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(The FASEB Journal. 2006;20:970-972.)
© 2006 FASEB

Plgf–/–eNos–/– mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia

Bruna Gigante*, Giulia Morlino*, Maria Teresa Gentile{dagger}, Maria Graziella Persico*,1 and Sandro De Falco*,1,2

* Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", Consiglio Nazionale delle Ricerche, Naples, Italy; and

{dagger} Instituto di Ricerche e Cura a Caratere 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

ABSTRACT

Neo-angiogenesis is a complex phenomenon modulated by the concerted action of several molecular factors. We have generated a congenic line of knockout mice carrying null mutations of both placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS), two genes that play a pivotal role in the regulation of pathological angiogenesis. In the present study, we describe the phenotype of this new experimental animal model after surgically induced hind-limb ischemia. Plgf–/–, eNos–/–, Plgf–/– eNos–/–, and wild-type C57BL/6J mice were studied. Plgf–/– eNos–/– mice showed the most severe phenotype: self-amputation, and death occurred in up to 47% of the animals studied; in ischemic legs, capillary density was severely reduced; macrophage infiltration and oxidative stress increased as compared to the other groups of animals. These changes were associated with an up-regulation of both inducible NOS (iNOS) expression and vascular endothelial growth factor (VEGF) protein levels in ischemic limbs, and to an increased extent of protein nitration. Our results demonstrate that the deletion of these two genes, Plgf, which acts in synergism with VEGF, and eNos, a downstream mediator of VEGF, determines a significant change in the vascular response to an ischemic stimulus and that oxidative stress within the ischemic tissue represents a crucial factor to maintain tissue homeostasis.—Gigante, B., Morlino, G., Gentile, M. T., Persico, M. G., De Falco, S. Plgf–/–eNos–/– mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia.


Key Words: hindlimb ischemia • macrophage infiltration • nitric oxide

IMPAIRED ANGIOGENESIS is one of the features involved in the pathogenesis of ischemic cardiovascular diseases (1) . Experimental and clinical studies are testing the efficacy of factors with known angiogenic properties in the treatment of myocardial infarction, stroke, and peripheral artery diseases (2) . Tissue hypoxia represents the common stimulus for the expansion of the vascular bed within ischemic tissues. However, the specificity of the response of every organ to hypoxia, the complex interplay among different angiogenic growth factors, and the multiple molecular signals ultimately leading to neo-angiogenesis and arteriogenesis, makes a clear dissection of the mechanisms underlying angiogenic disorders extremely hard. Gene knockout animal models of single factors have given crucial information to understand some aspects of this complex process (3) ; however, new genetic models are warranted to unveil the interplay between factors involved in pathological angiogenesis.

Characterization of placental growth factor (PlGF) null mice has clearly indicated that PlGF selectively modulates pathological angiogenesis. Plgf–/– mice, in fact, show an appropriate vascular response to physiological stimuli (4) and an impaired angiogenesis in a variety of disorders requiring vessel growth, such as cancer, ischemic conditions and wound healing (5) . PlGF acts in synergism with the central player of angiogenesis, the vascular endothelial growth factor (VEGF) and exerts its action through the binding and activation of VEGF receptor 1 (Flt-1) (6) . In vitro experiments have demonstrated that PlGF-activated Flt-1 is able to transphosphorylate and transactivate VEGF receptor 2 (Flk-1), normally recognized by VEGF, thus enhancing the response to VEGF stimulation (7) . Moreover, administration of recombinant PlGF, in vivo, amplifies VEGF-driven angiogenesis through the formation of mature and durable vessels in the ischemic heart and the enlargement of collateral arterioles in the ischemic limb with marked perfusional and functional improvement (8) , without resulting in the side effects such as edema and hypotension, usually observed after administration of recombinant VEGF-A (9) .

Endothelial NOS (eNOS) and its final by-product nitric oxide (NO) represent a downstream imperative for the angiogenic response elicited by VEGF (10 11 12) . NO plays a critical role in vascular biology, mediating many physiological functions including vascular tone and vascular permeability (13) . Deletion of eNOS gene, in fact, results in systemic hypertension (14 , 15) and pulmonary vasoconstriction (16) .

