This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 27/6/1426    most recent ATVBAHA.107.139642v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Weel, V. Articles by Quax, P. H.A. Search for Related Content PubMed PubMed Citation Articles by van Weel, V. Articles by Quax, P. H.A.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1426.)
© 2007 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Expression of Vascular Endothelial Growth Factor, Stromal Cell-Derived Factor-1, and CXCR4 in Human Limb Muscle With Acute and Chronic Ischemia

Vincent van Weel; Leonard Seghers; Margreet R. de Vries; Esther J. Kuiper; Reinier O. Schlingemann; Ingeborg M. Bajema; Jan H.N. Lindeman; Pien M. Delis-van Diemen; Victor W.M. van Hinsbergh; J. Hajo van Bockel; Paul H.A. Quax

From Gaubius Laboratory, TNO Quality of Life (V.v.W., L.S., M.R.d.V., J.H.N.L., P.H.A.Q.), Leiden, the Netherlands; Department of Surgery (V.v.W., L.S., J.H.N.L., P.H.A.Q.), Leiden University Medical Center, Leiden, the Netherlands; Department Ophtalmology (E.J.K., R.O.S.), Amsterdam Medical Center, Amsterdam, the Netherlands; Department of Pathology (I.B.), Leiden University Medical Center; Department of Pathology (P.M.D.-v.D.), VU University Medical Center, Amsterdam, the Netherlands; Department of Physiology (V.W.M.H.), Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherlands.

Correspondence to Dr P.H.A. Quax, Gaubius Laboratory TNO-Quality of Life, P.O. Box 2215, 2301CE Leiden, the Netherlands. E-mail paul.quax{at}tno.nl

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Vascular endothelial growth factor (VEGF)-induced stromal cell-derived factor-1 (SDF-1) has been implicated in angiogenesis in ischemic tissues by recruitment of CXCR4-positive bone marrow-derived circulating cells with paracrine functions in preclinical models. Here, evidence for this is provided in patients with peripheral artery disease.

Methods and Results— Expression patterns of VEGF, SDF-1, and CXCR4 were studied in amputated limbs of 16 patients. VEGF-A was expressed in vascular structures and myofibers. SDF-1 was expressed in endothelial and subendothelial cells, whereas CXCR4 was expressed in proximity to capillaries. VEGF-A, SDF-1, and CXCR4 expressions were generally decreased in ischemic muscle as compared with nonischemic muscle in patients with chronic ischemia (0.41-fold, 0.97-fold, and 0.54-fold induction [medians], respectively), whereas substantially increased in 2 patients with acute-on-chronic ischemia (3.5- to 65.8-fold, 3.9- to 19.0-fold, and 4.1- to 30.6-fold induction, respectively). Furthermore, these gene expressions strongly correlated with capillary area. Only acute ischemic tissue displayed a high percentage of hypoxia-inducible factor-1–positive nuclei.

Conclusions— These data suggest that VEGF and SDF-1 function as pro-angiogenic factors in patients with ischemic disease by perivascular retention of CXCR4-positive cells. Furthermore, these genes are downregulated in chronic ischemia as opposed to upregulated in more acute ischemia. The VEGF-SDF-1-CXCR4 pathway is a promising target to treat chronic ischemic disease.

In amputated limbs of patients with peripheral artery disease, expression patterns of VEGF, SDF-1, and CXCR4 suggest a coordinated role of these factors in ischemia-induced angiogenesis, which were downregulated in chronic ischemia versus upregulated in more acute ischemia. This pathway is a promising target to treat chronic ischemic disease.

Key Words: peripheral artery disease • angiogenesis • ischemia • VEGF • SDF-1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The stimulation of neovascularization using growth factors is a promising experimental treatment for arterial occlusive disease. Early results obtained from preclinical studies using angiogenic factors, in particular vascular endothelial growth factor (VEGF), were promising and led to great expectations. However, placebo-controlled clinical trials of therapeutic angiogenesis were inconsistent.^1–4 To improve angiogenic strategies, more information is required about cellular and molecular mechanisms involved in vascular growth in ischemic tissues.

