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* Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", Consiglio Nazionale delle Ricerche, Naples, Italy; and
Istituto di Ricerca e Cura a Carattere Scientifico Neuromed, Pozzilli (IS), Italy
2Correspondence: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", CNR, Via P. Castellino, 111, Naples 80131, Italy. E-mail: defalco{at}igb.cnr.it
SPECIFIC AIMS
Placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS) play a pivotal role in pathological angiogenesis. PlGF acts in synergism with the vascular endothelial growth factor (VEGF), whereas eNOS is one of the downstream targets of VEGF. To investigate the existence of a functional interaction between PlGF and eNOS, we have 1) generated a congenic line of knockout mice carrying both null mutations and 2) analyzed the phenotype of this new experimental animal model following surgically induced hind limb ischemia.
PRINCIPAL FINDINGS
1. Plgf–/– eNos–/– mice show defective neo-angiogenesis
Congenic (C57BL6/J) Plgf–/– and eNos–/– mice were intercrossed in our animal facility to establish Plgf–/– eNos–/– mice. For the present study, 3- to 4-month-old male wild-type C57BL6/J (n=10), Plgf–/– (n=12), eNos–/– (n=11), and Plgf–/– eNos–/– (n=19) mice were studied. To perform surgical unilateral hindlimb ischemia, right femoral artery was ligated and excised distal to the deep femoral artery and proximal to the bifurcation in saphenous and popliteal arteries. Seven days later, mice were sacrificed and both tibialis muscles from ischemic and contralateral limb were removed and processed for both immunohistochemical analyses, RNA and protein extraction.
Seven days after femoral artery ligation, wild-type and Plgf–/– mice did not show any macroscopic sign of ischemia, whereas eNos–/– mice consistently showed cyanosis of fingertip. Plgf–/– eNos–/– mice showed the most severe phenotype with cyanosis of fingertip (10.5%), gangrene of fingertip (21%), self-amputation of ischemic limb that occurred during the 7 days after surgery (16%) and death (31.5%), which was recorded and considered to be related to ischemia if occurred when the animal had fully recovered from the surgical procedure, usually 48–72 h after surgery.
To assess neo-angiogenesis in ischemic limbs, capillaries were stained with biotin-labeled lectin from Griffonia simplicifolia (Figure 1
A), and their density was measured in ischemic and contralateral limb in each animal and normalized by myocyte density (Figure 1B
). Neo-angiogenesis was expressed as ischemic/nonischemic (I/NI) capillary density ratio and vessels were counted as capillaries if the diameter were less than 10 µm. Plgf–/– mice showed a slight reduction of neo-angiogenesis compared to wild-type mice with I/NI capillary density ratio of 0.84 ± 0.06 and 0.94 ± 0.08, respectively (P=ns). As already described, eNos–/– mice showed a reduced neo-angiogenesis with a 0.71 ± 0.06 I/NI capillary density ratio. Plgf–/– eNos–/– mice showed a further reduction of I/NI capillary density ratio (0.59±0.03) with a significant impaired neo-angiogenesis when compared to wild-type and Plgf–/– mice (P<0.01).
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2. Plgf–/– eNos–/– mice show an increased macrophage infiltration and inducible NOS (iNOS) expression in ischemic limbs
Monocyte-macrophages presence was assessed in muscle sections by staining with a monoclonal antibody (mAb) anti-Mac-3 antigen. In ischemic limbs, macrophages infiltration area (%) increased from 2.5 ± 0.1 in wild-type, to 4.8 ± 0.3 and to 7.3 ± 0.2 in Plgf–/– and eNos–/– mice, respectively (P=ns vs. wild-type mice). In ischemic limbs from Plgf–/– eNos–/– mice a considerable macrophage cell infiltrate, with macrophages representing 15.1 ± 0.4% of the total section area (P<0.01 vs. wild-type and Plgf–/– mice and P<0.05 vs. eNos–/– mice) was observed.
