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Original Article

Therapeutic Effects of Autologous Bone Marrow Cells and Metabolic Intervention in the Ischemic Hindlimb of Spontaneously Hypertensive Rats Involve Reduced Cell Senescence and CXCR4/Akt/eNOS Pathways

Nigris, Filomena de PhD*; Balestrieri, Maria Luisa PhD*; Williams-Ignarro, Sharon MD; D'Armiento, Francesco P MD; Lerman, Lilach O MD, PhD§; Byrns, Russell PhD; Crimi, Ettore MD; Palagiano, Antonio MD*; Fatigati, Gennaro BS*; Ignarro, Louis J PhD; Napoli, Claudio MD, PhD, MBE, FACA*

Author Information
Journal of Cardiovascular Pharmacology: October 2007 - Volume 50 - Issue 4 - p 424-433
doi: 10.1097/FJC.0b013e31812564e4
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Abstract

INTRODUCTION

Peripheral arterial disease (PAD) most frequently presents with lower-limb exertional pain (ie, intermittent claudication), which may worsen to rest pain and/or ischemic ulceration.1 The prevalence of PAD increases with age and has been reported to be about 33.8% in persons aged 70 to 82 years.1 The management of patients with early stages of PAD consists of lifestyle modifications and pharmacotherapy to reduce risk factors. Hypertension represents a modifiable risk factor predisposing to PAD, with an odds ratio of 2.2 in men and 2.8 in women, conferring a very high cardiovascular risk.1 Unfortunately, about 30% of patients with critical-limb ischemia cannot be treated by a surgical or endovascular revascularization, and the only option for them is often amputation, especially in the presence of severe hypertension.1 Thus, there is a great need for alternative treatment to stimulate collateral artery growth and revascularization.2 Bone marrow consists of multiple cell populations, including endothelial progenitor cells (EPC), which can differentiate into endothelial cells and release several angiogenic factors.3-5 Through specific interactions with the CXCR4 receptor, the recruitment of EPC from bone marrow to peripheral blood leads to the formation of new vessels at the ischemic sites.6 Functional impairment of EPC associated with hypertension could constitute a limitation to their ability to repair. Local intramuscular autologous bone marrow cell (BMC) therapy4,7-9 or BMCs injected intravenously10-12 have been demonstrated to induce therapeutic angiogenesis in experimental ischemic limb models. The efficacy and safety of implantation in the ischemic muscle of autologous BMCs have been studied in patients with PAD.13,14 Specifically, using the same approach employed in some preclinical studies,4,7-9 the gastrocnemius muscle of the ischemic limb was injected with BMCs. Blood flow and pain-free walking time in legs injected with BMCs improved significantly.

Because tissue ischemia is associated with exceeding generation of oxygen radicals,15,16 negative effects attributable to perturbed shear stress,17,18 and microcirculatory damage,19,20 metabolic intervention with antioxidants and L-arginine, the precursor of NO, may promote beneficial effects in ischemia-induced angiogenesis beyond that provided by BMC therapy alone. Overall, the concomitant presence of hypertension can exacerbate oxidation-sensitive mechanisms activated by tissue ischemia.20,21 In hypertensive patients, the reduction of extracellular superoxide dismutase activity is partially responsible for the increased oxidative stress, as reflected by increased plasma nitrotyrosine and 8-isoprostanes.22-24 Moreover, there is a reduction of angiogenesis in the presence of hypertension.25

A plethora of studies have shown beneficial effects of antioxidants and L-arginine in vascular damage.16,18,26-30 We recently have demonstrated beneficial effects of autologous BMC therapy together with metabolic treatment in normotensive normocholesterolemic,11 hypercholesterolemic,12 or diabetic31 murine ischemic hindlimbs. To date, the impact of concurrent severe hypertension during experimental ischemic hindlimb is still unknown, even though the majority of patients with PAD also have elevated levels of blood pressure.1 Therefore, the goal of the present study was to investigate the effectiveness of intravenous therapy with autologous BMCs alone, or in combination with metabolic cotreatment afforded by antioxidants and L-arginine, in the hypertensive rat ischemic hindlimb model.

METHODS

Experimental Protocol and Hindlimb Ischemia Model

This study was conducted according to the Guidelines for Animal Experiments of the American Heart Association and in accordance with guidelines published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). Quality standards of the laboratories at the University of Naples (Italy) are in accordance with rules established from the Italian Ministery of Health and the European College of Laboratory Animal Medicine, and the laboratories at the University of California at Los Angeles, Calif, and the Mayo Clinic at Rochester, Minn are in accordance with standards of the Association for Assessment and Accreditation of Laboratory Animal Care.

The neovascularization capacity of BMCs alone or in cotreatment with 0.8% vitamin E added to the chow, 0.05% vitamin C added to the drinking water, and 5% of L-arginine in drinking water11,18,29,30 was investigated in a rat model of hindlimb ischemia32 in 4-week-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) (Charles River).30 Blood pressure was measured once a week using a tail-cuff probe connected to a pressure monitor.33 The hindlimb ischemic was induced, as described in detail elsewhere.11 Briefly, the proximal portion of the femoral artery, including the superficial and deep branches and the distal portion of the saphenous artery, were ligated with 7-0 silk suture. All arterial branches between the ligation were obliterated using an electric coagulator. The overlying skin was closed with three surgical staples.