A functional link between VEGF and eNOS has been established in vitro by Ziche and co-workers who first demonstrated that VEGF-induced angiogenesis was selectively blocked by eNOS inhibitors (11) . Murohara and co-workers demonstrated that eNos–/– mice showed a reduced neo-angiogenesis after surgically induced hindlimb ischemia, and this phenotype was not rescued by VEGF treatment (12) , indicating that eNOS is a downstream target of VEGF. More recently, it has been shown that VEGF up-regulates eNOS expression through the binding and activation of Flk-1/KDR receptor (17 18 19) , and NO itself modulates VEGF release from endothelial cells (20) , smooth muscle cells (21) , and macrophages (22) through an autocrine and/or paracrine loop.

Because of the involvement of Plgf and eNos genes in the regulation of neo-angiogenesis and the PlGF-dependent activity of VEGF in ischemic conditions, we have established a line of congenic C57BL/6J mice carrying both Plgf and eNos null mutations (Plgf–/– eNos–/–) to investigate the potential role in the angiogenic process of PlGF/eNOS interaction.

In the present study, we describe the phenotype of this new experimental animal model after surgically induced hindlimb ischemia.

MATERIALS AND METHODS

Animals
Congenic C57BL/6J Plgf–/– mice were generated by 11 backcrosses of 50% Sv129/ 50% Swiss Plgf–/– mice with C57BL/6J mice in our animal facility. Congenic C57BL/6J eNos–/– mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA, catalog number 002684). Plgf–/– and eNos–/– mice were intercrossed in our animal facility to establish congenic Plgf–/– eNos–/– mice.

For the present study, 3 to 4-month-old male mice carrying Plgf null mutation (Plgf–/–, n=12), eNos null mutation (eNos–/–, n=11) and both Plgf and eNos null mutation (Plgf–/–eNos–/–, n=19) were studied. A group of C57BL/6J (n=10) was taken as a control. The study was performed according to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No 85–23) and were approved by our Institutional Guidelines.

To perform surgical unilateral hind-limb ischemia, mice were anesthetized with 2, 2, 2-tribromoethanol (880 nmol/kg i.p.)(Sigma Aldrich, St. Louis, MO). Body weight (g) was recorded and, after induction of anesthesia, the right femoral artery was gently isolated, ligated and excised distal to the deep femoral artery and 0.5 cm proximal to the bifurcation in saphenous and popliteal arteries. Seven days later, body weight was recorded again, and mice were sacrificed by CO2 inhalation. Given the death or the self-amputation of ischemic legs in 9 out of 19 analyzed Plgf–/–eNos–/– mice (Table 1 ), the successive analyses were performed on the remaining 10 mice while for the other experimental animal groups, all of the operated mice were used. Both tibialis muscles from ischemic and contralateral limb were removed, cleaned, cut into two halves and either fixed in 4% paraformaldehyde and processed for immunohistochemical analyses or quickly frozen in liquid nitrogen and stored at –80°C for RNA and protein extraction.


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  		Table 1. Body weight, ischemic limb phenotype and death rate in wild-type, Plgf–/–, eNos–/– and Plgf–/–eNos–/– mice at baseline and 7 days after femoral artery ligation

The phenotype of ischemic limbs was defined as normal, if no signs of ischemia were detectable at physical examination, cyanotic, if cyanosis of fingertip was present, gangrenous, if gangrene of fingertip was present, and self-amputated, if self-amputation occurred during the 7 days after surgery. The death rate was also recorded and considered as related to ischemia if it occurred when the animal had fully recovered from the surgical procedure, usually 48–72 h after surgery.

Furthermore, to evaluate VEGF levels in ischemic muscles, an additional 10 Plgf–/– eNos–/– and 10 wild-type mice were studied two (n=5, for each group) and four (n=5, for each group) days after femoral artery ligation.