Recently, stem cells have been implicated to play a role in neovascularization.⁵ This has led to some promising results using autologous bone marrow transplantation for the stimulation of collateral artery growth in patients with peripheral artery disease.⁶ Furthermore, bone marrow mononuclear cells from patients with chronic ischemic heart disease have a reduced capacity to induce collateral formation in mice, which is paralleled by a reduced migratory response for bone marrow cells to stromal cell-derived factor 1 (SDF-1, also known as CXCL12) and VEGF.⁷

SDF-1 is implicated as a chemokine for CXCR4-positive stem cells.⁸ It was recently shown that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue.⁹ From the latter study, it was concluded that recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1–induced expression of SDF-1. More recently, it was demonstrated that bone marrow-derived circulating cells are retained in close proximity to angiogenic vessels by SDF-1 induced by VEGF in activated perivascular myofibroblasts in mice.¹⁰ Other investigators reported that cytokine-mediated release of SDF-1 from platelets constitute the major determinant of neovascularization through mobilization of nonendothelial CXCR4⁺VEGFR1⁺ hematopoietic progenitor cells.¹¹ Moreover, SDF-1 (gene) therapy enhances ischemia-induced vasculogenesis and angiogenesis in mice, and is associated with incorporation of bone marrow cells into the vasculature.^12,13

Here, expression patterns of VEGF, SDF-1, and CXCR4 were studied in relation to other angiogenic factors, such as VEGF-C, VEGF-D, and VEGF receptors 1 to 3 in amputated limbs of 16 patients with peripheral arterial disease, of which 14 had chronic ischemia, whereas 2 patients had acute-on-chronic ischemia. In both chronic and acute-on-chronic ischemia, SDF-1 was expressed in endothelial and subendothelial cells, whereas CXCR4 was expressed in proximity to capillaries. VEGF-A was expressed in vascular structures and myofibers. Interestingly, with an increased degree of ischemia, VEGF-A, SDF-1, and CXCR4 expressions were decreased or unchanged in patients with chronic ischemia, as opposed to substantially increased in both patients with acute-on-chronic ischemia, as described for the HIF-VEGF-VEGFR-2 pathway.¹⁴

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sample Collection From Patients
Samples of skeletal muscle were obtained after informed consent from 16 patients from November 2001 to June 2003, according to the guidelines of the Institutional Review Board. Patient characteristics are depicted in the Table. Patients underwent below-knee or above-knee amputation because of critical ischemia without possibilities for vascular reconstruction. All patients had chronic ischemia; however, patients 5 and 11 demonstrated sudden progression of ischemia by occlusion of a bypass graft leading to swift amputation, and were thus considered as acute-on-chronic ischemic cases. The former patient was re-admitted 1 month after urokinase thrombolysis of an occluded femoro-popliteal bypass graft with re-occlusion of the bypass. After an unsuccessful revascularization attempt, below-knee amputation was performed 1 week later. The latter patient showed sudden onset of rest pain caused by occlusion of a femoro-crural bypass graft 9 days postoperatively, which was followed by below-knee amputation 2 weeks later.

View this table: [in this window] [in a new window]   Patient Characteristics

To compare nonischemic with ischemic muscle within 1 patient, biopsies were performed at amputation level, representing relatively nonischemic muscle and, more distally, near the Achilles tendon (Soleus muscle) and between the toes (Interosseus, Extensor digitorum, or Adductor hallucis muscle), representing ischemic muscle (Table). Each biopsy was performed in duplo; one muscle sample was fixated in 4% formaldehyde and subsequently embedded in paraffin for immunohistochemistry, and one muscle sample was frozen in liquid nitrogen (LN₂) for RNA analysis. Because in 6 of 16 patients the biopsy samples could only be collected from 2 instead of 3 levels of the limb for various reasons, eg, previous foot amputation or extensive gangrene, comparisons of gene and protein expressions were performed between 2 levels for each patient to allow valid statistics, preferably between soleus and gastrocnemius muscle to maintain constant muscle types.

Immunohistochemistry
Immunohistochemistry was performed on 5-µm-thick paraffin-embedded sections of skeletal muscle using antibodies against CD31, CD34, SDF-1, CXCR4, VEGF-A, VEGF-C, and VEGF-D, and VEGF receptor-1, -2, and -3, and HIF-1 (for detailed methods please see http://atvb.ahajournals.org).

RNA Analysis
Total RNA was extracted from frozen muscle and reversed transcribed into cDNA. Real-time reverse-transcription polymerase chain reaction was performed using primers and probe sets for human SDF-1 and CXCR4 (Applied Biosystems), and human VEGF-A (designed with Perkin Elmer primer express software), normalized to GAPDH housekeeping gene (Perkin Elmer).