SHR and WKY animals were each randomized into four groups, maintaining the operators in a blinded fashion regarding treatments: (a) hindlimb ischemia treatment alone [controls, n = 12; the ischemic hindlimbs of these rats were injected with polyphosphate saline buffer (PBS) only]; (b) hindlimb ischemia and subsequent BMC therapy alone (n = 12); (c) hindlimb ischemia and BMC therapy plus metabolic intervention (n = 12); or (d) hindlimb ischemia and metabolic intervention alone (n = 10). After 24 hours of ischemia, 2 × 107 autologous BMCs diluted in PBS were injected intravenously. Here, we used an original technique for obtaining BMCs by aspiration from the femur of living rats, originally proposed by Verlinden et al34 and modified as previously described.11 After 1 day and 2 weeks, at the same hour interval (between 2:00 and 4:00 pm), rats were placed on a heating plate at 37°C to minimize temperature variation and ambient light. Excess hairs were removed from the limb, and blood flow was calculated in the foot and expressed as a ratio of ischemic to nonischemic leg by use of a laser Doppler blood-flow meter.11 The core temperature of the animals was kept stable during measurements.11 Histological and blood-flow analysis were performed in a blinded fashion regarding treatments.

Histology and Morphometric Analysis

Limb interstitial fibrosis in the semimembranous and adductor muscles was morphometrically assessed by using the Azan-Mallory staining, quantified by Qwin Leica Imaging System (Cambridge, UK), and expressed as a percentage of the total muscle section, as described elsewhere.11 Tissue vascularization was determined in 5-μM frozen sections of the adductor and semimembranous muscles from the ischemic and the nonischemic limbs by using a monoclonal antibody directed against CD31 (20 μL/mL, JC/70A, Dako, Carpinteria, Calif).11 Capillary densities were then calculated in randomly chosen fields of a definite area and expressed as the number of capillaries per myocyte relative to the individual nonischemic limb.11 T-lymphocytes and macrophages were detected by immunostaining with anti-CD3e monoclonal antibody (1:50 dilution; Santa Cruz Biotech, Santa Cruz, Calif) and the anti-F4/80 monoclonal antibody (1:500 dilution; Accurate Scientific, Westbury, NY), respectively.11,18,30 Proliferation-associated Ki67 (a proliferation-associated marker) was detected in CD31 immunostaining-positive cells by using the Ki-67 mouse IgG1 (1:50 dilution; Dako).11

Evaluation of Oxidative Stress and NOx Levels

Blood was collected at the time of sacrifice into Eppendorf tubes containing 1 mM Na2EDTA. Isoprostane 8-epi-PGF2a purified from plasma samples was measured using a commercially available immunoassay (Cayman Chemical, Ann Arbor, Mich).11,30 NO was measured as nitrite and nitrate (NOx), which are stable metabolites of NO, using the Griess reagent according to the manufacturer's instructions (Calbiochem).11,30 Concentration (mM) was calculated according to a standard curve calibration.

In Situ Detection of Apoptotic Cell Death

In situ detection of apoptotic cells, using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling method of fragmented DNA, was performed on cryostat section, as described previously.35

Isolation and Culture of Bone Marrow-Derived Endothelial Progenitor Cells

At sacrifice, in a subset of additional animals, bone marrow cells were obtained from the tibias and femurs of SHR (n = 5) and age-matched WKY rats (n = 5). Bone marrow-derived mononuclear cells (BM-MNCs) were isolated by density-gradient centrifugation with Histopaque 1083 (Sigma Chemical Co., St. Louis, Mo) and cultured in complete endothelial basal medium 2, as described previously.36

Senescence-Associated b-Galactosidase Activity Assay in BM-EPC

Senescence-associated b-galactosidase (SA-b-Gal) activity was measured as described previously.37 Briefly, bone marrow-derived endothelial progenitor cells (BM-EPCs) were fixed in 2% paraformaldehyde, incubated with fresh SA-b-Gal stain solution, and counterstained with 4′,6-diamino-phenylindole (0.2 μg/mL).

Telomeric Repeat Amplification Protocol Assay in BM-EPC

Quantitative analysis of telomerase activity, telomeric repeat amplification protocol assay, was performed using a TeloTAGGG PCR ELISAPLUS kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocol, as described previously.38

Western Blot Analysis

Total extracts from BM-EPC and arterial tissue11,18 (20-50 μg per lane) were loaded onto SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes, as described previously.18 Western blots were performed, using antibodies directed against phospho-Ak-Ser 473 and anti-Akt (1:50 dilution; Cell Signalling Technology, Inc., Beverly, Mass), anti-eNOS (1:500 dilution; Santa Cruz Biotechnology; also in arterial tissue homogenates), and antitubulin (GTU-88, 1:10.000 dilution; Sigma). BM-EPC detergent-insoluble membranes and detergent-soluble membranes were prepared as described previously36 and immunoblotted with CXCR4 antibodies (1:500 dilution; BD Biosciences Pharmingen, San Jose, Calif). Enhanced chemiluminescence was performed according to the instructions of the manufacturer (Amersham). Densitometry analysis was performed by LKB analyzer.18

Statistical Analysis

Histological analysis was performed in a blinded fashion. Comparisons among groups were analyzed by use of ANOVA. Post hoc range tests and pairwise multiple comparisons were performed with the t test (two sided) with Bonferroni adjustment. Probability values of P < 0.05 were considered statistically significant.

