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Cardiovascular Research 2006 69(4):925-935; doi:10.1016/j.cardiores.2005.12.005
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Copyright © 2006, European Society of Cardiology

Efficacy of systemic administration of SDF-1 in a model of vascular insufficiency: Support for an endothelium-dependent mechanism

Andrew N. Carra, Brian W. Howarda, Hsiao T. Yangb, Elaine Eby-Wilkensa, Paula Loosa, Alex Varbanova, Angela Qua, Jeffrey P. DeMutha, Michael G. Davisa, Alan Proiac, Ronald L. Terjungb and Kevin G. Petersa,*

aProcter and Gamble Pharmaceuticals, Cardiovascular Research Division, Health Care Research Center, 8700 Mason Montgomery Rd, Box 1064, Mason, Ohio, 45040, United States
bDepartment of Biomedical Sciences, University of Missouri, E102 Veterinary Medicine Bldg. Columbia, Missouri, 65211, United States
cDepartment of Pathology, Duke University Medical Center, Room 3078 Duke Hospital South, Durham, North Carolina, 27710, United States

* Corresponding author. Tel.: +1 513 622 0834; fax: +1 513 622 1433. Email address: peters.kg{at}pg.com

Received 23 September 2005; revised 30 November 2005; accepted 5 December 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Studies have reported that administration of stromal cell-derived factor-1 (SDF-1), the ligand for the G-protein coupled receptor CXCR4, increased collateral blood flow in a mouse model of vascular insufficiency via recruitment of endothelial precursor cells (EPC). The present study investigated the contribution of mature endothelial cells in the actions of SDF-1.

Methods: The regulation of SDF-1 and CXCR4 was examined in the rat cornea cauterization (CC) and aortic ring (AR) model. The functional significance of the SDF-1/CXCR4 pathway was explored in cultured endothelial cells, the AR model, and on collateral blood flow in a rat model of vascular insufficiency.

Results: In the present study, the CXCR4 transcript was dramatically upregulated in the rat CC and AR explants, systems containing and lacking bone marrow-derived EPCs, respectively. Addition of AMD3100, a selective CXCR4 antagonist, had no effect on vessel growth in the AR alone, but completely inhibited SDF-1 mediated increases in vascular sprouting. In cultured endothelial cells, SDF-1 alone or in combination with vascular endothelial growth factor (VEGF) significantly enhanced cell survival and migration. Finally, systemic administration of SDF-1 in a rat model of arterial insufficiency enhanced collateral blood flow above vehicle control and equal to that of VEGF after 2 weeks of treatment.

Conclusion: These studies support activation of the SDF-1/CXCR4 axis as a means to promote blood vessel growth and enhance collateral blood flow, at least in part, via direct effects on vascular endothelial cells.

KEYWORDS Angiogenesis; Blood flow; Collateral circulation; Endothelial factors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Acute or chronic interruptions in blood flow, as observed in myocardial infarction, peripheral artery disease (PAD) or certain types of stroke, result in inadequate tissue perfusion, cellular damage and loss of function. These debilitating diseases are associated with ominous morbidity and mortality, as well as a cost in the hundreds of billions of dollars. Pain in the legs upon walking is the most common symptom of PAD and, in severe cases, may deteriorate such that limb amputation is required. It has been estimated that ~27 million people in Europe and North America have PAD, including 16% of the population over 55 years of age [1]. With the current rise in the geriatric population in the United States and around the world, PAD stands to escalate as a formidable unmet medical need.

The best current approaches to treat occlusive cardiovascular disorders are interventional or surgical procedures to either reopen or to bypass the occlusion to re-establish blood flow to the ischemic area [2]. Given that PAD and other occlusive cardiovascular disorders are generally associated with the aging population with substantial co-morbidities, these procedures are considered high risk, leading a flurry of activity to identify alternative approaches. One alternative to invasive approaches to treat occlusive vascular disease is to promote the body's innate ability to develop collateral vessels that act as natural bypasses to improve and maintain blood flow.