Statistical Analysis
Results are expressed as mean±SEM or median with 95% CI. Comparisons between means were performed using 1-way ANOVA test with least significant difference post-hoc analysis. Comparisons of immunostaining intensity between different levels of limbs were performed with the Sign test (single-blinded), as described.¹⁵ Pearson correlations were used to study relationships. P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vessel Density and Size Parallel the Degree of Ischemia in Human Skeletal Muscle
Marked morphological features of skeletal muscle, characteristic for chronic ischemia, were observed in muscle biopsy samples derived from distal levels of the limb (most ischemic area) that were not apparent at the amputation level (relatively nonischemic area) (Figure 1A and 1B). They consisted of disorganized muscle composition, adipose cells within muscular tissue, regenerating and atrophic myofibers, and infiltrating inflammatory cells. Capillary density increased with the degree of ischemia (Figure 1C and 1D), which became significant at the level of interosseus muscle of the foot as compared with both amputation level and soleus muscle (239.6±29.5 as compared with 151±19.0 and 153.7±18.3 capillaries/mm², respectively; P=0.01; n=10; Figure 1E). In addition, area per capillary was increased in ischemic interosseus muscle as compared with at the amputation level (102.2±5.0 µm² as compared with 81.2±6.4 µm², respectively; P=0.03; n=10; Figure 1F).

View larger version (60K): [in this window] [in a new window]   Figure 1. Hematoxylin phloxin saffron and CD31 staining of gastrocnemius muscle at amputation level (A and C) and ischemic interosseus muscle (B and D). Patient 6. Magnification x150. E and F, Quantification of capillary density and area in muscle sections at the level of amputation, calf or foot, depicted as capillaries/mm2 and µm2, respectively (n=10 patients). *P<0.05, **P<0.01.

Expression Patterns of VEGFs and SDF-1 and Their Receptors in Human Ischemic Skeletal Muscle
VEGF-A was expressed in cytoplasm of myofibers, in endothelial cells, subendothelial pericytes, and adventitial (angiogenic) capillaries in ischemic muscle of nearly all patients (Figure 2A). Especially in muscle with acute-on-chronic ischemia, VEGF-A–expressing inflammatory cells were visible between myofibers (Figure 2B). In chronically ischemic muscle, atrophic myofibers, regenerating polynuclear myofibers, and satellite cells all strongly stained for VEGF-A (Figure 2C). There was no expression of VEGF-C in any muscle sample (Figure 2D), whereas expression was evident in control colon tissue (data not shown). VEGF-D was expressed in both nonischemic and ischemic muscle and was located in cytoplasm of myofibers, and was in close proximity to capillaries between myofibers (Figure 2E). VEGF receptor-1, -2, and -3 were expressed in nearly all microvessels adjacent to myofibers (Figure 2F to 2H).

View larger version (107K): [in this window] [in a new window]   Figure 2. A, VEGF-A staining of ischemic interosseus muscle (patient 10). Magnification x150. VEGF-A was expressed in myofibers (white arrow), adventitial capillaries (dotted arrow), and pericytes (black arrow). B, VEGF-A staining of acute-on-chronic ischemic interosseus muscle (patient 5) showing infiltrating VEGF-expressing cells. Magnification x150. C, VEGF-A staining of chronically ischemic interosseus muscle (patient 6) showing VEGF expression in atrophic myofibers associated with satellite cells (black arrows) and regenerating myofibers (white arrow). Magnification x200. Staining of ischemic muscle for VEGF-C (D, magnification x150), VEGF-D (E, magnification x150), VEGF-receptor 1 (F, magnification x200), VEGF-receptor 2 (G, magnification x200), and VEGF-receptor 3 (H, magnification x200).

SDF-1 antigen was present both in capillaries adjacent to myofibers (Figure 3A) and in larger vessels (Figure 3B and 3C). SDF-1 was located at endothelial cells, as indicated by co-localization with CD31 and CD34 endothelial staining (Figure 3D and 3E), as well as at subendothelial pericytes. CXCR4 was mainly expressed in proximity to capillaries between myofibers (Figure 3F). Only sporadically, CXCR4 expression was observed in the wall of larger vessels. This was most obviously encountered in relatively non-ischemic gastrocnemius muscle at the amputation level in a patient with chronically ischemic disease (Figure 3G). Importantly, CXCR4 expression was not observed in endothelial cells, indicating that there was no incorporation of CXCR4-positive cells in endothelium (Figure 3G, arrowheads). No staining was observed in negative control incubations for all antibodies used (supplemental Figure I, available online at http://atvb.ahajournals.org).