Real-time semiquantitative polymerase chain reaction (PCR) was performed to assess differences in eNOS and inducible nitric oxide synthase (iNOS) expression levels in ischemic as compared to contralateral muscles in the four experimental animal groups. ßbeta;-actin expression levels were used to normalize cDNA templates across different samples. eNOS expression level in non-ischemic skeletal muscles did not show any significant difference between wild-type and Plgf–/– mice. In ischemic muscle, eNOS expression was increased in wild-type (1.8-fold increase, P<0.05 vs. nonischemic muscles) and Plgf–/– mice (3.3-fold increase, P<0.01 vs. non-ischemic muscles).
iNOS expression level in non-ischemic muscles did not show any significant difference among wild-type, Plgf–/– and eNos–/– mice. Increase of iNOS expression was observed in Plgf–/– eNos–/– as compared to the expression levels observed in wild-type (3.45-fold increase) and eNos–/– mice (1.7-fold increase, both P<0.05). iNOS expression was consistently up-regulated in ischemic muscle. Ischemia was associated to a 2.58-fold increase in iNOS expression in wild-type, a 1.53-fold increase in Plgf–/– and 2.35-fold increase in eNos–/– mice (all P<0.05 vs. respective contralateral non-ischemic muscle). In Plgf–/– eNos–/– mice, iNOS expression showed a 3-fold increase vs. the contralateral non-ischemic muscle (P<0.01).
3. Increase of reactive oxygen species, protein nitration and VEGF levels in ischemic limbs of Plgf–/– eNos–/– mice
Tissue ischemia is often associated with an increased presence of reactive oxygen species (ROS). To detect the levels of oxygen radicals in the muscles of our experimental animal models, muscle sections were incubated with dihydro-ethidium (DHE) and after 30 min of incubation, fluorescence was detected with a 585-nm filter. In ischemic hindlimbs, fluorescence intensity, normalized on fluorescence intensity observed in contralateral non-ischemic limbs, was comparable in wild-type and Plgf–/– mice (1.20±0.07 and 1.17±0.06, respectively, P=ns) and tended to be higher in eNos–/– mice (1.4±0.08, P=0.06 vs. wild-type and Plgf–/– mice). Plgf–/– eNos–/– mice showed a consistent and significant increase in DHE fluorescence staining (1.76±0.07 P<0.01 vs. wild-type and Plgf–/– mice and P<0.05 vs. eNos–/– mice).
Given the high levels of oxygen radicals detected in the muscles of double null mice, we assessed the protein nitration levels in ischemic muscles by Western blot analysis, using a mAb anti-nitrotyrosine. The results were normalized against ßbeta;-tubulin. Protein nitration was expressed as ratio between arbitrary densitometric units of nitrotyrosine and ßbeta;-tubulin. No difference was observed among wild-type (1.36±0.03), Plgf–/– (1.40±0.23) and eNos–/– (1.28±0.30) mice (all P=ns); Plgf–/– eNos–/– mice, however, did show a significant increased protein nitration (2.03±0.23, P<0.05 vs. all of the other groups).
VEGF concentration (pg/mg of proteins) in ischemic muscles was evaluated by a quantitative ELISA. VEGF protein levels were similar in wild-type (47.25±1.58), Plgf–/– (60±4.44), and eNos–/– (41.87±1.58) mice, whereas Plgf–/– eNos–/– mice showed a significant increase of VEGF protein concentration (83.12±4.87, P<0.05 vs. wild-type and eNos–/–). VEGF protein concentration in ischemic muscles were also measured in additional 10 Plgf–/– eNos–/– and 10 wild-type mice, 2 (n=5, for each group) and 4 (n=5, for each group) days after femoral artery ligation. High levels of VEGF were detected in wild-type mice ischemic limbs at the early stage of ischemia (75.17±0.49 after 2 days), which decreased 4 (63.12±1.31) and 7 days (47.25±1.58) after femoral artery ligation. Notably, Plgf–/– eNos–/– mice showed VEGF protein levels similar to wild-type mice, 2 days after the ischemic stimulus (66.95±1.55), but, differently from the wild-type mice, VEGF concentration in ischemic skeletal muscles continued to increase 4 (73.21±1.11) and 7 days (83.12±4.87, P<0.05 vs. wild-type) after femoral artery ligation.
