Aging is an inevitable (“physiological”) cardiovascular risk factor. Understanding potential mechanisms of aging could lead to improved medical treatment of this increasing patient population. Studies in experimental models of hind limb ischemia reported impaired revascularization in old animals and demonstrated progression of the ischemic injury into skin ulcers and limb loss, in sharp contrast to the rapid flow recovery in younger animals.1–3 These severe ischemic symptoms were accompanied by decreased expression of vascular endothelial growth factor (VEGF) in hind limb muscle samples in the aging animals.2 Despite the loss of endogenous VEGF expression, this condition has been refractory to substitution of exogenously delivered VEGF in old animals,4 which was shown to be effective in other mouse hind-limb ischemia models using younger animals.5,6
Cardiovascular risk factors, including age, impair the functional integrity of the endothelium leading to reduced production of nitric oxide (EDNO) by endothelial nitric oxide synthase (ecNOS).7,8 EDNO plays a critical role in angiogenesis besides in the overall maintenance of vascular homeostasis.9–11 Animals with genetic deficiency of ecNOS (ecNOS-KO mice) develop severe ischemic necrosis following femoral artery ligation, which is refractory to exogenous VEGF treatment.11,12 This severe disease phenotype, which portrays the clinical symptoms of critical limb ischemia (CLI), is similar to that seen in old mice (>24 months old) without genetic deficiency of ecNOS.2,4
The similarity between the ischemic phenotype in ecNOS-KO mice and that seen in old wild-type mice provides (at least circumstantial) evidence that reduced EDNO production is indeed an important factor in the etiology of cardiovascular aging. However, other factors may also contribute to age-induced acceleration of ischemic limb damage.
To determine these factors without the confounding effect of age-dependent progressive EDNO impairment reported in wild-type animals,13 in the present study we investigated the effect of age on postischemic blood flow recovery and gross pathological changes in ecNOS-KO mice.
The main finding of the present study was that ischemic injury became more severe and postischemic revascularization deteriorated with age in ecNOS-KO animals, where progressive EDNO decrease cannot be the cause of the “aging process.” Significantly attenuated KDR expression in old compared to that seen in younger ecNOS-KO animals may provide a potential mechanistic explanation of the accelerated aging phenomenon seen in the absence of EDNO.
MATERIALS AND METHODS
Animals and Surgical Procedure
Male ecNOS-KO and C57BL/J6 mice (Jackson Laboratory, Bar Harbor, ME) at different ages (ecNOS-KO 3, 6, and 12 months old, and C57BL/J6 6 and 21 months old, respectively) were used for the experiments. The ecNOS-KO mice have been backcrossed to C57BL/J6 mice for >10 generations, therefore we used C57BL/J6 mice as control, when comparison needed to be made to wild-type animals. All mice were housed under controlled temperature (24°C) and lighting (14 : 10-h light-dark cycle) conditions with free access to food (normal chow) and water. The experiments were conducted according to protocols approved by the Animal Care Committee at Berlex Biosciences, in agreement with the recommendation of the American Association for the Accreditation of Laboratory Animal Care.
Before surgery, animals were anesthetized with 1.5% isoflurane inhalation. A skin incision (2 mm) was performed at the upper portion of the left hind limb overlying the femoral artery. Unilateral hind limb ischemia was established by resection of a proximal segment of the femoral artery leaving the femoral vein intact.12 Care was taken to leave the femoral nerve undamaged. Skin incisions were closed with 7.0-mm prolene nonabsorbable suture (Ethicon).
Hind limb blood flow perfusion was measured by using a laser Doppler perfusion imager (LDPI; Moor Instruments, Wilmington, DE) system. The LDPI uses a beam from a 2-mW helium-neon laser that sequentially scans a 7.5×7.5 cm tissue surface to a depth of 600 μm. The perfusion signal is split into 6 different intervals, and each is displayed as a separate color. Low or no perfusion was displayed as dark blue, whereas maximal perfusion was displayed as red. Temporal variations in tissue perfusion were displayed as a conventional plot after conversion to ASC II cord and exported to a spreadsheet software package. For each animal, perfusion flux (blood cells/unit area/unit time) was evaluated before the surgery to verify that the flow was similar in the 2 limbs, immediately after surgery to verify that the operated was ischemic, as well as serially at days 1, 3, 7, 10, 14, and 21 after induction of ischemia. In some instances the initial scan varied significantly from the subsequent scans and was not included in the data analysis. In such cases, scans were performed until the flux measurements were stable for 3 scans.
