Perioperative Iloprost and Endothelial Progenitor Cells in Uremic Patients With Severe Limb Ischemia Undergoing Peripheral Revascularization

Journal of Surgical Research 157, e129–e135 (2009) doi:10.1016/j.jss.2008.07.017

Perioperative Iloprost and Endothelial Progenitor Cells in Uremic Patients With Severe Limb Ischemia Undergoing Peripheral Revascularization Giuseppe Coppolino, MD,* Antoine Buemi, MD,†,1 Davide Bolignano, MD,* Antonio Lacquaniti, MD,* Michele La Spada, MD,† Francesco Stilo, MD,† Giovanni De Caridi, MD,† Francesco Benedetto, MD,† Saverio Loddo, MD,* Michele Buemi, MD,* and Francesco Spinelli, MD† *Department of Internal Medicine, University of Messina, Messina, Italy; and †Department of Vascular Surgery, University of Messina, Messina, Italy Submitted for publication April 15, 2008

The incidence of severe limb ischemia (SLI) is high among haemodialysis (HD) patients. Limb rescue rate after surgical revascularization is relatively poor compared with patients with normal renal function. Prostanoids are an interesting category as adjuvants to revascularization. New vessel growth develops not exclusively by proliferation of endothelial cells in vascular extremities but also by cells mobilized from the bone marrow (HSC), transformed into endothelial progenitor cells (EPC) contributing to both re-endothelialization and neovascularization. Basal number of HSC and EPC is significantly reduced in HD patients and correlated with a subsequent defective neovascularization. The aim of this study was to evaluate the effects of perioperative treatment with iloprost in uremic patients with acute ischemia of lower limbs, undergoing surgical revascularization, on endothelial progenitor cells, hypothesizing a possible biological mechanism induced by the prostanoids. A search was also made for vascular remodeling processes through the analysis of the concentrations of soluble adhesion molecules (i-CAM, v-CAM, e-selectin), biochemical markers of endothelial activation. Thirty HD patients with SLI undergoing peripheral revascularization were enrolled (15 were treated with iloprost and 15 with a placebo). Iloprost was administered as an intra-arterial bolus of 3000 ng over 1 to 3 min immediately after revascularization and in the same affected artery. Serum samples were taken before revascularization (T0), at 6 (T6) and 24 h (T24) after

infusion to measure sICAM-1, sE-selectin, and sVCAM-1, and for quantification of HSC and EPC. Progenitors were identified by specific surface markers CD34D, CD133D and VEGFR2D. Count was conducted using PROCOUNT performed in a TRUCOUNT tube and with a FACSort flow cytometer. Before revascularization, all patients showed a decreased number of HSC and EPC. After 6 h, HSC augmented significantly compared with T0 in both groups. The iloprost group attained a significant increase compared with the placebo group. HSC levels reduced drastically at T24. EPC augmented significantly compared with basal level after 24 h. In the iloprost group, the increase was considerable compared with the placebo group. A close negative correlation, assessed by Pearson coefficient (r), was found between HSC and EPC at T24 in the iloprost group (R [ 0.82 P < 0.01). Adhesion molecules had increased levels at T6 and T24 in both groups. Moreover, a close positive correlation, assessed by Pearson coefficient, was found between EPC and adhesion molecules in both groups but the iloprost group maintained a better statistical association. Revascularization stimulated HSC and EPC release from bone marrow but at a different time: HSC increased suddenly at 6 h and diminished to a minimal amount at T24, conversely, EPC increased significantly only at T24. Iloprost treatment was able to amplify this mechanism validating recent findings (North TE et al., [31]). Adhesion molecules as markers of endothelial activation and vascular development confirmed this tendency. Ó 2009 Elsevier Inc. All rights reserved.

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To whom correspondence and reprint requests should be addressed at the Department of Internal Medicine, University of Messina, Via Salita villa Contino 30, 98100 Messina, Italy. E-mail: [email protected].

Key Words: Iloprost; endothelial progenitor cells; uremic patients; peripheral revascularization.