View larger version (80K): [in this window] [in a new window]   Figure 3. Immunohistochemical stainings of ischemic interosseus muscle sample derived from patient 6. Staining for SDF-1 (A, magnification x150), staining of sequential sections of large vessels for SDF-1 (B, magnification x125, and C, higher magnification of the boxed area in B, magnification x400), for CD31 (D, magnification x125), and for CD34 (E, magnification x125). Staining for CXCR4 of muscle samples derived from patient 14. Ischemic interosseus muscle tissue (F, magnification x150), large vessel in gastrocnemius muscle tissue (G, magnification x200). Endothelial cells did not express CXCR4 (arrowheads).

Differential Expression of VEGF-A, SDF-1, and CXCR4 Between Acute and Chronic Ischemia
In chronically ischemic limbs, there was a decreased or unchanged RNA expression of VEGF-A, SDF-1, and CXCR4 in ischemic as compared with nonischemic tissues (fold inductions: VEGF-A, median 0.41 [95% CI, 0.18 to 0.85]; SDF-1, median 0.97 [95% CI, 0.44 to 1.36]; CXCR4, median 0.54 [95% CI, 0.18 to 1.23]; n=13; Figure 4). Significant downregulation (fold induction 0.5) occurred in 9 of 13 patients, 4 of 13 patients, and 8 of 13 patients for VEGF-A, SDF-1, and CXCR4, respectively. Only 2 of 13 patients with chronic limb ischemia showed significant upregulations (fold induction 2.0); 1 patient for both VEGF-A and CXCR4, but not SDF-1 (patient 9, 2.1-fold, 3.9-fold, and 1.4-fold induction, respectively; Figure 4), another patient for only CXCR4 (patient 6, 2.2-fold induction). On the contrary, in the acute-on-chronic ischemic limbs there was an overall increased RNA expression for VEGF-A, SDF-1, and CXCR4 in ischemic muscle, most evidently in patient 5, with 65.8-fold, 19.0-fold, and 30.6-fold inductions between ischemic and nonischemic muscle for VEGF, SDF-1, and CXCR4, respectively. For patient 11, these values were 3.5-fold, 3.9-fold, and 4.1-fold, respectively (Figure 4). In addition, there was a strong significant correlation between the ischemia-related changes of VEGF-A, SDF-1, and CXCR4 expressions within each patient (supplemental Table I). Moreover, VEGF-A, SDF-1, and CXCR4 expressions were correlated with capillary area (P=0.008, 0.010, and 0.014, respectively), but not with capillary density. No significant correlations with risk factors such as diabetes, smoking, hypertension, or dyslipidemia were observed. On a protein level, immunohistochemical staining intensity was not significantly different between ischemic and nonischemic muscle for the chronic ischemic group, for VEGF-A (9 and 3 signs, respectively; P=0.15), VEGF-D (5 and 7, respectively; P=0.77), VEGF-receptor 1 (6 and 7, respectively; P=1), VEGF-receptor 2 (9 and 4, respectively; P=0.27), VEGF-receptor 3 (7 and 6, respectively; P=1), SDF-1 (8 and 6, respectively; P=0.79), and CXCR4 (6 and 8, respectively; P=0.79). For the acute-on-chronic ischemia group, the number for 2 patients was too small to perform the Sign test for statistical comparison.

View larger version (18K): [in this window] [in a new window]   Figure 4. A, Box plot of mRNA expressions determined in muscle samples by real-time reverse-transcription polymerase chain reaction for VEGF-A, SDF-1, and CXCR4 in fold inductions between ischemic and nonischemic muscle (medians, N=15, *5=patient 5, o9=patient 9, o11=patient 11). B, Bar graph representing mRNA expressions of VEGF, SDF-1, and CXCR4 in fold inductions between ischemic and nonischemic muscle for each patient.