Stromal cell-derived factor-1 (SDF-1) is a member of the CXC subfamily of chemokine peptides. Unlike other chemokines that interact with multiple G-protein coupled receptors (GPCRs), SDF-1 mediates its effects through its only known receptor, CXCR4. Mounting evidence suggests that the SDF-1/CXCR4 axis may play a role in blood vessel growth and development. Mice lacking SDF-1 or CXCR4 exhibit malformations in the large vessels in the gastrointestinal tract [3]. Moreover, it was recently demonstrated that circulating endothelial precursor cells (EPCs) express CXCR4 and that local, intramuscular administration of SDF-1 coupled with systemic injections of isolated human EPCs improved collateral blood flow in mice with femoral artery resection [4]. In a second study, local gene transfer of an SDF-1 cDNA into the ischemic hindlimbs of bone marrow-transplanted mice enhanced EPC recruitment and improved blood flow [5]. Taken together, these studies demonstrate the potential utility of local administration of SDF-1 to improve collateral blood flow and underscore the importance of EPC recruitment in collateral development following the local administration of SDF-1.

In the present study, we show for the first time that CXCR4 is dynamically upregulated during angiogenesis in both intact rat cornea and in explanted rat aortic ring, the latter a model in which blood vessel development occurs in the absence of circulating EPCs. We further demonstrate that expression of CXCR4 in the aortic ring is localized to the endothelium of angiogenic sprouts and that, despite the absence of circulating EPCs, exogenous SDF-1 increased vascular sprouting in this model. In cultured human endothelial cells, we show that SDF-1 enhances responses which are prerequisite for blood vessel growth and development. These effects of SDF-1 are additive and in some cases synergistic with the effects of VEGF165, indicating cross-talk between the pathways or amplification of a common pathway. Finally, we show for the first time that systemic administration of SDF-1 in a model of arterial insufficiency enhances collateral blood flow. Taken together, these studies suggest that direct action of SDF-1 on differentiated ECs, in addition to recruitment of EPCs, contributes importantly to SDF-1 mediated angiogenesis and blood vessel remodeling. Furthermore, these studies provide evidence that approaches directed toward enhancing EC signaling via CXCR4 may be beneficial in PAD and other occlusive cardiovascular disease.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animal experiments
All animal work was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and the Institutional Animal Care and Use guidelines at the University of Missouri, Duke University and Procter and Gamble Pharmaceuticals.

2.2 Rat cornea model of angiogenesis
This model has been described in detail previously [6,7]. Cornea samples were dissected to include approximately 1–2 mm of conjunctiva and sclera along with the cornea; these specimens include the plexus of blood vessels at the corneoscleral limbus (limbal vessels) from which new blood vessels arise during corneal angiogenesis. Cornea samples were dissected approximately 1 mm inside of the corneoscleral limbus to exclude the limbal vessels; in the normal cornea these specimens are devoid of blood vessels.

2.3 Rat aortic ring assay of angiogenesis
The isolated rat aortic ring model of angiogenesis was performed based on previously described studies [8,9]. The endothelium of the vessels was removed ("denuded") in a subset of vessels by rubbing the vessel between thumb and forefinger [10]. SDF-1 and/or AMD3100 were added to the overlying media in treated wells. All images were acquired at 20 x magnification.

2.4 Total RNA isolation
Aortic samples were cut from the collagen gels at various days in culture and immediately immersed in TRIzol (Invitrogen), frozen on dry ice, and stored at –80 °C. Likewise, cornea and corneoscleral samples at various days post-cautery were excised and placed directly in TRIzol reagent, frozen on dry ice, and stored at –80 °C. To isolate RNA from cornea and corneoscleral samples, one 3 mm tungsten carbide bead (Qiagen) was added to each frozen sample and homogenization was immediately performed using the Mixer Mill 300 (Qiagen) by shaking twice at 20 shakes/s for 8 min each. Next, each sample was removed to a 1.5 ml pre-spun PLG heavy tube (Eppendorf). One hundred microliters of Chloroform (Sigma) were added, each sample was shaken vigorously for 15 s then immediately centrifuged for 10 min. at 9600 rpm (16,795 x g) using a 12200 rotor in a 4K15 centrifuge (Sigma). The upper aqueous phase was then transferred to another 1.5 ml tube followed by addition of 300 µl 70% EtOH. The samples were vortexed and passed through RNeasy columns placed on a Qiavac 24 vacuum manifold (Qiagen). The columns were washed with 700 µl RW1 and two aliquots of 500 µl RPE (Qiagen). All columns were then placed in 2 ml collection tubes and centrifuged 4 min at 9600 rpm as described above. The columns were removed to 1.5 ml centrifuge tubes and RNA was eluted in 90 µl DEPC water heated to 45 °C by centrifuging 4 min as described above. Optical density ratios (260/280) were then determined from 1.5 µl of each sample on an ND-1000, spectrophotometer (Nanodrop). Finally, 5 µg of each RNA sample were concentrated by vacuum centrifugation and resuspended in 10 µl DEPC water heated to 45 °C. RNA from aortic ring samples (about 20 rings/sample) was isolated similarly except the volume of Trizol was brought up to 1 ml before addition of the tungsten carbide bead, homogenization required 30 shakes/s, and the volume of Chloroform and 70% EtOH used was increased to 200 and 600 µl, respectively.