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INTRODUCTION

MATERIALS AND METHODS

In most developed countries, the incidence of severe limb ischemia is estimated to be 50 to 100 per 100,000 cases every year [1]. Current treatment options include medicines such as anticoagulants and antiplatelet drugs, intravascular treatments and bypass surgery. However, in patients with diffuse and distal peripheral severe stenosis, amputation of lower extremities is often required, especially in end stage renal disease (ESRD) patients. The prevalence and incidence of severe limb ischemia is high among patients with renal insufficiency and there are relatively low limb salvage rates after lower extremity surgical revascularization compared with those with normal renal function [2–4]. Results of preclinical studies have shown that angiogenic growth factors promote development of collateral arteries, which is called therapeutic angiogenesis [5]. Therapeutic angiogenesis is an important future therapeutic target for cardiovascular and peripheral ischemic diseases. Possible benefits from cardiovascular active therapies have recently been suggested in patients undergoing peripheral revascularization or major noncardiac surgical intervention [6]. New vessel growth develops not exclusively by the migration and proliferation of endothelial cells in vascular extremities but also by cells directly mobilized by the bone marrow, which are transformed into the so-called endothelial progenitor cells (EPC) contributing to both re-endothelialization and neovascularization. A reduced number of EPC was related to a higher cardiovascular risk and to a defective neovascularization. The basal number of progenitor cells is significantly lower in uremic patients than in the control population [7, 8]. This reduction was correlated to a defective neo-vascularization, and endothelial dysfunction [1, 9, 10]. Prostanoids represent a potentially interesting category as an adjuvant treatment of patients with severe limb ischemia undergoing peripheral revascularization [11]. Several ischemia-reperfusion studies described the use of prostaglandins for reduction of postischemic tissue injuries, and even recently both PGE1 and PGI2 appeared as potent inhibitors of reflow-paradox in a preclinical model of reperfusion injury [12, 13]. Encouraging results were obtained with the use of intraoperative and postoperative iloprost (lower incidence of major clinical events, more evident metabolic improvement by means of transcutaneous tensiometry) [14, 15]. The aim of this study was to evaluate the effects of perioperative treatment with iloprost in uremic patients with acute ischemia of lower limbs undergoing surgical revascularization, on endothelial progenitor cells, hypothesizing a possible biological mechanism induced by the prostanoids on vascular remodeling processes.

Patients and Study Design Between September 2006 and August 2008, 30 patients (15 in the placebo group and 15 in the iloprost group) undergoing haemodialysis for 3.5 to 4 h three times a wk [mean age 59.8 6 10.5 y, mean dialysis age 2.7 6 1.6 y, residual glomerular filtration rate (GFR) 2.3 6 0.6 mL/ min] were enrolled in a prospective study. Throughout the study, each patient complied with fluid and dietary restrictions, and maintained a constant ultrafiltration volume. Patients were on dialysis for primary interstitial nephritis, polycystic kidney disease, and glomerulonephritis. Patients were considered for inclusion if they presented with acute onset (less than 14 d) of symptoms suggestive of ischemia of lower limbs and were to be treated with surgical revascularization. Patients with lower limb ischemia due to trauma were excluded. Exclusion criteria included alcohol consumption, cigarette smoking or drug abuse, severe hypertension, heart or liver disease, thyroid disease, postural hypotension, diabetes mellitus, concomitant clinical conditions in which iloprost might increase the risk of bleeding, thrombocytopenia or thrombocytosis, and severe hepatic failure (cirrhosis). Iloprost (Endoprost; Italfarmaco S.p.A., Milan, Italy, under license of Schering AG, Berlin, Germany) or a placebo was administered as an intra-arterial bolus of 3000 ng over 1 to 3 min immediately after revascularization and in the same affected artery. An indwelling catheter was inserted into the antecubital vein to draw blood for blood drawing. Serum samples were taken immediately before revascularization (T0), and at 6 (T6), and 24 h (T24) after Iloprost infusion to measure sICAM-1 (ICAM-1 Predicta kit; Genzyme) and sE-selectin and sVCAM-1 (Bender Med System; Genzyme Corp., Cambridge, MA), and for quantification of EPCs. During infusion of experimental drugs, strict monitoring of blood pressure and heart rate was requested. Doppler examinations were performed before and after revascularization to assess the early outcome of surgery. The local Ethics Committee approved the study protocol.