CONCLUSIONS AND SIGNIFICANCE
In the present study, we report the generation of the first experimental animal model of defective angiogenesis determined by the combined deletion of Plgf and eNos. Neo-angiogenesis was severely impaired in Plgf–/– eNos–/– mice, suggesting that PlGF and eNOS interact in the modulation of this process in ischemic conditions. After surgically induced mild hindlimb ischemia, the Plgf–/– eNos–/– mice showed a heterogeneous ischemic phenotype ranging from the absence of macroscopic lesions to self-amputation and increased death rate occurring in 47% of the animals. This phenotypic heterogeneity resembles the one observed in clinical practice in patients, in which the same ischemic insult is associated with different clinical outcomes, and underscores the importance of the generation of new experimental animal models that more closely mimic polygenic human diseases.
Plgf–/–, eNos–/–, and Plgf–/– eNos–/– mice showed a different degree of impaired neo-angiogenesis in ischemic limbs. Our finding in eNos–/– mice is consistent with data already reported by Murohara and co-workers. Even though Plgf–/– mice represent an experimental animal model of defective angiogenesis in the site of ischemia in heart, retina, and skin, we did not observe an impaired neo-angiogenesis in Plgf–/– mice ischemic limbs, as compared to wild-type mice. Differences in the microenvironment of different tissues may influence the response to hypoxic and ischemic stimuli. In addition, our current experiments were performed in congenic mice; thus, modifier genes in the C57BL/6J genetic background may exert a protective effect in the modulation of neo-angiogenesis. Plgf–/– eNos–/– mice show a significant impaired neo-angiogenesis when compared to wild-type and Plgf–/– mice. The observation that an additional, although not significant, reduction in capillary density in Plgf–/– eNos–/– mice ischemic limbs, as compared to eNos–/– mice, is potentially responsible for a considerable phenotypic change strongly suggests the existence, in this experimental animal model, of a threshold capillary density value below which irreversible tissue damage may occur.
Plgf–/– eNos–/– mice also showed an increased macrophage infiltration, NO synthesis, and ROS content in the ischemic muscles. As a result of this unbalance between NO and ROS, we also observed an increased level of nitrated proteins ultimately leading to irreversible tissue damage. Excessive NO synthesis and the concomitant increase in cellular oxidative stress has been considered detrimental for cardiovascular function. More recently, both iNOS cellular source and tissue oxidative stress have been shown to be also beneficial on cardiovascular function.
The potential mechanism we can hypothesize, as outlined in Fig. 2
, is that in Plgf–/– eNos–/– mice, a defective neo-angiogenesis determines a prolonged and sustained ischemic stimulus, resulting in increased tissutal oxidative stress, which represents a driving force to further stimulate HIF-1
and VEGF through an hypoxia-independent mechanism, as supported by the finding of a constant increase in VEGF levels in ischemic Plgf–/– eNos–/– muscles, throughout the 7 days of ischemia. However, in the absence of both PlGF and eNOS, this compensatory mechanism starts a vicious cycle, in which oxidative stress driven by an excessive iNOS-mediated NO production, generates more oxidative stress. We cannot exclude, however, the possibility that oxidative stress might stimulate angiogenesis through a VEGF-independent mechanism.
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In conclusion, our study demonstrates that deletion of two genes involved in the regulation of pathological angiogenesis determines a dramatic change in the vascular response to an ischemic stimulus and underscores the importance of NO and the balance between ROS as crucial regulators of tissue homeostasis in physiological and pathological conditions.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4481fje
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