Immunohistochemical analysis
Histological transverse sections, 6 µm thick, obtained from paraffin-embedded tissue samples were used to assess capillary density, macrophage infiltration, and free oxygen radicals in ischemic and non-ischemic limbs. All measurements were performed on four different randomly chosen fields/sections on at least four serial sections for each animal. Capillaries were stained with biotin-labeled lectin from Griffonia simplicifolia (Sigma Aldrich). Signal was detected and amplified with Tyramide Signal Amplification kit (PSA) (NEN Life Science Products, Wellesley, MA, USA). Capillary density was assessed in ischemic and contralateral limb in each animal and normalized by myocyte density. Neo-angiogenesis was expressed as ischemic/nonischemic capillary density ratio. Vessels were counted as capillaries if the diameter was less than 10 µm.

Macrophages were identified with a monoclonal antibody (mAb) raised against Mac-3 antigen (Becton-Dickinson, Franklin Lakes, NJ, USA). Signal was detected and amplified with kit (Perkin Elmer, Norwalk, CT, USA). Macrophage infiltration within tissue was assessed by the evaluation of the Mac-3-positive staining area in each section and expressed as a percentage of the total area of each section analyzed.

Dihydro-ethidium (DHE) was used to evaluate the presence of superoxide anion (O2–) oxygen radicals. In the presence of O2–, DHE is oxidized to ethidium bromide, which binds the DNA. The fluorescence reaction was carried out by incubating muscle sections with DHE (2x10–6 mol/L) for 30' at room temperature. DHE fluorescence was detected with a 585 nm filter. Fluorescence intensity in each ischemic limb was normalized on fluorescence intensity detected in the respective contralateral nonischemic limb and expressed as arbitrary fluorescence units (AFU).

All images were recorded with a digital camera Leica DC480 (Leica), and morphometric and densitometric analyses were performed with QwinPro software (Leica). Slides were examined by two independent observers (BG, GM).

Real-time semi-quantitative polymerase chain reaction
Real-time semiquantitative polymerase chain reaction (PCR) was performed to assess differences in eNOS and iNOS expression levels in ischemic as compared to contralateral muscles. Total RNA was extracted from ischemic and contralateral muscles using the Trizol reagent (Invitrogen). ß-actin expression levels were used to normalize the cDNA templates across different samples. No differences were found, in fact, in ß-actin expression levels among different groups and experimental conditions.

Sense and antisense for eNOS, iNOS, and ß-actin primer sequences were the following: eNOS sense 5'-GGTGATGGCGAAGCGTGTGAAGGC-3', eNOS antisense 5'-CTAGACTCCTTCCTCTTGCGCCGCC-3' (position 1540–1564 and 1881–1905, respectively, GenBank accession number NM_008713); iNOS sense 5'-GCTTGGTGTTTGGGTGCCGGC-3', iNOS antisense 5'-CCTCGTGGCTTTGGGCTCCT CCA-3' (position 3198–3218 and 3591–3613, respectively, GenBank accession number NM_010927); ß-actin sense 5-' TCGTGCGTGACATCAAAGAG-3', ß-actin antisense 5'-GTCAGGCAGCTCATAGCTCT-3' (position 545–564 and 634–654, respectively, GenBank accession number M12481). Amplification reactions were carried out in a 96-well plate using 1 x SYBR Green dye reaction mixture (Applied Biosystems, Foster City, CA). Thermal cycling conditions consisted of 2 min at 50°C, 10 min at 95°C followed by 40 cycles with a 15-s denaturation step at 94°C and 1 min of annealing/extension step at 65°C. The synthesis of PCR products was verified on 1% agarose gel.

Data were analyzed using the comparative threshold cycle (CT) method (23) . The difference between eNOS and iNOS CT and ß-actin CT (DCT), was then calculated for each sample. eNOS and iNOS DCT values obtained in control muscles from wild-type mice were taken as baseline values and subtracted from the DCT values observed in ischemic muscles from wild-type mice and from contralateral nonischemic and ischemic muscles obtained from Plgf–/–, eNos–/– and Plgf–/–eNos–/– mice (DDCT). The relative quantification was then calculated using the formula 2–DDCT. Data are expressed as number of fold differences of eNOS and iNOS expression level in ischemic and contralateral nonischemic muscles compared with eNOS and iNOS expression level in wild-type contralateral, nonischemic, muscles (value assigned=1).

Protein extraction, Western blot analysis and ELISA assay
Protein extraction from muscles and ELISA assays for the determination of VEGF concentration were performed as previously reported (4) .