Interventions Interventions were performed using a standardized technique of percutaneous transluminal angioplasty (PTA). Technical success was defined as residual stenosis with less than 30% lumen reduction. PTA was performed in 50.5% patients on the femoro-popliteal artery, in 35.8% on the iliac artery, in 13.7% on the crural artery. Patients were matched for gender, dialytic age, and affected artery. Ten patients who had previously undergone diagnostic investigations were directly treated by revascularization by means of anterograde injection. In the remaining 20 cases, two different approaches were used: panoramic angiography of the aorta-iliac axis and lower extremity arteries via retrograde contralateral femoral access for diagnostic purposes and femoral anterograde injection to terminate the revascularization by means of subintimal infrapopliteal angioplasty.

Quantification of EPCs Fresh blood cytofluorimetry was used to quantify EPC. Cytofluorimetry is considered the gold standard for the quantitative enumeration of EPC: it is sensitive, accurate, and reproducible [16]. All peripheral blood specimens were collected and stored in 0.34 M K3EDTA anticoagulant and all processing was completed within 2 h. Peripheral blood progenitor cells were analyzed for the expression of cell surface antigens with direct three-color analysis using fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated and allophycocyanine (APC)-conjugated monoclonal antibodies (mAbs) by flow cytometry analysis (FACSCalibur; Becton Dickinson and Co., Franklin Lakes, NJ, http://www.bd.com), as reported elsewhere [17–19]. Briefly, before staining with specific monoclonal antibodies, cells were treated with foetal calf serum for 10 min, after which samples

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COPPOLINO ET AL.: ILOPROST AND EPC IN UREMIC PATIENTS were washed with a buffer containing phosphate-buffered saline and 0.5% bovine albumin; 50 mL of peripheral blood was then incubated with 10 mL of FITC-conjugated anti-human CD45 mAb (Becton Dickinson) and with 10 mL PE-conjugated anti-human CD34 mAb (Becton Dickinson), or with 5 mL of PE-conjugated anti-human CD34 mAb and 10 mL of APC-conjugated anti-human KDR mAb (R and D Systems Inc., Minneapolis, http://www.rndsystems.com), followed by incubation at room temperature for 15 min in the dark. 7-AAD (7-Amino-Actinomycin D- VIAPROBE; BD Pharmigen., San Diego, CA) was added to determine viable cells and exclude dead cells. To avoid cell loss, no wash was performed. Flow cytometric acquisition and analysis were performed on FACS VANTAGE (Becton Dickinson); the cytometer is equipped with a 488-nm argon laser and a 635 nm red-diode laser. The threshold was set on FITC fluorescence in a dot plot of CD45FITC vs side scatter (SSC) to exclude debris and ensure that all leukocyte populations and microbeads were included. The R6 region was created in an ungated dot plot of PE versus FITC fluorescence to include the microbeads. Acquisition was stopped when a minimum of 5000 events were present in this region. Gating strategies and sample analysis allowed the measurement of the absolute count of CD34þ and dual expression of CD34þVEGFR2þ cells. CD34þ cells were defined as haematopoietic stem cells (HSC), whereas CD34þ VEGFR2þ cells were defined as endothelial progenitor cells (EPC). Data were processed using the Macintosh CELLQuest software program (Becton Dickinson). The instrument setup was optimized daily by analyzing the expression of peripheral blood lymphocytes labelled with antiCD4 APC/CD8 PE/CD3 FITC/CD45 PerCP four-color combination. One trained operator, who was unaware of the patients’ clinical status, performed all the tests throughout the study.