For LDPI measurements, the hair of anesthetic mice was removed from both hind limbs before imaging. To minimize temperature variations during the scanning the mice were placed on a heating pad maintained at 37°C for 10 min before as well as during scanning. To make sure the blood flow has maximal perfusion in the measure area, the skin temperature of each hind limb will be kept at 35°C with heating light. For analysis, equal areas (±5%) of the control and ischemic limbs from the same anatomical region of the limbs were compared. Mean flux in the selected areas was computed using the Moor imaging software. Data are presented as percentage of mean blood flux in the operated ischemic limb relative to flux in the unoperated control limb. Ischemic tissue damage of the hind limb was evaluated by taking digital images of the legs at days of the LDPI measurement.
Gene Expression Analysis in Skeletal Muscle Samples
To ensure unbiased sampling and careful analysis of gene expression in the hind limb skeletal muscle, the entire tibialis and/or adductor muscles were removed from all animals in each investigated group (n=8) after sacrificing the mice. The muscles were cut into small pieces, and the entire muscle was weighed and homogenized in RLT lysis buffer (Qiagen, Valencia, CA). The muscle samples of the operated (ischemic) and the contralateral (nonischemic) limbs of 3- and 6-month-old ecNOS-KO mice and from 6-month-old control mice (C57BL/J6) were collected 4 days following surgery or at the end of the experimental period on day 21. The earlier time point was selected within the acute phase of the angiogenic response following surgical hind limb ischemia.5 The tissue pieces were homogenized in 600 μL RLT buffer using nickel beads (Qiagen) or disposable generator probes (Omni International, Warrento, VA). Total RNA was isolated by RNeasy kit with DNase I digestion (Qiagen). Relative abundance of Sonic hedgehog (Shh), VEGF receptor-2 (KDR), and von Willebrand factor (vWF) compared to control represented by 18S rRNA were measured by real-time quantitative polymerase chain reaction performed on ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA). Primers and probe for mouse KDR (GenBank accession number NM_01062) were upper primer, 5′ AGG TTT GCG TGC TCT TCA CA 3′ (nt 4892–4911), lower primer, 5′ CGT AAG AGT CCG GAA GGA ACT C 3′ (nt 4979–4958) and probe, FAM 5′ CGA GTT CCC TGT GGC GTT TCC TAC TCC TA 3′TAMRA (nt 4925–4953). For mouse Shh (GenBank accession number BC063087) were upper primer, 5′-GAGGTGCAAAGACAAGTTAAATGC-3′ (nt 303–326), lower primer, 5′-CGGTCACTCGCAGCTTCAC-3′ (nt 361–379) and probe, 5′-6FAM-TTGGCCATCTCTGTGATGAACCAGTGG-TAMRA-3′ (nt 328–354). For mouse vWF (GenBank accession number AJ238390): upper primer, 5′-CCGGAAGCGACCCTCAGA-3′ (nt 288–305), lower primer, 5′-CGGTCAATTTTGCCAAAGATCT-3′′ (nt 410–389), and probe, 5′-6FAM-TGGCCTCTACCAGTGAGGTTTTGAAGTACACAC-TAMRA-3′ (nt 350–378). Expressions were calculated against a standard curve with serial dilutions of total RNAs. 18S rRNA probe and primers (Taqman Ribosomal RNA Control Reagent, cat. no. P/N 4308329) were purchased from Applied Biosystems. Analysis was performed in quadruplicate for each sample. Values presented as a ratio of 18S expression in mouse skeletal muscle homogenates.
All results are expressed as mean ± standard error of the mean. Statistical significance was evaluated using unpaired Student test or ANOVA for comparisons between 2 means with the aid of computer software (Statistica 6.0). A value of P < 0.05 was considered statistically significant.