Finally, to test whether the difference in angiogenic expressions between acute and chronic ischemia is hypoxia-related, immunohistochemistry for HIF-1 was performed in a selection of patients with acute-on-chronic (n=2) and chronic (n=5) ischemia. Nuclear HIF-1 staining was observed in kidney carcinoma (positive control) and in part of cells of muscle specimens. HIF-1 nuclear staining was absent or limited at the amputation level, while it was very profound in muscle of acute hypoxic legs (patients 5, 11; nuclei of inflammatory cells brightly positive, those of endothelial cells and some muscle cells moderately positive). An intermediate staining (mainly inflammatory cells) was observed in chronic hypoxic specimens (supplemental Figure IIA). The percentage of HIF-1–stained nuclei of total nuclei strongly increased with the level of ischemia in more acute ischemic limbs (ratios between ischemic and amputation level 6.6 and 2.6 for patients 5 and 11, respectively), whereas no significant upregulation was observed in chronically ischemic limbs (ratios 0.9, 1.2, 0.6, 0.7, and 0.8 for patients 1, 4, 6, 8, and 9, respectively; supplemental Figure IIB).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we provide evidence that VEGF-A, SDF-1, and CXCR4 are expressed by or in close proximity to angiogenic vessels in ischemic muscle of patients with peripheral arterial disease. Expression patterns of these genes were highly correlated with each other and with capillary area, suggesting interactions and angiogenic functions, respectively.

Moreover, VEGF-A, SDF-1, and CXCR4 gene expressions were increased in acute-on-chronic limb ischemia, whereas they were generally decreased in chronic limb ischemia, as quantified by real-time polymerase chain reaction. Scoring of immunostained sections showed no significant regulation in protein expressions by ischemia. This may be explained by that groups were too small to determine differences using the Sign test, which is known as a crude statistical test. Another explanation may be that analysis of expression in a quantitative way based on immunohistochemistry is not reliable. Nevertheless, a recent study confirms our observation that expression of VEGF protein does not correlate with VEGF mRNA expression in an ischemic hind limb model in rats.¹⁶ In studies using similar muscle sampling methods from human ischemic legs as reported here, angiogenic expression patterns were highly variable, ranging from upregulation in ischemic tissues of HIF-1¹⁷ or VEGF,^18,19 to upregulation of FGF, but not VEGF,²⁰ to no regulation of pro-angiogenic factors,²¹ to restricted upregulation of the HIF-VEGF-VEGFR2 pathway in acute-on-chronic, but not chronic, ischemia.¹⁴ These inconsistencies may be explained by differences in methods used to determine expression, in studied tissue type (muscle or skin), in location of sampling (muscle type), and in patient selection. Here, expression of angiogenic factors was determined by mRNA analysis using reliable quantitative real-time polymerase chain reaction with an inpatient control and was, for the first time, correlated to the degree of ischemia as reflected by the amount of angiogenesis. VEGF, SDF-1, and CXCR4 expressions were highly correlated with capillary area, but not with capillary density, suggesting involvement of these factors in the enlargement of neovessels, as reported for VEGF.^22,23 Furthermore, locations of muscle biopsies within one level of the limbs were kept constant, if possible, to limit variations in expression caused by differences in muscle type. Finally, our cohort was divided in chronic ischemic or acute-on-chronic ischemic patients. In the latter group, VEGF, SDF-1, and CXCR4 upregulation in ischemic tissue was higher when muscle biopsy was performed 1 week as compared with 2 weeks after graft occlusion. Correspondingly, in previous mouse studies, SDF-1 and CXCR4 upregulations were transient after induction of hind limb ischemia.^24,25

In chronically ischemic muscle, VEGF-A mRNA expression was significantly downregulated, whereas both VEGF-A and VEGF-receptor 2 protein expression tended to be increased as compared with control muscle at the level of amputation, and were abundantly expressed in atrophic myofibers and satellite cells, as reported.¹⁸ One explanation may be that together with an increased VEGF-A expression in atrophic myofibers, there is a relative loss of muscular tissue that is replaced by fatty tissue. Furthermore, we hypothesize that in chronically ischemic muscle VEGF-A accumulates within atrophic myofibers, becomes dysfunctional, and leads to gene silencing or downregulation. This hypothesis is strengthened by our HIF-1 expression data, suggesting an inability of hypoxic tissues to express sufficient HIF-1, and thus downstream VEGF and SDF-1, in chronic ischemia as opposed to acute-on-chronic ischemia.

