pain or weakness (patients 2, 7, 8, 10, and 12) (Fig. 3C). The pain-free walking distance was assessed in four patients and prolonged in all of them after the carperitide administration (patient 2, 290 to 380 m; patient 7, 240 to 560 m; patient 8, 200 to 800 m; patient 10, 100 to 200 m). In another patient with a stair-climb test, the floor number of pain-free stair climbing was increased from five to seven.
Five patients had multiple foot ulcers, and dermatologists in our hospital had recommended foot amputation. Although the ulcers did not change in severity in two cases (patients 5 and 6), they improved in another three cases (patients 4, 11, and 13) for whom foot amputations could be avoided. A representative case is shown in Fig. 3, D–I.
Other changes observed during administration were as follows: hot sensation in lower extremities in eight patients (nos. 1, 2, 4, 5, 6, 7, 8, and 13), transient flush and slight nausea in one patient (no. 2), pitting edema in both feet in five patients on hemodialysis (nos. 1, 4, 6, 11, and 13), and an increase in menstrual bleeding in a patient (no. 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We showed here that carperitide, a recombinant human ANP, significantly accelerated blood flow recovery in a mouse model of ischemia-induced angiogenesis in both nondiabetic and diabetic conditions. The blood flow recovery in carperitide-treated diabetic mice was improved to a level similar to that in vehicle-treated nondiabetic mice. A histological analysis revealed that capillary density in the muscle of the ischemic limb was reduced in diabetic mice. The carperitide infusion significantly recovered capillary density in diabetic mice to the level in vehicle-treated nondiabetic mice. These observations indicate that carperitide can improve ischemia-induced angiogenesis, which accelerates blood flow recovery in diabetic conditions. We have shown that an increase of circulating BNP levels by targeted overexpression of the murine BNP gene in the liver or an overexpression of cGK throughout the body by the transgenic technology can accelerate the restoration of blood flow in limb ischemia experimentally generated by a femoral artery ligation, which results from the promotion of ischemia-induced angiogenesis through the activation of the ERK cascade (14). We have also shown that ANP at a physiological concentration induces proliferation and migration of ECs and enhances endothelial regeneration via activating ERK1/2 and phosphatidylinositol 3-kinase/Akt pathways in an in vitro wound healing assay using the cells from either coronary arteries or umbilical veins of humans (11). CNP, another member of the NP family, was shown to enhance migration of ECs and to accelerate reendothelialization in vein grafts after an arterial bypass surgery, although CNP inhibits proliferation and migration of vascular SMCs (16, 17). NPs use particulate GCs as their signaling receptors and share cGMP signaling pathways, especially signaling through cGKI, with NO, which activates soluble GC to generate cGMP (4). It is known that NO is a mediator of VEGF, which is a potent mitogen for vascular ECs and induces angiogenesis (19). A significant portion of VEGF-induced human EC proliferation is reportedly mediated by cGKI (20). In diabetes, hyperglycemia induces formation of reactive oxygen species, which decrease the bioavailability of NO (21). Taken together, deterioration of cGMP signaling appears to be a key process leading to the impaired angiogenesis and PAD in diabetes. In this study, the administration of carperitide could overcome the impairment of cGMP signaling in diabetic conditions, and it would be a new, therapeutic approach to PAD with diabetes. Because the urinary cGMP excretion rate is inversely correlated with the grade of Fontaine’s classification in PAD patients (5), an impairment of cGMP signaling appears to be a common feature of PAD. We therefore investigated the therapeutic potential of carperitide administration in PAD patients.
We did not assign participants to a vehicle-treated group for an ethical reason; most cases of participants had been treated with conventional therapies, which had not accomplished appreciable effects. The carperitide administration significantly increased ABI, effectively relieved symptoms including intermittent claudication and rest pain, and promoted the healing of foot ulcers in PAD patients. The dosage of carperitide we used in the human study was optimized for each patient according to the maximum permissible dosage, which is the highest dose possible without causing an excessive fall in systolic blood pressure, because sensitivity to exogenously administered ANP differs among patients depending, presumably, upon basal plasma ANP levels. Although doses of carperitide administration were lower than those usually given in the treatment of CHF, plasma cGMP levels were increased twice as much as basal levels, and relief from the characteristic signs and symptoms of PAD became possible. This observation suggests that a blood pressure fall would not limit the therapeutic use of carperitide for PAD patients.