2.5 Affymetrix GeneChip analysis
Five micrograms of total RNA were used to prepare biotin-labeled and fragmented cRNA, which was then hybridized to Affymetrix RAE230 A and B chips as described in the protocol provided in the Affymetrix GeneChip Expression Analysis Technical Manual. Transcript expression levels for CXCR4 and SDF-1 were generated using Affymetrix probe sets 1373661_a_at and 1369622_a_at, respectively. Similar data were obtained from additional probe sets (CXCR4: 1370097_a_at, 1389244_x_at; SDF-1: 1387655_at). Statistical analysis was based on the Affy signal (MAS 5.0 algorithm). The data from chip A and B were rescaled (normalized) based on the 100 common genes from both sets. Gene filtering was performed based on the minimum number of Affy Absent Calls per experimental condition. An ANOVA statistical model with log (base 2) of the Affy signal as a response was fitted for each non-filtered gene. The model parameter estimates were used to calculate Log Fold Change (LFC) between experimental conditions, and a corresponding uncertainty measure, standard error (SE). The ratio of LFC to SE is used to test statistically for significant differential gene expression. Statistical significance was imparted at P<0.0001. At this level of statistical significance, the chance of obtaining a false positive is less than 3 in 36,000 transcripts. For CXCR4 and SDF-1 multiple probe sets provided similar levels of significance.

2.6 Cell survival assay
Human Umbilical Vein Endothelial Cells were plated in 96-well tissue culture plates (20,000/well) for 2 h in serum containing media followed by 2 h of cell culture in serum-free media (DMEM/0.2% BSA). Serum starved endothelial cells were then incubated for 72 h (± growth factor) in DMEM/0.2% BSA. Cells remaining after treatment were assessed using the Cell Titer Glo Luminescent Cell Viability Assay reagent (Promega Corp.). Changes in luminescence were measured using a Victor V plate reader.

2.7 Cell migration assay
Human Umbilical Vein Endothelial Cells (HUVEC, Clonetics, Walkersville MD) were seeded at 50,000/well in a 24-well fibronectin coated fluoroblok migration plate (BD BioCoat Angiogenesis System, 3 µM pore size) in EBM (Cascade Biologics) supplemented with 0.1% BSA. Agonists were tested for their ability to induce a migration response by placing various concentrations in the lower chamber of the migration plate and incubated for 22 h at 37 °C/5% CO2. After incubation, cells on the agonist side of the filter were incubated with calcein AM (4 µg/ml) for 90 min and migrated cells detected by increased fluorescence (485/530) using a Victor V plate reader (Perkin Elmer/Wallac). Five percent fetal bovine serum (FBS) was used as a positive control.