To quantify the extent of nitrated proteins, Western blot analysis was performed using standard methods (24) . One hundred milligrams of each sample were loaded on SDS-polyacrylamide gels. Nitrated proteins were detected using a mAb antinitrotyrosine (clone A16, Upstate Biotechnology, Lake Placid, NY, USA), according to the manufacturer’s instructions. The results were normalized against ß-tubulin, detected on the same filter using anti-ß-tubulin polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Densitometry analyses were performed using the ImageQuant 5.2 software (Molecular Dynamics); protein nitration was expressed as ratio between arbitrary densitometric units (ADU) of nitrotyrosine and ß-tubulin.

Statistical analysis
Data are expressed as means ± SEM Differences in frequency of the different genotypes were tested with the {chi}2 test. Kolmogorov-Smirnov one-sample test was used to assess normality of the distribution of all variables. Differences among groups were tested by one-way ANOVA; Tukey HD test was used as post hoc test to identify which group differences account for the significant overall ANOVA. All calculations were carried out using Statistical Packages for the Social Sciences (SPSS) statistical package (ver. 12.1, Chicago, IL, USA).

RESULTS

Phenotypic characterization of double mutant mice
Plgf–/– eNos–/– mice were vital and fertile. However, the double mutant mice were born at a lower frequency than that expected by Mendel’s law: 4% vs. 6.25% (98 pups, c2=7,8, 8DF P=0.02) from Plgf±eNos± intercross.

Body weights at baseline and 7 days are reported in Table 1 after femoral artery ligation and the phenotypic characteristics of ischemic hindlimb in wild-type, Plgf–/–, eNos–/– and Plgf–/– eNos–/– mice 7 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 after induction of ischemia. During the 7 days after the intervention Plgf–/– eNos–/– mice lost body weight as compared to the other groups of mice and showed signs of severe ischemia characterized by cyanosis of fingertip (10.5%), gangrene of fingertip (21%) and self-amputation of ischemic limb (16%). Plgf–/– eNos–/– also showed an increased death rate (31.5%) (Table 1) .

Morphometric analysis
Immunohistochemical analysis was performed to assess neo-angiogenesis in ischemic limbs (Fig. 1 A). Plgf–/– mice showed a slight reduction of neo-angiogenesis compared to wild-type mice, 7 days after femoral artery ligation with an ischemic/non-ischemic capillary density ratio of 0.84 ± 0.06 and 0.94 ± 0.08, respectively, (P=ns). As already described (12) , eNos–/– mice showed a reduced neo-angiogenesis with a 0.71 ± 0.06 ischemic/non-ischemic capillary density ratio. Plgf–/– eNos–/– mice showed a further reduction of ischemic/non-ischemic capillary density ratio (0.59±0.03) with a significant impaired neo-angiogenesis when compared to wild-type and Plgf–/– mice (P<0.01) (Fig. 1) . The decreased ischemic/non-ischemic capillary density ratio of 0.13 shown by eNos–/– with respect to Plgf–/– mice (from 0.84±0.06 to 0.71±0.06), resulted in a slight difference in phenotype aggravation, indicating that a capillary ratio of almost 0.71 ± 0.06 is sufficient to prevent irreversible tissue damage. Notably, an additional reduction of 0.12 of ischemic/non-ischemic capillary density ratio observed in the Plgf–/– eNos–/– with respect to eNos–/– mice (from 0.71±0.06 to 0.59±0.03) resulted in the occurrence of a serious pathological phenotype and the appearance of limb gangrene and self-amputation. In ischemic limbs, in fact, absolute capillary density (vessels/mm2), was lower in Plgf–/– eNos–/– mice (270±3) as compared to eNos–/– (350±6), Plgf–/– (379±3, P<0.05 vs. Plgf–/– eNos–/–), and wild-type (433±7, P<0.01 vs. Plgf–/– eNos–/–) mice, confirming that it is the actual reduction in the absolute number of capillaries that significantly contributes to the severe phenotype observed in Plgf–/– eNos–/– mice (Table 1) .