RESULTS

Blood Pressure and Evaluation of Oxidative Stress, NOx Production, and Arterial eNOS Expression

As expected, systolic blood pressure was significantly higher in SHR groups (mean, 158 ± 9.4 mm Hg) than in WKY animals (mean, 102 ± 6.8 mm Hg; P < 0.001 versus SHR).

Under the present experimental conditions, untreated SHR rats (n = 12; rats that were not subjected to hindlimb ischemia) had 143 ± 55 pg/mL of isoprostanes and 20.4 ± 7.6 μM NOx, whereas untreated WKY rats had 126 ± 47 pg/mL of isoprostanes and 18.6 ± 8.2 μM NOx.

Plasma isoprostane levels in WKY ischemic hindlimb rats were significantly lower than SHR rats (P < 0.05). Metabolic intervention alone or cotreatment with BMC therapy and metabolic intervention resulted in a significant reduction of plasma isoprostane levels and an improvement of NO bioactivity in both SHR and WKY rats (P < 0.05) (Table 1). Moreover, metabolic treatment (MT) alone or in combination with BMC therapy significantly increased hindlimb arterial expression of eNOS in SHR and WKY rats (P < 0.05 versus respective group of ischemic hindlimb; Table 1). The eNOS-expression levels of untreated rats were 5.6 ± 2.0 in SHR rats and 5.2 ± 2.2 in WKY rats. The expression of iNOS did not change significantly among groups in SHR or WKY rats (not shown).

TABLE 1
TABLE 1:
Plasma Evaluation of Oxidative Stress, Nitrite and Nitrate (NOx) Production, and Arterial Endothelial Nitric Oxide Synthase (eNOS) Expression in the Various Study Groups of Spontaneously Hypertensive (SHR) and Wistar-Kyoto (WKY) Rats

Blood-Flow Recovery in Ischemic Hindlimbs

Blood-flow response to limb ischemia was significantly impaired in SHR rats in comparison with WKY rats (P < 0.05) (Fig. 1A and B). Under the defined experimental conditions at day 2 and after 2 weeks, a significant increase in blood flow after the injection of BMCs alone was observed in SHR and WKY rats compared with PBS infusion (Fig. 1A and B). Combination of BMC therapy with MT determined a noticeable improvement of blood flow in SHR and WKY rats compared with ischemic hindlimb (P < 0.01 by ANOVA) and BMC therapy (P < 0.05). These effects were more pronounced in WKY rats compared with SHR rats (P < 0.05). These data indicate that the restoration of perfusion in the ischemic hindlimbs was accelerated by intravenous BMC treatment alone and even further by combined therapeutic approach. The recovery of blood flow in the ischemic hindlimbs was not accelerated by MT alone.

FIGURE 1
FIGURE 1:
Evaluation of blood flow, capillary density, and Ki67 proliferative marker in the study groups of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Animals were divided into four groups: ischemia hindlimb treatment alone (IH, n = 12), ischemia hindlimb and subsequent bone marrow cell (BMC) therapy alone (IH + BMC, n = 12), ischemia hindlimb and BMC therapy plus metabolic intervention (IH + BMC + MT, n = 12), and ischemia hindlimb with metabolic intervention alone (IH + MT, n = 10). After treatment, (A) laser Doppler-derived blood flow (ischemic/nonischemic ratio) at day 2 and (B) at day 15, (C) capillary-density analysis (mean of number of positive cells per squared millimeter, counted in 10 different fields), and (D) percentage of Ki67-positive cells in CD31-positive cells were determined in a blinded fashion regarding treatments. No evidence for vascular rarefaction in the peripheral skeletal muscle before ligation was observed across groups, and the immediate postligation values were similar. Data are absolute values at the follow-up time point and are expressed as mean ± SD of single measurement in each rat of the study group. A, B: *P < 0.05 versus IH; **P < 0.01 versus IH; §P < 0.05 versus IH + BMC; #P < 0.05 versus SHR. C: *P < 0.05 versus IH; **P < 0.01 versus IH; §P < 0.05 versus IH + BMC; #P < 0.05 versus SHR, ‡P < 0.01 versus SHR. D: *P < 0.01 versus IH; §P < 0.05 versus IH + BMC; #P < 0.05 versus SHR.

Histological Evaluation of Angiogenesis, Inflammatory Cells, and Apoptosis

By using an antibody directed against CD31, histochemisty analysis has shown that the capillary density in the muscles of SHR was significantly decreased in comparison with WKY rats (P < 0.01 by ANOVA) and that the capillary densities of the SHR and WKY ischemic hindlimbs were markedly increased after the intravenous infusion of BMCs (P < 0.05). This effect was significantly amplified by infusion with BMCs combined with MT (P < 0.01 by ANOVA versus ischemic hindlimb; P < 0.05 versus BMC therapy; Fig. 1C). Analysis of the percentage of Ki67-positive cells, a marker of proliferation, shows that it persists significantly in both groups after BMC infusion alone or in combination with MT (P < 0.01 by ANOVA; Fig. 1D). Combination of BMCs with MT significantly improved cell proliferation compared with BMCs alone (P < 0.05). Finally, the effects of BMCs or BMCs combined with MT on capillary density and cell proliferation were consistently higher in WKY rats compared with SHR rats (P < 0.05). Neither capillary density nor cell proliferation was improved by MT alone.

