Journal of Vascular Surgery
Volume 51, Issue 1 , Pages 165-173, January 2010

Endothelial nitric oxide synthase affects both early and late collateral arterial adaptation and blood flow recovery after induction of hind limb ischemia in mice

  • Brian Park, MD

      Affiliations

    • Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Mass
    • ,
  • Ari Hoffman, MD

      Affiliations

    • University of California at San Francisco, San Francisco, Calif
    • ,
  • Yagai Yang, PhD

      Affiliations

    • University of California at San Francisco, San Francisco, Calif
    • ,
  • Jinglian Yan, PhD

      Affiliations

    • Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Mass
    • ,
  • Guodong Tie, PhD

      Affiliations

    • Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Mass
    • ,
  • Hossein Bagshahi, MD

      Affiliations

    • Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Mass
    • ,
  • Philip T. Nowicki, MD

      Affiliations

    • Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Mass
    • ,
  • Louis M. Messina, MD

      Affiliations

    • Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Mass
    • Corresponding Author InformationReprint requests: Louis M. Messina, MD, University of Massachusetts Medical School, 55 Lake Ave N, Worcester, MA 01655

Received 20 May 2009; accepted 12 August 2009. published online 02 November 2009.

Article Outline

Objective

The goals of this study were to determine if endothelial nitric oxide synthase (eNOS) affects both early and late collateral arterial adaptation and blood flow recovery after severe limb ischemia in a mouse model and to determine if eNOS-derived NO is necessary for recruitment of chemokine (C-X-C motif) receptor 4 (CXCR4)+ vascular endothelial growth factor receptor-1 (VEGFR1)+ hemangiocytes to the site of ischemia.

Methods

Two studies were completed. In the first, hind limb ischemia was induced by unilateral femoral artery excision in three groups: C57Bl6 (wild-type), eNOS−/−, and C57Bl/6 mice treated with NG-nitro-l-arginine methyl ester (L-NAME) from 1 day before excision through day 3 after excision (early L-NAME group). These groups were studied on day 3 after induction of ischemia. In the second study, hind limb ischemia was induced in C57Bl/6 mice (wild-type) and C57Bl/6 mice treated with L-NAME from days 3 through 28 after induction of ischemia. These groups were studied day 28 after ischemia induction. Dependent variables included hind limb perfusion, collateral artery diameter, and the number and location of hemangiocytes within the ischemic hind limb.

Results

In the first study, toe gangrene developed in the eNOS−/− and early L-NAME treatment groups by day 2. These groups demonstrated less blood flow recovery and smaller collateral artery diameter than the wild-type group. Hemangiocytes were present within the adventitia of collateral arteries in the wild-type group but were only sparsely present, in a random pattern, in the eNOS−/− and early L-NAME treatment groups. In the second study, the late L-NAME group showed less blood flow recovery and smaller collateral artery diameter on day 28 of ischemia than the wild-type group. Hemangiocytes were present in a pericapillary distribution in the wild-type group, but were present only sparsely in the late L-NAME treatment group.

Conclusion

Early (day 3) and late (day 28) adaptive responses to hind limb ischemia both require eNOS–derived NO. NO is necessary for normal hemangiocyte recruitment to the ischemic tissue.

Clinical Relevance

This study demonstrates that endothelial nitric oxide synthase (eNOS)-derived NO is requisite for both the early and late vascular recovery phases in response to hind limb ischemia. Moreover, it demonstrates that recruitment of hemangiocytes, a specific subset of vascular progenitor cells that display vascular endothelial growth factor receptor 1 (VEGFR1) and chemokine (C-X-C motif) receptor 4 (CXCR4) surface antigens, is dependent on the presence of NO. These findings enhance understanding of the basic biologic mechanisms in the recovery from ischemia. The need for eNOS-derived NO suggests that clinical strategies to enhance postocclusive flow recovery in peripheral artery disease might be more successful if eNOS or NO, or both, are applied as a part of the treatment paradigm. Hemangiocytes are an important part of the cellular response to ischemia. The present findings underscore their importance and suggest that preservation or enhancement of eNOS within the ischemic limb might improve the recruitment of these critical reparative cells to the site of ischemic injury.