ecNOS-KO Mice Exhibit Progressive Limb Loss and Accelerated Age-Dependent Impairment in Blood Flow Recovery Following Unilateral Femoral Artery Ligation
To elucidate the effect of aging in the absence of EDNO on postischemic revascularization we have performed unilateral femoral artery ligation in ecNOS-KO mice at 3, 6, and 12 months of age (n=8 mice in each group). The extent of initial acute ischemia, immediately after surgery (day 0), was similar in all groups. In the 3-month-old ecNOS-KO mice blood flow of the operated limb quickly, by day 7–10, returned to values, similar to that observed in the operated limb of 6-month-old control (C57BL/J6) mice (Figs. 1A, 2A). In the older ecNOS-KO groups blood flow recovery was significantly slower (Fig. 1A). In 6-month-old ecNOS-KO mice the severe prolonged ischemia resulted in toe necrosis and skin ulcers of the limb, whereas no gross pathological changes occurred in the 3-month-old group (Fig. 1B). In the 12-month-old ecNOS-KO group flow recovery was absent, and a complete loss of the operated limb occurred within 10 days after surgery (Figs. 1A, 1B). To assess age-related changes in control (C57BL/J6) mice a group of 21-month-old mice (n=5) were also subjected to unilateral hind limb ischemia. Flow recovery was somewhat impaired in the 21-month-old controls compared to the 6-month-old control group (Fig. 2A), but flow recovery in this old control group was still significantly better than in the 6-month-old ecNOS-KO mice (Fig. 1A). There was no visible ischemic tissue damage in any of the control animals studied (Fig. 2B).
Quantitation of Postischemic Revascularization in Mice With Severe Limb Necrosis
The severe ischemia resulted in limb loss in some of the 6-month-old and in all of the 12-month-old ecNOS-KO, therefore unbiased quantitation of angiographic pictures using anatomical reference points became impossible. Therefore, we chose a biochemical endpoint to quantify angiogenesis (ie, quantitative real-time-polymerase chain reaction analysis of the endothelial cell marker vWF mRNA using total RNA isolated from skeletal muscle samples. By the end of the ischemic recovery period a significant increase in vWF mRNA expression was detected in samples from the ischemic limb of the 3-month-old ecNOS-KO mice compared to the contralateral nonischemic samples (Fig. 3). In contrast, the level of 18S normalized vWF expression was significantly (*P<0.05) decreased in samples from the ischemic limbs of the 6-month-old mice compared with the samples from the contralateral nonischemic side indicating impaired angiogenesis in association with the severe ischemic injury (Fig. 3). The significant decrease (+P<0.05) in angiogenesis in the 6-month-old animals in comparison to the 3-month-olds indicates impaired age-related angiogenesis in ecNOS-KO mice.
Ischemia Associated Increase in KDR Expression is Absent in Older ecNOS-KO Mice With Severe Limb Necrosis
KDR expression was determined in the same samples used to measure vWF expression. In the 3-month-old ecNOS-KO mice KDR expression was significantly increased in the ischemic muscle samples compared with the contralateral, unoperated side (Fig. 4A). In 6-month-old mice, the level of KDR expression was significantly decreased (Fig. 4A). To determine changes in KDR expression independent of changes in endothelial cell numbers, we normalized KDR expression to vWF expression, which revealed a significantly enhanced KDR expression in the muscles harvested from the ischemic side of the 3-month-old animals in contrast to the 6-month-old ecNOS-KO mice developing the severe ischemic phenotype (Fig. 4B). The lack of postischemic KDR increase in the 6-month-old ecNOS-KO mice compared to 3-month-old samples resulted in a significant age-dependent decrease in the expression of the receptor (+P<0.05).
Postischemic Upregulation of Shh is Independent of EDNO
We have also investigated the expression pattern of Shh between samples collected from 6-month-old ecNOS-KO and C57BL/J6 mice (Fig. 5). We found no difference in the ischemia-induced changes in Shh expression in ecNOS-KO mice and that seen in age-matched control mice. There was also no statistical difference in basal Shh levels in samples obtained from ecNOS-KO and C57BL/J6 mice (Fig. 5).
The main findings of this study are as follows:
1. Significantly accelerated “age-dependent” impairment in ischemic limb injury and revascularization in ecNOS-KO mice compared with C57BL/J6 controls, confirming the important protective role of EDNO in this model.
2. The demonstration of progressive age-dependent impairment in postischemic revascularization and ischemic injury in ecNOS-KO mice, in the absence of progressive EDNO impairment.
3. Reduced postishemic expression of the KDR receptor in older ecNOS-KO mice compared to younger ecNOS-KOs, which may be one of the “EDNO-independent” factors contributing to age-induced aggravation of ischemic injury in older ecNOS-KO mice.
4. Unaltered ischemic regulation of Shh expression in ecNOS-KO mice compared with C57BL/J6 controls, which implies for the first time that hypoxic/ischemic upregulation of Shh is independent of EDNO.