Endothelial Progenitor Cells

At baseline and immediately before revascularization procedure, the number of HSC and EPC did not differ between the two groups. After 6 h, HSC increased significantly compared with basal measurement in both groups (3.12 6 1.29 versus 2.12 6 1.38 in the placebo group, 4.49 6 1.28 versus 2.22 6 1.38 in the iloprost group; P < 0.05). The group with iloprost infusion attained a significant increase compared with the placebo group (P < 0.05). HSC levels reduced drastically after 24 h in both groups (2.10 6 1.42 versus 3.12 6 1.29 in the placebo group, 1.07 6 1.42 versus 4.49 6 1.28 in the iloprost group; P < 0.05; Fig. 1A). EPC cells increased significantly compared with basal level in both groups after 24 h (0.29 6 0.059 versus 0.18 6 0.028 in the placebo group, 0.59 6 0.05 versus 0.18 6 0.028 in the iloprost group; P < 0.05; Fig. 1B). In the iloprost group, the increase was considerable compared with the placebo group (P < 0.05). Finally, a close negative correlation, assessed by Pearson’s coefficient (r), was found between HSC and EPC at 24 h in the iloprost group (R ¼ –0.82, P < 0.01).

Statistical Analysis

Adhesion Molecules

The circulating endothelial progenitor cell count was calculated as the absolute number of cells per micro liter (mL). The statistical analysis of variance of groups was performed using a 1-way ANOVA followed by Fisher’s test for the comparison between variance values in the single observation times. A value of P < 0.05 was considered statistically significant. Correlation between the number of HSCs and EPCs and the concentration of adhesion molecules was assessed by means of Pearson’s coefficient (r). All data were expressed as mean 6 SD. The SPSS 11.0 statistical package (SPSS Inc., Chicago., IL) and Microsoft Excel were used for tabulation and analysis. Graphs were constructed using Prism Statistical software (version 4.00; Graphpad, San Diego, CA).

Adhesion molecules level was similar at baseline in two groups. After 6 h, sE-selectin, sICAM-1 and sVCAM-1 increased significantly from basal measurement in both groups. Levels reduced drastically after 24 h (see Fig. 2A–C); in addition, a close positive correlation, assessed by Pearson’s coefficient (r), was found between EPC and the concentrations of all adhesion molecules in both groups. Interestingly, this correlation was better in the iloprost group (Table 2).

RESULTS

The most common symptom among those who underwent revascularization was rest pain. After treatment, pain was resolved in 90% of cases. Four patients (two in each group) underwent metatarsal and phalange minor amputations in the first weeks after revascularization. During the follow-up, five patients in the placebo group showed clinical signs of restenosis (reappearance of pain). No major complications occurred. Minor complications (inguinal pseudoaneurysm (n ¼ 1), distal embolization (n ¼ 1), and perforation (n ¼ 2) were treated endovascularly. We did not find statistically significant differences between the two groups for the following plasmatic parameters: hemoglobin, hematocrit, cholesterol, creatinine, urea, calcium, phosphorus, systolic and diastolic blood pressure, HDL cholesterol, triglycerides, and glucose (Table 1).

TABLE 1 Main Characteristics of Placebo and Iloprost Group Variables

Placebo group (n ¼ 15)

Iloprost group (n ¼ 15)

Age (y) Gender (M/F) Body mass index Kg/m2 Haemoglobin, g/dL Haematocrit % Cholesterol mg/dL Creatinine mg/dL Urea mg/dL Calcium mg/dL Phosphorus mg/dL Systolic blood oressure mmHg Dyastolic blood pressure mmHg HDL cholesterol mmol/L HDL-chol ratio Triglycerides mmol/L Glucose mmol/L

60.8 6 11.2 7/8 24.6 6 4.80 14.4 6 2.44 40.3 6 4.94 203 6 60.2 5.43 6 1.26 120 6 36 9.33 6 3.26 6.81 6 2.88 144 6 27 78 6 26 1.23 6 0.02 0.30 6 0.03 0.84 6 0.08 4.53 6 0.12

58.8 6 12.6 9/6 24.3 6 8.62 14.3 6 2.12 41.3 6 4.12 204 6 30.5 6.12 6 2.44 118 6 55 8.99 6 2.55 6.35 6 2.71 139 6 31 74 6 33 1.25 6 0.06 0.29 6 0.03 0.85 6 0.08 4.48 6 0.12

