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【摘要】
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 , 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.
【关键词】 peripheral artery disease angiogenesis ischemia VEGF SDF
Introduction
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
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.
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 (LN 2 ) 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
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 1 B). 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 1 D), 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/mm 2, respectively; P =0.01; n=10; Figure 1 E). In addition, area per capillary was increased in ischemic interosseus muscle as compared with at the amputation level (102.2±5.0 µm 2 as compared with 81.2±6.4 µm 2, respectively; P =0.03; n=10; Figure 1 F).
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 x 150. E and F, Quantification of capillary density and area in muscle sections at the level of amputation, calf or foot, depicted as capillaries/mm 2 and µm 2, 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 2 A). Especially in muscle with acute-on-chronic ischemia, VEGF-A–expressing inflammatory cells were visible between myofibers ( Figure 2 B). In chronically ischemic muscle, atrophic myofibers, regenerating polynuclear myofibers, and satellite cells all strongly stained for VEGF-A ( Figure 2 C). There was no expression of VEGF-C in any muscle sample ( Figure 2 D), 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 2 E). VEGF receptor-1, -2, and -3 were expressed in nearly all microvessels adjacent to myofibers ( Figure 2F to 2 H).
Figure 2. A, VEGF-A staining of ischemic interosseus muscle (patient 10). Magnification x 150. 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 x 150. 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 x 200. Staining of ischemic muscle for VEGF-C (D, magnification x 150), VEGF-D (E, magnification x 150), VEGF-receptor 1 (F, magnification x 200), VEGF-receptor 2 (G, magnification x 200), and VEGF-receptor 3 (H, magnification x 200).
SDF-1 antigen was present both in capillaries adjacent to myofibers ( Figure 3 A) and in larger vessels ( Figure 3B and 3 C). SDF-1 was located at endothelial cells, as indicated by co-localization with CD31 and CD34 endothelial staining ( Figure 3D and 3 E), as well as at subendothelial pericytes. CXCR4 was mainly expressed in proximity to capillaries between myofibers ( Figure 3 F). 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 3 G). Importantly, CXCR4 expression was not observed in endothelial cells, indicating that there was no incorporation of CXCR4-positive cells in endothelium ( Figure 3 G, arrowheads). No staining was observed in negative control incubations for all antibodies used (supplemental Figure I, available online at http://atvb.ahajournals.org).
Figure 3. Immunohistochemical stainings of ischemic interosseus muscle sample derived from patient 6. Staining for SDF-1 (A, magnification x 150), staining of sequential sections of large vessels for SDF-1 (B, magnification x 125, and C, higher magnification of the boxed area in B, magnification x 400), for CD31 (D, magnification x 125), and for CD34 (E, magnification x 125). Staining for CXCR4 of muscle samples derived from patient 14. Ischemic interosseus muscle tissue (F, magnification x 150), large vessel in gastrocnemius muscle tissue (G, magnification x 200). 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.
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
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-1 17 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.
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作者单位：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,