Figure 1
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  		Figure 1. A) Representative lectin staining of capillaries in nonischemic (NI) and ischemic (I) muscles of lower limbs of wild-type, Plgf–/–, eNOS–/–, and Plgf–/– eNos–/– mice. Scale bar represents 40 µm (x20 magnification). B) Capillary density in wild-type (n=10), Plgf–/– (n=12), eNos–/– (n=11), Plgf–/– eNos–/– (n=10) mice. Values are mean ± SEM and are expressed as ratio between capillary density in ischemic and non-ischemic muscles. ##P < 0.01 vs. wild-type and Plgf–/– mice.

In addition to a reduced neo-angiogenesis Plgf–/– eNos–/– ischemic limbs showed a profound rearrangement of muscle histology. In 60% of the serial sections examined, muscle bundles showed disarrayed myocytes with piled-up nuclei, interspersed in a trabecular extracellular matrix. These features were always absent in ischemic limbs from wild-type and Plgf–/– mice, but were present in 30% of the serial sections examined in eNos–/– mice (data not shown).

Monocyte-macrophages are recruited at site of neo-angiogenesis and play a crucial role in neo-angiogenesis (25) . To evaluate their contribution in our experimental model, we investigated their occurrence in ischemic and non-ischemic muscles. Macrophages were seldom detected in contralateral non-ischemic limbs, and no differences were detected among the different experimental groups (data not shown). In ischemic limbs (Fig. 2 ), however, 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–/– 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.


Figure 2
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  		Figure 2. A) Representative Mac-3 immunostaining of ischemic muscles of lower limbs of wild-type, Plgf–/–, eNos–/–, and Plgf–/– eNos–/– mice. Scale bar represents 40 µm (x20 magnification). B) Macrophage infiltration in ischemic muscles of lower limbs expressed as a percentage of Mac-3 positive-staining area with respect to the total section area. Values are mean ± SEM. The number of animals used for each group is as reported in Fig. 1 legend. ##P < 0.01 vs. wild-type and Plgf–/– mice. §P < 0.05 vs. eNos–/– mice.

eNOS and iNOS expression in ischemic tissues
Because NO plays a pivotal role during the angiogenic response, we evaluated by real-time PCR the expression levels of eNOS and iNOS in the four experimental animal groups. eNOS expression in non-ischemic skeletal muscles did not show any significant difference between wild-type and Plgf–/– mice. As expected, no PCR product was observed in eNos–/– and Plgf–/– eNos–/– muscles. In ischemic muscle, eNOS expression levels were increased in wild-type (1.8-fold increase, P < 0.05 vs. non-ischemic muscles) and Plgf–/– mice (3.3-fold increase, P<0.01 vs. nonischemic muscles) (Fig. 3 A).


Figure 3
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  		Figure 3. eNOS (A) and iNOS expression level (B) in nonischemic and ischemic muscles of lower limbs of wild-type, Plgf–/–, eNos–/–, and Plgf–/– eNos–/– mice. Data are expressed as fold increase vs. nonischemic wild-type muscles. The number of animals used for each group is as reported in Fig. 1 legend. *P < 0.05 vs. the respective nonischemic hind limbs; **P < 0.01 vs. the respective nonischemic hind-limbs; °P < 0.05 vs. wild-type and eNos–/–; ##P < 0.01 vs. wild-type and Plgf–/– mice; §§P < 0.01 vs. eNos–/– mice.

iNOS expression 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 3.0-fold increase vs. the contralateral nonischemic muscle (P<0.01) (Figure 3B ).

Reactive oxygen species detection
To detect the levels of oxygen radicals in the muscles of our experimental animal model, a DHE staining was performed. No difference in DHE fluorescence intensity was observed among contralateral non-ischemic muscles in the four different experimental groups. In ischemic hind-limbs (Fig. 4 ), 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).


Figure 4
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  		Figure 4. A) Representative DHE staining of ischemic muscles of lower limbs of wild-type, Plgf–/–, eNos–/–, and Plgf–/– eNos–/– mice (x20 magnification; scale bar represents 40 µM). B) DHE fluorescence intensity staining in ischemic muscles of lower limbs normalized on fluorescence intensity detected in the respective contralateral non-ischemic muscles. Data are expressed as arbitrary fluorescence units (AFU) ± SE. The number of animals used for each group is as reported in Fig. 1 legend. ##P < 0.01 vs. wild-type and Plgf–/– mice. §P < 0.05 vs. eNOS–/– mice.