2.8 Immunohistochemistry
Eight day aortic ring explants surrounded with intact collagen matrix were embedded in OCT compound and then frozen in liquid nitrogen-cooled isopentane for 1 min. Sections were placed on Gold Seal Ultrastick Microscope slides (Becton Dickinson). Sections were rehydrated with PBS, permeabilized for 30 min in 0.5% Triton X-100 in PBS, and then incubated for 2 h in 5% normal donkey serum to block non-specific antibody binding. Next, 1 µg/ml primary antibody (goat anti-CXCR4, Abcam, Inc.) in PBS containing 0.1% BSA was applied to the tissue and allowed to incubate overnight at 4 °C in a humidified chamber. Sections were rinsed in PBS and then incubated for 1 h in secondary antibody solution (Cy3 donkey anti-goat IgG: Jackson ImmunoResearch). These slides were then rinsed in 3 changes of PBS followed by 5% Donkey serum for 1–2 h. Slides were rinsed in PBS and incubated for 1–2 h in 1 µg/ml of the primary antibody solution (rabbit anti-human von Willebrand Factor-DAKOCytomation). Secondary antibody incubation was performed as before, except FITC-conjugated anti-rabbit IgG was employed. The slides were rinsed in 2 changes of PBS, one rinse with 0.1M Tris buffer (pH 8.2) and a cover slip added with Vectashield Mounting Medium with DAPI (Vector Laboratories). Images were taken with a Nikon Eclipse TE300 with RT Spot Camera and Image Pro Plus software.

2.9 Rat model of vascular insufficiency
Details of the rat model of vascular insufficiency have been previously described [11–13]. Briefly, peripheral arterial insufficiency was established in adult rats with bilateral occlusion of the femoral arteries inguinal ligament. In the same surgical procedure, a catheter connected to a 14 day osmotic pump (Alzet) was inserted into the iliac artery for infusion near the site of collateral expansion for systemic delivery of VEGF165, SDF-1{alpha} (R&D Systems) or vehicle (phosphate buffered saline, 10% sodium acetate, 1.6% glycerol, 0.02% sodium azide). All agents were supplied and administered in a manner blinded to the investigators. Blood flow was measured on day 16 to eliminate any acute effects of SDF-1 on blood flow. Muscle blood flow was determined in a blinded manner, utilizing radiolabeled microspheres during treadmill running, as described [11–13]. To ensure maximal vasodilation, animals were run on a treadmill (7° incline) at 20 m/min followed by second run at 25 m/min. Collateral blood flow was similar between the 2 different treadmill speeds.

2.10 Statistics
Values represent means ± standard error of the means (SEM). Statistical comparisons were made with Student's t test and one-way or two-way ANOVA where indicated, with significance imparted at P values<0.05, unless otherwise indicated.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 CXCR4 and SDF-1 are transcriptionally regulated during angiogenesis in vivo in the rat cornea
To explore the potential role of CXCR4 signaling during angiogenesis in vivo, expression profiles were obtained for both SDF-1 and CXCR4 in the rat cornea cautery (CC) model. In the rat CC model, angiogenesis is initiated by a silver/potassium nitrate cauterization of the normally avascular cornea [6]. In response to this stimulus, blood vessels arise from the surrounding limbal vasculature beginning on day 1 and extend to the site of injury (days 2–7). Between days 4 and 7, while some vessels regress, the remaining vessels continue to elongate towards the site of injury and differentiate into arterioles and venules (days 7–14) [14]. After day 7, there is further regression of blood vessels, but residual vessels remain through day 38. To examine transcriptional regulation of CXCR4 and SDF-1 during the angiogenic process, samples including the entire cornea and limbal vasculature (corneoscleral) were analyzed from days 0, 1, 2, 4, 7, 15, and 38 post-cautery. To distinguish expression changes occurring in the neovasculature from those occurring in the existing limbal vasculature, samples including the cornea but excluding the limbal vessels (cornea) were taken from days 0, 4, 7, and 15 post-cautery.

During angiogenesis in the CC model, CXCR4 transcript levels increased markedly in the corneoscleral (includes limbal vessels) at day 1 and continued to increase over time (~10-fold at day 7), but waned during vessel regression at day 15 (Fig. 1A). Importantly, CXCR4 transcript levels in the corneal samples (no limbal vessels) increased dramatically compared to baseline values suggesting that CXCR4 expression increased in the neovascular sprouts. In contrast to CXCR4, SDF-1 in the corneoscleral samples decreased at early time-points, but returned to baseline thereafter (Fig. 1B). However, SDF-1 transcript levels were increased in the corneal samples at days 4 and 7 consistent with the development of a gradient of SDF-1 expression in the cornea. Taken together, these results strongly suggest endothelial CXCR4 signaling could contribute to angiogenesis and vascular remodeling in the cornea. However, a contribution by inflammatory cells, resident corneal cells or circulating endothelial progenitor cells could not be ruled out in this model.