 

Critical limb ischemia secondary to peripheral arterial disease is a debilitating and potentially lethal disease. Critical limb ischemia is responsible for >150,000 amputations each year in the United States.1 In many patients, surgical or catheter-based revascularization procedures are not possible, and amputations are necessary. For these reasons, the development of molecular or cell-based therapies to enhance collateral artery enlargement and angiogenesis continues to be an area of substantial scientific and clinical interest.

Enlargement of the diameter of existing collateral arteries is the means by which blood flow downstream from the site of conduit artery occlusion is restored.2, 3 At least three mechanisms participate in this process: shear stress,4, 5 inflammation,6 and recruitment of bone marrow-derived vascular progenitor cells to areas of ischemia.7, 8, 9, 10 Nitric oxide (NO) is a component in each of these mechanisms.11, 12, 13, 14

During the early adaptive response to ischemia, increased shear stress within collateral arteries leads to NO-induced vasodilation that results in a transient, temporary increase in flow through the collaterals.11 During the late adaptive response to ischemia, NO participates in the recruitment of vascular progenitor cells that generate collateral artery remodeling, a process termed arteriogenesis, wherein net conductance across the collateral vessels is permanently increased.12, 13, 14

It is not universally accepted that NO derived from the endothelial isoform of nitric oxide synthase (eNOS) is requisite for arteriogenesis.11 In addition, the involvement of NO in the recruitment of hemangiocytes, a critically important subset of vascular progenitor cells,10, 15 has not been defined. This study was designed to determine if eNOS-derived NO is essential for both the early and late adaptive response to ischemia—to arteriogenesis—and to determine the importance of NO in hemangiocyte recruitment. We hypothesized that (1) eNOS-derived NO production is essential for collateral artery remodeling, or arteriogenesis, during the late adaptive response to ischemia and (2) recruitment of bone marrow-derived hemangiocytes to the ischemic hind limb vasculature requires the presence of eNOS-derived NO.

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Materials and methods 

Experimental animals and procedures 

The care of mice used in this study complied with the National Research Council's Guide for the Care and the Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts (Worcester, Mass).

C57Bl/6 mice and eNOS−/− mice (Jackson Laboratories, Bar Harbor, Me) were fed standard chow and maintained on a 12-hour light/dark cycle. Hind limb ischemia was generated by ligation of the femoral artery at the inguinal ligament and popliteal fossa, followed by excision of the artery and all branches.16 The procedure was done under 3% isoflurane anesthesia. Buprenorphine was administered during the immediate postoperative period.

Experimental protocols 

The early and late adaptive responses to acute hind limb ischemia were evaluated in separate studies. The early adaptive response to ischemia (vasodilation) was studied 3 days after unilateral femoral artery excision in three groups: C57Bl/6 mice (wild-type group), mice with targeted disruption of the eNOS loci (eNOS−/− group), and C57Bl/6 mice treated with NG-nitro-l-arginine methyl ester (L-NAME; 1 g/L) added to the drinking water 24 hours before femoral artery excision (early L-NAME group). The L-NAME treatment was continued until sacrifice on day 3 after induction of ischemia, a time selected because preliminary work indicated that mice in the eNOS−/− and early L-NAME groups uniformly developed limb gangrene by this time.

The late adaptive response to ischemia (arteriogenesis) was studied 28 days after induction of ischemia in two groups: C57Bl/6 mice (wild-type group) and C57Bl/6 mice treated with L-NAME (1 g/L in drinking water) beginning on day 3 after the induction of hind limb ischemia (late L-NAME group). The L-NAME treatment was continued until sacrifice. Day 28 after induction of hind limb ischemia was chosen as the time for study in the late adaptive response experiment because published reports indicate that postischemia arteriogenesis is evident at that time.3 The late adaptive response to ischemia was not studied in eNOS−/− mice because significant autoamputation consistently developed in these animals consistently by day 3 after induction of ischemia and we did not believe it was ethically appropriate to keep them alive >3 days.