We also have investigated interstitial fibrosis and infiltration of inflammatory cells. Macrophages and interstitial fibrosis were significantly lower in the WKY rat control group compared with SHR control rats (P < 0.05) (Table 2). In WKY rats, BMC therapy or a combination of BMCs with MT determined a consistent reduction of inflammatory cell number (P < 0.05) and interstitial fibrosis (P < 0.01 by ANOVA) compared with PBS-perfused ischemic hindlimbs. Reduction of interstitial fibrosis by BMC therapy alone or in combination with MT was significantly higher in WKY rats compared with SHR rats (P < 0.05). In both SHR and WKY rats, MT alone had a reducing effect only on interstitial fibrosis (P < 0.05 versus ischemic hindlimbs and P < 0.05 for WKY rats versus SHR rats).

TABLE 2
TABLE 2:
Computer-Assisted Morphometric Analysis of the Number of Inflammatory Cells (Number of Cells Per Squared Millimeter) Infiltrating Into the Ischemic Muscle, the Degree (%) of Muscular Interstitial Fibrosis, and Apoptosis (% of TdT-Positive Cells) in the Various Study Groups of Spontaneously Hypertensive (SHR) and Wistar-Kyoto (WKY) Rats

In SHR rats, the number of T cells was not significantly different among groups (Table 2). However, we have observed that infiltration of macrophages in ischemic muscle was significantly reduced in the group receiving cotreatment of BMCs and metabolic intervention (Table 2). Overall, a significant reduction of interstitial fibrosis was observed after BMC therapy alone (P < 0.01 by ANOVA), BMCs in combination with MT (P < 0.01), or MT alone (P < 0.05) compared with PBS infusion (Table 2). MT alone in SHR rats significantly reduced only interstitial fibrosis.

When apoptosis was assayed in WKY rats, we found that the percentage of apoptotic cells detected by TdT was significantly increased in ischemic hindlimbs versus nonischemic tissue (3.7 ± 2.6 versus 1.05 ± 0.4, P < 0.05). BMC treatment did not significantly alter the percentage of TdT-positive cells (Table 2). In contrast, MT significantly decreased the percentage of TdT-positive cells (P < 0.05). The same was true for SHR. Indeed, the percentage of apoptotic cells detected by TdT increased in ischemic hindlimbs versus nonischemic tissue (4.4 ± 3.1 versus 1.26 ± 0.48, P < 0.05). Moreover, the percentage of TdT-positive cells significantly decreased only after MT (P < 0.05) and not BMC infusion (Table 2). Both in WKY and SHR rats, the values of apoptotic cells detected after BMC therapy in combination with MT were comparable with those observed after MT alone.

Evaluation of BM-EPC Number and Functional Activity

When BM-MNCs isolated from SHR and WKY were cultured, the number of adherent BM-EPCs was significantly lower in SHR compared with WKY rats (P < 0.05) (Fig. 2A).

FIGURE 2
FIGURE 2:
Effect of bone marrow cell (BMC) infusion and metabolic treatment (MT) on bone marrow-derived endothelial progenitor cell (BM-EPC) number and functional activity. Animals were divided into four groups: ischemia hindlimb treatment alone (IH, n = 12), ischemia hindlimb and subsequent BMC therapy alone (IH + BMC, n = 12), ischemia hindlimb and BMC therapy plus metabolic intervention (IH + BMC + MT, n = 12), and ischemia hindlimb with metabolic intervention alone (IH + MT, n = 10). BM-EPC were isolated from each group of spontaneously hypertensive (SHR, n = 5) and Wistar-Kyoto (WKY rats, n = 5) at the sacrifice, and (A) cell number, (B) cellular senescence, and (C) telomerase activity were determined. Data are expressed as means ± SD. A: *P < 0.05 versus IH; #P < 0.05 versus respective group in SHR; ¶P < 0.05 versus IH group in WKY. B: *P < 0.05 versus IH; §P < 0.01 versus respective group in SHR; ¶P < 0.05 versus IH group in WKY; C: *P < 0.05 versus IH; **P < 0.01 versus IH; §P < 0.01 versus respective group in SHR; ¶P < 0.05 versus IH group in WKY.

In SHR and WKY rats, BMC treatment or MT alone did not significantly alter the number of DiLDL/lectin-positive cells compared with the ischemic hindlimbs in their respective groups (Fig. 2A). In contrast, BMCs in combination with MT determined a significant increase in DiLDL/lectin-positive BM-EPC (P < 0.05). Moreover, we observed that the rate of cellular senescence was significantly lower in WKY rats than in SHR (P < 0.01); again, only BMCs in combination with MT significantly inhibited the percentage of SA-b-Gal-positive cells (P < 0.05) (Fig. 2B). The number of senescent cells was inversely correlated to increased capillary density in the IH+BMC+MT group in SHR (R = −0.47, P < 0.01). When we measured the telomerase activity, we observed an activity significantly higher in WKY compared with SHR rats (P < 0.01) (Fig. 2C). In WKY rats, the telomerase activity was increased only by BMCs in combination with MT (P < 0.05), whereas in SHR rats it was significantly increased either by BMCs (P < 0.05) alone or in combination with MT (P < 0.01). Also, telomerase activity was positively correlated to increased capillary density in the IH-BMC-MT group of SHR (R = 0.48, P < 0.05).