VEGF-C has been restrictedly implicated in lymphangiogenesis, whereas VEGF-D is both a potent angiogenic and lymphangiogenic factor.²³ Correspondingly, in the present study VEGF-C was not expressed in ischemic muscle, whereas VEGF-D was abundantly expressed adjacent to capillaries. Interestingly, VEGF-receptor 3, often used for the detection of lymphatic endothelium, was expressed in nearly all microvessels throughout ischemic limbs, suggesting VEGF-receptor 3 expression on activated endothelial cells, as described.²⁶ The location of SDF-1 expression in vivo remains controversial. In some studies, SDF-1 expression was localized in endothelial cells,⁹ whereas others showed that SDF-1 mainly co-localized with peri-endothelial cells, probably of fibroblastic or smooth muscle nature.^10,27 Here, SDF-1 expression was located both in endothelial cells and in close proximity to the endothelium. Sequential sections stained for CD31 and CD34 confirmed endothelial localization. Furthermore, the contribution of incorporating bone marrow-derived cells to adult neovasculature is still debated, ranging from minor^28–31 to major,^32,33 in previous studies. Here, CXCR4-positive cells were only scarcely observed in arterial walls without evidence of incorporation in endothelium. However, there was a close relationship between CXCR4-positive cells and small capillaries between ischemic myofibers, suggesting peri-endothelial localization around newly sprouting vessels.

Finally, it should be noted that muscle biopsy samples collected at different levels of the amputated limbs not only differ in the degree of ischemia but also differ in muscular composition. For example, gastrocnemius muscle mainly consists of fast-twitch, glycolytic myofibers, whereas soleus muscle consists of slow-twitch, oxidative myofibers. A limitation of our methods may lie in that global gene expression varies between glycolytic and oxidative skeletal muscle, although not reported for SDF-1 and CXCR4.³⁴

In conclusion, we provide evidence in human ischemic skeletal muscle for a role of VEGF and SDF-1 in adult neovascularization via retention of CXCR4-positive cells. Moreover, VEGF, SDF-1, and CXCR4 were differentially expressed between acute-on-chronic and chronic ischemia. Future experiments should focus on differences in angiogenic expression profile between acute and chronic hypoxic conditions potentially leading to optimized angiogenic treatments.

	Acknowledgments

 
The authors acknowledge Kees van Leuven (TNO, Leiden) for his technical assistance.

Sources of Funding

This study was sponsored by the TNO-LUMC-VUMC tripartite angiogenesis program, the NHS Molecular Cardiology Program (M93.001), and the STW/DTPE Dutch program on Tissue Engineering (VGT.6747). P.v.D.D. was supported by the unrestricted AEGON First International Scholarship in Oncology.

Disclosures

None.

	Footnotes

 
Original received July 28, 2006; final version accepted February 14, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS, Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation. 2003; 107: 1359–1365.[Abstract/Free Full Text]

2. Rajagopalan S, Mohler ER III, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, Blebea J, Macko J, Kessler PD, Rasmussen HS, Annex BH. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation. 2003; 108: 1933–1938.[Abstract/Free Full Text]

3. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation. 2002; 105: 788–793.[Abstract/Free Full Text]

4. Kusumanto YH, van WV, Mulder NH, Smit AJ, van den Dungen JJ, Hooymans JM, Sluiter WJ, Tio RA, Quax PH, Gans RO, Dullaart RP, Hospers GA. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum Gene Ther. 2006; 17: 683–691.[CrossRef][Medline] [Order article via Infotrieve]

5. Shintani S, Murohara T, Ikeda H, Ueno T, Sasaki K, Duan J, Imaizumi T. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation. 2001; 103: 897–903.[Abstract/Free Full Text]

6. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H, Shimada K, Iwasaka T, Imaizumi T. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002; 360: 427–435.[CrossRef][Medline] [Order article via Infotrieve]

7. Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S. Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation. 2004; 109: 1615–1622.[Abstract/Free Full Text]

8. Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T, Nagler A, Ben Hur H, Many A, Shultz L, Lider O, Alon R, Zipori D, Lapidot T. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science. 1999; 283: 845–848.[Abstract/Free Full Text]

9. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med. 2004; 10: 858–864.[CrossRef][Medline] [Order article via Infotrieve]

10. Grunewald M, Avraham I, Dor Y, Bachar-Lustig E, Itin A, Yung S, Chimenti S, Landsman L, Abramovitch R, Keshet E. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell. 2006; 124: 175–189.[CrossRef][Medline] [Order article via Infotrieve]