Laser Doppler perfusion imaging 

A laser Doppler perfusion imager (LDPI; Moor Instruments Ltd, Devon, United Kingdom) was used to estimate blood flow within calf muscles. LDPI-derived data strongly correlate with calf muscle blood flow measured directly using microspheres, and the method is widely used to quantify perfusion in the rodent hind limb.16

Hair from limbs was removed by depilatory cream, and mice were placed on a heating plate at 37°C to minimize temperature variation. Studies were conducted under 1.5% isoflurane anesthesia. Data are presented as the ratio of perfusion to the ischemic hind limb and the contralateral nonischemic hind limb.

Immunostaining of thigh collateral arteries 

The thigh muscles were harvested on day 3 or 28 after induction of ischemia in the early or late adaptive response groups, respectively. Collateral arteries were identified by double staining with antibodies for CD31 (platelet endothelial cell adhesion molecule-1, a marker of endothelial cells) and smooth muscle actin (both antibodies from BD Biosciences, San Jose, Calif). Collateral artery diameter was measured in 10 randomly selected low-power fields using precalibrated microscope calipers (Carl Zeiss, Göttingen, Germany), and the average diameter for the ischemic and contralateral nonischemic hind limb was determined for each mouse. Collateral arterial diameter was expressed as a ratio of the diameters in ischemic and nonischemic hind limbs. All measurements were made in a blinded manner.

Immunostaining for hemangiocytes 

The presence of hemangiocytes was measured in thigh muscles on day 3 or 28 after the induction of ischemia in the early and late adaptive response groups, respectively. Hemangiocytes were identified by double staining for chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor receptor 1 (VEGFR1; both antibodies from BD Bioscience).10 The number of hemangiocytes was determined in 10 randomly selected high-power fields in ischemic and contralateral and nonischemic thigh muscle, and the average was calculated. Observations were made in a blinded manner.

Confocal microscopy 

A Leica DM IRBE confocal microscope was used (Leica Microsystems, Wetzler, Germany). Cy3-conjugated CXCR4 and fluorescein isothiocyanate-conjugated VEGFR1 antibodies (BD Bioscience) were used to identify hemangiocytes. Nuclei were identified by Draq5 staining (Biostatus Limited, Leicestershire, United Kingdom).

Nitrate/nitrite assay 

Serum samples were processed using a nitrate/nitrite (NOx) fluorometric assay kit (Cayman Chemical, Ann Arbor, Mich), which determines the concentration of NO2– and NO3–, the final products generated during NO metabolism in vivo. Fluorescence was detected using a Biotek Synergy 2 detector (BioTek Instruments Inc, Winooski, Vt).

Statistical analysis 

Differences among groups at each time point were analyzed by analysis of variance (ANOVA) for comparisons between three groups of mice, and the t test for comparisons between two groups, where appropriate. If the ANOVA f statistic was significant (P < .05), then post hoc Student-Newman-Keuls tests were done to determine the sites of significance at the P < .05 level threshold.

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Results 

Early adaptive response to hind limb ischemia 

The serum NOx concentration in the eNOS−/− group was 67% less than that noted in the wild-type group. Treatment of C57Bl/6 mice with L-NAME reduced serum NOx by 71% on day 1 and by 66% on day 3. These levels were similar to those noted in the eNOS−/− group (Fig 1).

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  • Fig 1. 

    Serum nitrite (NOx) concentration in wild-type, endothelial nitric oxide synthase (eNOS−/−) and early NG-nitro-l-arginine methyl ester (L-NAME) treatment groups. The serum NOx level was lower in the eNOS−/− mice than in the wild-type mice. L-NAME significantly reduced serum NOx levels ≤1 day after beginning L-NAME treatment, and the reduction was sustained throughout the duration of L-NAME treatment.

The eNOS−/− and early L-NAME treatment groups demonstrated significant toe necrosis by the second day after induction of ischemia, whereas wild-type mice did not (Fig 2, A). All three groups demonstrated a significant reduction of flow to the ischemic hind limb immediately after femoral artery excision, whereas flow to the contralateral hind limb did not change; consequently, the ischemic/nonischemic limb flow ratio decreased in all three groups. This circumstance remained unchanged in all three groups through day 2. On day 3, however, the wild-type group demonstrated an increase in flow to the ischemic hind limb, resulting in an ischemic/nonischemic flow ratio that was more than twofold greater than that noted in the eNOS−/− or L-NAME groups (Fig 2, B). The ischemic/nonischemic collateral artery diameter ratio was 45% lower in the eNOS−/− group and 55% lower in the early L-NAME group than in the wild-type group on day 3 (Fig 2, C).