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DISCUSSION Endothelial Progenitor Cells and Prostanoids

In contrast to the classical view of the endothelium as an inert lining for blood vessels, recent literature suggests a continuous turnover of endothelial cells, replaced by endotheliocytes adjacent to the lesions or mobilized from the bone marrow (HSC), which display both a hematopoietic and an endothelial differentiative potential and throughout the endothelial lineage are transformed into so-called endothelial progenitor cells (EPC). Bone marrow-derived endothelial progenitor cells (EPC) or angioblasts isolated from peripheral blood were shown to have incorporated into the sites of active angiogenesis [20]. While maturing in the circulation, HSC and EPC are characterized by the gradual expression and then disappearance of surface markers. In the present study, a search was made for two cell types: HSC and EPC [21–25]. Our findings confirm that basal number of progenitor cells in ESRD patients is significantly lower than in the control population [10]. This reduction was correlated with a subsequent

FIG. 1. Number of endothelial progenitor cells at two different maturative stages, HSC (A) and EPC (B) in Iloprost (large black-white squares) and placebo group (little black-white squares) before (T0), 6 h after (T6) and 24 h after (T24) Iloprost or placebo administration.*P < 0.05 versus basal dosage; yP < 0.05 versus placebo group; zP < 0.05 versus T6 measurement; xcorrelation with EPC: R¼ –0.82, P < 0.01.

defective neo-vascularization [26]. Angiogenesis is defined as the sprouting of blood vessels from pre-existing vascular structures by the recruitment of EPC to sites of new vessel formation. HSC and EPC were quantified in two groups of uremic patients with severe limb ischemia undergoing peripheral revascularization. The first group received perioperative iloprost administration, the second one only the intervention. In both groups, revascularization stimulated the release of progenitor cells from bone marrow. Other authors have already demonstrated an increase in stem cells about to undergo endothelial maturation following different

FIG. 2. Graphic representation of single endothelial adhesion molecules (ICAM, VCAM, and E-selectin) in Iloprost (large blackwhite squares) and placebo group (little black-white squares) before (T0), 6 h after (T6), and 24 h after (T24) Iloprost or placebo administration.*P < 0.05 versus basal dosage; yP < 0.05 versus placebo group; z P < 0.05 versus T6 measurement.

COPPOLINO ET AL.: ILOPROST AND EPC IN UREMIC PATIENTS

TABLE 2 Correlations (R) Between EPC and Adhesion Molecules at 24 Hours EPC at 24 h

sE-selectin

sICAM-1

sVCAM-1

Placebo group Iloprost group

R ¼ 0.59* R ¼ 0.81z

R ¼ 0.66y R ¼ 0.79*

R ¼ 0.71z R ¼ 0.83*

*P < 0.001. yP < 0.05. zP < 0.01.

pathological vascular stress inductor stimuli. Gill et al. demonstrated that the number of EPCs increased six hours after the trauma and that this increase persisted for 72 h after the event [27]. Wojakowski et al. [28] and Massa et al. [29] have shown that endothelial progenitor cells are mobilized within a few hours after acute myocardial infarction, the level of these cells remaining high in peripheral blood up to several days or weeks, compared with patients with stable angina, and healthy control subjects. Progenitor release had a different trend: HSC already increased 6 h after revascularization and diminished to a minimal amount after 24 h, conversely EPCs increased significantly only after 24 h. This feature could indicate that most HSC were released from bone marrow to promote neoangiogenesis and probably subsequently developed into EPC. Iloprost treatment compared with the placebo group amplified the described physiological mechanism leading to an enlarged HSC release after 6 h and an enhanced amount of EPC found after 24 h. Our data confirmed, in a human model, the recent discovery by Gensch et al. which recently demonstrated that treatment in mice with prostaglandin E1 was able to increase the number of endothelial progenitor cells circulating in the blood as well as in the bone marrow [30]. In agreement with us, North et al. demonstrated that PGE2 enhances the number of HSCs and multipotent progenitors in two vertebrate species, zebrafish and mice, and that chemicals which enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers [31]. Iloprost is known to modulate many of the mechanisms involved in inflammatory response and systemic damage following ischemia and reperfusion. The effects on platelet activation and blood clotting, the reduction of free radicals and cytokines production, and lower expression of intercellular adhesion molecules have been described in different patient populations. Prostaglandins of the E and prostacyclin series may dilate important vascular beds, directly improving local blood flow [32]. Several hypotheses can be put forward to explain the reduced number of this cell population in HD patients. One explanation could be that apoptosis is increased in