11. Jin DK, Shido K, Kopp HG, Petit I, Shmelkov SV, Young LM, Hooper AT, Amano H, Avecilla ST, Heissig B, Hattori K, Zhang F, Hicklin DJ, Wu Y, Zhu Z, Dunn A, Salari H, Werb Z, Hackett NR, Crystal RG, Lyden D, Rafii S. Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4(+) hemangiocytes. Nat Med. 2006; 12: 557–567.[CrossRef][Medline] [Order article via Infotrieve]

12. Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Ichiki T, Takeshita A, Egashira K. Gene transfer of stromal cell-derived factor-1alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation. 2004; 109: 2454–2461.[Abstract/Free Full Text]

13. Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, Murasawa S, Bosch-Marce M, Masuda H, Losordo DW, Isner JM, Asahara T. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation. 2003; 107: 1322–1328.[Abstract/Free Full Text]

14. Tuomisto TT, Rissanen TT, Vajanto I, Korkeela A, Rutanen J, Yla-Herttuala S. HIF-VEGF-VEGFR-2, TNF-alpha and IGF pathways are upregulated in critical human skeletal muscle ischemia as studied with DNA array. Atherosclerosis. 2004; 174: 111–120.[CrossRef][Medline] [Order article via Infotrieve]

15. Stuelten CH, Buck MB, Dippon J, Roberts AB, Fritz P, Knabbe C. Smad4-expression is decreased in breast cancer tissues: a retrospective study. BMC Cancer. 2006; 6: 25.[CrossRef][Medline] [Order article via Infotrieve]

16. Milkiewicz M, Hudlicka O, Shiner R, Egginton S, Brown MD. Vascular endothelial growth factor mRNA and protein do not change in parallel during non-inflammatory skeletal muscle ischaemia in rat. J Physiol. 2006; 577: 671–678.[Abstract/Free Full Text]

17. Ho TK, Rajkumar V, Ponticos M, Leoni P, Black DC, Abraham DJ, Baker DM. Increased endogenous angiogenic response and hypoxia-inducible factor-1alpha in human critical limb ischemia. J Vasc Surg. 2006; 43: 125–133.[CrossRef][Medline] [Order article via Infotrieve]

18. Rissanen TT, Vajanto I, Hiltunen MO, Rutanen J, Kettunen MI, Niemi M, Leppanen P, Turunen MP, Markkanen JE, Arve K, Alhava E, Kauppinen RA, Yla-Herttuala S. Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration. Am J Pathol. 2002; 160: 1393–1403.[Abstract/Free Full Text]

19. Choksy S, Pockley AG, Wajeh YE, Chan P. VEGF and VEGF receptor expression in human chronic critical limb ischaemia. Eur J Vasc Endovasc Surg. 2004; 28: 660–669.[CrossRef][Medline] [Order article via Infotrieve]

20. Palmer-Kazen U, Wariaro D, Luo F, Wahlberg E. Vascular endothelial cell growth factor and fibroblast growth factor 2 expression in patients with critical limb ischemia. J Vasc Surg. 2004; 39: 621–628.[CrossRef][Medline] [Order article via Infotrieve]

21. Favier J, Germain S, Emmerich J, Corvol P, Gasc JM. Critical overexpression of thrombospondin 1 in chronic leg ischaemia. J Pathol. 2005; 207: 358–366.[CrossRef][Medline] [Order article via Infotrieve]

22. van Weel V, Deckers MM, Grimbergen JM, van Leuven KJ, Lardenoye JH, Schlingemann RO, Nieuw Amerongen GP, van Bockel JH, van Hinsbergh VW, Quax PH. Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo. Circ Res. 2004; 95: 58–66.[Abstract/Free Full Text]

23. Rissanen TT, Markkanen JE, Gruchala M, Heikura T, Puranen A, Kettunen MI, Kholova I, Kauppinen RA, Achen MG, Stacker SA, Alitalo K, Yla-Herttuala S. VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res. 2003; 92: 1098–1106.[Abstract/Free Full Text]

24. Paoni NF, Peale F, Wang F, Errett-Baroncini C, Steinmetz H, Toy K, Bai W, Williams PM, Bunting S, Gerritsen ME, Powell-Braxton L. Time course of skeletal muscle repair and gene expression following acute hind limb ischemia in mice. Physiol Genomics. 2002; 11: 263–272.[Abstract/Free Full Text]

25. De Falco E, Porcelli D, Torella AR, Straino S, Iachininoto MG, Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi MC, Pesce M. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells. Blood. 2004; 104: 3472–3482.[Abstract/Free Full Text]