Protein nitration
An indirect measure of high levels of oxygen radicals is the increase of protein nitration. Given the high levels of oxygen radicals detected in the muscles of double-null mice, we assessed the protein nitration levels in ischemic muscles (Fig. 5 A). 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 the other groups). A representative Western blot analysis of two mice for each animal groups is reported in Fig. 5B .


Figure 5
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  		Figure 5. A) Densitometric analysis of Western blot analyses for nitrotyrosine ad ß-tubulin. Data represent the ratio between nitrotyrosine and ß-tubulin and are expressed as arbitrary densitometric units (ADU). The number of animals used for each group is as reported in Fig. 1 legend. ##P < 0.05 vs. wild-type, Plgf–/– and eNOS–/– mice. B) Representative Western blot analysis for nitrotyrosine (top) and ß-tubulin (bottom) of protein extracts from ischemic muscles of lower limbs of wild-type, Plgf–/–, eNos–/–, and Plgf–/– eNos–/– mice (n=2 for each experimental group).

VEGF levels in ischemic tissues
The persisting hypoxic conditions due to the reduced neo-angiogenesis sustain an increased expression of VEGF. At the same time, the massive macrophage infiltration in the ischemic limb due, in part, to the angiogenic stimulus, but also to the inflammatory status consequent to the reduced number of neovessels, represents a supplementary condition for VEGF production since macrophages themselves produce VEGF.

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 while Plgf–/– eNos–/– mice showed a significant increase of VEGF protein concentration (83.12±4.87, P<0.05 vs. wild-type and eNos–/–) (Fig. 6 A).


Figure 6
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  		Figure 6. A) Quantification of VEGF protein concentration in protein extracts from ischemic muscles of lower limbs of wild-type, Plgf–/–, eNos–/–, and Plgf–/– eNos–/– performed by ELISA assays. The number of animals used for each group is as reported in Fig. 1 legend. °P < 0.05 vs. wild-type and eNOS–/–. B) Time course of VEGF protein concentration in protein extracts from ischemic muscles of lower limbs of wild-type and Plgf–/– eNos–/– mice, performed by ELISA assays. n = 5 for each group at each time.

Consequently, further analysis was performed on Plgf–/– eNos–/– mice, evaluating the VEGF protein concentration in ischemic muscles also after 2 and 4 days after the ischemic stimulus. As control wild-type mice were analyzed under the same conditions (Fig. 6B ). As expected, high levels of VEGF (pg/ml) were detected in wild-type mice ischemic limbs at the early stage of ischemia (75.17±0.49 after 2 days), and decreased 4 days (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.

DISCUSSION

In the present study, we report the generation of the first experimental animal model of defective angiogenesis determined by the combined deletion of two genes, Plgf and eNos, that play a pivotal role in the regulation of neo-angiogenesis in pathological conditions. After surgically induced mild hindlimb ischemia wild-type and Plgf–/– mice did not present any macroscopic sign of ischemia while eNos–/– mice consistently showed a mild degree of ischemia manifested by cyanosis of fingertip. In contrast, in Plgf–/– eNos–/– mice, we observed a heterogeneous ischemic phenotype ranging from the absence of macroscopic lesions, as observed in Plgf–/– mice, to self-amputation and increased death rate occurring in 47% of the animals undergoing the surgical procedure. This phenotypic heterogeneity resembles the one observed in clinical practice in patients where the same ischemic insult is associated to different clinical outcomes and underscores the importance of the generation of new experimental animal models that more closely mimic polygenic human diseases (26) .

Plgf–/–, eNos–/– and Plgf–/– eNos–/– mice showed a different degree of impaired neo-angiogenesis in ischemic limbs. The ischemic/non-ischemic capillary density ratio, in fact, was slightly reduced in Plgf–/– mice as compared to wild-type mice and was progressively reduced in eNos–/– and Plgf–/– eNos–/– mice (Fig. 1) . The observation that even a further reduction in capillary density in Plgf–/– eNos–/– mice ischemic limbs, as compared to eNos–/– mice, is potentially responsible for a considerable phenotypic change from mild degree of ischemia to more severe phenotypic alterations, like gangrene and self-amputation, strongly suggests the existence in this experimental animal model of a threshold in capillary density value (between 0.71 and 0.59 in terms of ischemic/non-ischemic capillary density ratio, and between 350 and 270 in term of vessels/mm2) below which irreversible tissue damage may occur.