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  • Fig 2. 

    Responses of wild-type, endothelial nitric oxide synthase (eNOS−/−) and early NG-nitro-l-arginine methyl ester (L-NAME) treatment groups to hind limb ischemia. A, Effects of femoral artery excision on hind limb tissue integrity are shown in photographs that are representative of all animals in each group. The ischemic hind limb is shown on the left and white arrows in eNOS−/− and L-NAME mice denote evidence of tissue necrosis. B, Laser Doppler perfusion imager (LDPI) data are shown. Each mouse underwent 3 scans at each time point, and the ratio of the LDPI signal from the ischemic and nonischemic hind limb was calculated; the average ratio was used as a single data point for each mouse. C, Collateral artery diameter on day 3 after the induction of hind limb ischemia was determined as the average of the largest arteries identified within 10 randomly selected low-power fields for each mouse by a blinded observer. The ratio of the average diameter in the ischemic thigh to the average diameter in the nonischemic thigh was determined for each mouse and used as a single data point.

The number of CXCR4+ VEGFR1+ hemangiocytes within the ischemic thigh muscle was 86% less in the eNOS−/− and early L-NAME treatment groups than in the wild-type group (Fig 3, A). These cells were clearly aligned along collateral arteries in the ischemic thigh musculature in the wild-type group (Fig 3, B). Confocal microscopy revealed that hemangiocytes were primarily present within the adventitia of the collateral arteries within the ischemic thigh in the wild-type group (Fig 3, C).

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  • Fig 3. 

    Presence of hemangiocytes in the thigh musculature in wild-type, endothelial nitric oxide synthase (eNOS−/−), and early NG-nitro-l-arginine methyl ester (L-NAME) treatment groups on day 3 after induction of hind limb ischemia. A, Quantitation of hemangiocytes, defined as cells that colocalized chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor receptor (VEGFR) 1, was determined as the average number of hemangiocytes present in each mouse in 10 randomly selected high-power fields, determined by a blinded observer. B, In representative photomicrographs (original magnification ×200) of hemangiocyte immunostaining, VEGFR1 is stained green, CXCR4 is stained red, and nuclei are stained blue. A collateral vessel is present in each photomicrograph, evidenced by the linear cord of blue-stained nuclei. Note the presence of double-stained hemangiocytes clustered along the collateral artery in the ischemic thigh of the control mouse. C, Confocal microscopy of a collateral artery within the ischemic thigh of the wild-type group. The left photo is shown at original magnification ×200. The area delineated by the white square in the left photo is shown at original magnification ×400 in the right photo. Note the presence of a hemangiocyte in the adventitial area of a collateral artery. This adventitial distribution of hemangiocytes was commonly observed in the ischemic thigh muscle of wild-type mice, but it was never observed in eNOS−/− or early L-NAME treatment groups.

Late adaptive response to hind limb ischemia 

L-NAME treatment in the late adaptive response experiment was initiated on the third day after induction of hind limb ischemia, after the LDPI measurement was obtained. Blood flows to the ischemic and nonischemic hind limbs were similar in the wild-type and late L-NAME treatment groups through day 7. Beginning on day 14, blood flow to the ischemic hind limb became significantly greater in the wild-type group than the late L-NAME group, whereas flows to the contralateral nonischemic hind limb were unchanged; accordingly, the ischemic/nonischemic flow ratios in the L-NAME group were 33% and 44% less than of those in the wild-type group on days 14 and 28, respectively (Fig 4, A). The ischemic/nonischemic thigh collateral diameter ratio was 60% less in the late L-NAME group than the wild-type group on day 28 after induction of hind limb ischemia (Fig 4, B).

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  • Fig 4. 

    Response of wild-type and late NG-nitro-l-arginine methyl ester (L-NAME) treatment groups to hind limb ischemia. A, Laser Doppler perfusion imager (LDPI) data. Each mouse underwent 3 scans at each time point, and the ratio of the LDPI signal from the ischemic and nonischemic hind limb was calculated; the average ratio was used as a single data point for each mouse. B, Collateral artery diameter on day 28 after induction of hind limb ischemia was determined as the average of the largest arteries identified in each mouse within 10 randomly selected low-power fields by a blinded observer. The ratio of the average diameter in the ischemic thigh to the average diameter in the nonischemic thigh was determined for each mouse and used as a single data point.