It is reported that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase, is accumulated in patients with end-stage renal disease (ESRD) and a high plasma ADMA level is a strong indicator of risks for all-cause mortality and cardiovascular events (22). It might be speculated that responses to the carperitide administration are better in ESRD patients than in non-ESRD patients, because carperitide is supposed to restore cGMP signaling, which is impaired by ADMA, via an activation of GC-A. Considering heterogeneity of patients’ clinical characteristics, a larger number of participants will be needed to address this issue.
All patients, for whom exercise tolerance was evaluated, had been treated with conventional therapies using per os and per cutaneous medications under hospitalization and been encouraged to walk for at least 3 wk without any increases of pain-free walking distances. A 2-wk carperitide administration was then added to the conventional therapies and resulted in significantly improved exercise tolerance. The improvement, therefore, cannot be explained by a training effect only.
NPs have various biological effects on vascular functions other than the promotion of angiogenesis, and some of them appear favorable to treating PAD. NPs regulate vascular tone, and CNP, especially, is a candidate for endothelial-derived hyperpolarizing factor, which plays a fundamental role in the regulation of local blood flow and systemic blood pressure (23). In the clinical investigation of this paper, changes in symptoms and ABI appeared within a few days or a week of the administration. The effect of carperitide on symptoms in the early phase might be due to a vasodilatory action of ANP to some extent, because the changes appeared too early to be regarded as effects of vascular regeneration. On the other hand, the elongation of pain-free walking distance persisted after the cessation of the administration was ceased, and ABI remained elevated for several months after the end of administration. If the vasodilatory action of ANP is the only mechanism of the improvement, the effects of carperitide should disappear promptly at the cessation of the infusion, because the half life of ANP in circulation is a couple of minutes (24).
In patients with advanced arteriosclerosis, severe calcification of arterial walls in lower extremities can cause an overestimation of ankle blood pressure. Where vasodilators such as carperitide were used in such patients, ABI might be increased solely due to a decrease in brachial blood pressure. In this study, we observed slight decreases in brachial blood pressure, but we could observe increases in ankle blood pressure although the changes were not statistically significant. Increases in ABI, therefore, should not be false and should be, at least in part, the result of blood flow recovery.
The improvement in exercise tolerance and ABI might, therefore, be achieved by modifying vascular endothelial structure or promoting vascular regeneration. Plasma VEGF levels were not significantly elevated by the carperitide infusion in this study, indicating that VEGF is not an essential mediator of carperitide’s effects on PAD symptoms. It is reported that NPs elicit antiinflammatory and antithrombotic effects in animals (17, 25, 26), and further investigation will be needed to see whether such effects of NPs are clinically significant.
Carperitide is often used to treat CHF patients in Japan, and its safety is clinically proven. No critical side effects were observed in this study. An increase in menstrual bleeding observed in a participant could be accidental or a result of ANP’s vasodilatory action, because the symptom faded soon after the cessation of the infusion. There are, however, several reports indicating the physiological significance of CNP/GC-B signaling in the control of ovarian cycling (27, 28). A close observation would be needed where carperitide infusion would be applied to women of reproductive age for a long duration (more than 2 wk). Leg edema appeared in three patients, who were in relatively serious states of the foot disease. Many PAD patients develop postoperative edema after surgeries of revascularization (29), which indicates that they have circulatory inadequacy for autoregulating blood hydrostatic pressure. Because ANP reportedly plays an essential role in maintaining vascular permeability via GC-A on vascular ECs (30), edema might result from this direct action on vascular endothelium.
Conclusion
This study revealed that a long-duration diabetic condition impaired ischemia-induced angiogenesis and blood flow recovery in a mouse model of hind-limb ischemia and that ANP as a therapeutic agent for CHF can restore the ischemia-induced angiogenesis in diabetic mice. Based on this observation, we applied carperitide administration to 13 PAD patients and found that carperitide infusion at doses lower than those for CHF could safely improve signs and symptoms. Carperitide administration, therefore, can be a new therapeutic strategy for PAD, and it appears effective in patients for whom conventional therapies do not work well.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors of this manuscript have nothing to declare.
First Published Online November 8, 2007
Abbreviations: ABI, Ankle-brachial pressure index; ADMA, asymmetric dimethylarginine; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; cGK, cGMP-dependent protein kinase; cGKI, type I cGMP-dependent protein kinase; CHF, congestive heart failure; CNP, C-type natriuretic peptide; EC, endothelial cell; ESRD, end-stage renal disease; GC, guanylyl cyclase; NP, natriuretic peptide; PECAM, platelet endothelial cell adhesion molecule-1; SMC, smooth muscle cell; STZ, streptozotocin; VEGF, vascular endothelial growth factor.
Received August 7, 2007.
Accepted for publication November 1, 2007.
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