Effect of BMCs and MT on Akt Phosphorylation and eNOS Expression in BM-EPC

Akt and eNOS play an essential role in regulating EPC number38,39 and recruitment.40 When we examined the possible involvement of the PI3-K/Akt and eNOS pathways in the mechanism(s) regulating the BM-EPC functional activity, we found that p-Akt was significantly higher in WKY rats compared with their respective values in the SHR groups (P < 0.05) (Fig. 3A). Treatment with BMCs alone or in combination with MT determined a significant increase in Akt phosphorylation in BM-EPC from SHR and WKY rats in comparison with their respective values in control ischemic hindlimbs (P < 0.05).

FIGURE 3
FIGURE 3:
Evaluation of Akt phosphorylation and endothelial nitric oxide synthase (eNOS) expression in bone marrow-derived endothelial progenitor cells (BM-EPC). Total BM-EPC extracts from control animals (n = 5) (IH, lane A) and from animals treated with bone marrow cells (BMCs) alone (n = 5) (IH + BMC, lane B), BMCs and metabolic treatment (n = 5) (IH + BMC + MT, lane C), or metabolic treatment alone (n = 5) (IH + MT, lane D) were used to determine phosphorylation of (A) phosphorylated form of Akt, (B) Akt, and (D) eNOS-expression levels with specific antibodies as described in the Methods section. C, Akt/pAkt ratio was calculated on the basis of arbitrary unit values of Akt and phosphorylated Akt expression. Data are expressed as means ± SD. *P < 0.05 versus respective values in SHR groups; #P < 0.05 versus respective value in the IH group.

A significant decrease in Akt-expression levels was observed only in BM-EPC from WKY rats (P < 0.05 versus respective value in the control ischemic hindlimb; Fig. 3B).

When the Akt/p-Akt ratio was determined, a significant decrease was observed in BM-EPC from SHR and WKY rats after treatment with BMCs alone or in combination with MT (P < 0.05) (Fig. 3C).

Similar to p-Akt, eNOS expression in BM-EPC from WKY rats treated with BMCs, BMCs and MT, or MT alone was significantly higher compared with their respective values in the SHR groups (P < 0.05) (Fig. 3D). Moreover, only BMCs alone or in combination with MT determined a significant increase in BM-EPC eNOS expression in SHR and WKY rats in comparison with their respective values in the ischemic hindlimb group (P < 0.05) (Fig. 3D).

Effect of BMCs and MT on CXCR4 Expression in BM-EPC

The cell-surface expression of the CXCR4 chemokine receptor is one of the major determinants in the homing capacity of BMCs and EPC.41 In BM-EPC detergent-insoluble membranes (essentially made of rafts and caveolae; Fig. 4A), we found reduced levels of CXCR4 expression in SHR compared with WKY animals. These values were significantly higher in the soluble membranes of BM-EPC (Fig. 4B). Finally, BM-EPC from SHR and WKY animals receiving BMC treatment alone and in combination with MT had further increments of CXCR4-expression levels compared with their respective values in the ischemic hindlimb groups. CXCR4 expression in soluble membranes was positively correlated to increased capillary density in the IH + BMC + MT and IH + BMC groups of SHR (R = 0.64 and 0.53, P < 0.01, respectively).

FIGURE 4
FIGURE 4:
Effect of bone marrow cells (BMCs) and metabolic treatment (MT) on bone marrow-derived endothelial progenitor cell (BM-EPC) CXCR4-expression levels in BM-EPCs. Western blot analysis of (A) detergent-insoluble membranes and (B) detergent-soluble membranes of BM-EPC isolated from control rats (n = 5) (IH), rats treated with BMCs alone (n = 5) (IH + BMC), BMCs and metabolic treatment (n = 5) (IH + BMC + MT), or metabolic treatment alone (n = 5) (IH + MT) was performed with specific CXCR4 antibodies as described in the Methods section. Data are expressed as means ± SD. *P < 0.05 versus respective values in SHR groups; **P < 0.01 versus respective values in SHR groups; #P < 0.05 versus respective value in the IH group; §P < 0.01 versus respective value in the IH group.

DISCUSSION

We have shown that intravenous autologous BMCs alone or with metabolic intervention significantly ameliorated ischemia-induced angiogenesis in the hypertensive rat hindlimb by increasing blood flow, capillary density, and Ki67 proliferative marker, and by ameliorating BM-EPC functional activity (cell senescence and AKT/eNOS pathways) and homing capacity (via CXCR4).

This is a major advance regarding data already demonstrated in the healthy normotensive and normocholesterolemic,11 hypercholesterolemic,12 and diabetic31 murine models. Beneficial effects were associated with reduced macrophage infiltration in ischemic muscle and interstitial fibrosis as well as reduced systemic oxidative stress and increased NO/eNOS bioactivity in the presence of metabolic treatment. Specifically, metabolic treatment alone was effective in reducing systemic oxidative stress and increasing NO/eNOS bioactivity, whereas BMC therapy alone had no effects. By contrast, BMC treatment was more effective than metabolic treatment alone in improving ischemia-induced angiogenesis. Thus, it is conceivable that in this study the reduced oxidative stress and improved NO/eNOS bioactivity partially contributed to the improved ischemia-induced angiogenesis. Consistent with previous findings,27 angiogenesis in response to hindlimb ischemia was significantly attenuated during hypertension. In SHR, abnormalities in blood vessel growth and rarefaction of capillaries in the microcirculation have been documented.22,32,42-44 Damaged endothelial function in SHR45 and in hypertensive patients46 may contribute to a reduced angiogenesis in SHR compared with WKY. Accordingly, we have observed a significant defect in capillary density in SHR rats. Intravenous BMC infusion significantly ameliorated capillary density of SHR and WKY ischemic hindlimbs, and this effect was amplified by metabolic cotreatment.