Figure 1
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  		Fig. 1 Transcript levels of CXCR4 and SDF-1 in the cornea cauterization and rat aortic explant models. Time-course of CXCR4 (A, C) and SDF-1 (B, D) transcript levels in the cornea cauterization (A, B) and cultured aorta (C, D) models. Five or six corneoscleral samples (including limbal vessels) were taken at days 0, 1, 2, 4, 7, 15 and 38 post-cautery. Cornea samples (excluding limbal vessels) were taken at days 0, 4, 7, and 15 post-cautery. Five or six aortic samples were taken at days 0, 1, 2, 4, 7, 10 and 14 in culture from normal and endothelium-denuded samples. Expression data are presented as the fold-change compared to the respective day 0 value ± standard error of the mean. See Methods for statistics.

 
3.2 CXCR4 and SDF-1 are transcriptionally regulated during angiogenesis in aortic explants
To better understand the role endothelial CXCR4 signaling during angiogenesis, expression profiles of SDF-1 and CXCR4 were studied during angiogenesis in the rat aortic ring (AR) model, a system devoid of inflammatory cells or circulating endothelial precursor cells. In the AR model, neovascular sprouts extend spontaneously from segments of descending aorta embedded in collagen over the course of 14 days [15]. Vascular sprouting in this model recapitulates several characteristics of blood vessel growth and development in vivo, including degradation of the extracellular matrix, endothelial cell proliferation and migration, lumen formation and recruitment of pericytes and smooth muscle cells. For expression analysis, samples were taken from both intact and denuded (endothelium removed) AR samples at several time-points to determine the contribution of endothelial cells to the changes in SDF-1 and CXCR4 expression and to ensure a sampling of the entire process of vessel development.

Similar to the CC model, CXCR4 transcript levels were significantly upregulated in the nascent sprouts as early as day 1 (~20-fold) and continued to increase over time in culture (Fig. 1C). Denuded rings (no endothelium; see Methods) failed to produce vessel sprouting (data not shown) and exhibited little, if any, increase in CXCR4 levels with time in culture. CXCR4 levels at day 0 in the normal and denuded samples were considered below the limit of detection by the Affymetrix software, suggesting that CXCR4 expression in the quiescent rat aorta is low, but is quickly upregulated during sprout development. In contrast to CXCR4 expression, SDF-1 transcript levels decreased at day 1, returned to baseline at day 2 and then increased slightly thereafter (Fig. 1D). These findings in the rat AR parallel the expression pattern of both CXCR4 and SDF-1 in the rat CC and further support a role for endothelial CXCR4 signaling in angiogenesis and vascular remodeling.

3.3 CXCR4 expression is localized to neovascular endothelial cells in the rat aortic ring model
To verify that increases in CXCR4 transcript levels reflected endothelial expression in the rat aorta, we performed immunohistochemistry on fresh, frozen sections of aortic rings cultured for 7 days. CXCR4 positive staining was identified in the neovascular sprouts (Fig. 2A). To determine if CXCR4 labeled cells were endothelial in nature, sections were co-stained with von Willebrand factor (vWF), an endothelium-specific marker (Fig. 2B), and the nuclei stained with DAPI (Fig. 2C). An overlay of the images demonstrated that CXCR4 expression co-localized with vWF (Fig. 2D), indicating that CXCR4 is expressed in endothelial cells of the neovasculature. Staining with secondary antibody alone revealed autofluorescence in the aortic ring (also seen in Fig. 2A and B), but no staining of vascular sprouts (Fig. 2E).


Figure 2
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  		Fig. 2 CXCR4 is expressed in the rat aortic explant samples. Fresh, frozen cryo-sections of rat aortic explants (Day 7) were incubated with antibodies for CXCR4 (A), the endothelium-specific marker, von Willenbrand factor (B), or DAPI nuclear staining (C). An overlay of all three images (D) demonstrates that CXCR4 is expressed in endothelial cells in the rat aorta. The aortic explant (Ring) and a neovessel (Sprout) are indicated and autofluorescence was detected in the aortic ring in all samples. No signal (except autofluorescence) was observed with secondary antibody alone (E).