The number of CXCR4+ VEGFR1+ hemangiocytes present within the ischemic thigh muscles was 56% less in late L-NAME treatment group than in the wild-type group on day 28 after induction of hind limb ischemia, whereas the number of hemangiocytes present within the contralateral nonischemic thigh muscles was similar in both groups (Fig 5, A). Interestingly, the distribution of these cells was different than that observed in the early adaptive response study on day 3 after induction of hind limb ischemia. Thus, hemangiocytes were present between myofibers at the sites occupied by capillaries (Fig 5, B). Confocal microscopy confirmed the pericapillary location of these hemangiocytes (Fig 5, C).

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  • Fig 5. 

    Presence of hemangiocytes in the thigh musculature in control and late NG-nitro-l-arginine methyl ester (L-NAME) treatment groups 28 days after induction of hind limb ischemia. A, Quantitation of hemangiocytes, defined as cells that colocalized chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor receptor (VEGFR) 1, was determined as the average number of hemangiocytes present in each mouse in 10 randomly selected high-power fields, determined by a blinded observer. B, In representative photomicrographs of hemangiocyte immunostaining, VEGFR1 is stained green, CXCR4 is stained red, and nuclei are stained blue. In contrast to the data obtained at 3 days after the induction of hind limb ischemia (Fig 2, B), note that hemangiocytes are present in the areas between myocytes; that is, at the sites of capillaries (original magnification ×200). C, Confocal microscopy of the ischemic thigh of the wild-type group. The left-hand photo is shown at original magnification ×200. The area delineated by the white square in the left photo is shown at original magnification ×400 in the right photo. Note the presence of hemangiocytes in the pericapillary region.

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Discussion 

Adaptations within existing collateral arteries occur after femoral artery occlusion to restore vascular conductance and, hence, limb blood flow. The early adaptation is vasodilation, whereas the late adaption is arteriogenesis, the later being a process of collateral artery remodeling.3 Research has established that eNOS-derived NO mediates collateral artery vasodilation during the early adaptive response to ischemia; however, the role of eNOS-derived NO in arteriogenesis remains unclear.11, 17, 18, 19, 20, 21 The present studies support a role for eNOS-derived NO in both early and late collateral artery adaptations. In addition, this study provides the first evidence, to our knowledge, that eNOS is requisite for recruitment of hemangiocytes, a unique subset of bone marrow-derived vascular progenitor cells,10, 15 to the ischemic vasculature.

Occlusion of the femoral artery increases the rate of blood flow through pre-existing, small-diameter collateral arteries. This circumstance increases wall shear stress in these vessels, a mechanical stimulus that increases eNOS expression and activity, the latter through eNOS phosphorylation.5, 17 These events significantly enhance NO production and induce vasodilation of existing collateral arteries; new collateral arteries are not created during this process of arteriogenesis.3, 5, 17 The present findings are fully consistent with this process, as collateral artery diameter and perfusion within the ischemic hind limb were less on day 3 after induction of ischemia in the eNOS−/− and early L-NAME treatment groups than in the wild-type group.

It is known that the early postischemic, NO-induced vasodilation of collateral arteries is short-lived.3, 11, 18 Shear stress, the primary stimulus for this event, dissipates during vasodilation because shear stress is inversely proportional to the fourth power of the vessel radius.18 This temporary vasodilation gives way to collateral artery remodeling, or arteriogenesis, a process that permanently increases net collateral artery conductance.2, 3 Heretofore, it was not clear if eNOS-derived NO participates in the process of arteriogenesis. Yu et al19 and Lloyd et al20 demonstrated reduced postischemic arteriogenesis when eNOS activity was blocked or eliminated; in contrast, Mees et al11 failed to demonstrate an effect of eNOS-derived NO beyond 2 weeks after induction of ischemia. The present findings support a role for NO during arteriogenesis, because collateral artery diameter and blood flow within the ischemic hind limb were significantly less in the late L-NAME treatment group than in the wild-type group 28 days after the induction of ischemia. Collectively, our findings indicate that NO is an active participant in postischemic vascular adaptation for at least the first month after the induction of ischemia.