The reduction of oxidative stress and the improvement of NO/eNOS bioactivity elicited by antioxidants and L-arginine may have important, specific protective effects during hindlimb ischemia. Moreover, there is increasing evidence of multiple redox coregulator events affecting the transcription machinery in the arterial wall.47 In addition, because there are obvious signs of inflammation in the ischemic hindlimb,11,48,49 some of the beneficial effects seen in the present study can be attributed to the antinflammatory action (reduction of macrophage infiltration) elicited by antioxidants and L-arginine. Indeed, many signal-transduction pathways involved in hypertensive vascular inflammation and tissue injury20,21,25 are reduced by these compounds.

Because the major goal of the present study was to investigate recovery of blood flow and neoangiogenesis in hypertension, our work underscores the detailed role of the inflammatory pathways in the modulation of ischemia-induced angiogenesis and interstitial fibrosis. Nevertheless, our results open the way for therapeutic strategies aimed at decreasing oxidation-sensitive mechanisms and vascular inflammation together with activation of the active angiogenic process (sustained also by increased activity of Ki67 proliferative marker) in ischemic tissues during hypertension elicited by autologous BMCs and metabolic intervention.

BMC-based therapies have been used for several years to treat certain hematological diseases. In contrast to some previous studies, we did not select BMC subpopulations to promote higher rates of therapeutic neoangiogenesis.3,4,7-10 Following the successful protocol used previously,11,12,31 we have aspirated autologous BMCs in living hypertensive animals and intravenously reinjected the maximum dose of available cells alone or in cotreatment with a vasculoprotective metabolic treatment. The mechanism(s) by which BMC infusion led to a rescue of blood flow in ischemic hindlimbs is still unknown. Both apoptosis and necrosis are responsible for cell death after ischemia.50 However, we did not observe any significant reduction of apoptotic cells after BMC infusion; this indicates a lack of an antiapoptotic effect from this treatment alone. The percentage of TdT-positive cells significantly decreased only after metabolic treatment, both in SHR and WKY, suggesting the beneficial role that the reduction of oxidative stress elicited by metabolic treatment may have during hindlimb ischemia. It has been established that BMCs may function as supporting cells51 that can differentiate into endothelial cells52 after infusion with both macrophage colony-stimulating factor and granulocyte colony-stimulating factor, resulting in early recovery of blood flow in the ischemic limbs of mice.52 An important question with respect to the mechanism(s) by which BMC infusion led to a rescue of blood flow in ischemic hindlimbs is whether it ameliorates BM-EPC functional activity and homing. Imanishi et al38 have shown that BM-EPC derived from SHR showed lower differentiation and a higher rate of cellular senescence than that of BM-EPC from WKY. Importantly, we found that amelioration of BM-EPC functional activities is one of the mechanisms for the improved angiogenesis by the BMC treatment. Indeed, although the number and functional activity of BM-EPC from SHR rats were significantly lower than those of BM-EPC from WKY rats, BMC therapy alone was significantly effective in ameliorating cell number and reducing BM-EPC senescence only in WKY rats. Instead, BMC in combination with metabolic cotreatment consistently ameliorated cell number and reduced BM-EPC senescence, through telomerase activation, both in SHR and WKY rats.

The Akt pathway is required for statin-mediated increase of EPC number, suggesting an essential role in regulating hematopoietic progenitor-cell differentiation.39 Moreover, defective mobilization of BM-EPC has been shown to contribute to the impairment of ischemia-induced neovascularization in NOS3−/− mice.40 The concomitant increase of Akt phosphorylation and eNOS-expression levels in BM-EPC from animals treated with BMCs and metabolic intervention indicates that the Akt and eNOS pathway might be involved in the mechanism(s) regulating BM-EPC functional activity. Sbaa et al36 have shown a critical role of the structural protein caveolin for EPC mobilization from BM and the consecutive revascularization of ischemic tissues. CXCR4-receptor redistribution to rafts (at the leading edge of migrating cells) is followed by EPC internalization.36 Here, when we isolated detergent-insoluble membranes (essentially made of rafts and caveolae) from the rest of the cell extracts, we found reduced levels of CXCR4 expression in SHR compared with WKY animals, with values significantly higher in the soluble membranes compared with detergent-insoluble membranes. Interestingly, BMC treatment alone and in combination with metabolic treatment determined a consistent increase of CXCR4-expression levels in BM-EPC from SHR and WKY animals compared with their respective ischemic hindlimb control groups.

Some studies have suggested high neoangiogenic activity of mesenchymal cells10,53 or low-dose CD34+KDR+ cells.54 Therefore, we cannot exclude potential therapeutic effects of unknown BMC subpopulations that would be excluded a priori when selecting a unique population. Additional studies are in progress to address this important pathophysiologic issue.