 
3.4 SDF-1 enhanced sprouting in the rat aortic ring assay
To test the functional significance of endothelial CXCR4 expression during angiogenesis, the effects of exogenous SDF-1 were studied in the rat AR model. SDF-1 induced a dose-dependent increase in vascular sprouting measured by total vessel area and average vessel length (vessel extent) after 7 days in culture (Fig. 3A). To determine if the pro-angiogenic effect of SDF-1 in the AR assay was dependent on signaling through the CXCR4 receptor, AMD3100, a selective antagonist of CXCR4, was included in this assay (Fig. 3B) [16]. AMD3100 (10 µM) alone had no apparent effect on sprouting in the AR. However, both 10 and 100 µM (data not shown) AMD3100 completely suppressed SDF-1 mediated increases in vessel sprouting. Considering the genomics and IHC data demonstrating that CXCR4 is upregulated on endothelial cells during angiogenesis in the rat AR model, these data suggest that SDF-1 exerts its angiogenic effects via an endothelial-dependent mechanism.


Figure 3
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  		Fig. 3 SDF-1 enhances angiogenesis in the rat aortic ring model. (A) Rat aortic rings were cultured for 7 days in serum-free EBM alone or with increasing concentrations of SDF-1. The spontaneous growth of new vessels was significantly increased by inclusion of SDF-1 in the media in a concentration-dependent manner. Angiogenesis was quantitated by measuring the total vessel area (Area) and the average length of vessels (vessel extent) extending from the original vessel. (B) Aortic rings were treated with serum-free EBM alone (Vehicle), SDF-1, the CXCR4 antagonist AMD3100 or the combination of SDF-1 and AMD3100. Vessel growth was quantitated using automated imaging software developed in-house. Data are from n=5 aortic rings for all groups. *P<0.05 vs. control, one-way ANOVA, Tukey post hoc test. Images are at 20 x magnification.

 
3.5 SDF-1 stimulates endothelial cell survival and migration
To determine the cellular mechanisms by which endothelial CXCR4 signaling enhances angiogenesis, the effect of exogenous SDF-1 on endothelial cell survival and migration was studied. SDF-1 dose-dependently increased HUVEC cell survival and this effect was completely eliminated by CXCR4 blockade using AMD3100 (Fig. 4A). Addition of both SDF-1 and VEGF165 resulted in an increase in cell survival that appeared to be synergistic at higher concentrations of VEGF165 (Fig. 4B). Indeed, the maximal doses of VEGF165 and SDF-1 administered individually increased cell survival by 5.8- and 3.3-fold, respectively, while co-administration of both agents at these concentrations improved survival by 19.3-fold. Similar to the results in cell survival, SDF-1 increased endothelial cell migration in a dose-dependent manner and this effect was also inhibited by CXCR4 antagonist with AMD3100 (Fig. 5A). As observed in the endothelial cell survival assay, SDF-1 and VEGF165 co-administration resulted in a synergistic increase in cell migration with VEGF, SDF-1 or their co-administration increasing cell migration by 5.5-, 4.3- and 14.7-fold, respectively (Fig. 5B).


Figure 4
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  		Fig. 4 SDF-1 promotes endothelial cell survival. Cell survival was measured after 72 h in culture by quantitating the total amount of ATP remaining. Results are expressed as a percent of the ATP content of endothelial cells grown in the presence of fetal calf serum (FCS). (A) SDF-1 increased cell survival in a concentration-dependent manner (open bars) and this effect was eliminated in the presence of 10 µM AMD3100 (closed bars), a CXCR4 antagonist. (B) VEGF165 improved cell survival (open bars) and this effect was significantly enhanced by addition of SDF-1 at multiple concentrations (shaded bars). *P<0.05 vs. VEGF165 only at same dose, #P<0.05 vs. untreated cells, two-way ANOVA, Tukey post hoc test.

 

Figure 5
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  		Fig. 5 SDF-1 promotes migration in endothelial cells. Cell migration was measured in HUVEC culture in EBM+0.1% BSA as described in the Methods. (A) SDF-1 increased cell migration in a dose-dependent manner (open bars). SDF-1 effects on cell migration were blocked by 100 µM of the CXCR4 antagonist AMD3100. (B) VEGF165 increased cell migration (open bars) and this effect was augmented by addition of increasing concentrations of SDF-1 (filled bars). Data are plotted as the percent response to 5% FBS, used as a positive control. *P<0.05 vs. Baseline control, **P<0.05 vs. VEGF at same concentration, #P<0.05 vs. SDF-1 alone, two-way ANOVA, Tukey post hoc test.