An alternative means to evaluate the role of eNOS in postischemic arteriogenesis is by enhancing eNOS activity, a circumstance that might be predicted to increase collateral artery growth and blood flow recovery. This predication has been confirmed: the administration of adenovirus vectors encoding eNOS to the ischemic hind limb,21 overexpression of constitutively active eNOS in the ischemic hind limb,11 or dietary supplementation with L-arginine17 significantly improved postischemic arteriogenesis. Restitution of eNOS activity or administration of exogenous NO in the late L-NAME treatment group could provide additional support for the experimental hypothesis, if it restored arteriogenesis. However, coadministration of L-arginine and an arginine analogue such as L-NAME under in vivo conditions, or systemic administration of NO donor agents such as sodium nitroprusside, S-nitrosoglutathione, or 3-morpholinosydnonimine, could also generate confounding results because it would not be clear if these agents reached the ischemic hind limb.

Femoral artery excision establishes an inflammatory state within the ischemic tissue; monocytic6 and lymphocytic22 infiltration are requisite for postischemic arteriogenesis and angiogenesis. Activity of the Ca2+-independent isoform of nitric oxide synthase (iNOS) is increased in homogenates of ischemic hind limb muscle prepared 14 days after induction of ischemia; protein and messenger RNA expression of iNOS were not determined, nor was the specific localization of the iNOS enzyme within the tissue (personal communication, Jinglian Yan, Worcester, Mass, 2009).

The arginine analogue L-NAME interacts with the arginine binding sites of both eNOS and iNOS and, although then half maximal inhibitory concentration for L-NAME for each enzyme (3.1 μM for iNOS, 0.35 μM for eNOS), indicate a tenfold selectivity of eNOS > iNOS,24 it is likely that some L-NAME–induced inhibition of iNOS occurred in this study. In contemplating a possible role for iNOS-derived NO in arteriogenesis, consideration must be given to the site of NO production, as the exceedingly brief half-life of NO precludes a direct effect at a distance far removed from its point of origin. Additional studies of iNOS are warranted, with specific attention to the location and cellular source of iNOS expression within the ischemic hind limb; moreover, iNOS inhibition with 1400W, which has about a 105-fold specificity for iNOS > eNOS,23 might clarify the putative role of iNOS-derived NO in arteriogenesis.

An important and novel finding from the present experiments is that eNOS-derived NO is requisite for recruitment of hemangiocytes to the site of ischemic injury. Hemangiocytes, first described by Jin et al,10 represent a unique subset of bone marrow-derived progenitor cells that participate in postischemic vascular repair.10, 15 Hemangiocytes differ from endothelial progenitor cells based on surface antigens; thus, hemangiocytes express CXCR4 and VEGFR1, whereas endothelial progenitor cells express CD31, CD133, and VEGFR2.7, 8 It is well established that eNOS-derived NO participates in the recruitment of endothelial progenitor cells to the site of ischemic vascular injury.12, 13, 14

The present findings indicate a similar interaction for hemangiocytes. Specifically, hemangiocytes were evident in the adventitia of collateral arteries in the wild-type group by day 3 after induction of hind limb ischemia, whereas these cells were present only sporadically within the ischemic thigh when eNOS-derived NO was not present, that is, in the eNOS−/− or early L-NAME treatment groups. Mobilization and homing of CXCR4 hemangiocytes is also contingent on the chemokine stromal-derived factor-1α (SDF-1α),10 and an interaction between eNOS-derived NO and SDF-1α has been described;13 in this context, measurement of SDF-1α in the ischemic hind limb of eNOS−/− mice and the use of CXCR4 blocking antibodies in this group represent two potentially useful lines of further investigation.

Another novel finding from these experiments is the change in location of hemangiocytes during the course of postischemic vascular recovery: on day 3, these cells were present along collateral arteries, the site anticipated where these cells would be participating in arteriogenesis, whereas on day 28, these cells were present in a pericapillary location. Angiogenesis, defined as the de novo generation of new capillaries, is an essential part of postischemic vascular adaptation.3 The capillary/myofiber ratio, a marker for angiogenesis, was not measured in this study; however, the presence of hemangiocytes in the pericapillary regions between muscle cells might indicate that angiogenesis had occurred. Moreover, we propose that NO is requisite for recruitment of hemangiocytes to the per-capillary location, as evidenced by the significant reduction of pericapillary hemangiocytes in the late L-NAME treatment group.