CONCLUSION

We have shown that autologous BMC therapy, alone or with metabolic intervention, significantly ameliorated ischemia-induced angiogenesis in the hypertensive rat hindlimb. Beneficial effects were associated with increased blood flow, capillary density, and Ki67 proliferative marker, and ameliorated BM-EPC functional activity and homing capacity. Moreover, reduced macrophage infiltration in ischemic muscle and interstitial fibrosis, reduced systemic oxidative stress, and increased NO/eNOS bioactivity in the presence of metabolic treatment were observed. Because data from experimental models cannot be directly extrapolated to humans, a series of human studies need to be conducted to address safety and efficacy. This is particularly important for PAD, which is known to be progressive and rapid in hypertensive patients.1 In contrast, when considering mobilization of EPCs, the potential problems of inducing therapeutic angiogenesis by direct administration or gene transfer of angiogenic growth factors include a risk of neoplasia and plaque angiogenesis.2,4,55,56 It is critically important to confirm the safety of inducing therapeutic angiogenesis in patients, especially when considering long-term effects. Thus, human studies should validate the hypothesis that autologous BMCs, together with metabolic intervention, could be an effective clinical treatment for PAD in the concomitant presence of hypertension.

Funding Sources

This work was supported in part by National Research Funds to the II University of Naples, (C.N.) and international research grants (L.J.I. and L.O.L).

Conflict of Interest Disclosures

The authors have no conflicts of interest in connection with this article.