Our findings in eNos–/– mice are consistent with previous data of inhibition of neovascularization in the hindlimb ischemia model (12) and confirm the crucial role of eNOS in mediating VEGF effect on neovascularization reported by Ziche and co-workers in their seminal paper (11) where corneal neovascularization induced by VEGF or cells overexpressing VEGF was strongly reduced by eNOS pharmacological inhibition.

We did not observe a significantly impaired neo-angiogenesis in Plgf–/– mice ischemic limbs as compared to wild-type mice. It has been previously demonstrated that Plgf–/– mice show a reduced collateral growth proximal to the site of femoral artery ligation and a reduced neo-angiogenesis in the site of ischemia in heart, retina and skin (5) . First, differences in the microenvironment of different organs and tissues may influence the response of each tissue to hypoxic and ischemic stimuli; indeed a more pronounced effect on arteriogenesis of PlGF in ischemic hind limbs has also been reported (27) . Second, our current experiments were performed in congenic genetic background thus modifier genes in the C57BL/6J background may exert a protective effect in the modulation of neo-angiogenesis. It has been recently reported that 100% C57BL/6J strain express three times more Flk-1 receptor than 50% C57BL/6J / 50% 129Sv strain during embryonic life, demonstrating that differences in the genetic background may influence VEGF receptor expression levels and consequently VEGF and PlGF activity (28) .

Neo-angiogenesis was severely impaired in Plgf–/– eNos–/– mice, suggesting that PlGF and eNOS interact in the modulation of this process in ischemic conditions. Defective angiogenesis was associated to a profound rearrangement of muscle histology and to a massive increase in macrophage infiltration in Plgf–/– eNos–/– ischemic hind limbs. Macrophages are essential for a correct angiogenesis and arteriogenesis, and they can still be detected in sites of neo-angiogenesis 7 days after induction of hindlimb ischemia (25) (Fig. 2) . Macrophage infiltrate was still present, in fact, in wild-type, Plgf–/– and eNos–/– mice. In Plgf–/– eNos–/– mice, persistence of hypoxic/ischemic stimulus and the presence of necrotic myocytes as a consequence of more pronounced impairment of neo-angiogenesis most likely represent an additional and more prolonged stimulus to recruit macrophages and to sustain inflammation. In our experimental conditions, the presence of macrophages was associated to an up-regulation of iNOS expression (Fig. 3) , an increase in oxygen free radicals as detected by DHE staining (Fig. 4) , and as a consequence, an increase in protein nitration (Fig. 5) , all signs of severe chronic inflammation.

NO has a short half-life and, in physiological conditions, is synthesized in a pulsatile way by constitutive calcium-dependent NO synthases in endothelial (eNOS) and neuronal (nNOS) cells. In pathological conditions, such as endotoxemia, ischemia, or inflammation, activated-macrophages and smooth muscle cells express iNOS that produces, in a continuous fashion, large amount of NO (29) . NO reacts with oxygen free radicals, superoxide anion (O2–) and hydrogen peroxide (H2O2), produced by tissue metabolism; this reaction, usually minimal in physiological conditions (30) , catalyzes the formation of highly reactive intermediates such as peroxynitrite anions (ONOO–) and nitrogen dioxide (NO2) characterized by high cellular toxicity. Their half-life being extremely short, a footprint of their production is the presence of nitrated proteins that represent a sign of irreversible tissue damage. On average, the extent of protein nitration in Plgf–/– eNos–/– mice was significantly increased as compared to the other groups of mice (Fig. 5A ). However, we did observe discrete variability in the grade of nitration among the animals studied in each group (Fig. 5B ), and we cannot exclude that other posttranslational modifications such as 3-bromo-, 3-chloro- or 3-hydroxytyrosine also observed in chronic inflammation (31) may contribute to the observed tissue damage.