Most of the hemangiocytes seen within capillaries exhibited fragmentation of their nuclei, an observation that suggests apoptosis. It has been proposed that progenitor cells serve a paracrine effect; thus, these cells affect existing vascular cells, inducing repair and replication, rather than becoming permanently incorporated into existing vascular tissue.26

Interestingly, gangrene of the toes consistently occurred in eNOS−/− and early L-NAME–treated mice by day 3 after induction of hind limb ischemia but was never observed in late L-NAME–treated animals. One explanation for this finding is that the delay in initiating L-NAME treatment until day 3 after ischemia allowed sufficient time for NO-induced recruitment of hemangiocytes to pre-existing collateral arteries in a manner similar to that observed in the wild-type group studied on day 3. These cells have the potential to restructure the ischemic vasculature by direct12, 13, 14 and paracrine24 actions, improving vascular conductance and hence perfusion. Another explanation is that mice in the eNOS−/− and early L-NAME treatment groups were unable to generate the immediate eNOS-derived vasodilation of collateral arteries, a circumstance that would lead to a profound compromise in downstream perfusion.

We recognize two limitations to the interpretation of our results. First, the technique of femoral artery excision produces a sudden and very severe level of ischemia, whereas the disease process of critical limb ischemia in humans develops over a period of years.1 A more gradual, clinically duplicative model of femoral arterial occlusion has been described in rats that causes less inflammation and less tissue necrosis but lower levels of blood flow recovery than after acute occlusion.16 This model uses an ameroid constrictor, however, which is too large for use in the mouse hind limb.

Second, we did not use perfusion fixation at an in situ pressure during preparation of the thigh muscles before collateral artery diameter measurement; instead, this tissue was prepared at an effective pressure of 0 mm Hg. This approach certainly limited the diameter of collateral arteries at the time of measurement. The difference in collateral artery diameter among study groups was quite substantial, however, and collateral wall thickness did not vary among groups. It is thus unlikely that the use of perfusion fixation would have altered the outcome of collateral artery diameter.

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Conclusions 

The present findings are relevant to human peripheral arterial disease (PAD). PAD patients who develop intermittent claudication or critical limb ischemia have been shown to have endothelial dysfunction characterized by decreased NO bioavailability.25 This reduced NO bioavailability likely results in inadequate collateral artery enlargement in PAD. In addition, reduced NO bioavailability may impede the efficacy of molecular or chemical therapeutics in PAD. Clinical trials administering either recombinant VEGF26 or recombinant fibroblast growth factor-227 to PAD patients have demonstrated therapeutic efficacy only after 60 to 90 days of treatment. VEGF and recombinant fibroblast growth factor-2 both exert their effects by upregulation and activation of eNOS.28, 29 The present study indicates that eNOS-derived NO is requisite for acute adaptation to severe ischemia to prevent tissue necrosis and for sustained arteriogenesis. We propose that treatment with agents known to enhance eNOS activity may prove a useful adjunctive therapy in PAD. Only through a complete understanding of the specific and temporally distinct eNOS-related recovery responses can we hope to achieve effective molecular and cell-based therapies for human patients.

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Author contributions 


Conception and design: BP, LM

Analysis and interpretation: BP, LM

Data collection: BP, AH, YY, JY, GT, HB

Writing the article: BP, PN

Critical revision of the article: BP, PN, LM

Final approval of the article: LM

Statistical analysis: BP

Obtained funding: LM

Overall responsibility: LM

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We thank Jianming Li and Phong Dargon for assistance in obtaining confocal microscopy images.

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References 

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 This work was funded by HL75353 to Dr Messina.

 Competition of interest: none.

 The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a competition of interest.

PII: S0741-5214(09)01686-3

doi:10.1016/j.jvs.2009.08.045

Journal of Vascular Surgery
Volume 51, Issue 1 , Pages 165-173, January 2010

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