REFERENCES

1. Aronow WS. Management of peripheral arterial disease. Cardiol Rev. 2005;13:61-68.
2. Cao Y, Hong A, Schulten H, et al. Update on therapeutic neovascularization. Cardiovasc Res. 2005;65:639-648.
3. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964-967.
4. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003;9:702-712.
5. Asahara T, Kawamoto A. Endothelial progenitor cells for postnatal vasculogenesis. Am J Physiol Cell Physiol. 2004;287:C572-C579.
6. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines. Circulation. 2004;109:2487-2491.
7. Hamano K, Li TS, Kobayashi T, et al. The induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method. Surgery. 2001;130:44-54.
8. Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation. 2001;103:897-903.
9. Iwase T, Nagaya N, Fujii T, et al. Adrenomedullin enhances angiogenic potency of bone marrow transplantation in a rat model of hindlimb ischemia. Circulation. 2005;111:356-362.
10. Miranville A, Heeschen C, Sengenès C, et al. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation. 2004;110:349-355.
11. Napoli C, Williams-Ignarro S, de Nigris F, et al. Beneficial effects of concurrent autologous bone marrow cell therapy and metabolic intervention in ischemia-induced angiogenesis in the mouse hindlimb. Proc Natl Acad Sci USA. 2005;102:17202-17206.
12. de Nigris F, Williams-Ignarro S, Sica V, et al. Therapeutical effects of concurrent autologous bone marrow cell infusion and metabolic intervention in ischemia-induced angiogenesis in the hypercholesterolemic mouse hindlimb. Int J Cardiol. 2007;117:238-243.
13. Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic Angiogenesis using Cell Transplantation (TACT) Study Investigators. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002;360:427-435.
14. Higashi Y, Kimura M, Hara K, et al. Autologous bone-marrow mononuclear cell implantation improves endothelium-dependent vasodilation in patients with limb ischemia. Circulation. 2004;109:1215-1218.
15. Becker LB. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res. 2004;61:461-470.
16. de Nigris F, Lerman A, Ignarro LJ, et al. Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis. Trends Mol Med. 2003;9:351-359.
17. Fisher AB, Chien S, Barakat AI, et al. Endothelial cellular response to altered shear stress. Am J Physiol Lung Cell Mol Physiol. 2001;281:L529-L533.
18. de Nigris F, Lerman LO, Ignarro SW, et al. Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress. Proc Natl Acad Sci USA. 2003;100:1420-1425.
19. Tritto I, Ambrosio G. Spotlight on microcirculation: an update. Cardiovasc Res. 1999;42:600-606.
20. Cifuentes ME, Pagano PJ. Targeting reactive oxygen species in hypertension. Curr Opin Nephrol Hypertens. 2006;15:179-186.
21. Schulman IH, Zhou MS, Raij L. Nitric oxide, angiotensin II, and reactive oxygen species in hypertension and atherogenesis. Curr Hypertens Rep. 2005;7:61-67.
22. Jin L, Beswick RA, Yamamoto T, et al. Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats. J Physiol Pharmacol. 2006;57:343-357.
23. Biswas SK, Lopes de Faria JB. Hypertension induces oxidative stress but not macrophage infiltration in the kidney in the early stage of experimental diabetes mellitus. Am J Nephrol. 2006;26:415-422.
24. Zhou L, Xiang W, Potts J, et al. Reduction in extracellular superoxide dismutase activity in African-American patients with hypertension. Free Radic Biol Med. 2006;41:1384-1391.
25. Noon JP, Walker BR, Webb DJ, et al. Impaired microvascular dilatation and capillary rarefaction in young adults with a predisposition to high blood pressure. J Clin Invest. 1997;99:1873-1879.
26. Boak L, Chin-Dusting JP. Hypercholesterolemia and endothelium dysfunction: role of dietary supplementation as vascular protective agents. Curr Vasc Pharmacol. 2004;2:45-52.
27. Napoli C, Sica V, Pignalosa O, et al. New trends in anti-atherosclerotic agents. Curr Med Chem. 2005;12:1755-1772.
28. Cooke JP, Oka RK. Atherogenesis and the arginine hypothesis. Curr Atheroscler Rep. 2001;3:252-259.
29. Napoli C, Williams-Ignarro S, de Nigris F, et al. Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice. Proc Natl Acad Sci USA. 2004;101:8797-802.
30. Napoli C, Williams-Ignarro S, de Nigris F, et al. Physical training and metabolic supplementation reduce spontaneous atherosclerotic plaque rupture and prolong survival in hypercholesterolemic mice. Proc Natl Acad Sci USA. 2006;103:10479-10484.
31. Sica V, Williams-Ignarro S, de Nigris F, et al. Autologous bone marrow cell therapy and metabolic intervention in ischemia-induced angiogenesis in the diabetic mouse hindlimb. Cell Cycle. 2006;5:2903-2908.
32. Takeshita S, Tomiyama H, Yokoyama N, et al. Angiotensin-converting enzyme inhibition improves defective angiogenesis in the ischemic limb of spontaneously hypertensive rats. Cardiovasc Res. 2001;52:314-320.
33. Napoli C, Salomone S, Godfraind T, et al. 1,4-Dihydropyridine calcium channel blockers inhibit plasma and LDL oxidation and formation of oxidation-specific epitopes in the arterial wall and prolong survival in stroke-prone spontaneously hypertensive rats. Stroke. 1999;30:1907-1915.
34. Verlinden SF, van Es HH, van Bekkum DW. Serial bone marrow sampling for long-term follow up of human hematopoiesis in NOD/SCID mice. Exp Hematol. 1998;26:627-630.
35. Napoli C, Martin-Padura I, de Nigris F, et al. Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet. Proc Natl Acad Sci USA.. 2003;100:2112-2116.
36. Sbaa E, Dewever J, Martinive P, et al. Caveolin plays a central role in endothelial progenitor cell mobilization and homing in SDF-1-driven postischemic vasculogenesis. Circ Res. 2006;98:1219-1227.
37. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92:9363-9367.
38. Imanishi T, Kobayashi K, Hano T, et al. Effect of estrogen on differentiation and senescence in endothelial progenitor cells derived from bone marrow in spontaneously hypertensive rats. Hypertens Res. 2005;28:763-772.
39. Dimmeler S, Aicher A, Vasa M, et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI3-kinase/Akt pathway. J Clin Invest. 2001;108:391-397.
40. Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nature Med. 2003;9:1370-1376.
41. Shen H, Cheng T, Olszak I, et al. CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells. J Immunol. 2001;166:5027-5033.
42. le Noble FA, Stassen FR, Hacking WJ, et al. Angiogenesis and hypertension. J Hypertens. 1998;16:1563-1572.
43. Nakano N, Moriguchi A, Morishita R, et al. Role of angiotensin II in the regulation of a novel vascular modulator, hepatocyte growth factor (HGF), in experimental hypertensive rats. Hypertension. 1997;30:1448-1454.
44. le Noble JL, Tangelder GJ, Slaaf DW, et al. A functional morphometric study of the cremaster muscle microcirculation in young spontaneously hypertensive rats. J Hypertens. 1990;8:741-748.
45. Konishi M, Su C. Role of endothelium in dilator responses of spontaneously hypertensive rat arteries. Hypertension. 1983;5:881-886.
46. Panza JA, Quyyumi AA, Brush JE Jr, et al. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med. 1990;323:22-27.
47. de Nigris F, Lerman LO, Napoli C. New insights in the transcriptional activity and coregulator molecules in the arterial wall. Int J Cardiol. 2002;86:153-168.
48. Couffinhal T, Silver M, Zheng LP, et al. Mouse model of angiogenesis. Am J Pathol. 1998;152:1667-1679.
49. Arras M, Ito WD, Scholz D, et al. Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J Clin Invest. 1997;101:40-50.
50. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol. 2002;282:C227-C241.
51. Ziegelhoeffer T, Fernandez B, Kostin S, et al. Bone marrow-derived cells do not incorporate into the adult growing vasculature. Circ Res. 2004;94:230-238.
52. Minamino K, Adachi Y, Okigaki M, et al. Macrophage colony-stimulating factor (M-CSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization. Stem Cells. 2005;23:347-354.
53. Iwase T, Nagaya N, Fujii T, et al. Comparison of angiogenic potency between mesenchymal stem cells and mononuclear cells in a rat model of hindlimb ischemia. Cardiovasc Res. 2005;66:543-551.
54. Madeddu P, Emanueli C, Pelosi E, et al. Transplantation of low dose CD34+KDR+ cells promotes vascular and muscular regeneration in ischemic limbs. FASEB J. 2004;18:1737-1739.
55. Sensebe L, Deschaseaux M, Li J, et al. The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. Stem Cells. 1997;15:133-143.
56. Napoli C, Maione C, Schiano C, et al. Oxidation-Specific mechanisms and cardiovascular repair induced by autologous bone marrow cell infusion. Trends Mol Med. 2007;13:278-286.
Keywords:

bone marrow cell; hypertension; ischemic hindlimb; peripheral arterial disease; L-arginine; nitric oxide

© 2007 Lippincott Williams & Wilkins, Inc.