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progenitor cells. In HD patients, various cell lines and also endothelial progenitor cells show a marked tendency to undergo apoptosis. This behavior has been attributed to an increase in oxidative stress following an alteration in the balance between the production of oxidating substances and the activity of antioxidating systems. Known antiapoptotic stimuli, such as bcl-2, are reduced in patients on HD [33]. The presence of a mechanical endothelial damage in patients on ESRD was recently further confirmed by the circulating endothelial microparticles assay. Microparticles, membrane fragments, are shed by damaged or activated cells, mainly platelets and endothelial cells. In HD patients, circulating endothelial microparticles have been correlated with an impaired vascular function [34]. Importantly, EPC isolated from PGE1-treated mice were characterized by a reduced rate of apoptotic cell death. The inhibition of progenitor cell apoptosis could represent a novel mechanism of PGE1, and further studies are warranted to characterize the molecular pathway mediating this effect in more detail [27, 30, 35]. Adhesion Molecules, Endothelial Activation, and Prostanoids

In parallel with cell quantification, we measured the concentration of the three main endothelial-expressed cell adhesion molecules associated with leukocyte activation to understand if progenitor mobilization from bone marrow went together with the release of substances linked to active vascular development. As demonstrated by both in vivo and in vitro studies, in response to ipoxia and reoxygenation, endothelial cells produce chemotactic cytokines, such as interleukin (IL)-1, IL-8, and tumor necrosis factor (TNF)-a, and up-regulate their surface expression of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin, thus promoting the adhesion and the activation of leukocytes [36]. Recently Fox et al. demonstrated that thermal injury induced a rapid rise in EPCs that was proportional to the extent of the burn and significantly correlated with levels of angiogenic cytokines [37]. The adhesion of monocytic lineage cells on the vascular wall, which depends on their interaction with endothelial cells, is mediated by cell adhesion molecules, including ICAM-1, E-selectin, and VCAM-1. E-selectin is a member of the selectin family of cell adhesion molecules, and the intercellular cell adhesion molecule-1 (ICAM-1) and vascular endothelial cell adhesion molecule-1 (VCAM-1) belong to an immunoglobulin super family. The surface expression of these cell adhesion molecules is associated with their shedding into the peripheral circulation [38, 39]. Our results confirmed that revascularization induces a rapid increase in endothelial adhesion molecules. The concentration

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of adhesion molecules was increased in the iloprost group remaining at higher levels until 24 h and correlated directly with progenitor cell amount. Our findings appear to contradict those reported by other authors such as Mazzone et al. who showed that iloprost therapy reduces, in vivo, the inflammatory endothelial cell activation, and achieves a significant decrease of circulating adhesion molecules [40]. However, a significant direct inhibitory effect on these endothelial cells was seen only for concentrations that were about a 1000 times higher than those reached under therapeutic conditions. Furthermore, the decrease in adhesion molecules was observed after 72 h of treatment. In conclusion, adhesion molecule levels may be considered a marker of endothelial activation and remodeling. There is a close contact between bone marrow and vessels mediated by swift cytokine release in the blood circulation. The recruitment of EPCs and the release of adhesion molecules are severely impaired in uremic patients, thus suggesting new mechanisms underlying the increased cardiovascular risk in this population.

12. Rowlands TE, Gough MJ, Homer-Vanniasinkam S. Do prostaglandins have a salutary role in skeletal muscle ischemia-reperfusion injury? Eur J Vasc Endovasc Surg 1999;18:439.

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