Excessive NO synthesis and the concomitant increase in cellular oxidative stress has been considered detrimental for cardiovascular function (30) . However, recent experimental evidence suggests that iNOS cellular source may be the determinant for its beneficial or detrimental effect (32) . Experiments performed in vitro (33) and in vivo (34) suggest that oxidative stress induces VEGF and hypoxia inducible factor-1{alpha} (HIF-1{alpha}) in vascular smooth muscle cells, thus promoting neo-angiogenesis, through an hypoxia-independent mechanism and that antioxidant interventions reduce intraplaque neo-angiogenesis in hypercholesterolemic rabbits (35) . Moreover, selective iNOS inhibition deteriorates renal function in an experimental model of chronic glomerulonephritis (36) , thus suggesting that iNOS-mediated NO production may also exert a protective role.

Seven days after femoral artery ligation, increased tissue oxidative stress was associated to tissue damage and to an increase in proteins nitration in Plgf–/– eNos–/– mice. However, we were able to analyze only the animals that survived through the 7 days and that did not undergo self-amputation. Therefore we cannot exclude that limb salvage in these animals is related to the increased oxidative stress itself. Oxidative stress may represent, in fact, an additional mechanism to promote angiogenesis and to prevent severe ischemic lesions such as extensive gangrene and self-amputation. One potential mechanism we can hypothesize is that in Plgf–/– eNos–/– mice, a defective neo-angiogenesis determines a prolonged and sustained ischemic stimulus resulting in increased tissue oxidative stress, which represents a driving force to further stimulate HIF-1{alpha} and VEGF through an hypoxia-independent mechanism, as supported by the finding of a constant increase in VEGF muscle levels in ischemic Plgf–/– eNos–/– muscles, throughout the 7 days of ischemia (Figure 6B ). 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.

The concomitant deletion of Plgf and eNos may therefore strongly influence the ischemia-driven angiogenesis. In Plgf–/– mice, in fact, VEGF and eNOS up-regulation in ischemic muscles may stimulate angiogenesis. Indeed eNOS overexpression in transgenic mice was associated to increased capillary density and to improved perfusion in ischemic limbs (37) . In eNos–/– mice, PlGF, through its favorable action on arteriogenesis, may represent one of the factors exerting a protective effect against severe ischemia.

Another potential interesting finding observed in Plgf–/– eNos–/– mice is the increased expression of iNOS in contralateral non-ischemic limbs (Fig. 3B ). This increase was not associated with any particular finding in muscle histology and oxidative stress markers, and it may well represent a compensatory mechanism mostly due to eNOS absence in a more complex genetic background.

The establishment of new genetic animal models to study complex disorders, such as double knockout mice, might be of extreme importance to gain further insights in the pathophysiology of human diseases and to design new molecules that more selectively interfere with biological processes, as already have been suggested by results obtained in different experimental animal models (38 , 39) . However, one potential limitation of the polygenic experimental animal models is that genetic complexity per se may represent a stimulus for compensatory mechanisms that might have an impact on the observed phenotype. Further studies, in fact, conducted with selective eNOS and PlGF inhibitors are warranted to definitively validate our new experimental animal model.

In conclusion, our study demonstrates that deletion of two genes involved in the regulation of angiogenesis determines a dramatic change in the vascular response to an ischemic stimulus and underscores the importance of NO and the balance between reactive oxygen species as crucial regulators of tissue homeostasis in physiological and pathological conditions.

ACKNOWLEDGMENTS

The authors would like to thank Raffaele Improta, Ivan Solombrino, and Barbara Rossi for their technical assistance, Lieve Moons for help in lectin and MAC-3 staining, Dr. Claudio Arra and Dr. Andrea Affuso for their suggestions for animal care and handling, and Istituto Nazione Tumori "Fondazione G. Pascale" for animal housing. This work was supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC) (to M.G.P.) and BioGem s (s).c.a.r.l. (to M.G.P.). B.G. and R.I.’s salaries were provided by BioGem.

FOOTNOTES

1 These authors contributed equally to this work.

Received for publication July 7, 2005. Accepted for publication December 21, 2005.

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