Progressive EDNO Impairment-independent “Cardiovascular Aging”
Reduced collateral development has been demonstrated previously in 2-year-old mice compared to young animals.2 Impaired endothelial function and reduced availability of EDNO have been suggested to play a major role in this process.2 Our results confirmed the original hypothesis about the role of EDNO as an important factor in postischemic revascularization in mice. It was, however, unexpected that the lack of ecNOS accelerates the “aging process.” Severe limb ischemia resulting in symptoms such as ulceration and toe necrosis, which has been seen as early as the 6-month-old ecNOS-KO mice in our studies, was previously described in C57BL/J6 mice only beyond 2 years of age.2,4 In the present study, the oldest group of C57BL/J6 mice was the 21-month-old group, and although blood flow recovery in this group was impaired compared to the 6-month-old group, there has been no visible sign of ischemic toe and limb necrosis in these animals.
The severity of the ischemic damage and extent of limb necrosis in older ecNOS-KO mice limited the possibilities to evaluate postischemic revascularization using methods (ie, angiography and histology, which have been reported previously12,16). Instead of these methods we used an endothelial cell-specific marker gene, vWF, expression to quantitate angiogenesis from skeletal muscle homogenate. The results confirmed increased angiogenesis in 3-month-old ecNOS-KO mice, whereas in 6-month-old animals the severe ischemic disease phenotype corresponded with significantly impaired angiogenesis. Three-month-old ecNOS-KO mice showed no defect in postischemic blood flow recovery compared to wild-type controls, indicating that EDNO deficiency can be compensated by other factors in younger animals or that more prolonged absence of EDNO (>3 months) is needed for the development of deleterious effects on the revascularization potential.
Potential Role of Impaired KDR Regulation
To explore the potential mechanism(s) contributing to the phenomenon of accelerated “aging” we focused our investigation on determining the regulation of the VEGF receptor KDR expression. Earlier reports investigating the effect of surgical hind limb ischemia in different strains of mice indicated correlation between the expression level of the KDR receptor and the severity of ischemic injury.14 We found significantly elevated KDR levels, even after normalization to vWF expression, in the muscle samples from the ischemic limb (when compared to nonischemic contralateral limbs) of the 3-month-old ecNOS-KO mice.
KDR is a key receptor mediating the angiogenic effect of VEGF.17 Inhibition of the KDR receptor inhibits VEGF-induced endothelial cell proliferation, migration, and resistance to apoptosis via the activation of the serine/threonine kinase Akt/protein kinase B.18,19 This increase in KDR levels was absent in the ischemic muscles obtained from 6-month-old ecNOS-KO mice. The missing component of angiogenic growth factor signaling via KDR may have contributed to the development of the severe disease phenotype.
Upregulation of Shh in Response to Ischemia is NO-independent
The role of the hedgehog pathway has received attention recently as a potentially important regulator of angiogenesis and muscle regeneration following ischemia.15 Inhibition of Shh expression has been shown to result in decreased VEGF production and to impair postischemic revascularization in mice following surgical hind limb ischemia.15 The particular importance of this pathway in the aging process was first demonstrated by the therapeutic benefit of Shh protein administration in 2-year-old mice.4 The similarity of the ischemic pathology between the 2-year-old mice reported by Pola et al4 and our 6-month-old ecNOS-KO mice prompted us to evaluate potential changes in the ischemic regulation of Shh in the absence of EDNO in the ecNOS-KO mice.
We found significant hypoxia/ischemia induced Shh upregulation in all ischemic muscle samples, in both ecNOS-KO and wild-type mice. The mechanism by which ischemia upregulates Shh expression is presently unknown. Our findings, however, indicate that hypoxic/ischemic upregulation of the Shh gene does not require an intact ecNOS/EDNO system, and rule out the possibility that impaired ischemic Shh regulation contributed to the accelerated ischemic damage in the ecNOS-KO mice.
Reduced KDR levels likely result in impaired function of the Akt signal transduction pathway in response to VEGF stimulation. Inadequate levels of KDR could render this pathway insufficient to substitute for the missing antiapoptotic and proangiogenic effects of EDNO in ecNOS-KO mice. This could explain the rapid acceleration of the ischemic injury and impaired postischemic revascularization observed in the aging ecNOS-KO mice.
Older patients with chronic advanced peripheral arterial occlusive disease often exhibit reduced levels of angiogenic factors and impaired EDNO-mediated relaxation.20 The present study provides evidence that impairment of the components of angiogenesis could lead to severe pathology, similar to the clinical symptoms of CLI. Characterization of angiogenic growth factors and their receptors in older patients with endothelial dysfunction can possibly aid the design of a more efficient, individualized therapeutic strategy to slow the progression of peripheral cardiovascular disease.
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