 
3.6 Systemic delivery of SDF-1 augments collateral blood flow in vivo
Collectively, our data indicate that the SDF-1/CXCR4 pathway plays a direct functional role in angiogenesis by acting on endothelial cells. Others have shown that local administration of SDF-1 using plasmid vectors promotes collateral development suggesting that SDF-1 mediated recruitment of circulating endothelial progenitor cells is important for collateral vessel development [5]. To further investigate the relative importance of these two mechanisms, we tested the ability of systemically administered SDF-1 to augment collateral blood flow in a rat model of PAD.

Following bilateral femoral artery ligation, animals were divided into four treatment groups (12 animals/group) consisting of a vehicle group, a positive control group (VEGF165; 15 µg/kg/day) [9,17], a low dose SDF-1 group (5 µg/kg/day) and a high dose SDF-1 group (40 µg/kg/day). At day 16 post-ligation (to eliminate any acute effects of SDF-1 treatment) collateral blood flow measurements were performed during treadmill running at 20 m/min and subsequently at 25 m/min to ensure maximal vasodilation. Values obtained at each speed were similar suggesting that maximal collateral-dependent flow capacity was achieved (Table 1). Collateral blood flow to the calf muscle in the vehicle group exercised at 25 m/min was similar to previously reported values [9,18]. Blood flow in the VEGF165 and high dose SDF-1 groups were significantly higher than vehicle and SDF-1 low dose treatments at either the 20 or 25 m/min speeds (Table 1). These effects occurred in the absence of significant differences between the groups in heart rate or blood pressure (Table 2). Increases in collateral blood flow in this model are associated with an enlargement of the collateral vessel network in the thigh region [19,20]. This was observed in the present study by the significant increase in the angioscore, determined from X-rays of the collateral vessels, in the two groups that demonstrated improvements in collateral blood flow to the calf muscles (i.e., high-dose SDF-1 and VEGF groups). Further, there was a general correlation (r=0.61) between the angioscore and the directly measured collateral-dependent blood flow, obtained with microspheres (data not shown).


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  		Table 1 Hindlimb blood flow (ml/min/100 g tissue)

 

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  		Table 2 Blood pressure (mm Hg) and heart rate (bpm)

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Previous reports have suggested that activation of the SDF-1/CXCR4 system invokes mobilization and "homing" of EPCs to areas of ischemia and that these cells are responsible for the improvements in blood flow elicited by local administration of SDF-1 [4,5]. The present study demonstrates for the first time that systemic delivery of exogenous SDF-1 increased collateral-dependent blood flow to the calf muscles in a rat model of PAD. SDF-1 at the high dose was as effective as VEGF at increasing collateral-dependent blood flow. Improved blood flow in the SDF-1 high dose and VEGF treated rats occurred despite a modest decrease in peripheral blood pressure under resting or exercise conditions, suggesting that the improvements in blood flow in these groups may have been underestimated. Improvement of collateral blood flow in this study was accompanied by increased prominence of the collateral vasculature in the thigh, a process which involves remodeling of preexisting collateral vessels [21]. These findings demonstrate that the "homing" of EPCs to locally administered SDF-1 is not required for development and remodeling of collateral vessels and suggest that the action of systemically administered SDF-1 may be directly on the endothelium of preexisting collateral vessels.

4.1 Evidence for an endothelium-dependent mechanism
Data from several sources support a contribution of endothelial CXCR4 signaling in SDF-1 mediated angiogenesis and vascular remodeling. For example, cultured endothelial cells express CXCR4 and SDF-1 and addition of SDF-1 to the culture media promotes "angiogenic" endothelial responses such as chemotaxis, survival and capillary morphogenesis (tube formation) [22,23]. Data in the present study confirms and extends these findings by showing that SDF-1 mediated endothelial responses are blocked by AMD3100, a selective CXCR4 antagonist [16]. Moreover, we show that SDF-1 enhances VEGF mediated endothelial responses, an important finding considering SDF-1 has been shown to upregulate VEGF expression, while VEGF and bFGF administration increase expression of CXCR4 [4,22]. Thus, these pathways appear to be biologically interdependent. In addition, VEGF plays an important role in the angiogenic responses of the rat aortic ring [24] and the rat hindlimb models [25], suggesting that the action of SDF-1 at least in part reflects an amplification of the VEGF signaling cascade in endothelial cells in vitro and in vivo.