26. Valtola R, Salven P, Heikkila P, Taipale J, Joensuu H, Rehn M, Pihlajaniemi T, Weich H, deWaal R, Alitalo K. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am J Pathol. 1999; 154: 1381–1390.[Abstract/Free Full Text]

27. Ara T, Tokoyoda K, Okamoto R, Koni PA, Nagasawa T. The role of CXCL12 in the organ-specific process of artery formation. Blood. 2005; 105: 3155–3161.[Abstract/Free Full Text]

28. Peters BA, Diaz LA, Polyak K, Meszler L, Romans K, Guinan EC, Antin JH, Myerson D, Hamilton SR, Vogelstein B, Kinzler KW, Lengauer C. Contribution of bone marrow-derived endothelial cells to human tumor vasculature. Nat Med. 2005; 11: 261–262.[CrossRef][Medline] [Order article via Infotrieve]

29. Rajantie I, Ilmonen M, Alminaite A, Ozerdem U, Alitalo K, Salven P. Adult bone marrow-derived cells recruited during angiogenesis comprise precursors for periendothelial vascular mural cells. Blood. 2004; 104: 2084–2086.[Abstract/Free Full Text]

30. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002; 297: 2256–2259.[Abstract/Free Full Text]

31. Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R, Helisch A, Schaper W. Bone marrow-derived cells do not incorporate into the adult growing vasculature. Circ Res. 2004; 94: 230–238.[Abstract/Free Full Text]

32. Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z, Kolesnick R. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science. 2003; 300: 1155–1159.[Abstract/Free Full Text]

33. Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, Rafii S. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med. 2001; 193: 1005–1014.[Abstract/Free Full Text]

34. Campbell WG, Gordon SE, Carlson CJ, Pattison JS, Hamilton MT, Booth FW. Differential global gene expression in red and white skeletal muscle. Am J Physiol Cell Physiol. 2001; 280: C763–C768.[Abstract/Free Full Text]

This article has been cited by other articles:

				E. J. Suuronen, P. Zhang, D. Kuraitis, X. Cao, A. Melhuish, D. McKee, F. Li, T. G. Mesana, J. P. Veinot, and M. Ruel An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model FASEB J, May 1, 2009; 23(5): 1447 - 1458. [Abstract] [Full Text] [PDF]

				Y. Shiba, M. Takahashi, T. Hata, H. Murayama, H. Morimoto, H. Ise, T. Nagasawa, and U. Ikeda Bone marrow CXCR4 induction by cultivation enhances therapeutic angiogenesis Cardiovasc Res, January 1, 2009; 81(1): 169 - 177. [Abstract] [Full Text] [PDF]

				N. Krankel, R. G. Katare, M. Siragusa, L. S. Barcelos, P. Campagnolo, G. Mangialardi, O. Fortunato, G. Spinetti, N. Tran, K. Zacharowski, et al. Role of Kinin B2 Receptor Signaling in the Recruitment of Circulating Progenitor Cells With Neovascularization Potential Circ. Res., November 21, 2008; 103(11): 1335 - 1343. [Abstract] [Full Text] [PDF]

				V. Schachinger, A. Aicher, N. Dobert, R. Rover, J. Diener, S. Fichtlscherer, B. Assmus, F. H. Seeger, C. Menzel, W. Brenner, et al. Pilot Trial on Determinants of Progenitor Cell Recruitment to the Infarcted Human Myocardium Circulation, September 30, 2008; 118(14): 1425 - 1432. [Abstract] [Full Text] [PDF]

				J. J. Ohab and S. T. Carmichael Poststroke Neurogenesis: Emerging Principles of Migration and Localization of Immature Neurons Neuroscientist, August 1, 2008; 14(4): 369 - 380. [Abstract] [PDF]

				S. Dimmeler and A. Leri Aging and Disease as Modifiers of Efficacy of Cell Therapy Circ. Res., June 6, 2008; 102(11): 1319 - 1330. [Abstract] [Full Text] [PDF]

				S. Dimmeler Novel player in cell recruitment Blood, December 1, 2007; 110(12): 3821 - 3822. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 27/6/1426    most recent ATVBAHA.107.139642v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Weel, V. Articles by Quax, P. H.A. Search for Related Content PubMed PubMed Citation Articles by van Weel, V. Articles by Quax, P. H.A.