Further supporting a direct contribution of endothelial CXCR4 during angiogenesis, our data demonstrate for the first time that CXCR4 is markedly upregulated in the endothelium of neovessels that spontaneously sprout from explanted aortic rings. Importantly, in the rat AR model vascular sprouting occurs in the absence of EPCs, and denudation of the endothelium abrogates vascular sprouting. Collectively, our data and the data of others [22,26] showing that SDF-1 enhances vascular sprouting in the rat AR strongly support a role for direct endothelial CXCR4 signaling in SDF-1 mediated angiogenesis. Consistent with this possibility, AMD3100, a selective antagonist of SDF-1 at CXCR4, blocked SDF-1 mediated angiogenesis in the rat AR.

Interestingly, initial SDF-1 expression was very low in both the rat AR and the rat CC models of angiogenesis. In addition, AMD 3100 had no effect on basal sprouting in the rat AR. These findings together with the results in the rat PAD model demonstrating efficacy of systemically administered SDF-1 suggest that circulating SDF-1 rather than locally produced SDF-1 could be involved in angiogenesis and vascular remodeling in vivo. Again, these data imply that SDF-1 mediated recruitment of EPCs may not be required for angiogenesis and vascular remodeling in vivo.

An alternative mechanism for collateral remodeling is through amplification of endogenous endothelial signaling mediated by changes in shear stress [27–29]. Indeed, shear stress has been shown to stimulate ligand-independent activation of several RTKs including VEGFR2, TIE-2 and the insulin receptor [30,31]. Considering the cross-talk between the SDF-1 and VEGF receptor pathways it is possible that shear stress could contribute to SDF-1 mediated increases in collateral blood flow.

4.2 Potential role for EPCs
Systemic administration of SDF-1 has been shown to mobilize bone marrow-derived EPCs and hematopoietic stem cells [5,32,33]. Similarly, other angiogenic agents such as VEGF and angiopoietin-1, which have direct angiogenic effects on endothelial cells and proven efficacy in multiple models of vascular insufficiency, also stimulate mobilization of EPCs and hematopoietic stem cells [33]. Clearly the relative contribution of stem cell mobilization versus direct action on the endothelium deserves further study, especially in light of recent findings questioning the significance of EPCs in collateral development [34–36].

4.3 Implications for therapeutic angiogenesis with SDF-1
Our data suggest that systemic administration would be an acceptable approach for delivery of SDF-1 in patients with PAD as well as other forms of occlusive cardiovascular disease. In fact, this approach could have advantages over local delivery, as used in previous studies. In particular, systemic administration of SDF-1 should much more efficiently target the endothelium of remodeling collateral vessels that may be remote from the site of local administration of SDF-1. In addition, systemic administration may avoid potential hazards of local administration. For example, multiple local injections of a leukocyte chemo-attractant could result in inflammatory reactions or even ulceration, especially in patients with vascular insufficiency.

As with any novel therapy, use of SDF-1 or CXCR4 stimulating agents in patients with occlusive cardiovascular disease should be approached with caution. For example, recent data suggest that local expression of SDF-1 could play a role in the development of both primary atherosclerosis and transplant atherosclerosis [37]. Therefore, whether or not administration of SDF-1 would exacerbate preexisting atherosclerosis would need to be considered. However, at least one group has found that SDF-1 levels are actually decreased in patients with active coronary artery disease and that exogenous SDF-1 decreases markers of inflammation on cultured endothelial cells [38]. In conclusion, these data together with our data showing efficacy with no obvious adverse effects in the rat hindlimb model of vascular insufficiency suggest that systemic administration of SDF-1, or other CXCR4 agonists [39,40], could benefit patients with PAD and other forms of occlusive atherosclerotic cardiovascular disease without unacceptable side effects.


   Acknowledgements
 
We kindly thank Dr. Bruce Roberts for excellent technical assistance in completion of the rat cornea cauterization model.


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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